Coccidioidomycosis is a systemic mycoses caused by the dimorphic fungus Coccidioides immitis. Antigens currently used for immunologic assays are limited to coccidioidin (CDN), prepared as an autolysate of mycelia, and spherulin (SPH), an autolysate of spherule-phase cells. Numerous attempts have been made to purify immunoreactive antigens from CDN and SPH by using physicochemical procedures, with limited success. As an alternative approach, we developed polyclonal goat antisera and murine monoclonal antibodies (MAbs) for use as ligands in purifying antigens by solid-phase immunoadsorption. Three MAbs have been produced. One recognizes a carbohydrate epitope on the antigen that reacts with the tube precipitin (TP) antibody; one is directed against a peptide epitope on the antigen that reacts with the complement-fixing (CF) antibody; and one recognizes a peptide epitope on Antigen 2 (Ag2), a polymeric antigen which elicits T cell responses. The epitopes recognized by these MAbs are specific to C. immitis, as evidenced by the lack of reactivity of the antibodies with antigenic extracts from Histoplasma capsulatum and Blastomyces dermatitidis. Although the MAbs have proven to be effective ligands for the isolation of these antigens by solid-phase immunoadsorption, the efficacy of this approach is limited by the low yield of antigen. This problem can best be resolved by using recombinant DNA technology to identify, clone, and express the genes which encode these antigens, a strategy that is readily suitable for Ag2 and the CF antigen since these antigens have immunoreactive peptides.
Two specific aims are defined: (i) to identify, clone, and express the genes using a lambda ZAP II/cDNA library prepared from spherule-derived mRNA; and (ii) to evaluate and compare the immunologic reactivity of the recombinant and immunoaffinity-purified antigens. We will employ a bidirectional and complementary approach to identify the genes which encode the CF antigen and Ag2. One will be to use the MAbs, in conjunction with polyclonal goat antibodies, to identify fusion proteins produced by the lambda ZAP lI/cDNA-expression library; the other will be to use the MAbs to purify the antigens by solid-phase immunoadsorption and, on the basis of the N-terminus amino acid sequences of the purified antigens, develop oligonucleotide probes for identifying the genes in the cDNA library. Once the clones containing the CF and Ag2 peptides have been identified, studies will be done to subclone and express the genes in a Baculovirus system for large-scale production of nonfusion peptides. The recombinant peptides and immunoaffinity-purified antigens will be evaluated for their reactivity in enzyme-immunoadsorption assays, their ability to elicit T cell responses in infected mice, and their capacity to confer protection against challenge in mice. Successful completion of these aims will provide recombinant antigens for use in immunodiagnostic assays, for the development of a vaccine for coccidioidomycosis, and for studies of host-fungus interaction in this disease.
Showing the most recent 10 out of 13 publications
