Effect Of Cytokine Adjuvants On Antifertility That Induced By Dog Pellucida 3 DNA Vaccine In Mice

In the absence of valid control means,the number of stray animals worldwide has increased rapidly in recent decades,and the ways to restrict their populations effective and humane has become very urgent.In the process of mammalian fertilization,the zona pellucida-3(ZP3)glycoprotein on egg surface as the sperm receptor can bind to sperm and induce the sperm acrosome reaction,then complete the fertilization process.ZP3-specific antibody can block the binding site of ZP3 protein on oocyte surface,and inhibit sperm–egg interaction.ZP-based contraceptive vaccine is an ideal approach for human contraception and a humanistic control method for animals.To develop the ZP-based contraceptive vaccine for human application,we made an effort to improve the ZP3–specific antibodies immune response by selecting suitable immue adjuvants and proper vaccination route.DNA vaccine is considered as promising vaccine approach since its safe,cheap and ease to get and save,and it is broadly used for the development of anti-virus vaccine.In our previous study,adjuvant and sending way way can effectively stimulate the body to produce a comprehensive,effective and lasting immune response,Therefore,the dog ZP3 was used as the target antigen to prepare DNA vaccine,and to screen the molecular adjuvant to improve the effect of immunogenicity antifertility,so as to achieve the aim of controlling urban number of stray dogs.The efficacy of DNA vaccines may crucially depend upon their capacity to transduce dendritic cells(DCs).Granulocyte-macrophage colony-stimulating factor(GM-CSF)can influence the activation and maturation of antigen-presenting cells(APCs),particularly enhance the recruitment and function of DCs.In this study,mouse GM-CSF eukaryotic expression vector was constructed,then co-intramuscularly delivered with or 4 days prior to caine zona pellucida-3(CZP3)DNA vaccination.The results showed that GM-CSF improved the maturation of DCs,co-administration of GM-CSF and CZP3 enhanced the m RNA expressive levels of co-stimulation factors greatly.These data suggest that GM-CSF could promote the maturation of DCs and enhance its function on antigen presentation;GM-CSF could be used as the adjuvant of DNA vaccine to develop immunocontraceptive vaccine.Interleukin-5(IL-5)is a cytokine secreted by T helper 2(Th2)cells,that participates inthe regulation of antibody secretion.In order to investigate the possiblity of GM-CSF and IL-5 being used as immunization infertility adjuvants to improve the immunogenicity of canin-e zona pellucida 3 DNA vaccine,and futher enhance the immune effect and reduce the fertilit-y of stray dogs.The recombinant eukaryotic cytoplasmic pc DNA3-IL-5 and pc DNA3-GMCSF were combined with the zona pellucida 3 DNA vaccine pc DNA3-CZP3 to immunize fe-male BALB/C mice with electric pulses in a single or combined manner.The levels of Ig G and s Ig A(secretory Ig A)in serum and genital tract were measured by ELISA(enzyme-linked immuno sorbent assay),and the levels of IL-4 and IFN-? in serum.MTT assay was used to de-tect the proliferation of mouse spleen T cells.Frrowing rate and correlation analysis was per-formed at sixth weeks after all mice were coupled.The results showed that the level of s Ig A in Ig G and genital tract was extremely significant increased from 0 weeks to 6 weeks(P < 0.01),Among them,the effect of pc DNA3-IL-5,pc DNA3-GM-CSF,and pc DNA3-CZP3+ pc D-NA3-GM-CSF group was the most obvious.The levels of IL-4 and IFN-? in the serum of diff-erent immunized groups showed that the cytokine as immune adjuvant enhanced the humoral immune response and the cellular immune response intensity of the immunized mice in vary-ing degrees.Futhermore.the immunized groups pc DNA3-GM-CSF(P<0.01)and the pc DN-A3-GM-CSF+pc DNA3-IL-5 +pc DNA3-CZP3(P < 0.01)were significantly higher than the control group.And the proliferatio and of splenic T cells in pc DNA3-GM-CSF +pc DNA3-ZP-3,pc DNA3-IL-5+pc DNA3-CZP3 and pc DNA3-GM-CSF+pc DNA3-IL-5+ pc DNA3-CZP3 immunized group was significantly higher than that in control group(P <0.05),The result of of Spearman correlation analysis between the farrowing number and the antibody titers result showed the best negative correlation of pc DNA3-GM-CSF+pc DNA3-CZP3 immunized group,and the pc DNA3-GM-CSF+pc DNA3-IL-5+ pc DNA3 was the second.Compared with pc DNA3 and pc DNA3-CZP3,the number of litters decreased by 16 percent,27 percent,44 percent,and 68 percent,and 13 percent,33 percent,and 62 percent.So as to test the ability of serum CZP3 antibodies to bind to egg cells and the actual effect of this combination on fertilization,and to explore its association with fertility.Thus further experiments are needed,indirect-immunofluorescence and in vitro fertilization system.The results of indirect immunofluorescence showed that the fluorescence intensity and fluoresce-nce coverage of the surface of the mice eggs showed a positive correlation with the antibodylevel in the serum,in addition,the fertilization rate of immunized groups pc DNA3-CZP3+pc DNA3-IL-5,pc DNA3-CZP+pc DNA3-GM-CSF and pc DNA3-CZP3+ pc DNA3-GM-CSF+pc DNA3-IL-5 with good effect of antibody titter and egg binding were significant and extreme significant decreased.In the pc DNA3 and pc DNA3-CZP3 immune groups,the ratio was18 percent lower,23 percent,49 percent and 66 percent,and 17 percent,33 percent and 59 percent respectively.This is consistent with the mouse fertility rate.which was consistent with the results of fertility.Although these resul-ts are one-sided for explaining that the theory of antibody reduction of fertility by anti-body blocking effect,to some extent,it demonstrates the need to enhance the humoral immune resp-onse for antifertility.Conclusion: GM-CSF can promote antigen presented cells of mature,as adjuvant enhanced CZP3 DNA vaccine of humoral immunity and cellular immune response,usually significantly reduces the immune mice nests.When GM-CSF join IL-5 acts as the CZP3 DNA vaccine adjuvant further increases the antibody level and reduces the number of pups in the immune mice.The combined use of GM-CSF and IL-5 can be identified as the best strategy for future canine experiments.