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. 2018 Jul 3;15(1):46.
doi: 10.1186/s12977-018-0429-y.

CD8+ T cells specific for conserved, cross-reactive Gag epitopes with strong ability to suppress HIV-1 replication

Hayato Murakoshi  1 , Chengcheng Zou  1 , Nozomi Kuse  1 , Tomohiro Akahoshi  1 , Takayuki Chikata  1 , Hiroyuki Gatanaga  1   2 , Shinichi Oka  1   2 , Tomáš Hanke  3   4 , Masafumi Takiguchi  5
Affiliations

CD8+ T cells specific for conserved, cross-reactive Gag epitopes with strong ability to suppress HIV-1 replication

Hayato Murakoshi et al. Retrovirology. .

Abstract

Background: Development of AIDS vaccines for effective prevention of circulating HIV-1 is required, but no trial has demonstrated definitive effects on the prevention. Several recent T-cell vaccine trials showed no protection against HIV-1 acquisition although the vaccines induced HIV-1-specific T-cell responses, suggesting that the vaccine-induced T cells have insufficient capacities to suppress HIV-1 replication and/or cross-recognize circulating HIV-1. Therefore, it is necessary to develop T-cell vaccines that elicit T cells recognizing shared protective epitopes with strong ability to suppress HIV-1. We recently designed T-cell mosaic vaccine immunogens tHIVconsvX composed of 6 conserved Gag and Pol regions and demonstrated that the T-cell responses to peptides derived from the vaccine immunogens were significantly associated with lower plasma viral load (pVL) and higher CD4+ T-cell count (CD4 count) in HIV-1-infected, treatment-naive Japanese individuals. However, it remains unknown T cells of which specificities have the ability to suppress HIV-1 replication. In the present study, we sought to identify more T cells specific for protective Gag epitopes in the vaccine immunogens, and analyze their abilities to suppress HIV-1 replication and recognize epitope variants in circulating HIV-1.

Results: We determined 17 optimal Gag epitopes and their HLA restriction, and found that T-cell responses to 9 were associated significantly with lower pVL and/or higher CD4 count. T-cells recognizing 5 of these Gag peptides remained associated with good clinical outcome in 221 HIV-1-infected individuals even when comparing responders and non-responders with the same restricting HLA alleles. Although it was known previously that T cells specific for 3 of these protective epitopes had strong abilities to suppress HIV-1 replication in vivo, here we demonstrated equivalent abilities for the 2 novel epitopes. Furthermore, T cells against all 5 Gag epitopes cross-recognized variants in majority of circulating HIV-1.

Conclusions: We demonstrated that T cells specific for 5 Gag conserved epitopes in the tHIVconsvX have ability to suppress replication of circulating HIV-1 in HIV-1-infected individuals. Therefore, the tHIVconsvX vaccines have the right specificity to contribute to prevention of HIV-1 infection and eradication of latently infected cells following HIV-1 reactivation.

Keywords: CTL; Conserved epitope; Gag; HIV-1; Vaccine.

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Figures

Fig. 1
Fig. 1
Correlation of T-cell responses to Gag conserved peptides of tHIVconsvX. Correlation of T-cell responses to Gag conserved peptides of tHIVconsvX with pVL and CD4 count. T-cell responses to Gag peptide Pools 1, 2 and 3 derived from vaccine immunogen tHIVconsvX were enumerated using an IFN-γ ELISPOT assay in 200 HIV-1-infected Japanese individuals. Comparison of pVL and CD4 count between responders and non-responders to the Gag peptides was statistically analyzed using the Mann-Whitney test. The value in each figure represents the median of pVL and CD4 count
Fig. 2
Fig. 2
T-cell responses to 15-mer peptide pairs in Gag Pool 2. a The responses to individual pairs of 15-mer peptides in Gag Pool 2. Responses to peptide pairs in 50 responders to Pool 2 were analyzed by an IFN-γ ELISPOT assay. The dotted line at 200 SFU/106 CD8+ T cells indicates a threshold for a positive response. b Summary of responders and peptide pairs. Reported epitopes present in the pair of the 15-mer peptides according to the LANL-HSD. HLA+ responders are those with the matching restricting HLA allele for the reported epitope
Fig. 3
Fig. 3
T-cell responses to 15-mer peptide pairs in Gag Pool 3. a The responses to individual pairs of 15-mer peptides in Gag Pool 3. Responses to peptide pairs in 53 responders to Pool 3 were analyzed by an IFN-γ ELISPOT assay. The dotted line at 200 SFU/106 CD8+ T cells indicates a threshold for a positive response. b Summary of responders and peptide pairs. Reported epitopes included in the pair of the 15-mer peptides are shown according to the LANL-HSD. HLA+ responders are those with the matching restricting HLA allele for the reported epitope
Fig. 4
Fig. 4
Identification of novel T-cell Gag epitopes. a T-cell responses to peptide pairs in individuals KI-1020, KI-1102, and KI-1114, who did not have matching HLA alleles for previously reported epitopes. b HLA restriction. Responses by STCL stimulated with C1R or 721.221 cells expressing individual HLA molecule shared by the responders and pulsed with the peptide pair were analyzed by ICS assay. C052/053 and C054/055 peptides were analyzed by using the T cells derived from KI-1020, while C113/114 and C125/126 by using the T cells from KI-1114 and KI-1102, respectively. c Identification of 11-mer HIV-1 clade B overlapping peptides recognized by the HLA-A*26:02-, HLA-A*33:03-, or HLA-A*02:06-restricted T cells. The responses by STCL expanded with C052/053, C054/055, C113/114, or C125/126 peptide pairs to the corresponding stimulator cells pre-pulsed with 11-mer HIV-1 clade B-derived overlapping peptides across the parental 15-mer were analyzed in ICS assay. d Identification of optimal peptides. The STCL responses stimulated with peptide pairs C052/053, C113/114, or C125/126 to the corresponding stimulator cells C1R-A2602, C1R-A3303, or 721.221-A0206 pre-pulsed with individual truncated peptides were analyzed by ICS assay
Fig. 5
Fig. 5
Association of epitope-specific T-cell responses with clinical parameters in HIV-1-infected individuals with the restricting HLA. a T-cell responses to 6 epitope peptides were analyzed by using an IFN-γ ELISPOT assay. The differences in pVL or CD4 count between responders and non-responders to each epitope peptide in the individuals having HLA restriction molecules for the epitopes were statistically analyzed by using the Mann-Whitney test. b Correlation between a breadth of T-cell responses to 5 epitopes (AA9, TL8, WV8, RI8, and HR10) and pVL and CD4 count in 149 individuals carrying the HLA restriction molecules. The correlation was statistically analyzed using Spearman rank test
Fig. 6
Fig. 6
Ability of CTLs to recognize HIV-1-infected cells and to suppress HIV-1 replication in vitro. a Generation of the T-cell lines specific for TL8 or HR10 from PBMCs of HLA-B*40:02+ individual (KI-1391) or HLA-A*33:03+ one (KI-1320). The T-cell lines were established as shown in Materials and Methods. The T-cell lines established were stained with the specific tetramers. b Recognition of HIV-1-infected cells by CTLs specific for TL8 or HR10 epitopes. The T-cell lines stimulated with HIV-1 (NL4-3)-infected 721.221 cells (HIV+) expressing the corresponding HLAs or HLA-negative 721.221 cells, and IFN-γ production from the T cells was measured by the ICS assay. The proportions of 721.221-B4002, -A3303 and HLA-negative 721.221 cells infected with NL4-3 were 56.0, 59.7, and 64.1%, respectively. c Suppression of HIV-1 replication by the T-cell lines specific for TL8 or HR10. Primary CD4+ T-cells from healthy donors carrying the corresponding HLA alleles were infected with NL4-3, and then co-cultured with epitope-specific T cells at E:T ratios of 1:1 and 0.1:1. The concentration of p24 Ag in the culture supernatant was measured by using an enzyme-linked immunosorbent assay. The percentage of suppression was calculated as follows: (concentration of p24 without CTLs – concentration of p24 with CTLs) / concentration of p24 without CTLs × 100. The data are presented as the mean and SD (n = 3)
Fig. 7
Fig. 7
Recognition of mutant epitope peptides by T cells in HIV-1-infected individuals. a Frequencies of TL8 and HR10 mutant epitopes in Japanese individuals. The frequencies of mutant epitopes were investigated from 430 chronically HIV-1-infected Japanese individuals. b The CD8+ T-cell responses to the index and mutant epitope peptides in HIV-1-infected Japanese individuals were analyzed by using an IFN-γ ELISPOT assay. The results are shown as mean and SD (n = 3). c The responses of the T cell-lines specific for TL8 or HR10 to wild-type or mutant peptide-prepulsed 721.221 cells expressing the corresponding HLA alleles were analyzed by using the ICS assay. The results are shown as mean and SD (n = 3)
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