The Cryptococcus neoformans gene DHA1 encodes an antigen that elicits a delayed-type hypersensitivity reaction in immune mice.

When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5' rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.

Resistance to Coccidioides immitis in mice after immunization with recombinant protein or a DNA vaccine of a proline-rich antigen.

Two inbred strains of mice (BALB/c and C57BL/6) were vaccinated with either recombinant expression protein of a Coccidioides immitis spherule-derived proline-rich antigen (rPRA) in monophosphoryl lipid A-oil emulsion adjuvant or a DNA vaccine based on the same antigen. Four weeks after vaccination, mice were infected intraperitoneally with arthroconidia. By 2 weeks, groups of mice receiving saline or plasmids with no PRA insert exhibited significant weight loss, and quantitative CFUs in the lungs ranged from 5.9 to 6.4 log10. In contrast, groups of mice immunized with either rPRA or DNA vaccine had significantly smaller pulmonary fungal burdens, ranging from 3.0 to 4.5 log10 fewer CFUs. In vitro immunologic markers of lymphocyte proliferation and gamma interferon (IFN-gamma) release after splenocytes were stimulated with rPRA correlated with protection. Also, plasma concentrations of rPRA-specific total immunoglobulin G (IgG), IgG1, and IgG2a showed increases in vaccinated mice. These studies expand earlier work by demonstrating protection in mice which differ in H-2 background, by using an adjuvant that is potentially applicable to human use, and by achieving comparable protections with a DNA-based vaccine. Our in vitro results substantiate a Th1 response as evidenced by IFN-gamma release and increased IgG2a. However, IgG1 was also stimulated, suggesting some Th2 response as well. PRA is a promising vaccine candidate for prevention of coccidioidomycosis and warrants further investigation.

Proline-rich vaccine candidate antigen of Coccidioides immitis: conservation among isolates and differential expression with spherule maturation.

A proline rich antigen (PRA), which protects mice against Coccidioides immitis, has been analyzed for differential expression and variation among isolates. Northern blots of RNA from different stages of growth were probed with previously cloned cDNA and showed that mRNA for PRA increased as spherules transformed and matured from mycelia. This pattern corresponds to the relative potency of whole cell vaccines from similar preparations. The PRA gene was then cloned from a genomic library of the Silveira strain of C. immitis and its sequence analyzed. Eight other coccidioidal isolates, selected for diversity in geographic origin and resulting clinical disease, demonstrated heterogeneity in Southern blots and in sequences of polymerase chain reaction products. Silveira differed from other California isolates at 33 of 555 bases, whereas it differed from non-California isolates by <=2 bases. Only one of these substitutions affected protein sequence. Thus, tests or vaccines based on this gene are likely to cover most isolates.

Detecting serum antibodies to a purified recombinant proline-rich antigen of Coccidioides immitis in patients with coccidioidomycosis.

In previous work, antibodies in serum samples from patients with coccidioidomycosis were found to react with a proline-rich antigen (PRA) isolated from spherules of Coccidioides immitis, and the gene encoding this antigen was cloned. We expressed and purified recombinant PRA (rPRA) by removing the majority of amino acids contributed by the vector from the fusion protein. Purified rPRA reacted with serum IgG antibodies in 37 of 42 patients with culture-proven progressive pulmonary or extrapulmonary coccidioidal disease; specific antibodies in dilutions ranging from 1:40 to 1:102,400 were demonstrated (sensitivity, 88%). In contrast, for > 95% of patients without coccidioidomycosis reactivity of < 1:40 was demonstrated (specificity, 97%). Of 18 patients with primary self-limited coccidioidomycosis, none had detectable antibodies in serum samples collected up to 141 days after illness began. The association of antibodies to rPRA with progressive infection may have prognostic value.

Evaluation of the proline-rich antigen of Coccidioides immitis as a vaccine candidate in mice.

We have expressed the proline-rich antigen (PRA) from Coccidioides immitis in Escherichia coli and evaluated its potential as a vaccine candidate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the recombinant protein (rPRA) revealed two bands, which exhibited virtually identical primary amino acid sequences. T cells from rPRA-immunized BALB/c mice showed a significant in vitro proliferative response to rPRA. A small but statistically significant proliferative response was also induced by rPRA in T cells from mice immunized with whole-cell coccidioidal vaccines. BALB/c mice immunized with rPRA and challenged intraperitoneally with virulent C. immitis had a greatly reduced fungal burden in their lungs and spleens compared to unvaccinated mice. The number of organisms in the lungs was reduced 500-fold, and similar reductions were observed in the spleens of immunized mice. These studies support the continued development of rPRA as a candidate vaccine for prevention of coccidioidomycosis.