Vaccine-mediated immune responses to experimental pulmonary Cryptococcus gattii infection in mice

Abstract

Cryptococcus gattii is a fungal pathogen that can cause life-threatening respiratory and disseminated infections in immune-competent and immune-suppressed individuals. Currently, there are no standardized vaccines against cryptococcosis in humans, underlying an urgent need for effective therapies and/or vaccines. In this study, we evaluated the efficacy of intranasal immunization with C. gattii cell wall associated (CW) and/or cytoplasmic (CP) protein preparations to induce protection against experimental pulmonary C. gattii infection in mice. BALB/c mice immunized with C. gattii CW and/or CP protein preparations exhibited a significant reduction in pulmonary fungal burden and prolonged survival following pulmonary challenge with C. gattii. Protection was associated with significantly increased pro-inflammatory and Th1-type cytokine recall responses, in vitro and increased C. gattii-specific antibody production in immunized mice challenged with C. gattii. A number of immunodominant proteins were identified following immunoblot analysis of C. gattii CW and CP protein preparations using sera from immunized mice. Immunization with a combined CW and CP protein preparation resulted in an early increase in pulmonary T cell infiltrates following challenge with C. gattii. Overall, our studies show that C. gattii CW and CP protein preparations contain antigens that may be included in a subunit vaccine to induce prolonged protection against pulmonary C. gattii infection.

Figure 1. Protection afforded by immunization with C. gattii cell wall and cytoplasmic protein preparations.

BALB/c mice were mock-immunized with endotoxin free PBS or immunized with total cell wall proteins (CW), cytoplasmic proteins (CP), or a combined CW and CP protein preparation (CW/CP) in 50 µl of sterile PBS by nasal inhalation. Mice were subsequently challenged with 10⁴ CFU of C. gattii strain R265 intranasally. Survival was monitored twice daily and mice that appeared moribund or not maintaining normal habits (grooming) were sacrificed (A). Survival data shown are cumulative of 2 experiments each using 8 and 6 mice per experimental group. Asterisk indicates significantly different (**P<0.001) survival rates for each immunized group compared to mock-immunized mice as assessed by the Log-rank (Mantel-Cox) test Alternatively, lungs were excised at days 7, 14, and 21 post-challenge and the pulmonary cryptococcal burden quantified (B). Pulmonary fungal burden data are cumulative of 3 experiments using 4 mice per time point. Results are expressed as mean CFU per milliliter ± SEM. Asterisks indicate where significant decreases (*P<0.05), (**P<0.001) and (***P<0.0001) were observed compared to mock-immunized mice.

Figure 2. Pulmonary inflammatory leukocyte population at days 7, 14 and 21 post-infection.

BALB/c mice were mock-immunized with endotoxin free PBS or immunized with total CW proteins, CP proteins, or a combined CW and CP protein preparation in 50 µl of sterile PBS by nasal inhalation. Mice were subsequently given an intranasal challenged with 10⁴ CFU of C. gattii strain R265. The lungs were excised at days 7, 14 and 21 post-inoculation and a single cell suspension generated using enzymatic digestion. Leukocytes were labeled with antibodies against CD45⁺ leukocytes (A), F4/80⁺ macrophages (B), CD11c⁺/CD11b^int DCs (C), 1A8⁺ neutrophils (D), CD3⁺/CD4⁺ T cells (E), and CD3⁺/CD8⁺ T cells. Data shown are mean ± SEM of absolute cell numbers from three separate experiments using 4 mice per group per experiment. Asterisks (*) indicate significant increases (P<0.05) in leukocyte populations compared to the mock-immunized mice with PBS (control). PBS immunized  =  clear bars, CW immunized  =  gray bars, CP immunized  =  black bars, CW/CP immunized  =  hashed bars.

Figure 3. Evaluation of C. gattii specific antibody isotypes in mice during experimental pulmonary cryptococcosis.

BALB/c mice were mock-immunized with endotoxin free PBS or immunized with total CW proteins, CP proteins, or a combined CW and CP protein preparation in 50 µl of sterile PBS by nasal inhalation. Mice were immunized three times, with four week intervals between each immunization. Ten days following the final immunization, mice received an intranasal inoculation of 10⁴ CFU with C. gattii strain R265. Serum was obtained from mock-immunized mice and mice immunized with CW or CP proteins or a combined CW and CP protein preparation on days 7 and 14 post-secondary challenge. The isotypes of C. gattii CW-specific antibodies on day 7 (A) and CP-specific antibodies on day 7 (B) and the isotypes of C. gattii CW-specific antibodies on day 14 (C) and CP-specific antibodies on day 14 (D) were determined. Asterisks indicate significant increase (*P<0.05), (**P<0.001) and (***P<0.0001) in antibody isotypes compared to the mice immunized with PBS (control). The results shown are cumulative of three experiments using 4 mice per experiment.

Figure 4. 2-DE profile and immunoblot analysis of C. gattii cell wall and cytoplasmic proteins.

Protein components were separated in the first dimension using a pI 4-7 IPG (11cm) strip and in the second dimension using a 12% polyacrylamide gel. Following 2-DE, the proteins were stained with SYPRO-Ruby Red. (A) CW protein profile; (C) CP protein profile. Proteins in separate gels were transferred to PVDF membranes and incubated with pooled serum taken on day 14 post-challenge of mice immunized using a combination of CW and CP C. gattii proteins: (B), CW immunoblot; (D), CP immunoblot. Numbered spots represent immunogenic proteins whose corresponding identities are given in Tables 3 and 4.