Abstract: BACKGROUND: Due to low price, safety and effectiveness, anti caries DNA vaccine is expected to become an important means of human anti caries. Scholars are looking for an effective immune pathway to induce antibody production or a sustained-release system and adjuvant to protect vaccine titer, which has become a research focus in recent years. 
OBJECTIVE: To observe the immune effect of the sustained release hydrogel poly(lactic/glycolic acid)-polyethylene glycol-poly(lactic/glycolic acid) loaded with anti-caries gene vaccine pVAX1-SpaP/P. 
METHODS: The recombinant plasmid pVAX1-SpaP/P and the empty plasmid pVAX1 were loaded with poly(lactic/glycolic acid)-polyethylene glycol-poly(lactic/glycolic acid) hydrogel, respectively. Twenty-eight New Zealand white rabbits were randomly divided into seven groups. Quadriceps femoris injection, submandibular gland subcutaneous injection, oral administration, and nasal drip injection of hydrogel loaded with recombinant plasmid pVAX1-SpaP/P were performed in four groups. The remaining groups were the control groups. In the negative control group 1, quadriceps muscle was injected with hydrogel loaded with empty plasmid pVAX1. In the negative control group 2, hydrogel loaded with empty plasmid pVAX1 was orally taken. The quadriceps femoris of the blank control group was injected with saline. The immunization was enhanced once a week after the initial immunization. The second immunization was conducted one week apart, and immunized for the third time after an interval of two weeks. ELISA was used to detect anti-SpaP IgA antibody in saliva and anti-SpaP IgG antibody in serum before and after immunization. At 3 days after the end of the three immunizations, the expression of pVAX1-SpaP/P protein was observed by immunohistochemistry technique. 
RESULTS AND CONCLUSION: (1) Two weeks after immunization with hydrogels loaded with recombinant plasmid pVAX1-SpaP/P via different pathways, the levels of anti-SpaP IgA antibody in saliva and anti-SpaP IgG antibody in serum were significantly increased in rabbits. Anti-SpaP IgG antibody level reached a peak at 10 weeks; anti-SpaP IgA antibody level reached a peak at 6 weeks. Anti-SpaP IgG antibody levels in the oral group were higher than those in the other six groups at 6-12 weeks (P < 0.05). Anti-SpaP IgA antibody levels were higher in the submandibular gland group than those in the other six groups at 4-8 weeks (P < 0.05). (2) One week after immunization with hydrogels loaded with recombinant plasmid pVAX1-SpaP/P via different pathways, anti-SpaP IgA antibody levels increased and peaked at 8 weeks. The antibody levels were higher in the submandibular gland group than those in the other six groups at 1-6 weeks (P < 0.05). The antibody levels were higher in the oral group than those in the other six groups at 8-12 weeks (P < 0.05). Anti-SpaP IgG antibody level in rabbit saliva began to increase at 2 weeks after immunization, and reached a peak at 10 weeks. The antibody levels of the oral group were higher than those in the other six groups at 2, 3, and 6 weeks (P < 0.05). The antibody levels of the quadriceps femoris group at 10 weeks were higher than those of the other six groups (P < 0.05). (3) After immunization with hydrogels loaded with recombinant plasmid pVAX1-SpaP/P via different pathways, immunohistochemical staining showed that positive pVAX1-SpaP/P protein expression in the injection site tissues. (4) The results showed that the poly(lactic/glycolic acid)-polyethylene glycol-poly(lactic/glycolic acid) hydrogel loaded pVAX1-SpaP/P anti caries vaccine could induce specific antibody production in the four ways to induce the immune effect and maintain long time.

Key words: caries, surface protein antigen SpaP, gene vaccine, hydrogel, vaccines, immune response, Streptococcus mutans, target protein