Figure 2.

Amino acid changes in SIV Gag from r00032 post breakthrough are CD8+ and CD4+ T cell escape mutations. A) Schematic of the overlapping 15-mer peptides used to culture CD8+ and CD4+ T cell responses targeting the indicated regions of Gag, which exhibited pre- breakthrough substitutions. B) IFN-γ ELISpot measurements showing the response against SIVmac239 Gag_437-451LQ15 and the mutant peptide containing Gag K442R by a CD8+ T cell line. White bar = Response against peptide Gag_437-451LQ15. Black bar = Response against mutant peptide containing Gag K442R. Bars indicate the mean of duplicate wells with standard deviations shown. The point mutation is highlighted in grey and bold. C) IFN-γ ELISpot measurements showing the response against serial dilutions of SIVmac239 Gag_271-278LY8 and the mutant peptide containing Gag R276K by a Mamu-B*81:01-restricted CD8+ T cell line (top panel). Data are normalized to percent maximum IFN-γ-production and are the mean of duplicate wells. Intracellular cytokine staining showing the response against no stimulation, SIVmac239 Gag_273-287KK15, and the mutant peptide containing Gag R276K by a DRB1*12-restricted CD4+ T cell line (bottom panel). The point mutation is highlighted in grey and bold. D) Intracellular cytokine staining showing the response against no stimulation, SIVmac239 Gag_57-71CG15, and the mutant peptide containing Gag V63A by a DRB1*10:07-restricted CD4+ T cell line (top row) and a CD8+ T cell line (bottom row) Mamu-A*02 or Mamu-B*008:01. The point mutation is highlighted in grey and bold. E) Intracellular cytokine staining showing the response against no stimulation, SIVmac239 Gag_197-211QA15, and the mutant peptide containing Gag D205E by a DRB*W4:01-restricted CD4+ T cell line. The point mutation is highlighted in grey and bold. All data shown are representative of 3 or more independent experiments. N.D. = not determined. # = MHC-I restriction not Mamu-A1*002:01 or Mamu-B*008:01.