Generation And Immunoreactivity Of Recombinant Human Adenovirus Type 5 Co-Expressing RABV G Protein And SFTSV Gn Protein

BackgroundRabies is a highly lethal acute zoonotic diseases caused by rabies virus(RABV).More than 95%of rabies in China is spread by dogs,followed by cats.There is no effective treatment for rabies and the case fatality rate is almost 100%.The plan is to eliminate human rabies transmitted by dogs in our country by 2030.Vaccination of animal is the most effective way to prevent rabies,but the high cost of traditional rabies vaccines prevents the enforcement of compulsory immunization.Therefore,developing an affordable and efficacious vaccine for rabies control is in urgent need.Severe fever with thrombocytopenia syndrome(SFTS)is a new viral infectious zoonotic disease caused by Severe fever with thrombocytopenia syndrome virus(SFTSV)discovered in 2011.The disease is characterized by rapid onset,high fatality rate and wide distribution.Dogs,cats,cattle,sheep and other domestic and wild animals are all susceptible hosts of SFTSV.Recently,companion animals including dogs and cats have been given more attention for their close contact with humans,and considered as the main potential infection source of animal-to-human transmission.However,currently there is no approved vaccine and drug for SFTSV,so it is urgent to develop SFTSV vaccine to effectively control the spread of SFTS.In summary,both rabies and SFTS are serious zoonoses.Given that SFTSV and RABV share co-hosts and rabies vaccine is compulsory for cats/dogs in China,it is urgent to develop a safe,cheap and efficient bivalent vaccine that can prevent both RABV and SFTSV,in order to be able to cut off the animal transmission route and protect human and animals from infection.Previous studies have shown that adenovirus vector vaccines can induce strong humoral and cellular immunity in vivo and are widely used.In this study,recombinant human adenovirus type 5(Ad5-G-Gn)was constructed by inserting RABV G protein and SFTSV Gn protein with Ad5 as the vector.The biological characteristics and immune protection efficacy of Ad5-G-Gn were analyzed by in vitro cell experiment and animal experiment.Objectives1.RABV G protein gene and SFTSV Gn protein gene were inserted into Ad5 vector to construct recombinant human adenovirus Ad5-G-Gn expressing RABV G protein and SFTSV Gn protein.2.The biological characteristics of Ad5-G-Gn were analyzed in vitro cell experiments.The immunogenicity,Protective efficacy and the potential as a bivalent vaccine were verified by the animal immune challenge protection experiment.Methods1.Construction and identification of recombinant virus Ad5-G-Gn:Target genes RABV G and SFTSV Gn were recombined to human adenovirus type 5 through P2A gene connection.Recombinant virus Ad5-G-Gn expressing RABV G protein and SFTSV Gn protein was constructed,293T cells were inoculated with Ad5-G-Gn and Ad5-GFP to analyze the expression of RABV G protein and SFTSV Gn protein by immunofluorescence and Western blot.2.Mice immunity and virulence identification of Ad5-G-Gn:C57/BL6 male mice(6-8weeks)were randomly divided into 3 groups and immunized with Ad5-G-Gn,Ad5-GFP and DMEM respectively.The body weight,diet and mental state of mice were monitored with in 21 days after immunization.3.Detection of the ability of Ad5-G-Gn to induce specific neutralizing antibody:C57/BL6 mice serum were collected at 2,4,and 8 weeks after immunization,and the neutralizing antibody titers of RABV and SFTSV were detected by FAVN and FRNT test,respectively.4.Protective efficacy test of Ad5-G-Gn:4 weeks after immunization of C57/BL6 mice,intramuscular injection of RABV or SFTSV was performed.The clinical symptoms of RABV infected mice were observed to test the protective effect of Ad5-G-Gn on fatal RABV attack.The viral load of SFTSV in the spleen was tested by qRT-PCR to measure the rate at which SFTSV was cleared by Ad5-G-Gn.5.Detection of the ability of Ad5-G-Gn to induce lymph node recruitment/activation of DCs and B cells in mice:Flow cytometry assay was used to test the ability of Ad5-G-Gn to induce lymph node recruitment/activation of DCs(CD11c+CD86+,CD11c+MHC?+,CD11c+MHC?+)and B(CD19+CD40+)cells in BALB/c mice on day 3,6 and 9 after immunization.6.Detection of the ability of Ad5-G-Gn to induce mice spleen cells to secrete IFN-y and IL-4:4 weeks after immunization of BALB/c mice,the ability of Ad5-G-Gn to induce mice to produce IFN-? and IL-4 specific T cells under specific stimulation of RABV and SFTSV was tested by ELISpot kit.7.Detection of the ability of Ad5-G-Gn to induce specific antibody production in mice:The serum of the BALB/c mice were collected at week 2,4 and 8 after immunization,and the specific Ig/IgG2c/IgG1 titer under specific stimulation of RABV and SFTSV was tested by ELISA.Results1.Through IFA and Western blot experiment,the RABV G protein and SFTSV Gn protein were expressed correctly in Ad5-G-Gn.2.During the 21-day observation period,compared with the Mock group and Ad5-GFP group,Ad5-G-Gn group showed no significant difference in body weight,and the body weight of all three groups of mice showed an upward trend,with good growth state and no abnormal condition.3.The results of FAVN and FRNT experiments showed that Ad5-G-Gn could induce mice to produce high levels of RABV and SFTSV neutralizing antibodies,and the antibody average levels reached 27.72 IU/ml and 1:102 after 4 weeks of immunization,respectively.4.Experimental results of RABV infection showed that the Ad5-G-Gn could protect mice from virus infection by 100%,while the Mock group and the Ad5-GFP group were both 100%infected.SFTSV infected test results showed that the viral load of spleen in Ad5-G-Gn group was significantly lower than that in the Mock group and Ad5-GFP group.5.The results of flow cytometry showed that Ad5-G-Gn could recruit and/or activate more B and DCs cells in mice than the Mock group and the Ad5-GFP group.6.The results of ELISpot showed that under the RABV and SFTSV specific stimulation,the number of spot-forming cells(SFCs)produced by spleen cells of mice immuned with Ad5-G-Gn was significantly higher than that of the Mock group and Ad5-GFP group.7.The results of ELISA showed that Ad5-G-Gn could induce mice to produce high titers of specific RABV and SFTSV Ig,and Ad5-G-Gn could induce a strong Th1 immune response in mice after immunization of 4 weeks.Conclusion1.Recombinant human adenovirus type 5 Ad5-G-Gn expressing RABV G protein and SFTSV Gn protein was successfully constructed.2.Ad5-G-Gn is highly immunogenic that can induce strong humoral and cellular immunity in mice.It has 100%protection against the lethal attack of RABV and has strong protection against SFTSV infection.This indicates that Ad5-G-Gn has the potential to develop as a bivalent vaccine of RABV and SFTSV.