title: Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate.
abstract: Virus-like particles (VLPs) constitute a promising alternative as influenza vaccine. They are non-replicative particles that mimic the morphology of native viruses which make them more immunogenic than classical subunit vaccines. In this study, we propose HEK-293 cells in suspension culture in serum-free medium as an efficient platform to produce large quantities of VLPs. For this purpose, a stable cell line expressing the main influenza viral antigens hemagglutinin (HA) and neuraminidase (NA) (subtype H1N1) under the regulation of a cumate inducible promoter was developed (293HA-NA cells). The production of VLPs was evaluated by transient transfection of plasmids encoding human immunodeficiency virus (HIV) Gag or M1 influenza matrix protein. To facilitate the monitoring of VLPs production, Gag was fused to the green fluorescence protein (GFP). The transient transfection of the gag containing plasmid in 293HA-NA cells increased the release of HA and NA seven times more than its counterpart transfected with the M1 encoding plasmid. Consequently, the production of HA-NA containing VLPs using Gag as scaffold was evaluated in a 3-L controlled stirred tank bioreactor. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion and ultrafiltered by tangential flow filtration. Transmission electron micrographs of final sample revealed the presence of particles with the average typical size (150-200nm) and morphology of HIV-1 immature particles. The concentration of the influenza glycoproteins on the Gag-VLPs was estimated by single radial immunodiffusion and hemagglutination assay for HA and by Dot-Blot for HA and NA. More significantly, intranasal immunization of mice with influenza Gag-VLPs induced strong antigen-specific mucosal and systemic antibody responses and provided full protection against a lethal intranasal challenge with the homologous virus strain. These data suggest that, with further optimization and characterization the process could support mass production of safer and better-controlled VLPs-based influenza vaccine candidate.
authors.affiliation: Department of Chemical Engineering, Ecole Polytechnique de Montréal, Montréal, Québec, Canada; Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Alina
authors.initials: A
authors.lastname: Venereo-Sanchez
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Renald
authors.initials: R
authors.lastname: Gilbert
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Melanie
authors.initials: M
authors.lastname: Simoneau
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Antoine
authors.initials: A
authors.lastname: Caron
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Parminder
authors.initials: P
authors.lastname: Chahal
authors.affiliation: Human Health Therapeutics, National Research Council Canada, Ottawa, Ontario, Canada.
authors.forename: Wangxue
authors.initials: W
authors.lastname: Chen
authors.affiliation: Vaccine Program, Human Health Therapeutics, National Research Council Canada, Montréal, Québec, Canada.
authors.forename: Sven
authors.initials: S
authors.lastname: Ansorge
authors.affiliation: Centre for Vaccine Evaluation, Biologics and Genetic Therapies Directorate, HPFB, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Xuguang
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Chemical Engineering, Ecole Polytechnique de Montréal, Montréal, Québec, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Henry
authors.affiliation: Department of Bioengineering, McGill University, Montréal, Québec, Canada. Electronic address: amine.kamen@mcgill.ca.
authors.forename: Amine
authors.initials: A
authors.lastname: Kamen
mesh_terms: D000818:Animals; D005260:Female; D057809:HEK293 Cells; D006386:Hemagglutination Tests; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000276:immunology*; D006801:Humans; D000071497:Immunogenicity, Vaccine; D053118:Influenza A Virus, H1N1 Subtype; D007252:Influenza Vaccines / Q000276:immunology*; D007251:Influenza, Human / Q000517:prevention & control; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000276:immunology*; D009976:Orthomyxoviridae Infections / Q000517:prevention & control*; D014162:Transfection; D058425:Vaccines, Virus-Like Particle / Q000276:immunology*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000276:immunology
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; D007252:Influenza Vaccines; D058425:Vaccines, Virus-Like Particle; D054301:gag Gene Products, Human Immunodeficiency Virus; D009439:Neuraminidase
keywords: Bioreactor production; HEK-293; Influenza vaccine; Quantification; Stable cell line; Tangential flow filtration; Virus-like particles