title: Multiple amino acid residues confer temperature sensitivity to human influenza virus vaccine strains (FluMist) derived from cold-adapted A/Ann Arbor/6/60.
abstract: FluMist influenza A vaccine strains contain the PB1, PB2, PA, NP, M, and NS gene segments of ca A/AA/6/60, the master donor virus-A strain. These gene segments impart the characteristic cold-adapted (ca), attenuated (att), and temperature-sensitive (ts) phenotypes to the vaccine strains. A plasmid-based reverse genetics system was used to create a series of recombinant hybrids between the isogenic non-ts wt A/Ann Arbor/6/60 and MDV-A strains to characterize the genetic basis of the ts phenotype, a critical, genetically stable, biological trait that contributes to the attenuation and safety of FluMist vaccines. PB1, PB2, and NP derived from MDV-A each expressed determinants of temperature sensitivity and the combination of all three gene segments was synergistic, resulting in expression of the characteristic MDV-A ts phenotype. Site-directed mutagenesis analysis mapped the MDV-A ts phenotype to the following four major loci: PB1(1195) (K391E), PB1(1766) (E581G), PB2(821) (N265S), and NP(146) (D34G). In addition, PB1(2005) (A661T) also contributed to the ts phenotype. The identification of multiple genetic loci that control the MDV-A ts phenotype provides a molecular basis for the observed genetic stability of FluMist vaccines.
authors.affiliation: MedImmune Vaccines, Inc., 297 North Bernardo Avenue, Mountain View, CA 94043, USA. jinh@medimmune.com
authors.forename: Hong
authors.initials: H
authors.lastname: Jin
authors.forename: Bin
authors.initials: B
authors.lastname: Lu
authors.forename: Helen
authors.initials: H
authors.lastname: Zhou
authors.forename: Chienhui
authors.initials: C
authors.lastname: Ma
authors.forename: Jackie
authors.initials: J
authors.lastname: Zhao
authors.forename: Chin-fen
authors.initials: CF
authors.lastname: Yang
authors.forename: George
authors.initials: G
authors.lastname: Kemble
authors.forename: Harry
authors.initials: H
authors.lastname: Greenberg
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002642:Chick Embryo; D003080:Cold Temperature; D006801:Humans; D009980:Influenza A virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D007252:Influenza Vaccines / Q000737:chemistry* / Q000235:genetics; D007251:Influenza, Human / Q000821:virology; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012324:RNA-Dependent RNA Polymerase; D011995:Recombination, Genetic; D014162:Transfection; D014613:Vaccines, Attenuated / Q000737:chemistry / Q000235:genetics; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D007252:Influenza Vaccines; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; C057027:PB2 protein, Influenzavirus A; D014613:Vaccines, Attenuated; D014758:Viral Core Proteins; D014764:Viral Proteins; C077346:influenza virus polymerase basic protein 1; D012324:RNA-Dependent RNA Polymerase