An immunoreactive apoglycoprotein purified from Coccidioides immitis

Abstract

Deglycosylation of glycoproteins in a lysate of spherules of Coccidioides immitis has permitted purification and partial characterization of a proline-rich pronase-sensitive antigen. Moreover, soluble antigen specifically stimulated lymphocytes from persons with dermal delayed-type hypersensitivity to coccidioidal antigens. When related to reference coccidioidin by tandem two-dimensional immunoelectrophoresis, the antigen fused in the anodal region with a specific reference antigen (antigen 2). It did not show identity with coccidioidal antigens used in conventional serologic assays. Although immunoblots of the purified protein with monospecific rabbit antiserum showed a single antigen at 33 kDa, the parent spherule lysate bound the same antibody in a broad band between 70 and >200 kDa, which could be explained by microheterogeneity of glycosylation. Immunoelectron microscopy using affinity-purified human antibodies localized the antigen to the cell wall and internal septa of spherules. These findings suggest that the apoglycoprotein may be important in human immune responses to coccidioidal infection.

The Cryptococcus neoformans GeneDHA1 Encodes an Antigen That Elicits a Delayed-Type Hypersensitivity Reaction in Immune Mice

“…Deglycosylation of CneF-Cap67 with anhydrous hydrogen fluoride did not affect its ability to elicit a positive DTH response in mice immunized against C. neoformans. We included this deglycosylation step because it had been very helpful in separating protein antigens in extracts from another fungal pathogen, Coccidioides immitis (5). Although most of the carbohydrate is removed by anhydrous hydrogen fluoride, residual monosaccharides would be expected to be present after this treatment (19).…”

Evaluation of the Proline-Rich Antigen of Coccidioides immitis as a Vaccine Candidate in Mice

“…Recombinant protein was electrophoresed on a sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gel electrophoresis (PAGE) gel and stained with Coomassie blue or electroblotted to nitrocellulose or polyvinylidene difluoride membranes (Schleicher and Schuell, Keene, N.H.). Nitrocellulose blots were probed with a goat antiserum to spherule-derived PRA, which has previously been described (6). The N-terminal sequences of enterokinase-treated recombinant proteins were determined on the two bands seen with SDS-PAGE (see Results).…”

“…In order to biochemically purify this protein, a spherule extract was chemically deglycosylated, which removes most O-linked carbohydrate (17). We found that people with coccidioidomycosis make both T-cell and antibody responses to deglycosylated PRA (6,11). To further evaluate the antigenicity of this protein, we have expressed it in Escherichia coli.…”

The Public Health Need and Present Status of a Vaccine for the Prevention of Coccidioidomycosis

“…29 Subsequent efforts focused on the use and evaluation of purified fractions obtained from the cell wall as potential immunizing antigens. 30,31 Now, modern molecular biology, genomics, and an ever-increasing understanding of the immunologic response to coccidioidomycosis has raised the possibility of efficient antigen discovery, cloning, and production, leading coccidioidomycologists to renew a cooperative effort to identify and develop a safe and effective vaccine for this disease. 32 The Valley Fever Vaccine Project (VFVP) is an academic consortium that originated, in part, as a response to a major epidemic of coccidioidomycosis that plagued parts of California in the early 1990s.…”