Design Exploration Of PEDV Spike Protein Proline Mutation And Ferritin Nanoparticle Vaccine

Porcine Epidemic Diarrhea Virus(PEDV),as a member of the alphacoronavirus,can cause contagious enteric disease of pigs with the symptoms of vomiting,diarrhea and death by dehydration,resulting devastating impacts on the global swine industry.PEDV strains can be classified into G1 and G2 genotypes,with G2 being the predominant genotype in current outbreaks.The rapid mutation and challenging control of PEDV present difficulties.The available vaccines of PEDV primarily include inactivated and attenuated live vaccines.However,inactivated vaccines exhibit poor immunogenicity and incomplete inactivation,while attenuated live vaccines carry the risk of rebound.Therefore,the development of a safe,effective,and highly immunogenic vaccine is urgently needed.This study primarily focuses on the design and optimization of a PEDV subunit vaccine from three aspects:1.Compared with truncated proteins,the immunological efficacy of PEDV spike(S)protein trimer is superior.Based on the structure and regional division of PEDV spike(S)protein,we constructed and then expressed PEDV-S trimer and truncated protein using eukaryotic expression system.It was found that compared with various truncated proteins,the PEDV-S trimer could induce stronger neutralizing protection in immunized mice.Furthermore,the serum from mice immunized with the currently prevalent G2 subtype S protein also demonstrated neutralizing protection against the G1 subtype strain.2.The 2P(I1074P,L1075P)mutation enhances the expression level and stability of the PEDV-S protein.By comparing the structures of PEDV-S protein and SARS-Co V-2-S protein,we identified the proline(P)mutation sites that influence the conformational change of PEDVS protein from pre-fusion to post-fusion.We constructed and expressed proline mutant and wild-type(WT)proteins.Compared with the WT protein,the 2P mutant protein exhibits higher expression level and stability,making it suitable for further investigation.3,Ferritin nanoparticles can further enhance the immunogenicity of PEDV-S protein.Using 2P mutant protein as a model to modify ferritin nanoparticles,PEDV-S-Ferritin protein was constructed and expressed.Negative staining electron microscopy observation and nanoparticle size analysis demonstrated the presence of homogeneous nanoparticles.Compared with WT protein,2P-Ferritin protein could induce stronger neutralization protection in immunized mice,with a bias towards a Th1-type response,providing a theoretical foundation for clinical applications.In summary,this study focused on the subunit vaccine of PEDV,providing the theoretical basis and technical support for obtaining safer and more effective PEDV vaccines.