Recovery from coccidioidomycosis is associated with development of T-cell mediated immunity. However, the antigens that may elicit protective immunity are not known. In this project, I propose to use a novel technique to screen a large number of C. immitis proteins in an attempt to find candidates suitable for developing a vaccine. A Coccidioides cDNA expression library will be generated and screened for putative antigenic proteins with an anti-Coccidioides polyclonal serum by Dr. Cole. cDNA from positive clones will be isolated by us and subcloned on eukaryotes expression vectors. Following a procedure recently developed in our laboratory, recombinant naked DNA constructs will then be injected in mice. Immune responses are generated following this method that does not require the availability of purified antigen. T- and B-cell responses will be evaluated in collaboration with Dr. kirkland and the most promising antigens will be isolated and characterized. We will also focus our project on individual antigenic molecules that may play a role in immunity to Coccidioides. Since successful colonization of the host requires the transformation from arthroconidia to spherule, accompanied by a sudden increase in temperature, there is likely an increase in the expression of heat-shock proteins. These highly immunogenic molecules may be an interesting candidate for a vaccine. We have previously described that dnaJ, a heat-shock protein E. coli, is a target of strong immune responses. By comparing the conserved sequences of dnaJ equivalents in various species, we have designed ad hoc primers that have enabled us to obtain by polymerase chain reaction from Coccidioides, a DNA fragment that preliminary sequencing data suggest to be part of the Coccidioides equivalent of dnaJ. We plan to clone this protein and, in collaboration with Dr. Kirkland, to evaluate its immunogenicity This multi-target approach should lead to the identification of epitopes capable of inducing strong immune responses to be studied for the induction of protective immunity to Coccidioides immitis.
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