Identifying HLA supertypes by learning distance functions

MOTIVATION: The development of epitope-based vaccines crucially relies on the ability to classify Human Leukocyte Antigen (HLA) molecules into sets that have similar peptide binding specificities, termed supertypes. In their seminal work, Sette and Sidney defined nine HLA class I supertypes and claimed that these provide an almost perfect coverage of the entire repertoire of HLA class I molecules. HLA alleles are highly polymorphic and polygenic and therefore experimentally classifying each of these molecules to supertypes is at present an impossible task. Recently, a number of computational methods have been proposed for this task. These methods are based on defining protein similarity measures, derived from analysis of binding peptides or from analysis of the proteins themselves. RESULTS: In this paper we define both peptide derived and protein derived similarity measures, which are based on learning distance functions. The peptide derived measure is defined using a peptide-peptide distance function, which is learned using information about known binding and non-binding peptides. The protein derived similarity measure is defined using a protein-protein distance function, which is learned using information about alleles previously classified to supertypes by Sette and Sidney (1999). We compare the classification obtained by these two complimentary methods to previously suggested classification methods. In general, our results are in excellent agreement with the classifications suggested by Sette and Sidney (1999) and with those reported by Buus et al. (2004). The main important advantage of our proposed distance-based approach is that it makes use of two different and important immunological sources of information-HLA alleles and peptides that are known to bind or not bind to these alleles. Since each of our distance measures is trained using a different source of information, their combination can provide a more confident classification of alleles to supertypes.     

Predicted impact of the viral mutational landscape on the cytotoxic response against SARS-CoV-2

The massive assessment of immune evasion due to viral mutations that increase COVID-19 susceptibility can be computationally facilitated. The adaptive cytotoxic T response is critical during primary infection and the generation of long-term protection. Here, potential HLA class I epitopes in the SARS-CoV-2 proteome were predicted for 2,915 human alleles of 71 families using the netMHCIpan EL algorithm. Allele families showed extreme epitopic differences, underscoring genetic variability of protective capacity between humans. Up to 1,222 epitopes were associated with any of the twelve supertypes, that is, allele clusters covering 90% population. Next, from all mutations identified in ~118,000 viral NCBI isolates, those causing significant epitope score reduction were considered epitope escape mutations. These mutations mainly involved non-conservative substitutions at the second and C-terminal position of the ligand core, or total ligand removal by large recurrent deletions. Escape mutations affected 47% of supertype epitopes, which in 21% of cases concerned isolates from two or more sub-continental areas. Some of these changes were coupled, but never surpassed 15% of evaded epitopes for the same supertype in the same isolate, except for B27. In contrast to most supertypes, eight allele families mostly contained alleles with few SARS-CoV-2 ligands. Isolates harboring cytotoxic escape mutations for these families co-existed geographically within sub-Saharan and Asian populations enriched in these alleles according to the Allele Frequency Net Database. Collectively, our findings indicate that escape mutation events have already occurred for half of HLA class I supertype epitopes. However, it is presently unlikely that, overall, it poses a threat to the global population. In contrast, single and double mutations for susceptible alleles may be associated with viral selective pressure and alarming local outbreaks. The integration of genomic, geographical and immunoinformatic information eases the surveillance of variants potentially affecting the global population, as well as minority subpopulations.     

Advantage of rare HLA supertype in HIV disease progression

The highly polymorphic human leukocyte antigen (HLA) class I molecules help to determine the specificity and repertoire of the immune response. The great diversity of these antigen-binding molecules confers differential advantages in responding to pathogens, but presents a major obstacle to distinguishing HLA allele-specific effects. HLA class I supertypes provide a functional classification for the many different HLA alleles that overlap in their peptide-binding specificities. We analyzed the association of these discrete HLA supertypes with HIV disease progression rates in a population of HIV-infected men. We found that HLA supertypes alone and in combination conferred a strong differential advantage in responding to HIV infection, independent of the contribution of single HLA alleles that associate with progression of the disease. The correlation of the frequency of the HLA supertypes with viral load suggests that HIV adapts to the most frequent alleles in the population, providing a selective advantage for those individuals who express rare alleles.     

Widespread impact of HLA restriction on immune control and escape pathways of HIV-1

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.     

A HLA-DRB supertype chart with potential overlapping peptide binding function

HLA-DRB alleles are class II alleles that are associated with CD4+ T-cell immune response. DRB alleles are polymorphic and currently there are about 622 named in the IMGT/HLA sequence database. Each allele binds short peptides with high sensitivity and specificity. However, it has been suggested that majority of HLA alleles can be covered within few HLA supertypes, where different members of a supertype bind similar peptides showing distinct repertoires. Definition of DRB supertypes using binding data is limited to few (about 29) known alleles (< 5% of all known DRB alleles). Hence, we describe a strategy using structurally defined virtual pockets to group all known DRB alleles with regard to their overlapping peptide binding specificity.     

Population coverage by HLA class-I restricted cytotoxic T-lymphocyte epitopes

Vaccination using cytotoxic T-lymphocyte (CTL) epitopes has become a widely used immunization strategy, especially because their structure makes them an attractive alternative to the delivery of whole proteins as immunogens. Nonetheless, their use is limited, in particular because of their specificity, being recognized only by cognate HLA alleles. The potential for immunizing a substantial portion of an ethnically diverse population using a modest number of peptides has been aided by the identification of HLA supertypes. However, the derivation of epitopes is often guided by methods that do not guarantee cross-reactivity, so we consider the feasibility of providing vaccine coverage to a multi-ethnic population under different assumptions. In particular, two large datasets are used to estimate the number of peptides needed to provide > or =90% group-specific coverage of a multiethnic population, when specificity is assumed to be either to a single serologic or molecular type. These assumptions are evaluated utilizing a clinically important epitope repertoire derived from two human cytomegalovirus proteins, and data on the in vitro memory response elicited by these peptides is presented. In summary, our combined theoretical and empiric studies suggest that 90% coverage of some ethnic groups is attainable with 11 uniquely defined HLA-restricted CTL epitopes. The derivation of four or more additional CTL epitopes is needed to attain 90% coverage of Blacks or Asians, the minimally covered groups. Ninety percent coverage of all major ethnic groups in a multi-ethnic population appears feasible without relying on cross-reactivity, but may require two to three times more CTL epitopes than estimated for serologic data, homogenous populations, or HLA alleles grouped as supertypes.     

Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype

HLA diversity is seen as a major challenge to CTL vaccines against HIV. One current approach focuses on "promiscuous" epitopes, presented by multiple HLA alleles from within the same HLA supertype. However, the effectiveness of such supertype vaccines depends upon the functional equivalence of CTL targeting a particular epitope, irrespective of the restricting HLA. In this study, we describe the promiscuous HIV-specific CTL epitopes presented by alleles within the B7 supertype. Substantial differences were observed in the ability of CTL to select for escape mutation when targeting the same epitope but restricted by different HLA. This observation was common to all six promiscuous B7 epitopes identified. Moreover, with one exception, there were no significant differences in the frequency, magnitude, or immunodominance of the CTL responses restricted by different HLA alleles to explain these discrepancies. This suggests that the unique peptide/MHC complexes generated by even closely related HLA induce CTL responses that are qualitatively different. This hypothesis is supported by additional differences observed between CTL targeting identical epitopes but restricted by different HLA: first, the occurrence of distinct, HLA-specific escape mutation; second, the recruitment of distinct TCR repertoires by particular peptide/MHC complexes; and, third, significant differences in the functional avidity of CTL. Taken together, these data indicate that significant functional differences exist between CTL targeting identical epitopes but restricted by different, albeit closely related HLA. These findings are of relevance to vaccine approaches that seek to exploit HLA supertypes to overcome the problem of HLA diversity.     

Classification of A1- and A24-supertype molecules by analysis of their MHC-peptide binding repertoires

At the functional level, the majority of human leukocyte antigen (HLA) class I MHC variants can be classified into about ten different major groups, or supertypes, characterized by overlapping peptide binding motifs and repertoires. Previous studies have detailed the peptide binding specificity of the HLA A2, A3, B7, and B44 supertypes, and predicted, on the basis of MHC pocket structures, known motifs, or the sequence of T cell epitopes, the existence of the HLA A1 and A24 supertypes. Direct experimental validation of the A1 and A24 supertypes, however, has been lacking. In the current study, the peptide-binding repertoires and main anchor specificities of several common HLA A molecules (A*0101, A*2301, A*2402, A*2601, A*2902, and A*3002) predicted to be members of the A1 or A24 supertypes were analyzed and defined using single amino acid substituted peptides and a large peptide library. Based on the present findings, the A1 supertype includes A*0101, A*2601, A*2902, and A*3002, whereas the A24 supertype includes A*2301 and A*2402. Interestingly, A*2902 is associated with a motif and peptide binding repertoire that overlaps significantly with those of all of the A1- and A24-supertype molecules studied, representing—to our knowledge—the first report of significant cross-reactivity among molecules belonging to different supertypes.     

Identification and characterization of merozoite surface protein 1 epitope

Malaria is an important tropical infection which urgently requires intervention of an effective vaccine. Antigenic variations of the parasite and allelic diversity of the host are main problems in the development of an effective malaria vaccine. Cytotoxic T lymphocytes (CTL) directed against Plasmodium falciparum­derived antigens are shown to play an important role for the protection against malaria. The merozoite surface protein 1 (MSP1) is expressed in all the four life-cycle stages of Plasmodium falciparum and did not find any sequence similarity to human and mouse reference proteins. MSP1 is a known target of the immune response and a single CTL epitope binding to the HLA­A*0201 is available for merozoite form. Here, we report the results from the computational characterization of MSP1, precursor (1720 residue) and screening of highest scoring potential CTL epitopes for 1712 overlapping peptides binding to thirty four HLA class­I alleles and twelve HLA class­I supertypes (5 HLA­A and 7 HLA­B) using bioinformatics tools. Supertypes are the clustered groups of HLA class­I molecules, representing a sets of molecules that share largely overlapping peptide binding specificity. The prediction results for MSP1 as adhesin and adhesin-like in terms of probability is 1.0. Results also show that MSP1 has orthologs to other related species as well as having non allergenicity and single transmembrane properties demonstrating its suitability as a vaccine candidate. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

Promiscuous CTL recognition of viral epitopes on multiple human leukocyte antigens: biological validation of the proposed HLA A24 supertype

Multiple HLA class I alleles can bind peptides with common sequence motifs due to structural similarities in the peptide binding cleft, and these groups of alleles have been classified into supertypes. Nine major HLA supertypes have been proposed, including an A24 supertype that includes A*2301, A*2402, and A*3001. Evidence for this A24 supertype is limited to HLA sequence homology and/or similarity in peptide binding motifs for the alleles. To investigate the immunological relevance of this proposed supertype, we have examined two viral epitopes (from EBV and CMV) initially defined as HLA-A*2301-binding peptides. The data clearly demonstrate that each peptide could be recognized by CTL clones in the context of A*2301 or A*2402; thus validating the inclusion of these three alleles within an A24 supertype. Furthermore, CTL responses to the EBV epitope were detectable in both A*2301(+) and A*2402(+) individuals who had been previously exposed to this virus. These data substantiate the biological relevance of the A24 supertype, and the identification of viral epitopes with the capacity to bind promiscuously across this supertype could aid efforts to develop CTL-based vaccines or immunotherapy. The degeneracy in HLA restriction displayed by some T cells in this study also suggests that the dogma of self-MHC restriction needs some refinement to accommodate foreign peptide recognition in the context of multiple supertype alleles.     

Efficient peptide-MHC-I binding prediction for alleles with few known binders

MOTIVATION: In silico methods for the prediction of antigenic peptides binding to MHC class I molecules play an increasingly important role in the identification of T-cell epitopes. Statistical and machine learning methods in particular are widely used to score candidate binders based on their similarity with known binders and non-binders. The genes coding for the MHC molecules, however, are highly polymorphic, and statistical methods have difficulties building models for alleles with few known binders. In this context, recent work has demonstrated the utility of leveraging information across alleles to improve the performance of the prediction. RESULTS: We design a support vector machine algorithm that is able to learn peptide-MHC-I binding models for many alleles simultaneously, by sharing binding information across alleles. The sharing of information is controlled by a user-defined measure of similarity between alleles. We show that this similarity can be defined in terms of supertypes, or more directly by comparing key residues known to play a role in the peptide-MHC binding. We illustrate the potential of this approach on various benchmark experiments where it outperforms other state-of-the-art methods. AVAILABILITY: The method is implemented on a web server: http://cbio.ensmp.fr/kiss. All data and codes are freely and publicly available from the authors.     

Resistance-associated epitopes of HIV-1C—highly probable candidates for a multi-epitope vaccine

Earlier studies have identified a large number of immunogenic epitopes in HIV-1. Efforts are required to prioritize these epitopes in order to identify the best candidates for formulating an effective multi-epitope vaccine for HIV. We modeled 155 known cytotoxic T lymphocyte epitopes of HIV-1 subtype C on the 3D structure of HLA-A*0201, HLA-B*2705, and HLA-B*5101 using MODPROPEP, as these alleles are known to be associated with resistance to HIV/slow progression to AIDS. Thirty-six epitopes were identified to bind to all the three HLA alleles with better binding affinity than the control peptides complexed with each HLA allele but not to any of the HLA alleles reported to be associated with susceptibility to HIV infection/rapid progression to disease. As increase in stability of the epitope-HLA complex results in increased immunogenicity, the short-listed epitopes could be suitable candidates for vaccine development. Twenty of the 36 epitopes were polyfunctional in nature adding to their immunological relevance for vaccine design. Further, 9 of the 20 polyfunctional epitopes were found to bind to all three resistance-associated HLA alleles using an additional method, adding worth to their potential as candidates for a vaccine formulation for HIV-1C.     

Human Leukocyte Antigen Class I Supertypes and HIV-1 Control in African Americans

The role of human leukocyte antigen (HLA) class I supertypes in controlling human immunodeficiency virus type 1 (HIV-1) infection in African Americans has not been established. We examined the effects of the HLA-A and HLA-B alleles and supertypes on the outcomes of HIV-1 clade B infection among 338 African American women and adolescents. HLA-B58 and -B62 supertypes (B58s and B62s) were associated with favorable HIV-1 disease control (proportional odds ratio [POR] of 0.33 and 95% confidence interval [95% CI] of 0.21 to 0.52 for the former and POR of 0.26 and 95% CI of 0.09 to 0.73 for the latter); B7s and B44s were associated with unfavorable disease control (POR of 2.39 and 95% CI of 1.54 to 3.73 for the former and POR of 1.63 and 95% CI of 1.08 to 2.47 for the latter). In general, individual alleles within specific B supertypes exerted relatively homogeneous effects. A notable exception was B27s, whose protective influence (POR, 0.58; 95% CI, 0.35 to 0.94) was masked by the opposing effect of its member allele B*1510. The associations of most B supertypes (e.g., B58s and B7s) were largely explained either by well-known effects of constituent B alleles or by effects of previously unimplicated B alleles aggregated into a particular supertype (e.g., B44s and B62s). A higher frequency of HLA-B genotypic supertypes correlated with a higher mean viral load (VL) and lower mean CD4 count (Pearson''s r = 0.63 and 0.62, respectively; P = 0.03). Among the genotypic supertypes, B58s and its member allele B*57 contributed disproportionately to the explainable VL variation. The study demonstrated the dominant role of HLA-B supertypes in HIV-1 clade B-infected African Americans and further dissected the contributions of individual class I alleles and their population frequencies to the supertype effects.African Americans in the United States have been affected disproportionately by the human immunodeficiency virus type 1 (HIV-1) epidemic. They accounted for only 13% of the U.S. population but for 47% of AIDS cases diagnosed in the United States in 2006 (14, 17). An HIV-1 vaccine would be of enormous benefit to this subpopulation. One strategy for HIV-1 vaccine development seeks to capitalize on the recognition and destruction of HIV-1-infected cells by cytotoxic T lymphocytes (CTLs) (32, 33, 35).CTL recognition is critically dependent on binding, presentation, and cell surface display of a variety of antigenic peptides (epitopes) by extremely polymorphic human leukocyte antigen (HLA) molecules. As the relative importance of increasing numbers of HIV-1 epitopes for individual HLA class I molecules has been recognized (9, 16), the feasibility of developing a vaccine tailored to every epitope and HLA specificity has come to seem more remote. Conceptualization of epitope specificity in terms of broad groupings (supertypes) of HLA molecules may provide a rational but simpler approach to this challenge.Several efforts have succeeded at consolidating the huge spectrum of individual HLA class I alleles into four HLA-A and five HLA-B supertype categories (37-39), based on the ability of different HLA class I molecules to present similar epitopes. Unlike individual allele frequencies, which vary greatly across ethnic groups, all nine supertypes (comprising most, but not all, HLA-A and HLA-B alleles) are present in all human populations. This more uniform representation of allele groups may confer an advantage in the form of balancing selection (38).HLA alleles within one supertype that share epitope binding specificities might be expected to demonstrate similar associations with HIV-1 outcomes or vaccine response; conversely, alleles within a supertype that differ substantially in function might be expected to show differential responses to natural infection or vaccines designed on the basis of supertype (2). Functional heterogeneity of alleles within the supertypes could be due to differences in the class I alleles themselves (e.g., variable epitope avidity or tolerance to viral mutations), in host background (genetic epistasis), or in the virus (e.g., clade-related epitope specificities or viral escape).Previous work has detected associations between the HLA class I supertypes and HIV-1 outcomes for Caucasians with clade B infections (34, 44) and for native Africans with clade A or C infections (23, 28). We are unaware of studies among African Americans in the context of clade B HIV-1 infection or of any systematic attempt to tease apart the independent contributions of supertypes and their individual class I alleles to HIV-1 outcomes. Here we document the frequencies of the HLA class I alleles and supertypes in the Reaching for Excellence in Adolescent Care and Health (REACH) and HIV Epidemiologic Research Study (HERS) cohorts, and we report the relative effects of those alleles and supertypes on the degree of HIV-1 disease control.     

Broadly immunogenic HLA class I supertype-restricted elite CTL epitopes recognized in a diverse population infected with different HIV-1 subtypes

The genetic variations of the HIV-1 virus and its human host constitute major obstacles for obtaining potent HIV-1-specific CTL responses in individuals of diverse ethnic backgrounds infected with different HIV-1 variants. In this study, we developed and used a novel algorithm to select 184 predicted epitopes representing seven different HLA class I supertypes that together constitute a broad coverage of the different HIV-1 strains as well as the human HLA alleles. Of the tested 184 HLA class I-restricted epitopes, 114 were recognized by at least one study subject, and 45 were novel epitopes, not previously described in the HIV-1 immunology database. In addition, we identified 21 "elite" epitopes that induced CTL responses in at least 4 of the 31 patients. A majority (27 of 31) of the study population recognized one or more of these highly immunogenic epitopes. We also found a limited set of 9 epitopes that together induced HIV-1-specific CTL responses in all HIV-1-responsive patients in this study. Our results have important implications for the validation of potent CTL responses and show that the goal for a vaccine candidate in inducing broadly reactive CTL immune responses is attainable.     

Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism

 Herein, we review the epitope approach to vaccine development, and discuss how knowledge of HLA supertypes might be used as a tool in the development of such vaccines. After reviewing the main structural features of the A2-, A3-, B7-, and B44- supertype alleles, and biological data demonstrating their immunological relevance, we analyze the frequency at which these supertype alleles are expressed in various ethnicities and discuss the relevance of those observations to vaccine development. Next, the existence of five new supertypes (A1, A24, B27, B58, and B62) is reported. As a result, it is possible to account for the predominance of all known HLA class I with only nine main functional binding specificities. The practical implications of this finding, as well as its relevance to understanding the functional implication of MHC polymorphism in humans, are discussed.     

Definition of supertypes for HLA molecules using clustering of specificity matrices

Major histocompatibility complex (MHC) proteins are encoded by extremely polymorphic genes and play a crucial role in immunity. However, not all genetically different MHC molecules are functionally different. Sette and Sidney (1999) have defined nine HLA class I supertypes and showed that with only nine main functional binding specificities it is possible to cover the binding properties of almost all known HLA class I molecules. Here we present a comprehensive study of the functional relationship between all HLA molecules with known specificities in a uniform and automated way. We have developed a novel method for clustering sequence motifs. We construct hidden Markov models for HLA class I molecules using a Gibbs sampling procedure and use the similarities among these to define clusters of specificities. These clusters are extensions of the previously suggested ones. We suggest splitting some of the alleles in the A1 supertype into a new A26 supertype, and some of the alleles in the B27 supertype into a new B39 supertype. Furthermore the B8 alleles may define their own supertype. We also use the published specificities for a number of HLA-DR types to define clusters with similar specificities. We report that the previously observed specificities of these class II molecules can be clustered into nine classes, which only partly correspond to the serological classification. We show that classification of HLA molecules may be done in a uniform and automated way. The definition of clusters allows for selection of representative HLA molecules that can cover the HLA specificity space better. This makes it possible to target most of the known HLA alleles with known specificities using only a few peptides, and may be used in construction of vaccines. Supplementary material is available at .     

Anthrax Lethal Factor as an Immune Target in Humans and Transgenic Mice and the Impact of HLA Polymorphism on CD4+ T Cell Immunity

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4⁺ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.     

Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV''s structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies.     

Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.