The Study On Recombinant Interleukin 5 As Adjuvant Of Zona Pellucida DNA Contraceptive Vaccine In Mucosal Immunization

Interleukin-5(IL-5) is a Th2 type cytokine that functions as a homodimer and is secreted mainly by T-lymphocytes and to a lesser extent by mast cells. IL-5 has been shown to be critically involved in the differentiation of B-cells into antibody secreting plasma cells, induce antigen-specific IgA antibody responses, and to promote eosinophilia.To investigate the feasibility of enhancing mucosal immune response and improving antifertility effect using IL-5 as adjuvant in zona pellucida DNA contraceptive vaccine, the mouse interleukin-5 eukaryotic expression vector was constructed as adjuvant of contraceptive DNA vaccines. The cDNA corresponding to mIL-5, excluding 44~445 base sequences, was cloned in mammalian expression vector pVAX1, and the recombinant plasmid was named as pVAX1-mIL-5. Then in vitro transient expression was established by in vitro transfection of HeLa mammalian cells with pVAX1-mIL-5 plasmid DNA by lipofection. 30h after transfection, the expression of mIL-5 was confirmed by indirect immunofluorescence(IIF). Furthermore the vaccine pVAX1-pZP3α and recombinant pVAX1-mIL-5 were encapsulated with chitosan C390 to make nanoparticles. The determination of encapsulated efficiency of chitosan/pVAX1-pZP3α and chitosan/ pVAX1-mIL-5 nanoparticles shows that more than 95% DNA are encapsulated with chitosan. In the immunization study, the mice were divided into three groups, group A were immunized with pVAX1-pZP3α only, group B were immunized both with pVAX1-mIL-5 and pVAX1-pZP3α, group C were control mice. All mice were immunized three times (on week 0, 3, 6) by oral. Anti-ZP3 IgA antibody reseponses in serum and vaginal secretion were determined by ELISA. Results showed that Group B could be induced significantly a mucosal immune response higher than Group A, which characterized with IgAantibody reaction in the mice serum and vaginal secretions. The anti-ZP3 IgA antibody titer in the mice began to rise at week 2 and increased after boosting at week 4, and the highest titers were attained at week 10. The antifertility experiment showed that the antifertility rate is 50%, 100% and 0% in group A, B and C respectively. 13 weeks after first immunization, the mice were killed. The pathologial study of ovarian of immunized mice with infertility indicated that the ovarian structure is normal as the control mice, showing the normal follicles development with different stage follicles, normal corpus luteum. No lymphocyte filtration and other pathological changes were found during our experiments.In conclusion, interleukin-5 can enhance mucosal immune response and increase antifertility of zona pellucida DNA vaccine.