| Bovine Viral Diarrhoea(BVD)is a globally distributed infectious disease caused by Bovine Viral Diarrhoea Virus(BVDV),a single-stranded positive RNA virus that infects cattle,sheep,pigs,and other ruminants.BVDV causes not only clinical symptoms such as high fever,cough,diarrhoea and abortion in females but also persistent infection(PI)in calves,leading to immunosuppression.The disease is widespread,spreads quickly and lacks treatment,making it one of the most important animal diseases to prevent and control in the cattle industry.BVDV is classified as a Class II infectious disease in China and is a legally reported cause of disease by the World Organisation for Animal Health(WOAH).BVDV is of great economic importance to the cattle industry as it causes huge economic losses every year.Immunization is an effective method to prevent BVDV infection.Vaccination is paramount to prevent abortions in cows and the emergence of persistently infected calves.Vaccination requires inactivated or subunit vaccines with high safety profiles.However,due to the diversified subtypes of BVDV and their rapid variation,the preventive effects of inactivated vaccines are not desirable to prevalent strains and inactivated vaccines are not convenient to administrate intramuscularly;whereas subunit vaccines possess the advantages of a short production cycle and the possibility of fusion expression of superior proteins between subtypes.Therefore,it may be safer and more effective to develop a nasal nebulized E2 subunit vaccine with neutralizing activity.At the same time,the establishment of an E2-blocking ELISA antibody assay will facilitate better monitoring of vaccine efficacy.Details of the study are as follows:1.Construction and protein expression of BVDV E2,E2 Ft,and E2 Fc genesThe E2 gene sequence of BVDV XZ02 was registered as MF278652,and the transmembrane region was removed to form a truncated E2 to construct the BVDV E2,E2 Ft,and E2 Fc eukaryotic expression plasmids.The yield after purification was 1-3mg/100 m L.2.Immunization procedure and efficacy assessment of BVDV subunit vaccinesThe three subunit vaccines were emulsified with IMS 1313 VG adjuvant with a ratio of 1:1(Seppic,France)to a final protein concentration of 200 μg/m L.35 BVDV-negative calves aged 2-4 months were selected and vaccinated by intramuscular injection or nasal nebulization on days 1,21,and 35 of the trial.The positive control was BVDV inactivated vaccine.Serum,feces,and nasal mucosal swabs were collected after secondary immunization,and the Ig A and SIg A levels were measured.The results showed that the Ig A and SIg A levels in the blood(200-300 pg/m L)were higher than that in the mucosal(100-150 pg/m L).Both Ig A and SIg A levels were among the highest in the nasal mucosal immunized E2 Fc group(P < 0.01),and the nasal mucosal immunized E2 Ft and E2 were significantly different(P < 0.05).The ratios of CD4+ and CD8+ T cells in the mucosal immunized E2 Fc and E2 Ft groups were higher than that in the E2 group.Furthermore,the ratio of CD4+ T cells was higher than CD8+ T cells in both E2 Fc and E2 Ft groups,suggesting that the E2-Fc,E2 Ft,and inactivated vaccine induced a stronger BVDV-specific Th1-type cellular immune response than the E2 group.Lymphocyte proliferation experiments demonstrated that the highest lymphocyte stimulation index was observed in the E2 Fc immune and inactivated vaccine groups(p<0.001).In addition,the IFN-γ and IL-2 levels ranged from 150-250pg/m L in the mucosal immunized E2 Fc and E2 Ft groups,which was significantly higher than the IL-4 level.This indicated that mucosal immunized E2 Fc subunit vaccines significantly promote the Th1-type cellular immune response in calves.Specific antibodies produced in calves after immunized with E2,E2 Ft,and E2 Fc subunit vaccines were measured by ELISA.The results showed that the antibody levels induced by the E2 Ft and E2 Fc subunit vaccines were significantly higher than that of the E2 group along with the booster immunizations(P < 0.001).The antibody levels induced by mucosal-administrated E2 Ft and E2 Fc subunit vaccines were higher than those in the intramuscularly injected E2 Ft and E2 Fc groups but were not significantly different from those of the inactivated vaccine group.Neutralization assays and confocal microscopy results in bovine alveolar macrophages demonstrated that FcγRI transports E2 Fc fusion proteins across the mucosal barrier and that FcγRI and E2 Fc co-localizes in the cytoplasm and cell membrane of bovine macrophages,and that this antibody-like dimeric protein prolongs its half-life in vivo by binding to the receptor.The E2 Fc subunit vaccine-induced neutralizing antibodies up to1:64,which was superior to the E2 Ft subunit vaccine and comparable to the inactivated vaccine administered intramuscularly.3.Preparation of monoclonal antibody against BVDV XZ02 virusPurified BVDV-XZ02 virus was inactivated and emulsified with adjuvant in equal volumes and injected subcutaneously into 8-week-old female BALB/c mice at a dose of100 μg/each for 3 times with an interval of two weeks.Spleens of mice were taken for cell fusion on the third day after intraperitoneal shock immunization with 300 μL of inactivated virus.The E2 protein expressed by eukaryote was used as the coated antigen,and five monoclonal antibodies against E2 protein were screened out by indirect ELISA including B6,F10,F11,H12,and E8.All five monoclonal antibodies were identified as Ig G1 isoforms and the light chain were κ-chains by isotype detection kit.The monoclonal antibody summation assay demonstrated that all groups had AI values <10%,suggesting that the five monoclonal antibodies may act on the same antigenic epitopes.Through the analysis of antibody-blocking activity,the blocking rate of B6 was among the highest(87.68%).Therefore,horseradish peroxidase-labeled B6(HRP-B6)was used to explore the subsequent blocking ELISA detection method.4.Development of the BVDV E2 blocking ELISA antibody detection methodThe optimal concentrations of the antigen(3 mg/m L)and HRP-B6(1.4 mg/m L)monoclonal antibodies explored by square titration experiments were 1:1000 and 1:10000,and the critical value was 0.51 The BVDV-E2 blocking ELISA method was established.The five positive sera were tested for Mycobacterium bovis,Pasteurella bovis,bovine nodular rash,bovine infectious rhinotracheitis,and bovine foot-and-mouth disease without cross-reactivity;the compliance rate of the clinical samples was 94.35%;the intra-plate variation coefficient of the blocking ELISA was 2.93%,the inter-batch repeat was >15%and the sensitivity was 1:64.A total of 317 clinical serum samples from Wuhan and Tibet were detected,including 118 positive samples and 179 negative samples This detection method provides a guarantee for detecting the positive rate of BVDV in bovine serum and the immune effect of the vaccine.In this study,HEK293 cells were used to express three recombinant fusion proteins,BVDV-E2,BVDV-E2 Ft,and BVDV-E2 Fc and mixed with 1313 VG adjuvant to produce three subunit vaccines.The immune effects on calves were compared by mucosal immunization and intramuscular injection.The results showed that the efficacy of the E2 Fc subunit vaccine was better than that of E2 Ft and E2 in both mucosal and intramuscular immunization groups,and the overall efficacy of mucosal immunization was higher than that of intramuscular injection,confirming that the E2 Fc protein not only induced Ig A secretion,but also induced specific T cell immune response and Th1-type cellular immune response.E2 Fc protein bound to FcγRI on bovine alveolar macrophages to achieve transmucosal transport,prolonging the residence time of the fusion protein in vivo and promoting the production of specific antibodies.Further evidence is that the E2 Fc vaccine enhances cellular and humoral immune responses by mucosal immunization via nasal spray.In addition,the development of the BVDV E2 blocking ELISA detection method provides a strong guarantee for antibody monitoring of the BVDV vaccine. |
