| Lagurus lagurus is a wild pest mouse distributed in desert pasture of northern region in Xingjiang. It destroys the pasture and results in the environmental damages for both ecologically and economically, and severely demolishes the stock raising of Xinjiang. Conventional control ways which rely on poisoning, trapping and shooting can be ineffective in the long term, often lead to losses in non-target species, and cause severely environmental problems. Immunocontraception can block or prevent oocyte fertilization through immunological interference, and it is a more humane, species-specific and effective control method. DNA vaccine is conventionally delivered by muscular or subcutaneous injection, while this is not feasible for the overpopulation control of the widely distributed pest animals. Delivering bait will be feasible ways that control the population of pest mouse through mass vaccination.In mice, the oocyte is enveloped by an extracellular matrix termed zona pellucida (ZP), which is composed of three distinct glycoproteins-ZP1, ZP2 and ZP3. ZP3 acts as the primary sperm receptor, which can recognize and combine with sperm and induce acrosome reaction. ZP3 elicits antibodies that can inhibit sperm–oocyte binding and reduce fertility, it has been considered as a promising target antigen in immunocontraceptive vaccines.Because DNA vaccine has several advantages such as inducing humour immune response and cell immune response at the same time, simple preparation and cheap cost, it is also used to study the immunocontraceptive vaccine. But, the immunogenicity of DNA vaccine is not high, and its immune effect need be improved. So it is necessary to enhance the immune effect of DNA contraceptive vaccine through some methods such as adding adjuvant, change delivery route and optimizing plasmid vectors.To screen an molecule adjuvant that can improve the antifertility effect of DNA immunocontraceptive vaccine. We investigated the feasibility of mouse cytokine IL-31, IL-15, IL-33 and non-cytokine FAI3 as different molecular adjuvant, to enhance systemic and mucosal immune responses and improve antifertility effect of contraceptive DNA vaccine pcD-Lzp3 following the co-immunization. We have expored the immune effect that these three mouse cytokines and non-cytokine regulate the antifertility effects of DNA vaccine pcD-Lzp3.Cytokine IL-31 is a new cytokine that is mainly produced by activated CD4+ T helper type 2 (Th2) cells. IL-31 is important in T cell-mediated immune responses; it is involved in inflammation and degenerative skin diseases. In allergic contact dermatitis, IL-31 expression was enhanced and correlated with IL-4 and IL-13. IL-33 is a new member of IL-1-family cytokines. IL-33 mediates its biological effects via IL-1 receptor ST2, activates NFkB and MAP kinases, and drives production of TH2-associated cytokines from in vitro polarized TH2 cells. The expression pattern of human IL-33 appears restricted to epithelial cells from bronchus and small airways, fibroblasts, and smooth muscle cells, suggesting its potential involvement in mucosal immunity. DCs respond directly to IL-33 through the receptor ST2, IL-33-actived DCs trigger an atypical Th2-type response that produce IL-5 and IL-13, The IL-33 and DC interaction may represent a new pathway to initiate Th2-type immune response. IL-15 is a proinflammatory cytokine that enhance humoral and cellular immune responses by inducing proliferation, activation, and cytokine release from lymphocytes. IL-15 has been shown to promote DC maturation, and increased antibody titers when used as adjuvant. However, FAI is a fibrinogen–albumin–IgG receptor from group C streptococci, it is a novel multiple-ligand-binding protein that can bind to fibrinogen, albumin and IgG. The third gene fragment of FAI(415bp-702bp)- fai3 has the activity of mucosal adjuvant.At first, mouse IL-33 gene, IL-31 gene and IL-31 mutant gene were amplified by RT-PCR from testis and spleens of Balb/c mice. The recombinant plasmids pcD-mIL-15, pcD-mIL31,pcD-muIL31,pcD-mIL33 and pcD-fai3 were constructed successfully, and were expressed instantaneously in eukaryotic cell. The recombinant plasmids pcD-mIL-15, pcD-mIL31, pcD-muIL31, pcD-mIL33 and pcD-fai3 were encapsulated separately with chitosan to generate the different chitosan-DNA complexes. ICR mice were immunized with these chitosan-DNA complexes by intranasal route or oral route on day 0, 14, 28 and 42, respectively. Indirect ELISA was employed to determine the anti-LZP3 specific IgG, IgG1 and IgG2a antibody in sera, and the anti-LZP3 specific IgA in vaginal secretion and the feces.The results showed that the high levels of serum IgG and the low mean litter size were induced in the mice of intranasally co-immunized with chi-(pcD-Lzp3+pcD- muIL-31) or chi-(pcD-Lzp3+pcD-mIL-31). The lymphocyte proliferation results showed that the strong cell immune response was induced in the mice of these two co-immunized groups , and the high IgG2a levels suggested that Th1 immune respone was actived. Histological examinations on the ovaries from the infertile female mice of chi-(pcD-Lzp3+pcD-muIL-31) exhibited abnormal histological structure. In the co-immunized group of chi-(pcD-Lzp3+ pcD-mIL-31), the follicles were lacked in the ovaries of infertile mice, and the number of follicles were decreased markedly.The mice of intranasally co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) induced the highest levels of serum IgG , vaginal secreted IgA , fecal IgA and the lowest mean litter size. The lymphocyte proliferation results showed that the strongest cell immune response was induced in the mice of co-immunized with chi-(pcD-Lzp3+pcD-mIL-15). Histological examinations on the ovaries from the female mice of chi-(pcD-Lzp3+pcD-mIL-15) exhibited normal histological structure and normal follicles in different stage of development, without pathological changes specifically. The ovaries from the infertile female mice of chi-(pcD-Lzp3+pcD-mIL-33) showed the atretic follicle and the loss of oocyte . Our work demonstrated that the intranasal delivery of the molecular adjuvant mIL-33 with chi-pcD-Lzp3 could significantly enhance the antifertility effect of the Lzp3 DNA vaccine for contraception by enhancing both the systemic and mucosal immune responses.The mice of co-immunized group of chi-(pcD-Lzp3+pcD-fai3) produced the higher levels of serum IgG and fecal secreted IgA , and the low birth rate. Histological examinations on the ovaries from the female mice of chi-(pcD-Lzp3+pcD-fai3) exhibited normal histological structure and normal follicles in different stage of development, without pathological changes specifically. The level of IgG antibody in the infertile mice was significantly higher than in the fertile mice in the co-immunized group of chi-(pcD-Lzp3+pcD-mIL-33),chi-(pcD-Lzp3+pcD-fai3) , thus, there was a direct relationship between the levels of total IgG antibody and the infertility rate of mice among these groups.Delivering bait is a feasible, potentially effective way, it will be used as the delivering method of DNA vaccine pcD-Lzp3 in the future. We investigate the feasibility of fai3 and mIL-15 as mucosal adjuvant by oral route, to improve antifertility effect of DNA vaccine pcD-Lzp3. The above-mentioned chitosan-DNA complexes were add to mouse food, and made the food as the bait. Then, these baits were feed ICR mice on day 0, 14, 28 and 42, respectively.The ELISA results showed that the higher level of serum IgG and secreted IgA in feces, the lower mean litter size and birth rate were induced in the mice of co-immunized with chi-(pcD-Lzp3+pcD-fai3) through oral route. The level of serum IgG in the mice of chi-(pcD-Lzp3+pcD-mIL-15) was lower than that of chi-(pcD-Lzp3+pcD-fai3) co-immunized group, but the mice of this group induced the strong lymphocyte cell proliferation. Histological examinations also show the ovaries of immunized mice with the normal histological structure.In conclusion, mouse cytokine-mIL-33 as the molecular adjuvant co-immunized with pcD-Lzp3 through intranal route, could improve the antifertility effect of the Lzp3 DNA contraceptive vaccine by enhancing both the systemic and mucosal immune responses. Mouse cytokine mIL-31 and mIL-15 as the molecular adjuvant co-immunized with pcD-Lzp3, respectively, produced the lower level of IgG and antifertility effect than that of the co-immunized with chi-(pcD-Lzp3+pcD-mIL-33) . These results suggested that cytokine mIL-33 would have more better efficacy of molecular adjuvant than cytokine mIL-31 and mIL-15.Non-cytokine FAI3 as the molecular adjuvant co-immunized with pcD-Lzp3 intranally or orally, not only enhanced the systemic immune response, but also stimulated the mucosal immune response,and also can reduce the birth rate of mice. Thus, both mIL-33and FAI3 could act as molecular adjuvant, to improve the antifertility effect of DNA vaccine pcD-Lzp3 . |
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