Development And Immunogenicity Of Virus-like Particles Vaccine For Feline Panleukopenia

Feline panleukopenia virus(FPV)is an envelope-free,single-stranded DNA virus.Infection with FPV in felines causes a severe disease with typical symptoms of a decrease in white blood cells、diarrhea、and fever,which is called feline panleukopenia(FP).At present,vaccine immunization remains the primary measure to prevent FP.,and most of the FP vaccines used in China are conventional vaccines,which are still rely on imports.There is no domestically approved vaccine on the market yet.VP2 is the main component of the FPV coat protein,which is not only highly stable,but also can stimulate the production of specific antibodies in felines,and VP2 has gradually become the main target on novel FP vaccine research in recent years.It has been shown that VP2 can self-assemble into virus-like particles(VLPs),and the novel vaccines prepared from VLPs provide better safety than conventional vaccines and higher immunogenicity than conventional subunit vaccines.In this study,we expressed VP2 protein in prokaryotic cells and allowed it to self-assemble in dialysis solution.Combined VLPs with an adjuvant to prepare VLPs vaccines against the FPV strain 7 isolated in our laboratory.The safety and immunoprotection of the vaccine were verified in animals.The main contents of this study are as follows:1.Assembly and verification of FPV VLPsp ET30a-VP2 recombinant plasmid was constructed and transformed into p Tf16-BL21 receptor cells prepared from the p Tf16 chaperone protein plasmid.In the E.coli expression system,VP2 protein was substantially and solublly expressed.VP2 proteins were selfassembling into VLPs in a dialysis solution.It was found that the VLPs formed by the assembled VP2 protein had a very similar size and morphology to FPV,while the unassembled VP2 protein(N-VLPs)had a morphological size that was somewhat different from FPV.Furthermore,we confirmed the hemagglutination and antigenicity of VLPs were similar to that of natural virus particles through hemagglutination and indirect ELISA assays.2.Evaluation of the immune efficacy of FPV VLPs vaccine.VLPs and adjuvants were mixed to prepare a virus-like particle vaccine.The safety and immunogenicity of the vaccine were verified by animal tests,and it was found that the immunogenicity of VLPs was better than that of N-VLPs,and the effect was comparable to that of the commercial vaccine Fel-O-Vax-PCT.To explore the minimum immune dose of VLPs,the immunization effects of low,medium and high doses were compared,and it was found that the medium dose of VLPs might be the most appropriate immunization dose.To further verify whether the vaccine could protect kittens from FPV wild-type infection after immunization,challenge-protection were carried out on VLPs medium-dose immunization group,Fel-O-Vax-PCT immunization group,and normal saline immunization group,as well as a negative control group.It was found that kittens of normal saline group died from feline panleukopenia within 14 days after challenge,while the survival rate of kittens of the VLPs medium-dose immunization group and FelO-Vax-PCT immunization group and mock group was 100%,and the rectal swab and tissue viral load of kittens of the VLPs group were lower than those of the Fel-O-VaxPCT group.In conclusion,the vaccine could protect kittens from FPV infection,and the medium-dose VLPs vaccine was more effective in virus clearance compared with Fel-OVax-PCT.In addition,at the cellular level,it was verified that high-titer VLPs serum had a good neutralizing effect on different FPV strains,indicating that the VLPs vaccine had the potential to prevent different feline panleukopenia virus variants.