The Protective Mechanism Of Vibrio Vulnificus Hemolysin A Protein Vaccine In Vibrio Vulnificus Infected Mice

ObjectiveVibrio vulnificus hemolysin A（VvhA）is an extracellular protein which encoded by VvhA gene of V.vulnificus.VvhA has hemolytic activity and cytotoxicity.VvhA protein has strong antigenicity and sequence conservation.This protein was suitable for in vitro expression.In addition,VvhA protein immunization mice could produce VvhA antibodies which could neutralize toxins.Therefore,VvhA protein was suitable as candidate vaccine for vibrio vulnificus.VvhA protein as a candidate vaccine,the immunoprotective effect needs to be confirmed,and the mechanism of immunoprotection needs further exploration.In this study,VvhA protein vaccine was prepared by expressing and purifying VvhA protein,and BABL/c mouse model of VvhA protein vaccine was established.The mouse model infected with the standard strain of Vibrio vulnificus was used to evaluate the immune protective effect of VvhA protein vaccine through the survival rate,histopathology,serology and mass spectrometry imaging.This study will provide new experimental evidence for seeking higher vaccination protective effect.Methods1.The pET28a（+）-vvhA-Rosetta（DE3）was induced by IPTG to produce the recombinant VvhA fusion protein and purified using Ni-nitrilotriacetic acid resin.His-tagged proteins were identified by Western blot.2.VvhA vaccine was prepared and BALB/c mouse animal model immunized with VvhA vaccine was established.3.After intraperitoneal injection of lethal dose of Vibrio vulnificus,the VvhA immune protective effect was assessed by Serology、Pathology and Mass Spectrometry Imaging.4.In order to study the immune protective mechanism of VvhA vaccine,the response of Th（Th1、Th2、Th17、Th22、Tfh）and GC B cells were detected by flow cytometry.5.The effects of IL-21 on the immune protection of VvhA protein were studied by adjusting the level of IL-21 with immune blockade and enhancing methods.6.The levels of Tfh and GC B cells in lymph nodes of mice were detected by flow cytometry.After intraperitoneal injection of recombinant mouse IL-21 and mouse IL-21 antibody,we assessed the change of cytokines by Gene Sequencing to investigate the immune protective efficacy of VvhA vaccine.Results1.The VvhA protein was highly expressed in pET28a（+）-vvhA-Rosetta（DE3）after induced by IPTG.The target protein was purified by Ni-nitrilotriacetic acid resin system.The concentration of VvhA was2.3mg.ml^-1 by tested the OD_260/280 value.2.After BALB/c mouse animal model of VvhA protein immunization was established,and the lethal dose of V.vulnificus was injected intraperiton-eally.Pathological examination showed that the liver damage of mice immunized with VvhA was significantly reduced and the survival rate reached83.3%.The serological test results were consistent with the Pathology results（VvhA immunized:ALT:40U/L,AST:139U/L;control:ALT:164U/L,AST:653U/L）.3.After the mice were immunized with VvhA,the level of Th/Tfh/GCB cells were tested by flow cytometry.The result indicated that the level of Th1/Tfh/GC B cells were highly expressed in the mice immunized with VvhA.4.Increasing IL-21 function significantly increased the survival of mice immunized with VvhA.The survival rate reached 100%.In addition,increasing IL-21 significantly reduced organ damage in mice infected with V.vulnificus.5.Flow cytometry was used to detect the changes of Tfh cells and GC B cells in mouse lymph nodes.The results showed that the response of Tfh and GC B cells in mice with enhanced Il-21 function increased significantly.Whole Genome Sequencing（WGS）indicated that,IL-21 may induce CXCR5rise and the high-expressed genes involved in chemokine signaling pathway and the cytokine-cytokine receptors signaling pathway.Conclusions1.The VvhA protein vaccine was successfully prepared and the VvhA protein vaccine immunize BALB/c mouse model was established.2.VvhA protein as V.vulnificus vaccine has significant immune protection effect.3.The immune protection effect of the VvhA protein vaccine may be related to the up-regulation of Th1/Tfh/GC B cells.In addition,the mouse modle of IL-21 regulation was established.IL-21 induced up-regulation of CXCR5 expression.These up regulated genes involved in chemokine signaling pathway and cytokine-cytokine receptors signaling pathway to enhance VvhA protein immune protection.