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51 Influence of the Antigen and the Assay on the Characterisation of CTL Responses to HIV-1
E. Iglesias, A. Samri*, G. Kamkamidze, Y. Sun, G. Carcelain, C. Quillent-Gregoire, P. Debre, and B. Autran
Hosp. Pitié-Salpêtrière, Paris, France
Objectives: First, to compare the results of memory CTL analysis using chromium release and the effector CTL analysis using ex vivo ELISPOT assay. Second, by using recombinant vaccinia viruses (rVV) and single versus pooled optimal peptides, to determine whether the way of antigen presentation in those assays might influence the results.
Methods: CD8+ T-cell populations were analyzed with IFN-gamma ELISPOT assays using rVV expressing Env, Gag, Nef, Pol, Tat, and Vif genes or a panel of 44 known CTL HLA-matched peptides from Gag, Pol, Env, and Nef proteins from HIV-1 LAI. Seven chronically infected patients from the French Cohort IMMUNOCO and 7 long-term nonprogressors from the ALT cohort were analyzed.
Results: Chromium (Cr) release and ELISPOT assays using rVV are not different regarding their capacity for detecting CTL responses despite rare discrepancies. The sum of spot-forming cells (SFC)/106 cells for all peptides for individual proteins was calculated for all patients and compared with the number of spot obtained in the rVV ELISPOT assay. For all proteins the frequencies measured using peptides were always higher than those calculated using rVV. In contrast, the CTL frequencies obtained using pooled peptides were similar to the sum of SFC of the individual peptides for each protein. Finally, these observations were observed in both groups of patients.
Conclusions: (1) Cr release and rVV ELISPOT are comparable in their CTL detection capacity; (2) there are no differences among pools and single-tested optimal peptides in ELISPOT assay; (3) ELISPOT assays using peptides are more sensible than those using rVV.
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