Enhanced Immune Efficacy And Mechanism Of Targeted Anti-caries DNA Vaccines

Streptococcus mutans (S. mutans) has been strongly implicated as a causative organism of dental caries.Glucosyltransferases (GTFs) and A cell-surface protein (PAc) are two important virulence factors of S. mutans. They may mediate sucrose-independent or sucrose-dependent attachment of Streptococcus mutans to tooth surfaces, respectively. Thus, inhibiting both virulence factors is predicted to provide better protection against caries than inhibiting a single factor.Much attention has been paid to DNA vaccines since the report of Wolff. Compared with traditional vaccines, DNA vaccines have obvious advantages, such as a) stronger antigenicity, with the capacity to induce both cellular and humoral immune responses; b) long-term immune responses; and c) facilities for design, transport and storage. We had constructed an anti-caries DNA vaccine, pCIA-P, which encoded two highly conservative regions of PAc. Specific saliva anti-PAc SIgA could be induced in rats immunized with pCIA-P. In order to develop a highly efficient vaccine against caries, we had also constructed a fusion DNA vaccine, pGLUA-P, by cloning the GLU region of GTF into a DNA vaccine, pCIA-P.Usually, DNA vaccines display poor immunogenicity in large animals including human, which keep back the practical application of DNA vaccine. In 1998, Boyle first reported a targeted DNA vaccine encoding CTLA4 fused gene with human Ig and then immunued mice. CTLA4 expressed on the surface of activated T cell binds to CD80 and CD86 expressed by DC. As a result, specific antibody levels in BALB/c mice immunized with CTLA4-Ig were 10000-fold higher than those in mice immunized with non-targeted DNA vaccine at 2 weeks. Following DNA administration, DC can capture antigen and then undergo maturation. During maturation, DC can upregulate the expression of CD80 and CD86, which can bind to CTLA4, thus, it may enhance the capture of the targeted antigen, which facilitate the contact between the antigen and DC, and then matured DCs migrate to T cell areas of draining lymph nodes where they present antigen to CD4+ and CD8+ cells to initiate the specific immune response. CTLA4 targeted DNA vaccine possessed the strong ability to enhance the immune responses,which was related to the sizes of encoded antigen.The purposes of this report are to provide evidence that fewer caries lesions may be observed in rats following immuniztion with pGLUA-P compared with pCIA-P; to construct a targeted anti-caries DNA vaccine encoding CTLA4-Ig-GLU-A-P and check its immunogenicity and efficacy in monkeys or in rabbits compared with pGLUA-P immunized via systemic or mucosal route; to study the effects of the sizes of encoded antigen on the immunogenicity of targeted DNA vaccines; to study the mechanisms of targeted anti-caries DNA vaccine enhacing immune responses by constructing a stable cells expressing human B7-2 molecule and a targeted eukaryotic expressing plasmid labeled by green fluorescent protein (GFP).Part oneMethods (1) CHO (Chinese Hamster Ovary cell) cells were transfected with pGLUA-P with the use of Dosper (1,3-di-oleoyloxy-2-(6-carboxy-spermyl)-propyl-amid) and the expression of recombinant protein in cultured CHO cells was detected by specific anti-PAc or anti-GTF antibody with the use of irnmunofluorescence. (2) In order to check its anti-caries efficacy, gnotobiotic rats were immunized with pGLUA-P or pCIA-P via subcutaneous injection (s.v.) or intramuscular injection (i.m.). The specific antibodies in sera or saliva were detected by ELISA. The caries levels were scored using the Keyes method. (3) To extract and purify GTF-I in Escherichia coli, gtfB gene were amplified from pYNB13 and cloned into prokaryotic expression vector pQE30. The expression of recombinant glucosyltransferase-I was induced by IPTG in E. Coli JM109. The recombinant GTF-I were extracted and purified by Immobile Metal Ion Affinity Chromatography. Results (1) Many positive-staining cells with a red fluorescent color were found in the pGLUA-P-transfected CHO cells incubated with both anti-PAc IgG...