F-029. Multiparametric Flow Cytometry as a Method to Assess the Potential of Coccidioidal Antigens as Human Vaccines

L. A. Nesbit1, S. M. Johnson2, D. Pappagianis2, N. M. Ampel1;
1Univ. of Arizona, Tucson, AZ, 2Univ. of California, Davis, CA.

Background. Coccidioidomycosis is a rapidly growing problem in the southwestern United States. A vaccine could result in protection of individuals living or traveling through in endemic regions. Recently, multiparametric flow cytometry has been used to assess the potential of candidate vaccines in other diseases. We have explored this in human coccidioidomycosis using T27K, a complex coccidioidal preparation that is protective in mice. Methods. 2 x 106 peripheral blood mononuclear cells (PBMC) were incubated in AIM-V medium for 48 hr with 20 µg/ml of T27K and 1 µg/ml of anti-CD28 with 1µg/ml of anti-CD49d. During the final 12 hr of incubation, 3 µg/ml of Brefeldin A was added. Subsequently, non-adherent cells were stained with fluorescent antibodies directed against CD4, IFN-γ, IL-2, and TNF-α. A lymphocyte gate was established for 4.0 x 105 cells and the number of cells expressing intracellular cytokines counted. Results. PBMC from 7 coccidioidal immune donors incubated with T27K resulted in a significant increase in the frequency of CD4+ T lymphocytes expressing IFN-γ, IL-2 and TNF-α in various combinations compared to cells incubated alone (for all combinations, P = 0.02). As shown in the figure, there was a distinct subset of cells that simultaneously expressed all three cytokines (far left, +++). Conclusions. T27K, a known protective murine vaccine, produces a cytokine pattern consistent with a protective immune response in humans. Through empiric cytokine analysis of other antigens, this system can be used to assess other possible vaccine candidates.