The Study On The Enhanced Efficacy Of Contraceptive Mouse Zona Pellucida 3 Vaccine With Minimal Inflammatory Reactions To Ovary By Co-immunization Of DNA And Protein Technology

The mammalian zona pellucida (ZP) surrounding the growing oocytes and ovulated eggs is composed of glycoproteins, which mediate the initial spermatozoon–egg interaction of mammalian fertilization and is considered as a potential immunogen for the contraceptive vaccine. In mice, three sulfated glycoproteins (ZP1, ZP2, ZP3) are found within the ZP. Zona pellucida 3 (ZP3) acts as the primary sperm receptor, induces autoantibody that can prevent oocyte from fertilization and has been proposed as a vaccine candidate for the contraception in human. Due to the elicited autoreactive T cell inflammation that causes ovarian destruction, ZP3 based vaccine with removed T epitopes from the ZP3 is considered as a preferred approach. However, peptides with B epitopes as compared to proteins are less immunogenic and often lead to induce short term and reduced humoral responses. This study presents a novel strategy to eliminate the T cell inflammation while remains a high level of antibody by co-immunization of mZP3 DNA and protein vaccines, which resulted in a higher reduction rate of fertility in this group. The fertile rate fell from 100% in na?ve mice and 90% in pcDNA3 vector control to 30% in the co-immunized mice compared with 40%-60% in other immunized groups. ELISA analysis showed that mice in the co-immunized group had the highest OD values. There was a direct relationship between the levels of total IgG antibody and the rate of infertility in the co-immunized group because the mean level of IgG antibody in the infertile mice was significantly higher than in the fertile mice in this group. However, there was no correlation between the levels of total IgG antibody and the rate of infertility in other immunized group.Histological analysis showed that there were normal follicular developments of infertile mice in the co-immunized group; while other immunized groups of most infertile mice lacked mature follicles. There was a significant correlation between normal follicular development and the inhibition of T cell response in co-immunized mice. Meanwhile, co-immunization reduced the production of inflammatory cytokine, IFN-γ? and increased the productions of IL-10 and FoxP3 in CD4+ T cells, suggesting the anti-inflammation action may be via a T regulatory (TR) function.Since TR cells potently suppress immune responses and maintain the immunological self-tolerance, and the induced TR cells by the co-immunization protocol regulated the pathogenic T cell responses, which lead to avoid aberrant follicular development, we characterized and examined mechanism of TR cells by the use of autoimmune ovarian disease (AOD) as model. Autoimmune responses to ZP3 protein antigen are the important factor to induce AOD. Using AOD mice model induced by mZP3 in complete Freund's adjuvant (CFA), we tested the capacity of melioration AOD by the co-immunization of mZP3 protein and DNA vaccine. We showed here that AOD was profound diminished compared with other immunization methods and the melioration was correlated with the reduced T cell response and the inflammatory cytokine productions including IL-12 and IFN-γafter the co-immunization. This subtype TR cells highly expressed IL-10 and FoxP3. In order to conform which type cells to play an important role in ameliorating oophoritis from co-immunized group, CD25 profile within the CD4+ T cells was analyzed by FACS. We observed that CD4~+ T cells from all groups expressed a comparative level of CD25+. Anti-CD25 was adoptively transferred into co-immunized mice at the first co-immunization and the anti-VP1 served as control. Anti-CD25 did not impair the effect of co-immunization. The results showed that CD4~+CD25~- cells might be involved in ameliorating oophoritis. The CD4~+CD25~- subtype cells were purified from mice co-immunized with pcD-mZP3~+mZP3 or pcD-mZP3~+OVA (mismatch control) and were adoptively transferred intravenously into mice pre-immunized with mZP3 protein in CFA. The degree of oophoritis was decreased by transferring CD4~+CD25~- subtype cells from co-immunized mice but not mismatch controls. The above purified CD4~+CD25~- subtype cells were performed FACS. The production of FoxP3 and IL-10 was increased significantly in CD4+CD25- subtype cells from co-immunized mice but not mismatch controls. Furthermore, the meliorated AOD was apparently associated with the induction of mZP3 specific TR cells that exhibit a CD4~+/CD25~-/FoxP3~+ phenotype and expressed IL-10. This is the first time that an established autoimmune disease was treated by using the co-immunization of DNA and protein vaccines.In conclusion, we demonstrated that fertilities and mean litter sizes were significantly reduced in group of C57BL/6 mice co-immunized with pcD-mZP3~+mZP3 protein vaccines compared with na?ve mice and the mice immunized with pcDNA3 vector. Co-immunization not only induced the highest level of antibodies, little to none level of T cell proliferation specific to ZP3, but a strong correlation with the normal ovarian morphology. The impairment of T pathogenic T cells is correlated with the induction of IL-10 expressing CD4+ CD25- TR cells induced by the co-immunization.The results indicate that co-immunization of mZP3 DNA- and protein-based vaccines can reduce the fertility without interfere the normal follicular development and present a novel strategy to develop a contraceptive vaccine in human.