Development Of A ZmpB-Based Protein Vaccine Against Pneumococcal Infectious Diseases

ObjectiveStreptococcus pneumoniae is able to cause a variety of mucosal and invasive infectious diseases, including otitis media, sinusitis, pneumonia, bacteremia, and meningitis, especially in children, the elderly, or immuno-compromised individuals. Using antibiotics to control pneumococcal infections has become increasingly difficulty because of widespread and progressive antimicrobial resistance. Therefore, vaccination is a promising way to prevent the occurrence of pneumococcal diseases.Zinc metalloprotease B (ZmpB) is present in all isolated pneumococcal strains. The first 300 to 400 amino acids, at the N-terminal region of ZmpB, show 99% similarity between all pneumococcal serotypes examined, while the remainder of this protein displays high sequence variation. It was demonstrated that there were human B-cell epitopes in ZmpB by using a pneumococcal genome display library, and antigenic regions of ZmpB reacted with 77% of human adult sera, which is higher than the reactivity of PspA (60%), suggesting a broad recognition of ZmpB antigen. Therefore, ZmpB has the potential to be a candidate pneumococcal vaccine. Pneumococcal pneumolysin (Ply) and rDnaJ are candidate protein vaccines which have been shown to elicit protection against pneumococcal infection. In this study, we investigated the ability of immunization with recombinant ZmpB (rZmpB), alone or in combination with recombinant nontoxic Ply (DeltaA146 Ply), and rDnaJ to elicit protection against pneumococcal colonization and invasive infection in mice.MethodsRecombinant ZmpB (rZmpB) was cloned and expressed in Escherichia coli. The expression of ZmpB by different pneumococcal strains was detectable by Western blotting with anti-sera raised to recombinant ZmpB. To determine whether rZmpB or anti-ZmpB sera have a functional significance, we tested their ability to interfere with the adhesion of D39 to A549 cells. To study antigen-specific responses generated by intranasal immunization with rZmpB, both humoral and cell-mediated responses were investigated. Mouse splenocytes isolated from control and immunized mice were stimulated with rZmpB in vitro, the levels of cytokines IFN-γ, IL-10, and IL-17A in culture supernatants of splenocytes isolated from mice were detected by ELISA.. To evaluate protection against death afforded by mucosal immunization with rZmpB, invasive pneumococcal infection models by intranasal inoculation of different serotypes of S. pneumoniae were established in mice that mimic the natural route of pneumococcal infection. Furthermore, we evaluated the possibility that immunization with rZmpB in combination with rPly and rDnaJ would confer an additive protection against invasive pneumococcal infection in intranasal and intraperitoneal challenge experiments. Finally, we evaluated passive protection against invasive pneumococcal infection by antigen-specific antibodies.ResultsPurified rZmpB was >95% pure, as judged by SDS-PAGE, after it was stained with Coomassie brilliant blue R250. rZmpB migrated with the expected molecular size of approximately 90 kDa in SDS-PAGE. Anti-ZmpB polyclonal antibodies reacted with a band of approximately 210 kDa in fractions of all pneumococcal strains including CMCC 31436 (serotype 3), CMCC 31207 (serotype 6B), CMCC 31614 (serotype 14), and CMCC 31693 (serotype 19F) by western blot.. Both recombinant ZmpB protein and anti-ZmpB polyclonal antibodies significantly inhibited the adhesion of Streptococcus pneumoniae to A549 cells. In mucosal immunization studies, the levels of cytokines IFN-γ, IL-10, and IL-17A in culture supernatants of splenocytes isolated from rZmpB-immunized mice were significantly higher than those from control mice. mucosal immunization with recombinant ZmpB could significantly reduce pneumococcal lung colonization caused by S. pneumoniae serotypes 19F and 14 and significantly increase mice survival times following invasive pneumococcal challenge with different pneumococcal strains, including serotypes 2, 3, 6B, and 14. Furthermore, intraperitoneal immunization with recombinant ZmpB in combination with the recombinant pneumolysin mutant (DeltaA146 Ply) and heat shock protein 40 (DnaJ) could enhance the protection against pneumococcal infection compared to protection provided by single-protein antigens. Passive immunization with hyper-immune anti-sera against these three antigens also demonstrated that the combination of three hyper-immune anti-sera could provide better protection than single anti-sera.ConclusionsZmpB is a large protein present in all pneumococcal strains tested. This study indicates that immunization with ZmpB could protect against pneumococcal diseases, and a triple combination of recombinant ZmpB, Ply, and DnaJ provided better protection against invasive pneumococcal infection than single antigens. Therefore, our results suggest that ZmpB is a good candidate pneumococcal vaccine antigen.