Study On The Immune Effect Of Nano-glycyrrhizic Acid Combined With OMV From Rabbit Bordetella Bronchiseptica

Bordetella bronchiseptica(Bb)disease is known as a common chronic relapsing infection,which leads to huge economic losses in our rabbit-breeding industry.However,there is no satisfactory whole-cell inactivated vaccines to confer protection against the disease.In addition,conventional veterinary vaccine adjuvants have defects in safety as well as efficacy.All the above leads to a conclusion that it is urgent to develop novel vaccine candidates.In view of the success of outer membrane vesicle(OMV)vaccine against Neisseria meningitidis,we explored the preparation techniques of OMV from Bb and loaded OMV with nano-Glycyrrhizic acid(GAN),which was used as a vaccine adjuvant.We constructed GAN-OMV system and studied its immune effect in vivo and in vitro,to provide new ideas and experimental basis for the development of rabbit Bb novel subunit vaccines.1.Preparation and protein composition analysis of OMV from rabbit Bb.The objective of this study was to establish optimum preparation techniques of OMV from Bb.The strain FX-1 harboring various virulence factors was selected.And the preparation techniques of OMV were screened and optimized,including culture time,the addition of antibiotics and filtration method.Here we measured the characteristics of OMV by transmission electron microscope(TEM),scanning electron microscope(SEM)and dynamic light scattering(DLS),etc.We further analyzed the protein content and proteome of OMV by Bradford protein quantification,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and liquid chromatography-tandem mass spectrometry(LC-MS/MS).Our results showed that centrifugal ultrafiltration was a better preparation method.And the optimum culture time,cefalexin concentration and filtering method were18 h,64μg/m L and filtration though 0.45μm membrane once,respectively.Isolated OMV showed a nano-spherical particle with an average size of 75.66±11.84 nm.In addition,we identified that multiple virulence and infection-associated proteins were consistently enriched in the OMV.Together,our study concluded that optimum preparation techniques could significantly promote the production of OMV,which provided possibility and guidelines for the development of novel subunit vaccines.2.Immunological effect of GAN-OMV on macrophages.Fabrication of GAN-OMV was achieved by a mechanical extrusion process of two key components of GAN and OMV.And the characteristics of the prepared GAN-OMV subunit vaccine were evaluated,including shape,particle size and zeta potential,etc.Our results confirmed that GAN-OMV was successfully prepared and exhibited a core-shell structure.In this study,the effects of GAN-OMV were investigated on the immunological function of macrophage,such as macrophage proliferation,cytokine secretion and phagocytic function in vitro.Our study results demonstrated that GAN-OMV promoted macrophages proliferation and enhanced cytokines production such as IL-6,IL-10,IL-1βand TNF-α.In addition,confocal observation revealed that GAN-OMV worked on macrophages by entering macrophages through macropinocytosis-dependent and clathrin-mediated endocytotic pathways and being enriched on lysosomes in macrophages.3.Immunological effect of GAN-OMV on mice.In this study,immunization and challenge assay were performed on mice in four groups of GAN-OMV,Alum-OMV,OMV and blank control.The fabrication of GAN-OMV not only had a stable excipient effect but also for a better immunological performance.The mice immunized with GAN-OMV exhibited enhanced the level of lymphocytes and antigen-specific serum total Ig G and Ig G subclasses(Ig G1,Ig G2a)using blood routine and enzyme-linked immunosorbent assay(ELISA).Our experimental results also demonstrated that GAN-OMV significantly enhanced lymphocyte proliferation,boosted the frequency of B cells and the ratio of CD4~+to CD8~+T cells in spleen lymphocytes and induced the secretion of Th1 cytokines(IFN-γ),Th2 cytokines(IL-4)and Th17 cytokines(TNF-α、IL-6).These results suggested that GAN-OMV could stimulate strong humoral and cellular immune responses.Furthermore,the protective efficacy of GAN-OMV immunization against Bb infection was evaluated by serum bactericidal assay(SBA)and lungs for colonization assay.Our study results showed that the mice immunized with GAN-OMV exhibited higher level of bactericidal antibodies and lower level of Bb colonization in the lungs,from which could be told that the subunit vaccine based on GAN-OMV could prevent the infection of Bb.