A human immunodeficiency virus (HIV)-preventive vaccine will likely need to induce broad immunity that can recognize antigens expressed within circulating strains. To understand the potentially relevant responses that T-cell based vaccines should elicit, we examined the ability of T cells from early infected persons to recognize a broad spectrum of potential T-cell epitopes (PTE) expressed by the products encoded by the HIV type 1 (HIV-1) nef gene, which is commonly included in candidate vaccines. T cells were evaluated for gamma interferon (IFN-␥) secretion using two peptide panels: subtype B consensus (CON) peptides and a novel peptide panel providing 70% coverage of PTE in subtype B HIV-1 Nef. Eighteen of 23 subjects' T cells recognized HIV-1 Nef. In one subject, Nef-specific T cells were detected with the PTE but not with the CON peptides. The greatest frequency of responses spanned Nef amino acids 65 to 103 and 113 to 147, with multiple epitope variants being recognized. Detection of both the epitope domain number and the response magnitude was enhanced using the PTE peptides. On average, we detected 2.7 epitope domains with the PTE peptides versus 1.7 domains with the CON peptides (P ‫؍‬ 0.0034). The average response magnitude was 2,169 spot-forming cells (SFC)/10 6 peripheral blood mononuclear cells (PBMC) with the PTE peptides versus 1,010 SFC/10 6 PBMC with CON peptides (P ‫؍‬ 0.0046). During early HIV-1 infection, Nef-specific T cells capable of recognizing multiple variants are commonly induced, and these responses are readily detected with the PTE peptide panel. Our findings suggest that Nef responses induced by a given vaccine strain before HIV-1 exposure may be sufficiently broad to recognize most variants within subtype B HIV-1.

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The cellular immune system is characterized by flexibility with respect to epitope recognition at the level of peptide binding to HLA molecules and HLA-peptide complexes to T-cell receptors (TCRs). For epitopes recognized by cytotoxic T-lymphocytes (CTLs), amino acid substitutions at different positions have varying impact on recognition. By analyzing the frequencies of specific amino acid substitutions at each position in conjunction with HLA-peptide binding and immune-response data, we have developed new methods to predict cross-reactive recognition of epitope variants by CTLs. We derived position-specific substitution matrices (EPSSMs) through the analysis of known HLA ligands and achieved relatively accurate prediction of detrimental and tolerated amino acid substitutions. Initial analysis of amino acid substitutions in CTL epitopes with degenerate recognition showed strong position-specific preferences. This first systematic analysis further suggested that spatial constraint may be the major molecular factor determining the degenerate epitope recognition. As the data cumulates, we anticipate that eventually EPSSMs will be available for prediction of degenerate T-cell epitope recognition.

To understand how broad recognition of HIV-1 variants may be achieved we examined T-cell reactivity in newly-infected persons as well as vaccine recipients to a broad spectrum of potential Tcell epitope (PTE) variants containing conservative, semi-conservative and non-conservative amino acid substitutions. Among early-infected persons T-cells recognized epitope variants with one substitution at a significantly higher frequency versus those with two (P=0.0098) and three (P=0.0125) substitutions. Furthermore T cells recognized variants containing conservative substitutions at a higher frequency versus those containing semi-conservative (P=0.0029) and nonconservative (P<0.0001) substitutions. Similar effects were observed on recognition of variants by vaccine induced T-cells. Moreover even when variants were recognized, the IFN-γ and granzyme B responses as well as T-cell proliferation were of lower magnitude. Finally, we show that epitope distribution is strongly influenced by both processing preferences and amino acid entropy. We conclude that induction of broad immunity is likely to require immunogen sequences that encompass multiple variants. However, cost-effective design of peptide and sequence based vaccine immunogens that provide maximal coverage of circulating sequences may be achieved through emphasis on virus domains likely to be T-cell targets.