Purification And Experimental Immunogenicity For Recombinant Rabies Virus Expressing MERS-CoV S1 Protein

Middle East respiratory syndrome?MERS?is a highly contagious zoonotic disease caused by Middle East respiratory syndrome coronavirus?MERS-CoV?.Since the first case of MERS patients was diagnosed in 2012,it has received widespread attention of World Health Organization?WHO?because of its widely spread worldwide and high mortality.The first case of imported MERS in China was reported in 2015,which undoubtedly added challenges to China's MERS prevention and control,and also sounded the alarm.Epidemiological investigations showed that dromedary was an important intermediate host of MERS-CoV,and humans infected MERS-CoV by direct or indirect contact with diseased animals.Therefore,strengthening the new vaccine research for MERS,immuning the animal hosts such as dromedary is the key to prevention and control MERS.With the gradual upgrading of vaccine quality and safety requirements by vaccine regulatory authorities,vaccines that were simply and extensively produced were not been able to meet market demand.It is necessary to improve the purity of vaccines and to strengthen the research on the purification process of vaccines.Density gradient centrifugation and precipitation were the classic methods for virus separation and purification.The disadvantages include limited impurity removal effect,low purity of vaccine,poor safety,and large side effects,which make it impossible to scale up linearly for large-scale production.Some new separation and purification technologies,such as membrane filtration and chromatography,received extensive attention due to their highly degree of automation,ease of operation,and high recovery.Based on this,this study aimed to establish a suspension culture process for the recombinant rabies virus rSRV9-MERS_S1,study its concentration and purification method,and evaluate the immune effect of purified virus by animal test,thus laying a foundation for the research of MERS vaccine.1.Preliminary study on suspension culture of rSRV9-MERS_S11 The best MOI was determined by comparing the growth curve and virus titer of BHK 21 cells after different inoculation of multiple infections?MOI?.At the same time,parameters such as harvest time and number of harvests were optimized to determine the suspension culture conditions of the virus.The results showed that when MOI was 0.05 and cell density was 2×10⁶ cells/mL,the virus titer reached 2×10⁸ TCID₅₀/mL 48 h after inoculation.Then the cell supernatant was collected by centrifugation,and cell pellet was uniformly resuspended with the same volume of fresh medium.Afte continuely culturing for 48 h,the virus titer was up to 2×10⁹ TCID₅₀/mL.2.Study on concentration and purification of rSRV9-MERS_S11 After the harvested virus was inactivated,three purification methods(zinc acetate precipitation+Sepharose 4FF gel filtration chromatography,500KD hollow fiber ultrafiltration column+Sepharose 4FF gel filtration chromatography,500KD hollow fiber ultrafiltration column+Cellufine^TM Sulfate affinity chromatography)were used to screen a mild,high recovery and high purity purification method.The results showed that 500 KD hollow fiber ultrafiltration column+Sepharose 4FF gel filtration chromatography recovered the highest antigen content,50 mL of recombinant virus stock solution obtained 1.82 mg protein,and the removal rate of hybrid protein was97.02%;the purified virions had a better morphology under electron microscope.Considering the cost of production and other aspects,the method of concentration and purification of virus was finally determined.3.Study on experimental immunization of the purified rSRV9-MERS_S11 The purified virus were prepared by 500 KD hollow fiber ultrafiltration column+Sepharose 4FF gel filtration chromatography.At the same time,different doses?2?g,10?g,50?g?of purified virus and unpurified virus were used to immunize mice.The immunogenicity and immune effect of the purified virus were evaluated by measuring anti-MERS-CoV IgG antibody titer and anti-MERS-CoV neutralizing antibody level.The results showed that in terms of anti-MERS-CoV IgG antibody titer and anti-MERS-CoV neutralizing antibody level,the antibody level increased with the increase of the dose and number of immunization.The purified virus had a good immunogenicity and induced a good humoral immune response in mice.Purified virus induced mice to produce high levels of humoral immune responses earlier than unpurified viruses.