Study On The Protective Effect And Mechanism Of Fusion Protein DnaJ- â–³A146Ply Subcutaneous Immunization In Mice Against Streptococcus Pneumonia Infection

Objective: Streptococcus pneumonia is an important pathogen localized on human nasopharyngeal, which can cause serious diseases such as pneumonia, acute otitis media, meningitis, septicemia and so on. According to the WHO study, Streptococcus pneumonia is the main cause of death in children around the world, accounting for 16% of the children under 5 years old. Pneumococcal immunization is the most effective method to prevent Streptococcus pneumonia infection at present. Whatever, the vaccines of S.pneumoniae existing on market mostly based on the serotype-specific capsular polysaccharide; there are some disadvantages like limited serotype coverage, easy to convert serotype and not to be included in planned economy vaccines. Thus, research of a new subunit-protein vaccine that confers not dependent on the serotype, good protection effect and economy is imperative.DnaJ-△A146Ply was constructed with S.pneumoniae virulence factors DnaJ and △A146Ply by our team. △A146Ply is an potential mucosal adjuvants, and DnaJ-△A146Ply mucosal immunity in mice can induced Th17 cell response, which can stimulate sIgA secretion to resist the colonize of S.pneumoniae. Subcutaneous immunization is a routine, quick and safe way. Macrophages are necessary to clear the S.pneumoniae. So in this experimental study, we plan to research the protective effect and mechanism of fusion protein Dna- △ A146 Ply subcutaneous immunization in mice against Streptococcus Pneumonia infection, evaluate the subcutaneous adjuvant effect of △A146Ply, explore the molecular mechanism of immune response induced by pneumococcal fusion protein DnaJ-△A146Ply about macrophage, and to provide the basis to the preclinical research for DnaJ-△A146Ply vaccine.Methods: Prokaryotic expression DnaJ- △ A146 Ply reconstructed protein, of which LPS was removed after Ni+ column purification. LPS test kits and macrophage experiments were used to eliminate the interference of residual LPS. Recombinant proteins immunized C57BL/6 mice. A week after last injection, we constructed sepsis and colonize models, and evaluated the protective effect of proteins by observing the survival rates, bacterial capacity and pulmonary inflammatory infiltration. Knockout mice and antiserum experiments were used to explore the humoral and celluar immune response induced by DnaJ-△A146Ply; ELISA was used to detect the titer of specific antibody, the subtype of antibody and the cytokines of splenocyte and macrophage. The mRNA expression was measured by PT-PCR. The phosphorylation levels of Akt and NF-κB were evaluated by Western Blot.Results: Recombinant proteins with more than 85% purity and less than 0.1 EU/ug LPS were obtained. The residual of LPS did not affect the expression of TNF-α. The survival rate of DnaJ- △ A146 Ply was significantly higher than that of PBS control(P=0.0405), with a comparative rate to PPV23. The bacterial capacity of lung and nasopharyngeal in immune group decreased obviously(P﹤0.01)compared to control group, and the pulmonary inflammatory infiltration also decreased significantly.The results about humoral immune response: Mice were subcutaneous immunized with fusion protein DnaJ-△A146Ply.After that the survival rate of μMT-/- mice was lower than that of WT mice( P=0.0468),and nasopharyngeal in immune group increased obviously(P=0.0049);DnaJ-△A146Ply subcutaneous immunization induced mice to get specific IgG antibody more effectively than DnaJ, with a major subtype of IgG1, IgG2 and IgG2 b. What’s more, △A146Ply can assist DnaJ to produce more specific Ig G antibody( P < 0.01) quickly. The anti-serum of DnaJ- △A146Ply could help macrophage enhance phagocytosis VS negative control anti-serum(P=0.0160). In addition, the anti-sreum of DnaJ-△A146Ply were effective against the colonize and invasive infection of S.pneumoniae. The results about celluar immune response: DnaJ-△A146Ply subcutaneous immunization in mice irritated the spleen to produce significantly more cytokines IL-4、IFN-γ and IL-17 A than PBS control(P<0.05), while the IL-5 and IL-10 had no difference. The nasopharygeal colonize of IL-4-/-、IFN-γ-/-、IL-17A-/- mice were increased obviously than that of WT mice(P<0.05), without difference with PBS control. However, the survival rate only IL-4-/-、IFN-γ-/- mice had difference with WT mice(P<0.05), no IL-17A-/- mice. In TLR4-/- mice, the DnaJ- △ A146 Ply immune group counted more bacteria from nasal wash than in WT mice(P=0.014), while it was comparable to control group(P=0.2617); The titer of antibody was significantly lower than that of WT mice, the major subtype of antibody were also Ig G1、IgG2a and IgG2b; The cytokines of splenocyte IFN-γ、IL-4 and IL-17 A in TLR4-/- mice had no difference with their respective control group, and decreased than that of immune group in WT mice(P<0.05).DnaJ-△A146Ply significantly enhanced both the mRNA level and the protein expression of IL-6 and TNF-α in macrophage; It also advanced the phosphorylation levels of Akt and NF-κB of macrophage. And the inhibitors of Akt and NF-κB obviously decreased the expression of IL-6 and TNF-α.Conclusion: △A146Ply is a kind of potential subcutaneous adjuvants. DnaJ-△A146Ply subcutaneous immunization in mice by inducing humoral and cellular immune response resist Streptococcus Pneumoniae infection, and the protective effect of colonization depends on TLR4. Pneumococcal fusion protein DnaJ-△A146Ply induces the immune response of peritoneal macrophage through Akt and NF-κB signaling pathways.