| Hand, foot and mouth disease (HFMD) is a viral infectious disease that frequently occurs in infants and young children, the epidemic disease appears in majority of countries and regions around the world. Recently, large scale of HFMD outbreaked in the Asia-Pacific regions and caused significant morbidity and mortality. HFMD have been caused by Enterovirus which mainly due to Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16). CVA16 infection could lead to mild clinical symptoms, and more than 60% of cases were caused by CVA16. Whereas, EV71 often causes central nervous system complications including viral meningitis, encephalitis, poliomyelitis-like paralysis, pulmonary edema,myocarditis and death of infants and young children. In order for EV71 precaution, the inactivated vaccines were produced by three companies in mainland China, also proved highly efficient through III phase clinical trial conducted in 2013, but not listed yet. However, in-depth research is still needed due to the poor understanding for EV71/CVA16 antigenicity and epitopes.The EV71/CVA16 antigenicity and epitopes characteristic, especially identification of the neutralization epitopes is critically important for vaccine design. Several EV71 B celt epitopes were reported including the neutralization epitope SP70 (208-222aa) on EV71 VP1 and the broad spectrum neutralization epitope 141-155 aa on EV71 VP2, which were proved high titer neutralization and effective protection by appropriate carrier protein presentation. However, studies had shown that the epitopes on VP1 and VP2 belongs to linear neutralization epitope that had certain shortcomings for vaccine stability study. For CVA16 epitopes, little research was undergoing, mainly was 176-190aa antigen epitope of VP3. However the epitope was not able to induce rabbit neutralizing antibody, only induce neutralizing antibodies in mice.Ttherefore, CVA16 epitopes still need further comprehensive study. However, the discovery of three antigen epitopes is not sufficient for HFMD vaccine design, so the new antigen epitope in my thesis is meant for this.The purpose of the is to select a series of EV71 and CVA16 epitopes, respectively constructed into antigen presentation carrier by utilizeing prokaryotic expression system and immune animals, then detecting the immunogenicity and neutralizing epitopes of them. A novel antigen presentation platform, Norovirus P particle has high immunogenicity and can self-assembly into 20 nm 24 polymers particle. The surface Ioop2 allow different lengths of foreign fragments to insert, and increase immunogenicity of foreign antigen significantly by expanding antigen exposure. The carrier presentation avoids the shortcoming of poor immune response induced by unique small peptide.According to reports, no cross neutralizing antibodies and protection were found between EV71 and CVA16. We selected 23 feasible epitopes based on alignment of EV71 and CVA16 P1 using the MultAiin interface page, and then constructed them into Ioop2 of Norovirus P particle separately. We named the recombinant plasmid as PP-71-1, PP-71-2, PP-71-3……, according to their sites on P1. By SDS-PAGE and Western Blot, we proved the expression and high purity of the recombinant proteins. After three times of immune of mice, 12 chimeric protein were able to induce binding antibody,7 chimeric protein were able to nduce neutralizing antibody and only 71-6 (HYRAHARDGVFDYYT) can protect neotal mice against EV71 virus infection, located at 176-190 amino acid on EV71 VP3, whereas none of CVA16 epitopes can induce neutralizing antibody.For the identified neutralization epitope 71-6, we found that the PP-71-6 was equal to natural Norovirus P particles, which presenting 20 nm 24 polymers particles formation through structural analysis, indicating that the epitope was located at GH loop and canyon of EV71 viral pentamer. The comparison PP-71-6 with synthetic 71-6 peptide in ELISA and neutralization assays, we identified the 71-6 epitope (176-190 of VP3) acted as conformational neutralizing epitope.In conclusion, we identified a new EV71 neutralization epitope among 23 candidate epitopes utilizing Norovirus P particles, which showed efficient protection in vivo. This study provide theoretical basis for EV71/CVA16 virology and immunology research, and PP-71-6 chimeric protein act as promising vaccine candidate provides valuable design ideas for EV71 vaccine, the discovery of new conformational neutralization epitope will lay a foundation for the preparation of monoclonal antibody and antigen stability study. |
PDF Full Text Request