Chloroplast Biotechnology

Edited By: Henry Daniell and Keith Edwards

Virtual Issue: Chloroplast Biotechnology

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Introduction Introduction

Chloroplast Biotechnology Chloroplast transformation has several unique advantages. The highest levels of expression in the published literature for engineering agronomic traits or human therapeutic proteins were achieved using this concept. In addition, integration of foreign genes into the chloroplast genome offers transgene containment from pollen transmission because of maternal inheritance of the chloroplast genome in most crops. Chloroplast genetic engineering offers a number of other unique advantages including multi-gene engineering in a single transformation event, lack of gene silencing or position effects due to site specific transgene integration and minimal or lack of pleiotropic effects due to subcellular compartmentalization of toxic transgene products.

Tobacco served as the model system to confer herbicide, insect or disease resistance, drought or salt tolerance or phytoremediation.

More recently, chloroplast genomes of major crops including cotton and soybean, vegetables (carrot, cauliflower, cabbage, eggplant, lettuce, sugarbeet), tubers (potato), fruits (tomato) and trees (poplar) have been transformed. Significant progress has also been made in expressing vaccine antigens against human bacterial, viral and protozoan pathogens in chloroplasts and animal studies demonstrated their efficacy against pathogen or toxin challenge. Most importantly, oral delivery of vaccine antigens bioencapsulated in plant cells was shown to be more efficacious than injectable vaccines or in developing oral tolerance against autoimmune disorders.

Introduction Introduction

The Plant Biotechnology Journal is a leader in publication of articles in chloroplast biotechnology and has published the most number of articles in this filed (Plant Biotech Journal 17.87%; Plant Physiology 8.93%; Transgenic Research 12.84%; Plant Molecular Biology 8.37%).

In this special issue, articles review the history and progress made in chloroplast selectable markers and removal of marker genes, expression of biopharmaceuticals or manipulation of plastid fatty acid biosynthetic pathways. In addition, original articles report expression and evaluation of several human therapeutic proteins (insulin, HIV inhibitor cyanovirin, HIV-1 capsid protein p24, human papillomavirus L1 antigen, TGFβ3, HSA). Chloroplast transformation has also been used to confer phytoremediation or enhanced salt and cold tolerance or to express higher levels of foreign proteins in Chlamydomonas or in an immersion bioreactor.

These diverse applications of chloroplast biotechnology promises to advance this field towards commercial development. Recent funding to advance this concept by major pharmaceuticals (Bayer Pharma) or foundations (Bill and Melinda Gates Foundation or Juvenile Diabetes Research Foundation) augurs well for advancing inventions from laboratories to the clinic. Agronomic traits engineered via the chloroplast genome in soybean by Bayer Crop Science are advancing towards field studies. Production of low cost enzymes in chloroplasts of non-food/feed crops is ideal to produce biofuels from biomass. It is anticipated that this special issue on chloroplast biotechnology will be useful for the academic and industry researchers and regulatory agencies.

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Review Introduction

The chloroplast transformation toolbox: selectable markers and marker removal Anil Day, Michel Goldschmidt-Clermont Plastid transformation is widely used in basic research and for biotechnological applications. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. Selection of transgenic lines where all copies of the polyploid plastid genome are transformed requires efficient markers. A number of traits have been used for selection such as photoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolic inhibitors. Restoration of photosynthesis is an effective primary selection method in Chlamydomonas but can only serve as a screening tool in flowering plants. The most successful and widely used markers are derived from bacterial genes that inactivate antibiotics, such as aadA that confers resistance to spectinomycin and streptomycin. For many applications, the presence of a selectable marker that confers antibiotic resistance is not desirable. Efficient marker removal methods are a major attraction of the plastid engineering tool kit. They exploit the homologous recombination and segregation pathways acting on chloroplast genomes and are based on direct repeats, transient co-integration or co-transformation and segregation of trait and marker genes. Foreign site-specific recombinases and their target sites provide an alternative and effective method for removing marker genes from plastids.

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Review Introduction

Engineering plastid fatty acid biosynthesis to improve food quality and biofuel production in higher plants Marcelo Rogalski, Helaine Carrer The ability to manipulate plant fatty acid biosynthesis by using new biotechnological approaches has allowed the production of transgenic plants with unusual fatty acid profile and increased oil content. This review focuses on the production of very long chain polyunsaturated fatty acids (VLCPUFAs) and the increase in oil content in plants using molecular biology tools. Evidences suggest that regular consumption of food rich in VLCPUFAs has multiple positive health benefits. Alternative sources of these nutritional fatty acids are found in cold-water fishes.

However, fish stocks are in severe decline because of decades of overfishing, and also fish oils can be contaminated by the accumulation of toxic compounds. Recently, there is also an increase in oilseed use for the production of biofuels. This tendency is partly associated with the rapidly rising costs of petroleum, increased concern about the environmental impact of fossil oil and the attractive need to develop renewable sources of fuel. In contrast to this scenario, oil derived from crop plants is normally contaminant free and less environmentally aggressive. Genetic engineering of the plastid genome (plastome) offers a number of attractive advantages, including high-level foreign protein expression, marker-gene excision and transgene containment because of maternal inheritance of plastid genome in most crops. Here, we describe the possibility to improve fatty acid biosynthesis in plastids, production of new fatty acids and increase their content in plants by genetic engineering of plastid fatty acid biosynthesis via plastid transformation.

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Review Introduction

Chloroplast-derived vaccines against human diseases: achievements, challenges and scopes Andreas G. LĂśssl, Mohammad T. Waheed

Infectious diseases represent a continuously growing menace that has severe impact on health of the people worldwide, particularly in the developing countries. Therefore, novel prevention and treatment strategies are urgently needed to reduce the rate of these diseases in humans. For this reason, different options can be considered for the production of affordable vaccines. Plants have been proved as an alternative expression system for various compounds of biological importance. Particularly, plastid genetic engineering can be potentially used as a tool for cost-effective vaccine production. Antigenic proteins from different viruses and bacteria have been expressed in plastids. Initial immunological studies of chloroplast-derived vaccines have yielded promising results in animal models. However, because of certain limitations, these vaccines face many challenges on production and application level. Adaptations to the novel approaches are needed, which comprise codon usage and choice of proven expression cassettes for the optimal yield of expressed proteins, use of inducible systems, marker gene removal, selection of specific antigens with high immunogenicity and development of tissue culture systems for edible crops to prove the concept of low-cost edible vaccines. As various aspects of plant-based vaccines have been discussed in recent reviews, here we will focus on certain aspects of chloroplast transformation related to vaccine production against human diseases.

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Article Introduction

Enhanced chloroplast transgene expression in a nuclear mutant of Chlamydomonas Laure Michelet, Linnka Lefebvre-Legendre, Sarah E. Burr, Jean-David Rochaix, Michel Goldschmidt-Clermont Chloroplast transformation in microalgae offers great promise for the production of proteins of pharmaceutical interest or for the development of novel biofuels. For many applications, high level expression of transgenes is desirable. We have transformed the chloroplast of Chlamydomonas reinhardtii with two genes, acrV and vapA, which encode antigens from the fish pathogen Aeromonas salmonicida. The promoters and 5′ untranslated regions of four chloroplast genes were compared for their ability to drive expression of the bacterial genes. The highest levels of expression were obtained when they were placed under the control of the cis-acting elements from the psaA-exon1 gene. The expression of these chimeric genes was further increased when a nuclear mutation that affects a factor involved in psaA splicing was introduced in the genetic background of the chloroplast transformants. Accumulation of both the chimeric mRNAs and the recombinant proteins was dramatically increased, indicating that negative feedback loops limit the expression of chloroplast transgenes. Our results demonstrate the potential of manipulating anterograde signalling to alter negative regulatory feedback loops in the chloroplast and improve transgene expression.

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Article Introduction

Contained and high-level production of recombinant protein in plant chloroplasts using a temporary immersion bioreactor Franck Michoux, Niaz Ahmad, James McCarthy, Peter J. Nixon Chloroplast transformation is a promising approach for the commercial production of recombinant proteins in plants. However, gene containment still remains an issue for the large-scale cultivation of transplastomic plants in the field. Here, we have evaluated the potential of using tobacco transplastomic cell suspensions for the fully contained production of a modified form of the green fluorescent protein (GFP+) and, a vaccine antigen, fragment C of tetanus toxin (TetC). Expression of these proteins in cell suspension cultures (and calli) was much less than in leaves, reaching 0.5%–1.5% of total soluble protein (TSP), but still produced 2.4–7.2 mg/L of liquid culture. Much better expression levels were achieved with a novel protein production platform in which transgenic cell suspension cultures were placed in a temporary immersion bioreactor in the presence of Thidiazuron to initiate shoot formation. GFP+ yield reached 660 mg/L of bioreactor (33% TSP), and TetC accumulated to about 95 mg/L (8% TSP).

This new production platform, combining the rapid generation of transplastomic cell suspension cultures and the use of temporary immersion bioreactors, is a promising route for the fully contained low-cost production of recombinant proteins in chloroplasts.

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Article Introduction

Low-cost production of proinsulin in tobacco and lettuce chloroplasts for injectable or oral delivery of functional insulin and C-peptide Diane Boyhan, Henry Daniell Current treatment for type I diabetes includes delivery of insulin via injection or pump, which is highly invasive and expensive. The production of chloroplast-derived proinsulin should reduce cost and facilitate oral delivery. Therefore, tobacco and lettuce chloroplasts were transformed with the cholera toxin B subunit fused with human proinsulin (A, B, C peptides) containing three furin cleavage sites (CTB-PFx3). Transplastomic lines were confirmed for site-specific integration of transgene and homoplasmy. Old tobacco leaves accumulated proinsulin up to 47% of total leaf protein (TLP). Old lettuce leaves accumulated proinsulin up to 53% TLP. Accumulation was so stable that up to âˆź40% proinsulin in TLP was observed even in senescent and dried lettuce leaves, facilitating their processing and storage in the field. Based on the yield of only monomers and dimers of proinsulin (3 mg/g leaf, a significant underestimation), with a 50% loss of protein during the purification process, one acre of tobacco could yield up to 20 million daily doses of insulin per year. Proinsulin from tobacco leaves was purified up to 98% using metal affinity chromatography without any His-tag. Furin protease cleaved insulin peptides in vitro. Oral delivery of unprocessed proinsulin bioencapsulated in plant cells or injectable delivery into mice showed reduction in blood glucose levels similar to processed commercial insulin. C-peptide should aid in long-term treatment of diabetic complications including stimulation of nerve and renal functions. Hyperexpression of functional proinsulin and exceptional stability in dehydrated leaves offer a low-cost platform for oral and injectable delivery of cleavable proinsulin.

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Article Introduction

Optimization of the expression of the HIV fusion inhibitor cyanovirin-N from the tobacco plastid genome Zouhair Elghabi, Daniel Karcher, Fei Zhou,Stephanie Ruf, Ralph Bock Plants with transgenic plastid (chloroplast) genomes represent a promising production platform in molecular farming, mainly because of the plastids’ potential to accumulate foreign proteins to very high levels and the increased biosafety conferred by the maternal mode of plastid inheritance. Although some transgenes can be expressed to extraordinarily high levels, the expression of others has been unsuccessful. Lack of detectable transgene expression is usually attributable to either RNA instability or protein instability. Here, we have investigated the possibilities to improve the production of a pharmaceutical protein that is difficult to express in chloroplasts: the HIV-1 fusion inhibitor cyanovirin-N (CV-N). Testing various N-terminal and C-terminal fusions to peptide sequences from two proteins known to accumulate to high levels in transgenic plastids (GFP and the protein antibiotic PlyGBS), we show that both low mRNA stability and low protein stability contribute to the lack of detectable CV-N expression in chloroplasts. Both problems can be alleviated by N-terminal fusions to the CV-N coding region, thus highlighting a suitable strategy for optimization of plastid transgene expression.

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Article Introduction

Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability Oscar N. Ruiz, Derry Alvarez, Cesar Torres, Laura Roman, Henry Daniell Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy.

Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183 000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 ÎźM mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioniens in plants for mercury phytoremediation.

Chloroplast genetic engineering approach is useful to express metalscavenging proteins for phytoremediation.

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Article Introduction

A synthetic gene increases TGFβ3 accumulation by 75-fold in tobacco chloroplasts enabling rapid purification and folding into a biologically active molecule Martin F. Gisby, Philip Mellors, Panagiotis Madesis, Marianne Ellin, Hugh Laverty,Sharon O’Kane, Mark W. J. Ferguson, Anil Day Human transforming growth factor-β3 (TGFβ3) is a new therapeutic protein used to reduce scarring during wound healing. The active molecule is a nonglycosylated, homodimer comprised of 13-kDa polypeptide chains linked by disulphide bonds. Expression of recombinant human TGFβ3 in chloroplasts and its subsequent purification would provide a sustainable source of TGFβ3 free of animal pathogens. A synthetic sequence (33% GC) containing frequent chloroplast codons raised accumulation of the 13-kDa TGFβ3 polypeptide by 75-fold compared to the native coding region (56% GC) when expressed in tobacco chloroplasts. The 13-kDa TGFβ3 monomer band was more intense than the RuBisCO 15-kDa small subunit on Coomassie blue–stained SDS-PAGE gels. TGFβ3 accumulated in insoluble aggregates and was stable in leaves of different ages but was not detected in seeds. TGFβ3 represented 12% of leaf protein and appeared as monomer, dimer and trimer bands on Western blots of SDS-PAGE gels. High yield and insolubility facilitated initial purification and refolding of the 13kDa polypeptide into the TGFβ3 homodimer recognized by a conformationdependent monoclonal antibody. The TGFβ3 homodimer and trace amounts of monomer were the only bands visible on silver-stained gels following purification by hydrophobic interaction chromatography and cation exchange chromatography. N-terminal sequencing and electronspray ionization mass spectrometry showed the removal of the initiator methionine and physical equivalence of the chloroplast-produced homodimer to standard TGFβ3. Functional equivalence was demonstrated by near-identical dose–response curves showing the inhibition of mink lung epithelial cell proliferation. We conclude that chloroplasts are an attractive production platform for synthesizing recombinant human TGFβ3.

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Article

Immunogenicity of chloroplast-derived HIV-1 p24 and a p24-Nef fusion protein following subcutaneous and oral administration in mice Nuria Gonzalez-Rabade, Edward G. McGowan, Fei Zhou, Matthew S. McCabe, Ralph Bock, Philip J. Dix, John C. Gray, Julian K-C. Ma High-level expression of foreign proteins in chloroplasts of transplastomic plants provides excellent opportunities for the development of oral vaccines against a range of debilitating or fatal diseases. The HIV-1 capsid protein p24 and a fusion of p24 with the negative regulatory protein Nef (p24-Nef) accumulate to ∼4% and ∼40% of the total soluble protein of leaves of transplastomic tobacco (Nicotiana tabacum L.) plants. This study has investigated the immunogenicity in mice of these two HIV-1 proteins, using cholera toxin B subunit as an adjuvant. Subcutaneous immunization with purified chloroplast-derived p24 elicited a strong antigen-specific serum IgG response, comparable to that produced by Escherichia coli-derived p24.

Oral administration of a partially purified preparation of chloroplastderived p24-Nef fusion protein, used as a booster after subcutaneous injection with either p24 or Nef, also elicited strong antigen-specific serum IgG responses. Both IgG1 and IgG2a subtypes, associated with cell-mediated Th1 and humoral Th2 responses, respectively, were found in sera after subcutaneous and oral administration. These results indicate that chloroplast-derived HIV-1 p24-Nef is a promising candidate as a component of a subunit vaccine delivered by oral boosting, after subcutaneous priming by injection of p24 and/or Nef.

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Article

Improved heterologous protein expression in the chloroplast of Chlamydomonas reinhardtii through promoter and 5′ untranslated region optimization Beth A. Rasala, Machiko Muto, Joseph Sullivan, Stephen P. Mayfield Microalgae have the potential to be a valuable biotechnological platform for the production of recombinant proteins. However, because of the complex regulatory network that tightly controls chloroplast gene expression, heterologous protein accumulation in a wild-type, photosynthetic-competent algal chloroplast remains low. High levels of heterologous protein accumulation have been achieved using the psbA promoter/5′ untranslated region (UTR), but only in a psbA-deficient genetic background, because of psbA/D1-dependent auto-attenuation.

Here, we examine the effect of fusing the strong 16S rRNA promoter to the 5′ UTR of the psbA and atpA genes on transgene expression in the chloroplast of Chlamydomonas reinhardtii. We show that fusion of the 16S promoter had little impact on protein accumulation from the psbA 5′ UTR in a psbA-deficient genetic background. Furthermore, the 16S/psbA promoter/UTR fusion was silenced in the presence of wildtype levels of D1 protein, confirming that the psbA 5′ UTR is the primary target for D1-dependent auto-repression. However, fusion of the 16S promoter to the atpA 5′ UTR significantly boosts mRNA levels and supports high levels of heterologous protein accumulation in photosynthetic-competent cells. The 16S/atpA promoter/UTR drove LUXCT protein accumulation to levels close to that of psbA in a psbA− background, and drove expression of a human therapeutic protein to levels only twofold lower than the psbA 5′ UTR. The 16S/atpA promoter/UTR combination should have utility for heterologous protein production when expression from a photosynthetic-competent microalgal strain is required.

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Article

Tobacco chloroplast transformants expressing genes encoding dehydroascorbate reductase, glutathione reductase, and glutathione-S-transferase, exhibit altered anti-oxidant metabolism and improved abiotic stress tolerance Bénédicte Le Martret, Miranda Poage,Karen Shiel, Gregory D. Nugent, Philip J. Dix One approach to understanding the Reactive Oxygen Species (ROS)scavenging systems in plant stress tolerance is to manipulate the levels of antioxidant enzyme activities. In this study, we expressed in the chloroplast three such enzymes: dehydroascorbate reductase (DHAR), glutathione-S-transferase (GST) and glutathione reductase (GR).

Homoplasmic chloroplast transformants containing either DHAR or GST, or a combination of DHAR:GR and GST:GR were generated and confirmed by molecular analysis. They exhibited the predicted changes in enzyme activities, and levels or redox state of ascorbate and glutathione. Progeny of these plants were then subjected to environmental stresses including methyl viologen (MV)-induced oxidative stress, salt, cold and heavy metal stresses. Overexpression of these different enzymes enhanced salt and cold tolerance. The simultaneous expression of DHAR:GR and GST:GR conferred MV tolerance while expression of either transgene on its own didn’t. This study provides evidence that increasing part of the antioxidant pathway within the chloroplast enhances the plant’s ability to tolerate abiotic stress.

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Article

Tobacco plastidial thioredoxins as modulators of recombinant protein production in transgenic chloroplasts Ruth Sanz-Barrio, Alicia Fernández-San Millán, Patricia CorralMartínez, José M. Seguí-Simarro, Inmaculada Farran

Thioredoxins (Trxs) are small ubiquitous disulphide proteins widely known to enhance expression and solubility of recombinant proteins in microbial expression systems. Given the common evolutionary heritage of chloroplasts and bacteria, we attempted to analyse whether plastid Trxs could also act as modulators of recombinant protein expression in transgenic chloroplasts. For that purpose, two tobacco Trxs (m and f) with different phylogenetic origins were assessed. Using plastid transformation, we assayed two strategies: the fusion and the co-expression of Trxs with human serum albumin (HSA), which was previously observed to form large protein bodies in tobacco chloroplasts. Our results indicate that both Trxs behave similarly as regards HSA accumulation, although they act differently when fused or co-expressed with HSA. Trxs–HSA fusions markedly increased the final yield of HSA (up to 26% of total protein) when compared with control lines that only expressed HSA; this increase was mainly caused by higher HSA stability of the fused proteins. However, the fusion strategy failed to prevent the formation of protein bodies within chloroplasts. On the other hand, the co-expression constructs gave rise to an absence of large protein bodies although no more soluble HSA was accumulated. In these plants, electron micrographs showed HSA and Trxs co-localization in small protein bodies with fibrillar texture, suggesting a possible influence of Trxs on HSA solubilization. Moreover, the in vitro chaperone activity of Trx m and f was demonstrated, which supports the hypothesis of a direct relationship between Trx presence and HSA aggregates solubilization in plants co-expressing both proteins.

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Article

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes Mohammad T. Waheed, Nadja Thönes, Martin Müller, Syed W. Hassan, Johanna Gottschamel, Elke Lössl, Hans-Peter Kaul, Andreas G. Lössl Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs.

Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate.

Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate coadministration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.

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