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.

Keywords: Influenza; Live vaccine; Reassortants generation.

Fig. 1

Partial pyrograms of neuraminidase (NA) (A), PB2 (B), and NS (C) RNA from eggs after the first selective passage (eggs 21, 22, and 23 are shown) after co-infection of A/North Carolina/13/2014 and MDV. Green arrows indicate signature peaks of desired origin (NA—A/North Carolina/13/2014, internal genes—MDV), red arrows indicate signature peaks of genes on undesired origin in a mixed reassortants pool.

Fig. 2

Partial pyrograms of PB2 (A) and NS (B) gene RNA from eggs after the second selective passage (eggs 2–21, 2–22, and 2–23) after co-infection of A/North Carolina/13/2014 and MDV. Green arrows indicate signature peaks of desired origin (MDV), red arrows indicate signature peaks of A/North Carolina/13/2014 in a mixed reassortants pool.