How to Use Pharmacogenomic Information to Reduce the Risk of Experiencing Adverse Events

effective tools to prevent severe COVID19, hospitalisation, and death against all variants of concern, even if the quality of evidence greatly varies, depending on the vaccines considered. However, the Authors claim that the main questions remain the need of a booster dose, the waning immunity, and the not last long duration of immunity.

Moreover, the current vaccines appear not able to elicit an oral mucosal immunity, thus failing in limiting virus acquisition upon its entry through this route. In fact, the induction of mucosal front-line immunity has the potential to mitigate current and future respiratory virus epidemics and pandemics. In addition, since mucosal immunity maximises the individual protection against breakthrough infections, it contributes to decrease the disease severity and the risk for virus transmission upon infections .

Altogether, the immune response elicited by current COVID-19 vaccines, including both T-cell responses and neutralising antibody production, could be improved, within certain limits, by optimising the primary antigen sequence, the doses, the adjuvants, the immunisation regimes, the manufacturing processes, etc.

However, in my opinion, the main problem consists in the particular conformation of the protein S selected, as protective antigen, both in live or inert, attenuated immunogens, or as protein translated from mRNA-based vaccines.

As above discussed, the S protein shows a high binding affinity both to the angiotens in converting enzyme 2 (ACE2) receptor and to soluble ACE2.

The ACE2 is a multifunctional protein that plays a crucial role in several mechanisms, including: • regulation of renin-angiotensin, kinin-kallikrein systems, and amyloid peptides; • transport of neutral amino acids; • amino acid homeostasis regulation; • interaction with integrins.

Anyway, one of the main roles of ACE2 is to bind and inactive the potent vasopressine peptide angiotensin II (Ang II) by removing the C-terminal phenylalanine residue to yield Ang1–7.This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule.

According to antigen-antibody interaction theory, it is known that antibodies recognise antigens based on their structure as well as content.

At the same way, in biological systems, in addition to antibodies targeting antigens, all biomolecules, including enzymes catalysing their substrates, regulatory proteins binding DNA, and receptor-ligand complex systems, specifically recognise each other through the so-called mechanism “lock and key”. The ligand recognition can be very specific, allowing the binding to only a small part of an antigen or ligand (known as the epitope), and discriminating between highly similar epitopes.

In natural infections, when an immunocompetent animal is exposed to a T-dependent microbial antigen, it will develop an array of antibodies that each bind to a separate epitope of the antigen.

At the same way, the complex immune sensory system is able to discriminate self- from non-self-antigens, and autoimmune diseases represent the result of the breakdown in any of the mechanisms that maintain unresponsiveness to self (a state known as self-tolerance).

At light of these considerations, since that both S protein of SARS-Cov-2 and the human protein Ang-II, are capable of selectively recognising and binding the ACE 2 receptor, it can be supposed that S protein shares any sequence identity region with Ang-II.

In addition, S protein has been shown to share sequences similar to alveolar lung surfactant proteins, and this molecular mimicry can represent one of the main mechanisms of SARS-Cov-2 infection responsible for inducing the production of self-reactive antibodies in infected host Ref. Letter from Editor: On the molecular determinants of the SARSCov2 attack - Clinical Immunology 215 (2020) - 108426.

Consequently In any case, the limited immunogenic properties of the S protein used in COVID vaccines could be mainly due to any conformational similarity to Ang-II, and this similarity could explain both the numerous cases of infection occurring among the regularly vaccinated people and the number dramatically increasing of subjects who have been infected from SARS-Cov-2 for a second time.

This hypothesis can be easily tested by analysing, in a detailed and comparative manner, the conformational dynamics and the protein sequence alignment, to identify specific regions of similarity between S protein, Ang-II and other endogenous components, including surfactant proteins.

Altogether, these considerations should address the current studies both to further investigate the molecular characteristics of S protein potentially associated to its low immunogenicity and to identify other immunogenic SARSCoV-2 epitopes.

The reverse vaccinology approach allowed to select a further list of SARSCoV-2 antigens as promising candidates for vaccine development.

Among these, there are the virus nucleocapsid components and the membranegly coprotein, that have been reported to exhibit both pathogenic and immunogenic properties.

In particular, membrane glycoprotein, which is the most abundant viral

Mucosal COVID-19 vaccines represent a promise and challenge, mainly due to the needlefree administration, and the ability to induce both mucosal (IgA) and circulating (IgG and IgA) antibod- ies, as well as T-cell responses.

protein in SARS-CoV-2 and plays a crucial role in viral pathogenesis, has been reported to have a highly immunogenic domain, mainly consisting in its C-terminal region.

In addition, specific non-structural polyproteins (NSP), including NSP3, NSP4, and NSP6, involved in the viral adhesion and host invasion, appear to exhibit high protective immunogenicity. Currently, NSPs represent one of the most promising alternative COVID-19 vaccine candidates, since they have already been successfully used to induce protective immunity against other pathogens, including flaviviruses, hepatitis C virus, and HIV-1.

Peptide-based phage display technology can represent an inexpensive and versatile tool for large-scale screening methodology for the identification of potential vaccine candidates against SARS-Cov-2 as well as other microbial infections.

The ability to produce combinatorial peptide libraries with a highly diverse pool of randomised ligands, allows screening and selecting, through an affinity selection-based strategy called “biopanning”, a wide variety of targets with high antigenicity and immunogenicity. Also, peptide libraries can be panned against the antiserum of convalescent individuals, in order to identify both additional protective virus antigens and novel peptidomimetics of pathogen-related epitopes. Combined with bioinformatic approaches capable of identifying immunogenic epitopes, this strategy provides a promising framework for developing a more effective SARS-Cov-2 vaccine.

In addition, the phage-based vaccines can represent a valid strategy for vaccine design, due to being highly stable under harsh environmental conditions, and potent adjuvant capacities.

Phage display vaccines are obtained by expressing multiple copies of an antigen on the surface of immunogenic phage particles, thereby eliciting a powerful and effective immune response.

Recent advances consider the possibility of producing a peptide-directed phage particle that can be administered in an aerosolised form by inhalation. A combinatorial approach for liganddirected pulmonary delivery as a unique route for systemic targeting in vaccination showed to elicit, both in mice and nonhuman primates, a systemic and specific humoral response.

It is also hoped that efforts towards the development of mucosal vaccines consisting in peptide-directed phage particles leading to secretory IgA antibody production can provide a very strong first line of defense, by preventing the virus entry into the mucosa. It is becoming increasingly clear that local mucosal immune responses, that include, in addition to IgA antibodies, local mucosal IgG production, and cytotoxic T lymphocyte activation, are very important for protection against COVID-19 disease. However, one of the main challenges in designing phage display vaccines consists into assure that, following the insertion of the different SARS-CoV-2 antigen epitopes into the phage particles, the structure of these peptides is maintained intact as in the original three-dimensional conformation. Therefore, mucosal COVID-19 vaccines represent a promise and challenge, mainly due to the needle-free administration, and the ability to induce both mucosal (IgA) and circulating (IgG and IgA) antibodies, as well as T-cell responses. Mucosal immune responses could also contribute to reduce the frequency of asymptomatic SARS-CoV-2 positive individuals, who represent an important factor in triggering and sustaining infection chains.

In conclusion, the most efficient strategy to combat the current pandemic COVID-19 and save millions of human lives worldwide remains active immunisation. To date, current vaccines have been shown to reduce both mortality and the incidence of severe COVID19. Since, there are still some aspects, concerning their efficacy, immunogenicity and safety to be focused, the future studies will have toanalyse and comparegeneticvariants andmutations, and evaluate the conformational similarity degree between the viral S proteins and the human ACE2 receptor and other endogenous proteins, in addition to identify additional immunogenic epitopes and develop other alternative vaccines. References are available at www.pharmafocusasia.com

AUTHOR BIO

Maria Elsa Gambuzza works in Italian Ministry of Health. Maria has Degree in Biology and Postdegree in Medical Microbiology and Virology; Environmental Parassitology with PhD in: “Clinical Neuroscience; “Microbial Biotechnology“” Work’s medicine”. Maria has an experience in international prophylaxis of infectious diseases and management of pandemic emergencies. She has done research activities in molecular mimicry, and innovative vaccine strategies.

Luca Soraci works as a geriatrician and a Research Assistant at the Laboratory of Pharmacoepidemiology and Biostatistics at the National Institute for Research and Care of the Elderly (IRCCS INRCA), Italy. He has been collaborating in several studies concerning the role of SARS-CoV-2 and impact of COVID-19 in older patients with frailty and multimorbidity, as well as vaccination strategies for protecting such frail individuals. Other fields of interest include study of chronic kidney disease, multimorbidity, anticholinergic drug burden, and frailty.

Genetic testing can be used to reduce the risk of experiencing adverse reactions. Seldom is one single gene responsible for altering a drug’s benefitrisk balance. This is more often due to multiple genetic and non-genetic factors, thus increasing the complexity and limiting the utility of a widespread use of pharmacogenomics. Widespread use of genotyping and artificial intelligence can overcome these limits.

Giovanni Furlan, Fellow, International Society of Pharmacovigilance

Pharmacogenomics is the study of the genes, their structure, function, polymorphisms, transcription, and translation, how they interact with each other and with environment. The main goal of pharmacogenomics is to understand how genetic variants can alter a drug’s benefit-risk balance. Genetic differences that are likely to be of most relevance to drugs and that can alter their benefit-risk balance pertain to four broad categories: • Genes that affect proteins involved in the metabolism of a drug, its active metabolites, or their transport. Variants affecting these genes are mainly asso-

ciated with pharmacokinetic alterations and might require a drug dose adjustment • Genes that code for proteins that are intended or unintended drug targets and other pathways related to a drug’s pharmacological effect. Variants of these genes can shift the relationship between the drug dose and its effect, thus requiring a change in the administered drug dose • Genes not directly involved in the drug’s pharmacology that can alter an

“off-target” protein. In this instance idiosyncratic reactions (e.g., immune mediated adverse reactions) can occur • Genes that influence disease susceptibility or progression. Variants of these genes can become therapeutic targets.

These genetic variants are estimated to account for 15 per cent-20 per cent of the different desirable and undesirable responses that patients have following drug intake and for some drugs they can account for up to 95 per cent of interindividual variability.

Multiple genetic and non-genetic factors can increase the risk of experiencing an adverse reaction: the example of warfarin

It is rare that one single gene variant is responsible for altering the metabolism of a drug or for causing an idiosyncratic adverse reaction. More often, as in the case of warfarin, (a drug used for the prevention of thrombosis and thromboembolism) multiple genetic and environmental factors are involved. This drug is of particular interest since it has a narrow therapeutic index, the inter-individual variability of the dose required to achieve the required therapeutic effect can vary up to 20 times and an excessive dose increases the risk of bleeding, while a subtherapeutic dose increases the risk thromboembolism.

To identifywhich genetic variants are of importance, boththe metabolic steps and the mechanism of action of warfarin need to be considered.

As shown in figure 1, warfarin is a racemic mixture, and the S-enantiomer is more potent in inhibiting vitamin K epoxide reductase (VKORC1) as compared to the R-enantiomer. (Note: VKORC1 reduces vitamin K, an essential

cofactor of gamma-glutamylcarboxylase, the enzyme that converts hypofunctional clotting factors to functional). It is, therefore, not surprising that genetic variants affecting cytochrome 2C9 (CYP2C9), the enzyme that metabolises the S-enantiomer, have a greater influence in altering the dose of warfarin that is needed to achieve the therapeutic effect as compared to genetic variants of CYP1A2 and 3A4, since these cytochromes are responsible for the metabolism of the less potentR-enantiomer. The variability in the needed warfarin dose is partially explained by the more than 60 known variants affecting CYP2C9 and, even if not all these variants are important, their prevalence varies from one ethnicity to another. CYP2C9 *2 variant, for example, requires a lower warfarin dose, but its frequency is around 13-14 per cent in Caucasians and less than 1 per cent in Asian patients.CYP2C9*5,*6,*8 and *11 polymorphisms also require a lower warfarin dose, but their prevalence varies from 10 per cent in Africans to less than 1 per cent in Caucasians. There is another variant in a non-coding region of CYP2C9DNA that requires to reduce warfarin dose only in African Americans. The reason is not clear, but it could be that only in this population this variant is inherited together with another polymorphism that affects cytochrome metabolism.

Another very important genetic polymorphism reduces by twofold the expression of VKORC1 and, therefore, requires a lower dose of warfarin. The frequency of this allele is around 90 per cent in Asiatic patients, while it is only around 10 per cent in African Americans. Also, the gene coding for CYP4F2, that metabolises reduced vitamin K to hydroxy vitamin K, has a variant(CYP4F2*3) that lowers the concentration of this cytochrome thus increasing active vitamin K concentration and requiring a higher warfarin dose. As for the previously detailed variants, also this allele has a different prevalence in different ethnicities: it is presence ranges from 24 per cent in Caucasians to 7 per cent in African Americans.

Finally, calumelin, a protein that inhibits gamma-glutamyl carboxylase, is characterised by a variant that requires African Americans to take a lower warfarin dose to achieve the targeted therapeutic effect.

The multitude of variants that influence the required warfarin dose and whose prevalence varies in the different ethnicities, explains why traditional dosing algorithms, that only look at CYP2C9 *2 and *3 together with the above mentioned VKORC variant, might not be optimal for all patients regardless of their ethnicity. Algorithms should rather be adapted to consider which variants can influence the required drug dose in the various ethnicities. However, more precise dosing algorithms should also consider non-genetic factors. In fact, in the case of warfarin also elderly age, high weight, amount of vitamin K taken with the diet, drugdrug interactions involving the drug and tobacco smoking alter the required dose.

TERM DEFINITION GLOSSARY

Allele

A gene’s variant Enantiomer Either of the two molecules that are the mirror image of each other (and that form a racemic mixture) Gene variant A change in the most common DNA sequence Human leukocyte antigen A gene that codes for proteins responsible for the regulation of the immune system Odds ratio A statistical measure of the association between two events. Polymorphism The presence of variant forms of a DNA sequence Racemic mixture A mixture of two molecules that mirror images of each other and that are not superimposable

Reverse transcriptase inhibitor

A class of drugs that inhibit an enzyme (called reverse transcriptase) that syntheses DNA from an RNA template. This enzyme is necessary to viruses that insert a copy of their RNA into the host cell they invade. Therapeutic index The ratio of the amount of a drug that causes a therapeutic effect to the mount that causes toxicity Transcription The biological process of copying a segment of DNA into messenger RNA Translation The biological process of synthesizing a protein from messenger MRNA

Factors that influence the need for genetic testing

Since many genetic variants can influence the effect of a drug and it would be unpractical and expensive to perform genetic testing for all the involved alleles, the logical question is when genetic testing should be performed and for which variants. To answer this question, the strength of evidence associating an allele to an adverse event and the expected consequences of the adverse reaction need to be determined. Therefore, it is necessary to evaluatethe severity and seriousness of the adverse event associated with the genetic variants, since, for example, it is not worthwhile to investigate the contribution of a genetic polymorphisms to a mild localised erythema associated with a drug used for cancer. Other aspects that have to be considered are the magnitude of the adverse event increased frequency, the probability with which a genetic test will indicate the patients have the genetic variant of interest among those who actually have the variant (i.e., the analytical sensitivity), the accuracy with which a genetic test predicts the clinical disorder (i.e. the clinical validity of testing)and the different frequencies in the various ethnicities of all the genetic variants contributing to an adverse event.All these factors contribute to the genetic test positive predictive value that tells us what is the probability that a patient taking the drug of interest and with a positive test for a specific genetic allele will experience the adverse event.