Preparation And Immune Characteristics Of Dominant Genotypic Human Norovirus-like Particles

Human norovirus（HuNoV）is an important pathogen causing epidemic acute gastroenteritis.Lack of the mature in vitro culture system greatly limits the development of its vaccines and specific antibody drugs.The GII.4 HuNoV had been dominant in the world for the past decades,in recent years,the original non-epidemic strains such as GII.17 and GII.2[P16]had started to suddenly break out and cause epidemics,among which the variant strain GII.17 emerged at the end of 2014 and surpassed GII.4 to become the main epidemic strain in China and some Southeast Asian countries.Due to the long-term inability to achieve stable culture of HuNoV,the virus-like particles（VLP）based on the baculovirus expression system had become an alternative tool to resreach the evolution mechanism,antigenic diversity and outbreak mechanism of HuNoV.In this study,we took the dominant genotype of HuNoV as the research object,and analyzed their cross-protective effect by characterizing the antigen-antibody cross-reaction among each genotype.In addition,the broad-spectrum monoclonal antibodies were prepared and characterized based on capsid P protein.The specific experiment contents are as follows:1.Preparation and identification of dominant genotype HuNoV virus-like particles:we systematically characterized the genome structure and capsid protein function of the GII.2[P2]strain GZ2015-L335.The genome nucleotide sequence of the strain GZ2015-L335 was 7496 bp in size,and phylogenetic analysis showed that it was GII.2[P2]genotype.The results of SDS-PAGE and Western blot showed that the molecular weight of recombinant protein VP1 was about 58 k Da.We observed the protein could assemble into virus-like particles（VLP）with a diameter of approximately 30 nm by transmission electron microscopy.And the results of ELISA showed that the VLP had high binding activity with the antiserum of GII.2[P2]capsid P particles.In addition,the VLP showed a broad binding to both secretory salivary receptors,including A/B/O and non-secretory salivary receptors.2.Analysis of immune differences among HuNoV dominant genotypes:mice were immunized with VLP to prepare antiserum.The results of ELISA showed that the titers of each antiserum were above 10⁴;the results of cross-reaction showed that the GII.3 and GII.8 antiserum cross-reacted with heterotypic capsid P protein,while GII.4 and GII.17antiserum did not cross-react with capsid P protein;the results of cross-protection showed that the mice immunized with each type of VLP alone could only produce blocking antibodies against corresponding genotype,but there was no cross-blocking with other genotypes.3.Evaluation of HuNoV dominant genotype combined immune effect:preparation of the antiserum by co-immunized mice with VLP.The results of ELISA showed that the titers of the antiserum against each type of VLP were above 10⁵,showed that the combined immunization could induce more potent antibody responses in mice than immunization alone.The results of cross-reaction showed that the antiserum could interact with 13 capsid P proteins,indicated that the serum has a relatively broad binding activity.In addition,the results of cross-protection showed that the BT₅₀ of the four VLP receptors blocked by antiserum was 1,592（95%CI,1,016-2,333）,7,015（95%CI,2,460-20,796）,3,206（95%CI,857-9,204）and 1,355（95%CI,160-4,731）,respectively,indicated that the antiserum could simultaneously block the four antigens binding to HBGAs.4.Preparation and identification of HuNoV monoclonal antibody:A total of 12hybridoma cell lines that could stably secrete monoclonal antibodies were screened.The2A11 3D4 strain was purified and identified,and found that it had rubost antibody reactivity,with an antibody titer was 1:512,000 and broad binding activity to various HuNoV.This thesis systematically characterized the cross-reaction and cross-blocking activities of antiserum of HuNoV dominant genotypes,analyzed the cross-protective effect between common genotypes,and provided a theoretical basis for vaccine design and research.At the same time,the successful preparation of broad-spectrum monoclonal antibodies also provided an experimental conditions for the establishment of a rapid detection method for HuNoV.