| ObjectiveStreptococcus pneumoniae (S.pn) is one of the most important pathogens which induce seriously invasive infections and upper respiratory tract infections. Effective vaccines have become a meaningful approach with practical significance to prevent the infection of S.pn, so WHO recommends the introduction of pneumococcal conjugate vaccine into national immunization programs. Currently, pneumococcal vaccine has been among the world's most urgent vaccines.DnaJ belongs to the Hsp40 family, intraperitoneal immunization with recombinant DnaJ could induce protective efficiency against intranasal infection with serotype 3 of Strepococcus pneumoniae[1]. However, as immunogen, wether DnaJ could induce protection against infection of different pneumococcal serotypes is unknown. At the same time, there were no reports about the immunogenicity of DnaJ used in intranasal immnunization, protective efficiency against lethal infection with S.pn, as well as related immunologic mechanisms. So we carried out this research to study the protective efficiency of DnaJ and the mechnisms.MethodsWe got purified DnaJ from prokaryotic expression system, and immunized BALB/C mice with the recombinant protein to perpare DnaJ specific antisera. Polymerase chain reaction (PCR) and Western blot were used to analyze the expression and conversation of DnaJ in serotype 2, serotype 3, serotype 6B, serotype 14, serotype 19F and strain R6 of Streptococcus pneumoniae. BALB/C mice were intraperitoneally and intranasally immunized with recombinant protein, infected with Streptococcus pneumoniae through two ways according to the immunization to establish appropriate infection models to evaluated protective efficiency. We tested antibody titers, subtypes of IgG in sera and level of cytokines in culture supernatants of spleen cells from immunized mice. Serotype 19F of Streptococcus pneumoniae was introduced intranasally to immunized mice for colonization model, nasal wash and lung homogenates were collected to determine bacterial counts. Antibody adsorption test and passive protective test were applied to evaluate the protective efficiency of DnaJ specific antibodies; Incubated with DnaJ specfic antiserum, R6 was then primed to adhere to A549 cells to detect the function of antisera to inhibit adhesion in vitro. ResultsDnaJ fragments of Streptococcus pneumoniae was cloned into pET-32 (a) plasmid exactly. Expressive bacteria was induced with IPTG, and we obtained the recombinant protein in soluble manner, Ni-NTA affinity chromatography was applied to get purified protein, with the purity>90%. PCR and Western blot demonstrated that DnaJ was expressed in all the serotyes of S.pn used in this assay with a good conservative. Both intraperitoneal and intranasal immunization with DnaJ could protect host against lethal infection of different serotypes of S.pn with a good protective effect, protective efficiency from intraperitoneal immunization against serotype 2, serotype 3, serotype 6B and serotype 14 of S.pn were 50%,while for intranasal immunization, they came to 58.3%,66.7%,66.7%,50%. Intraperitoneal immunization with DnaJ protein stimulated high level of IgG1 in sera, and the titer came to 4.27×106; while intranasal immunization with DnaJ protein stimulated high level of IgG2b in sera, and the titer came to 2.13×106. Meanwhile, intranasal immunization also stimulated high levels of IL-10, IFN-γand IL-17A, doses were 239.11±168.77,55.86±39.25,765.68±159.11 (pg/ml), respectively. Results from anti-colonization experiment showed that DnaJ specific anti-sera could reduce the bacterial counts colonizing in nasopharynx and lung significantly (P <0.05). Immunized BALB/C mice with DnaJ specific antisera passively prolonged survival time and with survival rate increased significantly, which was 50%. After incubation with R6, antibody titers decreased greatly, while protective efficiency of antisera disappeared (P <0.05). Meanwhile, the antisera could inhibit the adhesion of S.pn to A549 cells in vitro.ConclusionsResults above showed that, DnaJ expressed in the all the strains of S.pn used in this assay with a good conservation. Intraperitoneal immunization with DnaJ protein could stimulate humoral immune response, and could protect hosts against lethal infection of the four pathogenic serotypes of S.pn, this protective effect was dependent on the specific antibody; intranasal immunization with DnaJ protein could stimulate humoral immune response as well as cellular immune response effectively, and had a good protective effect against intranasl infection of the four pathogenic serotypes of S.pn, at the same time, high level of IL-17A was stimulated in intranasal immunization, which helped hosts to resist nasopharyngeal colonization of S.pn effectively.
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