This is the first study, to our knowledge, to examine the relation of HCV clearance with HLA supertypes. Several different classification systems to define these supertypes have been proposed, based on different methods for determining which HLA molecules share similar binding affinities. Binding affinity is an important factor in determining the ability of T cells to recognize and respond to HCV antigens.4;5 In the current investigation, we examined multiple different well characterized supertypes defined by different laboratories, that were determined based on high-resolution HLA genotype data. Our results therefore provide important information regarding which if any of these supertype classification systems are biologically significant to HCV clearance. It is one of just a few studies that has used clinical outcomes and data to evaluate the relevance these proposed supertype definitions.
Supertypes are controversial since there is no agreed upon method for assessing the binding affinities of HLA molecules. Therefore, different research groups have reached different conclusions regarding the proper clustering of HLA alleles according to their binding affinities, into what are termed supertypes. The strongest association with HCV clearance in our study was with the HLA class II supertype DRB3, as defined by Greenbaum et al.,14
which had a significant inverse association with HCV clearance, even after correction for multiple tests using two separate methods. However, there is laboratory disagreement regarding the definition of the DRB3 supertype. The MHCcluster method, for example, did not find that DRB3 supertype alleles clustered together based on the sequence features defining their peptide binding grooves. We also note that the high-resolution HLA genotyping we conducted in this study population did not include genotyping of DRB3, DRB4 and DRB5 alleles – some of which are included in the DRB3 supertype14
– because these genes are not present in all individuals.
At least one prior study,20
for example, found that the DRB3*02 allele group was positively associated with HCV clearance in a population of 41 White participants, albeit, not among 52 Black participants. WIHS women are majority non-Hispanic Black, and it is not clear from that prior study if the DRB3*02:02 allele – which is included in the DRB3 supertype – was present in that study population.20
Nonetheless, the results of that prior study and of the current investigation suggest that important insights may be obtained from examining the relationship between DRB3, DRB4 and DRB5 alleles and HCV clearance in future studies.
Three supertypes were significantly associated with HCV clearance prior to adjustment for multiple tests, and had q
values of borderline significance when assessed using false discovery rate procedures. Each of these supertypes had a positive association with HCV viral clearance. The DR8 supertype reported by Lund et al.15
was especially noteworthy because the clustering of DR8 alleles was confirmed in MHCcluster analysis and its association with HCV clearance remained statistically significant in multivariable analysis. The DR8 supertype was also noteworthy because it included uncommon HLA alleles (in our population). Thus, it is only through examining these alleles within a supertype that their possible associations with HCV clearance could be recognized in the current analysis.
The association of DR1*01 with HCV clearance was not statistically significant in multivariable analysis, and only two of the three DR1*01 supertype alleles reported by Nielsen et al.16
clustered together in MHCcluster analysis. We therefore view the association of DR1*01 with HCV clearance with some skepticism, pending confirmation in other populations. Four of the eight alleles of the A supertype did not cluster together in MHCcluster analysis, and the association between the A supertype and HCV clearance was also non-significant in multivariable analysis. It is therefore unlikely that the A supertype has a biologic relationship with HCV clearance.
Suprisingly, no HLA class I supertypes were associated with HCV clearance in our study population, even in analyses that did not adjust for multiple testing. While this would seem to argue against the relevance of the HLA class I supertype classifications as they pertain to HCV, we note that HLA class I supertypes defined by Sette and Sidney,21
which we evaluated in the current analysis, were found to have strong associations with HIV RNA and CD4+ T cell levels in data reported by other groups.9;10;11
In addition to the possible associations of supertypes DRB3 and DR8 with HCV clearance, multivariable analysis also showed a significant relation of HCV clearance with two individual alleles, namely, DRB1*07:01 and B*57:03. Two other alleles that had been found to be associated with HCV clearance in our prior investigation6 were no longer significant after adjustment for HLA supertypes. It is notable that B*57:03 retained a significant independent association with HCV clearance in multivariable analysis while B*57:01 did not. While B*57:01 and B*57:03 differ only by two amino acids, recent research related to the interaction of B*57 molecules with the HIV-1 Gag epitope KF11 has shown that the amino acid sequence differences between B*57:01 and B*57:03 result in the recruitment of KF11-specific CD8+ T cells with very different T cell receptor (TCR) repertoires.22
Future studies are needed to determine whether CD8+ T cell responses to B*5701-restricted HCV epitopes also differ from those to B*5703-restricted HCV epitopes. We note, though, that power to detect significant associations for B*57:01 is lower than for B*57:03 in WIHS because B*57:01 is less prevalent in this majority non-Hispanic Black cohort.
Several limitations to the interpretation of these results must be considered. First, we note that there is no single accepted method or criteria for defining HLA supertypes. Thus, different scientific group have grouped different alleles into different supertypes. While we adopted an agnostic approach to the study of HLA supertypes, and assessed several different HLA supertype classification systems, it is possible none of these is wholly correct. The differences in methods used to define supertypes and peptide binding repertoires are reflected in both our calculation of percentage agreement between the studied supertypes and by our additional analysis using MHCcluster. Percentage agreement was high to very high (77% – 99%) between some, but not all, studied class I supertypes but was lower (37%–50%) between the class II supertypes. Further, MHCcluster provided additional alternative groups of HLA alleles that differed from the previously reported supertypes that, in the current study, had associations with HCV clearance. Second, the current study examined HCV antibody and HCV RNA data from the WIHS enrollment visit. Although most spontaneous HCV clearance occurs shortly after HCV infection, clearance of HCV RNA continues to occur over time in a small fraction of those with chronic HCV.23;24
Thus, it is possible that there is a small degree of misclassification in HCV viremia status that would be resolved with repeat HCV RNA testing. Lastly, we cannot exclude the possibility that the supertype associations identified in this study reflect associations of non-HLA genetic variants in linkage disequilibrium with HLA alleles.
Overall, our results suggest that the study of HLA supertypes may inform our understanding of the immunogenetic factors that influence host clearance of HCV infection. It is one of just a few studies that has used clinical outcomes and data to evaluate the relevance these proposed supertype definitions. In particular, if our results are confirmed by other groups, it would suggest that certain HLA class II binding pocket characteristics are associated with reduced (i.e., the binding pockets of the DRB3 supertype) or enhanced (i.e., DR8) host capacity to clear HCV infection, which could point to the types of peptide antigens most likely to be effective in a vaccine (one able to stimulate the immune system to clear HCV if exposed). From a research perspective, HLA supertypes may also be useful in allowing the assessment of uncommon HLA alleles. Thus, the current study suggests that studying HLA alleles according to their biologic characteristics could provide data that are relevant to larger groups of patients and provide new understanding of the role of HLA in health and disease. Conversely, the conflicting data regarding the relatedness of certain alleles suggests that methods for defining HLA binding pocket characteristics remain imperfect. Further research is needed to improve these methods, with clinical outcome studies used to adjudicate which definitions are most biologically relevant.