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32 Immune Recognition of Conserved Regions within Nef from HIV-1 Subtype C-Infected Individuals from Southern Africa
T. Mashishi*1, G. Hunt1, S. Loubser2, S. Nyoka1, W. Hide3, C. Williamson2, G. Ferrari5, A. Puren1, G. Ramjee4, S. A. Karim4, H. Cao6, H. Sheppard6, and C. Gray1
1Natl. Inst. for Virology, Johannesburg, South Africa; 2Univ. of Cape Town, South Africa; 3Univ. of Western Cape, South Africa; 4Med. Res. Council, Durban, South Africa; 5Duke Univ., Durham, NC, USA; and 6California Dept. of Hlth. Services, Berkeley, USA
Background: Analysis of the regulatory gene nef for sequence homology between subtypes is an important aim for vaccine development. We report on the nucleic acid and amino acid sequence analysis of Nef as well as Nef-specific CD8+ T-cell responses of subtype C HIV-1-infected individuals.
Methods: Full-length nef sequences were generated from 43 HIV-1 South Africans. Phylogenetic analysis and amino acid alignments of the sequences were used to determine the degree of intra- and intersubtype variability. A combination of the INFg ELISPOT assay and intracytoplasmic cytokine staining was used to identify regions within Nef that elicit CD8+ T-cell responses in 21 subtype C HIV-1-infected individuals from Southern Africa. A set of subtype B- and subtype C-based Nef overlapping peptides was used in the assays.
Results: Phylogenetic trees showed that the nef nucleotide sequences from South Africa as well as reference subtype C sequences from India, Botswana, and Brazil clustered together significantly (bootstrap value of 100%). The nucleotide sequences from the 43 amino acid sequences were aligned with a consensus C sequence to identify conserved and variable intraclade regions. In agreement with published data, all functional and structural motifs of Nef were highly conserved. The central region of Nef was significantly (p < 0.001) more conserved (amino acid variation of 12.2 + 7.1%) than either the N (17.0 + 9.6%) or C termini (16.6 + 6.1%). Both the central region and C terminus of Nef have been shown to be rich in CTL epitopes. Out of 21 subtype C-infected individuals that were screened for Nef responses, 13 (62%) gave a response to a peptide containing the QY-9 (QVPLRPMTY) epitope. HLA studies have shown that this epitope can be restricted by HLA- A2, A3, A11, and B35, which account for 39% of the South African population. Seven individuals (33.3%) gave responses to peptide sequences containing likely novel epitopes. All responses observed were directed to the highly conserved central region of Nef.
Conclusions: These data show that the central region of Nef is highly conserved across HIV-1 clades and that among recently infected individuals recognizing Nef, two-thirds focus on 1 immunodominant peptide within this region. These findings will be helpful in facilitating the choice of epitope-rich regions of Nef to be included into candidate vaccines.
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