title: In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays.
abstract: There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient invivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic invivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenzaA virus serodiagnosis.
authors.affiliation: National Reference Laboratory for Avian Influenza Virus, Friedrich-Loeffler-Institute, Federal Research Institute for Animal Health, D-17493 Greifswald, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Postel
authors.forename: Tobias
authors.initials: T
authors.lastname: Letzel
authors.forename: Frederik
authors.initials: F
authors.lastname: Müller
authors.forename: Ralf
authors.initials: R
authors.lastname: Ehricht
authors.forename: Philippe
authors.initials: P
authors.lastname: Pourquier
authors.forename: Malte
authors.initials: M
authors.lastname: Dauber
authors.forename: Christian
authors.initials: C
authors.lastname: Grund
authors.forename: Martin
authors.initials: M
authors.lastname: Beer
authors.forename: Timm C
authors.initials: TC
authors.lastname: Harder
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D016367:Baculoviridae / Q000235:genetics; D019921:Biotinylation; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D009980:Influenza A virus / Q000145:classification* / Q000276:immunology; D018613:Microscopy, Confocal; D011994:Recombinant Proteins / Q000378:metabolism*; D012698:Serologic Tests / Q000379:methods*; D012995:Solubility; D013045:Species Specificity
chemical_list: D000956:Antigens, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D011994:Recombinant Proteins