Novel Approaches to Vaccine Research

Novel Approaches to Vaccine Research

Editor

Kathleen L. Hefferon Cornell Research Foundation, Cornell University Ithaca, NY 14850, USA

Research Signpost, T.C. 37/661 (2), Fort P.O., Trivandrum-695 023 Kerala, India

Published by Research Signpost 2011; Rights Reserved Research Signpost T.C. 37/661(2), Fort P.O., Trivandrum-695 023, Kerala, India Editor Kathleen L. Hefferon Managing Editor S.G. Pandalai Publication Manager A. Gayathri Research Signpost and the Editor assume no responsibility for the opinions and statements advanced by contributors ISBN: 978-81-308-0449-1

Preface The quest for improved vaccines and novel delivery systems continues to be of paramount importance in global health. This book details the most recent research in these collective fields. In Novel Approaches to Vaccine Research, topics ranging from innovative approaches to eradicate polio to the future of smallpox vaccines are discussed. Novel tactics to prevent transmission of mucosal pathogens such as HPV and HIV are presented in detail. DNA vaccines tailored to combat emerging infectious diseases and cancers are described, as are improved vaccination strategies such as heterologous prime boost immunization. Transdermal delivery of vaccines and the use of adjuvants in vaccine delivery systems are reviewed in this book. Finally, the use of novel vaccine delivery systems including nanoparticle, plant and bacteriophage-based platforms for vaccine development are covered. The summation of these reviews, written by a collection of internationally renowned experts in their respective fields, provides a solid and valuable foundation for the field of vaccine research in general, and points toward future avenues of vaccine development for years to come.

Kathleen L. Hefferon

Contents

Chapter 1 Rapid response immunization with DNA vaccines against emerging infectious diseases and cancers Ronald B Moss, Cristina Bergamaschi and George Pavlakis

1

Chapter 2 Prophylactic human papillomavirus (HPV) vaccines Diane M Harper and Stephen L. Vierthaler

39

Chapter 3 Vaccination strategies for the eradication of polio Yash Paul

71

Chapter 4 The future of smallpox vaccines Andrew W. Artenstein

91

Chapter 5 Heterologous prime-boost immunization: HIV-specific systemic/mucosal immunity, cytokine milieu and CD8+ T cell avidity Charani Ranasinghe and Shubhanshi Trivedi

111

Chapter 6 Vaccine strategies to prevent mucosal transmission of HIV-1 Yongjun Sui and Jay A. Berzofsky

137

Chapter 7 Transdermal delivery of vaccines Sarika Namjoshi and Heather A.E. Benson

181

Chapter 8 Adjuvants and vaccine delivery systems Valerie A Ferro

199

Chapter 9 Nanoparticle-based vaccines Jean-Pierre Y. Scheerlinck

223

Chapter 10 Molecular pharming for plant-derived vaccines Kathleen Hefferon

243

Chapter 11 Bacteriophage-based platforms for vaccine development David S. Peabody and Bryce Chackerian

257

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 1-37 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

1. Rapid response immunization with DNA vaccines against emerging infectious diseases and cancers 1

Ronald B Moss1, Cristina Bergamaschi2 and George Pavlakis2

Executive Vice President, Clinical Development & Medical Affairs NexBio, Inc., 10665 Sorrento Valley Road, San Diego, CA 92121, USA; 2Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute at Frederick, Frederick, MD, USA

Abstract. Recent advances using plasmid DNA based vaccination against emerging infectious diseases and cancers suggest that this technology has substantial advantages over conventional vaccine approaches. This review will cover the latest progress in DNA vaccines with a focus on the potential utility of this approach for public health threats such as pandemic Influenza, HIV, and Cancer.

Plasmid DNA vaccines, as reviewed in this chapter, are an important rapid response approach to effectively stimulate both anti-pathogen and anti-tumor immune responses that can prevent and limit disease.

Background Almost 100 years after an influenza pandemic killed an estimated 50 – 100 million people worldwide, a new swine influenza strain (H1N1) appeared in 2009 and spread rapidly over time to over 74 countries as the World Health Organization (WHO) changed its alert system to full pandemic phase 6 (1). This Correspondence/Reprint request: Dr. Ronald B Moss, Executive Vice President, Clinical Development & Medical Affairs NexBio, Inc., 10665 Sorrento Valley Road, San Diego, CA 92121, USA E-mail: shotdoc92130@yahoo.com

2

Ronald B Moss et al.

pandemic has challenged public health officials worldwide with the difficult task of rapidly manufacturing a vaccine to a new strain of influenza. Such a public health threat exemplifies an important role for the use of plasmid DNA vaccines which may have some distinct advantages over conventional vaccines.

DNA vaccines represent a unique approach to immunization, one that utilizes modern molecular gene therapy to immunize and result in the in-vivo production of antigens. A host immune response is then generated similar to the human immune response to the antigen itself. However, by only requiring the genetic sequence, it is the most rapid method of developing and manufacturing vaccines. This review will focus on the origins of DNA vaccines and areas in public health where such an approach has distinct advantages to conventional vaccines approaches for both emerging infectious diseases and cancers.

Vaccines have played a critical role in curtailing deadly infectious diseases throughout history. For example, a recent study of the impact of preventative vaccines suggests that ever since the introduction of vaccines, the incidence of infectious diseases such as diphtheria, mumps, pertussis, tetanus, hepatitis A and B, Haemophilus influenza and varicella zoster has declined by more than 80% in the U.S (2). Furthermore, after the introduction of vaccines, large scale transmission of polio, measles, and rubella has been eliminated in the U.S and smallpox has been eradicated. Large scale trials of vaccines including the Salk polio vaccine field trial, resulted in dramatic decreases in the incidence of polio itself (3,4). Therapeutic vaccine approaches are also beginning to show promise in the control of cancers as well. DNA based tumor vaccines that target specific tumor antigens, for example, are approaching late stage clinical development and may complement conventional chemotherapy approaches.

Emerging infectious pathogens such as pandemic 2009 H1N1, Tuberculosis, HIV (human immunodeficiency virus), SARS coronavirus (severe acute respiratory syndrome virus), and avian influenza (H5N1) have all adapted evolutionary strategies to rapidly change their genetic compositions. Similarly, tumors have adapted to chemotherapies resulting in both resistance and growth in inaccessible sanctuaries.

Plasmid DNA vaccines, as reviewed in this chapter, may be an important option to effectively stimulate both anti-pathogen and anti-tumor immune responses in order to prevent and limit disease. This chapter will focus on basic aspects of DNA vaccines with a more detailed analysis of the use in the development of an effective AIDS and Cancer vaccine.

Historical aspects of DNA vaccines Almost 20 years ago, Felgner and collaborators at the University of Wisconsin, demonstrated that closed loops of double-stranded DNA (plasmids) which were injected into muscle tissue could be taken up by cells at the

DNA vaccination infectious disease cancer

3

administration site (transfection) resulting in the production (expression) of proteins not normally made by the host cell (5).

Shortly after these seminal observations, many including those led by Liu and colleagues at Merck Research Laboratories (6), Weiner and colleagues at University of Pennsylvania (7), Johnston and colleagues at University of Texas (8), Robinson and colleagues at University of Massachusetts (9), and Hoffman and colleagues at Naval Medical Research Center (10), demonstrated that immunization with DNA could result in the production of foreign proteins or antigens that stimulates immunity against the pathogen.

Recent clinical trials using DNA vaccines have been successfully conducted against various infectious pathogens including the malaria parasite, dengue viruses, cytomegalovirus (CMV), Ebola virus, seasonal influenza viruses, avian or pandemic influenza viruses, West Nile virus (WMV), SARS coronavirus, hepatitis B virus, and Human Immunodeficiency virus (HIV). In addition, because of the need for only the sequence of a gene, DNA vaccines are also being developed for vaccination against tumor antigens to treat and prevent cancer.

Proposed mechanism of DNA vaccines One of the greatest advantages of DNA vaccine is the ability to induce both humoral and cellular immune responses. After immunization with a DNA plasmid, transfected muscle cells may produce antigen or foreign proteins that then directly stimulate B cells of the immune system, which in turn produce antibodies as shown in figure 1 (11). Transfected muscle cells could possibly transfer the antigen to antigen presenting cells (as demonstrated by cross priming) which then transport the proteins via distinct pathways (the MHC I for CD8+T cells or MHC II for CD4+T cells) that result in the display of different processed fragments of antigens. Direct transfection of antigen presenting cells (such as dendritic cells) with subsequent processing and display of MHC-antigen complexes may also occur. The immune response generated by DNA vaccination is thought to be similar to the type induced by the pathogens themselves. Indeed, DNA vaccination generates antigens in their native form and with similar structure and function to antigens generated after natural infection.

DNA designer genes A plasmid used in DNA vaccination contains a gene encoding an antigen of the target pathogen or a tumor-associated antigen (immunogen gene).

Expression of the protein antigen is initiated in the host cell by a promoter, and

4

Ronald B Moss et al.

Figure 1. Proposed immune mechanism of DNA vaccines.

and “turned off� by a terminator (A polyadenylation signal sequence (polyA). Other genes such as the bacterial origin of replication sequence and an antibiotic resistance gene are incorporated for manufacturing purposes. The resulting plasmid is a stable, self-contained unit that can be manufactured by consistent and scalable bacterial fermentation and purification processes.

Manufacturing DNA vaccines Making DNA starts with E. coli cells which are transformed with the plasmid of interest. These cells are grown and stored frozen in a stock of vials called a Master Cell Bank. Growth of the E. coli is typically done via a fermentation process similar to that used in the manufacturing of certain alcoholic beverages. The recovery process then requires lysis of the cells, in order to release the plasmid retained within the E. coli cells. DNA is then purified using various chromatographic methods.

Vaccine production time Perhaps most relevant to emerging infectious diseases and cancers, DNA vaccines have the distinct advantage of a more rapid development time, are non- infectious, and have a well-defined manufacturing process. As the

DNA vaccination infectious disease cancer

5

antigens are in constant flux, the rapid manufacturing is important for lethal infections or aggressive tumors where timing of vaccination is critical. DNA vaccines contain no infectious components and can be produced safely without the handling of hazardous infectious agents. Furthermore, there is a well-defined analytical process for the manufacturing of all DNA vaccines, which is universally applicable to any DNA vaccine.

Safety Early in the development, there were numerous theoretical safety concerns regarding DNA vaccines. These concerns have dissipated over the years based on thousands of human subjects who have undergone DNA vaccination or plasmid-based gene therapy.

In pre-clinical safety studies in animals, the potential for DNA integration into the host genome has been shown to be negligible and several orders of magnitude below the spontaneous mutation rate that occurs naturally in mammalian genes and thus this is no longer consider a safety issue for DNA vaccines (12). DNA vaccines have not been observed to induce autoimmunity or anti-DNA antibodies in large scale clinical trials (13), providing the possibility of repeated successful administrations.

Numerous clinical trials of DNA vaccines have been carefully monitored to detect clinical, hematologic or biochemical abnormalities and no major adverse events have not been reported to date. Transient mild–moderate reactions at the injection site are commonly reported including pain, swelling and redness (14).

In summary, the potential risks of DNA vaccines appear to be minimal based on safety data from human clinical trials in thousands of subjects to date.

Advances in augmenting immune responses Over the years, much progress has been made in optimizing DNA vaccine immunogenicity. Recent progress has targeted many different aspects of DNA vaccination which has successfully resulted in the generation of stronger immune responses.

Naked DNA vaccines One of the earliest trials in humans of an unformulated or “naked� DNA vaccine was one that targeted the malaria parasite. Wang and colleagues at the Naval Medical Research Institute immunized 20 subjects with a plasmid that encoded naturally occurring forms of malaria proteins (15). In this trial, more

6

Ronald B Moss et al.

than half of the subjects were shown to have cells that can kill or lyse malaria infected cells (cytotoxic T cell or killer cells). In a more recent clinical trial, a DNA vaccine optimized for human expression and encoding modified forms of West Nile Virus proteins was studied by Martin and colleagues at the National Institute of Health (NIH) and demonstrated that the vaccine stimulated antibodies that inhibited the virus (neutralizing antibodies) in all individuals receiving the vaccination regimen (16). This unformulated DNA vaccine appears to induce a similar level of immune responses to those observed in vaccinated horses protected from WNV. In addition, a recent clinical trial by these same NIH researchers tested an optimized but unformulated DNA vaccine for SARS coronavirus and demonstrated neutralizing antibodies in all subjects who received three doses of the vaccine (17).

Modes of delivery The mode of delivery may also be a pertinent factor in eliciting the proper immune response after DNA immunization.

Needle and syringe is the predominant method to deliver both DNA vaccines as well as conventional vaccines. However, Roy and colleagues at PowderMed have used DNA precipitated onto gold particles which are driven into to skin with a blast of pressurized gas, and called this approach “particlemediated epidermal delivery� (PMED). In one clinical study by Roy and collaborators using PMED, individuals exhibited potent antibody responses to a hepatitis B DNA vaccine (18). In another study, Drape and colleagues at PowderMed also observed strong antibody responses to the influenza virus DNA vaccine with the PMED approach (19).

Another approach for the delivery of DNA vaccines is the use of needlefree devices. Needle-free injection of DNA vaccines has been utilized in numerous clinical studies and appears to be well-tolerated and may have some advantages of further augmenting the immune response to DNA vaccination. Nabel and colleagues at the NIH completed a recent study of an Ebola DNA vaccine also using this same needle-free device. They demonstrated that Ebola-specific antibody and CD4+ T-cell immune responses were elicited in all individuals who received the three-dose vaccination regimen (20).

Another novel mode of enhancing DNA vaccines has been a more invasive technique called electroporation. This method involves administration of brief electrical pulses of various voltages, after injection of a DNA vaccine, in order to enhance the uptake of DNA, presumably through the formation of transient pores in the muscle cell membrane. More

DNA vaccination infectious disease cancer

7

discussion on electroporation can be found in the section on DNA vaccines and AIDS. Some researchers have used plasmid DNA in what has been called a heterologous “prime-boost� vaccination approach. This method involves delivery of one or more plasmid DNA vaccine priming doses followed by a boost with a viral vector (such as adenovirus) which codes for the same antigens. In the prime-boost setting, DNA vaccination plays an important role in priming different types of T-cells (CD4+ and CD8+ T-cells) specific for various proteins. In a prime-boost study, Jacobson and colleagues, at the University of California at San Francisco, immunized subjects with a DNA vaccine for CMV who were then boosted with a live-attenuated CMV virus (Towne strain). Faster and stronger virus-specific T-cell responses were observed in the group of subjects that received the DNA and Towne strain compared with a group of subjects that received the Towne strain alone (21).

Adjuvanted DNA vaccines An area of potentially paramount importance for DNA vaccines is the addition of a formulations or adjuvant to further stimulate immunity.

Adjuvants are common to some licensed vaccines (Alum) and are included to potentiate the immune responses elicited by vaccination. For DNA vaccines, various delivery systems and adjuvants have been tested. One of the earliest promising adjuvants for plasmid DNA vaccines was poly-lactide coglycolide (PLG), cationic microparticles. Ulmer and colleagues at Chiron Corporation evaluated HIV DNA vaccines formulated with PLG microparticles and found strong antibody and T-cell responses in macaques (22). Poloxamers represent another class of adjuvants tested with DNA vaccines. Some poloxamers are nonionic block copolymers, and when combined with a cationic surfactant, bond with DNA to form small particles. A study by Wloch and colleagues examined DNA immunizations of human volunteers with CMV plasmids that encode for gB and pp65 genes, formulated with a specific poloxamer adjuvant. In that study CMV-specific T-cell responses were detected in a majority of CMV sero-negative individuals who were vaccinated (23). More recently studies of this same CMV vaccine in bone marrow transplant patients has demonstrated a decrease in viral replication in subjects vaccinated compared to a placebo control group (24). Because DNA vaccination occurred in an immune suppressed population, these results are particularly notable for the unique effects of a DNA vaccine. In such a difficult population for vaccination, the appropriate magnitude and breadth of

8

Ronald B Moss et al.

immune responses appears to have been generated and resulted in significant clinical impact.

Vaxfectin® is a cationic lipid- based adjuvant that bears a positive charge that binds electrostatically to negatively charged DNA. Studies in animals also demonstrated that Vaxfectin®-adjuvanted DNA vaccines can be protective against lethal viral challenges. For example, Webby and colleagues at St. Jude Children’s Research Hospital, immunized ferrets with three plasmids containing DNA components of the H5N1 pandemic influenza virus formulated with Vaxfectin® (25). After one or two immunizations, all animals were completely protected from lethal pandemic influenza virus challenge, while unvaccinated control animals died. Similarly, Griffin and colleagues at Johns Hopkins immunized juvenile and infant rhesus macaques by intramuscular and intradermal routes with measles antigen encoding plasmids formulated with Vaxfectin® (26). All of the vaccinated monkeys developed strong and durable neutralizing antibodies and they were challenged with high doses of measles virus after one year. All of the unvaccinated control animals developed viremia and became ill with rashes in contrast to the vaccinated animals which remained healthy and had no detectable virus levels. Lastly, the first human clinical trial of a DNA vaccine formulated with Vaxfectin® has been completed with plasmids encoding pandemic influenza virus antigens (H5N1). The preliminary results reported from this trial by Smith and colleagues at Vical Incorporated suggest that vaccination with H5 DNA plasmids formulated with this adjuvant were well-tolerated and stimulated strong H5 antibody responses in up to 67% of subjects (27). In addition, T cell responses were generated in a majority of subjects to the H5 antigen. The antibody response rate and safety profile observed in this trial are comparable to protein-based vaccines for pandemic influenza.

DNA vaccines for AIDS Although the initial results of DNA vaccination against HIV-1 and its analogous primate virus, Simian Immunodeficiency Virus (SIV) were encouraging, it was soon realized that the levels of immune responses achieved were low, even in mice. Further research uncovered the reason for this problem, i.e. inefficient expression of HIV and SIV proteins from standard expression vectors. HIV proteins are produced by unstable mRNAs and require the presence of a specialized virus RNA binding factor, Rev, for their export from the nucleus and for stabilization (28-31). In the absence of Rev, the expression of gag and env is very low. Even after addition of Rev protein, expression was not very high in several animal systems, especially mice, because Rev works very inefficiently in mouse cells. Therefore, an

DNA vaccination infectious disease cancer

9

alternative methodology for generating stable mRNAs was employed. The HIV coding regions of gag, pol and env were mutated to change multiple codons in a way that did not alter the expressed protein (32-36). This was possible by using synonymous codon substitutions. The result of these manipulations was a different mRNA encoding the same protein. This process was shown to improve expression of many proteins produced by unstable mRNAs, and has been called RNA optimization or codon optimization. Since many types of instability signals embedded into the wild type mRNA were eliminated, the mutagenized mRNA was able to be exported and translated using the default cellular export pathway. Elimination of mRNA embedded negative signals may happen by many combinations of codon changes, and it seems to reach optimal when mRNAs have a reasonably high GC content. The results of RNA/codon optimization were high expression of HIV proteins independent of the Rev protein, and also independent of the cells it was delivered. This allowed high expression of HIV plasmids in mice and more consistent results in DNA vaccination experiments (37-38).

Improvement of expression also led to improved viral vectors for HIV protein expression, because viruses that use the nuclear cell machinery for their expression (ie, adenoviruses, herpesviruses) also had the same restrictions for HIV protein expression, and benefited greatly by the use of “optimized� expression cassettes (37). These results led to almost universal adaptation of RNA/codon optimization in DNA vaccine projects and also in many gene therapy protocols, where foreign DNA expression is required.

DNA vaccines for AIDS: Induction of mucosal immune responses Since HIV-1 is acquired sexually, it is important that vaccines are able to protect the virus port of entry at the mucosal sites. DNA has not been considered a good inducer of mucosal immune response. DNA vaccination using specific mucosal vaccination protocols and prime/boost protocols has been shown to produce immune responses at mucosal sites. DNA vaccination was reported to induce systemic and mucosal immune responses in rhesus macaques when co-administered intradermally and intrarectally (40- 42). Upon intramuscular DNA administration in chimpanzees (43), the induction of HIV-1 Gag-specific IgA was reported in breast milk but not in mucosa. Vaccination with DNA alone via the intramuscular route in the absence of systemic or mucosal recombinant virus or protein boost did not result in detectable mucosal immune responses (44-47). In recent work, we have shown that efficient

10

Ronald B Moss et al.

intramuscular DNA vaccination against SIV by electroporation leads to the induction of cellular and humoral mucosal responses. Therefore, efficient DNA vaccination may be an excellent method to provide mucosal protection through a DNA vaccine.

DNA vaccines for AIDS: Improvements in DNA delivery Intramuscular DNA vaccination by needle and syringe in the absence of a heterologous boost has resulted in relatively low levels of systemic immune responses in primates and especially humans (48- 55). Despite the low immune responses, protection resulting in reduced viremia has been reported in DNA vaccinated macaques challenged with SIV or SHIV (55-61).

DNA Vaccines for AIDS: In vivo electroporation Substantial improvement in gene expression and immunogenicity of DNA vaccines has been achieved by in vivo electroporation (62-72), especially in macaques (68-73). Generation of high levels of immune responses in macaques after DNA electroporation was associated with protection against high dose challenge with pathogenic SIVmac251. Improved control of viremia both in the acute and chronic phase was demonstrated in groups of animals vaccinated using in vivo electroporation as DNA delivery method compared to the previously used needle/syringe DNA delivery (55, 73).

Therefore, a combination of improved expression and delivery has led to great improvements in immunogenicity and effectiveness of DNA vaccines against HIV/SIV in model systems.

DNA vaccines for AIDS: Human experiments The first attempts of DNA vaccination against AIDS in humans reported occasional or low immune responses (74, 75). Subsequent trials of a vaccine including gag, pol, nef and 3 env genes (49) with needle-free intramuscular injection showed a maximal response to Env after three vaccinations. Gag immunogenicity was poor. DNA also induced antibody against the eEnv. A second study in Uganda showed that 47% of vaccinees had CTL activity to HIV-1 Env B. Additionally, lymphoproliferative responses were observed in 14/15 vaccinees against p24 (76). These results are in general agreement with a compilation of Merck vaccine trials on gag DNA vaccine given twice (77).

This gag DNA/DNA regimen showed immunogenicity in 33% of recipients,

DNA vaccination infectious disease cancer

11

whereas a DNA/Ad5 regiment was positive in 55%. DNA/DNA elicited mainly a CD4 response, whereas Ad5/Ad5 elicited mainly a CD8 response.

Following several trials in macaques that showed increased immunogenicity by DNA electroporation, a recent study in humans also suggests that DNA electroporation may increase the immunogenicity of the DNA vaccine, although the overall immunogenicity of this DNA vaccine was low (17-33% responders by ELISPOT and no seroconversion (78)).

From these data, it is concluded that DNA immunization against AIDS requires more efficient vectors and procedures than recently applied, or use of DNA as a component of a more complex vaccine. DNA immunization remains attractive due to (a) its simplicity and flexibility, (b) the fact that it induces strong and broad cellular immune responses and (c) that it can be applied multiple times to boost the responses. In addition, DNA can be combined with many other vaccine modalities. The fact that DNA immunization appears to provide broad and varied responses to many antigens continues to prompt additional clinical trials.

Cancer DNA vaccines Cancer remains a leading cause of mortality worldwide, despite improvements in screening technologies and conventional therapies. Passive and active (vaccination) immunotherapy is a promising alternative strategy for cancer treatment. The success of antibody treatment and of adoptive cell transfer therapy in certain types of tumors has demonstrated the clinical effectiveness of passive immunotherapy (79, 80). A successful active vaccination will consist in the induction of tumor-specific effector cells able to target and kill cancer cells with a concurrent establishment of immunological memory to prevent recurrences.

Two major obstacles for cancer vaccination are the lack of strongly immunogenic tumor specific antigens and the presence of immunosupressive tumor microenvironment. Even if low level of spontaneous immunity occurs, peripheral tolerance can be induced by the presence of Tregs and myeloid-derived suppressor cells, abundant in tumor microenvironment (81, 82). Tumor itself can contribute to the creation of tolerogenic milieu through the downregulation of MHC-I molecules and the secretion of immunosuppressive molecules (83-85). Only a strategy with the ability both to break self-tolerance inducing high avidity and long lasting tumor immune responses and to counteract immune suppression will result in an effective cancer vaccine.

12

Ronald B Moss et al.

Cancer DNA vaccines: Tumor antigens Some tumors such as melanoma are characterized by the natural occurrence of tumor specific immune responses. The analysis of such cells represented pioneering work in the identification of tumor antigens (86). The introduction and constant improvement of genetic technology has further expanded the number of candidate tumor antigens, providing new targets for potential vaccine against different type of human cancers (87).

Tumor antigens can be classified on the basis of their source and structure. Among them, very few are tumor specific antigens. This group includes the idiotypic antigens of certain type of lymphoma (88) and mutated forms of onco-proteins, such as p53 (89) or ras (90). However, the majority of cancer vaccine studies have been performed using tumor-associated antigens (TAAs), found on both tumor and normal cells. In contrast to DNA vaccine against infectious diseases, the need to induce immunity against antigens shared by tumor and normal cells and, therefore, to break selftolerance represents the major challenge in the development of effective DNA cancer vaccines. Targeting TAAs with cancer vaccine has the potential adverse effect to induce immune responses against normal tissue leading to autoimmunity disorders.

TAAs can be cell-type specific differentiation antigens or oncofetal antigens. Example of lineage specific TAAs are: the melanoma antigens glycoprotein 100 (gp100), melanoma antigen recognized by T cells-1 (MART-1), tyrosinase (TYRP-1 and TYRP-2) that are expressed by normal melanocytes, pigmented cells of the retina as well as by melanoma cells (9196); or NY-ESO-1 found on several tumor types and in normal testis (97). An example of onco-fetal antigen is carcinoembryonic antigen (CEA) (98), associated with developing fetus and certain type of tumors such as colon cancer. TAAs can also be the results of modification in the structure of selfproteins (different glycosylation pattern for MUC-1 (99) or in the level of expression of self-proteins (HER2/neu) (100) during the transformation process in breast cancer.

Cancer DNA vaccines: Modification of tumor antigens included in DNA vaccine The versatility of DNA provides the advantage to alter the DNA-encoded antigens in order to improve the quality and quantity of the vaccine inducedimmune responses. Several modification have been reported to improve the otherwise poor immunogenicity of TAAs, including modification to attract

DNA vaccination infectious disease cancer

13

APCs and favor a more efficient uptake of the antigen by professional APCs, modification to exploit antigen processing and presentation, inclusion of heterologous immunogenic sequences and use of xenoantigens.

The presence of APCs at the site of antigen expression has been proposed to be critical for the induction of effective immune responses by DNA vaccine. Therefore, several groups have generated and tested cancer DNA vaccine encoding tumor antigens fused with molecules able to attract and activate APCs and to enhance antigen uptake by APCs. Among these, use of chemokines has revealed to be particularly effective in converting weak tumor antigens to potent immunogens. In one study, Biragyn et al. developed a DNA vaccine encoding the lymphoma specific idiotypic Id immunoglobulin (sFv, single chain molecules containing VH and VL domains linked to retain the native conformation) fused to monocyte chemotactic protein 3 (MCP-3). MCP-3 is a potent chemoattractant for monocytes and dendritic cells that express MCP-3 ligands, CCR1, CCR2 and CCR3 chemokine receptors. Immunization with DNA encoding MCP-3-sFv fusion targeted APCs for efficient receptor-mediated uptake and processing of sFv and resulted in protection against challenge with A20 tumor cells, through the induction of anti-tumor specific effector T-cells (for consistency) (101). In a different model, the immunogenicity of immature laminin receptor protein (OFA-iLRP, a non immunogenic embryonic antigen) was greatly enhanced by fusion with CCR6 ligands MIP3ι and β-defensin mDF2β. The CCR6 targeting promoted efficient antigen cross-presentation and induction of strong, long lasting tumor specific CD8+ T-cells and showed effectiveness in both prophylactic and therapeutic setting (102). DNA vaccine can also incorporate other molecules promoting the recruitment and activation of APCs, such as GM-CSF (103-104), CD40 ligand (105), the extracellular domain of flt-3 ligand (106) and CTLA-4 (107).

An optimal anticancer vaccine requires the cooperation of T CD4+ T helper cells and CD8+ CTL cells. Various strategies have been developed to manipulate the natural pathway of antigen processing and presentation, rerouting antigen to different intracellular sites, such as the lysosomalendosomal or proteasome compartment. Tumor antigens can be modified to promote their degradation through lysosomal-endosomal pathway, resulting in enhancement of MHC-II class presentation and priming of CD4+ T-cells.

An example is represented by the antigen fusion to the sequence encoding the lysosome-associated membrane protein-1 (LAMP-1) (70,73,108-109).

Immunization of mice with a vaccine encoding the chimeric human papilloma virus (HPV)-16 E7-LAMP-1 resulted in greater E7 specific antibodies titers and CTL responses in comparison to the vaccine containing

14

Ronald B Moss et al.

the unmodified E7 sequence (110). The enhanced immunogenicity also reflected in an increase in the therapeutic potency of the vaccine, since mice vaccinated with chimeric E7-LAMP-1 were able to reject established TC-1 tumors, whereas the vaccine encoding the unmodified E7 antigen was ineffective in reducing tumor burden (111). Efficient presentation in the context of MHC-II class can be also achieved by tumor antigen fusion to other targeting motifs of an endosomal or lysosomal protein, such as MAGE-A3 fusion to invariant chain or DC-LAMP (112).

Similarly, antigen presentation in association with MHC-I class can be promoted by fusion with sequences promoting proteasomal degradation of antigen, such as ubiquitin. Xiang et al. reported that an oral DNA vaccine (using Salmonella typhimurium as carrier) encoding the ubiquitin gene fused to gp10025-33 and TRP-2181-188 peptide epitopes induced high level of tumor specific CD8+ T-cells and was effective in rejecting tumor, after B16 melanoma cells challenge (113). A DNA plasmid encoding TRP-2 antigen fused to ubiquitin proved to be effective also in therapeutic settings (114).

Alternatively, a peptide sequence derived from beta-catenin providing an ubiquitination signal can be fused to DNA-encoded antigens (55).

Easy manipulation of DNA vaccine offers the possibility to activate CD4+ T helper cells for meaningful and persistence anti tumor response (115-116). CD4+ T helper cells orchestrate immune responses, helping B cells to produce antibodies, sustaining CD8+ T responses or suppressing immune responses, acting as Tregs. A strategy to evoke CD4 help with DNA vaccination consists in the incorporation of heterologous immunogenic sequence, such as microbial proteins (fragment C of tetanus toxin (FrC) (117119) or Pseudomonas aeroginosa exotoxin A (120)). Fusion of tumor antigens to these sequences results in general activation of T helper cells from a large anti-microbial immune repertoire that can potentially break tolerogenic pressure and help immune responses against weak tumor antigens. This strategy has been evaluated for the ability to promote antibody and CD4+ T cells responses against the tumor-specific Id immunoglobulin in a pre-clinical model of lymphoma. DNA-sFv revealed to be poorly immunogenic. In contrast, fusion of the sFv to FrC dramatically enhances the level of anti-Id antibodies, conferring protection against lymphoma (118).

This DNA vaccine is currently tested in a clinical trial in patients with follicular lymphoma. Fusion of tumor antigens to tetanus toxin sequence can be also used for induction of CTL responses. However, heterologous immuno-enhancing sequence has to be modified in order to retain the ability of activating T helper cells without providing immuno-dominant peptides that can compete with the weak tumor-derived epitopes (117). A DNA vaccine

DNA vaccination infectious disease cancer

15

encoding the colon carcinoma antigen gp70 was poorly immunogenic, even if fused to the full length FrC. However, DNA vaccine encoding a single domain of FrC fused to HLA-I restricted epitope of gp70 resulted in great induction of CTL responses and protection against CT26 carcinoma challenge (119). An alternative strategy is to substitute CD4+ T help with the provision of exogenous IL-15 (see below).

The use of DNA vaccine expressing tumor antigens derived from a different species (orthologous antigens) is an additional effective way of activating the immune system against the tumor. This approach called xenogenic vaccination has been proved to be effective in breaking tolerance and in inducing T cells or antibodies that can cross-react with the autologous tumor antigens. In the murine melanoma model, xenogenic immunization with naked DNA encoding human TRP-1 but not mouse TRP-1 induced TRP-1 specific humoral responses, areas of degipmentation of the skin and conferred protection against intravenous B16 tumor challenge (121). A similar effect was also observed after vaccination with DNA encoding human gp100 in mice (122-125). It has also been suggested that xenogenic vaccination with human gp100 in mice creates an altered epitope with higher affinity for MHC molecules, resulting in an enhanced immune outcome (123). Xenogenic vaccination may also generate non-specific help, such as T helper activation, DC activation or cytokine production. Based on this approach, a DNA vaccine encoding human tyrosinase has been approved by U.S. Department of Agriculture for the treatment of phase II and III oral melanoma in dogs (see below).

Cancer DNA vaccines: Modification of tumor microenvironment Cytokine administration in form of recombinant proteins or DNAs has been intensively used in immunotherapeutic approaches against cancer.

Cytokines can also be used as adjuvants to improve the immunogenicity of DNA vaccines. IL-12, IL-18, GM-CSF and IL-15 have all been tested in several cancer DNA vaccination models, showing the ability to shape the nature and increase the magnitude of tumor specific immune responses.

IL-12 plays a pivotal role in the initiation and maintenance of Th1 responses and induction of cellular immunity and has been extensively used as adjuvant in vaccination protocol for infectious diseases (126-127).

Co-delivery of DNA expressing IL-12 has been proved to be effective in enhancing the potency of different cancer DNA vaccine in both prophylactic and therapeutic setting. Among these, there are DNA vaccine encoding CEA for lung carcinoma (128-129) and DNA vaccine expressing TRP-2 or gp100

16

Ronald B Moss et al.

for melanoma (130). In addition to the positive effect in the induction long lasting immunity, IL-12 has been shown to arrest tumor growth (131), to promote tumor regression of established tumors (132) and to inhibit tumor angiogenesis (133). The expanding knowledge on the function and regulation of other cytokines in IL-12 family (IL-23, IL-27, IL-35) suggested the alternative or combinatorial use of these cytokines as additional possibilities to modulate the immune system in favor of anti-tumor immunity. A recent study demonstrated that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing metastatic neuroblastoma tumors, through the induction of anti-tumoral CTL responses and the inhibition of tumor-resident Tregs and Th17 cells (134).

Particularly promising is the use of GM-CSF as adjuvant in cancer DNA vaccines, because of its ability to recruits and activate DCs and to promote better antigen uptake and presentation. It has been showed that a DNA vaccine expressing gp100 together with GM-CSF promotes T-cell dependent protection against tumor challenge (124). A DNA vaccine expressing MAGE-1 and GM-CSF resulted in an increase level of antigen specific IgG titers that favor rejection of B16-MAGE tumor cells (135).

Similar results were obtained testing DNA vaccine expressing prostateassociated antigen (PSA) and HER2/Neu in prostate and breast cancer, respectively (136-137). Cancer DNA vaccines expressing GM-CSF are currently under evaluation in patients with melanoma or hormone refractory prostate cancer (138-139).

IL-18 is a pro-inflammatory cytokine involve in the induction and regulation of Th1 immunity. A study using DNA vaccine expressing MUC-1 together with IL-18 showed to be effective in breaking tolerance to MUC-1, conferring protective and therapeutic benefits in an experimental pulmonary metastastic model (140). IL-18 co-delivered with PSA-encoding DNA vaccine supported the enhancement of tumor specific T cells responses and tumor rejection in a murine prostate cancer model (141, 142).

IL-15 plays an important role in both innate and adaptive immune responses and may prove a good candidate vaccine adjuvant. Provision of IL-15 at the time of priming of immune responses increases the magnitude, longevity and functional avidity of antigen-specific memory CD8+ T-cells.

Inclusion of IL-15 in tumor vaccines supported a robust and long-term antigen-specific CD8+ T-cells in vivo (143) which could effectively protect the mice against tumor challenge (144). In a recent study, the co-expression of IL-15/IL-15RÎą with the NEU antigen by a DC vaccine also enhanced the anti-tumor effect in a transgenic mouse breast cancer model, by promoting antibody production (145). The use of IL-15 in cancer vaccination may

DNA vaccination infectious disease cancer

17

provide additional advantages by substituting for T CD4 help (145, 146). In absence of T CD4 help, antigen-specific CD8+ T-cells cells are short-lived and exhibit defective secondary responses because of TRAIL-mediated apoptosis. Exogenous provision of IL-15 induces “helpless” CD8+ T-cells cells to respond to antigen restimulation, mimicking T CD4 help in preventing TRAIL-mediated apoptosis and promoting the expression of anti-apoptotic molecule Bcl-xl (146). Similarly to IL-2, administration of IL-15 also results in non-specific stimulation of effector cells without promotion of Treg activity and has shown antitumor effects in murine models of established melanoma, colon carcinoma and spontaneous pancreatic tumor, when provided as either single chain IL-15 or IL-15/IL-15sRα complex (147150). The direct anti-tumor effect of IL-15 is mediated by multiple mechanisms. IL-15 stimulates both the innate immune system through the activation of NK cells and the adaptive immune system breaking selftolerance and promoting the proliferation and degranulation of tumor resident CD8+ T-cells directed toward tumor antigens. The effect appears to be long lasting and it is not counteracted by the concomitant induction of inhibitory regulatory elements, as the ratio effector/Tregs cell increases in tumor microenviroment (151).

One of the greatest problems related to the use of cytokines as adjuvants is their poor expression due to posttranscriptional and posttranslational restrictions. DNA technology has the advantage of providing methods of increasing the stability and secretion of cytokines (152-154).

Co-delivery of DNA-encoding chemokines such as MIP-1α and RANTES has also been tested for their ability to promote migration and activation of APCs and to enhance antigen presentation (155). Costimulation molecules such as B7.1 and B7.2 represent an additional class of adjuvants with potential use in cancer DNA vaccination.

Several studies investigating the use of cytokines as vaccine adjuvants have suggested the importance of dose, route and timing of administration to maximize positive effects while minimizing toxic and adverse ones.

Cytokines, chemokines and costimulation molecules sequences can be incorporated into the same DNA vector expressing tumor antigen or encoded by a separate but co-delivered vector, providing the advantage of localized adjuvant effect at the site of activation of immune responses.

The importance of the activation of innate immunity arm in the context of cancer vaccination has been demonstrated by the improved immunogenicity followed by the used of optimized backbone vector. Plasmid DNA derived from bacteria contain unmethylatd DNA CpG motifs that stimulate innate immunity and initiate and control adaptive immunity. CpG binds to Toll-like Receptor (TLR)-9 expressed by professional APCs,

18

Ronald B Moss et al.

promoting maturation of APCs and secretion of proinflammatory cytokines (IL-1, IL-12, TNF, IFN) (156). In addition, CpG signaling can also promote the suppression of inhibitory elements, inhibiting IL-10-producing Tregs activity and reverting the Tregs-induced tolerance (157, 158). Several preclinical models demonstrated the effectiveness of CpG oligodeoxynucleotide (ODNs) alone as prophylactic and therapeutic treatment of cancer (159, 160). When incorporated into the backbone of a plasmid DNA vaccine, CpG sequences increased tumor specific immune responses and confer protection against tumor, as demonstrated in murine melanoma model (161), in murine prostate carcinoma model (162) and in transgenic mammary carcinoma model (163). Therefore, engineering plasmid backbone with optimal CpG sequences is useful strategy to provide a suitable environment for immune activation against cancer. The use of TLR-9 agonist is currently tested in clinical trials in numerous tumors.

Cancer DNA vaccines: Path from preclinical studies to human clinical trials The original studies testing DNA vaccines for cancer were designed as immunization of mice with DNA encoding for foreign antigen (such as betagalactosidase, OVA or human CD4) followed by challenge with tumor cells, artificially transfected to express the same foreign antigen (164-166). The protection from tumor growth observed in these studies represented a proof of principle supporting the potential application of DNA vaccines in cancer therapy. However, these studies did not address the biggest challenge in the development of an effective cancer vaccine: the ability to break tolerance.

Therefore, the following studies aimed to evaluate the efficacy of DNA vaccine in inducing tumor specific immune responses to the level required for rejection of tumor in vivo. Several studies in mice showed preventive and therapeutic success after vaccination with DNA expressing melanoma antigens, such as gp100 (167) and TRP-2 (168), colon carcinoma antigen CEA (169) and breast cancer antigen HER2/neu (170). Immunization with these DNAs resulted in the development of humoral and cellular responses against tumor antigens that either conferred protection against tumor challenge or favor regression of established tumor. The encouraging results in preclinical studies allowed the translation of DNA vaccination for cancer into clinical trials. However, no cancer DNA vaccine has been yet approved for treatment of cancer in human in the U.S. Several phase I/II clinical trials using cancer DNA vaccines have been conducted in patients with melanoma, colorectal carcinoma, prostate carcinoma, B-cell lymphoma. Clinical studies have proved the safety and low

DNA vaccination infectious disease cancer

19

toxicity of DNA vaccines delivered at different sites and with different technologies, after repeated administrations and at high dose (up to 4 mg per injection). However, cancer DNA vaccines have been poorly immunogenic in humans, and with no clear benefits in term of clinical outcome.

Rosenberg et al. reported that both intramuscular and intradermal immunization with DNA expressing human gp100 failed to induce tumor specific immune responses in 22 patients with advance metastatic melanoma (171). The efficacy of a similar DNA vaccine was also evaluated in 26 patients with advance melanoma after intranodal delivery. In this trial, DNA vaccination resulted in detectable antigen specific immune responses in almost 50% of the treated patients but did not resulted in significant clinical responses (172). The lack of significant immunological and clinical benefits characterized other phase I studies testing DNA expressing MART-1 (173, 174). In a B-cell lymphoma trial, the immunogenicity of an intramuscular delivered DNA encoding for the specific idiotypic immunoglobulin fused to the xenogeneic constant region of murine IgG2a was evaluated. After immunization, the majority of the patients generated immune responses to the murine immunuglobulin carrier but no anti-Id antibodies were measured.

Only after subsequent series of immunization testing alternative delivery method (biojector), alternative route of administration (intradermal) and the adjuvant effect of GM-CSF encoding DNA, specific anti-Id humoral and cellular immune response were detected in 50% of the patients (175). Higher immune responses to a foreign antigen in comparison to TAAs were also found in a study performed in colorectal carcinoma patients. After vaccination with a dual expression plasmid encoding hepatitis B surface antigen (HBsAg) and CEA, 80% of treated patients developed anti-HBsAg antibody but only 4 of 17 patients developed lymphoproliferative responses to CEA. No objective clinical benefits from DNA vaccination were observed in the study (176).

The results from clinical trials suggested the necessity to optimize design and delivery of cancer DNA vaccines to fully explore the potential of this technology for the prevention and treatment of human cancer. In fact, some trials based on innovative DNA design as well as improved delivery methods showed more encouraging results. In a Phase I trial in hormone-refractory prostate cancer patients, the higher dose of PSA-encoding DNA together with GM-CSF and IL-2 resulted in development of both PSA-specific IgG antibody and PSA-specific CTLs. The patients who developed anti-PSA immune responses also exhibit stabilization of disease, measured as slower increase in the serum level of PSA (136,139). In a phase I/IIa trial in prostate cancer patients, DNA vaccine encoding prostatic acid phosphatase (PAP)

20

Ronald B Moss et al.

co-administered intradermally with GM-CSF was well tolerated, induced antigen-specific T-cell responses and resulted in an slower disease progression, assessed by increased PSA doubling (177). The immunogenicity of a novel DNA vaccine encoding a domain (DOM) of fragment C of tetanus toxin fused to a tumor-derived epitope from prostate-specific membrane antigen (PSMA) was evaluated in patients with recurrent prostate cancer after deliver with electroporation. DNA immunization via electroporation was well tolerated and associated with the development of strong and long lasting antibodies responses (178).

Possible explanations for the discrepancy in the immunological and clinical outcome between preclinical and clinical studies are also the timing of immunization and immunocompetence of the DNA vaccine recipient.

Most of the successful preclinical studies in mice were conducted in either preventive setting or in early and not yet vascularized tumors. In contrast, the patient population enrolled in DNA vaccine clinical trials consists of patients with advance metastatic disease resistant to conventional treatments, such as surgery, chemotherapy, radiotherapy or hormonal therapy. These patients are often immunocompromised and their response to DNA vaccination may be intrinsically sub-optimal.

Approved DNA vaccines DNA vaccines are already approved for animal health. The first DNA vaccine approved for animal health was one that protected horses against WNV. WNV is a mosquito-borne virus, which causes encephalitis or inflammation in the brain of infected animals and humans. Prior to approval of a vaccine, approximately one-third of the horses infected with WNV would die or be euthanized. The WNV DNA vaccine, developed by Fort Dodge Laboratories, was approved by the U.S Department of Agriculture (USDA) in 2005 after the demonstration of safety and efficacy (179).

A DNA vaccine has also been approved to prevent a fatal viral disease that afflicts salmon, called infectious hematopoietic necrosis virus. In mid2001, an epidemic occurred in Atlantic salmon killing up to 90% of the fish.

Scientists at Aqua Health, a unit of Novartis in Canada, conducted a field trial by immunizing millions of salmons with a single dose of DNA vaccine encoding for a protein of the virus (180). The vaccine was approved in 2005 based on the results of this trial that demonstrated that the vaccine, called Apex-IHN, protected the fish from death without adverse effects.

Additionally, a therapeutic DNA vaccine designed to treat dogs with skin cancer (melanoma) was granted conditional approval in 2007. This vaccine

DNA vaccination infectious disease cancer

21

was developed through a collaboration of Memorial Sloan-Kettering Cancer Center (MSKCC) and Merial Ltd. Canine melanoma is an aggressive form of cancer. Dogs with melanomas that have gone beyond initial stages typically have a lifespan of one to five months with conventional therapies. In addition, the cancer is often resistant to chemotherapy. In a study of the DNA vaccine conducted by MSKCC, many dogs who received the vaccinations lived beyond the average 13 month survival (181). Based on the significantly extended survival, the USDA approved this DNA vaccine in 2010. This is the first therapeutic vaccine approved by the U.S. government for the treatment of cancer in animals or humans.

Summary and conclusions Vaccination remains an important component of a rapid response to potential pandemics such as H1N1 influenza. In addition, distinct advantages for DNA vaccines have been discussed including the quest for an effective AIDS and cancer vaccine. The manufacturing time of conventional protein- based vaccines may be excessive and involve a relatively slow production time.

DNA vaccines, in contrast, have estimated vaccine production times that can be months earlier, as only the DNA sequence is required and the manufacturing process is standard (Figure 2) (182). DNA vaccines therefore have the unique benefit of large scale production for human use in a relatively streamlined period of time. In the case of potentially fatal emerging pathogens or tumors, reducing the production time of an effective vaccine may be critical in preventing spread of infection and death.

F/F=fill finish

Figure 2. Manufacturing timelines for DNA vaccines compared to egg-based protein vaccines.

22

Ronald B Moss et al.

In summary, plasmid DNA vaccines have made substantial progress demonstrating safety and the development of immunity to both pathogens and cancers. Future progress using plasmid DNA vaccines should provide an additional approachs for rapid vaccination against evolving microbes as well as tumor antigens.

Acknowledgements We thank Dr. Will Soo Hoo for work on the figures.

References 1.

Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team (2009) Emergence of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans. N Engl J Med, 2009, N Engl J M 10.1056.

2. Roush SW, Murphy TV; Vaccine-Preventable Disease Table Working Group.

Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States. JAMA.2007;298(18):2155-63.

3. Burke, DS, Lessons learned from the 1954 Field Trial of Poliomyelitis Vaccine Clinical Trials 2004; 1: 3-5.

4. Dutta, A., Epidemiology of poliomyelitis—Options and update, Vaccine 26 (2008) 5767-5773.

5. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, Felgner PL. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949 Pt 1): 1465-8.

6. Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, FeIgner PL, Dwarki VJ, Gromkowski SH,Deck RR, DeWitt CM, Friedman A, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein.

Science. 1993; 259(5102):1745-9.

7. Wang B, Ugen KE, Srikantan V, Agadjanyan MG, Dang K, Refaeli Y, Sato AI, Boyer J, Williams WV, Weiner DB. Gene inoculation generates immune responses against humanimmunodeficiency virus type 1. Proc Natl Acad Sci USA. 1993; 90(9):4156-60.

8. Tang DC, DeVit M, Johnston SA. Genetic immunization is a simple method for eliciting an immune response. Nature. 1992;356(6365):152-4.

9. Robinson HL, Hunt LA, Webster RG. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine. 1993; 11(9):957-60.

10. Martin T, Parker SE, Hedstrom R, Le T, Hoffman SL, Norman J, Hobart P, Lew D. Plasmid DNA malaria vaccine: the potential for genomic integration after intramuscular injection. Hum Gene Ther. 1999; 10(5):759-68.

11. Shedlock DJ, Weiner DB. DNA vaccination: antigen presentation and the induction of immunity. J Leukoc BioI. 2000; 68(6):793-806.

DNA vaccination infectious disease cancer

23

12. Glenting J, Wessels S. Ensuring safety of DNA vaccines. Microb Cell Fact. 2005; 4:26.

13. Liu MA, Wahren B, Karlsson Hedestam GB. DNA vaccines: recent developments and future possibilities. Hum Gene Ther. 2006;17(11):1051-61.

14. Klinman, D., Klaschik, S., Tross D., Shirota H., FDA guidance on prophylactic DNA vaccines: Analysis and recommendations. Vaccine (2009), doi:10.1016/ j.vaccine.2009.11.025.

15. Wang, R, Doolan, D., Le, TP., Hedstrom, RC, Coonaan, KM, Charpenvit, Y., Jones, TR, Hobart, P., Margalith, M., Ng, J., Weiss, Wr., Sedegah,M.,Taisne, CD., Norman, JA, Hoffman, Induction of antigen –specific cytotoxic T lymphocytes in humans by a malaria DNA vaccine, Science,1998,282,476-480.

16. Martin JE, Pierson TC, Hubka S, Rucker S, Gordon 11, Enama ME, Andrews CA, Xu Q, Davis BS, Nason M, Fay M, Koup RA, Roederer M, Bailer RT, Gomez PL, Mascola JR, Chang GJ, Nabel GJ, Graham BS. A West Nile virus DNA vaccine induces neutralizing antibody in healthy adults during a phase 1 clinical trial. J Infect Dis. 2007; 196(12):1732-40.

17. Martin JE, Louder MK, Holman LA, Gordon IJ, Enama ME, Larkin BD, Andrews CA, Vogel L, Koup RA, Roederer M, Bailer RT, Gomez PL, Nason M, Mascola JR, Nabel GJ, Graham BS; the VRC 301 Study Team. A SARS DNA vaccine induces neutralizing antibody and cellular immune responses in healthy adults in a Phase I clinical trial. Vaccine. 2008 Nov 25;26(50):6338-6343. Epub 2008 Sep 26.

18. Roy MJ, Wu MS, Barr LJ, Fuller JT, Tussey LG, Speller S, Culp J, Burkholder JK, Swain WF, Dixon RM, Widera G, Vessey R, King A, Ogg G, Gallimore A, Haynes JR, HeydenburgFuller D. Induction of antigen-specific CD8+ T cells, T helper cells, and protective levels of antibody in humans by particle-mediated administration of a hepatitis B virus DNA vaccine.Vaccine. 2000;19(7-8):764-78.

19. Drape,RJ, Macklin, MD, Barr, LJ,Jones, S, Haynes, JR, Dean, HJ, Epidermal DNA vaccines for influenza is immunogenic in humans, Vaccine, 2006, 24, 21, 4475-81.

20. Martin JE, Sullivan NJ, Enama ME, Gordon 11, Roederer M, Koup RA, Bailer RT,Chakrabarti BK, Bailey MA, Gomez PL, Andrews CA, Moodie Z, Gu L, Stein JA: Nabel GJ,Graham BS. A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine Immunol. 2006(11):126777. Epub 2006 Sep 20.

21. Jacobson MA, Adler SP, Sinclair E, Black D, Smith A, Chu A, Moss RB, Wloch MK, A CMV DNA vaccine primes for memory immune responses to liveattenuated CMV (Towne strain). Vaccine, 27 10 1540-1548, March 2009.

22. Derek O'Hagan, Manmohan Singh, Mildred Ugozzoli, Carl Wild2 Susan Barnett, Minchao Chen,1 Mary Schaefer, Barbara Doe, Gillis R. Otten, and Jeffrey B. Ulmer,Induction of Potent Immune Responses by Cationic Microparticles with Adsorbed Human Immunodeficiency Virus DNA Vaccines , Journal of Virology, October 2001, p. 9037-9043, Vol. 75, No. 19.

23. Wloch MK, Smith LR, Boutsaboualoy S, Reyes L, Han C, Kehler J, Smith HD, Selk L, Nakamura R, Brown JM, Marbury T, Wald A, Rolland A, Kaslow D,

24

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

Ronald B Moss et al.

Evans T, Boeckh M. Safety and immunogenicity of a bivalent cytomegalovirus DNA vaccine in healthy adultsubjects. J Infect Dis. 2008;197(12):1634-42.

Wilk, M, Chu, A Wloich, M, Gutterill, D., Smith, L., Rolland, A., Interim Analyss of a phase 2 trial of TransVax, a plasmid DNA immunotherapeutic for the control of Cytomegalovirus in Transplants, ICAAC, San Franciso, Abstract G1-1113a, 2009.

Lalor PA, Webby RJ, Morrow J, Rusalov D, Kaslow DC, Rolland A, Smith LR. Plasmid DNA-based vaccines protect mice and ferrets against lethal challenge with ANietharnl1203/04 (H5Nl) influenza virus. J Infect Dis. 2008; 197(12):1643-52.

Pan CH, Jimenez GS, Nair N, Wei Q, Adams RJ, Polack FP, Rolland A, Vilalta A, Griffin DE. Use of Vaxfectin Adjuvant with DNA Vaccine Encoding the Measles Virus Hemagglutinin and Fusion Proteins Protects Juvenile and Infant Rhesus Macaques against Measles Virus. Clin Vaccine Immunol. 2008;15(8): 1214-21. Epub 2008 Jun 4.

Smith, LR, Wloch,MK, Ye, M., Reyes,LR, Boutsaboualoy, S., Dunne,CE, Chaplin,JA, Rusalov,D, Rolland, AP, Fisher, CL,Al-Ibrahim,MS, Kabongo,ML, Steigbigel,R, Belshe,RB, Kitt,ER, Chu, AH, Moss, RB, Phase 1 clinical trials of the safety and immunogenicity of adjuvanted plasmid DNA vaccines encoding influenza A virus H5 hemagglutinin,Vaccine. 2010 Mar 16;28(13):2565-72.

Epub 2010 Jan 29.

Felber, B. K., and G. N. Pavlakis. 1993. Molecular biology of HIV-1: positive and negative regulatory elements important for virus expression. AIDS 7 Suppl 1:S51-62.

Pavlakis, G. N., and B. K. Felber. 1990. Regulation of expression of human immunodeficiency virus. New Biol 2:20-31.

Felber, B. K., M. Hadzopoulou-Cladaras, C. Cladaras, T. Copeland, and G. N. Pavlakis. 1989. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A 86:14951499.

Hadzopoulou-Cladaras, M., B. K. Felber, C. Cladaras, A. Athanassopoulos, A. Tse, and G. N. Pavlakis. 1989. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol 63:1265-1274.

Schneider, R., M. Campbell, G. Nasioulas, B. K. Felber, and G. N. Pavlakis.

1997. Inactivation of the human immunodeficiency virus type 1 inhibitory elements allows Rev-independent expression of Gag and Gag/protease and particle formation. J Virol 71:4892-4903.

Afonina, E., M. Neumann, and G. N. Pavlakis. 1997. Preferential binding of poly(A)-binding protein 1 to an inhibitory RNA element in the human immunodeficiency virus type 1 gag mRNA. J Biol Chem 272:2307-2311.

Nasioulas, G., A. S. Zolotukhin, C. Tabernero, L. Solomin, C. P. Cunningham, G. N. Pavlakis, and B. K. Felber. 1994. Elements distinct from human immunodeficiency virus type 1 splice sites are responsible for the Rev dependence of env mRNA. J Virol 68:2986-2993.

DNA vaccination infectious disease cancer

25

35. Schwartz, S., M. Campbell, G. Nasioulas, J. Harrison, B. K. Felber, and G. N. Pavlakis. 1992. Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. J Virol 66:7176-7182.

36. Schwartz, S., B. K. Felber, and G. N. Pavlakis. 1992. Distinct RNA sequences in the gag region of human immunodeficiency virus type 1 decrease RNA stability and inhibit expression in the absence of Rev protein. J Virol 66:150-159.

37. Qiu, J. T., B. Liu, C. Tian, G. N. Pavlakis, and X. F. Yu. 2000. Enhancement of primary and secondary cellular immune responses against human immunodeficiency virus type 1 gag by using DNA expression vectors that target Gag antigen to the secretory pathway. J Virol 74:5997-6005.

38. Qiu, J. T., R. Song, M. Dettenhofer, C. Tian, T. August, B. K. Felber, G. N. Pavlakis, and X. F. Yu. 1999. Evaluation of novel human immunodeficiency virus type 1 Gag DNA vaccines for protein expression in mammalian cells and induction of immune responses. J Virol 73:9145-9152.

39. Fitzgerald, J. C., G. P. Gao, A. Reyes-Sandoval, G. N. Pavlakis, Z. Q. Xiang, A. P. Wlazlo, W. Giles-Davis, J. M. Wilson, and H. C. Ertl. 2003. A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag. J Immunol 170:1416-1422.

40. Wang, S. W., P. A. Kozlowski, G. Schmelz, K. Manson, M. S. Wyand, R. Glickman, D. Montefiori, J. D. Lifson, R. P. Johnson, M. R. Neutra, and A. Aldovini. 2000. Effective induction of simian immunodeficiency virus-specific systemic and mucosal immune responses in primates by vaccination with proviral DNA producing intact but noninfectious virions. J Virol 74:10514-10522.

41. Wang, S. W., F. M. Bertley, P. A. Kozlowski, L. Herrmann, K. Manson, G. Mazzara, M. Piatak, R. P. Johnson, A. Carville, K. Mansfield, and A. Aldovini.

2004. An SHIV DNA/MVA rectal vaccination in macaques provides systemic and mucosal virus-specific responses and protection against AIDS. AIDS Res Hum Retroviruses 20:846-859.

42. Fuller, D. H., P. A. Rajakumar, L. A. Wilson, A. M. Trichel, J. T. Fuller, T. Shipley, M. S. Wu, K. Weis, C. R. Rinaldo, J. R. Haynes, and M. Murphey-Corb.

2002. Induction of mucosal protection against primary, heterologous simian immunodeficiency virus by a DNA vaccine. J Virol 76:3309-3317.

43. Bagarazzi, M. L., J. D. Boyer, M. A. Javadian, M. A. Chattergoon, A. R. Shah, A. D. Cohen, M. K. Bennett, R. B. Ciccarelli, K. E. Ugen, and D. B. Weiner.

1999. Systemic and mucosal immunity is elicited after both intramuscular and intravaginal delivery of human immunodeficiency virus type 1 DNA plasmid vaccines to pregnant chimpanzees. J Infect Dis 180:1351-1355.

44. Belyakov, I. M., J. D. Ahlers, G. J. Nabel, B. Moss, and J. A. Berzofsky. 2008.

Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization. Virology 381:106-115.

45. Eo, S. K., M. Gierynska, A. A. Kamar, and B. T. Rouse. 2001. Prime-boost immunization with DNA vaccine: mucosal route of administration changes the rules. J Immunol 166:5473-5479.

26

Ronald B Moss et al.

46. Yang, K., B. J. Whalen, R. S. Tirabassi, L. K. Selin, T. S. Levchenko, V. P. Torchilin, E. H. Kislauskis, and D. L. Guberski. 2008. A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection. J Immunol 180:6159-6167.

47. Jeyanathan, M., J. Mu, K. Kugathasan, X. Zhang, D. Damjanovic, C. Small, M. Divangahi, B. J. Petrof, C. M. Hogaboam, and Z. Xing. 2008. Airway delivery of soluble mycobacterial antigens restores protective mucosal immunity by single intramuscular plasmid DNA tuberculosis vaccination: role of proinflammatory signals in the lung. J Immunol 181:5618-5626.

48. Tavel, J. A., J. E. Martin, G. G. Kelly, M. E. Enama, J. M. Shen, P. L. Gomez, C. A. Andrews, R. A. Koup, R. T. Bailer, J. A. Stein, M. Roederer, G. J. Nabel, and B. S. Graham. 2007. Safety and immunogenicity of a Gag-Pol candidate HIV-1 DNA vaccine administered by a needle-free device in HIV-1-seronegative subjects. J Acquir Immune Defic Syndr 44:601-605.

49. Graham, B. S., R. A. Koup, M. Roederer, R. T. Bailer, M. E. Enama, Z. Moodie, J. E. Martin, M. M. McCluskey, B. K. Chakrabarti, L. Lamoreaux, C. A. Andrews, P. L. Gomez, J. R. Mascola, and G. J. Nabel. 2006. Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine. J Infect Dis 194:1650-1660.

50. Mwau, M., I. Cebere, J. Sutton, P. Chikoti, N. Winstone, E. G. Wee, T. Beattie, Y. H. Chen, L. Dorrell, H. McShane, C. Schmidt, M. Brooks, S. Patel, J. Roberts, C. Conlon, S. L. Rowland-Jones, J. J. Bwayo, A. J. McMichael, and T. Hanke.

2004. A human immunodeficiency virus 1 (HIV-1) clade A vaccine in clinical trials: stimulation of HIV-specific T-cell responses by DNA and recombinant modified vaccinia virus Ankara (MVA) vaccines in humans. J Gen Virol 85:911-919.

51. Kutzler, M. A., and D. B. Weiner. 2008. DNA vaccines: ready for prime time?

Nat Rev Genet 9:776-788.

52. Bansal, A., B. Jackson, K. West, S. Wang, S. Lu, J. S. Kennedy, and P. A. Goepfert. 2008. Multifunctional T-cell characteristics induced by a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine regimen given to healthy adults are dependent on the route and dose of administration. J Virol 82:6458-6469.

53. Jaoko, W., F. N. Nakwagala, O. Anzala, G. O. Manyonyi, J. Birungi, A. Nanvubya, F. Bashir, K. Bhatt, H. Ogutu, S. Wakasiaka, L. Matu, W. Waruingi, J. Odada, M. Oyaro, J. Indangasi, J. Ndinya-Achola, C. Konde, E. Mugisha, P. Fast, C. Schmidt, J. Gilmour, T. Tarragona, C. Smith, B. Barin, L. Dally, B. Johnson, A. Muluubya, L. Nielsen, P. Hayes, M. Boaz, P. Hughes, T. Hanke, A. McMichael, J. Bwayo, and P. Kaleebu. 2008. Safety and immunogenicity of recombinant low-dosage HIV-1 A vaccine candidates vectored by plasmid pTHr DNA or modified vaccinia virus Ankara (MVA) in humans in East Africa.

Vaccine 26:2788-2795.

54. Wilson, C. C., M. J. Newman, B. D. Livingston, S. MaWhinney, J. E. Forster, J. Scott, R. T. Schooley, and C. A. Benson. 2008. Clinical phase 1 testing of the safety and immunogenicity of an epitope-based DNA vaccine in human

DNA vaccination infectious disease cancer

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

27

immunodeficiency virus type 1-infected subjects receiving highly active antiretroviral therapy. Clin Vaccine Immunol 15:986-994.

Rosati, M., A. von Gegerfelt, P. Roth, C. Alicea, A. Valentin, M. Robert-Guroff, D. Venzon, D. C. Montefiori, P. Markham, B. K. Felber, and G. N. Pavlakis. 2005.

DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge. J Virol 79:8480-8492.

Wilson, N. A., J. Reed, G. S. Napoe, S. Piaskowski, A. Szymanski, J. Furlott, E. J. Gonzalez, L. J. Yant, N. J. Maness, G. E. May, T. Soma, M. R. Reynolds, E. Rakasz, R. Rudersdorf, A. B. McDermott, D. H. O'Connor, T. C. Friedrich, D. B. Allison, A. Patki, L. J. Picker, D. R. Burton, J. Lin, L. Huang, D. Patel, G. Heindecker, J. Fan, M. Citron, M. Horton, F. Wang, X. Liang, J. W. Shiver, D. R. Casimiro, and D. I. Watkins. 2006. Vaccine-induced cellular immune responses reduce plasma viral concentrations after repeated low-dose challenge with pathogenic simian immunodeficiency virus SIVmac239. J Virol 80:5875-5885.

Barouch, D. H., T. M. Fu, D. C. Montefiori, M. G. Lewis, J. W. Shiver, and N. L. Letvin. 2001. Vaccine-elicited immune responses prevent clinical AIDS in SHIV(89.6P)-infected rhesus monkeys. Immunol Lett 79:57-61.

Egan, M. A., W. A. Charini, M. J. Kuroda, J. E. Schmitz, P. Racz, K. TennerRacz, K. Manson, M. Wyand, M. A. Lifton, C. E. Nickerson, T. Fu, J. W. Shiver, and N. L. Letvin. 2000. Simian immunodeficiency virus (SIV) gag DNAvaccinated rhesus monkeys develop secondary cytotoxic T-lymphocyte responses and control viral replication after pathogenic SIV infection. J Virol 74:7485-7495.

Muthumani, K., M. Bagarazzi, D. Conway, D. S. Hwang, K. Manson, R. Ciccarelli, Z. Israel, D. C. Montefiori, K. Ugen, N. Miller, J. Kim, J. Boyer, and D. B. Weiner. 2003. A Gag-Pol/Env-Rev SIV239 DNA vaccine improves CD4 counts, and reduce viral loads after pathogenic intrarectal SIV(mac)251 challenge in Rhesus Macaques. Vaccine 21:629-637.

Mossman, S. P., C. C. Pierce, A. J. Watson, M. N. Robertson, D. C. Montefiori, L. Kuller, B. A. Richardson, J. D. Bradshaw, R. J. Munn, S. L. Hu, P. D. Greenberg, R. E. Benveniste, and N. L. Haigwood. 2004. Protective immunity to SIV challenge elicited by vaccination of macaques with multigenic DNA vaccines producing virus-like particles. AIDS Res Hum Retroviruses 20:425-434.

Koopman, G., D. Mortier, S. Hofman, N. Mathy, M. Koutsoukos, P. Ertl, P. Overend, C. van Wely, L. L. Thomsen, B. Wahren, G. Voss, and J. L. Heeney.

2008. Immune-response profiles induced by human immunodeficiency virus type 1 vaccine DNA, protein or mixed-modality immunization: increased protection from pathogenic simian-human immunodeficiency virus viraemia with protein/DNA combination. J Gen Virol 89:540-553.

Aihara, H., and J. Miyazaki. 1998. Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 16:867-870.

Mathiesen, I. 1999. Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Ther 6:508-514.

Prud'homme, G. J., Y. Glinka, A. S. Khan, and R. Draghia-Akli. 2006.

Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases. Curr Gene Ther 6:243-273.

28

Ronald B Moss et al.

65. Rizzuto, G., M. Cappelletti, D. Maione, R. Savino, D. Lazzaro, P. Costa, I. Mathiesen, R. Cortese, G. Ciliberto, R. Laufer, N. La Monica, and E. Fattori.

1999. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci U S A 96:6417-6422.

66. Wang, Z., P. J. Troilo, X. Wang, T. G. Griffiths, S. J. Pacchione, A. B. Barnum, L. B. Harper, C. J. Pauley, Z. Niu, L. Denisova, T. T. Follmer, G. Rizzuto, G. Ciliberto, E. Fattori, N. L. Monica, S. Manam, and B. J. Ledwith. 2004.

Detection of integration of plasmid DNA into host genomic DNA following intramuscular injection and electroporation. Gene Ther 11:711-721.

67. Widera, G., M. Austin, D. Rabussay, C. Goldbeck, S. W. Barnett, M. Chen, L. Leung, G. R. Otten, K. Thudium, M. J. Selby, and J. B. Ulmer. 2000. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol 164:4635-4640.

68. Otten, G., M. Schaefer, B. Doe, H. Liu, I. Srivastava, J. zur Megede, D. O'Hagan, J. Donnelly, G. Widera, D. Rabussay, M. G. Lewis, S. Barnett, and J. B. Ulmer.

2004. Enhancement of DNA vaccine potency in rhesus macaques by electroporation. Vaccine 22:2489-2493.

69. Otten, G. R., M. Schaefer, B. Doe, H. Liu, J. Z. Megede, J. Donnelly, D. Rabussay, S. Barnett, and J. B. Ulmer. 2006. Potent immunogenicity of an HIV-1 gag-pol fusion DNA vaccine delivered by in vivo electroporation. Vaccine 24:4503-4509.

70. Rosati, M., A. Valentin, R. Jalah, V. Patel, A. von Gegerfelt, C. Bergamaschi, C. Alicea, D. Weiss, J. Treece, R. Pal, P. Markham, E. T. A. Marques, J. T. August, A. Khan, R. Draghia-Aklie, B. K. Felber, and G. N. Pavlakis. 2008. Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation. Vaccine 26:5223-5229.

71. Luckay, A., M. K. Sidhu, R. Kjeken, S. Megati, S. Y. Chong, V. Roopchand, D. Garcia-Hand, R. Abdullah, R. Braun, D. C. Montefiori, M. Rosati, B. K. Felber, G. N. Pavlakis, I. Mathiesen, Z. R. Israel, J. H. Eldridge, and M. A. Egan. 2007.

Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol 81:5257-5269.

72. Hirao, L. A., L. Wu, A. S. Khan, D. A. Hokey, J. Yan, A. Dai, M. R. Betts, R. Draghia-Akli, and D. B. Weiner. 2008. Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques. Vaccine 26:3112-3120.

73. Rosati, M., C. Bergamaschi, A. Valentin, V. Kulkarni, R. Jalah, C. Alicea, V. Patel, A.S. von Gegerfelt, D.C. Montefiori, D.J. Venzon, A.S. Khan, R. DraghiaAkli, K.K. Van Rompay, B.K. Felber, and G.N. Pavlakis. 2009. DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge. Proc Natl Acad Sci U S A 106:15831-15836.

74. MacGregor, R. R., J. D. Boyer, K. E. Ugen, K. E. Lacy, S. J. Gluckman, M. L. Bagarazzi, M. A. Chattergoon, Y. Baine, T. J. Higgins, R. B. Ciccarelli, L. R. Coney, R. S. Ginsberg, and D. B. Weiner. 1998. First human trial of a DNA-

DNA vaccination infectious disease cancer

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

86.

87.

88.

29

based vaccine for treatment of human immunodeficiency virus type 1 infection: safety and host response. J Infect Dis 178:92-100.

MacGregor, R. R., J. D. Boyer, R. B. Ciccarelli, R. S. Ginsberg, and D. B. Weiner. 2000. Safety and immune responses to a DNA-based human immunodeficiency virus (HIV) type I env/rev vaccine in HIV-infected recipients: follow-up data. J Infect Dis 181:406.

Eller, M. A., L. A. Eller, M. S. Opollo, B. J. Ouma, P. O. Oballah, L. Galley, C. Karnasuta, S. R. Kim, M. L. Robb, N. L. Michael, H. Kibuuka, F. WabwireMangen, B. S. Graham, D. L. Birx, M. S. de Souza, and J. H. Cox. 2007.

Induction of HIV-specific functional immune responses by a multiclade HIV-1 DNA vaccine candidate in healthy Ugandans. Vaccine 25:7737-7742.

Asmuth, D. M., E. L. Brown, M. J. DiNubile, X. Sun, C. del Rio, C. Harro, M. C. Keefer, J. G. Kublin, S. A. Dubey, L. S. Kierstead, D. R. Casimiro, J. W. Shiver, M. N. Robertson, E. K. Quirk, and D. V. Mehrotra. Comparative cell-mediated immunogenicity of DNA/DNA, DNA/adenovirus type 5 (Ad5), or Ad5/Ad5 HIV-1 clade B gag vaccine prime-boost regimens. J Infect Dis 201:132-141.

Vasan, S., S. J. Schlesinger, Y. Huang, A. Hurley, A. Lombardo, Z. Chen, S. Than, P. Adesanya, C. Bunce, M. Boaz, R. Boyle, E. Sayeed, L. Clark, D. Dugin, C. Schmidt, Y. Song, L. Seamons, L. Dally, M. Ho, C. Smith, M. Markowitz, J. Cox, D. K. Gill, J. Gilmour, M. C. Keefer, P. Fast, and D. D. Ho. Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine. PLoS One 5:e8617.

Carter, P.J. 2006. Potent antibody therapeutics by design. Nat Rev Immunol 6:343-357.

Rosenberg, S.A., N.P. Restifo, J.C. Yang, R.A. Morgan, and M.E. Dudley. 2008.

Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 8:299-308.

Curiel, T.J. 2007. Tregs and rethinking cancer immunotherapy. J Clin Invest 117:1167-1174.

Whiteside, T.L. 2008. The tumor microenvironment and its role in promoting tumor growth. Oncogene 27:5904-5912.

Bubenik, J. 2004. MHC class I down-regulation: tumour escape from immune surveillance? (review). Int J Oncol 25:487-491.

Munn, D.H. 2006. Indoleamine 2,3-dioxygenase, tumor-induced tolerance and counter-regulation. Curr Opin Immunol 18:220-225.

Munn, D.H., and A.L. Mellor. 2007. Indoleamine 2,3-dioxygenase and tumorinduced tolerance. J Clin Invest 117:1147-1154.

Rosenberg, S.A. 1997. Cancer vaccines based on the identification of genes encoding cancer regression antigens. Immunol Today 18:175-182.

Cheever, M.A., J.P. Allison, A.S. Ferris, O.J. Finn, B.M. Hastings, T.T. Hecht, I. Mellman, S.A. Prindiville, J.L. Viner, L.M. Weiner, and L.M. Matrisian. 2009.

The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin Cancer Res 15:5323-5337.

Stevenson, G.T., and F.K. Stevenson. 1975. Antibody to a molecularly-defined antigen confined to a tumour cell surface. Nature 254:714-716.

30

Ronald B Moss et al.

89. Theobald, M., J. Biggs, D. Dittmer, A.J. Levine, and L.A. Sherman. 1995.

Targeting p53 as a general tumor antigen. Proc Natl Acad Sci U S A 92:1199311997.

90. Abrams, S.I., P.H. Hand, K.Y. Tsang, and J. Schlom. 1996. Mutant ras epitopes as targets for cancer vaccines. Semin Oncol 23:118-134.

91. Bakker, A.B., M.W. Schreurs, A.J. de Boer, Y. Kawakami, S.A. Rosenberg, G.J. Adema, and C.G. Figdor. 1994. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med 179:1005-1009.

92. Kawakami, Y., S. Eliyahu, C.H. Delgado, P.F. Robbins, K. Sakaguchi, E. Appella, J.R. Yannelli, G.J. Adema, T. Miki, and S.A. Rosenberg. 1994. Identification of a human melanoma antigen recognized by tumor-infiltrating lymphocytes associated with in vivo tumor rejection. Proc Natl Acad Sci U S A 91:6458-6462.

93. Kawakami, Y., S. Eliyahu, C.H. Delgado, P.F. Robbins, L. Rivoltini, S.L. Topalian, T. Miki, and S.A. Rosenberg. 1994. Cloning of the gene coding for a shared human melanoma antigen recognized by autologous T cells infiltrating into tumor. Proc Natl Acad Sci U S A 91:3515-3519.

94. Brichard, V., A. Van Pel, T. Wolfel, C. Wolfel, E. De Plaen, B. Lethe, P. Coulie, and T. Boon. 1993. The tyrosinase gene codes for an antigen recognized by autologous cytolytic T lymphocytes on HLA-A2 melanomas. J Exp Med 178:489-495.

95. Wang, R.F., P.F. Robbins, Y. Kawakami, X.Q. Kang, and S.A. Rosenberg. 1995.

Identification of a gene encoding a melanoma tumor antigen recognized by HLAA31-restricted tumor-infiltrating lymphocytes. J Exp Med 181:799-804.

96. Wang, R.F., E. Appella, Y. Kawakami, X. Kang, and S.A. Rosenberg. 1996.

Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J Exp Med 184:2207-2216.

97. Chen, Y.T., M.J. Scanlan, U. Sahin, O. Tureci, A.O. Gure, S. Tsang, B. Williamson, E. Stockert, M. Pfreundschuh, and L.J. Old. 1997. A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening. Proc Natl Acad Sci U S A 94:1914-1918.

98. Zaremba, S., E. Barzaga, M. Zhu, N. Soares, K.Y. Tsang, and J. Schlom. 1997.

Identification of an enhancer agonist cytotoxic T lymphocyte peptide from human carcinoembryonic antigen. Cancer Res 57:4570-4577.

99. Zrihan-Licht, S., H.L. Vos, A. Baruch, O. Elroy-Stein, D. Sagiv, I. Keydar, J. Hilkens, and D.H. Wreschner. 1994. Characterization and molecular cloning of a novel MUC1 protein, devoid of tandem repeats, expressed in human breast cancer tissue. Eur J Biochem 224:787-795.

100. Gullick, W.J., S.B. Love, C. Wright, D.M. Barnes, B. Gusterson, A.L. Harris, and D.G. Altman. 1991. c-erbB-2 protein overexpression in breast cancer is a risk factor in patients with involved and uninvolved lymph nodes. Br J Cancer 63:434-438.

101. Biragyn, A., K. Tani, M.C. Grimm, S. Weeks, and L.W. Kwak. 1999. Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity. Nat Biotechnol 17:253-258.

DNA vaccination infectious disease cancer

31

102. Biragyn, A., R. Schiavo, P. Olkhanud, K. Sumitomo, A. King, M. McCain, F.E. Indig, G. Almanzar, and D. Baatar. 2007. Tumor-associated embryonic antigenexpressing vaccines that target CCR6 elicit potent CD8+ T cell-mediated protective and therapeutic antitumor immunity. J Immunol 179:1381-1388.

103. Tao, M.H., and R. Levy. 1993. Idiotype/granulocyte-macrophage colonystimulating factor fusion protein as a vaccine for B-cell lymphoma. Nature 362:755-758.

104. Syrengelas, A.D., T.T. Chen, and R. Levy. 1996. DNA immunization induces protective immunity against B-cell lymphoma. Nat Med 2:1038-1041.

105. Xiang, R., F.J. Primus, J.M. Ruehlmann, A.G. Niethammer, S. Silletti, H.N. Lode, C.S. Dolman, S.D. Gillies, and R.A. Reisfeld. 2001. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J Immunol 167:4560-4565.

106. Hung, C.F., K.F. Hsu, W.F. Cheng, C.Y. Chai, L. He, M. Ling, and T.C. Wu. 2001. Enhancement of DNA vaccine potency by linkage of antigen gene to a gene encoding the extracellular domain of Fms-like tyrosine kinase 3-ligand.

Cancer Res 61:1080-1088.

107. Boyle, J.S., J.L. Brady, and A.M. Lew. 1998. Enhanced responses to a DNA vaccine encoding a fusion antigen that is directed to sites of immune induction.

Nature 392:408-411.

108. Valentin, A., P. Chikhlikar, V. Patel, M. Rosati, M. Maciel, K.H. Chang, P. Silvera, B.K. Felber, G.N. Pavlakis, J.T. August, and E.T. Marques. 2009.

Comparison of DNA vaccines producing HIV-1 Gag and LAMP/Gag chimera in rhesus macaques reveals antigen-specific T-cell responses with distinct phenotypes. Vaccine 27:4840-4849.

109. Marques, E.T., Jr., P. Chikhlikar, L.B. de Arruda, I.C. Leao, Y. Lu, J. Wong, J.S. Chen, B. Byrne, and J.T. August. 2003. HIV-1 p55Gag encoded in the lysosomeassociated membrane protein-1 as a DNA plasmid vaccine chimera is highly expressed, traffics to the major histocompatibility class II compartment, and elicits enhanced immune responses. J Biol Chem 278:37926-37936.

110. Wu, T.C., F.G. Guarnieri, K.F. Staveley-O'Carroll, R.P. Viscidi, H.I. Levitsky, L. Hedrick, K.R. Cho, J.T. August, and D.M. Pardoll. 1995. Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens. Proc Natl Acad Sci U S A 92:11671-11675.

111. Lin, K.Y., F.G. Guarnieri, K.F. Staveley-O'Carroll, H.I. Levitsky, J.T. August, D.M. Pardoll, and T.C. Wu. 1996. Treatment of established tumors with a novel vaccine that enhances major histocompatibility class II presentation of tumor antigen. Cancer Res 56:21-26.

112. Bonehill, A., C. Heirman, S. Tuyaerts, A. Michiels, K. Breckpot, F. Brasseur, Y. Zhang, P. Van Der Bruggen, and K. Thielemans. 2004. Messenger RNAelectroporated dendritic cells presenting MAGE-A3 simultaneously in HLA class I and class II molecules. J Immunol 172:6649-6657.

113. Xiang, R., H.N. Lode, T.H. Chao, J.M. Ruehlmann, C.S. Dolman, F. Rodriguez, J.L. Whitton, W.W. Overwijk, N.P. Restifo, and R.A. Reisfeld. 2000. An

32

Ronald B Moss et al.

autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci U S A 97:5492-5497.

114. Zhang, M., C. Obata, H. Hisaeda, K. Ishii, S. Murata, T. Chiba, K. Tanaka, Y. Li, M. Furue, B. Chou, T. Imai, X. Duan, and K. Himeno. 2005. A novel DNA vaccine based on ubiquitin-proteasome pathway targeting 'self'-antigens expressed in melanoma/melanocyte. Gene Ther 12:1049-1057.

115. Janssen, E.M., E.E. Lemmens, T. Wolfe, U. Christen, M.G. von Herrath, and S.P. Schoenberger. 2003. CD4+ T cells are required for secondary expansion and memory in CD8+ T lymphocytes. Nature 421:852-856.

116. Shedlock, D.J., and H. Shen. 2003. Requirement for CD4 T cell help in generating functional CD8 T cell memory. Science 300:337-339.

117. Rice, J., T. Elliott, S. Buchan, and F.K. Stevenson. 2001. DNA fusion vaccine designed to induce cytotoxic T cell responses against defined peptide motifs: implications for cancer vaccines. J Immunol 167:1558-1565.

118. King, C.A., M.B. Spellerberg, D. Zhu, J. Rice, S.S. Sahota, A.R. Thompsett, T.J. Hamblin, J. Radl, and F.K. Stevenson. 1998. DNA vaccines with single-chain Fv fused to fragment C of tetanus toxin induce protective immunity against lymphoma and myeloma. Nat Med 4:1281-1286.

119. Rice, J., S. Buchan, and F.K. Stevenson. 2002. Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen. J Immunol 169:3908-3913.

120. Hung, C.F., W.F. Cheng, K.F. Hsu, C.Y. Chai, L. He, M. Ling, and T.C. Wu. 2001. Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 61:3698-3703.

121. Weber, L.W., W.B. Bowne, J.D. Wolchok, R. Srinivasan, J. Qin, Y. Moroi, R. Clynes, P. Song, J.J. Lewis, and A.N. Houghton. 1998. Tumor immunity and autoimmunity induced by immunization with homologous DNA. J Clin Invest 102:1258-1264.

122. Schreurs, M.W., A.J. de Boer, C.G. Figdor, and G.J. Adema. 1998. Genetic vaccination against the melanocyte lineage-specific antigen gp100 induces cytotoxic T lymphocyte-mediated tumor protection. Cancer Res 58:2509-2514.

123. Gold, J.S., C.R. Ferrone, J.A. Guevara-Patino, W.G. Hawkins, R. Dyall, M.E. Engelhorn, J.D. Wolchok, J.J. Lewis, and A.N. Houghton. 2003. A single heteroclitic epitope determines cancer immunity after xenogeneic DNA immunization against a tumor differentiation antigen. J Immunol 170:5188-5194.

124. Rakhmilevich, A.L., M. Imboden, Z. Hao, M.D. Macklin, T. Roberts, K.M. Wright, M.R. Albertini, N.S. Yang, and P.M. Sondel. 2001. Effective particlemediated vaccination against mouse melanoma by coadministration of plasmid DNA encoding Gp100 and granulocyte-macrophage colony-stimulating factor.

Clin Cancer Res 7:952-961.

125. Neal, Z.C., M.K. Bates, M.R. Albertini, and H. Herweijer. 2007. Hydrodynamic limb vein delivery of a xenogeneic DNA cancer vaccine effectively induces antitumor immunity. Mol Ther 15:422-430.

DNA vaccination infectious disease cancer

33

126. Trinchieri, G., S. Pflanz, and R.A. Kastelein. 2003. The IL-12 family of heterodimeric cytokines: new players in the regulation of T cell responses.

Immunity 19:641-644.

127. Trinchieri, G. 2003. Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol 3:133-146.

128. Chakrabarti, R., Y. Chang, K. Song, and G.J. Prud'homme. 2004. Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulate DNA vaccination against carcinoembryonic antigen (CEA). Vaccine 22:1199-1205.

129. Song, K., Y. Chang, and G.J. Prud'homme. 2000. IL-12 plasmid-enhanced DNA vaccination against carcinoembryonic antigen (CEA) studied in immune-gene knockout mice. Gene Ther 7:1527-1535.

130. Tuting, T., A. Gambotto, A. DeLeo, M.T. Lotze, P.D. Robbins, and W.J. Storkus.

1999. Induction of tumor antigen-specific immunity using plasmid DNA immunization in mice. Cancer Gene Ther 6:73-80.

131. Lucas, M.L., and R. Heller. 2003. IL-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma. DNA Cell Biol 22:755-763.

132. Lucas, M.L., L. Heller, D. Coppola, and R. Heller. 2002. IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol Ther 5:668-675.

133. Spadaro, M., E. Ambrosino, M. Iezzi, E. Di Carlo, P. Sacchetti, C. Curcio, A. Amici, W.Z. Wei, P. Musiani, P.L. Lollini, F. Cavallo, and G. Forni. 2005. Cure of mammary carcinomas in Her-2 transgenic mice through sequential stimulation of innate (neoadjuvant interleukin-12) and adaptive (DNA vaccine electroporation) immunity. Clin Cancer Res 11:1941-1952.

134. Salcedo, R., J.A. Hixon, J.K. Stauffer, R. Jalah, A.D. Brooks, T. Khan, R.M. Dai, L. Scheetz, E. Lincoln, T.C. Back, D. Powell, A.A. Hurwitz, T.J. Sayers, R. Kastelein, G.N. Pavlakis, B.K. Felber, G. Trinchieri, and J.M. Wigginton. 2009.

Immunologic and therapeutic synergy of IL-27 and IL-2: enhancement of T cell sensitization, tumor-specific CTL reactivity and complete regression of disseminated neuroblastoma metastases in the liver and bone marrow. J Immunol 182:4328-4338.

135. Sun, X., L.M. Hodge, H.P. Jones, L. Tabor, and J.W. Simecka. 2002.

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination. Vaccine 20: 1466-1474.

136. Pavlenko, M., A.K. Roos, A. Lundqvist, A. Palmborg, A.M. Miller, V. Ozenci, B. Bergman, L. Egevad, M. Hellstrom, R. Kiessling, G. Masucci, P. Wersall, S. Nilsson, and P. Pisa. 2004. A phase I trial of DNA vaccination with a plasmid expressing prostate-specific antigen in patients with hormone-refractory prostate cancer. Br J Cancer 91:688-694.

137. Yo, Y.T., K.F. Hsu, G.S. Shieh, C.W. Lo, C.C. Chang, C.L. Wu, and A.L. Shiau.

2007. Coexpression of Flt3 ligand and GM-CSF genes modulates immune responses induced by HER2/neu DNA vaccine. Cancer Gene Ther 14:904-917.

34

Ronald B Moss et al.

138. Cassaday, R.D., P.M. Sondel, D.M. King, M.D. Macklin, J. Gan, T.F. Warner, C.L. Zuleger, A.J. Bridges, H.G. Schalch, K.M. Kim, J.A. Hank, D.M. Mahvi, and M.R. Albertini. 2007. A phase I study of immunization using particlemediated epidermal delivery of genes for gp100 and GM-CSF into uninvolved skin of melanoma patients. Clin Cancer Res 13:540-549.

139. Miller, A.M., V. Ozenci, R. Kiessling, and P. Pisa. 2005. Immune monitoring in a phase 1 trial of a PSA DNA vaccine in patients with hormone-refractory prostate cancer. J Immunother 28:389-395.

140. Shi, F.F., G.R. Gunn, L.A. Snyder, and T.J. Goletz. 2007. Intradermal vaccination of MUC1 transgenic mice with MUC1/IL-18 plasmid DNA suppresses experimental pulmonary metastases. Vaccine 25:3338-3346.

141. Kim, J.J., J.S. Yang, K. Dang, K.H. Manson, and D.B. Weiner. 2001.

Engineering enhancement of immune responses to DNA-based vaccines in a prostate cancer model in rhesus macaques through the use of cytokine gene adjuvants. Clin Cancer Res 7:882s-889s.

142. Marshall, D.J., K.A. Rudnick, S.G. McCarthy, L.R. Mateo, M.C. Harris, C. McCauley, and L.A. Snyder. 2006. Interleukin-18 enhances Th1 immunity and tumor protection of a DNA vaccine. Vaccine 24:244-253.

143. Rubinstein, M.P., A.N. Kadima, M.L. Salem, C.L. Nguyen, W.E. Gillanders, and D.J. Cole. 2002. Systemic administration of IL-15 augments the antigen-specific primary CD8+ T cell response following vaccination with peptide-pulsed dendritic cells. J Immunol 169:4928-4935.

144. Yang, Z., L. Wang, H. Wang, X. Shang, W. Niu, J. Li, and Y. Wu. 2008. A novel mimovirus vaccine containing survivin epitope with adjuvant IL-15 induces longlasting cellular immunity and high antitumor efficiency. Mol Immunol 45:16741681.

145. Steel, J.C., C.A. Ramlogan, P. Yu, Y. Sakai, G. Forni, T.A. Waldmann, and J.C. Morris. Interleukin-15 and its receptor augment dendritic cell vaccination against the neu oncogene through the induction of antibodies partially independent of CD4 help. Cancer Res 70:1072-1081.

146. Oh, S., L.P. Perera, M. Terabe, L. Ni, T.A. Waldmann, and J.A. Berzofsky. 2008.

IL-15 as a mediator of CD4+ help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis. Proc Natl Acad Sci U S A 105:5201-5206.

147. Dubois, S., H.J. Patel, M. Zhang, T.A. Waldmann, and J.R. Muller. 2008.

Preassociation of IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on proliferation of NK and CD8+/CD44high T cells and its antitumor action. J Immunol 180:2099-2106.

148. Epardaud, M., K.G. Elpek, M.P. Rubinstein, A.R. Yonekura, A. BellemarePelletier, R. Bronson, J.A. Hamerman, A.W. Goldrath, and S.J. Turley. 2008.

Interleukin-15/interleukin-15R alpha complexes promote destruction of established tumors by reviving tumor-resident CD8+ T cells. Cancer Res 68:2972-2983.

149. Kobayashi, H., S. Dubois, N. Sato, H. Sabzevari, Y. Sakai, T.A. Waldmann, and Y. Tagaya. 2005. Role of trans-cellular IL-15 presentation in the activation of

DNA vaccination infectious disease cancer

35

NK cell-mediated killing, which leads to enhanced tumor immunosurveillance.

Blood 105:721-727.

150. Stoklasek, T.A., K.S. Schluns, and L. Lefrancois. 2006. Combined IL-15/IL15Ralpha immunotherapy maximizes IL-15 activity in vivo. J Immunol 177:6072-6080.

151. Weiss, J.M., T.C. Back, A.J. Scarzello, J.J. Subleski, V.L. Hall, J.K. Stauffer, X. Chen, D. Micic, K. Alderson, W.J. Murphy, and R.H. Wiltrout. 2009. Successful immunotherapy with IL-2/anti-CD40 induces the chemokine-mediated mitigation of an immunosuppressive tumor microenvironment. Proc Natl Acad Sci U S A 106:19455-19460.

152. Bergamaschi, C., M. Rosati, R. Jalah, A. Valentin, V. Kulkarni, C. Alicea, G.M. Zhang, V. Patel, B.K. Felber, and G.N. Pavlakis. 2008. Intracellular interaction of interleukin-15 with its receptor alpha during production leads to mutual stabilization and increased bioactivity. J Biol Chem 283:4189-4199.

153. Jalah, R., M. Rosati, V. Kulkarni, V. Patel, C. Bergamaschi, A. Valentin, G.M. Zhang, M.K. Sidhu, J.H. Eldridge, D.B. Weiner, G.N. Pavlakis, and B.K. Felber.

2007. Efficient systemic expression of bioactive IL-15 in mice upon delivery of optimized DNA expression plasmids. DNA Cell Biol 26:827-840.

154. Bamford, R.N., A.P. DeFilippis, N. Azimi, G. Kurys, and T.A. Waldmann. 1998.

The 5' untranslated region, signal peptide, and the coding sequence of the carboxyl terminus of IL-15 participate in its multifaceted translational control. J Immunol 160:4418-4426.

155. Sumida, S.M., P.F. McKay, D.M. Truitt, M.G. Kishko, J.C. Arthur, M.S. Seaman, S.S. Jackson, D.A. Gorgone, M.A. Lifton, N.L. Letvin, and D.H. Barouch. 2004. Recruitment and expansion of dendritic cells in vivo potentiate the immunogenicity of plasmid DNA vaccines. J Clin Invest 114:1334-1342.

156. Krieg, A.M. 2002. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20:709-760.

157. Yang, Y., C.T. Huang, X. Huang, and D.M. Pardoll. 2004. Persistent Toll-like receptor signals are required for reversal of regulatory T cell-mediated CD8 tolerance. Nat Immunol 5:508-515.

158. Urry, Z., E. Xystrakis, D.F. Richards, J. McDonald, Z. Sattar, D.J. Cousins, C.J. Corrigan, E. Hickman, Z. Brown, and C.M. Hawrylowicz. 2009. Ligation of TLR9 induced on human IL-10-secreting Tregs by 1alpha,25-dihydroxyvitamin D3 abrogates regulatory function. J Clin Invest 119:387-398.

159. Lonsdorf, A.S., H. Kuekrek, B.V. Stern, B.O. Boehm, P.V. Lehmann, and M. Tary-Lehmann. 2003. Intratumor CpG-oligodeoxynucleotide injection induces protective antitumor T cell immunity. J Immunol 171:3941-3946.

160. Krieg, A.M. 2007. Development of TLR9 agonists for cancer therapy. J Clin Invest 117:1184-1194.

161. Schneeberger, A., C. Wagner, A. Zemann, P. Luhrs, R. Kutil, M. Goos, G. Stingl, and S.N. Wagner. 2004. CpG motifs are efficient adjuvants for DNA cancer vaccines. J Invest Dermatol 123:371-379.

162. Ren, J., L. Zheng, Q. Chen, H. Li, L. Zhang, and H. Zhu. 2004. Coadministration of a DNA vaccine encoding the prostate specific membrane

36

Ronald B Moss et al.

antigen and CpG oligodeoxynucleotides suppresses tumor growth. J Transl Med 2:29.

163. Aurisicchio, L., D. Peruzzi, A. Conforti, S. Dharmapuri, A. Biondo, S. Giampaoli, A. Fridman, A. Bagchi, C.T. Winkelmann, R. Gibson, E.R. Kandimalla, S. Agrawal, G. Ciliberto, and N. La Monica. 2009. Treatment of mammary carcinomas in HER-2 transgenic mice through combination of genetic vaccine and an agonist of Toll-like receptor 9. Clin Cancer Res 15:1575-1584.

164. Irvine, K.R., J.B. Rao, S.A. Rosenberg, and N.P. Restifo. 1996. Cytokine enhancement of DNA immunization leads to effective treatment of established pulmonary metastases. J Immunol 156:238-245.

165. Condon, C., S.C. Watkins, C.M. Celluzzi, K. Thompson, and L.D. Falo, Jr. 1996.

DNA-based immunization by in vivo transfection of dendritic cells. Nat Med 2:1122-1128.

166. Wang, B., M. Merva, K. Dang, K.E. Ugen, W.V. Williams, and D.B. Weiner.

1995. Immunization by direct DNA inoculation induces rejection of tumor cell challenge. Hum Gene Ther 6:407-418.

167. Nawrath, M., J. Pavlovic, R. Dummet, J. Schultz, B. Strack, J. Heinrich, and K. Moelling. 1999. Reduced melanoma tumor formation in mice immunized with DNA expressing the melanoma-specific antigen gp100/pmel17. Leukemia 13 Suppl 1:S48-51.

168. Bronte, V., E. Apolloni, R. Ronca, P. Zamboni, W.W. Overwijk, D.R. Surman, N.P. Restifo, and P. Zanovello. 2000. Genetic vaccination with "self" tyrosinaserelated protein 2 causes melanoma eradication but not vitiligo. Cancer Res 60:253-258.

169. Conry, R.M., A.F. LoBuglio, F. Loechel, S.E. Moore, L.A. Sumerel, D.L. Barlow, and D.T. Curiel. 1995. A carcinoembryonic antigen polynucleotide vaccine has in vivo antitumor activity. Gene Ther 2:59-65.

170. Chen, Y., D. Hu, D.J. Eling, J. Robbins, and T.J. Kipps. 1998. DNA vaccines encoding full-length or truncated Neu induce protective immunity against Neuexpressing mammary tumors. Cancer Res 58:1965-1971.

171. Rosenberg, S.A., J.C. Yang, R.M. Sherry, P. Hwu, S.L. Topalian, D.J. Schwartzentruber, N.P. Restifo, L.R. Haworth, C.A. Seipp, L.J. Freezer, K.E. Morton, S.A. Mavroukakis, and D.E. White. 2003. Inability to immunize patients with metastatic melanoma using plasmid DNA encoding the gp100 melanomamelanocyte antigen. Hum Gene Ther 14:709-714.

172. Tagawa, S.T., P. Lee, J. Snively, W. Boswell, S. Ounpraseuth, S. Lee, B. Hickingbottom, J. Smith, D. Johnson, and J.S. Weber. 2003. Phase I study of intranodal delivery of a plasmid DNA vaccine for patients with Stage IV melanoma. Cancer 98:144-154.

173. Triozzi, P.L., W. Aldrich, K.O. Allen, R.R. Carlisle, A.F. LoBuglio, and R.M. Conry. 2005. Phase I study of a plasmid DNA vaccine encoding MART-1 in patients with resected melanoma at risk for relapse. J Immunother 28:382-388.

174. Weber, J., W. Boswell, J. Smith, E. Hersh, J. Snively, M. Diaz, S. Miles, X. Liu, M. Obrocea, Z. Qiu, and A. Bot. 2008. Phase 1 trial of intranodal injection of a

DNA vaccination infectious disease cancer

37

Melan-A/MART-1 DNA plasmid vaccine in patients with stage IV melanoma. J Immunother 31:215-223.

175. Timmerman, J.M., G. Singh, G. Hermanson, P. Hobart, D.K. Czerwinski, B. Taidi, R. Rajapaksa, C.B. Caspar, A. Van Beckhoven, and R. Levy. 2002.

Immunogenicity of a plasmid DNA vaccine encoding chimeric idiotype in patients with B-cell lymphoma. Cancer Res 62:5845-5852.

176. Conry, R.M., D.T. Curiel, T.V. Strong, S.E. Moore, K.O. Allen, D.L. Barlow, D.R. Shaw, and A.F. LoBuglio. 2002. Safety and immunogenicity of a DNA vaccine encoding carcinoembryonic antigen and hepatitis B surface antigen in colorectal carcinoma patients. Clin Cancer Res 8:2782-2787.

177. McNeel, D.G., E.J. Dunphy, J.G. Davies, T.P. Frye, L.E. Johnson, M.J. Staab, D.L. Horvath, J. Straus, D. Alberti, R. Marnocha, G. Liu, J.C. Eickhoff, and G. Wilding. 2009. Safety and immunological efficacy of a DNA vaccine encoding prostatic acid phosphatase in patients with stage D0 prostate cancer. J Clin Oncol 27:4047-4054.

178. Low, L., A. Mander, K. McCann, D. Dearnaley, T. Tjelle, I. Mathiesen, F. Stevenson, and C.H. Ottensmeier. 2009. DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer. Hum Gene Ther 20:1269-1278.

179. http://www.wyeth.com/animalhealth 180. http://www.ah.novartis.com/aqua/en/index.shtml 181. http://fightcaninecancer.wordpress.com/2010/01/11/new-canine-melanoma-vaccine/ 182. Forde, GM, Rapid-response vaccines- Does DNA offer a solution, Nature Biotechnology, 23, 9, 2005, 1059-1062

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 39-69 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

2. Prophylactic human papillomavirus (HPV) vaccines Diane M Harper1,2 ,3 and Stephen L. Vierthaler1,2

University of Missouri-Kansas City School of Medicine, 1Departments of Obstetrics and Gynecology, 2Community and Family Medicine, 3Biomedical and Health Informatics, Kansas City, MO 64139, USA

Introduction Many HPV associated diseases have been well recognized for centuries.

Common hand and feet warts are caused most often by HPV types 1, 2 and 4; flat warts are most often caused by HPV types 3 and 10. Epidermodysplasia verruciformis, a rare skin cancer, is associated with several HPV types including 5, 8, 9, 12, 14, 15, 17 [1]. Currently there are 120 recognized papillomaviruses that are specific to humans [2]. Papillomaviruses are both species and tissue specific; for instance, canine papillomaviruses causing dog warts do not cause human warts and human hand warts do not cause human genital warts [3]. There are over 40 HPV types that cause human anogenital infections. These types are classified as low, intermediate and high risk HPV types. The trichotomous differentiation is based on the magnitude of the odds ratio association for cervical cancer from epidemiologic studies [4]. HPV 16 is the most commonly associated type for squamous cell carcinoma (SCC) of the cervix causing between 40-60% of SCC [5]. HPV 16 and 18 share nearly equal responsibility for most of the adenocarcinomas of the cervix, together Correspondence/Reprint request: Dr. Diane M Harper, University of Missouri-Kansas City School of Medicine Truman Medical Center Lakewood, 7900 Lee's Summit Road, Kansas City, MO 64139, USA E-mail: diane.m.harper@gmail.com

40

Diane M Harper & Stephen L. Vierthaler

causing about 80% of the adenocarcinomas [6]. Cervical cancer is the second most common cancer among women worldwide where its burden is four fold higher in countries without organized cytology screening [7]. In countries with cervical cytology screening, the incidence of cervical cancer is already well below what the two vaccines can achieve with the current cytology screening programs.

Relevant high risk HPV types Prevalence by HPV types varies widely across the world [8] and by disease state. Table 1 indicates that relatively few women in the general population are infected with HPV 16/18 [9]. Among women who participate in organized cytologic screening, approximately 8% have abnormal cytology results annually. Over half of the abnormal cytology results are atypical squamous cells of undetermined significance (ASC-US) [10]. About 40% of ASC-US results are associated with a broad range of high risk HPV (hrHPV) types [11].

Among women with definitive cytologic abnormalities, the contribution by HPV 16/18 increases with lesion severity. The cytologic abnormality most consistent with HPV infection is the low grade squamous intraepithelial lesion. Approximately 3% of screened women have an LSIL cytology result; among women with LSIL, 35% are infected with HPV 16 and 18 [12]. The cytologic abnormality most consistent with integrated HPV and oncogenic transformation is the high grade squamous intraepithelial lesion (HSIL).

Approximately 0.5% of screened women have an HSIL cytology result; HPV 16/18 are associated with about 50% of HSIL cytology results [13]. Less than 1 in 1000 screened women have cancerous cytology results. Prevention of HPV 16/18 infections by prophylactic vaccination may eliminate about 10% of ASC-US, about 25% of LSIL, about 50% of HSIL and about 70% of cervical Table 1. HPV genotype distribution among women with increasingly severe cervical abnormalities.

Normal women [9]

Women with LSIL [12]

Women with HSIL [13]

Women with squamous cell cervical carcinoma [5]

Women with adenocarcinoma of the cervix [5, 6]

HPV 16

1.8%

27%

45%

54%

31%-43%

HPV 18

0.7%

9%

7%

13%

32%-38%

HPV 31

0.7%

12%

9%

4%

2-3%

HPV 33

0.5%

8%

7%

4%

1%-2%

HPV 45

0.5%

5%

2%

4%

4%-7%

Prophylactic human papillomavirus (HPV) vaccines

41

cancer results. In absolute numbers, prophylactic vaccination provides an overall 17% reduction in abnormal cytology results [14].

Accomplishment of cervical cytology programs The current incidence of cervical cancer in countries with organized cytology screening ranges from 4/100,000 women in Finland to 8/100,000 women in the US [15, 16]. The lowest incidence that cytology screening can accomplish is 2-3/100,000 [17], and the current incidence of cervical cancer in countries without any cytology screening is between 50-80/100,000 women [18].

The reduction from 50-80/100,000 women to the 4-8/100,000 levels was accomplished by repeated cytology screens over a 20 year period of time [19, 20], when at least 70% of the female population above 20 years old participated [21, 22]. When less than 70% of the population participated in routine screening, the incidence of cervical cancer significantly increased in as little as five years [19, 23]. This lesson will become important for HPV vaccinated adolescents.

Cervical cytology screening is cost effective under the following conditions: intervals of every 3-5 years for women older than 20 years, using cytology with HPV triage testing after ASC-US cytology, continuing screening without any stopping age (screening until death), and assuming greater than 70% population participation [24, 25]. Adding a stopping age for a finite number of lifetime screens, as has been done in many European countries, improves the cost effectiveness of cytology screening [26].

Cytology screening programs have been very successful secondary prevention techniques for reducing cervical cancer.

Limitations of screening programs There is no screening or vaccination program, other than for smallpox, that has eradicated a human disease. Ethically, while it is noble to attempt to reduce, eliminate and make extinct infectious diseases, it is not feasible to eradicate HPV, the cause of cervical cancer [27, 28]. Cervical cytology can detect most abnormalities, but even with 100% population coverage, cervical cytology can only reduce the incidence of cervical cancer, at lowest, to 2-3/100,000 women [17]. Cytology requires repeated screenings; when women willfully stop screening, the incidence of cervical cancer increases [23]. About a quarter of women who develop cervical cancer have regularly participated in cytology screening programs with normal results [28]. The psychological process of screening and having an abnormal result provokes a decreased quality of life; this is worsened when the cytology result is falsely

42

Diane M Harper & Stephen L. Vierthaler

positive [29]. Treatment for cervical intraepithelial neoplasias grade 2 and 3 (CIN 2/3) by excisional methods can lead to increased future reproductive morbidity [30-34]. Finally, HPV infections occur as a field effect with autoinoculation and immune suppression leading to recurrent anogenital cancers on average 10 years after the initial CIN 2/3 lesion [35, 36].

HPV vaccines The principal of prophylactic HPV vaccination is neutralization of the HPV virion prior to basal cell entry preventing the infection that may lead to cervical cancer. Basic work shows that high titers of antibodies effectively block both receptors necessary for HPV virion engulfment [37]. The HPV virion presents both a L1 and a L2 outer protein coat for antigen recognition (Figure 1). The L1 proteins are more immunogenic, but are also type dependent,

Figure 1. Model of HPV infection and antibody neutralization [37]. Figure not drawn to scale.

Panel a) depicts HPV infection.

Step 1.There must be scarification of the epithelium all the way to the basement membrane (BM). There can be no HPV infection without the full layer of epithelium being denuded; likewise, there can be no HPV infection if the BM is breached (clinically seen as bleeding from the capillary network immediately below the BM).

Step 2. The denuded BM now exposes available receptors (denoted as heparin sulfate proteoglycan (HSPG) for the L1 proteins on the virion. The L1 protein coat binds to the available receptor. Step 3. This binding causes a conformational change in the outer surface of the HPV coat exposing the L2 protein subsurface. Step 4. The L2 protein surface is now cleaved in a special way (denoted as the action of furin/proprotein

Prophylactic human papillomavirus (HPV) vaccines

43

Figure 1. Legend continued convertase (PC 5/6)) resulting in the exposure of two new epitopes: a crossneutralizing L2 epitope and a previously hidden L1 region. Step 5. Cell receptors on the basal cell bind to the L1 epitope and the cross neutralizing L2 epitope to allow HPV engulfment into the basal cell where transport to the nucleus ensues.

Panel b) depicts neutralization of HPV infection by high antibody concentrations induced by prophylactic vaccine to L1 VLPs 16/18. Both the BM and basal cell receptors are blocked from their tasks.

Step 1. Scarification of the epithelium exposing the BM and its HSPG receptors. Step 2.

Cervicovaginal mucous anti-L1 HPV 16/18 antibodies transudated from the capillary network below the BM in high concentrations bind to the abundant L1 virion sites.

With these sites blocked, the HSPG receptor cannot bind the virion. Step 3. The abundant antibody bindings prevent any conformational change in the outer virion coat. Step 4. Without conformational change, there is no substrate for L2 cleavage.

Step 5. The abundant antibody bindings have blocked infection because the basal cell receptors are unable to bind to the HPV virion and no engulfment occurs.

Panel c) depicts neutralization of HPV infection by low antibody concentrations as when antibody titers are waning. The BM receptor remains fully functional, but the basal cell receptor is blocked.

Step 1. Scarification of the epithelium exposing the BM and its HSPG receptors. Step 2.

The denuded BM now exposes available receptors (denoted as HSPG) for the L1 proteins on the virion. Cervicovaginal mucous anti-L1 HPV 16/18 antibodies transudated from the capillary network below the BM in low concentrations occasionally bind to a few L1 epitopes. Step 3. This binding causes a conformational change in the outer surface of the HPV coat exposing the L2 protein subsurface, but the new epitope exposures do not become functional. Step 4. The induced anti-L1 antibodies prevent stable engagement of the basal cell surface receptor. Step 5.

Without stable basal cell surface receptor binding to the L1 protein coat, virion engulfment cannot occur, hence infection is blocked.

Panel d) depicts neutralization of HPV infection by anti-L2 antibodies, not currently incorporated into the prophylactic vaccines. Because anti- L2 antibodies are not type specific L2 proteins may be included in future generations of the prophylactic vaccines. The BM receptor remains fully functional, but the basal cell receptor is blocked.

Step 1. Scarification of the epithelium exposing the BM and its HSPG receptors. Step 2.

The L1 protein coat binds to the available HSPG receptors. Step 3. This binding causes a conformational change in the outer surface of the HPV coat exposing the L2 protein subsurface. Step 4. The anti-L2 antibodies transudate from the capillary network beneath the basement membrane and prevent stable engagement of the HPV virion with the basal cell surface receptor. Step 5. Without stable basal cell surface receptor binding, virion engulfment cannot occur, hence infection is blocked.

44

Diane M Harper & Stephen L. Vierthaler

whereas the L2 proteins are pan-protective but induce much lower antibody titers [38]. Both prophylactic HPV vaccines contain L1 virus like particles (VLPs). Figure 1 details the mechanism of infection and the mechanism of protection by prophylactic vaccines.

Cervarix contains 20 micrograms each of HPV 16 and HPV 18 L1 VLPs. The AS04 adjuvant was developed to mimic the Toll-like receptor 4 agonist to enhance both cellular and humoral immune responses effecting, in part, stronger and more enduring antibody responses than traditional aluminum salt adjuvants both in animal models and in human studies [4043]. AS04 contains 500 micrograms of aluminum hydroxide and 50 micrograms of 3-O-desacyl-4'-monophosphoryl lipid A (MPL) which is a lipopolysaccharide of Salmonella minnesota R595 [39]. AS04 has been found safe and tolerable in trials and in post marketing surveillance of a Hepatitis B vaccine adjuvanted with AS04 (Fendrix) used in both those with and without an impaired immune system [40-42]. There are additional manufacturing components used in the Baculovirus expression system of Trichoplusia ni insect cells, to result in the final 0.5 ml dose of Cervarix (Table 2) [44].

Gardasil contains 120 micrograms of antigenic L1 VLPs to HPV 16, 18, 6 and 11. This high dose is thought to be the reason for the demyelinating autoimmune neurologic side effects reported after Gardasil, occurring more often in girls than boys [45-52]. The adjuvant, whose general purpose is to maximize duration of vaccine efficacy, is a proprietary aluminum mixture.

There are additional manufacturing components used along with a yeast expression system, to result in the final 0.5 ml dose of Gardasil (Table 2) [53]. Yeast allergies preclude administration of Gardasil.

Table 2. Vaccine composition of a 0.5 ml dose of HPV vaccine.

Prophylactic human papillomavirus (HPV) vaccines

45

Efficacy of HPV vaccines Naive population efficacies in 15/16-26 year old females Table 3 shows that both Cervarix and Gardasil are quite effective in preventing both persistent infection and CIN 2+ caused by HPV 16/18 [54-60].

Table 3. Significant Efficacies in Females 16-26 years old by Endpoint, Population and Time of Follow Up.

Cervarix: 6 month persistent infection is defined as the detection of DNA from the same HPV type in two consecutive cervical cytology samples collected over any 6-month period; 12-month persistence as detection of the same HPV type in all available cytology samples collected over any 12-month period.

Gardasil: Persistent infection means PCR detection of the same HPV type on two occasions at least 4 months apart.

CIN 1+ means a composite endpoint of CIN 1, CIN 2, CIN 3, adenocarcinoma in situ or worse CIN 2+ means a composite endpoint of CIN 2, CIN 3, adenocarcinoma in situ or worse

46

Diane M Harper & Stephen L. Vierthaler

Table 3. Footnote continued ATP-E means according to protocol for efficacy all women who met eligibility criteria and complied with the protocol; who received three injections; whose baseline Pap was normal, ASCUS or LSIL, who were seronegative to HPV 16 and 18 and DNA negative for HPV 16/18 at baseline; cases counted starting one day after the third vaccination.

ATP-E* means those in the ATP-E but HPV type assignment algorithm was used to resolve causation when multiple HPV types were present.

TVC means total vaccinated cohort: women with at least one injection, seropositive or negative, PCR positive or negative for one or more HPV types at baseline, regardless of Pap result; case counting first day after first injection.

TVC-E means total vaccinated cohort for efficacy: women with at least one injection, baseline Pap of normal, ASCUS or LSIL, HPV 16/18 DNA negative at baseline, case counting began the day after the first injection.

PPii means per-protocol: seronegative and PCR negative to HPV 6, 11, 16, 18 at baseline, remained PCR negative for vaccine relevant type through one month post dose 3 and received three doses within one year; cases were counted starting one month after three doses given.

PE means previously exposed population: women who were seropositive for HPV 6/11/16/18 and were PCR negative for HPV 6/11/16/18 at baseline.

PPSP means per protocol susceptible population: for those women who were seronegative and PCR negative for the vaccine related HPV types at study entry regardless of entry cytology results, remained PCR negative for the vaccine relevant HPV types through one month after receipt of the third injection; cases counted one month after three doses given.

USP means unrestricted susceptible population: for those women who were seronegative and PCR negative for the vaccine related HPV types at study entry regardless of entry cytology results, received one or more injections, cases counted from the first day after the first injection.

MITT-3 means the third definition of modified intent to treat in which subjects are included regardless of serostatus or baseline HPV status, regardless of baseline Pap test, and cases counted after 1 day after Month 1.

ITT means intention to treat population: regardless of serostatus or HPV DNA PCR status to vaccine relevant HPV types at study entry, regardless of entry cytology, regardless of timing and number of injections received, cases were counted from day 1 after the first injection.

NPS means naive population simulation: women were seronegative to HPV 6/11/16/18 and PCR negative to HPV 6/11/16/18/31/33/35/39/45/51/52/56/58/59 at baseline, had a normal Pap at baseline, received at least one injection, had at least one follow up visit; cases counted after day 1.

The efficacy for Cervarix is established for at least 8.4 years. The efficacy for Gardasil is established for at least 5 years. Additional protection, as detailed in Table 4, is recognized by the FDA for Cervarix to include CIN 2+ lesions caused by HPV 31, 33 and 45 [61]; Gardasil does not have recognized cross protection against other oncogenic HPV types [62]. Cervarix and Gardasil both reduce the proportion of abnormal cytology screens by about 15% in the population of women continuing to be screened (Table 5) in the proportions anticipated by epidemiologic type distributions described above [59, 63].

Gardasil, under maximal beneficial assumptions of 100% efficacy and lifetime duration, could lower the population incidence of cervical cancer to 14/100,000 women; Cervarix under identical assumptions could lower the population incidence of cervical cancer to 9.5/100,000 women. This will be a substantial

Prophylactic human papillomavirus (HPV) vaccines

47

Table 4. Cross protection offered by the vaccines.

Infection definitions: 6-month definition required the detection of the same HPV type in two consecutive cervical samples, with no negative sample in between, over a minimum of 5 months; CIN 2+ means cervical intraepithelial neoplasia grades 2 and 3 and adenocarcinoma in-situ.

Lesions may be co-infected with HPV 16/18.

TVC means total vaccinated cohort: women with at least one injection, seropositive or negative, PCR positive or negative for one or more HPV types at baseline, regardless of Pap result; case counting first day after first injection.

TVC-N means TVC naive: women who received ≥1 vaccine dose with normal cytology, seronegative for HPV-16/18 and DNA negative for 14 oncogenic HPV types at baseline ((16, 18, 31. 33, 35, 39,45, 51, 52, 56, 58, 59, 66, and 68); case counting starting first day after first injection. Subjects were followed for an average 44.3 months after first injection.

USP-N means unrestricted susceptible population approximating the sexually naive female population where women at baseline had normal cytology and were seronegative and DNA negative for 6, 11, 16, 18; and were DNA negative, regardless of serostatus, for 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59. Subjects were followed for a mean of 3.6 years (43.2 months) after first injection.

Persistent infection means one of the following: detection of the same HPV type in cervicovaginal/anogenital swabs at ≥ 2 consecutive visits spaced ≥6 months apart (± 1 mo window) OR the presence of cervical/genital disease associated with the type-specific HPV DNA detected in cervicovaginal or anogenital swab samples at the visit directly before or after biopsy.

CIN 2+ means a composite endpoint of cervical intraepithelial neoplasia grades 2 and 3 and adenocarcinoma in-situ. Lesions may be co-infected with HPV 16/18.

48

Diane M Harper & Stephen L. Vierthaler

Table 5. Reduction in Abnormal Cytology irrespective of HPV causation over 3-4 years.

Population

ASCUS LSIL HSIL All abnormal cytology

TVC-N TVC-N TVC-N TVC-N

Cervarix [63] Vaccine Efficacy (96.1% Confidence Intervals) 20% (9, 29) 24% (14, 33) 54% (5, 79) 11% (5, 16)

Gardasil [59] Population Vaccine Efficacy (95% Confidence Intervals) USP-N 22% (9, 36) USP-N 17% (9, 24) USP-N 45% (4, 69) USP-N 17% (10, 24)

ASCUS means atypical squamous cells of undetermined significance irrespective of high risk HPV type association.

LSIL means low grade squamous intraepithelial lesion irrespective of HPV type association.

HSIL means high grade squamous intraepithelial lesion irrespective of HPV type association.

TVC-N means TVC naive: a TVC subset including women 15-25 years old who received ≼1 vaccine dose with normal cytology, seronegative for HPV-16/18 and DNA negative for 14 oncogenic HPV types at baseline ((16, 18, 31. 33, 35, 39,45, 51, 52, 56, 58, 59, 66, and 68); case counting starting first day after first injection. Subjects were followed for an average 39.5 months after first injection.

USP-N means unrestricted susceptible population approximating the sexually naive female population where women 16-26 years old at baseline had normal cytology and were seronegative and DNA negative for 6, 11, 16, 18; and were DNA negative, regardless of serostatus, for 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59; received at least one injection and had at least one follow up datum; case counting started after day 1; subjects were followed for a mean of 3.6 years (43.2 months) after first injection.

accomplishment for women's health in countries whose incidence of cervical cancer is currently much higher than the lowest achievable vaccine incidences.

Efficacy in 15/16-26 year old females who were seropositive/DNA negative for the vaccine relevant types at baseline Table 6 details the data showing that there is high efficacy for both vaccines in preventing vaccine HPV type relevant disease in women who have already had prior exposure but are currently without these HPV type specific infections. In women without current infections, Cervarix has a 44 month 89% efficacy against CIN 2+ caused by HPV 16/18 in women who have seroconverted to past HPV 16/18 infection [64]; Gardasil has a 40 month 100% efficacy against condyloma caused by HPV 6/11/16/18 in women who are currently without infection, but seroconverted to past HPV 6/11/16/18 infections [58]. It is important to note in the seropositive/DNA negative populations that HPV vaccines have the same efficacy for those women above 18 years as they do for those very young adolescents.

Prophylactic human papillomavirus (HPV) vaccines

49

Table 6. Vaccine Efficacy in women 15-25 years old who were seropositive/DNA negative for vaccine relevant types at baseline.

Infection definitions: 6-month definition required the detection of the same HPV type in two consecutive cervical samples, with no negative sample in between, over a minimum of 5 months; a 12-month definition required the detection of the same HPV type at consecutive assessments, with no negative samples in between, over a minimum of 10 months.

ATP-E means according to protocol for efficacy all women who met eligibility criteria and complied with the protocol; who received three injections; whose baseline Pap was normal, ASCUS or LSIL, cases counted starting day after the third vaccination.

ATP-E* means those in the ATP-E but HPV type assignment algorithm was used to resolve causation when multiple HPV types were present.

Endpoint definition: Pathology panel consensus diagnosis of lesion with HPV 6, 11, 16 or 18 DNA detected in an adjacent section of the same lesion in frozen tissue adjacent to the lesion, OR in a swab of the lesion, AND in cervicovaginal samples obtained at the visit antecedent to the biopsy visit, with the latter condition not required if vaccine HPV type DNA was detected in an adjacent section of the same lesion.

Exposed population means 16-26 year old women seropositive and DNA negative to the same HPV type at baseline as found in the vaccine type endpoint.

Efficacy with less than three doses of vaccine Early studies indicate that Cervarix is 100% effective for a median of 3 years against HPV 16/18 persistent infection after only one dose [65]. There are no efficacy studies for less than three doses of Gardasil, but early research indicates that two doses of Gardasil induce similar antibody titers 24 months

50

Diane M Harper & Stephen L. Vierthaler

after the first injection as three doses - but only for HPV 16, 6 and 11. The anti HPV 18 titers are significantly less after two doses than after three doses [66].

Efficacy in naive women 24-45 years In older naive women seronegative and DNA negative for the HPV 16, 18 and 6, 11, Gardasil showed 85% efficacy at preventing HPV 16/18 infections, Table 7. Cervarix: less than 3 doses efficacy [65].

Endpoint

Population

Vaccine Efficacy (95% CI)

Median time of follow up

2 doses

TVC*

84% (50, 96)

3 yrs

1 dose

TVC*

100% (67, 100)

3 yrs

One-year persistence was defined as two positive tests for the same HPV type in visits 10+ months apart in women negative at enrollment for that HPV type with no intervening negatives and whose infection occurred after randomization. TVC* means total vaccinated cohort of women who were HPV 16/18 DNA negative at baseline regardless of serostatus, regardless of entry cytology and who had at least one follow up datum. Attack rate of HPV 16/18 infection in the control arm was consistently 4.5-5/100 women over the study.

Table 8. Gardasil efficacy in older women 24-45 years [67].

Ages (years)

Endpoint

24-34

Population

Vaccine Efficacy (95% CI)

Follow up time

86% (64, 96)

44 mo

100% (83, 100)

44mo

82% (36, 97)

44 mo

86% (42, 99)

44 mo

85% (68, 94)

44 mo

95% (80, 99)

44 mo

PP HPV 16/18 related endpoint HPV 6/11 related endpoint

35-45

PP HPV 16/18 related endpoint HPV 6/11 related endpoint

Combined 24-45

PP HPV 16/18 related endpoint HPV 6/11 related endpoint

PP means per protocol population who are women seronegative to the relevant vaccine HPV type at day 1 and PCR negative to that type in cervicovaginal swabs or biopsy samples, or both, from day 1 to month 7. Additionally, women must have received all three vaccinations within 1 year,

Prophylactic human papillomavirus (HPV) vaccines

51

Table 8. Footnote continued and have one or more follow-up visits after month 7. Protocol violators were generally not included. Cases were counted starting at month 7.

Endpoint definition: the combined incidence of vaccine-type-related infection of at least 6 months’ duration or disease of cervical intraepithelial neoplasia or external genital lesions.

Infection definition: Infection of 6 months or more duration was defined as detection of the same HPV type in cervicovaginal or anogenital swabs at two or more consecutive visits spaced at least 6 months apart (1 month visit windows); or presence of cervical or genital disease associated with the relevant type with type-specific HPV DNA detected in cervicovaginal or anogenital swabs at the visit directly before or after the biopsy sample was taken. Of the ten cases in Gardasil arm, 9 were persistent infection and one was disease.

Disease was defined as a tissue sample diagnosed as cervical, vulvar, or vaginal intraepithelial neoplasia; adenocarcinoma in situ; genital warts with type-specific HPV DNA detected. A case was defined as a consensus diagnosis by the pathology panel of one of these end-point events, with vaccine related HPV DNA detected in an adjacent histologic section of the same biopsy specimen regardless of co-infected HPV types.

and 95% efficacy in preventing HPV 6/11 infections [67]. There are no data at this time for CNI 2+ disease prevention in this older naive population.

Likewise, there are no efficacy data available at this time for Cervarix in this population, but the immunogenicity data show that 25-55 year old women mount a peak antibody response to Cervarix that is at least 50 fold higher than natural infection induces (Figure 2) and this response stays many fold higher than natural infection titers through month 48 for both HPV 16 and HPV 18 [68-70].

Efficacy in naive women 16-24 years for non-cervical outcomes Noncervical outcomes comprise less than 8% of all costs for HPV associated diseases, hence contribute only a minor portion to HPV associated cancer control [71]. Vulvar and vaginal cancer precursors caused by HPV 16/18 were quite effectively prevented in this population for 44 months (Table 9); likewise, external genital condyloma caused by any HPV type were prevented 83% of the time for 44 months [72].

Efficacy in naive males 16-24 years In heterosexual males who were, at baseline, seronegative to HPV 6, 11, 16 and 18 and PCR DNA negative to 14 HPV types tested (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) there was an 85% efficacy against external genital condyloma caused by any HPV type that lasted for at least 29 months after Gardasil (Table 10) [73-75].

52

Diane M Harper & Stephen L. Vierthaler

Figure 2. Antibody titers in women 15-55 years of age seronegative at baseline in the ATP cohort over time [68]. GMT means geometric mean titre and is shown with 95% confidence intervals; EU/mL = ELISA units per milliliter. Arrows indicate the vaccination time points (Months 0, 1 and 6). Seropositivity is defined as ≼8 EU/mL for anti- HPV-16 and ≼7 EU/mL for anti-HPV-18. Natural infection is from the GMTs in women seropositive, DNA negative for HPV-16 and HPV-18 from the PATRICIA trial [70].

In males who have sex with males (MSM), the epidemiology of HPV associated anal cancers differs from that documented in heterosexual men.

98% of anal cancers in MSM are attributed to oncogenic HPV infections and in heterosexual men only 75% of anal cancers are attributed to oncogenic HPV infections [76]. It is not surprising, therefore, that anal intraepithelial neoplasia grade 2/3 caused by HPV 6, 11, 16, 18 was shown to be prevented in 74% of MSM for at least 29 months after Gardasil, but its efficacy against AIN 2+ was not an endpoint in heterosexual male trials where the prevalence of disease is much less. (Table 11) [77, 78].

Prophylactic human papillomavirus (HPV) vaccines

53

Table 9. Gardasil efficacy for non cervical outcomes.

Endpoint Population VIN 2/3 caused by HPV 16/18 VaIN 2/3 caused by HPV 16/18 VIN 2/3 caused by any HPV type§ VaIN 1 caused by HPV 6/11/16/18 VaIN 1 caused by any HPV VIN 1 caused by HPV 6/11/16/18† Condyloma caused by HPV 6/11/16/18 Condyloma caused by any HPV type

PP PP ITT PP ITT PP PP PP

Vaccine Efficacy (95% CI)

Average time of follow up

Reference

100% (56, 100)

44 mo

60

100% (50, 100)

44 mo

60

50% (9, 73)

44 mo

60

100% (64, 100)

42 mo

72

31% (4, 51)

42 mo

72

100% (74, 100)

42 mo

72

99% (96, 100)

44 mo

60

83% (74, 89)

44 mo

60

VaIN 1 means vaginal intraepithelial neoplasia grade 1 VIN 1 means vulvar intraepithelial neoplasia grade 1 VIN 2/3 means a composite endpoint of vulvar intraepithelial neoplasia grades 2 or 3 VaIN 2/3 means a composite endpoint of vaginal intraepithelial neoplasia grades 2 or 3 PP means Per Protocol: women were seronegative at baseline and PCR negative for each type Day 1 through Month 7, regardless of entry cytology; cases were counted from the first day after 30 days after the third injection.

ITT means intention to treat population: regardless of serostatus or HPV DNA PCR status to vaccine relevant HPV types at study entry, regardless of entry cytology, regardless of timing and number of injections received, cases were counted from day 1 after the first injection.

PE means previously exposed population: women who were seropositive for HPV 6/11/16/18 and were PCR negative for HPV 6/11/16/18 at baseline. †VIN 1 caused by any HPV was not statistically significant § VaIN 2/3 caused by any HPV type was not statistically significant.

Immunogenicity of HPV vaccines Immune titers induced by Cervarix and Gardasil have been measured in different proprietary assays whose results are not comparable among HPV types nor between brands of vaccine. To abrogate these continued discussions about non-comparable titers, head to head trials were performed with the competitive Luminex assay (cLIA) and the enzyme linked immunosorbent assay (ELISA) to measure induced antibody titers one month after the third

54

Diane M Harper & Stephen L. Vierthaler

Table 10. Efficacy against condyloma in heterosexual men.

Endpoint Population

Vaccine

Average

Efficacy

time of

(95% CI)

follow up

Reference

Condyloma caused by HPV 6/11/16/18

PPE

89% (66-98)

29 mo

74

HPV 6*

PPE

84% (47, 97)

29 mo

74

HPV 11*

PPE

91% (38, 100)

29 mo

74

any HPV type

GHN

85% (62, 96)

29 mo

73

HPV 6/11/16/18

PPE

86% (73, 93)

29 mo

75

HPV 6

PPE

88% (66, 97)

29 mo

75

HPV 11

PPE

93% (57, 100)

29 mo

75

HPV 16

PPE

79% (56, 91)

29 mo

75

HPV 18

PPE

96% (76, 100)

29 mo

75

Persistent infection caused by

PPE means per protocol for efficacy in male trials: seronegative and PCR negative to the relevant HPV type at Day 1, Free of infection with the relevant HPV type through Month 7, Received all 3 doses; cases were counted from the first day after 30 days after the third injection.

Persistent infection means PCR detection of the same HPV type on two occasions at least 4 months apart GHN = Generally HPV Na誰ve (received at least 1 dose of vaccine or placebo; seronegative to 6, 11, 16, and 18 and PCR negative to all 14 types tested (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59); cases counted starting at Day 1). * Male condyloma caused by HPV 16 or 18 were not statistically significant.

dose of HPV vaccine. Figure 3 shows that regardless of assay system used, Cervarix induces several fold higher antibody titers than does Gardasil for both HPV 16 and 18 [79].

Anti-HPV 16 geometric mean titers were measured for Gardasil over the 5 years of the trial [80]. Titers were one hundred fold higher than natural infection titers at peak (Figure 4) followed by a titer plateau at ten-fold higher than natural infection titers through 5 years. Of the 100% of women who seroconverted after 3 doses of Gardasil, 14% completely lost measurable antibody titers to HPV 16 by 8.5 years (Table 12) [81]. Cervarix also induces peak anti-HPV 16 titers over a hundred fold above natural infection titers in

Prophylactic human papillomavirus (HPV) vaccines

55

Table 11. Efficacy in MSM subpopulation.

Endpoint

Vaccine

Average

Efficacy

time of

Population

(95% CI)

follow up

HPV 6

PPE

92 (47, 100)

29 mo

HPV 11

PPE

NS

HPV 16

PPE

94 (60, 100)

29 mo

78

HPV 18

PPE

100 (52, 100)

29 mo

78

Reference

Persistent infection

AIN 2+ caused by HPV 6/11/16/18

PPE

74 (9, 95).

78 78

29 mo

77

Persistent infection means PCR detection of the same HPV type on two occasions at least 4 months apart.

AIN 2+ means a composite endpoint of anal intraepithelial neoplasia grades 2 and 3.

PPE means per protocol for efficacy: seronegative and PCR negative to the relevant HPV type at Day 1, Free of infection with the relevant HPV type through Month 7, Received all 3 doses; cases were counted from the first day after 30 days after the third injection.

Figure 3. Comparative anti-HPV 16 and anti-HPV 18 titers measured in two assays for Cervarix and Gardasil [79]. Immunogenicity comparisons between Cervarix and Gardasil one month after 3rd dose (mo 7) in 18-45 year old women: ELISA vs. cLIA measurement systems. ELISA, Enzyme-linked immunosorbent assay in ELISA units/ml (EU/ml), cLIA, competitive Luminex immunoassay in milliMerck units/ml (mMerckU/ml).

56

Diane M Harper & Stephen L. Vierthaler

Figure 4. Anti-HPV 16 geometric mean titers (GMT) over 5 years [80]. Vaccine induced anti-HPV 16 titers after 3 doses of Gardasil in women 16-26 years of age.

Gardasil has induced sustained antibody titers for HPV 16 more than ten-fold higher than natural infection titers at 5 years after initial vaccination with 98.8% seropositivity maintained measured by the competitive Luminex immunoassay (cLIA) in milliMerck units/ml (mMU/ml). GMT, geometric mean titer; HPV, human papillomavirus; NIT, natural infection titer.

Table 12. Percentage loss of measurable antibody titers by time after initial Gardasil injection.

HPV type

Month 18 [80]

Month 36 [80]

Month 60 [80]

8.5 years [81]

6

2

6

10

-

11

2

4

9

-

16

0

0

1

14

18

14

24

35

-

Antibody titers measured by cLIA

100% of women as well, and maintains a robust response through 8.4 years with no loss of women who seroconverted (Figure 5) [82, 83].

Anti-HPV 18 geometric mean titers after 3 doses of Gardasil returned to natural infection titers by 18 months (Figure 6). At this time 14% of Gardasil recipients did not have any measurable anti-HPV 18 titers [80]. The loss of

Prophylactic human papillomavirus (HPV) vaccines

57

Figure 5. Sustained anti-HPV-16 titers continue through 8.4 years among ATP cohort for immunogenicity [82, 83].

GMT means geometric mean titres for anti-HPV-16 neutralising antibodies by pseudovirion based neutralisation assay (PBNA). *Horizontal line represents the neutralising antibody level in women from a phase III efficacy study who had cleared a natural infection before enrollment.

ATP cohort for immunogenicity means women who met all eligibility criteria, were seronegative at baseline for HPV 16/18, DNA negative for 14 oncogenic HPV types at baseline, normal cytology, complied with study procedures in preceding and current studies and had data available for at least one vaccine antibody blood sample.

PRE means pre-vaccination.

Figure 6. Anti HPV 18 geometric mean titers (GMT) over 5 years [58, 80]. Vaccine induced anti-HPV 18 titers afetr 3 doses of Gardasil in women 16-26 years of age.

Gardasil has induced antibody titers for HPV 18 measured by the competitive Luminex immunoassay (cLIA) in milliMerck units/ml (mMU/ml) that return to natural infection titer levels at 18 months [80] after initial vaccination with significant continued loss of seropositivity over time (Table 12). Natural infection titers do not prevent future type specific infections [58], leaving the woman at risk for new infections. GMT, geometric mean titer; HPV, human papillomavirus; NIT, natural infection titer.

58

Diane M Harper & Stephen L. Vierthaler

Figure 7. Sustained anti HPV 18 titers continue through 8.4 years among ATP cohort for immunogenicity [82, 83].

GMT means geometric mean titres for anti-HPV-18 neutralising antibodies by pseudovirion based neutralisation assay (PBNA). *Horizontal line represents the neutralising antibody level in women from a phase III efficacy study who had cleared a natural infection before enrollment.

ATP cohort for immunogenicity means women who met all eligibility criteria, were seronegative at baseline for HPV 16/18, DNA negative for 14 oncogenic HPV types at baseline, normal cytology, complied with study procedures in preceding and current studies and had data available for at least one vaccine antibody blood sample.

PRE means pre-vaccination.

anti-HPV 18 titers accelerated with time; by 3 years, 24% had lost anti-HPV 18 titers, and by 5 years, 35% of women vaccinated with Gardasil had no further measurable anti-HPV 18 titers [80]. Vaccinating very young adolescents who then approach a very high attack rate of HPV 18 more than 5 years after vaccination may not be protected from abnormal cytologies and their cancers caused by HPV 18.

Cervarix induces anti-HPV 18 titers in 100% of women which stay fourfold higher than natural infection titers at 8.4 years with 100% of women retaining measurable antibody titers [82, 83].

Anti-HPV 6 and anti-HPV 11 geometric mean titers return to natural infection titers 18 months and 12 months, respectively, after the three dose Gardasil series (Figure 8), with up to 10% of women losing measurable titers to HPV 6 or 11 by five years after vaccination (Table 12) [80].

Safety of HPV vaccines In general, at this time, Gardasil and Cervarix are considered safe for most women by US federal and international regulatory bodies. Both vaccines have local injection site reactions that may remain painful, red or swollen for up to 2-3 days after injection. Compatibility of the HPV vaccines with other childhood/adolescent vaccines have been extensively detailed elsewhere, and despite limited follow up are recommended by the regulatory

Prophylactic human papillomavirus (HPV) vaccines

a)

59

b) Figure 8. Anti HPV 6 and anti HPV 11 geometric mean titers (GMT) over 5 years [58, 80]. Induced anti-HPV 6 and anti-HPV 11 titers after three doses of Gardasil in women 16-26 years of age. a) HPV 6 titers overlap with natural infection titers at 18 months after initial vaccination. At natural infection titers, women are susceptible for new infections of HPV 6 [58]. b) HPV 11 titers overlap with natural infection titers at 12 months after initial vaccination. At natural infection titers, women are susceptible for new infections of HPV 11 [58].

authorities [84]. There are extensive case reports in the literature indicating possible autoimmune demyelinating side effects from Gardasil [46-52, 85-95]; these adverse events are being monitored.

60

Diane M Harper & Stephen L. Vierthaler

Cost effectiveness of HPV vaccination Modeling shows that the duration of HPV vaccine efficacy must last at least 15 years for cervical cancers to be prevented. If there is less duration, cervical cancers are merely postponed, not prevented [96]. Any booster injections needed will make vaccination even more costly both because of the cost of an additional dose of cold-storage vaccine and the cost of the population recall and implementation.

At the current pricing of HPV vaccines, US$108.72 and $96.08 for one dose of Gardasil and Cervarix, respectively, [97], regardless of limited or lifetime duration of vaccine efficacy, cervical cancer screening is more cost effective than either HPV vaccination alone or HPV vaccination in combination with cervical cancer screening for the 9-26 year age range of vaccinees [96, 98-110]. The most critical parameter in cost effectiveness analyses is the continued coverage of screening [111]. If less than 70% of women participate in routine cytology screening, even with HPV vaccination, the incidence of cervical cancer will increase. If less than 70% of women participate in routine cytology screening, even with HPV vaccination, the incremental cost effectiveness ratio of HPV vaccination increases making vaccination less cost effective and more expensive. If the duration of HPV vaccination efficacy is not at least 15 years and there is less than 70% of women participating in routine cytology screening, then there is no benefit of HPV vaccination in that population.

Conclusions Using the US and its racial/ethnic populations as an example, HPV vaccination programs will have marginal benefit, if any, depending on the current incidence of cervical cancer already attained. Figure 9 shows the most recently reported incidence rates of cervical cancer in the US by race/ethnicity [112]. These screening rates represent the current participation rate of women in the US. The lowest incidence that Gardasil can achieve is 14/100,000 women, a higher rate than what the Pap screening system has already accomplished for every US racial/ethnic population documented by the CDC. It is clear that complete vaccination of the US female population with Gardasil will not lower the incidence of cervical cancer in the US for women of any racial/ethnic populations beyond what the current participation in screening has already accomplished. While a second generation nonovalent Gardasil vaccine is in trials now, the lowest incidence of cervical cancer potentially achievable if there is 100% efficacy for all seven oncogenic types with lifetime duration of efficacy is still only 9.3/100,000 women.

Prophylactic human papillomavirus (HPV) vaccines

61

15

Incidence per 100,000 women

14 12.8

11.1

10

9.5 7.9

8

7.5 6.4

5

0

American Asian/Pacific Indian Islander

White

Black

Hispanic

Overall

Gardasil vaccine

Cervarix vaccine 2

Figure 9. Incidence of Cervical Cancer in the US [111]. Incidence of Cervical Cancer in the US currently achieved by Pap screening compared to the lowest possible incidence of cervical cancer the HPV vaccines can achieve assuming 100% vaccine coverage and lifetime duration of 100% vaccine efficacy.

The lowest incidence that Cervarix can achieve is 9.5/100,000 women.

Cervarix may be able to lower the incidence of cervical cancer for Black and Hispanic populations in the US if their participation in cytology screening programs does not improve. One dose vaccination with Cervarix is a very promising method of lowering cervical cancer incidence with the minimum of costs and logistics of implementation. Similar changes in national incidences of cervical cancer will be noted globally.

On a patient level, both HPV vaccines can reduce the prevalence of HPV infections in the population and the proportion of cytology screens that are abnormal. By reducing the number of CIN 2/3 lesions developing, vaccination will prevent individual women from excisional surgical procedures which is a large step forward in women's health. In countries with cervical cancer incidence rates above 9.5/100,000 or 14/100,000, the HPV vaccines are expected to reduce cervical cancer incidence proportionally.

References 1.

2.

Tyring S. Human papillomavirus infections: epidemiology, pathogenesis, and host immune response. J Am Acad Dermatol 2000;43(1 Pt 2): S18-S26.

Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen H, de Villiers EM. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology. 2010;401:70-9.

62

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Diane M Harper & Stephen L. Vierthaler

de Villiers EM, Fauquet C, Broker TR, Bernard HU, zur Hausen H. Classification of papillomaviruses. Virology. 2004;324:17-27.

Munoz N, Bosch FX, de SanJose S, Herrero R, Castellsague X, Shah KV, Snijders PJ, Meijer CJ. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348;518-527.

Clifford GM, Smith JS, Plummer M, Munoz N, Franceschi S. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis.

British Journal of Cancer. 2003;88:63-73.b Castellsagué X, Díaz M, de Sanjosé S, Muñoz N, Herrero R, Franceschi S, Peeling RW, Ashley R, Smith JS, Snijders PJ, Meijer CJ, Bosch FX; International Agency for Research on Cancer Multicenter Cervical Cancer Study GroupWorldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors: implications for screening and prevention. J Natl Cancer Inst.

2006 Mar 1;98(5):303-15.

Parkin DM. Bray F. Ferlay J. Pisani P Global cancer statistics, 2002. CA: a Cancer Journal for Clinicians. 2005;55:74-108.

Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, Schiffman MH, Moreno V, Kurman R, Shah KV, International Biological Study on Cervical Cancer Study Group. Prevalence of human papillomavirus in cervcila cancer: a Worldwide; perspective. J Natl Cancer Inst 1995;87:796-802.

Clifford GM, Gallus S, Herrero R, Muñoz N, Snijders PJF, Vaccarella S, Anh PTH, Ferreccio C, Hieu NT, Matos E, Molano M, Rajkumar R, Ronco G , de Sanjosé S, Shin HR, Sukvirach S, Thomas JO, Tunsakul S, Meijer CJLM, Franceschi S,and the IARC HPV Prevalence Surveys Study Group. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005; 366: 991-98.

Eversole GM, Moriarty AT, Schwartz MR, Clayton AC, Souers R, Fatheree LA, Chmara BA, Tench WD, Henry MR, Wilbur DC. Practices of participants in the College of American Pathologists interlaboratory comparison program in cervicovaginal cytology, 2006. Arch Pathol Lab Med. 2010;134:331-5.

Stany MP, Bidus MA, Reed EJ, Kaplan KJ, McHale MT, Rose GS, Elkas JC. The prevalence of HR-HPV DNA in ASC-US Pap smears: A military population study. Gynecol Oncol. 2006 Apr;101(1):82-5.

Clifford GM, Rana RK, Franceschi S, Smith JS, Gough G, Pimenta JM. Human papillomavirus genotype distribution in low-grade cervical lesions: comparison by geographic region and with cervical cancer. Cancer Epidemiol Biomarkers Prev 2005;14(5):1157-64.

Clifford GM, Smith JS, Aguado T, Franceschi S. Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis British Journal of Cancer. 2003;89:101-105.a Schiffman M. Integration of human papillomavirus vaccination, cytology, and human papillomavirus testing.Cancer (Cancer Cytopathol) 2007:111:145-53.

Arbyn M, Autier P, Ferlay J. Burden of cervical cancer in the 27 member states of the European Union: estimates for 2004. Ann Oncol 18(8):1423-5, 2007.

Prophylactic human papillomavirus (HPV) vaccines

63

16. http://seer.cancer.gov/FastStats/html/CERVIX.html#mort [accessed October 1, 2010].

17. Sawaya GF, Grimes DA. New technologies in cervical cytology screening: a word of caution. Obstet Gynecol 1999;94:307-310.

18. Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer 2006; 118:3030-44.

19. Finnish Cancer Registry, www.cancerregistry.fi/tilastot. Accessed Sept. 2010a.

20. Finnish Cancer Registry, www.cancerregistry.fi/joukkltarkastus. Accessed Sept.

2010b.

21. Quinn, M, Babb P, Jones J, Allen E. Effect of screening on incidence of and mortality from cancer of cervix in England: evaluation based on routinely collected statistics. BMJ 318:904-8, 1999.

22. Peto J, Gilham C, Fletcher O, Matthews FE. The cervical cancer epidemic that screening has prevented in the UK. Lancet 364(9430):249-56, 2004.

23. Harper DM, Nieminen P, Paavonen J, Lehtinen M. Cervical cancer incidence can increase despite HPV vaccination. Lancet Infect Dis. 2010;10:594-5.

24. Goldhaber-Fiebert JD, Stout NK, Salomon JA, Kuntz KM, Goldie SJ. Costeffectiveness of cervical cancer screening with human papillomavirus DNA testing and HPV 16/18 vaccination. J Natl Cancer Inst 100:308-20, 2008.

25. Neumann PJ, Sandberg EA, Bell CM, Stone PW, Chapman RH. Are pharmaceuticals cost-effective? A review of the evidence. Health Aff Millwood).

2000; 19 (2): 92-109.

26. Anttila A, Ronco G, Clifford G, Bray F, Hakama M, Arbyn M, Weiderpass E. Cervical cancer screening programmes and policies in 18 European countries. Br J Cancer. 2004 August 31; 91(5): 935-941.

27. Caplan AL. Is disease eradication ethical? Lancet. 2009;373:2192-3.

28. Herbert A, Anshu, Culora G, et al. Invasive cervical cancer audit: why cancers developed in a high-risk population with an organised screening programme.

BJOG. 2010;117(6):736-745.

29. Rogstad KE. The psychological impact of abnormal cytology and colposcopy.

BJOG. 2002;109(4):364-368.

30. Arbyn M, Kyrgiou M, Simoens C, et al. Perinatal mortality and other severe adverse pregnancy outcomes associated with treatment of cervical intraepithelial neoplasia: meta-analysis. BMJ. 2008;337:a1284.

31. Noehr B, Jensen A, Frederiksen K, Tabor A, Kjaer SK. Loop electrosurgical excision of the cervix and risk for spontaneous preterm delivery in twin pregnancies. Obstet Gynecol. 2009;114(3):511-515.

32. Sjøborg KD, Vistad I, Myhr SS, et al. Pregnancy outcome after cervical cone excision: a case-control study. Acta Obstet Gynecol Scand. 2007;86(4):423-428.

33. Jakobsson M, Gissler M, Paavonen J, Tapper AM. Loop electrosurgical excision procedure and the risk for preterm birth. Obstet Gynecol. 2009;114(3):504-510.

34. Acharya G, Kjeldberg I, Hansen SM, Sørheim N, Jacobsen BK, Maltau JM. Pregnancy outcome after loop electrosurgical excision procedure for the management of cervical intraepithelial neoplasia. Arch Gynecol Obstet.

2005;272(2):109-112.

64

Diane M Harper & Stephen L. Vierthaler

35. Edgren G, Sparen P. Risk of anogenital cancer after diagnosis of cervical intraepithelial neoplasia: a prospective population-based study. Lancet Oncol.

2007;8(4):311-316.

36. Kalliala I, Anttila A, Pukkala E, Nieminen P. Risk of cervical and other cancers after treatment of cervical intraepithelial neoplasia: retrospective cohort study.

BMJ 2005;331:1183-5.

37. Day PM, Kines RC, Thompson CD, Jagu S, Roden RB, Lowy DR, Schiller JT. In vivo mechanisms of vaccine-induced protection against HPV infection. Cell Host & Microbe. 2010;8(3):260-70.

38. Jagu S, Kwak K, Garcea RL, Roden RB. Vaccination with multimeric L2 fusion protein and L1 VLP or capsomeres to broaden protection against HPV infection.

Vaccine. 2010 Jun 17;28(28):4478-86.

39. Garcon N, Van Mechelen M, Wettendorff M. Development and evaluation of AS04, a novel and improved adjuvant system containing PL and aluminium salt.

In: Schijns VEJC, O'Hagan DT, editors. Immunopotentiators in modern vaccines.

London: Elsevier Academic Press, 2006:161-77.

40. Boland G, Beran J, Lievens M et al. Safety and immunogenicity profile of an experimental hepatitis B vaccine adjuvanted with As04. Vaccine 2004; 23: 316-20.

41. Thoelen S, De Clercq N, Tornieporth N. A prophylactic hepatitis B vaccine with a novel adjuvanted system. Vaccine 2001;19:2400-3.

42. Tong NK, Beran J, Kee SA, et al. Immunogenicity and safety of an adjuvanted hepatitis B vaccine in pre-hemodialysis and hemodialysis patients. Kidney Int 2005;68:2298-303.

43. Giannini SL, Hanon E, Moris P, et al. Enhanced humoral and memory B cellular immunity using HPV 16/18 L1 VLP vaccine formulated with teh MPL/aluminium salt combination (AS04) compared to aluminium salt only.

Vaccine 2006;24:5937-49.

44. Cervarix [Package Insert]. Rixensart, Belgium: GlaxoSmithKline Biologicals, 2009.

45. Klein SL, Jedlicka A, Pekosz A. The Xs and Y of immune responses to viral vaccines. Lancet Infect Dis. 2010;10(5):338-349.

46. Sutton I, Lahoria R, Tan I, Clouston P, Barnett M. CNS demyelination and quadrivalent HPV vaccination. Mult Scler 2009;15:116-9.

47. Wildemann B, Jarius S, Hartmann M, Regula JU, Hametner C. Acute disseminated encephalomyelitis following vaccination against human papillomavirus. Neurology 2009;72:2132-3.

48. Huang E, Strober J, Lomen-Hoerth C. Demyelination and Severe Loss of Spinal Motor Neurons following HPV vaccination. Abstract number WIP-19. American Neurological Association 134th Annual Meeting. October 13, 2009. Report of motor neuron disease after HPV vaccine-http://www.medscape.com/ viewarticle/ 711461 [accessed October 3, 2010].

49. McCarthy JE, Filiano J. Opsoclonusmyoclonus after human papillomavirus vaccine in a pediatric patient. Parkinsonism Relat Disord 2009;15:792-4.

Prophylactic human papillomavirus (HPV) vaccines

65

50. Cohen SM. Multiple evanescent white dot syndrome after vaccination for human papillomavirus and meningococcus. J Pediatr Ophthalmol Strabismus 2009;25: 1-3.

51. Khalifa YM, Monahan PM, Acharya NR. Ampiginous choroiditis following quadrivalent human papilloma virus vaccine. Br J Ophthalmol. 2010 Jan; 94(1):137-9.

52. Blitshteyn S. Postural tachycardia syndrome after vaccination with Gardasil. Eur J Neurol. 2010 Jul;17(7):e52.

53. Gardasil [Package Insert]. Whitehouse Station, NJ: Merck & Co., Inc. 2009.

54. De Carvalho N, Teixeira J, Roteli-Martins CM, Naud P, De Borba P, Zahaf T, Sanchez N, Schuind A. Sustained efficacy and immunogenicity of the HPV16/18 AS04-adjuvanted vaccine up to 7.3 years in young adult women. Vaccine.

2010 Aug 31;28(38):6247-6255.

55. Paavonen J, Naud P, Salmer贸n J, Wheeler CM, Chow SN, Apter D, Kitchener H, Castellsague X, Teixeira JC, Skinner SR, Hedrick J, Jaisamrarn U, Limson G, Garland S, Szarewski A, Romanowski B, Aoki FY, Schwarz TF, Poppe WA, Bosch FX, Jenkins D, Hardt K, Zahaf T, Descamps D, Struyf F, Lehtinen M, Dubin G; HPV PATRICIA Study Group, Greenacre M. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet. 2009 Jul 25;374(9686):301-14.

56. The GlaxoSmithKline Vaccine HPV-023 Study Group. Sustained efficacy and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 8.4 years. Abstract #632 Presented at ESPID, May 4-8, 2010 57. Villa LL, Costa RL, Petta CA, et al. High sustained efficacy of a prophylactic quadrivalent human papillomavirus types 6/11/16/18 L1 virus-like particle vaccine through 5 years of follow-up. Br J Cancer 2006;95:1459-66.

58. Olsson WE, Kjaer SK, Sigurdsson K, et al. Evaluation of quadrivalent HPV 6/11/16/18 vaccine efficacy against cervical and anogenital disease in subjects with serological evidence of prior vaccine type HPV infection. Human Vaccines 2009; 5:10:696-704 59. Mu帽oz N, Kjaer SK, Sigurdsson K, et al. Impact of human papillomavirus (HPV)-6/11/16/18 vaccine on all HPV-associated genital diseases in young women. J Natl Cancer Inst 2010;102(5):325-39.

60. http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProd ucts/UCM111274.pdf [accessed October 4, 2010].

61. Romanowski B for PATRICIA Study Group. Efficacy of the HPV-16/18 AS04adjuvanted vaccine against non-vaccine oncogenic HPV types: end-of-study results. Presented at 26th International Papillomavirus Conference, Montreal Canada, July 3-8, 2010.

62. Brown DR, Kjaer SK, Sigurdsson K, et al. The impact of quadrivalent human papillomavirus (HPV; types 6, 11, 16, and 18) L1 virus-like particle vaccine on infection and disease due to oncogenic non-vaccine HPV types in generally HPV-naive women aged 16-26 years. J Infect Dis 2009;199:926-35.

66

Diane M Harper & Stephen L. Vierthaler

63. Paavonen J on behalf of the HPV PATRICIA Study Group. Efficacy of HPV 16/18 AS04-adjuvanted vaccine against abnormal cytology, colposcopy, colposcopy referrals and cervical procedures. Presentation # SS 4-1. EuroGin. Monte Carlo, Monaco February 17-20, 2009.

64. Poppe W, Paavonen J, Naud P, Salmerón J, Chow SN, Apter D, Kitchener H, Castellasgué X, Teixeira J, Skinner SR, Hedrick J, Jaisamrarn U, Limson G, Garland S, Szarewski A, Romanowski B, Akoi F, Schwarz TF, Hardt K, Zahaf T, Descamps D, Struyf F, Lehtinen M, Dubin G, for the HPV PATRICIA Study Group. Vaccine efficacy with/without evidence of prior HPV-16/18 infection: analysis of PATRICIA, a phase III trial with AS04-adjuvanted HVP 16/18 vaccine. Presented at ESGO poster #115 Belgrade, Serbia October 11-14, 2009 65. Kreimer AR, Rodriguez AC, HildesheimA, Herrero R, Porras C, Schiffman M, Gonzalez P, Solomon D, Jimenez S, Schiller J. Proof-of-Principle: Efficacy of fewer than 3-doses of a bivalent HPV 16/18 vaccine against incident persistent HPV infection in Guanacaste, Costa Rica. Presented at 26th International Papillomavirus Conference, Montreal, Canada, July 3-8, 2010.

66. Dobson S, Dawar M, Kollmann T, et al. P-690: A two dose HPV vaccine schedule in girls: Immunogenicity at 24 months. Presented at 26th International Papillomavirus Conference, Montreal Canada, July 3-8, 2010.

67. http://www.cdc.gov/vaccines/recs/acip/downloads/min-feb10.txt [accessed October 3, 2010] 68. Schwarz TF, Spaczynski M, Schneider A, Wysocki J, Galaj A, Perona P, Poncelet S, Zahaf T, Hardt K, Descamps D, Dubin G on behalf of the HPV Study Group for Adult Women. Immunogenicity and tolerability of an HPV-16/18 AS04-adjuvanted prophylactic cervical cancer vaccine in women aged 15–55 years. Vaccine 27 (2009) 581-587.

69. Schwarz TF, Spaczynski M, Schneider A, Wysocki J, Galaj A, Schulze K, Poncelet S, Catteau G, Thomas F, Descamps D. Long-term persistence of immune response to HPV-16/18 AS04-adjuvanted cervical cancer vaccine in women aged 15-55 years. Abstract O-6.3 Presented at AOGIN, New Delhi, India, March 26-28, 2010.

70. Paavonen J, Jenkins D, Bosch FX, et al: Efficacy of a prophylactic adjuvanted bivalent L1 virus-like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: an interim analysis of a phase III double-blind, randomised controlled trial. Lancet 2007; 369: 2161–2170.

71. Hu D, Goldie S. The economic burden of noncervical human papillomavirus disease in the United States. Am J Obstet Gynecol 2008;198:500.e1-7.

72. FUTURE I/II Study Group, et al. Four year efficacy of prophylactic human papillomavirus quadrivalent vaccine against low grade cervical, vulvar, and vaginal intraepithelial neoplasia and anogenital warts: randomised controlled trial. BMJ. 2010;341:c3493.

73. http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMateri als/../VaccinesandRelatedBiologicalProductsAdvisoryCommittee/UCM184997.p pt [accessed October 3, 2010].

Prophylactic human papillomavirus (HPV) vaccines

67

74. http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProd ucts/UCM190978.pdf [accessed October 3, 2010].

75. http://www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/ApprovedProd ucts/UCM190977.pdf [accessed October 3, 2010].

76. http://www.who.int/hpvcentre/en/ [accessed October 2, 2010].

77. Palefsky J. Efficacy of the quadrivalent HPV vaccine to prevent anal intraepithelial neoplasia among young men who have sex with men. Presented orally at 26th International Papillomavirus Conference, July 3-8, 2010, Montreal, Canada.

78. Palefsky, J, for The Male Quadrivalent HPV Vaccine Efficacy Trial Team.

Quadrivalent HPV vaccine efficacy against anal intraepithelial neoplasia in men having sex with men. Presentation SS19-2. EuroGin, Feb 17-20, 2010. Monte Carlo, Monaco.

79. Dessy F, Poncelet S, Xhenseval V, MĂŠric D, Dubin G on behalf of the HPV-010 Study Group. Comparative evaluation of the immunogenicity of two prophylactice HPV cervcial cancer vaccines by Merck's competitive Luminex immunoassay (cLIA) and GSK's binding ELISA. Presentation # P IM-5.

EuroGin, Monte Carlo, Monaco, Feb 17-20, 2009.

80. Olsson SE, Villa LL, Costa RL, et al. Induction of immune memory following administration of a prophylactic quadrivalent human papillomavirus (HPV) types 6/11/16/18 L1 virus-like particle (VLP) vaccine. Vaccine 2007;25:4931-4939.

81. Rowhani-Rahbar A, Mao C, Hughes JP, et al. Longer term efficacy of a prophylactic monovalent human papillomavirus type 16 vaccine. Vaccine 2009;27: 5612-5619.

82. De Carvalho N, Teixeira J, Roteli-Martins CM, Naud P, De Borba P, Zahaf T, Sanchez N, Schuind A. Sustained efficacy and immunogenicity of the HPV16/18 AS04-adjuvanted vaccine up to 7.3 years in young adult women. Vaccine.

2010 Aug 31;28(38):6247-6255.

83. The GlaxoSmithKline Vaccine HPV-023 Study Group. Sustained efficacy and immunogenicity of the HPV-16/18 AS04-adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 8.4 years. Abstract #632 Presented at ESPID, May 4-8, 2010.

84. Harper DM. Currently approved prophylactic HPV vaccines. Expert Rev Vaccines. 2009 Dec;8(12):1663-79.

85. Kang LW, Crawford N, Tang ML, et al. Hypersensitivity reactions to human papillomavirus vaccine in Australian schoolgirls: retrospective cohort study.BMJ 2008; 337:a2642.

86. Debeer P, De Munter P, Bruyninckx F, Devlieger R. Brachial plexus neuritis following HPV vaccination. Vaccine 2008;26: 4417-9.

87. Lower J. Two unclear cases of death. Can we still recommend HPV vaccination?

MMW Fortschritte der Medizin 2008;150:6.

88. Lawrence G, Gold MS, Hill R, et al. Annual report: surveillance of adverse events following immunisation in Australia, 2007.Commun Dis Intell 2008; 32: 371-87.

89. Ojaimi S, Buttery JP, Korman TM. Quadrivalent human papillomavirus recombinant vaccine associated lipoatrophy. Vaccine 2009;27:2876-78.

68

Diane M Harper & Stephen L. Vierthaler

90. Marsee DK, Williams JM, Velazquez EF. Aluminum granuloma after administration of the quadrivalent human papillomavirus vaccine. Report of a case. Am J Dermatopathol 2008;30:622-4.

91. Studdiford J, Lamb K, Horvath K, et al. Development of unilateral cervical and supraclavicular lymphadenopathy after human papillomavirus vaccination.

Pharmacotherapy 2008; 28: 1194-7.

92. Brotherton JM, Gold MS, Kemp AS, et al. Anaphylaxis following quadrivalent human papillomavirus vaccination. CMAJ 2008;179:525-33.

93. Das A, Chang D, Biankin AV, Merrett ND. Pancreatitis following human papillomavirus vaccination. Med J Aust. 189(3):178, 2008.

94. Pugnet G, Ysebaert L, Bagheri H, et al. Immune thrombocytopenia purpura following human papillomavirus vaccination. Vaccine 2009;27:3690.

95. Katoulis AC, Liakou A, Bozi E, et al. Erythema multiforme following vaccination for human papillomavirus. Dermatology 2010;220:60-62.

96. Barnabas RV, Laukkanen P, Koskela P, Kontula O, Lehtinen M, Garnett GP. Epidemiology of HPV 16 and cervical cancer in Finland and the potential impact of vaccination: Mathematical modeling analyses. PLoS Med 3(5):e138, 2006.

97. http://www.cdc.gov/vaccines/programs/vfc/cdc-vac-price-list.htm [accessed October 2, 2010].

98. Chesson HW, Ekwueme DU, Saraiya M, Markowitz LE. Cost-effectiveness of human papillomavirus vaccination in the United States. Emerg Infect Dis 14(2):244-51, 2008.

99. Coupe VMH, van Ginkel J, de Melker HE, Snijders PJF, Meijer CJLM, Berkhof J. HPV16/18 vaccination to prevent cervical cancer in The Netherlands: Modelbased cost-effectiveness. Int J Cancer 124:970-978, 2009.

100. de Kok IMCM, van Ballegooijen M, Habbema JDF. Cost-effectiveness analysis of human papillomavirus vaccination in the Netherlands. J Natl Cancer Inst 101:1083-92, 2009.

101. Diaz M, Kim JJ, Albero G, Smith JSS, Clifford G, Bosch FX, Goldie SJ. Health and economic impact of HPV 16 and 18 vaccination and cervical cancer screening in India. Br J Cancer 99(2):230-8, 2008.

102. Ginsberg GM, Edejer TTT, Lauer JA, Sepulveda C. Screening, prevention and treatment of cervical cancer — a global and regional generalized costeffectiveness analysis. Vaccine 27(43):6060-79, 2009.

103. Goldhaber-Fiebert JD, Stout NK, Salomon JA, Kuntz KM, Goldie SJ. Costeffectiveness of cervical cancer screening with human papillomavirus DNA testing and HPV 16/18 vaccination. J Natl Cancer Inst 100:308-20, 2008.

104. Goldie SJ, Diaz M, Constenla D, Alvis N, Andrus JK, Kim SY. Mathematical models of cervical cancer prevention in Latin America and the Caribbean.

Vaccine 26S:L59-L72, 2008.

105. Goldie SJ, Kohli M, Grima D, Weinstein MC. Wright TC, Bosch FX, Franco E. Projected clinical benefits and cost effectiveness of a human papillomavirus 16/18 vaccine. J Natl Cancer Inst 96:604-15, 2004.

106. Jit M, Choi YH, Edmunds WJ. Economic evaluation of human papillomavirus vaccination in the United Kingdom. BMJ 337:a769, 2008.

Prophylactic human papillomavirus (HPV) vaccines

69

107. Kim JJ, Goldie SJ. Health and economic implication of HPV vaccination in the United States. N Engl J Med 359:821-32, 2008.

108. Rogoza RM, Ferko N, Bentley J, Meijer CJ, Berkhof J, Wang KL, Downs L, Smith JS, Franco EL. Optimization of primary and secondary cervical cancer prevention strategies in an era of cervical cancer vaccination: a multi-regional health economic analysis. Vaccine 26S:F46-58, 2008.

109. Techakehakij W, Feldman RD. Cost-effectiveness of HPV vaccination compared with Pap smear screening on a national scale: a literature review. Vaccine 26:6258-65, 2008.

110. Thiry N, De Laet C, Hulstaert F, Neyt M, Huybrechts M, Cleemput I. Cost effectiveness of human papillomavirus vaccination in Belgium: Do not forget about cervical cancer screening. Int J Technol Assess Health Care 25(2):161-70, 2009.

111. Berkhof J et al. Modeling the influence of screening uptake on the future incidence of cervical cancer and the cost-effectiveness of HPV vaccination. Oral Presentation #464. IPV Conference Montreal, July 3-8, 2010.

112. http://apps.nccd.cdc.gov/uscs/cancersbyraceandethnicity.aspx. [accessed Oct 3, 2010].

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 71-89 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

3. Vaccination strategies for the eradication of polio Yash Paul Consultant Pediatrician, Maharaja Agrasen Hospital, Vidhyadhar Nagar Jaipur-302023, India

Abstract. Global polio eradication program through OPV was initiated in 1988, in India it was launched in 1995. Global polio eradication was expected to occur by end of the year 2000. Many remedial interventions like increase in the quantity of P3 vaccine viruses, increase in the number of vaccination rounds were taken.

Oral polio vaccine has effectively reduced the incidence of polio globally and in India many states have been polio free for a long time while occasional polio cases are occurring in some states. On the other hand more than 98% of polio cases occurring in India are being reported from Uttar Pradesh and Bihar, two states which constitute approximately 32% of the country's population.

Monovalent OPV1 and monovalent OPV3 have also been introduced as additional tools in Uttar Pradesh and Bihar. The current polio scenario indicates that current trivalent and monovalent oral polio vaccines cannot eradicate polio from Uttar Pradesh and Bihar because some children from these two states show poor response to OPV. But, the experts project poor vaccine coverage as the reason for failure of eradication program. Increased vaccine coverage in these two states has not resulted in decline in polio incidence. On the other hand polio incidence has increased.

Correspondence/Reprint request: Dr. Yash Paul, A-D-7, Devi Marg, Bani Park, Jaipur-302016, India E-mail: dryashpaul2003@yahoo.com

72

Yash Paul

Thus, there is an urgent need to identify the precise reason(s) for failure of the program in different parts of the world, so that appropriate remedial intervention be taken for different areas.

Vaccination strategies for the eradication of polio In 1988, World Health Assembly, during its 41st meeting, passed resolution 28, declaring that "World Health Organization (WHO) takes initiative for global eradication of polio exclusively by OPV by the year 2000." This resolution is known as WHA-41.28 (World Health Assembly 1988). The task appeared easy and goal attainable because man being the only reservoir for polioviruses and availability of two vaccines (i) trivalent oral polio vaccine (tOPV) and (ii) enhanced inactivated polio vaccine (eIPV).

Initial deadline for global polio eradication was year 2000, which has not been achieved. Though number of countries reporting polio cases has come down from 125 to four countries namely India, Pakistan, Afghanistan and Nigeria, even increased efforts and additional strategies and vaccines have failed to eradicate wild poliovirus. An attempt is made to analyse the reasons for failure of the polio eradication program. Remedial measures can be taken if reasons for failure of the program have been identified.

Was OPV the right choice?

OPV was chosen as a tool for eradication because of the following reasons: 1.

2.

3.

4.

Ease of administration, as it is administered orally.

Low cost.

Being a live vaccine, it was believed to confer long-lasting immunity.

Quick acting vaccine, because of induction of intestinal mucosal immunity (Secretory IgA), immunity being achieved in a matter of days rather than months (Paul JR. 1971), which meant that it could be used in the event of local epidemic.

5. Through secondary spread of live attenuated vaccine virus inducing immunity in close contacts, additional benefit to the community could occur, this issue of collective interest was considered a great beneficial point.

6. Simultaneous administration of OPV to eligible population in campaign mode can effectively block person-to-person transmission of wild poliovirus.

The scientific information regarding limitations of OPV available at the time of launching of global polio eradication in 1988 was as follows:

Vaccination strategies for the eradication of polio

73

1. It can cause vaccine associated paralytic poliomyelitis (VAPP) in the vaccine recipients.

2. Secondary spread of mutant neurovirulent vaccine polioviruses can cause VAPP in close contacts.

3. Some children, specially from tropical and developing countires, show poor response to OPV. But, it was presumed that number of VAPP cases would be small and repeated doses of OPV given to the children during pulse polio campaigns would over come the problem of poor seroconversion in these children. Thus, the policy makers were right in opting for OPV in 1988.

Did we make adequate efforts?

The author shall discuss this issue in the Indian perspective. Pulse polio immunization was started in India in 1995. October 2 and November 14 were assigned for two rounds of pulse polio immunization every year. OPV was initially administered in these campaigns to the children upto two years of age, but later OPV was administered to all the children upto five years of age.

There was a rapid decline in polio incidence in many parts of the country and some states became polio free by 1997 as can be seen in Table 1. But by 1999 it had become clear that polio eradication would not occur by year 2000. The vaccine being administered at that time had 1,000,000 vaccine virus particles of P1; 100,000 vaccine virus particles of P2 and 500,000 vaccine virus particles of P3 per dose of two drops of OPV. In 1999, the quantity of P3 vaccine virus particles was increased from 500,000 to 600,000 particles per dose.

There are 35 states and union territories in India, Uttar Pradesh and Bihar, two states have approximately 32% of the country's population but majority of polio cases are occurring in these two states (Table 1). In 2005, monovalent OPV1 (mOPV1) and later monovalent OPV3 (mOPV3), which are said to be about 3 times more potent than trivalent OPV (tOPV) were introduced in Uttar Pradesh. In 2006 monovalent polio vaccines were introduced as additional tool in Bihar also. In 2007, the number of vaccination rounds in endemic areas was increased to a round every month. All these interventions can be said to be different steps taken in the right direction.

However, despite all these efforts, polio has not been eradicated from Uttar Pradesh and Bihar, on the contrary, polio incidence has risen since 2006.

OPV has eradicated polio from most parts of the world, also from most parts of India, shows that it is an effective vaccine. But, despite concerted efforts polio cases are still occuring in four countries, with sporadic cases mostly by imported wild polioviruses in some more countries. Similarly, in

74

Yash Paul

Table 1. Number of polio cases in different states from 1998 - 2008 as on 27th March, 2010.

India majority of polio cases are occurring in Delhi, Uttar Pradesh and Bihar with occasional cases being reported from other parts.

Factors which have adverse effect on polio eradication 1. Infrastructure facilities Shortage of health workers and facilities, and short supply of vaccine may result in poor vaccine coverage.

2. Social factors Some religious groups or ethnic groups show resistance or reluctance to vaccination because of some beliefs or misinformation which may result in low vaccine coverage.

Vaccination strategies for the eradication of polio

75

3. Environmental factors Poor sanitation and poor hygienic practices help in transmission of causative organisms where spread of infection occurs by faeco-oral route, and overcrowding results in increased chances of spread of air-borne infections.

Poliovirus spreads by both methods, thus, poor sanitation, poor hygienic habits and overcrowding help spread of causative organism, and have adverse effect on polio eradication.

4. High birth rate High birth rate in the community results in increased population of vulnerable individuals who may lead to high transmission of the disease.

5. Vaccine failure In case adequate antibodies to provide protection are not generated after appropirate numbers of doses of a vaccine, it is called a case of vaccine failure. Factors for poor antibody generation by OPV may be in the vaccine and/or in the host.

A. The vaccine The potency of OPV can be affected during manufacture (less chances), transportation or storage (probably), because very efficient cold-chain system is required as OPV happens to be a very heat labile vaccine. During hot summer seasons, electric power is supplied in most parts of India for few hours a day, which may adversely affect the potency of OPV. B. The host Multiple factors in the vaccinee may be responsible for poor response to the vaccine. In the vaccine recipients, the presence of other enteroviruses, malnutrition, immunosuppression due to disease or drugs can be reasons for a poor response to OPV. It is a well-known fact that, in India, and many other countries, doctors, practioners of other systems of medicine and those in fraudulent medical role administer corticosteroids even for trivial ailments (Paul Y, 2007a). Some genetic factors could be responsible for poor response shown to OPV in some children.

6. Mutation in vaccine polioviruses Attenuated vaccine polioviruses are genetically unstable and undergo mutations in the intestinal tract of OPV recipients (Minor PD et al. 1988,

76

Yash Paul

Minor PD et al. 1989). Sometime these vaccine viruses re-acquire neurotoxicity and cause paralysis. It is called vaccine associated paralytic poliomyelitis (VAPP). If it occurs in vaccine recipients it is called recipient VAPP, and if it occurs in close contact of vaccine recipient because of secondary spread of mutant neurotoxic vaccine polioviruses it is called contact VAPP. Though incidence of such cases is extremely low, but it varies in different populations.

Though VAPP has long been recognized, The Global Polio Eradication Initiative currently categorizes vaccine-derived polioviruss (VDPVs) as: 1. Circulating VDPVs (cVDPVs) which emerge in areas with inadequate OPV coverage.

2. Primary immunodeficiency-associated VDPVs (iVDPVs) 3. Ambiguous VDPVs (aVDPVs).

Circulating VDPVs have caused outbreaks in different countries: in 2000 in Hispaniola, in 2001 in Philippines, in 2002 in Madagasca and in 2004 in China (Heymann DL, et al. 2006). Persons with immunodeficiency conditions are known to excrete iVDPV for a long period, which may further spread the disease.

Why has OPV failed in Uttar Pradesh and Bihar in India It can be said that all the factors that have an adverse effect on polio eradication exist in India; in varying degrees in different parts of the country, but not exclusively in Uttar Pradesh and Bihar. Therefore, why do most polio cases occur in Uttar Pradesh and Bihar, while many states have been polio free for a long period and few polio cases occur periodically in other states? It is pertinent to state that the majority of polio cases that are being reported in other states occur in individuals who have migrated from Uttar Pradesh and Bihar (Paul Y. 2009).

A high birth rate in Uttar Pradesh 32.8 per 1000 population and Bihar 31.9 per 1000 population (Mittal SK, Mathew JL 2007) is projected as a reason for failure of eradication of polio from these two states. Dadra and Nagar Haveli, Meghalaya, Madhya Pradesh, and Rajasthan have crude birth rates of 34.9, 28.5, 31.2 and 31.2 per 1000 population respectively. Dadra and Nagar Haveli, and Meghalaya have been polio free for a long time, while occasional cases occur in Madhya Pradesh and Rajasthan (Table 1). Thus, potency of vaccine, vaccine coverage and the hosts may be making substantial contribution in different response to same vaccine in different parts of the country.

Vaccination strategies for the eradication of polio

77

Vaccine The nation has been repeatedly assured that the vaccine being administered to the children is of high potency. Experts had stated: "Hence, it is very reassuring to note that the Oral Polio Vaccine (OPV) used in the country is adequately potent"(Polio Eradication Strategies in India 2006).

Vaccine coverage Vaccine coverage has been variable in different states due to different factors. Vaccine coverage in Uttar pradesh and Bihar during the early phase of polio eradication program in fact, had been very low although records showed high vaccine coverage. It was due to many factors which need not be discussed here. Vaccine coverage has increased appreciably since 2006 because of social mobilisation by the national celebrities and religious leaders, particularly in the Muslim community.

Hosts It was known since long that children in tropical and developing countries respond poorly to OPV (Lee LH, et al. 1964; Poliomyetitis Commission 1964; Drozodov SG et al. 1969; Melnick JL, 1978). Poor seroconversion had been reported from India during 1970s(John TJ 1972; John TJ, Jayabal P, 1972; John TJ 1976). But, precise reasons for poor response were not known. The problem of non-responders to hepatitis B vaccine is known since 1980s and measles vaccine since 1990s(Poland GA, Jacobson RM 1998). In 2004 Newport, et al. (Newport MJ et al. 2004) reported role of genetic factors in antibody response to OPV. Grassly et al. estimated that per dose vaccine efficacy of trivalent OPV for type 1 was 9% (6-13%) for Uttar Pradesh, 18% (9-26%) for Bihar and 21%(15-27%) for rest of India; for type 3, efficacy was 9%(3-15%) for Uttar Pradesh, 22%(4-36%) for Bihar and 21%(8-33%) for rest of India (Grassly NC et al. 2006). Thus, children from Uttar Pradesh show poor response to OPV type 1 and 3, while children from Bihar show slightly poor response to OPV type 1 but better response to OPV type 3. This different response to different strains in OPV by children from these two states cannot be due to environmental factors alone. It is thus possible that due to some genetic factors children from different populations show different response to OPV (Paul Y. 2007b). In a study that was conducted in Pakistan, the immunological response observed for polio vaccine type 3 was statistically significantly higher than those

78

Yash Paul

observed in The Gambia and Oman, but was lower than these observed in Thailand(du Chatelet IP et al. 2003).

As has been stated earlier that different factors which have adverse effects on polio eradication exist all over the country in varying degrees.

Three factors viz., vaccine potency, vaccine coverage and host response to OPV can explain the differences observed in polio incidence in different parts of the country.

It has also been stated earlier that OPV being administered all over the country has been found to be of good potency. In case low potency vaccine is provided to any region(s) time and again polio incidence would be very high in that area(s) only. Thus, presently the number of polio cases in comparison to the eligible population in any area of Uttar Pradesh and Bihar rules out the role of poor quality of OPV playing any big role in continuation of occurrence of polio cases in these two states. It is also being observed that among the polio cases occurring in other states majority of polio cases occur in those children who have migrated from these two states. This also rules out any major contribution due to low potency of OPV. As stated earlier that poor response to OPV had been reported from India (John TJ, 1972, John TJ, Jayabal P, 1972; John TJ 1976), but it would be interesting to note that incidence of vaccine failure is on rise. A study from Delhi for 1989 to 1994 period had noted that in 1989 14% polio cases had received three or more doses of OPV, while 22.9% of polio cases in 1994 had received three or more doses of vaccine before onset of disease (Ahuja B, et al. 1996). This author had reported that in Rajasthan, during 1999, 25% of polio cases had received 6 or more doses (Paul Y. 2000) and, during 2000, 58% polio cases had received 6 or more doses of vaccine (Paul Y. 2002).

According to the data made available by WHO and NPSP, incidence of vaccine failure is on rise as can be seen in Table 2.

Environmental factors influence the speed of spread of the disease, but do not influence the quantity of antibodies generated by the vaccine.

Antibody titre generated by OPV is affected by the potency of vaccine, number of doses of vaccine administered and host respose. As stated earlier that vaccine being administered all over the country is of good potency, thus number of OPV doses administered to a child and the host factors influence the antibody levels in the children. Number of eligible population receiving vaccine i.e., vaccine coverage, number of doses received and response by the vaccinees influence the out-come of polio eradication program. Thus, success or failure of eradication program depends on vaccine coverage and response by the hosts.

The immune individuals who develop immunity following vaccination or natural infection act as barriers in the spread of wild polio viruses in the

Vaccination strategies for the eradication of polio

79

Table 2. Number of OPV doses received by polio cases, 1998-2009.

community. If proportion of such persons reaches about 90%, chances of further spread of wild polioviruses become very low. This forms the basis for mass vaccination campaign. Thus, for polio eradication the proportion of immune population is important.

The problem of poor immunogenicity in the Indian children was known.

The policy makers laid stress on vaccine coverage only. No studies were done during the early phase of polio eradication campaign regarding the seroconversion rates. Study by Kohler et al. (2002) provided the indirect evidence of poor seroconversion in the Indian children. The risk of VAPP is highest with the first dose of OPV (Strebel PM et al. 1992; Nkowane BM et al. 1984; Andrus JK et al. 1995). According to Kohler et al. study (2002) during 1999 in India there were 181 VAPP cases, out of these 60 cases were recipient VAPP cases who had developed paralysis following administration of OPV. Among these 60 cases nine children (15%) developed paralysis following the first dose of OPV, four children (6.7%) after second dose, 15 children (25%) after the third dose and 32 children (53.3%) after the fourth or higher dose. National Polio Surveillance Project data for 2000 from Rajasthan showed that 15 VAPP cases had occurred, but all after the subsequent dose of OPV (Paul Y. 2002). "Why few children in India developed VAPP following first dose of OPV? Plausible explanation for this observation could be that in India the first dose of OPV is given soon after birth or by 6 weeks of age, and the persistent maternal antibodies prevent development of paralysis by mutant neurovirulent vaccine polioviruses as well as by wild polioviruses. Onset of paralysis after subsequent OPV dose, especially after 4th and higher dose indicates that although maternal antibodies had declined or disappeared, OPV administered had failed to generate antibodies levels required for protection. A child who develops paralysis after twelth dose of OPV proves that previous 11 doses of OPV had not generated adequate antibodies, suggesting that children were showing poor response to OPV"(Paul Y. 2008b).

80

Yash Paul

Why did children from different areas show different responses to the same OPV? As had been stated earlier that all the factors which have adverse effect on polio eradication exist all over the country in different degrees in different places. Even if it is presumed that all these adverse factors existed in high proportion in Bihar and Uttar Pradesh, still it can not explain that why 98% of polio cases were occurring in these two states which have about 32% of national population, specially after introduction of special remedial interventions in these two states. As already stated the answer was provided

by Grassly et al. that the per dose vaccine efficacy of tOPV for type 1 was 9% (6 - 13%) for Uttar Pradesh, 18% (9 - 26%) for Bihar and 21% (15 27%) for the rest of India; for type 3, eficacy was 9% (3 - 15%) for Uttar Pradesh, 22% (4 - 36%) for Bihar and 21% (8 - 33%) for the rest of India(Grassley NC et al. 2006). The different response to different strains in OPV shown by children from these two states cannot be due to environmental factors alone, thus suggesting that some genetic factors could be responsible for different response to different strains of OPV by children(Paul Y. 2007b).

Do genes play any role in response to vaccine?

There is overwhelming evidence that genes play important role in resistance or susceptibility to particular diseases. Similarly lately investigators have found role of genetic factors in response to vaccines.

Human leukocyte antigen (HLA) genes are located on the short arm of human chromosome 6 and function by producing proteins that bind with antigenic peptide fragments (produced by processing of foreign protein antigens) and display these to T cells, thereby stimulating an immune response. Thus, the immune response to foreign antigens is at least in part under genetic control by the HLA genes and is intimately related to antigen: HLA binding and presentation (Poland GA, Jacabson RM, 1998). For the most part, class I HLA molecules present processed peptides to CD8+ (suppressor) cells, and the class II molecules present peptides to CD4+ (helper) cells. For both class I and II mechanisms, only those processed peptides that can bind within the peptide binding grooves of the HLA molecule can be presented. Through this mechanism, there is allele-specific restriction of peptide binding and, therefore, restricted antigen presentation limited to specific HLA alleles (Suh WK et al. 1994, Monaco JJ, 1995, Long EO, et al. 1989, Jackson MR, Peterson PA, 1993).

Gm and Km genes code for the antigenic determinants of the heavy and light chains of immunoglobulins, respectively. Gm groups are localized to the constant region of the Îł heavy chain and thus are limited to IgG classes and

Vaccination strategies for the eradication of polio

81

each are exclusive to one of the four IgG subclauses. Gm allotypes are located on chromosome 14. Many of the Gm factors are inherited together.

The Km factors are present on the light chains and thus are represented in all classes of immunoglobulins including IgG, IgM, IgD, IgA, and IgE. The Km allotypes are located on chromosomes 2 (Poland GA, Jacobson RM, 1998).

Investigators have found significant variation in Gm and Km allotypes among different races in human population (Siegrist CA, Lombert PM, 1998; Rosen FS, 1990; Reith W, Mach B, 2001; Singh N et al. 1997).

Role of genetic factors During mass vaccination program, different results have been observed in different parts of the world and also in different parts of India to the same vaccines. This difference in decline in polio incidence could be due to different resistance to wild polioviruses and/or different response to OPV in different ethnic groups. It is well-known and established fact that different individuals respond differently to a disease, drug or vaccine. This could be genetic in origin, as it has been observed that Africans are more susceptible to tuberculosis, some populations like Indians have higher incidence of diabetes.

Till few decades back in India Tribal populations lived in close circles and intermixing of different populations occurred rarely. Although, at present there is no segregation of populations on ethnic or religious basis, still some ethnic groups form major part of the population in different parts of India due to geographical and cultural factors. The author had proposed the hypothesis that some genetic factors could be responsible for different response to OPV in India (Paul Y. 2007b): The polio scenario in India and neighboring countries can be seen in two maps. Horizontal line denote high incidence, vertical line denote low incidence and clear areas represent polio free areas.

1. Tibet, China, Nepal are situated to north of India; Myanmar and Bangladesh are situated to east of India have mongoloid and negrito ethnic population as majority, where polio eradication occurred quickly.

2. Pakistan and Afghanistan do not have mongoloid or negrito ethnic population in any appreciable number, are situated to west of India and are still reporting polio cases.

3. The states and union territories where decline in polio incidence occurred rapidly have higher mongoloid and/or negrito ethnic populations or had been Portuguese or French colonies before becoming part of independent India.

82

Yash Paul

4. Delhi, Uttar Pradesh and Bihar have never been polio free despite addition of monovalent oral poliovaccines type 1 and type 3 and increased vaccine coverage and more doses of vaccine being administered.

5. There are few states where occasional polio cases are occurring. Many of these cases occur in those children who have migrated from Uttar Pradesh or Bihar.

The polio scenario can be seen in the two maps.

Map 1 - 1996 Map 2 - 2001 onwards.

The experts do not think that genetic factors play any role in poor response shown by some children to OPV. It would be pertinent to state that some time back the experts did not consider vaccine failure as a contributing factor in India. In year 2003 the author had raised the issue of vaccine failure

by OPV (Paul Y. 2003 a, Paul Y. 2003 b). Project Manager of National Polio Surveillance Project, WHO, India (Wenger J. 2003) had stated "Thus, the outbreak of 2002, and the problems of polio eradication were not caused by

Figure 1. States of India and neighboring countries.

Vaccination strategies for the eradication of polio

83

Figure 2. States of India and neighboring countries.

failure of OPV or occurrence of VAPP but failure to vaccinate children adequately. The number of polio cases between March and July 2003 in these states (Uttar Pradesh and Bihar) is at its lowest ever. Successes like these clearly demonstrate that polio eradication will succeed in India." Chairman Polio Eradication Committee of the Indian Academy of Pediatrics (John TJ. 2003) had stated: "I disagree with Dr. Paul's audacious prediction that India will not succeed to eradicate polio unless his three directives are followed.

His first directive to take appropriate remedial measures for the high incidence of vaccine failure is superfluous".

In 2005 again the author (Paul Y, 2005) had stated: "On the other hand, it can be said that the present eradication program (with OPV) ensures that polio cases will continue to occur owing to vaccine failure and due to mutant vaccine polioviruses."

As stated earlier Grassly et al. (Grassly NC et. al. 2006) had observed in their study that children from Uttar Pradesh show poor response to OPV1 and OPV3, and children from Bihar show slightly poor response to OPV1, but show superior response to OPV3. This different response to different vaccine

84

Yash Paul

components by two different populations cannot be due to environmental factors or malnutrition and intercurrent infections in the hosts which may specifically improve response of hosts from Bihar to OPV3. It clearly suggests some genetic factors could be responsible for this (Paul Y, 2007 b).

According to polio incidence India can be divided in three groups.

1. Those states where no polio case has been reported 2001 onwards. There are fourteen such states (Table 1). There were 265 polio cases in the year 2000, and 268 cases in 2001, but there were 1600 polio cases in 2002, still no polio case was reported from these states. Among the seven north-east states also called 'seven-sisters' Assam is the only state which has not eradicated polio. Among these seven states Assam has highest 'non-local' or migratory population.

2. Those states where polio incidence has declined slowly, few cases occur, but not every year.

3. Those states which have not become polio free, polio cases occur every year. There are three such states namely Delhi, Uttar Pradesh and Bihar.

Uttar Pradesh and Bihar are endemc states, Delhi needs to be considered as special state because it is the capital of India, and thus people from all over the country are found here. Among the polio cases occurring in Delhi most of them have migrated from Uttar Pradesh and Bihar. This also suggests that children from these two states show poor response to OPV.

Need for re-appraisal of polio eradication strategy Public health programs focus intervention at level of population. Such programs are organised for the benefit of whole or some specific section of population. For example under five children are target population for global polio eradication. It is presumed that pre-launch evaluation of the global polio eradication program regarding feasibility, acceptibility and relative benefits and harms which would accrue from the program had been done.

There is also a need for periodic ongoing evaluation of the program to assess if the program is providing high benefits. In case it is found that less benefit and/or more harm is occurring, then honest re-evaluation of the whole program should be done; and if needed the program be modified, suspended for some time or even abandoned.

It should be acknowledged that trivalent OPV has reduced the global burden of polio incidence but has not eradicated polio despite many remedial interventions and extensions of polio eradication deadline. It should be considered an achievement that polio cases are occurring in some limited

Vaccination strategies for the eradication of polio

85

areas only, but policy makers may have to take some tough decisions. The continuation of occurrence of polio cases in some areas despite addition of monovalent oral polio vaccines type 1 and type 3 could be due to some deficiency in vaccine potency, low vaccine coverage, or some factors in the vaccine recipients for poor response to vaccine or combination of all these factors. Another issue of concern for the policy makers should be the observation that incidence of vaccine failure is on rise as can be seen in Table 2.

There is an urgent need to identify the precise reasons for failure to eradicate polio, so that appropriate remedial measures be taken. Blaming people for poor vaccine coverage as the only reason for failure of eradication program and extending deadline repeatedly should be stopped.

What needs to be done?

Multitude factors like financial resources, human resources, infrastructure facilities, potency of vaccine, number of doses received by individuals, number of immunized persons, nutritional status, immune status and genetic factors in hosts all play role in success or failure of polio eradication program. Over crowding, poor sanitation help in rapid spread of the disease, but do not play a direct role on antibody formation. On the other hand potency of vaccine, immune status and genetic factors influence the antibody formation in response to the vaccine. These factors play role in different combinations and proportion in different populations and settings.

Thus, it is necessary to identify the factors which have significant effect on the program for different areas, to introduce right interventions. For example if vaccine coverage is poor, it needs to be improved, but, if vaccine coverage is good and children have been administered appropriate number of doses of vaccine, that would suggest that some host factors are playing negative role.

In such situation it is necessary to identify the factors which are responsible for poor antibody generation like malnutrition, inter-current infections specially by enteroviruses, immunosuppression or genetic factors for remedial interventions. At this point of time no remedial interventions are available if poor response to OPV is due to some genetic factors.

If poor vaccine coverage and less than appropriate doses of vaccine are found to be the major contributing factors in failure of polio eradication, all efforts should be made to improve vaccine coverage. In case vaccine coverage has improved but, polio incidence has not declined then there is a definite need to change the strategy and vaccine as is the situation in Uttar Pradesh and Bihar in India.

In the past vaccine coverage in these two states especially among Muslim children was very poor, although official figures showed high vaccine

86

Yash Paul

coverage. For last 3-4 years vaccine coverage has tremendously improved due to efforts of celebrities, religious and social leaders. During this period monovalent OPV vaccines have been added and number of vaccination rounds increased as stated earlier, but, polio incidence has risen instead of showing decline. But policy makers intend to introduce bivalen oral polio vaccine. This shows that experts are not willing to face the facts and accept the reality or are living in a make believe world of their own.

In the May 2008 issue of Indian Pediatrics the WHO experts stated: "do we need a change in the polio eradication strategies? The strategies have eliminated polio from all but two states in India (and more than 120 countries globally)" and that "Uttar Pradesh may have already interrupted poliovirus type 1 transmission and also appears to be on track to probably interrupt type 3 transmission in 2008"(Sutter RL et al. 2008). It is an irony that polio scenario in India has shown a sea change since then. In the week ending June 7, 2008, one case of polio by P1 had appeared in Uttar Pradesh. But in 2008 out of 75 polio cases by P1, which occurred in India 62 polio cases had occurred in Uttar Pradesh. Even during 2009 as on March 27, 2010, 741 polio cases had occurred in 2009 in India and out of 80 polio cases by P1 in India, 34 polio cases had occurred in Uttar Pradesh. The experts predicted interruption of wild polio-virus type 3 in Uttar Pradesh in 2008, but there were 243 polio cases by P3 in Uttar Pradesh in 2008, and as on March 27, 2010, 569 polio cases by P3 have occurred.

It would be prudent for the policy makers to acknowledge the limitations of OPV, despite the fact that OPV has drastically reduced polio incidence.

There is need for finding the precise reasons for failure of polio eradication program in these four countries, which may differ not only from country to country, but, also for different parts of a country. The author would suggest following approach for Uttar Pradesh and Bihar in India (Paul Y. 2010): (i) Find the reasons for poor response, identify such children, take remedial interventions and then administer OPV. Does not appear to be feasible. (ii) Indentify poor responders, to be administered IPV selectively and good responders be administered OPV. Does not appear to be feasible. (iii) Administer IPV to all eligible children from Bihar and Uttar Pradesh.

"The policy makers will have to redefine the strategy for rest of India.

Polio cases are not occurring in many states for a long time following OPV administration only. Because of constraints of resources OPV may be continued in these states, but, sooner or later OPV will have to be discontinued all over India to stop VAPP cases and avoid any risk to the community by VDPVs"(Paul Y. 2009).

Vaccination strategies for the eradication of polio

87

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

Ahuja B, Gupta VK, Tyagi A. (1996). Paralytic poliomyelitis (1989 - 1994): report from a Sentinel Centre. Indian Pediatr 33: 734-745.

Andrus JK, Strebel PM, de Ruadros CA, Olive JM. (1995). Risk of vaccineassociated paralytic poliomyelitis in Latin America 1989 - 1991. Bull World Health Oran 73: 33-40.

Drozodov SG, Cockburn WC. (1969). The state of poliomyelitis in the World. In: Proceedings of the 1st international conference on vaccines against Viral and Rickettsial diseases in Man. Washington, DC: Pan American Health Organization: pp. 198-209. du Chatelet IP, Merchant AT, Fisher-Hoch S, et al. (2003). Serological response and poliovirus excretion following different combined oral and inactivated poliovirus vaccines immunization schedules. Vaccine 21: 1710-1718.

Global Polio Eradication Initiative. Annual Report 2008. World Health Organization, 2009.

Grassly NC, Frazer C, Wenger J, Deshpande JM, Sutter RW, Heymann DL, et al.

(2006). New strategies for elimination of polio from India. Science 314: 1150-1153.

Heymann DL, Sutter RW, Aylward RB. (2006). Polio eradication: interrupting transmission, towards a polio free world. Future Virol 1: 181-188.

Jackson MR, Peterson PA. (1993). Assembly and intra-cellular transport of MHC class I molecule. Annu Rev Cell Biol, 9: 207-235.

John TJ, Jayabal P. (1972 a). Oral polio vaccination of children in tropics. The poor seroconversion rates and the absence of viral interference. Am J Epidemiol 69: 263 - 269.

John TJ. (1972 b). Problems with oral polio vaccines in India. Indian Pediatr 9: 252-256.

John TJ. (1976). Antibody response of infants in tropics to five doses of oral poliovaccine. BMJ 1: 811-812.

John TJ. (2003). Polio eradication in India (Reply). Indian Pediatr 40: 1102-1104.

Kohler KA, Bannerjee K, Hlady WG, Andrus JK, Sutter RW. (2002). Vaccine associated paralytic poliomyelitis in India during 1999: decreased risk despite massive use of oral polio vaccine. Bull WHO 80: 210-216.

Lee LH, Wenner HA, Rosen L. (1964). Prevention of poliomyelitis in Singapore by live vaccine. BMJ 1: 4077-4080.

Long EO, Jacobson S. (1989). Pathways of viral antigen processing and presentation to CTL: defined by the mode of virus entry. Immunol Today 10: 45 - 48.

Melnick JL. (1978). Advantages and disadvantages of killed and live poliomyelitis vaccine. Bull WHO 56: 21 - 38.

Minor PD, Dunn G. (1988) The effect of sequences in the 5' non-coding region on the replication of polioviruses in the humangut. J Gen Virol 69: 1091-1096.

Minor PD, Dunn G, Evans DMA, Magrath DI, John A, et al. (1989) The temperature sensitivity of the Sabin type 3 vaccine strain of polioviruses:

88

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Yash Paul

molecular and structural effects of a mutation in the caspid protein VP3. J Gen Virol 70: 1117-1123.

Mittal SK, Mathew JL. (2007). Polio eradication in India: the way forward.

Indian J Pediatr 74: 153-160.

Monaco JJ. (1995). Pathways for the processing and presentation of antigens to T cells. J Leukoc Biol 57: 543-547.

Newport MJ, Geotghebuer T, Weiss HA, Whittle H, Siegrist CA, Marchant A. (2004). Genetic regulation of immune responses to vaccines in early life. Genes Immun 5: 122-129.

Nkowane BM, Wassilak SGF, Orenstein WA, et al. (1987). Vaccine-associated paralytic poliomyelitis. United States: 1973 through 1984. JAMA 257: 1335-1340.

Paul JR. (1971). A History of Polio Myelitis, New Haven: Yale University Press 1971.

Paul Y. (2000). Polio Eradication Strategy: Need for Re-appraisal. Indian Pediatr 37: 913-916.

Paul Y. (2002). Accuracy of the National Polio Surveillance Project Data in Rajasthan. Indian J Pediatr 69: 667-673.

Paul Y. (2003 a). Can polio be eradicated from India through present Polio Eradication Program? BMJ (South-Asia edn.) 19: 499-501.

Paul Y. (2003 b). Polio eradication in India. Indian Pediatr 40: 1100-1101.

Paul Y. (2005). Let us face the facts and accept the reality. Indian Pediatr 42: 728-729.

Paul Y. (2007a). What needs to be done for polio eradication in India. Vaccine 25: 6431 - 6436.

Paul Y. (2007 b). Role of genetic factors in polio eradication: New challenge for policy makers. Vaccine 25: 8365-8371.

Paul Y. (2008 a). OPV cannot eradicate polio from India: do we need any further evidence? Vaccine 26: 2058-2061.

Paul Y. (2008 b). Why has polio eradication program failed in India? Indian Pediatr 45: 381-388.

Paul Y. (2009). Oral polio vaccines and their role in polio eradication in India.

Expert Rev Vaccines 8(1): 35-41.

Paul Y. (2010). Polio eradication in India: Have we reached the dead end?

Vaccine 28: 1061-1062.

Poland GA, Jacobson RM. (1998). The genetic basis for variation in antibody response to vaccines. Curr Opin Pediatr 10: 208-215.

Polio Eradication Strategies in India: Recommendations under IAP Action Plan 2006. Indian Pediatr 2006; 43: 1057-1063.

Poliomyelitis Commission, Western Region Ministry of Health, Nigeria (1966).

Poliomyelitis vaccination in Ibadan, Nigeria during 1964 with oral vaccine. Bull WHO 34: 865-876.

Reith W, Mach B. (2001). The bare lympocyte syndrome and regulations of MHC expression. Annu Rev Immunol 19: 331-373.

Rosen FS. (1990). Genetic deficiences in specific immune responses. Semin Hematol 27: 333-341.

Vaccination strategies for the eradication of polio

89

40. Siegrist CA, Lamber PH. (1998). Maternal immunity and infant responses to immunization: factors influencing infant responses. Dev Biol stand 95: 133-139.

41. Singh N, Agrawal S, Rastogi AK. (1997). Infectious diseases and immunity special reference to major histocompatibility complex. Emerg Infect Disease 3: 41-49.

42. Strebel PM, Sutter RW, Cochi SL, et al. (1992). Epidemiology of poliomyelitis in the United State one decade after the last reported case of indigenous wild virusassociated disease. Clin Infect Dis 14: 568-579.

43. Suh WK, Cohen-Deyle ME, Fruh K, Wang K, Peterson PA, Williams DB. (1994). Interaction of MHC Class I molecules with the transporter associated with antigen processing. Science 264: 1322-1326.

44. Sutter RL, Jafari H, Aylward B. (2008). IAP Recommendations on Polio Eradication and Improvement of Routine Immunization. Indian Pediatrics 45: 353-355.

45. Wenger J. (2003). Polio eradication in India. BMJ (South-Asia edn.) 19: 684-687.

46. World Health Assembly 1988. Global Eradication of Poliomyelitis by the year 2000: resolution of the 41st World Health Assembly. Geneva (Switzerland): World Health Organization, (Resolution WHA 41.28).

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 91-110 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

4. The future of smallpox vaccines Andrew W. Artenstein Physician-in-Chief, Department of Medicine, Director, Center for Biodefense and Emerging Pathogens, Memorial Hospital of Rhode Island, Professor of Medicine and Community Health, The Warren Alpert Medical School of Brown University, USA

Abstract. Smallpox, eradicated as a cause of natural disease through an intensive global vaccination effort in the latter part of the twentieth century using live vaccinia virus, has resurfaced as a possible agent of bioterrorism. For this reason, there is renewed interest in smallpox vaccines. Although live vaccinia virus, a related orthopoxvirus, has a long and successful clinical track record as an effective smallpox vaccine, its use is associated with uncommon yet serious adverse events. This has led to a surge of recent research into newer generation smallpox vaccines with the potential of improved safety profiles yet retained efficacy.

For nearly two centuries after Jenner’s landmark experiments with smallpox vaccination in 1798, the general strategy of engendering vaccineinduced protection against smallpox remained largely unchanged. Jenner’s work provided the first scientifically rigorous demonstration that cowpox could be used in lieu of variolation, the deliberate intradermal inoculation of virulent variola virus into uninfected hosts; the former approach became widespread in the Western world by the early part of the nineteenth century, resulting in relative control of epidemic smallpox [1]. At some point, most likely in the late Correspondence/Reprint request: Dr. Andrew W. Artenstein, Department of Medicine, Memorial Hospital of RI, 111 Brewster Street, Pawtucket, RI 02860, USA. E-mail: artenstein@brown.edu

92

Andrew W. Artenstein

nineteenth or early twentieth centuries and for unknown reasons, the nature of the vaccine virus changed. The “new” vaccine virus, known as vaccinia and later proven to be a distinct species of orothopoxvirus, was neither a natural cause of human nor animal disease. The origins of vaccinia have never been completely elucidated; the virus may have either emerged as the result of genetic hybridization between cowpox and variola viruses, from selection via multiply passaged cowpox under laboratory culture conditions, or as the virologic vestige of a previously extant orthopoxvirus species, such as horsepox [2,3]. Nonetheless, intradermal vaccinia, like cowpox, provided robust cross-protective immunity against smallpox, as evidenced by its proven track record of effectiveness [4].

Smallpox was eradicated as a cause of natural human disease after an intensive global campaign in the 1960s and 1970s by the World Health Organization (WHO) and sponsoring countries using live vaccinia virus [5].

The last naturally acquired case of smallpox occurred in Somalia in 1977 [5]; the last known human case occurred in 1978 as a result of inadvertent laboratory exposure [5,6]. The routine use of smallpox vaccination essentially ceased in the early1970s, although it continued to be administered in selected populations into the late twentieth century. In late 2002, the U.S. reinstituted large-scale vaccination of its military forces and civilian health care workers using live vaccinia in response to a perceived threat of bioterrorism involving smallpox. Concurrently, other nations have developed extensive stockpiles of vaccines; it is estimated that current capabilities would be sufficient to vaccinate approximately ten percent of the world’s population [7]. Concerns over bioterrorism, along with safety issues regarding the use of live vaccinia, have galvanized research into novel strategies of vaccination against this ancient scourge of nature and current biothreat agent. The risk-versus-reward discussion of pre-exposure vaccination against smallpox has been recently presented [8]; this work will review the status of new smallpox vaccine products.

First generation smallpox vaccines “First generation” smallpox vaccines, derived from Jenner’s original formulation and comprising a variety of live vaccinia viruses [9], possess a proven track record of clinical effectiveness highlighted by their success in the global eradication campaign of the 1970s [5]. While immune determinants of protection against smallpox remain incompletely understood, the historical record provides ample data concerning a clinical correlate of protection in humans; observations from the use of variola, cowpox, and subsequently vaccinia viruses document the direct relationship between a

The future of smallpox vaccines

93

vaccine-associated major cutaneous reaction, or “take”, and protection against smallpox [5,10,11]. The protection appears to be of long duration [12] and to correlate with the presence of neutralizing antibodies [5,9]. The cellular arm of the immune response is also known to have a significant role in containing vaccinia [13] and, by extrapolation, variola; smallpox vaccination induces robust and durable vaccinia-specific cytotoxic T-lymphocytes (CTL) and gamma interferon production by T cells in naïve recipients, and these may correlate with neutralizing antibody responses [14].

The original production method of first generation vaccines involved scarification of calf, sheep, or water buffalo skin and viral isolation from skin scrapings containing pus, serum and extruded lymph [5,15]. The resultant liquid suspension of vaccine or “wet” lymph contained viable bacteria, largely skin commensals that were minimized by the use of glycerol and later phenol in processing [5]. By the 1950s liquid vaccine lymph preparations had largely been replaced by lyophilized preparations that enhanced preservation of vaccinia virus viability [5]. Vaccine production by animal scarification was abandoned more than twenty-five years ago and because smallpox had been eradicated, essentially no first generation vaccine has been manufactured since then. This led to the view in 2001 that the stockpiled supply was insufficient to cope with a potential large-scale bioterrorist threat.

The stockpile consisted of lymph-derived vaccinia, largely the last production lots of Dryvax-brand smallpox vaccine, manufactured by Wyeth Laboratories using the New York City Board of Health (NYCBOH) strain of vaccinia.

Multiple studies have since demonstrated that existing stockpiles can be expanded by diluting vaccine; lymph-derived, live vaccinia products retain surrogate clinical efficacy at ten-fold dilutions in both vaccinia-naïve and vaccinia-experienced subjects [8,15,16].

Second generation smallpox vaccines Second generation smallpox vaccines (Table 1), in which full-strength vaccinia virus is grown in tissue culture rather than in the skin of large mammals, possess theoretical advantages conferred by this modern manufacturing technique: improved sterility, lowered risk of contamination by adventitious agents [17], viral genetic homogeneity, and relative ease of large scale, consistent production. ACAM1000, a clonal isolate derived from Dryvax and grown in human diploid lung cells (MRC-5), demonstrates similar immunogenicity and cutaneous efficacy at comparable doses to the Dryvax gold standard in animal models and demonstrates an improved safety profile in preclinical neurovirulence studies in suckling mice and rhesus macaques [18,19]. ACAM2000, derived from the ACAM1000 master virus

94

Andrew W. Artenstein

Table 1. Smallpox vaccines and vaccine candidates, 2008.

Platform

Product

Parent

Rationale for its use

strain FIRST GENERATION Lymph-derived

Dryvax (Wyeth)

vaccinia virus

NYCBH (New

Historical experience in

York City

U.S. through the era of

Board of

routine use

Health) SPSV (Sanofi-Pasteur

NYCBH

smallpox vaccine)

Produced in 1956-1957 and used in U.S. program of that era; in frozen storage since

Elstree-RIVM (master

Lister

Historical experience in

seed stock held at the

Intensified Smallpox

National Institute of

Eradication Programme

Public Health in The Netherlands—RIVM) SECOND GENERATION Replication

ACAM2000

NYCBH

Defined manufacturing

competent tissue-

(Acambis): cloned

process; reduced theoretical

cultured vaccinia

virus grown in Vero

risk of adventitious agents

virus

cells

compared with lymphderived vaccine; less neurovirulent in animal models

Elstree-BN (Bavarian-

Lister

Nordic)

Defined manufacturing process; reduced theoretical risk of adventitious agents compared with lymphderived vaccine

THIRD GENERATION Replication

LC16m8 vaccine:

competent,

derived from 53 serial

Japanese children in 1973-

highly attenuated

passages in rabbit

1975; better safety profile

vaccinia virus

kidney cells;

than traditional live

temperature sensitive,

vaccinia, less neurovirulent

small-plaque

in animals, but unproven

phenotype due to

clinical efficacy

mutation in B5R gene

Lister

Experience in >100,000

The future of smallpox vaccines

95

Table 1. Continued Ankara

Theoretically improved

Replication

Modified vaccinia

deficient, highly

Ankara (MVA):

safety profile, especially for

attenuated

derived from >570

those in whom live vaccinia

vaccinia virus

serial passages in

is contraindicated. Used in

chicken embryo

120,000 primary vaccinees

fibroblasts:

in Germany in 1970s;

IMVAMUNE

unproven clinical efficacy

(Bavarian-Nordic); TBC-MVA (Therion) NYVAC (Sanofi-

Copenhagen

Theoretically improved

Pasteur): attenuated by

safety profile, especially for

the deletion of 18 open

those in whom live vaccinia

reading frames from a

is contraindicated

plaque-cloned vaccinia isolate dVV-L: derived from

Lister

Theoretically improved

deletion of UDG gene

safety profile and can be

needed for viral

manufactured in cell line

replication

that complements UDG deficiency, thus increased capacity for rapid production

Subunit vaccines

Recombinant proteins;

Vaccinia

Theoretically improved

Plasmid DNA

viruses,

safety profile

different sources

by three additional passages in Vero cells [20], has nearly identical biological characteristics to those of its progenitor in animals [21].

Randomized phase II and III clinical trials in which nearly 1100 vaccinia-na誰ve subjects were vaccinated with ACAM2000, demonstrated its non-inferiority compared to first generation Dryvax at similar vaccinia virus inocula, using cutaneous responses (i.e. takes) as an efficacy endpoint; ACAM2000 did not meet the non-inferiority measure using geometric mean neutralizing antibody titers (GMT) on day 30 after vaccination as another efficacy endpoint [20, 22]. In vaccinia-experienced subjects, ACAM2000 only met the non-inferiority threshold for the GMT endpoint but not for cutaneous responses [22]. Nonetheless, in August 2007, ACAM2000 became

96

Andrew W. Artenstein

the inaugural second generation smallpox vaccine to be licensed for human use by the U.S. Food and Drug Administration (FDA), leading to the delivery of 192.5 million doses to the U.S. government for stockpiling purposes [23].

The vaccine received the following clinical indication: “active immunization against smallpox disease for persons deemed to be at high risk for smallpox infection” [22]. ACAM2000 is not expected to be commercially distributed in the U.S. in order to minimize its use and therefore its attendant risk [24].

CCSV, another second generation vaccine grown in MRC-5 cells, compared favorably with Dryvax in a single-center study of 150 vaccinia-naïve and 100 vaccinia-experienced subjects [25]. However, this agent was apparently “deselected” by the manufacturer for further advancement.

Third generation smallpox vaccines Despite the theoretical advantages conferred by second generation vaccines, they comprise replication competent, virulent vaccinia viruses and therefore are potentially associated with a number of the uncommon but welldescribed serious adverse events of first generation smallpox vaccines [10].

Alternative candidates based on attenuated vaccinia strains, third generation vaccines, may offer more favorable therapeutic ratios.

LC16m8, a replication competent, highly attenuated vaccinia strain, derives from fifty-three serial passages of a Lister strain isolate in rabbit kidney cells [26]. LC16m8 appears to be less neurovirulent in animals than unattenuated Lister strain vaccinia [27,28]; its use in more than 100,000 Japanese children in the 1970s demonstrated take rates and neutralizing antibody responses similar to those of lymph-derived, live smallpox vaccines [28,29]. However, the vaccine was never formally field-tested, as smallpox was no longer an epidemic threat in Japan at the time.

Recently, LC16m8 was shown to engender complete protection in both a rabbit model using intradermal rabbitpox challenge and a mouse model using aerosolized ectromelia (i.e. mousepox) virus [30]. In the mouse model, LC16m8-vaccinated animals developed higher vaccinia-specific neutralizing antibody titers, enhanced neutralization of intracellular mature virus (IMV), and comparable capacity to neutralize extracellular enveloped virus (EEV) as compared with Dryvax-vaccinated animals [30]. The latter finding is reassuring in that the B5R gene, required for EEV formation yet deleted during the attenuation process in LC16m8, is a neutralizing antibody target.

Additional data suggest that LC16m8 may be a safer alternative to unattenuated vaccine strains in immunocompromised hosts. While comparable protection is noted between LC16m8 and Dryvax in a BALB/c

The future of smallpox vaccines

97

mouse vaccinia challenge model [31], LC16m8 is non-lethal to SCID mice [32,33]. Combined data from trials involving nearly 1700 vaccinia-na誰ve subjects demonstrate ninety-five percent take rates and ninety percent neutralizing antibody seroconversions with LC16m8 [28,34], similar to those reported with first and second generation vaccines in na誰ve individuals [20].

MVA, a replication defective, highly attenuated vaccinia was initially used as a priming vaccine followed by first generation smallpox vaccination in more than 120,000 primary vaccinees in Germany in the 1970s [35]. It is attenuated via 570 serial passages in chicken embryo fibroblasts leading to DNA deletions in approximately fifteen percent of its genome, including genes related to host range and immune evasion; thus MVA is generally replication incompetent in mammalian cells [36]. It has been advanced as a third generation alternative vaccine of potential utility in immunocompromised hosts in whom live vaccinia vaccines are generally contraindicated [37].

Theoretically though, MVA may regain the potential for growth in certain mammalian cell lines due to reversions at the nucleotide level [36].

Unlike replication-competent vaccinia, MVA does not result in stereotypical neurovirulence upon intracerebral inoculation of suckling mice and may protect against subsequent intracerebral live vaccinia challenge [36].

Additionally, MVA is not associated with detectable viral replication in irradiated mice and rabbits and protects irradiated mice against live vaccinia challenge [36]. Immunosuppressed cynomolgus macaques demonstrate no significant clinical, hematological or pathological abnormalities following inoculation with high-dose MVA by multiple routes, although vaccinial genomes are detectable by polymerase chain reaction (PCR) from tissues in the majority of macaques [38].

MVA is immunogenic and protective in both normal and variably immunosuppressed mice [39,40]. However, animals clearly require multiple and higher doses of MVA to achieve comparable antibody titers to those induced by replication-competent vaccinia [39], and immunosuppressed macaques may fail to develop MVA-specific IgG responses despite high vaccine doses [38]. In comparisons of first generation vaccinia, LC16m8, and MVA, the latter appears to be the least immunogenic, requiring 100-fold more virus to produce similar response levels [32].

MVA protects cynomolgus macaques from lethal intravenous [41] or respiratory [42] monkeypox challenges. Such studies confirm data in mice that high-dose MVA or priming with MVA followed by vaccination with first generation vaccinia virus is necessary to generate immune responses and protection analogous to those observed with replication-competent vaccinia virus alone [41-44]. Multiple doses of MVA in macaques appear to elicit humoral immune responses of similar magnitude and breadth as those seen using single doses of live vaccinia virus [45]. In some cases MVA-immunized

98

Andrew W. Artenstein

animals, while protected against lethal disease, develop pox lesions following viral challenge; thus, this product may not abrogate the transmission potential of orthopoxviruses.

In humans MVA induces neutralizing antibodies in only fifty percent of naïve subjects receiving a single dose; whereas eighty percent seroconvert after two doses [46]. The magnitude and duration of humoral immune responses are dose-dependent; the proportion of subjects with neutralizing antibodies diminishes by at least half within three months after the second dose [46]. Vaccinia-experienced subjects demonstrate more rapid seroconversion or a boosting response and more durable antibody levels after a single dose of MVA [46]. When employed as a priming vaccine in vaccinia-naïve subjects, MVA induces a “modified-take skin reaction” with or without a vesicle upon Dryvax challenge three months later, similar to cutaneous responses observed in vaccinia-experienced subjects primed with MVA or given Dryvax alone [47]. Priming with multiple doses of MVA decreases cutaneous viral shedding after Dryvax challenge in naïve subjects.

Neutralizing antibody titers are comparable among the vaccinated groups; higher vaccinia-specific CD8+ CTL are noted in those receiving multiple doses of MVA than in those given one dose of MVA or Dryvax alone [47].

In summary, MVA modifies the cutaneous reactogenicity of live vaccinia without altering its immunogenicity, and multiple MVA priming doses may enhance immune responses to live vaccinia products. However, MVAvaccinated individuals have never been subjected to variola challenge, thus potential efficacy against smallpox can only be extrapolated.

Other attenuated, replication-defective, vaccine candidates may show promise as priming agents in immunocompromised hosts. NYVAC, derived from the Copenhagen vaccine strain of vaccinia and attenuated by the deletion of eighteen nonessential open reading frames [48,49], modulates the effects of Dryvax when used as a priming agent in immunodeficient rhesus macaques [50] yet fails to protect macaques with AIDS against a lethal, intravenous monkeypox challenge [51]. A replication-defective derivative of the Lister strain of vaccinia, bioengineered by deleting the gene encoding for an essential replication cycle enzyme, uracil-DNA-glycosylase (UDG) [52], has similar pre-clinical characteristics to MVA, but theoretically is unable to revert to virulence because it only grows in permanent cell lines capable of complementing the enzyme deletion [52,53].

Next generation smallpox vaccines Subunit products are also under investigation as alternative smallpox vaccines. Limited, preclinical data support the immunogenicity and protective

The future of smallpox vaccines

99

effect of a vaccinia envelope protein, H3L, in BALB/c mice; passive transfer of H3L-neutralizing antibodies also appears protective [54]. Multiple immunizations with combinations of three outer membrane proteins of IMV (e.g. L1, A27) and EEV (e.g. A33, B5), or with combinations of the genes encoding these proteins, are protective in mice and macaque models [55,56].

The latter approach prevents viremia in immunized, challenged monkeys [56]. Animals primed with plasmid DNA encoding the four proteins, then boosted with the analogous proteins, survive lethal monkeypox challenge with significantly milder disease than those immunized with the proteins alone [57]. Innovative approaches, such as the expression of these genes in tobacco plants, may lead to economically advantageous manufacturing methods for such products [58].

Safety of smallpox vaccines Substantial volumes of safety data have accumulated with first generation vaccines through their historical use in widespread smallpox vaccination, the intensified eradication program, and post-eradication vaccination exemplified by the recent U.S. military and civilian healthcare worker programs. Surveillance data from the late 1960s in the U.S. showed serious complications of smallpox vaccination in approximately four per 100,000 individuals with an overall risk of death of one per million primary vaccinations [59-61]. The rate of serious adverse events may be strainrelated; a retrospective meta-analysis describes a six-fold increased risk of death with the Lister as compared with the NYCBOH strains [62].

Serious, albeit rare, complications of vaccination are well-documented and occur with higher frequency in primary vaccinees or those with immunologic abnormalities [59,60,63] (Table 2). Postvaccinial encephalitis, a rare disorder of the central nervous system that generally occurs in children younger than five years of age during the second week following vaccination, is associated with a high mortality rate or severe neurological impairment [10,64]. Other serious adverse events are associated with specific predispositions: progressive vaccinia, a frequently fatal complication of smallpox vaccination in immunocompromised hosts, involves regional and metastatic spread of vaccinia virus as a consequence of the inability to contain the localized infection; and eczema vaccinatum, characterized by extension of the local vaccinia infection to other cutaneous areas actively or remotely affected by atopic dermatitis [10,61].

A number of other complications of smallpox vaccination, including generalized vaccinia, congenital vaccinia, inadvertent inoculation, and bacterial superinfection [5,61,65,66] are all potential causes of severe morbidity (or

100

Andrew W. Artenstein

Table 2. Noteworthy adverse events after smallpox vaccination, U.S., December 2002 – June 2004 [107].

Events and rates among 628,414 DoD

Events and rates among 39,566 DHHS

vaccinees* Event type

vaccinees [61]

No. of

Rate per

events

No. of

Rate per

Historical

million DoD events

million

rate per million

vaccinees

DHHS

vaccinees

vaccinees

[56,57,60]

Moderate or Serious Post-Vaccinial

1

1.6

1

26

2.6—8.7†

83 #

132

21 #

531

100 [62]

0

0

0

0

2–35 †

0

0

0

0

1—7 †

Encephalitis Acute myopericarditis Eczema vaccinatum Progressive vaccinia Mild or Temporary Generalized

40

64

3

77

45—212 †

1

1.6

0

0

NA

73 §

116

24 §

607

606†

47

75

0

0

8—27†

vaccinia, mild Erythema multiforme major Inadvertent inoculation, self Vaccinia transfer to contact Abbreviations: DoD, United States Department of Defense; DHHS, United States Department of Health and Human Services; NA, not available * Primarily composed of uniformed military personnel plus some DoD civilian employees; a minority of this total was healthcare workers † Based on adolescent and adult smallpox vaccination from 1968 studies (both primary and revaccination) # DoD events include 4 biopsy-confirmed, 73 probable, and 6 suspected cases; DHHS events include 0 confirmed, 5 probable, and 16 suspected cases. § DoD events include 59 inadvertent inoculations of the skin and 14 of the eye; DHHS events include 21 inadvertent inoculations of the skin and 3 of the eye.

The future of smallpox vaccines

101

mortality in the case of congenital vaccine) in vaccinees or their close contacts [10,61]. The incidence of serious adverse events expected in modern mass vaccinations using first generation vaccinia viruses could potentially be significantly higher than historical levels due to a larger population of individuals with vaccine contraindications and a larger proportion of vaccinia-naïve individuals in the population [67,68]. That this higher risk did not materialize in contemporary, post-eradication programs was likely due to rigorous, risk-based contraindication screening and extensive education. In the setting of a smallpox outbreak, though, fewer exemptions might be granted. Thus, a major focus of newer vaccine approaches is to improve upon safety while maintaining efficacy.

Live vaccinia virus vaccines are also associated with a high incidence of local and systemic symptoms. The majority of vaccinia-naïve subjects experience local symptoms related to the vaccination site and as many as forty percent experience mild to moderate constitutional symptoms, such as headache, myalgias, malaise, or fever [10]. Data from both the Lister/Elstree [62,69] and the NYCBOH strains [10,59,70] of vaccinia virus confirm the higher incidence of local and systemic adverse events in primary vaccinees, as compared with re-vaccinees [37]. While immunogenicity and efficacy in primary vaccinees are not apparently affected by diluting unattenuated vaccinia viruses up to ten-fold, fever, systemic symptom score, and missed activities are significantly mitigated [71].

The rates of adverse events in the ongoing DoD vaccination program (Table 2) are below historically anticipated levels [72-75] for a number of reasons including careful screening to exclude those at predictably higher risk, enhanced vaccine education, and a generally healthy population pool.

Ten military subjects with undiagnosed HIV infection, all with CD4+ counts above 280 cells/mm3, were inadvertently vaccinated and tolerated the local vaccinia infection without untoward clinical sequelae [76]. In the concurrent DHHS program seven cases involving the well described, serious complications of smallpox vaccination were reported: one subject experienced suspected post-vaccinial encephalitis, three with confirmed or suspected generalized vaccinia, and three subjects experienced ocular auto-inoculation (Table 2) [77,78] The relative dearth of “expected” vaccine complications in these programs is likely multi-factorial: more rigorous screening for contraindications than during the era of routine vaccine use, a lower overall denominator of vaccinees than during past mandated routine vaccination, limiting vaccines to adults, and possible reporting differences [79].

Cardiac complications of first generation smallpox vaccines were reported, albeit infrequently, during the era of routine use. Five cases of myopericarditis were described in association with the NYCBOH strain in

102

Andrew W. Artenstein

the U.S. [77]; data from Finland and Australia involving non-NYCBOH vaccinia strains support rates as high as one case per 10,000 vaccinees [80] and 1.6 per million [81], respectively. Up to three percent of Swedish military recruits were found to have nonspecific, asymptomatic T-wave changes on electrocardiogram following smallpox vaccination in the 1960s [82,83].

Nonetheless, a retrospective review of death certificates in New York City during a four-month period in 1947 in which six million people were vaccinated against smallpox using the NYCBOH strain failed to show a significant increase in cardiac deaths attributable to vaccination [84].

In the recent, post-eradication vaccination programs, two forms of cardiac complications associated with smallpox vaccination were recognized: ischemic events and myopericarditis. The U.S. military identified twenty-four subjects with ischemic events within four weeks of vaccination; the civilian program identified ten [77,78,85,86]. Of these, nineteen experienced myocardial infarction, three of whom died. Both the military and civilian rates of ischemic events were within the range expected for an age-matched population, and all occurred in vaccinia experienced individuals [86,87].

Additionally, four cases of dilated cardiomyopathy in the military cohort and three cases in the civilian cohort, all but one in revaccinees, were recognized between one and seven months after vaccination [77].

Despite the lack of a clear causal relationship between ischemic cardiac events and smallpox vaccination, the Centers for Disease Control and Prevention (CDC) promulgated new recommendations regarding cardiac pre-screening, surveillance, and vaccine contraindications for pre-outbreak smallpox vaccination based on the temporal associations [66]. Vaccine deferral on the basis of known heart disease or multiple cardiac risk factors was not associated with a clear reduction in ischemic cardiac events [87].

The U.S. DoD identified 140 cases of myopericarditis during its first two program years, largely in male, Caucasian, primary vaccinees [78], representing a rate of approximately 1.2 per 10,000—similar to historical rates in Finnish conscripts [80]. The rate in the civilian DHHS vaccination program in which twenty-one cases were identified was similar if only probable cases were considered, but was approximately 5.5 per 10,000 [85] if both suspected and probable cases were included. Both rates were higher than age-matched, unvaccinated individuals, and since cases cluster in the second week after vaccination, the appropriate conclusion is that primary smallpox vaccination of adults using first generation vaccinia is associated with a hitherto unrecognized, increased risk of myopericarditis.

Second generation vaccines, ACAM2000 [20] and CCSV [25], show no significant differences in local or systemic adverse events as compared with Dryvax. While none of the rare but well described, serious adverse events

The future of smallpox vaccines

103

related to smallpox vaccines have been noted with these vaccines to date, small sample sizes preclude a relative risk determination. Seven of 2983 (0.2%) vaccinia-na誰ve subjects who received ACAM2000 and three of 868 (0.3%) who received Dryvax during recent phase II and III trials were identified as cases of suspected vaccine-induced myopericarditis [15,20,22].

These rates extrapolate to approximately five-fold higher than those noted in the U.S. DoD and DHHS efforts, possibly as a result of rigorous, active surveillance for cardiac complications informed by the findings of these post-eradication vaccination programs [8,65].

Although no statistically significant differences were observed in the rates of myopericarditis between those who received ACAM2000 versus Dryvax, the phase III trials of ACAM2000 were prematurely terminated on this basis. Because myopericarditis cases have occurred in subjects who had received first generation or second generation vaccines, this complication appears to be directly or indirectly related to vaccinia virus and unlikely to be related to an adventitious agent introduced in the processing of lymph. The higher incidence of myopericarditis observed in both treatment groups in the ACAM2000 studies, as compared with the government-sponsored vaccination programs, likely results from active surveillance using routine assessments of cardiac symptoms, cardiac enzymes and electrocardiograms designed to identify asymptomatic individuals or cases involving only mild or transient symptoms.

The prototypical third generation vaccines, LC16m8 and MVA, lack large-scale human safety evaluations. LC16m8 was noted to be well tolerated in both an open-label study involving 1529 adult primary vaccinees and 1692 adult re-vaccinees [33] and in comparison with Dryvax in 153 vaccinia-na誰ve volunteers; [88] neither vaccinia-associated serious adverse events nor cardiovascular complications were noted, although cardiac evaluations in the former study were only performed thirty days after vaccination, an insensitive timeframe for assessing cardiac complications of smallpox vaccines. In openlabel study one primary vaccinee developed acute sensorineural deafness, and one reported chest pain ascribed by the authors to musculoskeletal causes, with no further information provided [89].

MVA appears to be associated with dose-related, local reactions in the majority of recipients; these self-limited events have not led to discontinuation of subjects from phase I studies [46]. In a small study of vaccinia-na誰ve individuals with either a history of atopic dermatitis or with active atopic dermatitis, groups in which first generation vaccinia vaccines are traditionally contraindicated, all subjects receiving MVA reported mild to moderate local reactogenicity but no serious adverse reactions [90]. MVAprimed subjects exhibit decreased reactogenicity and minimal systemic

104

Andrew W. Artenstein

symptoms following Dryvax challenge as compared with placebo-primed subjects, supporting a modulating effect of MVA in the context of safety, similar to that seen in efficacy studies. No vaccinia-associated serious adverse events or cardiac complications have been observed with MVA to date, although cardiac evaluations are uniformly lacking [47]. One recent study using serial electrocardiogram assessments found new, transient ST-segment or T-wave abnormalities in 2% and 15% of volunteers who received multiple doses of MVA followed by Dryvax; however, there did not appear to be discernible differences with the control group [91].

Conclusion New generation smallpox vaccines, specifically second generation (tissue culture-derived vaccinia) and third generation (highly attenuated vaccinia) vaccines potentially have similar efficacy to that of first generation smallpox vaccines. Second generation vaccines, like first generation ones, are associated with a significant risk of myopericarditis that substantially limits their utility in a pre-event setting. Third generation products may possess improved safety profiles, but this has yet to be proven in adequately powered studies or experience with large numbers of vaccinees. Highly attenuated, replication-defective vaccinia, MVA, sacrifices degrees of immunogenicity and efficacy for its theoretically improved safety profile. Multi-dose requirements for some third generation products limit their utility in outbreak settings.

The risks associated with currently licensed vaccines probably do not justify their pre-event use in groups with a very low perceived risk of smallpox exposure. New generation vaccines that are demonstrated to have significantly improved safety profiles after adequate human studies may alter the risk-versus-benefit assessment. Newer generation smallpox vaccines that employ highly attenuated and/or non-replicative forms of vaccinia or subunit vaccine approaches, some with promising pre-clinical data, may provide significantly safer, effective alternatives over the next five years that will enhance biodefense. Viral subunit strategies, in particular, may provide a flexible platform in the future upon which to build capabilities for protection against genetically altered forms of smallpox.

Acknowledgement The author wishes to acknowledge Ms. Katherine Bollesen for administrative assistance with the manuscript.

The future of smallpox vaccines

105

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

Artenstein AW. Smallpox In: Vaccines: A biography. Artenstein AW (Ed.).

Springer (2009).

Baxby D. The origins of vaccinia virus. J Infect Dis. 136(3), 453-455 (1977).

Baxby D. Edward Jenner’s inquiry; a bicentenary analysis. Vaccine. 17, 301-307 (1999).

Downie AW. Jenner’s cowpox inoculation. Br Med J. 2(4726), 251-256 (1951).

Fenner F, Henderson DA, Arita I, Zdenêk J, Ladnyi ID. Smallpox and its Eradication. World Health Organization, Geneva, (1988).

Wade N. New smallpox case seems lab-caused. Science. 201(4359), 893 (1978).

Wortley PM, Schwartz B, Levy PS, Quick LM, Evans B, Burke B. Healthcare workers who elected not to receive smallpox vaccination. Am J Prev Med. 30(3), 258-265 (2006).

Artenstein AW, Grabenstein JD. Smallpox vaccines for biodefense: Need and feasibility. Expert Rev Vaccines. 7(8), 1225-1237 (2008).

Kennedy RB, Ovsyannikova IG, Jacobson RM, Poland GA. The immunology of smallpox vaccines. Curr Opin Immunol. 21, 314-320 (2009).

Henderson DA, Inglesby TV, Bartlett JG et al. Smallpox as a biological weapon.

JAMA. 281, 2127-2137 (1999).

Breman JG, Henderson DA. Diagnosis and management of smallpox. N Engl J Med. 346, 1300-1308 (2002).

Taub DD, Ershler WB, Janowski M, et al. Immunity from smallpox vaccine persists for decades: A longitudinal study. Am J Med. 121(12), 1058-1064 (2008).

Redfield RR, Wright DC, James WD et al. Disseminated vaccinia in a military recruit with human immunodeficiency virus (HIV) disease. N Engl J Med. 316, 673-676 (1987).

Ennis FA, Cruz J, Demkowicz Jr. WE et al. Primary induction of human CD8 cytotoxic T lymphocytes and interferon-γ- producing T cells after smallpox vaccination. J Infect Dis. 185, 1657-1659 (2002).

Artenstein AW. New generation smallpox vaccines: A review of preclinical and clinical data. Rev Med Virol. Published online in advance of print: Wiley InterScience (www.interscience.wiley.com) (2008).

Frey SE, Newman FK, Yan L et al. Response to smallpox vaccine in persons immunized in the distant past. JAMA. 289, 3295-3299 (2003).

Murphy FA, Osburn BI. Adventitious agents and smallpox vaccine in strategic national stockpile. Emerg Infect Dis. 11(7), 1086-1089 (2005).

Weltzin R, Liu J, Pugachev KV et al. Clonal vaccinia virus grown in cell culture as a new smallpox vaccine. Nat Med. 9, 1125-1130 (2003).

Monath TP, Caldwell JR, Mundt W et al. ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain) – a second-generation smallpox vaccine for biological defense. Int J Infect Dis. 8(S2), S31-S44 (2004).

Artenstein AW, Johnson C, Marbury TC et al. A novel, cell culture-derived smallpox vaccine in vaccinia-naive adults. Vaccine. 23, 3301-3309 (2005).

106

Andrew W. Artenstein

21. Acambis, Inc. Investigator’s Brochure for ACAM2000 smallpox vaccine.

Cambridge, MA. (August 2002).

22. ACAM2000 Smallpox (vaccinia) vaccine, live package insert. www.fda.gov/ Cber/label/acam2000LB.pfd (Accessed June, 2008).

23. FDA approves second-generation smallpox vaccine. www.fda.gov/bbs/topics/ NEWS/2007/NEW01693.html (Accessed April, 2008).

24. ACAM2000 (smallpox vaccine). www.fda.gov/ohrms/dockets/AC/07/slides/ 2007-4292S2_5.ppt (Accessed June, 2008).

25. Greenberg RN, Kennedy JS, Clanton DJ et al. Safety and immunogenicity of new cell-cultured smallpox vaccine compared with calf-lymph derived vaccine: A blind, single-centre, randomized controlled trial. Lancet. 365, 389-409 (2005).

26. FDA approves second-generation smallpox vaccine. Available at: http://www. fda.gov/bbs/topics/NEWS/2007/NEW01693.html. Accessed 4 September 2007.

27. Wiser I, Balicer RD, Cohen D. An update on smallpox vaccine candidates and their role in bioterrorism related vaccination strategies. Vaccine. 25, 976-984 (2007).

28. Kenner J, Cameron F, Empig C et al. LC16m8: An attenuated smallpox vaccine.

Vaccine. 24, 7009-7022 (2006).

29. Empig C, Kenner JR, Perret-Gentil M et al. Highly attenuated smallpox vaccine protects rabbits and mice against pathogenic orthopoxvirus challenge. Vaccine.

24, 3686-3694 (2006).

30. Kidokoro M, Tashiro M, Shida H. Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8. PNAS. 102, 1452-1457 (2005).

31. Meseda CA, Mayer AE, Kumar A et al. Comparative evaluation of the immune responses and protection engendered by LC16m8 and Dryvax smallpox vaccines in a mouse model. Clin Vaccine Immunol. 16, 1261-1271 (2009).

32. Yokote H, Shinmura Y, Satou A et al. Safety and efficacy study of attenuated smallpox vaccine LC16m8 in animals. Presented at: 2006 ASM Biodefense Research Meeting for the American Society for Microbiology. Washington DC, USA, 15-18 February 2006.

33. Mayr A, Stickl H, Müller HK et al. The smallpox vaccination strain MVA: Marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defense mechanism (author’s transl).

Zentralbl Bakteriol [B]. 167, 375-390 (1978).

34. Saito T, Fujii T, Kanatani Y et al. Clinical and Immunological response to attenuated tissue-cultured smallpox vaccine LC16m8. JAMA. 301(10), 1025-1033 (2009).

35. Stittelaar KJ, Kuiken T, de Swart RL et al. Safety of modified vaccinia virus Ankara (MVA) in immune-suppressed macaques. Vaccine. 19, 3700-3709 (2001).

36. Wyatt LS, Earl PL, Eller LA et al. Highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic vaccinia virus challenge. PNAS. 101, 4590-4595 (2004).

37. Meseda CA, Garcia AD, Jumar A et al. Enhanced immunogenicity and protective effect conferred by vaccination with combinations of modified vaccinia virus

The future of smallpox vaccines

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

107

Ankara and licensed smallpox vaccine Dryvax in a mouse model. Virology. 339, 164-175 (2005).

Earl PL, Americo JL, Wyatt LS et al. Immunogenicity of a highly attenuated MVA smallpox vaccine and protection against Monkeypox. Nature. 428, 182-185 (2004).

Stettelaar KJ, van Amerongen G, Kondova I et al. Modified vaccinia virus Ankara protects macques against respiratory challenge with Monkeypox virus. J Virol. 79, 7845-7851 (2005).

McCurdy LH, Rutigliano JA, Johnson TR et al. Modified vaccinia virus Ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin-4. J Virol. 78, 12471-12479 (2004).

Abdalrhman I, Gurt I, Katz E. Protection induced in mice against a lethal orthopox virus by the Lister strain of vaccinia virus and modified vaccinia virus Ankara (MVA). Vaccine. 24, 4152-4160 (2006).

Vollmar J, Arndtz N, Eckl KM et al. Safety and immunogenicity of IMVAMUNE, a promising candidate as a third generation smallpox vaccine.

Vaccine. 24, 2065-2070 (2006).

Parrino J, McCurdy LH, Larkin BD et al. Safety, immunogenicity and efficacy of modified vaccinia Ankara (MVA) against Dryvax® challenge in vaccinia-naïve and vaccinia-immune individuals. Vaccine. 25, 1513-1525 (2007).

Nájera JL, Gómez CE, Domingo-Gil E et al. Cellular and biochemical differences between two attenuated poxvirus vaccine candidates (MVA and NYVAC) and role of the C7L gene. J Virol. 80, 6033-6047 (2006).

Grandpre LE, Duke-Cohan JS, Ewald BA et al. Immunogenicity of recombinant modified vaccinia Ankara following a single or multi-dose vaccine regimen in rhesus monkeys. Vaccine. 27, 1549-1556 (2009).

Edghill-Smith Y, Venzon D, Karpova T et al. Modeling a safer smallpox vaccination regimen, for human immunodeficiency virus type 1-infected patients, in immunocompromised macaques. J Infect Dis. 188, 1181-1191 (2003).

Edghill-Smith Y, Bray M, Whitehouse CA et al. Smallpox vaccine does not protect macaques with AIDS from a lethal monkeypox virus challenge. J Infect Dis. 191, 372-381 (2005).

Ober BT, Brűhl P, Schmidt M et al. Immunogenicity and safety of defective vaccinia virus Lister: Comparison with modified vaccinia virus Ankara. J Virol.

76, 7713-7723 (2002).

Coulibaly S, Brűhl P, Mayrhofer J et al. The nonreplicating smallpox candidate vaccines defective vaccinia Lister (dVV-L) and modified vaccinia Ankara (MVA) elicit robust long-term protection. Virology. 341, 91-101 (2005).

Davies DH, McCausland MM, Valdez C et al. Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice. J Virol. 79, 11724-11733 (2005).

Fogg C, Lustig S, Whitbeck C et al. Protective immunity to vaccinia virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions. J Virol. 78, 10230-10237 (2004).

Hooper JW, Thompson E, Wilhelmsen C et al. Smallpox DNA vaccine protects nonhuman primates against lethal Monkeypox. J Virol. 78, 1133-1143 (2004).

108

Andrew W. Artenstein

53. Pulford DJ, Gates A, Bridge SH et al. Differential efficacy of vaccinia virus envelope proteins administered by DNA immunization in protection of BALB/c mice from a lethal intranasal poxvirus challenge. Vaccine. 22, 3358-3366 (2004).

54. Lane JM, Ruben RL, Neff JM et al. Complications of smallpox vaccination, 1968: National surveillance in the United States. N Engl J Med. 281, 1201-1208 (1969).

55. Lane JM, Ruben FL, Neff JM et al. Complications of smallpox vaccination, 1968: Results of ten statewide surveys. J Infect Dis. 122, 303-309 (1970).

56. Fulginiti VA, Papier A, Lane JM et al. Smallpox vaccination: A review, Part II. Adverse events. Clin Infect Dis. 37, 251-271 (2003).

57. Kretzschmar M, Wallinga J, Teunis P et al. Frequency of adverse events after vaccination with different vaccinia strains. PLoS Med. 3, 1341-1351 (2006).

58. Rigano MM, Manna C, Giulini A et al. Plants as biofactories for the production of subunit vaccines against bio-security-related bacteria and viruses. Vaccine. 27, 3463-3466 (2009).

59. Lane JM, Millar JD. Risks of smallpox vaccination complications in the United States. Am J Epidemiol. 93, 238-240 (1971).

60. Miravalle A, Roos KL. Encephalitis complicating smallpox vaccination. Arch Neurol. 60, 925-928 (2003).

61. Center for Disease Control and Prevention. Surveillance guidelines for smallpox vaccine (vaccinia) adverse reactions. MMWR. 55(RR01): 1-16 (2006).

62. Neff JM, Lane JM, Fulginiti VA et al. Contact vaccinia-transmission of vaccinia from smallpox vaccination. JAMA. 288, 1901-1905 (2002).

63. Kemper AR, Davis MM, Freed GL. Expected adverse events in a mass smallpox vaccination campaign. Eff Clin Pract. 5, 84-90 (2002).

64. Lane MJ, Goldstein J. Evaluation of 21st-century risks of smallpox vaccination and policy options. Ann Intern Med. 138, 488-493 (2003).

65. Auckland C, Cowlishaw A, Morgan D et al. Reactions to smallpox vaccine in na誰ve and previously-vaccinated individuals. Vaccine. 23, 4185-4187 (2003).

66. Baggs J, Chen RT, Damon IK et al. Safety profile of smallpox vaccine: Insights from the laboratory worker smallpox vaccination program. Clin Infect Dis. 40, 1133-1140 (2005).

67. Couch RB, Winokur P, Edwards KM et al. Reducing the dose of smallpox vaccine reduces vaccine-associated morbidity without reducing vaccination success rates or immune responses. J Infect Dis. 195, 826-832 (2007).

68. Grabenstein JD, Winkenwerder W. US military smallpox vaccination program experience. JAMA. 289, 3278-3282 (2003).

69. Sejvar JJ, Labutta RJ, Chapman LE et al. Neurologic adverse events associated with smallpox vaccination in the United States, 2002-2004. JAMA. 294, 27442750 (2005).

70. Lewis FS, Norton SA, Bradshaw D et al. Analysis of cases reported as generalized vaccinia during the US military smallpox vaccination program, December 2002 to December 2004. J Am Acad Dermatol. 55, 23-31 (2006).

71. Tasker SA, Schnepf GA, Lim M et al. Unintended smallpox vaccination of HIV1-infected individuals in the United States military. Clin Infect Dis. 38, 13201322 (2004).

The future of smallpox vaccines

109

72. Casey CG, Iskander JK, Roper MH et al. Adverse events associated with smallpox vaccination in the United States, January-October 2003. JAMA. 294, 2734-2743 (2005).

73. Poland GA, Grabenstein JD, Neff JM. The US smallpox vaccination program: A review of a large modern era smallpox vaccination implementation program.

Vaccine. 23, 2078-2081 (2005).

74. Strikas RA, Neff LJ, Rotz L et al. US civilian smallpox preparedness and response program, 2003. Clin Infect Dis. 46(Suppl 3), 157-167 (2008).

75. McMahon AW, Zinderman C, Ball R et al. Comparison of military and civilian reporting rates for smallpox vaccine adverse events. Pharmacoepidemiol Drug Saf. 16(6), 597-604 (2007).

76. Karjalainen J, Heikkila J, Nieminen MD et al. Etiology of mild acute infectious myocarditis. Relation to clinical features. Acta Med Scand. 213, 65-73 (1983).

77. MacAdam D, Whitaker W. Cardiac complications after vaccination for smallpox.

Br Heart J. 2, 1099-1100 (1962).

78. Ahlborg B, Linroth K, Nordgren B. ECG-changes without subjective symptoms after smallpox vaccination of military personnel. Acta Med Scand Suppl. 464, 127-134 (1966).

79. Helle EP, Koskenvuo K, Heikkila J et al. Myocardial complications of immunizations. Ann Clin Res. 10, 280-287 (1978).

80. Thorpe LE, Mostashari F, Karpati AM et al. Mass smallpox vaccination and cardiac deaths, New York City, 1947. Emerg Infect Dis. 10, 917-920 (2004).

81. Casey CG, Iskander JK, Roper MH, Mast EE, Wen XJ, Török TJ. Adverse events associated with smallpox vaccination in the United States, January-October 2003.

JAMA. 294(21), 2734-2743 (2005).

82. Swerdlow DL, Roger MH, Morgan J et al. Ischemic cardiac events during the Department of Health and Human Services Smallpox Vaccination Program, 2003. Clin Infect Dis 46(Suppl 3), 234-241 (2008).

83. Eckart RE, Shry EA, Atwood JE et al. Smallpox vaccination and ischemic coronary events in healthy adults. Vaccine. 25, 8359-8364 (2007).

84. Halsell JS, Riddle JR, Atwood JE et al. Myopericarditis following smallpox vaccination among vaccinia – naïve US military personnel. JAMA. 289, 32833289 (2003).

85. Fujii T, Kannatani Y, Matsumura T et al. Clinical evaluation of attenuated smallpox vaccine LC16m8 in Japan. Presented at: 2006 ASM Biodefense Research Meeting for the American Society for Microbiology. Washington DC, USA, 15-18 February 2006.

86. Kenner JR, Gurwith M, Luck A et al. Safety and immunogenicity of attenuated smallpox vaccine, LC16m8: results from a phase 2 study. Presented at: 2006 ASM Biodefense Research Meeting for the American Society for Microbiology.

Washington DC, USA, 15-18 February 2006.

87. von Sonnenburg F, Perona P, Schunk M et al. First clinical experience with a third generation smallpox vaccine in subjects with atopic dermatitis. Presented at: 2006 ASM Biodefense Research Meeting for the American Society for Microbiology. Washington DC, USA, 15-18 February 2006.

110

Andrew W. Artenstein

88. Parrino J, Graham BS. Smallpox vaccines: Past, present, and future. J Allergy Clin Immunol. 118, 1320-1326 (2006).

89. McCurdy LH, Larkin BD, Martin JE et al. Modified vaccinia Ankara: Potential as an alternative smallpox vaccine. Clin Infect Dis. 38, 1749-1753 (2004).

90. Artenstein, AW. Bioterrorism and biodefense. In: Infectious Diseases, second edition. Cohen J, Powderly WG (eds.), Mosby: London, 99-107 (2003).

91. Sano J, Chaitman BR, Swindle J et al. Electrocardiography screening for cardiotoxicity after modified vaccinia Ankara vaccination. Am J Med. 122(1), 79-84 (2009).

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 111-136 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

5. Heterologous prime-boost immunization: HIV-specific systemic/mucosal immunity, cytokine milieu and CD8+ T cell avidity Charani Ranasinghe and Shubhanshi Trivedi

Molecular Mucosal Vaccine Immunology Group, Department of Immunology The John Curtin School of Medical Research, The Australian National University Canberra ACT 2601, Australia

Abstract. Development of effective vaccines against human immunodeficiency virus (HIV), tuberculosis (TB) and malaria has been a difficult task. We and others, have shown that heterologous prime-boost immunization has the ability to generate excellent T cell mediated immunity (CMI) and some B cell immunity in animal models. Studies have shown that the route of delivery, the recombinant vector combinations, the order of vector delivery (prime or the boost) can markedly modulate the vaccine-specific effector and memory T cell immunity. Due to the disappointing outcomes of many HIV-1 systemic vaccine trials (vaccines delivered to blood), there is now a great drive to develop novel vaccine strategies that induce immunity at the mucosae, which is the primary site of infection. More and more studies are now demonstrating that not only the magnitude of immunity measured by IFN-γ production, the ‘quality’ or ‘avidity’ of T cells as well as the poly-functionality of T cells (ability to produce other cytokine and chemokines such as IL-2, TNF-α, CCL3, CCL5) play an important role in protective immunity.

Correspondence/Reprint request: Dr. Charani Ranasinghe, Molecular Mucosal Vaccine Immunology Group Department of Immunology, The John Curtin School of Medical Research, The Australian National University, Canberra ACT 2601, Australia. E-mail: Charani.Ranasinghe@anu.edu.au

112

Charani Ranasinghe & Shubhanshi Trivedi

In this report we mainly aim to discuss the pitfalls, successes and future of systemic and mucosal prime-boost immunization strategies in relation to HIV-1 vaccine development.

Introduction The development of various vaccines against infectious diseases has significantly improved the quality of human life (Salk et al., 1954, Bazin, 2001, Frazer and Cox, 2006). However, still over 9.5 million people die each year due to infectious diseases especially HIV/AIDS, TB, malaria and nearly all live in developing countries (WHO 2008). Hence, there is an urgent need to develop effective treatments specifically vaccination strategies to combat these diseases. The discovery of recombinant DNA (rDNA) immunization in early 1990s offered hope for many diseases for which traditional vaccine delivery approaches were ineffective (Pardoll and Beckerleg, 1995, Lu et al., 2008). These vaccines showed significant advantages over other immunization strategies mainly due to vectors being non-replicative, noninfectious, non-integrating, stable and easier to prepare at lower cost.

Furthermore, Fuller and colleagues for the 1st time demonstrated that subsequent gene gun delivery of DNA vaccines (example; HIV-1 envelope glycoprotein - gp120) could induce both humoral and cellular immunity in small animal models (Fuller and Haynes, 1994). Despite these successes, other rDNA delivery methods such as intra muscular (i.m.) strategies intravenous (i.v.) were unable to generate comparable immune responses to gene gun delivery and also DNA vaccines alone have failed to elicit effective protective immunity against highly pathogenic organisms such as HIV-1, TB. Despite these failures rDNA vaccines soon became an excellent priming modality during the advent of prime boost vaccination (Leong et al., 1995, Ramsay et al., 1997).

Prime-boost immunization Prime-boost approach involves priming of immune response with one vaccine and subsequent boosting with either same vaccine referred to as homologous prime-boost immunization or with same antigen delivered using another vector known as heterologous prime-boost immunization (Figure 1).

The latter immunization strategy, where same encoded vaccine antigens are presented to the immune system using two different vectors for example rDNA and recombinant fowl pox virus (rFPV) have shown to generate enhanced T cell mediated immunity compared to the homologous primeboost strategy (Figure 2). This is mainly due to its ability to circumvent the

Heterologous prime-boost immunization

113

Figure 1. Schematic diagram of heterologous prime-boost immunization, eliciting expansion of HIV-specific T cells (red) following FPV-HIV or VV-HIV booster immunization.

Figure 2. T cell responses generated by homologous and heterlogous prime-boost immunization. BALB/c Mice n=4-5 were immunized three weeks apart with i) 4x DNA-HIV (expressing gag/pol genes) – white bars, ii) 2x FPV-HIV – grey bars or iii) 2x DNA-HIV/ 2x FPV-HIV – black bars, 4 weeks post booster immunization, spleen (systemic) and genito-rectal (mucosal) T cell responses were evaluated by IFN-γ ELIspot assay following HIV-specific overlapping 15 mer gag peptide pool (NIH reference laboratory). Data represent a pooled value.

anti-vector immunity, which can dampen the immunity to encoded vaccine antigens (Leong et al., 1995, Ramsay et al., 1997). One of the first demonstrations of heightened T and B cell immunity using prime-boost immunization was reported when model antigen hemagglutin (HA) gene of

114

Charani Ranasinghe & Shubhanshi Trivedi

influenza virus was used in a rDNA prime and rFPV boost immunization strategy (Leong et al., 1995, Ramsay et al., 1997, Ramshaw and Ramsay, 2000). Furthermore, tetrameric peptide-MHC complex studies ex-vivo and lytic assays have revealed that rDNA/rFPV prime-boost immunization can generate high levels of antigen-specific cytotoxic T lymphocytes (CTL) that recognize target cells expressing low levels of specific antigen (T cells of high avidity) and are more capable of eliminating target cells as compared to mice given either vector alone (Estcourt et al., 2002).

Since these findings, range of DNA prime-boost vaccine modalities or combinations have been tested against many diseases and pathogens. For example; recombinant DNA-HIV envelope glycoprotein (gp120) followed by gp120 protein subunit boost was shown to generate good antibody and CTL immune responses to vaccine antigens (Barnett et al., 1997). Heterologous HIV-1, rDNA/rFPV prime-boost immunization regime was shown to generate enhanced systemic and mucosal CTL responses in mice (Figure 2) and macaques. Moreover in macaques these vaccines elicited protection against pathogenic challenge (Kent et al., 1998, Kent et al., 2005). An HIV rDNA prime followed by recombinant modified vaccinia virus ankara (rMVA) boost immunization also showed protective immunity against a highly pathogenic HIV intra rectal (i.r.) challenge in a rhesus macaque model seven months post booster immunization (Amara et al., 2001). These observations generated great optimism for a potential human HIV-1 DNA prime-boost vaccine.

In the field of tuberculosis vaccine development, compared to homologous prime-boost regimes, significantly higher IFN-Îł secreting CD4+ T-cell responses were observed in guinea pigs following rDNA prime and rMVA boost immunization with vectors expressing Antigen 85A (Ag85A) and a fusion protein of 6-kDa early secretory antigenic target (ESAT-6) and Ag85B. This vaccine regime also offered better protection than BCG alone (Williams et al., 2005). One of our studies has also demonstrated that rDNA prime-Mycobacterium bovis BCG boost vaccination strategy in cattle could induce significantly enhanced protection against bovine tuberculosis, compared to BCG alone (Skinner et al., 2003). Another study in calves, rDNA/BCG strategy was shown to induced increased CD4+ T cell responses, specific antibody responses and superior protection (10-100-fold) against an intra tracheal challenge with virulent M. bovis compared to the rDNA or BCG alone strategies (Cai et al., 2006). Hill and co-workers also showed that rDNA/rMVA prime-boost can generate high level T cell responses against Plasmodium falciparum vaccine antigens in both mice, non-human primates (Hill et al., 2000) and also in humans (Moorthy et al., 2004). Moreover, it was demonstrated that DNA-LACK/MVA-LACK (Leishmania homologue of

Heterologous prime-boost immunization

115

the mammalian receptor for activated C kinase) immunization can generate greater IFN-γ and TNF-α expression by CD8+ T cells and protective immunity against Leishmaniasis major in mice (Perez-Jimenez et al., 2006).

A recent L. infantum safety trial in dogs using 1 mg rDNA expressing tryparedoxin peroxidase (TRYP) followed by 108 pfu of MVA-TRYP was shown to induce enhanced IFN-γ production and IGg2A responses against vaccine antigens compared to LACK rDNA/rMVA prime-boost immunization (Carson et al., 2009). Recently, in cancer vaccine development, rDNA prime and Venezuelan equine encephalitis virus like-replicon particles (VRP) boost encoding the six-transmembrane epithelial antigen of the prostate (STEAP) was shown to induce high IFNγ, TNFα and IL-2 against STEAP epitopes and significantly delay prostate tumor growth when compared to either of the vaccine modality alone (Garcia-Hernandez Mde et al., 2007). Furthermore, due to the poor outcomes in humans with rDNA vaccination, in the recent past viral-viral and viral-protein prime- boost immunization strategies have also been investigated against diseases such as Malaria, hepatitis B, TB, and HIV-1 respectively (Anderson et al., 2004, Moorthy et al., 2004, Hutchings et al., 2005, Vordermeier et al., 2009, Ranasinghe et al., 2006, Corbett et al., 2008).

Many studies have clearly demonstrated the ability of heterologous prime-boost immunization strategies to elicit excellent cell mediated immunity against viral, bacterial, parasitic or tumor antigens that offer protective immunity in animal models. Therefore, in this chapter we mainly aim to discuss prime-boost HIV-1 vaccines, roadblocks in HIV vaccine development, the value of prime-boost immunization strategies, and ways to improve prime-boost vaccination strategies to enhance both mucosal and systemic immunity to HIV-1.

Difficulties in HIV-1 vaccine development In 1984, after HIV was confirmed to cause AIDS (Barre-Sinoussi et al., 1983, Broder and Gallo, 1984) it was declared by US Health and human services that a vaccine would be available within two to three years. Today, more than two decades have passed since the identification of HIV-1 and finding a suitable vaccine has been a complex task. Some of the difficulties associate in the development of an HIV-1 vaccine can be identified as: (i) The extraordinary diversity of HIV-1, due to divergent clades, hampering the design of a universal vaccine (Osborn, 1995), ii) HIV-1 envelop being heavily glycosylated conferring considerable energy barriers for B-cells to recognize neutralizing epitopes (Kwong et al., 1998), including viruses ability to evade antibody-mediated neutralization by conformational masking

116

Charani Ranasinghe & Shubhanshi Trivedi

of receptor-binding sites (Kwong et al., 2002) and iii) also not clearly understanding the immune correlates of protection in humans, have made it difficult to design vaccines that generate sustained B and T cell immunity (Barouch, 2008). Furthermore, the ability of HIV to lay dormant in infected cells, sheltered from both anti-retroviral therapy and from host immune response (Saksena and Haddad, 2003, Craigo et al., 2004, Alexaki et al., 2008, Haynes and Shattock, 2008), plus viral escape following treatment (Barouch et al., 2002) has also added another layer of complexity to HIV-1 vaccine development.

Prime-boost approach for an HIV-1 vaccine Many of the current human HIV-1 vaccine trial have failed to elicit protective immunity, Unfortunately, studies that have generated excellent immune responses in small animal models and non-human primates have failed to translate effectively into humans (Kelleher et al., 2006, Jaoko et al., 2008, Asmuth et al.). Thus, it is thought that total prevention of HIV-1 infection with a vaccine could possibly be a difficult task, and a successful strategy such as prime-boost immunization at least has a greater potential to control plasma viral load and virus-infected cell numbers and retard the disease progression.

HIV-1 human clinical trial - Where are we now In early 2000 VAXGen sponsored “VAX004�, a phase III double-blind, randomized placebo-controlled clinical trial utilizing vaccine that contained recombinant HIV glycoprotein 120 (rgp120) derived from 2 different B clade strains. In total 5403 individuals confirmed as HIV-1 uninfected at entry, participated in the trial, which consisted of 7 vaccinations using rgp120 with alum or placebo alum only. 4331 individuals completed the trial (2887 rgp120, 1426 control), but despite all efforts the vaccine was found to have no overall protective efficacy. These results further indicated that, only targeting an antibody response might not be sufficient to protect against HIV-1 infections in humans (Flynn et al., 2005, Pitisuttithum et al., 2006). Due to the poor outcomes with vaccines that induce neutralizing antibody responses, researches for the past decade have also been more focused on testing T cell based vaccines for HIV-1. Following promising CMI and protective immunity with HIV-1 gag/pol rDNA/ rFPV prime-boost immunization strategy in small animal models and in non-human primates, our group was involved in a randomized, placebo-controlled phase-1 clinical trial using of 1 mg DNA prime (pHIS-HIV-B) and recombinant fowl pox virus (rFPV-HIV-B) boost

Heterologous prime-boost immunization

117

vaccine for HIV-1. Unlike animal studies, immunity elicited to vaccine antigens in humans were rather disappointing, there were no significant differences in HIV-specific T cell immune response at nine weeks in vaccine recipients compared to placebo group. On the contrary, the trial clearly demonstrated that the rDNA and rFPV were extremely safe in humans (Kelleher et al., 2006). Many other rDNA prime followed by recombinant poxvirus boost studies, conducted around the world at the same time, also reported similar outcomes in humans (Jaoko et al., 2008). We believe that the differences in immunogenicity between humans and animal models could have been related to the relatively low dose of rDNA vaccine used or the cytokine milieu they induce.

Merck HIV vaccine trials network (HVTN-502 and HVTN-503) or STEP trial was conducted in 2007, involving 3000 uninfected volunteers at high risk, using three recombinant adenoviral (AD5) vectors expressing HIV-1 clade B Gag, Pol and Nef. Individuals were vaccinated three times, with the last two vaccinations 6 months apart. The aim of this proof-of-concept study was to evaluate whether the vaccine prevented HIV infection and whether the vaccine reduced the amount of virus in those who developed the infection. In September 2007 trial was unexpectedly terminated as the vaccine neither prevented HIV infection nor reduced the virus in infected individuals. In addition, in subjects with pre-existing AD5 neutralizing titers, greater number of HIV-1 infections was observed. The STEP study had its impact on Phambilli trial (HVTN-503) in South Africa and it was also terminated (Sekaly, 2008, Hanke, 2008, Bansal et al., 2010). Interestingly, a recent composite study from the Merck Phase I human trial, using 5mg DNA HIV-1 B clade gag prime followed by 1x1010 vp/dose of rAD5 DNA HIV-1 B clade gag boost immunization, revealed that even though both CD+ and CD8+ gag T cell responses were observed following heterologous prime-boost immunization, no significant differences were detected compared to rDNA/rDNA (which predominantly generated a CD4+ response) or rAd5/rAD5 alone (which elicited CD8+ responses) (Asmuth et al.). These observations, together with our recent findings suggest that rather than the dose of rDNA used, may be the cytokine milieu a vaccine induces could play a more important role in generating effective immunity especially in humans (Ranasinghe and Ramshaw, 2009b).

Interestingly, improved prime-boost approaches have once again become the most effective strategy to elicit T cell mediated immunity (Bansal et al., 2010). Alternative prime-boost protocol, involving multigene, polyvalent DNA prime-protein boost HIV-1 formulation DP6-001 in a phase 1 clinical trial demonstrated high titer serum antibody responses against HIV-1 antigens and robust cross subtype HIV-1 specific T cell immunity. This trial

118

Charani Ranasinghe & Shubhanshi Trivedi

involved 3x rDNA vaccine priming followed by two protein booster immunizations. But unfortunately, trial was terminated due to the generation of skin reactogenicity in volunteers (Wang et al., 2008). Another recent Phase 1 clinical trial using 2x i.m. (4.2 mg) DNA prime and 2x i.m. (107.7 pfu/ml) New York Vaccinia Virus (NYVAC; a highly attenuated strain of Vaccinia virus) boost strategy expressing Gag/Pol/Nef and Env from HIV-1 clade C isolate, has been highly immunogenic in humans. (McCormack et al., 2008).

Interestingly, this vaccine strategy induced poly-functional CD4+ and CD8+ T cells with broad T cell responses to at least 4 epitopes, with 91% of vaccinees eliciting strong T cell responses towards Env, and 48% showing similar responses against Gag, Pol, Nef antigens. Moreover, these T cell responses persisted up to 72 weeks in 70% of vaccinees. (Harari et al., 2008).

In 2009, the largest HIV-1 vaccine study ever conducted in humans known as the RV144 or Thai vaccine trial was conducted. More than 16402 HIV-negative volunteers participated in this randomized combination recombinant canarypox vector vaccine (ALVAC HIV [VCP1521]) prime followed by a recombinant glycoprotein 120 subunit vaccine (AIDSVAX B/E) boost trial. Results of this RV144 trial were relatively modest with 31.2% reduction in rates on infection suggesting that prime-boost combination could induces protective immune responses (Rerks-Ngarm et al., 2009) that were not observed with either vector alone (Autran et al., 2008, Pitisuttithum et al., 2006). However, it is thought that such a weak vaccine is unlikely to be practical as it involves six injections over four visits and failed to reduce viral loads in those who did became infected (Bateman, 2009, Bansal et al.). Several ongoing clinical trials, preclinical studies and this recent RV144 prime-boost proof-of-concept trial gives us hope that a globally effective HIV-1 vaccine may be possible with prime-boost immunization strategy and has offered renewed optimism for the future.

Mucosal HIV vaccine – Possible strategy for a successful HIV-1 vaccine Majority of human pathogens including HIV-1 initiates encounter via the mucosal barrier (Freihorst and Ogra, 2001, Kallenius et al., 2007). The gastrointestinal tract and genito-rectal mucosa are the primary sites of HIV-1 infection and subsequent virus replication and CD4+ T cell depletion (Veazey et al., 1998, Belyakov et al., 2001a, Veazey et al., 2001). Thus, more and more studies have now established that an ideal HIV vaccine should induce anti-HIV-1 neutralizing antibody and also, good cell mediated immunity (CMI) at mucosal surface, where the virus is 1st encountered. It is

Heterologous prime-boost immunization

119

thought that such approach would better control viral replication and prevent systemic dissemination of virus (Belyakov and Berzofsky, 2004).

Furthermore, as systemic vaccine trials (vaccines delivered via intramuscular (i.m.) route to blood) in humans have elicited poor outcomes, currently the potential importance of alternative HIV-1 vaccines strategies, such as mucosal immunization, as well as why systemic HIV-1 vaccines have performed poorly in humans need to be evaluated.

Differences in mucosal and systemic immunization strategies Purely systemic immunization strategies i.m. and intravenous (i.v) although induce good systemic T cell responses, rarely induce long lasting or optimal mucosal immunity. In contrast, mucosal immunization has the ability to trigger immunity at local and distant mucosae (Kozlowski and Neutra, 2003, Mitchell et al., 1998, Ogra et al., 2001, Kallenius et al., 2007). We have observed that following purely systemic immunization (i.m/i.m) of rFPV and rVV encoding antigen of AE clade HIV-1 in a prime-boost strategy, strong systemic but poor mucosal immune responses were generated (Ranasinghe et al., 2006). This is most likely due to systemic immunization not inducing T cells with recognized mucosal homing markers such as ι4β7 and CCR9 that enable T cells to migrate to mucosae (Cromwell et al., 2000, Stenstad et al., 2006, Kiyono and Fukuyama, 2004) and also not inducing mucosal secretory IgA antibodies that play a major role in viral clearance (Freihorst and Ogra, 2001). In another study, i.m immunization with recombinant replication defective adenovirus expressing mycobacterial antigen Ag85A (rAd-Mtb-Ag85A) vaccine despite strong systemic responses failed to provide protection from Mycobacterium tuberculosis challenge (Santosuosso et al., 2005). Similarly, in a systemic DNA prime live vaccine boost study, despite elevated systemic IFNγ responses failed to show protection against Mycobacterium bovis aerosol challenge (Skinner et al., 2003). Also systemic rDNA prime rMVA boost immunization, even though generated SIV specific CTL showed no protection in macaques from intrarectal pathogenic SIVmac25 challenge (Hanke et al., 1999). These studies clearly demonstrate the importance of mucosal vaccine strategies that induce strong sustained mucosal immunity to protect against mucosal challenge.

Mucosal DNA delivery and vaccine adjuvants In order to enhance immunogenicity and target DNA vaccines to mucosal surfaces, several mucosal adjuvants and vaccine delivery systems have been tested. Cholera toxin (CT) and structurally similar E.coli thermolabile

120

Charani Ranasinghe & Shubhanshi Trivedi

enterotoxin (LT) are most widely used mucosal adjuvants and inducer of excellent mucosal humoral immunity (Imaoka et al., 1998, Kubota et al., 1997). But due to toxicity, both CT and LT are not suitable for use in human mucosal vaccines. Therefore, a number of nontoxic mutants of CT and LT are currently being investigated (Peppoloni et al., 2003, Baudner and Giudice, 2010). A double mutant CT has shown to induce enhanced OVAspecific immune responses in both mucosal and systemic lymphoid tissue with no central nervous system toxicity (Hagiwara et al., 2006). When macaques were immunized i.r. with an HIV synthetic-peptide vaccine with mutant LT as an adjuvant (R192G), compared to subcutaneous (s.c.) immunization effective viral clearance was observed in intestine, the major site of virus replication, following an i.r. pathogenic SHIV-Ku2 challenge (Belyakov et al., 2001b). In another study co-administration of alphagalactosylceramide (Îą-Galcer) as an adjuvant with a CTL-inducing HIV envelop peptide has shown to induce antigen specific IFN-Îł producing cells in both systemic and mucosal compartments (Courtney et al., 2009).

Furthermore, in an elegant study Klavinskis et al. showed that plasmid DNA delivered with cationic lipid complexes (i.e. 1, 2-dimyristyloxypropyl3 dimethylhydroxyethyl ammonium bromide) could induce CTL responses to encoded antigens in genito-rectal nodes, cervical nodes and spleen (Klavinskis et al., 1999). Interestingly, in our studies we have found that liposomal encapsulated DNA-gagpol i.n. prime following by a i.m. FPVgagpol boost was unable to generate strong sustained immunity in mice and macaques compared i.n. FPV-gag/pol prime followed by an i.m. DNAgag/pol (Ranasinghe et al Manuscript submitted) (Kent et al., 2005). In this study we also found that the rFPV is an excellent mucosal delivery vector, inducing protective immunity following a pathogenic CCR5-tropic SHIV (SF162P3) intra vaginal challenge (Kent et al., 2005). Systemic priming with DNA and subsequent mucosal boosting with liposome encapsulated hepatitis B surface antigen (HBsAg) protein has also shown enhanced humoral and T cell immune response as compared to non-encapsulated protein boost (Yang et al., 2008). Moreover, several cytokines and chemokines such as IL-6, IL-12, IL-15, CCL5 (RANTES), XCL1 (lymphotectin) have also been tested as potential mucosal adjuvants to enhance mucosal immunity (Ramsay et al., 1994, Lillard et al., 1999, Lillard et al., 2001, Kim et al., 2000).

Different delivery vectors and vaccine regimes In recent years, a number of different vectors and immunization regimes have been tested in prime-boost vaccination approach against HIV-1. A number of pivotal studies in mice and macaques suggest that mucosal prime-

Heterologous prime-boost immunization

121

boost strategies play a significant role in controlling HIV infection. Recent studies in our laboratory indicate that intranasal (i.n.) HIV-FPV prime followed by i.m HIV-VV (vaccinia virus) boost can generate robust long term CD4+ and CD8+ systemic and mucosal T-cell responses to HIV-1 antigen in BALB/c mice as compared to purely systemic (i.m/i.m) immunization regime (Ranasinghe et al., 2006, Ranasinghe et al., 2007).

Similarly, Belyokov and co-workers have also demonstrated that i.m. or i.r. rDNA prime followed by mucosal rMVA or rAD boost can generate effective mucosal HIV specific CD8+ CTL in gut (Belyakov et al., 2008). Aerosol vaccination with HIV-Clade C vaccine DNA prime followed by NYVAC-C boost elicited antigen specific genito-rectal immune response as well as systemic immunity, despite the low uptake of vaccine by mucosal tissue in macaques (Corbett et al., 2008). They also showed that, aerosol vaccination with rNYVAC and rMVA was safe, with no pathology in brain or lung, and would be a simple and cost effective delivery against mucosal transmitted pathogens (Corbett et al., 2008).

Despite the difficulties in developing effective orally deliverable vaccines due to low pH, presence of mucins and proteases in gut, oral vaccines are attractive alternatives due their ease of administration and potential of immune response in both mucosal and systemic compartments.

It has been shown that delivery of antigens to the intestine can induce enhanced mucosal cellular immunity (Ko et al., 2009, Wang et al., 2009).

More interestingly, clinical trials of transgenic plant based vaccines for hepatitis B, has shown to resist the harsh gut environment and generate enhanced serum antibody against HBsAg (Lugade et al., 2010, Thanavala et al., 2005). Plant-based vaccines for HIV-1 have been evaluated and thought to have the potential, to be used as mucosal booster vaccination in a prime-boost modality (Webster et al., 2005, Matoba, 2009 #574). In a homologous rAD5 HIV-gp140B oral or ileum prime followed by rAD5 i.m. booster regimen in mice, intra ileum priming and intramuscular boosting was shown to induced 100-fold higher CD8+ T-cell immunity in small intestine as well as in systemic compartment as compared to oral route. This study showed the combination of low pH environment, presence of proteases as well as mucosal glycocalyx in gastrointestinal tract are major obstacles for oral vaccines (Wang et al., 2009). When Rhesus macaques were rDNA prime/oral Lesteria boosted with SIV-Gag, elevated numbers of SIV-specific mucosal CD8+ T cells that expressed ι4β7 homing-markers were reported (Neeson et al., 2006). Furthermore, potential use of attenuate Salmonella and Shigella in an oral prime-boost strategy that could generate good HIV-1 Env neutralizing antibodies and CTL immunity were also discussed (Devico et al., 2002). In a recent study, using attenuated L. monocytogenes-gag oral, i.r. or

122

Charani Ranasinghe & Shubhanshi Trivedi

intra vaginal prime followed by AD5-gag i.m. booster immunization was shown to induce higher Gag-specific CD8+ T cells in spleen and vaginal lamina propria in mice (Li et al., 2008). We have also evaluated the efficacy of oral delivery of rFPV, although orally primed mice generated systemic Gag as well as Pol 1 antigen specific IFN-γ responses, low mucosal T cell responses were observed in genito-rectal lymph nodes. Moreover, to induce good systemic immunity high doses of purified rFPV were required (Ranasinghe et al., 2006).

Recently Xu et al. demonstrated the potential use of novel recombinant mumps virus (rMuV) expressing HIV-1 Gag (rMuVgag) and highly attenuated form of recombinant vesicular stomatistis virus also expressing HIV-1 Gag (rVSVN4CT1gag1), in a prime-boost immunization in Rhesus macaques. Single priming with rMuVgag s.c. route and boosting after 8 weeks with rVSVN4CT1gag1 i.m. route elicited peak gag-specific CMI with 30003500 IFN-γ spot forming units per 106 peripheral blood lymphocytes. In contrast, lower responses were observed with the inverse vaccination strategy.

However, when long-term immune responses were analyzed by intracellular cytokine staining percentage of gag-specific CD4+ and CD8+ T cells expressing IFN-γ, TNF-α and IL-2 were higher in rVSVN4CT1 i.m. prime followed by a rMuVgag s.c. booster immunization (Xu et al., 2009). The safety of rMuV vector in adult humans with pre-existing MuV-specific immune response due to childhood vaccination or natural infection is unclear. In another study, when mice were primed with rBCG/HIV-1gagE s.c. route and boosted i.v. with highly attenuated replication deficient recombinant vaccinia virus DIs strain expressing same HIV-1 A/E gag gene (rDIs/HIV-1gagE) prolonged CTL responses as well as memory T cells were detected compared to intradermal (i.d) prime-boost immunization (Promkhatkaew et al., 2009). All these findings clearly indicate that a rational combination of viral vectors, the order in which the vectors are delivered and routes of delivery should be seriously taken into consideration when designing effective mucosal and systemic prime-boost vaccine strategies against diseases like HIV-1.

Role of CD8+ T cell avidity and protective immunity Most of the human HIV-1 vaccine trials have failed to establish true correlates of protection. We and others, have found that many co-stimulatory molecules or molecular adjuvants (IL-12, GM-CSF, 41BBL), although enhance the magnitude of T cell immunity are ineffective in controlling infection (Boyer et al., 2000, Demberg et al., 2008, Ranasinghe and Ramshaw, 2009a). These findings increasingly suggest that not only the magnitude but also the “quality” or “avidity” of the T cell response generated

Heterologous prime-boost immunization

123

against vaccine antigens may be particularly important in protection against pathogenic organisms (Rowland-Jones et al., 2001, Belyakov et al., 2006, Ranasinghe et al., 2007, Belyakov et al., 2007). Unfortunately, not many HIV-1 vaccine trials have evaluated the avidity of T cells immunity generated following vaccination. It is well established that CTLs of higher avidity are capable of recognizing low concentrations of antigen essential for rapid clearance of infection in-vivo. While low-avidity CTLs can only recognize peptide-MHC complex at high antigen concentrations and are ineffective at controlling infection (Alexander-Miller, 2005, Alexander-Miller et al., 1996).

High avidity CD8+ T cells are generated during early stages of pathogen infection but subsequently low-avidity CD8+ T cells have shown to persist in chronic infections (Alexander-Miller, 2000). More and more studies now postulate that rather than the magnitude of T cell immunity, avidity and polyfunctional CD8+ T cell responses may also be important in generating protective immunity (Rowland-Jones et al., 2001, Seder et al., 2008, Palucka et al., 2009). It is thought that high avidity CTL are capable of generating multiple cytokines and chemokines following activation compared to low avidity T cells. Following vaccination, polyfunctional CD8+ T cells also referred to as multifunctional CD8+ T cells can produce two or more cytokines or chemokines upon peptide or antigen stimulation. The generation of polyfunctional CD8+ T cells that secrete IFN-Îł, IL-2 and TNFÎą is considered as hallmark of protective immunity (Ahmed and Gottschalk, 2009, Duvall et al., 2006, Ferre et al., 2009, McCormack et al., 2008, Sekaly, 2008, Imrie et al., 2007).

In our laboratory, we have evaluated the avidity of HIV-specific CD8+ T cell responses generated following different immunization regimes. We have shown that following FPV-HIV/VV-HIV prime-boost immunization, mucosal (i.n./i.n. or i.n./i.m.) immunization can generate higher avidity HIVspecific CD8+ T cells compared to pure systemic (i.m./i.m.) immunization with broader cytokine/chemokine profiles. We have also found that i.n. HIVFPV prime i.m HIV-VV boost immunization generated superior quality T cells compared with i.m HIV-DNA prime/ i.n HIV-FPV boost immunization strategy and these animals were better protected against mucosal influenzaHIV challenge (Ranasinghe et al Manuscript submitted). Belyakov et al., have also compared immunization strategies that induce mucosal as well as systemic HIV-specific CTL in mice after DNA-rgp160env priming followed by recombinant MVA or AD5 gp160env booster immunization. They have shown that systemic rDNA/ rMVA prime-boost strategy in macaques generated high avidity CTLs in systemic compartment but no protective immunity in mucosa. In contrast, combined mucosal/systemic prime-boost strategy was able to induce high number of mucosal high avidity CD8+ T

124

Charani Ranasinghe & Shubhanshi Trivedi

cells that offered greater protection following mucosal pathogenic challenge (Belyakov and Ahlers, 2009, Belyakov et al., 2006, Belyakov et al., 2008).

Thus, these studies clearly indicate that i) prime-boost immunization is an effective modality for induction of high-avidity CD8+ T cells, ii) but route of vaccine delivery and the vector combination can play a critical role in the induction of high or low avidity CD8+ T cells.

Why mucosal immunization induces high avidity CTLs? Our recent findings have demonstrated that a mucosal versus systemic heterologous FPV-HIV/ VV-HIV prime-boost immunization can influence not only the magnitude but also the avidity of T cell immunity generated to vaccine antigens and protective capacity (Table 1). Intriguingly, we have for the 1st time demonstrated a hierarchical HIV-specific CD8+ T cell avidity profile for an immunodominant HIV epitope (KdGag197-207), depending on the route of vaccine delivery (i.n./i.n. > i.n./i.m. > i.m./i.m.) (Table 1). We have also established that mucosal immunization generates HIV-specific CD8+ T cells with lower IL-4 and IL-13 cytokine production but much higher in avidity Table 1. Magnitude of CD8+ T cell response, avidity and protection following i.n./i.m. FPV-HIV/VV-HIV prime- boost immunization in BALB/c and IL-13-/- mice.

Delivery

Mice

Magnitude

IL-4/IL-13 in

CTL

route

strain

of response

expression HIV-

avidity

(IFN-γ)

specific CTL

Protection

i.n./i.n.

BALB/c

Low

Low

High

+++

i.n./i.m.

BALB/c

High

Medium

Medium

++

i.m./i.m.

BALB/c

High

High

Low

+

i.n./i.n.

IL-13-/-

Low

Absent

High

+++++

i.n./i.m.

IL-13-/-

High

Absent

High

++++±

i.m./i.m.

IL-13-/-

High

Absent

High

++++±

BALB/c mice (n=4-5) were prime-boost immunized i.n./i.m. with FPV-HIV/VV-HIV (expressing gag/pol genes). 14 days post booster immunization spleens were harvested, single cell suspensions were prepared and magnitude of systemic CD8+ T cell responses were measured by IFN-γ ELIspot, the expression of IL-4 & IL-13 by KdGag197-205-specific T cells by single cell multiplex RT-PCR and avidity of CTL by KdGag197-205-specific tetramer dissociation (Ranasinghe et al., 2007). 6 weeks post booster immunization protection was evaluated following an intranasal influenza virus encoding an HIV-KdGag197-207 challenge, (+) indicates the level of protection, evaluated by weight gain or loss.

Heterologous prime-boost immunization

125

than those generated following systemic (i.m.) immunization (Ranasinghe et al., 2007). Our studies are further substantiate the findings by Kelso and co-workers where they for the 1st time demonstrated that IL-4 can be expressed by subset of CD8+ cells in-vitro and they play a role in CD8+ T cell avidity (Kelso and Groves, 1997, Kienzle et al., 2004). In the early 90’s IL-4 expressing CD8+ T cell subset was also reported following chronic HIV-1 infection (Maggi et al., 1994) and recently similar observations were reported following dengue infection (Imrie et al., 2007). When FPV-HIV/ VV-HIV prime-boost immunization was performed on IL-13-/- & IL-4-/mice (gene-knock out mice), these animals showed markedly enhanced effector CD8+ T cell avidity (Ranasinghe and Ramshaw, 2009b). Data indicated that in a mouse model complete loss of IL-13 enhanced effector and memory CTL avidity (Table 1), where as IL-4 was necessary in the cell milieu (produced by other cells not CD8+) to maintain effective memory T cell immunity and IFN-γ production (Table 2) (Ranasinghe and Ramshaw, 2009b). Furthermore, our studies also showed that transcription factor STAT6 was also involved in regulation of memory T cell avidity (Table 2) (Ranasinghe and Ramshaw, 2009b). Interestingly, it is also thought that duration of antigen stimulation at lymphoid tissue (i.e. nasal-associated lymphoid tissue) may alter the avidities of CTL clones generated (Yoshizawa et al., 2001). An interesting study by Bullock and co-workers showed that antigen density presented by dendritic cells (DC) could markedly influence avidity of memory CD8+ T cells, and lower antigen concentrations generate in high avidity memory CTL (Bullock et al., 2003). By manipulating the turnover of a viral protein Gray et.al. have shown that level of peptide presentation is a critical factor in selective expansion of high versus low avidity CD8+ T cells (Gray et al., 2004). These studies clearly demonstrated that Th1/Th2 cytokine milieu and most likely the mode of antigen uptake and presentation via mucosa or nasal associated lymphoid tissue (by microfold cells Table 2. Effector and memory CTL avidity following i.n./i.m. FPV-HIV/VV-HIV prime-boost immunization in BALB/c, IL-4-/-, 13-/- and STAT6-/- mice.

Effector T cell

Effector CTL

Memory T cell

Memory CTL

IFN-γ(a)

avidity

IFN-γ(b)

avidity

BALB/c

632±18

++

978±205

++

IL-4-/-

235±178

++++

470±109

++±

IL-13-/-

488±58

++++±

1055±378

+++++

STAT6-/-

968±86

++±

1485±261

+++++

Mice strain

126

Charani Ranasinghe & Shubhanshi Trivedi

(M cells) and dendritic cells (DCs)), compared to systemic vaccination play a significant role in CTL avidity.

Summary and concluding remarks Many studies have shown that heterologous prime-boost vaccination strategies can generate impressive immune response against a variety of encoded antigens. As majority of human pathogens enter through mucosae, novel heterologous prime-boost delivery approaches and molecular adjuvants that could enhance magnitude, avidity and induce polyfunctional effector/ memory T cells at mucosae need to be evaluated. Also, better immunological markers (biomarkers) and improved immunological techniques that can evaluate protective mucosal immune parameters need to be developed. We believe that, when developing T cell vaccines, understanding the fundamentals of “how & why� the different vaccine vectors, routes of vaccine delivery can modulate antigen presentation in different tissues and the cytokine milieu they induce can generate high or low avidity T cells, will enable the development of effective vaccines in the future. Taken together the Thai HIV-1 clinical vaccine trial and many other recent promising results obtained with prime-boost vaccine strategies, we believe prime-boost immunization still offers great hope for a future HIV-1 vaccine.

Financial disclosure/ acknowledgements This work was supported by The Australian National Health and Medical Research Council project grant award 525431 (CR).

Conflict of interest The authors declare no financial or commercial conflict of interest.

References 1.

2.

3.

4.

Ahmed, N. & Gottschalk, S. (2009) How to design effective vaccines: lessons from an old success story. Expert Rev Vaccines., 8, 543-6.

Alexaki, A., Liu, Y. & Wigdahl, B. (2008) Cellular reservoirs of HIV-1 and their role in viral persistence. Curr HIV Res., 6, 388-400.

Alexander-Miller, M. A. (2000) Differential expansion and survival of high and low avidity cytotoxic T cell populations during the immune response to a viral infection. Cell Immunol., 201, 58-62.

Alexander-Miller, M. A. (2005) High-avidity CD8+ T cells: optimal soldiers in the war against viruses and tumors. Immunol Res., 31, 13-24.

Heterologous prime-boost immunization

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

127

Alexander-Miller, M. A., Leggatt, G. R., Sarin, A. & Berzofsky, J. A. (1996) Role of antigen, CD8, and cytotoxic T lymphocyte (CTL) avidity in high dose antigen induction of apoptosis of effector CTL. J Exp Med., 184, 485-92.

Amara, R. R., Villinger, F., Altman, J. D., Lydy, S. L., O'neil, S. P., Staprans, S. I., Montefiori, D. C., Xu, Y., Herndon, J. G., Wyatt, L. S., Candido, M. A., Kozyr, N. L., Earl, P. L., Smith, J. M., Ma, H. L., Grimm, B. D., Hulsey, M. L., Miller, J., Mcclure, H. M., Mcnicholl, J. M., Moss, B. & Robinson, H. L. (2001) Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science, 292, 69-74.

Anderson, R. J., Hannan, C. M., Gilbert, S. C., Laidlaw, S. M., Sheu, E. G., Korten, S., Sinden, R., Butcher, G. A., Skinner, M. A., Hill, A. V. & Dunachie, S. J. (2004) Enhanced CD8+ T cell immune responses and protection elicited against Plasmodium berghei malaria by prime boost immunization regimens using a novel attenuated fowlpox virus Prime-boost strategies for malaria vaccine development. J Immunol, 172, 3094-100.

Asmuth, D. M., Brown, E. L., Dinubile, M. J., Sun, X., Del Rio, C., Harro, C., Keefer, M. C., Kublin, J. G., Dubey, S. A., Kierstead, L. S., Casimiro, D. R., Shiver, J. W., Robertson, M. N., Quirk, E. K. & Mehrotra, D. V. (2010) Comparative cell-mediated immunogenicity of DNA/DNA, DNA/adenovirus type 5 (Ad5), or Ad5/Ad5 HIV-1 clade B gag vaccine prime-boost regimens. J infect Dis, 201, 132-41.

Autran, B., Murphy, R. L., Costagliola, D., Tubiana, R., Clotet, B., Gatell, J., Staszewski, S., Wincker, N., Assoumou, L., El-Habib, R., Calvez, V., Walker, B. & Katlama, C. (2008) Greater viral rebound and reduced time to resume antiretroviral therapy after therapeutic immunization with the ALVAC-HIV vaccine (vCP1452). Aids., 22, 1313-22.

Bansal, G. P., Malaspina, A. & Flores, J. (2010) Future paths for HIV vaccine research: Exploiting results from recent clinical trials and current scientific advances. Curr, 12, 39-46.

Barnett, S. W., Rajasekar, S., Legg, H., Doe, B., Fuller, D. H., Haynes, J. R., Walker, C. M. & Steimer, K. S. (1997) Vaccination with HIV-1 gp120 DNA induces immune responses that are boosted by a recombinant gp120 protein subunit. Vaccine., 15, 869-73.

Barouch, D. H. (2008) Challenges in the development of an HIV-1 vaccine.

Nature., 455, 613-9.

Barouch, D. H., Kunstman, J., Kuroda, M. J., Schmitz, J. E., Santra, S., Peyerl, F. W., Krivulka, G. R., Beaudry, K., Lifton, M. A., Gorgone, D. A., Montefiori, D. C., Lewis, M. G., Wolinsky, S. M. & Letvin, N. L. (2002) Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature., 415, 335-9.

Barre-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T., Chamaret, S., Gruest, J., Dauguet, C., Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. & Montagnier, L. (1983) Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science., 220, 868-71.

128

Charani Ranasinghe & Shubhanshi Trivedi

15. Bateman, C. (2009) 'Breakthrough' Thai HIV vaccine trial controversy. S Afr Med J., 99, 774, 776-7.

16. Baudner, B. C. & Giudice, G. D. (2010) Determining the activity of mucosal adjuvants. Methods, 626, 261-85.

17. Bazin, H. (2001) [Members of the Vaccine Central Committee: a group of men deserving their native land and even humanity]. Bull Acad Natl Med., 185, 749-65.

18. Belyakov, I., M., Hel, Z., Kelsall, B., Kuznetsov, V. A., Ahlers, J. D., Nacsa, J., Watkins, D. I., Allen, T. M., Sette, A., Altman, J., Woodward, R., Markham, P. D., Clements, J. D., Genoveffa, F., Srober, W. & Berzofsky, J. A. (2001a) Mucosal AIDS Vaccine Reduces Disease and Viral Load in Gut Reservoir and Blood After Mucosal Infection of Macaques. Nature Medicine, 7, 1320-1326.

19. Belyakov, I. M. & Ahlers, J. D. (2009) Comment on "trafficking of antigenspecific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination". J Immunol., 182, 1779-80.

20. Belyakov, I. M., Ahlers, J. D., Nabel, G. J., Moss, B. & Berzofsky, J. A. (2008) Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization. Virology., 381, 106-15.

21. Belyakov, I. M. & Berzofsky, J. A. (2004) Immunobiology of mucosal HIV infection and the basis for development of a new generation of mucosal AIDS vaccines. Immunity, 20, 247-53.

22. Belyakov, I. M., Hel, Z., Kelsall, B., Kuznetsov, V. A., Ahlers, J. D., Nacsa, J., Watkins, D. I., Allen, T. M., Sette, A., Altman, J., Woodward, R., Markham, P. D., Clements, J. D., Franchini, G., Strober, W. & Berzofsky, J. A. (2001b) Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques. Nat Med, 7, 1320-6.

23. Belyakov, I. M., Isakov, D., Zhu, Q., Dzutsev, A. & Berzofsky, J. A. (2007) A novel functional CTL avidity/activity compartmentalization to the site of mucosal immunization contributes to protection of macaques against simian/human immunodeficiency viral depletion of mucosal CD4+ T cells. J Immunol., 178, 7211-21.

24. Belyakov, I. M., Kuznetsov, V. A., Kelsall, B., Klinman, D., Moniuszko, M., Lemon, M., Markham, P. D., Pal, R., Clements, J. D., Lewis, M. G., Strober, W., Franchini, G. & Berzofsky, J. A. (2006) Impact of vaccine-induced mucosal high-avidity CD8+ CTLs in delay of AIDS viral dissemination from mucosa.

Blood., 107, 3258-64 25. Boyer, J. D., Cohen, A. D., Ugen, K. E., Edgeworth, R. L., Bennett, M., Shah, A., Schumann, K., Nath, B., Javadian, A., Bagarazzi, M. L., Kim, J. & Weiner, D. B. (2000) Therapeutic Immunisation of HIV-infected Chimpanzees using HIV-1 Plasmid Antigens and Interleukin-12 Expressing Plasmids. AIDS, 14, 1515-22.

26. Broder, S. & Gallo, R. C. (1984) A pathogenic retrovirus (HTLV-III) linked to AIDS. N Engl J Med., 311, 1292-7.

Heterologous prime-boost immunization

129

27. Bullock, T. N., Mullins, D. W. & Engelhard, V. H. (2003) Antigen density presented by dendritic cells in vivo differentially affects the number and avidity of primary, memory, and recall CD8+ T cells. J Immunol., 170, 1822-9.

28. Cai, H., Yu, D. H., Hu, X. D., Li, S. X. & Zhu, Y. X. (2006) A combined DNA vaccine-prime, BCG-boost strategy results in better protection against Mycobacterium bovis challenge. DNA Cell Biol., 25, 438-47.

29. Carson, C., Antoniou, M., Ruiz-Arguello, M. B., Alcami, A., Christodoulou, V., Messaritakis, I., Blackwell, J. M. & Courtenay, O. (2009) A prime/boost DNA/Modified vaccinia virus Ankara vaccine expressing recombinant Leishmania DNA encoding TRYP is safe and immunogenic in outbred dogs, the reservoir of zoonotic visceral leishmaniasis. Vaccine., 27, 1080-6.

30. Corbett, M., Bogers, W. M., Heeney, J. L., Gerber, S., Genin, C., Didierlaurent, A., Oostermeijer, H., Dubbes, R., Braskamp, G., Lerondel, S., Gomez, C. E., Esteban, M., Wagner, R., Kondova, I., Mooij, P., Balla-Jhagjhoorsingh, S., Beenhakker, N., Koopman, G., Van Der Burg, S., Kraehenbuhl, J. P. & Le Pape, A. (2008) Aerosol immunization with NYVAC and MVA vectored vaccines is safe, simple, and immunogenic. Proc Natl Acad Sci U S A., 105, 2046-51.

31. Courtney, A. N., Nehete, P. N., Nehete, B. P., Thapa, P., Zhou, D. & Sastry, K. J. (2009) Alpha-galactosylceramide is an effective mucosal adjuvant for repeated intranasal or oral delivery of HIV peptide antigens. Vaccine., 27, 3335-41.

32. Craigo, J. K., Patterson, B. K., Paranjpe, S., Kulka, K., Ding, M., Mellors, J., Montelaro, R. C. & Gupta, P. (2004) Persistent HIV type 1 infection in semen and blood compartments in patients after long-term potent antiretroviral therapy.

AIDS Res Hum Retroviruses., 20, 1196-209.

33. Cromwell, M. A., Veazey, R. S., Altman, J. D., Mansfield, K. G., Glickman, R., Allen, T. M., Watkins, D. I., Lackner, A. A., Johnson, R. P., Vogel, T. U., Fuller, D. H., Mothe, B. R., Steffen, S., Boyson, J. E., Shipley, T., Fuller, J., Hanke, T., Sette, A., Moss, B. & Mcmichael, A. J. (2000) Induction of mucosal homing virus-specific CD8(+) T lymphocytes by attenuated simian immunodeficiency virus. Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen. J Virol, 74, 8762-6.

34. Demberg, T., Boyer, J. D., Malkevich, N., Patterson, L. J., Venzon, D., Summers, E. L., Kalisz, I., Kalyanaraman, V. S., Lee, E. M., Weiner, D. B. & RobertGuroff, M. (2008) Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirusSIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge. J Virol., 82, 10911-21.

35. Devico, A. L., Fouts, T. R., Shata, M. T., Kamin-Lewis, R., Lewis, G. K. & Hone, D. M. (2002) Development of an oral prime-boost strategy to elicit broadly neutralizing antibodies against HIV-1. Vaccine., 20, 1968-74.

36. Duvall, M. G., Jaye, A., Dong, T., Brenchley, J. M., Alabi, A. S., Jeffries, D. J., Van Der Sande, M., Togun, T. O., Mcconkey, S. J., Douek, D. C., Mcmichael, A. J., Whittle, H. C., Koup, R. A. & Rowland-Jones, S. L. (2006) Maintenance of HIV-specific CD4+ T cell help distinguishes HIV-2 from HIV-1 infection. J Immunol., 176, 6973-81.

130

Charani Ranasinghe & Shubhanshi Trivedi

37. Estcourt, M. J., Ramsay, A. J., Brooks, A., Thomson, S. A., Medveckzy, C. J. & Ramshaw, I. A. (2002) Prime-boost immunization generates a high frequency, high-avidity CD8(+) cytotoxic T lymphocyte population. Int Immunol., 14, 31-7.

38. Ferre, A. L., Hunt, P. W., Critchfield, J. W., Young, D. H., Morris, M. M., Garcia, J. C., Pollard, R. B., Yee, H. F., Jr., Martin, J. N., Deeks, S. G. & Shacklett, B. L. (2009) Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control. Blood., 113, 3978-89.

39. Flynn, N. M., Forthal, D. N., Harro, C. D., Judson, F. N., Mayer, K. H. & Para, M. F. (2005) Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis., 191, 654-65.

40. Frazer, I. H. & Cox, J. (2006) Finding a vaccine for human papillomavirus.

Lancet., 367, 2058-9.

41. Freihorst, J. & Ogra, P. L. (2001) Mucosal immunity and viral infections. Ann Med, 33, 172-7.

42. Fuller, D. H. & Haynes, J. R. (1994) A qualitative progression in HIV type 1 glycoprotein 120-specific cytotoxic cellular and humoral immune responses in mice receiving a DNA-based glycoprotein 120 vaccine. AIDS Res Hum Retroviruses., 10, 1433-41.

43. Garcia-Hernandez Mde, L., Gray, A., Hubby, B. & Kast, W. M. (2007) In vivo effects of vaccination with six-transmembrane epithelial antigen of the prostate: a candidate antigen for treating prostate cancer. Cancer Res., 67, 1344-51.

44. Gray, P. M., Parks, G. D. & Alexander-Miller, M. A. (2004) Modulation of CD8+ T cell avidity by increasing the turnover of viral antigen during infection.

Cell Immunol., 231, 14-9.

45. Hagiwara, Y., Kawamura, Y. I., Kataoka, K., Rahima, B., Jackson, R. J., Komase, K., Dohi, T., Boyaka, P. N., Takeda, Y., Kiyono, H., Mcghee, J. R. & Fujihashi, K. (2006) A second generation of double mutant cholera toxin adjuvants: enhanced immunity without intracellular trafficking. J Immunol., 177, 3045-54.

46. Hanke, T. (2008) STEP trial and HIV-1 vaccines inducing T-cell responses.

Expert Rev Vaccines., 7, 303-9.

47. Hanke, T., Samuel, R. V., Blanchard, T. J., Neumann, V. C., Allen, T. M., Boyson, J. E., Sharpe, S. A., Cook, N., Smith, G. L., Watkins, D. I., Cranage, M. P. & Mcmichael, A. J. (1999) Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using a multiepitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen. J Virol, 73, 7524-32.

48. Harari, A., Bart, P. A., Stohr, W., Tapia, G., Garcia, M., Medjitna-Rais, E., Burnet, S., Cellerai, C., Erlwein, O., Barber, T., Moog, C., Liljestrom, P., Wagner, R., Wolf, H., Kraehenbuhl, J. P., Esteban, M., Heeney, J., Frachette, M. J., Tartaglia, J., Mccormack, S., Babiker, A., Weber, J. & Pantaleo, G. (2008) An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses. J Exp Med., 205, 63-77.

Heterologous prime-boost immunization

131

49. Haynes, B. F. & Shattock, R. J. (2008) Critical issues in mucosal immunity for HIV-1 vaccine development. J Allergy Clin Immunol., 122, 3-9; quiz 10-1 Epub 2008 May 12.

50. Hill, A. V., Reece, W., Gothard, P., Moorthy, V., Roberts, M., Flanagan, K., Plebanski, M., Hannan, C., Hu, J. T., Anderson, R., Degano, P., Schneider, J., Prieur, E., Sheu, E. & Gilbert, S. C. (2000) DNA-based vaccines for malaria: a heterologous prime-boost immunisation strategy. Dev Biol (Basel). 104, 171-9.

51. Hutchings, C. L., Gilbert, S. C., Hill, A. V. & Moore, A. C. (2005) Novel protein and poxvirus-based vaccine combinations for simultaneous induction of humoral and cell-mediated immunity. J Immunol, 175, 599-606.

52. Imaoka, K., Miller, C. J., Kubota, M., Mcchesney, M. B., Lohman, B., Yamamoto, M., Fujihashi, K., Someya, K., Honda, M., Mcghee, J. R. & Kiyono, H. (1998) Nasal Immunisation of Nonhuman Primates with Simian Immunodeficiency Virus p55gag and Cholera Toxin Adjuvant Induces Th1/Th2 Help for Virus-Specific Immune Responses in Reproductive Tissues. The Journal of Immunology, 161, 5952-5958.

53. Imrie, A., Meeks, J., Gurary, A., Sukhbataar, M., Kitsutani, P., Effler, P. & Zhao, Z. (2007) Differential functional avidity of dengue virus-specific T-cell clones for variant peptides representing heterologous and previously encountered serotypes. J Virol., 81, 10081-91.

54. Jaoko, W., Nakwagala, F. N., Anzala, O., Manyonyi, G. O., Birungi, J., Nanvubya, A., Bashir, F., Bhatt, K., Ogutu, H., Wakasiaka, S., Matu, L., Waruingi, W., Odada, J., Oyaro, M., Indangasi, J., Ndinya-Achola, J., Konde, C., Mugisha, E., Fast, P., Schmidt, C., Gilmour, J., Tarragona, T., Smith, C., Barin, B., Dally, L., Johnson, B., Muluubya, A., Nielsen, L., Hayes, P., Boaz, M., Hughes, P., Hanke, T., Mcmichael, A., Bwayo, J. & Kaleebu, P. (2008) Safety and immunogenicity of recombinant low-dosage HIV-1 A vaccine candidates vectored by plasmid pTHr DNA or modified vaccinia virus Ankara (MVA) in humans in East Africa. Vaccine., 26, 2788-95.

55. Kallenius, G., Pawlowski, A., Brandtzaeg, P. & Svenson, S. (2007) Should a new tuberculosis vaccine be administered intranasally? Tuberculosis (Edinb). 87, 257-66.

56. Kelleher, A. D., Puls, R. L., Bebbington, M., Boyle, D., Ffrench, R., Kent, S. J., Kippax, S., Purcell, D. F. J., Thomson, S., Wand, H., Cooper, D. A. & Emery, S. (2006) A Randomised, Placebo-controlled Phase I Trial of DNA Prime, Recombinant Fowlpox Virus Boost Prophylactic Vaccine for HIV-1. AIDS, 20, 294-297.

57. Kelso, A. & Groves, P. (1997) A single peripheral CD8+ T cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions. Proc Natl Acad Sci U S A., 94, 8070-5.

58. Kent, S. J., Dale, C. J., Ranasinghe, C., Stratov, I., De Rose, R., Chea, S., Montefiori, D. C., Thomson, S., Ramshaw, I. A., Coupar, B. E., Boyle, D. B., Law, M., Wilson, K. M. & Ramsay, A. J. (2005) Mucosally-administered humansimian immunodeficiency virus DNA and fowlpoxvirus-based recombinant

132

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

Charani Ranasinghe & Shubhanshi Trivedi

vaccines reduce acute phase viral replication in macaques following vaginal challenge with CCR5-tropic SHIV(SF162P3). Vaccine, 23, 5009-5021.

Kent, S. J., Zhao, A., Best, S. J., Chandler, J. D., Boyle, D. B. & Ramshaw, I. A. (1998) Enhanced T-cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowlpox virus. J Virol, 72, 10180-8.

Kienzle, N., Baz, A. & Kelso, A. (2004) Profiling the CD8low phenotype, an alternative career choice for CD8 T cells during primary differentiation. Immunol Cell Biol., 82, 75-83.

Kim, J. J., Yang, J. S., Dentchev, T., Dang, K. & Weiner, D. B. (2000) Chemokine gene adjuvants can modulate immune responses induced by DNA vaccines. J Interferon Cytokine Res., 20, 487-98.

Kiyono, H. & Fukuyama, S. (2004) NALT- versus peyer's-patch mediated mucosal immunity. Nat. Immunol., 4, 699-710.

Klavinskis, L. S., Barnfield, C., Gao, L. & Parker, S. (1999) Intranasal immunization with plasmid DNA-lipid complexes elicits mucosal immunity in the female genital and rectal tracts. J Immunol, 162, 254-62.

Ko, S. Y., Cheng, C., Kong, W. P., Wang, L., Kanekiyo, M., Einfeld, D., King, C. R., Gall, J. G. & Nabel, G. J. (2009) Enhanced induction of intestinal cellular immunity by oral priming with enteric adenovirus 41 vectors. J Virol., 83, 748-56.

Kozlowski, P. A. & Neutra, M. R. (2003) The role of mucosal immunity in prevention of HIV transmission. Curr Mol Med, 3, 217-28.

Kubota, M., Miller, C. J., Imaoka, K., Kawabata, S., Fujihashi, K., Mcghee, J. R. & Kiyono, H. (1997) Oral Immunisation with Simian Immunodeficiency virus p55gag and Cholera Toxin Elicits both Mucosal IgA and Systemic IgG Immune Responses in Nonhuman Primates. Journal of Immunology, 5321-9.

Kwong, P. D., Doyle, M. L., Casper, D. J., Cicala, C., Leavitt, S. A., Majeed, S., Steenbeke, T. D., Venturi, M., Chaiken, I., Fung, M., Katinger, H., Parren, P. W., Robinson, J., Van Ryk, D., Wang, L., Burton, D. R., Freire, E., Wyatt, R., Sodroski, J., Hendrickson, W. A. & Arthos, J. (2002) HIV-1 evades antibodymediated neutralization through conformational masking of receptor-binding sites. Nature., 420, 678-82.

Kwong, P. D., Wyatt, R., Robinson, J., Sweet, R. W., Sodroski, J. & Hendrickson, W. A. (1998) Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature., 393, 648-59.

Leong, K. H., Ramsay, A. J., Morin, M. J., Robinson, H. L., Boyle, D. B. & Ramshaw, I. A. (1995) Molecular approaches to the control of infectious diseases. Vaccine 95, Brown F, Chanock H, Norrby E, Eds., 327-331.

Li, Z., Zhang, M., Zhou, C., Zhao, X., Iijima, N. & Frankel, F. R. (2008) Novel vaccination protocol with two live mucosal vectors elicits strong cell-mediated immunity in the vagina and protects against vaginal virus challenge. J Immunol., 180, 2504-13.

Heterologous prime-boost immunization

133

71. Lillard, J. W., Jr., Boyaka, P. N., Hedrick, J. A., Zlotnik, A. & Mcghee, J. R. (1999) Lymphotactin acts as an innate mucosal adjuvant. J Immunol., 162, 1959-65.

72. Lillard, J. W., Jr., Boyaka, P. N., Taub, D. D. & Mcghee, J. R. (2001) RANTES potentiates antigen-specific mucosal immune responses. J Immunol., 166, 162-9.

73. Lu, S., Wang, S. & Grimes-Serrano, J. M. (2008) Current progress of DNA vaccine studies in humans. Expert Rev Vaccines., 7, 175-91.

74. Lugade, A. A., Kalathil, S., Heald, J. L. & Thanavala, Y. (2010) Transgenic plant-based oral vaccines. Immunol Invest., 39, 468-82.

75. Maggi, E., Giudizi, M. G., Biagiotti, R., Annunziato, F., Manetti, R., Piccinni, M. P., Parronchi, P., Sampognaro, S., Giannarini, L., Zuccati, G. & Romagnani, S. (1994) Th2-like CD8+ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J Exp Med., 180, 489-95.

76. Mccormack, S., Stohr, W., Barber, T., Bart, P. A., Harari, A., Moog, C., Ciuffreda, D., Cellerai, C., Cowen, M., Gamboni, R., Burnet, S., Legg, K., Brodnicki, E., Wolf, H., Wagner, R., Heeney, J., Frachette, M. J., Tartaglia, J., Babiker, A., Pantaleo, G., Weber, J., Harari, A., Bart, P. A., Stohr, W., Tapia, G., Garcia, M., Medjitna-Rais, E., Burnet, S., Cellerai, C., Erlwein, O., Barber, T., Moog, C., Liljestrom, P., Wagner, R., Wolf, H., Kraehenbuhl, J. P., Esteban, M., Heeney, J., Frachette, M. J., Tartaglia, J., Mccormack, S., Babiker, A., Weber, J. & Pantaleo, G. (2008) EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

Vaccine., 26, 3162-74.

77. Mitchell, E. A., Bergmeier, L. A., Doyle, C., Brookes, R., Hussain, L. A., Wang, Y. & Lehner, T. (1998) Homing of mononuclear cells from iliac lymph nodes to the genital and rectal mucosa in non-human primates. Eur J Immunol, 28, 3066-74.

78. Moorthy, V. S., Imoukhuede, E. B., Keating, S., Pinder, M., Webster, D., Skinner, M. A., Gilbert, S. C., Walraven, G. & Hill, A. V. (2004) Phase 1 evaluation of 3 highly immunogenic prime-boost regimens, including a 12-month reboosting vaccination, for malaria vaccination in Gambian men. J Infect Dis., 189, 2213-9.

79. Neeson, P., Boyer, J., Kumar, S., Lewis, M. G., Mattias, L., Veazey, R., Weiner, D. & Paterson, Y. (2006) A DNA prime-oral Listeria boost vaccine in rhesus macaques induces a SIV-specific CD8 T cell mucosal response characterized by high levels of alpha4beta7 integrin and an effector memory phenotype. Virology., 354, 299-315.

80. Ogra, P. L., Faden, H. & Welliver, R. C. (2001) Vaccination strategies for mucosal immune responses. Clin Microbiol Rev, 14, 430-45.

81. Osborn, J. E. (1995) HIV: the more things change, the more they stay the same.

Nat Med., 1, 991-3.

82. Palucka, K., Ueno, H., Fay, J. & Banchereau, J. (2009) Harnessing dendritic cells to generate cancer vaccines. Ann N Y Acad Sci., 1174, 88-98.

83. Pardoll, D. M. & Beckerleg, A. M. (1995) Exposing the immunology of naked DNA vaccines. Immunity., 3, 165-9.

134

Charani Ranasinghe & Shubhanshi Trivedi

84. Peppoloni, S., Ruggiero, P., Contorni, M., Morandi, M., Pizza, M., Rappuoli, R., Podda, A. & Del Giudice, G. (2003) Mutants of the Escherichia coli heat-labile enterotoxin as safe and strong adjuvants for intranasal delivery of vaccines.

Expert Rev Vaccines., 2, 285-93.

85. Perez-Jimenez, E., Kochan, G., Gherardi, M. M. & Esteban, M. (2006) MVALACK as a safe and efficient vector for vaccination against leishmaniasis.

Microbes Infect., 8, 810-22 Epub 2006 Jan 13.

86. Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., Martin, M., Van Griensven, F., Hu, D., Tappero, J. W. & Choopanya, K. (2006) Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis., 194, 1661-71 Epub 2006 Nov 3.

87. Promkhatkaew, D., Matsuo, K., Pinyosukhee, N., Thongdeejaroen, W., LeangAramgul, P., Sawanpanyalert, P. & Warachit, P. (2009) Prime-boost vaccination using recombinant Mycobacterium bovis BCG and recombinant vaccinia virus DIs harboring HIV-1 CRF01_AE gag in mice: influence of immunization routes.

Southeast Asian J Trop Med Public Health., 40, 273-81.

88. Ramsay, A. J., Husband, A. J., Ramshaw, I. A., Bao, S., Matthaei, K. I., Koehler, G. & Kopf, M. (1994) The role of interleukin-6 in mucosal IgA antibody responses in vivo. Science., 264, 561-3.

89. Ramsay, A. J., Leong, K. H. & Ramshaw, I. A. (1997) DNA vaccination against virus infection and enhancement of antiviral immunity following consecutive immunization with DNA and viral vectors. Immunol Cell Biol., 75, 382-8.

90. Ramshaw, I. A. & Ramsay, A. J. (2000) The prime-boost strategy: exciting prospects for improved vaccination. Immunol Today, 21, 163-5.

91. Ranasinghe, C., Medveczky, J. C., Woltring, D., Gao, K., Thomson, S., Coupar, B. E. H., Boyle, D. B., Ramsay, A. J. & I.A., R. (2006) Evaluation of fowlpoxvaccinia virus prime-boost vaccine strategies for high-level mucosal and systemic Iimmunity against HIV-1. Vaccine, 24, 5881-5895.

92. Ranasinghe, C. & Ramshaw, I. A. (2009a) Genetic heterologous prime-boost vaccination strategies for improved systemic and mucosal immunity. Expert Rev Vaccines., 8, 1171-81.

93. Ranasinghe, C. & Ramshaw, I. A. (2009b) Immunisation route-dependent expression of IL-4/IL-13 can modulate HIV-specific CD8(+) CTL avidity. Eur J Immunol., 39, 1819-30.

94. Ranasinghe, C., Turner, S. J., Mcarthur, C., Sutherland, D. B., Kim, J. H., Doherty, P. C. & Ramshaw, I. A. (2007) Mucosal HIV-1 Pox Virus Prime-Boost Immunization Induces High-Avidity CD8+ T Cells with Regime-Dependent Cytokine/Granzyme B Profiles. J Immunol., 178, 2370-9.

95. Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., Chiu, J., Paris, R., Premsri, N., Namwat, C., De Souza, M., Adams, E., Benenson, M., Gurunathan, S., Tartaglia, J., Mcneil, J. G., Francis, D. P., Stablein, D., Birx, D. L., Chunsuttiwat, S., Khamboonruang, C., Thongcharoen, P., Robb, M. L., Michael, N. L., Kunasol, P. & Kim, J. H. (2009) Vaccination with ALVAC and

Heterologous prime-boost immunization

135

AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med., 361, 2209-20 Epub 2009 Oct 20.

96. Rowland-Jones, S. L., Pinheiro, S., Kaul, R., Hansasuta, P., Gillespie, G., Dong, T., Plummer, F. A., Bwayo, J. B., Fidler, S., Weber, J., Mcmichael, A. J. & Appay, V. (2001) How importnat is the 'quality' of the Cytotoxic T Lymphocyte (CTL) Response in Protection against HIV Infection? Immunology Letters, 79, 15-20.

97. Saksena, N. K. & Haddad, D. N. (2003) Viral reservoirs an impediment to HAART: new strategies to eliminate HIV-1. Curr Drug Targets Infect Disord., 3, 179-206.

98. Salk, J. E., Bazeley, P. L., Bennett, B. L., Krech, U., Lewis, L. J., Ward, E. N. & Youngner, J. S. (1954) Studies in human subjects on active immunization against poliomyelitis. II. A practical means for inducing and maintaining antibody formation. Am J Public Health Nations Health., 44, 994-1009.

99. Santosuosso, M., Zhang, X., Mccormick, S., Wang, J., Hitt, M. & Xing, Z. (2005) Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen. J Immunol., 174, 7986-94.

100. Seder, R. A., Darrah, P. A. & Roederer, M. (2008) T-cell quality in memory and protection: implications for vaccine design. Nat Rev Immunol., 8, 247-58.

101. Sekaly, R. P. (2008) The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? J Exp Med., 205, 7-12.

102. Skinner, M. A., Buddle, B. M., Wedlock, D. N., Keen, D., De Lisle, G. W., Tascon, R. E., Ferraz, J. C., Lowrie, D. B., Cockle, P. J., Vordermeier, H. M. & Hewinson, R. G. (2003) A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis.

Infect Immun., 71, 4901-7.

103. Stenstad, H., Ericsson, A., Johansson-Lindbom, B., Svensson, M., Marsal, J., Mack, M., Picarella, D., Soler, D., Marquez, G., Briskin, M. & Agace, W. W. (2006) Gut associated lymphoid tissue primed CD4+ T cells display CCR9 dependent and independent homing to the small intestine. Blood.

104. Thanavala, Y., Mahoney, M., Pal, S., Scott, A., Richter, L., Natarajan, N., Goodwin, P., Arntzen, C. J. & Mason, H. S. (2005) Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci U S A., 102, 3378-82.

105. Veazey, R. S., Demaria, M., Chalifoux, L. V., Shvetz, D. E., Pauley, D. R., Knight, H. L., Rosenzweig, M., Johnson, R. P., Desrosiers, R. C. & Lackner, A. A. (1998) Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection. Science., 280, 427-31.

106. Veazey, R. S., Gauduin, M. C., Mansfield, K. G., Tham, I. C., Altman, J. D., Lifson, J. D., Lackner, A. A. & Johnson, R. P. (2001) Emergence and kinetics of simian immunodeficiency virus-specific CD8(+) T cells in the intestines of macaques during primary infection. J Virol., 75, 10515-9.

107. Vordermeier, H. M., Villarreal-Ramos, B., Cockle, P. J., Mcaulay, M., Rhodes, S. G., Thacker, T., Gilbert, S. C., Mcshane, H., Hill, A. V., Xing, Z. & Hewinson,

136

Charani Ranasinghe & Shubhanshi Trivedi

R. G. (2009) Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infect Immun., 77, 3364-73.

108. Wang, L., Cheng, C., Ko, S. Y., Kong, W. P., Kanekiyo, M., Einfeld, D., Schwartz, R. M., King, C. R., Gall, J. G. & Nabel, G. J. (2009) Delivery of human immunodeficiency virus vaccine vectors to the intestine induces enhanced mucosal cellular immunity. J Virol., 83, 7166-75.

109. Wang, S., Kennedy, J. S., West, K., Montefiori, D. C., Coley, S., Lawrence, J., Shen, S., Green, S., Rothman, A. L., Ennis, F. A., Arthos, J., Pal, R., Markham, P. & Lu, S. (2008) Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers. Vaccine., 26, 3947-57.

110. Webster, D. E., Thomas, M. C., Pickering, R., Whyte, A., Dry, I. B., Gorry, P. R. & Wesselingh, S. L. (2005) Is there a role for plant-made vaccines in the prevention of HIV/AIDS? Immunol Cell Biol., 83, 239-47.

111. Williams, A., Hatch, G. J., Clark, S. O., Gooch, K. E., Hatch, K. A., Hall, G. A., Huygen, K., Ottenhoff, T. H., Franken, K. L., Andersen, P., Doherty, T. M., Kaufmann, S. H., Grode, L., Seiler, P., Martin, C., Gicquel, B., Cole, S. T., Brodin, P., Pym, A. S., Dalemans, W., Cohen, J., Lobet, Y., Goonetilleke, N., Mcshane, H., Hill, A., Parish, T., Smith, D., Stoker, N. G., Lowrie, D. B., Kallenius, G., Svenson, S., Pawlowski, A., Blake, K. & Marsh, P. D. (2005) Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb). 85, 29-38.

112. Xu, R., Nasar, F., Megati, S., Luckay, A., Lee, M., Udem, S. A., Eldridge, J. H., Egan, M. A., Emini, E. & Clarke, D. K. (2009) Prime-boost vaccination with recombinant mumps virus and recombinant vesicular stomatitis virus vectors elicits an enhanced human immunodeficiency virus type 1 Gag-specific cellular immune response in rhesus macaques. J Virol., 83, 9813-23.

113. Yang, K., Whalen, B. J., Tirabassi, R. S., Selin, L. K., Levchenko, T. S., Torchilin, V. P., Kislauskis, E. H. & Guberski, D. L. (2008) A DNA vaccine prime followed by a liposome-encapsulated protein boost confers enhanced mucosal immune responses and protection. J Immunol., 180, 6159-67.

114. Yoshizawa, I., Soda, Y. & Mizuochi, T. (2001) Enhancement of Mucosal Immune Responses Against Hiv-1 Gag by DNA Immunization. Vaccine, 19, 2995-3003.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 137-180 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

6. Vaccine strategies to prevent mucosal transmission of HIV-1 Yongjun Sui and Jay A. Berzofsky Vaccine Branch, National Cancer Institute, National Institutes of Health 10 Center Drive, Bethesda, MD 20892, USA

Abstract. The development of an effective human immunedeficiency virus (HIV-1) vaccine remains a critically important goal to curb the global AIDS epidemic. Though no HIV-1 vaccine is available to date, we have accumulated significant amounts of information on the induction of humoral and cellular immune responses against HIV-1 infection. Novel strategies of antigen design and vaccination approaches have been developed, which have allowed us to further investigate the protective mechanisms and develop more effective HIV-1 vaccines. This review highlights the recent advances on mucosal transmission and pathogenesis of HIV-1/SIV, and strategies for its prevention by HIV-1/SIV vaccines.

I. Introduction: Current HIV-1 vaccine clinical trials To date, the clinical trials to test the efficacy of HIV-1 vaccines have had only limited success. As previous exposure to most pathogens will lead to generation of neutralizing antibodies, and thus protect against the disease, the first logical approach was to investigate purified recombinant gp120 for Correspondence/Reprint request: Dr. Yongjun Sui & Dr. Jay Berzofsky, Vaccine Branch, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA E-mail: suiy@mail.nih.gov, berzofsj@mail.nih.gov

138

Yongjun Sui & Jay A. Berzofsky

generating neutralizing antibodies. Unfortunately, the first HIV-1 vaccine to enter the efficacy trials, a monomeric gp120 protein based on laboratoryadapted strains of HIV-1, was unable to induce neutralizing antibodies to commonly transmitted HIV-1 strains and failed to afford protection in two efficacy trials (Flynn et al., 2005; Mascola et al., 1996; Moore et al., 1995; Pitisuttithum et al., 2006). This marked failure of the first generation of HIV-1 vaccine was a disappointment. Over the years, numerous attempts to improve antibody-based vaccine efficacy were unsuccessful (Burton et al., 2004).

Given the difficulty in generating broadly reactive neutralizing antibodies, the HIV-1 vaccine field has turned its attention towards the development of T-cell vaccines. This shift was also encouraged by studies showing the critical role of CD8+ T cells in containing HIV or SIV infections (Jin et al., 1999; Schmitz et al., 1999). A variety of strategies and vectors have been used to elicit cellular immunity in the macaque models and some of them showed promising results (Amara et al., 2001; Barouch et al., 2000; Casimiro et al., 2005; Letvin et al., 2006; Shiver et al., 2002). In addition, the presence of vaccine-induced T cell responses before challenge was shown to correlate with improved viral control upon infection (Letvin, 2006). However, the recent STEP phase IIb clinical trial using Adenovirus type 5 (Ad5) expressing Gag, Pol, and Nef of HIV was a clear failure (McElrath et al., 2008). This cell-mediated vaccine regimen did not prevent HIV-1 infection or reduce the viral level, even though it was immunogenic, and induced a measurable IFNÎł ELISPOT response in 75% of vaccine recipients.

Furthermore, in the vaccinated individuals with high prior immunity to Ad5, there was a trend observed towards higher risk of HIV-1 infection compared to the placebo recipients (McElrath et al., 2008). In exploratory analyses, irrespective of baseline Ad5 antibody titer, the hazard ratio of HIV-1 infection was higher in Ad5 seropositive men and uncircumcised men, but not in Ad5 seronegative or circumcised men (McElrath et al., 2008).

Despite many disappointments in HIV-1 vaccine clinical trials, a recent clinical trial of an AIDS vaccine conducted in Thailand (RV144 trial) demonstrated 30% reduction in acquisition (Rerks-Ngarm et al., 2009), which represents the first success in decades of effort. This HIV-1 vaccine involves four injections of ALVAC-HIV (vCP1521), which is a recombinant canarypox vector vaccine that has been genetically engineered to express subtype E HIV-1 gp120 linked to transmembrane anchoring portion of gp41 (strain LAI), and HIV-1 gag and protease (LAI strain), and two-booster injections of AIDSVAX B/E, which is a recombinant glycoprotein gp120 subunit vaccine. Though a phase 3 trial of AIDSVAX B vaccine involving injection-drug users showed no effect on HIV-1 acquisition (Pitisuttithum et al., 2006), the combinations surprisingly mediated modest protection

Mucosal HIV vaccine development

139

against largely heterosexual transmission. The RV144 vaccine induced binding antibody in almost all the vaccinees, lymphoproliferation in the large majority, and measurable antigen-specific CD8+ T cell responses in only about 15% of the vaccinees. In the subjects who did acquire HIV-1 infection, vaccination did not affect viremia or the CD4+ T cell count. These are different from the preclinical non-human primate data, which we will discuss later. Overall, this trial might provide important guidance to the field.

II. HIV-1 infection and mucosal immunity Despite many years of intensive study, the immunologic correlations of protection from HIV-1 infection and/or progression remain incompletely understood, greatly hampering the efforts towards developing effective HIV-1 vaccines. A growing body of evidence suggests that mucosal immunity plays an intimate and fundamental role in HIV-1 transmission and disease development. One problem with these clinical trials was that mucosal immunity was not assessed and its contribution to protection has yet to be determined. Recently, much emphasis has been put on the research in this field.

HIV-1 infection has been considered a disease of the mucosal immune system due to the fact that mucosal surfaces of the genital and gastrointestinal (GI) tracts play an important role in the pathogenesis of HIV-1 infections.

They serve as the major portal of entry for HIV-1, with about 85% of HIV-1 transmission through mucosal surfaces, either gastrointestinal or genital. The mucosal tissue also contains the majority of the body’s lymphocytes (Brenchley et al., 2004; Douek et al., 2003; Veazey et al., 1998; Veazey and Lackner, 1998), and consequently, the immunological subsystem of the gut is a major reservoir for HIV and SIV replication (Veazey et al., 1998; Veazey and Lackner, 2004), that seeds the bloodstream and the rest of the body, and one of the first places where CD4+ T cells are depleted by the infection (Brenchley et al., 2004).

The mucosal tissues involved in the sexual transmission of HIV include cervical-vaginal, colorectal, foreskin and oral epithelium, as well as associated lymphoid tissue such as tonsils (Kozlowski and Neutra, 2003; Maher et al., 2004). The HIV target cells in the mucosal layers are mainly CD4+ T cells, monocyte /macrophages and DCs including Langerhans cells (Miller and Shattock, 2003). DCs play a critical role in mediating HIV transmission in the mucosal tissues. They are at the front line of encountering HIV, and could extend their dendrites between epithelial tight junctions in the small intestinal, rectal and vaginal epithelia to the lumen, capture HIV, and carry it to regional lymph nodes (Cameron et al., 1992). Besides being

140

Yongjun Sui & Jay A. Berzofsky

directly infected by HIV, even in the absence of viral replication, they can also capture and transfer HIV to CD4+ T cells via synapses.

The fact that transmission of HIV-1 occurs primarily via the mucosal route suggests that HIV-1 vaccines may need to elicit mucosal immune responses. Effective induction of mucosal immunity often occurs in the mucosa-associated lymphoid tissues (MALT), which contains all necessary immunopotent cells. M cells take up the antigens from the lumen. DCs could extend their dendrites to the lumen to sample antigen as well. Subsequently, DCs and macrophages process and present the antigen to T and B cells to induce humoral and cellular mucosal immune responses. These antigenspecific T and B cells then emigrate through the blood and home to distant mucosal effector sites such as lamina propria of the GI tract, respiratory and reproductive tracts.

Virus-specific CD8+ T cells, which persist throughout the chronic phase of infection, have been documented in the gastrointestinal tract (Critchfield et al., 2007), as well as throughout the female reproductive tract, including endo-and ectocervix, vaginal mucosa, uterine endometrium and fallopian tubes (Evans et al., 2001). Significant efforts have been focused on development of mucosal immunity in the genital tract. The study performed

by Reynolds et al. revealed that the virus-specific CD8+ T-lymphocyte response in the genital tact is "too late and too little" to clear infection and prevent CD4+ T-lymphocyte loss (Reynolds et al., 2005). According to this study, the female reproductive tract is a poor inductive site for SIV antigenspecific cellular responses. Though robust responses in cervicovaginal tissues and uterus were documented, they appeared only several days after the peak of virus production. Furthermore, the response in gut-associated lymphoid tissue (GALT) was surprisingly low or undetectable, possibly resulting from the severe and sustained depletion of CD4+ T lymphocytes in the GALT. However, the documentation of robust responses in female reproductive tissues suggested that vaccines that rapidly induce potent HIV/SIV-specific cellular immune responses might be able to prevent acquisition of HIV-1 infection by the mucosal route of transmission. Indeed, a pre-existing stable population of effector-cytotoxic CD8+ T cells at the mucosal site with minimal systemic expansion of T cells at the time of challenge has been shown to be able to provide significant protection from vaginal SIV challenge (Genesca et al., 2008). Meanwhile, it has to be kept in mind that the menstrual cycle affects the cervicovaginal immunological responses dramatically. CTL activity is suppressed during the post-ovulatory phase.

This might provide a window of opportunity for HIV infection and dissemination (Yeaman et al., 1998).

Mucosal HIV vaccine development

141

III. The early events of HIV infection in the mucosal tissues Given the fact that the majority of HIV transmission occurs via mucosal surfaces, elucidating the early events of HIV infection occurring in the mucosal tissues is critical to our understanding of the host-pathogen relationship and to exploitation of the knowledge in developing effective HIV vaccines and/or microbicides to combat HIV/AIDS. Macaque models have been widely used to demonstrate the early events of the SIV infection in the genital mucosa, and significant progress has been made, which potentially could be used for effective vaccine development.

After intravaginal inoculation of rhesus macaques with SIV, the virus was detected in dendritic or Langerhans cells, and CD4+ T lymphocytes in the lamina propria region located under the vaginal epithelium within 4 h of inoculation, in the draining internal iliac lymph nodes within 2 days, and in the blood within 5 days (Hu et al., 2000; Miller et al., 1992; Spira et al., 1996). In another study, after intravaginal infection with SIV, the virus was first observed in the endocervical tissue with viral loads there increasing on days 3–7 post infection, while SIV was first found in the systemic compartment of distant lymph nodes and bone marrow on day 12 (Zhang et al., 1999). The intensive studies performed by Haase et al further revealed the potential viral vulnerability at the mucosal portal of entry (Haase, 2010; Miller et al., 2005; Li et al., 2009). SIV viral infection occurs very fast, and virus crosses the mucosal epithelial barrier within hours of mucosal exposure.

However, the virus infects only a small founder population, and a local expansion is necessary during the first week of infection in order to generate sufficient virus and infected cells. Furthermore, continuous seeding from an expanding source of production is likely critical for the later establishment of a productive infection throughout the systemic lymphoid tissues. This weakness of the virus at the point of entry provides an opportunity for prevention strategies. If founder populations of infected cells do not expand sufficiently and continuously to establish a self-propagating infection due to microbicides or vaccine-induced immune mechanisms present at mucosal front lines (Haase, 2010; Miller et al., 2005), HIV/SIV transmission might be prevented. Indeed, Li et al showed that glycerol monolaurate−a widely used antimicrobial compound with inhibitory activity against proinflammatory cytokines−can protect rhesus macaques from acute infection despite repeated intravaginal exposure to high doses of SIV (Li et al., 2009).

The genetic bottleneck during HIV transmission was first suggested in 2004 (Derdeyn et al., 2004), and confirmed later. In a study of individuals from Zambia and Rwanda that were infected by two distinct viral genetic subtypes, Haaland et al compared viral sequences encoding the envelope

142

Yongjun Sui & Jay A. Berzofsky

from newly infected individuals and their spouses, as well as newly infected individuals infected by someone other than their spouse (Haaland et al., 2009). They found that in the majority of cases, HIV-1 infection is initiated by a single viral variant from complex quasispecies in the transmitting partner. The genital mucosa provides a natural barrier to infection by multiple genetic variants of HIV-1, but that this barrier can be lowered by inflammatory genital infections (Haaland et al., 2009). Inflammatory genital infections moderate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV-1. Keele et al. identified and characterized the transmitted and early founder virus envelopes in primary HIV-1 infection, and found that 78 of 102 subjects had evidence of productive clinical infection by a single virus, and 24 others had evidence of productive clinical infection by no more than two to five viruses (Keele et al., 2008). However, once the virus is replicating in lymphatic tissues, usually beginning in the second week of infection, HIV/SIV is highly adapted to evade immune clearance mechanisms. The virus has access to more susceptible target cells in the LNs, and viral replication explodes and then quickly disseminates.

Viral level peaks at the end of week 2 and declines to relatively stable levels at week 4. By that time, the viral reservoir is established, CD4 T cell depletion occurs and other pathological processes will set the stage and eventually lead to progression to AIDS (Haase, 2010; Zhang et al., 1999).

Thus reduction in viral loads, delay in disease progression should be a fallback strategy (Graham, 2009), but prevention of establishment of systemic infection must be the primary goal.

IV. The concept of a mucosal HIV-1 vaccine Because of the unique role mucosal tissues play in HIV transmission and pathogenesis, the induction of antiviral immune responses in these tissues would greatly impact viral transmission and infection. As discussed above, the first week of infection is critical. To prevent HIV from disseminating, the time window is limited to only a few days to one week. Within the first few days of exposure to virus, mucosal vaccines against HIV-1 must induce effective antigen-specific immune responses at the point of entry to nip the infection in the bud before it disseminates. Failing that, a pre-existing mucosal immunity that can keep the major mucosal reservoir of HIV replication in check may be able to slow or prevent the development of AIDS. An ideal mucosal vaccine would be one inducing both HIV-specific antibodies, especially IgG and IgA with broad neutralization activity, and long–lasting polyfunctional CD8+ and CD4+ T cell responses of high breadth

Mucosal HIV vaccine development

143

and magnitude at the mucosal sites to defend against HIV replication.

Substantial efforts have been made to induce effective immune responses directly at the mucosal sites where HIV infection occurs. A number of studies have shown that reduction of viremia resulted from virus-specifc CTL responses, mainly mediated by mucosal CD8+ T cells in rhesus macaque models (Belyakov et al., 2001; Belyakov et al., 2006; Genesca et al., 2008).

Indeed, studies from our laboratory showed that mucosal immunization of macaques with an HIV/SIV peptide vaccine was more effective against SHIV challenge than systemic vaccination (Belyakov et al., 2001) and that a peptide-prime/poxviral boost approach that induced a strong CTL mucosal response could impact dissemination of intrarectally administered pathogenic SHIV-ku2 (Belyakov et al., 2006). This is also highlighted by the recent observation that vaginal CD8+ T Cells mediated protection of rhesus macaques against vaginal SIV challenge (Genesca et al., 2008).

Though HIV-specific T cell responses may prevent and/or reduce viral replication, it is widely believed that antibodies able to neutralize HIV infection will also be important components of an effective vaccine.

Especially specific antibodies to HIV envelope that inactivate virus at the mucosal surfaces involved in sexual contact are of interest for the design of a vaccine against HIV-1.

The protective role of antibodies in the prevention of mucosal infection has been demonstrated in the macaque model. Systemically given human neutralizing monoclonal antibodies of the IgG1 subtype have been shown to be able to protect against mucosal SHIV infection (Mascola et al., 2000). A vaginally applied monoclonal antibody to HIV-1 gp120 protected macaques from SHIV infection through the vagina (Veazey et al., 2003). In a recent study, macaques receiving systemic (intramuscular) immunizations, alone or in combination with mucosal (intranasal) immunizations of gp120, were protected from infection, with no detectable plasma viral RNA, provirus, or seroconversion to nonvaccine viral proteins, and better preservation of intestinal CD4+ T cells (Barnett et al., 2008). Overall, the studies suggested that either systemic (which can transudate to the mucosa)or mucosal IgG antibodies could protect some mucosal surfaces from pathogenic challenge.

Furthermore, Bogers et al. found in their study that serum neutralizing antibodies against the challenge virus appeared to correlate with protection (Bogers et al., 2008) but higher serum levels likely result in higher IgG levels reaching the mucosa. However, HIV vaccines to date have not elicited broadly neutralizing antibodies across clades.

In addition to neutralizing antibodies, some non-neutralizing antibodies could effectively inhibit HIV replication through Ab-dependent cellular cytotoxicity (ADCC) (Holl et al., 2006) and/or Ab-dependent cell-mediated

144

Yongjun Sui & Jay A. Berzofsky

viral inhibition (ADCVI) (Florese et al., 2009; Xiao et al., 2010). In a recent recombinant adenovirus vaccine study assessing the contributions of nonneutralizing antibodies, Xiao et al found that the gp140 protein -boosted group exhibited significantly greater antibody activities mediating ADCC and ADCVI in sera, and transcytosis inhibition in rectal secretions. Furthermore, ADCC and ADCVI activities were directly correlated with antibody avidity, suggesting the importance of antibody maturation for functionality. Both ADCVI and % ADCC killing pre-challenge were significantly correlated with reduced acute viremia (Xiao et al., 2010). In addition, antibody in rectal secretions was significantly correlated with transcytosis inhibition, which also strongly correlated with reduced viremia post-challenge. A recent elegant study performed by Burton et al. also found that there was a dramatic decrease in the ability of a broadly neutralizing antibody to protect macaques against SHIV challenge when Fc receptor (FcR) and complement-binding activities were engineered out of the antibody. No loss of antibody protective activity was associated with the elimination of complement binding alone, indicating the critical role of FcR binding. These data suggested that the induction of strong humoral immune responses including both neutralizing and non-neutralizing inhibitory (FcR binding) antibodies at mucosal sites might mediate better protection against HIV transmissions (Hessell et al., 2007).

Reports on the existence of such HIV-specific mucosal antibody responses, which might mediate resistance against HIV mucosa transmission in humans, were controversial. HIV-1-specific IgA has been reported in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative individuals, and IgA from these sites has been shown to neutralize HIV-1 (Devito et al., 2000; Hirbod et al., 2008). In addition, these IgA antibodies were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner (Devito et al., 2000). However, in another cohort of HIV exposed uninfected individuals, no significant vaginal IgA or IgG responses against HIV-1 or HIV-2 were detected, and none of the vaginal secretions tested displayed any HIV-1 neutralizing activity (Dorrell et al., 2000).

V. Test of mucosal HIV-1 vaccine concept using an HIV/SIV peptide vaccine with adjuvant Our lab has been working on developing a mucosal HIV vaccine by using HIV/SIV peptides as a prototype vaccine. Use of peptide vaccines (Elsawa et al., 2004) afforded ease of manipulation, but the strategy was

Mucosal HIV vaccine development

145

intended to develop principles applicable to any type of vaccine construct, and the epitopes could be expressed in DNA vaccines (Doria-Rose and Haigwood, 2003; Liu, 2003; Liu and Ulmer, 2000) or viral (Moss, 1991) or bacterial (De Berardinis and Haigwood, 2004; Paterson and Johnson, 2004; Sadoff et al., 1988) vectors (see below).

To overcome MHC polymorphism, we located multideterminant regions of HIV-1 gp160 containing overlapping epitopes presented by several class II MHC molecules (Hale et al., 1989). The “cluster peptides� spanning such clusters of epitopes were recognized by T cells with multiple class II MHC molecules in both mice and humans (Berzofsky et al., 1991). Base on this, vaccine peptides PCLUS3-18 and PCLUS6-18, consisting of cluster peptides colinearly synthesized with a CTL epitope, P18 (Ahlers et al., 1993), were synthesized and were shown to induce high titers of neutralizing antibodies (Ahlers et al., 1993) and P18-specific CTL in mice (Shirai et al., 1994).

These helper-CTL epitope peptide constructs have served as prototype vaccines for investigating HIV/SIV vaccine strategies.

We first demonstrated in the mouse model that CD8+ CTL that must be present in the mucosal site of exposure could achieve long-lasting immune resistance to mucosal viral transmission. The resistance was ablated by depleting CD8+ cells in vivo and required CTL in the mucosa, whereas systemic (splenic) CTL are shown to be unable to protect against mucosal challenge (Belyakov et al., 1998a). Though systemic immunization was found to induce some mucosal immunity, the expression of the mucosal homing receptors was transient (Masopust et al., 2010), and therefore the tissue must be seeded with memory T cell precursors shortly after activation.

In addition, it was not clear that the mucosal immunity induced by systemic immunization was as great as would be achieved by mucosal immunization.

Given the mucosal transmission of HIV-1, and also to test the concept of whether a mucosal or systemic route of vaccine delivery is optimal to induce protective immunity, we immunized the macaques with same peptide vaccine either intrarectally administrated or subcutaneously administrated (Belyakov et al., 2001). Strong CTL responses were observed in the colon and mesenteric LN of the intrarectally immunized animals and in the axillary LN in the subcutaneously-immunized group (Belyakov et al., 2001). These responses correlated with the level of T-helper responses as well. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely both in blood and intestine in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, mucosal immunization may be more effective than systemic at reducing viral load and preventing disease

146

Yongjun Sui & Jay A. Berzofsky

because it more effectively induces CTL in the gut mucosa, where much viral replication takes place.

In the mouse model, we also found that high avidity CTL were much more effective at clearing virus than low avidity CTL specific for the same epitope and MHC molecule (Alexander-Miller et al., 1996). High avidity CTL could kill cells sooner after they were infected, when little viral protein had been synthesized and before much viral progeny was made (Derby et al., 2001), and so were more effective at controlling the virus infection. The greater efficacy of high avidity CTL in clearing virus was confirmed in an LCMV model (Gallimore et al., 1998), and extended to killing tumors in other labs (Yee et al., 1999; Zeh III et al., 1999), and our own (Derby et al., 2001). Taken together, the data from the murine studies clearly showed that the quality of the CTL, not just quantity, is critical in vaccine efficacy, and induction of high avidity CTL is an important goal.

Therefore, in the second primate study performed in the lab, we tested the hypothesis that mucosal CTLs of sufficiently high avidity and quantity can actually affect dissemination from the initial mucosal nidus of infection (Belyakov et al., 2006). In addition to the previously used CTL epitopes presented by Mamu-A*01, two Tat and one Vif epitope (Allen et al., 2000) were synthesized with PCLUS3 at the N-terminus to expand the breadth of the vaccine. All these peptides were used to prime with mutant Escherichia coli labile toxin LT (R192G), a synergistic combination of cytokines hGMCSF, hIL-12, and D-type CpG oligodeoxynucleotide (ODN) as mucosal adjuvant. A replication-deficient poxvirus vector vaccine, NYVAC (expressing SIV239 gag & pol, HIV-1 env IIIB) was used as a boost. We found that this peptide-prime/poxviral boost vaccine induced high levels of high-avidity mucosal CTLs, which impacted the dissemination of intrarectally administered pathogenic SHIV-ku2 in macaques. Furthermore, such protection correlated better with mucosal than with systemic CTLs and particularly with levels of high-avidity mucosal CTLs. These findings suggest that reduction in viral load was dependent primarily on high avidity CTL in the mucosa, and demonstrate that mucosal CTL can impact the initial nidus of infection and reduce it to the point that dissemination to the blood is delayed, if not eliminated (Belyakov et al., 2006).

The first two cohorts of macaques in our studies were challenged with pathogenic SHIV. Since SHIV viruses were not considered to reflect the actual biological circumstances of HIV-1 infection and transmission in humans (Feinberg and Moore, 2002), we used SIVmac as intrarectal challenge virus in our third macaque HIV/SIV peptide vaccine study (Sui et al., 2010). The purpose of this study was to test the protective efficacy of using different molecular adjuvants (Sui et al., 2010). Adjuvant plays a

Mucosal HIV vaccine development

147

pivotal role to enhance and direct vaccine-specific immunity (Ahlers et al., 2003; Ahlers et al., 1997a; Belyakov et al., 2000). Certain molecular adjuvants such as Toll-like receptor (TLR) agonists and cytokines can improve the quantity and quality of antigen-specific T cell responses (Kwissa et al., 2007; Wille-Reece et al., 2006), and our lab discovered a synergistic adjuvant effect of a combination of TLR2, 3 and 9 agonists to augment the functional avidity (Zhu et al., 2010). We and others also found that IL-15 is a strong cytokine adjuvant, as it promotes the homeostatic expansion of CD8+ memory T cells, and the induction of higher avidity, longer-lived T cells (Boyer et al., 2007; Kutzler et al., 2005; Oh et al., 2004; Oh et al., 2008).

Combinations of these adjuvants might therefore improve the magnitude and quality of vaccine-induced responses. Indeed, in an intra-colorectally administrated peptide primed/MVA boosted mucosal vaccine regimen, the macaques immunized with a triple combination of TLR2, 3, and 9 agonists and IL-15 demonstrated better protection against intrarectal pathogenic SIVmac251 challenge, whereas animals receiving vaccine without adjuvant, or with either TLR agonists or IL-15 alone, did not get protected, showing similarly high peak and set-point plasma viral loads (Sui et al., 2010).

Furthermore, a surprising threshold-like effect existed for vaccine-induced adaptive immune control of viral load, and only antigen-specific polyfunctional CD8+ T cells correlated with protection, not tetramer+ T cells, demonstrating the importance of T-cell quality (Sui et al., 2010).

Although most interest in molecular adjuvants has been to improve adaptive immunity, in this study we also investigated their ability to induce protective innate immunity. An adjuvant only control group, given TLR agonists and IL-15 without vaccine, was included during immunization. After challenge, we found that adjuvant-alone without vaccine antigen impacted the intrarectal SIVmac251 challenge outcome by demonstrating a certain level of protection. The protection was correlated with surprisingly long-lived APOBEC3G (A3G)-mediated innate immunity; in addition, even among animals receiving vaccine with adjuvants, viral load correlated inversely with A3G levels. Overall, this study suggested that strategic use of molecular adjuvants could provide better mucosal protection through induction of both innate and adaptive immunity (Sui et al., 2010).

VI. Vaccine strategies to induce mucosal and systemic immunity In reality, most candidate HIV vaccines induce both mucosal and systemic immunity, involving antigen-specific T cell and antibodies, and in some of the cases, innate immunity as well. HIV antigens delivered by viral vectors, in the form of DNA, and as recombinant proteins have been

148

Yongjun Sui & Jay A. Berzofsky

extensively explored, and could provide high-grade of protection in the macaque models. Heterologous prime-boost regimens, usually involving different viral vectors that are sequentially paired with each other or with DNA or recombinant protein, turned out to be the most promising approach.

Based on different vaccine delivery vector platforms, we summarize and highlight the recent advances in developing an HIV vaccine, mainly focusing on the variety of vaccine constructs that have been used to induce mucosal and systemic immunity against HIV/SIV. a. Viral vectors One of the novel immunization approaches is to use viral vectors to deliver HIV-1 antigen. Viral vectors have several attractive features, including the ability to induce strong humoral and cellular -mediated immunity. Poxvirus- and adenovirus-vectored HIV vaccines have been the most extensively studied in the clinic. Two potential problems of this strategy are the pre-existing anti-vector immunity in the targeted populations, as well as antigenic competition from the vector’s own viral antigens (though replicating rAd vectors do not produce much antigenic competition).

1. Poxvirus as an HIV vaccine vector Poxvirus vectors have been the most intensively studied live recombinant vector (Moss, 1991), and numerous studies have demonstrated their ability to induce mucosal immune responses against different antigens (Gherardi and Esteban, 2005). However, recombinant poxvirus-based vaccines induced only moderate immunogenicity in clinical trials. Therefore, these have been widely used as a booster component in heterologous prime–boost regimens, which we will discuss later.

Recombinants based on the attenuated modified vaccinia virus Ankara (MVA) vector were effective in stimulating HIV-specific immunity in the genital and rectal tracts following mucosal delivery, and also controlling SIV (Negri et al., 2001; Nilsson et al., 2002; Ourmanov et al., 2000; Seth et al., 2000) and SHIV (Barouch et al., 2001; Earl et al., 2002) viremia and disease progression. However, despite MVA’s highly attenuated nature, repeated immunization with MVA still elicited anti-vector immunity that limited the effectiveness of later vaccination (Sharpe et al., 2001). Thus, the canarypox (ALVAC) and fowlpox (FPV) viral vectors are being evaluated in clinical trials. They are naturally replication-deficient in humans, and do not seem to elicit antibodies in humans to their outer structural proteins that would be neutralizing, and humans do not have pre-existing immunity. Several

Mucosal HIV vaccine development

149

ALVAC-SIV studies have been performed in macaque models (Pal et al., 2002; Pal et al., 2006b). In contrast to the RV144 clinical trial, which conferred modest protection in acquisition, but failed to affect the viremia or the CD4+ T cell count in people who did become infected (Rerks-Ngarm et al., 2009), ALVAC-SIV did not prevent infection from SIVmac251 challenge. Nevertheless, a decrease in virus load during primary infection and protection from CD4 loss during both acute and chronic phases of infection were observed. A trend for enhanced survival of the vaccinated macaques was also observed. Neither boosting the ALVAC-SIV-gpe with gp120 immunizations nor administering the vaccine by a combination of mucosal and systemic immunization routes increased significantly the protective effect of the ALVAC-SIV-gpe vaccine (Pal et al., 2002). In fact, Stevceva et al found that both mucosal and systemic routes of immunization with live, attenuated NYVAC/ SIV(gpe) recombinant vaccine result in gag-specific CD8+ T-cell responses in mucosal tissues of macaques (JV 2002). In one of these studies, Pal et al evaluated the protective efficacy of two human ALVAC-HIV-1 recombinant vaccines expressing Gag, Pol, and gp120 (vCP250) or Gag, Pol, and gp160 (vCP1420) in a prime-boost regimen against a high-dose mucosal exposure to the pathogenic neutralizationresistant variant SHIV(KU2) virus (Pal et al., 2006b). They found that systemic immunization with these HIV vaccines decreased viral load levels not only in blood but unexpectedly also in mucosal sites and protected macaques from peripheral CD4+ T-cell loss. This protective effect was stronger when the gp120 antigen was included in the vaccine. Inclusion of recombinant Tat protein in the boosting phase along with the Env protein did not contribute further to the preservation of CD4+ T cells (Pal et al., 2006b).

In retrospect, the macaque study whose results most closely parallel the RV144 human clinical trial was the low-dose oral challenge studies in infant macaques (Van Rompay et al., 2005). Multiple immunizations of ALVACSIV or MVA-SIV vaccines were given to infant macaques during the first 3 weeks of life. Then these infant macaques were challenged with repeated daily oral inoculations with virulent SIVmac251 at 4 weeks of age. The authors observed that significantly fewer ALVAC-SIV-immunized infants were infected compared with unimmunized infants. When the monkeys that were not infected during infancy were rechallenged and infected later, ALVAC-SIV- and MVA-SIV-vaccinated animals had reduced viremia compared with unimmunized animals (Van Rompay et al., 2005).

2. Adenovirus as a HIV vaccine vector Replicating adenoviruses are attractive vehicles for mucosal delivery also. They target mucosal inductive sites, and can infect dividing and non-

150

Yongjun Sui & Jay A. Berzofsky

dividing cells, with no integration into the host genome (Robert-Guroff, 2007). Robert-Guroff’s group has demonstrated in both macaque (Patterson et al., 2004) and chimpanzee (Peng et al., 2005) models that replicating adenovirus recombinant vaccines induced potent cellular and humoral immune responses. Furthermore, replicating adenovirus-SIV/HIV priming/subunit boosting regimens elicited protective efficacy against SHIV89.6P (Demberg et al., 2007; Patterson et al., 2008) and SIVmac251 (Malkevitch et al., 2006; Patterson et al., 2004), Replication-defective rAd5 vectors were shown to elicit high-magnitude cytotoxic T-lymphocyte responses against HIV (Casimiro et al., 2003; Santra et al., 2005; Shiver et al., 2002) in macaque models as well. Persistent HIV-1 gag-specific CD8+ T-cell responses, including the secretion of interferon-γ, interleukin-2 and tumor necrosis factor-ι, were induced in more than 50% of the study volunteers in human clinical trials (Girard et al., 2006). Replicationincompetent Ad5 vaccine vector expressing Gag, used either alone or as a booster inoculation after priming with a DNA vector, has been shown to elicit effective anti-immunodeficiency-virus immunity (Shiver et al., 2002). After challenge with a pathogenic SHIV, the animals immunized with Ad5 vector exhibited the most pronounced attenuation of the virus infection (Shiver et al., 2002). However, only the DNA prime-Ad5-boosted, but not the Ad5prime-Ad5-boost Mamu-A*01(+) cohort exhibited a notable reduction in peak plasma viral load and early set-point viral burdens against SIVmac239 challenge (McDermott et al., 2005). Consistent with the monkey data, the MRKAd5 HIV-1 Gag/pol/Nef candidate vaccine did not prevent infection, and had no impact on early plasma viral load reduction in those who received the vaccine compared to the placebo recipients (McElrath et al., 2008). In addition, a pre-existing higher level of Ad5 immunity appeared to increase the risk of infection in recipients of vaccine rather than placebo, though technically it was not significant (with p=0.07). Rare serotype adenoviral vectors, such as Ad26 and Ad35, have thus been developed as potential alternative. Liu et al showed that an improved T cell-based vaccine regimen utilizing two serologically distinct adenovirus vectors, such as Ad26/Ad5, or Ad35/Ad5, elicited augmented magnitude, breadth, and polyfunctionality of cellular immune responses as compared with the homologous rAd5 regimen, which subsequently afforded substantially improved protective efficacy against SIVmac251 challenge (Liu et al., 2008). b. DNA vaccines Since the first attempts in the early 1990s (Fynan et al., 1993; Robinson et al., 1993; Ulmer et al., 1993), genetically engineered DNA has been

Mucosal HIV vaccine development

151

demonstrated to be able to be used as a vaccine and elicit immune responses.

This simple and safe approach induces not only humoral immunity but also cellular immunity (Graham et al., 2006). A large amount of data has been generated in preclinical model systems, and several technical improvements including gene optimization strategies, improved RNA structural design, novel formulations and immune adjuvants, and more effective delivery approaches have been tested. For example, the use of species-specific codon optimization and optimization for RNA nuclear export results in increased protein production on a per-cell basis, leading to enhanced T-cell responses and antibody induction (Kumar et al., 2006; Ramakrishna et al., 2004; Rosati et al., 2005; Yan et al., 2007). These methods, when combined, induced augmented levels of immune responses.

One big problem with DNA vaccines is the low efficiency for in vivo transfection. The most common route of immunization used in DNA vaccine studies is the intramuscular route. Other approaches such as gene gun delivery to the skin (Fynan et al., 1993; Oran and Robinson, 2003), direct transfections of APCs (Porgador et al., 1998), microneedle application apparatus, which could bypass the stratum corneum layer of the skin, and thus reach Langerhans cells — the APCs of the skin (Vandervoort and Ludwig, 2008), and Dermavir, which is a patch containing plasmid DNA and can be topically administered and delivered into epidermal Langerhans cells (Calarota et al., 2007; Cristillo et al., 2007), have been tested for delivering DNA. However, none of these approaches alone achieved levels of protection comparable to those of other immunization modalities. A significant effort has been made to increase efficacy. Among them, electroporation is one of the most impressive strategies, which has been extensively studied in large animals including non-human primates. Recent studies by several groups have demonstrated that in vivo expression levels improved markedly using this approach — levels increased by several fold over plasmid injection alone (Hirao et al., 2008; Luckay et al., 2007). For example, Rosati et al showed that using electroporation as a delivery approach, DNA-only vaccination induced high immune responses and provided protection after challenge with SIVmac251 by lowering the levels of both acute and chronic viral loads (Rosati et al., 2009). The level of protection was similar to that in other successful vaccine modalities applied to this model. However, human trials have indicated that the magnitude of immune responses after most DNA vaccination remains low (Bodles-Brakhop et al., 2009; Lu, 2008). As a whole, the DNA vaccines have not been very immunogenic in humans by themselves, although the newest optimized DNA vaccines (Rosati et al., 2009) have not yet been tested in humans. Prime-boost regimes with other vaccine delivery vectors have been needed to confer more effective protection against HIV infections.

152

Yongjun Sui & Jay A. Berzofsky

c. Prime-boost modality Since a single modality vaccine has often been inadequate to induce effective immune responses against HIV, combinations of heterologous immunization approaches have been attempted.

1. DNA prime-viral vector boost DNA prime-viral vector boost regimens have been widely tested in macaque models. One study utilizing a DNA prime-recombinant adenovirus boost vaccine encoding a Gag-Pol-Nef and Env was performed by Seaman et al. The vaccinated monkeys demonstrated broad cellular and humoral immune response after immunization, which conferred protection following challenge with the pathogenic SHIV 89.6P (Seaman et al., 2005). In another study to evaluate the antibody responses, Mascola et al. found that a single rAd5 immunization elicited anti-Env antibody responses, but there was little boosting with subsequent rAd5 immunizations. In contrast, rAd5 boosting of DNA-primed monkeys resulted in a rapid rise in antibody titers, including the development of anti-HIV-1 neutralizing antibodies (Nab) (Mascola et al., 2005). DNA/rAd immunization with multiple diverse Env immunogens was shown to enhance the breadth of NAb responses against HIV-1. This suggested that Env immunogens could prime for anamnestic NAb responses against a heterologous challenge virus (Seaman et al., 2007). Besides antibody responses, a DNA prime/rAd boost regimen induced polyfunctional T cell responses as well (Cox et al., 2008; Koup et al., 2010). For example, Koup et al found that compared to DNA or rAd5 vaccine alone, a DNAprime/rAd5 boost regimen induced 7-fold higher magnitude Env-specific HIV-1-specific CD8+ T-cell responses, which expressed multiple functions and were predominantly long-term central or effector memory T cells (Koup et al., 2010).

Heterologous prime/boost regimens with DNA priming and MVA boosting have also been demonstrated to induce high levels of immune responses and thus protect HIV infections. Amara et al. showed that DNA priming followed by a recombinant MVA booster controlled a highly pathogenic immunodeficiency virus challenge in a rhesus macaque model (Amara et al., 2001). Similarly, two DNA inoculations at 0 and 8 weeks and a single rMVA booster at 24 weeks effectively controlled an intrarectal challenge administered 7 months after the booster (Amara et al., 2001).

Combined parenteral and mucosal administration of a DNA prime -MVA boost vaccine regimen was shown to be able to further enhance cellular immunity and systemic control of SHIV infection (Makitalo et al., 2004).

Mucosal HIV vaccine development

153

This study found that the breadth and magnitude of the induced immune responses correlated with protective efficacy. Consistent with the MVA boost, Harari et al showed that a DNA prime and NYVAC boost vaccine regimen was also highly immunogenic (Harari et al., 2008). T cell responses could be detected in 90% of vaccinees and was superior to responses induced

by NYVAC C alone (33% of responders). Furthermore, the vaccine-induced T cell responses were polyfunctional, broad and durable. The vaccineinduced T cell responses were strongest and most frequently directed against Env (91% of vaccines), but about half of the vaccinees also demonstrated responses against Gag-Pol-Nef (Harari et al., 2008). It was also found that even intrarectal as well as systemic DNA could prime for a recombinant viral mucosal boost to elicit mucosal immunity (Belyakov et al., 2008).

2. DNA prime-protein boost A DNA prime followed by a traditional subunit protein boost is one of the most simple and effective vaccine modalities to elicit antibody responses.

The immunogenicity of an HIV-1 vaccine comprised of Env antigens was first evaluated in rhesus macaques (Letvin et al., 1997; Pal et al., 2006a; Pal et al., 2005; Rasmussen et al., 2002). Pal et al showed that DNA vaccines encoding four Env antigens from multiple HIV-1 subtypes and HIV-1 Gag antigen from a single subtype elicited a persistent level of binding antibodies to gp120 (Pal et al., 2006a). These responses were markedly enhanced following boosting with homologous gp120 proteins in QS-21 adjuvant irrespective of the route of DNA immunization (Pal et al., 2006a). These sera neutralized both homologous and, to a lesser degree, heterologous HIV-1 isolates. Four of the six immunized animals were completely protected following rectal challenge with a SHIV, whereas the virus load was reduced in the remaining animals compared to na誰ve controls. Thus this modility elicits anti-HIV-1 immune responses capable of protecting macaques against mucosal transmission of an R5 tropic SHIV isolate. d. Other modalities Classical vaccine strategies have failed to control HIV replication. Over the years, several new modalities have been developed to elicit long-lasting anti-HIV immune responses, as the induction of strong and persistent memory antibody and/or CD8+ T cell responses has been considered one of the hallmarks of successful vaccination. Persistent vectors such as CMV, lentiviral vectors and BCG have been tried in macaque models.

154

Yongjun Sui & Jay A. Berzofsky

1. Human cytomegalovirus (CMV) as a vaccine vector Most current T cell vaccine strategies use nonpersistent vectors that produce antigen temporarily, and induce central memory T cell (TCM cell)based immune responses. It has been proposed that vaccines designed to maintain differentiated effector memory T cell (TEM cell) responses at viral entry sites might improve efficacy (Hansen et al., 2009). In contrast to TCM cells, which have limited immediate effector function and seem to develop too slowly to prevent the initial viral replication in nonhuman primate models, TEM cells are the predominant type of T cell in mucosal effector sites, and ready to fight and contain the small founder populations at the mucosal sites, when the virus is probably most susceptible to immune control. Hansen et al. therefore developed SIV protein-encoding vectors based on rhesus cytomegalovirus (RhCMV), which upon infection will establish a lifelong persistence (Hansen et al., 2009). The CMV vector based HIV vaccine was characterized by prolonged viral gene expression, infecting multiple cell types throughout the body, and most importantly, inducing highfrequency and highly TEM-biased CD4+ and CD8+ T cell responses. This study found that after immunization with recombinant RhCMV/SIV constructs, the macaques developed robust anti-SIV cellular immune responses with a notable skewing towards an effector memory versus a central memory phenotype (Hansen et al., 2009). The vaccinated animals were then challenged by low-dose repeated intrarectal challenge of SIVmac239 virus. Compared to control ones, the vaccinated animals showed increased resistance to acquisition of SIVmac239 infection with four out of 12 macaques not getting infected. These data suggest that vaccines capable of generating and maintaining HIV-specific TEM cells might be able to impair viral replication at its earliest stage, and thus decrease the incidence of HIV acquisition after sexual exposure.

2. Lentiviral vectors as an HIV vaccine delivery platform Because of its persistence, a lentiviral vector is also an attractive vehicle as an HIV vaccine delivery platform. It transduces nondividing cells, including dendritic cells, enables sustained transgene expression and thus the induction of high proportions of specific cytotoxic T cells and long-lasting memory T cells. A recent pilot study from Beignon et al. showed that lentiviral vector-based prime/boost vaccination conferred protection against SIVmac251 challenge in macaques (Beignon et al., 2009). Macaques were immunized twice by low-dose subcutaneous injections of HIV-1-derived lentiviral vectors encoding a nonsecreted SIVmac239 Gag protein. A strong

Mucosal HIV vaccine development

155

and diverse cellular immunity was elicited in macaques, which resulted in a significant protection against early viral replication after a massive SIVmac251 challenge. Furthermore, the vaccine conferred protection against the severe depletion of the CD28+ CD95+ memory CD4+ T-cell compartment, as well as total CD4+ T cells. In other studies, the immunogenicity of an HIVbased lentiviral vector, VRX1023, was tested in a mouse model. VRX1023 contains the 5′ and 3′ long terminal repeats, unmodified Gag-Pol sequence, Rev response element (RRE), and Rev sequence from the HIV-1 NL4-3 molecular clone. This vector induced significant mucosal and systemic cellular and humoral responses against HIV after sub-cutaneous injection (Lemiale et al., 2010). A VRX1023-primed/Ad5 HIV-boosted vaccine regimen induced a robust, and long-lasting antigen-specific immune response, which was superior to either Ad5/Ad5 or VRX1023/VRX1023 homologous immunizations. In addition, vector-specific neutralizing antibodies were also significantly reduced (Asefa et al., 2010).

3. Recombinant Bacille Calmette-Guérin (BCG) as a vaccine vector Recombinant Bacille Calmette-Guérin (BCG) has been proposed as an HIV vaccine vector because of its safety, persistence, and the ability to induce long-lasting memory. A number of studies with SIV and HIV expressed in BCG vectors have been conducted in non-human primates.

Early attempts by Yasutomi et al showed that immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys (Yasutomi et al., 1993). The same group further demonstrated that a combined immunization modality using a recombinant BCG-SIV Gag construct for priming, and peptide formulated in liposome for boosting, elicited a greater p11C-specific CTL response than did a single immunization with peptide-liposome alone. This single epitope vaccine, however, did not protect against intravenous SIV challenge (Yasutomi et al., 1995). A single simultaneous inoculation of all four recombinants elicited SIV-specific IgA and IgG antibody, and cellular immune responses, including CTL and helper T cell proliferation (Leung et al., 2000). Mederle et al explored immune responses induced in cynomolgus macaques to rBCGSIV (Mederle et al., 2003), a mixture of three recombinant BCG strains expressing the SIVmac251 nef, gag and env genes. CTL responses targeted against the three antigens and IFN-γ secretion were observed after intradermal inoculation. Anti-SIV IgAs in the rectum were elicited after a rectal or oral boosting. However, the protection was limited, with all the animals getting infected upon SIVmac251 rectal challenge. Using an rBCG-

156

Yongjun Sui & Jay A. Berzofsky

SIVgag vector to protect against SHIV challenge turned out to be more effective. Ami et al. found that a rBCG-SIVgag-primed/ replication-deficient vaccinia virus-SIV-boosted regimen induced high levels of IFN-Îł spotforming cells specific for SIV Gag. This combination regimen protected the macaques from mucosal challenge with pathogenic SHIV (Ami et al., 2005).

Someya et al showed that immunization with an rBCG Env V3 vaccine elicits a strong, type-specific V3 NAb response in rhesus macaques. While this response was not sufficient to provide protection against a pathogenic SHIV challenge, it was able to significantly reduce the viral load in macaques following challenge with a nonpathogenic SHIV (Someya et al., 2005). In one recent macaque study, high frequency SIV-specific cellular responses were observed in the rBCG-SIV-primed/ recombinant adenovirus 5 (rAd5)SIV boosted macaques. In addition, these cellular responses were predominantly polyfunctional CD8+ T cells with greater magnitude and more persistence than those generated after vaccination with rAd5 alone (Cayabyab et al., 2009). e. Mosaic HIV vaccine targeting virus diversity Globally, HIV-1 is extraordinarily variable, and this diversity poses a major obstacle to AIDS vaccine development (Gaschen et al., 2002). The mosaic vaccine approach, which applies an in silico algorithm to select vaccine immunogens that provide maximal coverage of potential T cell epitopes for a viral population, was shown to be able to target the diversity of HIV viruses. Fischer et al first proposed the concept of mosaic HIV vaccine (Fischer et al., 2007), and recently, Barouch et al (Barouch et al., 2010) and Santra (Santra et al., 2010) et al demonstrated independently that mosaic vaccines could elicit broad cellular immune responses and confer enhanced immune coverage of diverse HIV strains in macaques. Barouch et al.

(Barouch et al., 2010) used Ad26 as the delivery vector, and compared the breadth and magnitude of epitope-specific CD8+ and CD4+ T lymphocyte responses elicited by bivalent mosaic, consensus and natural sequence HIV antigens in rhesus macaques. Consistent with the theoretical prediction, mosaic HIV-1 gag, pol, and env antigens augment both the breadth and depth of the epitope-specific cellular immune responses as compared to consensus and natural sequence HIV antigens. Santra et al (Santra et al., 2010) immunized the macaques with a DNA prime/recombinant vaccinia virus boost regimen. The HIV gag and nef vaccine constructs expressed either polyvalent mosaic or consensus proteins. The magnitudes of the vaccineinduced Gag-specific and Nef-specific antibody and ELISPOT responses were comparable between the groups. Notably, mosaic immunogens induced

Mucosal HIV vaccine development

157

CD8+ T lymphocytes with greater cross-reactivity than the consensus immunogen; they responded to more epitopes of each viral protein, and to more variant sequences of each epitope. Taken together, mosaic HIV vaccines might provide a solution to cope with the genetic diversity of HIV in a population. An alternative is to identify highly conserved epitopes that the viruses cannot afford to mutate. f. Strategies to induce antibody-mediated protection An antigen that could elicit broad neutralization antibodies against HIV is the key for HIV vaccine development. However, very little progress has been made in searching for such antigens. Adoptively transferred broad neutralizing antibodies could protect the macaques from intravenous, vaginal and rectal challenges of SHIV virus (Baba et al., 2000; Mascola et al., 1999; Mascola et al., 2000; Shibata et al., 1999). One big problem was the high antibody concentration used to achieve the protective effect, which turned out to be at least two orders of magnitude greater than in vitro neutralization concentrations (Nishimura et al., 2002; Parren et al., 2001). This calculation, though having several caveats, raised the concern that sterilizing protection solely via antibodies might not be achievable. However, a recent study by Hessel et al showed that plasma concentration of antibody corresponding to relatively modest neutralization titers could protect macaque from repeated low-dose R5 SHIV challenge, via a mucosal route, more closely mimicking the case for the vast majority of HIV-1 infections worldwide (Hessell et al., 2009). Even before this study, DeVico et al showed that a full-length single chain (FLSC) containing BaLgp120 and rhesus macaque CD4 domains 1 and 2, induced strong antibody responses to CD4-induced epitopes (DeVico et al., 2007). Animals vaccinated with a FLSC demonstrated significant protection against a rectal SHIV challenge compared to naive animals. Furthermore, this control of viral infection correlated with stronger antibody responses to CD4 inducible epitopes, but not to anti-CD4 responses, anti-gp120 binding or neutralization antibodies. This study thus confirmed the concept that vaccines capable of inducing only antibody responses could also achieve prophylactic efficacy against mucosal challenge (DeVico et al., 2007). Further proof of antibody-only mediated protection came from a study performed by Johnson et al using an alternative to more traditional strategies (Johnson et al., 2009).

Immunoadhesin constructs containing variable heavy and variable light chains from the SIV gp120-specific Fab clones, which had been derived directly from SIV-infected macaques (Johnson et al., 2003), a rhesus IgG2 Fc fragment, and also a linker linking all these components together, were expressed by adeno-associated virus (AAV) vector to passively deliver

158

Yongjun Sui & Jay A. Berzofsky

protective antibody constructs. Nine rhesus macaques were immunized with AAV vectors carrying the immunoadhesin constructs. Six of them were protected against SIVmac316 challenge, and all nine were protected from AIDS, whereas all six of the controls became infected and four of them died over the course of experiments. Although considerable hurdles remain, this gene transfer technology successfully generated long-lived, protective anti-SIV biological activity in the serum, independent of the adaptive immune system.

VII. Conclusions and future perspectives To develop an effective HIV-1 vaccine, we are still facing tremendous challenges: to identify the ideal antigens, adjuvant, and delivery vehicles which can induce high quantity and quality immune responses both in the systemic and at the mucosal sites, to overcome the genetic diversity of HIV-1 virus etc. Most urgently, we still lack knowledge regarding what kind of immune responses should be induced in order to prevent infection and/or disease progress. Therefore, a better understanding of the underlying mechanisms of immune responses to control the viral transmission and pathogenesis will be needed to inform the design of next generation of HIV-1 vaccines.

Considering the many issues that need to be addressed, it becomes clear that not all of the questions can be answered by the study of HIV itself (Virgin and Walker, 2010). We therefore combined the information and knowledge from other fields and proposed a push-pull model to develop an HIV-1 vaccine to protect against HIV-1 transmission and progress (figure 1).

This model includes four components: epitope enhancement to optimize the antigens (Ahlers et al., 2001; Ahlers et al., 1997b; Okazaki et al., 2006; Okazaki et al., 2003), molecular adjuvants such as cytokines/TLRs ligands to improve the quality of the responses (Ahlers et al., 2003; Oh et al., 2003a; Oh et al., 2004; Oh et al., 2008; Zhu et al., 2010; Zhu et al., 2008a), agents to block negative regulators such as PD-1/TGF-β (Takaku et al., 2009 (2010); Terabe et al., 2009; Velu et al., 2008), and approaches to induce in situ or guide the anti-HIV CD4+/CD8+ T cells and antibodies trafficking to the gut or vaginal mucosal compartments.

First, the viral antigen itself may not bind optimally to MHC molecules of the host. After all, viruses evolved to evade the immune system, not to be good vaccines. We ought to be able to do better than the natural viral sequence. Thus, we would start by mapping epitopes and modifying their sequences to increase the affinity for common MHC (HLA in humans or Mamu in macaques) molecules, without altering the surface of the peptide

Mucosal HIV vaccine development

159

Figure 1. Push-pull model to induce optimized immune responses for HIV vaccine development. This model includes four components: epitope enhancement to optimize the antigens, molecular adjuvants such as cytokines/TLRs to improve the quality of the responses, approaches to target to the appropriate tissue, and blockade of negative regulators such as PD-1/TGF-β to allow the immune responses to achieve their full potential. (Drawing courtesy of Dr. Masaki Terabe.)

presented by the MHC molecule to the T cell receptor. We call this process “epitope enhancement.� Although we have carried out epitope enhancement primarily with peptides for convenience, enhanced epitopes can be incorporated in any vaccine construct, such as DNA, viral vector, bacterial vector, or recombinant protein, as well as peptide. Such epitope enhancement can increase the immunogenicity of epitopes and induce responses to epitopes that would otherwise be subdominant.

We then propose to push or drive the response not just toward increased magnitude, but also in a particular direction to improve the quality for a particular purpose, such as a certain cytokine balance or increased CD8+ T cell avidity for clearing virus-infected cells. We had previously shown that high avidity CD8+ T cells are more effective than low avidity ones for the same peptide-MHC complex at clearing virus infections (Alexander-Miller et al., 1996). We accomplish this by use of defined molecular adjuvants, such as cytokines, chemokines, TLR ligands and costimulatory molecules like CD40L (or agonistic anti-CD40 antibodies). For example, we have found that a combination of three costimulatory molecules can increase CD8+ T cell avidity (Oh et al., 2003b), as can the expression of IL-15 by the vaccine vector (Oh et al., 2004). IL-15 can also increase CTL longevity, even in the absence of CD4+ T cell help (Oh et al., 2003a; Oh et al., 2008). We have also seen that CD40L synergizes with GM-CSF, as does IL-12 with GM-CSF, to increase CD8+ T cell response magnitude (Ahlers et al., 2002; Ahlers et al.,

160

Yongjun Sui & Jay A. Berzofsky

1997a). More recently, we have seen that a double combination of ligands for TLRs 2/6 and 3 or TLRs 9 and 3 can increase the magnitude of the CD8+ T cell response (Zhu et al., 2008a) and that a triple combination of all three increases protection against a viral infection by increasing T cell avidity as well, probably through increased production of IL-15 by dendritic cells (Zhu et al., 2010).

Pushing the response in the right functional direction is not enough, however, because we have to direct it to the right tissue, also. In the case of HIV, which is naturally transmitted through mucosa, this means directing T cell migration to the relevant GI or genital mucosa. Although there is some evidence that systemic (e.g. IM) immunization can induce some mucosal immunity (Baig et al., 2002; Barnett et al., 2008; Kaufman et al., 2008; Vogel et al., 2003), this does not mean that systemic immunization is equally effective as mucosal immunization. Although very few studies have compared mucosal and systemic immunization with the same vaccine, we have done this in both mice and macaques, and found consistently that the mucosal route gives much better mucosal T cell immunity than systemic immunization with the same vaccine (Belyakov et al., 1998a; Belyakov et al., 1998b; Belyakov et al., 2001). Of course, some viral vector vaccines may accomplish the same thing by taking advantage of the tropism of the virus, such as adenovirus used by Kaufman et al (Kaufman et al., 2008), to home to mucosa even if it is not delivered by a mucosal route, but that is just another way to get the vaccine to the mucosa. Further, protection against a mucosal virus challenge does not necessarily imply mucosal immunity. For example, it is well known that an IM vaccine can protect against influenza, which is mucosally transmitted, but this does not imply that mucosal immunity was induced. The protection afforded by conventional “flu shots� is purely antibody-mediated.

For T cell immunity, it is clear that to achieve sterilizing immunity against HIV, it is necessary to have T cells present at the local site of transmission to eradicate the initial nidus of infection before it disseminates.

Once virus disseminates to the blood stream, it is too late for T cells to do more than control an HIV or SIV infection, but it is unlikely that T cells could eradicate it. Therefore, there is just a narrow window in time and space when the initial infection is isolated in the mucosa and vulnerable to eradication by T cells, which would have to be present locally in the mucosa at the site of transmission (Haase, 2010). Therefore, we believe that if T cell immunity is the goal, it will be essential to direct the vaccine to the appropriate mucosa. This could be accomplished by directly administering it there or targeting it there using some targeting method. We and others have found that intrarectal immunization is the most effective for inducing

Mucosal HIV vaccine development

161

colorectal immunity (Belyakov et al., 1998b) and induces vaginal immunity as well (Zhu et al., 2008b). Recently, we have developed a method to deliver antigens to the colonic mucosa via a more convenient oral route, using nanoparticles with protective coatings to bypass the stomach and small intestine, where oral tolerance might be induced (Zhu et al., manuscript submitted). Alternatively, one can use viral vector tropisms to target the antigens, as noted above, or can artificially upregulate mucosal homing molecules on the T cells to direct them to the relevant mucosa (Greenberg paper in press, etc.). Of note, we have found that all major vaccine modalities can be delivered by the intra-colorectal route without loss of immunogenicity, including protein and peptides (Belyakov et al., 1998b), adenoviral and poxviral vectors (Belyakov et al., 2008; Belyakov et al., 2006; Belyakov et al., 1998c; Sui et al., 2010), and DNA (Belyakov et al., 2008), as well as cytokine and TLR ligand adjuvants (Sui et al., 2010; Zhu et al., 2010).

Pushing and steering the immune response are important, but are not sufficient if there are brakes holding back the response. Therefore, the other component of the push-pull approach is to “pull” the response by removing the brakes, i.e. negative regulatory mechanisms. We have focused on regulatory type II NKT cells that suppress immune responses by making IL-13 that induces myeloid cells to make TGF-beta (Ambrosino et al., 2007; Berzofsky and Terabe, 2008; Fichtner-Feigl et al., 2008; Park et al., 2004; Terabe and Berzofsky, 2008; Terabe et al., 2000; Terabe et al., 2003; Terabe et al., 2005). Indeed we found that blocking IL-13 or TGF-β enhanced vaccine responses (Ahlers et al., 2002; Takaku et al., 2009 (2010); Terabe et al., 2009). Of course, we also may need to eliminate or reduce Foxp3+ T reg cells and myeloid derived suppressor cells (MDSC). Anti-TGF-β may also be inhibiting the induction of inducible T reg cells. Furthermore, we may need to block negative regulatory molecules on the T cells themselves, such as PD-1 or CTLA-4 or their ligands (like PD-L1 and PD-L2) (Day et al., 2006; Finnefrock et al., 2009; Onlamoon et al., 2008; Petrovas et al., 2006; Petrovas et al., 2007; Trautmann et al., 2006; Velu et al., 2008). (However, in contrast to PD-1, blockade of CTLA-4 has been associated with T cell activation leading to increased viral load (Beck et al., 2006; Cecchinato et al., 2008; Yang et al., 2007), potentially limiting its use). Because these mechanisms may interact with each other, it may not be necessary to block all of them at the same time. However, as multiple inhibitory mechanisms may co-exist, it is likely that it will be necessary to block more than one. For example, it was shown that blocking CTLA-4 at the same time as depleting Treg cells was effective in a cancer model (Sutmuller et al., 2001).

Furthermore, in an LCMV infection model, blockade of PD-1 and LAG-3 simultaneously and synergistically improved T cell responses and diminished

162

Yongjun Sui & Jay A. Berzofsky

viral load in vivo (Blackburn et al., 2009). While concerns have been raised about blockade of negative regulators because mice genetically deficient in some of these cells or molecules may be prone to autoimmune disease (Bergman et al., 2001), and some autoimmune side effects have been seen during anti-CTLA-4 treatment in patients (Beck et al., 2006; Yang et al., 2007), it is much less likely that one would risk autoimmune side effects if the blockade or depletion is transient, just during the time of vaccine induction of the immune response, in contrast to lifelong absence in a knockout mouse. There is enough evidence in animal model systems to suggest the efficacy of several of these approaches, and enough evidence that just inducing T cells may not be sufficient if they are kept in an inactive or exhausted state or kept away from the target tissue by regulatory cells, that it will also be necessary to include this “pull� side of the equation to achieve a maximal vaccine response.

Overall, then, we think that HIV has developed sufficiently clever ways of evading and suppressing the immune system that we will need to include all of these components of a push-pull vaccine strategy, in addition to use of conserved epitopes that the virus cannot afford to mutate without loss of fitness, if we are going to succeed in making a broadly effective vaccine against HIV and other viruses that cause chronic infections, as well as against cancer.

Acknowledgments We thank Masaki Terabe for helping to prepare the figure, and Barney Graham, Marjorie Guroff, and Genoveffa Franchini for critical reading of the manuscript and helpful suggestions.

References 1.

2.

3.

4.

Ahlers, J.D., Belyakov, I.M., and Berzofsky, J.A. (2003) Cytokine, chemokine and costimulatory molecule modulation to enhance efficacy of HIV vaccines.

Current Molecular Medicine 3; 285-301.

Ahlers, J.D., Belyakov, I.M., Terabe, M., Koka, R., Donaldson, D.D., Thomas, E., and Berzofsky, J.A. (2002) A push-pull approach to maximize vaccine efficacy: abrogating suppression with an IL-13 inhibitor while augmenting help with GM-CSF and CD40L. Proc Natl Acad Sci U S A 99; 13020-13025.

Ahlers, J.D., Belyakov, I.M., Thomas, E.K., and Berzofsky, J.A. (2001) High affinity T-helper epitope induces complementary helper and APC polarization, increased CTL and protection against viral infection. J Clin Invest 108; 1677-1685.

Ahlers, J.D., Dunlop, N., Alling, D.W., Nara, P.L., and Berzofsky, J.A. (1997a) Cytokine-in-adjuvant steering of the immune response phenotype to HIV-1

Mucosal HIV vaccine development

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

163

vaccine constructs: GM-CSF and TNFa synergize with IL-12 to enhance induction of CTL. JImmunol 158; 3947-3958.

Ahlers, J.D., Pendleton, C.D., Dunlop, N., Minassian, A., Nara, P.L., and Berzofsky, J.A. (1993) Construction of an HIV-1 peptide vaccine containing a multideterminant helper peptide linked to a V3 loop peptide 18 inducing strong neutralizing antibody responses in mice of multiple MHC haplotypes after two immunizations. JImmunol 150; 5647-5665.

Ahlers, J.D., Takeshita, T., Pendleton, C.D., and Berzofsky, J.A. (1997b) Enhanced immunogenicity of HIV-1 vaccine construct by modification of the native peptide sequence. ProcNatlAcadSciUSA 94; 10856-10861.

Alexander-Miller, M.A., Leggatt, G.R., and Berzofsky, J.A. (1996) Selective expansion of high or low avidity cytotoxic T lymphocytes and efficacy for adoptive immunotherapy. ProcNatlAcadSciUSA 93; 4102-4107.

Allen, T.M., Mothe, B.R., Sidney, J., Jing, P., Dzuris, J.L., Liebl, M.E., Vogel, T.U., O'Connor, D.H., Wang, X., Wussow, M.C., et al. (2000) CD8(+) lymphocytes from simian immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule mamu-A*01: implications for vaccine design and testing.

Journal of Virology 75; 738-749.

Amara, R.R., Villinger, F., Altman, J.D., Lydy, S.L., O'Neil, S.P., Staprans, S.I., Montefiori, D.C., Xu, Y., Herndon, J.G., Wyatt, L.S., et al. (2001) Control of a mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vaccine. Science 292; 69-74.

Ambrosino, E., Terabe, M., Halder, R.C., Peng, J., Takaku, S., Miyake, S., Yamamura, T., Kumar, V., and Berzofsky, J.A. (2007) Cross-regulation between type I and type II NKT cells in regulating tumor immunity: A new immunoregulatory axis. J Immunol 179; 5126-5136.

Ami, Y., Izumi, Y., Matsuo, K., Someya, K., Kanekiyo, M., Horibata, S., Yoshino, N., Sakai, K., Shinohara, K., Matsumoto, S., et al. (2005) Primingboosting vaccination with recombinant Mycobacterium bovis bacillus CalmetteGuerin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity. J Virol 79; 12871-12879.

Asefa, B., Korokhov, N., and Lemiale, F. (2010) Heterologous HIV-based lentiviral/adenoviral vectors immunizations result in enhanced HIV-specific immunity. Vaccine 28; 3617-3624.

Baba, T.W., Liska, V., Hofmann-Lehmann, R., Vlasak, J., Xu, W., Ayehunie, S., Cavacini, L.A., Posner, M.R., Katinger, H., Stiegler, G., et al. (2000) Human neutralizing monoclonal antibodies of the IgG1 subtype protect against mucosal simian-human immunodeficiency virus infection. Nat Med 6; 200-206.

Baig, J., Levy, D.B., McKay, P.F., Schmitz, J.E., Santra, S., Subbramanian, R.A., Kuroda, M.J., Lifton, M.A., Gorgone, D.A., Wyatt, L.S., et al. (2002) Elicitation of simian immunodeficiency virus-specific cytotoxic T lymphocytes in mucosal compartments of rhesus monkeys by systemic vaccination. J Virol 76; 1148411490.

164

Yongjun Sui & Jay A. Berzofsky

15. Barnett, S.W., Srivastava, I.K., Kan, E., Zhou, F., Goodsell, A., Cristillo, A.D., Ferrai, M.G., Weiss, D.E., Letvin, N.L., Montefiori, D., et al. (2008) Protection of macaques against vaginal SHIV challenge by systemic or mucosal and systemic vaccinations with HIV-envelope. AIDS 22; 339-348.

16. Barouch, D.H., O'Brien, K.L., Simmons, N.L., King, S.L., Abbink, P., Maxfield, L.F., Sun, Y.H., La Porte, A., Riggs, A.M., Lynch, D.M., et al. (2010) Mosaic HIV-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys. Nat Med 16; 319-323.

17. Barouch, D.H., Santra, S., Kuroda, M.J., Schmitz, J.E., Plishka, R., BucklerWhite, A., Gaitan, A.E., Zin, R., Nam, J.H., Wyatt, L.S., et al. (2001) Reduction of simian-human immunodeficiency virus 89.6P viremia in rhesus monkeys by recombinant modified vaccinia virus Ankara vaccination. J Virol 75; 5151-5158.

18. Barouch, D.H., Santra, S., Schmitz, J.E., Kuroda, M.J., Fu, T.M., Wagner, W., Bilska, M., Craiu, A., Zheng, X.X., Krivulka, G.R., et al. (2000) Control of viremia and prevention of clinical AIDS in rhesus monkeys by cytokineaugmented DNA vaccination. Science 290; 486-492.

19. Beck, K.E., Blansfield, J.A., Tran, K.Q., Feldman, A.L., Hughes, M.S., Royal, R.E., Kammula, U.S., Topalian, S.L., Sherry, R.M., Kleiner, D., et al. (2006) Enterocolitis in patients with cancer after antibody blockade of cytotoxic Tlymphocyte-associated antigen 4. J Clin Oncol 24; 2283-2289.

20. Beignon, A.S., Mollier, K., Liard, C., Coutant, F., Munier, S., Riviere, J., Souque, P., and Charneau, P. (2009) Lentiviral vector-based prime/boost vaccination against AIDS: pilot study shows protection against Simian immunodeficiency virus SIVmac251 challenge in macaques. J Virol 83; 10963-10974.

21. Belyakov, I.M., Ahlers, J.D., Brandwein, B.Y., Earl, P., Kelsall, B.L., Moss, B., Strober, W., and Berzofsky, J.A. (1998a) The Importance of local mucosal HIVspecific CD8+ cytotoxic T lymphocytes for resistance to mucosal-viral transmission in mice and enhancement of resistance by local administration of IL-12. JClinInvest 102 (12); 2072-2081.

22. Belyakov, I.M., Ahlers, J.D., Clements, J.D., Strober, W., and Berzofsky, J.A. (2000) Interplay of cytokines and adjuvants in the regulation of mucosal and systemic HIV-specific cytotoxic T lymphocytes. J Immunol 165; 6454-6462.

23. Belyakov, I.M., Ahlers, J.D., Nabel, G.J., Moss, B., and Berzofsky, J.A. (2008) Generation of functionally active HIV-1 specific CD8+ CTL in intestinal mucosa following mucosal, systemic or mixed prime-boost immunization. Virology 381; 106-115.

24. Belyakov, I.M., Derby, M.A., Ahlers, J.D., Kelsall, B.L., Earl, P., Moss, B., Strober, W., and Berzofsky, J.A. (1998b) Mucosal immunization with HIV-1 peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes and protective immunity in mice against intrarectal recombinant HIV-vaccinia challenge. ProcNatlAcadSciUSA 95; 1709-1714.

25. Belyakov, I.M., Hel, Z., Kelsall, B., Kuznetsov, V.A., Ahlers, J.D., Nacsa, J., Watkins, D., Allen, T.M., Sette, A., Altman, J., et al. (2001) Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques. Nature Medicine 7; 1320-1326.

Mucosal HIV vaccine development

165

26. Belyakov, I.M., Kuznetsov, V.A., Kelsall, B., Klinman, D., Moniuszko, M., Lemon, M., Markham, P.D., Pal, P., Clements, J.D., Lewis, M.G., et al. (2006) Impact of Vaccine-induced Mucosal High Avidity CD8+ CTLs in Delay of AIDS-viral Dissemination from Mucosa. Blood 107; 3258-3264.

27. Belyakov, I.M., Wyatt, L.S., Ahlers, J.D., Earl, P., Pendleton, C.D., Kelsall, B.L., Strober, W., Moss, B., and Berzofsky, J.A. (1998c) Induction of mucosal CTL response by intrarectal immunization with a replication-deficient recombinant vaccinia virus expressing HIV 89.6 envelope protein. JVirol 72; 8264-8272.

28. Bergman, M.L., Cilio, C.M., Penha-Goncalves, C., Lamhamedi-Cherradi, S.E., Lofgren, A., Colucci, F., Lejon, K., Garchon, H.J., and Holmberg, D. (2001) CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice. J Autoimmun 16; 105-113.

29. Berzofsky, J.A., Pendleton, C.D., Clerici, M., Ahlers, J., Lucey, D.R., Putney, S.D., and Shearer, G.M. (1991) Construction of peptides encompassing multideterminant clusters of HIV envelope to induce in vitro T-cell responses in mice and humans of multiple MHC types. JClinInvest 88; 876-884.

30. Berzofsky, J.A., and Terabe, M. (2008) NKT cells in tumor immunity: opposing subsets define a new immunoregulatory axis. J Immunol 180; 3627-3635.

31. Blackburn, S.D., Shin, H., Haining, W.N., Zou, T., Workman, C.J., Polley, A., Betts, M.R., Freeman, G.J., Vignali, D.A., and Wherry, E.J. (2009) Coregulation of CD8+ T cell exhaustion by multiple inhibitory receptors during chronic viral infection. Nat Immunol 10; 29-37.

32. Bodles-Brakhop, A.M., Heller, R., and Draghia-Akli, R. (2009) Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments. Mol Ther 17; 585-592.

33. Bogers, W.M., Davis, D., Baak, I., Kan, E., Hofman, S., Sun, Y., Mortier, D., Lian, Y., Oostermeijer, H., Fagrouch, Z., et al. (2008) Systemic neutralizing antibodies induced by long interval mucosally primed systemically boosted immunization correlate with protection from mucosal SHIV challenge. Virology 382; 217-225.

34. Boyer, J.D., Robinson, T.M., Kutzler, M.A., Vansant, G., Hokey, D.A., Kumar, S., Parkinson, R., Wu, L., Sidhu, M.K., Pavlakis, G.N., et al. (2007) Protection against simian/human immunodeficiency virus (SHIV) 89.6P in macaques after coimmunization with SHIV antigen and IL-15 plasmid. Proc Natl Acad Sci U S A 104; 18648-18653.

35. Brenchley, J.M., Schacker, T.W., Ruff, L.E., Price, D.A., Taylor, J.H., Beilman, G.J., Nguyen, P.L., Khoruts, A., Larson, M., Haase, A.T., et al. (2004) CD4+ T Cell Depletion during all Stages of HIV Disease Occurs Predominantly in the Gastrointestinal Tract. J Exp Med 200; 749-759.

36. Burton, D.R., Desrosiers, R.C., Doms, R.W., Koff, W.C., Kwong, P.D., Moore, J.P., Nabel, G.J., Sodroski, J., Wilson, I.A., and Wyatt, R.T. (2004) HIV vaccine design and the neutralizing antibody problem. Nat Immunol 5; 233-236.

37. Calarota, S.A., Weiner, D.B., Lori, F., and Lisziewicz, J. (2007) Induction of HIV-specific memory T-cell responses by topical DermaVir vaccine. Vaccine 25; 3070-3074.

166

Yongjun Sui & Jay A. Berzofsky

38. Cameron, P.U., Freudenthal, P.S., Barker, J.M., Gezelter, S., Inaba, K., and Steinman, R.M. (1992) Dendritic cells exposed to human immunodeficiency virus type-1 transmit a vigorous cytopathic infection to CD4+ T cells. Science 257; 383-386.

39. Casimiro, D.R., Chen, L., Fu, T.M., Evans, R.K., Caulfield, M.J., Davies, M.E., Tang, A., Chen, M., Huang, L., Harris, V., et al. (2003) Comparative immunogenicity in rhesus monkeys of DNA plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene. J Virol 77; 6305-6313.

40. Casimiro, D.R., Wang, F., Schleif, W.A., Liang, X., Zhang, Z.Q., Tobery, T.W., Davies, M.E., McDermott, A.B., O'Connor, D.H., Fridman, A., et al. (2005) Attenuation of simian immunodeficiency virus SIVmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing Gag. J Virol 79; 15547-15555.

41. Cayabyab, M.J., Korioth-Schmitz, B., Sun, Y., Carville, A., Balachandran, H., Miura, A., Carlson, K.R., Buzby, A.P., Haynes, B.F., Jacobs, W.R., et al. (2009) Recombinant Mycobacterium bovis BCG prime-recombinant adenovirus boost vaccination in rhesus monkeys elicits robust polyfunctional simian immunodeficiency virus-specific T-cell responses. J Virol 83; 5505-5513.

42. Cecchinato, V., Tryniszewska, E., Ma, Z.M., Vaccari, M., Boasso, A., Tsai, W.P., Petrovas, C., Fuchs, D., Heraud, J.M., Venzon, D., et al. (2008) Immune activation driven by CTLA-4 blockade augments viral replication at mucosal sites in simian immunodeficiency virus infection. J Immunol 180; 5439-5447.

43. Cox, K.S., Clair, J.H., Prokop, M.T., Sykes, K.J., Dubey, S.A., Shiver, J.W., Robertson, M.N., and Casimiro, D.R. (2008) DNA gag/adenovirus type 5 (Ad5) gag and Ad5 gag/Ad5 gag vaccines induce distinct T-cell response profiles. J Virol 82; 8161-8171.

44. Cristillo, A.D., Lisziewicz, J., He, L., Lori, F., Galmin, L., Trocio, J.N., Unangst, T., Whitman, L., Hudacik, L., Bakare, N., et al. (2007) HIV-1 prophylactic vaccine comprised of topical DermaVir prime and protein boost elicits cellular immune responses and controls pathogenic R5 SHIV162P3. Virology 366; 197-211.

45. Critchfield, J.W., Lemongello, D., Walker, D.H., Garcia, J.C., Asmuth, D.M., Pollard, R.B., and Shacklett, B.L. (2007) Multifunctional human immunodeficiency virus (HIV) gag-specific CD8+ T-cell responses in rectal mucosa and peripheral blood mononuclear cells during chronic HIV type 1 infection. J Virol 81; 5460-5471.

46. Day, C.L., Kaufmann, D.E., Kiepiela, P., Brown, J.A., Moodley, E.S., Reddy, S., Mackey, E.W., Miller, J.D., Leslie, A.J., DePierres, C., et al. (2006) PD-1 expression on HIV-specific T cells is associated with T-cell exhaustion and disease progression. Nature 443; 350-354.

47. De Berardinis, P., and Haigwood, N.L. (2004) New recombinant vaccines based on the use of prokaryotic antigen-display systems. Expert Rev Vaccines 3; 673-679.

48. Demberg, T., Florese, R.H., Heath, M.J., Larsen, K., Kalisz, I., Kalyanaraman, V.S., Lee, E.M., Pal, R., Venzon, D., Grant, R., et al. (2007) A replicationcompetent adenovirus-human immunodeficiency virus (Ad-HIV) tat and Ad-HIV

Mucosal HIV vaccine development

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

167

env priming/Tat and envelope protein boosting regimen elicits enhanced protective efficacy against simian/human immunodeficiency virus SHIV89.6P challenge in rhesus macaques. J Virol 81; 3414-3427.

Derby, M.A., Alexander-Miller, M.A., Tse, R., and Berzofsky, J.A. (2001) High avidity CTL exploit two complementary mechanisms to provide better protection against viral infection than low avidity CTL. J Immunol 166; 1690-1697.

Derdeyn, C.A., Decker, J.M., Bibollet-Ruche, F., Mokili, J.L., Muldoon, M., Denham, S.A., Heil, M.L., Kasolo, F., Musonda, R., Hahn, B.H., et al. (2004) Envelope-constrained neutralization-sensitive HIV-1 after heterosexual transmission. Science 303; 2019-2022.

DeVico, A., Fouts, T., Lewis, G.K., Gallo, R.C., Godfrey, K., Charurat, M., Harris, I., Galmin, L., and Pal, R. (2007) Antibodies to CD4-induced sites in HIV gp120 correlate with the control of SHIV challenge in macaques vaccinated with subunit immunogens. Proc Natl Acad Sci U S A 104; 17477-17482.

Devito, C., Broliden, K., Kaul, R., Svensson, L., Johansen, K., Kiama, P., Kimani, J., Lopalco, L., Piconi, S., Bwayo, J.J., et al. (2000) Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells. J Immunol 165; 5170-5176.

Doria-Rose, N.A., and Haigwood, N.L. (2003) DNA vaccine strategies: candidates for immune modulation and immunization regimens. Methods 31; 207-216.

Dorrell, L., Hessell, A.J., Wang, M., Whittle, H., Sabally, S., Rowland-Jones, S., Burton, D.R., and Parren, P.W. (2000) Absence of specific mucosal antibody responses in HIV-exposed uninfected sex workers from the Gambia. AIDS 14; 1117-1122.

Douek, D.C., Picker, L.J., and Koup, R.A. (2003) T cell dynamics in HIV-1 infection. Annu Rev Immunol 21; 265-304.

Earl, P.L., Wyatt, L.S., Montefiori, D.C., Bilska, M., Woodward, R., Markham, P.D., Malley, J.D., Vogel, T.U., Allen, T.M., Watkins, D.I., et al. (2002) Comparison of vaccine strategies using recombinant env-gag-pol MVA with or without an oligomeric Env protein boost in the SHIV rhesus macaque model.

Virology 294; 270-281.

Elsawa, S.F., Rodeberg, D.A., and Celis, E. (2004) T-cell epitope peptide vaccines. Expert Rev Vaccines 3; 563-575.

Evans, T.G., Bonnez, W., Rose, R.C., Koenig, S., Demeter, L., Suzich, J.A., O'Brien, D., Campbell, M., White, W.I., Balsley, J., et al. (2001) A Phase 1 study of a recombinant viruslike particle vaccine against human papillomavirus type 11 in healthy adult volunteers. J Infect Dis 183; 1485-1493.

Feinberg, M.B., and Moore, J.P. (2002) AIDS vaccine models: challenging challenge viruses. Nat Med 8; 207-210.

Fichtner-Feigl, S., Terabe, M., Kitani, A., Young, C.A., Fuss, I., Geissler, E.K., Schlitt, H.J., Berzofsky, J.A., and Strober, W. (2008) Restoration of tumor immunosurveillance via targeting of interleukin-13 receptor-alpha 2. Cancer Res 68; 3467-3475.

168

Yongjun Sui & Jay A. Berzofsky

61. Finnefrock, A.C., Tang, A., Li, F., Freed, D.C., Feng, M., Cox, K.S., Sykes, K.J., Guare, J.P., Miller, M.D., Olsen, D.B., et al. (2009) PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination. J Immunol 182; 980-987.

62. Fischer, W., Perkins, S., Theiler, J., Bhattacharya, T., Yusim, K., Funkhouser, R., Kuiken, C., Haynes, B., Letvin, N.L., Walker, B.D., et al. (2007) Polyvalent vaccines for optimal coverage of potential T-cell epitopes in global HIV-1 variants. Nat Med 13; 100-106.

63. Florese, R.H., Demberg, T., Xiao, P., Kuller, L., Larsen, K., Summers, L.E., Venzon, D., Cafaro, A., Ensoli, B., and Robert-Guroff, M. (2009) Contribution of nonneutralizing vaccine-elicited antibody activities to improved protective efficacy in rhesus macaques immunized with Tat/Env compared with multigenic vaccines. J Immunol 182; 3718-3727.

64. Flynn, N.M., Forthal, D.N., Harro, C.D., Judson, F.N., Mayer, K.H., and Para, M.F. (2005) Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J Infect Dis 191; 654-665.

65. Fynan, E.F., Webster, R.G., Fuller, D.H., Haynes, J.R., Santoro, J.C., and Robinson, H.L. (1993) DNA vaccines: protective immunization by parental, mucosal and gene-gun inoculation. ProcNatAcadSciUSA 90; 11478-11482.

66. Gallimore, A., Dumrese, T., Hengartner, H., Zinkernagel, R.M., and Rammensee, H.G. (1998) Protective immunity does not correlate with the hierarchy of virusspecific cytotoxic T cell responses to naturally processed peptides. J Exp Med 187; 1647-1657.

67. Gaschen, B., Taylor, J., Yusim, K., Foley, B., Gao, F., Lang, D., Novitsky, V., Haynes, B., Hahn, B.H., Bhattacharya, T., et al. (2002) Diversity considerations in HIV-1 vaccine selection. Science 296; 2354-2360.

68. Genesca, M., Skinner, P.J., Hong, J.J., Li, J., Lu, D., McChesney, M.B., and Miller, C.J. (2008) With minimal systemic T-cell expansion, CD8+ T Cells mediate protection of rhesus macaques immunized with attenuated simian-human immunodeficiency virus SHIV89.6 from vaginal challenge with simian immunodeficiency virus. J Virol 82; 11181-11196.

69. Gherardi, M.M., and Esteban, M. (2005) Recombinant poxviruses as mucosal vaccine vectors. J Gen Virol 86; 2925-2936.

70. Girard, M.P., Osmanov, S.K., and Kieny, M.P. (2006) A review of vaccine research and development: the human immunodeficiency virus (HIV). Vaccine 24; 4062-4081.

71. Graham, B.S. (2009) What does the report of the USMHRP Phase III study in Thailand mean for HIV and for vaccine developers? Clin Exp Immunol 158; 257-259.

72. Graham, B.S., Koup, R.A., Roederer, M., Bailer, R.T., Enama, M.E., Moodie, Z., Martin, J.E., McCluskey, M.M., Chakrabarti, B.K., Lamoreaux, L., et al. (2006) Phase 1 safety and immunogenicity evaluation of a multiclade HIV-1 DNA candidate vaccine. J Infect Dis 194; 1650-1660.

73. Haaland, R.E., Hawkins, P.A., Salazar-Gonzalez, J., Johnson, A., Tichacek, A., Karita, E., Manigart, O., Mulenga, J., Keele, B.F., Shaw, G.M., et al. (2009)

Mucosal HIV vaccine development

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

169

Inflammatory genital infections mitigate a severe genetic bottleneck in heterosexual transmission of subtype A and C HIV-1. PLoS Pathog 5; e1000274.

Haase, A.T. (2010) Targeting early infection to prevent HIV-1 mucosal transmission. Nature 464; 217-223.

Hale, P.M., Cease, K.B., Houghten, R.A., Ouyang, C., Putney, S., Javaherian, K., Margalit, H., Cornette, J.L., Spouge, J.L., DeLisi, C., et al. (1989) T cell multideterminant regions in the human immunodeficiency virus envelope: toward overcoming the problem of major histocompatibility complex restriction.

IntImmunol 1; 409-415.

Hansen, S.G., Vieville, C., Whizin, N., Coyne-Johnson, L., Siess, D.C., Drummond, D.D., Legasse, A.W., Axthelm, M.K., Oswald, K., Trubey, C.M., et al. (2009) Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge. Nat Med 15; 293-299.

Harari, A., Bart, P.A., Stohr, W., Tapia, G., Garcia, M., Medjitna-Rais, E., Burnet, S., Cellerai, C., Erlwein, O., Barber, T., et al. (2008) An HIV-1 clade C DNA prime, NYVAC boost vaccine regimen induces reliable, polyfunctional, and long-lasting T cell responses. J Exp Med 205; 63-77.

Hessell, A.J., Hangartner, L., Hunter, M., Havenith, C.E., Beurskens, F.J., Bakker, J.M., Lanigan, C.M., Landucci, G., Forthal, D.N., Parren, P.W., et al.

(2007) Fc receptor but not complement binding is important in antibody protection against HIV. Nature 449; 101-104.

Hessell, A.J., Poignard, P., Hunter, M., Hangartner, L., Tehrani, D.M., Bleeker, W.K., Parren, P.W., Marx, P.A., and Burton, D.R. (2009) Effective, low-titer antibody protection against low-dose repeated mucosal SHIV challenge in macaques. Nat Med 15; 951-954.

Hirao, L.A., Wu, L., Khan, A.S., Hokey, D.A., Yan, J., Dai, A., Betts, M.R., Draghia-Akli, R., and Weiner, D.B. (2008) Combined effects of IL-12 and electroporation enhances the potency of DNA vaccination in macaques. Vaccine 26; 3112-3120.

Hirbod, T., Kaul, R., Reichard, C., Kimani, J., Ngugi, E., Bwayo, J.J., Nagelkerke, N., Hasselrot, K., Li, B., Moses, S., et al. (2008) HIV-neutralizing immunoglobulin A and HIV-specific proliferation are independently associated with reduced HIV acquisition in Kenyan sex workers. AIDS 22; 727-735.

Holl, V., Peressin, M., Decoville, T., Schmidt, S., Zolla-Pazner, S., Aubertin, A.M., and Moog, C. (2006) Nonneutralizing antibodies are able to inhibit human immunodeficiency virus type 1 replication in macrophages and immature dendritic cells. J Virol 80; 6177-6181.

Hu, J., Gardner, M.B., and Miller, C.J. (2000) Simian immunodeficiency virus rapidly penetrates the cervicovaginal mucosa after intravaginal inoculation and infects intraepithelial dendritic cells. J Virol 74; 6087-6095.

Jin, X., Bauer, D.E., Tuttleton, S.E., Lewin, S., Gettie, A., Blanchard, J., Irwin, C.E., Safrit, J.T., Mittler, J., Weinberger, L., et al. (1999) Dramatic rise in plasma viremia after CD8+ T cell depletion in simian immunodeficiency virus-infected macaques. JExpMed 189; 991-998.

170

Yongjun Sui & Jay A. Berzofsky

85. Johnson, P.R., Schnepp, B.C., Zhang, J., Connell, M.J., Greene, S.M., Yuste, E., Desrosiers, R.C., and Clark, K.R. (2009) Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nat Med 15; 901-906.

86. Johnson, W.E., Sanford, H., Schwall, L., Burton, D.R., Parren, P.W., Robinson, J.E., and Desrosiers, R.C. (2003) Assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities. J Virol 77; 9993-10003.

87. Kaufman, D.R., Liu, J., Carville, A., Mansfield, K.G., Havenga, M.J., Goudsmit, J., and Barouch, D.H. (2008) Trafficking of antigen-specific CD8+ T lymphocytes to mucosal surfaces following intramuscular vaccination. J Immunol 181; 4188-4198.

88. Keele, B.F., Giorgi, E.E., Salazar-Gonzalez, J.F., Decker, J.M., Pham, K.T., Salazar, M.G., Sun, C., Grayson, T., Wang, S., Li, H., et al. (2008) Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection. Proc Natl Acad Sci U S A 105; 7552-7557.

89. Koup, R.A., Roederer, M., Lamoreaux, L., Fischer, J., Novik, L., Nason, M.C., Larkin, B.D., Enama, M.E., Ledgerwood, J.E., Bailer, R.T., et al. (2010) Priming immunization with DNA augments immunogenicity of recombinant adenoviral vectors for both HIV-1 specific antibody and T-cell responses. PLoS One 5; e9015.

90. Kozlowski, P.A., and Neutra, M.R. (2003) The role of mucosal immunity in prevention of HIV transmission. Curr Mol Med 3; 217-228.

91. Kumar, S., Yan, J., Muthumani, K., Ramanathan, M.P., Yoon, H., Pavlakis, G.N., Felber, B.K., Sidhu, M., Boyer, J.D., and Weiner, D.B. (2006) Immunogenicity testing of a novel engineered HIV-1 envelope gp140 DNA vaccine construct.

DNA Cell Biol 25; 383-392.

92. Kutzler, M.A., Robinson, T.M., Chattergoon, M.A., Choo, D.K., Choo, A.Y., Choe, P.Y., Ramanathan, M.P., Parkinson, R., Kudchodkar, S., Tamura, Y., et al.

(2005) Coimmunization with an optimized IL-15 plasmid results in enhanced function and longevity of CD8 T cells that are partially independent of CD4 T cell help. J Immunol 175; 112-123.

93. Kwissa, M., Amara, R.R., Robinson, H.L., Moss, B., Alkan, S., Jabbar, A., Villinger, F., and Pulendran, B. (2007) Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus. J Exp Med 204; 2733-2746.

94. Lemiale, F., Asefa, B., Ye, D., Chen, C., Korokhov, N., and Humeau, L. (2010) An HIV-based lentiviral vector as HIV vaccine candidate: Immunogenic characterization. Vaccine 28; 1952-1961.

95. Letvin, N.L. (2006) Progress and obstacles in the development of an AIDS vaccine. Nat Rev Immunol 6; 930-939.

96. Letvin, N.L., Mascola, J.R., Sun, Y., Gorgone, D.A., Buzby, A.P., Xu, L., Yang, Z.Y., Chakrabarti, B., Rao, S.S., Schmitz, J.E., et al. (2006) Preserved CD4+ central memory T cells and survival in vaccinated SIV-challenged monkeys.

Science 312; 1530-1533.

Mucosal HIV vaccine development

171

97. Letvin, N.L., Montefiori, D.C., Yasutomi, Y., Perry, H.C., Davies, M.E., Lekutis, C., Alroy, M., Freed, D.C., Lord, C.I., Handt, L.K., et al. (1997) Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination. Proc Natl Acad Sci U S A 94; 9378-9383.

98. Leung, N.J., Aldovini, A., Young, R., Jarvis, M.A., Smith, J.M., Meyer, D., Anderson, D.E., Carlos, M.P., Gardner, M.B., and Torres, J.V. (2000) The kinetics of specific immune responses in rhesus monkeys inoculated with live recombinant BCG expressing SIV Gag, Pol, Env, and Nef proteins. Virology 268; 94-103.

99. Li, Q., Skinner, P.J., Ha, S.J., Duan, L., Mattila, T.L., Hage, A., White, C., Barber, D.L., O'Mara, L., Southern, P.J., et al. (2009) Visualizing antigenspecific and infected cells in situ predicts outcomes in early viral infection.

Science 323; 1726-1729.

100. Liu, J., O'Brien, K.L., Lynch, D.M., Simmons, N.L., La Porte, A., Riggs, A.M., Abbink, P., Coffey, R.T., Grandpre, L.E., Seaman, M.S., et al. (2008) Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 457; 87-91.

101. Liu, M.A. (2003) DNA vaccines: a review. J Intern Med 253; 402-410.

102. Liu, M.A., and Ulmer, J.B. (2000) Gene-based vaccines. Mol Ther 1; 497-500.

103. Lu, S. (2008) Immunogenicity of DNA vaccines in humans: it takes two to tango.

Hum Vaccin 4; 449-452.

104. Luckay, A., Sidhu, M.K., Kjeken, R., Megati, S., Chong, S.Y., Roopchand, V., Garcia-Hand, D., Abdullah, R., Braun, R., Montefiori, D.C., et al. (2007) Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol 81; 5257-5269.

105. Maher, D., Wu, X., Schacker, T., Larson, M., and Southern, P. (2004) A model system of oral HIV exposure, using human palatine tonsil, reveals extensive binding of HIV infectivity, with limited progression to primary infection. J Infect Dis 190; 1989-1997.

106. Makitalo, B., Lundholm, P., Hinkula, J., Nilsson, C., Karlen, K., Morner, A., Sutter, G., Erfle, V., Heeney, J.L., Wahren, B., et al. (2004) Enhanced cellular immunity and systemic control of SHIV infection by combined parenteral and mucosal administration of a DNA prime MVA boost vaccine regimen. J Gen Virol 85; 2407-2419.

107. Malkevitch, N.V., Patterson, L.J., Aldrich, M.K., Wu, Y., Venzon, D., Florese, R.H., Kalyanaraman, V.S., Pal, R., Lee, E.M., Zhao, J., et al. (2006) Durable protection of rhesus macaques immunized with a replicating adenovirus-SIV multigene prime/protein boost vaccine regimen against a second SIVmac251 rectal challenge: role of SIV-specific CD8+ T cell responses. Virology 353; 83-98.

108. Mascola, J.R., Lewis, M.G., Stiegler, G., Harris, D., VanCott, T.C., Hayes, D., Louder, M.K., Brown, C.R., Sapan, C.V., Frankel, S.S., et al. (1999) Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies. J Virol 73; 4009-4018.

172

Yongjun Sui & Jay A. Berzofsky

109. Mascola, J.R., Sambor, A., Beaudry, K., Santra, S., Welcher, B., Louder, M.K., Vancott, T.C., Huang, Y., Chakrabarti, B.K., Kong, W.P., et al. (2005) Neutralizing antibodies elicited by immunization of monkeys with DNA plasmids and recombinant adenoviral vectors expressing human immunodeficiency virus type 1 proteins. J Virol 79; 771-779.

110. Mascola, J.R., Snyder, S.W., Weislow, O.S., Belay, S.M., Belshe, R.B., Schwartz, D.H., Clements, M.L., Dolin, R., Graham, B.S., Gorse, G.J., et al.

(1996) Immunization with envelope subunit vaccine products elicits neutralizing antibodies against laboratory-adapted but not primary isolates of human immunodeficiency virus type 1. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group. J Infect Dis 173; 340-348.

111. Mascola, J.R., Stiegler, G., VanCott, T.C., Katinger, H., Carpenter, C.B., Hanson, C.E., Beary, H., Hayes, D., Frankel, S.S., Birx, D.L., et al. (2000) Protection of macaques against vaginal transmission of a pathogenic HIV-1/SIV chimeric virus by passive infusion of neutralizing antibodies. Nat Med 6; 207-210.

112. Masopust, D., Choo, D., Vezys, V., Wherry, E.J., Duraiswamy, J., Akondy, R., Wang, J., Casey, K.A., Barber, D.L., Kawamura, K.S., et al. (2010) Dynamic T cell migration program provides resident memory within intestinal epithelium. J Exp Med 207; 553-564.

113. McDermott, A.B., O'Connor, D.H., Fuenger, S., Piaskowski, S., Martin, S., Loffredo, J., Reynolds, M., Reed, J., Furlott, J., Jacoby, T., et al. (2005) Cytotoxic T-lymphocyte escape does not always explain the transient control of simian immunodeficiency virus SIVmac239 viremia in adenovirus-boosted and DNA-primed Mamu-A*01-positive rhesus macaques. J Virol 79; 15556-15566.

114. McElrath, M.J., De Rosa, S.C., Moodie, Z., Dubey, S., Kierstead, L., Janes, H., Defawe, O.D., Carter, D.K., Hural, J., Akondy, R., et al. (2008) HIV-1 vaccineinduced immunity in the test-of-concept Step Study: a case-cohort analysis.

Lancet 372; 1894-1905.

115. Mederle, I., Le Grand, R., Vaslin, B., Badell, E., Vingert, B., Dormont, D., Gicquel, B., and Winter, N. (2003) Mucosal administration of three recombinant Mycobacterium bovis BCG-SIVmac251 strains to cynomolgus macaques induces rectal IgAs and boosts systemic cellular immune responses that are primed by intradermal vaccination. Vaccine 21; 4153-4166.

116. Miller, C.J., Li, Q., Abel, K., Kim, E.Y., Ma, Z.M., Wietgrefe, S., La FrancoScheuch, L., Compton, L., Duan, L., Shore, M.D., et al. (2005) Propagation and dissemination of infection after vaginal transmission of simian immunodeficiency virus. J Virol 79; 9217-9227.

117. Miller, C.J., McChesney, M., and Moore, P.F. (1992) Langerhans cells, macrophages and lymphocyte subsets in the cervix and vagina of rhesus macaques. Lab Invest 67; 628-634.

118. Miller, C.J., and Shattock, R.J. (2003) Target cells in vaginal HIV transmission.

Microbes Infect 5; 59-67.

119. Moore, J.P., Cao, Y., Qing, L., Sattentau, Q.J., Pyati, J., Koduri, R., Robinson, J., Barbas, C.F., 3rd, Burton, D.R., and Ho, D.D. (1995) Primary isolates of human immunodeficiency virus type 1 are relatively resistant to neutralization by

Mucosal HIV vaccine development

173

monoclonal antibodies to gp120, and their neutralization is not predicted by studies with monomeric gp120. J Virol 69; 101-109.

120. Moss, B. (1991) Vaccinia virus: a tool for research and vaccine development.

Science 252; 1662.

121. Negri, D.R., Baroncelli, S., Michelini, Z., Macchia, I., Belli, R., Catone, S., Incitti, F., ten Haaft, P., Corrias, F., Cranage, M.P., et al. (2001) Effect of vaccination with recombinant modified vaccinia virus Ankara expressing structural and regulatory genes of SIV(macJ5) on the kinetics of SIV replication in cynomolgus monkeys. J Med Primatol 30; 197-206.

122. Nilsson, C., Sutter, G., Walther-Jallow, L., ten Haaft, P., Akerblom, L., Heeney, J., Erfle, V., Bottiger, P., Biberfeld, G., and Thorstensson, R. (2002) Immunization with recombinant modified vaccinia virus Ankara can modify mucosal simian immunodeficiency virus infection and delay disease progression in macaques. J Gen Virol 83; 807-818.

123. Nishimura, Y., Igarashi, T., Haigwood, N., Sadjadpour, R., Plishka, R.J., Buckler-White, A., Shibata, R., and Martin, M.A. (2002) Determination of a statistically valid neutralization titer in plasma that confers protection against simian-human immunodeficiency virus challenge following passive transfer of high-titered neutralizing antibodies. J Virol 76; 2123-2130.

124. Oh, S., Berzofsky, J.A., Burke, D.S., Waldmann, T.A., and Perera, L.P. (2003a) Coadministration of HIV vaccine vectors with vaccinia viruses expressing IL-15 but not IL-2 induces long-lasting cellular immunity. Proc Natl Acad Sci U S A 100; 3392-3397.

125. Oh, S., Hodge, J.W., Ahlers, J.D., Burke, D.S., Schlom, J., and Berzofsky, J.A. (2003b) Selective induction of high avidity CTL by altering the balance of signals from antigen presenting cells. J Immunol 170; 2523-2530.

126. Oh, S., Perera, L.P., Burke, D.S., Waldmann, T.A., and Berzofsky, J.A. (2004) IL-15/IL-15R alpha-mediated avidity maturation of memory CD8+ T cells. Proc Natl Acad Sci U S A 101; 15154-15159.

127. Oh, S., Perera, L.P., Terabe, M., Ni, L., Waldmann, T.A., and Berzofsky, J.A. (2008) IL-15 as a mediator of CD4+ help for CD8+ T cell longevity and avoidance of TRAIL-mediated apoptosis. Proc Natl Acad Sci U S A 105; 52015206.

128. Okazaki, T., Pendleton, C.D., Sarobe, P., Thomas, E.K., Harro, C., Schwartz, D., Iyengar, S., and Berzofsky, J.A. (2006) Epitope-enhancement of a CD4 HIV epitope toward the development of the next generation HIV vaccine. Journal of Immunol 176; 3753-3759.

129. Okazaki, T., Pendleton, D.C., Lemonnier, F., and Berzofsky, J.A. (2003) Epitope-enhanced conserved HIV-1 peptide protects HLA-A2-transgenic mice against virus expressing HIV-1 antigen. J Immunol 171; 2548-2555.

130. Onlamoon, N., Rogers, K., Mayne, A.E., Pattanapanyasat, K., Mori, K., Villinger, F., and Ansari, A.A. (2008) Soluble PD-1 rescues the proliferative response of simian immunodeficiency virus-specific CD4 and CD8 T cells during chronic infection. Immunology 124; 277-293.

174

Yongjun Sui & Jay A. Berzofsky

131. Oran, A.E., and Robinson, H.L. (2003) DNA vaccines, combining form of antigen and method of delivery to raise a spectrum of IFN-gamma and IL-4producing CD4+ and CD8+ T cells. J Immunol 171; 1999-2005.

132. Ourmanov, I., Brown, C.R., Moss, B., Carroll, M., Wyatt, L., Pletneva, L., Goldstein, S., Venzon, D., and Hirsch, V.M. (2000) Comparative efficacy of recombinant modified vaccinia virus Ankara expressing simian immunodeficiency virus (SIV) Gag-Pol and/or Env in macaques challenged with pathogenic SIV. J Virol 74; 2740-2751.

133. Pal, R., Kalyanaraman, V.S., Nair, B.C., Whitney, S., Keen, T., Hocker, L., Hudacik, L., Rose, N., Mboudjeka, I., Shen, S., et al. (2006a) Immunization of rhesus macaques with a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine elicits protective antibody response against simian human immunodeficiency virus of R5 phenotype. Virology 348; 341-353.

134. Pal, R., Venzon, D., Letvin, N.L., Santra, S., Montefiori, D.C., Miller, N.R., Tryniszewska, E., Lewis, M.G., VanCott, T.C., Hirsch, V., et al. (2002) ALVACSIV-gag-pol-env-based vaccination and macaque major histocompatibility complex class I (A*01) delay simian immunodeficiency virus SIVmac-induced immunodeficiency. J Virol 76; 292-302.

135. Pal, R., Venzon, D., Santra, S., Kalyanaraman, V.S., Montefiori, D.C., Hocker, L., Hudacik, L., Rose, N., Nacsa, J., Y., E.-S., et al. (2006b) Systemic Immunization with an ALVAC-HIV-1/Protein Boost Vaccine Strategy Protects Rhesus Macaques from CD4+ T-Cell Loss and Reduces Both Systemic and Mucosal SHIVKU2 RNA Levels. Journal of Virology 80; 3732-3742.

136. Pal, R., Wang, S., Kalyanaraman, V.S., Nair, B.C., Whitney, S., Keen, T., Hocker, L., Hudacik, L., Rose, N., Cristillo, A., et al. (2005) Polyvalent DNA prime and envelope protein boost HIV-1 vaccine elicits humoral and cellular responses and controls plasma viremia in rhesus macaques following rectal challenge with an R5 SHIV isolate. J Med Primatol 34; 226-236.

137. Park, J.M., Terabe, M., van den Broeke, L.T., Donaldson, D.D., and Berzofsky, J.A. (2004) Unmasking immunosurveillance against a syngeneic colon cancer by elimination of CD4+ NKT regulatory cells and IL-13. International J of Cancer 114; 80-87.

138. Parren, P.W., Marx, P.A., Hessell, A.J., Luckay, A., Harouse, J., Cheng-Mayer, C., Moore, J.P., and Burton, D.R. (2001) Antibody protects macaques against vaginal challenge with a pathogenic R5 simian/human immunodeficiency virus at serum levels giving complete neutralization in vitro. J Virol 75; 8340-8347.

139. Paterson, Y., and Johnson, R.S. (2004) Progress towards the use of Listeria monocytogenes as a live bacterial vaccine vector for the delivery of HIV antigens. Expert Rev Vaccines 3; S119-134.

140. Patterson, L.J., Beal, J., Demberg, T., Florese, R.H., Malkevich, N., Venzon, D., Aldrich, K., Richardson, E., Kalyanaraman, V.S., Kalisz, I., et al. (2008) Replicating adenovirus HIV/SIV recombinant priming alone or in combination with a gp140 protein boost results in significant control of viremia following a SHIV89.6P challenge in Mamu-A*01 negative rhesus macaques. Virology 374; 322-337.

Mucosal HIV vaccine development

175

141. Patterson, L.J., Malkevitch, N., Venzon, D., Pinczewski, J., Gomez-Roman, V.R., Wang, L., Kalyanaraman, V.S., Markham, P.D., Robey, F.A., and Robert-Guroff, M. (2004) Protection against Mucosal Simian Immunodeficiency Virus SIV(mac251) Challenge by Using Replicating Adenovirus-SIV Multigene Vaccine Priming and Subunit Boosting. J Virol 78; 2212-2221.

142. Peng, B., Wang, L.R., Gomez-Roman, V.R., Davis-Warren, A., Montefiori, D.C., Kalyanaraman, V.S., Venzon, D., Zhao, J., Kan, E., Rowell, T.J., et al. (2005) Replicating rather than nonreplicating adenovirus-human immunodeficiency virus recombinant vaccines are better at eliciting potent cellular immunity and priming high-titer antibodies. J Virol 79; 10200-10209.

143. Petrovas, C., Casazza, J.P., Brenchley, J.M., Price, D.A., Gostick, E., Adams, W.C., Precopio, M.L., Schacker, T., Roederer, M., Douek, D.C., et al. (2006) PD-1 is a regulator of virus-specific CD8+ T cell survival in HIV infection. J Exp Med 203; 2281-2292.

144. Petrovas, C., Price, D.A., Mattapallil, J., Ambrozak, D.R., Geldmacher, C., Cecchinato, V., Vaccari, M., Tryniszewska, E., Gostick, E., Roederer, M., et al.

(2007) SIV-specific CD8+ T cells express high levels of PD1 and cytokines but have impaired proliferative capacity in acute and chronic SIVmac251 infection.

Blood 110; 928-936.

145. Pitisuttithum, P., Gilbert, P., Gurwith, M., Heyward, W., Martin, M., van Griensven, F., Hu, D., Tappero, J.W., and Choopanya, K. (2006) Randomized, double-blind, placebo-controlled efficacy trial of a bivalent recombinant glycoprotein 120 HIV-1 vaccine among injection drug users in Bangkok, Thailand. J Infect Dis 194; 1661-1671.

146. Porgador, A., Irvine, K.R., Iwasaki, A., Barber, B.H., Restifo, N.P., and Germain, R.N. (1998) Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization. J Exp Med 188; 10751082.

147. Ramakrishna, L., Anand, K.K., Mohankumar, K.M., and Ranga, U. (2004) Codon optimization of the tat antigen of human immunodeficiency virus type 1 generates strong immune responses in mice following genetic immunization. J Virol 78; 9174-9189.

148. Rasmussen, R.A., Hofmann-Lehman, R., Montefiori, D.C., Li, P.L., Liska, V., Vlasak, J., Baba, T.W., Schmitz, J.E., Kuroda, M.J., Robinson, H.L., et al. (2002) DNA prime/protein boost vaccine strategy in neonatal macaques against simian human immunodeficiency virus. J Med Primatol 31; 40-60.

149. Rerks-Ngarm, S., Pitisuttithum, P., Nitayaphan, S., Kaewkungwal, J., Chiu, J., Paris, R., Premsri, N., Namwat, C., de Souza, M., Adams, E., et al. (2009) Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 361; 2209-2220.

150. Reynolds, M.R., Rakasz, E., Skinner, P.J., White, C., Abel, K., Ma, Z.M., Compton, L., Napoe, G., Wilson, N., Miller, C.J., et al. (2005) CD8+ Tlymphocyte response to major immunodominant epitopes after vaginal exposure to simian immunodeficiency virus: too late and too little. J Virol 79; 9228-9235.

176

Yongjun Sui & Jay A. Berzofsky

151. Robert-Guroff, M. (2007) Replicating and non-replicating viral vectors for vaccine development. Curr Opin Biotechnol 18; 546-556.

152. Robinson, H.L., Hunt, L.A., and Webster, R.G. (1993) Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11; 957-960.

153. Rosati, M., Bergamaschi, C., Valentin, A., Kulkarni, V., Jalah, R., Alicea, C., Patel, V., von Gegerfelt, A.S., Montefiori, D.C., Venzon, D.J., et al. (2009) DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge. Proc Natl Acad Sci U S A 106; 15831-15836.

154. Rosati, M., von Gegerfelt, A., Roth, P., Alicea, C., Valentin, A., Robert-Guroff, M., Venzon, D., Montefiori, D.C., Markham, P., Felber, B.K., et al. (2005) DNA vaccines expressing different forms of simian immunodeficiency virus antigens decrease viremia upon SIVmac251 challenge. J Virol 79; 8480-8492.

155. Sadoff, J.C., Ballou, W.R., Baron, L.S., Majarian, W.R., Brey, R.N., Hockmeyer, W.T., Young, J.F., Cryz, S.J., Ou, J., Lowell, G.H., et al. (1988) Oral Salomonella typhimurium vaccine expressing circumsporozoite protein protects against malaria. Science 240; 336-338.

156. Santra, S., Liao, H.X., Zhang, R., Muldoon, M., Watson, S., Fischer, W., Theiler, J., Szinger, J., Balachandran, H., Buzby, A., et al. (2010) Mosaic vaccines elicit CD8+ T lymphocyte responses that confer enhanced immune coverage of diverse HIV strains in monkeys. Nat Med 16; 324-328.

157. Santra, S., Seaman, M.S., Xu, L., Barouch, D.H., Lord, C.I., Lifton, M.A., Gorgone, D.A., Beaudry, K.R., Svehla, K., Welcher, B., et al. (2005) Replication-defective adenovirus serotype 5 vectors elicit durable cellular and humoral immune responses in nonhuman primates. J Virol 79; 6516-6522.

158. Schmitz, J.E., Kuroda, M.J., Santra, S., Sasseville, V.G., Simon, M.A., Lifton, M.A., Racz, P., Tenner-Racz, K., Dalesandro, M., Scallon, B.J., et al. (1999) Control of viremia in simian immunodeficiency virus infection by CD8+ lymphocytes. Science 283; 857-860.

159. Seaman, M.S., Leblanc, D.F., Grandpre, L.E., Bartman, M.T., Montefiori, D.C., Letvin, N.L., and Mascola, J.R. (2007) Standardized assessment of NAb responses elicited in rhesus monkeys immunized with single- or multi-clade HIV1 envelope immunogens. Virology 367; 175-186.

160. Seaman, M.S., Xu, L., Beaudry, K., Martin, K.L., Beddall, M.H., Miura, A., Sambor, A., Chakrabarti, B.K., Huang, Y., Bailer, R., et al. (2005) Multiclade human immunodeficiency virus type 1 envelope immunogens elicit broad cellular and humoral immunity in rhesus monkeys. J Virol 79; 2956-2963.

161. Seth, A., Ourmanov, I., Schmitz, J.E., Kuroda, M.J., Lifton, M.A., Nickerson, C.E., Wyatt, L., Carroll, M., Moss, B., Venzon, D., et al. (2000) Immunization with a modified vaccinia virus expressing simian immunodeficiency virus (SIV) Gag-Pol primes for an anamnestic Gag-specific cytotoxic T-lymphocyte response and is associated with reduction of viremia after SIV challenge. J Virol 74; 25022509.

Mucosal HIV vaccine development

177

162. Sharpe, S., Polyanskaya, N., Dennis, M., Sutter, G., Hanke, T., Erfle, V., Hirsch, V., and Cranage, M. (2001) Induction of simian immunodeficiency virus (SIV)specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity. J Gen Virol 82; 2215-2223.

163. Shibata, R., Igarashi, T., Haigwood, N., Buckler-White, A., Ogert, R., Ross, W., Willey, R., Cho, M.W., and Martin, M.A. (1999) Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys. Nat Med 5; 204-210.

164. Shirai, M., Pendleton, C.D., Ahlers, J., Takeshita, T., Newman, M., and Berzofsky, J.A. (1994) Helper-CTL determinant linkage required for priming of anti-HIV CD8+ CTL in vivo with peptide vaccine constructs. JImmunol 152; 549-556.

165. Shiver, J.W., Fu, T.M., Chen, L., Casimiro, D.R., Davies, M.E., Evans, R.K., Zhang, Z.Q., Simon, A.J., Trigona, W.L., Dubey, S.A., et al. (2002) Replicationincompetent adenoviral vaccine vector elicits effective anti-immunodeficiencyvirus immunity. Nature 415; 331-335.

166. Someya, K., Cecilia, D., Ami, Y., Nakasone, T., Matsuo, K., Burda, S., Yamamoto, H., Yoshino, N., Kaizu, M., Ando, S., et al. (2005) Vaccination of rhesus macaques with recombinant Mycobacterium bovis bacillus CalmetteGuerin Env V3 elicits neutralizing antibody-mediated protection against simianhuman immunodeficiency virus with a homologous but not a heterologous V3 motif. J Virol 79; 1452-1462.

167. Spira, A.I., Marx, P.A., Patterson, B.K., Mahoney, J., Koup, R.A., Wolinsky, S.M., and Ho, D.D. (1996) Cellular targets of infection and route of viral dissemination after an intravaginal inoculation of simian immunodeficiency virus into rhesus macaques. J Exp Med 183; 215-225.

168. Sui, Y., Zhu, Q., Gagnon, S., Dzutsev, A., Terabe, M., Vaccari, M., Venzon, D., Klinman, D., Strober, W., Kelsall, B., et al. (2010) Innate and adaptive immune correlates of vaccine and adjuvant-induced control of mucosal transmission of SIV in macaques. Proc Natl Acad Sci U S A 107; 9843-9848.

169. Sutmuller, R.P.M., Van Duivenvoorde, L.M., Van Elsas, A., Schumacher, T.N.M., Wildenberg, M.E., Allison, J.P., Toes, R.E.M., Offringa, R., and Melief, C.J.M. (2001) Synergism of cytotoxic T lymphocyte-associated antigen 4 blockade and depletion of CD25+ regulatory T cells in antitumor therapy reveals alternative cytotoxic T lymphocyte responses. J Exp Med 194; 823-832.

170. Takaku, S., Terabe, M., Ambrosino, E., Peng, J., Lonning, S., McPherson, J.M., and Berzofsky, J.A. (2009 (2010)) Blockade of TGF-beta enhances tumor vaccine efficacy mediated by CD8(+) T cells. Int J Cancer 126; 1666-1674.

171. Terabe, M., Ambrosino, E., Takaku, S., O'Konek, J.J., Venzon, D., Lonning, S., McPherson, J.M., and Berzofsky, J.A. (2009) Synergistic enhancement of CD8+ T cell-mediated tumor vaccine efficacy by an anti-transforming growth factorbeta monoclonal antibody. Clin Cancer Res 15; 6560-6569.

172. Terabe, M., and Berzofsky, J.A. (2008) The role of NKT cells in tumor immunity. Adv Cancer Res 101; 277-348.

178

Yongjun Sui & Jay A. Berzofsky

173. Terabe, M., Matsui, S., Noben-Trauth, N., Chen, H., Watson, C., Donaldson, D.D., Carbone, D.P., Paul, W.E., and Berzofsky, J.A. (2000) NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nature Immunology 1; 515-520.

174. Terabe, M., Matsui, S., Park, J.-M., Mamura, M., Noben-Trauth, N., Donaldson, D.D., Chen, W., Wahl, S.M., Ledbetter, S., Pratt, B., et al. (2003) Transforming Growth Factor-β production and myeloid cells are an effector mechanism through which CD1d-restricted T cells block Cytotoxic T Lymphocyte-mediated tumor immunosurveillance: abrogation prevents tumor recurrence. J Exp Med 198; 1741-1752.

175. Terabe, M., Swann, J., Ambrosino, E., Sinha, P., Takaku, S., Hayakawa, Y., Godfrey, D.I., Ostrand-Rosenberg, S., Smyth, M.J., and Berzofsky, J.A. (2005) A nonclassical non-Va14Ja18 CD1d-restricted (type II) NKT cell is sufficient for down-regulation of tumor immunosurveillance. J Exp Med 202; 1627-1633.

176. Trautmann, L., Janbazian, L., Chomont, N., Said, E.A., Gimmig, S., Bessette, B., Boulassel, M.R., Delwart, E., Sepulveda, H., Balderas, R.S., et al. (2006) Upregulation of PD-1 expression on HIV-specific CD8+ T cells leads to reversible immune dysfunction. Nat Med 12; 1198-1202.

177. Ulmer, J.B., Donnelly, J.J., Parker, S.E., Rhodes, G.H., Felgner, P.L., Dwarki, V.J., Gromkowski, S.H., Deck, R.R., DeWitt, C.M., Friedman, A., et al. (1993) Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259; 1745-1749.

178. Van Rompay, K.K., Abel, K., Lawson, J.R., Singh, R.P., Schmidt, K.A., Evans, T., Earl, P., Harvey, D., Franchini, G., Tartaglia, J., et al. (2005) Attenuated poxvirus-based simian immunodeficiency virus (SIV) vaccines given in infancy partially protect infant and juvenile macaques against repeated oral challenge with virulent SIV. J Acquir Immune Defic Syndr 38; 124-134.

179. Vandervoort, J., and Ludwig, A. (2008) Microneedles for transdermal drug delivery: a minireview. Front Biosci 13; 1711-1715.

180. Veazey, R.S., DeMaria, M., Chalifoux, L.V., Shvetz, D.E., Pauley, D.R., Knight, H.L., Rosenzweig, M., Johnson, R.P., Desrosiers, R.C., and Lackner, A.A. (1998) Gastrointestinal Tract as a Major Site of CD4+ T Cell Depletion and Viral Replication in SIV Infection. Science 280; 427-431.

181. Veazey, R.S., and Lackner, A.A. (1998) The gastrointestinal tract and the pathogenesis of AIDS. AIDS 12; S35-S42.

182. Veazey, R.S., and Lackner, A.A. (2004) Getting to the guts of HIV pathogenesis.

J Exp Med 200; 697-700.

183. Veazey, R.S., Shattock, R.J., Pope, M., Kirijan, J.C., Jones, J., Hu, Q., Ketas, T., Marx, P.A., Klasse, P.J., Burton, D.R., et al. (2003) Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 9; 343-346.

184. Velu, V., Titanji, K., Zhu, B., Husain, S., Pladevega, A., Lai, L., Vanderford, T.H., Chennareddi, L., Silvestri, G., Freeman, G.J., et al. (2008) Enhancing SIVspecific immunity in vivo by PD-1 blockade. Nature.

Mucosal HIV vaccine development

179

185. Virgin, H.W., and Walker, B.D. (2010) Immunology and the elusive AIDS vaccine. Nature 464; 224-231.

186. Vogel, T.U., Reynolds, M.R., Fuller, D.H., Vielhuber, K., Shipley, T., Fuller, J.T., Kunstman, K.J., Sutter, G., Marthas, M.L., Erfle, V., et al. (2003) Multispecific vaccine-induced mucosal cytotoxic T lymphocytes reduce acutephase viral replication but fail in long-term control of simian immunodeficiency virus SIVmac239. J Virol 77; 13348-13360.

187. Wille-Reece, U., Flynn, B.J., Lore, K., Koup, R.A., Miles, A.P., Saul, A., Kedl, R.M., Mattapallil, J.J., Weiss, W.R., Roederer, M., et al. (2006) Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. J Exp Med 203; 1249-1258.

188. Xiao, P., Zhao, J., Patterson, L.J., Brocca-Cofano, E., Venzon, D., Kozlowski, P.A., Hidajat, R., Demberg, T., and Robert-Guroff, M. (2010) Multiple vaccineelicited non-neutralizing anti-envelope antibody activities contribute to protective efficacy by reducing both acute and chronic viremia following SHIV89.6P challenge in rhesus macaques. J Virol. 84; 7161-7173 189. Yan, J., Yoon, H., Kumar, S., Ramanathan, M.P., Corbitt, N., Kutzler, M., Dai, A., Boyer, J.D., and Weiner, D.B. (2007) Enhanced cellular immune responses elicited by an engineered HIV-1 subtype B consensus-based envelope DNA vaccine. Mol Ther 15; 411-421.

190. Yang, J.C., Hughes, M., Kammula, U., Royal, R., Sherry, R.M., Topalian, S.L., Suri, K.B., Levy, C., Allen, T., Mavroukakis, S., et al. (2007) Ipilimumab (antiCTLA4 antibody) causes regression of metastatic renal cell cancer associated with enteritis and hypophysitis. J Immunother 30; 825-830.

191. Yasutomi, Y., Koenig, S., Haun, S.S., Stover, C.K., Jackson, R.K., Conard, P., Conley, A.J., Emini, E.A., Fuerst, T.R., and Letvin, N.L. (1993) Immunization with recombinant BCG-SIV elicits SIV-specific cytotoxic T lymphocytes in rhesus monkeys. J Immunol 150; 3101-3107.

192. Yasutomi, Y., Koenig, S., Woods, R.M., Madsen, J., Wassef, N.M., Alving, C.R., Klein, H.J., Nolan, T.E., Boots, L.J., Kessler, J.A., et al. (1995) A vaccineelicited, single viral epitope-specific cytotoxic T lymphocyte response does not protect against intravenous, cell-free simian immunodeficiency virus challenge. J Virol 69; 2279-2284.

193. Yeaman, G.R., White, H.D., Howell, A., Prabhala, R., and Wira, C.R. (1998) The mucosal immune system in the human female reproductive tract: potential insights into the heterosexual transmission of HIV. AIDS Res Hum Retroviruses 14 Suppl 1; S57-62.

194. Yee, C., Savage, P.A., Lee, P.P., Davis, M.M., and Greenberg, P.D. (1999) Isolation of high avidity melanoma-reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol 162; 2227-2234.

195. Zeh III, H.J., Perry-Lalley, D., Dudley, M.E., Rosenberg, S.A., and Yang, J.C. (1999) High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J Immunol 162; 989-994.

196. Zhang, Z.-Q., Schuler, T., Zupancic, M., Wietgrefe, S., Staskus, K.A., Reimann, K.A., Reinhart, T.A., Rogan, M., Cavert, W., Miller, C.J., et al. (1999) Sexual

180

Yongjun Sui & Jay A. Berzofsky

transmission and propagation of SIV and HIV in resting and activated CD4+ T cells. Science 286; 1353-1357.

197. Zhu, Q., Egelston, C., Gagnon, S., Sui, Y., Belyakov, I.M., Klinman, D.M., and Berzofsky, J.A. (2010) Using 3 TLR ligands as a combination adjuvant induces qualitative changes in T cell responses needed for antiviral protection in mice. J Clin Invest 120; 607-616.

198. Zhu, Q., Egelston, C., Vivekanandhan, A., Uematsu, S., Akira, S., Klinman, D.M., Belyakov, I.M., and Berzofsky, J.A. (2008a) Toll-like receptor ligands synergize through distinct dendritic cell pathways to induce T cell responses: Implications for vaccines. PNAS 105; 16260–16265.

199. Zhu, Q., Thomson, C.W., Rosenthal, K.L., McDermott, M.R., Collins, S.M., and Gauldie, J. (2008b) Immunization with adenovirus at the large intestinal mucosa as an effective vaccination strategy against sexually transmitted viral infection.

Mucos Immunol 1; 78-88.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 181-197 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

7. Transdermal delivery of vaccines Sarika Namjoshi and Heather A.E. Benson Curtin Health Innovation Research Institute, School of Pharmacy, Curtin University Perth, WA, Australia

Abstract. The skin is the largest and most accessible organ of the body. Vaccine administration to the skin offers many advantages including ease of access, reduced spread of blood-bourne diseases, and the potential for generation of both systemic and mucosal immune response. This chapter provides an overview of the current research and development of transdermal delivery of vaccines. The skin as a site of vaccine delivery, its barrier properties and the approaches to overcome these barriers are covered. Included are formulation approaches such as liposomes, physical penetration enhancers such as electroporation, and technologies that create micron-sized pores in the skin, such as microneedles.

Introduction The World Health Organisation estimates that 32% of Hepatitis B Virus infections, 40% of Hepatitis C Virus infections and 5% of Human Immunodeficiency Virus infections in developing countries are attributable to unsafe injection practices (Organization, 2004). The development of needlefree immunization methods has thus become an important goal in global health care. Dermal vaccination or transcutaneous immunisation is a needlefree method of vaccine delivery which has the potential to reduce the risk of Correspondence/Reprint request: Dr. Sarika Namjoshi, Dermcare-vet, Springwood, QLD 4127, Australia E-mail: sarika@dermcare.com.au

182

Sarika Namjoshi & Heather A.E. Benson

needle-borne diseases, improve access to vaccination by simplifying procedures (trained personnel and use of sterile equipment not required) and assist in the implementation of multiple boosting and multivalent vaccine regimes.

Skin as a site for vaccine delivery The skin has multiple barrier properties to minimize water loss from the body and prevent the permeation of environmental contaminants into the body. These barriers can be considered as physical, enzymatic and immunological.

Physical barrier The epidermis is in a constant state of renewal, with formation of a new cell layer of keratinocytes at the stratum basale, loss of their nucleus and other organelles to form desiccated, proteinaceous corneocytes on their journey towards desquamation, which occurs from the skin surface, at the same rate as formation, in normal skin. The outermost layer, the stratum corneum, consists of a brick wall like structure of corneocytes in a matrix of intercellular lipids, with desmosomes acting as molecular rivets between the corneocytes. The stratum corneum presents an effective physical barrier to the permeation of large molecules such as vaccines. This is the first barrier property that must be overcome to provide effective transdermal vaccine delivery.

Enzymatic barrier The skin possesses many enzymes capable of hydrolyzing peptides and proteins. These are involved in the keratinocyte maturation and desquamation process (Zeeuwen, 2004), formation of natural moisturizing factor (NMF) and general homeostasis (Hachem et al., 2005). Their potential to degrade topically applied vaccine antigens should be considered.

Immunological barrier When the skin is damaged, environmental contaminants can access the epidermis to initiate an immunological response. This includes (i) epithelial defence as characterized by antimicrobial peptides (AMP) produced by keratinocytes – both constitutively expressed (e.g. human beta defensin 1 (hBD1), RNAse 7 and psoriasin) and inducible (e.g. hBD 2-4 and LL-37); (ii) innate-inflammatory immunity, involving expression of pro-inflammatory

Transdermal delivery of vaccines

183

cytokines and interferons; and (iii) adaptive immunity based on antigen presenting cells, such as epidermal Langerhans and dendritic cells, mediating T-cell responses (Meyer et al., 2007). For example, Langerhans cells initiate specific immune responses by processing and presenting antigen fragments to native T cells in the lymph nodes (Romani et al., 2003). This promotes the generation of both systemic (IgG and IgM) and mucosal (IgA) humoral immune responses (Gockel et al., 2000). Thus transdermal delivery targets the vaccine to the skin, thereby promoting its contact with Langerhans cells and potentially reducing the required dose of vaccine (Babiuk et al., 2000).

Many approaches have been investigated to overcome the skins barrier properties in order to deliver antigens via the skin. They range from formulation approaches such as liposomes, to minimally invasive technologies that create channels in the skin, such as microneedles. These are summarized in Figure 1 and are described in the following sections.

All methods aim to overcome the stratum corneum barrier and target vaccine to immune-responsive cells such as Langerhans cells.

Figure 1. Immunization by dermal routes: primary delivery methods under investigation and/or development. A Liquid-jet injection. B Epidermal powder immunization. C Topical application of vaccines to the epidermis, via: a hair follicles, b tape stripping to remove the stratum corneum, c thermal or radio-wave-mediated ablation of the stratum corneum, d colloidal carriers such as microemulsions and liposomes increase dermal absorption, e low-frequency ultrasound as an adjuvant and to increase skin penetration, f topically applied adjuvants to induce a potent immune responses, g electroporation of the stratum corneum, h shallow microneedles that penetrate into the epidermis (Mitragotri, 2005).

184

Sarika Namjoshi & Heather A.E. Benson

A. Liquid-jet injection Needle-free injection devices Liquid jet injectors use a high-velocity jet (typically 100 to 200 m/s) to deliver molecules through the skin into the subcutaneous or intramuscular region. Jet injectors can be broadly classified into multi-use nozzle jet injectors (MUNJIs) and disposable cartridge jet injectors (DCJIs), depending on the number of injections carried out with a single device (Mitragotri, 2006).

Commercially available liquid jet injectors consists of a power source (compressed gas or spring), piston, drug or vaccine-loaded compartment and an application nozzle, with typical orifice size in the range of 150 to 300 亮m (Mitragotri, 2006). Upon actuation the power source pushes the piston to rapidly increase the pressure within the drug-loaded compartment, thereby forcing the drug solution through the orifice as a high velocity liquid jet. When the jet impacts on the skin it creates a hole allowing the liquid to enter the skin.

The process of hole formation and liquid jet deposition occurs within microseconds. The deposited liquid can then disperse within the tissues to illicit an immune response. An example of a liquid jet injector is shown in Figure 2.

Applications of liquid-jet injectors have been focused on delivery of macromolecules that do not passively permeate the skin. Commercially available devices include the Antares Vision速 and Choice速 (Antares, Minneapolis) that deliver a variable dose of insulin; V-Go Mini-Ject system for insulin (Valeritas, Parsippany, NJ); Biojector 2000 (Bioject, Tualatin, OR); PenJet (PenJet Corp., Santa Monica, CA) for smallpox vaccination; Injex (HNS International, Anaheim, CA) for administration of insulin and human growth hormone; Zeneo (Crossject, Paris, France).

Figure 2. Antares Vision速 jet propulsion delivery system (Antares Pharma, Mineapolis, USA).

Transdermal delivery of vaccines

185

Needle-free injection has been shown to increase immune responses to both conventional and DNA-based vaccines. For example, seroconversion rates and antibody titres elicited in humans, by a hepatitis A vaccine or a trivalent influenza vaccine, were found to be increased by at least 10% when using needle-free injections compared to needle and syringe administration (Williams et al., 2000). Clinical studies have shown that the number of responders and the mean antibody response were comparable to or better as compared to needle injection, possibly due to better tissue distribution of the vaccine (Parent du Chatelet et al., 1997, Williams et al., 2000). Recently, the Centers for Disease Control and Prevention (CDC) presented positive clinical data for the Biojector® 2000 administration of influenza vaccination (Gomez et al., 2010). They reported that intradermal vaccination by jet injection, permitted reduced "dose-sparing" amounts of vaccine, increasing the speed and avoiding the risks and discomfort of the traditional "Mantoux" needle method commonly used for tuberculosis skin testing. Vet Jet® is a transdermal jet-injector for administration of Purevax® (Merial, Duluth, GA), a non-adjuvanted leukemia vaccination for cats.

Despite the potential advantages of jet injectors, the uptake of the technology has been limited due to variable reactions, including pain and bruising, at the site of administration. Some studies reported higher levels of pain associated with jet injectors as compared to standard injections whereas others have found no difference between the two methods (Jackson et al., 2001, Sarno et al., 2000). Arora et al recently reported a novel pulsed micro-jet system designed to reduce the adverse effects at the site of administration (Arora et al., 2007). In this case a piezoelectric transducer is used to control the delivery volumes (2–15 nL), jet diameters (50–100 μm) and injection velocity (>100 m/s) thus minimizing pain and tissue damage. In order to see future vaccine applications of the jet-injector approach, further technology development will be required to provide effective pain-free delivery at reasonable cost.

B. Epidermal powder immunization Powder injectors were first used for DNA and RNA transfection into plants (Klein et al., 1988). The technique has subsequently been investigated for transdermal protein delivery, gene therapy and vaccination (Sartorelli et al., 1997, Sarphie et al., 1997, Chen et al., 2001a, Burkoth et al., 1999, Chen et al., 2000, Chen et al., 2001b). The device design principles are similar to liquid injectors, with a powder compartment and compressed carrier gas, such as helium. Upon actuation, the particles are carried by the gas, to impact the skin surface at high velocity, puncturing micron-sized holes in the epidermis to facilitate skin deposition (Kendall et al., 2004).

186

Sarika Namjoshi & Heather A.E. Benson

Humoral and cell mediated immune response following vaccination with jet propelled particles (including influenza, hepatitis B, rabies) has been demonstrated in animal studies (Fuller et al., 2006). Clinical studies have also been undertaken, with immune responses generated against influenza (Drape et al., 2006) and malaria (McConkey et al., 2003). A commercial example is the Particle Mediated Epidermal Delivery (PMED速) technology, initially developed at Oxford University, U.K. and currently owned by Pfizer. PMED delivers DNA vaccines into the skin in a dry powder formulation of microscopic gold particles and is currently in development for a range of vaccines.

Powder injectors offer advantages over liquids in terms of formulation and stability issues. Initial safety studies suggest that the powder injectors are reasonably well tolerated, and the particle bombardment offers advantages with regard to Langerhans cell targeting and immune system activation.

Research is continuing on technology development to better understand and optimize particle and formulation characteristics, and on clinical evaluation.

Kendall recently published an excellent review of ballistic administration to the skin (Kendall, 2010).

C. Topical application In addition to the systems that bombard the skin with liquid or solid vaccines, a number of other methods have been investigated that can be applied to the skin, to reduce the stratum corneum barrier, and/or carry vaccine into the skin (as summarised in Figure 1). Topical applications range from non-invasive formulation based approaches (e.g. colloidal carriers), energy based approaches (ultrasound or sonophoresis, and electroporation), stratum corneum ablation and minimally invasive approaches (such as microneedles).

Topical adjuvants Topical administration of the vaccine with adjuvants, such as cholera toxin, has been shown to induce strong systemic and mucosal immune responses. The adjuvant activates the Langerhans cells in the skin thus priming the immune response to the co-administered vaccine (Belyakov et al., 2004). A number of animal studies have provided positive immune responses for vaccines including HIV (Belyakov et al., 2004), Japanese encephalitis(Cheng et al., 2009), Helicobacter pylori (Hickey et al., 2009b) and Chlamydia infections(Hickey et al., 2009a). A human study has confirmed the potential of this administration method. Volunteers were administered patches containing recombinant Escherichia coli colonization factor CS6, either with heat-labile enterotoxin adjuvant or patches containing

Transdermal delivery of vaccines

187

CS6 alone (Guerena-Burgueno et al., 2002). There were no responses to the CS6 alone patch, whilst strong IgG and IgA immune responses were found in volunteers who received the adjuvant combination patch. A recent placebocontrolled clinical study involving 500 healthy volunteers, also demonstrated the effectiveness of applying an adjuvant patch to induce an immune response, though in this case the vaccine was not applied topically (Glenn et al., 2009). In this case, an adjuvant patch containing heat labile Escherichia coli enterotoxin was placed at the site of an intramuscular H5N1 vaccine injection and compared to vaccine injection alone. The adjuvant patch significantly enhanced the immune response to the H5N1 vaccine, with a 73% seroprotection rate. Other potential adjuvants are also being investigated (Cheng et al., 2009, Karande et al., 2009, Hickey et al., 2009a, Hickey et al., 2009b).

Colloidal carriers The rationale for the use of colloidal carriers is that compounds with unfavourable permeation characteristics can be packaged within carriers that will permeate the skin. Whilst there has been considerable research in the application of liposomes and lipid particle carriers, there is no conclusive evidence that these carriers can permeate the skin intact.

Nanoparticles and nanocarriers Nanoparticles and microparticles are polymeric particles in the nanometer and micrometre size range respectively. Compounds can be incorporated into the particles in the form of a solid dispersion or a solid solution, or bound to the particle surface by physical adsorption and chemical binding, thus allowing the particles to act as carriers or as adjuvants for the vaccine. Whilst there have been sporadic reports of nanoparticle based skin delivery, the general consensus is that nanoparticles administered to the skin do not permeate the intact stratum corneum, but may accumulate in hair follicles (Alvarez-Roman et al., 2004, Graf et al., 2009, Larese et al., 2009, Baroli, Baroli et al., 2007). Consequently their potential utility for passive transdermal vaccine delivery is limited.

Liposomes and elastic vesicles Liposomes consist of multiple bilayers of phospholipids capable of solubilising both lipophilic and hydrophilic compounds within their structure.

Despite early promise that they could act as skin permeation carriers,

188

Sarika Namjoshi & Heather A.E. Benson

evidence of their permeation across the stratum corneum intact has not emerged. Alteration of the composition including incorporation of surfactants, provides elastic or deformable to liposomes that are claimed to be capable of deforming in shape so as to “squeeze through� narrow pores in the stratum corneum (Cevc, 2004). Gupta et al. evaluated the potential of elastic vesicle transfersomes, non-ionic surfactant vesicles (niosomes) and liposomes in non-invasive delivery of tetanus toxoid (TT) (Gupta et al., 2005). Topically administered TT containing transfersomes, elicited an immune response (anti-TT-IgG) equivalent to intramuscularly alum-adsorbed TT-based immunization. Strong cellular and humoral immune responses were also reported following transcutaneous immunization with HBsAg DNAcationic deformable liposome complex (Wang et al., 2007) and Hepatitis B surface antigen-loaded ethosomes (Mishra et al., 2008). Whilst this is an active research area for the permeation enhancement of small molecules (Cevc and Vierl, 2010), vaccine development is more limited, and there are significant formulation and stability considerations with these systems.

Energy based approaches Exposure of the skin to energy in the form of electrical pulses or ultrasonic waves can disrupt the stratum corneum barrier to increase permeability. This approach has been extensively investigated for drugs and macromolecules, and to a lesser extent for vaccine delivery.

Electroporation Electroporation involves the administration of electrical pulses to create transient pores in the skin and thus increase the skin permeability to drugs and macromolecules. Delivery of DNA vaccines into muscle or skin tissue with electroporation systems generated robust immune responses in a number of disease models including influenza (H5N1 and H1N1) (Chen et al., 2008, Laddy et al., 2008), human papillomavirus (Benencia et al., 2008), and HIV (Liu et al., 2008, Hirao et al., 2008a, Rosati et al., 2008, Hirao et al., 2008b, Cristillo et al., 2008). Inovio Biomedical Corporation (Blue Bell, PA) has developed a series of hand-held, cordless electroporation devices that have been used in many vaccine delivery studies (Bodles-Brakhop et al., 2009).

Recently, the electroporation technique itself, independent of DNA delivery, was shown to recruit and trigger cells involved in antigen presentation and immune response (Chiarella et al., 2008). This adjuvant-like property is likely to enhance the continued development and success of electroporation based DNA vaccines and immunotherapeutics. The current literature on

Transdermal delivery of vaccines

189

electroporation enhanced DNA vaccine delivery was recently reviewed by Bodles-Brakhop and colleagues (Bodles-Brakhop et al., 2009).

Ultrasound or sonophoresis Low frequency sonophoresis involves application of ultrasound waves at frequencies between 20 to 100 kHz to the skin surface to reduce the stratum corneum barrier and thereby increase skin permeability (Ogura et al., 2008).

Treatment protocols have involved concurrent ultrasound administration and pretreatment prior to the application of a drug solution or patch. Low frequency ultrasound (20 kHz) was used to deliver a tetanus toxoid, illiciting a robust immune response in mice (Tezel et al., 2005). IgG antibody titres generated were similar for 1.3 g of toxoid delivered by ultrasound to the skin and 10 g administered by subcutaneous injection. The authors proposed that the immune response may be partially mediated by ultrasonic activation of Langerhans cells. A number of clinical studies of low frequency ultrasound (not vaccine related) have been reported. A commercial ultrasound device, SonoPrep, for administration of local anesthetic, was launched in 2004 but withdrawn in 2007.

Thermal ablation or microporation Thermal ablation generates micron-size holes in the stratum corneum by selectively heating small areas of the skin surface to hundreds of degrees. The heat is applied for micro- to milliseconds so that heat transfer to the viable tissues is avoided, thus minimising pain and damage. Using this technique, a 100-fold increase in reported gene expression was obtained following application to mice of an adenovirus vaccine carrying a melanoma antigen, when compared to application to intact skin (Bramson et al., 2003).

Commercially available examples are the PassPort速 system by Altea Therapeutics Corp (Altanta, GA) and the ViaDerm速 device by TransPharma Ltd (Israel). Both devices have been tested with a range of small and macromolecules. The PassPort system was utilized in the vaccine study described above and the company has a development focus in the vaccine area (www.alteatherapeutics.com).

Microneedles Microneedles consist of pointed micro-sized projections, fabricated into arrays with up to a hundred needles, that penetrate through the stratum corneum to create microscopic holes, thus providing delivery pathways for

190

Sarika Namjoshi & Heather A.E. Benson

vaccines and drugs. A number of different microneedle systems have been investigated including: solid microneedles that pierce the skin to increase permeability allowing the vaccine solution to then be applied via the skin surface; solid microneedles coated with dry powder vaccine for dissolution in the skin; microneedles composed of polymer with encapsulated vaccine for rapid or controlled release in the skin, and hollow microneedles through which the vaccine solution can be infused into the skin (Figure 3).

Solid or insoluble microneedles are generally composed of metal such as titanium or silicone. The microneedles permeabilize the skin by forming micron-sized holes though the stratum corneum. The microneedle array is then removed and a drug/vaccine containing patch is applied. This approach is termed “poke & patch�. Coated microneedles have an insoluble core coated with drug that dissolves off within the skin (Gill and Prausnitz, 2007); the so

Figure 3. Microneedles for transdermal delivery: (a) solid microneedles for permeabilizing skin via formation of micron-sized holes, (b) solid microneedles coated with dry drug or vaccine, (c) polymeric microneedles with encapsulated drug or vaccine, (d) hollow microneedles. (Arora et al., 2008).

Transdermal delivery of vaccines

191

called “coat & poke” approach. Polymer microneedles contain the drug or vaccine in a solid solution of needle that dissolves, swells or degrades on skin insertion, then releasing the drug or vaccine (Park et al., 2006, Kolli and Banga, 2008, Li et al., 2009, Lee et al., 2008). Insoluble hollow microneedles create holes through which the drug solution can pass into the skin (Wang et al., 2006): the “poke & flow” approach. Of these, the development of insoluble solid and hollow microneedles is most advanced for vaccine delivery.

Microneedles are seen as an attractive option for vaccine delivery although to date, most data is based on animal studies. A number of animal based studies have demonstrated immune responses achieved by microneedle administration in excess of that achieved by conventional injections. This includes administration of influenza vaccine tested in mice (Alarcon et al., 2007), ChimeriVaxTM-JE for yellow fever tested in primates (Dean et al., 2005), plasmid DNA encoding hepatitis B surface antigen, and recombinant protective antigen of Bacillus anthracis for anthrax tested in rabbits (Mikszta et al., 2006). In addition, the combination of microneedles and electroporation has been investigated. 4pox DNA vaccine was administered by skin electroporation using plasmid DNA-coated microneedle arrays (Hooper et al., 2007). Mice vaccinated with the 4pox DNA vaccine mounted robust antibody responses against the four immunogens-of-interest, including neutralizing antibody titers that were greater than those elicited by the traditional live virus vaccine administered by scarification. Moreover, vaccinated mice were completely protected against a lethal (>10 LD50) intranasal challenge with vaccinia virus strain IHD-J. This was the first vaccine study in which microneedle-mediated electroporation has been used to immunize animals.

Clinical studies generally report no significant adverse effects from microneedles, including minimal erythema and pain, most likely because the projections are not long enough to reach nerve endings in the deeper tissue (Prausnitz, 1999, Prausnitz, 2004). A number of small and large pharmaceutical companies, including 3M (St Paul, MN), Becton Dickinson (Franklin Lakes, NJ), Zosano Pharma (Fremont, CA), Corium (Menlo Park, CA), Valeritas (Bridgewater, NJ), Nanopass Technologies Ltd (Nes Ziona, Israel) are actively developing microneedle technologies. A recent review of the literature on microneedle development for vaccine delivery is available from one of the leading scientists in the area (Prausnitz et al., 2009).

Summary, future directions There is considerable interest and research effort towards the development of transdermal vaccine delivery. This chapter provides an overview of the main approaches that have been investigated to overcome the

192

Sarika Namjoshi & Heather A.E. Benson

stratum corneum barrier in order to provide sufficient vaccine in the tissues.

Development of some of these techniques is actively being pursued and there is considerable promise that a new transdermal vaccine delivery method may emerge. There are some major considerations that must be met if transdermal vaccine delivery is to be successful. First, the advantage that some delivery technologies activate Langerhans cells to stimulate an immune response and act as an adjuvant, is a considerable advantage. However, if the delivery technology is causing substantial cell damage and inflammation, this may negate any positive adjuvant effect and also cause local adverse effects.

Therefore a thorough understanding of the delivery parameters and fine control of the technology is essential. Second, if transdermal vaccination is to be a reality for mass immunization, the unit cost per vaccination must be low.

References 1.

Alarcon, J. B., Hartley, A. W., Harvey, N. G. and Mikszta, J. A. (2007) Preclinical evaluation of microneedle technology for intradermal delivery of influenza vaccines. Clin Vaccine Immunol, 14, 375-81.

2. Alvarez-Roman, R., Naik, A., Kalia, Y. N., Guy, R. H. and Fessi, H. (2004) Skin penetration and distribution of polymeric nanoparticles. J Control Release, 99, 53-62.

3. Arora, A., Hakim, I., Baxter, J., Rathnasingham, R., Srinivasan, R., Fletcher, D. A. and Mitragotri, S. (2007) Needle-free delivery of macromolecules across the skin by nanoliter-volume pulsed microjets. Proc Natl Acad Sci U S A, 104, 4255-60.

4. Arora, A., Prausnitz, M. R. and Mitragotri, S. (2008) Micro-scale devices for transdermal drug delivery. Int J Pharm, 364, 227-36.

5. Babiuk, S., Baca-Estrada, M., Babiuk, L. A., Ewen, C. and Foldvari, M. (2000) Cutaneous vaccination: the skin as an immunologically active tissue and the challenge of antigen delivery. J Control Release, 66, 199-214.

6. Baroli, B. (2010) Penetration of nanoparticles and nanomaterials in the skin: fiction or reality? J Pharm Sci, 99, 21-50.

7. Baroli, B., Ennas, M. G., Loffredo, F., Isola, M., Pinna, R. and Lopez-Quintela, M. A. (2007) Penetration of metallic nanoparticles in human full-thickness skin. J Invest Dermatol, 127, 1701-12.

8. Belyakov, I. M., Hammond, S. A., Ahlers, J. D., Glenn, G. M. and Berzofsky, J. A. (2004) Transcutaneous immunization induces mucosal CTLs and protective immunity by migration of primed skin dendritic cells. J Clin Invest, 113, 998-1007.

9. Benencia, F., Courreges, M. C. and Coukos, G. (2008) Whole tumor antigen vaccination using dendritic cells: comparison of RNA electroporation and pulsing with UV-irradiated tumor cells. J Transl Med, 6, 21.

10. Bodles-Brakhop, A. M., Heller, R. and Draghia-Akli, R. (2009) Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments. Mol Ther, 17, 585-92.

Transdermal delivery of vaccines

193

11. Bramson, J., Dayball, K., Evelegh, C., Wan, Y. H., Page, D. and Smith, A. (2003) Enabling topical immunization via microporation: a novel method for pain-free and needle-free delivery of adenovirus-based vaccines. Gene Ther, 10, 251-60.

12. Burkoth, T. L., Bellhouse, B. J., Hewson, G., Longridge, D. J., Muddle, A. G. and Sarphie, D. F. (1999) Transdermal and transmucosal powdered drug delivery.

Crit Rev Ther Drug Carrier Syst, 16, 331-84.

13. Cevc, G. (2004) Lipid vesicles and other colloids as drug carriers on the skin.

Advanced Drug Delivery Reviews: Breaking the Skin Barrier, 56, 675-711.

14. Cevc, G. and Vierl, U. (2010) Nanotechnology and the transdermal route: A state of the art review and critical appraisal. J Control Release, 141, 277-99.

15. Chen, D., Endres, R. L., Erickson, C. A., Weis, K. F., McGregor, M. W., Kawaoka, Y. and Payne, L. G. (2000) Epidermal immunization by a needle-free powder delivery technology: immunogenicity of influenza vaccine and protection in mice. Nat Med, 6, 1187-90.

16. Chen, D., Periwal, S. B., Larrivee, K., Zuleger, C., Erickson, C. A., Endres, R. L. and Payne, L. G. (2001a) Serum and mucosal immune responses to an inactivated influenza virus vaccine induced by epidermal powder immunization. J Virol, 75, 7956-65.

17. Chen, D., Weis, K. F., Chu, Q., Erickson, C., Endres, R., Lively, C. R., Osorio, J. and Payne, L. G. (2001b) Epidermal powder immunization induces both cytotoxic T-lymphocyte and antibody responses to protein antigens of influenza and hepatitis B viruses. J Virol, 75, 11630-40.

18. Chen, M. W., Cheng, T. J., Huang, Y., Jan, J. T., Ma, S. H., Yu, A. L., Wong, C. H. and Ho, D. D. (2008) A consensus-hemagglutinin-based DNA vaccine that protects mice against divergent H5N1 influenza viruses. Proc Natl Acad Sci U S A, 105, 13538-43.

19. Cheng, J. Y., Huang, H. N., Tseng, W. C., Li, T. L., Chan, Y. L., Cheng, K. C. and Wu, C. J. (2009) Transcutaneous immunization by lipoplex-patch based DNA vaccines is effective vaccination against Japanese encephalitis virus infection. J Control Release, 135, 242-9.

20. Chiarella, P., Massi, E., De Robertis, M., Sibilio, A., Parrella, P., Fazio, V. M. and Signori, E. (2008) Electroporation of skeletal muscle induces danger signal release and antigen-presenting cell recruitment independently of DNA vaccine administration. Expert Opin Biol Ther, 8, 1645-57.

21. Cristillo, A. D., Weiss, D., Hudacik, L., Restrepo, S., Galmin, L., Suschak, J., Draghia-Akli, R., Markham, P. and Pal, R. (2008) Persistent antibody and T cell responses induced by HIV-1 DNA vaccine delivered by electroporation. Biochem Biophys Res Commun, 366, 29-35.

22. Dean, C. H., Alarcon, J. B., Waterston, A. M., Draper, K., Early, R., Guirakhoo, F., Monath, T. P. and Mikszta, J. A. (2005) Cutaneous delivery of a live, attenuated chimeric flavivirus vaccine against Japanese encephalitis (ChimeriVax)-JE) in non-human primates. Hum Vaccin, 1, 106-11.

23. Drape, R. J., Macklin, M. D., Barr, L. J., Jones, S., Haynes, J. R. and Dean, H. J. (2006) Epidermal DNA vaccine for influenza is immunogenic in humans.

Vaccine, 24, 4475-81.

194

Sarika Namjoshi & Heather A.E. Benson

24. Ekwueme, D. U., Weniger, B. G. and Chen, R. T. (2002) Model-based estimates of risks of disease transmission and economic costs of seven injection devices in sub-Saharan Africa. Bull World Health Organ, 80, 859-70.

25. Fuller, D. H., Loudon, P. and Schmaljohn, C. (2006) Preclinical and clinical progress of particle-mediated DNA vaccines for infectious diseases. Methods, 40, 86-97.

26. Gill, H. S. and Prausnitz, M. R. (2007) Coated microneedles for transdermal delivery. J Control Release, 117, 227-37.

27. Glenn, G. M., Thomas, D. N., Poffenberger, K. L., Flyer, D. C., Ellingsworth, L. R., Andersen, B. H. and Frech, S. A. (2009) Safety and immunogenicity of an influenza vaccine A/H5N1 (A/Vietnam/1194/2004) when coadministered with a heat-labile enterotoxin (LT) adjuvant patch. Vaccine, 27 Suppl 6, G60-6.

28. Gockel, C. M., Bao, S. and Beagley, K. W. (2000) Transcutaneous immunization induces mucosal and systemic immunity: a potent method for targeting immunity to the female reproductive tract. Mol Immunol, 37, 537-44.

29. Gomez, V., Palomeque, F. S., Feris, J., Fernรกndez, J., Sรกnchez, J., Moro, P. L., Mota, C. S., Sรกnchez, V., Guzmรกn, G., Bridges, C. B., Bresee, J. S., Friede, M., Zehrung, D. L., Chen, R. T. and Weniger, B. G. 2010. Still-blinded Safety and Immunogenicity Data from a Trial of Reduced-dose, Intradermal, Influenza Vaccination by Needle-free Jet Injection. In 13th Annual Conference on Vaccine Research, P14. Bethesda, MD: www.nfid.org/conferences/vaccine10.

30. Graf, C., Meinke, M., Gao, Q., Hadam, S., Raabe, J., Sterry, W., Blume-Peytavi, U., Lademann, J., Ruhl, E. and Vogt, A. (2009) Qualitative detection of single submicron and nanoparticles in human skin by scanning transmission x-ray microscopy. J Biomed Opt, 14, 021015.

31. Guerena-Burgueno, F., Hall, E. R., Taylor, D. N., Cassels, F. J., Scott, D. A., Wolf, M. K., Roberts, Z. J., Nesterova, G. V., Alving, C. R. and Glenn, G. M. (2002) Safety and immunogenicity of a prototype enterotoxigenic Escherichia coli vaccine administered transcutaneously. Infect Immun, 70, 1874-80.

32. Gupta, P. N., Mishra, V., Rawat, A., Dubey, P., Mahor, S., Jain, S., Chatterji, D. P. and Vyas, S. P. (2005) Non-invasive vaccine delivery in transfersomes, niosomes and liposomes: a comparative study. Int J Pharm, 293, 73-82.

33. Hachem, J.-P., Man, M.-Q., Crumrine, D., Uchida, Y., Brown, B. E., Rogiers, V., Roseeuw, D., Feingold, K. R. and Elias, P. M. (2005) Sustained serine proteases activity by prolonged increase in pH leads to degradation of lipid processing enzymes and profound alterations of barrier function and stratum corneum integrity. J Invest Dermatol, 125, 510-520.

34. Hickey, D. K., Aldwell, F. E. and Beagley, K. W. (2009a) Transcutaneous immunization with a novel lipid-based adjuvant protects against Chlamydia genital and respiratory infections. Vaccine, 27, 6217-25.

35. Hickey, D. K., Aldwell, F. E., Tan, Z. Y., Bao, S. and Beagley, K. W. (2009b) Transcutaneous immunization with novel lipid-based adjuvants induces protection against gastric Helicobacter pylori infection. Vaccine, 27, 6983-90.

36. Hirao, L. A., Wu, L., Khan, A. S., Hokey, D. A., Yan, J., Dai, A., Betts, M. R., Draghia-Akli, R. and Weiner, D. B. (2008a) Combined effects of IL-12 and

Transdermal delivery of vaccines

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

195

electroporation enhances the potency of DNA vaccination in macaques. Vaccine, 26, 3112-20.

Hirao, L. A., Wu, L., Khan, A. S., Satishchandran, A., Draghia-Akli, R. and Weiner, D. B. (2008b) Intradermal/subcutaneous immunization by electroporation improves plasmid vaccine delivery and potency in pigs and rhesus macaques. Vaccine, 26, 440-8.

Hooper, J. W., Golden, J. W., Ferro, A. M. and King, A. D. (2007) Smallpox DNA vaccine delivered by novel skin electroporation device protects mice against intranasal poxvirus challenge. Vaccine, 25, 1814-23.

Jackson, L. A., Austin, G., Chen, R. T., Stout, R., DeStefano, F., Gorse, G. J., Newman, F. K., Yu, O. and Weniger, B. G. (2001) Safety and immunogenicity of varying dosages of trivalent inactivated influenza vaccine administered by needle-free jet injectors. Vaccine, 19, 4703-9.

Karande, P., Arora, A., Pham, T. K., Stevens, D., Wojicki, A. and Mitragotri, S. (2009) Transcutaneous immunization using common chemicals. J Control Release, 138, 134-40.

Kendall, M., Mitchell, T. and Wrighton-Smith, P. (2004) Intradermal ballistic delivery of micro-particles into excised human skin for pharmaceutical applications. J Biomech, 37, 1733-41.

Kendall, M. A. (2010) Needle-free vaccine injection. Handb Exp Pharmacol, 193-219.

Klein, T. M., Fromm, M., Weissinger, A., Tomes, D., Schaaf, S., Sletten, M. and Sanford, J. C. (1988) Transfer of foreign genes into intact maize cells with highvelocity microprojectiles. Proc Natl Acad Sci U S A, 85, 4305-4309.

Kolli, C. S. and Banga, A. K. (2008) Characterization of solid maltose microneedles and their use for transdermal delivery. Pharm Res, 25, 104-13.

Laddy, D. J., Yan, J., Kutzler, M., Kobasa, D., Kobinger, G. P., Khan, A. S., Greenhouse, J., Sardesai, N. Y., Draghia-Akli, R. and Weiner, D. B. (2008) Heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus DNA antigens. PLoS One, 3, e2517.

Larese, F. F., D'Agostin, F., Crosera, M., Adami, G., Renzi, N., Bovenzi, M. and Maina, G. (2009) Human skin penetration of silver nanoparticles through intact and damaged skin. Toxicology, 255, 33-7.

Lee, J. W., Park, J. H. and Prausnitz, M. R. (2008) Dissolving microneedles for transdermal drug delivery. Biomaterials, 29, 2113-24.

Li, G., Badkar, A., Nema, S., Kolli, C. S. and Banga, A. K. (2009) In vitro transdermal delivery of therapeutic antibodies using maltose microneedles. Int J Pharm, 368, 109-15.

Liu, J., Kjeken, R., Mathiesen, I. and Barouch, D. H. (2008) Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation. J Virol, 82, 5643-9.

McConkey, S. J., Reece, W. H., Moorthy, V. S., Webster, D., Dunachie, S., Butcher, G., Vuola, J. M., Blanchard, T. J., Gothard, P., Watkins, K., Hannan, C.

196

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

Sarika Namjoshi & Heather A.E. Benson

M., Everaere, S., Brown, K., Kester, K. E., Cummings, J., Williams, J., Heppner, D. G., Pathan, A., Flanagan, K., Arulanantham, N., Roberts, M. T., Roy, M., Smith, G. L., Schneider, J., Peto, T., Sinden, R. E., Gilbert, S. C. and Hill, A. V. (2003) Enhanced T-cell immunogenicity of plasmid DNA vaccines boosted by recombinant modified vaccinia virus Ankara in humans. Nat Med, 9, 729-35.

Meyer, T., Stockfleth, E. and Chrstophers, E. (2007) Immune response profiles in human skin. Br J Dermatol, 157, 1-7.

Mikszta, J. A., Dekker, J. P., 3rd, Harvey, N. G., Dean, C. H., Brittingham, J. M., Huang, J., Sullivan, V. J., Dyas, B., Roy, C. J. and Ulrich, R. G. (2006) Microneedle-based intradermal delivery of the anthrax recombinant protective antigen vaccine. Infect Immun, 74, 6806-10.

Mishra, D., Mishra, P. K., Dubey, V., Nahar, M., Dabadghao, S. and Jain, N. K. (2008) Systemic and mucosal immune response induced by transcutaneous immunization using Hepatitis B surface antigen-loaded modified liposomes. Eur J Pharm Sci, 33, 424-33.

Mitragotri, S. (2005) Immunization without needles. Nat Rev Immunol, 5, 905-16.

Mitragotri, S. (2006) Current status and future prospects of needle-free liquid jet injectors. Nat Rev Drug Discov, 5, 543-8.

Ogura, M., Paliwal, S. and Mitragotri, S. (2008) Low-frequency sonophoresis: current status and future prospects. Adv Drug Deliv Rev, 60, 1218-23.

Parent du Chatelet, I., Lang, J., Schlumberger, M., Vidor, E., Soula, G., Genet, A., Standaert, S. M. and Saliou, P. (1997) Clinical immunogenicity and tolerance studies of liquid vaccines delivered by jet-injector and a new single-use cartridge (Imule): comparison with standard syringe injection. Imule Investigators Group.

Vaccine, 15, 449-58.

Park, J. H., Allen, M. G. and Prausnitz, M. R. (2006) Polymer microneedles for controlled-release drug delivery. Pharm Res, 23, 1008-19.

Prausnitz, M. R. (1999) A practical assessment of transdermal drug delivery by skin electroporation. Advanced Drug Delivery Reviews, 35, 61-76.

Prausnitz, M. R. (2004) Microneedles for transdermal drug delivery. Adv Drug Deliv Rev, 56, 581-7.

Prausnitz, M. R., Mikszta, J. A., Cormier, M. and Andrianov, A. K. (2009) Microneedle-based vaccines. Curr Top Microbiol Immunol, 333, 369-93.

Romani, N., Holzmann, S., Tripp, C. H., Koch, F. and Stoitzner, P. (2003) Langerhans cells - dendritic cells of the epidermis. APMIS, 111, 725-40.

Rosati, M., Valentin, A., Jalah, R., Patel, V., von Gegerfelt, A., Bergamaschi, C., Alicea, C., Weiss, D., Treece, J., Pal, R., Markham, P. D., Marques, E. T., August, J. T., Khan, A., Draghia-Akli, R., Felber, B. K. and Pavlakis, G. N. (2008) Increased immune responses in rhesus macaques by DNA vaccination combined with electroporation. Vaccine, 26, 5223-9.

Sarno, M. J., Blase, E., Galindo, N., Ramirez, R., Schirmer, C. L. and TrujilloJuarez, D. F. (2000) Clinical immunogenicity of measles, mumps and rubella vaccine delivered by the Injex jet injector: comparison with standard syringe injection. Pediatr Infect Dis J, 19, 839-42.

Transdermal delivery of vaccines

197

65. Sarphie, D. F., Johnson, B. and al., e. (1997) Bioavailability following transdermal powdered delivery (TPD) of radiolabeled inulin to hairless guinea pigs. J Control Release, 47, 61–69.

66. Sartorelli, P., Aprea, C., Bussani, R., Novelli, M. T., Orsi, D. and Sciarra, G. (1997) In vitro percutaneous penetration of methyl-parathion from a commercial formulation through the human skin. Occup Environ Med, 54, 524-5.

67. Tezel, A., Paliwal, S., Shen, Z. and Mitragotri, S. (2005) Low-frequency ultrasound as a transcutaneous immunization adjuvant. Vaccine, 23, 3800-7.

68. Wang, J., Hu, J. H., Li, F. Q., Liu, G. Z., Zhu, Q. G., Liu, J. Y., Ma, H. J., Peng, C. and Si, F. G. (2007) Strong cellular and humoral immune responses induced by transcutaneous immunization with HBsAg DNA-cationic deformable liposome complex. Exp Dermatol, 16, 724-9.

69. Wang, P. M., Cornwell, M., Hill, J. and Prausnitz, M. R. (2006) Precise microinjection into skin using hollow microneedles. J Invest Dermatol, 126, 1080-7.

70. Williams, J., Fox-Leyva, L., Christensen, C., Fisher, D., Schlicting, E., Snowball, M., Negus, S., Mayers, J., Koller, R. and Stout, R. (2000) Hepatitis A vaccine administration: comparison between jet-injector and needle injection. Vaccine, 18, 1939-43.

71. World Health Organization (2004) Safety of Injections: Global Facts & Figures 72. Zeeuwen, P. L. (2004) Epidermal differentiation: the role of proteases and their inhibitors. Eur J Cell Biol, 83, 761-73.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 199-222 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

8. Adjuvants and vaccine delivery systems Valerie A Ferro University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences 161 Cathedral Street, Glasgow, G4 0RE, UK

Abstract. Adjuvants and delivery systems form an important part of vaccine formulations, particularly since the newer generation of peptide- and protein-based vaccines require immune potentiation to improve their efficacy. However, very few adjuvants have approval for use in humans. This review examines the range of adjuvants that are used in vaccines on the market, such as mineral salts, oilin-water emulsions, microbial- and plant-based derivatives.

Examples of adjuvants and delivery systems in development that have been or are close to being tested in clinical trials are also described (including immunostimulatory factors, gold particles and lipid vehicles). Furthermore, technologies that are designed for mucosal route administration are considered, including particulates and non-particulates. Overall, it is clear that there is a need for a greater understanding of the immune system in order to improve the safety and efficacy of vaccination using adjuvants.

I. Introduction Vaccination is considered an important public health intervention in preventing infectious disease. Historically, a notable success and eradication of disease is exemplified by smallpox (Kennedy et al., 2009). A list of current Correspondence/Reprint request: Dr. Valerie A Ferro, University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street, Glasgow, G4 0RE, UK. E-mail: v.a.ferro@strath.ac.uk

200

Valerie A Ferro

vaccine preventable diseases shows the range and extent of conditions that are under control (http://www.cdc.gov/vaccines/vpd-vac/default.htm), while diseases such as AIDS and malaria continue to present challenges (Awasthi, 2008). When considering our recent and current global health status, new demands are being placed on vaccine R&D to achieve greater safety and efficacy (Wolf et al., 2010), to target emerging pathogens often with rapidresponse (Stephenson, 2005), to protect against biological warfare (Kennedy et al., 2009), and to tackle non-infectious conditions such as (cancer, fertility, allergy, lifestyle) (Ferro and Mordini, 2004). Therefore, parallel technological advances have been necessary, not least of all in the adjuvant and delivery fields (Brunner et al., 2010). This chapter will examine progress in the field and the impact of novel approaches to vaccine research.

1. What are adjuvants?

As defined by the European Medicines Evaluation Agency (EMEA), adjuvants are substances that are expected to enhance and accelerate the efficacy and longevity of a specific immune response to antigens, with a minimum of long-term harmful effects themselves (EMEA, 2005). They may be capable of directing the immune response, reducing the amount of antigen or doses required and improving responses in cases where immunity may be compromised (eg the elderly, newborns). In addition, to promoting an appropriate immune response, the expectations are that vaccine formulations will have a long shelf-life with minimum cold storage and stability issues, be cheap to manufacture and be biodegradable. In 2006, the EMEA made an attempt to further distinguish between adjuvants and immunomodulators, stating that an adjuvant is part of a vaccine formulation, while an immunomodulator is a substance that can be administered separately or at a different time point (EMEA, 2006). Furthermore, an adjuvant helps the immune response by local action with an antigen, while an immunomodulator has a systemic action. In this review, no distinction is made between the two, since in the main - principles applying to adjuvants are applicable to immunomodulators.

Not all vaccines require co-administration with an adjuvant – these include live vaccines or those based on whole attenuated pathogens (Guy, 2007). The reasons are due to complexity of whole organisms, the numbers of epitopes that they present and their relative size. Subunit protein - and peptide-based vaccines are considered to be safer, but are poorly immunogenic and therefore require potent adjuvants to activate the immune system. Similarly, vaccines against intracellular pathogens need to be capable of activating strong cellular responses, including T helper (Th) 1 cells and

Immunogenicity

201

cytotoxic T lymphocytes (CTLs). Thus, the new generation of vaccines necessitate the use of adjuvants, balanced with strict regulation, as they come in many forms and are expected to have compatibility with a wide range of substances. This makes it difficult to apply standardised approaches to assessment of efficacy and safety. The evaluation, licensing, control and monitoring of adjuvants is carried out by National Regulatory Authorities (NRA), which are required to have critical knowledge on the increasing number of new products, technical issues, manufacturing processes and surveillance of efficacy and problems once the formulated adjuvants are administered to the end-user. The World Health Organisation (WHO) consolidates key regulatory information and communicates them to the worldwide community of researchers, NRA and manufacturers. The WHO produces various documents, which provide a useful resource of regulatory expectations (pre- and post-clinical evaluation) (WHO, 2004).

2. Regulatory requirements Barriers to new adjuvant approvals include: safety, tolerability, preclinical/clinical screening and expense. In order that researchers do not waste time, effort and money in this field of research, it is important that regulatory considerations are made early on (Carter et al., 2006). Laboratory studies define the physical, chemical and biological characteristics of the adjuvant/vaccine combination, while pre-clinical testing is performed in animals and informs the clinicians of potential toxic/reactogenic effects. Preclinical testing standards have an impact later on and form justification for subsequent clinical Phase I, Phase II and Phase III studies. Pre-clinical screening is important, but only offers a guide as to what may occur in the clinical testing stage. A notable and recent tragic example of this is the testing of the drug TGN1412 in 2006 (Liedert et al., 2007). In clinical trials, adjuvants and vaccines require additional special attention compared with other therapeutics because they are given to healthy infants and children, are administered to prevent disease and not to treat it, and there must be consistency from batch-to-batch. Post-marketing surveillance continues to collect data on safety and effectiveness in large numbers of recipients and adverse events. Consideration has to be given to local tolerance which may be influenced by route of administration, induction of hypersensitivity and anaphylaxis, pyrogenicity, systemic toxicity, reproductive toxicity, genotoxicity and carcinogenicity. With the use of adjuvants, one goal is also to reduce the amount of antigen and together with delivery systems enable fewer administrations to be made. Evaluation of adjuvants necessitates demonstration of antigen compatibility, consistency in association of the

202

Valerie A Ferro

adjuvant and antigen with time and over a range of temperatures, adjuvant component purity and degradation. The combination with antigen may also result in more severe local reactions. As with any therapeutic products, the risk to benefit ratio needs to be assessed. In the case of adjuvants, the latest emphasis leans towards safety over efficacy when a vaccine is administered to a healthy population, while in high risk groups or for example in cancer vaccines a higher level of toxicity may be acceptable (Chamberlain and Gronvall, 2007).

A major problem in adjuvant evaluation is that it is not always possible to predict compatibilities and potential pitfalls without going into late preclinical or early clinical (or sometimes even post-clinical) stages. This is particularly so, when there are no relevant bioassays, animal models or reagents available for particular disease models. For example when testing an influenza vaccine, it is not possible to evaluate the infection challenge in a common mouse strain; the virus needs to be adapted by multiple lung passages, before it becomes lethal for mice (Barnard, 2009). The most common influenza animal model is the ferret as it exhibits many symptoms seen in the human (nasal discharge and fever), however, there are very few ferret specific reagents available for antibody and cytokine assessment. Apart from mice, animals used to specifically assess adjuvants include guinea pigs for quality control and hypersensitivity and rabbits for toxicity (Leenaars et al., 1995; Maurer, 2007; Alving, 2002). Even if an appropriate model is found, there may still be a disparity in correlating events in animals with human responses and a better understanding of the infection is required. This can be exemplified by lessons learnt from animal models used to test a schistosome vaccine (Coulson and Kariuki, 2006). In mice, some strategies used to test vaccine candidate efficacy could be detrimental if tested in clinical trial without going into nohuman primate systems first.

II. Adjuvant classification and range Adjuvants have been in use since the early 20th century and yet their classification into groups continues to pose difficulty due to the diversity of the substances with often-unknown mechanisms of action. Mechanisms of action can include: stabilisation of 3-D epitope conformation, generation of depot effects, targeting and interaction with antigen-presenting cells, stimulation of Th1/Th2 or CTL responses (Edelman, 2000). Adjuvants can be broadly classified into a) immune potentiators that act on the immune system to promote a response against an antigen and b) delivery systems that present antigens to the immune system in an appropriate manner (Reed et al., 2009).

Immunogenicity

203

In addition, some adjuvants enable slow release of antigen to protect from a bolus effect or rapid elimination from the circulation, while others act by causing local inflammatory reaction that stimulates innate immunity. Another classification system works on the basis of whether they are particulate/nonparticulate or their mechanism of action (Cox and Coulter, 1997). Edelman (2000) has collated a comprehensive list of different classes of adjuvants.

Examples of adjuvants are outlined below, some of which are found in approved human vaccines, while others are examples that are in development.

1. Mineral salts Mineral salt adjuvants include: aluminium, calcium, iron and zirconium.

Aluminium salt-based adjuvants were introduced in the 1930s and until recently in the US, were the only approved adjuvants for human use. Since 2009, Cervarix® vaccine that includes AS04 (a combination of alum and monophosphoryl lipid A, MPL) has also been approved. Alum is a generic term applied to the various forms of aluminium-based salts, including aluminium oxy-hydroxide, aluminium hydroxy-phosphate or aluminium hydroxy-sulphate employed since the application of the potassium alumprecipitated proteins described by Alexander Glenny in 1926. However, alum should specifically refer to the water-soluble aluminium potassium sulphate to which antigen in phosphate buffer is added and precipitated through addition of a basic solution, since other salts labelled “alum” have different properties (Lindblad, 2004). Depending on the aluminium salt used and conditions used for adsorbing the protein antigen, two binding processes may be involved: electrostatic interaction or ligand exchange (Brewer, 2006).

Antigen adsorbed onto alum results in Th2 responses as well as production of IL-4/IL-5 cytokines and IgG/IgE antibodies (Lindblad, 2004). As such it has been used successfully in tetanus, diphtheria and pertussis vaccines (eg Wyeth Lederle’s Acel-Imune® DTaP), where protection against infection is dependent on the generation of neutralising antibodies. Other examples of licensed human vaccines containing alum include RECOMBIVAX HB® (a recombinant Hepatitis B vaccine) and MENJUGATE® (Meningococcal Group C conjugate vaccine). A more complete list of vaccines with aluminium salts can be found at http://www.scribd.com/doc/17365604/ VaccineCDCInfo. Aluminium salt-based adjuvants are not suitable for diseases such as HIV, tuberculosis and malaria as they are not effective in inducing Th1 and CTL responses therefore; alternative adjuvants are required.

204

Valerie A Ferro

The advantages of alum lie in its ability to enhance antibody titres and provide long-lasting effects, to provide antigen stability, relative cheapness and ease of manufacture. The disadvantages include variations in adsorption of antigens to different salts, inability to mount Th1 or CTL responses that are required for intracellular pathogens and issues with accidental freezing which results in loss of potency (Lindblad and Schonberg, 2010). Adverse effects of aluminium can be local (injection site pain and sometimes necrosis, inflammation and lymphadenopathy), as well as systemic (nausea, fever, immunotoxicity) (Gupta et al., 1993; Petrovsky and Aguilar, 2004). It is also less well tolerated when administered by subcutaneous or intradermal routes and so is mainly used intramuscularly.

The mechanism of action is still not fully understood, but several theories have been proposed over the years, in particular the concept that formation of a depot facilitates slow release of antigen and may be involved in maintenance of memory (Lambrecht et al., 2009). Recently, it has been demonstrated that retention of antigen does not necessarily cause immunopotentiation (Noe et al., 2010). Other theories include: local inflammation causing recruitment of appropriate immune cells (Goto et al., 1997), uptake of the insoluble particles by antigen presenting cells (Morefield et al., 2005), cytotoxicity to macrophages (Petrovsky et al., 2007) and activation of th NALP3 inflammasome pathway which contributes to the immediate innate response at the injection site as well as induction of adaptive cellular immunity (reviewed by De Gregorio et al., 2008). Li et al.

(2007) compared the cytokine responses induced by three aluminium-based products in the presence of toll-like receptor (TLR4 or TLR2) agonists stimulated human peripheral blood mononuclear cells (PBMC); these were Imject Alum (from Pierce), Alhydrogel (Sigma-Aldrich) and Adju-Phos (Brenntag). The agonists and adjuvants alone were unable to stimulate IL1-β from the PBMC, yet in the presence of Alhydrogel and Adju-Phos IL1-β was released in a dose-dependent manner. The adjuvants were also able to stimulate release of IL-18 (consistent with Th2 activity of alum), IL-10 (a Th2 cytokine) and inhibit IP-10 (a Th1 cell associated chemokine). Studies like this are important to increase our knowledge about the mechanism of action and there seems to be resurgence in activity in this area as our understanding of the role and complexity of the cytokine network in immunity progresses. Most recent focus has been on the involvement of alum-based adjuvants and the NOD-like receptor pathway (NLRP3). A greater understanding of this role, or indeed establishment of the mechanism of action of alum-based adjuvants, will enable more specific targeting of the required immune pathways and will be important for future developments.

Immunogenicity

205

2. Oil-in-water emulsions The “gold standard” adjuvant in terms of potency is Freund’s adjuvant, based on oil-in-water droplets. The incomplete adjuvant (FIA) consists of a water-in-oil emulsion of mineral oil (Marco 52, 85%) and the surfactant Arlacel A (mannide mono-oleate, 15%), while the complete adjuvant (FCA) has 500μg heat-killed Mycobacterium tuberculosis per ml of mixture. Into these adjuvants, aqueous dissolved antigen is emulsfied. However, perceived extreme toxicity and side-effects precludes its use in human and veterinary vaccines (Yamanaka et al., 1992). FCA is also banned or restricted in use in experimental animals in many countries and has been replaced by TiterMax™, which uses copolymer CRL89-41 instead of the mycobacterium employed in FCA (Zhou and Afshar, 1995). Muramyl dipeptide (MDP) derived from the mycobacterial cell wall complex and other synthetic analogues continue to be investigated, but they are still believed to be too reactogenic for use in humans (Moschos et al., 2004). Montanides (ISA51 and ISA720, developed by Seppic) are similar to FIA and are composed of a highly purified mannide-mono-oleate emulsifier used 3 times higher percentage-wise than the surfactant in FIA (Miller et al., 2005). They induce humoral and cell-mediated immunity with various antigens (Ganne, 1994).

However, as with other bacterial derived products, they are associated with costly formulation, stringent culture facilities and they have been shown to produce unacceptable local reactions (Toledo et al., 2001).

Other oil-in-water emulsions include MF59™, AS02 and AS03, Adjuvant 65 and Lipovant. The mechanism of action of these emulsions is thought to be due to their particulate and irritant properties, which result in local inflammation and macrophage responses (Tritto et al, 2009). These adjuvants generate a better IgG1:IgG2a balanced response than alum and because they are able to produce antibodies against haemagglutinin and induce CD8+ T-cell responses, they are used in influenza vaccines (eg GSK’s Influenza A Prepandrix vaccine). MF59™ consists of 4.3% squalene (a natural organic compound derived from shark liver oil), stabilized by Tween™80 and Span 85 (Tagliabue and Rappouli, 2008). It is approved in Europe and is found in several influenza vaccines manufactured by Novartis (eg Fluad®). MF59™ has also been shown to be safe and well tolerated in newborn infants and is compatible with a range of antigens including those from HIV (Cunningham et al., 2001).

Studies have examined the use of oil-in-water emulsions in mucosal delivery of vaccines (Shahiwala and Amiji, 2008; Bielinska et al., 2008) and in the future there is likely to be a rise in approved adjuvants via the

206

Valerie A Ferro

parenteral route being applied to the mucosal routes, as well as development of novel oil-in-water emulsions designed for mucosal applications.

Nevertheless, structural fluidity and emulsion stability issues may mean that oil-in-water adjuvants will be restricted to therapeutic rather than prophylactic vaccines.

3. Microbial derivatives The immune system recognises regular patterns pathogen-recognition receptors, including: toll-like receptors (TLRs), C-type lectins, CD14, scavenger receptors, the NOD-like receptor family and the RIG-1-like receptor family (Holzi et al., 2008). The pathogen-associated molecular patterns (PAMPs) that bind TLRs are mainly associated with adjuvants. The first to be approved for human use was MPL™ (monophosphoryl lipid A), found in Fendrix® (a Hepatitis B vaccine, approved by the EU in 2005).

MPL™ was developed by Corixa Corp.; later acquired by GlaxoSmithKline (GSK). MPL™ is an endotoxin derivative from Salmonella Minnesota R595 and is a component of AS02 and AS04 (GSK’s proprietary adjuvants which are mixed with QS-21 and alum, respectively). AS04 is used in the Human Papilloma Virus (Cervarix, which has been approved), Herpes Simplex Virus (Simplirix, currently in Phase III trials), and malaria (Mosquarix, currently Phase II trials) vaccines. A recent clinical trial was carried out in renal insufficiency patients administered hepatitis vaccines containing either AS02 or AS04. They found that 3 doses of the AS02 vaccine gave a more rapid and persistent protection in these patients, compared with the 4 dose licensed AS04 vaccine (Surquin et al., 2010). Other MPL-based adjuvants include AS01B and AS02A, which have been evaluated against a range of intracellular diseases (Waitumbi et al., 2009; Garcon et al., 2007) and MPL™ has also been licensed in Europe and Canada for treatment of allergies (Pollinex® Quattro, Allergy Therapeutics). The mechanism of action of MPL™ is through stimulating TLR4, inducing pro-inflammatory cytokines (TNF-α, IL-2 and IFN-γ) and stimulating Th1 responses (Thompson et al., 2005). Macrophages and dendritic cells are recruited and activated with the ability to effectively present antigen to T and B lymphocytes, directing maturation of the lymphocytes into antigen-specific effeector and memory cells (Baldridge et al., 2006). In terms of manufacturing issues, there are problems with consistency of formulation, and cost, although on the positive side, it is highly stable.

Another immunostimulatory adjuvant, which is comprised of synthetic oligodeoxynucleotides, containing unmethylated CpG, acts through TLR9

Immunogenicity

207

and activates dendritic cells and release of pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12, IFN-α and IFN-γ). Since CpG induces Th1 and CTL responses it has been investigated against viral infections, cancer and allergy.

CpG based adjuvants such as CPG7907 and CPG 7909 (Coley Pharmaceuticals) have been tested with an alum-based Hepatitis B vaccine (Gupta and Cooper, 2008; Pichichero, 2008) and in conjunction with Monatide ISA-51 (Karbach et al., 2010). In clinical trials, adverse effects have included injection site reactions, flu-like symptoms, and headaches (Cooper et al., 2004) probably due to stimulation of TNF-α, which remains a barrier to acceptance of CpG adjuvants in prophylactic vaccines. Other concerns include the possibility of the role of TLR9 signalling in autoimmune conditions (Vollmer and Krieg, 2009).

Outer membrane vesicle (OMV) vaccine formulations contain particles made from bacterial outer membranes, and may represent an alternative to attenuated live microorganisms. An example is the VA-MENGOC-BC® vaccine (Finlay Institute, Cuba) that consists of purified OMV from serogroup B meningococcus and purified capsular polysaccharides from serogroup C meningococcus, adsorbed in an aluminium hydroxide gel.

Other bacterial products include toxins produced by bacteria, such as cholera toxin (CT) and heat labile toxin (LT, from Escherichia coli) that induce toxin-neutralizing antibodies and is the basis of modified toxins being used as adjuvants; they are particularly useful as mucosal adjuvants (Lycke, 2005; Fingerut et al, 2005). Recently, a clinical trial carried out on 500 healthy adults receiving two intramuscular doses of A/Vietnam/1194/2004 A/H5N1 vaccine (5μg, 15μg or 45μg) or saline, 21 days apart (Glenn et al., 2009). With each of vaccine dose, a 50μg LT adjuvant patch was applied over the injection site at either the second or both immunisations. The adjuvant patch significantly enhanced the immune response, and enabled a 73% seroprotection rate after a single dose. However, the two-dose patch plus injection regimen resulted in more robust responses. Thus, future directions in vaccine approaches may see combination administration routes as above or as in heterologous prime-boost regimens using different delivery systems (reviewed by Lu, 2009).

4. Natural products Despite the intense activity in natural product research aimed at drug discovery, in comparison, little work appears to be targeted at adjuvant discovery. Yet, the best example of a plant-derived adjuvant is Quil A and the purified fraction QS-21™ (originally patented by Cambridge Biotech Corp., and acquired by Antigenics Inc.). Quil A consists of saponins (triterpenoid

208

Valerie A Ferro

glycosides) derived from the bark of the South American soap bark tree, Quillaja saponaria. QS-21™ induces Th1 cytokines and IgG2a antibodies as well as stimulating CTL responses. Numerous clinical trials have been conducted using QS-21™ in cancer vaccines and infectious disease, including HIV-1, influenza, herpes, malaria, and hepatitis B (Evans et al., 2001; Mbawuike et al., 2007; Ballou, 2009; Vandepapaliere et al., 2008), as well as in Alzheimer immunotherapy (http://www.antigenics.com/products/tech/ qs21/). The main problem with QS-21™, on its own, is injection site pain, granulomas and haemolysis – hence it is not used in human vaccines (Rajput et al., 2007). In addition, it is difficult to purify to homogeneity, has doselimiting toxicity, and chemical instability; thus other chemically stable synthetic entities are being developed and evaluated (Adams et al., 2010).

More often however, the Quil A saponins have been used in combination with other adjuvants such as immunostimulatory complexes (ISCOMs), initially developed by Isotec and is used in an equine vaccine licensed for use in Europe (Equilis® Prequenza Te, Intervet). Other plant saponins are also under investigation (reviewed by Sun et al., 2009a; Song and Hu, 2009).

ADVAX is a natural plant-based adjuvant, from dahlia tubers, developed

by Vaxine Pty Ltd (Australia) and is composed of nanocrystalline particles of the polysaccharide inulin (Cooper and Carter, 1986). Gamma- and deltaisoforms of inulin have the ability to enhance humoral and cellular immune responses without toxicity and have been used for renal function testing (so are safe for human use). Inulin is heat stable with a long shelf life, and is biodegradable (being excreted in urine). When crystallized in the presence of an Alhydrogel suspension and transformed to gamma inulin, it forms electron-dense ovoids (Algammulin) that adsorb antigen and activate complement (Cooper and Steele, 1991). ADVAX is currently being evaluated in humans against HIV-1 antigens (Vasan et al., 2010).

Chitosan is a soluble polymer derived from chitin, which is commonly found in invertebrate exoskeletons and fungi and has received attention in inclusion in delivery systems since it is biocompatible and biodegradable (Kafetzopoulos et al., 1993). It is also useful in mucosal vaccines and has been reviewed recently by Arca et al. (2009).

The examples given above show how natural products can be successfully developed as adjuvants. Schepetkin and Quinn (2006), described the immunomodulatory activity of polysaccharides from plants, fungi and algae that could be investigated further. Nature provides an extensive resource, yet this is an area that has not been fully exploited. Instead of spending significant resources on R&D of pure, defined chemical entities, it may be possible to utilize traditional medicines as dietary supplements to

Immunogenicity

209

enhance vaccination, and this could be particularly beneficial to the developing world in keeping costs down.

5. Endogenous immunostimulatory factors Since cytokines are key regulators of the immune system, it is pertinent that they have been examined for their suitability as natural adjuvants.

However, manipulating the cytokine network is complex and depends on timing, length of exposure, cells targeted and interaction with other players.

Cytokines have been used to influence the immune type response, to induce long-lasting memory or to strengthen the response in co-administration with other adjuvants. For example, the rationale for using IL-12 is that bacterial products and intracellular parasites induce the cytokine from macrophages, monocytes, dendritic cells and B lymphocytes. It increases interferon gamma (IFN-γ) and TNF-α by NK and Th cells, has an important role in inducing Th1 from naive Th0 cells and generating CTLs, it increases secretion of IL2 and when administered mucosally is able to redirect Th2-tpe responses to a Th1-type response (Stevceva et al., 2007). However, systemic administration is less favourable than mucosal administration. Other cytokines that have been examined include GM-CSF, IL-2, IL-18, IL-15, IL-17, IFN-γ and TNF-γ to name just a few. Similarly, chemokines such as IL-8, RANTES, CCL19 and CCL21 have also been tested. Kornbluth and Stone (2006) provide a comprehensive list of cytokines and chemokines that have been examined. A disadvantage in using cytokines is their short half-life. However, this has been overcome by covalently attaching them to polyethylene glycol, which has the added advantage of lowering the dose required and thus reducing adverse events (Stevceva et al., 2007). Probably, the greatest advantage of studying the potential of cytokines and chemokines is that it has led to a greater understanding of how the immune system is regulated, and in particular, has highlighted the difference between “mice and men”.

6. Inert vehicles (gold) A delivery system that is used in epidermal powder immunisation (EPI) enables antigens to be delivered on 1.5-2.5 micron gold particles to the epidermis, using a needle-free system. An example of gold particle technology was extensively developed by Powderject Ltd and after a series of acquisitions was taken up by Pfizer in 2006. The system known as particlemediated epidermal delivery (PMED) has been tested with a hepatitis B and influenza virus DNA vaccine (Moss, 2009).

210

Valerie A Ferro

7. Lipid particulates Numerous lipid-based systems are currently being investigated for their use in vaccine delivery. They include liposomes (Bangham, 1972), immune stimulating complexes (ISCOMs) (reviewed by Sun et al., 2009b), archaeosomes (reviewed by Krishnan and Sprott, 2008), cochleates (reviewed

by Rao et al., 2007) and non-ionic surfactant vesicles (NISV) (Brewer and Alexander, 1994). a. Liposomes. Liposomes are comprised of fluid bilayer membranes capable of encapsulating a wide range of proteins and peptides, but because they lack inherent adjuvant properties on their own, they require the addition of immunostimulatory molecules, resulting in ISCOMs (Perrie et al., 2008).

Furthermore, they are susceptible to attack from extremes of pH or enzymatic degradation. b. ISCOMs. the preferred components are 0.5% Quil A/QS21, 0.1% cholesterol and 0.1% phosphatidylethanolamine with antigen added in phosphate-buffered saline. The matrix component without antigen is known as ISCOMATRIX® adjuvant (reviewed by Skene and Sutton, 2006). They generate protective immunity including CTL responses, IFN-γ and IL12 to a range of antigens, but the characteristics of the protein influences suitability for association with the adjuvant. They are generally administered parenterally, although they have been examined in mucosal immunisation as well. They reduce the haemolytic activity and toxicity of Quil A and enable less antigen to be used. In a human influenza trial two ISCOM vaccines resulted in quicker titres with a higher proportion of the vaccines showing CTL responses compared with conventional influenza vaccines (Rimmelzwaan et al., 2000). c. Archaeosomes. consist of liposomes made from the polar ether lipids of Archaea. They enhance the recruitment and activation of antigen presenting cells, and deliver antigen to both MHC pathways for antigen presentation, without eliciting inflammatory responses. Systemic administration of antigen-containing archaeosomes elicit strong and sustained antigen-specific antibody responses comparable, to those obtained with Freund's adjuvant, as well as to intracellular pathogens. They also induce antigen-specific cellmediated immunity, including CTL responses. Krishnan and Sprott (2008) summarise the characteristics of archaesomes: • •

High stability due to the ether archaeal polar lipid composition Ability for activating DC costimulation

Immunogenicity

• • •

211

Ability to direct both antibody and cell mediated immunity Capacity to induce CD8+ T cell responses Capability of inducing long memory response

d. Cochleates. are stable phospholipid-calcium precipitates derived from the interaction of anionic lipid vesicles with divalent cations. They have a defined multi-layered structure, consisting of a lipid bilayer sheet, rolled into a spiral with a hydrophobic internal space. It is believed that the calcium ions maintain the rolled form, through ionic interaction. Antigens contained in the interior of the cochleate structure remain intact, even in harsh environments, thus making them useful for mucosal administration (Gould-Fogerite et al., 1998; Acevedo et al., 2009). e. Non-ionic surfactant vesicles (NISV). are produced in a similar manner to liposomes. However, where liposomes are considered delivery systems, NISV have inherent adjuvant properties. They are also stable in air and do not require special storage (Brewer and Alexander, 1994). They have been used with a wide variety of antigens, including those of viral and noninfectious origin (Mohamedi et al, 2000; Ferro and Stimson, 1998). They induce specific local and systemic responses, and can be modified for mucosal use (Conacher et al., 2001; Mann et al, 2004).

III. Mucosal adjuvants Mucosal immunisation offers a convenient and attractive approach to stimulating both local and systemic immunity, while targeting pathogens or conditions that require mucosal as well as systemic protection. One way of targeting the pathogens that attack the mucosa are by boosting Th17 cells, produced in the presence of IL-23 and IL-17 (Khader et al., 2009). In addition, mucosal immunisation is attractive for use in mass-vaccination programmes, such as those carried out in the developing world (Giudice and Campbell, 2006), to reduce cross-infection from contaminated needles and decontamination costs (Brody, 2004) and where self-administration (or the use of untrained medical personnel) is of cost-benefit. Mucosal administration can be achieved through many different sites although the most practical routes are intranasal and oral. However, vaccine components are easily degraded, particularly during passage through the gut and the vaccine can get diluted in the large surface of the gastrointestinal tract. The nasal mucosa is very attractive due to the absence of acidity and enzymes. Furthermore, the small surface area requires a low amount of antigen and adjuvant, with medical devices already developed for delivering drugs (eg aerosol sprayers).

212

Valerie A Ferro

The first oral vaccine was produced nearly 50 years ago, yet few mucosal vaccines exist on the market - mainly because it is difficult to stimulate strong mucosal IgA immune responses (Holmgren et al., 2005). A recent review of the market described less than 10 mucosal vaccines, most of which used live, attenuated organisms (Mann et al, 2009a). Thus the hurdle remains - achieving efficacy while maintaining safety. The goal for many researchers working on mucosal vaccines is to move away from the use of live organisms and some examples are given below.

1. Bilosomes Bilosomes are based on NISV containing bile salts to stabilise the vesicles for oral delivery as first described by Conacher et al. (2001). The concept arose from experiments designed to observe the effect of gastric and intestinal conditions on antigen contained in lipid vesicles. Bovine serum albumin and a measles peptide were used to demonstrate that deoxycholate incorporated into NISV prevented degradation and loss of antigen from the vesicles. The bilosomes induce both cell mediated (IL-2) and antibody (local and systemic) responses. A range of antigens has subsequently been examined including: influenza haemagglutinin (Mann et al., 2004), tetanus toxoid (Mann et al., 2006) and hepatitis B surface antigen (Shukula et al., 2010).

They can be made by different methods, including the use of chloroform to dissolve the lipids, followed by evaporation of the solvent to form a thin film, which can then be hydrated. This method is not suitable for industrial scale manufacture as there is an environmental impact of chloroform evaporation, as well as residual chloroform effects to be considered. Attempts were therefore made to change the protocol, using a lipid melt method, followed by homogenisation to introduce an improved manufacturing process without the need for solvent (Mann et al., 2004). A recent paper described a further attempt to reduce the number of steps required for lipid melt manufacture (Bennett et al., 2009) and demonstrates how modifications can have a profound effect on efficacy. Briefly, a 5:4:1 molar ratio of lipids 1-monopalmitoyl glycerol, cholesterol and dicetyl phosphate were melted in a round bottomed flask using an oil bath at 120 째C, then hydrated as follows. a) 3-Step method. Addition of 0.025 M carbonate buffer, pH 9.7 (preheated to 60 째C). The mixture was homogenised for 2 min, followed by addition of 10mM bile salt solution (preheated to 60 째C) and homogenised for 8 min at 8000 rpm. The resultant mixture was then cooled to 30 째C over 2 h in a water bath, followed by the addition of carbonate buffer

Immunogenicity

213

containing antigen (preheated to 30 °C) and homogenised for 1 min at 8000 rpm. b) 1-Step method. 100 mM bile salt dissolved in 0.025 M carbonate buffer, pH 9.7, and antigen (preheated to 60 °C) was added to the lipids, followed by homogenisation for 13 min at 8000 rpm, then cooled to 30 °C over 2 h in a water bath with occasional agitation.

In order to improve shelf life, both formulations were also lyophilised and characterisation of all four formulations were carried out.

Both methods produce bilosomes of approximately 150-200nm.

Lyophilisation increased the size of the vesicles 4 fold, but improved the long-term stability and antigen entrapment. However, the 1-step method was inadequate in inducing an antibody response against New Caledonian haemagglutinin. Physical measurements indicated that both the 1-step and 3-step vesicles had similar characteristics, yet efficacy was compromised.

The lipid-melt method produces a more uniform population of vesicles and would be the preferred option by regulatory authorities. However, the size and uniformity of the vesicles has been shown to influence the Th1/Th2 balance and subsequent efficacy (Mann et al., 2009b). In a ferret infection challenge model, animals were given influenza antigen in two oral bilosome formulations (chloroform film hydration and lipid melt), resulting in predominantly large or small vesicles, respectively. Efficacy was compared with an intramuscular injection of Mastaflu®.

A)

B)

Freeze fracture electron microscopy, shows bilosomes made by A) a traditional chloroform film hydration method, compared with B) a lipid-melt method. Imaging was carried out using a LEO912 energy filtering electron microscope at 80 kV.

214

Valerie A Ferro

This brief collation of experiments with respect to the bilosome demonstrates how simple modifications can be made to comply with regulatory restrictions, with significant impact on therapeutic outcomes.

Thus, when designing an adjuvant or delivery system, it is important to give early consideration to down stream events such as ease of manufacture and practicality of use in a clinical setting.

10

10

9

9

8

8

7

7

Activity 2 Activity 1 Nasal Discharge Sneezing

6 5 4

Activity 2 Activity 1 Nasal Discharge Sneezing

6 5 4

3

3

2

2

1

1

0

0

1

2

3

4

5

1

Days Post-infection

2

3

4

5

Days Post-infection

A) Chloroform film hydration: large vesicles

B) Lipid melt: small vesicles

10 9 8 7 Activity 2 Activity 1 Nasal Discharge Sneezing

6 5 4 3 2 1 0 1

2

3

4

5

Days Post-infection

C) Intramuscular injection

Each animal (n=6) was administered 45Îźg per dose (x4) and then challenged with 106.5 TCID50 virus. The symptoms were noted daily and the health scores refer to (1 point each for sneezing, purulent discharge from external nares, decreased activity or play [activity 1] and 2 points for no spontaneous activity or decreased alertness [activity 2]. This numerical score correlates to human symptoms (scale: very mild, mild, moderate or severe).

2. Polymeric technologies There are other technologies that likewise do not contain whole, albeit attenuated organisms. These target mucosal sites, such as Peyer’s patches via

Immunogenicity

215

M cells; for example by incorporating M cell-specific lectins, adhesins or immunoglobulins (Vyas and Gupta, 2007). In addition, biodegradable polymers that can aid mucoadhesion have proven invaluable. In this way, chitosan has been used to good effect in nasal delivery, not only as a slowrelease agent, but also in inducing strong immune responses (Sayin et al., 2009). Other polymers include dextran, poly (lactide-co-glycolide) and polylactic acid. Mishra et al. (2010) recently reviewed developments and progress in these polymeric systems.

3. Non-particulate technologies One innovative delivery approach to reducing manufacturing costs and improving storage and distribution has been to construct plants that express vaccine antigens. Mice fed with CT-B and LT-B expressed in raw potato induced potent serum IgG and mucosal IgA responses against CT-B/LT-B, while conferring protection from toxin challenge (Haq et al., 1995; Arakawa et al., 1998). Studies have also been carried out in humans (Tacket, 2005).

However, use of potato and maize systems showed rapid antigen degradation in the digestive tract, and consistent dosing was difficult to achieve (Tacket, 2007). On the other hand, a transgenic rice system, known as MucoRice targets M cells and protects antigen in the harsh environment of the gastrointestinal tract (Takahashi et al., 2009).

Summary, future directions Vaccination remains an important healthcare intervention, however, the emphasis needs to move to the design of improved adjuvants, with greater safety and efficacy. It is no longer acceptable for adjuvants to be tarnished with the reputation of being “the immunologist’s dirty little secret” (Woodman and Blackman, 2005). It is important that we have a better understanding of the regulation of the immune system so that adjuvants and delivery systems can be designed with a targeted approach. Thus, in the future we are more likely to see adjuvants where the mechanism of action is a major priority rather than an after thought. The “Holy Grail” in vaccination is via the mucosal route. Therefore, future directions are likely to see further progress in this field, with more products reaching the market. In recent years, a number of mucosal vaccines have reached the counter, but have as quickly disappeared due to inherent problems associated with reversion of pathogens. Continued development of technologies that do not depend on live organisms is therefore critical and increasing our understanding of mucosal immune sites is clearly going to drive the next generation of vaccines.

216

Valerie A Ferro

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Acevedo, R., Callicó, A., del Campo, J., González, E., Cedré, B., González, L., Romeu, B., Zayas, C., Lastre, M., Fernández, S., Oliva, R., García, L., Pérez, J.L., Pérez, O. (2009) Intranasal administration of proteoliposome-derived cochleates from Vibrio cholerae O1 induce mucosal and systemic immune responses in mice. Methods. 49; 309-15.

Adams, M.M., Damani, P., Perl, N.R., Won, A., Hong, F., Livingston, P.O., Ragupathi, G., Gin, D.Y. (2010) Design and synthesis of potent quillaja saponin vaccine adjuvants. J. Am. Chem. Soc. 132; 1939-45.

Alving, C.R. (2002) Design and selection of vaccine adjuvants: animal models and human trials. Vaccine. 20 Suppl 3; S56-64.

Arakawa, T., Yu, J., Chong, D.K., Hough, J., Engen, P.C., Langridge, W.H. (1998) A plant-based cholera toxin B subunit-insulin fusion protein protects against the development of autoimmune diabetes. Nat. Biotechnol. 16; 934-8.

Arca, H.C., Günbeyaz, M., Senel, S. (2009) Chitosan-based systems for the delivery of vaccine antigens. Expert Rev. Vaccines. 8; 937-53.

Awasthi, S. (2008) Next Generation of Human Vaccines: What Does the Future Hold? Hum. Vaccin., 4; 344-6.

Baldridge, J., Myers, K., Johnson, D., Persing, D., Cluff, C., Hershberg, R. (2006) Monophosphoryl Lipid A and Synthetic Lipid A Mimetics as TLR4-based Adjuvants and Immunomodulators. In Vaccine Adjuvants: Immunological and Clinical Principles, eds CJ Hackett and DA Harn, p235-255. Humana Press Inc., USA. Ballou, W.R. (2009) The development of the RTS, S malaria vaccine candidate: challenges and lessons. Parasite Immunol. 31; 492-500.

Bangham, A.D. (1972) Lipid bilayers and biomembranes. Annu. Rev. Biochem.

41; 753-76.

Barnard, D.L. (2009) Animal models for the study of influenza pathogenesis and therapy. Antiviral Res. 82; A110-22.

Bielinska, A.U., Janczak, K.W., Landers, J.J., Markovitz, D.M., Montefiori, D.C., Baker, J.R. (2008) Nasal immunization with a recombinant HIV gp120 and nanoemulsion adjuvant produces Th1 polarized responses and neutralizing antibodies to primary HIV type 1 isolates. AIDS Res. Hum. Retroviruses. 24; 271-81.

Bennett, E., Mullen, A.B., Ferro, V.A. (2009) Translational modifications to improve vaccine efficacy in an oral influenza vaccine. Methods. 49; 322-7.

Brewer, J.M. (2006) (How) do aluminium adjuvants work? Immunol. Lett. 102; 10-5.

Brewer, J.M., Alexander, J. (1994) Studies on the adjuvant activity of non-ionic surfactant vesicles: adjuvant-driven IgG2a production independent of MHC control. Vaccine. 12; 613-9.

Brody, S. (2004) Declining HIV rates in Uganda: due to cleaner needles, not abstinence or condoms. Int. J. STD. AIDS. 15; 440-1.

Brunner, R., Jensen-Jarolim, E., Pali-Schöll, I. (2010) The ABC of clinical and experimental adjuvants - a brief overview. Immunol. Lett. 128; 29-35.

Immunogenicity

217

17. Carter, K.C., Ferro, V.A., Alexander, J., Mullen, A.B. (2006) Translation of an experimental oral vaccine formulation into a commercial product. Methods. 38; 65-8.

18. Chamberlain, A., Gronvall, G.K. (2007) Immune-boosting adjuvants. Biosecur.

Bioterror. 5; 202-5.

19. Conacher, M., Alexander, J., Brewer, J.M. (2001) Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine. 19; 2965-74.

20. Cooper, P.D., Carter, M. (1986) Anticomplementary action of polymorphic “solubility forms” of particulate inulin. Mol. Immunol. 23; 895-901.

21. Cooper, P.D, Steele, E.J. (1991) Algammulin: A new vaccine adjvant comprising gamma inulin particles containing alum. Vaccine. 9; 351-7.

22. Cooper, C.L., Davis, H.L., Morris, M.L., Efler, S.M., Krieg, A.M., Li, Y., Laframboise, C., Al-Adhami, M.J., Khaliq, Y., Seguin, I., Cameron, D.W. (2004) Safety and immunogenicity of CPG 7909 injection as an adjuvant to Fluarix influenza vaccine. Vaccine. 22; 3136-43.

23. Coulson, P.S., Kariuki, T.M. (2006) Schistosome vaccine testing: lessons from the baboon model. Mem. Inst. Oswaldo Cruz. 101 Suppl 1; 369-72.

24. Cox, J.C., Coulter, A.R. (1997) Adjuvants - a classification and review of their modes of action. Vaccine. 15; 248-56.

25. Cunningham, C.K., Wara, D.W., Kang, M., Fenton, T., Hawkins, E., McNamara, J., Mofenson, L., Duliege, A.M., Francis, D., McFarland, E.J., Borkowsky, W. Pediatric AIDS Clinical Trials Group 230 Collaborators. (2001) Safety of 2 recombinant human immunodeficiency virus type 1 (HIV-1) envelope vaccines in neonates born to HIV-1-infected women. Clin. Infect. Dis. 32; 801-7.

26. De Gregorio, E., Tritto, E., Rappuoli, R. (2008) Alum adjuvanticity: unraveling a century old mystery. Eur. J. Immunol. 38; 2068-71.

27. Edelman, R. (2000) An Overview of Adjuvant Use. In Vaccine Adjuvants: Preparation Methods and Research Protocols, ed DT O’Hagan, pp1-27, Humana Press, USA. 28. EMEA 2005: Guidelines on adjuvants in vaccines for human use.

EMEA/CHMP/VEG/134716/2004.

29. EMEA 2006: Explanatory note on immunomodulators for the guideline on adjuvants in vaccines for human use. EMEA/CHMP/VWP/244894/2006.

30. Evans, T.G., McElrath, M.J., Matthews, T., Montefiori, D., Weinhold, K., Wolff, M., Keefer, M.C., Kallas, E.G., Corey, L., Gorse, G.J., Belshe, R., Graham, B.S., Spearman PW, Schwartz D, Mulligan MJ, Goepfert P, Fast P, Berman P, Powell M, Francis, D. (2001) QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans. Vaccine.

19; 2080-91.

31. Ferro, V.A., Mordini, E. (2004) Peptide vaccines in immunocontraception. Curr.

Opin. Mol. Ther. 6; 83-9.

32. Ferro, V.A., Stimson, W.H. (1998) Investigation into suitable carrier molecules for use in an anti-gonadotrophin releasing hormone vaccine. Vaccine. 16; 1095-102.

218

Valerie A Ferro

33. Fingerut, E., Gutter, B., Meir, R., Eliahoo, D., Pitcovski, J. (2005) Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast. Vaccine. 23; 4685-96.

34. Ganne, V., Eloit, M., Laval, A., Adam, M., Trouve, G. (1994) Enhancement of the efficacy of a replication-defective adenovirus-vectored vaccine by the addition of oil adjuvants. Vaccine. 12; 1190-6.

35. Garçon, N., Chomez, P., Van Mechelen, M. (2007) GlaxoSmithKline Adjuvant Systems in vaccines: concepts, achievements and perspectives. Expert Rev. Vaccines. 6; 723-39.

36. Giudice, E.L., Campbell, J.D. (2006) Needle-free vaccine delivery. Adv. Drug Deliv. Rev. 58; 68-89.

37. Glenn, G.M., Thomas, D.N., Poffenberger, K.L., Flyer, D.C., Ellingsworth, L.R., Andersen, B.H., Frech, S.A. (2009) Safety and immunogenicity of an influenza vaccine A/H5N1 (A/Vietnam/1194/2004) when coadministered with a heat-labile enterotoxin (LT) adjuvant patch. Vaccine. 27 Suppl 6; G60-6.

38. Gould-Fogerite, S., Kheiri, M.T., Zhang, F., Wang, Z., Scolpino, A.J., Feketeova, E., Canki, M., Mannino, R.J. (1998) Targeting immune response induction with cochleate and liposome-based vaccines. Adv. Drug Deliv. Rev. 32; 273-287.

39. Goto, N., Kato, H., Maeyama, J., Shibano, M., Saito, T., Yamaguchi, J., Yoshihara, S. (1997) Local tissue irritating effects and adjuvant activities of calcium phosphate and aluminium hydroxide with different physical properties.

Vaccine. 15; 1364-71.

40. Gupta, R.K., Relyveld, E.H., Lindblad, E.B., Bizzini, B., Ben-Efraim, S., Gupta, C.K. (1993) Adjuvants - a balance between toxicity and adjuvanticity. Vaccine.

11; 293-306.

41. Gupta, K., Cooper, C. (2008) A review of the role of CpG oligodeoxynucleotides as toll-like receptor 9 agonists in prophylactic and therapeutic vaccine development in infectious diseases. Drugs R D. 9; 137-45.

42. Guy, B. (2007) The perfect mix: recent progress in adjuvant research. Nat. Rev. Microbiol. 5; 505-17.

43. Haq, T.A., Mason, H.S., Clements, J.D., Arntzen, C.J. (1995) Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science. 268; 714-6.

44. Holmgren, J., Adamsson, J., Anjuère, F., Clemens, J., Czerkinsky, C., Eriksson, K., Flach, C.F., George-Chandy, A., Harandi, A.M., Lebens, M., Lehner, T., Lindblad, M., Nygren, E., Raghavan, S., Sanchez, J., Stanford, M., Sun, J.B., Svennerholm, A.M., Tengvall, S. (2005) Mucosal adjuvants and anti-infection and anti-immunopathology vaccines based on cholera toxin, cholera toxin B subunit and CpG DNA. Immunol. Lett. 97; 181-8.

45. Hölzl, M.A., Hofer, J., Steinberger, P., Pfistershammer, K., Zlabinger, G.J. (2008) Host antimicrobial proteins as endogenous immunomodulators. Immunol.

Lett. 119; 4-11.

46. Kafetzopoulos, D., Martinou, A., Bouriotis, V. (1993). Bioconversion of chitin to chitosan: Purification and characterization of chitin deacetylase from Mucor rouxii. Proc. Natl. Acad. Sci. USA. 90; 2564-8.

Immunogenicity

219

47. Karbach, J., Gnjatic, S., Bender, A., Neumann, A., Weidmann, E., Yuan, J., Ferrara, C.A., Hoffmann, E., Old, L.J., Altorki, N.K., Jäger, E. (2010) Tumorreactive CD8+ T-cell responses after vaccination with NY-ESO-1 peptide, CpG 7909 and Montanide ISA-51: association with survival. Int. J. Cancer. 126; 909-18.

48. Kennedy, R.B., Ovsyannikova, I.G., Jacobson, R.M., Poland, G.A. (2009) The immunology of smallpox vaccines. Curr. Opin. Immunol. 21; 314-20.

49. Khader, S.A., Gaffen, S.L., Kolls, J.K. (2009) Th17 cells at the crossroads of innate and adaptive immunity against infectious diseases at the mucosa. Mucosal Immunol. 2; 403-11.

50. Kornbluth, R.S., Stone, G.W. (2006) Immunostimulatory combinations: designing the next generation of vaccine adjuvants. J. Leukoc. Biol. 80; 1084-102.

51. Krishnan, L., Sprott, G.D. (2008) Archaeosome adjuvants: immunological capabilities and mechanism(s) of action. Vaccine. 26; 2043-55.

52. Lambrecht, B.N., Kool, M., Willart, M.A., Hammad, H. (2009) Mechanism of action of clinically approved adjuvants. Curr. Opin. Immunol. 21; 23-9.

53. Leenaars, P.P., Hendriksen, C.F., Koedam, M.A., Claassen, I., Claassen, E. Comparison of adjuvants for immune potentiating properties and side effects in mice. (1995) Vet. Immunol. Immunopathol. 48; 123-38.

54. Li, H., Nookala, S., Re, F. (2007) Aluminium hydroxide adjuvants activate caspase-1 and induce IL-1beta and IL-18 release. J. Immunol. 178; 5271-6.

55. Liedert, B., Bassus, S., Schneider, C.K., Kalinke, U., Löwer, J. (2007) Safety of phase I clinical trials with monoclonal antibodies in Germany - the regulatory requirements viewed in the aftermath of the TGN1412 disaster. Int. J. Clin.

Pharmacol. Ther. 45; 1-9.

56. Lindblad, E.B. (2004) Aluminium adjuvants - in retrospect and prospect.

Vaccine. 22; 3658-68.

57. Lindblad, E.B., Schonberg, N.E. (2010) Aluminium Adjuvants: Preparation, Application, Dosage and Formulation with Antigen. In Vaccine Adjuvants, pp4158, ed G Davis, Humana Press, USA. 58. Lu S. (2009) Heterologous prime-boost vaccination. Curr. Opin. Immunol. 21; 346-51.

59. Lycke, N. (2005) Targeted vaccine adjuvants based on modified cholera toxin.

Curr. Mol. Med. 5; 591-7.

60. Mann, J.F., Ferro, V.A., Mullen, A.B., Tetley, L., Mullen, M., Carter, K.C., Alexander, J., Stimson, W.H. (2004) Optimisation of a lipid based oral delivery system containing A/Panama influenza haemagglutinin. Vaccine. 22; 2425-9.

61. Mann JF, Scales HE, Shakir E, Alexander J, Carter KC, Mullen AB, Ferro VA. (2006) Oral delivery of tetanus toxoid using vesicles containing bile salts (bilosomes) induces significant systemic and mucosal immunity. Methods. 38; 90-5.

62. Mann, J.F., Acevedo, R., del Campo, J., Pérez, O., Ferro, V.A. (2009a) Delivery systems: a vaccine strategy for overcoming mucosal tolerance? Expert Rev. Vaccines. 8; 103-12.

63. Mann, J.F., Shakir, E., Carter, K.C., Mullen, A.B., Alexander, J., Ferro, V.A. (2009b) Lipid vesicle size of an oral influenza vaccine delivery vehicle

220

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

Valerie A Ferro

influences the Th1/Th2 bias in the immune response and protection against infection. Vaccine. 27; 3643-9.

Mbawuike, I., Zang, Y., Couch, R.B. (2007) Humoral and cell-mediated immune responses of humans to inactivated influenza vaccine with or without QS21 adjuvant. Vaccine. 25; 3263-9.

Maurer, T. (2007) Guinea pigs in hypersensitivity testing. Methods. 41; 48-53.

Miller, L.H., Saul, A., Mahanty, S. (2005) Revisiting Freund's incomplete adjuvant for vaccines in the developing world. Trends Parasitol. 21; 412-4.

Mishra, N., Goyal, A.K., Tiwari, S., Paliwal, R., Paliwal, S.R., Vaidya, B., Mangal, S., Gupta, M., Dube, D., Mehta, A., Vyas, S.P. (2010) Recent advances in mucosal delivery of vaccines: role of mucoadhesive/biodegradable polymeric carriers. Expert Opin. Ther. Pat. 2010 Mar 29. [Epub ahead of print].

Mohamedi, S.A., Brewer, J.M., Alexander, J., Heath, A.W., Jennings, R. (2000) Antibody responses, cytokine levels and protection of mice immunised with HSV-2 antigens formulated into NISV or ISCOM delivery systems. Vaccine. 18; 2083-94.

Morefield, G.L., Sokolovska, A., Jiang, D., HogenEsch H., Robinson, J.P., Hem, S.L. (2005) Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro. Vaccine. 23; 1588-95.

Moschos, S.A., Bramwell, V.W., Somavarapu, S., Alpar, H.O (2005).

Comparative immunomodulatory properties of a chitosan-MDP adjuvant combination following intranasal or intramuscular immunisation. Vaccine. 23; 1923-30.

Moss, R.B. (2009). Prospects for control of emerging infectious diseases with plasmid DNA vaccines. J. Immune Based Ther. Vaccines. 7; 3.

Noe, S.M., Green, M.A., Hogenesch, H., Hem, S.L. (2010) Mechanism of immunopotentiation by aluminum-containing adjuvants elucidated by the relationship between antigen retention at the inoculation site and the immune response. Vaccine. 2010 Mar 5. [Epub ahead of print].

Perrie, Y., Mohammed, A.R., Kirby, D.J., McNeil, S.E., Bramwell, V.W. (2008) Vaccine adjuvant systems: enhancing the efficacy of sub-unit protein antigens.

Int. J. Pharm. 364; 272-80.

Petrovsky, N., Aguilar, J.C. (2004) Vaccine adjuvants: current state and future trends. Immunol. Cell Biol. 82; 488-96.

Pichichero, M.E. (2008) Improving vaccine delivery using novel adjuvant systems. Hum. Vaccin. 4; 262-70.

Rajput, Z.I., Hu, S., Xiao, C., Arijo, A.G. (2007). Adjuvant effects of saponins on animal immune responses. J. Zhejiang Univ. Sci. B. 8; 153-161.

Rao, R., Squillante, E., Kim, K.H. (2007) Lipid-based cochleates: a promising formulation platform for oral and parenteral delivery of therapeutic agents. Crit.

Rev. Ther. Drug Carrier Syst. 24; 41-61.

Reed, S.G., Bertholet, S., Coler, R.N., Friede, M. (2009) New horizons in adjuvants for vaccine development. Trends Immunol. 30; 23-32.

Rimmelzwaan, G.F., Nieuwkoop, N., Brandenburg, A., Sutter, G., Beyer, W.E., Maher, D., Bates, J., Osterhaus, A.D. (2000) A randomized, double blind study in

Immunogenicity

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

221

young healthy adults comparing cell mediated and humoral immune responses induced by influenza ISCOM vaccines and conventional vaccines. Vaccine. 19; 1180-7.

Sayin, B., Somavarapu, S., Li, X.W., Sesardic, D., Senel, S., Alpar OH. (2009) TMC-MCC (N-trimethyl chitosan-mono-N-carboxymethyl chitosan) nanocomplexes for mucosal delivery of vaccines. Eur. J. Pharm. Sci. 38; 362-9.

Shahiwala, A., Amiji, M.M. (2008) Enhanced mucosal and systemic immune response with squalane oil-containing multiple emulsions upon intranasal and oral administration in mice. J. Drug Target. 16; 302-10.

Shukla A, Katare OP, Singh B, Vyas SP. (2010) M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes. Int. J. Pharm. 385; 47-52.

Skene, C.D., Sutton, P. (2006) Saponin-adjuvanted particulate vaccines for clinical use. Methods. 40; 53-9.

Song, X., Hu, S. (2009) Adjuvant activities of saponins from traditional Chinese medicinal herbs. Vaccine. 27; 4883-90.

Stephenson, I. (2005) Are we ready for pandemic influenza H5N1? Expert Rev. Vaccines. 4; 151-5.

Stevceva, L., Moniuszko, M., Ferrari, M.G. (2006) Utilizing IL-12, IL-15 and IL-7 as Mucosal Vaccine Adjuvants. Lett. Drug Des. Discov. 3; 586-592.

Sun, H.X., Xie, Y., Ye, Y.P. (2009a) Advances in saponin-based adjuvants.

Vaccine. 27; 1787-96.

Sun, H.X., Xie, Y., Ye, Y.P. (2009b) ISCOMs and ISCOMATRIX. Vaccine. 27; 4388-401.

Surquin, M., Tielemans, C.L., Kulcsár, I., Ryba, M., Vörös, P., Mat, O., Treille, S., Dhaene, M., Stolear, J-C., Kuriyakose, S.O., Leyssen, M.X., Houard, S.A. (2010) Rapid, enhanced, and persistent protection of patients with renal insufficiency by AS02(V)-adjuvanted hepatitis B vaccine. Kidney Int. 77; 247-55.

Tacket, C.O. (2005) Plant-derived vaccines against diarrheal diseases. Vaccine.

23; 1866-9.

Tacket, C.O. (2007) Plant-based vaccines against diarrheal diseases. Trans. Am. Clin. Climatol. Assoc. 118; 79-87.

Tagliabue, A., Rappuoli, R. (2008) Vaccine adjuvants: the dream becomes real.

Hum. Vaccin. 4; 347-9.

Takahashi, I., Nochi, T., Yuki, Y., Kiyono, H. (2009) New horizon of mucosal immunity and vaccines. Curr. Opin. Immunol. 21; 352-8.

Thompson, B.S., Chilton, P.M., Ward, J.R., Evans, J.T., Mitchell, T.C. (2005).

Low toxicity versions of LPS, MPL® adjuvant and RC529 are efficient adjuvants for CD4+ T cells. J Leukoc. Biol. 78; 1273-80.

Toledo, H., Baly, A., Castro, O., Resik, S., Laferté, J., Rolo, F., Navea, L., Lobaina, L., Cruz, O., Míguez, J., Serrano, T., Sierra, B., Pérez, L., Ricardo, M.E., Dubed, M., Lubián, A.L., Blanco, M., Millán, J.C., Ortega, A., Iglesias, E., Pentón, E., Martín, Z., Pérez, J., Díaz, M., Duarte, C.A. (20001) A phase I clinical trial of a multi-epitope polypeptide TAB9 combined with Montanide ISA 720 adjuvant in non-HIV-1 infected human volunteers. Vaccine. 19; 4328-36.

222

Valerie A Ferro

96. Tritto, E., Mosca, F., De Gregorio, E. (2009) Mechanism of action of licensed vaccine adjuvants. Vaccine. 27; 3331-4.

97. Vandepapelière, P., Horsmans, Y., Moris, P., Van Mechelen, M., Janssens, M., Koutsoukos, M., Van Belle, P., Clement, F., Hanon, E., Wettendorff, M. Garçon, N., Leroux-Roels, G. (2008) Vaccine adjuvant systems containing monophosphoryl lipid A and QS21 induce strong and persistent humoral and T cell responses against hepatitis B surface antigen in healthy adult volunteers.

Vaccine. 26; 1375-86.

98. Vasan, S., Schlesinger, S.J., Huang, Y., Hurley, A., Lombardo, A., Chen, Z., Than, S., Adesanya, P., Bunce, C., Boaz, M., Boyle, R., Sayeed, E., Clark, L., Dugin, D., Schmidt, C., Song, Y., Seamons, L., Dally, L., Ho, M., Smith, C., Markowitz, M., Cox, J., Gill, D.K., Gilmour, J., Keefer, M.C., Fast, P., Ho, D.D. (2010) Phase 1 safety and immunogenicity evaluation of ADVAX, a multigenic, DNA-based clade C/B' HIV-1 candidate vaccine. PLoS One. 5; e8617.

99. Vollmer, J., Krieg, A.M. (2009) Immunotherapeutic applications of CpG oligodeoxynucleotide TLR9 agonists. Adv. Drug Deliv. Rev. 61; 195-204.

100. Vyas, S.P, Gupta, P.N. (2007) Implication of nanoparticles/microparticles in mucosal vaccine delivery. Expert Rev. Vaccines. 6; 401-18.

101. Waitumbi, J.N., Anyona, S.B., Hunja, C.W., Kifude, C.M., Polhemus, M.E., Walsh, D.S., Ockenhouse, C.F., Heppner, D.G., Leach, A., Lievens, M., Ballou, W.R., Cohen, J.D., Sutherland, C.J. (2009) Impact of RTS,S/AS02(A) and RTS,S/AS01(B) on genotypes of P. falciparum in adults participating in a malaria vaccine clinical trial. PLoS One. 4; e7849.

102. WHO (2004). Guidelines on clinical evaluation of vaccines: regulatory expectations. WHO Technical Report Series No. 924.

103. Wolf, J.J., Kaplanski, C.V., Lebron, J.A. (2010) Nonclinical safety assessment of vaccines and adjuvants. Methods Mol. Biol. 626; 29-40.

104. Woodman, D.L., Blackman, M.A. (2005) Vaccine development: baring the “dirty little secret”. Nature Med. 11; 715-716.

105. Yamanaka, M., Hiramatsu, K., Hirahara, T., Okabe, T., Nakai, M., Sasaki, N., Goto, N. (1992) Pathological studies on local tissue reactions in guinea pigs and rats caused by four different adjuvants. J. Vet. Med. Sci. 54; 685-92.

106. Zhou, E.M., Afshar, A. (1995) Comparison of Freund's adjuvant and TiterMax in inducing anti-idiotype to idiotypic antibodies against pseudorabies virus antigens.

Vet. Immunol. Immunopathol. 48; 113-22.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 223-241 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

9. Nanoparticle-based vaccines Jean-Pierre Y. Scheerlinck Centre for Animal Biotechnology, Faculty of Veterinary Science The University of Melbourne, Parkville, 3010 Vic., Australia

Abstract. Most modern vaccines require the use of adjuvants for the induction of effective immune responses. There is currently a large range of adjuvants and vaccine delivery systems in development, aiming at activating antigen presenting cells. One of the most promising developments in the field is the use of nanoparticles as a vaccine delivery system. Nanoparticles have remarkable intrinsic adjuvant properties, which can be harnessed and combined with more classical adjuvant. The resulting vaccine delivery systems is very effective at inducing immune responses, while at the same time limiting the negative side effects often associated with the use of classical adjuvants on their own. Here we review the field of nanoparticle-based vaccines and focus on the possible mechanisms by which these modern adjuvants achieve their extraordinary effectiveness.

Introduction Many of the modern vaccines require the use of adjuvants to be effective.

The function of these adjuvants is to increase and/or modulate the immune response to the vaccine antigen. As more recombinant vaccines become the Correspondence/Reprint request: Dr. Jean-Pierre Y. Scheerlinck, Centre for Animal Biotechnology, Faculty of Veterinary Science, The University of Melbourne, Parkville, 3010 Vic., Australia E-mail: j.scheerlinck@unimelb.edu.au

224

Jean-Pierre Y. Scheerlinck

mainstream, new forms of adjuvants are urgently required. Indeed, many of the killed or attenuated vaccines have intrinsic adjuvant activities, while most of the recombinant vaccines need some form of adjuvant to be effective.

There are many ways in which adjuvants can modulate immune responses, and in several cases adjuvants rely on a mix of different mechanisms. These include, but are not limited to, triggering of Toll-like receptors (TLR), activating the inflammasome, acting as a depot for the antigen, and targeting specific antigen presenting cell (APC) populations involved in immune induction. Other delivery systems force the antigen into particular antigen processing pathways to induce particular types of immune responses.

In developing new adjuvants it is essential to maximise immunogenicity (i.e. the ability to induce effective immune responses to the vaccine antigen), while at the same time minimising reactogenicity (i.e. the side effects often in the form of inflammation associated with the use of adjuvants). Nanoparticles have become an increasingly researched form of vaccine adjuvant for a range of pathologies. In some cases these nanoparticles are chemically inert and therefore have a low toxicity or reactogenicity, while at the same time remaining nevertheless quite effective at inducing a wide range of cellular and humoral immune responses. In this context it is essential to remember that most vaccines aim at inducing potent immune memory responses, whether high primary immune responses are induced or not [1]. Indeed, only a small number of vaccines use concealed antigens, which are not exposed to the immune system during infection, such as the Bm86 antigen in the TickGuard series of vaccine against Boophilus microplus in cattle [2]) and as such rely on pre-existing antibodies for protection. The majority of vaccines, however, rely on the induction of a long-term immune memory that is restimulated following exposure to the vaccine antigen(s) or closely related antigens. This exposure results in an effective immune restimulation of the circulating memory cells leading to a rapid and potent immune response that controls the infection. Hence it is essential to induce potent immune responses that are resistant to manipulation by the pathogen during infection [1]. In this review recent progress in nanoparticle-based vaccines will be discusses, while larger particles in the Îźm range will only be considered for comparison purpose.

Inert nanoparticles Nanoparticles made from inert material with varying degrees of biodegradability, including lipids, polystyrene, gold, poly (D,L-lactic-coglycolic acid) (PLGA) and poly(Îł-glutamic acid) (Îł-PGA), have been shown to posses adjuvant activity. These particles, at least in part, work through the

Nanoparticle-based vaccines

225

targeting of the antigen to APCs, including dendritic cells (DC) and Langerhans cells [3]. While the mechanism of this targeting is as yet poorly understood it is clear that the precise size of the nanoparticles is critical to their effectiveness as only nanoparticles with a quite narrow size range of 4050 nm are preferentially taken up by APCs [4]. Indeed, 40-100 nm sized particles were preferentially taken up by Dec205+ dendritic cells (DCs) compared to larger 1 µm beads, which were preferred by F4/80+ macrophages in vivo [5]. However, there is also a report suggesting that larger particles (200-1000 nm) are taken up and transported to the lymph node by DCs (i.e. CD11c+, Dec205+) while smaller (20-100 nm) particles are not taken-up as well by these DCs and instead drain to the lymph node as free particles until they are taken up be a number of cells within the lymph node [6]. While at first sight this may seem contradictory, the “small particles” used by the second group are significantly smaller (20 nm) than these used previously (40-100nm). It is therefore possible that the smaller particles (20 nm) behaves in this system as “soluble antigen” rather than particles which might require a diameter of at least 40 nm to be viewed by the immune system as particles.

Interestingly, this size range (40-100 nm) corresponds to the size of many viruses and hence one can speculate that the immune system has evolved a way of specifically perceiving particles of this size as a threat [7]. Viewed in this light, size might be considered as a special case pathogen associated molecular pattern (PAMP) alongside a large list of PAMPs. These PAMPs includ amongst others CpG motives in viral/bacterial DNA [8], lipopolysaccharide (LPS), single stranded RNA or apoptotic cells that are able to provide a “danger signal” [9] and hence trigger immune responses and act as adjuvants when supplemented to vaccines.

It is still unclear where the interaction between the DCs and the nanoparticles takes place. The site of injection, generally the skin contains Langerhans cells and DCs and therefore the particles can be taken up by these cells at the site of injection. However, lymphatic capillaries in the skin are typically 10-80 μm in diameter [10] and are therefore expected to carry the nanoparticles directly to the lymph node via afferent lymph. Thus the nanoparticles have a second chance of interacting with the DCs in the draining lymph node.

One of the attractions of these adjuvants is that they rely on basic mechanisms for targeting the antigen to APCs and hence they can be used across species [11,12] and are therefore useful not only for human but also for a range of veterinary vaccines [13]. This is in contrast to other strategies relying on specific receptor interactions which may be more species specific.

Indeed, it would be unusual for example that, antibodies targeting surface

226

Jean-Pierre Y. Scheerlinck

receptors on the surface of the APCs of one species would cross-react with the surface receptor of cells from another unrelated species. Other adjuvants such as CpG-oligonucleotides (CpG-ODN) have also been shown to be species specific [14]. Hence, for each species a different reagent needs to be tested in the target species, while for inert nanoparticles this does not appear to be a requirement. Obviously, it is possible to combine receptor mediated targeting with nanoparticles use [15], but one would then have to optimise the targeting strategy for every species individually in a similar way as when no nanoparticles are used.

The skin represents an attractive surface through which to delivery vaccines [16]. However, the most superficial layer of the skin (i.e. stratum corneum), is impermeable to molecules larger than 500 Dalton [17] and limits the vaccine reaching the epidermis and APCs below it. One way to gain access is to treat the skin with cyanacrylate allowing the nanoparticles to cross the skin and be taken up by Langerhans cells (i.e. skin resident DCs).

This leads to T cell activation and effectively induction of immune responses to the nanoparticles-associated antigen. Hair follicles play an important role in this mode of vaccination by allowing the particles to get into the skin [18].

This approach was more effective with small nanoparticles (40nm) compared to larger ones (>500nm), which remained largely in the top layer of the hair follicle [18]. More recently skin application of nanoparticles has been used in humans, demonstrating the effective induction of T cell responses to an influenza vaccine [19]. This approach is quite different to the blasting of nanoparticles into the skin through the use of a gene gun, which is often used for the delivery of DNA vaccines (see section on DNA vaccine delivery below).

While inert nanoparticles do not appear to negatively influence the host, inducing immune responses with minimal reactogenicity (i.e. local tissue damage) [11,12], there is some concern that these particles would remain in the body long after delivery and that this would be seen as a long-term problem in humans. To circumvent this possible problem, biodegradable nanoparticles have been generated and tested. For example, 250 nm poly(ÎłPGA) showing promising adjuvant properties using an ovalbumin based mouse model [20].

The mechanism by which inert nanoparticles exercise their adjuvant activity is still quite speculative. Particle of sizes ranging between 20 and 200 nm are typically taken up by caveolae, clathrin-coated vesicles (pits) or their independent receptors [21]. It has been suggested that nanoparticles are able to activate NFÎşB leading to the release of pro-inflammatory cytokines [22].

However, a mix of particle sizes was used and since larger (titanium) particles are also able to activate NFÎşB, it is not possible to unequivocally conclude that the observed effect is linked to the size of particles describe

Nanoparticle-based vaccines

227

here. More recently, the inflammasome has been linked to the ability of alum to function as an adjuvant following phagocytosis [23] and the destabilisation of the phagosome [24]. The surface charge of the nanoparticles seems to play a role in the internalisation process [25,26], with positively-charged carboxylated poly-L-lysine-coated nanoparticles being more readily taken up by human DC compared to negatively charged and bovine serum albumin coated nanoparticles [27].

The ability of nanoparticles to preferentially target APC populations has been harnessed to activate natural killer T (NKT) cells [28]. In these experiments 500-1000 nm poly-lactic acid nanoparticles were combined with the potent NKT cell antigen α-galactosylceramide. In doing so the αgalactosylceramide was presented to the NKT cells by CD11c+ DCs at the expense of presentation by B cells leading to NKT cell activation instead of anergy. Thus by targeting the antigen to specific APCs the outcome of the immunisation protocol was dramatically altered. This example also illustrates how nanoparticles can be modified to incorporate additional immunostimulatory signals. This is considered in the next section.

Nanoparticles incorporating known immuno-stimulatory signals While nanoparticles, by virtue of their size, are able to stimulate immune responses, there is a wide range of additional modifications to these nanoparticles aimed at improving their effectiveness and/or changing the characteristics of the immune response induced.

Synthetic particles with additional immuno-stimulatory signals Inert biocompatible particles such as PLGA particles can be used on their own as vaccine delivery devices by virtue of their ability to protect the antigen from degradation. However, they are much more effective when modified to incorporate “danger signals”. In a recent study ~200 nm PLGA particles modified with LPS have been shown to be a more effective adjuvant compared to the inert particles on their own [29]. The enhanced adjuvant activity has been linked to the activation of the inflammasome leading to the production of IL-1β in a similar way as proposed for Alum [23].

Dendrimers are hyper-branched polymers with a well defined structure which increase in size with each new branching generation [30]. While generally small, with a size approximating a large protein, it is possible to obtain particles of about 10 nm in about 10 branching generations [31]. As such they bridge the size gap between soluble proteins and nanoparticles.

228

Jean-Pierre Y. Scheerlinck

Dendrimers are generally considered inert and do not have intrinsic adjuvant properties [30], possibly due to their small size compared to the inert nanoparticles discussed previously, which are typically 50-200 nm in size and have adjuvant activity though their ability to target the antigen to DCs and possibly activate DCs following uptake. As such these dendrimers have been used mostly in combination with conventional adjuvants. However, dendrimers allow the combination of peptides, carbohydrates and immunostimulatory molecules [30] and are therefore considered in this section. One such method is to incorporate lipids into the construct using a lipid polylysine core peptide, which has the effect of increasing the immunogenicity of the peptides [32,33]. The mechanism leading to increase immunogenicity is unknown but one could speculate that the lipid part of the construct would lead the small lipid polylysine core peptide conjugated to the antigens to aggregate into larger complexes. It is also possible to introduce bacterial lipoprotein structures that interact with TLR 2 [34].

ISCOMs and ISCOM-based adjuvants Quil A is a component derived from the bark of the Quillaja saponaria tree well known for its adjuvant properties. Components derived from Quil A can readily be formulated with phospholipids and cholesterol to form hollow particles with a diameter of approximately 40 nm. Antigen with a hydrophobic substructure can be incorporated into these nanoparticles to form immuno-stimulating complexes (ISCOMs) [35]. The precise immunological characteristics of these ISCOMs can be changed by modifying the sub-fractions of Quil A used [36].

One of the challenges in formulating ISCOMs is the requirement for hydrophobic substructures in the antigen incorporated into the nanoparticles.

This requirement is necessary in order for the antigen to intercalate into the hydrophobic core of the nanoparticles composed of cholesterol and phospholipids, in a similar way to membrane associated proteins. This represents a challenge as many proteins with hydrophobic substructures are difficult to express as recombinant proteins and/or are insoluble, making the production of these antigens often a difficult task. Many different strategies have been developed to address this problem by using soluble proteins and adding linkers to incorporate the antigen into the ISCOMs (reviewed in [7]).

Alternatively, the antigen can also simply be mixed with preformed “empty� (i.e. antigen-free) ISCOMs refer to as ISCOMATRIXTM. In such a situation the ISCOMATRIXTM is not bound to the antigen, one can therefore expect that at least some adjuvant will influence cells that are not exposed to the antigen. Nevertheless, ISCOMATRIXTM is able to promote the production of

Nanoparticle-based vaccines

229

humoral and T helper immune responses to antigen mixed with it. However, at least for some antigens the ability to induce cytotoxic immunity is largely compromised, leading to the speculation that in the cases where cytotoxic immunity is induced there is a stronger association between the ISCOMATRIXTM adjuvant and the antigen due to the physicochemical properties of the antigen [37]. Such an association would occur post-mixing of the antigen and the ISCOMATRIXTM and is not easily assessed particularly in vivo. However, in the absence of such an association it is difficult to envisage how the antigen can enter the MHC class I pathway, which is a pre-requisite for the induction of cytotoxic T cells.

Virus-like particles Virus-like particles (VLPs) are self-assembling nanoparticles composed of one or several recombinant viral proteins expressed in vitro resulting in the formation of non-infective nanoparticles of 20 nm to 100 nm in size.

Currently VLPs-based vaccines on the market include the Hepatitis B vaccine and the human papilloma virus (HPV) L1 vaccine, with many others in various stages of development [38]. The Hepatitis B vaccine is composed of the surface antigen of this virus (HBsAg), which was more recently supplemented with the additional preS1 and preS2 sequences.

VLPs are listed in this section rather than in the section dealing with inert nanoparticles because they are composed of a complex mixture of compounds that arguably have the potential to be immuno-stimulatory even if no co-stimulatory molecules are added during their preparation. Some of these components include proteins and lipoproteins derived from the expression system in which the recombinant viral proteins making up the VLPs, are expressed [38]. Thus differences in the properties of VLPs can result through the expression of the viral proteins in different expression systems. Due to their ability to produce large amounts of recombinant proteins at a relatively low cost and their ability to glycosilate the expressed proteins, large scale in vitro culture expression systems using yeast, baculovirus and mammalian cells remain popular. More recently, plant expression systems are also being used [39] and VLPs produced by tobacco plants have been shown to be immunogenic following oral delivery [40].

While the VLP-based vaccines on the market are purified prior to injection, the ability to deliver a VLP-vaccine orally opens the door to using unpurified plant extracts expressing the VLPs. Indeed, while at least theoretically there is no need for purification of these plant-derived VLPs because the impurities could be digested, it remains a major challenge to assure quality control and consistency without purification steps, and the

230

Jean-Pierre Y. Scheerlinck

regulatory authorities controlling human use of vaccine are likely to, at least initially, require strict quality control measures that would be difficult to achieve without purification of the VLPs. In the veterinary field these controls are much less restrictive and the vaccines are much more pricesensitive suggesting that un-purified plant derived vaccines might first be implemented for veterinary vaccines.

In addition, there is some evidence that at least some viral proteins (for example influenza hemagglutinin, [41]) are immuno-stimulatory, and since VLPs by definition contain viral proteins it is possible that in some cases these would act (even if unintentionally) as adjuvants. The ability to express several proteins at the same time and have them incorporated into the same VLPs [42], allows for the production of VLPs not only containing several antigens, but also to add proteins with known immuno-stimulatory properties.

These immuno-stimulatory molecules can be derived from pathogens such as influenza hemagglutinin or cholera toxin B [43]. Thus immunogenic semisynthetic 150 nm nanoparticles can be produced by mixing viral proteins such as influenza hemagglutinin with lipids and antigens derived from other pathogens [44]. Such virosomes take advantage of the receptor binding and immunological properties of the hemagglutinin to generate potent immune response and have been used in humans [41]. They can also be derived from the immune system of the organism being vaccinated, with immunoregulatory molecules such as granulocyte-macrophage colony stimulating factor (GM-CSF) and CD40L significantly enhancing the immunogenicity of the VLPs in which they are incorporated [45].

Nanoparticles for DNA vaccine delivery The use of nanoparticles for the delivery of DNA vaccines has a long history with the early discovery that, DNA-coated gold nanoparticles can be used for the ballistic delivery of these vaccines through the skin using a genegun. The particle size of the nanoparticles used for this application is typically larger than 600 nm in diameter with one of the most widely used sizes being 1000nm (i.e. right at the upper limit of the nanoparticle range).

Ballistic delivery of nanoparticles The ballistic delivery of DNA through gold coated nanoparticles has been successful in a wide range of species including mice [46], cattle [47], sheep [48,49], guinea-pigs [50], horses [51], pigs [52] and primates [53]. The level of expression of the DNA vaccine in the skin of different species was variable [54] and it is therefore not clear that extrapolation of DNA

Nanoparticle-based vaccines

231

vaccination protocols between species, even relatively genetically related species, is possible [53]. In most cases where different species were used the delivery parameters were optimised for each species as variations between species are common although not necessarily large. While not many studies compare the different routes of DNA delivery it appears that the optimal route for DNA vaccination could be species-dependent. For example, it is widely recognised that gene-gun delivery of the DNA is very effective in rodents, while in sheep intra-muscular DNA immunisation was found to be more effective in a side-by-side comparison [48]. In large animals it was often found that DNA vaccination following gene-gun delivery (or indeed many other routes of delivery) induced poor primary immune responses.

However, there was clear evidence that the immune memory was primed since a subsequent protein boost (or infective challenge) resulted in a rapid and enhanced immune response. However, as noted previously, for most vaccines the presence of an effective immune memory response that can be restimulated upon subsequent encounter with the antigen during infection is critical to the effectiveness of the vaccination strategy rather than the presence of high primary immune responses [1].

More recently, modified PLGA nanoparticles have been used as a delivery system for DNA to the skin following gene-gun delivery [55]. These particles are generally smaller and can be modified to contain nanodots so that they can more easily be tracked in vivo, showing that they are present in Langerhans cells and that they migrate to the lymph node.

Injected nanoparticles Beside the use of nano- or microparticles for the ballistic delivery of DNA vaccines using a gene-gun or similar device, nanoparticles have also been used to augment the delivery of DNA vaccines injected into the skin.

Indeed, DNA vaccines combined with 128 nm cationic nanoparticles resulted in a 250 times increase in the IgG titre to the encoded antigen compared to the delivery of the DNA vaccine without the nanoparticles [56]. In another study, poly-L-lysine coated nanoparticles were complexed with the DNA vaccine resulting in the production of antigen-specific immune responses that included immunity to an antigen expressed by tumor cells, leading to their rejection in a mouse model [26]. Another approach has been to use ~300 nm cationized gelatin coated nanoparticles to deliver immuno-stimulatory CpG-ODN, thus combining the adjuvant properties of the nanoparticles with these of the CpG-ODN. The CpG-ODN from classes B and C associated with nanoparticles also increased interferon-Îą in primary human plasmacytoid DCs [57].

232

Jean-Pierre Y. Scheerlinck

Nanoparticles are also used to protect the DNA vaccines from degradation by encapsulating the plasmid DNA in poly (D,L-lactic-coglycolic acid) also referred to as PLGA and widely used in human surgical applications [58]. The resulting vaccine delivery system not only protects the vaccine from degradation but also favours uptake by macrophages. As a result these macrophages now express genes encoded by the encapsulated DNA [59]. Another way of protecting the DNA is to use stabilized plasmid lipid particles (SPLP), which are lipid based 140nm sized neutral charge particles that are taken up by APCs [60]. Liposomal nanoparticle (LN) can also be used to encapsulate CpG-ODN. The CpG-ODN in these nanoparticles acts as an adjuvant by activating the APCs, presumably through TLR9, while the SPLP delivers the DNA and transfects these cells to express the encoded antigen. The result is a dramatic increase of the immune response following intravenous administration of SPLP and LN CpG-ODN [60].

An interesting development is the combination of DNA vaccines delivered as a DNA tattoo (i.e. using a an oscillating multi-needle tattoo device to deliver the DNA to the skin) with DNA complexed with cationic DOTAP–DOPE liposomes or with cationic poly(amidoamine) to form lipoplex and polyplex nanoparticles [61]. In this study the authors found that while the lipoplex and polyplex nanoparticles were effective at transfecting cells in vitro, this effectiveness did not translate in vivo. However, they were able to dramatically improve the in vivo results by neutralising the positive charge of the nanoparticles using PEGylation. They speculate that the positively charged nanoparticles interact with the negatively charged extracellular matrix preventing the DNA coated nanoparticles from reaching their targets in vivo [61].

In a combination of DNA delivery system and a VLP, DNA virosomes have been produced by solubilising the virus with dicaproylphosphatidylcholine and incorporating the DNA using cationic lipids resulted in the production of particles that protected the DNA from nucleases [62].

Nanoparticles for vaccine delivery to mucosal surfaces The induction of immune responses at mucosal surfaces is a rapidly expanding area of research. Indeed, a large number of infections enter the body through mucosal surfaces, which form the majority of the exposed surfaces in the host. As a result, if one is able to block this early entry of the pathogen into the body, infection would be prevented early on. Such early intervention is extremely beneficial as it prevents the pathogen from being established and therefore limits its opportunity to proliferate and hence become more difficult to eradicate. However, the induction of mucosal

Nanoparticle-based vaccines

233

immune responses remains difficult and the use of nanoparticles for this purpose has been investigated at a number of levels.

ISCOMs were discussed above as a form of nanoparticles supplemented with immuno-stimulatory components purified from Quil A. These ISCOMs are very immunogenic when delivered through injection and are also effective at inducing immune responses following oral delivery [63]. It has been proposed that the induction of immunity to the orally delivered vaccine adjuvanted by ISCOMs, as opposed to the induction of tolerance as is often the case following oral delivery, is the result of DC activation in the mesenteric lymph nodes. The DC-activation can be a consequence of the particulate nature of the ISCOMs and/or the Quil A components and provides the necessary signals for immune induction [63].

More recently, ISCOMATRIXTM was mixed with antigen and delivered directly to the lung of experimental sheep [64]. In these experiments it was possible to induce strong mucosal and systemic immune responses using minute amounts of influenza antigen. In the absence of the adjuvant no such responses were observed suggesting that even though influenza has intrinsic adjuvant activities (and in humans is currently used un-adjuvanted), the addition of nanoparticles-based adjuvants had a dramatic effect. In a followup study it was also demonstrated that this effect is not limited to influenza antigens but was also present when recombinant antigens from cytomegalovirus and Helicobacter pylori were used [65].

Nanoparticles produced from poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) and designated as PLA-PEG have been delivered to the nasal cavity of rats and shown to be efficiently taken up, as they were recovered in many organs, but mostly in the lymph nodes [66]. The smaller (i.e. 200 nm) particles were more effectively taken-up compared to larger particles.

However, it is also likely that some of the antigen recovered in the various organs is the result of uptake through the airways and the digestive systems, since nasal delivery as performed by the authors, does not limit the spread of the preparation to other organs and only a small fraction of antigen may be taken up into the nasal mucosa. Indeed, in a sheep model where antigen uptake in the lymphatic system was measured directly, it was found that only a very small proportion of the antigen migrates through the mucosa into the lymphatics and the majority of the antigen is swallowed [67]. In a more recent study more sophisticated 100nm PLA-PEG nanoparticles were shown to be effective at inducing immune responses to hepatitis B antigen following nasal delivery [68]. However, here also the authors could not determine the relative contribution of oral versus nasal components of the observed effect, as nasal delivery will inevitably also result in some of the preparation being swallowed [67]. In this context it is worthwhile noting that the delivery of these particles via oral route [69], yielded results virtually identical to these previously

234

Jean-Pierre Y. Scheerlinck

published by the same group following intranasal delivery [68]. Thus the results obtained in the first study could just as easily be explained by the oral spill-over of the vaccine into the gastrointestinal system following intranasal delivery, highlighting the difficulties in interpreting nasal delivery studies.

Another advantage of using nanoparticles for the delivery of DNA vaccines is that these particles can protect the DNA from degradation allowing for oral delivery. For example, 289 to 500 nm nanoparticles coated with amino-pegylated poly(methyl vinyl ether-co-maleic anhydride) associated with DNA were well tolerated following oral delivery. In addition, these nanoparticles crossed the cellular membrane of the gastrointestinal mucosa and allowed for the delivery of intact DNA [70].

Immune responses induced by exosomes Exosomes are small (50-90 nm) particles produced by a range of cells [71], including follicular DCs [72] and platelets [73], composed of lipids and proteins. While the precise role of these nanoparticles is yet to be elucidated they seem to be involved in protein sorting, including the release of unnecessary proteins during reticulocyte maturation, and play a role in the regulation of immune responses [74]. Indeed, in vivo derived exosomes containing tumour antigens induce antigen-specific immune response with anti-tumor activity, suggesting that antigens associated with exosomes are very immunogenic [75]. Exosomes can be recovered from a wide range of body fluids including urine [76], malignant tumor effusions [77], blood [78], afferent lymph and cerebral fluid [79]. As such they could also play a role in the communication between cells at distal location [78], a function that is not easily performed by many of the other immune regulatory mechanisms.

One of the interesting aspects of exosomes is that they can contain antigen that have been processed by DCs. For example, in an early study it was shown that Toxoplasma gondii-pulsed DCs produce exosomes that contain antigen and are able to induce both cellular and humoral immune responses [80]. In addition, peptides can be delivered to DCs via exosomes which appear to effectively take-up these particles [81] and peptide/MHC complexes within exosomes have been shown to effectively induce MHC restricted immune responses [82].

Exosomes have therefore, now been recognised as potent inducers of immune responses, and can be considered as sophisticated adjuvants. These properties have been harnessed for the treatment of tumors with promising clinical trials results in melanoma patients [83]. More recently the immunogenic effects of exosomes have been further improved by the addition of cytokines such as GM-CSF [84]. Interestingly, more recent

Nanoparticle-based vaccines

235

advances have demonstrated that exosomes are also able to induce NK cell activation hereby explaining earlier results suggesting that an T cellindependent tumor regression is possible [85]. Thus exosomes can induce both MHC restricted and non-MHC restricted immune responses in vivo.

Concluding remarks The use of nanoparticles as adjuvants and vaccine delivery systems has received much attention in the recent past due to the combination of the generally low reactogenicity and high immunogenicity of these adjuvants. The size of the nanoparticles seems to play a critical role in targeting the antigen to specific cell populations contributing to the immunogenicity. At the same time, the ability to further modulate the immune responses has been explored by including immuno-modulators ranging from classical adjuvants to specific TLR activators to cytokines. These immuno-modulators are therefore also targeted to the most potent APCs allowing them to induce effective immune responses. Fig 1 Intrinsic adjuvant activity

Size-mediated DC targeting Size as a PAMP

Antigen Inflammasome activation

Nanoparticle (10-1000 nm)

Antigen with adjuvant properties TLR activation

Antigen

Quil A Immunomodulator Combination with classical adjuvants

Cytokines and immune mediators

Antigen-presentation and cellular communication network Antigen Antigen presenting cell

Antigen-containing exosomes

Figure 1. Schematic representation of different forms of nanoparticle-based vaccines and their proposed mode of action. Nanoparticles have intrinsic adjuvant activity which can be supplemented by a number of additional more classical adjuvants.

Exosomes produced by antigen presenting cells can also act as vaccine delivery systems particularly for therapeutic vaccines requiring immune responses against weak antigens such as cancer derived molecules.

236

Jean-Pierre Y. Scheerlinck

shows a schematic of the different forms of nanoparticle-based vaccine delivery systems and their proposed mode of action.

Acknowledgement JPS is supported by a research grant of the Australian Research Council.

References 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

Scheerlinck, J.-P.Y. and Yen, H.H. (2010) Defining immune memory resilience: implications for vaccine development. Expert Rev Vaccines 9 (4), 351-353.

Willadsen, P. and Kemp, D.H. (1988) Vaccination with 'concealed' antigens for tick control. Parasitol Today 4 (7), 196-198.

Mottram, P.L. et al. (2007) Type 1 and 2 Immunity Following Vaccination Is Influenced by Nanoparticle Size: Formulation of a Model Vaccine for Respiratory Syncytial Virus. Mol Pharm 4 (1), 73-84.

Fifis, T. et al. (2004) Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol 173 (5), 3148-3154.

Xiang, S.D. et al. (2006) Pathogen recognition and development of particulate vaccines: does size matter? Methods 40 (1), 1-9.

Manolova, V. et al. (2008) Nanoparticles target distinct dendritic cell populations according to their size. Eur J Immunol 38 (5), 1404-1413.

Scheerlinck, J.P. and Greenwood, D.L. (2008) Virus-sized vaccine delivery systems. Drug Discov Today.13 (19-20), 882-887.

Klinman, D.M. et al. (1996) CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc Natl Acad Sci U S A 93 (7), 2879-2883.

Gallucci, S. and Matzinger, P. (2001) Danger signals: SOS to the immune system. Current Opinion in Immunology 13 (1), 114-119.

Swartz, M.A. et al. (2008) Lymphatic drainage function and its immunological implications: From dendritic cell homing to vaccine design. Semin Immunol 20 (2), 147-156.

Scheerlinck, J.P. et al. (2006) Systemic immune responses in sheep, induced by a novel nano-bead adjuvant. Vaccine 24 (8), 1124-1131.

Greenwood, D.L. et al. (2008) Vaccination against foot-and-mouth disease virus using peptides conjugated to nano-beads. Vaccine 26 (22), 2706-2713.

Scheerlinck, J.P. and Greenwood, D.L. (2006) Particulate delivery systems for animal vaccines. Methods 40 (1), 118-124.

Chaung, H.C. (2006) CpG oligodeoxynucleotides as DNA adjuvants in vertebrates and their applications in immunotherapy. Int Immunopharmacol 6 (10), 1586-1596.

Nanoparticle-based vaccines

237

15. Klippstein, R. and Pozo, D. (2010) Nanotechnology-based manipulation of dendritic cells for enhanced immunotherapy strategies. Nanomedicine. 6 (4), 523-529.

16. Combadiere, B. and Mahe, B. (2008) Particle-based vaccines for transcutaneous vaccination. Comp Immunol Microbiol Infect Dis 31 (2-3), 293-315.

17. Bos, J.D. and Meinardi, M.M. (2000) The 500 Dalton rule for the skin penetration of chemical compounds and drugs. Exp Dermatol 9 (3), 165-169.

18. Vogt, A. et al. (2006) 40 nm, but not 750 or 1,500 nm, nanoparticles enter epidermal CD1a+ cells after transcutaneous application on human skin. J Invest Dermatol 126 (6), 1316-1322.

19. Vogt, A. et al. (2008) Transcutaneous anti-influenza vaccination promotes both CD4 and CD8 T cell immune responses in humans. J Immunol 180 (3), 1482-1489.

20. Uto, T. et al. (2009) Modulation of innate and adaptive immunity by biodegradable nanoparticles. Immunol Lett 125 (1), 46-52.

21. Pelkmans, L. (2005) Secrets of caveolae- and lipid raft-mediated endocytosis revealed by mammalian viruses. Biochim Biophys Acta 1746 (3), 295-304 22. Zhou, Y.M. et al. (2003) Oxidative stress and NFkappaB activation in the lungs of rats: a synergistic interaction between soot and iron particles. Toxicol Appl Pharmacol 190 (2), 157-169.

23. Eisenbarth, S.C. et al. (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453 (7198), 1122-1126.

24. Hornung, V. et al. (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol 9 (8), 847-856.

25. Josephson, L. et al. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates. Bioconjug Chem 10 (2), 186191.

26. Minigo, G. et al. (2007) Poly-l-lysine-coated nanoparticles: A potent delivery system to enhance DNA vaccine efficacy. Vaccine 25 (7), 1316-1327.

27. Thiele, L. et al. (2001) Evaluation of particle uptake in human blood monocytederived cells in vitro. Does phagocytosis activity of dendritic cells measure up with macrophages? J Control Release 76 (1-2), 59-71.

28. Thapa, P. et al. (2009) Nanoparticle formulated alpha-galactosylceramide activates NKT cells without inducing anergy. Vaccine 27 (25-26), 3484-3488.

29. Demento, S.L. et al. (2009) Inflammasome-activating nanoparticles as modular systems for optimizing vaccine efficacy. Vaccine 27 (23), 3013-3021.

30. Heegaard, P.M. et al. (2009) Dendrimers for Vaccine and Immunostimulatory Uses. A Review. Bioconjug Chem. 21 (3), 405-418.

31. D'Emanuele, A. et al. (2006) Dendrimers. Encyclopedia of Pharmaceutical Technology: Third Edition, 872 - 890.

238

Jean-Pierre Y. Scheerlinck

32. Olive, C. et al. (2003) Potential of lipid core peptide technology as a novel selfadjuvanting vaccine delivery system for multiple different synthetic peptide immunogens. Infect Immun 71 (5), 2373-2383.

33. Olive, C. et al. (2005) Protection against group A streptococcal infection by vaccination with self-adjuvanting lipid core M protein peptides. Vaccine 23 (17-18), 2298-2303.

34. Spohn, R. et al. (2004) Synthetic lipopeptide adjuvants and Toll-like receptor 2-structure-activity relationships. Vaccine 22 (19), 2494-2499.

35. Morein, B. et al. (1984) Iscom, a novel structure for antigenic presentation of membrane proteins from enveloped viruses. Nature 308 (5958), 457-460.

36. Hu, K.F. et al. (2005) The immunomodulating properties of human respiratory syncytial virus and immunostimulating complexes containing Quillaja saponin components QH-A, QH-C and ISCOPREP703. FEMS Immunol Med Microbiol 43 (2), 269-276.

37. Pearse, M.J. and Drane, D. (2004) ISCOMATRIX adjuvant: a potent inducer of humoral and cellular immune responses. Vaccine 22 (19), 2391-2395.

38. Grgacic, E.V. and Anderson, D.A. (2006) Virus-like particles: passport to immune recognition. Methods 40 (1), 60-65.

39. Santi, L. et al. (2006) Virus-like particles production in green plants. Methods 40 (1), 66-76.

40. Santi, L. et al. (2008) An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles. Vaccine 26 (15), 1846-1854.

41. Moser, C. et al. (2007) Influenza virosomes as a combined vaccine carrier and adjuvant system for prophylactic and therapeutic immunizations. Expert Rev Vaccines 6 (5), 711-721.

42. Vieira, H.L. et al. (2005) Triple layered rotavirus VLP production: kinetics of vector replication, mRNA stability and recombinant protein production. J Biotechnol 120 (1), 72-82.

43. Ludwig, C. and Wagner, R. (2007) Virus-like particles-universal molecular toolboxes. Curr Opin Biotechnol 18 (6), 537-545.

44. Gluck, R. et al. (2005) Adjuvant and antigen delivery properties of virosomes.

Curr Drug Deliv 2 (4), 395-400.

45. Skountzou, I. et al. (2007) Incorporation of glycosylphosphatidylinositolanchored granulocyte- macrophage colony-stimulating factor or CD40 ligand enhances immunogenicity of chimeric simian immunodeficiency virus-like particles. J Virol 81 (3), 1083-1094.

46. Bennett, A.M. et al. (1999) Gene gun mediated vaccination is superior to manual delivery for immunisation with DNA vaccines expressing protective antigens from Yersinia pestis or Venezuelan Equine Encephalitis virus. Vaccine 18 (7-8), 588-596.

47. Braun, R.P. et al. (1999) Particle-mediated DNA immunization of cattle confers long-lasting immunity against bovine herpesvirus-1. Virology 265 (1), 46-56.

Nanoparticle-based vaccines

239

48. De Rose, R. et al. (2002) Efficacy of DNA vaccination by different routes of immunisation in sheep. Vet Immunol and Immunopathol 90 (1-2), 55-63.

49. Niesalla, H. et al. (2009) Systemic DNA immunization against ovine lentivirus using particle-mediated epidermal delivery and modified vaccinia Ankara encoding the gag and/or env genes. Vaccine 27 (2), 260-269.

50. Oliveira, E. et al. (2005) Analysis of the immune response against mixotope peptide libraries from a main antigenic site of foot-and-mouth disease virus.

Vaccine 23 (20), 2647-2657.

51. Lunn, D.P. et al. (1999) Antibody responses to DNA vaccination of horses using the influenza virus hemagglutinin gene. Vaccine 17 (18), 2245-2258.

52. Olsen, C.W. (2000) DNA vaccination against influenza viruses: a review with emphasis on equine and swine influenza. Vet Microbiol 74 (1-2), 149-164.

53. McCluskie, M.J. et al. (1999) Route and method of delivery of DNA vaccine influence immune responses in mice and non-human primates. Mol Med 5 (5), 287-300.

54. Hengge, U.R. et al. (1996) Expression of naked DNA in human, pig, and mouse skin. J Clin Invest 97 (12), 2911-2916.

55. Lee, P.W. et al. (2010) Multifunctional core-shell polymeric nanoparticles for transdermal DNA delivery and epidermal Langerhans cells tracking.

Biomaterials 31 (8), 2425-2434.

56. Cui, Z. and Mumper, R.J. (2003) The effect of co-administration of adjuvants with a nanoparticle-based genetic vaccine delivery system on the resulting immune responses. Eur J Pharm Biopharm 55 (1), 11-18.

57. Zwiorek, K. et al. (2008) Delivery by cationic gelatin nanoparticles strongly increases the immunostimulatory effects of CpG oligonucleotides. Pharm Res 25 (3), 551-562.

58. Greenland, J.R. and Letvin, N.L. (2007) Chemical adjuvants for plasmid DNA vaccines. Vaccine 25 (19), 3731-3741.

59. Mok, H. and Park, T.G. (2008) Direct plasmid DNA encapsulation within PLGA nanospheres by single oil-in-water emulsion method. Eur J Pharm Biopharm 68 (1), 105-111.

60. Wilson, K.D. et al. (2009) The combination of stabilized plasmid lipid particles and lipid nanoparticle encapsulated CpG containing oligodeoxynucleotides as a systemic genetic vaccine. J Gene Med 11 (1), 14-25.

61. van den Berg, J.H. et al. (2010) Shielding the cationic charge of nanoparticleformulated dermal DNA vaccines is essential for antigen expression and immunogenicity. J Control Release 141 (2), 234-240.

62. de Jonge, J. et al. (2007) Cellular gene transfer mediated by influenza virosomes with encapsulated plasmid DNA. Biochem J 405 (1), 41-49.

63. Mowat, A.M. (2005) Dendritic cells and immune responses to orally administered antigens. Vaccine 23 (15), 1797-1799.

240

Jean-Pierre Y. Scheerlinck

64. Wee, J.L. et al. (2008) Pulmonary delivery of ISCOMATRIX influenza vaccine induces both systemic and mucosal immunity with antigen dose sparing. Mucosal Immunol 1 (6), 489-496.

65. Vujanic, A. et al. Combined mucosal and systemic immunity following pulmonary delivery of ISCOMATRIX adjuvanted recombinant antigens. Vaccine 28 (14), 2593-2597.

66. Vila, A. et al. (2005) PLA-PEG particles as nasal protein carriers: the influence of the particle size. Int J Pharm 292 (1-2), 43-52.

67. Yen, H.H. et al. (2006) A sheep cannulation model for evaluation of nasal vaccine delivery. Methods 38 (2), 117-123.

68. Jain, A.K. et al. (2009) Synthesis, characterization and evaluation of novel triblock copolymer based nanoparticles for vaccine delivery against hepatitis B. J Control Release 136 (2), 161-169.

69. Jain, A.K. et al. (2010) PEG-PLA-PEG block copolymeric nanoparticles for oral immunization against hepatitis B. Int J Pharm 387 (1-2), 253-262.

70. Yoncheva, K. et al. (2008) Development of bioadhesive amino-pegylated poly(anhydride) nanoparticles designed for oral DNA delivery. J Microencapsul 25 (2), 82-89.

71. Thery, C. et al. (2001) Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. J Immunol 166 (12), 7309-7318.

72. Denzer, K. et al. (2000) Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface. J Immunol 165 (3), 1259-1265.

73. Robertson, C. et al. (2006) Cellular prion protein is released on exosomes from activated platelets. Blood 107 (10), 3907-3911 74. de Gassart, A. et al. (2003) Lipid raft-associated protein sorting in exosomes.

Blood 102 (13), 4336-4344 75. Zeelenberg, I.S. et al. (2008) Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses. Cancer Res 68 (4), 1228-1235 76. Pisitkun, T. et al. (2004) Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci U S A 101 (36), 13368-13373.

77. Andre, F. et al. (2002) Malignant effusions and immunogenic tumour-derived exosomes. Lancet 360 (9329), 295-305.

78. Caby, M.P. et al. (2005) Exosomal-like vesicles are present in human blood plasma. Int Immunol 17 (7), 879-887.

79. Vella, L.J. et al. (2008) Enrichment of prion protein in exosomes derived from ovine cerebral spinal fluid. Vet Immunol Immunopathol 124 (3-4), 385-393.

80. Aline, F. et al. (2004) Toxoplasma gondii antigen-pulsed-dendritic cell-derived exosomes induce a protective immune response against T. gondii infection. Infect Immun 72 (7), 4127-4137.

Nanoparticle-based vaccines

241

81. Montecalvo, A. et al. (2008) Exosomes As a Short-Range Mechanism to Spread Alloantigen between Dendritic Cells during T Cell Allorecognition. J Immunol 180 (5), 3081-3090.

82. Andre, F. et al. (2004) Exosomes as potent cell-free peptide-based vaccine. I. Dendritic cell-derived exosomes transfer functional MHC class I/peptide complexes to dendritic cells. J Immunol 172 (4), 2126-2136.

83. Escudier, B. et al. (2005) Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial. J Transl Med 3 (1), 10.

84. Dai, S. et al. (2008) Phase I clinical trial of autologous ascites-derived exosomes combined with GM-CSF for colorectal cancer. Mol Ther 16 (4), 782-790.

85. Viaud, S. et al. (2009) Dendritic cell-derived exosomes promote natural killer cell activation and proliferation: a role for NKG2D ligands and IL-15Ralpha. PLoS One 4 (3), e4942.

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 243-255 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

10. Molecular pharming for plant-derived vaccines Kathleen Hefferon Cornell Research Foundation, Cornell University, Ithaca, NY 14850, USA

Abstract. Vaccine proteins and other biopharmaceuticals can be quickly and inexpensively generated in whole plants or plant tissue culture. Plant-derived vaccines negate any concern with regard to biological contamination which arise in animal cell culture systems, and present new hope for developing countries, where poor medical infrastructure, lack of needles or refrigeration and high cost make administration of vaccines much more difficult. In addition to this, proteins derived from plants have a select advantage over proteins produced in bacterial expression systems due to their ability to undergo post-translational modifications.

This chapter reviews the current status of molecular pharming in plants with particular emphasis on the potential impact for developing countries. Examples of vaccine proteins which have been produced in plants and the results of their respective preliminary clinical trials are described. The chapter concludes with a description of the steps involved in the large scale production of vaccines and other therapeutic proteins.

Correspondence/Reprint request: Dr. Kathleen Hefferon, Cornell Research Foundation, Cornell University Ithaca, NY 14850, USA. E-mail: klh22@cornell.edu

244

Kathleen Hefferon

Introduction The concept of using plants as production platforms for the expression of vaccine and other therapeutic proteins, or molecular pharming, originated as a potential answer to the need for biopharmaceuticals in developing countries that are safe, inexpensive and easy to store or transport. It has been demonstrated that plant-derived vaccines can be orally administered through direct consumption of edible plant tissue and effectively evoke a mucosal immune response [1]. Plant expression systems offer a select advantage over bacterial systems as they possess the inherent ability to generate proteins that undergo the same post-translational modifications as their mammalianderived counterparts. The quantities of plant-derived biopharmaceuticals produced vary depending on the specific plant production platform, and have generated yields as great as 45% of a plant cell’s total soluble protein, yet at a fraction of the cost of mammalian cell culture systems. For example, while up to 250 mg of biopharmaceutical protein per liter have been observed in some plant cell culture systems, the cost of purification of plant-derived vaccines can be as little as one hundredth of the cost of the same protein produced in mammalian cells [2].

The driving force behind molecular pharming One of the principal driving forces behind the development of molecular pharming has been its feasiblility for providing relief to Third World countries. Infectious diseases are responsible for over 2 million preventable deaths a year. Furthermore, one fifth of the world’s infants remain unimmunized as a result of limitations on vaccine production, distribution and delivery. With their low cost and lack of need for medical infrastructure, needles or refrigeration, vaccine proteins produced from plant tissues could very well provide a much needed solution. In addition to this, plant-derived vaccines could potentially play a lead role against diseases that are less prominent and whose treatments are poorly financed, such as dengue fever, hookworm and rabies.

For many infectious diseases, the mode of entry is through the mucosal system, such as the bronchial airways and the gastrointestinal tract (gut).

However, since many antigens do not last for long in the harsh environment of the digestive tract, it is difficult to deliver proteins to the intestinal immune system by direct oral consumption. A select advantage to plant-made vaccines is that the plant tissue can provide protection and prevent degradation of the antigen while it passes through the gut. A further problem revolves around the fact that since many antigens do not become recognized

Molecular pharming for plant-derived vaccines

245

as foreign in the GI tract, they have poor immunogenic properties. Adjuvants, however, are capable of altering the immunogenic context by which an antigen may be encountered by the body. Cholera toxin subunit B (CT-B), for example, can modify the cellular environment for highly efficient antigen presentation. CT-B can function as an adjuvant for coadministered antigens, or as an efficient transmucosal carrier molecule and delivery system for plant-derived subunit vaccines [3]. In the latter case, proteins which exhibit weak immunogenicity can be coupled to CT-B as part of a fusion protein and be expressed in plant tissue, thus increasing their antigenicity within the gut.

The majority of human therapeutic proteins are glycoproteins and while plants in general possess the intrinsic ability to produce glycoproteins, they differ in some respects from those derived from their mammalian counterparts. These differences in glycosylation motifs between plant and mammalian derived therapeutic proteins may lead to an increase in allergenicity or even an undesirable immune response. New advances in glycobiology have enabled the further ‘humanization’ of plant-derived vaccines and immunoglobulins by altering glycosylation pathways found in plants [4]. This includes the generation of transgenic plants which possess mammalian glycosylation machinery, for example, or the production of ‘knockout’ plants which lack plant-specific glycosylases or other enzymes involved in plant-specific post-translational modifications. Additional characteristics which may be constructed in plants used for production of vaccine proteins include a signal peptide which retains proteins within the endoplasmic reticulum [5].

Technologies used to produce vaccine proteins in plants There are two basic methods by which to express proteins in plants. Plant cells can be stably transformed and regenerated into transgenic plants.

Alternatively, proteins can undergo transient expression in mature plants using Agroinfection or virus expression vectors.

Stable plant transformation Today, the two most popular methods by which to generate transgenic plants are Agrobacterium-mediated transformation and biolistic delivery.

Agrobacterium-mediated transformation was originally based on the fact that Agrobacterium tumefaciens could induce tumors in plants by transferral of bacterial genetic material into the genome of infected plant cells [6]. Further studies conducted in the 1970’s indicated that large plasmids within several virulent strains of Agrobacteria known as the ‘tumor-inducing’ or Ti

246

Kathleen Hefferon

plasmids were responsible for tumorigenesis. It was this potential of using Ti plasmids as vectors to introduce foreign genes into plant cells that led to the first transgenic plants [7]. Another transformation technique, known as biolistic delivery, was developed in response to the restricted host range of Agrobacterium with respect to monocots such as corn and rice. This procedure involves high velocity microprojectiles (microcarriers consisting of subcellular-sized tungsten particles coated with the desired DNA of interest) which are then ‘shot’ using a ‘gene gun’ into plant tissue [8].

While many nuclear transformed plants have been shown to stably produce vaccine proteins, plastid or chloroplast engineering is a new and upcoming technology which in many cases produce proteins of interest at much greater levels, making them very attractive expression platforms for vaccine production [9]. Since each plant cell may contain several hundred chloroplasts, the protein of interest is produced at much greater levels than via nuclear transformation. Furthermore, plastids are not contained in the pollen of plants, so there is no need for concern regarding issues such as cross-pollination or outcrossing of plastid transformed plants with their weedy relatives. Since the plastid genome is bacterial in organization, several genes can be stacked under a single promoter, facilitating the ability to produce several proteins at once in the transgenic plant. Examples of vaccines produced in plants by plastid transformation include cholera toxin CTB, anthrax PA, canine parvovirus, tetanus toxin and plague vaccine [10].

Table 1. Examples of Vaccine Proteins produced in Transgenic Plants.

Molecular pharming for plant-derived vaccines

247

Transient expression of vaccine proteins in plants Plant virus expression vectors have been used extensively for vaccine production. Two types of expression systems based on plant RNA viruses have been developed for production of immunogenic peptides and proteins in plants: epitope presentation systems (short antigenic peptides fused to the CP that are displayed on the surface of assembled virus particles) and polypeptide expression systems (these systems express the entire recombinant protein) [11-13]. Plant viruses with DNA genomes, such as geminiviruses, have been explored as potential protein expression systems, as they can accumulate to extremely high copy numbers in inoculated cells, resulting in greatly elevated levels of gene expression. The geminivirus CP gene can be replaced with a foreign gene and the resulting recombinant virus displays increases of viral DNA as great as 300,000 copies per cell, indicating that foreign protein expression can be enhanced enormously [14].

Plant expression vectors have the advantage of producing a high yield of vaccine proteins over a short time period (as little as 1-2 weeks), but can be restrictive in terms of host plants which are available. Examples of plant viruses which have been engineered to produce vaccines in plants include tobacco mosaic virus, potato virus X, cucumber mosaic virus, cowpea mosaic virus and alfalfa mosaic virus.

A few years ago, an agricultural biotech company known as Icongenetics, Inc. developed a method for transfecting plants with recombinant virus vectors [15]. Using this ‘magnifection’ approach, an A. tumefaciens suspension could be infiltrated into the intercellular space of all mature leaves of a tobacco plant, resulting in virus infection that is more synchronous and faster than systemic infection. This agroinfiltration technique has gained momentum as a very effective means by which to produce large quantities of vaccine protein over a short time period. For example, the GreenVax Project of the US is currently utilizing a plant-based expression system to produce the H1N1 swine flu vaccine rapidly and in large quantities, to be stockpiled and on hand for potential future pandemics [16].

Clinical trials of therapeutic proteins produced in plants The number of different vaccines produced in plants at the point when this chapter was written is large and the studies are robust. A number of these studies have the entered clinical trial stage. The results of preliminary clinical studies for plant-made vaccines against Hepatitis B Virus, LT-B, Norwalk Virus, Human Papillomavirus and Measles Virus, as well as antibodies against Rabies Virus are described in the following section.

248

Kathleen Hefferon

Hepatitis B virus (HBV) Hepatitis B Virus is well known as an infectious disease that is responsible for significant mortality in the Third World. The surface antigen of Hepatitis B Virus, HBsAg, is one of the most popular antigens selected for vaccine development. A selective advantage of HBsAg as a plant-derived vaccine is its capacity to form intact immunogenic virus-like particles (VLPs) in transgenic plant tissue which have been demonstrated to be as highly immunogenic as the yeast-derived HBsAg currently used as an injectable vaccine [17]. This study involved feeding mice five grams of transgenic potato tubers containing HBsAg once a week for a total period of three weeks. Mice who were fed transgenic tubers exhibited an anti-HBsAg antibody response a few days after the first two doses, then peaked four weeks after the third dose, and finally returned to baseline levels eleven weeks after the third dose. On the other hand, the mice used as controls who were fed nontransgenic potato exhibited no elevated anti-HBsAg antibody response. Mice who were orally administered yeast-derived HBsAg exhibited a poor antibody response [18]. The strong primary antibody response which was exhibited in mice who were fed the transgenic potato tubers was thought to be the result of antigen protection by encapsulation within the plant tissue.

Digestion of the potato within the intestinal tract delayed antigen release, leading to a more robust immune response. The fact that the HBsAg is presented to the immune system in the form of VLPs further renders the antigen more immunogenic than the yeast-derived vaccine [19].

More recently a Phase 1 human clinical trial was performed to assess the possibility of using transgenic potato tubers expressing HBsAg. A doubleblind, placebo controlled study involved feeding 100 gram doses of uncooked transgenic potato tubers expressing approximately 8.5 Âľg/g HBsAg to previously vaccinated individual volunteers. Out of sixteen volunteers, ten who ingested three doses of the transgenic potato tubers exhibited increased levels of serum anti-HBsAg titres. No such increase was observed in volunteers who ate the nontransformed potatoes included as negative controls [20]. This initial study provided the proof of concept that plants can in fact provide vaccine proteins which are both efficiacious and safe.

Childhood diarrheal diseases Enterotoxigenic E. coli (ETEC) and Norwalk Virus (NV) are easily two of the most devastating diarrheal diseases targeting children of developing countries. Human clinical trials involved feeding healthy adult volunteers transgenic potato, soybean or corn expressing either the heat labile and highly

Molecular pharming for plant-derived vaccines

249

immunogenic B subunit of LT (LT-B) or the coat protein (CP) of NV in a randomized, double-blind fashion. In either case, both humoral and systemic immune responses were directly induced and mice could be partially protected against challenge by each pathogen [21].

Besides their direct role in evoking immune responses, LT-B and NV CP can also act as vaccine delivery vehicles and carry other antigens in the form of virus like particles. For example, norovirus capsid protein expressed in plant cells can assemble into virus-like particles (NVLPs) that mimic the antigenic structure of authentic virions. Oral and nasal delivery of NVLPs have been demonstrated to efficiently produce antibodies at distal mucosal sites, and as a consequence, NVLPs could be used to deliver an antigen in the form of a chimeric fusion protein [22].

Human papilloma virus Human Papillomavirus is a major causative agent of cervical cancer in women, particularly in developing countries. Vaccines which are currently available are both costly and difficult to distribute in these countries. Using a mouse model, a plant-derived vaccine against human papillomavirus L1 capsid protein has been developed. The Li CP can also be assembled in plants in the form of VLPs. Initial studies by Biemelt et al. (2003) demonstrated that half of mice fed transgenic potatoes expressing HPV VLPs developed L1-specific antibodies [23]. A plant-optimized version of the L1 capsid protein of HPV was later introduced into tobacco potato plants, and accumulated higher levels of VLPs. Mice fed potato tubers expressing this altered version of L1 elicited a significant enhanced serum antibody response.

Measles virus Measles Virus, contracted through the respiratory tract, causes several hundred times more fatalities in the third world than in developing nations, with over 30 million cases of measles reported in 2004. The virus is highly contagious and is hard to control in areas which have inadequate refrigeration, medical infrastructure, and syringes required for conventional vaccine administration.

In a preliminary heterologous prime boost vaccination study, a DNA measles vaccine used in conjunction with a plant-derived antigen booster has been demonstrated to evoke a substantial IgG immune response. In a later study, the MV-H protein was expressed in lettuce and proven to be immunogenic in mice. The nature of the immune response appeared to depend upon the manner in which the MV-H antigen is presented to the immune system [24].

250

Kathleen Hefferon

Rabies virus Rabies is responsible for over 50,000 deaths a year, particularly in South East Asia and Africa. Unfortunately, however, because it is not a major cause of mortality in developed countries, rabies does not receive substantial financial support. Treatment of rabies infection includes immunization as well as the topical application of rabies-specific antibodies at the bite wound. These antibodies are able to neutralize the virus, thus providing passive protection.

Unfortunately, antibodies against rabies are costly and difficult to produce in large quantities. The large-scale production of inexpensive plant-derived monoclonal antibodies (Mabs) would be a significant global health benefit.

Anti-rabies human monoclonal antibodies (Mab) have been developed in tobacco plants by Ko et al., (2003), and exhibit an anti-rabies virus neutralizing activity and affinity comparable to HRIG, its mammalianderived counterpart [25]. Furthermore, the plant-derived Mab selected glycosylation pattern, while differing from its’ mammalian counterpart, had no negative effect on its relative neutralizing and protective efficacy.

Allergies, oral tolerance and dose response relationships to plant made vaccines Several plant-derived vaccines have been analyzed for their potential to induce oral tolerance to common allergies. Rice plants were transformed with mouse T cell epitope peptides corresponding to Japanese Cedar pollen allergens. Oral tolerance was induced in mice who consumed transgenic rice prior to systemic challenge. This plant-derived tolerance strategy was also shown to successfully suppress asthma-based allergies. Transgenic narrow leaf lupin plants have been generated which express the allergen sunflower seed albumin (SSA). Oral consumption of SSA induced an antigen-specific IgG2a antibody response. A delayed-type hypersensitivity response was also prevented from taking place [26].

There are also concerns that orally administered plant-derived vaccines could be responsible for unwantingly promoting tolerance to vaccines or allergies to co-administered food proteins. As a result, consumption of a plant-derived vaccine by accident may affect a person’s later response to similar antigens, reducing the ability of the immune system to eliminate infection and leading to vaccine inefficiency. Corn-derived LT-B has been used as a model system to address these concerns. The maximum nonstimulatory dose of an orally administered plant-derived vaccine antigen in mice has been determined [27]. Similarly, the threshold level of orally administered plant-derived LT-B which did not stimulate detectable levels of

Molecular pharming for plant-derived vaccines

251

antibody but could nonetheless induce immune priming was identified. The relationship between oral administration of plant-derived antigens and the immune response is being actively pursued by several research groups.

Scale-up and commercialization of plant-derived therapeutic proteins One significant advantage of the use of plant expression systems for molecular pharming is the high stability of recombinant proteins produced in plants. Expression of a therapeutic protein can be targeted to a particular plant organ or tissue, where it can be stored for months or even years in some cases without substantial loss and with protein stability maintained.

Transgenic crops expressing biopharmaceuticals are also more amenable to upscaling or downscaling by simply adjusting the acreage available. Crops can be easily harvested and plant material processed on a large scale.

Procedures to extract protein from plant tissues have already been developed, and are in general simple and inexpensive. Production of recombinant proteins in plants can be as low as 2-10% of microbial fermentation systems and 0.1% of the cost of mammalian cell culture systems. Since some of these plant products are planned to be administered as food products, such as edible vaccines, the time and expense of a purification process can be much reduced or even virtually eliminated.

The extraction and purification of proteins from organisms or biological tissue can be a laborious and expensive process, and often represents the principal reason why vaccines and other therapeutic agents reach costs that become unattainable for many. It is therefore more economically feasible for the amount of vaccine or therapeutic protein expressed in a plant to be produced at industrial-scale levels. For example, the typical yield of biopharmaceuticals produced in a plant-based system is 0.1-1.0% of total soluble protein, a value which is competitive with other expression systems [28]. The huge quantity of generated plant biomass can compensate for any low protein yield. This unlimited scalability is of tremendous advantage, even under containment conditions, as it is possible to grow large amounts of crops under immense greenhouse facilities. For example, 250 acres of greenhouse space would be sufficient to grow a sufficient number of transgenic potato plants to satisfy the demand for the Hepatitis B Virus vaccine in Southeast Asia. In many instances, a particular biopharmaceutical is required in large quantities, such as monoclonal antibodies used as topical mucosal treatments.

Guy’s 13 Mab against Streptococcus mutans and HIV protein microbicides are very much needed at an inexpensive cost and in large quantities for the

252

Kathleen Hefferon

developing world. Both of these criteria can be met using field-grown biopharmaceuticals as compared to other production platforms.

Plant production platforms for molecular biopharming There are several alternatives to using field grown transgenic plants as production platforms for molecular pharming. One of the most easily accessible is the use of plant cell culture. One advantage to the use of plant tissue culture for the production of biopharmaceuticals over the use of plants grown in open fields is the fact that reservations regarding containment or regulatory approval no longer have to be addressed. For example, since variations in soil quality and unexpected alterations in weather patterns may make it difficult to maintain the good manufacturing practice conditions required for the uniform production of biopharmaceuticals in plants grown in the field, suspensions of plant cells are instead grown in precisely controlled environments. In many plant cell culture systems, the cells are grown continuously and the therapeutic product secreted into the media, resulting in downstream processing which is much less expensive. One disadvantage is that there is a much higher prevalence of genetic instability of transformants in plant cell culture systems when compared to field grown transgenic plants. A number of different plant cell culture systems have been developed for molecular pharming; these include hairy root cultures, mosses, various aquatic plant species such as duckweed and kelp, and several types of algae [29-31].

As different technologies associated with the generation of plant-derived biopharmaceuticals continues to advance, expression levels of these proteins are also rapidly increasing. As mentioned earlier, protein purification from plant tissues tends to be easier and significantly less expensive than purification procedures regularly used by their mammalian and bacterial counterparts. In certain cases, plant-derived biopharmaceuticals, such as topically applied monoclonal antibodies, need only be partially purified, rendering them even more cost effective [32]. The first commercialized plantderived vaccine, poultry vaccine against Newcastle Disease and produced in tobacco cell culture was marketed by Dow AgriSciences in 2006 [33]. Many other plant-derived therapeutic proteins are completing clinical trials and approach market release.

Concluding remarks This chapter has set out to describe the many advantages to manufacturing biopharmaceuticals, specifically vaccine proteins, using a plant-based production platform. As mentioned above, the costs of

Molecular pharming for plant-derived vaccines

253

manufacturing and processing plant-derived vaccines are significantly lower than those attributed to mammalian and prokaryotic cell-based production systems. Production for plant-derived proteins has been estimated to be only 2-10% of the cost of microbial fermentation systems and 0.1% of the cost of mammalian cell cultures. Scalability limitations of transgenic animal and fermentation systems also make molecular pharming an attractive alternative.

Unlike animal or bacterial cell fermentor systems, it is not a difficult or expensive process to rapidly up or downscale the number of plants grown which express a specific vaccine protein. The generation of vaccine proteins in plants also circumvents the problem of potential contamination by viruses, prions and other biological contaminants, which can pose a significant problem in mammalian cell culture systems.

It was originally believed that plant vaccines could be produced in local fields and routinely consumed locally as a whole fruit or vegetable in rural communities of developing countries. However, it soon became apparent that it was not feasible to obtain plant tissue containing the vaccine protein at consistent enough levels for oral consumption. It is far more likely that the final product is packaged in the form of a capsule or liquid suspension for oral delivery, rather than directly consumed at an unknown and variable concentration. Several levels of regulatory control exist for the production of plant-derived vaccines for the international marketplace, and the approval process for a given plant-derived vaccine is far more extensive than any followed by traditional vaccines sold commercially today.

Plant-derived vaccines provide an opportunity to develop safer, more effective and less expensive vaccination strategies. This year, the GreenVax Project of the US has employed a plant-based expression system to produce the H1N1 swine flu vaccine rapidly and in large quantities, to be stockpiled and on hand for potential future pandemics (13). Not only can plant-made vaccines provide a tremendous asset to combat newly emerging infectious diseases, they can also provide tremendous opportunities to help those who require them the most, the rural poor in developing countries. Plant-derived vaccines have arrived just in the nick of time.

References 1.

2.

Thanavala, Y., Huang Z & Mason H. Plant-derived vaccines : a look back at the highlights and a view to the challenges on the road ahead. Expert Review on Vaccines 5(2), 249-260 (2006).

McDonald, K.A. et al. Production of human alpha-1-antitrypsin from transgenic rice cell culture in a membrane bioreactor. Biotechnology Progress 21(3), 728-34 (2005).

254

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Kathleen Hefferon

Limaye, A. et al. Receptor-mediated oral delivery of a bioencapsulated green fluorescent protein expressed in transgenic chloroplasts into the mouse circulatory system. The FASEB Journal 20 (7), 959-961 (2006).

Bardor M, et al. Analytical strategies to investigate plant N-glycan profiles in the context of plant-made pharmaceuticals. Current Opinion in Structural Biology 16(5), 576-83 (2006).

Gomord, V., Chamberlain, P., Jeffries, R. and Faye, L. Biopharmaceutical production in plants: problems, solutions and opportunities. Trends in Biotchnology 24(4): 147-149. (2005).

Gelvin, SB (2003). Improving plant genetic engineering by manipulating the host. TRENDS in Biotechnology 21(3): 95-98.

Tzfira, T, Citovsky, V. (2006). Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current Opinion in Biotechnology 17: 147-154.

Christou, P. (1997). Rice Transformation: bombardment. Plant Molecular Biology 35: 197-203.

Daniell, H. and Dhingra, A. Multigene engineering: dawn of an exciting new era in biotechnology. Current Opinion in Biotechnology. 13: 1326-1341 (2002).

Daniell, H., Khan, M.S. and Allison, L. Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology. Trends in Plant Sciences 76; 84-91.

Fitchen, J., Beachy, R.N., Hein, M.B. (1995). Plant virus expressing hybrid coat protein with added murine epitope elicits autoantibody response. Vaccine, 13: 1051-7.

Johnson, J., Lin, T., Lomonosoff, G. (1997) Presentation of heterologous peptides on plant viruses: genetics, structure and function. Annu Rev Phytopathol. 35, 67-86.

Fernandez-Fernandez, M.R., Martinez-Torrecuadrada, J.L., Casal, J.I., Garcia, J.A. (1998) Development of an antigen presentation system based on plum pox potyvirus. FEBS Lett. 427, 229-235.

Timmermans, MCP, Das, OP, and Messing, J: (1994). Geminiviruses and their uses as extrachromosomal replicons. Ann Rev Plant Phy Plant Mol Biol., 45: 79-112.

Gleba, Y., Marillonnet, S., and Klimyuk, V. Engineering viral expression vectors for plants: the full virus and the deconstructed virus strategies. Current Opinion in Plant Biology. 7: 182-188. (2004) Naik, G. Teasing Vaccines from Tobacco. Wall Street Journal. Feb 24, 2010. online.wsj.com/.../SB10001424052748703503804575083611168442980.html Kong, Q., Richter, L., Yang, Y.F., Arntzen, C., Mason, H.S., Thanavala, Y. (2001) Oral administration with hepatitis B surface antigen expressed in transgenic plants. PNAS 98(20) 11539-11544.

Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, Goodwin P, Arntzen CJ, Mason HS. Immunogenicity in humans of an edible vaccine for hepatitis B. Proc Natl Acad Sci U S A. (2005) 102(9):3378-82.

Thanavala, Y. and Lugade, A.A. Oral transgenic plant-based vaccine for hepatitis B Immunologis Research. Vol 46, No 1-3 (2010).

Molecular pharming for plant-derived vaccines

255

20. Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat Biotech.

2000;18:1167-71.

21. Moravec, T., Schmidt, M.A., Herman, E.M. and Woodford-Thomas, T. Production of Escherichia coli heat labile toxin (LT)B subunit in soybean seed and analysis of its immunogenicity as an oral vaccine. Vaccine 25 (9): 16471657. (2007) 22. Herbst-Kralovetz M, Mason HS, Chen Q. Norwalk virus-like particles as vaccines. Expert Review Vaccines. 2010 Mar;9(3):299-307 23. Biemelt, S., Sonnewald, U., Galmbacher, P. Willmitzer, L. asnd Muller, M. Production of human pappillomavirus type 16 virus-like particles in transgenic plants. J. Virol. 77(17):9211-9220. (2003) 24. Webster, D.E., Smith, S.D., Pickering, R.J., Strugnell, RA., Dry, I.B. and Wesselingh, S.L. Measles virus hemagglutinin protein expressed in transgenic lettuce induces neutralizing antibodies in mice following mucosal vaccination.

Vaccine 24(17): 3544-3548 (2006).

25. Ko, K. & Koprowski, H. Plant biopharming of monoclonal antibodies. Virus Research 111(1), 93-100 (2005).

26. Smart, V. et al. A plant-based allergy vaccine suppresses experimental asthma via an IFN-gamma and CD4+CD45RBlow T cell-dependent mechanism. Journal of Immunology 171(4), 2116-2126 (2003).

27. Beyer, A.J. et al. Low-dose exposure and immunogenicity of transgenic maize expressing the Escherichia coli heat-labile toxin B subunit. Environmental Health Perspectives 115(3), 354- 360 (2007).

28. Twyman, R.M., Stoger, E., Schillberg, S., Christou, P. and Fischer, R. (2002) Molecular farming in plants: host systems and expression technology. TRENDS in Biotechnology 21(12), 570-579.

29. Guillon, S.M., Tremoulliaux-Guiller, J., Kumar Pati, P., Rideau, M. and Gantet, P. (2006). Harnassing the potential of hairy roots: dawn of a new era. TRENDS in Biotechnology, 24 No. 9; 403-409.

30. Borisjuk, N.V., Borisjuk, L.G., Logendra, S., Petersen, F., Gleba, Y., and Raskin, I. (1999) Production of recombinant proteins in plant root exudates. Nat. Biotechnol. 17(9); 466-469.

31. Gao, J., Hooker. B.S., and Anderson, D.B. (2004) Expression of functional human coagulation factor XIII A-domain in plant cell suspension s and whole plants. Protein Expr. Purif. 37(1); 89-96.

32. Sparrow, P.A.C., Judith A. Irwin, J.A., Phil J. Dale, P.J., Twyman, R.M. and Ma, J.K.C. (2007) Pharma-Planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals Transgenic ResearchVolume 16, Number 2; 147-161.

33. www.dowagro.com/newsroom/corporatenews/2006/20060131b.htm

Research Signpost 37/661 (2), Fort P.O. Trivandrum-695 023 Kerala, India

Novel Approaches to Vaccine Research, 2011: 257-285 ISBN: 978-81-308-0449-1 Editor: Kathleen L. Hefferon

11. Bacteriophage-based platforms for vaccine development David S. Peabody and Bryce Chackerian Department of Molecular Genetics and Microbiology University of New Mexico School of Medicine Albuquerque, New Mexico

Vaccines based on virus-like particles Traditional viral vaccines, based on attenuated or inactivated virus preparations, are among the most successful and cost-effective public health interventions ever devised. However, both these technologies have problems that can raise significant barriers to vaccine development and implementation in today’s regulatory climate. For example, attenuated vaccines can cause disease in immunodeficient or pregnant individuals. On occasion, attenuated viruses can revert to a more virulent forms and lead to vaccine-related disease outbreaks. The production of inactivated vaccines presents manufacturing challenges; it requires the handling of large volumes of a virulent pathogen and runs the risk of incomplete inactivation. Furthermore, the process of inactivation itself, particularly when using chemical agents, may alter the structure of the virus, affecting the quality of the immune response.

Moreover, the manufacture of both attenuated and inactivated vaccines relies on an ability to produce large quantities of virus, and, for many viruses, the lack of tissue culture systems that allow for efficient propagation of virus is a Correspondence/Reprint request: Dr. David S. Peabody, Department of Molecular Genetics and Microbiology University of New Mexico School of Medicine, Albuquerque, New Mexico.

E-mail: dpeabody@salud.unm.edu

258

David S. Peabody & Bryce Chackerian

substantial barrier to using either of these methods. The advent of recombinant techniques in the 1980s provided a means to avoid some of these problems by allowing the preparation of subunit vaccines that consist of isolated viral antigens. Subunit vaccines generally have good safety profiles, but vaccines based on individual proteins or peptides are generally far less effective than whole virus preparations, because they lack the highly multivalent, repetitive structure characteristic of highly immunogenic viral antigens. Thus, they generally require more frequent and larger doses of antigen in combination with potent adjuvants.

Many viral structural proteins have the ability to self-assemble into virus-like particles (VLPs), which resemble virions, but, lacking viral genomes, are non-infectious. As vaccines, VLPs combine many of the advantages of subunit and whole-virus antigens. VLPs can be produced by recombinant technologies, using expression systems (such as bacteria, yeast, or insect cells) that can generate large amounts of antigen protein without relying on the ability of the parental virus to replicate. Because of their particulate structure, VLPs can be purified easily by density gradient centrifugation or size exclusion chromatography. Lacking viral nucleic acids, VLPs cannot replicate, and are therefore intrinsically safer than attenuated viruses, but like attenuated and inactivated viruses, the regularity of VLP structure presents viral epitopes as dense, highly repetitive arrays. These structures strongly stimulate B cells and induce high titer antibody responses. Multivalent antigens such as VLPs can activate B cells at much lower concentrations than monomeric antigens [1-4] and do not usually require coadministration of exogenous adjuvants in order to induce strong antibody responses [5, 6].

Importantly, VLPs make excellent vaccines against the virus from which they were derived. The Hepatitis B virus (HBV) and Human Papillomavirus (HPV) vaccines are two examples of clinically approved VLP-based vaccines. Both of these vaccines safely and consistently induce high titer, durable antibody responses in humans [7, 8]. VLP-based vaccines for immunoprophylaxis against a number of other human virus infections are in clinical and preclinical development (reviewed by [9].

Using recombinant VLPs to induce antibody responses against heterologous antigens So far VLPs have been used mainly to produce vaccines against the viruses from which they are derived, but they show great promise for presentation of epitopes from other sources, even when those targets are normally poorly immunogenic. Linking target antigens, either genetically or chemically, to the surfaces of VLPs causes them to be displayed at high

Bacteriophage-based platforms for vaccine development

259

density. This high-density display, in turn, dramatically enhances the ability of these antigens to induce antibody responses. An impressive accumulation of data demonstrates the effectiveness of VLP presentation as a method for boosting antibody responses to diverse molecules. One illustrative example is the anti-smoking vaccine developed recently by Cytos Biotechnology that targets nicotine. In a phase I clinical study nicotine-conjugated VLPs were well-tolerated and induced high titer nicotine-specific IgG responses in immunized subjects [10]. Thus a normally non-immunogenic substance becomes strongly immunogenic when displayed as a dense repetitive array on a VLP. Even more impressively, VLP display can be used to overcome the mechanisms of B cell tolerance to induce antibody responses against selfantigens. Self-antigens displayed on VLPs are inherently immunogenic at low doses and without exogenous adjuvants. For example, when one selfantigen was conjugated to VLPs, it induced IgG titers 1,000-times higher than when the self-antigen was simply linked to a foreign T helper epitope [11]. Moreover, when displayed on the VLP, the self-antigen was as immunogenic as a foreign antigen presented in the same context. The magnitude of the anti-self IgG responses correlated with the density at which the self-antigen was displayed on the VLP surface, with maximum antibody responses induced when antigens were displayed with a spacing of 50 – 100 Angstroms [12, 13]. These results indicate that B cell recognition of “foreignlike” multivalent structural elements overwhelms the mechanisms that normally maintain B cell tolerance. VLP-display of self-antigens has been successfully used to target molecules that are involved in the pathogenesis of a variety of chronic diseases, including Alzheimer’s, hypertension, and certain cancers. Many of these vaccines have shown clinical efficacy in animal models and several are currently in clinical trials; see [14] for review.

Bacteriophages for vaccine discovery Bacteriophages and VLPs This review is about the potential use of bacteriophages as platforms for discovery and presentation of peptide and protein vaccine immunogens. Of course, bacteriophages themselves are not, strictly speaking, VLPs, since they are actually infectious for their bacterial hosts. However, when used as platforms for immunogenic presentation of peptides and proteins they may be regarded as effectively equivalent to VLPs, because they lack infectivity for animal cells. Bacteriophages offer several practical advantages. They can typically be produced rapidly and in large amounts, and are easily purified. In many cases their structures are understood in great detail, and their molecular

260

David S. Peabody & Bryce Chackerian

biology and genetics have been so thoroughly characterized that extensive technical resources exist for genetic and biochemical manipulation. All this facilitates the engineered presentation of specific peptide and protein antigens on the bacteriophage surface in highly immunogenic formats. In phage display a foreign sequence is genetically fused to one of the structural proteins of the virus in such a way that it is presented at the virus surface.

Sometimes the goal is simply to display a specific previously identified peptide epitope or protein antigen in high copy number on the surface of the viral particle where it acquires the potent immunogenicity characteristic of a multivalent particulate antigen. But phage display offers another important capability – to identify epitopes in complex random sequence libraries by affinity selection using antibody targets. The epitopes thus identified may themselves be regarded as vaccine candidates. Here we discuss several bacteriophage systems that have been employed in one or both of these modes.

We begin with a brief discussion of the best-characterized and most widely used phage display system, the one based on the filamentous phages. However, it has been discussed so extensively before, that here we prefer to concentrate on other phage-based platforms. Some of them have so far been little used for vaccine purposes, but each, in our view, offers significant potential advantages.

The filamentous phages The phage display method first appeared in the mid-1980's and relied on fusion of foreign peptides to structural proteins of the single-strand DNA phages like f1, fd and M13, called filamentous phages because of their long, thin, helical capsids. Phage display is an extraordinarily powerful and versatile technology that enables the affinity selection of novel binding functions from large populations of randomly generated peptide sequences.

From a sufficiently complex library, phage bearing peptides with practically any desired binding activity can be physically isolated by affinity selection, and, since each particle carries in its genome the genetic information for its own replication, the selectants can be amplified in bacteria. As we will see, other bacteriophages have been exploited for peptide/protein presentation, but filamentous phages remain the basis of the most popular affinity-selection systems in general use. The technology is highly developed. Random sequence peptide libraries, kits for affinity selection, and an extensive collection of display vectors adapted for a variety of specific applications are readily available from both commercial and academic sources. The method has served to identify peptides with affinity for a huge variety of biological receptors, and even for non-biological substances (e.g. metals). For all their

Bacteriophage-based platforms for vaccine development

261

advantages, however, the filamentous phages have certain limitations for vaccine development. In particular, for reasons explained below, they aren’t very well suited for display at the very high densities that yield maximum immunogenicity, so, although they excel at epitope identification, they don’t usually make very good immunogens.

In the two most commonly utilized modes, peptides are presented either polyvalently by fusion to pVIII, the protein making up the long helical portion of the virus, and present in about 2700 copies, or at low valency by fusion to pIII, a protein found in only a few copies at one end of the particle (Figure 1). Typically, peptides are fused to the N-termini of these proteins where they are accessible on the surface of the virus. Display on pVIII is most useful for affinity-selection of a spectrum of peptides with a broad range of affinities for their targets. This is because polyvalent display makes it difficult for affinity selection to distinguish intrinsically strong interactions from multiple weak ones (i.e. affinity vs. avidity). Display on pIII, on the other hand, lowers valency and allows selection of stronger interactions. For this reason an initial round selection using pVIII display may be followed by subsequent rounds of pIII display as a means of isolating first a relatively complex population of peptides having some affinity for the receptor, followed by a kind of “affinity maturation” process.

Figure 1. Schematic illustrations of the structures of the bacteriophages described in the text. (after Figure 1 in ref. [71]).

262

David S. Peabody & Bryce Chackerian

Even when presented on pVIII, however, a foreign peptide is not present at the very high densities required for maximum immunogenicity. This is because viruses with even short peptide-pVIII fusions (e.g. 4 or 5 amino acids) are severely crippled, and can be propagated only when the fusion protein is expressed at low levels, which drastically limits virus yield, or when co-expressed with wild-type pVIII to produce mosaic particles, which typically reduces display density to between 1% and 15%) [15]. This necessitates that synthetic versions of the affinity-selected epitopes be conjugated to an appropriate carrier before immunization. However, the affinities of peptides for their selection targets are frequently dependent on their structural contexts, and often fail to maintain the affinity they showed when present on the phage particle [15]. Thus, epitopes optimized by affinity selection in one structural context frequently fail to maintain the favored conformation in their new environments. Sidhu et al. have attempted to solve the valency problem by isolating pVIII mutants more tolerant of fusions, and succeeded in increasing somewhat the display density of two different proteins [16], but reliable high-density display on filamentous phage remains problematic.

Affinity selection using antibody targets is a powerful means for identification of linear epitopes in phage-displayed random sequence and antigen fragment libraries. It would seem straightforward to develop a vaccine by identifying the relevant peptide epitopes by affinity selection and then utilizing them directly as immunogens to elicit the desired antibody response. Indeed, epitopes identified by affinity selection are often able to elicit a relevant antibody response, but they are generally unable to elicit high titer responses, especially when fused to pIII. This was well illustrated by the first use of recombinant phage immunogens reported by del Cruz et al. [17].

Fusion to pVIII yields somewhat stronger antibody responses, but even then the display density is simply too low to reliably yield a high titer response [18, 19]. This is not due to some intrinsic lack of immunogenicity of filamentous phages per se, since high titer responses to phage proteins themselves are readily induced. Moreover, high-density chemical conjugation of peptides to filamentous phages increases their immunogenicity [20].

Lambda The bacteriophage lambda virion consists of a 50nm icosahedral head attached to a flexible 150 nm tail (Figure 1). Its head is comprised of two proteins, gpE, which is primarily responsible for the structure of the icosahedron, and gpD. Formation of a so-called prohead is initated when the connector (a complex of 12 copies of gpB and 10 copies of gpC) nucleates

Bacteriophage-based platforms for vaccine development

263

the assembly of 415 copies of gpE. The connector sits at the 5-fold portal vertex, the point through which DNA will be packaged once formation of the prohead is complete, and which will join head to tail in the fully assembled virus. As DNA is encaspidated through the portal, the prohead expands and becomes capable of associating with the second major head protein, gpD, 405-420 copies of which bind to the capsid surface as trimers. The gpD protein is normally essential for lambda growth, but it turns out to be nonessential in cases when the phage DNA has deletions that reduce its genome to less than 82% of wild-type size. This quasi-dispensability, and gpD's accessibility on the head's outer surface originally suggested its possible utility as a site for display of foreign peptides and proteins. As we will describe below, fusions to gpD are the primary means of peptide and protein display on lambda phage. A second display site is provided by the major tail protein, gpV. Thirty-six gpV hexamers make up the shaft of the tail, which is joined to the head through the connector (Figure 1). As much as the C-terminal third of gpV can be deleted with no apparent effect on infectivity, originally giving rise to the idea that it might tolerate genetic fusion of foreign peptides and proteins.

Display on the phage tail. In the first reported attempts at lambda phage display, Maruyma et al. fused two large foreign sequences (E. coli Ă&#x;-galactosidase, 465kDa, and Bauhinia purpura lectin, 120kDa) to the C-terminus of a truncated gpV [21]. Each fusion was constructed with an amber codon at its junction, meaning that the fusion protein was only produced in the presence of an amber-suppressing tRNA, and, therefore, at levels dictated by the efficiency of nonsense suppression (typically a few percent). The tails thus produced were mosaics, mixtures of gpV and the gpV-fusions, with the foreign proteins present only at low copy numbers – in some cases at an average of less than one per particle. Growth in strains with higher efficiency suppressors revealed phage growth defects, indicating that the presence of sufficient amounts of the fusion proteins interfered with tail assembly. In work reported about the same time. Dunn also expressed mixtures of gpV and gpV-fusions, but this time the gpV was expressed from a plasmid, and resulted in mosaic tails with the gpV expressed from the phage [22]. The ratio of wild-type gpV to gpV-fusion in phage particles depended on the relative expression levels of the two proteins and on their relative abilities to be incorporated into the assembling tail. A small peptide was well tolerated, but attempts to present a large protein (i.e. Ă&#x;galactosidase) confirmed that high-density display of foreign proteins was inhibitory of growth. Thus it seems that the tail protein is unsuited for multivalent display of large proteins, and for this reason is unlikely to prove

264

David S. Peabody & Bryce Chackerian

useful for highly immunogenic presentation of antigens, although it remains an under-explored possibility as a site for presentation of peptides.

Display on the phage head. Early efforts to utilize gpD fused an eight amino acid peptide (hormone AII) and a 65 amino acid protein (protein G) to the gpD N-terminus on plasmids, which were then introduced into a lambda lysogen defective for gpD. A large deletion of the genome, made growth of the virus independent of gpD, so the assembling particle was free to bind a quantity of gpD-fusion that depended on its availability and on the tolerance of the particle to the fusion protein. The resulting phage particles were shown to display the foreign sequences on their surfaces at, or very near, the maximum possible density of 405 copies per particle [23]. Similar efforts by Mikawa et al. to display Ă&#x;-galactosidase, protein A, and Ă&#x;-lactamase were also successful, and, at least in the cases of protein A and Ă&#x;-lactamase, the display densities were high enough to saturate the gpD binding sites on the phage head [24]. Yang et al. also demonstrated that several proteins with masses up to about 60kDa could be incorporated at saturating densities on the lambda particle [25]. Gupta et al. similarly displayed various size fragments of the HIV p24 protein as gpD fusions, but found that the copy numbers they attained seemed to correlate roughly with the size of the fused sequence and varied from a high of 350 for a 72 amino acid fragment, to 210 copies for a 156 amino acid piece, to a low of 154 copies of the 231 amino acid protein [26].

In fact, for many proteins, display at the maximal possible gpD density has proven problematic, especially with full-length genomes where gpD is necessary for phage viability. In such cases the general strategy has been to produce mosaic phage whose heads contain a mixture of wild-type and fusion gpD's. Several strategies for incorporating the fusions at lower, more tolerable densities exist. First, the wild-type and fusion proteins can be coexpressed from different genes, or they can be expressed from a single gene with a suppressible nonsense codon at the fusion junction. Zanghi et al., for example, showed that the failure of the phage to incorporate a fusion of gpD to the tenth fibronectin type III domain could be corrected by coexpression with wild-type gpD [27]. In this case both wild-type and fusion proteins were produced from plasmids. Second, one can utiliize a non-gpD-requiring lambda deletion mutant. Since gpD itself can be omitted, the mutant virus produces particles that are free to incorporate as much of the gpD-fusion as they can handle.

When maximum immunogenicity is the goal, very high display density is obviously a desirable trait, but it is unclear how often a new protein can be expected to present a problem. Unfortunately, lambda's tolerance of gpD fusions seems to have been assessed only in a relatively limited number of

Bacteriophage-based platforms for vaccine development

265

specific cases, and since failures are most likely under-reported in the literature, it is difficult to arrive at a definite conclusion. The ability of gpD to tolerate a wide variety of foreign sequences, such as those encountered in a complex random sequence library, seems never to have been systematically evaluated. Indeed the reasons for such failures are not even altogether clear although it is likely that some fusions are simply too bulky to bind the capsid without steric interference. When density limitations exist, they seem to apply primarily to large proteins, and lambda seems to have little problem displaying peptides and relatively small proteins at saturating density. And even with problematic proteins the display density is likely sufficient for good immuogenicity, although this has been explicitly tested in very few cases (see below).

Lambda possesses the capacity for construction of complex random sequence peptide and antigen fragment libraries from which vaccine targets could be isolated by affinity selection, thus potentially incorporating epitope identification (by affinity selection) and immunogenic display capabilities into a single platform. Such library-based applications have perhaps been discouraged by the fact that lambda cloning is a bit less convenient and substantially less efficient than cloning in plasmids or filamentous phage. Not only is the genome inconveniently large, but even moderately efficient cloning required, until recently, that ligation products be subjected to in vitro packaging reactions for introduction into bacteria by infection. These shortcomings have been largely overcome, however, by the work of Sternberg and Hoess [23] and by Gupta et al. [26], who developed systems for the efficient Cre/Lox-mediated recombination of gpD fusions from plasmids into the genome of a superinfecting lambda phage. Libraries can be constructed first in plasmids and then introduced into the lambda genome by recombination, without the necessity for in vitro packaging of ligation products.

Despite these advances, when compared to filamentous phages lambda has been little utilized for display generally, and has found even less application specifically for immunogenic presentation of foreign peptides and proteins. The literature offers little information regarding the immunogenicity of lambda recombinants, but there is no reason to believe that foreign proteins presented at high density should be any less immunogenic than the phage itself. This expectation was borne out recently by Gamage et al. who created a lambda-based vaccine for a frequently fatal malady of young pigs called post-weaning multisystemic wasting disease, caused by Porcine Circovirus 2. They fused relevant portions of the PCV2 capsid protein to gpD, isolated the particles, and immunized animals [28]. The recombinant phage induced a high titer antibody response that neutralized the virus in an

266

David S. Peabody & Bryce Chackerian

in vitro infectivity assay. No results were reported for protection of animals, but these results encourage the further development of lambda as a platform for immunogen presentation.

Lambda as a vehicle for delivery of DNA vaccines So far we have emphasized the direct presentation of surface-displayed foreign proteins and peptides on the lambda phage particle. However, somewhat greater energy seems to have been devoted to development of the virus as a delivery vehicle for DNA vaccines [29-33]. Here vaccine work overlaps with efforts to delivery DNA for gene therapy purposes. It is well known that naked DNA injected into animals can find its way into a sufficient number of cells that the foreign protein is expressed and induces an immune response. DNA vaccines have a number of potential advantages [34], but unfortunately, their application has been hampered by the relative inefficiency of DNA entry into mammalian cells. Several different technologies, including tissue electroporation and gene guns have been employed as means of overcoming this barrier. Recent experiments, however, show that DNA is delivered more efficiently when first cloned into the lambda genome, and then presented to cells inside the intact phage particle.

Packaging DNA within the viral particle renders it resistant to degradation, and, surprisingly, facilitates its entry into cells. The efficiency of entry, although much enhanced compared to naked DNA, is still relatively poor, however, and some effort has been devoted to display on the phage surface of proteins or peptides that enhance DNA entry and expression by destabilizing the membrane, by binding specific cellular receptors, or by favorably influencing the fate of the particle once it enters the cell. For example, phage displaying a peptide able to bind the cell surface ÎąvĂ&#x;3 integrin of K562 cells, yielded a 100-fold enhancement of cellular uptake [35]. Unfortunately, this translated into only a 3-fold enhanced expression of the foreign protein, indicating the importance not only of improving particle uptake, but also of appropriately directing the intracellular fate of the DNA. Zanghi et al. have attempted to accomplish both these goals simultaneously by displaying a binding peptide for the cell surface protein, CD40 (on gpV), and a peptide target for ubiquitylation (on gpD), thus hoping to enhance entry both into the cell and into the nucleus [36]. In similar work, Piersanti et al. have exploited the Adenovirus penton base protein, which normally plays a role in cell binding, entry and endosomal escape. They fused it to gpD and demonstrated rather dramatic improvements of lambda-mediated marker gene transduction into cell lines in culture [37]. Similarly taking advantage the HIV Tat protein's cell entry and nuclear localization activities, Eguchi et al. fused a

Bacteriophage-based platforms for vaccine development

267

Tat peptide to lambda gpD and also demonstrated large increases in effiiency of gene delivery to cells in culture [38]. Further work along these lines may eventually result in modified lambda phage that deliver DNA vaccines with high efficiency. Combining lambda's capacity for DNA delivery with its ability to directly display epitopes on its surface seems a particularly promising strategy [33]. It has also been possible to take advantage of the phage display capabilities of lambda to immunologically identify a relevant antigen in a bacterial genome library, and then deliver the DNA via the phage particle [39].

T4 phage Bacteriophage T4 is an example of one of the many phages with weirdly complicated structures. Despite its structural complexity, the simplicity of its cultivation and its amenability to genetic dissection have made it an important model system in molecular biology, and it has been extensively studied from biochemical, genetic and structural points of view. Peculiarities of its structure and assembly offer unique advantages for display of peptides and proteins, including the capacity for high-density presentation of large proteins, and even of hetero-oligomeric protein complexes. This is accomplished by fusion to either of two non-essential capsid-associated proteins called Hoc and Soc. The T4 virion (see Figure 1) consists of a prolate icosahedral head, a whiskered collar, and a contractile tail with fibers. The 166 Kbp doublestranded DNA genome is contained within the head, and is injected through the tail into a susceptible host at the moment of infection. The head contains more than a dozen proteins, but the icosahedral shell itself is made up of only three essential components: The major capsid protein, gp23, is present in 930 copies (155 hexamers) and makes up the bulk of the capsid surface. The minor capsid protein, called gp24, is present at 55 copies per particle and is encountered as pentamers at the vertices of the head. A third protein, gp20, is responsible for formation of a dodecameric head-tail connector at one of the vertices. Interestingly, the outer surface of the capsid is covered by two additional proteins: Hoc (highly immunogenic outer capsid protein) and Soc (small outer capsid protein). The functions of these two accessory proteins are only poorly understood, and although virally encoded, neither is necessary for phage viability. Hoc associates with capsids at the centers of gp23 hexamers (155 Hoc/particle), while Soc (up to 810 copies) binds the capsid as a trimer in locations where three gp23 hexons meet. Neither protein associates with the head until the mature capsid is formed, and, importantly, the purified Soc and Hoc proteins may be assembled in vitro onto purified

268

David S. Peabody & Bryce Chackerian

Soc-less/Hoc-less particles. They may also be supplied in vivo by expression from the T4 genome itself, or in trans from a plasmid. Each of the two proteins binds independently.

As early as 1996 Ren et al. reported the immunogenic display of peptides and proteins on T4 particles [40]. Their experience illustrates several commonly exploited features of the T4 system. Specifically, a 43 amino acid segment of HIV gp120 corresponding to the V3 loop, and the poliovirus VP1 protein (312-amino acids) were fused to the C-terminus of Soc. Attempts to supply the fusion proteins from plasmids in infected cells were frustrated when the proteins failed to fold correctly when produced at high levels, but two alternative strategies were successful. In the first, the misfolded proteins were extracted from inclusion bodies, refolded, and then bound to capsids in vitro. In the second, the Soc fusion genes were introduced directly into the T4 genome by recombination. Both methods resulted in surface display of the foreign proteins, but the display density varied. The V3 peptide was present at about 300 copies per particle, a density sufficient to make it strongly immunogenic, while polio VP1 was found at between only 25 and 100 copies.

In similar early experiments, Jiang et al. fused 36 amino acids of the Neisseria meningitidis PorA protein (a class I porin) to both the Hoc and Soc proteins [41]. High-density surface expression was attained whether the PorA sequence was expressed as a Hoc fusion in the T4 genome, or as a Soc fusion from a plasmid. In each case the recombinant proteins associated with the head in more or less the expected amounts, were accessible to PorA-specific antibodies, and elicited a high titer antibody response in immunized mice.

More recently Cao et al. [42] produced a T4-based vaccine that protects chickens against infection by very virulent Infectious Bursal Disease Virus (vvIBDV). An attenuated vaccine strain of vvIBDV is already available, but like many other attenuated viruses it is cumbersome to produce in quantity, and carries with it the risk of reversion to the virulent form. A T4-based alternative was produced by fusion of the 441-amino acid vvIBDV VP2 protein to the C-terminus of Soc. The fusion was constructed initially on a plasmid and then introduced into the T4 genome by recombination in vivo.

The resulting recombinant particles were reactive with anti VP2 antibodies and induced an anti VP2 antibody response in immunized chickens.

Importantly, immunization with T4 protected chickens against infection by vvIBDV. Both mice and pigs were also protected from Foot and Mouth Disease Virus (FMDV) infection when immunized orally or by injection with T4 phages decorated with soc fusions to the capsid precursor protein P1, or to the viral 3C proteinase. Orally delivered foot-and-mouth disease virus capsid

Bacteriophage-based platforms for vaccine development

269

protomer vaccine displayed on T4 bacteriophage surface: 100% protection from potency challenge in mice. Z.J. Rena, C.J. Tianc, Q.S. Zhuf, M.Y. Zhaoa, A.G. Xine, W.X. Niea, S.R. Lingd, M.W. Zhub, J.Y. Wub, H.Y. Lanb, Y.C. Caoc, Y.Z. Bi. Vaccine (2008) 26, 1471—1481.

The highest display densities reported so far for T4 were achieved by taking advantage of the ability to saturate both hoc and soc binding sites on the capsid by in vitro binding of fusion proteins to Hoc/Soc-defective phage particles. In the culmination [43] of a series [43-47] of increasingly complicated experiments, it was demonstrated that virtually the entire T4 capsid surface could be decorated with fusion proteins. In this case, all three components of anthrax toxin, Lethal Factor, Protective Antigen, and Edema Factor, were included. Lethal Factor was fused to the N-terminus of Soc, while Protective Antigen was joined to its C-terminus, thus simultaneously displaying two proteins from a single Soc recombinant. Meanwhile, Edema Factor was fused to Hoc's N-terminus. The purified Hoc-less/Soc-less capsid was then incubated with the proteins until it became fully saturated, thus displaying a total of 1775 copies of foreign protein on the T4 surface. It was also possible to assemble the three components of the anthrax toxin taking advantage of naturally occuring noncovalent interactions between the anthrax toxin components. Lethal Factor was first linked to the T4 surface by fusion to Soc or Hoc, and then Protective Antigen (PA) heptamers and Edema Factor (EF) were bound through their normal interactions with Lethal Factor (LF) and with one another [46]. This presumably has the advantage of presenting the anthrax toxin as a hetero-oligomeric complex in its native conformation. Immunization of mice with T4 phage displaying PA, LF, and EF without exogenous adjuvant elicited antibodies against all three anthrax antigens [44]. Relative to display of PA alone, co-display of all three antigens enhanced antibody titers against PA three-fold.

Finally, it should be noted that the T4 platform recently demonstrated its utility for elicitation of antibody responses to a tumor-associated self-antigen [48]. Given the increasing therapeutic importance of monoclonal antibodies directed against self-antigen targets, and in view of the high costs and inconvenience of their use, it makes sense to consider possible vaccine alternatives. Now it seems T4 can be added to the self-antigen vaccine armamentarium.

That T4 has found only very limited application for display of random sequence libraries probably has mostly to do with practical limits to library complexity. It is typically more cumbersome to clone into a big genome like T4’s, and the lack of a practical and efficient in vitro packaging system further complicates matters. For these reasons, it has not found much application for affinity selection from complex random sequence or antigen

270

David S. Peabody & Bryce Chackerian

fragment libraries. However, T4 excels as a system for the high-density, highly immunogenic display of full-length proteins and their complexes.

T7 phage Outside the filamentous phages, T7 is probably the most extensively used for display and affinity selection, and for good reasons: it is easily and rapidly propagated, cloning foreign DNA is straightforward, and commercial kits and reagents facilitate every step of the process. Various T7 strains have been engineered for cloning fusions at convenient restriction sites. As with lambda phage, efficient cloning requires the ligation of foreign DNA to the two “arms” of phage DNA, followed by an in vitro packaging reaction, and subsequent infection of E. coli, but the components of the assembly reaction are commercially available and are reported to result in more than 108 recombinant plaques per µg of DNA. T7’s suitability for display applications is a consequence of its structure.

It has an icosahedral head built from 415 copies of the gene 10 protein, which forms the 60 hexamers that make up the capsid faces, and the pentamers that make up 11 of 12 vertices. The 12th vertex is occupied by a head-tail connector, which is joined in turn to a short tail with six tail fibers (see Figure 1). As with other dsDNA phages, a prohead is assembled first from the capsid protein, this time with the assistance of a scaffold protein. The prohead is then filled with the 40Kbp genome through a portal at the connector vertex, after which the scaffold dissociates, and the tail and fibers join to the connector, forming the mature virion. The head is a natural mosaic consisting of two forms of the gene 10 capsid protein. The major form, called 10A, has 344 amino acids. The other, 10B, is identical to 10A from the N-terminus to amino acid 341, where a programmed translational frameshift bypasses the usual stop codon and adds an extra C-terminal segment that gives the 10B protein a length of 397 amino acids. Because the frameshift occurs at a relatively low frequency, 10B is produced in smaller amounts and makes up less of the capsid than 10A, but either protein can assemble into capsids all by itself, indicating a tolerance to C-terminal extensions that originally inspired the fusion of foreign peptides/proteins at this site.

The T7 capsid readily displays 415 copies of peptides smaller than about 50 amino acids long at the 10A C-terminus, but longer sequences interfere with capsid assembly unless present at reduced density. For this reason, several vectors have been created that deliberately limit display to low (0.1-1 copies per particle) and medium (1-15 copies) densities. This is accomplished by mutating the gene 10 translation initiation codon in the T7 genome to reduce expression of the fusion protein. Meanwhile, wild-type gene 10

Bacteriophage-based platforms for vaccine development

271

protein is provided in trans from a plasmid, thus allowing the phage to grow normally. The resulting particle is a mosaic that incorporates mostly wildtype gene 10 protein and only a few copies of the fusion. However, since the fusion is encoded within the T7 genome, the genotype/phenotype linkage essential for affinity selection is preserved. Its relative intolerance of long fusions makes T7 less suited than, say, T4 for presentation of large proteins at the high densities necessary for really potent immunogenicity, but it is fully capable of small peptide presentation at the capsid-saturating density of 415 copies. The flexibility to present peptides either at high or low valency can be an important advantage of T7 as a platform for the discovery and immunogenic display of vaccine epitopes. We have already noted that high valency display complicates the affinity selection of peptides with high affinity for their selection targets (e.g. a mAb). But by appropriate vector choice, T7 offers the possibility of valency control over a wide range.

Peptides may be presented at low densities to facilitate the selection of highaffinity interactions by bio-panning, and then at high density for immunization purposes by the simple means of changing vectors.

Despite its obvious strengths as a display platform for affinity selection, the literature contains relatively few accounts of attempts to utilize it as a combined epitope discovery and presentation platform. In summary, T7 is less useful than T4 for engineered display of big peptides and proteins, but represents an excellent, and, so far, under-utilized resource for epitope discovery and immunogenic display.

Virus-like particles of single-strand RNA phages Desirable properties of a VLP-display platform For maximum utility we think a VLP peptide display system should possess the following characteristics. First, to ensure high immunogenicity foreign peptides should be displayed as dense repetitive arrays at sites accessible on the VLP surface. Second, since display will normally be accomplished by genetic insertion, a display site must be identified that is highly tolerant of widely diverse peptides. Unfortunately, the effects on protein folding of such insertions, even in surface loops, are largely unpredictable and result frequently in folding failures. Third, it would be a great advantage if the VLP could encapsidate the nucleic acid encoding the viral protein-peptide fusion, thus enabling recovery of affinity-selected sequences from complex peptide libraries by nucleic acid amplification. The reader will already have noted the extent to which the other phage display systems described here satisfy these criteria. Here we describe a system based

272

David S. Peabody & Bryce Chackerian

on VLPs of single-strand RNA phages that we believe offers significant advantages for discovery, optimization and immunogenic display of peptide epitopes.

The single-strand RNA bacteriophages are a group of viruses found widely distributed in nature. Several have been characterized in great detail in terms of genome sequence, molecular biology, and capsid structure and assembly. MS2 is perhaps the best-studied member of the group and has been the focus of most of the work performed in the author’s laboratories, although recent work also exploits a related phage called PP7 [49]. MS2 has a 3569nucleotide single-strand RNA genome that encodes only four proteins: maturase, coat, lysis and replicase. The viral particle is comprised of 180 coat polypeptides, one molecule of maturase, and one copy of the RNA genome.

Thus, in comparison to the other phages described here, the RNA phages are surprisingly simple, and since the coat protein itself is entirely responsible for formation of the icosahedral shell, the MS2 VLP can be produced from plasmids as the product of a single gene.

RNA phages and their VLPs have been utilized in two major modes for immunogenic display of epitopes, namely chemical conjugation and genetic fusion. Each has its advantages and drawbacks. The capsid presents a repetitive array of reactive groups that may be utilized with commercially available reagents to cross-link an immunogen to the particle surface. Nonimmunogenic haptens and even self-antigens can elicit strong antibody responses when presented in such a manner. For example, the RNA phage QĂ&#x; has been extensively utilized by the Swiss company called Cytos to produce a number of vaccine candidates. This approach offers several important advantages. Both peptide and non-peptide immunogens can be presented in dense repetitive arrays, full length polypeptide antigens can be conjugated to the particle in their native conformations, thus avoiding the folding and assembly problems that sometimes accompany genetic fusion to viral structural proteins, and a wide range of molecular sizes is compatible with chemical conjugation. In a way, this is analogous to the T4 display system described above, in that the antigen is attached to the capsid after its synthesis. Several vaccines utilizing this approach are now in clinical development. The successes and limitations of this chemical conjugation approach have been discussed thoroughly in recent reviews [50, 51] and the subject is not treated further here.

Genetic insertion of a foreign sequence into a VLP structural protein enables its display on the VLP surface in favorable cases. This technique is limited to peptide targets, but successful incorporation of an epitope into a VLP guarantees that the antigen will be displayed in a uniform structural environment and at high density. This technique also has advantages from a

Bacteriophage-based platforms for vaccine development

273

manufacturing standpoint; chimeric particles can be purified using the same well-established methods used to purify unmodified parental VLPs. Genetic display of targets on VLPs can also be used to develop highly effective vaccines. For example, recombinant VLPs comprised of the HBV core antigen (HBcAg) were among the first particles used for display of heterologous antigens [52]. HBcAg VLP-based vaccines have been shown to protect animal models from infection by malaria [53] and influenza A virus [54]. Nevertheless, there are substantial barriers to generating chimeric VLPs. Most importantly, in order for a site of peptide insertion to be useful for display, it must be present on the surface of the VLP and insertions cannot interfere with protein folding or VLP assembly. In some cases, proper folding and presentation can be accomplished by replacing exposed, immunodominant viral epitopes (these are predominantly loop structures) with the target epitope. Because many epitopes in their native environments are also found in surface loops, such sites are natural candidates for peptide display. However, the effects of peptide insertion are notoriously difficult to predict, and all too often result in protein folding failures (~50% for HBcAg particles [55]). For instance, peptides with high hydrophobicity, a high Ă&#x;-strand index, a large volume, or a strong positive charge impeded the assembly of HBcAg chimeric particles [56, 57]. In some cases, particle assembly can be restored by flanking the insertions with acidic residues (e.g. glutamic acid), by altering the specific insertion site, and/or by making additional modifications to the C-terminus of core protein [57].

Not withstanding these considerations, genetic fusion can offer the important advantage of an affinity-selection capability. This is the approach emphasized in our laboratories and the one we focus on here. At the outset it should be understood that, unlike the other phage-based display systems, ours does not employ the infectious virus, but rather a VLP produced by expression of coat protein from a plasmid. The single-strand RNA genome of MS2 is complexly folded, dynamic structure. It is generally intolerant of arbitrary alterations, and therefore not very amenable to genetic manipulation. On the other hand, VLPs expressed from plasmids are easily subjected to genetic engineering. Furthermore, they can be synthesized in bacteria at high levels, are easily purified, and, crucially, they can be engineered to encapsidate the mRNA molecules that encode them. The particle is structurally simple, consisting of a single coat protein that selfassembles into a T=3 icosahedral particle whose structure is understood at high resolution [58, 59]. MS2 has been extensively studied, and a collection of convenient genetic and biochemical tools facilitate VLP engineering [60-63]. For example, a simple colony color assay for coat protein’s translational repressor activity provides a convenient means of determining

274

David S. Peabody & Bryce Chackerian

whether any particular manipulation (e.g. a peptide insertion) disrupts coat protein folding, and a straightforward gel electrophoresis assay rapidly assesses whether a recombinant VLP properly assembles.

It is always possible to display specific, already identified epitopes on the surface of RNA phage. But, it is also possible to conduct affinity selections using, for example, monoclonal or polyclonal antibodies, as a means of epitope discovery and optimization. We describe here the basic outlines of a procedure for affinity selection of peptide epitopes. We have produced plasmid vectors that facilitate the construction of complex VLP peptide libraries. The VLPs are synthesized in bacteria and are subjected to affinity selection (e.g. by biopanning). Since each VLP encapsidates the RNA sequence that encodes it, affinity selected sequences can be reverse transcribed, amplified by PCR and then recloned for synthesis of VLPs to be used in a subsequent round of selection. The procedure is similar to that used in other phage display systems, but relies on amplification of selected sequences in vitro instead of propagation of an infectious phage. Although the amplification step is less convenient, the MS2 VLP platform has advantages, which we discuss in some detail below. They include the possibility of more efficient discovery and optimization of mimotopes capable of eliciting a desired immune response.

Figure 2. A. The structure of the MS2 coat protein dimer as seen edge-on. The two polypeptide chains are colored red and green, with the three amino acids of each of the AB-loops shown in gold space-fill. B. The dimer as it would be viewed from outside the VLP. Note the proximity of N- and C-termini. C. The structure of the VLP, again with the AB-loops in gold to emphasize their spatial arrangement and the densely repitive nature of their presentation on the particle surface.

Bacteriophage-based platforms for vaccine development

275

Engineering coat protein to tolerate foreign peptide insertions MS2 coat protein is a 129 amino acid polypeptide that folds as a dimer.

Its two subunits are intimately intertwined in the manner shown in Figure 3A & B. In addition to its structural role, coat protein is also a gene regulatory protein. As it accumulates during the infection cycle the coat protein dimer binds a small hairpin at the start of the replicase cistron in viral RNA and shuts off replicase translation. The VLP itself is formed when ninety coat protein dimers assemble into the structure illustrated in Figure 3C. The RNA binding site resides on the surface of the coat protein ß-sheet oriented toward the VLP’s interior. Coat protein offers several potential sites for display of foreign peptides, but our work has concentrated mainly on insertions in the AB-loop, which consists of a 3-residue ß-turn connecting coat protein’s A and B ß-strands (Figure 3A). Peptides inserted here are highly exposed on the VLP surface, and since many epitopes in their native environments are found in surface loops, this seems a natural location for display.

Early efforts to display foreign peptides on MS2 VLPs were complicated by the unfortunate tendency of AB-loop insertions to interfere with coat protein folding. For example, Mastico et al. were successful in displaying six different peptides in the AB-loop, but in five of the six cases it was necessary

Figure 3. The general features of pDSP62. The plasmid utilizes the T7 promoter and transcription terminator sequences to express high levels of the MS2 coat protein single-chain dimer. The upstream half of the single-chain dimer is a synthetic “codonjuggled” sequence that deviates from the wild-type by the presence of the maximum possible number of silent nucleotide substitutions. It confers resistance to kanamycin, replicates as a plasmid from the ColE1 origin, or as a phagemid from the M13 origin in the presence of the M13CM1 helper.

276

David S. Peabody & Bryce Chackerian

to express the protein at reduced temperature or to purify the misfolded protein from inclusion bodies and refold it in vitro [64]. We confirmed and extended this result, first by insertion of a few specific peptides [65, 66], and then by construction of libraries of random-sequence peptide insertions [65].

When tested for the ability to repress translation in the colony color assay described above, none of the specific peptides we tested, and only about 2% of random-sequence clones, were able to repress Ă&#x;-galactosidase translation.

Although coat protein folding is not normally tolerant of AB-loop insertions, we fortunately found a simple means to suppress these defects.

Inspection of the protein's 3-dimensional structure (Figure 3B) reveals the close physical proximity of the N-terminus of one chain to the C-terminus of the other. We covalently linked these ends by duplicating the coat sequence and fusing the two copies into one long reading frame, thus producing "dimers" whose two halves are synthesized as a single polypeptide chain.

This so-called single-chain dimer retains all the functions of normal coat protein; it represses translation, and assembles normally into a VLP, but is considerably more resistant to thermal and chemical denaturation than the wild-type protein, and dramatically more tolerant of destabilizing amino acid substitutions (for examples see refs. [67] and [66]). Our hopes that subunit fusion might render the protein resistant to the effects of AB-loop insertions were confirmed when we constructed libraries of random sequence insertions of 6, 8 or 10 amino acids and subjected them to tests for translational repression and VLP assembly. The results dramatically illustrated the importance of subunit fusion for successful peptide display. Whereas the conventional coat protein tolerated only about 2% of random sequence peptide insertions, the single chain dimer produced VLPs for about 98% of random 6mers, about 90% of 8mers, and around 80% of 10mers. Thus although the success rate declined somewhat as the size of the insert increased, the vast majority of AB-loop insertions were tolerated.

Immunogenicity The ability of peptides displayed on MS2 single-chain dimer VLPs to elicit antibodies has been assessed in a number of specific cases. For example, mice immunized with VLPs displaying a peptide from the V3 loop of HIV or with a peptide from the second extracellular loop (ECL2) of CCR5 produced antisera with titers in the 104-105 range [65]. Both recombinant VLPs elicited antibodies that efficently bound the V3 and ECL2 peptides in their native environments. More recently, we employed VLPs of a related phage, PP7, to display a peptide from the L2 protein of Human Papillomavirus (HPV) [49]. As expected, it elicited high titer antibody

Bacteriophage-based platforms for vaccine development

277

responses in mice, but more importantly, it protected them from genital infection by HPV pseudoviruses derived from two highly divergent viral strains (specifically, HPV16 and 45), raising the possibility of a single vaccine, which unlike the exisitng HPV vaccines, would provide protection against a broad range of HPV isolates though the use of a single immunizing particle.

RNA packaging and affinity selection It has long been assumed that the same RNA hairpin bound by coat protein to repress replicase translation is also responsible for specific genome encapsidation, presumably by nucleating capsid assembly on the RNA. We find, however, that coat protein expressed from a plasmid assembles into VLPs that contain large amounts of coat-specific RNA without the need for the packaging hairpin [49, 65]. Packaging of coat-specific RNA establishes the genotype/phenotype linkage that makes affinity selection possible.

Affinity selection Examples already cited above demonstrate the ability of RNA phage VLPs to display specific, previously identified epitopes in a highly immunogenic format, and to elicit a neutralizing antibody response. But RNA phage VLPs also make possible the identification and optimization of epitopes by affinity selection against antibody targets. Affinity selections have now been successfully conducted against several antibody targets using the MS2 VLP platform. For example, we constructed a random sequence 8-mer peptide library and subjected it to biopanning using a monoclonal antibody for anthrax protective antigen known to recognize a five-amino acid epitope, ASFFD [68]. The VLP-associated RNA was subjected to reverse transcription and polymerase chain reaction and cloned. Sequence analysis of some first and second round selectants reveals a family of peptides whose consensus sequence matched that of the ASFFD epitope. A few individual VLP selectants were produced in E. coli and purified. ELISA shows that they do indeed bind the F20 monoclonal antibody. Moreover, immunization of mice with the selectants elicits antibodies that react with the epitope in its native environment on protective antigen.

A plasmid vector for construction of high complexity libraries In work still unpublished we created a plasmid vector called pDSP62 that embodies the following features.

278

David S. Peabody & Bryce Chackerian

Kanamycin resistance. Unlike ampicillin, selections using kanamycin allow for efficient elimination in liquid culture of the non-transformed cells present after electroporation.

An M13 origin of replication and a helper phage. Our first random sequence peptide libraries were constructed by use of PCR to introduce random sequences in the coat protein sequence, which was then cloned into the appropriate plasmid vector. But methods that involve ligation are inconvenient to scale up to levels that result in very high library complexities.

Therefore, we make random insertions using a very efficient method for sitedirected mutagenesis described by Kunkel [69]. Others have used it to produce filamentous phage libraries in the 1011 complexity range [70]. It relies on the ability of plasmids containing an M13 replication origin to produce single-stranded circular DNA in the presence of an M13 helper phage. When grown in a dut-, ung- host the DNA incorporates some dUTP in place of dTTP. A mismatched oligonucleotide primer is annealed to this DNA template, is elongated using DNA polymerase, and then ligated to produce closed circular DNA. Subsequent transformation of any ung+ strain results in strongly preferential propagation of the mutant strand. The primer extension mutagenesis reaction can be conducted on relatively large quantities of DNA (e.g. 20ug), enough to readily generate on the order of 1011 individual recombinants by electroporation [70]. To facilitate the application of this method, pDSP62 contains an M13 origin of replication. Since the usual helper phages (e.g. M13KO7) confer resistance to kanamycin they are unsuited to the propagation of pDSP62. We therefore also constructed a chloramphenicol resistant M13 helper phage we call M13CM1.

A synthetic "codon-juggled" coat gene. Our method relies on the specific insertion of peptides in one of the two AB-loops of the coat protein singlechain dimer. Library construction using the primer extenstion method described above requires the ability to direct primer annealing specifically to only one half of the single-chain dimer sequence. For this reason, pDSP62 the upstream copy of the coat sequence consistes of a "codon-juggled" version that introduces the maximum possible number of silent nucleotide substitutions, making it possible to direct primer annealling specifically to the desired half of the single-chain dimer, while preserving the wild-type amino acid sequence.

Valency control We assume that selection of peptides having the highest affinity for a given monoclonal antibody will provide the best molecular mimics of the native antigen, and that these are the most likely to induce a relevant antibody

Bacteriophage-based platforms for vaccine development

279

response. Ideally one conducts the first round of selection using multivalent display, thus obtaining a relatively complex population including all peptides having some minimal affinity for the target. Reducing the display valency in subsequent rounds increases the stringency of affinity selection. To achieve lower densities we constructed a mutant that places a nonsense codon on the junction between the two halves of the dimer. This means that ribosomes translating the coat sequence will normally terminate after synthesis of the first half of the single-chain dimer, that is, after synthesis of the wild-type form of coat protein. It, of course, will proceed to assemble into VLPs. In the presence of the appropriate nonsense suppressor tRNA, however, some percentage of ribosomes will translate through the stop codon to produce the single-chain dimer, which co-assembles with the wild-type protein to form mosaic VLPs. Since the peptide insertion resides only in the downstream half of the sequence, only the single-chain dimer displays it. Therefore the average display valency of the VLPs is determined by the efficiency of nonsense suppression, which depends in turn on the expression level of the suppressor. We constructed a synthetic alanine-inserting suppressor tRNA and expressed it from a second plasmid. We estimate that VLPs present about three peptides per particle on average with the suppressor we normally use, but this can be adjusted over a wide range. Experiments now underway are determining the best valencies for selection of high affinity interactions, but preliminary results already show that reduction of display valency has a large effect on affinity selection stringency (unpublished results).

Mimotopes Using filamentous phage display it has been possible in favorable cases to utilize affinity-selection to isolate so-called mimotopes, molecular mimics of epitopes. Sometimes it has been possible to mimic the structures of complex, conformational epitopes, and occasionally the peptides thus identified are even able to elicit antibodies that react with the original epitope in its native environment. However, in many cases these antibodies react well with the immunizing synthetic peptide, but do not recognize the epitope in its native environment. When inserted into the coat protein AB-loop, RNA phage VLPs present peptides in a conformationally constrained manner. Perhaps more importantly, the MS2 VLP platform allows both affinity selection and immunization to be carried out on a single structural framework, without the necessity of transferring a peptide optimized in one structural environment to a different environment on a more immunogenic platform for vaccination. The use of RNA phage VLPs may, therefore, increase the frequency with which mimotopes able to induce a desired response can be identified.

280

David S. Peabody & Bryce Chackerian

Conclusions Although display on filamentous phages has been extensively utilized for epitope mapping, and, to a lesser extent, for presentation of peptide and protein immunogens, the bacteriophages and phage-derived VLPs described above represent an under-utilized resource for epitope discovery and immunogenic display. Their particulate and molecularly repetitive nature ensures the high immunogenicity of the antigens they display, while simultaneously offering the potential for epitope discovery and optimization by affinity-selection.

Reference 1.

2.

3.

4.

5.

6.

7.

8.

9.

Brunswick M, Finkelman FD, Highet PF, Inman JK, Dintzis HM, Mond JJ: Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation. J Immunol 1988, 140(10):3364-3372.

Dintzis HM, Dintzis RZ, Vogelstein B: Molecular determinants of immunogenicity: the immunon model of immune response. Proc Natl Acad Sci U S A 1976, 73(10):3671-3675.

Dintzis RZ, Middleton MH, Dintzis HM: Inhibition of anti-DNP antibody formation by high doses of DNP-polyacrylamide molecules; effects of hapten density and hapten valence. J Immunol 1985, 135(1):423-427.

Milich DR, Chen M, Schodel F, Peterson DL, Jones JE, Hughes JL: Role of B cells in antigen presentation of the hepatitis B core. Proc Natl Acad Sci U S A 1997, 94(26):14648-14653.

Harro CD, Pang YY, Roden RB, Hildesheim A, Wang Z, Reynolds MJ, Mast TC, Robinson R, Murphy BR, Karron RA et al: Safety and immunogenicity trial in adult volunteers of a human papillomavirus 16 L1 virus-like particle vaccine. J Natl Cancer Inst 2001, 93(4):284-292.

Zhang LF, Zhou J, Chen S, Cai LL, Bao QY, Zheng FY, Lu JQ, Padmanabha J, Hengst K, Malcolm K et al: HPV6b virus like particles are potent immunogens without adjuvant in man. Vaccine 2000, 18(11-12):1051-1058.

Poovorawan Y, Chongsrisawat V, Theamboonlers A, Leroux-Roels G, Kuriyakose S, Leyssen M, Jacquet JM: Evidence of protection against clinical and chronic hepatitis B infection 20 years after infant vaccination in a high endemicity region. J Viral Hepat.

Romanowski B, de Borba PC, Naud PS, Roteli-Martins CM, De Carvalho NS, Teixeira JC, Aoki F, Ramjattan B, Shier RM, Somani R et al: Sustained efficacy and immunogenicity of the human papillomavirus (HPV)-16/18 AS04adjuvanted vaccine: analysis of a randomised placebo-controlled trial up to 6.4 years. Lancet 2009, 374(9706):1975-1985.

Roy P, Noad R: Virus-like particles as a vaccine delivery system: myths and facts. Hum Vaccin 2008, 4(1):5-12.

Bacteriophage-based platforms for vaccine development

281

10. Maurer P, Jennings GT, Willers J, Rohner F, Lindman Y, Roubicek K, Renner WA, Muller P, Bachmann MF: A therapeutic vaccine for nicotine dependence: preclinical efficacy, and Phase I safety and immunogenicity. Eur J Immunol 2005, 35(7):2031-2040.

11. Chackerian B, Lowy DR, Schiller JT: Conjugation of a self-antigen to papillomavirus-like particles allows for efficient induction of protective autoantibodies. J Clin Invest 2001, 108(3):415-423.

12. Chackerian B, Lenz P, Lowy DR, Schiller JT: Determinants of autoantibody induction by conjugated papillomavirus virus-like particles. J Immunol 2002, 169(11):6120-6126.

13. Jegerlehner A, Storni T, Lipowsky G, Schmid M, Pumpens P, Bachmann MF: Regulation of IgG antibody responses by epitope density and CD21mediated costimulation. Eur J Immunol 2002, 32(11):3305-3314.

14. Chackerian B: Virus-like particles: flexible platforms for vaccine development. Expert Reviews of Vaccines 2007, 6:381-390.

15. Van Houten NE, Scott JK: Phage Libraries for Developing Antibody-Targeted Diagnostics and Vaccines. In: Phage Display in Biotechnology and Drug Discovery. Edited by Sidhu S: Taylor and Francis Group; 2005: 165-254.

16. Sidhu SS, Weiss GA, Wells JA: High copy display of large proteins on phage for functional selections. J Mol Biol 2000, 296(2):487-495.

17. de la Cruz VF, Lal AA, McCutchan TF: Immunogenicity and epitope mapping of foreign sequences via genetically engineered filamentous phage. J Biol Chem 1988, 263(9):4318-4322.

18. Greenwood J, Willis AE, Perham RN: Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens. J Mol Biol 1991, 220(4):821-827.

19. Willis AE, Perham RN, Wraith D: Immunological properties of foreign peptides in multiple display on a filamentous bacteriophage. Gene 1993, 128(1):79-83.

20. Saphire EO, Montero M, Menendez A, van Houten NE, Irving MB, Pantophlet R, Zwick MB, Parren PW, Burton DR, Scott JK et al: Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J Mol Biol 2007, 369(3):696709.

21. Maruyama IN, Maruyama HI, Brenner S: Lambda foo: a lambda phage vector for the expression of foreign proteins. Proc Natl Acad Sci U S A 1994, 91(17):8273-8277.

22. Dunn IS: Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. J Mol Biol 1995, 248(3):497-506.

23. Sternberg N, Hoess RH: Display of peptides and proteins on the surface of bacteriophage lambda. Proc Natl Acad Sci U S A 1995, 92(5):1609-1613.

24. Mikawa YG, Maruyama IN, Brenner S: Surface display of proteins on bacteriophage lambda heads. J Mol Biol 1996, 262(1):21-30.

282

David S. Peabody & Bryce Chackerian

25. Yang F, Forrer P, Dauter Z, Conway JF, Cheng N, Cerritelli ME, Steven AC, Pluckthun A, Wlodawer A: Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD. Nat Struct Biol 2000, 7(3):230237.

26. Gupta A, Onda M, Pastan I, Adhya S, Chaudhary VK: High-density functional display of proteins on bacteriophage lambda. J Mol Biol 2003, 334(2):241254.

27. Zanghi CN, Lankes HA, Bradel-Tretheway B, Wegman J, Dewhurst S: A simple method for displaying recalcitrant proteins on the surface of bacteriophage lambda. Nucleic Acids Res 2005, 33(18):e160.

28. Gamage LN, Ellis J, Hayes S: Immunogenicity of bacteriophage lambda particles displaying porcine Circovirus 2 (PCV2) capsid protein epitopes.

Vaccine 2009, 27(47):6595-6604.

29. Clark JR, March JB: Bacteriophage-mediated nucleic acid immunisation.

FEMS Immunol Med Microbiol 2004, 40(1):21-26.

30. Jepson CD, March JB: Bacteriophage lambda is a highly stable DNA vaccine delivery vehicle. Vaccine 2004, 22(19):2413-2419.

31. March JB, Clark JR, Jepson CD: Genetic immunisation against hepatitis B using whole bacteriophage lambda particles. Vaccine 2004, 22(13-14):16661671.

32. Clark JR, March JB: Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Trends Biotechnol 2006, 24(5):212-218.

33. Clark JR, March JB: Bacterial viruses as human vaccines? Expert Rev Vaccines 2004, 3(4):463-476.

34. March JB: The viability of DNA vaccines--marcus evans conference. 3-5 February 2004, London, UK. IDrugs 2004, 7(3):226-228.

35. Lankes HA, Zanghi CN, Santos K, Capella C, Duke CM, Dewhurst S: In vivo gene delivery and expression by bacteriophage lambda vectors. J Appl Microbiol 2007, 102(5):1337-1349.

36. Zanghi CN, Sapinoro R, Bradel-Tretheway B, Dewhurst S: A tractable method for simultaneous modifications to the head and tail of bacteriophage lambda and its application to enhancing phage-mediated gene delivery. Nucleic Acids Res 2007, 35(8):e59.

37. Piersanti S, Cherubini G, Martina Y, Salone B, Avitabile D, Grosso F, Cundari E, Di Zenzo G, Saggio I: Mammalian cell transduction and internalization properties of lambda phages displaying the full-length adenoviral penton base or its central domain. J Mol Med 2004, 82(7):467-476.

38. Eguchi A, Akuta T, Okuyama H, Senda T, Yokoi H, Inokuchi H, Fujita S, Hayakawa T, Takeda K, Hasegawa M et al: Protein transduction domain of HIV-1 Tat protein promotes efficient delivery of DNA into mammalian cells.

J Biol Chem 2001, 276(28):26204-26210.

39. March JB, Jepson CD, Clark JR, Totsika M, Calcutt MJ: Phage library screening for the rapid identification and in vivo testing of candidate genes for a DNA vaccine against Mycoplasma mycoides subsp. mycoides small colony biotype. Infect Immun 2006, 74(1):167-174.

Bacteriophage-based platforms for vaccine development

283

40. Ren ZJ, Lewis GK, Wingfield PT, Locke EG, Steven AC, Black LW: Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4. Protein Sci 1996, 5(9):18331843.

41. Jiang J, Abu-Shilbayeh L, Rao VB: Display of a PorA peptide from Neisseria meningitidis on the bacteriophage T4 capsid surface. Infect Immun 1997, 65(11):4770-4777.

42. Cao YC, Shi QC, Ma JY, Xie QM, Bi YZ: Vaccination against very virulent infectious bursal disease virus using recombinant T4 bacteriophage displaying viral protein VP2. Acta Biochim Biophys Sin (Shanghai) 2005, 37(10):657-664.

43. Li Q, Shivachandra SB, Zhang Z, Rao VB: Assembly of the small outer capsid protein, Soc, on bacteriophage T4: a novel system for high density display of multiple large anthrax toxins and foreign proteins on phage capsid. J Mol Biol 2007, 370(5):1006-1019.

44. Shivachandra SB, Li Q, Peachman KK, Matyas GR, Leppla SH, Alving CR, Rao M, Rao VB: Multicomponent anthrax toxin display and delivery using bacteriophage T4. Vaccine 2007, 25(7):1225-1235.

45. Shivachandra SB, Rao M, Janosi L, Sathaliyawala T, Matyas GR, Alving CR, Leppla SH, Rao VB: In vitro binding of anthrax protective antigen on bacteriophage T4 capsid surface through Hoc-capsid interactions: a strategy for efficient display of large full-length proteins. Virology 2006, 345(1):190198.

46. Li Q, Shivachandra SB, Leppla SH, Rao VB: Bacteriophage T4 capsid: a unique platform for efficient surface assembly of macromolecular complexes. J Mol Biol 2006, 363(2):577-588.

47. Sathaliyawala T, Rao M, Maclean DM, Birx DL, Alving CR, Rao VB: Assembly of human immunodeficiency virus (HIV) antigens on bacteriophage T4: a novel in vitro approach to construct multicomponent HIV vaccines. J Virol 2006, 80(15):7688-7698.

48. Ren SX, Ren ZJ, Zhao MY, Wang XB, Zuo SG, Yu F: Antitumor activity of endogenous mFlt4 displayed on a T4 phage nanoparticle surface. Acta Pharmacol Sin 2009, 30(5):637-645.

49. Caldeira JD, Medford A, Kines RC, Lino CA, Schiller JT, Chackerian B, Peabody DS: Immunogenic display of diverse peptides, including a broadly cross-type neutralizing human papillomavirus L2 epitope, on virus-like particles of the RNA bacteriophage PP7. Vaccine 2010, 28(27):4384-4393.

50. Chackerian B: Virus-like particles: flexible platforms for vaccine development. Expert Rev Vaccines 2007, 6(3):381-390.

51. Jennings GT, Bachmann MF: The coming of age of virus-like particle vaccines. Biol Chem 2008, 389(5):521-536.

52. Clarke BE, Newton SE, Carroll AR, Francis MJ, Appleyard G, Syred AD, Highfield PE, Rowlands DJ, Brown F: Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 1987, 330(6146): 381-384.

284

David S. Peabody & Bryce Chackerian

53. Schodel F, Wirtz R, Peterson D, Hughes J, Warren R, Sadoff J, Milich D: Immunity to malaria elicited by hybrid hepatitis B virus core particles carrying circumsporozoite protein epitopes. J Exp Med 1994, 180(3):10371046.

54. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W: A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med 1999, 5(10):1157-1163.

55. Jegerlehner A, Tissot A, Lechner F, Sebbel P, Erdmann I, Kundig T, Bachi T, Storni T, Jennings G, Pumpens P et al: A molecular assembly system that renders antigens of choice highly repetitive for induction of protective B cell responses. Vaccine 2002, 20(25-26):3104-3112.

56. Karpenko LI, Ivanisenko VA, Pika IA, Chikaev NA, Eroshkin AM, Veremeiko TA, Ilyichev AA: Insertion of foreign epitopes in HBcAg: how to make the chimeric particle assemble. Amino Acids 2000, 18(4):329-337.

57. Billaud JN, Peterson D, Barr M, Chen A, Sallberg M, Garduno F, Goldstein P, McDowell W, Hughes J, Jones J et al: Combinatorial approach to hepadnavirus-like particle vaccine design. J Virol 2005, 79(21):13656-13666.

58. Golmohammadi R, Valegard K, Fridborg K, Liljas L: The refined structure of bacteriophage MS2 at 2.8 A resolution. J Mol Biol 1993, 234(3):620-639.

59. Ni CZ, Syed R, Kodandapani R, Wickersham J, Peabody DS, Ely KR: Crystal structure of the MS2 coat protein dimer: implications for RNA binding and virus assembly. Structure 1995, 3(3):255-263.

60. Peabody DS: Translational repression by bacteriophage MS2 coat protein expressed from a plasmid. A system for genetic analysis of a protein-RNA interaction. J Biol Chem 1990, 265(10):5684-5689.

61. Peabody DS, Al-Bitar L: Isolation of viral coat protein mutants with altered assembly and aggregation properties. Nucleic Acids Res 2001, 29(22):E113.

62. Peabody DS: The RNA binding site of bacteriophage MS2 coat protein. Embo J 1993, 12(2):595-600.

63. Lim F, Peabody DS: Mutations that increase the affinity of a translational repressor for RNA. Nucleic Acids Res 1994, 22(18):3748-3752.

64. Mastico RA, Talbot SJ, Stockley PG: Multiple presentation of foreign peptides on the surface of an RNA-free spherical bacteriophage capsid. J Gen Virol 1993, 74 ( Pt 4):541-548.

65. Peabody DS, Manifold-Wheeler B, Medford A, Jordan SK, do Carmo Caldeira J, Chackerian B: Immunogenic display of diverse peptides on virus-like particles of RNA phage MS2. J Mol Biol 2008, 380(1):252-263.

66. Peabody DS: Subunit fusion confers tolerance to peptide insertions in a virus coat protein. Arch Biochem Biophys 1997, 347(1):85-92.

67. Peabody DS, Chakerian A: Asymmetric contributions to RNA binding by the Thr(45) residues of the MS2 coat protein dimer. J Biol Chem 1999, 274(36):25403-25410.

68. Gubbins MJ, Berry JD, Corbett CR, Mogridge J, Yuan XY, Schmidt L, Nicolas B, Kabani A, Tsang RS: Production and characterization of neutralizing

Bacteriophage-based platforms for vaccine development

285

monoclonal antibodies that recognize an epitope in domain 2 of Bacillus anthracis protective antigen. FEMS Immunol Med Microbiol 2006, 47(3): 436-443.

69. Kunkel TA, Bebenek K, Mcclary J: Efficient Site-Directed Mutagenesis Using Uracil-Containing DNA. Methods in Enzymology 1991, 204:125-139.

70. Sidhu SS, Lowman HB, Cunningham BC, Wells JA: Phage display for selection of novel binding peptides. Methods Enzymol 2000, 328:333-363.

71. Casjens, S., and Hendrix, R. (1988) Control Mechanisms in dsDNA Bacteriophage Assembly, in The Bacteriophages, Vol. 1, Plenum Press, New York and London.