Construction And Immunogenicity Of Recombinant AAV9 Expressing Glycoprotein Of Rabies Virus

Rabies is an ancient and acute zoonotic disease with high mortality,which is caused by rabies virus(RABV)infection.There are about 55,000 human rabies deaths in the world annually;China is a high incidence region of rabies.Rabies virus is a neurotropic virus of family Rhabdoviridae and genus Lyssavirus.The fatality rate of rabies is nearly 100%once patients appear clinical signs and symptoms.The main intervention strategy in rabies control is vaccination.Adeno-associated virus type 9(AAV9)is defective virus belonging to family Parvoviridae and genus Dependovirus.AAV9 has low immunogenicity and toxicity,and it is effective in crossing the blood-brain barrier.In this study,the primary protective antigen G protein gene of rabies virus was cloned into AAV9 to generate the recombinant adeno-associated virus rAAV9-GFP-RABVG.The expression,immunogenicity and protectability of G protein in the rAAV9-GFP-RABVG were evaluated by cell culture in vitro and animal immunological and challenge protection experiments.OBJECTIVE1.Adeno-associated virus type 9 was used as vectors to express RABV glycoprotein G to construct the recombinant adeno-associated virus rAAV9-GFP-RABVG.2.To detect G protein expression by cell culture in vitro.To evaluate immunogenicity and protectability of rAAV9-GFP-RABVG by animal immunological experiments.Providing the reference for the development of new recombinant viral vector rabies vaccine.METHODS1.293T cells were infected with rAAV9-GFP-RABVG,and G protein expression were detected by indirect immunofloresence assay and Western blot.2.Female BALB/c mice(6-8 weeks old)were vaccinated with rAAV9-GFP-RABVG in skeletal muscle of hind legs.Body weights,diet and coat color of mice were monitored daily for 21 days.The safety of the recombinant virus was preliminary evaluated by monitoring change of body weight and status of mice.3.Female BALB/c mice(6-8 weeks old)were vaccinated with rAAV9-GFP-RABVG in skeletal muscle of hind legs.Blood samples were collected at 2,4,8,12 and 24 weeks after immunization to isolate peripheral serum.Fluorescent antibody virus neutralization(FAVN)test was uesed to detect rabies virus neutralizing antibody(VNA)titer and the duration and variation of VNA.4.Flow cytometry was performed to assess the effects of rAAV9-GFP-RABVG on the recruitment and/or activation of B,T and DC cells in mice.5.ELISpot kit was uesed to determine the ability of IFN-? and IL-4 secretion in mouse splenocytes,which induced by rAAV9-GFP-RABVG in the presence of specific stimulant.6.ELISA kit was uesed to determine the ability of IL-2,IL-4,IL-10 and IFN-?secretion in mouse splenocytes,which induced by rAAV9-GFP-RABVG in the presence of specific stimulant.7.Challenge protection experiment.Female BALB/c mice(6-8 weeks old)were vaccinated with rAAV9-GFP-RABVG in skeletal muscle of hind legs,at 21 days after immunization,mice were challenged with HuPBN3 in skeletal muscle of opposite hind legs.Clinical signs of rebies and survival were observed daily for 21 days,brain tissues in dead mice were isolated for RT-PCR to determine whether rAAV9-GFP-RABVG can protect mice from lethal doses of rabies virus.RESULTS1.RABV G protein expression in rAAV9-GFP-RABVG infected cells were detected by indirect immunofloresence assay and Western blot,and the molecular weights is 70kD corresponding to the expected size.2.Compared with mock group and AAV9-GFP control group,mice in the rAAV9-GFP-RABVG recombinant virus group showed slight body weight changes and showed no abnormal behavior or neurological symptoms.3.Detection of VNA titers in immunized mouse serum showed that rAAV9-GFP-RABVG could induce strong and sustained VNA compared with AAV9-GFP control group.VNA titer was 13.66 IU/ml at 2 weeks after immunization.VNA titers reached the peak at 8 weeks after immunization and maintained high level at 24 weeks after immunization.4.Flow cytometry showed that rAAV9-GFP-RABVG could induce more recruitment and/or activation of B cells(CD19+ and CD40+)and DCs cells(CDllc+ and CD80+,CDllc+ and CD86+,CD11c+ and MHC ?)in mice compared with mock group and AAV9-GFP control group.Significantly more IFN-?-or IL-4-secreting CD4+ T cells and IFN-?-secreting CD8+ T cells were detected in splenocytes of mice immunized with rAAV9-GFP-RABVG.5.Results of IFN-? and IL-4 ELISpot assays showed similar trends.Compared with mock group and AAV9-GFP control group,spot forming cells(SFCs)were significantly higher in splenocytes of mice immunized with rAAV9-GFP-RABVG.6.ELISA showed that rAAV9-GFP-RABVG induced significantly secretion of IL-2,IL-4,IL-10 and IFN-? in splenocytes compared with mock group and AAV9-GFP control group.7.Results of challenge protection experiment showed that typical clinical signs of rebies were observed in mice of mock group and AAV9-GFP control group,and the survival rate was 0%at the end of the observation period.No clinical signs of rebies were observed in mice of rAAV9-GFP-RABVG recombinant virus group,and the survival rate was 100%at the end of the observation period.RT-PCR results of brain tissue in dead mice of mock group and AAV9-GFP control group showed dead mice died of rabies.CONCLUSION1.The recombinant adeno-associated virus rAAV9-GFP-RABVG expressing rabies virus G protein was constructed successfully.2.The rAAV9-GFP-RABVG recombinant virus induced a specific immune response against rabies virus in mice including strong humoral and cellular immune response.It provided mice with full protection against lethality rabies virus.It was demonstrated that the recombinant virus has the potential of a new rabies vaccine candidate.