Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif

Abstract

Virus-specific cytotoxic T-lymphocyte (CTL) responses are critical in the control of human immunodeficiency virus type 1 (HIV-1) infection and will play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes that are efficiently recognized by CTL is the first step in the development of future vaccines. Here we describe the immunological characterization of a number of novel HIV-1-specific, HLA-A2-restricted CTL epitopes that share a high degree of conservation within HIV-1 and a strong binding to different alleles of the HLA-A2 superfamily. These novel epitopes include the first reported CTL epitope in the Vpr protein. Two of the novel epitopes were immunodominant among the HLA-A2-restricted CTL responses of individuals with acute and chronic HIV-1 infection. The novel CTL epitopes identified here should be included in future vaccines designed to induce HIV-1-specific CTL responses restricted by the HLA-A2 superfamily and will be important to assess in immunogenicity studies in infected persons and in uninfected recipients of candidate HIV-1 vaccines.

FIG. 1

Peptide-specific CD8⁺ T-cell responses to HLA-A2 binding peptides as measured by IFN-γ Elispot assay. CD8⁺ T-cell responses were tested for 23 different peptides at two different time points for each subject studied and are shown as mean frequencies. Only subjects with positive responses (>50 SFC/10⁶ PBMC [SFC/Mill PBMC]) are shown. (a) Individuals with chronic HIV-1 infection (n = 18). (b) Individuals with acute HIV-1 infection (n = 6). The maximum number of epitopes targeted was six, by subject SF17697.

FIG. 2

CD8⁺ T-cell frequencies against described optimal CTL epitopes for the corresponding HLA type were measured by intracellular IFN-γ staining on flow cytometry for two individuals (AC04 [A] and AC13 [B]). Responses to the negative control without peptide (a) and five positive responses to Vpr-59 (b) and four other described optimal CTL epitopes for the corresponding HLA type (c to f) are demonstrated, and percentages of activated CD8⁺ T cells are shown. FITC, fluorescein isothiocyanate; PerCP, peridinin chlorophyll protein.

FIG. 3

Percent specific lysis of target cells pulsed with the Vpr-59 peptide or no peptide (control) by epitope-specific clones in chromium release assay. Results for subject AC13 (a) and subject AC04 (b) are shown at different effector-to-target ratios (E:T). In panel c, determination of HLA restriction using Vpr-59-specific CTL clones in a chromium release assay is shown for individual AC13. Targets were either pulsed with no peptide (white bars) or pulsed with the relevant peptide Vpr-59 (black bars). The HLA class I types of the targets used were as follows: A∗0201/3, B7/60; A28/29, B14/44; and A34/68, B57/71 (matching HLA class I alleles are shown in bold).

FIG. 4

Percent specific lysis of HLA-A2-positive, TAP-competent (T1) (a) and TAP-deficient (T2) (b) B-cell line target cells by Vpr-59-specific CTL clones. T1 and T2 B-cell lines were infected with the molecular HIV-1 clone NL4-3 HIV (+NL4-3). The control target cells used in the assay were uninfected T1 and T2 cells and infected T1 and T2 cells pulsed with the Vpr-59 peptide (+NL4-3+peptide). Results are shown at different effector-to-target ratios (E:T) on day 4 after infection, following chromium 51 labeling of the cells, as described previously (85).