Construction And Immunological Study On Feline Herpesvirus Bacterial-Like Particles And Gene Deletion Vaccine

Feline viral rhinotracheitis（FVR）,which is widely prevalent worldwide,is an acute contact infectious disease mainly manifests as an upper respiratory tract infection caused by feline herpesvirus 1（FHV-1）.FHV-1 mainly infects cats,tigers,leopards,and other felines,with a mortality rate of up to 50%in infected kittens.Given the high infectivity and prevalence of FVR,vaccination is the optimal measure to prevent FHV-1 infection in felines.Currently,the commercially inactivated FVR vaccine is usually used in combination with the feline infectious rhinoconjunctivitis and feline panleukopenia syndrome vaccines,but these triple vaccines have a relatively poor protective effect against FHV-1 infections.However,commercialized attenuated live vaccines retain the complete virulence gene of FHV-1,which can easily lead to latent infections after animal vaccination and pose a risk of the return of virulence.Therefore,the development of a safe and effective novel FVR vaccine is crucial for the prevention of FHV-1.In this study,two novel vaccines for FVR were developed using Gram-positive enhancer matrix（GEM）surface display technology and CRISPR/Cas9 gene editing technology,respectively.The immunological effects of the two candidate vaccines were evaluated,which laid the foundation for the development of a novel vaccine against FVR with high safety and immunogenicity.1.Construction and characterization of FHV-1 bacterial-like particlesIn this study,bacterial-like particles（BLPs）displaying FHV-1 glycoprotein were prepared using GEM-PA surface display technology.Three recombinant baculoviruses expressing gB-PA3,gC-PA3,and gD-PA3 fusion proteins were constructed using the insect cell-baculovirus expression system,and the expression of three fusion proteins were identified by indirect immunofluorescence（IFA）and Western blot.The results showed that the insect cells infected with recombinant baculoviruses could successfully express three fusion proteins.BLPs were prepared by combining the three fusion proteins with GEM,and the BLPs were identified by transmission electron microscopy,immunofluorescence and Western blot.The results showed that the FHV-1 gB-PA3,gC-PA3,and gD-PA3 fusion proteins were successfully displayed on the surface of GEM.In summary,three BLPs displaying FHV-1 gB,gC,and gD proteins were successfully prepared in this study,which were named gB-GEM,gC-GEM,and gD-GEM,respectively.2.Experimental immunological study of FHV-1 bacterial-like particles vaccineIn this study,mice were intramuscularly immunized with four BLPs named gB-GEM,gC-GEM,gD-GEM,and gB&gC&gD-GEM（gB-GEM,gC-GEM,and gD-GEM mixed in a 1:1:1 ratio of protein amount）mixed with Gel02 to evaluate the immune response.The results of humoral immunization showed that four BLPs could stimulate the production of neutralizing antibodies against FHV-1,and the neutralizing antibody produced by mice in the gB&gC&gD-GEM group could reach a maximum potency of1:2^4.5,which was significantly higher than other groups.The cellular immune responses were detected by flow cytometry and ELISpot,and the results showed that there was a significant activation of both dendritic cells（DC）and B cells in the inguinal lymph nodes in the vaccine groups.The number of splenocytes secretingIFN-γand IL-4 in the vaccine group was significantly increased.The secretion of Th1-associated cytokines（IL-2,IFN-γ,and TNF-α）and Th2-associated cytokines（IL-4,IL-6,and IL-10）in the supernatants of the spleen lymphocytes in the vaccine groups was increased,among these,the secretion of six cytokines in the gB&gC&gD-GEM group was significantly higher than that of the control group.The proportion of activated B cells and central memory T cells in the spleen of the vaccine group was elevated to different degrees.Based on the immune response in mice,we selected gB&gC&gD-GEM mixed with Gel02 for the immunization of cats,and the results showed that neutralizing antibodies against FHV-1 could be detected at the 6th week after the first immunization and the FHV-1 neutralizing antibody was still up to 1:2^3.5 at 17th week after the first immunization.In summary,FHV-1 gB&gC&gD-GEM can induce good immune responses in mice and cats.3.Construction and characterization of FHV-1 TK,gI,and gE gene deletion virusesIn this study,we constructed TK single gene-deletion virus（FHVΔTK）,gI/gE double gene-deletion virus（FHVΔgI&gE）and TK/gI/gE triple gene-deletion virus（FHVΔTK&gI&gE）by knocking out the FHV-1 virulence genes TK,gI,and gE usingCRISPR/Cas9 gene editing technology.Compared with the parental virus FHV-1,there was no significant change in the viral titers of FHVΔTK and FHVΔgI&gE,but the titers of FHVΔTK&gI&gE were significantly decreased.The area of plaque formed by FHVΔTK,FHVΔgI&gE,and FHVΔTK&gI&gE was significantly smaller than that of the parental virus FHV-1,and the area of the plaque formed by FHV FHVΔTK&gI&gE was significantly smaller than that of FHVΔTK.PCR characterization of the three recombinant viruses from different generations showed that FHVΔTK,FHVΔgI&gE,and FHVΔTK&gI&gE had well genetic stability.In summary,three recombinant FHV-1,named FHVΔTK,FHVΔgI&gE,and FHVΔTK&gI&gE,were successfully obtained.4.The immunological study of FHV-1 TK,gI,and gE gene deletion virusesIn this study,the pathogenicity of FHVΔTK,FHVΔgI&gE,and FHVΔTK&gI&gE were evaluated.Cats were intranasally inoculated with three gene deletion viruses(10^6.5TCID₅₀ per cat),and the pathogenicity was evaluated according to the clinical symptoms and detoxification.The results of pathogenicity showed that cats in the FHVΔgI&gE and FHVΔTK&gI&gE groups had a shorter duration of detoxification and milder clinical symptoms than those in the FHV-1 group,which showed higher safety.To further evaluate the protection effect of the gene deletion viruses,cats were immunized with FHVΔgI&gE,FHVΔTK&gI&gE,and Fel-O-Vax（commercial inactivated vaccine）by subcutaneous injection,with a booster at 3rd week,and then challenged with 10⁵ TCID₅₀ FHV-1 by intranasal inoculation at 4th week after the booster immunization.The results showed that,compared with the control group,the FHVΔgI&gE,and FHVΔTK&gI&gE groups had significantly reduced clinical symptoms,viral loads in eye and nasal swabs,pathological damage to lungs and tracheas,and viral loads in the trigeminal ganglia and lungs.In summary,FHVΔgI&gE and FHVΔTK&gI&gE had good immunogenicity and can protect cats from FHV-1challenge.In this study,an FHV-1 BLPs gB&gC&gD-GEM prepared could induce good immunity in mice and cats.Gene deletion vaccines FHVΔgI&gE,and FHVΔTK&gI&gE have good safety and immunogenicity and can significantly reduce the clinical severity after cats are infected with FHV-1.The preparation of the FHV-1bacterial-like particles vaccine and gene deletion vaccine lays the foundation for the development of a novel vaccine against FHV-1.