Generation And Immunoreactivity Of Recombinant Adenovirus Expressing SFTSV Gn Protein

BackgroundSevere fever with thrombocytopenia syndrome(SFTS),a zoonotic infectious disease caused by SFTS virus(SFTSV).Cases of SFTS were first reported in China in 2009,subsequently in South Korea,Japan,Vietnam and other countries.The fatality rate of this disease can reach 31%,It has the characteristics of rapid onset,high infectivity,wide infection spectrum and wide distribution,which seriously threatens human health and animal husbandry safety.There are currently no approved therapies and SFTSV vaccines for human,companion animals,or livestock.It was found that SFTSV glycoproteins(Gn and Gc)play an important role in the formation of virus particles and adhesion to new target cells.There are a wide range of neutralizing epitopes in subdomains II and III of SFTSV Gn head,which can produce higher levels of specific neutralizing antibodies.In addition,some researchers collected the serum of severe SFTS patients,and found that the serum of survivors contained SFTSV Gn specific IgG antibodies,but Gc specific antibodies were not detected.The results showed that neutralizing antibodies to SFTSV Gn were highly correlated with patient survival.In this study,Gn was selected as an important target for SFTSV vaccine research.Currently,adenovirus is widely used in vaccine development as a gene delivery vector with the advantages of high titer,good safety,and induction of strong mucosal,humoral and cellular immune responses in the organism.Therefore,this study aims to construct recombinant adenovirus type 5 vector vaccine expressing SFTSV Gn,and analyze its biological characteristics,protective properties and induced immune responses through in vitro and animal experiments.Objective(1)Recombinant human adenovirus type 5 vaccine Ad5-Gn expressing SFTSV Gn protein was successfully constructed.(2)The biological characteristics of Ad5-Gn were detected by in vitro cell experiments,and the pathogenicity,protection and immunogenicity of Ad5-Gn were evaluated by mice animal experiments,which laid a foundation for further development of SFTSV genetic engineering vaccine.Methods(1)Construction and identification of Ad5-Gn:The ORF region of SFTSV Gn gene was inserted into human type 5 adenovirus vector by enzyme digestion and ligation to obtain recombinant adenovirus Ad5-Gn by Adeasy packaging system.Ad5Gn,Ad5-GFP and DMEM were inoculated into cells,and the expression of SFTSV Gn was identified by IFA and western blot.(2)Immunization procedures and safety evaluation of Ad5-Gn:SPF grade BALB/c mice were randomly divided into Ad5-Gn group,Ad5-GFP group and blank group(n=6 mice/group)and respectively immunized with Ad5-Gn,Ad5-GFP and DMEM.The diet,behavior,body weight and mental status of mice were monitored continuously within 21 days after immunization to evaluate the safety of recombinant adenovirus Ad5-Gn and Ad5-GFP.(3)Detection of antibody level induced by Ad5-Gn in mice vivo:Serum of 6-8 weeks old female BALB/c mice was collected at 2w,4w and 8w of immunization,and neutralization antibody of SFTSV was detected by fluorescent reduction neutralization test.The proportion of specific antibody subtypes,especially IgG1 and IgG2a,induced by Ad5-Gn was determined by enzyme-linked immunosorbent assay.(4)Evaluation of the protective effect of Ad5-Gn against SFTSV infection:IFNAR-/-BALB/c mice were infected with SFTSV by intramuscular injection of 2×105FFU in the left hind limb after 4 weeks of immunizing.The behavior,weight,diet and mental status of the mice were observed within 21 days after infection,and the clinical symptom rating scale,survival curve and body weight change curve were drawn.The liver and spleen tissues were taken for histopathological identification,and the liver,spleen,brain and lung tissues were taken for real-time quantitative fluorescence PCR(qRT-PCR)to detect SFTSV RNA load.(5)Detection of recruitment/activation of DCs and B cells in mice induced by Ad5-Gn:On the 3rd,6th and 9th day after immunizing female BALB/c mice,the inguinal lymph nodes and peripheral blood cells of the mice were collected and isolated,and the recruitment/activation of DCs and B cells in mice induced by Ad5-Gn were detected by flow cytometry.(6)Detection of the ability of Ad5-Gn to induce specific T cell production in mice:After immunizing BALB/c mice for 4 weeks,mice spleen lymphocytes were collected to detect the number of Ad5-Gn produce IFN-y and IL-4 specific T cells by ELISpot.Flow cytometry was used to analyze the production of specific CD8+T cells and CD4+T cells that secreted IFN-y and IL-4 in the spleen of mice induced by Ad5-Gn.Results(1)Recombinant adenovirus Ad5-Gn were successfully constructed,and SFTSV Gn were successfully expressed by immunofluorescence assay at the cellular level.Western blot proved that Ad5-Gn could successfully express SFTSV Gn,and the size(61 KDa)was consistent with the expected.(2)During the observation period of 21 days after immunization,the mice in the Ad5-Gn group showed an upward trend in body weight,smooth hair,normal diet and good mental status,which were basically the same as those in the Ad5-GFP group and the blank group.(3)Neutralizing antibody experiments demonstrated that Ad5-Gn could induce BALB/c mice to produce high levels of SFTSV neutralizing antibodies,up to 1:373.3.ELISA assay showed that Ad5-Gn could induce high levels of IgG1 and IgG2a specific antibodies in mice,which were biased towards Th1 immune response.(4)SFTSV infection protection assay showed that Ad5-Gn protected mice from lethal challenge with SFTSV.SFTSV virus RNA was discovered in spleen,liver,brain and lung tissues of mice by qRT-PCR in Ad5-GFP group and blank group,but hardly detected in Ad5-Gn group(<0.1 TCID50/mg).The spleen and liver of Ad5-GFP group and blank group showed obvious pathological changes,while Ad5-Gn group showed no obvious abnormalities.(5)Flow cytometry analysis showed that Ad5-Gn could induce mice to recruit/activate more DC cells and B cells than Ad5-GFP group and blank group.(6)The ELISpot assay showed that Ad5-Gn induced mice spleen lymphocytes to produce more specific T lymphocytes secreting IFN-y and IL-4 cytokines under the stimulation of SFTSV intact virus particles or SFTSV Gn protein.The results of flow cytometry proved that the proportion of CD8+T cells,CD4+Thl and Th2 cells generated by spleen lymphocytes in Ad5-Gn group was higher than that in Ad5-GFP group and blank group.ConclusionReplication defect recombinant adenovirus Ad5-Gn expressing SFTSV Gn protein with good safety was successfully constructed in this study.Ad5-Gn has good immunogenicity,and a single dose of Ad5-Gn can induce mice to rapidly produce high levels of neutralizing antibodies against SFTSV,which completely protects mice against lethal SFTSV challenge.Further studies indicated that Ad5-Gn could rapidly induce mice to effectively activate and recruit DCs and B cells,induce mice to generate more specific CD8+ T cells and CD4+ T cells,and enhance cellular and humoral immunity in mice.