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76 A Panel of MHC Class I Restricted Viral Peptides for Use as a Control for Vaccine Trial ELISPOT Assays
E. Kuta*1, J. Currier1, E. Turk1, L. Loomis-Price1, G. Ferrari2, S. Janetzki3, D. Birx1, and J. Cox1
1The US Military HIV Res. Program, Rockville, MD, USA; 2Duke Univ., Durham, NC, USA; and 3Zellnet Consulting, New York, NY, USA
Background: As progress is made toward developing a safe and effective HIV vaccine, there is a need for a robust, sensitive assay to evaluate vaccine immunogenicity in Phase II/III trials. The enzyme-linked immunospot (ELISPOT) assay fits these criteria and is a technology that is readily transferable, can be used with fresh and cryopreserved specimens, and is amenable to high throughput screening. There is a need for reagents that can be used as positive controls and for optimizing and standardizing the assay.
Methods: We selected a panel of twenty-three 10- to 11-mer viral (Flu, CMV, and EBV) epitopes recognized by CD8-positive T cells and presented by 11 class I HLA-A and HLA-B types whose cumulative frequencies represent >100% of Caucasian individuals. We examined interferon-gamma (IFN-gamma) secretion in PBMC incubated with the peptides using a modified ELISPOT assay.
Results: IFN-gamma secretion was detected in 16/18 (89%) HIV-1 seronegative and 14/20 (70%) HIV-1 seropositive individuals. Release of IFN-gamma in response to the pool of peptides was CD8+ T-cell mediated and HLA restricted. Stimulation of PBMC with individual peptides or the pool of 23 peptides led to the expansion of T cells capable of killing target cells expressing the appropriate viral antigen. IFN-gamma secretion by PBMC stimulated with the peptide pool could be detected by intracellular cytokine (ICC) staining in the CD8+ T-cell population. Leucopheresis (LP) samples provide a convenient source of cryopreserved PBMC for testing interassay, interlab, and interperson variability. One LP sample was tested using different protocols, reagents, and laboratory personnel; the range of IFN-gamma secreting cells for 17 different assays was 1,428(2,888/106 PBMC. When using an automated ELISPOT reader, the size, shape, and intensity of the peptide-specific IFN-gamma secreting cells could be identified, while excluding artifacts or background spots. Overall, the peptide-specific spots were superior to PHA-specific spots for use as controls in the ELISPOT assay.
Conclusions: The panel of peptides described here will provide a valuable reagent for QC of ELISPOT, ICC, and CTL assays for use in vaccine trials.
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