Immune Effect Evaluation Of A Vesicular Stomatitis Virus-based Vaccine Against Coxsackievirus B4

Research background and purposeCoxsackievirus B（CV-B）is divided into six serotypes.The CV-B group has a wide distribution of viruses.Humans have a greater chance of infection.The virus enters the human body and replicates in specific tissues or organs.Causes lesions in the heart,pancreas,muscles,and bones.Coxsackievirus B4（CV-B4）is a common and typical virus in the CV-B group.Studies have shown that the CV-B4 virus is one of the major pathogenic factors that cause insulin-dependent diabetes in infants and young children,and can also cause neonatal infection with myocarditis and encephalitis.Compared with other enteroviruses,CV-B4 virus has a higher mortality rate.At present,there is no vaccine to prevent and control CV-B4 infection and there is a lack of effective therapeutic drugs.At present,genetic engineering vaccines have become increasingly sophisticated in their research and development,and have better safety than traditional inactivated vaccines.Considering the increase in demand for CV-B4 vaccines,in this study,a recombinant CV-B4 vaccine was constructed using Vesicular stomatitis virus（VSV）as a vector and Western Blot and indirect immunofluorescence were used to validate the recombinant vaccine.The expression level of the protein was compared with the established CV-B4 infection model of BALB/c suckling mice and compared with the traditional CV-B4 inactivated vaccine,and the protective effect of the CV-B4 genetic engineering vaccine on suckling mice was further evaluated.The expected period was CV-Prevention and control of the B4 virus provides a safer and more effective vaccine,reducing the infection rate of the CV-B4 virus.In this experiment,the recombinant vector vaccine VSV-VP1 containing the CV-B4isolate LY114F VP1 fragment constructed with VSV as the vector was verified by Western Blot and indirect immunofluorescence to verify the successful construction of the recombinant vaccine.Furthermore,based on the previously established CV-B4 animal infection model,the levels of Ig G and Ig A produced in BALB/c adult mice after intranasal immunization,subcutaneous immunization,and subcutaneous immunization with CV-B4inactivated vaccine were compared between VSV-VP1 vaccine and immunized BALB.Survival rate,histopathological changes and expression of cytokines in pregnant mice after c-pregnancies,evaluation of CV-B4 genetically engineered vaccines and traditional inactivated vaccines in order to prevent the selection of vaccine against CV-B4 infection and application for reference.Research methods1.Preparation and in vivo pathogenicity of CV-B4 isolatesThe virus total RNA was extracted from the feces of the children and the CV-B4 virus was initially identified and sequenced.For samples positive for CV-B4,proliferation was performed on Vero cells to determine the viral load.The selected 1-day-old BALB/c suckling mice were infected intramuscularly with 10⁷ TCID₅₀/head of CV-B4 virus fluid.In vivo pathogenicity of CV-B4 virus was analyzed by animal survival rate and clinical score.The CV-B4 virus fluid was injected into adult male BALB/c mice to observe changes in blood glucose and glucose tolerance in adult rats.2.Verification of VP1 protein expression in VSV-VP1 recombinant vaccineTo verify the correct expression of VPV protein of VSV-VP1 recombinant vaccine protein,two experiments were performed to verify:Western Blot detection and indirect immunofluorescence.The VSV-VP1 recombinant vaccine coated with CV-B4 VP1 was used to infect Vero cells.CV-B4 virus solution was used as a control to collect the cell fluids for SDS-PAGE.Western Blot was used to compare the CV-B4 VP1 and CV-B4 in recombinant vaccines.The size of the target protein;Vero cells were infected with VSV-VP1,CV-B4 multi-antibody serum was incubated,and DAPI staining was performed to observe whether the CV-B4 VP1 protein was normally expressed under fluorescence microscope.3.Comparison of Immune Effects of VSV-VP1 and Inactivated CV-B4 VaccineBALB/c male adult mice were randomly divided into 4 groups:CV-B4 inactivated immunization group,VSV-VP1 subcutaneous immunization group,VSV-VP1 intranasal immunization group,and blank control group.Each mouse was immunized at equal doses.The ocular sera of the mice were collected by centrifugation at 0,1 w,2 w,3 w,4 w,5 w,and 6 w after immunization to detect the Ig G content,and the feces of the mice were collected to measure the Ig A content.In order to further verify the efficacy of the two vaccines,a mother-borne antibody protection experiment was performed.The newly-pregnant adult female rats were randomly divided into 5 groups,namely CV-B4 inactivated group,VSV-VP1 intranasal immunization group,VSV-VP1 subcutaneous immunization group,negative control group and blank control group.The pregnant mice in the control group were injected with PBS in equal doses.For one-day-old pups born,an equal dose of intramuscular injection of CV-B4virus was used.Daily record of suckling body weight and clinical symptoms.Microscopic examination of pathological changes in suckling mouse tissue.The blood of suckling mice infected with CV-B4 virus was detected by ELISA on the first,third and fifth days.The supernatant was collected by centrifugation at 4°C overnight,and the cytokines IL-6,IL-10and IFN-γin plasma were measured.And the expression level of TNF-αto preliminary understand the role of the above cytokines in CV-B4 infection.Research results1.SDS-PAGE gel electrophoresis showed that the CV-B4 VP1 protein was successfully expressed in the VSV-VP1 vaccine.At the same time,the fluorescence signal of CV-B4 VP1 protein expression was also observed by indirect immunofluorescence assay.2.Comparison of VSV-VP1 genetic engineering vaccine and CV-B4 inactivated vaccine against adult mice showed that VSV-VP1 intranasal immunization produced a lower level of Ig G after immunization than VSV-VP1 recombinant vaccine and CV-B4inactivation.The vaccine produces high levels of Ig G.3.HE staining of the heart,lungs,hindlimbs and brains of the neonatal mice infected with the VSV-VP1 genetic engineering vaccine and the CV-B4 inactivated vaccine.The results showed that the CV-B4 virus had more damage to the brain and muscles.Large,mainly caused by brain,hindlimb lymphocyte infiltration,edema,and fibrous tissue proliferation,and the emergence of muscle bundle fracture.In the positive control group,neurons showed degeneration and necrosis,stromal inflammatory edema and a large number of lymphocytic infiltration in the choroid plexus of the ventricle.The detection results of plasma cytokines IL-6,IL-10,IFN-γand TNF-αshowed that the mean value of cytokines in each group of VSV-VP1 intranasal immunization group was lower than that of VSV-VP1 subcutaneous immunization group and CV-B4inactivated group and positive control group,and the difference was statistically significant.Conclusion1.One-day-old BALB/c suckling mice were selected and infected with 107TCID50/c V-B4 virus via intramuscular injection.After 3 days of infection,typical symptoms of the central nervous system,stable onset time,and lethality were observed.Highly characterized CV-B4 infection model;2.Western blotting and indirect immunofluorescence showed that the VPV-VP1-coated CV-B4 VP1 fragment was successfully expressed;3.The immune effects of VSV-VP1 vaccine and subcutaneously immunized CV-B4vaccine against suckling mice by intranasal immunization and subcutaneous immunization have different degrees of protection,especially the intranasal immunization of VSV-VP1vaccine.After immunization,it exerts a strong immune effect on the body.The results of the experimental study on maternal antibody protection showed that the protective effect of intranasal immunization against VSV-VP1 vaccine was better.Based on the above findings,the CV-B4 genetic engineering vaccine constructed with VSV as a vector has a safe and highly effective immunoprotective effect,and will have a broader development prospect in the future.