Evaluation And Application Of Immunogenicity Of Brucella Outer Membrane Vesicles(OMVs)

Brucellosis is a zoonotic disease caused by Brucella that can cause abortions in livestock and eventhe fever,arthritis and meningitis in human.At present,the animal Brucella vaccines are mainly attenuated vaccines,but there is the disadvantage of strong virulence that can easily lead to miscarriage and low protection of those vaccines.In view of the successful development of the Neisseria meningitidis outer membrane vesicle(OMVs)vaccine.This project explored the preparation methods of Brucella species OMVs and evaluated the immunogenicity and immunoprotective properties of OMVs and their components in a mouse model.This project explored the preparation methods of Brucella OMVs,Then the immunogenicity and immunoprotective properties of OMVs and components were evaluated in a mouse model.It laid the foundation for the study of a new subunit vaccine for Brucella.Objects:(1)Taking the epidemic strain 043 of Brucella melitensis from Xinjiang as the research object to extract and purify the outer membrane vesicles(OMVs),identify the morphology of the OMVs and analyze their components.To explore the effect of OMVs on the expression of macrophage cytokines and its effect on bacterial adhesion internalization.(2)To establish a serological evaluation method for OMVs-associated outer membrane protein(OMP),recombinant outer membrane protein and lipopolysaccharide core protein,and to obtain proteins with strong specificity and reactogenicity.(3)To analyze the immunogenicity and immunoprotection of Brucella OMVs and its'related proteins.Methods:(1)OMVs were extracted by ultracentrifugation,their morphology and size were observed by transmission electron microscopy,protein composition of OMVs was identified by SDS-PAGE.After macrophages were stimulated by OMVs,the expression of cytokines was detected by RT-PCR,its effect on cell adhesion and internalization was evaluated by CFU count.(2)The OMP was extracted and identified.Some recombinant proteins were expressed and purified that were BP26,OMP10,GMD,and ManB proteins.Then the reactivity of these proteins were verified by Western Blot.proteins with high specificity and reactivity were screened by indirect ELISA.(3)Mice immunized with OMVs,OMP,BP26,OMP10 and BP26+OMP10 double antigens that were immunized with Freund's adjuvant and nano-adjuvant(LDH)respectively.The specific antibody level was detected by indirect ELISA,and the level of cellular immunity was evaluated by flow cytometry detection of T lymphocyte subsets and detection of IFN-?and IL-4 expression levels by ELISA.After14 days of booster immunization,mice were challenged with Brucella M5 strain at 2×10 ~6CFU.After 15 days of challenge,histopathological observations and spleen bacteria load tests were performed to evaluate the immune protection.Results:(1)The size of brucella 043 OMVs was between 20-200nm with an average of60.5nm,and its protein composition was mainly around 22,29,51KD.OMVs can significantly induce transcriptional expression of IFN-?,IL-6,IL-12,TNF-?and IL-10 genes produced by mouse macrophages,and IFN-?and IL-6 are expressed early.OMVs can also significantly enhance adhesion and internalization of RAW264.7 cells to brucella.(2)Brucella OMP and BP26,OMP10,GMD and ManB proteins were successfully obtained,and the optimal ELISA reaction conditions for different antigens were established by tandem titration.The Brucella OMVs,OMP,BP26,and OMP10 with good reaction were screened.(3)The results of the immunoassay showed that the experimental animals could produce specific antibodies and reached the highest level on the first 21 days.The antibodies increased significantly after reinduction.High levels of IFN-?were detected in splenic lymphocytes of both Freund's adjuvant and nano-adjuvant immunized groups,but IL-4 levels were not significantly different from those of PBS group,indicating that both OMVs and their related proteins could induce Strong Th1-type immune response.The results of lymphocyte subsets showed that each group of antigens could induce the proliferation of non-specific T lymphocytes.Compared with OMP,BP26 and OMP10,under the action of nano-adjuvant,OMVs induced cell immunity very well,promoted the expression of CD4+T cells,and induced a stronger Th1-type immune response.Mice immunized with Brucella could induce pathological changes of spleen,liver,lung and kidney in varying degrees.The OMVs and BP26 in the nano-adjuvant group had slight pathological changes.The lesions in the other experimental groups were obvious and severe.OMP10 and OMVs lesions were milder in the Freund'adjuvant group.The results of challenge experiments showed that the OMVs spleen index was the lowest in the nano-adjuvant group,and the difference was extremely significant compared with the non-immune group(p<0.01),while the spleen bacteria load was significantly different from the non-immune group(p<0.01);The OMP10 spleen index and CFU count were the lowest in the adjuvant group,which was significantly higher than the non-immune difference(p<0.01).Conclusion:(1)The method of extraction of Brucella OMVs was successfully established and its morphology and size were identified.(2)Brucella OMVs can induce strong Th1-type immune responses in RAW264.7 cells and promote adhesion and internalization of RAW264.7 cells to bacteria.(3)An indirect ELISA method for serological evaluation was established and optimized,and immunodominant antigens with specificity and better reactogenicity were screened.(4)The immunization and challenge experiments of mice confirmed the immunogenicity and protective effects of OMVs,and the nano-adjuvants had a higher immune-enhancing effect on OMVs.