The Immunocontraceptive Effect Of Zone Pellucida 3 (ZP₃) DNA Vaccines

Lagurus lagurus is a dominant species of wild pest mouse in Xinjiang grassland, which contributes to a great loss of thousands upon thousands pasture because of its overpopulation every year. In order to prevent and control the population of the species, various approaches were undertaken, in which the immunocontraceptive technology exhibiting great promising because of its advantage compared to physical or chemical poisons based traditional methods in efficacy, specificity and safety. Therefore, a DNA vaccine based immunocontraceptive vaccine for Lagurus zona pellucida 3 was developed to prevent fertilization of Lagurus lagurus in the study.The mammalian oocyte is surrounded by an extracellular translucent envelope termed the zona pellucida (ZP). The ZP mediates the initial recognition and binding events of sperm to the oocyte in a relatively species specific manner, thus playing a vital role in fertilization. In most species, ZP is composed of three biochemically and immunologically distinct glycoproteins, ZP1, ZP2, and ZP3, which can recognize and combine with sperm and induce acrosome reaction. It has been widely concentrated that fertilization could be successfully prevented when the combination of ZP3 and sperm was blocked by monoclonal antibodies both in vivo and in vitro, so ZP3 glycoprotein has been considered a good candidate for developing an immunocontraceptive vaccine.In the studies, to control the population of the pasture bandicoot Lagurus lagurus, zona pellucida 3(zp3) was selected as the candidate antigen in immunocontraceptive vaccine to prevent fertilization of Lagurus lagurus, involving the following four aspects:1 Confirming the species specificity of immunocontraception vaccines To investigate the species-specific immunocontraception effects of mzp3 and lzp3 vaccines, we constructed four corresponding immunocontractive recombinant plasmids:pcDNA3-mzp(3pcD-M),pcDNA3-lzp(3pcD-L),pcDNA3-aat-comp-mzp3 (pcD-ACM) and pcDNA3-aat-comp-lzp3 (pcD-ACL) using zona pellucida 3 gene of Lagurus lagurus (lzp3) and Mus musculus (mzp3) .After the verifying of the zp3 genes expression in livers of mice with the introduction of hydrnamic transfection instead of traditional Hela cell transfection by RT-PCR,NIH mice were immunized with zp3 DNA vaccines.The result of ELISA showed that all of the recombinants could elicit high titer and specific anti-ZP3 antibody, which exhibit binding the ZP of oocytes from immunized mice in immunofluorescence assay.Antifertility experiment showed that mzp3 and lzp3 groups both enhanced contraceptive effects (p<0.05), especially the group of pcD-ACM (p<0.01). And there were no disruption of follicular development in pcD-M,pcD-L and pcD-ACL groups but not in group of pcD-ACM. The present studys proved that L. lagurus zona pellucida 3 gene (lzp3) and M. musculus zona pellucida 3 gene (mzp3) had immunocontraception effects and primarily indicated that there were no species specificity between lzp3 and mzp3 in NIH mice.2 Optimizing the immunocontraceptive Vaccines carrierTo investigate the immunocontraceptive effect of Lagurus lagurus zona pellucida 3 gene (lzp3) fused by Mycobacterium tuberculosis HSP70 and C-terminal of Hsp70, we constructed corresponding immunocontractive recombinant lasmids: pcDNA3-lzp3-HSP70 (pcD-L-HSP70) and pcDNA3-lzp3-HSP70C (pcD-L-HSP70C) using zona pellucida 3 gene of Lagurus lagurus (lzp3). Meanwhile, pcDNA3-lzp3(pcD-L)and pcDNA3-aat-comp-lzp3-C3d3(pcD-ACLC), which were proven to be effective immunocontraceptive vaccines by our lab previously ,were studied as control. After the verifying of the zp3 genes expression in livers of mice with the introduction of hydrnamic transfection instead of traditional Hela cell transfection by RT-PCR, NIH mice were immunized with zp3 DNA vaccines.Results of ELISA showed that all of the recombinants in mice could elicit specificity of anti-ZP3 antibody; which could combine the ZP of oocytes from immunized mice in immunofluorescence assay. MTS assay showed that the lymphocytes in all groups were proliferated by the stimulation of recombinant Lzp3 with no disruption of follicular in ovaries; especially the groups of pcD-ACLC and pcD-L-HSP70C (p<0.01). And antifertility experiment showed that besides pcD-L-HSP70 all three other groups enhanced sterile effects (p<0.05), especially the groups of pcD-ACLC and pcD-L-HSP70C (p<0.01). The present studys proved that L. lagurus zona pellucida 3 gene (lzp3) fused by C-terminal of Mycobacterium tuberculosis Heat chock protein HSP70 had a good immunocontraception effects while L. lagurus zona pellucida 3 gene (lzp3) fused by Mycobacterium tuberculosis Heat chock protein HSP70 had a poor effect on controlling bearing. So C-terminal of Mycobacterium tuberculosis Heat chock protein HSP70 might be a superior candidate to deliver the adjuvant function in lzp3 DNA immunocontraceptive vaccination rather than the complete HSP70 molecule.3 Confirming the route of immunocontraceptive vaccinesTo effectively control the bearing of the vertebrate pests Lagurus lagurus in XingJiang,We investigate the immunocontraceptive effect of chitosan-DNA vaccines pcDNA3-lzp3(pcD-L)and pcDNA3-aat-comp-lzp3-C3d3 (pcD-ACLC) , which were proven to be effective immunocontraceptive vaccines by our lab previously, by a quick oral delivery. After the verifying of the zp3 genes expression,which were packaged by chitosan or not, in livers of mice with the introduction of hydrnamic transfection instead of traditional Hela cell transfection by RT-PCR, NIH mice were immunized with intramuscular and oral delivery respectively. MTS assay showed that the lymphocytes in all groups were proliferated by the stimulation of recombinant Lzp3 with no disruption of follicular in ovaries, especially the groups of oral immunity (p<0.01) ; ELISA results showed that all serum of the oral groups almost elicited specificity of anti-ZP3 IgG and IgA antibody in serum and there were not in intramuscular administration ,While a increased levels of SIgA were successfully generated in fecal extracts of oral immune mice; Antifertility experiment showed that oral and intramuscular immunity could decreased the mean quantity of bearing, though they were not enough; Derect immunofluorescence results showed that the zona pellucidas of sterile mice could give off green irradiance in UV. The present studys primarily presumed chitosan–DNA vaccine pcD-L and pcD-ACLC had a possibilitity to decrease the level of bearing.4 Deternmination the profile of IgG isotypes response elicited by the zona pellucida 3 gene (zp3) immunitionTo investigate the possible immunocontraceptive mechanism, we detected the IgG isotype of collected serum immunised by DNA, protein and DNA-protein vaccines with isotype kit. The results indicated that all kinds of serum were IgG1/IgG2a>1, namely Th2 humoral immune reaction, which heavily decreased the oophoritis probability .