Study On The Subunit Dual Vaccine Of Swine Fever And Porcine Epidemic Diarrhea

At present,Classical swine fever（CSF）and Porcine epidemic diarrhea（PED）are still the focus of disease prevention the pig industry in China.Sows often need to be vaccinated with traditional CSFV vaccine 3 to 4 times,3 to 5 times or more PEDV vaccine per year.To reduce the frequency of sow vaccination and immune stress,and reduce the risk of African swine fever virus（ASFV）infection caused by excessive contact between personnel and pigs.In this study,a subunit dual vaccine（named E2-S1）based on CSFV protective antigen E2 and PEDV protective antigen S1 was prepared,and the vaccine was comprehensively evaluated by Balb/c mice,rabbits and pigs,and the results showed the E2-S1 subunit dual vaccine can be used for effective prevention and control of CSFV and PEDV.1.In this study,a suspension Hek-293F cell was successfully domesticated,and CSFV protective protein E2,PEDV protective protein S1 and E2-S1 fusion protein were successfully expressed in Hek-293F cells through transient transfection expression technology.The purified E2,S1 and E2-S1 recombinant proteins were emulsified with ISA 201VG adjuvant to prepare vaccines,and immunized Balb/c mice with E2,S1 and E2-S1 vaccines at a dose of 10μg,respectively.After using the indirect ELISA method detects serum antibodies,it was found that the E2-S1 immunization group can simultaneously produce high levels of E2 and S1 antibodies（Ig G）,which are significantly higher than the E2 or S1 immunization group.The immunized mouse serum was tested for CSFV and PEDV neutralizing antibodies,and it was found that the E2-S1 immunization group could simultaneously produce high levels of CSFV and PEDV neutralizing antibodies,with titers of 1357 and 3413,respectively（neutralizing titer 1:1357,1:3413）,The CSFV neutralizing antibody titer of the E2 immunization group was 599（neutralization titer 1:599）,and the PEDV neutralization titer of the S1 immunization group was 1536（neutralization titer 1:1536）,E2-S1 can produce higher levels of neutralizing antibodies than E2 or S1 alone.The good immune effect of E2-S1 shows that there is good compatibility between E2 and S1 protein.E2 and S1 may be intramolecular adjuvants for each other.After fusion expression,mutual immunity enhancement phenomenon appears,the mechanism needs to be further studied.In addition,the cytokine test results show that E2,S1and E2-S1 vaccines can induce high levels of IL-4,but almost no IFN-γ,the E2,S1 and E2-S1vaccines mainly elicited humoral immune response mainly regulated by Th2 cells.2.The E2-S1 protein was emulsified with ISA 201VG adjuvant to prepare vaccine,and immunized 4 New Zealand rabbits at a dose of 20μg,4 New Zealand rabbits were immunized with commercial E2 subunit vaccine and PBS respectively,as the vaccine and negative control group.Using the American IDEXX swine fever antibody detection kit to detect rabbit immune serums,it was found that E2 antibody blocking rate of E2-S1 vaccine immunization group and the commercial E2 subunit vaccine immunization group could both reach more than 90%,indicating that the E2-S1 vaccine has good immune effect.Rabbit serum was tested for CSFV neutralizing antibody,and it was found that the neutralizing antibody titer of the E2-S1immunization group was 4245（neutralizing titer 1:4245）,which was higher than the commercial E2 subunit vaccine group of 1641（neutralizing titer）1:1641).After challenge with the C strain CSFV,all rabbits in the PBS group showed typical fever and could not be protected.The rabbits in the E2-S1 immunization group and the commercial E2 subunit vaccine immunization group did not appear typical fever,it showed that the E2-S1 vaccine could produce the same complete protective effect as the commercial E2 subunit vaccine.In addition,the E2-S1 protein and ISA 201VG adjuvant were emulsified to prepare a vaccine,followed by immunization of 15 pregnant sows with an immunization dose of 100μg.Using the American IDEXX swine fever antibody detection kit and the indirect ELISA method established by the E0 protein to detect E2 and E0 antibodies,28 days after the first immunization,the average blocking rate of E2 antibodies increased from 24%to 78%,28 days after the second immunization,the average blocking rate of E2 antibody increased to 86%,however,the average OD450 value of the E0 antibody in the tracking serum is around 1.0,indicating that E2-S1 vaccine can also produce high levels of E2 antibody after being used for clinical pig immunization,which has good immunogenicity.3.The purified E2-S1 recombinant protein was emulsified with ISA 201VG adjuvant to prepare a vaccine,and immunized PED negative sows at a dose of 100μg.Pigs were simultaneously immunized with an equal volume of PBS as a control group.Before the challenge,Ig G,Ig A,and SIg A antibodies in the sera of piglets and sows and colostrum were detected by indirect ELISA established by S1 protein and the IDEXX porcine epidemic diarrhea Ig A antibody kit,respectively.And finding that high levels of SIg A antibodies（S/P value of 1.2）and Ig G antibodies（OD450 of 3.1）can be produced in colostrum of E2-S1immunization group,high levels of Ig A antibodies（S/P value of 1.0）and Ig G antibodies（OD450 of 3.2）were also produced in the serum of sows in E2-S1 immunization group,but the corresponding Ig G,Ig A and SIg A antibodies were not detected in the PBS immunization group.Before the challenge,the serum PEDV neutralizing antibody titers of sows and piglets in E2-S1 immunization group were detected,and it was found that they could reach 717 and 346（neutralization titers 1:717,1:346）,but the neutralizing antibodies of PBS immunization group are not detectable.The 4-day-old piglets from the E2-S1 and PBS immunization group were challenged with PEDV,and it showed that the piglets in the PBS group survived 0/5,the E2-S1vaccine immunized piglets survived 3/5,indicating that E2-S1 vaccine was effective for the piglets with a protection rate of 60%,and the E2-S1 vaccine has a good protective effect.4.In this study,Eu³⁺-DTTA labeling reagent and Eu³⁺nanosphere were used to label E2and S1 proteins,respectively,and dissociation-enhanced time-resolved fluorescence immunoassay method E2-DELFIA and nano-microsphere time-resolved methods fluorescence immunoassay method S1-nano-TRFIA based on the principle of double antigen sandwich were established.The coincidence rate,specificity and sensitivity of E2-DELFIA and IDEXX-CSFV-ELISA were 92.96%,96.19%and 91.06%,respectively.The coincidence rate,specificity and sensitivity of S1-nano-TRFIA and PEDV-S1-ELISA methods were 93.82%,90.59%and 96.47%,respectively.E2-DELFIA and S1-nano-TRFIA methods can not only evaluate the level of E2 and S1 protein antibodies before and after the immunization of E2-S1vaccine,but also these two methods have the advantages of short incubation time and simple operation,which are suitable for high-throughput detection.In summary,the E2-S1 subunit dual vaccine has good immune and protective effects and is expected to be developed into a commercial vaccine for CSFV eradication and PEDV effective prevention.E2 and S1 proteins may act as intramolecular adjuvants for each other,and the mutual immunity enhancement phenomenon is worthy of further study.The established E2-DELFIA and S1-nano-TRFIA methods can accurately and quickly evaluate E2 and S1antibodies,and provide rapid diagnostic tools for immune evaluation of E2-S1 vaccines.