15th International AIDS Conference Bangkok, Thailand - July 11-16, 2004

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10070)

Farah BM, Ogola S, Anzala O, Indangasi J, Tarragona A, Gibiansky L, Bwayo J
Kavi, Niarobi, Kenya

BACKGROUND: Phase I safety and immunogenicity vaccine trial designed to stimulate CTL responses started in Nairobi (Kenya) in January 2001. Thus, it is imperative to establish a standardized, sensitive approach to measure HIV-1 specific CD8+ T lymphocyte activities that can be employed in field trial settings and used to compare the relative immunogenicities of various vaccine regimens. We chose to focus on the development and validation of an IFN-γ Elispot assay for the screening and quantification of HIV-1 specific CD8+ T-cells.

METHODS: To establish optimal conditions for the performance of IFN-γ ELISPOT assay in healthy volunteers fresh and cryopreserved PBMC samples purified from single leukapheresis were analyzed. Samples from 9 volunteers were divided into two aliquots each. One aliquot from each pair was analyzed fresh using the ELISPOT assay, the other one was frozen, stored for one week and then analyzed using the same ELISPOT assay. To investigate the assay variability and define criteria for positive response, PBMC from 30 HIV-1 seronegative individuals were analyzed. The inter-and intra-operator variability were investigated.

RESULTS: Paired t-tests for the PHA, peptide pools and Flu, EBV,CMV (FEC) data has not revealed any significant differences between the fresh and frozen samples data. Analysis of variance (ANOVA model with OPERATOR and DAY effects) did not reveal significant (at α≥0.05 level) operator or day effects. Variability of SFU counts in the no-response wells (negative control -ve, pools 1, 2,3 4, 9 and 90) was about CV=91%, whereas variability in the wells with positive response was about CV=11% and CV=17% for PHA and FEC, respectively.

CONCLUSION: Results of the validation procedure confirmed excellent concordance in the ability to detect positive responses in cryopreserved and freshly isolated PBMCS. Variability inherent in the methodology is acceptably low and that reproducibility among operators is high.

040711
A10070

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