Evidence of CD8+ T-cell-mediated selective pressure on human immunodeficiency virus type 1 nef in HLA-B*57+ elite suppressors

Abstract

Elite suppressors (ES) are human immunodeficiency virus type 1 (HIV-1)-infected patients who maintain viral loads of <50 copies/ml without treatment. The observation that the HLA-B*57 allele is overrepresented in these patients implies that HIV-1-specific CD8(+) T cells play a key role in suppressing viral replication. We have previously shown that while CD8(+) T-cell escape mutations are rarely seen in proviral Gag sequences in resting CD4(+) T cells from peripheral blood, they are present in every clone amplified from the low levels of free virus in the plasma of HLA-B*57(+) ES. In this study, we compared the pattern of mutations in Nef sequences amplified from peripheral blood CD4(+) T cells and from plasma virus. We show that Nef mutations are present in plasma virus but are rare in the cellular sequences and provide evidence that these plasma Nef variants represent novel escape mutants. The results provide further evidence of CD8(+) T-cell-mediated selective pressure on plasma virus in ES and suggest that there must be ongoing HIV-1 replication in spite of the very low viral loads seen for these patients.

FIG. 1.

Phylogenetic analyses of nef sequences obtained via amplification of nef from both free plasma virus and resting CD4⁺ T cells. All reactions were carried out under limiting dilution conditions and were clonal. Only sequences derived from independent PCRs are shown on the trees. Phylogenetic analyses were carried out on the sequences using both classical maximum likelihood and Bayesian approaches. Only maximum likelihood trees are shown; however, both methods yielded identical topologies. The precision of the phylogenetic reconstructions was analyzed via bootstrap replication, with bootstrap support values of greater than 50% illustrated on the trees. M group ancestral sequences were used as outgroups. For each patient studied, colored triangles represent sequences obtained from free plasma virus, while colored circles represent proviral sequences derived from resting CD4⁺ T cells. The colors of the circles and triangles correspond to the time points at which the sequences were obtained. Time of entry refers to the time the first sequence was obtained.

FIG. 2.

Sequence analysis of region of Nef containing all four HLA-B*57-restricted epitopes. KF9 (Nef 82-90) is shaded in gray, HW9 (Nef 116-124) is shaded in pink, the overlapping epitope YT9 (Nef 120-128) is encased in a box, and the epitope GF14 (Nef 130-143) is shaded in green. The epitope Nef 106-119 is targeted by ES3 and is shown encased in a box. Where available, both plasma and proviral sequences are shown with the date when sample was obtained (month/year) as well as the number of independent clones analyzed. The first sequence shown for each subject is the consensus clade B sequence. Homology to this sequence is indicated by dots.

FIG. 3.

IFN-γ responses to variants of epitope KF9. Proviral variants are shown in blue and plasma variants are shown in pink for ES8 (A) and ES3 (B). For ES7 (C) and ES9 (D), the consensus clade B variants are shown in blue and autologous variants are shown in pink.

FIG. 4.

IFN-γ secretion from cell lines in response to variants of epitope KF9. Cell lines specific for KF9 variants KAAVDLSHF (A) and KAALDLSHF (B) were stimulated with peptides. Proviral variants are shown in blue and plasma variants are shown in pink.

FIG. 5.

IFN-γ responses to autologous variants of Nef epitopes. (A and B) The response to consensus clade B (blue) versus autologous (pink) variants of Nef 116-124 is shown for ES9 (A) and ES8 (B). (C) The response to consensus clade B (blue) versus autologous (pink) variants of Nef 120-128 is shown for ES8. (D) The response to proviral (blue) versus plasma (pink) variants of Nef 105-119 is shown for ES3.