Definition of a New HLA B*52-Restricted Rev CTL Epitope Targeted by an HIV-1-Infected Controller

Abstract

The analysis of T-cell responses in HIV-1-infected controllers may contribute to a better understanding of the protective components of the immune system. Here, we analyzed the HIV-1-specific T-cell response in a 59-year-old HIV-1-infected controller, infected for at least seven years, who presented with low viral loads ranging from <20 copies/mL to 200 copies/mL and normal CD4 counts of >800 cells/µL. In γ-IFN-ELISpot assays using freshly isolated PBMCs, he displayed a very strong polyclonal T-cell response to eight epitopes in Gag, Nef and Rev; with the dominant responses directed against the HLA-B*57-epitope AISPRTLNAW and against a so-far-unknown epitope within Rev. Further analyses using peptide-stimulated T-cell lines in γ-IFN-ELISpot assays delineated the peptide RQRQIRSI (Rev-RI8) as a newly defined HLA-B*52-restricted epitope located within a functionally important region of Rev. Peptide-stimulation assays in 15 HLA-B*52-positive HIV-1-infected subjects, including the controller, demonstrated recognition of the Rev-RI8 epitope in 6/15 subjects. CD4 counts before the start of antiviral therapy were significantly higher in subjects with recognition of the Rev-RI8 epitope. Targeting of the Rev-RI8 epitope in Rev by CTL could contribute to the positive association of HLA-B*52 with a more favorable course of HIV-1-infection.

Keywords: CTL; HIV-1 infection; HLA B*52; Rev; elite controller; epitope.

Figure 1

(a) Recognition of HLA-matching HIV-1 peptides by freshly isolated PBMCs from controller #1. A total of 2 × 10⁵ PBMCs were incubated directly after isolation for 40 h with peptides corresponding to HLA-I-restricted epitopes in γ-IFN ELISpot assay at a final concentration of 20 µg/mL. The mitogen phytohaemagglutinin (PHA) served as a positive control. w/o: PBMCs without peptide. (b) Recognition of HIV-1 peptides by specific T-cell lines from controller #1. PBMCs were incubated with peptides corresponding to HLA-I-restricted epitopes and IL2. Ten to fourteen days after stimulation, 50 μL of outgrowing CTL were tested in γ-IFN ELISpot assay for peptide recognition at a final concentration of 20 μg/mL Peptide-specific SFUs, after subtraction of the background without peptide, are presented. Peptides: Nef-QK10: QVPLRPMTYK, Nef-HW9: HTQGYFPDW, Rev07-11: RRRRWRERQRQIRSI, Gag-AK9: ATLYCVHQK, Gag-AL15: ACQGVGGPGHKARVL, Gag-pool 9 (containing 5 overlapping 11 amino acid long peptides including peptide EKAFSPEVIPMFSAL, further sequences given in Section 2.2), Gag-AW10: AISPRTLNAW, Gag-CK10 CQGVGGPGHK, Gag-TW10: TSTLQEQIGW, Gag-KF11: KAFSPEVIPMF.

Figure 2

Mapping of the minimal epitope targeted by Rev-07–11-specific T-cells in controller #1. PBMCs were stimulated with Rev-07-11 peptide and IL-2 and outgrowing cells were tested after 10–14 days for recognition of overlapping and truncated peptides at a final peptide concentration of 20 µg/mL in γ-IFN-ELISpot assays. Shown are spot-forming units (SFUs) per 1:10⁵ cells. Peptide-stimulated T-cell lines without peptides served as negative controls. (a) Analysis of recognition of the overlapping 15-mer peptides Rev07-10, Rev07-11, Rev07-12 and Rev07-13. (b) Fine mapping of the epitope with truncated peptides delineated Rev-RI8 as the minimal epitope (marked in red).

Figure 3

Analysis of HLA-restriction of the Rev epitope Rev-RI8. (a) A total of 1 × 10⁵ cells of the Rev-RI8-specific T-cell line from controller #1 were incubated in a γ-IFN ELISpot assay with peptide Rev-RI8 and blocking anti-CD4- or anti-CD8-antibodies. w/op: cells without peptide; w/P: cells with peptide Rev-RI8 (b) A total of 1 × 10⁵ cells of the Rev-RI8-specific T-cell line from controller #1 were incubated in a γ-IFN ELISpot assay with 1 × 10⁵ allogeneic Rev-RI8-peptide-loaded B-LCLs sharing single HLA alleles with controller #1. Shown are spot-forming units (SFUs) after incubation of T-cells with Rev-RI8-loaded B-LCL after subtraction of background, which was assessed by co-incubating T-cell lines with B-LCLs without addition of the peptide. Shown are the HLA alleles of the B-LCLs shared by the T-cell line (HLA-I type of controller #1 (A*11, B*057, B*52, C*06, C*12).

Figure 4

Analysis of peptide-sensitizing capacity of peptide Rev-RI8. A total of 1 × 10⁵ cells each from Rev-RI8-specific T-cell lines from the three HLA-B*52-positive subjects, #1, #2 and #5, were incubated with serial dilutions of peptide Rev-RI8 in γ-IFN ELISpot assays. Shown are adjusted spot-forming units (SFUs) after subtraction of the background. In this graph, 100 % represents the highest frequencies of SFUs determined for the individual T-cell line. The 50% peptide sensitizing activity (EC₅₀) was calculated using non-linear curve regression analysis. EC₅₀ values: subject #1: 2.2 µg/mL, subject #2: 1.8 µg/mL, subject #5: 2.9 µg/mL; mean: 2.3 µg/mL.

Figure 5

Recognition of peptide Rev-RI8 by HLA-B*52-positive HIV-1-infected subjects. PBMCs were incubated with peptide Rev-RI8 and IL2. After 10–14 days, 50 μL of outgrowing CTL were tested for peptide recognition at a final peptide concentration of 20 μg/mL.

Figure 6

Pre-ART CD4 counts in HLA-B*52+ subjects. HIV-1-infected HLA-B*52+ subjects with detection of Rev-RI8-specific T-cells (responders) displayed significantly higher CD4+ T-cells (median 602/µL, range 263–928) than subjects without recognition of Rev-RI8 (non-responders, median CD4+ T-cells: 192/µ, range 5–517), Mann–Whitney-U-Test: p = 0.012 (p = 0.0290, if #1 is omitted). For the only untreated subject (controller #1) the CD4 count at the time point of ELISpot analysis of Figure 1a was used (CD4 928/µL). Significance with p < 0.05 is indicated by *.