Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice

Abstract

Streptococcus pneumoniae is an important human pathogen causing both mucosal (otitis media and pneumonia) and systemic (sepsis and meningitis) diseases. Due to increasing rates of antibiotic resistance, there is an urgent need to improve prevention of pneumococcal disease. Two currently licensed vaccines have been successful in reducing pneumococcal disease, but there are limitations with their use and effectiveness. Another approach for prevention is the use of live attenuated vaccines. Here we investigate the safety and protection induced by live attenuated strains of S. pneumoniae containing combinations of deletions in genes encoding three of its major virulence determinants: capsular polysaccharide (cps), pneumolysin (ply), and pneumococcal surface protein A (pspA). Both the cps and ply/pspA mutants of a virulent type 6A isolate were significantly attenuated in a mouse model of sepsis. These attenuated strains retained the ability to colonize the upper respiratory tract. A single intranasal administration of live attenuated vaccine without adjuvant was sufficient to induce both systemic and mucosal protection from challenge with a high dose of the parent strain. Immunization with cps mutants demonstrated cross-protective immunity following challenge with a distantly related isolate. Serum and mucosal antibody titers were significantly increased in mice immunized with the vaccine strains, and this antibody is required for full protection, as microMT mice, which do not make functional, specific antibody, were not protected by immunization with vaccine strains. Thus, colonization by live attenuated S. pneumoniae is a potentially safe and less complex vaccine strategy that may offer broad protection.

FIG. 1.

Attenuation of S. pneumoniae: comparison of the level of attenuation of various S. pneumoniae type 6A live vaccines over a 9-day period postinoculation. Data are based on a minimum of 10 animals in each group. Statistical differences compared to the parent strain were determined by the Kaplan-Meier log-rank test. ⋄, 6A; ▴, 6Aply; •, 6ApspA; □, 6Aply/pspA; ○, 6Acps; asterisk, PBS control.

FIG. 2.

Colonization of attenuated S. pneumoniae: comparison of the abilities of various live vaccine strains to colonize the mouse nasopharynx at day 9 (A) and day 2 (B) postinoculation. Density of pneumococci in upper respiratory tract lavage fluid is shown by the mean log CFU/ml ± the standard error of the mean. n = 4 to 9 mice per group per time point. The dotted line indicates the limit of detection. Statistical differences were determined using the Mann-Whitney test with comparison to the corresponding parent strain. *, P < 0. 0286; **, P = 0.004; ***, P = 0.0008.

FIG. 3.

Protection induced by live attenuated vaccine strains. (A) Survival rates of C57BL/6 mice immunized with 6Aply/pspA (▴; n = 8), 6Acps (*; n = 15), or vehicle-only control (•; n = 14) following intranasal challenge with the 6A parent strain. Statistical differences compared to the vehicle control were determined by the Kaplan-Meier log-rank test. (B) Colonization density at day 9 postinoculation of mice challenged with the type 6A parent strain (solid bars) or type 4 parent strain (hatched bars) after immunization with the indicated live vaccine. The 6Acps/ply vaccine was delivered in two doses prior to challenge. Values represent the means of 6 to 14 mice/group ± the standard errors of the means. The dotted line indicates the limit of detection. Colonization density was analyzed in surviving mice. Eight mice in the mock-immunized group and one animal in the 6Acps group that did not survive were not included in the analysis. Statistical differences were determined using the Mann-Whitney test with comparisons to the corresponding mock-immunized group. **, P < 0.0083.

FIG. 4.

Cross-protection induced by live attenuated vaccine strains. (A) Survival rates of C57BL/6 mice immunized with 4cps in a single dose (▪; n = 12) versus two doses (▴; n = 12), compared to the vehicle-only control (•; n = 15), following intranasal challenge with the type 6A isolate. Statistical differences compared to the vehicle control were determined by the Kaplan-Meier log-rank test. (B) Colonization density at day 9 postinoculation of mice challenged with the type 4 parent isolate (solid bars) or type 6A parent isolate (hatched bars) after immunization with the indicated live vaccine of the other serotype. One dose of the vaccine strain was administered except where a second dose is indicated (2×). Values represent the means of 6 to 12 mice/group ± the standard errors of the means. The dotted line indicates the limit of detection. Colonization density was analyzed in surviving mice. Eight mice in the mock-immunized group and four animals in the 4cps single-dose group that did not survive were not included in the analysis. Statistical differences were determined using the Mann-Whitney test with comparisons to the corresponding mock-immunized group. *, P < 0.0278; **, P = 0.0028.

FIG. 5.

ELISA titers of IgG and IgA: levels of IgG in serum against type 6A (A) or type 4 (B) (whole bacteria) from mice immunized with the indicated live vaccine. Values shown are the GMT ± standard errors of the means (SEMs) following one or two (2X) doses of the vaccine strain. Symbols in panel A: *, P = 0.0293; **, P < 0.01. Symbols in panel B: *, P = 0.0121; **, P = 0.007. (C) Levels of IgA against type 6A (whole bacteria) in nasal wash samples from mice immunized with the indicated live vaccine. Values shown are GMT/μg total protein ± SEM. *, P < 0.02. Statistical differences were determined using the Mann-Whitney test, with comparisons to the mock-immunized control group.

FIG. 6.

Mechanism of protection. (A) Survival rates of μMT mice (□; n = 10), MHC II^−/− mice (*; n = 8), and wild-type C57BL/6 mice (▾; n = 15) immunized with 6Acps, μMT mice mock immunized with PBS (⧫; n = 7), or C57BL/6 mice mock immunized with PBS (○; n = 14) and challenged with the 6A parent strain. (B) Colonization density at day 9 postinoculation of mice challenged with the 6A parent strain after immunization with 6Acps. Values represent the means of 8 to 15 mice/group ± the standard errors of the means. Statistical differences were determined using the Mann-Whitney test with comparisons to the wild-type C57BL/6 mice. **, P = 0.0083.