Role of Escape Mutant-Specific T Cells in Suppression of HIV-1 Replication and Coevolution with HIV-1

Abstract

The accumulation of HIV-1 escape mutations affects HIV-1 control by HIV-1-specific T cells. Some of these mutations can elicit escape mutant-specific T cells, but it still remains unclear whether they can suppress the replication of HIV-1 mutants. It is known that HLA-B*52:01-restricted RI8 (Gag 275 to 282; RMYSPTSI) is a protective T cell epitope in HIV-1 subtype B-infected Japanese individuals, though 3 Gag280A/S/V mutations are found in 26% of them. Gag280S and Gag280A were HLA-B*52:01-associated mutations, whereas Gag280V was not, implying a different mechanism for the accumulation of Gag280 mutations. In this study, we investigated the coevolution of HIV-1 with RI8-specific T cells and suppression of HIV-1 replication by its escape mutant-specific T cells both in vitro and in vivo HLA-B*52:01⁺ individuals infected with Gag280A/S mutant viruses failed to elicit these mutant epitope-specific T cells, whereas those with the Gag280V mutant one effectively elicited RI8-6V mutant-specific T cells. These RI8-6V-specific T cells suppressed the replication of Gag280V virus and selected wild-type virus, suggesting a mechanism affording no accumulation of the Gag280V mutation in the HLA-B*52:01⁺ individuals. The responders to wild-type (RI8-6T) and RI8-6V mutant peptides had significantly higher CD4 counts than nonresponders, indicating that the existence of not only RI8-6T-specific T cells but also RI8-6V-specific ones was associated with a good clinical outcome. The present study clarified the role of escape mutant-specific T cells in HIV-1 evolution and in the control of HIV-1.IMPORTANCE Escape mutant-specific CD8⁺ T cells were elicited in some individuals infected with escape mutants, but it is still unknown whether these CD8⁺ T cells can suppress HIV-1 replication. We clarified that Gag280V mutation were selected by HLA-B*52:01-restricted CD8⁺ T cells specific for the GagRI8 protective epitope, whereas the Gag280V virus could frequently elicit GagRI8-6V mutant-specific CD8⁺ T cells. GagRI8-6V mutant-specific T cells had a strong ability to suppress the replication of the Gag280V mutant virus both in vitro and in vivo In addition, these T cells contributed to the selection of wild-type virus in HLA-B*52:01⁺ Japanese individuals. We for the first time demonstrated that escape mutant-specific CD8⁺ T cells can suppress HIV-1 replication and play an important role in the coevolution with HIV-1. Thus, the present study highlighted an important role of escape mutant-specific T cells in the control of HIV-1 and coevolution with HIV-1.

Keywords: CTL; HIV-1; HLA-B*52:01; coevolution; escape mutation.

FIG 1

Recognition of RI8-6S and RI8-6A mutant epitopes by HLA-B*52:01-restricted RI8-6T-specific T cells. (A) Association of HLA-B*52:01 with mutations at Gag280 in 390 HIV-1 subtype B-infected Japanese individuals. Gag280S, Gag280A, Gag280V, and Gag280T were found in 26, 10, 12, and 51 HLA-B*52:01⁺ individuals, respectively; Gag280S, Gag280A, Gag280V, Gag280I, and Gag280T in were found 8, 8, 45, 6, and 224 HLA-B*52:01⁻ individuals, respectively. (B) T cell responses to RI8-6T peptide or its mutants. (C) Response (left) and identification (right) of RI8-specific T cells in PBMCs from KI-809 were analyzed by using the ELISPOT assay and RI8-6T tetramer, respectively. (D and E) Recognition of RI8-6S or -6A mutant epitopes by RI8-6T-specific T cell clones. T cell responses to 721.221-B*52:01 cells prepulsed with RI8-6T, RI8-6S, or RI8-6A peptide at various concentrations (D) and to those infected with NL43-Gag280T (wild-type), -Gag280S, or -Gag280A (E) were analyzed by performing ICS assays. The frequencies of p24 antigen-positive cells among 721.221-B*52:01 cells infected with NL43-Gag280T, -Gag280S, and -Gag280A and 721.221 cells infected NL43-Gag280T were 59.6%, 54.6%, 59%, and 59%, respectively (E). (F) Binding affinity of RI8 and its mutant peptides to HLA-B*52:01. (G) Longitudinal sequence analysis at Gag280 in 3 HLA-B*5201⁺ Japanese individuals. (H) T cell responses to RI8 and RI8-6A peptides in 6 Gag280A-infected and those to RI8 and RI8-6S in 6 Gag280S-infected individuals. Results are given as means with SD (n = 3). The dotted line indicates the threshold for a positive response (B, C, and H). Statistical analysis was performed by using the unpaired t test (D to F). *, P < 0.05; **, P < 0.01; ****, P < 0.0001. WT, wild type.

FIG 2

Recognition of RI8-6V mutant epitope by HLA-B*52:01-restricted RI8-6T-specific T cells. (A) Longitudinal sequence analysis at Gag280 in an HLA-B*5201⁺ Japanese individual. (B) Identification of RI8-specific T cells among PBMCs from KI-906 before and after the emergence of the Gag280V mutant virus. (C and D) Recognition of RI8-6V mutant epitope by RI8-6T-specific T cell lines. T cell responses to 721.221-B*52:01 cells prepulsed with RI8-6T or -6V peptide (C) and to those infected with NL43-Gag280T or -Gag280V (D) were analyzed. Frequencies of p24-positive cells among 721.221-B*52:01 cells infected with NL43-Gag280T and -Gag280V and 721.221 cells infected with -Gag280T were 60.8%, 58.0%, and 66.0%, respectively (D). (E) Ability of RI8-6T-specific T cells to suppress the replication of Gag280T and Gag280V virus. Results are given as means with SDs (n = 3). Statistical analysis was performed by using the unpaired t test (B to D). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Ability of RI8-6V-specific T cells to recognize RI8-6V-infected cells and to suppress RI8-6V replication. (A) Longitudinal sequence analysis at Gag280 in an HLA-B*5201⁺ Japanese individual infected with HIV-1 subtype B. (B) Identification by tetramer staining of RI8-specific T cells in PBMCs from KI-855 infected with a mixture of Gag280T and Gag280V viruses in December 2010. (C and D) Recognition of RI8-6T or -6V epitope by T cell clones established from the RI8-6V tetramer⁺ or RI8-6T tetramer⁺ T cell population. Responses of these clones to 721.221-B*52:01 cells prepulsed with RI8-6T or -6V peptide (C) and to those infected with NL43-Gag280T or -Gag280V were analyzed by using the ICS assay (D). The frequencies of p24 antigen-positive cells among 721.221-B*52:01 cells infected with NL43-Gag280T and Gag280V were 35.3% and 28.9%, respectively, whereas those of 721.221 infected with NL43-Gag280T and -Gag280V were 30.3% and 34.8%, respectively (D). (E) Staining of RI8-6V-specific, RI8-6T-specific, and cross-reactive T cell clones with both HLA-B*52:01-RI8-6T and HLA-B*52:01-RI8-6V tetramers. (F) Ability of RI8-6V-specific, cross-reactive, and RI8-6T-specific T cell clones to suppress the replication of Gag280-6T and -6V viruses. Results are given as means with SD (n = 3). percent suppression of HIV-1 replication is presented. Statistical analysis was performed by using the unpaired t test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (C, D, and F).

FIG 4

Detection of RI8-6V-specific T cells and their ability to suppress RI8-6V replication in Gag280V-infected HLA-B*5201⁺ Japanese individuals. (A) T cell responses to RI8-6V (6V) or RI8-6T (6T) epitopes were analyzed by performing the IFN-γ ELISPOT assay. The blue shading indicates a positive response in the ELISPOT assay (>200 spots/10⁶ CD8⁺ T cells). NA, not analyzed. (B) Identification by tetramer staining of RI8-specific T cells among PBMCs from 5 Gag280V-infected individuals. PBMCs were stained with HLA-B*52:01-RI8-6T and HLA-B*52:01-RI8-6V tetramers. (C) Ability of RI8-6V-specific T cells to suppress the replication of Gag280-6T and -6V viruses. RI8-6V-specific T cell lines were induced from PBMCs of 4 individuals by stimulating the PBMCs with RI8-6V peptide and culturing them for 14 days. The frequencies of RI8-6V-specific and RI8-6T-specific T cells were measured by staining with both HLA-B*52:01-RI8-6T and HLA-B*52:01-RI8-6V tetramers (left). Activated CD4⁺ T cells from an HLA-B*52:01⁺ individual infected with NL43-Gag280T or -Gag280V were cocultured with RI8-6V-specific T cells at an E:T ratio of 0.1:1 (right). Results are given as means with SD (n = 3). Percent suppression of HIV-1 replication is presented. Statistical analysis was performed by using the unpaired t test. ***, P < 0.001; ****, P < 0.0001.

FIG 5

Comparison of clinical outcomes among individuals infected with HIV-1 having different Gag 280 mutations and between T cell responders to RI8 and nonresponders. (A) Comparison of CD4 counts for individuals infected with Gag280T (wild type), Gag280V, or Gag280S/Gag280A virus among 99 subtype B-infected HLA-B*52:01⁺ Japanese individuals. (B) Association of T cell responses to RI8-6T/6V with clinical outcome. Comparison of CD4 count and pVL between T cell responders and nonresponders to RI8-6T/6V among 95 subtype B-infected HLA-B*52:01⁺ Japanese individuals is shown. Statistical analysis was performed by using the Mann-Whitney test. The value indicated by the red line in each graph represents the median of the CD4 count.

FIG 6

Summary of this study.