AEGiS-15IAC: The use of viral peptides as a quality control for ELISPOT assays in HIV-1 vaccine trials in Kenya.

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

The use of viral peptides as a quality control for ELISPOT assays in HIV-1 vaccine trials in Kenya.

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. A10065)

Farah BM, Ogola S, Indangasi J, Oyaro M, Anzala O, Omosa-Manyonyi G, Jaoko W, Gillmour J, Bwayo J
Kavi, Nairobi, Kenya

BACKGROUND: ELISPOT assay has been used both for answering basic research questions and for immunogenicity assessments of HIV-1 vaccine candidates in clinical trials. As progress is made towards developing a safe and effective HIV vaccine, there must be an assay that is robust and sensitive and the ELISPOT is one such assay. However the reagents that can be used as positive control must be optimized and standardized. Although mitogens such as PHA provide a qualitative answer to whether the assay works, they do not test antigen-specific T cell activation. The use of antigen-specific stimulation rather than mitogen stimulation is also good for standardizing the IFN-γ secretion kinetics. We describe the use of a pool of Flu, EBV and CMV (FEC) peptides to elicit release of IFN-γ from T- cells of volunteers in HIV-1 vaccine trials.

METHOD: A panel of 22, 8-10 mer epitopes recognized by CD8+ T cells and presented by 12 class I HLA-A and HLA-B types were selected. We examined the secretion of IFN-γ in PBMC incubated with the peptides using a modified ELISPOT assay in healthy African volunteers enrolled in four double-blinded, placebo controlled HIV-1 vaccine studies at eight time points.

RESULTS: IFN-γ secretion was detected in 46% in first study, 73% in second study, 73% in third study and 58% in fourth study. Release of IFN-γ response to pool of peptides was CD8+ T cell mediated and HLA restricted and the responses were consistent at each time point.

CONCLUSION: FEC peptide pool is useful for standardization, quality control and optimization of the ELISPOT assay in vaccine trials. The FEC pool in addition to PHA is useful as a control for ELISPOT assay in vaccine trials where HLA type is not necessarily already known at the time of the assay. FEC pool allows different laboratories to determine the extent of inter-assay and inter-laboratory variations in vaccine trials.

Keywords: AEGIS, AIDS Vaccines, CD8-Positive T-Lymphocytes, Peptides, Enzyme-Linked Immunosorbent Assay, HIV-1, Quality Control, Lymphocyte Activation, Epitopes, Antigens, Kenya, virology, immunology

040711
A10065