Human T-cell lines and clones that recognize Histoplasma capsulatum antigens will be developed to complement those isolated from mice. The surface phenotype, lymphokine production, response of cloned T cells to heterologous fungal antigens, and major histocompatibility complex restriction of proliferation, will be studied. Anti-idiotypic antibodies to human T-cell clones will be produced; using these antibodies, the surface receptor on T cells for Histoplasma antigens will be characterized biochemically. In parallel, Histoplasma yeast antigens from the cell wall/membrane that trigger proliferation by human and murine T-cell clones will be isolated and characterized. Yeasts will be disrupted and cell wall/membrane solubilized. this material will be subjected to SDS-PAGE and transferred to nitrocellulose. Stained bands will be cut, dissolved, and particle suspensions tested for immunoreactivity with T-cell clones. ONce the immunogenic fractions(s) has been identified, it will be purified by either preparative gel electrophoresis or gel chromatography followed by high performance liquid chromatography. The carbohydrate composition of the antigen will be analyzed by gas chromatography, and the nature of the antigen recognized by human and murine T cell clones will be determined by chemical and enzymatic degradative procedures. The gene encoding the protein portion of the H antigen will be cloned and inserted into an expression vector to produce a recombinant H antigen. The amino terminus of deglycosylated H will be amino acid sequenced. Oligonucleotide probes will be synthesized and used to screen by Southern blot analysis an established genomic library from strain G217B yeasts. Rapid sequencing of DNA will be performed. mRNA will be isolated and Northern blot analysis using nick-translated DNA performed to estimate message size. The sequence of genomic DNA will be analyzed for coding regions. The gene will be inserted into an expression vector and recombinant H antigen tested for immunoregulatory activity. These studies will identify purified yeast antigens recognized by T cells and provide a new approach (i.e., molecular cloning) for the isolation of H. capsulatum antigens. These antigens may be useful in the development of vaccines or as immunomodulatory agents for the treatment of histoplasmosis.
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