title: Human monoclonal ScFv that bind to different functional domains of M2 and inhibit H5N1 influenza virus replication.
abstract: BACKGROUND
  Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.
  
  
  METHODS
  Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.
  
  
  RESULTS
  HuScFv derived from four huscfv-phagemid transformed E. coli clones (no. 2, 19, 23 and 27) showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.
  
  
  CONCLUSIONS
  HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants.
authors.affiliation: Department of Immunology, Graduate Program in Immunology, Mahidol University, Bangkok 10700, Thailand.
authors.forename: Tippawan
authors.initials: T
authors.lastname: Pissawong
authors.forename: Santi
authors.initials: S
authors.lastname: Maneewatch
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-In
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.forename: Fonthip
authors.initials: F
authors.lastname: Dong-din-on
authors.forename: Jeeraphong
authors.initials: J
authors.lastname: Thanongsaksrikul
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Songserm
authors.forename: Pongsri
authors.initials: P
authors.lastname: Tongtawe
authors.forename: Kunan
authors.initials: K
authors.lastname: Bangphoomi
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D000998:Antiviral Agents / Q000302:isolation & purification* / Q000378:metabolism; D002642:Chick Embryo; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D053124:Influenza A Virus, H5N1 Subtype / Q000276:immunology* / Q000502:physiology*; D019151:Peptide Library; D011485:Protein Binding; D057127:Single-Chain Antibodies / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000276:immunology* / Q000378:metabolism; D014779:Virus Replication; D000086082:Viroporin Proteins
chemical_list: D000914:Antibodies, Viral; D000998:Antiviral Agents; D019151:Peptide Library; D057127:Single-Chain Antibodies; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins