High frequency of HIV mutations associated with HLA-C suggests enhanced HLA-C-restricted CTL selective pressure associated with an AIDS-protective polymorphism

Abstract

Delayed HIV-1 disease progression is associated with a single nucleotide polymorphism upstream of the HLA-C gene that correlates with differential expression of the HLA-C Ag. This polymorphism was recently shown to be a marker for a protective variant in the 3'UTR of HLA-C that disrupts a microRNA binding site, resulting in enhanced HLA-C expression at the cell surface. Whether individuals with "high" HLA-C expression show a stronger HLA-C-restricted immune response exerting better viral control than that of their counterparts has not been established. We hypothesized that the magnitude of the HLA-C-restricted immune pressure on HIV would be greater in subjects with highly expressed HLA-C alleles. Using a cohort derived from a unique narrow source epidemic in China, we identified mutations in HIV proviral DNA exclusively associated with HLA-C, which were used as markers for the intensity of the immune pressure exerted on the virus. We found an increased frequency of mutations in individuals with highly expressed HLA-C alleles, which also correlated with IFN-γ production by HLA-C-restricted CD8(+) T cells. These findings show that immune pressure on HIV is stronger in subjects with the protective genotype and highlight the potential role of HLA-C-restricted responses in HIV control. This is, to our knowledge, the first in vivo evidence supporting the protective role of HLA-C-restricted responses in nonwhites during HIV infection.

Figure 1. Protective effect of the -35 SNP in the Han Chinese population

(A) Mean viral load (log10 cp/ml) and (B) median CD4 counts (cells/ml blood) in HIV-infected individuals. Patient samples were grouped based on their genotype at -35. Statistical analyses was performed using the Kruskal-Wallis rank sum test and a two-tailed t-test (C) Enrichment of the -35CC genotype in the HIV-positive cohort. The HIV-negative cohort was used as baseline and relative frequencies of each genotype were compared with those of the HIV-positive cohort. Statistical analysis was performed using a chi-square test. The p-value applies to a comparison of CC and TT rates in cases (HIV+) versus controls (HIV-). Numbers are listed in Table I.

Figure 2. -35CC subjects have lower viremia and higher CD4 counts than TT individuals

Groups were defined according to the frequencies of subjects with (A) HIV-1 viral load <2000 or >10 000 viral RNA copies per ml plasma and (B) CD4 counts <200, <350 and >500 cells/ml blood which represent different clinical stage of HIV-1 disease progression. (C) Subjects with mVL <2000 exhibit higher CD4 counts than individuals with mVL >10 000. Each dot represents a patient. Statistical analysis was performed using a two-tailed t-test. P = 0.004.

Figure 3. Selection of C-mut

Numbers of patients showing C-mut (black) or not (white). Six mutations associated with HLA-C exclusively were found in individuals expressing the corresponding HLA-C alleles. Substitutions and LD between HLA-C alleles associated and -35 SNP are shown below the graph. When substitutions were found inside known epitopes, the epitope sequences are shown above the corresponding bars. C-mut # 3 (KWSKCSMIGWPRVRERMR), #4 (TAPPEESFRFGEETTTPSQK) and #6 (WQLDCTHVEGKIILVAVH) were found in unpublished epitopes.

Figure 4. Association between C-mut and -35 SNP

(A) Increased frequency of C-mut in presence of the CC genotype. Subjects were categorised in three groups based on their genotype at -35 (TT, CT and CC). The total numbers of C-mut in each group were counted and the frequencies were calculated based on numbers listed in Table 1 (some subjects showed more than one C-mut). (B) Distribution of -35 SNP in the cohort. Frequencies of subject with C-mut are shown in black and frequencies of individuals not exhibiting C-mut (rest of the cohort) are shown in white (Table 1). The likelihood to show C-mut is five times higher in ‘CC’ than in ‘TT’ subjects (black). (C) CC subjects exhibit an increased frequency of substitutions in HIV pro DNA associated with HLA-C but not with HLA-A or –B alleles. Ratios of subjects with the CC genotype versus individuals with the TT genotype are shown for each HLA Class I association. Statistical analyses were performed using a chi-square test using numbers from Table 1. In C, the p-value applies to a comparison between the numbers of CC versus TT subjects with A-mut, B-mut or C-mut.

Figure 5. LD between -35 SNP and HLA-alleles

(A) -35 SNP is in LD with some HLA-C alleles. Allelic frequencies of the different HLA-C alleles are shown according to the genotypes at -35: CC (black), CT (grey) and TT (white). Annotations above each bar indicate which of the ‘C’ or ‘T’ allele at -35 is associated with HLA-C alleles. Significant associations are represented by *. HLA-Cw*03 subtypes are differentially associated with -35 SNP: Cw*03:02 associates with ‘C’ whereas Cw*03:03 is in LD with ‘T’. (B) Frequencies of the main protective HLA-alleles in subjects showing C-mut. HLA-B*51 is the only allele enriched in patients with C-mut (black). Frequencies of subjects without C-mut are shown in white. (C) Increased frequency of C-mut in CC subjects in absence of HLA-B*51. Analysis described in Fig. 4A was repeated after exclusion of all HLA-B*51 subjects showing C-mut. Statistical analysis was performed using a Chi-square test. In addition, a logistic regression analysis adjusting for HLA-B*51 showed that its presence did not affect the results.

Figure 6. C-mut are escape mutations and markers for immunogenic epitopes

(A) Magnitude of IFN-γ responses to 18-mer HIV-1 consensus peptides. PBMCs from patients expressing HLA-C alleles associated with each C-mut were used in an ELISpot assay. Each bar represents one patient. HLA-C type of each subject is shown above the bars; the HLA-C alleles associated with C-mut are in bold (Fig. 2). Two to five responding patients were identified for each peptide. (B) TNF-α and IFN-γ production is CD8+ T cell mediated, HLA-Cw*01:02-restricted and peptide-specific. A short-term CTL line was re-stimulation with VL8-pulsed .221HLACw*01:02 transfectants (left). Untransfected VL8-pulsed .221 cells were used as control (right). Numbers indicate percentage of positive cells. Gated on CD3+ T cells. (C and D) S173T and T470A substitutions are escape mutations. Peptide titration assay with CTL line or PBMCs in presence of decreasing concentrations of adapted (white) and nonadapted (black) (C) VL-8 or (D) TAPPEESFRFGEETTTPSQK peptides in an ELIspot assay. Data representative of two independent experiments. Statistical analyses were performed using a two-tailed t-test. Significant differences are indicated by the presence of *.

Figure 7. 3′UTR variant associates with protection and C-mut in the Han Chinese population

(A) Mean viral load (log10 cp/ml) and (B) median CD4 counts (cells/ml blood) in HIV-infected individuals in relation to 263 del/ins. (C) Increased frequency of C-mut in CC subjects. Frequencies of C-mut in the cohort in relation to 263del/ins. (D) Frequencies of subjects with (black) or without (white) C-mut. Subjects were grouped based on the presence of 263del/ins. Frequencies of individuals are shown. Statistical analyses were performed using Kruskal-Wallis rank sum test and chi-square test.