Comprehensive analysis of human immunodeficiency virus type 1 (HIV-1)-specific gamma interferon-secreting CD8+ T cells in primary HIV-1 infection

Abstract

Human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) T cells provide an important defense in controlling HIV-1 replication, particularly following acquisition of infection. To delineate the breadth and potency of these responses in patients upon initial presentation and before treatment, we determined the fine specificities and frequencies of gamma interferon (IFN-gamma)-secreting CD8(+) T cells recognizing all HIV-1 proteins in patients with primary infection. In these subjects, the earliest detected responses were directed predominantly against Nef, Tat, Vpr, and Env. Tat- and Vpr-specific CD8(+) T cells accounted for the greatest frequencies of mean IFN-gamma spot-forming cells (SFC). Nef-specific responses (10 of 21) were more commonly detected. A mean of 2.3 epitopes were recognized with various avidities per subject, and the number increased with the duration of infection (R = 0.47, P = 0.031). The mean frequency of CD8(+) T cells (985 SFC/10(6) peripheral blood mononuclear cells) correlated with the number of epitopes recognized (R = 0.84, P < 0.0001) and the number of HLA-restricting alleles (R = 0.79, P < 0.0001). Neither the total SFC frequencies nor the number of epitopes recognized correlated with the concurrent plasma viral load. Seventeen novel epitopes were identified, four of which were restricted to HLA alleles (A23 and B72) that are common among African descendents. Thus, primary HIV-1 infection induces strong CD8(+)-T-cell immunity whose specificities broaden over time, but their frequencies and breadth do not correlate with HIV-1 containment when examined concurrently. Many novel epitopes, particularly directed to Nef, Tat, and Env, and frequently with unique HLA restrictions, merit further consideration in vaccine design.

FIG. 1.

Mapping and characterization of HIV-1-specific T-cell response in acute infection. The HIV-1-specific T-cell response from subject 1282 was determined with cryopreserved PBMCs by IFN-γ Elispot assay. The bars depict the average number of IFN-γ spot-forming cells (SFC) per well containing 200,000 PBMCs (± standard error). (A) Initial mapping of the HIV-1-specific T-cell response to peptide pools spanning Gag, Pol, Env, Nef, and Tat. Eight previously defined HLA-A2-restricted (pool) and one HLA-A31-restricted (RLRDLLLVTR) optimal CTL epitopes were also tested. Phytohemagglutinin (PHA)-stimulated cells served as the positive control, and cells stimulated with irrelevant peptides (Neg) or no peptide served as negative controls. (B) Determination of the T-cell subset responsible for the IFN-γ secretion. T-cell-enriched (CD4⁺) or depleted (CD4⁻) cell populations were tested in an IFN-γ Elispot assay with Nef pool 1. Staphylococcal enterotoxin B (SEB) instead of phytohemagglutinin stimulation was used as a positive control in this assay. (C) Fine mapping of the T-cell response to Nef with individual 20-mers (1 to 10) included in Nef pool 1. The amino acid sequence of the positive 20-mer peptide Nef 8 is shown. (D) Determination of the optimal Nef epitope. Twelve overlapping 9-mers spanning Nef 8 were tested in the Elispot assay. The amino acid sequence of the recognized 9-mer Nef8-12 (AL9) is shown. (E) Peptide titration of the Nef-AL9 epitope. Standard IFN-γ Elispot was carried out with the indicated peptides at 0.64, 3.2, 16, 80, 400, 2,000, and 10,000 ng/ml. The regression curve is drawn with the Sigmoidal Fit tool in the Origin 6.0 software. (F) HLA class I restriction of the Nef-AL9 epitope. Six mismatched B-LCLs were pulsed with (black bars) or without (white bars) the Nef-AL9 peptide. The class I alleles matching subject 1282 (A2 and A31; B44 and B60; and Cw3 and Cw16) are indicated for each B-LCL. IFN-γ secretion was determined by Elispot assay after incubating 1282 PBMCs with these peptide-pulsed B-LCLs.

FIG. 2.

Novel and previously defined CD8⁺-T-cell epitopes in acute and early infection. (A) Clustering of CD8⁺-T-cell epitopes in Nef and gp41. Gag-, Env-, and Nef-specific CD8⁺ epitopes revealed in this study are illustrated. The amino acid (a.a.) numeration of each protein is shown. The previously known epitopes are shown in solid bars, and the newly defined epitopes are shown in open bars. (B) Broad CD8⁺ response to eight HIV-1 proteins. CD8⁺ responses to each HIV-1 protein in color coding are illustrated for each subject. Subjects are presented in the order of day postinfection (dpi) when the T-cell analysis was performed. The frequency (C) and breadth (D) of CD8⁺ responses were analyzed based on the size of the protein. The mean frequency and number of epitopes were calculated for each HIV-1 protein and subjects recognizing epitopes within the particular protein, then divided by the size of the protein in number of amino acids, and multiplied by 100.

FIG. 3.

Magnitude of CD8⁺ IFN-γ T cells correlates with breadth but not functional avidity of primary response. The total frequency of virus-specific CD8⁺ T cells is plotted against the number of epitopes (A) and number of restricting HLA class I alleles (B) for each subject. (C) The frequency of virus-specific CD8⁺ T cells was plotted against the functional avidity (EC₅₀) for each epitope. The EC₅₀ value is shown in a log₁₀ scale. The R and P values are indicated for each graph.

FIG. 4.

Breadth and magnitude of CD8⁺-T-cell response in relation to duration of infection and plasma viral load. (A) The HIV-1-specific CD8⁺-T-cell response broadens with the duration of infection. The number of HIV-1-specific CD8⁺-T-cell epitopes was plotted for each subject against the sampling time, indicated as days postinfection (dpi). (B) No correlation between the frequency of CD8⁺ responses and the plasma viral load. The total HIV-1-specific CD8⁺-T-cell frequency was plotted against the plasma HIV-1 RNA load (in log₁₀ scale) for each subject. The R and P values are indicated for each graph.

FIG. 5.

HLA class I presentation of virus-specific epitopes in acute and early infection. Twelve HLA-A, 18 HLA-B, and 12 HLA-C alleles were represented in this study cohort of 21 subjects. These alleles are indicated on the x axis with the specific allele number. The frequency for each allele is shown as the total number of subjects having the allele (white bars) and number of subjects recognizing HIV-1 epitopes restricted to the particular class I allele (black bars).