To our knowledge this is the first large scale “reverse genomics” study in which the results of a genetic analysis were used to directly inform the selection and subsequent testing of particular viral antigens. Overall, we were able to provide immunological support for 190 HLA associated polymorphisms in subtype B HIV-1 as being sites of direct
T cell recognition in vivo
based on ex vivo
IFNγ responses in the appropriate HLA background. This was 58% of the HLA associations tested in the study, representing an increase from only 35% that could have been explained by well characterised published CD8 T cell epitopes alone, prior to any cellular testing. For nine high probability epitopes there was a sufficiently frequent HLA type to show that the most likely HLA restriction of the epitopic response in the cohort matched that of the prediction, and there was sufficient frequency of testing and responses in at least 40% of cases to give the best level of evidence for immunoreactivity. A further set of possible novel epitopes was defined with responses rates of less than 40% but immunoreactivity in at least one individual with the predicted restricting HLA allele. It is notable however, that even well characterised published epitopes which have been used as a standard to validate genetic associations and as reagents in immunological studies had a mean response rate of only 33%. We therefore applied a higher standard of evidence for immunogenicity to potential novel epitopes compared with that observed for known epitopes in this study. The fact that cellular responsiveness was correlated with sequence match of the testing antigen to autologous virus, as shown in other studies (40
) further confirms that viral diversity does influence the specificity of cellular responses within the individual. These data in general provide experimental evidence of a direct biological basis for 190 strongly HLA associated subtype B HIV-1 polymorphisms proteome-wide as sites of HIV-1 adaptation to HLA restricted T cell responses and should serve to guide further epitope characterisation and viral escape studies.
HIV-1 Nef was associated with the greatest number of epitopes which elicited IFNγ over the whole cohort and within individuals. This intense immunogenicity is in keeping with the extreme levels of HLA allele-specific selection in Nef shown in several population-based genetics studies (14
) and mirrors the distribution of well characterised epitopes defined by cellular studies. As the majority of putative epitopes were tested in parallel with their “HLA-adapted” pair, we were also able to determine if any functional consequences of polymorphism within epitopes were apparent in a screening ELISpot assay. Marked reductions in IFNγ responses associated with the polymorphisms were seen in a proportion of cases supporting a role for loss of TCR engagement or HLA-peptide binding in vivo
in these examples. There were also instances in which the HLA-adapted or “escaped” version of the epitope elicited equivalent or higher magnitude responses than the non-adapted versions. In a screening ELISpot with excess peptide concentrations, it is possible that such reactivity patterns result from T cell cross-reactivity, though this appeared to be more likely to occur with Nef epitopes, compared with other proteins and it is not clear why TCR clonotypes specific for Nef epitopes should be inherently more cross-reactive than other TCRs. Furthermore, we did not find that responses which appeared more inherently cross-reactive, as indicated by lack of match with autologous viral sequences were more likely to respond to the adapted epitopes. The general determinants of T cell recognition of viral variants have been explored in other studies (42
). It is important to emphasise we have tested specific epitope pairs based on population-signals of adaptation. In all these specific instances of positive responses to HLA-adapted epitopes, there was strong statistical evidence of the adapted residue being enriched in vivo
in the selecting HLA-context in the original HLA associations analysis, suggesting that in the cellular studies here, either the true differences in peptide avidity were not apparent at excess peptide concentrations and would diverge with serial peptide dilutions, or alternatively that inducing immune responses to adapted variants provides some selective advantage to HIV-1 in vivo
The formation of neo-epitopes as a result of T cell escape has been described in longitudinal studies (44
) but our data suggest this could be reasonably common phenomena. We have described cases of HLA selection leading to high avidity, neo-epitope-specific responses in chronic progressive HIV infection (45
) and have argued that this could represent a way for HIV mutations to promote “bad” immunodominance patterns in chronic infection and drive HIV evolution, not necessarily away from all immune recognition but to enhanced but ineffective recognition of a narrow range of epitopes. In this study, there were several extremely complex patterns of HLA associated polymorphisms in Nef leading to formation of new epitope targets for the same and new HLA alleles which were partially overlapping or distant from the original epitope. Given this combination of high variability with high density of reactive epitopes, including reactivity to many overlapping HLA adapted variants, it is not surprising that Nef epitopes as a proportion of all reactive epitopes are relatively inflated and the IFNγ responses to Nef dominates over all others when considered at a population level. If these Nef responses lead to a relative reduction in targeting of more structurally or functionally constrained proteins such as Gag or Pol in-vivo
, where viral adaptations are more likely to incur fitness costs, then Nef-dominated immunity is conceivably more advantageous to the virus than the host. Since these immunodominance patterns characterise chronic infection where immune control has manifestly failed, recapitulating such immune hierarchies by a vaccine immunogen would seem empirically undesirable, particularly for therapeutic vaccines which could serve to boost this inflation. It is not known whether broad poly-specific vaccine-induced responses prior to viral exposure could block, not block or even enhance particular transmitting viral variants, though this data will emerge as more polyvalent strategies in preventative vaccines advance to clinical trials. Computational strategies which are based on conservation or are polyvalent but seek to minimise the inclusion of rare or unfavourable epitopes or are based on acute transmitted founder viruses may overcome this issue. This set of immunological data could be useful to help in scoring algorithms used to computationally optimize inclusion of important circulating acute variants and perhaps help in excluding particular variants that appear prone to interference or immunodominance phenomena in vivo
Despite the large size of our study cohort, the extreme polymorphism of HLA molecules still limits the degree to which the HLA allele restriction of many responses could be defined analytically and limited stored cellular material on our study cohort subjects precluded further experimental studies. As we assigned a higher ranking to known or high probability HLA restrictions for those epitopes with overlapping HLA restrictions, our study is also inherently conservative, with a bias against assignment of novel epitope responses when there are limited numbers of individuals with that HLA. Furthermore, the use of an epitope prediction program trained on characteristics of known epitopes will inevitably tend to predict epitopes more similar to known epitopes and therefore the 507 associations for which no proximal epitope was predicted cannot be absolutely excluded as sites of true immune selection, particularly given the low mean response rate of even known epitopes shown here. However the additional peptide synthesis, sample and assay requirement of assessing all possible epitopic regions and variants spanning all associations is prohibitive at a practical level.
The challenges of translating the findings of genetic HLA polymorphism association studies to the functional, cellular level are considerable and include the extreme polymorphism of HLA and HIV-1 as discussed above (which necessitates large sample sizes), availability of samples and subjects for immunological testing, limitations in amount and quality of cryopreserved sample material (particularly from pre-treatment time-points), the general heterogeneity of T cell responses between subjects and over time, limitations of ex vivo
based assays and single biomarkers such as IFNγ, and the false discovery rate of associations arising from any genetic associations study. Nevertheless, we were able to expand the base of immunological support for a number of subtype B HIV-1 polymorphisms being sites of immune selection. Apart from providing positive evidence for immune reactivity, the absence of any reactivity for some peptides can also be useful in studies of secondary or compensatory mutational networks. Indeed this study would suggest that only the minority of HLA-HIV polymorphisms (given a q-value cut-off of 0.2, with adjustment for viral phylogeny) can be explained by primary escape or co-targeting of multiple epitopes and many others are more likely secondary mutations affecting structurally or functionally interdependent residues. Mapping the mutational networks or genetic haplotypes in HIV-1 which determine viral fitness under diverse host environments will reveal more about the importance of specific residues in HIV replication and pathogenesis. The information provided here on mutations which are highly HLA allele specific but not within or near epitopes could help those modelling or studying such co-variation networks for both vaccine research and identifying novel ligands for antiviral drugs. More HLA association population-based studies will continue to be done in new and genetically diverse populations (46
) and in larger populations, and the output from several studies have already been used as presumptive sites of viral escape in a number of secondary analyses. However, there is little value in generating vast numbers of hypotheses across these studies unless they are systemically tested and validated at the functional level where possible. The results of such testing can then be used to refine mapping of primary viral escape and compensatory pathways, iterate and validate analytical approaches to genetic studies, and understand the links between HIV polymorphism, adaptation and immunogenicity.