Construction And Characterization Of Chimeric Virus-like Particles Containing The Multiple Epitopes Of Enterovirus 71(EV71) And Coxsackievirus A16(CA16)

Enterovirus 71(EV71)and Coxsachievirus 16(CA16)are the main pathogens of hand,foot-mouth disease(HFMD)and infect under 5 years old children.Children co-infected with EV71 and CA16 viruses led to severe neurological damage or death.Co-infection of two virusesresults in the emergence of recombinant viruses.To control and prevent HFMD,developmentof the bivalent vaccines against EV71 and CA16 is necessary.The virus-like particles as a new vaccine strategy exhibited the advantages such as highly immunogenic,non-infectious,and scale production.Currently,the VLPs strategies were employed for new vaccine development,such as papillomavirus vaccine and hepatitis B virus vaccine.Based on the self-assembly of hepatitis B virus core(HBc),we developed chimeric virus-like particles that simultaneously displaying conserved antigen epitopes of EV71 and CA16.Mice were used as animal models to evaluate the immunogenicity and protective efficiency of the chimeric VLPs vaccines.Based on baculovirus expression system,we constructed novel recombinant chimeric enterovirus VLPs based on EV71 VLPs by replacing the single or multiple antigen epitopes of CA16 corresponding EV71 regions.The results shown as follows:First,the three chimeric VLPs were constructed,respectively.The residues between As 175 and Ser81 of HBc were substituted by the fusion fragment of EV71 SP70 with conserved epitope PEP91 of CVA16 VP1 protein to generate the chimeric tHBc/SP,Then,the fusion fragment tHBc/SPA was obtained by fusing the CD4+T cell dominant antigen epitope A3 of EV71 VP2 protein to the C-terminus of tHBc/SP.The chimeric fragments were cloned into pET28 to generate the plasmid ptHBc/SP and ptHBc/SPA,respectively.The plasmid pHBc was used as a control.After transduction of the recombinant plasmid into E.coli,the recombinant protein was expressed,purified and self-assembled into VLPs,respectively.Second,the immugenicity and protective efficiency of the chimeric VLPs were investigated in BALB/c mice.Immunization with the chimeric VLPs induced epitope-or virus-specific IgG and neutralization antibodies against EV71 and CA16 in mice.Compared with inactivated EV71,the Th1 cytokine(IFN-?,IL-2)induced by chimeric VLPs were significantly increased,and the responses of Th2 cytokine(IL-4,IL-10)were decreased.Neonatal mice born to the immunized dams with the recombinant particles were completely protected from lethal EV71 and partially protected from CA16 infection Co-expression of the conserved human MHC class I CD4+ T cell epitope(aa248-263 of VP2)did not improve antiviral immunity of the chimeric VLP vaccine in mice.Our results demonstrate that the experimental combination vaccines comprised of EV71 and CA16 epitopes induce both humoral and cellular immune responses and therefore support further preclinical and clinical development of a bivalent VLP vaccine targeting both CA16 and EV71.Third,the three recombinant transfer plasmids were constructed.The p10 promoter of the plasmid pFBDM was replaced with the CMV promoter to reduce 3CD expression and to increase P1 expression driven by the pH promoter.And then the EV71 SP70 fragment was substituted with the corresponding fragment of CA16 to construct recombinant transfer plasmid pFBDM P1/SP70.Similarly,CA16 SP70,VP2136-150 VP3176-190 and VP448-62 were used to replace the corresponding fragment of EV71 to construct recombinant transfer plasmid pFBDM P1/4M-C3CD,respectively.The recombinant baculovirus BacP1-C3CD,BacP1/SP70-C3CD and BacPl/4M-C3CD were generated using MultiBac system.The recombinant protein was expressed in infected Sf9 cells,separately.After SDS-PAGE,Western blot analysis and purification of recombinant proteins,three kinds of recombinant VLPs were further observed by electron microscopy.These results laid the foundation for new vaccine candidates for control and prevention of children HFMD.