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.

Project Start
2000-09-01
Project End
2001-08-31
Budget Start
Budget End
Support Year
6
Fiscal Year
2000
Total Cost
$121,281
Indirect Cost
Name
Veterans Medical Research Fdn/San Diego
Department
Type
DUNS #
933863508
City
San Diego
State
CA
Country
United States
Zip Code
92161
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Mirbod-Donovan, Fariba; Schaller, Ruth; Hung, Chiung-Yu et al. (2006) Urease produced by Coccidioides posadasii contributes to the virulence of this respiratory pathogen. Infect Immun 74:504-15
Xue, Jianmin; Hung, Chiung-Yu; Yu, Jieh-Juen et al. (2005) Immune response of vaccinated and non-vaccinated mice to Coccidioides posadasii infection. Vaccine 23:3535-44
Hung, Chiung-Yu; Seshan, Kalpathi R; Yu, Jieh-Juen et al. (2005) A metalloproteinase of Coccidioides posadasii contributes to evasion of host detection. Infect Immun 73:6689-703
Johannesson, Hanna; Townsend, Jeffrey P; Hung, Chiung-Yu et al. (2005) Concerted evolution in the repeats of an immunomodulating cell surface protein, SOWgp, of the human pathogenic fungi Coccidioides immitis and C. posadasii. Genetics 171:109-17
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Johannesson, Hanna; Vidal, Pilar; Guarro, Josep et al. (2004) Positive directional selection in the proline-rich antigen (PRA) gene among the human pathogenic fungi Coccidioides immitis, C. posadasii and their closest relatives. Mol Biol Evol 21:1134-45
Delgado, Nelson; Xue, Jianmin; Yu, Jieh-Juen et al. (2003) A recombinant beta-1,3-glucanosyltransferase homolog of Coccidioides posadasii protects mice against coccidioidomycosis. Infect Immun 71:3010-9

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