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

Abstract

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.

FIG. 1

Protective efficacy of vaccine in mice immunized twice (4 weeks apart) with saline (control) or an rPRA or DNA vaccine and challenged 4 weeks after a boost with viable arthroconidia. Two weeks later, mice were sacrificed and tissues (lung and spleen) were removed and processed as described in Materials and Methods. The box plot data represent CFU (log₁₀) counts per organ. Each box represents percentiles 25 to 75, and the line inside indicates the 50th percentile. Caps represent the 10th and 90th percentiles. Ribi Adj. denotes the MPL-oil emulsion.

FIG. 2

Proliferative responses of splenocytes from immunized mice to antigens. Splenocytes from BALB/c (filled bars) or C57BL/6 (hatched bars) mice immunized with saline, rPRA (with or without adjuvant), or DNA were tested in vitro by using rPRA (10 μg/ml in CM). Each bar represents the mean (± the standard error of the mean) SI. The numbers in parentheses are the numbers of experiments done.

FIG. 3

Production of IFN-γ in stimulated splenocytes from immune mice. Spleen cells from C57BL/6 mice immunized twice (4 weeks apart) with saline, rPRA (with or without adjuvant), or DNA were tested in vitro 4 weeks after boosting. Below the bars are the stimuli used (medium control [C], BSA [B], and rPRA [P]). Cells were incubated for 3 days before they were harvested, and IFN-γ was quantitated by using an ELISA kit.

FIG. 4

Total-IgG and IgG subclass responses of immunized BALB/c mice. Mice were immunized as described in Materials and Methods and terminally bled, and plasma samples were tested for PRA-specific total IgG (open bars), IgG1 (rising diagonal bars), and IgG2a (falling diagonal bars) by ELISA. Results represent reciprocal titers (1/dilution).