Validation of Coccidiodes Target Antigens for Immunotherapy

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We have shown that we can reverse antigen-specific anergy in lymphocytes from patients with disseminated coccidioidomycosis by incubating them with dendritic cells (DC) pulsed with C. immitis spherule lysate. A Thl cytokine profile and cellular proliferation were observed in response to the spherule lysate. Since spherule lysates are unlikely to be approved as an antigen preparation for clinical use, the overall objectives of this project are to validate recombinant antigens in our DC system that have been shown to be protective in murine models against lethal challenge with coccidioides fungus. We hypothesize that DC pulsed with defined recombinant antigens from Coccidioides spherules in the presence of adjuvant will elicit a protective TH1 cytokine profile and reverse coccidioidal anergy in lymphocytes from patients with disseminated coccidioidomycosis. Specific aims to test this hypothesis are to validate recombinant coccidioides antigens, Ag2/PRA (proline rich antigen), CSA (coccidioides specific antigen) and Ag2/PRA-CSA chimeric fusion protein by evaluating DC phenotype and function from nafve, healthy immune donors and from patients with disseminated coccidioidomycosis. While determining how recombinant antigens affect DC generation, phenotype and function, in the presence and absence of monophosphoryl Lipid A (MPL) adjuvant, lymphocyte subsets that respond to antigen-pulsed DC will be analyzed for cytokines secretion and cell surface markers. Chemokine receptors on DC as well as central and effector memory lymphocyte markers will be analyzed by flow cytometry. Data obtained from these studies with recombinant antigens will be evaluated and compared with that obtained with T27K coccidioides spherule lysate to determine if the lymphocyte responses fit a protective profile. The overall goal of this project is to position us for a clinical trial using immunotherapy with recombinant coccidoides antigens for the treatment of disseminated coccidioidomycosis that is refractive to anti-fungal treatment.