A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia

Abstract

PspA and pneumolysin are two important vaccine candidates, able to elicit protection in different models of pneumococcal infection. The high immunogenic potential of PspA, combined with a possible adjuvant effect of pneumolysin derivatives (due to their ability to interact with TLR-4) could greatly improve the immunogenicity and coverage of a protein-based pneumococcal vaccine. A chimeric protein including the N-terminal region of PspA in fusion with the pneumolysin derivative, PlD1, has been shown to induce high antibody levels against each protein, and protect mice against invasive challenge. The aim of the present study was to investigate the cellular response induced by such vaccine, and to evaluate protection in a murine model of lobar pneumococcal pneumonia. Pneumococcal pneumonia was induced in BALB/c mice by nasal instillation of a high dose of a serotype 14 strain with low virulence. Airway inflammation was confirmed by total and differential cell counts in BAL and by histological analysis of the lungs, and bacterial loads were measured 7 days after challenge. Cytokine levels were determined in the bronchoalveolar fluid (BALF) of mice immunized with rPspA-PlD1 fusion after challenge, by flow cytometry and ELISA. After challenge, the mice developed lung inflammation with no invasion of other sites, as demonstrated by histological analysis. We detected significant production of TNF-α and IL-6 in the BALF, which correlated with protection against pneumonia in the group immunized with rPspA-PlD1. In conclusion, we found that the rPspA-PlD1fusion is protective against pneumococcal pneumonia in mice, and protection is correlated with an early and controlled local inflammatory response. These results are in agreement with previous data demonstrating the efficacy of the fusion protein against pneumococcal sepsis and reinforce the potential of the rPspA-PlD1 protein chimera as a promising vaccine strategy to prevent pneumococcal disease.

Fig 1. Mouse model of pneumococcal pneumonia.

(A) Bacterial counts were determined in the lungs of nonimmunized mice on days 5 and 7 after intranasal inoculation with pneumococcal strains St 245/00 (serotype 14) and P854 (serotype 19F). Values were compared for each strain after 5- and 7 days using Student t test (*p<0,05). (B). Leucocyte infiltrates in the BALF were calculated for different time points after intranasal challenge with pneumococcal strain St 245/00. Statistical analysis was performed using one way ANOVA with Dunnet’s posttest. *p<0,05 in comparison with cells counts at 0 hours.

Fig 2. Lung colonization by Streptococcus pneumoniae in mice immunized with rPspA_PlD1.

Mice immunized with 3 doses of rPspA, rPlD1, or the fusion protein in Alum were challenged intranasally with 5x10⁶ CFU of St 245/00. Bacterial counts in the lungs of immunized and control mice (injected with Alum diluted in PBS) are shown after 2–48 h (A) or seven days (B). (*p<0.05 for the same immunization group at different time points and #p<0.05 for immunized x control mice).

Fig 3. Induced immune responses in lungs and BALF.

(A) Cellular infiltrate in the BALF following pneumococcal challenge. Immunized and control mice were euthanized at different time points after intranasal inoculation of St 245/00 and the total cell counts in the BALF were determined and compared among immunization groups and times points. Statistical analysis was performed using ANOVA with Tukey’s and Dunnet’s posttests. (*) p<0,05 between samples from the same immunization group at different times after challenge; (#) p<0,05 in comparison with the control (Alum) at the same time point; (o) p<0,05 when comparing immunization with rPspA-PlD1 versus isolated proteins at the same time point. (B and C) Histological analysis of lung tissue after pneumococcal challenge. 1 μm sections of the left lung lobe from mice immunized with the hybrid (B) and from control mice (C) after 48 h were stained with hematoxylin-eosin, showing slight and moderate levels of inflammation, respectively (Original magnification, x400).

Fig 4. Cytokine production by immunized mice after pneumococcal challenge.

Production of TNF-α (A) and IL-6 (B) in the BALF of immunized mice was detected by ELISA and compared with control mice (receiving adjuvant in PBS). *p<0.05 for the same group at different time periods; #p<0.05 as compared with the control group;°p<0.05 for the rPspA-PlD1 group as compared to the individual proteins.