Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives

et al. (2013) Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives. PLoS ONE 8(3): e59605. doi:10.1371/journal.pone.0059605 Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives Cibelly Goulart 0 Thais Raquel da Silva 0 Dunia Rodriguez 0 Walter Rodrigo Politano 0 Luciana C. C. Leite 0 Michelle Darrieux 0 Bernard Beall, Centers for Disease Control & Prevention, United States of America 0 1 Centro de Biotecnologia, Instituto Butantan, Sa o Paulo, Brazil, 2 Programa de Po s-Graduac a o Interunidades em Biotecnologia-USP-IPT-IB, Sa o Paulo, Brazil, 3 Laborato rio de Biologia Celular e Molecular, Universidade Sa o Francisco , Braganc a Paulista , Brazil 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 coadministered 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. - Funding: This work was supported by FAPESP (Fundacao de Amparo a` Pesquisa do Estado de Sao Paulo) and Fundacao Butantan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript Competing Interests: The authors have declared that no competing interests exist. Streptococcus pneumoniae is a major human pathogen, accounting for over 10% of total deaths in children under the age of five [1]. Despite the well established efficacy of conjugate vaccines against invasive disease, the high production costs involved in the conjugation processes limit their implementation in lower income countries, in which the burden of pneumococcal diseases is highest. Also, due to the limited number of polysaccharides included in the formulations, the extent of vaccine coverage tends to decrease as less prevalent serotypes emerge. In fact, serotype replacement has been observed after the introduction of PCV7 in different populations [2,3]. Finally, serotype replacement is associated with the emergence of antibiotic resistant clones [4], reinforcing the need for cost-effective strategies that confer broad protection, such as protein-based vaccines. PspA and Pneumolysin (Ply) are among the most well studied pneumococcal proteins; their contribution to virulence has been demonstrated with mutant strains lacking either one or both proteins, which have shown reduced fitness in different models of colonization, lung infection and bacteremia [5]. Mutant strains were cleared more rapidly from the lungs and blood of mice when compared to wild type counterparts [5,6] and deposited more C3 in vitro [7]. Furthermore, the combination of both mutations had an additive effect on C3 deposition and pneumococcal clearance [6], suggesting that these proteins contribute synergistically to bacterial evasion of innate immune responses [6,7]. Recombinant forms of PspA and Pneumolysin derivatives (Pds) have been investigated as potential vaccine candidates in different animal models, with promising results. The N-terminal region of PspA, which is responsible for inhibiting complement deposition on the bacterial surface [8,9] and contains most of the immunogenic epitopes of the molecule [10], confers protection against invasive infection [1113], lobar pneumonia [14] and colonization [15,16]. Furthermore, it has been recently demonstrated that maternal immunization with PspA protects the offspring against pneumococcal infection [17]. The N-terminus of PspA, however, exhibits structural and serological variability [18]. Based on the observation that different PspA molecules induce antibodies with distinct degrees of cross-reactivity [19,20] and cross-protection [11,21], it has been suggested that PspAbased anti-pneumococcal vaccines should include more than one molecule in order to extend coverage. The potential of PspA as a vaccine candidate has been further supported by human clinical trials, which have demonstrated the induction of antibodies with high cross-reactivity against heterologous molecules [21], which can passively protect mice against fatal pneumococcal infection [21]. Pneumolysin (Ply) is a cholesterol dependent cytolysin with several biological effects, such as activation of classical complement pathway [22], induction of apoptosis in numerous cells types [23,24], impairment of ciliary function in the lungs and induction of oxidative burst by neutrophils [22]. In fact, the instillation of purified Ply in the lungs is sufficient to reproduce many aspects of pneumococcal pneumonia in rats (reviewed in [22]). Furthermore, Ply has been shown to interact with TLR-4 [25] and to induce TLR-4 independent activation of the NLRP3 inflammasome, contributing to host protection against pneumococcal pneumonia [26] and lethal infection [25]. Since Ply is toxic in its native form, several detoxified forms named pneumolysoids (Pds) have been produced, by sitedirected mutagenesis or chemical detoxification, and evaluated for their immunogenicity and protective effect in different animal models, with variable results, including protection in rhesus macaques [14,2733]. Of those toxoids, the best characterized are PdB, carrying a Trp-Phe substitution at position 433 [30], and PdT, a triple mutant containing Asp-385 to Asn, Cys-428 (...truncated)