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65 Detection of HIV-1 Cross-Clade and Clade-Specific CD8 Responses from Thai and US HIV-1-Infected Individuals Using a Modified Interferon-gamma ELISPOT Assay
J. Currier*1, M. DeSouza2, P. Chanbancherd2, D. Birx1, and J. Cox1
1The US Military HIV Res. Program, Rockville, MD, USA and 2Armed Forces Inst. of Pathology, Bangkok, Thailand
Background: Detection of cross-clade and clade-specific CD8 responses to HIV-1 has been traditionally performed using chromium release cytotoxicity assays following in vitro stimulation with specific antigen. An inherent disadvantage of this assay is that it relies upon the in vitro outgrowth of antigen-specific CD8 cells, which may skew the relative frequencies of these cells in the PBMC. Here, a modified interferon-gamma ELISPOT assay has been used to directly measure the frequency of cross-clade and clade-specific CD8 responses from PBMC of North Americans and Thais.
Methods: Recombinant vaccinia vectors expressing Gag, Env, and Nef gene products from HIV-1 clades A, B, C, D, F, G, H, and CRF_01 were used to infect autologous B-LCL cells from HIV-1 clade B- and CRF_01-infected donors. Antigen-specific responses from PBMC cocultured for 24 hours with infected autologous B-LCL were measured directly using the interferon-gamma ELISPOT assay. CD8 dependence of the responses was verified by immunomagnetic bead depletion. Fine specificity of the cross-clade responses was further mapped using overlapping synthetic peptides in a matrix format.
Results: High frequencies (>2,000 SFU/106 PBMC) of HIV-1 cross-clade and clade-specific CD8 responses were detectable directly from the PBMC of infected North Americans and Thais. In some cases the infecting clade was not predictive of the clade toward which a response is directed or of the relative magnitude of the responses. Mapping of the fine specificity of the cross-clade responses with synthetic peptides revealed that the response was limited to a single epitope. Using a combination of the recombinant vaccinia-infected B-LCL and the overlapping peptide matrix assays, broadly cross-clade-specific responses could be mapped to the epitope level in 3 days.
Conclusions: The application of B-LCL infected with recombinant vaccinia vectors expressing HIV-1 gene products from a range of different clades has been successful for determining the cross-clade specificity of CD8 responses both qualitatively and quantitatively.
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