Identification of antigens of coccidioides immitis . Which stimulated immune t lymphocytes

: T-cell mediated immune response to coccidioidomycosis has been shown to be the principal mechanism of resistance to this respiratory fungal disease in experimental animals. In this study, a Coccidioides immitis antigen-specific murine T-cell line was used to identify macromolecules capable of eliciting an immune mouse T-cell proliferative response. The murine T-cell line was selected on the basis of its strong positive response to a soluble conidial wall fraction (SCWF), which had previously been shown to be reactive in humoral and cellular immunoassays. An antigen-specific T-cell line rather than T-cell clones was used to identify multiple antigens. The T-cell immunoblot method was employed first to identify immunoreactive sub-fractions of the SCWF, and then to identify T-cell fusion proteins (FPs) obtained from a cDNA expression library constructed in lambda gt11. The library was screened with anti-SCWF. The nucleotide sequence of a 0.2 kilobase cDNA insert encoding a FP which elicited vigorous T-cell response was determined. A construct of this insert was subcloned into the pET expression vector system and 6.5-kilodalton (kDa) recombinant protein (RP) expressed in Escherichia coli was isolated. The RP and FP were shown to be homologous on the basis of identify of their amino acid sequences. Antibody raised in guinea pigs against the RP recognized a 59-kDa native protein of the mycelial culture filtrate produced by three separate strains of C. immitis, and reacted with the cell wall of arthroconidia as detected by immunofluorescence microscopy. In this study we have identified and partly characterized a potentially important T-cell stimulating antigen of C. immitis.