Comprehensive epitope analysis of human immunodeficiency virus type 1 (HIV-1)-specific T-cell responses directed against the entire expressed HIV-1 genome demonstrate broadly directed responses, but no correlation to viral load

Abstract

Cellular immune responses play a critical role in the control of human immunodeficiency virus type 1 (HIV-1); however, the breadth of these responses at the single-epitope level has not been comprehensively assessed. We therefore screened peripheral blood mononuclear cells (PBMC) from 57 individuals at different stages of HIV-1 infection for virus-specific T-cell responses using a matrix of 504 overlapping peptides spanning all expressed HIV-1 proteins in a gamma interferon-enzyme-linked immunospot (Elispot) assay. HIV-1-specific T-cell responses were detectable in all study subjects, with a median of 14 individual epitopic regions targeted per person (range, 2 to 42), and all 14 HIV-1 protein subunits were recognized. HIV-1 p24-Gag and Nef contained the highest epitope density and were also the most frequently recognized HIV-1 proteins. The total magnitude of the HIV-1-specific response ranged from 280 to 25,860 spot-forming cells (SFC)/10(6) PBMC (median, 4,245) among all study participants. However, the number of epitopic regions targeted, the protein subunits recognized, and the total magnitude of HIV-1-specific responses varied significantly among the tested individuals, with the strongest and broadest responses detectable in individuals with untreated chronic HIV-1 infection. Neither the breadth nor the magnitude of the total HIV-1-specific CD8+-T-cell responses correlated with plasma viral load. We conclude that a peptide matrix-based Elispot assay allows for rapid, sensitive, specific, and efficient assessment of cellular immune responses directed against the entire expressed HIV-1 genome. These data also suggest that the impact of T-cell responses on control of viral replication cannot be explained by the mere quantification of the magnitude and breadth of the CD8+-T-cell response, even if a comprehensive pan-genome screening approach is applied.

FIG. 1.

Correlation between peptide matrix and individual peptide approach. The number of responses to peptides detected by the matrix approach on the x axis is compared to the number of responses detected by the use of individual peptides as the current gold standard (y axis) for Gag (upper panel) and Nef (lower panel). Linear regression lines, correlation coefficients, and P values are given in the plots as calculated by linear regression analysis (n = 20).

FIG. 2.

Peptide recognition across the entire expressed HIV-1 genome. The 504 individual overlapping peptides are represented on the x axis, and the corresponding percentage of study subjects with a response to the individual peptide are represented on the y axis. The horizontal bar indicates the corresponding regions for the individual peptides. The sequences of the three most frequently targeted peptides, located in Nef and p24Gag, are shown in the text boxes.

FIG. 3.

Frequency of recognition of the individual proteins and protein subunits. Panels a to d summarize the percentages of individuals with responses to at least one peptide for the individual protein or subunit, stratified by study groups as indicated. Panels e to h show amino acid-adjusted scores for protein recognition. The amino acid-adjusted score is defined as the frequency of recognition of the individual protein divided by protein length in amino acids. (a and e) Acute treated; (b and f) acute post-STI; (c and g) chronic treated; (d and h) chronic untreated.

FIG. 4.

Comparison of breadth and magnitude between the different study groups. Panel a summarizes the number of epitopic regions targeted per individual in the different study groups, while panel b indicates the total magnitude of the CD8-T-cell response to the entire expressed genome per individual (measured by Elispot and given in SFC/10⁶ PBMC). For both panels the median number of regions targeted and median magnitude for each group are indicated as horizontal black bars. P values were calculated using a two-tailed t test.

FIG. 5.

Correlation between total magnitude and breadth of the HIV-1-specific CD8⁺-T-cell response. The top panel shows the correlation between total magnitude of the HIV-1-specific CD8⁺-T-cell response in SFC/10⁶ PBMC (as measured by Elispot) to the total number of epitopic regions targeted per individual. The bottom panel shows the association between the total magnitude of the HIV-1-specific CD8⁺-T-cell response and the number of protein subunits recognized per study subject.

FIG. 6.

Relative contributions of individual protein subunits to the total magnitude of the HIV-1-specific CD8-T-cell response. Each pie chart shows the mean contribution of the individual protein subunits to the total magnitude of response directed against all of HIV-1 for one of the four study groups.

FIG. 7.

Correlation between viral load and breadth and magnitude of the total HIV-1-specific CD8-T-cell response. The association between HIV-1 RNA and the number of epitopic regions targeted per individual (top panel), the number of protein subunits recognized (middle panel), and the total HIV-1-specific magnitude as measured by Elispot (in SFC/million PBMC) (bottom panel) is depicted. Viral load in plasma (HIV-1 RNA copies/ml) was determined at the time of T-cell analysis for each individual. P values shown in each panel were determined by the Spearman rank correlation. The solid line represents a regression line, and all graphic representation was performed using Graphpad Prism 3. Plasma viral loads of <50 were expressed as 49 for statistical analysis.