title: Human monoclonal single chain antibodies (HuScFv) that bind to the polymerase proteins of influenza A virus.
abstract: Current anti-influenza drugs target the viral neuraminidase or inhibit the function of the ion channel M2 protein. Not only is the supply of these drugs unlikely to meet the demand during a large influenza epidemic/ pandemic, but also has an emergence of drug resistant influenza virus variants been documented. Thus a new effective drug or antiviral alternative is required. The influenza virus RNA polymerase complex consists of nucleoproteins (NP) that bind to three polymerase subunits: two basic polymerases, PB1 and PB2, and an acidic polymerase (PA). These proteins play a pivotal role in the virus life cycle; thus they are potential targets for the development of new anti-influenza agents. In this study, we produced human monoclonal antibodies that bound to the influenza A polymerase proteins by using a human antibody phage display library. Complementary DNA was prepared from the total RNA of a highly pathogenic avian influenza (HPAI) virus: A/duck/Thailand/144/2005(H5N1). The cDNA synthesized from the total virus RNA was used as template for the amplification of the gene segments encoding the N-terminal halves of the PB1, PB2 and PA polymerase proteins which encompassed the biologically active portions of the respective proteins. The cDNA amplicons were individually cloned into appropriate vectors and the recombinant vectors were introduced into Escherichia coli bacteria. Transformed E. coli clones were selected, and induced to express the recombinant proteins. Individually purified proteins were used as antigens in bio-panning to select the phage clones displaying specific human monoclonal single chain variable fragments (HuScFv) from a human antibody phage display library constructed from Thai blood donors in our laboratory. The purified HuScFv that bound specifically to the recombinant polymerase proteins were prepared. The inhibitory effects on the biological functions of the respective polymerase proteins should be tested. We envisage the use of the HuScFv in their cell penetrating version (transbodies) as an alternative influenza therapeutic to current anti-virus drugs.
authors.affiliation: Faculty of Allied Health Sciences, Thammasat University, Rangsit Campus, Pathumthani, Thailand.
authors.forename: Umaporn
authors.initials: U
authors.lastname: Thathaisong
authors.forename: Santi
authors.initials: S
authors.lastname: Maneewatch
authors.forename: Kasem
authors.initials: K
authors.lastname: Kulkeaw
authors.forename: Kanyarat
authors.initials: K
authors.lastname: Thueng-In
authors.forename: Ornutcha
authors.initials: O
authors.lastname: Poungpair
authors.forename: Potjanee
authors.initials: P
authors.lastname: Srimanote
authors.forename: Thaweesak
authors.initials: T
authors.lastname: Songserm
authors.forename: Pongsri
authors.initials: P
authors.lastname: Tongtawe
authors.forename: Pramuan
authors.initials: P
authors.lastname: Tapchaisri
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000918:Antibody Specificity; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D053124:Influenza A Virus, H5N1 Subtype / Q000201:enzymology / Q000276:immunology*; D019151:Peptide Library; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase