Immunobiological activity of recombinant H antigen from Histoplasma capsulatum

The H antigen from Histoplasma capsulatum is one of the known principal antigens of this fungus, but information regarding its identity and its participation in cellular immunity is largely unavailable. Therefore, we sought to determine both the nature of this antigen and the nature of its involvement in the cell-mediated immune response. The antigen was isolated from histoplasmin and digested with selected proteinases, and the cleavage products were subjected to reverse-phase high-performance liquid chromatography. Amino acid sequences of protein fragments were obtained by Edman degradation. A fragment of the gene encoding the H antigen was isolated by using degenerate primers in the PCR. This fragment was used to screen a genomic library, and the full-length gene was isolated and sequenced. The deduced amino acid sequence revealed homology to extracellular beta-glucosidases. A cDNA was generated by reverse transcription PCR and cloned into the expression vector pET 19b. Recombinant H antigen was isolated from inclusion bodies of Escherichia coli and tested for its ability to elicit and induce an in vitro cell-mediated immune response in BALB/c mice. Recombinant antigen stimulated splenocytes from mice immunized with viable yeast cells or with antigen suspended in adjuvant. Mice inoculated with H antigen were not protected against either a sublethal or a lethal inoculum of yeast cells. Thus, H antigen stimulates a cell-mediated immune response but does not induce a protective response to H. capsulatum.