Intranasal Immunization with the Commensal Streptococcus mitis Confers Protective Immunity against Pneumococcal Lung Infection

Abstract

Streptococcus pneumoniae is a bacterial pathogen that causes various diseases of public health concern worldwide. Current pneumococcal vaccines target the capsular polysaccharide surrounding the cells. However, only up to 13 of more than 90 pneumococcal capsular serotypes are represented in the current conjugate vaccines. In this study, we used two experimental approaches to evaluate the potential of Streptococcus mitis, a commensal that exhibits immune cross-reactivity with S. pneumoniae, to confer protective immunity to S. pneumoniae lung infection in mice. First, we assessed the immune response and protective effect of wild-type S. mitis against lung infection by S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4). Second, we examined the ability of an S. mitis mutant expressing the S. pneumoniae type 4 capsule (S. mitis TIGR4cps) to elicit focused protection against S. pneumoniae TIGR4. Our results showed that intranasal immunization of mice with S. mitis produced significantly higher levels of serum IgG and IgA antibodies reactive to both S. mitis and S. pneumoniae, as well as enhanced production of interleukin 17A (IL-17A), but not gamma interferon (IFN-γ) and IL-4, compared with control mice. The immunization resulted in a reduced bacterial load in respiratory tissues following lung infection with S. pneumoniae TIGR4 or D39 compared with control mice. With S. mitis TIGR4cps, protection upon challenge with S. pneumoniae TIGR4 was superior. Thus, these findings show the potential of S. mitis to elicit natural serotype-independent protection against two pneumococcal serotypes and to provide the benefits of the well-recognized protective effect of capsule-targeting vaccines.IMPORTANCEStreptococcus pneumoniae causes various diseases worldwide. Current pneumococcal vaccines protect against a limited number of more than 90 pneumococcal serotypes, accentuating the urgent need to develop novel prophylactic strategies. S. pneumoniae and the commensal Streptococcus mitis share immunogenic characteristics that make S. mitis an attractive vaccine candidate against S. pneumoniae In this study, we evaluated the potential of S. mitis and its mutant expressing pneumococcal capsule type 4 (S. mitis TIGR4cps) to induce protection against S. pneumoniae lung infection in mice. Our findings show that intranasal vaccination with S. mitis protects against S. pneumoniae strains D39 (serotype 2) and TIGR4 (serotype 4) in a serotype-independent fashion, which is associated with enhanced antibody and T cell responses. Furthermore, S. mitis TIGR4cps conferred additional protection against S. pneumoniae TIGR4, but not against D39. The findings highlight the potential of S. mitis to generate protection that combines both serotype-independent and serotype-specific responses.

Keywords: Streptococcus mitis; Streptococcus pneumoniae; commensals; infection; pneumonia; vaccines.

FIG 1

IgG responses after intranasal immunization of mice with S. mitis. Mice were immunized with S. mitis, and nasal wash, BALF, and serum samples were collected to analyze IgG responses. (A) Reactivity of the sera from the immunized mice with S. mitis and S. pneumoniae serotypes by SDS-PAGE in gradient gels and by immunoblotting. Each lane was loaded with 50 µg of protein from the indicated bacterial species. (B) Levels of streptococcus-reactive IgG in antisera by whole-cell ELISA. The sera were diluted 1:1,000 and nasal wash and BALF 1:10. The data are shown as means ± standard deviations (SD) and pooled from the results of two independent experiments with 4 mice in each group. Each symbol represents data from an individual mouse, and the horizontal bars are the mean values for the groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (unpaired Student's t test.)

FIG 2

IgA responses to intranasal immunization of mice with S. mitis. Mice were immunized with S. mitis or PBS, and nasal wash, BALF, and serum samples were collected to analyze IgA responses. (A) Reactivity of the sera from the immunized mice with S. mitis and S. pneumoniae serotypes by SDS-PAGE in gradient gels and by immunoblotting. Each lane was loaded with 50 µg of protein from the indicated bacterial species. (B) Levels of streptococcus-reactive IgA antibodies in sera by whole-cell ELISA. The sera were diluted 1:1,000 and nasal wash and BALF 1:10. The data are shown as means ± SD and were pooled from the results of two independent experiments with 4 mice in each group. Each symbol represents data from an individual mouse, and the horizontal bars are mean values for the groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant (unpaired Student's t test).

FIG 3

Cytokine responses in mice immunized intranasally with S. mitis. Samples were collected from the nasal wash, BALF, and sera from the immunized mice, and the levels of cytokines were measured using ELISA. The data are shown as means ± SD and were pooled from the results of two independent experiments with 4 mice in each group. Each symbol represents data from an individual mouse, and the horizontal bars are mean values for the groups. **, P < 0.01; ns, not significant (unpaired Student's t test).

FIG 4

Production of cytokines by lung CD4⁺ T cells after S. mitis immunization. IL-17A, IFN-γ, and IL-4 production in CD4⁺ T cells isolated from the lungs was analyzed by flow-cytometric intracellular-cytokine staining. (Top) Cells were gated on CD3⁺ CD4⁺ T cells. (Bottom) Representative flow cytometric dot plot images (left) and a summary of the percentages of cytokine-producing CD4⁺ T cells (right). The data are shown as means ± SD and were pooled from the results of two independent experiments with 4 mice in each group. Each symbol represents data from an individual mouse, and the horizontal bars are mean values for the groups. ***, P < 0.001; ns, not significant (unpaired Student's t test).

FIG 5

Production of cytokines by splenic CD4⁺ T cells after S. mitis immunization. IL-17A, IFN-γ, and IL-4 production in CD4⁺ T cells isolated from the spleen was analyzed by flow-cytometric intracellular-cytokine staining. Cells were gated on CD3⁺ CD4⁺ T cells as described in the legend to Fig. 4. Representative flow-cytometric images (left) and a summary of the percentages of cytokine-producing CD4⁺ T cells (right) are shown. The data are shown as means ± SD and were pooled from the results of two independent experiments with 4 mice in each group. Each symbol represents data from an individual mouse, and the horizontal bars are mean values for the groups; ns, not significant (unpaired Student's t test).

FIG 6

Testing protective immunity to S. pneumoniae elicited by S. mitis or its mutant. CD1 mice were intranasally inoculated with S. mitis or S. mitis expressing the S. pneumoniae TIGR4 capsule (S. mitis TIGR4cps) or with PBS (sham) at days 0, 14, and 21 and then subjected to intranasal challenge with S. pneumoniae strain TIGR4 or D39 a week after the last immunization. The mice were sacrificed 24 h following the challenge infection, and the nasal wash, BALF, and lungs were collected for analysis of bacterial CFU. (A) Pneumococcal load in S. mitis- or S. mitis TIGR4cps-immunized mice following TIGR4 challenge. (B) Bacterial burden in mice immunized with S. mitis and subjected to D39 infection. The data are shown as means ± SD and were pooled from the results of two independent experiments with 4 mice in each group for S. pneumoniae TIGR4 challenge, whereas one experiment with 4 mice was performed for S. pneumoniae D39 challenge. Each symbol represents data from an individual mouse, and the horizontal bars are mean values for the groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. One-way ANOVA and the Tukey test were used to compare multiple groups and an unpaired Student t test for comparing two groups.