Construction And Immunogenicity Study Of Recombinant Human Adenovirus Type 5 Expressing F And H Proteins Of Canine Distemper Virus

Canine distemper(CD)is an acute,febrile,highly infectious animal disease caused by canine distemper virus(CDV),which has a very high incidence rate and mortality.CDV can not only infect common canine and feline animals,but also infect various wild animals such as raccoons,weasels,skunks,bearcats,and seals.It can be seen that CD has caused serious harm to the breeding and conservation of dogs,wild animals,and rare protected animals.Vaccine immunization is one of the effective methods for preventing canine distemper,but there is currently no canine distemper vaccine applied to wild animals.Currently,the canine vaccine is a live attenuated vaccine for canine distemper,and the use of live attenuated vaccines has a potential risk of virulence reversion to some extent.So developing a safer and more effective canine distemper vaccine is of great significance.Human adenovirus type 5 is widely used as a viral vector in vaccine development,and adenovirus vectors have good safety;can simultaneously express multiple genes;High viral titer;It can stimulate the body to produce efficient immune responses through various pathways.Therefore,this study used replication-deficient human adenovirus type 5 as a vector and combined with Gateway technology to construct recombinant adenoviruses expressing CDV F and CDV H,respectively,and studied their immunogenicity.We used seamless cloning technology to connect the green fluorescent protein gene(EGFP)and CDV11F/CDV11 H gene into the entry vector p ENTRTM/D-TOPO,respectively,to obtain the recombinant entry plasmids p ENTR-EGFP-CDV11 F and p ENTR-EGFP-CDV11 H.Subsequently,EGFP-CDV11 F and EGFP-CDV11 H were inserted into the adenovirus backbone vector to obtain recombinant plasmids p Ad-EGFP-CDV11 F and p Ad-EGFP-CDV11 H.The correctly identified recombinant adenovirus plasmids were transfected into 293 A cells after PacⅠ enzyme linearization to rescue the recombinant adenoviruses rAd-EGFP-CDV11 F and rAd-EGFP-CDV11 H,and observed through fluorescence microscopy and transmission electron microscopy Indirect immunofluorescence and protein immunoblotting were used to identify the recombinant adenovirus.Recombinant adenoviruses rAd-EGFP-CDV11 F and rAd-EGFP-CDV11 H were immunized separately and in combination through intramuscular injection(i.m.)and intranasal injection(i.n.)routes in mice,and the changes in anti CDV neutralizing antibodies,specific binding antibodies,and antibody subtypes in mice were determined on days 7,21,and 35 after immunization;Detection of alveolar secretory Ig A antibody levels,spleen lymphocyte IL-4,and IFN-γ at 35 dpv Secretion levels and cytokines(IL-2、IL-4、IL-6、IL-10、IFN-γ、TNF-α)Secretion level.The results showed that the rAd-EGFP-CDV11F/rAd-EGFP-CDV11 H infected 293 A and Vero cells showed green fluorescence signals through fluorescence microscopy and transmission electron microscopy,and the typical icosahedron structure was observed under transmission electron microscopy;Through indirect immunofluorescence and Western blot analysis,the recombinant adenovirus was identified to be specifically recognized by anti CDV F/H protein antibodies after infecting 293 A cells and Vero cells,displaying specific bands at approximately 78KDa/81KDa;The rAd-EGFPCDV11F/rAd-EGFP-CDV11 H titers respectively reached 1.62×109 IFU/m L and 1.5×109 IFU/m L。The results of immunizing mice with recombinant adenoviruses rAd EGFP CDV11 F and rAd EGFP CDV11 H showed that in terms of intramuscular injection immunity,the highest neutralizing antibody titer of CDV in the i.m.H immune group was 64,and the titer of specific Ig G antibody in the i.m.F and i.m.H immune groups can reach 1:4096 to 1:25600,with Ig G2a/Ig G1 <1.After immunization,spleen lymphocytes IL-4,IL-10,and IFN-γ secretion of cytokines was significantly increased.In terms of nasal immunization,the highest titer of CDV neutralizing antibodies in mice immunized with i.n.H can reach 16,while the titer of specific Ig G antibodies in mice immunized with i.n.F and i.n.H can reach1:6400 to 1:25600,with Ig G2a/Ig G1 <1.The titer of SIg A in mouse alveolar lavage fluid ranges from1:200 to 1:800.After immunization,the spleen lymphocytes IL-2,IL-6,IL-10,IFN-γ and TNF-αecretion of cytokines was significantly increased.The above results indicate that the recombinant adenovirus vector vaccine can induce strong Th1 and Th2 type immune responses through intramuscular injection and intranasal immunization.In addition,the recombinant adenovirus rAdEGFP-CDV11 H intranasal immunization can induce a good mucosal immune response dominated by SIg A.The results of intramuscular injection and oral immunization with recombinant adenoviruses rAd EGFP CDV11 F and rAd EGFP CDV11 H showed that the highest neutralizing antibody titer of CDV in the i.m.CDV11 H immunized group was 1:16,while the specific Ig G antibody titer of i.m.CDV11 H immunized group was 1:12800.The above results indicate that recombinant adenovirus can induce a certain humoral immune response in dogs after intramuscular injection and oral immunization.In summary,two recombinant adenoviruses were successfully constructed and obtained,which express CDV11 F and H proteins,respectively,namely rAd-EGFP-CDV11 F and rAd-EGFP-CDV11 H.In BALB/c mice,rAd-EGFP-CDV11 F and rAd-EGFP-CDV11 H were safe and immunogenic via both intramuscular injection and intranasal inoculation.They induced humoral and cellular immune responses in mice,of which mucosal immunity were aroused thorough intranasal inoculation.Two recombinant adenoviruses can induce humoral immune responses in beagles by intramuscular injection and oral immunization.rAd-EGFP-CDV11 F and rAd-EGFP-CDV11 H are potential candidate vaccine for CDV.