The Preparation Of Inactivated Vaccines For Porcine Circovirus Type 2 (PCV-2)(Yangzhou Strain) Through PK-15 Cells Suspension Culture Process And Its Quality Evaluation Research

Porcine Circovirus Disease(PCVD)is kind of diseases caused by Porcine Circovirus type 2(PCV2).PCV2 was found to be associated with post-weaning multisystemic wasting syndrome(PMWS)and mainly cause the lymph node enlargement,which can make the function of lymph immune system depressed.The disease is prevalent in the world and cause great losses to the swine industry worldwide.There is no effective drugs against this disease.For PCV-2,vaccination with the high-quality vaccines,is still one of the important methods for its prevention and control.'Thus,the quality of the vaccine is directly responsible for the effect of vaccination.Currently,the production of the majority of vaccines used for domestic animals including the PCV-2 vaccine still use traditional cell culture process with roller bottle.The traditional technology had several drawbacks including batch differences,unstable quality and low viral titers,which can not meet the needs for the animal infectious disease prevention.In this study,we used cell bioreactor technology to produce PCV-2 and optimized the production process,providing reliable information for the large-scale cultivation of PCV-2,and the quality evaluation research on the application of the technology of preparation of vaccine.1.Optimal Propagation Conditions of PCV-2 YZ Strain on PK-15 Cells in BioreactorIn order to obtain higher titers of PCV-2 YZ strain by the establishment of PK-15 cell microcarrier suspension culture process,key parameters for cells inoculation,the reactor speed,PCV-2 inoculated time and quantity etc.were optimized in NBC-7.5L bioreactor.The results showed that the growth status of PK-15 cells was the best under following conditions:calf serum;1 ×109 cells per ml;the reactor speed was more than 40 rpm.In addition there was a positive correlation between cell growth and glucose consumption.The best viral inoculation time was 7 days after cell culture.The viral titer was highest on 72 h after inoculation of virus of 0.1 MOI with DMEM medium containing 2%calf serum.This study provided a technically key parameter basis for large-scale production of PCV-2 YZ strain vaccine.2.Screening of Inactivation Conditions of Porcine Circovirus Type 2 YZ StrainTo study the best inactivated condition of porcine circovirus type 2 YZ strain by binary ethylenimine(BEI)and formaldehyde.A strain of YZ was inactivated by BEI with final concentrations of 0.2 ‰?0.5 ‰?1.0 ‰ and formaldehyde with 2.0‰ as a control incubated at 32 ? at different time.The results showed that 0.2‰ BEI could not completely inactivate PCV2 at 32 ? for 72 h while 0.5‰ BEI could inactivate PCV2 completely at 32?for 48 h.Considering the complexity of large production,the optimal inactivated scheme was 1.0‰ BEI on PCV2 virus at 32 ? for 48 h.Vaccines inactivated by BEI or formaldehyde were safety to piglets except that the BEI inactivated vaccine could stimulate immune response more quickly than formaldehyde inactivated vaccine.3.Safety and immune efficacy tests for the vaccineThe qualified vaccine of PCV2(Lot number YH-001,YH-002,YH-003)were inoculated into both piglets and pregnant sows by one dose,two doses and one dose with repeat inoculation.The results of post inoculation twenty-one days in pigs showed that there were no abnormal responses among all inoculated animals with the dose described above,and there was no significant differences in weight between experimental pigs and control ones.Thus,the vaccines were safety to both piglets and pregnant sows.Piglets and pregnant sows were first vaccinated with vaccines(YH-001?YH-002 and YH-003)and subsequently suffered from boost-vaccination in a three-weekinterval.The pigs were challenged with PCV-2 YZ strain virus containing 4×105.0TCID50 on 2 weeks post boost-vaccination.The results showed that the vaccine group of pigs can get 100%(5/5)protecting pigs from viral challenge,while control pigs were diseased with 100%morbility.The onset of clinical symptoms of the pigs were elevated for body temperature and average relative weight gain,which were significantly different from those of the control group,and the 28d challenged pigs with virus viremia and immunohistochemistry positive.4.Study on growth and decline of antibodies of PCV-2 vaccine in pigsThe piglets and pregnant sows were immunized with the qualified PCV-2 vaccine with different dose of antigen.The results showed that the pigs could be protected from viral challenge if IFA titer of antibodies ?1:500,which also indicates that the titers of antibodies in pigs were positive correlated to protection rate of vaccine.The results of growth and decline of antibodies of PCV-2 vaccine indicated that the immune response could be stimulated quickly with high-level IFA titer(1:600?1:900).The IFA antibody titers could arrive at 1:600?900 post 2 two-weeks after boost-vaccination.The high-level antibody titer could last at least 8 months as shown that the IFA antibody titers were>1:500 post six-weeks and were between 1:420 and 1:480 after vaccination.