Construction And Immunogenicity Of A Novel Bivalent Recombinant Adenovirus Vector Vaccine Induces Immune Protection Against SARS-CoV-2 And Influenza A Virus H3N2

ObjectiveTo evaluate the immunological protective effect of a novel recombinant bivalent vaccine against SARS-Co V-2 Beta variant and influenza A virus H3N2 in a mouse model.Methods1.Construction of HAd V5-S1-2A-HAHere we construct an adenovirus 5(Ad5)-based bivalent vaccines designed to Spike 1(S1)from SARS-Co V-2(B.1.351)and hemagglutinin(HA)from H3N2 Cambodia strain(A/Cambodia/e0826360/2020),which are named as HAd V5-S1-2A-HA.Furthe rmore,Virus amplification,purification,titration,and in vitro expression verification were carried out.2.Immunization and challenge1)ELISA,hemagglutination inhibition test,pseudovirus neutralization test and ELISPOT assay were used for Low-dose group(1×10~8vp/mouse),high-dose group(5×10~9vp/mouse)and control group detection of humoral and cellular immunological response in BALB/c female mice after a single immunizing by intramuscular injection 2 weeks,then challenge protections against H3N2-X31,H1N1-PR8 and SARS-Co V-2(B.1.351)were performed in high-dose group and control group mice 3-6weeks post immunization.2)ELISA,hemagglutination inhibition test,pseudovirus neutralization test and ELISPOT assay were used for Low-dose group,high-dose group and control group detection of humoral and cellular immunological response in BALB/c female mice after a single-dose intranasal administration 2 weeks,then challenge protections against H3N2-X31 and H1N1-PR8 were performed in Low-dose group,high-dose group and control group mice 3 weeks post immunization,and against SARS-Co V-2(B.1.351)in high-dose group and control group 4 weeks.3)After a single immunizing by intramuscular injection 3 weeks,boosting immunization by intranasal administration;after a single-dose intranasal administration 4 weeks,boosting immunization by intramuscular injection,the boosting-dose is 5×10~9vp/mouse.ELISA,hemagglutination inhibition test,pseudovirus neutralization test and ELISPOT assay were used for detection of humoral,mucosal and cellular immunological response in BALB/c female mice after a boosting immunization 2 weeks.Priming low-dose by intramuscular injection and boosting by intranasal administration(I.M.1×10~8vp+I.N.)in mice,then challenge protections against H3N2-X31 and H1N1-PR8 were performed in mice 3 weeks post-boost,and against SARS-Co V-2(B.1.351)4 weeks.Results1.After a single immunizing by intramuscular injection of HAd V5-S1-2A-HA immunization,HA-specific humoral immunity and S1-specific cellular immune responses could be detected in the low-dose group,while the high-dose group induced robust dual-antigen(S1,HA)-specific humoral and cellular immune responses,and could completely protect mice against H3N2-X31 challenge and extenuate SARS-Co V-2(B.1.351)infection based on reduction of viral load in lung of mice,delayed death occurs in immunized mice with high-dose group of HAd V5-S1-2A-HA after H1N1-PR8 virus infection.2.After a single-dose intranasal administration of HAd V5-S1-2A-HA immunization,the low-dose group could hardly induce dual-antigen(S1,HA)-specific humoral and cellular immune responses,but the high-dose group could,and could almost protect mice against H3N2-X31 challenge and extenuate SARS-Co V-2(B.1.351)infection based on reduction of viral load in lung of mice,delayed death occurs in immunized mice with high-dose group of HAd V5-S1-2A-HA after H1N1-PR8 virus infection.3.The dual-antigen(S1,HA)-specific humoral and cellular immune responses were significantly enhanced after boosting.The I.M.1×10~8vp+I.N.immunization strategy could completely protect mice against H3N2-X31 challenge and extenuate SARS-Co V-2(B.1.351)infection based on reduction of viral load in lung of mice,delayed death occurs in immunized mice after H1N1-PR8 virus infection,and more than the time for the death of mice after a single injection of immunization.ConclusionIn this study,the recombinant bivalent vaccine HAd V5-S1-2A-HA constructed induced antigen-specific humoral and cellular immune responses in mouse.Antigen-specific humoral,mucosal and cellular immune responses were significantly enhanced in different combined immunization strategies.Furthermore it also induced immune protection against SARS-Co V-2 and H3N2 challenge in mice.Here we also design different immunization strategies,in order to evade the influence of pre-existing NAb responses to Ad5.Therefore,HAd V5-S1-2A-HA has good application prospects for further research and development.