Construction And Immunogenicity Of Virus-like Particles Of Porcine Epidemic Diarrhea Virus

Porcine Epizootic Diarrhea(PED)is a newborn piglets intestinal infectious disease caused by pathogens of Porcine Epizootic Diarrhea Virus(PEDV).Piglets are mainly caused acute watery diarrhea,vomiting and dehydration,eventually resulting in newborn piglets mortality.The mortality rate reached to 80%-100%.Adult pigs are not caused death generally.The disease was first discovered in Europe in the 1970 s,and later in Asian countries,the first time in the United States broke out in 2013.It is one of the greatest infectious diseases in hindering on China's pig industry development in recent years.Because of the new variant of PEDV,the newborn piglets can not be protectedagainst PEDV infection and death by attenuated and inactivated vaccines prepared from the classical vaccine strain.So it is urgent to exploit a new vaccine to cope with the outbreak of PEDV.Virus-like Particles(VLPs)are hollow virus shells that are assembled by one or several structural proteins of the virus.It does not contain viral nucleic acids and VLPs have no risks of reversion to virulence;the shape and size of the VLPswere similar to those of the na?ve virus.So,it maintained more perfect immunogenicity.In this study,we used baculovirus expression system to successfully construct a recombinant baculovirus containing the main structural protein genes of PEDV epidemic strain to produce PEDV VLPs,and eveluated its immunogenicity.The recombinant plasmids pFastBac-Dual-2S,p FastBac-Dual-2M and the pFastBac-Dual-2E containing respectively PEDV Spike protein(S),Membrane protein(M)and Envelop Protein(E)gene were constructed successfully,and the recombinant baculovirus rpFastBac-Dual-2S,rpFastBac-Dual-2M and rpFastBac-Dual-2E were rescued in Sf9 cells.PEDV VLPs was produced by means of coinfection Sf9 cells.The morphology and composition of PEDV VLPs were identified by transmission electron microscopy observation,indirect immunofluorescence assay and Western Blotting respectively.The results showed that PEDV VLPs containing the major structural proteins S,M and E proteins of PEDV were harvest with the morphology similar to the naive virus.Immunogenicity of PEDV VLPs was analyzed by intramuscular injection in mice.The results showed that PEDV VLPs could induce the higher level of anti-PEDV neutralizing antibody by serum microneutralization(SN)assays,which showed a significant difference compared with the control group.And we think that PEDV VLPs have good immunogenicity.Furthermore,IFN-? and IL-4 were detected in spleen lymphocytes induced by immunized PEDV VLPs through ELISpot assay.The double staining of CD4+ T cells and CD8+ T cells showed that CD4+ T cells and CD8+ T cells secreted higher IFN-? and IL-4 levels,the difference was significant by comparing with the control group.Results indicated that PEDV VLPs can induce cellular immunity and humoral immunity in mice.Therefore,the development of PEDV VLPs based on the exploitation of PEDV vaccine has the potential prospects.PEDV mainly caused gastrointestinal lesions of newborn piglets,so we think that vaccination should be able to induce mucosal immunity of pregnant sows and piglets can obtain enough mucosal immune antibody s IgA.Therefore,we constructed the recombinant plasmids pFastBac-Dual-2CTB of expressing the CTB fusion protein genes on the basis of PEDV VLPs,and further rescued the recombinant baculovirus rp FastBac-Dual-2CTB.Sf9 cells were coinfected by rpFastBac-Dual-2CTB with rpFastBac-Dual-2S,rp FastBac-Dual-2M and rpFastBac-Dual-2E.The chimeric CTB PEDV VLPs was harvested.The morphology and the components of the harvested chimeric PEDV VLPs with CTB was identified through observation transmission electron microscopy,indirect immunofluorescence assay and Western Blotting respectively.The results showed that we obtained chimeric PEDV VLPs with CTB,which are similar to the naive virus in morphology.Chimeric PEDV VLPs with CTB was used to immunize mice and pigs respectively.Serum neutralizing antibody analysis showed that a high level of anti-PEDV neutralizing antibody was secreted in the serum of mice and pigs.The higher level of sIgA antibody was also detected in the intestine fluid of the mice and colostrum of the pig.The resulted demonstrated that chimeric PEDV VLPs with CTB not only induce the systemic immune response,but also stimulate the local humoral immune response.The results of ELISpot analysis and double staining intracellular factor showed that chimeric PEDV VLPs with CTB could stimulate the splenic lymphocytes to secrete higher levels of IFN-? and IL-4 in oral immunization mice.Results indicated that chimeric PEDV VLPs with CTB can induce the body to produce a strong cellular and humoral immune response.In conclusion,PEDV VLPs produced by insect-baculovirus expression system possess a perfect immunogenicity,which shows that the vaccine on the basis of PEDV VLPs have a broad development space and bright prospect.In this study,we selected CTB which could stimulate mucosal immunity,as the molecular adjuvant of mucosal immunization vaccine,and constructed chimeric PEDV VLPs with CTB for oral immunization,and achieved well-deserved immunological effect.This study provides a theoretical basis and data reference for the exploitation of anti-PEDV mucosal immunization vaccine.