Protection against Lethal Murine Coccidioidomycosis by a Soluble Vaccine from Spherules

Abstract

The formaldehyde-killed, whole-spherule vaccine, which is protective against lethal challenge of laboratory animals withCoccidioides immitis, was fractionated. It yielded a soluble, multicomponent, subcellular fraction termed the 27K vaccine. This vaccine, when it was accompanied by adjuvant, protected mice against lethal intranasal and intravenous challenge withC. immitis.

Characteristics of the Protective Subcellular Coccidioidal T27K Vaccine

“…The soluble, aqueous antigen, T27K, was prepared from thimerosalinactivated mature endosporulating spherules (TKS) of C. posadasii strain Silveira as previously described. 9,10 In brief, mature endosporulating spherules, were inactivated with thimerosal (1:10,000 final concentration), washed with endotoxin-free Cellgro PBS, pH 7.4 (Mediatech, Herndon, VA, USA), and stored at 4 • C in phosphate-buffered saline (PBS) + thimerosal (1:10,000 concentration) until use. Spherules were mechanically disrupted with glass beads in a bead beater (Biospec Products, Bartlesville, OK, USA) and then centrifuged for 30 min at 2 K × g. The supernatant was collected and subjected to additional centrifugation for 30 min at 27 K × g. This supernatant was then collected, passed through 0.2-m pore-size filter, concentrated using a stirred pressure cell (Millipore, Billerica, MA, USA) fitted with a YM10 (10,000 MWCO) membrane, dialyzed against water, lyophilized, and stored at -20 • C.…”

“…Later, the 27K vaccine provided protection against respiratory (IN) challenge equal to that provided by the FKS vaccine. 10 However, the formalin-derived 27K preparation did not fractionate well by traditional methods, such as polyacrylamide gel electrophoresis (PAGE), and this limited the ability to isolate and identify the protective components. Zimmermann et al 11 then showed that a similar vaccine prepared from thimerosal-inactivated spherules (T27K) also offered protection, but was more readily fractionated, perhaps because of less chemical cross-linking.…”

Molecular Cloning and Expression of a cDNA Encoding a Coccidioides posadasii 1,2‐α‐Mannosidase Identified in the Coccidioidal T27K Vaccine by Immunoproteomic Methods

“…The coccidioidal T27K vaccine was prepared from thimerosal-killed, mechanically disrupted mature endosporulating spherules as previously described. 5 The Silveira strain of C. posadasii, formerly known as the non-California C. immitis, was used. 9…”

“…1 For instance, it has been demonstrated that formaldehyde-killed whole spherules and mechanically disrupted spherules protect mice against respiratory challenge with the organism. [2][3][4][5] The development of a safe and effective human vaccine will most likely require the isolation of the protective component(s) from these crude preparations. The coccidioidal T27K vaccine is a soluble, subcellular fraction from the SE phase of C. posadasii consisting mainly of protein and carbohydrate which has been shown to protect mice against intranasal challenge with C. posadasii.…”

“…The coccidioidal T27K vaccine is a soluble, subcellular fraction from the SE phase of C. posadasii consisting mainly of protein and carbohydrate which has been shown to protect mice against intranasal challenge with C. posadasii. 5 Recent efforts have focused on the molecular characterization of the crude T27K vaccine so that potential protective antigens can be identified. [6][7][8] In this study, we report the identification of a class I 1,2-␣-mannosidase protein detected in the coccidioidal T27K vaccine using immunoproteomic methods.…”