Functional properties and epitope characteristics of T-cells recognizing natural HIV-1 variants

Abstract

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 T-cell 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 non-conservative (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-gamma 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.

Figure 1. Effects of numbers and conservation of amino acid substitutions on recognition of T-cell epitope variants in subjects with primary HIV-1 infection (A, B) and in HIV-1 vaccine recipients (C, D)

PBMC were stimulated with HIV-1 PTE peptide sets in a matrix format and tested for IFN-γ production using an ELISpot assay. Responses were examined to the Nef protein in subjects with primary infection and to Gag, Env and Pol in the vaccinees. Responses > 3-fold background and > 50 SFC/10⁶ cells above background were scored positive. All responses were confirmed at the single peptide level. Shown is the frequency of recognition of epitope variants containing one, two and three amino acid substitutions; and conservative, semi-conservative and non-conservative substitutions.

Figure 2. IFN-γ and Granzyme B responses to optimal epitope variants in the Nef protein

Standard IFN-γ and granzyme B ELISpot assays were performed by stimulating with the optimal epitopic peptides and variants at serial concentrations between 10,000 and 0.1 ng/ml. Shown here are PBMC responses from subjects 1188 (WF9 epitope variants) (A) and 1692 (RW8 epitope variants) (B). The SFC frequencies are plotted against the log₁₀ peptide concentration. CON B sequence for each epitope is indicated on the top. Identity of autologous sequence to amino acid in the CON sequence is indicated by a dot.

Figure 2. IFN-γ and Granzyme B responses to optimal epitope variants in the Nef protein

Standard IFN-γ and granzyme B ELISpot assays were performed by stimulating with the optimal epitopic peptides and variants at serial concentrations between 10,000 and 0.1 ng/ml. Shown here are PBMC responses from subjects 1188 (WF9 epitope variants) (A) and 1692 (RW8 epitope variants) (B). The SFC frequencies are plotted against the log₁₀ peptide concentration. CON B sequence for each epitope is indicated on the top. Identity of autologous sequence to amino acid in the CON sequence is indicated by a dot.

Figure 3. T-cell proliferation in response to stimulation with Nef epitope variants relative to IFN-γ and granzyme B responses

Standard IFN-γ and granzyme B ELISpot assays were performed by stimulating with the optimal epitopic peptides and variants at serial concentrations between 10,000 and 0.1 ng/ml. Shown here are PBMC responses from subjects 1238 (AL9 epitope variants) (A) and 1212 (AL9 epitope variants) (B). The SFC frequencies are plotted against the log₁₀ peptide concentration. Autologous sequence where available is shown and identity of autologous sequence to amino acid in the CON sequence is indicated by a dot. The decrease in CFSE signal was used to monitor proliferation. Positive and negative controls included cells incubated with anti-CD3 mAb (30 ng/mL) and anti-CD28 mAb (1 µg/mL), and with medium alone.

Figure 3. T-cell proliferation in response to stimulation with Nef epitope variants relative to IFN-γ and granzyme B responses

Standard IFN-γ and granzyme B ELISpot assays were performed by stimulating with the optimal epitopic peptides and variants at serial concentrations between 10,000 and 0.1 ng/ml. Shown here are PBMC responses from subjects 1238 (AL9 epitope variants) (A) and 1212 (AL9 epitope variants) (B). The SFC frequencies are plotted against the log₁₀ peptide concentration. Autologous sequence where available is shown and identity of autologous sequence to amino acid in the CON sequence is indicated by a dot. The decrease in CFSE signal was used to monitor proliferation. Positive and negative controls included cells incubated with anti-CD3 mAb (30 ng/mL) and anti-CD28 mAb (1 µg/mL), and with medium alone.

Figure 4. Epitope distribution in the Nef protein relative to entropy and cleavage scores

Amino acid entropy (A) and proteasomal cleavage scores (B) were compared between 50 optimal epitopes versus all possible 9-mers spanning the nef sequence. Epitope distribution relative to entropy and cleavage scores is shown from the N- to C-terminus (C). Entropy and C-terminus cleavage scores for targeted optimal epitopes and the group of all Nef subtype B 9-mers were compared by the use of Wilcoxon Signed Rank Test.

Figure 4. Epitope distribution in the Nef protein relative to entropy and cleavage scores

Amino acid entropy (A) and proteasomal cleavage scores (B) were compared between 50 optimal epitopes versus all possible 9-mers spanning the nef sequence. Epitope distribution relative to entropy and cleavage scores is shown from the N- to C-terminus (C). Entropy and C-terminus cleavage scores for targeted optimal epitopes and the group of all Nef subtype B 9-mers were compared by the use of Wilcoxon Signed Rank Test.