title: Implementation of new approaches for generating conventional reassortants for live attenuated influenza vaccine based on Russian master donor viruses.
abstract: Cold-adapted influenza strains A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69, originally developed in Russia, have been reliable master donors of attenuation for preparing live attenuated influenza vaccines (LAIV). The classical strategy for generating LAIV reassortants is robust, but has some disadvantages. The generation of reassortants requires at least 3 passages under selective conditions after co-infection; each of these selective passages takes six days. Screening the reassortants for a genomic composition traditionally starts after a second limiting dilution cloning procedure, and the number of suitable reassortants is limited. We developed a new approach to shorten process of preparing LAIV seed viruses. Introducing the genotyping of reassortants by pyrosequencing and monitoring sequence integrity of surface antigens starting at the first selective passage allowed specific selection of suitable reassortants for the next cloning procedure and also eliminate one of the group selective passage in vaccine candidate generation. Homogeneity analysis confirmed that reducing the number of selective passages didn't affect the quality of LAIV seed viruses. Finally, the two-way hemagglutination inhibition test, implemented for all the final seed viruses, confirmed that any amino acid substitutions acquired by reassortants during egg propagation didn't affect antigenicity of the vaccine. Our new strategy reduces the time required to generate a vaccine and was used to generate seasonal LAIVs candidates for the 2012/2013, 2014/2015, and 2015/2016 seasons more rapidly.
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-G16, 1600 Clifton Road, Atlanta, GA 30333, United States; Battelle, Atlanta, GA 30329, United States.
authors.forename: Svetlana
authors.initials: S
authors.lastname: Shcherbik
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-G16, 1600 Clifton Road, Atlanta, GA 30333, United States; Battelle, Atlanta, GA 30329, United States.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Pearce
authors.affiliation: Institute of Experimental Medicine, Department of Virology, St. Petersburg, Russia.
authors.forename: Irina
authors.initials: I
authors.lastname: Kiseleva
authors.affiliation: Institute of Experimental Medicine, Department of Virology, St. Petersburg, Russia.
authors.forename: Natalie
authors.initials: N
authors.lastname: Larionova
authors.affiliation: Institute of Experimental Medicine, Department of Virology, St. Petersburg, Russia.
authors.forename: Larisa
authors.initials: L
authors.lastname: Rudenko
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-G16, 1600 Clifton Road, Atlanta, GA 30333, United States.
authors.forename: Xiyan
authors.initials: X
authors.lastname: Xu
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-G16, 1600 Clifton Road, Atlanta, GA 30333, United States.
authors.forename: David E
authors.initials: DE
authors.lastname: Wentworth
authors.affiliation: Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, MS-G16, 1600 Clifton Road, Atlanta, GA 30333, United States. Electronic address: tbousse@cdc.gov.
authors.forename: Tatiana
authors.initials: T
authors.lastname: Bousse
mesh_terms: D000818:Animals; D002642:Chick Embryo; D006385:Hemagglutination Inhibition Tests; D053121:Influenza A Virus, H2N2 Subtype / Q000276:immunology; D009981:Influenza B virus / Q000276:immunology; D007252:Influenza Vaccines / Q000191:economics / Q000276:immunology; D008969:Molecular Sequence Data; D016865:Reassortant Viruses; D012426:Russia; D014773:Virology / Q000379:methods*
chemical_list: D007252:Influenza Vaccines
keywords: Influenza; Live vaccine; Reassortants generation