Enchanced Mucosal Immune Efficacy Of Targeted Anti-caries DNA Vaccine

Streptococcus mutans has been strongly implicated as a cariogenic organism, it has been reported that vaccine immunization was a promising method against dental caries. DNA vaccination has been developed recently and known for its ability to induce both humoral and cellular immune responses when delivered into animals. We have already constructed an anti-caries DNA vaccine, pCIA-P, which encoding two high-conservative regions of PAc and a fusion anti-caries DNA vaccine, fusing the A-P fragment of PAc and GLU region of GTF-I, could induce protective anti-caries immune responses. In order to induce high immune response, a targeted fusion anti-caries DNA vaccine carrying the signal peptide and extracellular of human CTLA-4 gene and human Fc and hinge regions of IgG1 was also constructed. Many studies had demonstrated that the three kinds of anti-caries DNA vaccine all could induce protective anti-caries immune responses. Among them, the targeted fusion anti-caries DNA vaccine was the most efficient approach against dental caries.Mucosal immunity is the first line of defense against pathogens to prevent infection. An important factor for protective mucosal immunity is the introduction of specific secretory IgA (S-IgA). A specific immune response against dental caries is also mainly provided by S-IgA antibodies, which are generated by the common mucosal immune system (CMIS). They act as the first line of defense against dental caries by blocking of adhesin-receptor interaction. The best and more effective way to induce the mucosal immunity was immunization via mucosal route. It has been shown nasal or oral immunization with DNA vaccine cannot elicite significant Ag-specific salivary IgA antibody responses. Therefore induction of anti-caries specific mucosal immunity appears to be an important strategy in our study.The efficacy of orally and nasally administrated 'naked' DNA vaccine is low due to the factors like the degradation of the DNA in thegastrointestinal (GI) tract and low uptake by the gut associated lymphoid tissue (GALT). Several studies have showed that by associating the vaccine with a number of microparticlate drug carrier systems, the uptake by M-cell is enhanced and the degradation of the vaccine in the GI tract is prevented. Chitosan, the deacetylated form of chitin, is a biodegradable polysaccharide from crustscean shells. Due to its good biocompatibility and toxicity profile, it has been widely used in pharmaceutical research and in industry as a carrier for drug delivery and as biomedical material for artificial skin and wound healing bandage applications. It is also able to open the tight junctions and allow paracellular transport across theepithelium. In our study, in order to effectively induce specific mucosal immunity, we' d like to utilize chitosan as a quickly acting absorption enhancing agent, to construct a chitosan-embedded gene vaccine, and the morphology of the complex was observed and its potential to induce specific immune responses by chitosan-DNA was carefully studied.Human β-defensin (HBD) are small cationic antimicrobial peptides produced by the epithelial cells, which directly kill a range of bacteria (both gram-positive and gram-negative species), fungi and some viruses. Besides antimicrobial activity, they may also play a role in links to the innate and adaptive immune responses. β-defensin appears to attract immature dendritic cells and CD4/CD45RO (memory) T cells and CD8 T cells via binding to the chemokine receptor, CCR6. So, here β-defensin was used as mucosal adjuvant to induce the mucosal immunity of our chitosan- pGJA-P vaccine. In order to induce β-defensin in the local area, several species of oral bacteria were examined for its ability to modulate hBD-2 and -3 gene expression in cultured normal human gingival epithelial cells (HGE). The bacteria inducing the most high-level β-defensin will provide antigen candidated to construct novel union DNA vaccine to peridontal disease and caries.There were three parts in this study:Part one Immunization against dental caries with Targeted fusion anticaries DNA vaccine in gnotobiotic hamsters from cariesMethods: Hamsters, infected with S. mutans at 20 days of age, were immunized with pGJA-P/CI, pGJA-P/VAX, pGLUA-P/CI by intramuscularinjection (i.m.) or intranasal (i.n.) under design, Control hamsters either immunized with plasmid pCI, pVAX or distilled water. All the rats were boosted 2 weeks later. ELISA determined the antibody responses induced by the vaccines. Keyes caries score was used to evaluate the anti-caries effectiveness of the vaccines at the terminal study. Result: As for the antibody reactions and caries scores, there were significantly (P<0.01) differences between hamsters immunized with DNA vaccine and non-immunized hamsters. Hamsters immunized with pGJA-P/CI or pGJA-P/VAX by nasal had the significantly (P<0. 01) highest level of specific salivary anti-PAc IgA antibodies and their Keyes scores were significantly (P<0.01) lower than others. There were no significant difference between the organ weight and organ index of hamsters immunized with plasmid and control. Conclusion: Intranasal routes displayed the higher mucosal antibody response and better efficacy against caries. Intranasal immunized with targeted fusion anticaries DNA vaccine appears to be a promising approach against dental caries. FDA approved the vector pVAX to use in clinic, so pGJA-P/VAX was used in the following study.Part two Enchanced Mucosal Immune Efficacy of Targeted Anti-caries DNA Vaccines with chitosan mucosal delivery systemI Synthesis and characterization of chitosan-DNA microparticles as mucosal delivery system:Methods: (1) Chitosan-DNA complexes were prepared by embedding plasmid DNA pGJA-P with chitosan. Three factors (including the concentration of DNA and sodium sulfate, the ratio of N/P) influencing preparation were optimized by orthogonal test. The morphology of the complex was observed by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). (2) Protection to plasmid DNA from DNAse I degradation provided by chitosan was examined. (3) In order to study the transfection effecacy, chitosan- pEGFP and chitosan-pGJA-P were dropped onto single layers of CHO or NIH3T3 cells repectively. Expression of recombinant protein in NIH3T3 cells transfected by CS-pGJA-P microparticles was detected by histrochemistry. (4) Release studies were done in similar gastro liquid (SGL) and similar intestinal liquid (SIL) at 37° C. Results: (1) Chitosan microparticles being prepared under optimal conditions of50 mM Na2SO4, 200H-g/ml DNA at an N/P ratio of 4, was spherical with uniform diameter of about 4 - 6 Mm. (2) When incubated with 28U/ml DNAse I at 37 °C, naked plasmid DNAshowed significant degradation in 15 min, whereas the plasmid DNA recovered from chitosan-DNA complex after the same treatment remained intact. (3) Many green fluorescent protein or positive brown colors were found in the Hela cells transfected with chitosan- pEGFP or chitosan-pGJA-P without the help of Lipofectamine-2000. (4) When incubated with SIL, after a release of about 10% in the first 60 min, no DNA was released during the following 180 min. Whereas incubated with SGL, it exhibited a small burst release about 11% in the first 60 minute, and then slow release at a constant but different rate. Conclusions: Chitosan was found to be able to protect DNA and enhance the correct expression of DNA in eukaryotic cell. Chitosan microparticles showed suitable characteristics in vitro for mucosal vaccination and were therefore a promising carrier system for DNA vaccine mucosal delivery.II Mucosal immunization with anti-caries DNA vaccine on Chitosan microparticles by oral and nasal route:Methods: CS-pGJA-P microparticles were prepared using a complex coacervation process as before. Forty-eight 4-week-old BALB/c mice were randomly divided into 8 groups (tablel). Immunizations were boosted 2 weeks later. Saliva and sera were collected in 0, 2, 4, 6, 8 and 10 weeks. Specific antibody responses were assayed by ELISA. 10 weeks later, the mice were killed and the splenocytes of mice were cultured respectively, the concentration of IFN-Y, IL-4 in the supernatant of splenocytes was assayed by quantitative "sandwich" ELISA. Result: (1) Both CS-pGJA-P and "naked" pGJA-P could induce GTF-specific antibody responses in serum and saliva, and the induced specific antibody titers were notably higher than those of control mices (P<0. 01). Local and systemic immune responses in mice immunized with CS-pGJA-P by two mucosal routes were significantly higher than those in mice treated with "naked" pGJA-P. The levels of serum anti-GTF IgG of group A, E (1:3162 by nasal, 1:1479 by oral), going up to peak at 6 weeks, were significantly higher than those of group B, F (1:63, 1:100 respectively) (both P<0.01); The level of salivary anti-GTF IgA of group A, E (1:202, 1:128), going up to peak at8 weeks, were significantly higher than those of group B, F (1: 6, 1:5 respectively) (both P<0. 01). (2) The detection results of IFN- Y and IL-4 in supernatants of splencytes of each group were compared. The concentrations of IFN- y in supernatant of splenocyte of mice treated with pGJA-P alone (group B, F) were higher than those of mice immunized with CS-pGJA-P(group A, E) and control group(P<0. 01). While the concentrations of IL-4 in the group A, E were higher than those in the group B. F (P<0. 01), Conclusions: Our findings suggested that Chitosan-pGJA-P may be more efficient than naked pGJA-P to induce specific immune responses against S. mutans by oral or nasal immunization. Furthermore, Chitosan-pGJA-P potentiated Th2-type immune response, which was efficient against dental caries. The anti-caries DNA vaccine chitosan mucosal delivery system could be a promising approach against dental caries.Part three Induction of human beta-defensin mRNAs by oral bacteria in primary gingival epithelial cellsMethods: Fusobacterium nuc lea turn (F.n) , Streptococcus gordonii (S. g) Streptococcus mutans (S.ni) , Streptococcus sorbinus iS. s) Porphyromonas gingival is CP.g) and Actinobacillus actinomycetemcomitans {A. a) were cultured and the cell wall of each bacterial were prepared. The primary gingival epithelial cells islating from the health were co-incubated with live bacteria at a bacteria/cell ratio of 100: 1, or treated with the cell wall at lOOng/ml. Total RNA extracted and gene expression of hBD-1, 2,3 was determined by RT-PCR, P-actin gene as an internal invariant control. Results: (1) HBD-1 mRNA was detected in unistimulated and stimulated cultures of HGE, and was not siginificantly modulated by any stimulats. (2) HBD-2 mRNA was up-regulated expression. It was induced by both viable commensal and pathogenic bacterial and cell wall of F.n , S. g, S. m andS. s. The molecular level of HBD-2 in cell induced by F. n, A. a, P. g, S. g, S. mandS. s was 0.66 + 0.03,0. 61+0. 06,0.85+0.07, 0. 55+0. 07,0. 44+0.12,0. 58+0. 04 respectively compared with the internal invariant control. The level of induction with lives P. gingivalisvas the highest than that of other live bacterial or cell wall (p<0.05, p<0.01). (3) The increased hBD-3 gene expression was dependent upon viable bacteria, and not their cell wall, thehighest level of hBD-3 induction was observed in the cell stimulated with the A. a, P. g and S. m. (0.96 + 0.16,1.02+0.11,0.54+0.23, respectively) Compared with other level, there was a siginificantly different (p<0. 05, p<0. 01) .Conclusions: P. gingivalis were more effective in up-regulating hBD-2, HBD-3 than other commensals and pathogens, which can afford antigen candidate for our further new DNA vaccine.