Construction Of Human Norovirus Capsid Protein Antigen Library And GⅡ.17 Evolution Mechanism Research

Norovirus(NoV)is an important pathogen that causes non-bacterial gastroenteritis.The lack of cell culture systems or small animal models for human diseases severely hinder the development of vaccines and therapeutic agents.NoV has rich genetic diversity,but it is difficult to form a cross-protection between different genotypes of viruses.Therefore,analyzing the molecular mechanism of cross-reactivitity between different genotypes will provide important theoretical support for virus vaccine design.In the past few decades,GⅡ.4 was the predominant genotype causing the outbreak.However,the emergence of GⅡ.17 Kawasaki variants surpassed GⅡ.4 and triggered a new epidemic peak in the cold season of 2014/2015.Therefore,exploring the molecular mechanism of the epidemic mechanism of NoV novel variants will provide new strategies for virus prevention and control.This thesis took the representative strains of NoV common genotypes as research objects.By characterizing the immunogenicity of the VP1 protein of each genotype strain,the relationship between virus immunogenicity and genetic diversity was analyzed and characterized.In addition,the molecular mechanism of capsid protein mutations affecting the prevalence of GⅡ.17 recent variants was also studied.Specific contents as follows:1.Construction of human Norovirus capsid protein antigen library.Collecting and sorting out NoV genome database information,selecting 9 strains of GI(GI.1-GI.9)and 19 strains of GⅡ(GⅡ.1-GⅡ.22,excluding swine-derived GⅡ.11,GⅡ.18 and GⅡ.19)as representative strains.28 different genotypes of capsid P proteins were prepared by E.coli prokaryotic expression system.2.Immune diversity of human norovirus capsid P protein.ELISA results of antisera produced from immunized mice showed that the titer of each genotype antisera could reach at least 10~5.And then evaluate the broad-spectrum combination of different genotype antisera.The results showed that 73.9%antisera had weak broad spectrum;GⅡ.5 antisera could cross-react with seven capsid P proteins,which preliminary showed that GⅡ.5 has the potential as a multivalent vaccine.In addition,P protein sequence alignment and evolutionary analysis showed that only 16.5%sites were conserved.The conserved sites 423-431 aa,462-474 aa and517-531 aa may be the cause of cross-reactivity between genotypes.3.Research on the evolutionary mechanism of GⅡ.17 NoV based on cross-immunity.The representative strains of GⅡ.17 four clusters(A-D)of were selected to prepare capsid P protein.Mice immunized with P particle to obtain antiserum.Saliva-receptor binding assay showed that the recent variants(GⅡ.17 C and GⅡ.17 D)have stronger binding ability to A/B/O type HBGA and PGM than previous strains(GⅡ.17 A and GⅡ.17 B),which indicated that the receptor binding sites of GⅡ.17 may changed during the evolution.The cross-reactivity assay showed that antisera of previous strains(GⅡ.17 A and GⅡ.17 B)reacted with recent variants(GⅡ.17 C and GⅡ.17 D)at high OD values from 0.8 to 1.16,while recent variant antisera cross-reacting with previous strains were weak,with OD values between 0.26 and 0.56.The cross-protection assay indicated that the antisera of previous strains had no inhibitory effect on recent variants.Finally,mutations at amino acids 353-363,373-384,394-404 and 444-454 were important sites that affected the immune cross-reactivity in GⅡ.17 NoV.These data indicated that recent pandemic variants GⅡ.17 C and GⅡ.17 D avoided the herd immunity effect of previous GⅡ.17A and GⅡ.17 B strains through changes in receptor binding capacity and antigenic variation.This thesis took the capsid protein VP1 of NoV common genotypes as research objects,systematically characterized the immune diversity,clarified the molecular mechanism of immune cross-reactivity between genotypes,and provided a theoretical basis for vaccine development.At the same time,taking the evolutionary process of the GⅡ.17 capsid proteins as an entry point,rationally designing capsid protein mutants to analyze the impact of key amino acid mutations on immunogenicity,and systematically explainning the difference in receptor binding function of variants of different clusters and changes in immunogenicity,which provided new insights for virus prevention and control strategies.