Vaccination of mice with recombinant nucleic acid vaccine encoding the integral membrane protein Sj23 and cytokine IL-12 elicits specific immune responses against schistosoma japonica

Abstract

To develop a Schistosoma japonica integral membrane protein Sm23 or Sj23 combined with murine IL-12 DNA-base vaccine against schistosomiasis. Plasmids pVIVO2-Sj23 and pVIVO2-IL12-Sj23, expressing the integral membrane protein Sj23 of Schistosoma japonica and/or murine IL-12 were constructed. The plasmid pVIVO2-IL12-Sj23 was transfected into the human embryonic kidney cells of the line 293. RT-PCR was used to detect the expression of Sj23 mRNA in the 293 cells. Indirect immunofluorescence test was used to detect the expression of Sj23 protein. Fifty BALB/c mice were randomly divided into 5 equal groups to be injected with pVIVO2-IL12-Sj23 plasmid DNA, pVIVO2-Sj23 plasmid DNA, pVIVO2-IL12 plasmid DNA, pVIVO2 blank vector, and normal saline respectively into the quadriceps muscle of thigh. Four weeks after each mouse were infested with 40 +/- 2 cercariae of Schistosoma japonica. Six weeks after the infestation the mice were killed to calculate the load of schistosoma and the amount of eggs per gram (EPG) of liver so as to calculate the worm reduction rate and egg reduction rate after the vaccination. Before immunization, 4 weeks after immunization, and 6 weeks after immunization blood samples were collected from the caudal veins of mice. With soluble egg antigen (SEA) and adult worm antigen (AWA) ELISA was used to detect the serum IgG level. Western blotting was used to detect the serum specific anti-Sj23 IgG level. Six weeks after the cercaria challenge single splenocyte suspension was prepared. Splenocytes were cultured with SEA, and concanavalin A (ConA). ELISA was used to detect the levels of IL-4 and IFN-gamma in the supernatant. Flow cytometry was used to analyze the subgroups of splenocyte. + Forty-eight hours after the transfection, RT-PCR and indirect immunofluorescence test showed expression of Sj23 mRNA and protein in the HEK-293 cells. The worm reduction rate was 45.53% and the egg reduction was 58.35% in the pVIVO2-IL12-Sj23 group, significantly higher than those in the monovalent vaccine pVIVO2-Sj23 group (27.23% and 33.93% respectively, both P <0.05). ELISA and Western blotting analysis showed that the level of IgG specific for Sj23 significantly increased 4 weeks after vaccination in the pVIVO2-IL12-Sj23 and pVIVO2-Sj23 groups without significant difference between these 2 groups (P > 0.05). After stimulation of ConA and SEA the level of Th1 type cell factor IFN-gamma was higher and the level of the Th2 type cellular factor IL-4 was low in the supernatant of suspension of splenocytes of the pVIVO2-IL12-Sj23 group. FCM showed the percentages of CD4+ and CD8+ subgroups of the murine splenocytes of all experimental groups were all significantly lower than those of the normal mice (all P < 0.001 approximately 0.02). However, there was no significant difference in the CD4+/CD8+ ratio among the experimental groups (all pVIVO2-IL12-Sj23 is sufficient to elicit significant levels of protective immunity against Schistosoma japonica challenge infection. IL-12, a cytokine and a gene adjuvant, is able to induce Th1 responses and hence the protective immunity.