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. 2006 Apr 18:7:8.
doi: 10.1186/1471-2172-7-8.

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response

Jeffrey R Currier  1 , Unchalee Visawapoka, Sodsai Tovanabutra, Carl J Mason, Deborah L Birx, Francine E McCutchan, Josephine H Cox
Affiliations

CTL epitope distribution patterns in the Gag and Nef proteins of HIV-1 from subtype A infected subjects in Kenya: use of multiple peptide sets increases the detectable breadth of the CTL response

Jeffrey R Currier et al. BMC Immunol. .

Abstract

Background: Subtype A is a major strain in the HIV-1 pandemic in eastern Europe, central Asia and in certain regions of east Africa, notably in rural Kenya. While considerable effort has been focused upon mapping and defining immunodominant CTL epitopes in HIV-1 subtype B and subtype C infections, few epitope mapping studies have focused upon subtype A.

Results: We have used the IFN-gamma ELIspot assay and overlapping peptide pools to show that the pattern of CTL recognition of the Gag and Nef proteins in subtype A infection is similar to that seen in subtypes B and C. The p17 and p24 proteins of Gag and the central conserved region of Nef were targeted by CTL from HIV-1-infected Kenyans. Several epitope/HLA associations commonly seen in subtype B and C infection were also observed in subtype A infections. Notably, an immunodominant HLA-C restricted epitope (Gag 296-304; YL9) was observed, with 8/9 HLA-CW0304 subjects responding to this epitope. Screening the cohort with peptide sets representing subtypes A, C and D (the three most prevalent HIV-1 subtypes in east Africa), revealed that peptide sets based upon an homologous subtype (either isolate or consensus) only marginally improved the capacity to detect CTL responses. While the different peptide sets detected a similar number of responses (particularly in the Gag protein), each set was capable of detecting unique responses not identified with the other peptide sets.

Conclusion: Hence, screening with multiple peptide sets representing different sequences, and by extension different epitope variants, can increase the detectable breadth of the HIV-1-specific CTL response. Interpreting the true extent of cross-reactivity may be hampered by the use of 15-mer peptides at a single concentration and a lack of knowledge of the sequence that primed any given CTL response. Therefore, reagent choice and knowledge of the exact sequences that prime CTL responses will be important factors in experimentally defining cross-reactive CTL responses and their role in HIV-1 disease pathogenesis and validating vaccines aimed at generating broadly cross-reactive CTL responses.

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Figures

Figure 1
Figure 1
Phylogenetic trees of the gag (A) and nef (B) proviral DNA sequences obtained from the cohort. Circled are the DNA sequences of the isolates from which the screening peptide sets were deduced. The large cluster of sequences in both trees represents subtype A. Bootstrap values are shown.
Figure 2
Figure 2
Magnitude of CTL responses measured for each subject in response to either Gag (A) or Nef (B) peptide sets (Con = consensus; Iso = isolate). For each study subject all detected ELIspot responses against each peptide set were totaled (expressed as SFC/106 PBMC) and plotted beside each other.
Figure 3
Figure 3
Frequency of response histograms for all peptides from each Gag peptide set. All peptide responses scored as positive (by cut-off criteria described in Methods) are shown, including those obtained for adjacent overlapping peptides. Histograms are shown for (A) consensus subtype A, (B) subtype A (CRF01_AE) isolate, (C) subtype C isolate, and (D) subtype D isolate. Peptides are numbered sequentially along the x-axis.
Figure 4
Figure 4
Frequency of response histograms for all peptides from each Nef peptide set. All peptide responses scored as positive (by cut-off criteria described in Methods and Materials) are shown, including those obtained for adjacent overlapping peptides. Histograms are shown for (A) subtype A isolate, (B) subtype C isolate, and (C) subtype D isolate. Peptides are numbered sequentially along the x-axis.
Figure 5
Figure 5
Venn diagram display of responses (epitopes) detected with either Gag (A) or Nef (B) peptide sets. Each overlapping region is annotated with the number of responses detected for that particular relationship. For example, the central region in panel A, which is bounded by all four circles, shows that 22 responses were detected by all four Gag peptide sets.
Figure 6
Figure 6
A. The YL9 response is HLA-CW*0304 restricted. CFC assays for IFNγ up-regulation were performed using 15-mer peptide pulsed (solid bars) or sham-pulsed (open bars), partially HLA-matched BLCL as antigen-presenting cells for PBMC. Media alone, and media containing SEB (hatched bars), served as negative and positive controls respectively. The SEB response has been truncated for clarity and the frequency of responding cells appended beside the bar. All panels were gated based upon the CD3+CD8+CD69HIGH cell population in the PBMC. Only BLCL possessing the HLA- CW*0304 allele (boxed) can present the peptide. The parent 15-mer (peptide #74 from A-consensus peptide set), and the minimal 9-mer peptide (YL9) added alone to the PBMC stimulated an equivalent frequency of CD8+ T cells. B. A CTL line generated by in vitro stimulation with peptide #74 from the A-Gag consensus peptide set efficiently lysed autologous BLCL pulsed with the same peptide, but not adjacent peptides #73 or #75, in a standard chromium release assay. Autologous BLCL pulsed with the predicted minimal epitope (9-mer YL9), and three variants of this epitope detected in the same cohort, were also lysed. Symbols for the effector to target ratios are denoted below the figure panel. C. Effector cells from the re-stimulated CTL line from above were able to lyse YL9-pulsed allogeneic BLCL, which express different alleles from the HLA- CW03 family. For each BLCL tested sham- and peptide pulsed targets are shown at a range of E:T ratios from 20:1 down to 2.5:1. The symbols for the E:T ratios are the same as used in panel 6B. The left-most BLCL on the panel are the autologous cells (HLA-CW*0304 positive), and right-most BLCL are negative for HLA- CW03 alleles. D. Sequences of the peptides used for the minimal epitope mapping and variant peptide testing.
Figure 7
Figure 7
CTL response of two HLA-CW*0304 positive subjects (A. KNH1237; B. KNH1263) against YL9 peptide and three variants as measured by IFNγ ELIspot assay. Peptides were titrated through a concentration range of 10-5 to 10-11 M as denoted. The dashed line denotes the YL9 index peptide for clarity.
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