F-031. Isolation and Evaluation of a Glycoprotein from the Coccidioidal Vaccine T27K

S. M. Johnson1, N. M. Ampel2, L. A. Nesbit2, C. N. Miller1, D. Pappagianis1;
1Univ. of California, Davis, CA, 2Univ. of Arizona, Tucson, AZ.

Background: The coccidioidal vaccine T27K is a complex preparation derived from mature endosporulating spherules. This preparation engenders protection against lethal Coccidioides infection in the mouse model and specifically stimulates peripheral blood mononuclear cells (PBMC) from immune human donors as demonstrated by release of IL-2. Previous studies have shown this preparation to be highly glycosylated and following deglycosylation, the stimulatory capacity was reduced. We have therefore sought to isolate and evaluate the glycoproteins from the T27K. Methods: Components of the T27K were reduced and denatured and then separated by continuous elutriation electrophoresis (CCE) using the BioRad Prep Cell. Fractions, 1 ml, were collected and samples of each electrophoresed and the gels stained with Gel Code Blue. Fractions containing the same molecular weight components as judged following electrophoresis were pooled. Glycoproteins were then isolated by affinity chromatography with conconavalin A (ConA) lectin. Presence of glycosylation was confirmed by Periodic Acid Schiff (PAS) staining following electrophoresis. One glycoprotein migrating at approximately 60 kDa was initially identified and isolated. This protein as well as T27K was incubated with immune and non-immune PBMC and the IL-2 release measured. The protein’s identity was determined using mass spectrometry (LC MS/MS). Results: Following CCE and ConA lectin affinity chromatography, a component migrating as a single band at approximately 60 kDa was obtained. This band, stained with PAS and was terminally mannosylated as indicated by its binding to ConA. When immune cells were stimulated with this protein, IL-2 was specifically released. Mass spectrometry identified the protein as the heat shock protein 60. Conclusions: A glycoprotein, identified using mass spectrometry as heat shock protein 60, was isolated from the T27K using CCE and ConA lectin affinity chromatography. This protein retained its stimulatory capacity when incubated with human immune PBMC. This protein represents a vaccine candidate and will be evaluated in the future.