| Objective Streptococcus pneumoniae (S.pn) is mainly localized onthe human respiratory tract can cause a range of serious diseases; such asotitis media, pneumonia, meningitis, septicemia, and so on. Pneumococcalinfectious disease has a higher morbidity and mortality all over the world,especially for children under5years old and olders. As the developmentthe antibiotic resistance strains of Streptococcus pneumoniae, vaccineplays an important role in prevent the infectious disease of Streptococcuspneumoniae. Currently available pneumococcal vaccines are all based onthe serotype-specific capsular polysaccharides; the limited serotypecoverage can be an issue. Therefore, the research of an affordable vaccinethat confers comprehensive protection from pneumococcal infectiousdisease remains hot.Protein vaccine is a class of very promising new vaccine, inducingstrong immunogenicity, non-serotype-dependent, and simple productionprocess. Streptococcus pneumoniae DnaJ (Hsp40) is a member of a familyof heat shock protein40. Many reports have shown that intraperitoneal orintranasal immunization with recombinant DnaJ induced a strikingprotective immune response and protected BALB/C mice against focaland lethal infections with different serotypes of S.pneumoniae.Pneumolysin (Ply) is a conserved53KDa Streptococcus pneumoniae surface expression protein with hemolytic activity. Attenuated Ply protein(ΔA146Ply) has been used as a potential vaccine candidate in animalmodels; intranasal immunization with recombinant ΔA146Ply can inducea striking protective immune response and reduces the colonization ofdifferent serotypes of S.pneumoniae on the nasopharynx and lung. Thereare also some reports suggested that Ply can be used as a potentialmucosal adjuvant for combination use with other proteins, inducinghigher levels of specific IgG and IgA. Therefore, in this study we willevaluate the potential adjuvant effect of ΔA146Ply; identify the protectionefficacy and immune mechanism of immunization with recombinantDnaJ-ΔA146Ply fusion protein in C57BL/6mice model; and then assessits feasibility as a vaccine candidate protein.Method The Streptococcus pneumoniae DnaJ (full length), attenuatedPly protein (ΔA146Ply), DnaJ-ΔA146Ply and ΔA146Ply-DnaJ fusionprotein were expressed in Escherichia coli by prokaryotic expressiontechnology. Then the proteins were purified by Ni-NTA affinitychromatography and removed the mixed endotoxin. After that, the purifiedrecombinant proteins were immunized C57BL/6mice to prepare specificpolyclonal antiserum. Use specific anti-DnaJ and anti-ΔA146Plypolyclonal antiserum to verify the epitope of two fusion proteins byWestern blot. Western blot analysis was also used to recognize whether thespecific antiserum of anti-DnaJ-ΔA146Ply anti-ΔA146Ply-DnaJ fusionprotein can measure the DnaJ and Ply protein in different serotypes ofStreptococcus pneumoniae, respectively.In this study, groups of C57BL/6mice were mucosal immunized withpurified recombinant DnaJ-ΔA146Ply or ΔA146Ply-DnaJ fusion protein,respectively. Antibody titers of specific IgG and its subtype in serum andspecific IgA in saliva were measured by ELISA after immunization. The levels of IL-4, IFN-γ, IL-10and IL-17A in the splenocytes supernatantswere detected by ELISA Kits. We also observed the histological change inanimals after immunization by HE staining of lung tissue, then detected thetotal IgE levels in serum by ELISA Kit. Streptococcus pneumoniae CMCC31207(6B serotype) and CMCC31693(19F serotype) were used toconstruct focal infection model in C57BL/6mice, and then enumerate thebacteria loads in nasal wash and lung. Streptococcus pneumoniae D39(2serotype) and CMCC31436(3serotype) were used to construct lethalinfection model in C57BL/6mice, and then monitor the survival of eachmouse for consecutive21days. Purified recombinant DnaJ-ΔA146Plyfusion protein were mucosal immune to C57BL/6mice (WT) andIL-17A-deficient C57BL/6mice (IL-17A KO), respectively. Antibodytiters of specific IgG in serum and specific IgA in saliva were measured byELISA after immunization. Streptococcus pneumoniae CMCC31693(19Fserotype) was used to construct focal infection model, and then enumeratethe bacteria loads in nasal wash and lung.Result We have successfully constructed pET-28(a)-DnaJ-ΔA146Plyand pET-28(a)-ΔA146Ply-DnaJ recombinant plasmid, and theDnaJ-ΔA146Ply or ΔA146Ply-DnaJ fusion protein with>90%puritywere successfully prepared; and the endotoxin levels are not higher than0.1EU/μg. Western blot results have shown that DnaJ-ΔA146Ply andΔA146Ply-DnaJ fusion protein both can be identified by anti-DnaJ andanti-ΔA146Ply polyclonal antisera. While anti-DnaJ-ΔA146Ply andΔA146Ply-DnaJ antisera can not only identify specific recombinant DnaJand ΔA146Ply, even can identify the natural DnaJ and Ply proteins indifferent serotypes of Streptococcus pneumoniae.Compared with DnaJ alone, the IgG and IgA antibody titers (P <0.01)in both serum and saliva were effectively stimulate by DnaJ-ΔA146Ply fusion protein mucosal immunization, and IgG1, IgG2a, and IgG2b werethe major antibody subtypes in the serum samples. Also, DnaJ-ΔA146Plyfusion protein increased specific IgG in sera ahead of time compared withthe control group. Different serotypes of Streptococcus pneumoniaechallenge experiments have shown that mucosal immunization withDnaJ-ΔA146Ply fusion protein can significantly reduce6B and19Fserotype pneumococcal colonization on nasopharyngeal (P <0.05) andlung (P <0.001), and also can improve survival time of C57BL/6miceinfected by lethal dose of D39(2serotype)(P <0.001) and CMCC31436(3serotype)(P <0.001), the protection efficacy was comparable withPPV23. Meanwhile, HE staining of the lung tissue after immunizationsuggested that: the lung inflammatory changes of mice immunizedDnaJ-ΔA146Ply fusion protein were weaker than CT group, and the totalserum IgE levels were also significantly lower than CT group.The amount of IL-17A produced in splenocytes supernantant fromDnaJ-A146Ply immunized mice were significantly higher than DnaJ or A146Ply vaccinated mice (P <0.05). Compared with wild-type mice(WT), IL-17A-deficient mice (IL-17KO) cannot stimulate the productionof high titers of IgA (P <0.05) in saliva after mucosal immunization withDnaJ-ΔA146Ply fusion protein, and not reduce19F serotypeStreptococcus pneumoniae in colonization nasopharyngeal (P <0.001) andlung (P <0.001).Conclusion This study firstly demonstrated that ΔA146Ply hasmucosal adjuvant function, and can induce higher levels of specific IgGand sIgA in C57BL/6mice when were fusion expressed with DnaJ. Ascompared with the CT adjuvant, this protein adjuvant was safer.DnaJ-ΔA146Ply fusion protein can be used as a new pneumococcalvaccine candidate protein, it can significantly reducing colonization of different serotypes of Streptococcus pneumoniae in nasopharyngeal andlung; and DnaJ-A146Ply can protect immunized mice from challengewith lethal doses of S.pneumoniae strains which is comparable to thatachieved by PPV23, and the protection is not serotype-dependent.Mucosal immunization with DnaJ-ΔA146Ply fusion protein can stimulateTh17immune response generating higher levels of IL-17A. These resultsdemonstrate that IL-17A-mediated immune response is important forDnaJ-A146Ply elicited production of antigen specific sIgA andprotection against pneumococcal infections. In summary, we believe thatDnaJ-ΔA146Ply fusion protein is a better pneumococcal vaccinecandidate protein, mainly through IL-17A to play its role in immuneprotection. |
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