Study On The Effects Of A Dual-promoter DNA Vaccine Against Dental Caries

Dental caries is a worldwide disease,which jeopardizes the public health.Since it was confirmed as the result of a ubiquitous bacterial infection,etiological studies as well as immunation strategies have been pursued in order to deal with this infectious disease.Now,substantial evidence has been provided that dental caries is caused mainly by mutans streptococci(MS),especially S.mutans,which has a close association with the onset and progress of this disease.The related virulence factors of S.mutans have been extensively studied and employed as the potential candidate antigens for anti-caries vaccines and immunization.To lead to dental caries,MS must first colonize the tooth surface,which relies on several cell-surface proteins of S.mutans,such as the adhesin antigen ?/?(also designated as PAc or P1)and glucosyltransferases(GTFs).For initial colonization,MS need to adhere to saliva-coated tooth surface,which is mediated by PAc via the saliva-binding region(SBR).This process is followed by the accumulation of MS on tooth surface,which depends on extracellular water-insoluble glucans synthesized by GTFs.GTFs have two functional domains:an N-terminal catalytic sucrose-binding domain(CAT),and a C-terminal glucan-binding region(GBR).Experiments have showed that antibodies targeting the functional regions of these virulence factors can significantly inhibit the colonization of MS on tooth surface and reduce the lesion of dental caries.In previous studies,we have cloned gene segments encoding SBR and GBR(sbr and gbr for short),and constructed several plasmids harboring sbr and/or gbr segment.In order to explore for efficient ways of improving the effect of immunization,we designed the experiment.In this study,we employed expression vector pCMVnir,which harbors dual promoter CMV-nirB,to express the cloned antigens.Delivered by attenuated Salmonella Typhimurium strain SL3261,DNA vaccine pCN-SS/SG was expected to enhance the expression level of the cloned antigens compared with nirB used alone,and acquire a satisfied mucosal immune response.First,we expressed the cloned antigens in both SL3261 vector and CHO cells under the control of CMV-nirB promoters to test the function of the dual promoter system.Then,we explored the immunogenicity of anti-caries vaccine pCN-SS/SG by gastrointestinally administering the transformed Salmonella strain SL3261/pCN-SS/SG to BALB/c mice.Finally,we evaluated the effectiveness of this immune regime by comparing the immune responses and S.mutans reduction between the experimental groups.Methods:Part ?.The immune responses induced by Salmonella-based mucosal vaccine pCN-SS/SG in BALB/c mice1.Recombinant SBR(rSBR)and GBR(rGBR)were expressed in the JM109(DE3)cells using plasmids pTriEx-4-SBR and pTriEx-4-GBR respectively,and purified with HisTrapTM kit(Amersham).Then,six-week-old BALB/c mice were immunized subcutaneously with SBR or GBR to prepare the mouse anti-SBR or mouse anti-GBR serums.2.Plasmids pCN-SS/SG and pNir-SS/SG were used to transform S.typhimurium strain SL3261 and CHO cells,respectively.Antigen expression was detected after the transformants were anaerobically cultured overnight,or transfected CHO cells were incubated in DMEM medium for 48 h.3.Plasmid persistence in SL3261 during the bacterial growth in vitro was determined by culturing the transformed bacteria for approximately 100 generations.(5 days)without antibiotic selection.To determine the plasmid stability in vivo,mice were sacrificed at different time points post-immunization,and spleens,livers,and,intestinal tracts were collected and homogenized in PBS,respectively.The stability of the plasmid was evaluated by the ratio of the number of cells containing the plasmid to the total number of cells.4.BALB/c mice were orally immunized with Salmonella clones containing pCN-SS/SG or pNir-SS/SG plasmid on day 0 and at week 16.The levels of specific IgG,IgG2a,IgGl and IgA antibodies to SBR and GBR in serum and saliva samples were determined by standard ELISA at different time points post-immunization.Th1/Th2 cytokine secretion by splenocytes from immunized mice was also detected.Part ?.Inhibition of S.mutans colonization in BALB/c mice1.S.Typhimurium SL3261 was transformed with pCN-SS/SG,pNir-SS/SG and pCMVnir,respectively.Groups of BALB/c mice were orally immunized with one of the SL3261 strains containing pCN-SS/SG,pNir-SS/SG or pCMVnir.Among all the immunized mice,the pNir-SS/SG-immunized group was used as positive control and the pCMVnir-immunized group was used as negative control.2.Two weeks after the booster immunization,groups of mice were orally challenged with S.mutans strain UA159.The oral swabs were taken from individual mouse to check the bacteria before and after the inoculation.3.S.mutans infection levels were assessed by swabbing mice at weekly intervals for 8 weeks,beginning 1 week after the last infection.Results:1.The cloned antigens can be stably and separately expressed under the control of CMV-nirB promoter both in vector SL3261 and eukaryotic cells.With signal peptide,the expressed antigens can be excreted extracellularly and remain soluble.2.Plasmid persistence showed that pCN-SS/SG could stably persist during the growth of S.Typhimurium SL3261/pCN-SS/SG in vitro,and this in vitro stability could translate into the in vivo one,for S.Typhimurium SL3261/pCN-SS/SG could grow and persist in host reticuloendothelial system for up to 4 weeks.3.Antigen-specific serum IgG and salivary IgA antibody responses were induced in mice after immunization with SL3261/pCN-SS/SG.These responses were further enhanced after the booster immunization and remained high until the end of the experiment.Also,These antibody responses were significantly higher(P<0.01)in pCN-SS/SG-immunized mice than in pNir-SS/SG-immunized group.4.IgG subclass analysis revealed that antigen-specific serum IgG2a and IgGl responses were induced in mice by SL3261/pCN-SS/SG.Meanwhile,cytokine assay also showed a significant increase in both Thl(IFN-y)and Th2 cytokines(IL-4 and IL-10)in SL3261/pCN-SS/SG-immunized mice upon stimulation with SBR and GBR,indicating a Thl/Th2-mixed type of immune response to the cloned antigens.5.Following challenge,mice receiving Salmonella clones containing pCN-SS/SG or pNir-SS/SG plasmid showed a significant(P<0.01)reduction in S.mutans colonization compared to the sham-immunized and unimmunized controls.In addition,mice immunized with SL3261/pCN-SS/SG had significantly lower(P<0.05)levels of S.mutans than those receiving SL3261/pNir-SS/SG at weeks 3-8 after challenge,exhibiting a over 99%reduction in S.mutans colonization compared to the unimmunized control.Conclusion:1.DNA vaccine pCN-SS/SG could properly express target genes in both Salmonella vector SL3261 and eukaryotic cells under the control of CMV-nirB promoter.2.Delivered by attenuated Salmonella,mucosal-based vaccine(pCN-SS/SG)with the CMV-nirB dual promoter is more effective in inhibiting S.mutans colonization than nirB promoter used alone.In summary,the dual-promoter formula in the Salmonella-based DNA vaccine pCN-SS/SG was successful in inducing a mucosal immune response against S.mutans.Compared with nirB promoter used alone,the CMV-nirB promoter system is more efficient in inducing salivary IgA and consequently more effective in inhibiting S.mutans colonization.The dual promoter system(CMV-nirB)as a novel strategy showed a promising prospect for taking advantage of mucosal immunization against dental caries.