Development And Immune Effect Evaluation Of Inactivated Vaccine Against Porcine Deltacoronavirus (Strain HNZK-04)

Porcine Deltacoronavirus（PDCo V）can cause infection of pigs at all stages,mainly causing watery diarrhea,vomiting and severe dehydration in piglets,and seriously endangering the intestinal health and growth performance of pigs.Recent studies have found that the disease can lead to mixed infection of a variety of porcine intestinal coronaviruses.The pig industry suffered serious economic losses for the reason.Vaccination is an effective way to prevent the disease,but there is no relevant vaccine in China.In order to effectively prevent and control PDCo V,this project carried out experimental researches from the aspects of suspension domestication of LLC-PK1 cells,screening of vaccine adjuvants,optimization of vaccine production process,and systematic evaluation of vaccine safety and effectiveness,aiming to develop a safe and efficient vaccine.The main research contents are as follows:1.Suspension acclimation of LLC-PK1 cells.LLC-PK1 cells were acclimated in serum-free suspension by gradually decreasing serum and increasing suspension medium.Cells adapted to suspension growth environment were monoclonal and PDCo V was inoculated into monoclonal cell lines.According to the titer of PDCo V,the PDCo V highly adaptive cell line LLC-PK1Sa was optimized from the monoclonal cell line.2.Screening of immune adjuvants for inactivated vaccine.The vaccine was prepared by combining PDCo V inactivated antigen with ISA201,GEL02,IL-1β,Cp G ODN2395 and MPLA adjuvant,and the piglets were immunized twice,14 days apart.Blood samples were collected at different times after immunization,and serum was sterile isolated.Serum Ig G,Ig G1,Ig G2a antibody levels and IL-4,IFN-γcytokine expression levels were detected by ELISA.Serum neutralizing antibody titers were detected by cell neutralization test.Vaccine adjuvants that could induce humoral immune response and improve cellular immune response and produce high level of neutralizing antibody titers were screened.The results confirmed that the combination of ISA201 and Cp G ODN2395 could significantly enhance the immune effect of PDCo V inactivated antigen.3.Optimization of the production process of inactivated vaccine.The proliferation conditions of PDCo V in suspended cells LLC-PK1Sa were optimized in10L bioreactor according to the single factor experiment method.The safety test and immunogenicity test were used to compare different antigen inactivation technologies and antigen matching technologies.The results showed that the optimal conditions for PDCo V proliferation were as follows:initial cell density 3.5×10⁶cells/m L,dose MOI=0.06,TPCK trypsin concentration 7.5μg/m L,36 hours after infection.The optimal inactivation conditions were as follows:BEI final concentration of 5 mmol/L,32℃for 42 hours.After inactivation,the residual BEI is neutralized with the same amount of sodium thiosulfate to ensure complete inactivation and no residue.The efficacy of PDCo V vaccine can be ensured by using PDCo V virus with the content of inactivated provirus not less than 10^7.0TCID₅₀/0.1m L,and the vaccine can be prepared according to the mass ratio of antigen to adjuvant of 1∶1.4.Safety evaluation of inactivated vaccines.A single dose was inoculated through the neck muscle of target animals at the minimum age of use.Target animals at the minimum age of use and sows at the age of 75-80 days of gestation were inoculated through the neck muscle route for single dose repeated inoculation and one overdose inoculation.The safety of the vaccine was evaluated by observing and recording clinical symptoms and effects on sow performance.Mice and guinea pigs were injected intraperitoneally.The abnormal toxicity of the vaccine was evaluated by observing the clinical symptoms.The results showed that the three batches of vaccine did not cause local or systemic adverse reactions in piglets and pregnant sows,and had no significant effect on the performance of pregnant sows,indicating that the vaccine was safe for piglets and pregnant sows.There were no adverse effects on the growth performance of mice and guinea pigs,indicating that the vaccine had no exogenous toxic substances or unsafe factors.5.Evaluation of the effectiveness of inactivated vaccines.By means of immune challenge protection and determination of serum neutralizing antibody titer,the relationship between serum antibody level and challenge protection,the minimum immune dose to piglets and pregnant sows,the rule of antibody fluctuation,duration of immunization and immunization program were studied.The results showed that when the serum neutralizing antibody was≥1∶32,it could provide more than 80%protection to the pigs.The minimum immune dose was 0.5 m L/head for piglets and1.5 m L/head for pregnant sows.Piglets and pregnant sows developed immunity from21 to 28 days after active immunization,and the duration of immunity was 7 months.The duration of passive immunization was from birth to 28 days of age.The optimal immunization program was as follows:first immunization of sows at 75-80 days of gestation,and reinforcement immunization at an interval of 14 days,2.0 m L/head;Piglets born from the immunized sows were actively immunized at 28 days of age,with an additional immunization of 1.0 m L/head after 14 days.Piglets born from unimmunized sows were actively immunized at 3 days of age,with an additional immunization of 1.0 m L/head at an interval of 14 days.