Coccidioidomycosis is a disease in which T-cell mediated immunity has been shown to play a critical role in host defense. Both clinical and experimental data support this conclusion. An unusual feature of this mycosis is that high titers of antibody, as detected by complement fixation, are a poor prognostic sign. Therefore, determination of which C. immitis antigens stimulate T-cell responses rather than antibody responses is essential for subsequent isolation of macromolecules of the organism that elicit immunoprotection. In the proposed research, we have used a novel molecular approach to the systematic identification of the recombinant T-cell reactive proteins (RTPs), an evaluation of the immunoprotective properties of these macromolecules in a murine model of coccidioidomycosis. Results of our earlier studies have indicated that potent T-cell reactive proteins are expressed during 1) transition of the saprobic to parasitic phase, 2) isotropic growth of spherules, and 3) endosporulation. On this basis, we will construct three corresponding cDNA expression libraries with mRNA isolated from the above parasitic phases. The libraries will be screened with existing antiserum raised against cell wall and whole cell preparations which have been shown to be T-cell reactive in immune lymph node proliferation (ILNP) assays. Reactive clones are isolated and the C. immitis cDNA of each is ligated into the pCMV mammalian expression vector. BALB/c mice are immunized subcutaneously with the pCMV plus cDNA insert which expresses the C. immitis protein. Mice are monitored by ELISA for production of the antibody against crude C. immitis antigen and then sacrificed for evaluation of their T-cell reactivity in ILNP assays. The cDNA of reactive clones is subcloned into a prokaryotic expression vector (e.g., pSE40), and the RTP is purified by immunoaffinity chromatography using the corresponding specific murine antiserum obtained as sacrifice, as above. The RTP is further tested for reactivity in murine ILNP assays and T-cell lines, an in patient lymphocyte proliferation assays. The selected cDNAs are used to screen the original expression library, or a C. immitis genomic library, to isolate the full-length gene. The cDNA from that gene will be used for expression o the RTP for further evaluation of its reactivity as above. Ultimately, this approach will yield multiple RTPs which can be evaluated for immunoprotection in mice against C. immitis challenge. Immunoprotective recombinant proteins obtained by this approach are qualified candidates for a human vaccine against coccidioidomycosis.
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