Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage.

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.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage

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.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis.

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Immunization with PhtD truncated fragments reduces nasopharyngeal colonization by Streptococcus pneumoniae.

Despite the undeniable success of polysaccharide vaccines against Streptococcus pneumoniae infections, there is a consensus on the scientific field that this approach should be revised in order to overpass the problems related with these formulations, such as serotype replacement and high production costs. The study of conserved pneumococcal proteins or its truncated fragments has emerged as a serotype independent alternative. In this work, we have characterized the immune response elicited by systemic immunization of mice with the Histidine triad protein D (PhtD) and its' amino and carboxyl terminal fragments. The proteins were shown to be immunogenic and protective against pneumococcal colonization, with increased IL-17 production, and induction of antibodies able to limit pneumococcal adhesion to human respiratory cells. Antiserum against PhtD_Nter, but not C_ter or PhtD, promoted an increase in bacterial phagocytosis in vitro. Interestingly, antibodies against the PhtD_Nter displayed cross-reactivity with two other pneumococcal proteins, PspA and PspC, due to sequence similarities in the proline rich region of the molecules. On a whole, our results support the inclusion of PhtD, and more specifically, its N-terminal fragment, in a multicomponent serotype independent vaccine.

Conjugation of PspA4Pro with Capsular Streptococcus pneumoniae Polysaccharide Serotype 14 Does Not Reduce the Induction of Cross-Reactive Antibodies.

Current pneumococcal vaccines are composed of bacterial polysaccharides as antigens, plain or conjugated to carrier proteins. While efficacious against vaccine serotypes, epidemiologic data show an increasing incidence of infections caused by nonvaccine serotypes of Streptococcus pneumoniae The use of pneumococcal surface protein A (PspA) as a carrier protein in a conjugate vaccine could help prevent serotype replacement by increasing vaccine coverage and reducing selective pressure of S. pneumoniae serotypes. PspA is present in all pneumococcal strains, is highly immunogenic, and is known to induce protective antibodies. Based on its sequence, PspA has been classified into three families and six clades. A PspA fragment derived from family 2, clade 4 (PspA4Pro), was shown to generate antibodies with a broad range of cross-reactivity, across clades and families. Here, PspA4Pro was modified and conjugated to capsular polysaccharide serotype 14 (PS14). We investigated the impact of conjugation on the immune response induced to PspA4Pro and PS14. Mice immunized with the PS14-mPspA4Pro conjugate produced higher titers of anti-PS14 antibodies than the animals that received coadministered antigens. The conjugate induced antibodies with opsonophagocytic activity against PS14-carrying strains, as well as against a panel of strains bearing PspAs from five clades (encompassing families 1 and 2) bearing a non-PS14 serotype. Furthermore, mice immunized with PS14-mPspA4Pro were protected against nasal colonization with a nonrelated S. pneumoniae strain bearing PspA from clade 1, serotype 6B. These results demonstrate that the cross-reactivity mediated by PspA4Pro is retained following conjugation, supporting the use of PspA4 as a carrier protein in order to enhance pneumococcal vaccine coverage and encourage its further investigation as a candidate in future vaccine designs.

Role of Streptococcus pneumoniae Proteins in Evasion of Complement-Mediated Immunity.

The complement system plays a central role in immune defense against Streptococcus pneumoniae. In order to evade complement attack, pneumococci have evolved a number of mechanisms that limit complement mediated opsonization and subsequent phagocytosis. This review focuses on the strategies employed by pneumococci to circumvent complement mediated immunity, both in vitro and in vivo. At last, since many of the proteins involved in interactions with complement components are vaccine candidates in different stages of validation, we explore the use of these antigens alone or in combination, as potential vaccine approaches that aim at elimination or drastic reduction in the ability of this bacterium to evade complement.