MHC Supertypes Research Articles

Immunogenetics, sylvatic plague and its vectors: insights from the pathogen reservoir Mastomys natalensis in Tanzania

Yersinia pestis is a historically important vector-borne pathogen causing plague in humans and other mammals. Contemporary zoonotic infections with Y. pestis still occur in sub-Saharan Africa, including Tanzania and Madagascar, but receive relatively little attention. Thus, the role of wildlife reservoirs in maintaining sylvatic plague and spillover risks to humans is largely unknown. The multimammate rodent Mastomys natalensis is the most abundant and widespread rodent in peri-domestic areas in Tanzania, where it plays a major role as a Y. pestis reservoir in endemic foci. Yet, how M. natalensis’ immunogenetics contributes to the maintenance of plague has not been investigated to date. Here, we surveyed wild M. natalensis for Y. pestis vectors, i.e., fleas, and tested for the presence of antibodies against Y. pestis using enzyme-linked immunosorbent assays (ELISA) in areas known to be endemic or without previous records of Y. pestis in Tanzania. We characterized the allelic and functional (i.e., supertype) diversity of the major histocompatibility complex (MHC class II) of M. natalensis and investigated links to Y. pestis vectors and infections. We detected antibodies against Y. pestis in rodents inhabiting both endemic areas and areas considered non-endemic. Of the 111 nucleotide MHC alleles, only DRB*016 was associated with an increased infestation with the flea Xenopsylla. Surprisingly, we found no link between MHC alleles or supertypes and antibodies of Y. pestis. Our findings hint, however, at local adaptations towards Y. pestis vectors, an observation that more exhaustive sampling could unwind in the future.

Identification of immunodominant T cell epitopes induced by natural Zika virus infection.

Zika virus (ZIKV) is a flavivirus primarily transmitted by Aedes species mosquitoes, first discovered in Africa in 1947, that disseminated through Southeast Asia and the Pacific Islands in the 2000s. The first ZIKV infections in the Americas were identified in 2014, and infections exploded through populations in Brazil and other countries in 2015/16. ZIKV infection during pregnancy can cause severe brain and eye defects in offspring, and infection in adults has been associated with higher risks of Guillain-Barré syndrome. We initiated a study to describe the natural history of Zika (the disease) and the immune response to infection, for which some results have been reported. In this paper, we identify ZIKV-specific CD4+ and CD8+ T cell epitopes that induce responses during infection. Two screening approaches were utilized: an untargeted approach with overlapping peptide arrays spanning the entire viral genome, and a targeted approach utilizing peptides predicted to bind human MHC molecules. Immunoinformatic tools were used to identify conserved MHC class I supertype binders and promiscuous class II binding peptide clusters predicted to bind 9 common class II alleles. T cell responses were evaluated in overnight IFN-γ ELISPOT assays. We found that MHC supertype binding predictions outperformed the bulk overlapping peptide approach. Diverse CD4+ T cell responses were observed in most ZIKV-infected participants, while responses to CD8+ T cell epitopes were more limited. Most individuals developed a robust T cell response against epitopes restricted to a single MHC class I supertype and only a single or few CD8+ T cell epitopes overall, suggesting a strong immunodominance phenomenon. Noteworthy is that many epitopes were commonly immunodominant across persons expressing the same class I supertype. Nearly all of the identified epitopes are unique to ZIKV and are not present in Dengue viruses. Collectively, we identified 31 immunogenic peptides restricted by the 6 major class I supertypes and 27 promiscuous class II epitopes. These sequences are highly relevant for design of T cell-targeted ZIKV vaccines and monitoring T cell responses to Zika virus infection and vaccination.

EPIPOX: Immunoinformatic Characterization of the Shared T-Cell Epitome between Variola Virus and Related Pathogenic Orthopoxviruses.

Concerns that variola viruses might be used as bioweapons have renewed the interest in developing new and safer smallpox vaccines. Variola virus genomes are now widely available, allowing computational characterization of the entire T-cell epitome and the use of such information to develop safe and yet effective vaccines. To this end, we identified 124 proteins shared between various species of pathogenic orthopoxviruses including variola minor and major, monkeypox, cowpox, and vaccinia viruses, and we targeted them for T-cell epitope prediction. We recognized 8,106, and 8,483 unique class I and class II MHC-restricted T-cell epitopes that are shared by all mentioned orthopoxviruses. Subsequently, we developed an immunological resource, EPIPOX, upon the predicted T-cell epitome. EPIPOX is freely available online and it has been designed to facilitate reverse vaccinology. Thus, EPIPOX includes key epitope-focused protein annotations: time point expression, presence of leader and transmembrane signals, and known location on outer membrane structures of the infective viruses. These features can be used to select specific T-cell epitopes suitable for experimental validation restricted by single MHC alleles, as combinations thereof, or by MHC supertypes.

Effects of heterozygosity and MHC diversity on patterns of extra-pair paternity in the socially monogamous scarlet rosefinch

Extra-pair copulation without apparent direct benefits is an evolutionary puzzle that requires indirect fitness benefits to females to explain its ubiquity in socially monogamous mating systems. Using wild scarlet rosefinches (Carpodacus erythrinus), we tested if genetic benefits in the form of global (microsatellite) heterozygote advantage, adaptive genes (major histocompatibility complex), or complementary genes (using both markers) were responsible for female extra-pair mate choice, while considering that the benefits of mate choice may be conditional on female genotype. We found no evidence for assortative or relatedness-based mating (complementary genes), but higher MHC diversity, microsatellite heterozygosity, and condition were significantly related to male extra-pair paternity (EPP) success. In contrast, female probability of having extra-pair offspring decreased with increasing heterozygosity. Interestingly, extra-pair and within-pair males had higher heterozygosity than their female mates and extra-pair males had higher MHC supertype diversity. The only genetic difference between extra-pair and within-pair offspring was lower variance in MHC allelic diversity within extra-pair offspring, providing limited support for indirect genetic fitness benefits for the markers tested. Offspring had both higher neutral heterozygosity and number of MHC supertypes than adults, as well as significant identity disequilibrium, potentially suggesting that mates are chosen to increase offspring diversity in the period of the present study. Overall, our results point to an EPP heterozygote advantage for males, especially when involving less heterozygous females, and suggest that heterozygosity effects on reproduction may differ between the sexes.

Mhc‐linked survival and lifetime reproductive success in a wild population of great tits

Major histocompatibility complex (Mhc) genes are frequently used as a model for adaptive genetic diversity. Although associations between Mhc and disease resistance are frequently documented, little is known about the fitness consequences of Mhc variation in wild populations. Further, most work to date has involved testing associations between Mhc genotypes and fitness components. However, the functional diversity of the Mhc, and hence the mechanism by which selection on Mhc acts, depends on how genotypes map to the functional properties of Mhc molecules. Here, we test three hypotheses that relate Mhc diversity to fitness: (i) the maximal diversity hypothesis, (ii) the optimal diversity hypothesis and (iii) effect of specific Mhc types. We combine mark-recapture methods with analysis of long-term breeding data to investigate the effects of Mhc class I functional diversity (Mhc supertypes) on individual fitness in a wild great tit (Parus major) population. We found that the presence of three different Mhc supertypes was associated with three different components of individual fitness: survival, annual recruitment and lifetime reproductive success (LRS). Great tits possessing Mhc supertype 3 experienced higher survival rates than those that did not, whereas individuals with Mhc supertype 6 experienced higher LRS and were more likely to recruit offspring each year. Conversely, great tits that possessed Mhc supertype 5 had reduced LRS. We found no evidence for a selective advantage of Mhc diversity, in terms of either maximal or optimal supertype diversity. Our results support the suggestion that specific Mhc types are an important determinant of individual fitness.

Abstract 3529: A direct mass spectrometry approach for HPV T cell epitope identification

Abstract To rationally design therapeutic cancer vaccines, it is important to know which T cell epitopes are present on cancer cells. Malignant transformation affects the cellular antigen processing machinery, thus not every epitope derived from intracellular proteins is necessarily presented by MHC molecules on the cancer cell surface. Up to now, T cell epitopes have mostly been defined by indirect methods, such as screening peptide libraries for MHC binders, and subsequent lymphocyte reactivity and cytotoxicity assays. However, all these assays with externally added peptides do not answer the question whether a candidate epitope is naturally processed and presented on the malignant cell. This can now be elucidated by using highly sensitive mass spectrometry approaches. For this study, we chose high-risk HPV-driven cancers as a model system. The induction and maintenance of the malignant phenotype in these tumors are dependent on two viral oncoproteins, E6 and E7, which are therefore present in all stages of HPV-driven cancers. We here aimed to assess the presence of T cell epitopes restricted by the major MHC class I supertypes, namely HLA-A1, A2, A3, A11, A24, B7, and B15, on HPV16-transformed cells. Prospective epitopes from the HPV16 E6 and E7 proteins for the above MHC supertypes were identified by high-throughput in silico epitope predictions, employing several web-based prediction algorithms. These peptides were tested for MHC binding in cellular binding assays on B-LCL of the respective HLA type. Finally, MHC-peptide complexes were immunoprecipitated from HPV16-transformed cells, and the presence of candidate epitopes was analyzed by nano-HPLC-MS2 and MS3 mass spectrometry. Binding of the majority of predicted peptides to the respective HLA allele was verified in the cellular assays. Several novel HPV16 MHC binders were identified. We optimized the immunoprecipitation protocol for epitope yield. Mass spectrometry analysis was successfully established and validated for HLA-A2, and is currently performed for the other supertypes. In conclusion, we show that ascertaining the actual cellular presentation of HPV T cell epitopes is feasible. As the included HLA supertypes have a cumulative population prevalence of >95%, the resulting set of peptides may be used for the formulation of a widely applicable therapeutic HPV vaccine, or for immunomonitoring purposes in other HPV immunotherapy studies. * These authors contributed equally to this study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3529. doi:1538-7445.AM2012-3529

Mice, men and MHC supertypes

Evaluation of: Tan TG, Mui E, Cong H et al. Identification of T. gondii epitopes, adjuvants, and host genetic factors that influence protection of mice and humans. Vaccine 28(23), 3977–3989 (2010).Mice are still the most used model organism in initial phases of vaccine design. Bioinformatics is becoming increasingly more important in vaccine development, both for the design of novel simplified epitope-based vaccines and also in order to understand the specific immune response of selected vaccine formulations. Toxoplasma gondii, an intracellular parasite, causes severe neurologic and ocular disease in congenitally infected and immunocompromised individuals. No protective vaccine exists against human toxoplasmosis. However, studies with mice have revealed immunodominant cytotoxic T lymphocyte epitopes originating from type II strains. These verified epitopes might be useful in human vaccines as the peptide binding repertoire of H-2Ld MHC and MHCs belonging to the HLA-B07 supertype are very similar. Here, the results obtained using these epitopes in BALB/c as well as transgenic HLA-B*0702 mice are discussed. The stunning results obtained from the use of refined computational methods for the prediction of cytotoxic T lymphocyte epitopes are also discussed. The results highlight some important issues regarding both the use of mice but also the choice of bioinformatics methods in vaccine development.

Compatibility counts: MHC-associated mate choice in a wild promiscuous primate

The mechanisms and temporal aspects of mate choice according to genetic constitution are still puzzling. Recent studies indicate that fitness is positively related to diversity in immune genes (MHC). Both sexes should therefore choose mates of high genetic quality and/or compatibility. However, studies addressing the role of MHC diversity in pre- and post-copulatory mate choice decisions in wild-living animals are few. We investigated the impact of MHC constitution and of neutral microsatellite variability on pre- and post-copulatory mate choice in both sexes in a wild population of a promiscuous primate, the grey mouse lemur (Microcebus murinus). There was no support for pre-copulatory male or female mate choice, but our data indicate post-copulatory mate choice that is associated with genetic constitution. Fathers had a higher number of MHC supertypes different from those of the mother than randomly assigned males. Fathers also had a higher amino acid distance to the females' MHC as well as a higher total number of MHC supertypes and a higher degree of microsatellite heterozygosity than randomly assigned males. Female cryptic choice may be the underlying mechanism that operates towards an optimization of the genetic constitution of offspring. This is the first study that provides support for the importance of the MHC constitution in post-copulatory mate choice in non-human primates.

An evolutionary approach to major histocompatibility diversity based on allele supertypes

Human leukocyte antigens are traditionally classified by serologic or molecular techniques into a bewildering variety of alleles. It is generally believed that this allelic diversity is maintained by selection pressures for inbreeding avoidance and/or maximal immune system diversity. While the usual antigen-based classification of individual alleles may be most appropriate in the artificial situation of tissue transplantation, we hypothesize that a functional classification based on allele supertypes may represent a more biologically relevant way to view MHC diversity in the contexts of mate choice and disease pathogenesis. Furthermore, immune system diversity could be quantitatively estimated by calculating a Supertype Diversity Index (SDI) which is the number of different MHC supertypes possessed by an individual. This hypothesis generates a number of testable predictions. First, it predicts that a reduced inherited diversity of MHC allele supertypes may predispose to the development of malignancies because of a decreased native ability to present different tumor-associated antigens. Furthermore, specific autoimmune diseases may be associated with the presence or absence of a particular MHC supertype rather than a particular MHC haplotype. In transplant medicine, it is possible that unmatched alleles may trigger a weaker foreign antigen response if they are matched by allele supertype. Finally, there have been several studies documenting dissortative mating in humans for dissimilar MHC alleles. We predict that natural selection should favor maximization of the heterozygosity of allele supertypes instead of the heterozygosity of individual alleles and that the previously observed dissortative mating may actually be an adaptive strategy to maximize allele supertype diversity.

Neutral versus adaptive genetic variation in parasite resistance: importance of major histocompatibility complex supertypes in a free-ranging primate

Current discussions in evolutionary ecology and conservation genetics focus on the relative importance of using selective neutral markers or markers of coding genes to identify adaptive and evolutionary relevant processes. Genetic diversity might be particularly important in immune genes (e.g., in genes of the major histocompatibility complex, MHC), which are influencing pathogen and parasite resistance. We investigated the effects of neutral versus adaptive genetic variation in parasite resistance in a natural population of fat-tailed dwarf lemurs (Cheirogaleus medius). No association between neutral overall individual genetic diversity and parasite load could be detected. In 149 individuals, we identified 50 MHC class II alleles of the functionally important duplicated DRB locus. The investigation of the functional importance of immune gene (MHC) diversity and parasite selection in natural populations is often problematic due to extensive polymorphism in the MHC genes and restrictions in available sample sizes. Here, for the first time we applied an approach that has been developed in human medical studies. Eleven MHC class II supertypes were identified based on shared antigen-binding similarities. The number of individual MHC supertypes had no influence on the nematode burden. However, we found evidence for a specific MHC supertype (supertype 1) that was linked to infected individuals, a higher number of different nematode infections and high intensity of infection per individual. Moreover, one rare MHC supertype (supertype 7) was revealed to be advantageous with respect to parasite burden. Thus, our results add evidence to the small body of studies that show significant associations between specific MHC constitutions and naturally occurring parasites in the complexity of natural populations.

Production of Functionally Active Recombinant HLA Molecules Representing the Supertypes B62 and B58 and the Generation of Corresponding Peptide‐Binding Assays and Tetramers

Rationale: Major histocompatibility complex class I (MHC I) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic CD8+ T cells. The goal of the human MHC project is to predict the binding strength of any given peptide/MHC complex. This prediction allows the design of peptide‐based vaccines. The prediction requires representative binding data from MHC alleles from all the nine HLA supertypes. Here, we describe the genetic construction, protein production and purification as well as the establishment‐binding assays for two recombinant MHC supertype alleles, HLA‐B*1501 and HLA‐B*5801. Methods: Using the Quikchange Multisite Directed Mutagenesis Kit (Stratagene), codon‐optimized genes encoding HLA‐B*1501 and HLA‐B*5801 are created. The two MHC I molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. The binding (KD) of natural T‐cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (RIAs) and enzyme‐linked immunosorbent assays (ELISAs). The MHC molecules are biotinylated during expression. Results: The expression of MHC I resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of KD for various peptide ligands ranging from strong binders (KD < 50 nM) to low binders (KD > 5 µM). Tetramerization is visualized by SDS‐PAGE. Conclusion: An effective method for the production of highly pure MHC I molecules has been applied to HLA‐B*1501 and HLA‐B*5801, and RIA and ELISA binding assays for those alleles have been established, showing the binding of various peptide ligands to the MHC I molecules.