Regulation On The Immune Response Induced By Anti-Caries Vaccine At Salivary Glands

Dental caries is still a common disease which affected human health. It was showed that the incidence of caries in China is higher than50%. It is agreed that promotion of specific antibody levels against cariogenic bacteria Streptococcus mutans (S. mutans) in saliva can effectively prevent dental caries. Studies on immunization against dental caries began in the seventies of the last century. So far, however, data have shown that specific antibody in saliva induced by vaccination against dental caries is not sufficient for effectively controlling the disease.Studies have shown that specific IgA antibody secreting cells (IgA+ASCs) generated through mucosally inoculating could migrate to lamina propria of respiratory tract, gastro-intestinal tract and exocrine glands such as salivary glands. It is clear that oral antigen is captured by antigen-presenting cells in intestinal tracts and then transported to the Peyer's patch (PP, which was defined as one of mucosal immune-inducing tissues or sites) and sensitizes related antigen-specific immune cells. Some sensitized B cells by class switch recombination (CSR) differentiate into antigen-specific IgA+ASCs and subsequently migrate to the mucosal immune effector sites (including salivary glands) from bloodstream. In the effector sites, they produce and secrete IgA playing a protective role.Chemotactic chemokine CCL28is crucial in guiding homing of IgA+ASCs in blood circulation selectively to mucosal immune effector sites. CCL28produced by salivary gland epithelial cells can be secreted into the salivary gland acinar lumina as well as transported to the free surface of vein endothelial cells through presently unknown mechanism where it interacts with chemokine receptor CCR10on IgA+ASCs and then initiates the cells homing to the salivary glands. Moreover, histidine-rich CCL28carboxy-terminal exerts the strong ability of killing G-/G+bacteria and some fungi through changing the permeability of the cell wall envelope. Therefore, it is clear that CCL28plays an important role in the oral defense.S. mutans is associated with the initiation and progression of dental caries formation. The functional domain of AgⅠ/Ⅱ of the microbe responsible for initial adherence is localized on the N-terminal one-third of the molecule, which contains an alanine-rich repeat region and is termed saliva-binding region (SBR). Studies have suggested its importance in the initial adherence of S. mutans to saliva-coated tooth surfaces and subsequent development of dental caries. Because its importance in the pathogenesis of the caries, SBR have been became a significant candidate for anti-caries vaccine.In this study, we have constructed a eukaryotic expression plasmid harboring the gene encoding CCL28and investigated its immune enhancing effects through retrograde perfusion of the recombinant plasmid to the rat salivary glands. At the same time, we immunized the animals with our previously constructed dual-promoter expression plasmid pCN-SSIE which acts as the DNA vaccine. And then, we detected the immune effects induced by the combined applications of intragastric administration of the plasmid pCN-SSIE and retrograde perfusion of the plasmid encoding CCL28to the rat salivary glands. We explore the way to increase the levels of both IgA and CCL28in saliva in order to provide new ideas to enhance the immune responses of anti-caries vaccines.Material and Method:Part I:Human CCL28gene fragment obtained by PCR amplification was inserted into the eukaryotic expression vector pCMV-3Tag-8. The recombinant plasmid pCMV-3Tag-8-CCL28was testeded by restriction enzyme digestion and gene sequencing. The successfully constructed plasmid pCMV-3Tag-8-CCL28was transfected into293cells and then the expression of CCL28protein in the supernatant after transfection was detected by western blot. Streptococcus mutans UA159(S. mutans UA159) was cultured at37℃in the anaerobic condition. Microbial cells in logarithmic growth phase were resuspended at1×106cells/ml in fresh BHI medium or293conditions and the activity of S. mutans UA159was observed under the fluorescence microscopy. The zinc/pDNA solution was prepared, which contained175μg/ml pCMV-3Tag-8-CCL28and3.6mM Zn2+in sterile water. Rat parotid glands and saliva were extracted at0,2,7and14days after retrograde perfusion zinc/pDNA solution to parotid ducts. After immunization, the levels of CCL28and SIgA in parotid glands were detected by RT-PCR and immunofluorescence; the expressions of CCL28and SIgA in saliva were tested by enzyme-linked immunosorbent assay (ELISA). Bacterial suspensions of S. mutans UA159were prepared and then were inoculated into the sterile male rats. Using zinc chloride solution, pCMV-3Tag-8and zinc/pCMV-3Tag-8-CCL28respectively immunized the gnotobiotic rats by retrograde perfusion to the parotid glands. One week later, the dental plaque was collected and investigated Streptococcus mutans by real-time quantitative PCR.Part Ⅱ:The plasmid pCN-SSIE was purified and transformed into attenuated salmonella by CaCl2method. Rats were immunized by the attenuated salmonella transferred with pCN-SSIE at a concentration of bacteria to1010/ml. After3weeks, the zinc/pCMV-3Tag-8-CCL28solutions were administered into parotid glands through retrograde perfusion. The levels of CCL28in parotid glands and the expressions of CCL28and SIgA in saliva were detected. The levels of CCR10in small intestinal mucosa and salivary glands were detected.Results:Part Ⅰ:We have successfully constructed eukaryotic expression plasmid pCMV-3Tag-8-CCL28containing CCL28gene. Results of experiments in vitro showed that this plasmid secreted CCL28protein in eukaryotic cells and elicited bactericidal activity. The administration of plasmid through retrograde perfusion to the parotid gland could effectively increase the expressions of CCL28in the parotid gland and in saliva, and enhanced the levels of SIgA in saliva; the amount of S. mutans in gnotobiotic rats dramatically decreased. Part II:After1week, the expressions of CCL28in the parotid gland and in saliva and SIgA in saliva were significantly increased via the strategy of combined immunization; the expressions of CCR10in in small intestinal mucosa and salivary glands increased.Conclusions:1. The construction of the eukaryotic expression plasmid harboring the gene encoding CCL28was successful. Experiments in vitro and in vivo confirmed that CCL28protein possessed strong antibacterial and bactericidal functions. Salivary duct retrograde perfusion with the plasmid constructed in the present study made the transfection of the salivary gland with the gene encoding CCL28a success.2. Rats were treated by retrograde perfusion of plasmid pCMV-3Tag-8-CCL28in the parotid ducts and immunized throuth oral delivery of attenuated Salmonella transformed with pCN-SSIE. The levels of both CCL28in the parotid glands and specific anti-SBR SIgA in saliva were significantly increased. The expressions of CCR10in salivary glands were also enhanced. These results showed that much more IgA+ASCs migrated to the salivary glands and secreted SIgA. This strategy has a synergistic effect on inducing mucosal immune responses against cario-pathagens.