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Southwest Tanzania is affected by an HIV-1 epidemic consisting of subtypes A, C, and D, and their recombinant forms. This study was designed to assess whether the Gag- and Nef-specific T-cell response is biased towards recognizing the infecting subtype.The infecting subtypes were characterized with a Multi-hybridization assay that discriminates between subtypes A, C and D. The interferon-γ ELISPOT assay was used to detect the Gag- and Nef-specific T-cell responses in freshly isolated peripheral blood mononuclear cells in 56 seropositive patients. To study the HIV-specific T-cell responses, isolate-based Gag and Nef peptide sets representative of the locally occurring subtypes were used. The results were analysed at the total protein and single peptide level.In the study population, 35% were infected with a pure C subtype, 24% and 23% with ACD or AC recombinant forms, respectively. The total magnitude (P < 0.01) and breadth (P < 0.01) of the Gag-specific T-cell response detected with the subtype C-Gag peptide set was significantly greater than that detected with either the subtype A-Gag or D-Gag peptide sets. No significant difference was observed in the Nef-specific response. In 85% of responses targeting the most immunodominant Gag epitopes with subtype-specific sequence differences, the best recognized epitope variant corresponded to the infecting subtype.The Gag-specific T-cell response had a preference for recognizing peptides related to the infecting subtype.