A Recombinant Multivalent Vaccine (rCpa1) Induces Protection for C57BL/6 and HLA Transgenic Mice against Pulmonary Infection with Both Species of Coccidioides

Abstract

Coccidioidomycosis is caused by Coccidioides posadasii (Cp) and Coccidioides immitis (Ci), which have a 4-5% difference in their genomic sequences. There is an urgent need to develop a human vaccine against both species. A previously created recombinant antigen (rCpa1) that contains multiple peptides derived from Cp isolate C735 is protective against the autologous isolate. The focus of this study is to evaluate cross-protective efficacy and immune correlates by the rCpa1-based vaccine against both species of Coccidioides. DNA sequence analyses of the homologous genes for the rCpa1 antigen were conducted for 39 and 17 clinical isolates of Cp and Ci, respectively. Protective efficacy and vaccine-induced immunity were evaluated for both C57BL/6 and human HLA-DR4 transgenic mice against five highly virulent isolates of Cp and Ci. There are total of seven amino acid substitutions in the rCpa1 antigen between Cp and Ci. Both C57BL/6 and HLA-DR4 mice that were vaccinated with an rCpa1 vaccine had a significant reduction of fungal burden and increased numbers of IFN-γ- and IL-17-producing CD4+ T cells in the first 2 weeks post challenge. These data suggest that rCpa1 has cross-protection activity against Cp and Ci pulmonary infection through activation of early Th1 and Th17 responses.

Keywords: HLA-DR4 mice; T-cell immunity; Valley fever; coccidioidomycosis; cross-protection; vaccine.

Figure 1

GCP-rCpa1 vaccination results in protection against multiple clinical isolates of Cp. (A) GCP-rCpa1 vaccine construct illustration. (B) Vaccination and immunological analysis timeline. (C) The daily bodyweight change (%) of C57BL/6 mice that were subcutaneously vaccinated with either the GCP-rCpa1 vaccine (circle) or GCP-MSA mock (triangle), or unvaccinated (square), then separately challenged with approximately 90–120 viable spores isolated from one of the three Cp clinical isolates (C735, Silveira, or 3488; representative of two independent studies, n = 8 mice per group). Mouse bodyweight at the time of challenge was set as 100%. Data points represent the average of percentage bodyweight measured daily for 14 days. Error bars represent the ±SD per timepoint. (D) Colony-forming units (CFUs) of whole tissue (lung and spleen) were determined by plate culture homogenates of unvaccinated, mock, and vaccinated mice at 14 DPC (n = 8 per group). The data are presented by whisker box plots of log₁₀ CFUs detected from plated cultures. Asterisks and daggers indicate statistically significant differences between CFU values of the GCP-rCpa1-vaccinated versus unvaccinated mice (*, p < 0.05) and the vaccinated versus mock groups (†, p < 0.05), as determined by ordinary one-way ANOVA comparing percent weight change at 14 DPC. Statistical differences in fungal burden were conducted using Kruskal–Wallis multiple comparison tests.

Figure 2

The GCP-rCpa1 vaccine confers cross-protection against isolates of Ci. C57BL/6 mice were vaccinated with the GCP-rCpa1 vaccine (circle), mock immunized with GCP-MSA (triangle), and unvaccinated (square) using the same schedule shown in Figure 1B. (A) Representative daily bodyweight changes (%) of two independent studies using the three groups of mice that were separately challenged with approximately 90–120 spores isolated from two Ci clinical isolates (RS and 2394). Mouse bodyweight at the time of challenge was set as 100% (n = 8 mice per group). Data points represent the average of percentage bodyweight measured daily for 14 days. Mice challenged with Cp-C735 served as a reference for comparison. (B) CFUs were determined by plate culture of lung and spleen homogenates of unvaccinated, mock, and vaccinated mice at 14 DPC (n = 8 mice per group). The data are presented as whisker box plots of CFUs (log₁₀). Unvaccinated mice that were challenged with Ci-RS presented significantly reduced CFUs in the lungs and spleen compared with those challenged with isolate Cp-C735. These data suggest that Ci-RS is less virulent. All groups of GCP-rCpa1-vaccinated mice had reduced CFUs in the lungs and spleen compared with the mock groups. Asterisks and daggers indicate statistically significant differences between CFU values of the GCP-rCpa1-vaccinated versus nonvaccinated mice (*, p < 0.05) and the vaccinated versus mock groups (†, p < 0.05), as determined by ordinary one-way ANOVA comparing percent weight change at 14 DPC. Statistical differences in fungal burden were conducted using Kruskal–Wallis tests.

Figure 3

The GCP-rCpa1 vaccine induced the acquisition of Th1 and/or Th17 cells in the lungs of C57BL/6 mice that were challenged with Cp isolates. Pulmonary Th1, Th17, and Th2 cells expressing IFN-γ-, IL-17a, and IL-5 were evaluated using intracellular cytokine assays. The gating strategy is illustrated in (A). The absolute numbers (A–D) of gated, specific-cytokine-producing cells per whole lung were determined at 7 and 14 DPC. A separate group of mice was vaccinated with GCP-rCpa1 (black bars), injected with GCP-MSA (mock; white bars), or unvaccinated (dotted bars) using the protocol shown in Figure 1B. Three weeks after the final vaccination, mice were separately challenged with a lethal dose of spores isolated from Cp-C735 (B), Cp-Silveira (C), and Cp-3488 (D). Asterisks and daggers in panels B to D indicate significantly higher absolute numbers of the respective T-cell phenotypes in the lungs of the GCP-rCpa1-vaccinated versus nonvaccinated mice (*, p < 0.05) and the vaccinated versus mock groups (†, p < 0.05). Four mice per group per timepoint were used. The results are presented as mean values ±SEM and were analyzed using ordinary one-way ANOVA.

Figure 4

The GCP-rCpa1 vaccine induced acquisition of Th1 and/or Th17 cells in the lungs of C57BL/6 mice that were challenged with Ci isolates. Pulmonary Th1 and Th17 in the lungs of Ci-RS (A) and Ci-2394 (B) were enumerated, as described in Materials and Methods. Statistical significance for both Th1 and Th17 responses was observed in both Ci isolates (RS and 2394, (A,B)) at 7 DPC comparing unvaccinated with GCP-rCpa1 vaccinated mice. Total cell numbers of both Th1 and Th17 cells were significantly elevated in the GCP-rCpa1 vaccinated mice compared with mock and control mice at 7 DPC (*, p < 0.05), GCP-rCpa1-vaccinated versus unvaccinated mice (†, p < 0.05), and GCP-rCpa1-vaccinated versus mock mice. Four mice per group per timepoint were utilized. The results are presented as mean values ±SEM and were analyzed using ordinary one-way ANOVA.

Figure 5

The GCP-rCpa1 vaccine elicits cross protection and a mixed Th1 and Th17 response in the lungs of HLA-DR4 transgenic mice. HLA-DR4 (DRB1*04:01 allele) transgenic mice were immunized and then separately challenged with Cp-C735, Cp-3488, and Ci-2394. HLA-DR4 mice were subcutaneously vaccinated with the GCP-rCpa1 vaccine (circle), mock vaccinated (triangle, GCP-MSA), or unvaccinated (square) and then separately challenged with approximately 90–120 viable spores isolated from Cp isolates (C735 or 3488) or Ci (2394) (10 mice per group). Whisker box plots presented CFU (log₁₀) values from whole lungs at 9 days post challenge (A). Pulmonary Th1 and Th17 cells were evaluated by intracellular cytokine staining assays. Asterisks and daggers indicate statistically significant differences between CFU values of the GCP-rCpa1-vaccinated versus nonvaccinated mice (*, p < 0.05) and the vaccinated versus mock groups (†, p < 0.05). Differences in fungal burden were determined using Kruskal–Wallis tests. (B) Both Th1 and Th17 cells were significantly elevated for GCP-rCpa1-vaccinated mice compared with mock mice (GCP-MSA) that were challenged with Cp-C735 (B), Cp-3488 (C), and Ci-2394 (D). Ordinary one-way ANOVA was used to evaluate vaccinated versus mock groups (†, p < 0.05), and eight mice per group were used for flow cytometry studies.