Impact of HLA-B*52:01-Driven Escape Mutations on Viral Replicative Capacity

Abstract

HLA-B*52:01 is strongly associated with protection against HIV disease progression. However, the mechanisms of HLA-B*52:01-mediated immune control have not been well studied. We here describe a cohort with a majority of HIV C-clade-infected individuals from Delhi, India, where HLA-B*52:01 is highly prevalent (phenotypic frequency, 22.5%). Consistent with studies of other cohorts, expression of HLA-B*52:01 was associated with high absolute CD4 counts and therefore a lack of HIV disease progression. We here examined the impact of HLA-B*52:01-associated viral polymorphisms within the immunodominant C clade Gag epitope RMTSPVSI (here, RI8; Gag residues 275 to 282) on viral replicative capacity (VRC) since HLA-mediated reduction in VRC is a central mechanism implicated in HLA-associated control of HIV. We observed in HLA-B*52:01-positive individuals a higher frequency of V280T, V280S, and V280A variants within RI8 (P = 0.0001). Each of these variants reduced viral replicative capacity in C clade viruses, particularly the V280A variant (P < 0.0001 in both the C clade consensus and in the Indian study cohort consensus p24 Gag backbone), which was also associated with significantly higher absolute CD4 counts in the donors (median, 941.5 cells/mm³; P = 0.004). A second HLA-B*52:01-associated mutation, K286R, flanking HLA-B*52:01-RI8, was also analyzed. Although selected in HLA-B*52:01-positive subjects often in combination with the V280X variants, this mutation did not act as a compensatory mutant but, indeed, further reduced VRC. These data are therefore consistent with previous work showing that HLA-B molecules that are associated with immune control of HIV principally target conserved epitopes within the capsid protein, escape from which results in a significant reduction in VRC.IMPORTANCE Few studies have addressed the mechanisms of immune control in HIV-infected subjects in India, where an estimated 2.7 million people are living with HIV. We focus here on a study cohort in Delhi on one of the most prevalent HLA-B alleles, HLA-B*52:01, present in 22.5% of infected individuals. HLA-B*52:01 has consistently been shown in other cohorts to be associated with protection against HIV disease progression, but studies have been limited by the low prevalence of this allele in North America and Europe. Among the C-clade-infected individuals, we show that HLA-B*52:01 is the most protective of all the HLA-B alleles expressed in the Indian cohort and is associated with the highest absolute CD4 counts. Further, we show that the mechanism by which HLA-B*52:01 mediates immune protection is, at least in part, related to the inability of HIV to evade the HLA-B*52:01-restricted p24 Gag-specific CD8⁺ T-cell response without incurring a significant loss to viral replicative capacity.

Keywords: C clade; CTL; HLA; HLA-B*52:01; Indian; compensatory mutation; escape mutation; human immunodeficiency virus; p24 Gag; viral replicative capacity.

FIG 1

Frequency of B*52:01-restricted mutations and the association between CD4 count and HLA-B alleles (n = 138). (A) Association between HLA-B alleles and CD4 counts. Vertical bar represents the median CD4 counts, boxes indicate the IQR, and brackets are the maximum and minimum values. The median CD4 count is marked on the top of each bar. A Kruskal-Wallis test was applied (P = 0.004). Here, the individual P value was corrected by Bonferroni’s correction (P value threshold of 0.0038). The asterisk represents a statistically significant difference after Bonferroni correction for multiple tests between the absolute CD4 count in individuals expressing a particular HLA allele and in the remaining cohort. (B) Comparison of the percentages of the wild type (WT) and V280X variants (where X is T, S, or A) at Gag-280 in HLA-B*52:01-positive (B52+ve) and -negative (B52−ve) individuals. P < 0.0001 (Fisher’s exact test); q = 0.006. (C) CD4 counts of each V280X mutant in HLA- B*52:01-positive and -negative individuals. Bar represents the median of CD4 counts. A Mann-Whitney U test and Steel-Dwass test were performed for post hoc analysis (P < 0.05 of V280A compared to results with the wild type). **, P < 0.01; ***, P < 0.001.

FIG 2

Frequency of Gag K286R and the CD4 count of each mutant group (n = 138). (A) Frequency of Gag V280X, K286R, and V280X/K286R variants in HLA-B*52:01-positive and -negative individuals. (B) Comparison of the percentages of K286R in wild-type (WT) and V280X viruses in HLA-B*52:01-positive and -negative individuals. (C) CD4 count in wild-type and V280X viruses with/without K286R in HLA-B*52:01-positive and -negative individuals. The bar represents the median of the CD4 count. Fisher’s exact test for frequency difference and a Mann-Whitney U test and Steel-Dwass test post hoc analysis for CD4 counts were applied. ns, not significant.

FIG 3

Maximum likelihood phylogenetic tree of p24 Gag proviral sequences of HIV-1 group M, N, P, and O. The C clade sequences are shown in purple (n = 138), B clade sequences are in blue, and A clade sequences are in green. Subjects recruited in our cohort are numbered with the prefix “IN” and highlighted with dots. Reference Indian sequences randomly selected from 1993 to 2002 are highlighted in purple. Other reference sequences are shown in black. All of the reference sequences are from the Los Alamos database (http://www.hiv.lanl.gov/).

FIG 4

Viral replicative capacity (VRC) of T280X/R286K B clade and V280X/K286R C clade mutant viruses. (A) Relative VRC of the consensus sequence normalized to that of the B clade wild type (WT) (NL4-3). (B) Relative VRC of B clade viruses normalized to that of the B clade wild type (NL4-3). (C) Relative VRC of C clade viruses normalized to that of the C clade wild type [SK-254(M)]. (D) Relative VRC of IC viruses normalized to that of the IC wild type. All bars are shown as means with standard deviations. Patterned bars represent the R286K/K286R mutant. Clades are indicated according to the color legend [blue, NL4-3; yellow, SK-254(M); green, Indian C clade consensus (IC)]. One-way ANOVA multiple comparison and Dunnett’s correction were used for calculating P values. Asterisks indicate P values for comparison of the results with the mutant virus to those with the wild type (ns, not significant [P ≥ 0.05]; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 5

Identification and VRC of the developed variants during viral inoculation. (A) Positions of the developed variants in Gag HXB2. (B) VRC of L135F/V280S/K286R and I223T/V280A/K286R variants in IC. All bars are shown as means with standard deviations. Striped bars represent developed mutants. One-way ANOVA multiple comparison and Dunnett’s correction were used for calculating P values. Asterisks indicate P values for results with the mutants compared to those with the wild type. (ns, not significant [P ≥ 0.05]; ***, P < 0.001; ****, P < 0.0001).