Study Of Enhanced Immune Response Of Anti-caries DNA Vaccine Via Co-delivery Of Plasmid DNA Expressing CCL19and CCL17

Dental caries remains one of the most common infectious diseases affecting humans in the world. According to dozens of experimental data, our group has proven that anti-caries DNA vaccine is a promising strategy for inducing potent immune responses against dental caries. Although DNA vaccination often works well in small animal models, it displays poor efficiency in larger animals and human. Thus, it is a critical and urgent task to find ways to improve the immunogenicity of DNA vaccines. We have shown that DCs targeted anti-caries DNA vaccines induced accelerated and increased antibody responses compared to nontargeted vaccines. The expression profiles of immunologically relevant genes of these two DNA vaccines transfected DCs were investigated by gene array analysis. Overall, the expression levels of28genes were up-regulated in targeted anti-caries DNA vaccines transfected DCs compared to nontargeted vaccines transfected DCs. Notably, we identified the specific up-regulation of mRNA expression level of chemokine CCL19and CCL17. which may play an important role in the development of a more effective dental caries vaccine.The role of chemokines played in immune system is often through the chemokine receptors expressed on targeted cells. CCR7. the receptor of CCL19. is mainly expressed on mature DCs and distinct T and B cell subpopulations. CCR.7and its ligand CCL19are recognized as essential molecules for the directional migration of mature DCs and the establishment of a functional microenvironment to prime naive T cells. CCL17can attract CCR4positive cells, which are mainly T helper(Th)2cells, and therefore appears to be important in the development of an acquired Th2response. In addition, it is reported that CCL17is required in CCL19-CCR7dependent migration of dendritic cells.This study is based on our previous result of gene array analysis. We assessed whether co-delivery CCL19. CCL17along with anti-caries DNA vaccine could elevate the efficiency of immune responses, and explained the underlying mechanism involved in the enhanced immune response.Three parts are included in the study:Part oneThe plasmid encoding CCL19-GFP fusion protein, designated as pCCL19/GFP. was constructed by inserting the murine cel19gene into the GFP-expressing vector pAcGFP1-N1. The chemotactic effect of the CCL19-GFP fusion protein expressed by pCCL19/GFP on DCs was assessed in vitro and in vivo by Transwell and flow cytometric analysis, respectively. Mice were intramuscularly immunized with anti-caries DNA vaccine pCIA-P or with pCIA-P plus pCCL19/GFP. Serum anti-PAc IgG levels were assessed by an enzyme-linked immunosorbent assay (ELISA). Splenocytes derived from immunized mice were restimulated with rPAc protein for48h. and IFN-y and IL-4production was determined by ELISA. The presence of pCCL19/GFP in the spleen and draining lymph nodes after intramuscular immunization was assessed by PCR. pCCL19/GFP protein expression in these tissues was analyzed under the microscope and by flow cytometry. Results:CCL19-GFP fusion protein demonstrated strong chemotactic activity on DCs in vitro. Genetic co-transfer of CCL19increased the levels of serum PAc-specific IgG. Splenocytes from pCIA-P plus pCCL19/GFP vaccinated mice produced significantly higher IFN-y levels compared to pCIA-P-vaccinated mice. Following muscular immunization, pCCL19/GFP was shown to exist and express CCL19-GFP fusion protein in the spleen and draining lymph nodes. Administration of CCL19DNA increased the number of mature DCs in secondary lymphoid tissues.Part twoThe plasmid encoding CCL17-GFP fusion protein, designated as pCCL17/GFP, was constructed by inserting the murine cel17gene into the GFP-expressing vector pAcGFP1-N1. Mice were intramuscularly immunized with anti-caries DNA vaccine pCIA-P or with pCIA-P plus pCCL17/GFP. Serum anti-PAc IgG levels were assessed by ELISA. Splenocytes derived from immunized mice were restimulated with rPAc protein for48h. and IFN-y and IL-4production was determined by ELISA. The number of DCs in the spleen and draining lymph nodes after intramuscular immunization with pCCL17/GFP was assessed by flow cytometric analysis. Results: Genetic co-transfer of CCL17accelerated and increased antibody responses. However, splenocytes from pCIA-P plus pCCL17/GFP vaccinated mice did not significantly change IFN-γ and IL-4levels compared to pCIA-P-vaccinated mice. Administration of CCL17DNA increased the number of mature DCs in secondary lymphoid tissues. Part threeThe plasmid pCCL19/VAX and pCCL17/VAX were constructed by inserting the murine cel19and cel17gene into the vector pVAX1. BALB/c mice were intranasally immunized with anti-caries DNA vaccine pCIA-P plus pCCL19/VAX, pCCL17/VAX or both. Serum and saliva anti-PAc antibody levels were determined by using ELISA. Cariogenic diet and S. mutans were used to set up the experimental caries models. We analyzed the effects of pCCL19/VAX, pCCL17/VAX or both on the serum PAc-specific IgG and saliva PAc-specific IgA antibody responses, the colonization of S. mutans on rat teeth, and the caries levels. Results:pCCL19/VAX, pCCL17/VAX promoted the production of PAc-specific IgG in serum and SIgA in saliva of mice by intranasal immunization. The combination of pCCL19/VAX and pCCL17/VAX lead higher antibody levels than pCCL19/VAX or pCCL17/VAX alone. Co-delivery of pCCL19/VAX, pCCL17/VAX in experimental caries models displayed significantly fewer S. mutans colonization and caries score, meanwhile enhanced saliva PAc-specific IgA and serum PAc-specific IgG antibody responses. In addition, the combination of pCCL19/VAX and pCCL17/VAX was more effective than pCCL19/VAX or pCCL17/VAX alone. Furthermore, we found that enhanced PAc-specific IgA responses in saliva were associated with the inhibition of S. mutans colonization of tooth surfaces and endowed better protection w ith significant fewer caries lesions.