title: Egg-adaptive mutations in H3N2v vaccine virus enhance egg-based production without loss of antigenicity or immunogenicity.
abstract: The recently detected zoonotic H3N2 variant influenza A (H3N2v) viruses have caused 343 documented cases of human infection linked to contact with swine. An effective vaccine is needed for these viruses, which may acquire transmissibility among humans. However, viruses isolated from human cases do not replicate well in embryonated chicken eggs, posing an obstacle to egg-based vaccine production. To address this issue, we sought to identify egg-adaptive mutations in surface proteins that increase the yield of candidate vaccine viruses (CVVs) in eggs while preserving their immunizing effectiveness. After serial passage of a representative H3N2v isolate (A/Indiana/08/2011), we identified several egg-adaptive combinations of HA mutations and assessed the egg-based replication, antigenicity, and immunogenicity of A/Puerto Rico/8/34 (H1N1, PR8)-based 6+2 reverse genetics CVVs carrying these mutations. Here we demonstrate that the respective combined HA substitutions G1861V+N2461K, N1651K+G1861V, T1281N+N1651K+R762G, and T1281N+N1651K+I102M, all identified after egg passage, enhanced the replication of the CVVs in eggs without substantially affecting their antigenicity or immunogenicity. The mutations were stable, and the mutant viruses acquired no additional substitutions during six subsequent egg passages. We found two crucial mutations, G186V, which was previously defined, and N246K, which in combination improved virus yield in eggs without significantly impacting antigenicity or immunogenicity. This combination of egg-adaptive mutations appears to most effectively generate high egg-based yields of influenza A/Indiana/08/2011-like CVVs.
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA. Electronic address: subrata.barman@stjude.org.
authors.forename: Subrata
authors.initials: S
authors.lastname: Barman
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA. Electronic address: john.franks@stjude.org.
authors.forename: John
authors.initials: J
authors.lastname: Franks
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA. Electronic address: jasmine.turner@stjude.org.
authors.forename: Jasmine C
authors.initials: JC
authors.lastname: Turner
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA; Viral Infectious Disease Research Center, Korea Research Institute of Bioscience & Biotechnology (KRIBB) , 125 Gwahak-ro, Youseong-gu, Daejeon, 305-806, South Korea. Electronic address: syoon@kribb.re.kr.
authors.forename: Sun-Woo
authors.initials: SW
authors.lastname: Yoon
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA. Electronic address: robert.webster@stjude.org.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Webster
authors.affiliation: Division of Virology, Department of Infectious Diseases, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Mail Stop 330, Memphis, TN, 38105-3678, USA. Electronic address: richard.webby@stjude.org.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Webby
mesh_terms: D000222:Adaptation, Physiological; D019943:Amino Acid Substitution; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002642:Chick Embryo; D005289:Ferrets; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000254:growth & development / Q000276:immunology / Q000378:metabolism; D053122:Influenza A Virus, H3N2 Subtype / Q000235:genetics* / Q000254:growth & development / Q000276:immunology* / Q000378:metabolism; D008297:Male; D009154:Mutation; D012692:Serial Passage; D013552:Swine; D014776:Virus Cultivation; D014779:Virus Replication
chemical_list: D000914:Antibodies, Viral; D019267:Hemagglutinin Glycoproteins, Influenza Virus
keywords: Antigenicity; Egg adaptation; Hemagglutinin H3 mutations; Immunogenicity; Influenza A virus; Vaccine virus; Variant virus; Zoonosis