Immunological Evaluation Of Norovirus Tetravalent Vaccine And Identification Of G?.4 Norovirus Blocking Antibody Epitope

Noroviruses(NoV)has become the leading cause of viral acute gastroenteritis worldwide with the large-scale use of rotavirus vaccine.NoV has multiple genotypes and different genotypes are highly variable.The easily emerging of new strains poses a great threat to human health.Currently,there are no vaccines and effective drugs available for the prevention and curation of norovirus infections.To develop a broad-spectrum vaccine against norovirus,we carried out two parts of work:(1)Immunological evaluation of norovirus tetravalent vaccine(2)Analyze the epitope of blockade antibody against the major norovirus epidemic genotype GII.4.Previously,our laboratory has obtained the highly expressed yeast strains that express GI.1,GII.3,GII.4 and GII.17 VLPs,which represent norovirus GI genome and GII genome.To evaluate the immunogenicity of norovirus vaccine,on the basis of previous work,we prepare GI.1,GII.3,GII.4,GII.17 virus-like particles respectively by using yeast recombinant expression system.The electron microscopy images showed that four genotypes of NoV VLPs are present typical spherical particles.Monovalent(GI.1 or GII.3 or GII.4 or GII.17),bivalent(GI.1 & GII.4)or tetravalent(GI.1 & GII.3 & GII.4 & GII.17)vaccine candidates immune mice respectively,the result shows that the tetravalent vaccine group can induced immune antibody response in mice comparable to that of the monovalent and bivalent vaccine groups.And as the increase of immunization times,the specific antibody response increases along.The blocking assay shows tetravalent vaccine group can induce balanced blockade antibodies which can block all of the four genotypes VLPs from binding to attachment cofactors PGM.It indicates that there is no mutual interference among GI.1,GII.3,GII.4 and GII.17 genotypes in combined immunization and the combined tetravalent virus-like particle vaccine can be used as a promising vaccine candidate for the prevention of the infections causing by polytype norovirus.GII.4 is the main causative,also the highest variable genotypes of norovirus.To analysis the blockade epitope of GII.4,we analyze the blocking reaction of GII.4 MAbs against time-ordered GII.4 VLPs by using the antibody blocking assay.The result shows that the blockade antibody GII.4-M5 can block GII.4-2009,GII.4-2012 S and GII.4-2012 H but not GII.4-2006 a or GII.4-2006 b from binding to attachment cofactors PGM.Then we used GII.4-2006 b and GII.4-2012 H as templates to construct a series of chimeric VLPs which recombine at different regions within P2 domain by homologous recombination technique.Through analyzing the antibody responses of blockade antibody GII.4-M5 to different GII.4 chimeric VLPs,we conclude that GII.4-M5 recognize the 361-375 amino acids,located on the top of VP1,highly exposed and close to the binding pocket of HBGA overlapping at site 374.It indicates that the amino acids at 361-375 of GII.4 VLP are the key blockade epitope of GII.4,the major pathogenic genotype of norovirus.The above results proved that the tetravalent vaccine based on virus-like particles is a promising candidate vaccine with broad spectrum protection against norovirus.The key blockade epitope(amino acids at 361-375 on VP1)of the major pathogenic genotype of norovirus were also identified.