Preparation And Immune Effect Of Recombinant Adenovirus Oral Vaccine Expressing Rabies Virus Glycoprotein

Rabies is a zoonotic infectious disease caused by the invasion of Rabies virus into the central nervous system of humans or animals.Once the patient has clinical symptoms,the case fatality rate exceeds 99.9%.The World Health Organization has advised that oral rabies vaccination of animals can prevent the spread of rabies virus in terrestrial carnivore populations and reduce the risk of infection of livestock and humans.In China,there are a large number of free-range dogs,cats,stray dogs and wild animals.It is difficult to intensively vaccinate,and the price of injectable vaccines is high.The injection process is cumbersome and it is impossible to form an effective immune circle for wild animals.At the same time,there is no vaccine on the Chinese market that induces the body to produce protective rabies virus neutralizing antibodies through oral immunization.Therefore,it is extremely important to study a safe,efficient and convenient new genetic engineering oral vaccine for rabies.The recombinant adenovirus Ad Hu5-tRVG-hrGFP expressing three copies of the rabies virus glycoprotein gene is a recombinant rabies vaccine strain that can be vaccinated by the oral route.Sodium Alginate is a natural polysaccharide with the stability,viscosity and safety required for pharmaceutical preparations,and has been widely used in the field of medicine.In this study,Ad Hu5-tRVG-hrGFP was used as a model virus,sodium alginate was used as a slow-release material,and the dosage form was selected to make sodium alginate pellets with a simple process to prepare an oral vaccine of Ad Hu5-tRVG-hrGFP strain.The immune response of mice is studied to provide a better reference for the prevention and treatment of rabies.First,the human embryonic kidney transformed cells（Ad5-293）were cultured and the Ad Hu5-tRVG-hrGFP strain was identified and expanded.With reference to the Ad Hu5-tRVG-hrGFP strain-specific identification primer（t RVG-FP/RP）designed by Dr.Tan Yeping,the DNA of the strain was extracted,PCR amplified and identified.The result of agarose gel electrophoresis of PCR amplification products showed that a specific target band was amplified with a size of about 1,780 bp,which was consistent with the expected result.Recombinant adenovirus strain Ad Hu5-tRVG-hrGFP was cultured by Ad5-293 cells,a total of 80 m L of virus liquid was harvested,and its titer was determined to be 10^7.1 FFU/m L by TCID₅₀ method.Secondly,the preparation process of sodium alginate pellets was explored by single factor experiment and orthogonal experiment.The effects of sodium alginate solution concentration,sodium alginate solution to virus liquid volume ratio,calcium chloride solution concentration and curing time on the encapsulation efficiency,drug loading,drop acceleration and drop distance of the pellets were investigated through single factor experiments.Through L9（3⁴）orthogonal experiment,the best preparation process conditions of pellets were obtained.Orthogonal experiment results show that the best preparation process is:the concentration of sodium alginate solution is 2%,the volume ratio of sodium alginate solution to virus solution is 1∶2,the concentration of calcium chloride solution is 0.4 mol/L,the curing time is 30 min,drops The distance is 8 cm and the drop acceleration is 2.5 m L/min.The results of range analysis showed that the influencing factors of pellet quality were:sodium alginate solution concentration（AGS）>calcium chloride solution concentration（Ca Cl₂）>sodium alginate solution to virus volume ratio>curing reaction time.The encapsulation rate of the pellet aggregate prepared under this process condition was 78.86%,and the drug loading was 15.16%.The titer of the non-lyophilized pellet virus Ad Hu5-tRVG-hrGFP prepared by this process was determined by the TCID₅₀method to be 10^5.5 FFU/m L.The TCID₅₀ method was used to determine the tolerance of artificial gastric juice of unfreeze-dried pellets.The experiment showed that the viral titer of the pellets began to slightly decrease when treated with simulated gastric juice for 2 h,suggesting that the pellets have the potential to resist gastric acid.Finally,mice were orally immunized with pellets,and the fluorescent antibody virus neutralization test was used to detect the serum antibody neutralizing antibody titer to observe the immune effect of oral vaccine-induced mice.Immunogenicity experiments showed that:At 21 days,75%of the mice with Ad Hu5-tRVG-hrGFP virus produced protective anti-rabies antibodies,and 50%of mice without oral freeze-dried Ad Hu5-tRVG-hrGFP pellets produced protective anti-rabies antibodies.The challenge protection experiment showed that the protection rate of mice induced by direct oral administration of Ad Hu5-tRVG-hrGFP virus solution after 21 days against the challenge of rabies virus standard challenge strain CVS-24 was 100%.After 21 days of hr GFP pellets,the protection rate of mice induced by antibodies against CVS-24 challenge was 50%.The above experimental results show that Ad Hu5-tRVG-hrGFP is used as a model and sodium alginate is a sustained-release material,and the recombinant adenovirus sodium alginate sustained-release pellets have the potential of oral vaccines;this study provides a new oral vaccine for rabies reference.