Combination of immune and viral factors distinguishes low-risk versus high-risk HIV-1 disease progression in HLA-B*5701 subjects

Abstract

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.

Fig 1

Sequence variation in the four Gag p24 HLA-B*5701-restricted epitopes (ISW9, KF11, TW10, and QW9) and the CypA-binding loop for HRPs (A) and LRPs (B). HXB2 (top) is used as the reference sequence. Amino acid residues defining the epitopes and the compensatory mutations for TW10 in the CypA-binding loop are marked in boldface. The relative positions for each epitope and the CypA-binding loop are in parentheses.

Fig 1

Sequence variation in the four Gag p24 HLA-B*5701-restricted epitopes (ISW9, KF11, TW10, and QW9) and the CypA-binding loop for HRPs (A) and LRPs (B). HXB2 (top) is used as the reference sequence. Amino acid residues defining the epitopes and the compensatory mutations for TW10 in the CypA-binding loop are marked in boldface. The relative positions for each epitope and the CypA-binding loop are in parentheses.

Fig 2

Likelihood mapping of HIV-1 Gag p24 sequences from different HLA-B*5701 subjects. Each dot represents the likelihoods of the three possible unrooted trees for a set of four sequences (quartets) selected randomly from the data set (see Materials and Methods). Dots close to the corners or the sides represent tree-like and network-like phylogenetic signals, respectively. The central area of the likelihood map, highlighted by a smaller triangle inside the map, represents a star-like signal. The higher the percentage of the dots (out of 10,000 random quartets) in the center of the triangle (given at the base of each map), the higher the phylogenetic noise in the data due to low genetic diversity and/or homoplasy. The spread of the data points in the remaining areas of the triangle carries little information, as long as these data points are equally distributed in each of the three corners (see Fig. S3 in the supplemental material). (A) Likelihood mapping of HIV-1 sequences sampled from HRP subjects. (B) Likelihood mapping of HIV-1 sequences sampled from LRP subjects.

Fig 3

Maximum likelihood gag p24 genealogies (using only unique sequences) for HRPs (A to C) and LRPs (D to F). Strains sampled at different time points (estimated weeks from infection) are indicated by colored dots according to the legend in the figure. Branch lengths are drawn to scale in nucleotide substitutions per site according to the bar at the bottom of each tree. Statistical support for specific branches are indicated by one (bootstrap value of >65% or aLRT; P > 0.75) or two (bootstrap value of >65% and aLRT; P > 0.75) asterisks. Amino acid replacements along supported internal branches are indicated; the numbering refers to Gag amino acid positions in HXB2, which was used as the reference. Residues for which there is statistically significant evidence (P > 95%) of positive selection are highlighted in boldface.

Fig 4

Median nucleotide substitution rate and 95% HPD intervals of HIV-1 Gag p24 in six longitudinally sampled HLA-B*5701 subjects. Substitution rates are given in nucleotide substitutions/site/year along the x axis and were estimated by Bayesian inference, assuming either a strict or relaxed molecular clock depending on the best-fitting model of each subject (Table 3). Substitution rates in the HRP group (P1 to P3) are significantly higher (P = 0.025 by Mann-Whitney U-test) than those in the LRP group (P4 to P6).

Fig 5

Functional discrepancies of CD8⁺ T cell responses to wild-type HLA-B*5701-restricted epitopes in HRPs compared to LRPs. (A) Flow cytometry plots illustrating polyfunctional CD8⁺ T cell responses to the TW10 epitope for representative HRP and LRP subjects. (B) Pie charts representing the proportion of CD8⁺ T cell functions induced by the four wild-type HLA-B*5701-restricted epitopes (average response of the three time intervals for each subject). One to four functions are illustrated by the colors yellow, orange, red, and blue, respectively. The round bars surrounding the pie charts indicate diverse functions as indicated by the pie chart arcs. P values refer to permutation tests performed to compare the differences between the pie charts. (C) Frequency comparison of the functions of HLA-B*5701-restricted CD8⁺ T cell responses in HRPs (blue) and LRPs (red) for each of the 14 potential combinations. The bars represent means and interquartile ranges (IQR). Significant differences between the bars for HRPs and LRPs are represented by an asterisk, which indicates P < 0.05 using Student's t test. (D) Pie charts demonstrating the functional diversity of longitudinal CD8⁺ T cell responses to the four HLA-B*5701-restricted wild-type epitopes between HRPs and LRPs at each of the three time intervals (I₁ to I₃). The round bars surrounding the pie charts indicate various functions as indicated by the pie chart arcs. P values refer to permutation tests performed to compare the differences between the pie charts.

Fig 6

Functional discrepancies of QW9- and TW10-specific CD8⁺ T cell responses between HRPs and LRPs. (A) Pie charts representing the fraction of CD8⁺ T cell responses to each specific HLA-B*5701-restricted wild-type epitope. One to four functions are illustrated by the colors yellow, orange, red, and blue, respectively. Permutation tests were performed to compare the differences between the pie charts. (B) Representative flow cytometry plots demonstrating CD8⁺ T cells producing IFN-γ, IL-2, and MIP-1β simultaneously (colored events represent those cells producing MIP-1β among gated IFN-γ/IL-2 double-positive cells). (C) Frequency comparison of the functional profile of QW9- and TW10-specific polyfunctional CD8⁺ T cell responses in HRPs (blue) and LRPs (red) for each of the functional combinations. The bars represent the means and IQR. Significant differences between the bars are represented by an asterisk, which indicates P < 0.05 using Student's t test. (D) Pie charts demonstrating polyfunctional activity of CD8⁺ T cell responses to the four HLA-B*5701-restricted major autologous variants between HRPs and LRPs at each of the three time intervals (I₁ to I₃). The round bars surrounding the pie charts indicate various functions as indicated by the pie chart arcs. P values refer to permutation tests performed to compare the differences between the pie charts.

Fig 7

IL-2 production by CD8⁺ T cells is associated with CD4⁺ T cell counts. (A) Box-and-whisker plots (Tukey) demonstrating the longitudinal production of IL-2 for HRPs (left) or LRPs (right). P values (**, P < 0.01) were obtained from a one-way analysis of variance and a nonparametric Kruskal-Wallis test with Dunn's multiple comparison tests to compare all pairs of columns. (B) Correlation analysis between the average production of IL-2 and CD4 counts or VL for all subjects using the Spearman nonparametric test.