Generation And Application Of Monoclonal Antibodies Against Neuraminidase Of Novel Influenza A(H7N9)Virus

Since 2013,a novel reassortant H7N9 virus associated with severe human infection has emerged in eastern China.Its continuous circulation in poultry in China has caused sporadic human infections annually.As of 28 May 2017,1514 laboratory confirmed cases were reported.In the current 5th wave,a highly pathogenic H7N9 virus(HP-H7),with the HA antigenicity distinct from the previous,were detected in patients and poultry.Furthermore,an extended geographic spread of the virus was also observed.With the guidance of rapid diagnosis tests for H7N9 virus,early antiviral treatment and effective management would be timely implemented,which were essential to clinical practice and controlling the outbreak.Neuraminidase(NA),one of the antigens on the surface of influenza virus,has been shown to cleave off the sialic acid residues and allows virus particles to release from the infected cells.The monomer of NA tetramer was divided into the cytoplasmic,transmembrane,stalk and globular head domains,which include the antigenic epitopes and enzymatic active sites.Some antibodies induced by these sites inhibit the NA’s enzymatic activity.The neuraminidase-inhibitory antibodies with protective effective effects in vivo and in vitro,could sharply inhibit replication efficacy and reduce the severity of infection.In this study,the mouse-derived monoclonal antibodies(mAbs)against neuraminidase(NA)of influenza A(H7N9)virus were generated using hybridoma technology,and their biological functions were investigated.Based on their characteristics,we then chose two N9-specific mAbs,3C1 and 3E9 and established a double antibody sandwich enzyme linked immunosorbent assay(DAS ELISA)for the detection of N9.Results:I.Generation and characterization of mAbs against N9 of H7N9 subtype influenza virus1.Twenty-five mAbs against NA of H7N9 were obtained from the hybridomas generated by mouse immunization and cell fusion and screened by N9-binding test.2.Reassortant viruses with different NA subtypes,rgH6N2,rgH6N9 and rgH9N2 containing seasonal NAs from A/Brisbane/10/2007(H3N2),or avian N9 of A/AH1/2013(H7N9)and avian N2 of 33982/2009(H9N2),respectively,as well asA/Environment/Jiangxi/28/2009(H11N9)were used to evaluate the neuraminidase inhibitory effect of 25 mAbs.The mAbs demonstrated differential inhibition effects on NA activity of the viruses.The IC50 values against rgH6N9 and H11N9 were<1.6～>100μg/ml,15～>100μg/ml were for avian N2 and>100pg/ml for seasonal N2.3.A detailed functional identification of 1G8,3C4 and 4E8 was carried out.They demonstrated different epitope-recognizing with N9.3C4 and 4E8 exhibited neuraminidase inhibitory activity,with a IC50 of 1.45μg/ml and 8.65μg/ml,respectively.II.Establishment of N9 antigen detecting method for avian Influenza virus1.The optimization of Ab pairing was based on the rapid testing using the fluorescent-conjugation antibody pairing platform.N9-specific Abs.3C1 and 3E9,showed the highest binding affinity to N9 compared with others and hence were developed and assembled into the DAS ELISA.2.DAS-ELISA demonstrated high sensitivity in two genetic N9 lineages.Its limit of detection is 0.125HAU/50μl for live virus,AH1/2013 and A/Environment/Jiangxi/28/2009(H11N9)and 6.25ng/ml for N9 protein of A/Anhuil/2013(H7N9,AH1/2013),respectively.3.The DAS-ELISA showed negative signal with rgH6N1,rgH6N2 and rgH9N2 even at a high virus titer of 8HAU/50μL,demonstrating high specificity.4.We examined the DAS ELISA for the original clinic throat swabs from five H7N9 patients which was identified by Q-PCR.The positive signal was observed in two H7N9 specimens,while all were negative by using commercial Flu A and H7 subtype rapid antigen tests.Conclusions1.Twenty-five anti-N9 mAbs were obtained.Twenty-two of them demonstrated inhibitory effects on N9 activity,with IC50 of<1.6～15μg/ml.2.A DAS ELISA for detecting infectious H7N9 virus or N9 protein was developed.High quality of performance was found in testing N9,virus and clinical specimens.