Abstract: Objective: To compare the immune effects of poly (lactic/glycolic acid)-polyethylene glycol-poly (lactic/glycolic acid) hydrogel sustained-release anti-caries gene vaccine pVAX1-GbpA/GBD through different immunization routes. Methods: The temperature-sensitive biodegradable hydrogel PLGA-PEG-PLGA was mixed with recombinant plasmid pVAX1-GbpA/GBD and blank control. Twenty-eight New Zealand rabbits were randomly divided into seven groups (4 in each group). Plasmid pVAX1-GbpA/GBD was applied through intramuscular injection, oral administration, subcutaneous injection, or intranasal drip to rabbits. The control groups were intramuscular injection of pVAX1, oral administration of pVAX1 and hydrogel, and intramuscular injection of saline. All rabbits were immunized three times. After the first-time immunity, these rabbits need interval one week to strengthen the immunity by the same dosage, and then interval two weeks to strengthen the immunity 2nd time. The antibody responses induced by the vaccines were detected by indirect ELISA method. And the expression of recombinant protein was detected by immunohistochemistry technique. Results: (1) After 2 weeks of immunization, the levels of specific IgA antibody and IgG antibody in the saliva of 4 groups with anti caries gene vaccine increased significantly (P<0.05). The IgA anti GTF antibody level reached the highest in the 6th week, and the IgA anti GTF antibody level in the oral administration group was higher than those in the other 6 groups in the 4th week. The level of IgA anti GTF antibody in the submandibular gland injection group was higher than those in the other 6 groups at 6 and 10 weeks. The levels of IgA anti GTF antibody in the oral administration group and submandibular gland injection group were higher than those in the other groups at 8 and 12 weeks (P<0.05), however, no significant difference between them (P>0.05). (2) The levels of specific IgA antibody and IgG antibody in the serum of caries prevention gene vaccine group increased significantly 2 weeks after immunization (P<0.05). The level of IgG anti GTF antibody in the oral administration group was higher than those in the other 6 groups at 3, 6, and 10 weeks (P<0.05). The level of IgG anti GTF antibody in the submandibular gland injection group was higher than those in the other 6 groups at 4 and 8 weeks (P<0.05). (3) Immunohistochemical staining showed that the positive expression of pvax1 gbpA/GBD target protein was observed in the immune sites of four groups of anti caries gene vaccine group. The staining of pvax1 gbpA/GBD protein was obvious and strongly expressed in the cytoplasm of epithelial cells in the small intestinal mucosa of oral vaccine group and in the cytoplasm of ductal region of submandibular gland injection group. Conclusion: Through 4 immunization ways, the hydrogel sustained-release pVAX1-GbpA/GBD vaccine can induce effective systemic and mucosal immune response and maintain long time. The antibody levels induced by oral administration and submandibular gland injection are better.

Key words: glucan bind protein, gene vaccine, hydrogel, caries, immune