The Research Of Coxsackievirus B 5 Cold Adapted Attenuated Live Vaccine

Coxsackie virus B 5(Coxsakievirus B5,CVB5)is an enterovirus that infects children under 5 years of age.It causes hand foot and mouth disease,and diarrhea in infants,and even complications such as aseptic meningitis,myocarditis,I type diabetes.This threads the health of infants worldwide.However,there is no effective treatment update,and there is no vaccine to prevent CVB5 infection.Therefore,the development of CVB5 vaccine is urgent.The purpose of this study is to prepare CVB5 attenuated vaccine by cold-passage method,and to evaluate its safety and efficacy in animal experiments.In order to obtain CVB5 temperature-sensitive strain,we carry out the method of cold-passage method.CVB5 virus stored in our lab was filtered and inoculated in the Vero cell,and cultured at 37oC.The harvest was the wild virus,named JZ-WT.Then the virus was passaged 50 generations at 33oC.The virus at passage 10,20,30,40,50were named JZ-P10,JZ-P20,JZ-P30,JZ-P40,and JZ-P50,respectively.The titers of virus harvested at every 10 generation were measured at 33 and 37oC.The temperature sensitivity was determined by comparing the titers between different generations and temperatures.The JZ-P50 temperature-sensitive(ts)strain was evaluated for its safety and efficacy.The virus was purified and filtered.The safety evaluation is based on the clinical features of CVB5-infected neonatal BALB/c,such as the body weight changes,weakness,hunchbacked,paralysis,and death,as well as the individual,organ and pathological sections.The efficacy of ts strain was assessed by the viral immunogenicity and protection.The total and neutralizing antibodies in the serum of infected-mice was measured for the immunogenicity.The mother mice were immunized with ts JZ-P50 and the newborn mice were challenged with wild-type CVB5.The body-weight loss and death rates of the infected-mice were evaluated for the protection of ts CVB5 strain.Finally,CVB5 genome of different passages were sequenced.The mutations of nucleotide and amino acid in viral genome analyzed for the molecular mechanism of CVB5 temperature-sensitivity,attenuation.The results showed that:(1)from the 20 generation,CVB5 virus were gradually adapted to grow at 33oC,and decreased its adaptability to grow at 37oC,(2)from 40to 50,the ratio of the titer at 33 and 37oC was more than 100 times,indicated that the virus had become a temperature sensitive type from the 40th passage,(3)from the CPE of infected Vero cells,Hela cells and KMB17 cells,CVB5 grew suitably in Vero cells.The results of the safety assessment of CVB5 ts strains by animal experiments were suggested that,(1)after three injected doses,the weight of JZ-WT group mice were decreased gradually at 10~6TCID50/mouse,while the weight in the negative control and the JZ-P50 group mice continued to increase slowly,(2)from the clinical index,organ weight in the negative control group and the JZ-P50 infected group were significantly higher than those of the wild JZ-WT infected group,(3)from the histopathological section,no damage was observed in the tissue of JZ-P50 infected mice.Based on the above results,it indicated that the CVB5 strain was attenuated.The results of the efficacy evaluation showed that,(1)the titer of serum total antibody against CVB5 in the three doses immunized mice was 1:500,the neutralization antibody titer was 1:10,and the total antibody titer of the single immunized mice was 1:50,and the neutralization antibody was not detected,(2)the protective ability of passive immune mice was detected,and it was found that the neonatal mice challenged with CVB5 wild-type strain in the JZ-P50 immunized-mother mice group were all survived,but the neonatal mice challenged with CVB5 wild-type strain in the PBS-immunized-mother mice group were dead in 11 days.This proved that the attenuated strain has a good protective effect on mice model.Genome sequencing of a serial of cold-passage strains showed that many sites of nucleotides and amino acids occurred mutations which was involved in viraltemperature sensitivity and attenuation.These mutant sites included nucleotides C106T,T664C,A754T,T800C,A1322G,G1514T,T1515G,G1517A,T1519C,C2821A,A2827T,T2870C,G3133A,G3543A,A3574G,A3672G,T4476A,G6592A,amino acids Q4L,Q257H,W258G,I259T,T693N,E695V,G797D,E934K,K944R,M977V,S1245T,G1950E.In this experiment,we obtained a serials of CVB5 cold-passage strains,and further confirmed that these strains were cold-adapted,te mperature-sensitive,attenuated,safe and protective.This laid a foundation for the pilot and large-scale culture of the CVB5vaccine.