Fig 1.
Protection induced by CAF01-adjuvanted monovalent subunit vaccine.
(A) Schematic of vaccination strategy. WT BALB/c and C57BL/6 mice received a prime and two biweekly boosts with the indicated CAF01-adjuvanted vaccines. Mice were challenged with C. neoformans two weeks after the last boost, and then monitored for 70d for survival. The figure was created with BioRender.com. (B) Survival curves of BALB/c mice. (C) Survival curves of C57BL/6 mice. SQ, subcutaneous. OT, orotracheal. Unvac, unvaccinated mice. CAF01, mice receiving CAF01 without antigens. n denotes the total number of mice in the experimental group; each group included at least 2 independent experiments. ****, P < 0.0001; NS, P > 0.05, comparing the experimental group with Unvac mice.
Fig 2.
CAF01-adjuvanted combination vaccines enhance the protection of mice against experimental cryptococcosis.
WT BALB/c mice received three SQ vaccinations with either 2-Ag or 4-Ag combination vaccines, following by orotracheal challenge with C. neoformans. Mice were monitored until death or study termination at 70 DPI for survival (A) and change in body weight (B). Data are from two independent experiments, each with 4-5 mice per group. Significant survival compared with unvaccinated mice was observed for both vaccinated groups in panel A (2-Ag, P = 0.0009; 4-Ag, P <0.0001). WT C57BL/6 mice or CARD9-/- mice on the C57BL/6 background received 2-Ag vaccines following the same schedule. Mice were then monitored until 70 DPI for survival (C). Significant survival was observed in panel C, when comparing survival of vaccinated WT C57BL/6 and CARD9-/- mice (P = 0.0006), or comparing vaccinated and unvaccinated WT C57BL/6 mice (P = 0.0003). Unvac, unvaccinated. 2-Ag, vaccinated with CAF01-Cda1+Cda2. 4-Ag, vaccinated with CAF01-Cda1+Cda2+Blp4+Cpd1Δ.
Fig 3.
Fungal control and immune cell recruitment following CAF01-adjuvanted combination vaccines and/or infection.
BALB/c mice received three biweekly vaccines with either 2-Ag or 4-Ag combination vaccines, followed by pulmonary challenge with C. neoformans. Control mice were left unvaccinated. Mice were euthanized at specified timepoints, and individual lungs were collected for analysis. Lung weights (A), fungal burdens (B), lung leukocytes numbers (C), CD4+ T cells (D) and CD8+ T cells (E) were determined as in Methods. Vac, vaccinated. Un/Unvac, unvaccinated. 2-Ag, vaccination with CAF01-Cda1+Cda2. 4-Ag, vaccination with CAF01-Cda1+Cda2+Blp4+Cpd1Δ. DPI, day post infection. Data are from two independent experiments, each with 3–5 mice per group. Each dot represents the value obtained from an individual mouse. CFUs are presented with individual values and medians. The dotted line in B represents the inoculum of 2 x 104 yeast cells. Lung weights, and numbers of leukocytes, CD4+ T cells and CD8+ T cells are presented with means ± SEM. The results of the statistical comparison between groups are shown in S3 Table.
Fig 4.
Analysis of ex vivo antigen-stimulated CD4+ T activation and cytokine production following CAF01-adjuvanted combination vaccination and infection.
BALB/c mice were vaccinated subcutaneously thrice at biweekly intervals with either 2-Ag (Cda1+Cda2) or 4-Ag (Cda1+Cda2+Blp4+Cpd1Δ) combination vaccines adjuvanted in CAF01. Two weeks after the last boost, mice were orotracheally challenged with C. neoformans. Mice were euthanized at 0 DPI (uninfected), 10 DPI, or 70 DPI. Controls included unvaccinated mice euthanized at 0 DPI or 10 DPI. Lungs were harvested and single-cell suspensions were prepared. Leukocytes were cultured in complete media supplemented with amphotericin B and stimulated with HK C. neoformans or left unstimulated (Unstim) for 18 h. Then the cells were collected, stained, and analyzed by polychromatic FACS, as described in Methods. The numbers of CD4+ T cells expressing the activation marker CD154 (Fig 4A, 4E and 4I), or producing the intracellular cytokines IFNγ (Fig 4B, 4F and 4J), TNFα (Fig 4C, 4G and 4K), and IL-17 (Fig 4D, 4H and 4L) following ex vivo stimulation were determined. Unvac, unvaccinated. DPI, days post infection. HK, heat-killed. Data are from two independent experiments, each with 3–5 mice per group. Data are presented as means ± SEM. Each dot represents the value obtained from an individual mouse. Statistical comparisons between groups are shown in S4 Table.
Fig 5.
Analysis of polyfunctional lung CD4+ T cells following ex vivo stimulation with HK Cryptococcus.
Lung leukocytes were prepared from the same sets of mice (10 DPI) as described in Fig 4. Leukocytes were cultured in complete media supplemented with amphotericin B and stimulated with HK C. neoformans for 18 h. Brefeldin A was added during the last 4 h of culture. Then the cells were collected, stained, and analyzed by polychromatic FACS. After Boolean gating analysis in FlowJo software, the frequencies of the seven possible CD4+ T cell subpopulations expressing any combination of the IFNγ, TNFα, and IL-17 cytokines were determined. Additionally, the corresponding numbers of each subpopulation were subsequentially enumerated. Unvac, unvaccinated. 2-Ag, vaccination with CAF01-Cda1+Cda2. 4-Ag, vaccination with CAF01-Cda1+Cda2+Blp4+Cpd1Δ. Data are from two independent experiments, each with 3–5 mice per group. Each dot represents the value obtained from an individual mouse. Data are presented as means ± SEM. The bar graphs show the cell numbers of each subpopulation. Means of cytokine-producing CD4+ T cells were compared. P < 0.05 was considered significantly different. * denotes a significant comparison with Unvac group, # denotes a significant comparison between 2-Ag and 4-Ag groups. Pie charts represent the fraction of each subpopulation in cytokine-expressing CD4+ T cells. Pie chart color-coding for each subpopulation is shown below the bar graph.
Fig 6.
Analysis of ex vivo antigen-stimulated CD8+ T cytokine production following CAF01-adjuvanted combination vaccination and infection.
Lung leukocytes were prepared from the same set of mice as described in Fig 4. Leukocytes were cultured in complete media supplemented with amphotericin B and stimulated with HK C. neoformans or left unstimulated (Unstim) for 18 h. Brefeldin A was added during the last 4 h of culture. Then the cells were collected, stained, and analyzed by polychromatic FACS. The numbers of CD8+ T cells producing the intracellular cytokines IFNγ (Fig 6A, 6D and 6G), TNFα (Fig 6B, 6E and 6H), and IL-17 (Fig 6C, 6F and 6I) following ex vivo stimulation were determined. Unvac, unvaccinated. DPI, days post infection. 2-Ag, vaccination with CAF01-Cda1+Cda2. 4-Ag, vaccination with CAF01-Cda1+Cda2+Blp4+Cpd1Δ. HK, heat-killed. Data are from two independent experiments, each with 3–5 mice per group, and are presented as means ± SEM. Each dot represents the value obtained from an individual mouse. Statistical comparisons between groups are shown in S5 Table.
Fig 7.
IFNγ production by ex vivo stimulated lung leukocytes, splenocytes, and PBMCs following vaccination and/or infection.
BALB/c mice were vaccinated subcutaneously thrice at biweekly intervals with either 2-Ag (Cda1+Cda2) or 4-Ag (Cda1+Cda2+Blp4+Cpd1Δ) combination vaccines adjuvanted in CAF01. Two weeks after the last boost, mice were orotracheally challenged with C. neoformans. Mice were euthanized at 0 DPI (uninfected), 10 DPI, or 70 DPI. Controls included unvaccinated mice euthanized at 0 DPI or 10 DPI. Lung leukocytes, splenocytes and PBMCs were prepared as described in Methods. Cells were cultured in complete media supplemented with amphotericin B and stimulated with HK C. neoformans or left unstimulated (Unstim) for either 18 h (lung) or 3 days (spleen and PBMC). Supernatants were collected and analyzed for IFNγ by ELISA. Antigen-stimulated production for lungs (Fig 7A, 7D and 7G), spleens (Fig 7B, 7E and 7H), and PBMCs (Fig 7C, 7F and 7I) are presented as means ± SEM; statistical comparisons between groups are shown in S6 Table. Each dot represents the value obtained from an individual mouse except for PBMCs where each dot represents technical duplicates of pooled PBMCs from each independent experiment. Correlations between lung CFUs and lung leukocytes IFNγ levels (Fig 7J), or splenocytes IFNγ production (Fig 7K) following HK Cn stimulation were analyzed with simple linear regression (10 DPI). The results are presented with best-fit line and confidence bands. The Pearson correlation was used for statistical analysis. A P value < 0.05 was considered statistically significant. Unvac, unvaccinated. DPI, days post infection. HK, heat-killed. Data are from two independent experiments, each with 3–5 mice per group.