Generation Of Human Single-chain Variable Fragment Antibodies Against Noroviruses And Identification Of Antigen Mimic Epitopes

Background and ObjectiveNoroviruses（NoVs）remain the leading viral cause of acute gastroenteritis（AGE）worldwide in all age groups,which is an important public health issue.Based on amino acid diversity of the complete VP1 gene,NoVs are divided into ten genogroups.GII.4 NoV has been the predominant genotype of NoV-related AGEs worldwide since the 1990s.However,in the 2014～2016 NoV epidemic seasons,the GII.17 NoV variant emerged and replaced the GII.4 genoclusters as the dominant strain.Meanwhile,in 2015～2018 seasons,GII.4/2012 Sydney new variants caused NoV-related AGE outbreaks in many countries.GII.6 NoV infections are usually sporadic,whereas,in recent years,the incidence of GII.6 variants showed an obvious increasing trend in China and all over the world.Due to the genetic and antigenic diversity of NoV,along with lack of a mature cell culture system and suitable small animal infection models,there is no licensed vaccine or antibody drug for NoVs.Therefore,combined with human single-chain variable fragment（scFv）antibodies against No Vs from an outbreak caused by GII.17 NoV,scFv antibodies were produced using peripheral blood mononuclear cells（PBMC）from persons infected with GII.6 NoV at the stage of recovery and NoV antigen mimic epitopes were preliminarily identified through phage display technology,which will lay a foundation for the development of therapeutic antibodies and further studies of neutralizing epitopes aganist No Vs.Methods1.Construction of human phage antibody libraryPBMC from peripheral blood of patients at the convalescent phase were collected and isolated,total RNA was extracted and then reverse transcribed into cDNA.Variable heavy and light chains of antibodies were amplified by PCR,and were assembled into scFv by splice overlap extension PCR,then was cloned to phagemid pCANTAB-5E to construct a phage antibody library.The recombinant rate and diversity of the antibody library were determined by colony PCR and DNA sequencing.2.Bio-panning of human NoV phage antibody libraryNoV P particle was taken as an antigen to screen out specific scFv antibodies by 4 rounds of ’Glycine-hydrochloric acid elution’.Phage-ELISA was used to identify their binding specificity.3.Preparation and biological characterization of human scFv antibodies aganist NoVsUsing the positive phages as templates,scFv were amplified to construct recombinant plasmids,then expressed by prokaryotic system and purified by Glutathione Sepharose 4 Fast Flow resin.ELISA was used to determined their specificity and neutralization.4.Preliminary identification of NoV antigen mimic epitopesThrough phage display technology,human scFv antibodies aganist NoV as the coated protein,phage display 12-peptide library was used for 3 rounds of bio-panning.The specificity of phage clones was identified by Phage-ELISA,and the ability to compete with NoV P proteins was identified by ELISA.The amino acid sequences that mimic the epitopes were determined by sequencing.Results1.Construction of human phage antibody libraryA primary human phage scFv antibody library was successfully constructed,with a capacity of 8.8×106 PFU/mL.The library showed a good recombination rate and diversity.2.Bio-panning of human NoV phage antibody libraryPhage clones specifically bound to P proteins were enriched by bio-panning.10 strains of phage scFv antibodies with good affinity to NoV P protein were successfully obtained.3.Preparation and biological characterization of human scFv antibodies aganist NoVs10 strains of human scFv antibodies aganist NoVs were obtained through prokaryotic expression.These antibodies could specifically bind to GII.4,GII.6 and GII.17 NoV P proteins.In addition,all of them showed strong ability to block the binding of GII.4 P protein to HBGA receptors,5 strains had rather strong blockade ability to GII.6 and 4 had rather weak blockade ability aganist GII.17.4.Preliminary identification of NoV antigen mimic epitopesNoV antigen mimic epitopes were preliminarily identified through phage display technology.27 different polypeptide sequences were obtained,all of which had a good affinity with scFv antibodies,and had competitive inhibitory effect on NoV P proteins,and 2 consensus sequences were found,MGLDLWENFQVL and FHWPSYYLTPWV,containing three peptides with high homology,WXXPXY,YXXXXXXXLXPXXWV and MGXDXW,suggesting they mimic the epitopes binding of NoVs to scFv antibodies.Conclusions1.A human phage scFv antibody library was successfully constructed using PBMC from patients infected with GII.6 NoV during convalescence,and specific anti-NoV phage scFv antibodies were sceened out by bio-panning.2.Human scFv antibodies aganist NoVs were prepared by the prokaryotic expression system.10 strains of scFv antibodies showed high affinity to GII.4,GII.6 and GII.17 P proteins,and had different degree of abilities to block the binding of the three NoV strains to the HBGA receptors.3.NoV antigen mimic epitopes were preliminarily identified through phage display technology.Two consensus sequences were found to have high afinity with scFv antibodies aganist NoVs,and competitive inhibitory effects on the binding of NoV P proteins to scFv antibodies.Two sequences with high homology to GII.4,GII.6 and GII.17 NoV VP1 regions were found,WXXPXY and YXXXXXXXLXPXXWV,suggesting that they might be the common conformational epitopes of different NoV strains;and a sequence was also found to be with highly homologous to the GII.6 VP1 region:MGXDXW,resided in 402～407,suggesting that it be a specific epitope of GII.6 NoV.