Immunization with a combination of three pneumococcal proteins confers additive and broad protection against Streptococcus pneumoniae Infections in Mice

Abstract

Pneumococcal polysaccharide-based vaccines are effective in preventing pneumococcus infection; however, some drawbacks preclude their widespread use in developing and undeveloped countries. Here, we evaluated the protective effects of ATP-dependent caseinolytic protease (ClpP), pneumolysin mutant (DeltaA146 Ply), putative lipoate-protein ligase (Lpl), or combinations thereof against pneumococcal infections in mice. Vaccinated mice were intraperitoneally and/or intranasally challenged with different pneumococcal strains. In intraperitoneal challenge models with pneumococcal strain D39 (serotype 2), the most striking protection was obtained with the combination of the three antigens. Similarly, with the intranasal challenge models, (i) additive clearance of bacteria in lungs was observed for the combination of the three antigens and (ii) a combination vaccine conferred complete protection against intranasal infections of three of the four most common pneumococcal strains (serotypes 14, 19F, and 23F) and 80% protection for pneumococcal strain 6B. Even so, immunity to this combination could confer protection against pneumococcal infection with a mixture of four serotypes. Our results showed that the combination vaccine was as effective as the currently used vaccines (PCV7 and PPV23). These results indicate that system immunization with the combination of pneumococcal antigens could provide an additive and broad protection against Streptococcus pneumoniae in pneumonia and sepsis infection models.

FIG. 1.

Protection elicited by Lpl, ClpP, and ΔA146 Ply against pneumococcal lung colonization. BALB/c mice were immunized with the indicated antigens and intranasally challenged with S. pneumoniae as follows: 1.5 × 10⁶ CFU of the 31614 strain (A) and 10⁷ CFU of the 31693 strain (B). The lung colonization of individual mice is shown at day 5 after challenge, indicating the median CFU per lung (horizontal lines). The broken line on the y axis indicates no lung infection. Detection limitation, 100 CFU. In panel B, the L, P, and C represent Lpl, ΔA146 Ply, and ClpP, respectively.

FIG. 2.

Protection of vaccinated mice from pneumococcal strain D39 infections. (A) BALB/c mice were challenged intraperitoneally with strain D39 at an LD₅₀ of 150 (2.0 × 10⁴ CFU; combination versus PPV23, P = 0.5206; combination versus control, P < 0.0001) (A) or intranasally with strain D39 at an LD₅₀ of 10³ (7.5 × 10⁶ CFU; combination versus PPV23, P = 0.9720; combination versus control, P < 0.0001) (B). The results are represented in survival curves and analyzed by the log-rank test. n refers to the number of animals. For all experiments, control protein-immunized mice served as negative controls.

FIG. 3.

Protection conferred by combination against multiple pneumococcal strain infections. BALB/c mice were immunized with the combination of Lpl plus ClpP plus ΔA146 Ply and intranasally challenged with 2.0 × 10⁸ CFU of strain 31759 (PS type 23F; P < 0.0001) (A); 1.5 × 10⁸ CFU of strain 31693 (PS type 19F; P < 0.0001) (B); 1.0 × 10⁸ CFU of strain 31614 (PS type 14; P < 0.0001) (C); 2.0 × 10⁸ CFU of strain 31207 (PS type 6B; PCV7 versus combination, P = 0.1189; combination versus control, P < 0.0001; PCV7 versus control, P = 0.0012) (D); 7.5 × 10⁶ CFU of strain 31436 (PS type 3; combination versus PPV23, P = 0.7873; combination versus control, P < 0.0001; PPV23 versus control, P = 0.0005) (E); or with a mixture of serotype 6B (2.0 × 10⁸ CFU), serotype 14 (1.0 × 10⁸ CFU), serotype 23F (2.0 × 10⁸ CFU), and serotype 19F (1.5 × 10⁸ CFU) (combination versus PCV7, P = 0.1823; combination versus control, P < 0.0001; PCV7 versus control, P = 0.0005) (F). Survival was monitored for 21 days after challenge. The results are represented in survival curves and analyzed by the log-rank test. n refers to the number of animals. For all experiments, control protein immunized mice served as negative controls.

FIG. 4.

Serotype-specific pneumococci responsible for the death of mice infected with a mixture of four strains. M, marker. Strains 31207 (serotype 6B), 31693 (serotype 19F), 31759 (serotype 23F), and 31614 (serotype 14) are indicated. Lanes 1, 2, and 3 denote the negative control, PCV7, and the combination vaccine, respectively.

FIG. 5.

Pneumococcal sepsis survival studies using passively intraperitoneally transferred antisera in BALB/c mice. (A) Passive transfer of antisera or diluted (1:15) antisera immediately, followed by challenge with D39 strain at a 6-LD₅₀ dose (600 CFU) (log-rank; undiluted versus control, P < 0.0001; 1/15 diluted, P < 0.0001; median survival, diluted and undiluted sera, undefined; control, 1.0 day). (B) Passive transfer of control antisera adsorbed with R6 (control), combination antisera adsorbed with R6, and combination antisera adsorbed with R6 mutants, immediately followed by a D39 challenge at a 6-LD₅₀ dose (600 CFU). Control antisera served as the controls (wt-R6 adsorbed versus R6 mutant adsorbed, P = 0.0023). The results are represented in survival curves and were analyzed by using the log-rank test.