Immunologic pressure within class I-restricted cognate human immunodeficiency virus epitopes during highly active antiretroviral therapy

Abstract

Cytotoxic T lymphocytes (CTL) and highly active antiretroviral therapy (HAART) are known to exert strong evolutionary pressures on the virus population during human immunodeficiency virus (HIV) infection. However, it is not known whether CTL responses continue to substantially affect viral evolution during treatment. To study the effect of immunologic pressure on viral sequences during HAART, we identified 10 targeted HIV-specific CD8+-T-cell epitopes in five treatment-naive patients, sequenced each epitope in plasma-derived viruses, and then identified evidence of immunologic pressure at these epitopes by comparing the frequency of viral variants in plasma to the frequency of the CD8+-T-cell response for each variant identified. For one of the five patients, evidence of viral evolution was found during therapy. The sequence of the CTL-targeted epitope changed from an apparent escape variant prior to the initiation of therapy, to the sequence that is best recognized by the CTL response after the initiation of therapy, and then finally to a new escape variant during continued therapy. These data show that CTL-mediated pressure can continue to affect viral evolution after the initiation of HAART, even when treatment drives the viral load below detectable levels, and suggest that antiretroviral therapy may preferentially inhibit those virus variants that escape the CTL response.

FIG. 1.

Epitope-specific CD8⁺ T-cell responses and autologous virus sequences from subject F. CD8⁺ T-cell responses to screening peptides and peptide variants are shown to the left in each graph. Epitope frequencies, determined by nucleic acid sequencing of viral clones, are shown to the right in each graph. Screening peptides and variant amino acid sequences are shown on the abscissa. None of the sequenced clones contained the screening peptide EIYKRWII. Sequencing data and response data for the epitope IRLRPGGKK and its variant are from week 12 of therapy. Sequencing data are from 12 weeks and response data are from week 16 of therapy for EIYKRWII and its variants. An asterisk indicates that no nucleotide sequence corresponding to the screening epitope EIYKRWII was found in any viral sequence determined either prior to or after the initiation of therapy.

FIG. 2.

Plot of viral load and CD8⁺ IFN-γ production in response to TAFTIPSI, TAFTIPST, and TAFTIPSM in patient R. CD8⁺-T-cell responses to individual peptides and viral loads are plotted against the time after the initiation of treatment. Responses to either TAFTIPSI, TAFTIPST, or TAFTIPSM were measured at the same time. The arrows and text indicate the numbers of viral clones sequenced and the sequence found at Pol 295-302 at the indicated times. The peptide concentration in all assays was 2 μg/ml.

FIG. 3.

Contour plots showing peptide-specific CD3 and CD8 down-regulation in CD8⁺ T cells from patient R in response to TAFTIPSI and other autologous viral epitopes 19 weeks after the initiation of HAART. PBMC were gated sequentially on small lymphocytes (a) and on small lymphocytes and CD3 expression (b). Plots show CD3 or CD8 surface staining versus intracellular IFN-γ staining. Cells were incubated in the absence or presence of added peptide, as indicated. The peptide concentration was 2 μg/ml throughout. The geometric mean fluorescence for CD3 or CD8 expression in responding CD8⁺ T cells is given in the lower right corner of each contour plot.

FIG. 4.

Plot of data showing frequency of CD8⁺-T-cell IFN-γ production in response to various concentrations of TAFTIPSI, TAFTIPST, and TAFTIPSM. Plot a shows a sigmoidal fit of CD8⁺ IFN-γ production in response to various concentrations of TAFTIPSI (○) and TAFTIPST (•). The half-saturation point for TAFTIPSI was 15 nM and that for TAFTIPST was 90 nM. Responses shown are from PBMC prepared from blood drawn during week 14 of treatment. Plot b shows CD8⁺ IFN-γ production in response to various concentrations of TAFTIPSI (○) and TAFTIPSM (•). Responses shown are from blood drawn during week 25 of treatment.

FIG. 5.

Peptide-induced surface expression of HLA-A2 and -B51 in T2 cells. T2 cells were incubated for 9 h in RPMI supplemented with 100 U of penicillin/ml, 100 μg of streptomycin sulfate/ml, 1.7 mM sodium glutamate, and 10 μg of β2-microglobulin/ml, exposed to NLVPVATV, TAFTIPSI, TAFTIPST, TAFTIPSM, and IRLRPGGKK at the indicated concentrations, and then stained with anti-HLA-A,B,C (W6/32). Results are shown as mean geometric fluorescence intensities versus peptide concentrations.

FIG. 6.

Clade B consensus sequence and consensus sequences for plasma viruses from weeks 0, 14, and 19 of HAART for subject R. Twenty-four, 16, and 18 plasmids containing amplified viral cDNAs were sequenced, respectively. Dots indicate identity with the clade B consensus sequence. The locations of common drug resistance mutations for zidovudine (V118I), lamivudine (M184V), and nevirapine (K103N, Y181C/I, and Y188C/L/H) are circled in the consensus B clade sequence.