Immunopeptidomics for next-generation bacterial vaccine development

Mass spectrometry-based immunopeptidomics screens allow the identification of bacterial antigens presented on infected cells.These antigens can easily be encoded in next-generation nucleic acid-based vaccines as novel tools to tackle increasing antimicrobial resistance.Mainly infection models with Mycobacterium and Chlamydia have been screened so far, but recent advances in both immunopeptidomics and mRNA vaccine technology prelude an increase in screens on intracellular bacterial infection models. Antimicrobial resistance is an increasing global threat and alternative treatments substituting failing antibiotics are urgently needed. Vaccines are recognized as highly effective tools to mitigate antimicrobial resistance; however, the selection of bacterial antigens as vaccine candidates remains challenging. In recent years, advances in mass spectrometry-based proteomics have led to the development of so-called immunopeptidomics approaches that allow the untargeted discovery of bacterial epitopes that are presented on the surface of infected cells. Especially for intracellular bacterial pathogens, immunopeptidomics holds great promise to uncover antigens that can be encoded in viral vector- or nucleic acid-based vaccines. This review provides an overview of immunopeptidomics studies on intracellular bacterial pathogens and considers future directions and challenges in advancing towards next-generation vaccines. Antimicrobial resistance is an increasing global threat and alternative treatments substituting failing antibiotics are urgently needed. Vaccines are recognized as highly effective tools to mitigate antimicrobial resistance; however, the selection of bacterial antigens as vaccine candidates remains challenging. In recent years, advances in mass spectrometry-based proteomics have led to the development of so-called immunopeptidomics approaches that allow the untargeted discovery of bacterial epitopes that are presented on the surface of infected cells. Especially for intracellular bacterial pathogens, immunopeptidomics holds great promise to uncover antigens that can be encoded in viral vector- or nucleic acid-based vaccines. This review provides an overview of immunopeptidomics studies on intracellular bacterial pathogens and considers future directions and challenges in advancing towards next-generation vaccines. With the advent of antibiotics in the first half of the 20th century, many bacterial diseases lost their devastating dominance over humankind. Commonplace outbreaks of the plague, cholera, and many other bacterial diseases claimed a death toll in the hundreds of millions. A combination of improved sanitary standards, elevated living conditions, and the availability of antibiotics massively reduced these outbreaks; however, (over)use of antibiotics has accelerated the emergence of antimicrobial resistance (AMR). Annual AMR-related deaths are predicted to skyrocket from 700 000 in 2019 to 10 million globally in 2050 [1.Mohammed A. et al.No Time to Wait: Securing the Future from Drug-resistant Infections. UN Interagency Coordination Group on Antimicrobial Resistance (IACG), 2019Google Scholar]. Hence, the World Health Organization (WHO) and the US Centers for Disease Control and Prevention (CDC) have identified pathogenic bacteria for which the AMR situation is particularly dire, including several intracellular pathogens such as Salmonella, Shigella spp. and Mycobacterium tuberculosis (Mtb) [2.Centers for Disease Control and Prevention Antibiotic Resistance Threats in the United States, 2019. CDC, 2019Crossref Google Scholar,3.Tacconelli E. et al.Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis.Lancet Infect. Dis. 2018; 18: 318-327Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar]. In addition to stringent antibiotic stewardship and fast development of novel antibacterial drugs by initiatives like the AMR Action Fund [4.Clancy C.J. Nguyen M.H. Buying Time: The AMR Action Fund and the State of Antibiotic Development in the United States 2020.Open Forum Infect. Dis. 2020; 7ofaa464Crossref PubMed Scopus (0) Google Scholar], vaccines are regarded as highly effective tools to mitigate resistance (recently reviewed in [5.Micoli F. et al.The role of vaccines in combatting antimicrobial resistance.Nat. Rev. Microbiol. 2021; 19: 287-303Crossref PubMed Scopus (1) Google Scholar]). The prophylactic use of bacterial vaccines prevents infections, thereby reducing the need for antibiotic prescription and minimizing selective drug pressure [5.Micoli F. et al.The role of vaccines in combatting antimicrobial resistance.Nat. Rev. Microbiol. 2021; 19: 287-303Crossref PubMed Scopus (1) Google Scholar]. In contrast to antibiotics, antibacterial vaccines remain effective against their target pathogen over time. Furthermore, high vaccination rates create a herd immunity that protects susceptible individuals who cannot be effectively vaccinated, like the elderly, immunosuppressed, or chronically sick people [5.Micoli F. et al.The role of vaccines in combatting antimicrobial resistance.Nat. Rev. Microbiol. 2021; 19: 287-303Crossref PubMed Scopus (1) Google Scholar]. Several antibacterial vaccines have been utilized successfully in the past and are routinely used nowadays, typically providing a high degree of immunity and safety. These include vaccines against tetanus, diphtheria, and pertussis, against Haemophilus influenzae type B (Hib), pneumococcus, as well as meningococcus [6.Mendoza N. et al.Existing antibacterial vaccines.Dermatol. Ther. 2009; 22: 129-142Crossref PubMed Scopus (11) Google Scholar,7.Poolman J.T. Expanding the role of bacterial vaccines into life-course vaccination strategies and prevention of antimicrobial-resistant infections.NPJ Vaccines. 2020; 5: 84Crossref PubMed Scopus (0) Google Scholar]. There are also vaccines against typhoid fever, anthrax, and cholera that are administered in, and for those traveling to, endemic areas. In contrast, for tuberculosis (TB) as a major global health burden, the only readily available and inexpensive vaccine, Bacillus Calmette–Guérin (BCG), does not offer efficient and reliable protection against all forms of the disease [8.Kernodle D.S. Decrease in the effectiveness of Bacille Calmette–Guerin vaccine against pulmonary tuberculosis: a consequence of increased immune suppression by microbial antioxidants, not overattenuation.Clin. Infect. Dis. 2010; 51: 177-184Crossref PubMed Scopus (0) Google Scholar, 9.Mangtani P. et al.Protection by BCG vaccine against tuberculosis: a systematic review of randomized controlled trials.Clin. Infect. Dis. 2014; 58: 470-480Crossref PubMed Scopus (439) Google Scholar, 10.Rana A. et al.Recent Trends in system-scale integrative approaches for discovering protective antigens against mycobacterial pathogens.Front. Genet. 2018; 9: 572Crossref PubMed Scopus (3) Google Scholar]. Efforts to bolster the protection afforded by BCG – by using it in combination with MVA85A (modified vaccinia Ankara 85A) – have not yielded significant improvements [11.Kashangura R. et al.Effects of MVA85A vaccine on tuberculosis challenge in animals: systematic review.Int. J. Epidemiol. 2015; 44: 1970-1981Crossref PubMed Scopus (14) Google Scholar]. Most of the aforementioned vaccines target extracellular bacteria that replicate outside host cells, while only the tuberculosis and typhoid fever vaccines target intracellular pathogens. Being protected from the humoral, and parts of the cellular immune response, as well as several antibiotics inside host cells, intracellular bacteria often present a particular clinical challenge rendering vaccine development even more crucial [12.Kamaruzzaman N.F. et al.Targeting the hard to reach: challenges and novel strategies in the treatment of intracellular bacterial infections.Br. J. Pharmacol. 2017; 174: 2225-2236Crossref PubMed Scopus (47) Google Scholar]. Many intracellular pathogens are also capable of persisting in host cells by entering a dormant state such as Mtb, Salmonella, Bartonella, uropathogenic Escherichia coli or Brucella [13.Byndloss M.X. Tsolis R.M. Chronic bacterial pathogens: mechanisms of persistence.Microbiol. Spectr. 2016; 4https://doi.org/10.1128/microbiolspec.VMBF-0020-2015Crossref PubMed Scopus (21) Google Scholar]. Many recent antibacterial vaccines, like the ones against Hib, meningococcus, and streptococcus, are conjugate vaccines with bacterial polysaccharides bound to carrier proteins. Conjugate vaccines achieve high protective efficacy, even in young children, but suffer from relatively long development times and are mainly targeted against extracellular bacteria [5.Micoli F. et al.The role of vaccines in combatting antimicrobial resistance.Nat. Rev. Microbiol. 2021; 19: 287-303Crossref PubMed Scopus (1) Google Scholar,7.Poolman J.T. Expanding the role of bacterial vaccines into life-course vaccination strategies and prevention of antimicrobial-resistant infections.NPJ Vaccines. 2020; 5: 84Crossref PubMed Scopus (0) Google Scholar,14.Avci F. et al.Glycoconjugates: What it would take to master these well-known yet little-understood immunogens for vaccine development.mSphere. 2019; 4e00520-19Crossref PubMed Scopus (6) Google Scholar]. For intracellular pathogens, the development of effective vaccines has long been held back by a lack of antigen knowledge along with the inability of most vaccine platforms to elicit strong cytotoxic responses. The latter can now be overcome by next-generation vaccines, including viral vector, DNA, and mRNA vaccines that induce both humoral and cytotoxic immune responses and allow relatively fast development times (Box 1, and reviewed in detail in [15.Zhang C. et al.Advances in mRNA vaccines for infectious diseases.Front. Immunol. 2019; 10: 594Crossref PubMed Scopus (175) Google Scholar, 16.Gary E.N. Weiner D.B. DNA vaccines: prime time is now.Curr. Opin. Immunol. 2020; 65: 21-27Crossref PubMed Scopus (32) Google Scholar, 17.Sasso E. et al.New viral vectors for infectious diseases and cancer.Semin. Immunol. 2020; 50: 101430Crossref PubMed Scopus (3) Google Scholar]). All three approaches introduce the genetic information encoding the actual antigen into host cells for intracellular antigen synthesis, resulting in elevated cytotoxic immune responses.Box 1Next-generation nucleic acid-based vaccinesViral vectors (such as adenoviral vectors) encode the antigen(s) to be delivered, which are typically surface-exposed antigens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, within a nonpathogenic viral vehicle. While viral vectors are a flexible platform, allowing the rapid adaption to new antigen targets of emerging pathogens, they might suffer from pre-existing immunity against the vector, neutralizing the vaccine before cellular uptake. This is a particular problem for booster vaccinations using the same viral vector and also the relatively costly cell-based production of viral vectors present a challenge [17.Sasso E. et al.New viral vectors for infectious diseases and cancer.Semin. Immunol. 2020; 50: 101430Crossref PubMed Scopus (3) Google Scholar]. In the case of DNA vaccines, the antigen-encoding DNA is introduced directly or lipid-encapsulated into host cells. Advantages of DNA vaccines include high stability and absence of pre-existing immunity, while reduced expression efficiency has been an issue due to the need of nuclear import before transcription into antigen-encoding mRNA and nuclear export prior to antigen translation [16.Gary E.N. Weiner D.B. DNA vaccines: prime time is now.Curr. Opin. Immunol. 2020; 65: 21-27Crossref PubMed Scopus (32) Google Scholar]. In contrast, mRNA-based vaccines introduce the antigenic information via mRNA to omit nuclear import, allowing direct antigen translation, and like DNA vaccines, they are not subject to pre-existing immune defenses. While it has been known since the nineties that exogenous transcribed mRNA can be used to express proteins in vivo [18.Wolff J.A. et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Google Scholar], only in recent years has mRNA emerged as a promising vaccination platform technology. In vitro transcribed (IVT) mRNA showed its utility as a vaccine format to promote prophylactic protection against infections with viruses such as Zika [19.Richner J.M. et al.Vaccine mediated protection against Zika virus-induced congenital disease.Cell. 2017; 170 (e212): 273-283Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar] and influenza [20.Bahl K. et al.Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses.Mol. Ther. 2017; 25: 1316-1327Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar]. A number of modifications to the vector used to produce the mRNA, as well as to the synthetic mRNA itself, have further ameliorated the biologic properties of the IVT mRNA [21.Steinle H. et al.Concise review: application of in vitro transcribed messenger RNA for cellular engineering and reprogramming: progress and challenges.Stem Cells. 2017; 35: 68-79Crossref PubMed Scopus (0) Google Scholar]. In the past year, the SARS-CoV-2 pandemic greatly accelerated the clinical entry of next-generation vaccines, especially mRNA vaccines conferring over 90% protection against coronavirus disease 2019 (Covid-19) [22.Baden L.R. et al.Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.N. Engl. J. Med. 2021; 384: 403-416Crossref PubMed Scopus (1346) Google Scholar,23.Polack F.P. et al.Safety and efficacy of the BNT162b2 mRNA covid-19 Vaccine.N. Engl. J. Med. 2020; 383: 2603-2615Crossref PubMed Scopus (2180) Google Scholar] This has proven that mRNA-based vaccines have evolved to safe, well-tolerated and very effective vaccines against viral infections. However, their potential in the context of intracellular bacterial infections remains largely unexplored [24.Maruggi G. et al.Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens.Vaccine. 2017; 35: 361-368Crossref PubMed Scopus (41) Google Scholar, 25.Xue T. et al.RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection.Infect. Immun. 2004; 72: 6324-6329Crossref PubMed Scopus (0) Google Scholar, 26.Lorenzi J.C. et al.Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis.BMC Biotechnol. 2010; 10: 77Crossref PubMed Scopus (27) Google Scholar]. mRNA vaccines therefore represent a promising alternative to conventional vaccines based on their high potency, capacity for short development time, as well as their potential for low-cost manufacture and safe administration [27.Pardi N. et al.mRNA vaccines – a new era in vaccinology.Nat. Rev. Drug Discov. 2018; 17: 261-279Crossref PubMed Scopus (803) Google Scholar]. Viral vectors (such as adenoviral vectors) encode the antigen(s) to be delivered, which are typically surface-exposed antigens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, within a nonpathogenic viral vehicle. While viral vectors are a flexible platform, allowing the rapid adaption to new antigen targets of emerging pathogens, they might suffer from pre-existing immunity against the vector, neutralizing the vaccine before cellular uptake. This is a particular problem for booster vaccinations using the same viral vector and also the relatively costly cell-based production of viral vectors present a challenge [17.Sasso E. et al.New viral vectors for infectious diseases and cancer.Semin. Immunol. 2020; 50: 101430Crossref PubMed Scopus (3) Google Scholar]. In the case of DNA vaccines, the antigen-encoding DNA is introduced directly or lipid-encapsulated into host cells. Advantages of DNA vaccines include high stability and absence of pre-existing immunity, while reduced expression efficiency has been an issue due to the need of nuclear import before transcription into antigen-encoding mRNA and nuclear export prior to antigen translation [16.Gary E.N. Weiner D.B. DNA vaccines: prime time is now.Curr. Opin. Immunol. 2020; 65: 21-27Crossref PubMed Scopus (32) Google Scholar]. In contrast, mRNA-based vaccines introduce the antigenic information via mRNA to omit nuclear import, allowing direct antigen translation, and like DNA vaccines, they are not subject to pre-existing immune defenses. While it has been known since the nineties that exogenous transcribed mRNA can be used to express proteins in vivo [18.Wolff J.A. et al.Direct gene transfer into mouse muscle in vivo.Science. 1990; 247: 1465-1468Crossref PubMed Google Scholar], only in recent years has mRNA emerged as a promising vaccination platform technology. In vitro transcribed (IVT) mRNA showed its utility as a vaccine format to promote prophylactic protection against infections with viruses such as Zika [19.Richner J.M. et al.Vaccine mediated protection against Zika virus-induced congenital disease.Cell. 2017; 170 (e212): 273-283Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar] and influenza [20.Bahl K. et al.Preclinical and clinical demonstration of immunogenicity by mRNA vaccines against H10N8 and H7N9 influenza viruses.Mol. Ther. 2017; 25: 1316-1327Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar]. A number of modifications to the vector used to produce the mRNA, as well as to the synthetic mRNA itself, have further ameliorated the biologic properties of the IVT mRNA [21.Steinle H. et al.Concise review: application of in vitro transcribed messenger RNA for cellular engineering and reprogramming: progress and challenges.Stem Cells. 2017; 35: 68-79Crossref PubMed Scopus (0) Google Scholar]. In the past year, the SARS-CoV-2 pandemic greatly accelerated the clinical entry of next-generation vaccines, especially mRNA vaccines conferring over 90% protection against coronavirus disease 2019 (Covid-19) [22.Baden L.R. et al.Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.N. Engl. J. Med. 2021; 384: 403-416Crossref PubMed Scopus (1346) Google Scholar,23.Polack F.P. et al.Safety and efficacy of the BNT162b2 mRNA covid-19 Vaccine.N. Engl. J. Med. 2020; 383: 2603-2615Crossref PubMed Scopus (2180) Google Scholar] This has proven that mRNA-based vaccines have evolved to safe, well-tolerated and very effective vaccines against viral infections. However, their potential in the context of intracellular bacterial infections remains largely unexplored [24.Maruggi G. et al.Immunogenicity and protective efficacy induced by self-amplifying mRNA vaccines encoding bacterial antigens.Vaccine. 2017; 35: 361-368Crossref PubMed Scopus (41) Google Scholar, 25.Xue T. et al.RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection.Infect. Immun. 2004; 72: 6324-6329Crossref PubMed Scopus (0) Google Scholar, 26.Lorenzi J.C. et al.Intranasal vaccination with messenger RNA as a new approach in gene therapy: use against tuberculosis.BMC Biotechnol. 2010; 10: 77Crossref PubMed Scopus (27) Google Scholar]. mRNA vaccines therefore represent a promising alternative to conventional vaccines based on their high potency, capacity for short development time, as well as their potential for low-cost manufacture and safe administration [27.Pardi N. et al.mRNA vaccines – a new era in vaccinology.Nat. Rev. Drug Discov. 2018; 17: 261-279Crossref PubMed Scopus (803) Google Scholar]. The question remains, though, of which bacterial antigen(s) to encode into these vaccine platforms. In contrast to viruses, the potential antigen palette for pathogenic bacteria is significantly more diverse, with typically several thousand genes per bacterium rendering the choice of the correct antigen a challenging task. This is illustrated by the limited coverage of most intracellular bacterial pathogens in the Immune Epitope Database (IEDB, www.iedb.org) as depicted in Figure 1 [28.Vita R. et al.The Immune Epitope Database (IEDB): 2018 update.Nucleic Acids Res. 2019; 47: D339-D343Crossref PubMed Scopus (351) Google Scholar]. Vaccine development has come a long way since Pasteur postulated his classical vaccinology approach of isolating, inactivating, and injecting disease-causing agents to protect patients. Reverse vaccinology, as spearheaded by Rino Rappuoli in 2000, utilizes full pathogen genomes to infer protein sequences followed by in silico predictions to identify secreted and exposed surface proteins as suitable vaccine candidates [29.Rappuoli R. Reverse vaccinology.Curr. Opin. Microbiol. 2000; 3: 445-450Crossref PubMed Scopus (426) Google Scholar]. Recombinantly expressed in E. coli or other systems, the candidate antigens’ immunogenicity is evaluated in animal models prior to clinical studies. This way, a serogroup B meningococcal vaccine, as well as vaccine candidates against group B streptococcal infection, Chlamydia, antibiotic-resistant Staphylococcus aureus, and Streptococcus pneumoniae were successfully developed [30.Sette A. Rappuoli R. Reverse vaccinology: developing vaccines in the era of genomics.Immunity. 2010; 33: 530-541Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar]. Building on the initial reverse vaccinology platform, reverse vaccinology 2.0 was gradually implemented in the past decade harnessing contemporary platforms like B cell repertoire deep sequencing, structure-based antigen design, and mass spectrometry-based proteomics [31.Rappuoli R. et al.Reverse vaccinology 2.0: Human immunology instructs vaccine antigen design.J. Exp. Med. 2016; 213: 469-481Crossref PubMed Scopus (37) Google Scholar]. Immunoproteomics, as a collection of techniques that readily allow the detection of antigenic peptides or proteins, has benefitted greatly from recent quantum leaps in mass spectrometry (MS) and data analysis [32.Fulton K.M. et al.Immunoproteomics methods and techniques.Methods Mol. Biol. 2019; 2024: 25-58Crossref PubMed Scopus (1) Google Scholar]. Methods for investigating extracellular antigens include variants of immunocapture MS for circulating immune complexes (CICs), electroimmunoprecipitation of antibody–antigen complexes, and multiple affinity protein profiling (MAPPing) [32.Fulton K.M. et al.Immunoproteomics methods and techniques.Methods Mol. Biol. 2019; 2024: 25-58Crossref PubMed Scopus (1) Google Scholar]. Also, serological proteome analysis (SERPA) as well as array-based approaches have been utilized for antigen analysis [32.Fulton K.M. et al.Immunoproteomics methods and techniques.Methods Mol. Biol. 2019; 2024: 25-58Crossref PubMed Scopus (1) Google Scholar]. All of these methods, however, require antibody secretion and can therefore only detect targets of the humoral immune response. Antibody-independent detection of antigens can be realized with another type of immunoproteomic methodology known as immunopeptidomics. Immunopeptidomics aims to detect antigens as peptides that are presented on cell surfaces via major histocompatibility complexes (MHCs), commonly termed immunopeptides, MHC-associated peptides, or MHC ligands. These immunopeptides are antigen fragments of 8–25 amino acids that are loaded intracellularly onto MHC complexes before transportation to the cell surface for T cell inspection. The two classes of MHC molecules, MHC class I and II, facilitate presentation of antigens from cytosolic and vacuolar/extracellular antigens, respectively, and they bind different T cell populations with distinct functionalities. While MHC II expression is restricted mainly to professional antigen-presenting cells (APCs), MHC I is present on all nucleated cells and binds CD8 cytotoxic T cells to initiate cell death in compromised cells. The resulting cytotoxic immunity is particularly relevant for many intracellular pathogens and is therefore specifically spotlighted in this review (Box 2).Box 2Cellular immunity based on MHC presentationThe MHC class I pathway features proteasomal digestion of cytosolic intracellular antigens with optional further trimming by other proteases, such as endoplasmic reticulum aminopeptidase 1 and 2 (ERAP1 and 2), to a peptide length of around 8–12 amino acids, enabling loading onto MHC I complexes in the endoplasmic reticulum (ER). Introduction into the ER occurs via the transporter associated with antigen processing (TAP) before binding to MHC class I molecules and transportation of the entire complex to the cell surface via the Golgi apparatus for CD8 T cell surveillance and elimination of pathogen-infected or malignant cells. In healthy cells, class I peptides result from defective ribosomal products (DRiPs) or retiree proteins favoring abundant, short-lived proteins [33.Bassani-Sternberg M. et al.Mass spectrometry of human leukocyte antigen class I peptidomes reveals strong effects of protein abundance and turnover on antigen presentation.Mol. Cell. Proteomics. 2015; 14: 658-673Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar]. While standard proteasomes facilitate continuous monitoring of correct protein synthesis, immunoproteasomes occur in immune cells or upon immune stimulation. 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In infection, PCPS could benefit immunity by circumventing minor antigen mutations providing stable epitopes for persistent immune responses [48.Paes W. et al.Contribution of proteasome-catalyzed peptide cis-splicing to viral targeting by CD8(+) T cells in HIV-1 infection.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 24748-24759Crossref PubMed Scopus (16) Google Scholar].Human MHC molecules are termed human leukocyte antigens (HLAs) and both MHC I and MHC II are each divided into classical and nonclassical HLAs. The three classical MHC I genes (HLA-A, HLA-B, HLA-C) are codominantly expressed with thousands of diverse alleles using variable peptide-binding specificities facilitating presentation of a broad antigen spectrum. Allele frequencies vary greatly, with a dozen alleles covering the majority of most local, homogeneous populations, while different ethnic groups and distant geographical regions present with diverging dominant alleles, having substantial implications for antigen prioritization and vaccine development [49.Gonzalez-Galarza F.F. et al.Allele frequency net database (AFND) 2020 update: gold-standard data classification, open access genotype data a