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

Abstract

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.

FIG. 1

SDS-PAGE and immunoblot analysis of rPRA. Samples of rPRA were electrophoresed on 12.5% polyacrylamide gels and stained with Coomassie blue (A), or samples were transferred to nitrocellulose and proteins were detected with monospecific goat antiserum and alkaline phosphatase-conjugated secondary antibody (B). (A) Coomassie blue-stained gel. Lane 1, isopropylthiogalactoside-induced bacterial cell lysate; lane 2, eluate from Ni-NTA column; lane 3, thrombin-cleaved rPRA; lane 4, Ni-NTA-purified thrombin-cleaved rPRA. (B) Representative immunoblot. Lane 1, isopropylthiogalactoside-induced bacterial cell lysate; lane 2, isopropylthiogalactoside-induced bacterial cell lysate from empty vector; lane 3, biochemically purified spherule-derived PRA.

FIG. 2

Proliferative response of lymph node T cells from mice immunized with PRA plotted versus antigen concentration in vitro. The means and standard deviations of three determinations are shown. Open symbols represent PRA; filled symbols represent mycelial F+L.

FIG. 3

Proliferative response of lymph node T cells from mice immunized with CFA alone (open bars), formalin-killed spherules (stippled bars), or live, attenuated C. immitis followed by formalin-killed spherules (solid bars). The maximal response for each antigen is shown. The concentration of PRA and mycelial F+L was 25 μg/ml. The formalin-killed spherules were at a concentration of 10⁵/ml. Means and standard deviations of three determinations are shown.

FIG. 4

A box plot representation of the numbers of CFU found in the lungs and spleens of immune or control mice 14 days after infection with C. immitis. Control mice were immunized with CFA alone. The box indicates the 25th and 75th percentiles of the data; the line represents the 50th percentile. The capped bars represent the 10th and 90th percentiles. Symbols above and below the box indicate individual values above the 90th or below the 10th percentile.