| Aims:The strategy to construct anti-caries DNA vaccine with targeting to cytotoxic T lymphocyte antigen 4 (CTLA-4) which binds to B7 molecule expressed on the surfaces of antigen-presenting cells(APC), is proved to be an effective way to enhancement of immune response and protective effect compared with untargeted DNA vaccine. But the mechanism of CTLA-4 fusion anticaries DNA vaccine to increase the antigen immune reponse and protection for caries challenge is still unclear.Methods:We constructed targeted anti-caries DNA vaccine encoding Streptococcus mutans antigens fused to murine CTLA4,untargeted anticaries DNA vaccine encoding Streptococcus mutans antigens fused to human CD5. Mice were given targeted, untargeted anti-caries DNA vaccine and empty plasmids pCI. Then, immune response, the numbers of AFC in CLNs and lymphcytes proliferation were determined. DCs in immune effector tissues were tested by FACS.The gene expression changes of DCs in different groups were tested by gene array. The candidate genes which were selected form gene array were confirmed by Real-time PCR. Results1. Ag-specific antibody responsesThe salivary anti-PAc and anti-GLU IgA antibody level of Group pmGJA-P were both significantly higher than that of Group pCDA-P at day 14 and 28.The anti-PAc and anti-GLU IgG levels in serum samples of mice vaccinated with pmGJA-P showed significantly higher than those of mice vaccinated with pCDA-P at day 14 and 28.Mice given pmGJA-P displayed increasing numbers of PAc and GLU specific IgA AFCs in CLNs. Elevated numbers of anti-PAc and anti-GLU IgG AFCs were detected in spleen of the group immunized with pmGJA-P compared with group with pCDA-P. Mice immunized with pCI as a control group did not exhibit any anti-Ag AFCs.2. Lymphocytes proliferative and cytokine responsesBoth splenic and CLNs lymphocytes from mice treated with pmGJA-P and pCDA-P showed significantly difference proliferative responses than pCI group. There was no significantly difference between Group pmGJA-P and pCDA-P.Mice immunized pmGJA-P and pCDA-P had higher numbers of PAc and GLU specific IFN-y and IL-4 producing cells in their CLNS and spleens as compared with control groups. The numbers of PAc and GLU specific IFN-y producing cells of CLNs and spleens from mice immunised pmGJA-P were no significantly different in comparison to mice given pCDA-P. Spleens and CLNs from pmGJA-P immunised mice contained elevated numbers of Ag specific IL-4 producing cells had significantly greater numbers of cytokine-secreting than mice given pCDA-P.CLNs or spleen from the mice given pmGJA-P exhibited significantly higher levels of IL-4 and IL-5 mRNA expression when compared with mice given pCDA-P. PAc or GLU stimulated lymphocytes from the spleen and CLNs of mice given pmGJA-P or pCDA-P contained significantly increased levels of IFN-y and IL-4 mRNA when compared with mice immunized nasally with pCI. In addition, IFN-γmRNA level was no significant different observed in mice given pmGJA-P and pCDA-P.3. DCs subtype in mucosal tissues test by FACSOur results observed major increases in numbers of CD11C+DCs in CLNs and spleen of mice given pmGJA-P when compared with mice given pCDA-P or pCI. Further, higher levels of MHC II, CD80, and CD86 were expressed by CD11C+DCs from mice given pmGJA-P compared with mice given pCDA-P or pCI.There was no significant difference in numbers of CD11c+DCs and costimulator molecule expression on CD11c+DCs between mice immunized pCDA-P and pCI. 4. Gene expression differenceWe defined a selective group of 8 genes that were significantly different expression for at least in one group mice given pmGJA-P or pCDA-P compared with mice given empty plasmids. In selected 8 genes, 7 genes were conformed by Real-time PCR.These genes were involved in DCs mature, Ag uptake, presentation, survival.ConclusionThe machenism of targeted CTLA-4 fusion DNA to enhance immunity may relate to increasing capture of antigen to stimulate DCs mature,migaration,antigen processing,and survival.
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