title: Direct random insertion of an influenza virus immunologic determinant into the NS1 glycoprotein of a vaccine flavivirus.
abstract: A live chimeric vaccine virus against Japanese encephalitis (JE), ChimeriVax-JE, was used to define methods for optimal, random insertion of foreign immunologic determinants into flavivirus glycoproteins. The conserved M2e peptide of influenza A virus was randomly inserted into the yellow fever-specific NS1 glycoprotein of ChimeriVax-JE. A technique combining plaque purification with immunostaining yielded a recombinant virus that stably expressed M2e at NS1-236 site. The site was found permissive for other inserts. The insertion inhibited NS1 dimerization in vitro, which had no significant effect on virus replication in vitro and immunogenicity in vivo. Two different NS1-specific monoclonal antibodies and a polyclonal antibody efficiently recognized only the NS1 protein dimer, but not monomer. Adaptation of the virus to Vero cells resulted in two amino acid changes upstream from the insert which restored NS1 dimerization. Immunized mice developed high-titer M2e-specific antibodies predominantly of the IgG2A isotype indicative of a Th1-biased response.
authors.affiliation: Sanofi pasteur, Discovery US, 38 Sidney Street, Cambridge, MA 02139, USA.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Rumyantsev
authors.forename: Zhen-xi
authors.initials: ZX
authors.lastname: Zhang
authors.forename: Qing-sheng
authors.initials: QS
authors.lastname: Gao
authors.forename: Nicolas
authors.initials: N
authors.lastname: Moretti
authors.forename: Nathan
authors.initials: N
authors.lastname: Brown
authors.forename: Harold
authors.initials: H
authors.lastname: Kleanthous
authors.forename: Simon
authors.initials: S
authors.lastname: Delagrave
authors.forename: Farshad
authors.initials: F
authors.lastname: Guirakhoo
authors.forename: Marc S
authors.initials: MS
authors.lastname: Collett
authors.forename: Konstantin V
authors.initials: KV
authors.lastname: Pugachev
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000917:Antibody Formation / Q000276:immunology; D002522:Chlorocebus aethiops; D004664:Encephalitis Virus, Japanese / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D005416:Flavivirus / Q000276:immunology*; D022321:Japanese Encephalitis Vaccines / Q000276:immunology*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*; D053060:West Nile Virus Vaccines / Q000276:immunology
chemical_list: D000914:Antibodies, Viral; C445780:ChimeriVax; D000939:Epitopes; D022321:Japanese Encephalitis Vaccines; C100315:NS1 protein, Flavivirus; D014614:Vaccines, Synthetic; D017361:Viral Nonstructural Proteins; D014765:Viral Vaccines; D053060:West Nile Virus Vaccines