Production And Identification Of Human Anti-H7N9 Avian Influenza Virus IgG Antibody

In March 2013,Chinese Center for Disease Control and Prevention(CDC)published the first three confirmed novel H7N9 avian influenza cases,up to now the virus has caused hundreds of human infection and has showed high mortality rate[1].Avian influenza virus gene is highly mutable and this requires us to maintain sharp vigilance to prevent pandemic event.M2 protein blockers and neuraminidase inhibitors are the first-line drugs towards Avian influenza virus,however it has been confirmed that H7N9 avian influenza virus is completely resistant to M2 protein blockers and 10%of the virus is resistant to neuraminidase inhibitors[1-2].In recent years,new antibody drugs developed by genetic engineering showed perfect prospect in avian influenza prevention and control,with the continue improvement of the technology,the emergence of fully human antibody development technology can make up for deficiencies of the early genetically engineered antibodies[3].Hemagglutinin(HA)is a homotrimer protein on avian influenza virus surface,it could induce avain influenza virus to the host cell membrane receptors and result in membrane fusion[4].HA is composed of globular HA1 subunit and stalk-like HA2 subunit linked by disulfide bonds,both subunits carry multiple antibody binding sites,and the binding sites on HA2 subunit are more conserved than the ones on HA1 subunit[5].Neutralizing antibodies targeting some binding sites on HA can effectively block the viral infection mechanism and induce a variety of in vivo antiviral immune responses and has a good application prospect in the prevention and treatment of avian influenza[6].A human immune Fab phage-display library could be used to select HA specific Fab antibodies,by construct full molecule expression vectors the full molecule neutralizing antibodies against H7N9 HA could be expressed through eukaryotic expressing system.This research could provide a alternative therapeutic antibody in therapies to counter the H7N9 virus and bring new ideas to the prophylaxis and treatment of emerging avain influenza viruses.Methods1.Panning of human anti-H7N9 Fab phage antibody libraryA/Shanghai2/2013(H7N9)HA protein was used to screen the human Fab phage library,after six rounds of combining-eluting-amplifying,specific bacterial clones were randomly selected.The Fab clones which were positive in phage-ELISA were sequenced to obtain the variable region sequences.2.Construction and identification of recombinant eukaryotic expression vectors of human anti-H7N9 HA IgG antibodyThe sequences of the variable regions of anti-H7N9 Fab were compared with human genome sequence database(http://www.imgt.org/).Mutations in the nucleic acid sequence gene were repaired at the beginning of FR1 and at the end of the FR4 according to homology.The eukaryotic expression vectors pFUSE-CHIg-hGl and pFUSE-CLIg-hκ,which carry type IgG1 human heavy chain and light chain(Kappa)constant regions respectively,were double digested.The primers of heavy and light chain were designed according to the principle of In-Fusion PCR and the variable regions of the antibody were amplified and the PCR products were cloned into the eukaryotic expression vectors.After transformed into E.coli DH5α,the positive clones were sequenced.3.Expression and purification of human anti-H7N9 HA IgG antibodyEukaryotic recombinant plasmids of correct sequences were transfected into 293F cells.Supernatant of transfected cells was collected 6 days later and the filtered supernatant was purified using AKTA purifier by Hitrap Protein A prepacked column.30 kD ultrafiltration centrifugal tube was used to replace antibody buffer by PBS,the concentration was filtered using 0.22 μm filter membrane and was subpackaged.4.Analysis of mmunological characteristics of human anti-H7N9 HA IgG antibodyThe binding specificity of anti-H7N9 IgG were analyzed by enzyme-linked immuno sorbent assay(ELISA),and western blot experiments.5.Identification of the neutralizing ability of human anti-H7N9 HA IgG antibodyCalculate the TCIDso value of H7N9 avian influenza virus in MDCK cells using Reed-Muench’s method and conduct microneutralization assay accordingly to acquire the neutralizing titer of the IgG antibody.Prophylactic effect of the antibody against H7N9 virus was tested in embryonated chicken eggs.Hemagglutination inhibition test was conducted to identify the HA subunit where the antibody binding site locate.Results1.Panning of human anti-H7N9 Fab phage antibody libraryFive positive clones(1A4、2F2、4C1、5B2、6A7)were obtained through phage-ELISA,the clones were sequenced and the sequences were analyzed.One Fab clone 6 A7 was chose and the sequences were demonstrated to be variable region’s genes of human immunoglubulin,including VH gene and Vκ at the length of 3 51 bp and 339 bp,respectively.2.Construction and identification of recombinant eukaryotic expression vectors of human anti-H7N9 HA IgG antibodyAfter sequence alignment and primer designthe VH gene and Vκ gene were cloned into digested vectors and eukaryotic expression plasmids were constructed by In-Fusion PCR.The plasmids were transformed into E.coli DH5α and the sequences were right.3.Expression and purification of human anti-H7N9 HA IgG antibodyThe human anti-H7N9 HA IgG antibody was successfully expressed in 293F eukaryotic expression system.The expressed antibody showed high yield and the purified product was good in quality.The purified protein was detected by SDS-PAGE and the result showed a 55 kD band and a 25 kD band,which were the heavyand light chain of the IgG antibody respectively.4.Analysis of mmunological characteristics of human anti-H7N9 HA IgG antibodyIn the ELISA test,the IgG antibody could specifically bind to inactivated H7N9 virus even at the concentration of 0.039 μg/mL and showed high dose-effect relationship.Western blot test demonstrated that the antibody could specifically bind to H7N9 HA antibody.5.Identification of the neutralizing ability of human anti-H7N9 HA IgG antibodyThe TCID50 value of H7N9 avian influenza virus in MDCK cells was tested using Reed-Muench’s method and the neutralizing titer of the IgG antibody was 15.6μg/mL.Prophylactic effect of the antibody against H7N9 virus in embryonated chicken eggs showed that the protection effect of the IgG antibody was positively associated to its concentration and could protect 100%of the eggs when the dosage was 200μg.Hemagglutination inhibition test indicated that the IgG may bind to the HA2 subunit of hemagglutinin.Conclusions1.Successfully selected an Fab antibody that have broad neutralizing effect on H7N9 virus from a human immune Fab phage-display library,the eukaryotic expression vectors of human anti H7N9 HA IgG antibody was constructed and the purified antibody showed good biological activity.2.Preliminary proved that the human anti H7N9 HA IgG had good neutralizing activity towards H7N9 virus and the targeting epitope may locate on the HA2 subunit,thus the antibody may have broad spectrum neutralization activity.