Translation of HLA-HIV associations to the cellular level: HIV adapts to inflate CD8 T cell responses against Nef and HLA-adapted variant epitopes

Abstract

Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 individuals from the same cohort, which gave rise to 874 HLA-HIV associations in genetic analyses, taking into account autologous viral sequences and individual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA-HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for diversity coverage approaches in HIV vaccine design.

Figure 1. Summary of immunological investigations of 874 HLA-HIV associations in the study

Figure 2. Distribution of epitopes predicted (A), tested and targeted (B) across the HIV-1 proteome

(A)Numbers of putative and known epitopes around sites of HLA associated HIV adaptation are shown for each protein. (B)The distribution of epitopes tested and eliciting positive IFNγ responses spanning the nine viral proteins is shown. The numbers of epitopes are adjusted for varying lengths of different proteins by dividing them by the number of codons in each protein.

Figure 3. Magnitude of epitope-specific IFNγ responses (median) across all HIV-1 proteins for putative, A-list and B-list epitopes

The plots display the median magnitude of all non-zero IFNγ responses for each epitope. Protein medians are annotated; N^I/N^T ≡ number epitopes/number epitopes tested. No responses were detected against the four VPU peptides tested.

Figure 4. Immunogenic epitopes identified in IFNγ ELISpot assays

Epitopes that were tested in five or more individuals and had a positive response rate of at least 40% are shown on the x-axis with the number of responders (■) and non-responders (□) shown on the y-axis. The restricting HLA allele is shown above each bar. The majority of epitopes were identified from the central region of Nef.

Figure 5. Summary of results of testing in IFNγ ELISpot assays

Figure 6. Proportions of responders and median magnitude of IFNγ responses for putative and known epitopes tested in five or more individuals

The plots indicate sample medians (heavy white lines), interquartile ranges (limits of black boxes) and value ranges (limits of whiskers). Median (IQR) of number of individuals tested per epitope are: putative epitopes = 10 (6-16), A list epitopes = 10 (6-16) and B list epitopes = 10.5 (7-13); Median (IQR) of numbers of responses per epitope are: putative epitopes = 2 (1-5), A list epitopes = 5 (2-9) and B list epitopes = 4 (2-7). The proportions of responding individuals with epitope specific responses ≥ 100 SFUs/10⁶ PBMCs (A) and median magnitudes of all non-zero IFNγ responses (B) elicited by putative epitopes were significantly lower in comparison to known epitopes with HLA-HIV associations (Mann-Whitney test).

Figure 7. Comparison of responses to non-adapted (■) and adapted (□) epitopes

Number of positive IFNγ responses to adapted and non-adapted epitopes across the HIV-1 proteome. Numbers are adjusted for varying protein length by dividing by number of codons.

Figure 8. Marked inflation of Nef epitopes and Nef-specific IFNγ responses associated with HIV diversity

The relative proportions of protein-specific epitopes in a single monovalent subtype B HIV-1 strain (A) compared with the proportions if all possible variants are included (B). C shows the proportion of protein-specific IFNγ responses derived from this study.