Generation And Immunoreactivity Of Recombinant Human Adenovirus Type 5 Co-expressing RABV G Protein And CDV H Protein

Rabies virus(RABV)and canine distemper virus(CDV)are the most harmful pathogens in canine populations and other carnivores.Rabies is caused by RABV with high fatality.About 59,000 human die of rabies from 150 countries each year,with 95%of the cases occurring in Asia and Africa.99%of rabies human cases are caused by canines which infected with the RABV,especially in underdeveloped rural areas.China has set a goal that eliminate human rabies transmitted by canines by 2030.According to data published in 2011,China spent 10 billion yuan on rabies vaccines,mainly for people vaccination and receiving antiserum therapy after being bitten,which is more than 80%of all rabies vaccines produced globally.According to World Health Organization(WHO),vaccinating 70%of dogs for 7 consecutive years can eliminate the rabies transmission.We needs to improve the vaccine coverage of dogs and prevent the transmission of rabies from the source.Canine distemper is a highly pathogenic and highly infectious disease caused by CDV.CDV infects a broad host range of carnivores worldwide with severe immunosuppression and multi-systemic clinical signs including respiratory,gastrointestinal and central nervous system symptoms.CDV infects a broad host range of carnivores worldwide including wolves,foxes,raccoons,kinkajous,red pandas,black bears,giant pandas,ferrets,minks,spotted hyenas,lions and tigers,even in non-human primates.Some studies have shown that Paget malformed osteitis is significantly associated with CDV infection.The fatality rate in animals infected with CDV is 30%-80%,with almost 100%in ferrets.Canine distemper is highly contagious which spread by direct contact between sick dogs and healthy dogs,as well as by airborne droplets in the respiratory tract.Canine distemper is one of the most serious viral infectious diseases in dog breeding industry,which seriously affects the development of fur breeding industry such as foxes and minks.Dogs are the main source of canine distemper virus infection in foxes,minks and other animals.Canine distemper vaccine must be immunized to dogs,foxes,mink,etc.It is necessary to develop a safe,effective and cheap canine distemper vaccine.Rabies virus and canine distemper virus have many common hosts,including dogs,raccoons,pandas,weasels and non-human primates.Vaccination is the most conventional and effective approach for preventing against RABV or CDV.Live attenuated vaccines and vaccinia vector virus vaccines have been commercially produced to protect carnivores from canine distemper.For RABV,the use of traditional live attenuated rabies vaccines raised safety concerns about reversion of virulence and they are no longer available commercially in China,while the live-attenuated CDV vaccines might retain residual virulence in highly susceptible species.Therefore,the development of safe and efficient bivalent attenuated vaccines for rabies and canine distemper is needed.The replication-defective adenovirus human type5(Ad5)vector has been widely used in vaccine research with advantages of safe,efficacious and capable of expressing a variety of exogenous genes simultaneously.Ad5-based human vaccine expressing Ebola virus glycoprotein has been approved for new drug registration in China in 2017.In 2020,the recombinant novel coronavirus vaccine using human type 5 adenovirus as the carrier has entered clinical trial phase ?.In this study,the defective human adenovirus type 5 was used as the carrier to express the RABV glycoprotein and CDV hemagglutinin protein,and the recombinant adenovirus rAd5-G-H was obtained.The protective effect of the recombinant virus was analyzed through experiments in vitro,animal immunity and animal challenge experiments.Objective1.Replication-competent recombinant human type-5 adenovirus was used as vector to expressing the RABV G protein and CDV H protein to construct the recombinant adenovirus rAd5-G-H.2.We will detect RABV G protein and CDV H protein by cell culture in vitro.We will evaluate the immunogenicity and protection of rAd5-G-H using mouse and fox animal models.Our study will provide scientific basis for the development of new combined rabies and canine distemper vaccines.Methods1.The recombinant replication-defective rAd5-G-H co-expressing the RABV G protein and CDV H protein is generated.The morphology and size of rAd5-G-H and rAd5-GFP were observed using a transmission electron microscopy with negative staining technique.,We use PCR and immunofluorescence assay(IFA)to confirm the expression of RABV G and CDV H.2.6-8 weeks old female BALB/c mice were intramuscularly(i.m.)immunized with rAd5-G-H.The mice were observed daily for signs of disease,bodyweight changes or death until 3 weeks after vaccination.3.6-8 weeks old female BALB/c mice and 6 weeks old silver foxes were i.m.immunized with rAd5-G-H.Blood samples were collected from mice and foxes.Animal sera were tested for RABV neutralizing antibodies(VNA)using fluorescent antibody virus neutralization(FAVN)test and CDV VNA using fluorescent reduction neutralization test(FRNT).4.At 3,6 and 9 days after vaccination,inguinal lymph nodes were collected from the immunized mice.At 7 and 14 days after vaccination,peripheral blood was collected from the immunized mice.Flow cytometry was carried out to quantify the immune cells in inguinal lymph nodes.5.At 4 weeks after vaccination,the lymphocytes from spleen isolated from the immunized mice were stimulated with the purified inactive RABV and CDV Secreted IFN-y or IL-4 were quantitated using ELISpot assay.6.At 4 weeks after vaccination,the mice were challenged with 100 fold of 50%intramuscular lethal dose(100 IMLD50)of street rabies virus HuNPB3 strain by i.m and foxes in each group were challenged i.m.with 100 fold of 50%lethal dose(100 LD50)of CDV wild strain CDVQL.Results1.A recombinant replication-incompetent adenovirus serotype5(rAd5-G-H)carrying ORFs of SRV9 G and CDV-11 H genes was generated.The inserted nucleotide components were confirmed by PCR.The morphology of rAd5-G-H and rAd5-GFP in the electron microscope were fairly homogeneous icosahedral with an average diameter of 70?100 nm.IFA indicated that the RABV G protein and CDV H protein expressed in the Vero cells infected by rAd5-G-H.2.All the mice showed no abnormal behavior or neurological symptoms after vaccination.There was no significant difference in body weight changes among all three groups.3.rAd5-G-H can induced strong RABV and CDV VNA in mice and foxes,which suggested that rAd5-G-H has strong immunogenicity.4.In comparison of groups of rAd5-GFP and DMEM,more DCs(CD11c+CD86+,CD11c+MHCI+,CD11c+MHC11+)and B cells(CD19+CD40+)were significantly recruited and/or activates in inguinal lymph nodes of the mice immunized with rAd5-G-H at 3,6 and 9 days post infection.More B cells(CD19+CD40+)were significantly detected in peripheral blood of the mice immunized with rAd5-G-H at 7 and 14 days post infection.5.From ELISpot assays,rAd5-G-H induced significantly RABV and CDV specific IFN-y and IL-4 spot forming cells(SFCs)in immunized mice which indicated that rAd5-G-H elicited both the Thl and Th2 cell-mediated immune responses in immunized mice.6.rAd5-G-H could completely protect immune mice against the lethal challenge of RABV,and completely protect foxes against the lethal challenge of CDV.ConclusionIn this study,the recombinant replication-defective virus rAd5-G-H,co-expressing the RABV glycoprotein and the CDV hemagglutinin,was generated.A single dose immunity of rAd5-G-H induced robust and rapid humoral and cell-mediated immunity against RABV and CDV,rAd5-G-H can protect mice from a lethal RABV challenge and protect foxes from a lethal CDV challenge.The rAd5-G-H may be potentially as a bivalent candidate vaccine protecting animals from RABV and CDV.