Development Of Oral Rabies Vaccine For Wildlife

Rabies virus belongs to Rhabdoviridae-Lyssavirus,infect all warm blooded animals and popular all over the world,human once infected the virus,there is no effective treatment,the death rate is almost 100%.Although There is no detailed rabies epidemic data of wild animals in China,human rabies was caused by the bite of wild animals and the rabies virus was isolated from the dead wild animals happened now and then.It indicates that rabies epidemic exists in the wild animals in China.According to the experience of Europe and other developed countries,the effective way to eliminate the wild animal rabies is to put on oral rabies vaccine in the field.But in China,there are only inactivated vaccines for dogs by intramuscular injection,not suitable for wild animals.Therefore,the research of large-scale production of oral rabies vaccine for wild animals is of great importance to the prevention and control of wildlife rabies in china.On account of above contents,the following experiments have been carried out in this study:1.Screening of attenuated rabies virus strains.Experiment content:?The strain was passaged continuously in rat brain.A strain of rabies virus CNX8601 isolated from the Ningxia area of China was continuously passaged in rat brain,virus latency,virus titer and peripheral pathogenicity were detected.·?The strain was passaged continuously in Vero cells.The rat brain adapted CNX-M strain was continuously passaged on Vero cells,and the latent period,virus titer and peripheral pathogenicity of the strain were detected.? Screening and purification of attenuated strains.5 attenuated strains were screened by plaque assay,and their primary pathogenicity and immunogenicity were evaluated.2.Culture of highly immunogenic rabies virus in bioreactor.Test contents:? High density culture of Vero cells in bioreactor.The Vero cells were cultured in NBS 40L bioreactor and carrier,and the cell culture conditions were explored.14 batch experiments were carried out to determine the parameters such as pH,Do,temperature,medium perfusion rate and so on.?Culture rabies virus by bioreactor.Using vero cells as a substrate,harvest virus liquid flowly by NBS 40L bioreactor.Through 9 batches test,to determine the parameters of the time of add virus and virus multiplicity of infection(MOI),virus harvest time and so on.?Establishment of fluorescence method for detection of virus titer.3.Preparation and characterization of liposome attenuated live vaccine.Experiment contents:? Prepare of vaccine.The blank liposomes were prepared by reverse evaporation method.1:1 ratio of blank liposome and live attenuated vaccine blended and freeze-dried to prepare the attenuated live liposome vaccine.?The morphology of liposome encapsulated virus particles was observed by electron microscopy,and the size distribution and particle size were detected.4.Observation of the immune effect of liposome attenuated live vaccine in mice.Test content:preparation of live attenuated vaccine and live attenuated Liposome Vaccine,oral immunization was carried out in mice respectively,the production of neutralizing antibodies and cellular immune were detected in mice.5.Dose of liposomal oral attenuated live vaccine.Test content:Beagle dogs as animal model,chicken head as the bait,2ml,4ml and 8ml oral dose of immune Liposome Vaccine,detection of seroconversion rate,serum levels of neutralizing antibodies and immune persistence.Through the above experiments,the following results are obtained:1.Rabies virus attenuated strain screening results showed:?The CNX8601 strain was passaged continuously for 50 generations in rat brain,named CNX-M,the incubation period is 11?14 days starting,stable to 5-7 days;the virus titer could reach 5.671gLD50/0.03ml,subcutaneous pathogenicity is 1.51gLD50/0.03ml,adapted the street virus to fixed virus.?The rat brain fixed virus adaptation by Vero cells to 50 generations,named CNX-V,the virus titer increased gradually,can reach 6.31gLD50/0.03ml,subcutaneous pathogenicity is 1.51gLD50/0.03ml,brain fixed virus strain adapted to Vero cell culture.Plaque purification and rat brain transmission were performed using CNX-V strains to enhance viral titer,a attenuated strain was screened out,named CNX-V-5,Mice orally took 0.2ml once,no mice pathogenicity,neutralizing antibody reached 1.5IU/ml,and the positive rate was 90%.2.Bioreactor cultures of highly immunogenic rabies viruses determined the following process parameters:?The inoculation density of Vero cells was 4?6 ×106/ml,and the parameters of cell culture stage were pH7.2,dissolved oxygen 70%,stirring speed 100RPM and glucose maintained at 0.5.?The Multiplicity of Infection of CNX-V-5 strain was 0.1.The parameters of virus culture stage were pH7.4,dissolved oxygen 70%,stirring speed 100RPM and glucose maintained at 0.3.3.Preparation and characterization of liposome attenuated live vaccine,Transmission electron microscopy showed that the liposomes prepared in this method were uniform in size and round or oval in shape.The average size of rabies virus liposome was about 14.25 m,and the average encapsulation efficiency was 68.6 ±3.68%.4.In vivo immune effect of mice showed:?None of the 32 mice showed any morbidity,indicating that the attenuated vaccine did not cause pathogenicity in mice.?Compared with the neutralizing antibody:the neutralizing antibody were stably maintained 3IU for 1?6 months in the liposome attenuated live vaccine group compared with the antibody remained at 2IU in inactivated vaccine group,and the difference was statistically significant.? T lymphocyte subsets:the proportion of CD3+ and CD4+ in the attenuated live vaccine group was higher than that in the attenuated live vaccine group.The CD4+/CD8+ difference between the two groups was also significant,P<0.05.?NK cell activity:compared with live attenuated vaccine group and live attenuated vaccine group,t=0.301,P>0.05,the difference was not significant.?IL-2 test:liposome attenuated live vaccine group compared with live attenuated vaccine group,t=2.55,P<0.05,there was significant difference.?IFN-? detection:compared with live attenuated vaccine,the attenuated live vaccine group was t=3.33,P<0.01,the difference was very significant.5.The dose of liposome attenuated live vaccine was demonstrated:?The seroconversion rate of observation:the conversion rate of 2ml group:the 1 months was 100%,the 3th month was 80%,to the 6th month was 60%,and to the 15th month was 30%;4ml group:the first 3 months were 100%,the first 12 months were 90%,the 15th month was 80%;8ml group:the first 9 months were 100%,the 15th month was 90%.?Neutralizing antibody level:8ml dose group was the highest,4ml dose gtoup was middle,2ml dose group was the lowest,and the neutralizing antibody levels decreased prolong the time in the 3 groups.Through the above results,the following conclusions are drawn:1.Successful screened and adapted rabies virus attenuated vaccine strain:CNX-V-5,vaccine made by this strain can induce neutralizing antibody and failed to pathogenic to mice and dogs;2.Established the rabies virus production process by bioreactor,the establishment of the process provided the technical support for veterinary freeze-dried liposomal oral live attenuated vaccine of large-scale culture,also will reduce the production cost for future large-scale use.3.The preparation method of blank liposome was established.The liposome solution was mixed with the virus harvest liquid and freeze-dried to prepare the freeze-dried live attenuated rabies vaccine.The encapsulation efficiency of liposomes was about 68%,and the average size was 14.25 ? m.4.As an adjuvant of live attenuated rabies vaccine,liposome played an important role in neutralizing antibody and cellular immunity in mice.5.Take the dogs as animal model,the dosage and delivery bait of freeze-dried live attenuated rabies liposome oral vaccine were preliminarily explored,test results showed that with chicken head as bait,4ml and 8ml immune dose produced good immune effect.Innovation point of the thesis1.Obtained the first candidate vaccine of attenuated rabies vaccine with independent intellectual property rights and good immunogenicity for the production of wild animal vaccines in china.2.Complete the Vero cell bioreactor high-density culture research,establish Vero cell high-density culture technology,Vero cell culture density>107/ml,is 10 times of the conventional culture cell density.3.The liposome was firstly used as an adjuvant in oral rabies vaccine,and proved that it has significant humoral and cellular immune enhancement through oral immunization.4.For the first time,the industrialized production of oral rabies vaccine for wild animals has been made,which fills the gap of rabies vaccine for wild animals in china.