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Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Streptococcus pneumoniae is a human pathogen that colonizes the naso and/or oropharynx and can cause otitis, pneumonia, bacteremia and meningitis. To broaden the protection against pneumococcus, several pneumococcal proteins have been investigated as vaccine candidates. In this study we analyzed the immunological response induced by mouse subcutaneous immunization with a fusion of the Polyamine transport protein D (PotD) and a pneumolysin derivative (PdT), resulting in a hybrid rPotD-PdT protein. Immunization of mice with rPotD-PdT induced increased production of nitric oxide, indicating a higher innate immune response. In agreement, immunization of mice with the hybrid protein was more immunogenic than the individual proteins or their combination, eliciting higher antibody levels. The anti-rPotD-PdT IgG displayed increased binding onto the pneumococcal surface. Furthermore, the anti-rPotD-PdT antisera promoted superior opsonophagocytosis as compared with the other tested formulations. However, despite that the encouraging results in vitro, immunization with the hybrid was not sufficient to induce protection against sepsis with a highly virulent pneumococcal strain. taken together, the results suggest that hybrid proteins are an interesting strategy, able to promote improved immune responses, but the inclusion of other antigens may be necessary to promote protection against invasive infections caused by this bacterium. [ABSTRACT FROM AUTHOR]

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge. [ABSTRACT FROM AUTHOR]