Rescue Of Recombinant Virus And Biological Characteristics Of Mineralized Rabies Vaccine

Rabies is a lethal zoonoses infectious disease caused by Rabies virus?RABV?that mainly affects the central nervous system.RV can infect humans and all warm-blooded animals and cause rabies.Once clinical symptoms occurred,the mortality rate reaches almost 100%,about 55 000 people die of the disease each year in the world,mainly due to bites from animals such as dogs and cats.In this study,the reverse genetic manipulation was used to insert the green fluorescent protein?GFP?gene between the N and P genes of the rabies virus vector,and the recombinant rabies virus rSAD-GFP carrying the GFP gene was rescued on Vero cells,and the biological properties of recombinant virus were studied.By measuring the virus growth curve,the results showed that the growth of the recombinant virus and the parent virus in Vero cells was basically the same,and there was no significant difference;the recombinant virus was serially passaged in Vero cells for several generations,no loss of exogenous genes was found,indicating that the foreign Genes can be stably inherited in rabies virus.BALB/C mice were infected with the rescued recombinant virus SAD-GFP by intracerebral injection.The pathogenicity of the recombinant virus was evaluated by this test.There was no visible clinical symptom and death after the recombinant virus was challenged.The rescued recombinant virus belongs to the attenuated strain.Based on the successful construction of the rSAD-GFP strain,we used this plasmid to construct a recombinant plasmid containing the mineralized gene W6,and a series of adjustments were made to the insertion position of the exogenous mineralization gene,and different recombinant plasmids were obtained.Transfection of Vero E6 cells failed to rescue the recombinant virus.The location of the mineralized gene has an impact on the rescue of the virus,and the rabies virus contains a capsule,which is also an obstacle in the rescue process,and studies have found that the presence of continuous aspartic acid?D?or glutamic acid?E?leads to the insertion of strong negatively charged peptides,in this case the virus is also very difficult to save successfully.Mesoporous nanosilica is a kind of hollow solid particle,and there are many small holes like honeycomb holes on the outside.This honeycomb structure makes it possible for silica particles to pass hydrogen bonds,ionic bonds,electrostatic interactions,and hydrophobic forces.In combination with proteins,mesoporous silica can be loaded with viral particle antigens and can protect viral antigens.In this study,we attempted to use mesoporous silica as an in vitro mineralization candidate adjuvant.The recombinant rabies virus rSAD-GFP inactivated antigen was mixed with mesoporous silica nano silica at a certain ratio to study mesoporous silica as effects of vaccine adjuvants on the physicochemical properties of recombinant rabies vaccines,and determination of glycoprotein content in vaccines by M-ABT?antigen content assay method?.The BALB/C mouse model was used to further evaluate the immune effects of the inactivated antigens and vaccine of inactivated antigens with mesoporous nano-SiO₂ as an adjuvant.Anti-RABV G protein IgG antibody was detected in serum by ELISA method,and serum neutralizing antibody was detected by RFFIT.At the same time,Splenocytes on days 21st and 28th were used for ELISPOT assays to analyze the secretion levels of IL-4 and IFN-?;on the 28th day,mice were challenged with 30 LD₅₀ CVS to evaluate the efficacy of vaccine protection.The results show that the mesoporous nano-SiO₂ adjuvant can induce cellular immunity and humoral immunity.The results of the challenge protection experiment showed that the protection rate of pure antigen can reach 70%,and the protection rate of antigen mixed with adjuvant can reach 100%.The study of mesoporous nano-SiO₂ as an in vitro mineralization and adjuvant has laid a foundation for the research of new rabies vaccines.