Sabin Inactivated Polio Vaccine (SV-1 Human Deploid Cell) Preclincal Studies

In 1894, The most massive epidemic situation of polio was borken in America according to records. After that,people do much more research on this disease. In the 1950s Thomas Weller and Frederick Robbins first successfully developed poliovirus cultivation technique in live cells. Jonas Salk sucessfully developed the first inactivated polio vaccine in the next year depend on homas Weller and Frederick Robbins's research (hereinafter referred to as IPV).6 years latter, Albert Sabin developed the first attenuated polio vaccine (hereinafter referred to as OPV), because of low price, many country take it in to EPI. In 1970 China choose the OPV into EPI. In 2000,the western pacific was confirmed as Polio-free areas by WHO,including China. But the VAPP and cVDPVs caused by OPV could threaten human health and even affect public health and social stability. In addition, the wild poliovirus is endemic in Afghanistan and Pakistan. It is very easy to import the virus to China. For instance, the imported polio virus has circulated in XinJiang province in 2011. WHO reported that Polio eradication is very urgency for public health in the worldwide On May 26,2012. And WHO would do a integrated and detailed strategy for the last stage of Polio eradication.The main objective of the strategy for the last stage is polio eradication. The strategy include polio eradication and cVDPVs eradication. As the member of WHO, It is our responsibility to implement the strategy. While there is only one sIPV manufacture in China, and can not supply demand of market.This study was focus on developing a new inactivated polio vaccine by using a much more safer cell human lung fibroblast cell (hereinafter referred to as SV-1) as the cell substrate. We inoculated the poliovirus on SV-1, proliferation, when 90% CPE was found we harvest the virus. After clarification, ultra-filtration, density gradient centrifugation and inactivation, we got the three sub-sero type polio bulk. Finnally through dose proportion, the sIPV vaccine was obtained.Test results showed that the inoculum concentration of three types of poliovirus is 0.01-0.1 MOI, culturing with 199 culture medium containing 0.5% newborn bovine serum, when 75?100% CPE was found we harvest the virus.antigen content is high and yield stability in the harvest. Then dealing with the harvest fluid by the 4-level clarification filter, two stage of ultrafiltration concentration, then centrifugating the ultrafiltration fluid by sucrose density gradient centrifugation, the centrifugation parameters selected was 30% of the first sucrose concentration,55% of the second sucrose concentration,30000 rpm of centrifugal rotational speed.after centrifugating 10 hours,collecting poliovirus in the different zone. Test results showed that average removal rate of protein of type three is greater than 99%, the average recovery rate of D-antigen is greater than 98.5%. The experimental results of inactivated process show that the three type of poliovirus all can inactivated within 80 hours by 90 ?g/ml and 60 ?g/ml of the final formaldehyde concentration completely, the inactivated time of 90 ?g/ml of the formaldehyde concentration is less than the inactivated time of 60 ?g/ml of the formaldehyde concentration.The inactivated process be determined to the ultra-centrifugate fluid inactivated by 90 ?g/ml of the final formaldehyde concentration under the condition of 37 ×1.0 ? for 6 days, after filtered using 0.2 ?m filter, then continue inactivating for 6 days, a total of inactivated time is 12 days.The formulation of trivalent vaccine process and the results of experimental of immunogenicity showed that the M199 solution containing glycine can be as a protectant, The doses of monovalent concentrate of type ?, ?, ? respectively choose 30/40/30 DU per 0.5 ml, Inoculating on experiment animals, the diluted multiples of the medium positive seroconversion rate of type ?, ?, ? is 3.4/3.8/3.3 in sIPV(SV-1) respectively. The dose of type ?, ?, ? in IPV(SV-1) satisfy respectively the requirements of 3?9 times dilution of the dose about the medium positive seroconversion rate of the neutralizing antibody. in accordance with the results of long-term immunogenicity study showed that for Sabin strains inactivated polio vaccine (SV-1 human diploid cells) of confirmation immune dose, based immune of 3 needle is superior to 1 or 2 needle, and neutralizing antibody levels till 84 days still maintain a high level, and have immune protection effect.For Sabin strain inactivated polio (SV-1 human diploid cells), preclinical studies have shown that production process is feasible and stable. The purity of sIPV (SV-1) is high, sIPV (SV-1) is safe and effective, and sIPV (SV-1) is suitable for the volume production.