Antigenic Relatedness Between GII.4 And GII.17 Noroviruses And Generation Of Human Monoclonal Antibodies Against GII.17

Background and ObjectiveHuman norovirus(NoV)have become the leading cause of epidemic and sporadic acute gastroenteritis worldwide.NoV infects people of all ages and causes a mild-to-moderate self-limited illness in previously healthy individuals.However,disease can become life threatening in children,the elderly,and the immunocompromised.NoV was estimated to cause 684 million episodes of diarrheal disease and 212,000 deaths for all ages annually.NoV is extremely antigenically diverse.Currently,NoVs can be classified into 10 genogroups(GI-GX)and at least 49 different genotypes.Since the mid-1990s,GII.4 have caused the majority of outbreaks,with new strains emerging every 2-3 years.However,it was noted that a novel GII.17 variant emerged as a predominant NoV,causing major epidemics in China and several other Asian countries during the epidemic seasons of 2014-2016,replacing the previously predominant GII.4/Sydney 2012 variant.Due to lack of cell culture and small animal infection models,there is currently no licensed vaccine to prevent NoV infection.Vaccine efforts have focused on GII.4 virus-like particles or P particle subunits.It is urgent to explore cross-blockade activity between GII.4 and GII.17 NoVs,and whether GII.17 needs to be considered for future vaccine strategies against NoV.NoV is extremely genetic and antigenic diversity.The discovery of broadly neutralizing monoclonal antibodies against conserved epitopes may spur great efforts on universal epitope vaccine design.Here we report 2 study of AGE outbreaks caused by GII.17 variant.We determined cross-blockade activity between GII.4 and GII.17 NoVs,constructed NoV human antibody library and generated human anti-NoV single-chain variable antibody with neutralizing activity,which may serve as a strategy for designing a new epitope vaccine and therapeutic measures for HuNoV.Methods1.Outbreak investigationStool and blood samples were collected,and stool suspension were detected by one-step real-time reverse transcription-PCR assay.RT-PCR was performed for further genotyping.The positive PCR products were sequenced and genetic identity of the viruses was determined.2.Detection of NoV-specific antibodiesP particles were used as coating antigen,and NoV-specific antibodies in serum(plasma)were detected by ELISA.3.Cross-blockade assayDue to lack of cell culture and small animal infection models,an HBGA-based blockade assay served as a surrogate for neutralization.A blockade assay to measure the ability of serum antibodies to block NoV P particles binding to saliva was developed and optimized.A saliva sample,which showed good binding to the following P particles was coated on a microtiter plate.Sera from NoV-infected patients were used to determine cross-blockade against the HBGA attachment of NoV.The 50%blocking titer(BT50),defined as the maximal dilution(folds)of a serum sample that showed at least 50%blockade(OD)compared with the positive control,was determined for each serum sample.4.Construction of NoV human antibody libraryPBMC from 13 patients were isolated and then reverse transcribed to cDNA.ScFv were amplified by overlap PCR.The phage antibody library was constructed by ligating the scFv into pCANTAB 5E phagemid vector followed by transformation into TGI electrocompetent cells by electroporation.5.Bio-panning of antibodies by phage displayThe rescue of phage library was done with M13KO7 helper phage.The phages were subjected to five rounds of enrichment by bio-panning.Antibodies reactivity to GII.17 were tested using ELISA.6.Generation of human anti-NoV scFv antibodiesThe scFv sequences were cloned into the expression vector pGEX-4T-1.The recombinant expression plasmid pGEX-4T-1-scFv was transformed into BL21 competent cells and induced by IPTG.ScFv antibodies were purified using Glutathione Sepharose 4 Fast Flow resin.The biological activity of scFv antibodies were tested by ELISA.Results1.No cross-blockade activity between GII.4 and GII.17The first outbreak of acute gastroenteritis in a hospital(GII.17 outbreak-1)was caused by the GII.17 variant.Six convalescent-phase sera from the symptomatic individuals(n=10)showed increased antibody titers against the new GII.17 variant with a≥4-fold increase.It was noted that no blockade antibody titers against GII.4 NoV were seen among the convalescent-phase sera with a≥4-fold increase.Sera from GII.4 NoV-infected patients did not cross-block against GII.17 NoV-attachment factor interaction.Our data indicated no cross-blockade activity between GII.4 and GII.17,using human sera from patients after NoV infection.2.GII.17 infected patients whose plasma antibodies exhibited potent and neutralizing activityThe second outbreak of acute gastroenteritis in a troop(GII.17 outbreak-2)was also caused by the GII.17 variant.GII.17 NoV-specific antibody titer was 1:25600-1:409600 in convalescent-phase plasma from GII.17 infected patients.Nine convalescent-phase sera from the symptomatic individuals showed blockade antibody titers of≥200 against GII.17 NoV-attachment factor interaction3.Construction and bio-panning of NoV human antibody librariesPBMCs isolated from GII.17 NoV infected patients whose plasma antibodies exhibited strongly binding and neutralizing activity were used to construct a human anti-NoV scFv phage library of 2.4×107CFU/mL.By extensive bio-panning of the library with specific antigen P particle,we identified 11 GII.17 specific scFv monoclonals.4.Generation and preliminary identification of human anti-NoV scFvThe construction of recombinant expression plasmid pGEX-4T-1-scFv was successful,and scFv was expressed and purification by prokaryotic expression system successfully.We identified 9 GII.17 specific scFv and 5 neutralizing scFv.Conclusions1.From GII.17 outbreak-1,our data suggested no cross-blockade activity between GII.4 and GII.17.Thus,GII.17 may be an important active antigenic type or immunotype that needs to be considered for future vaccine strategies against human NoV.2.Based on GII.17 outbreak-2,we constructed a human anti-NoV scFv phage library using PBMC from GII.17 NoV infected patients and GII.17-specific scFv antibodies were identified from phage library.3.We isolated and characterized human scFv antibodies that have good binding and neutralizing activity.These antibodies can used to identify neutralizing epitopes,which may provide insight into the strategy for designing a new epitope vaccine and therapeutic measures for HuNoV.