The Effects Of Mucosal Adjuvant On FimH And Its Variants

Dental caries is one of the most common chronic infectious diseases which seriously affects people's quality of life.Streptococcus mutans(S.mutans)is considered to be a major pathogen closely related to the destruction of soft and hard tissues on the surface of teeth.Cell surface protein antigen c(PAc)is a kind of glycoproteins existing on the surface of bacteria,which is the main virulence factor of bacteria and can mediate the sucrose independent adhesion of S.mutans to tooth surface.PAc has been used in different experimental systems to induce the production of specific antibodies and inhibit the colonization of cariogenic microorganisms.However,there is currently no vaccine on the market,mainly because of the low ability to induce and maintain protective antibody levels.Most peptide antigens,such as PAc,tend to produce a lower immune response without the help of an effective mucosal vaccine adjuvant.Therefore,searching for novel mucosal adjuvants aimed at enhancing the specific immune response of anti-caries vaccine is an urgent problem to be solved.FimH is an adhesin that exists at the end of type I pili on the surface of most intestinal bacteria and can specifically bind to cell receptors.Fim H mediates the specific adhesion between bacteria and susceptible host cells.Studies have shown that the Fim H of different bacteria has the mutation of individual sequences in the process of evolution.The variation on the genome sequence,known as gene polymorphism,is a very important factor determining individual phenotype,which will lead to amino acid replacement and thus change the adhesion phenotype.High adhesion mutation can enhance the ability of bacteria to adhere to host cells,which is the adaptive evolution of bacteria.The gene mutation in the coding region will lead to the change of biological characters,which has great research value.As a pathogen associated molecular pattern(PAMP),Fim H variants may be used as more effective adjuvants in modern vaccines.On this basis,our study selected single amino acid substitutions containing natural mutations in clinical strains and random mutations based on thepublished literature.Purified Fim H and its mutants were first detected to induce the phenotypic maturation of DC2.4 in vitro,and the animal model were used to further verify their immune enhancement effects as adjuvants in vivo.Part I: Construction and purification of Fim H and its mutants Objective: To screen non-synonymous mutations generated by spontaneous mutations in clinical strains and random mutations in the public literature that may have significance for the function of Fim H as well as constructe the mutant plasmids and purify the recombinant proteins.Methods: Site-directed mutagenesis kit was used to carry out single-point mutation on four mutation sites of Fim H,and the mutated plasmids were verified by sequencing.The correct plasmids p K12,p A25 P,p G73 R,p A106 and p T158 P were transformed into Escherichia coli(E.coli)competent cells,which were induced to express recombinant proteins with appropriate concentration of IPTG,and the recombinant proteins were purified by affinity chromatography.Results: The plasmids of p A25 P,p G73 R,p A106 and p T158 P with single amino acid mutation were correctly constructed.The recombinant proteins were successfully expressed and purified.The proteins were identified by coomase brilliant blue staining and Western Blot,indicating the correct expression of the target protein.The purified protein was shown as a single band in SDS-PAGE.Conclusion: Fim H variants with single amino acid mutation were successfully constructed and purified.Part II: The effect of Fim H and its mutants on the phenotypic and functional maturation of DC2.4Objective: To investigate the effect of Fim H and its mutants on DC2.4 maturation and the role of TLR4 in the activation process in vitro.Methods: The CCK8 was used to observe the effect of Fim H and its variants on the survival rate of cells.Purified Fim H and its variants were used to treat DC2.4 cells,and the expression of costimulatory molecules,MHCII molecules and TLR4 molecules on the cell surface were detected by flow cytometry.Real-time quantitative PCR and ELISA were used to detect the m RNA levels and secretion of cytokines.In addition,Farwestern and western methods were used to verify the interaction between Fim H and its variants with TLR4.Fim H and its mutants were labeled with fluorescent dye and incubated with DC2.4 cells to detect the uptake of the proteins.Results: The CCK8 results showed that Fim H and its mutates had no obvious cytotoxicity in the dose range from 1?g/ml to10?g/ml.A106 T and T158 P significantly upregulated the expression levels of CD40,CD80,MHCII and TLR4 on the cell surface.In addition,A106 T and T158 P significantly increased the m RNA and secretion levels of IL-6 and TNF-?.Farwestern and western results confirmed the interaction of Fim H and its mutants with TLR4.The results of flow cytometry showed that DC2.4 cells were more readily to phagocytize A106 T and T158 P.Conclusion: A106 T and T158 P promoted the phenotype maturation of DC2.4 and the secretion of cytokines through TLR4 signals.In addition,DC2.4 were more readily to phagocytize and internalize A106 T and T158 P proteins.Part III: Fim H and its mutants enhance the mucosal immune response of anti-caries vaccine Objective: To explore whether Fim H and its mutants as adjuvants can enhance the antibody production of vaccines,the proliferation of spleen lymphocytes and effect the type of immune response.Methods: K12+PAc,A106T+PAc and T158P+PAc were immunized by buccal mucosa,MPL was used as a positive control,and serum Ig G(Ig G1,Ig G2a)and saliva Ig A levels were detected by ELISA.Meanwhile,spleen cells of immunized mice were isolated and cultured in vitro.After retreatment with PAc,spleen cell proliferation was detected by MTS.Results: T158P+PAc significantly increased serum and salivary antibody levels.Serum level of Ig G1 isotype was significantly higher than that of Ig G2 a,indicating that T158 P co-immunization can produce Th2-biased immune response.Moreover,the proliferation of spleen lymphocytes was significantly enhanced in T158P-immunized mice.Conclusion: T158 P as an adjuvant can enhance the mucosal immune response of anti-caries vaccine.Part IV: The evaluation of the protective efficacy and safety of Fim H and its mutants as adjuvants on anti-caries vaccineObjective: To explore whether co-immunization of Fim H and its variants in rats can prevent the formation of dental caries and have side effects.Methods: K12+PAc,A106T+PAc and T158P+PAc were immunized by buccal mucosa,MPL was used as a positive control,and S.mutans Ingbritt was inoculated to establish a bacterial rat model.Specific antibody level was tested by ELISA,and caries score was used to detect caries prevention.HE staining was performed on the main organs of rats to observe the histological changes.Results: T158P+PAc can significantly increase the level of specific antibodies and reduce the formation of dental caries.No obvious mass heteromorphism or other abnormal manifestations were found in the main organs of the immunized rats.Conclusion: T158 P,as an effective mucosal adjuvant,can enhance the protective efficacy of the anti-caries vaccine without obvious side effects.