Research On Enhancement Of CpG ODN On The Efficacy Of Recombinant FMDV Vaccine

Foot-and-mouth disease (FMD) is a highly contagious disease caused by the infection of Foot-and-mouth disease virus (FMDV). VP1 epitope is the major immuno-dominant epitope on FMDV to elicit protective humoral immunity.Vaccination is the most effective way for the prevention of FMD. In the past, attenuated and inactivated viral vaccine was commonly used. However, with the development of molecular biology, more and more genetic engineered vaccines are being developed, such as DNA vaccine, subunit vaccine and live vector vaccine.Adjuvant can be used to improve immunogenecity of antigen and to modulate avidity and specificity of antibody. Now the commonly used vaccine adjuvant includes aluminum salt, oil adjuvant, liposome, ISCOMs and molecular adjuvant.In recent years among the molecular adjuvant, CpG ODN has been widely used and displayed potential immuno-stimulatory effect. Beside of its ability to induce nonspecific immune activation, CpG ODN can also promote antigen-specific immune responses. It can induce Th1-bias immune response even under the combination with Th2-bias adjuvant. Compared with other vaccine adjuvants, CpG ODN can assist antigen to induce more antibodies rapidly and to generate cytotoxic T lymphocyte. Furthermore antigen dose could be reduced after administering CpG ODN.In order to test whether CpG ODN can enhance the efficacy of recombinant VP1 protein, we immunized guinea pigs with recombinant VP1 protein vaccine with CpG ODN or Montanide ISA 206. This article was divided in to the following parts:In the first part, we applied 3H-TdR incorporation and cell count to detect the activity of CpG ODN designed in our lab on swine PBMC and splenocyte of guinea pig. At last, F1 was selected as the optimal CpG ODN because of the strongest activity in swine even the modest activity in guinea pigs.In the second part, we immunized guinea pigs with recombinant VP1 protein vaccine with CpG ODN or Montanide ISA 206 on day 0, 48 via i.m. and detected the antibody level of sera by ELISA. Guinea pigs in two groups were immunized with recombinant VP1 protein, either with or without F1. Guinea pigs in other two groups immunized with recombinant VP1 protein vaccine emulsified with Montanide ISA 206 in the presence or absence of F1.We used two antigens of swine type O inactivated FMDV or swine recombinant VP1 protein to coat and detected the antibody level of sera by ELISA. When two different antigens were used to coat, the antibody titer of mice immunized by recombinant VP1 protein emulsified with 206 oil raised obviously on day 14 after the first immunization, then reached to the highest level on day 28 and respectively decreased to the foot on day 35 and 42. The sera antibody titer of other groups increased unobviously after the first immunization. On day 7 after the second immunization, the sera antibody titer of all groups raised unobviously when coated with swine type O inactivated FMDV, but when coated with recombinant VP1 protein, the antibody induced by recombinant VP1 protein vaccine emulsified with Montanide ISA 206 in the presence or absence of CpG F1 raised remarkably, reach to 4500 and 2500 respectively.The above results indicated that CpG ODN and Montanide ISA 206 used together as vaccine adjuvant did not generate anticipated synergistic effect and CpG ODN decreased the effect of Montanide ISA 206. To exploit the interaction of the two adjuvants, we carry out further research in vitro.The splenocytes of guinea pigs were treated with CpG F1, Montanide ISA 206 or both, and the morphology of the cells was observed under microscope followed by counting the alive cells. The splenocytes stimulated by CpG F1 appeared integrated cell membrane, lucent cytoplasm and undilated. Debris appeared in the splenocytes stimulated by Montanide ISA 206. The splenocytes stimulated by Montanide ISA 206 in the presence of CpG F1 appeared to be dilated and emerged apoptosis. The numbers of alive cells treated with CpG F1, Montanide ISA 206 or both are about 4.2x105, 1.7x105 and 1.3x105 respectively. So the living state of the splenocytes treated with CpG F1 is better than that of the splenocytes treated with Montanide ISA 206 or both. The living state of the splenocytes stimulated by Montanide ISA 206 in the presence of CpG F1 was the worst. CpG F1 and Montanide ISA 206 applied together as vaccine adjuvant did not generate anticipated synergistic effect. This phenomenon might associate with the type of the CpG ODN, the way of the immunization, the dose of the CpG ODN and the aggravated cell injury on the efficacy of the both adjuvants. So the further study would be considered the above factors.We concluded that the efficacy of swine recombinant VP1 protein vaccine can be enhanced by applying Montanide ISA 206 alone. CpG F1 and Montanide ISA 206 used together as vaccine adjuvant did not generate anticipated synergistic effect and CpG F1 decreased the effect of Montanide ISA 206. The experiment in vitro also indicated that the splenocytes were damaged under the effect of CpG F1 and Montanide ISA 206.