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.

Quality Assessment of Virus-Like Particles at Single Particle Level: A Comparative Study

“…Interestingly, Gag-eGFP VLPs do not achieve the expected organized structure as Gag VLPs probably due to the eGFP fusion that alters the native budding process [59]. These differences could not be detected by TEM due to the interference of negative staining with the samples [11,36,60]. To our knowledge, this is the first time were Gag-eGFP VLP intraparticle organization is observed.…”

Integrase Defective Lentiviral Vector as a Vaccine Platform for Delivering Influenza Antigens

“…Pseudotype-based influenza genes delivery represents an alternative to successfully express HA in mammalian cells providing an efficacious vaccine when tested in chickens and mice ( 61 , 62 ). More recently, Venereo-Sanchez et al showed that HIV-Gag-based VLP displaying H1N1 induced a sustained immune response which provided full protection after lethal challenge with the homologous virus strain in mice ( 63 ). Of note, IDLVs used in our report were pseudotyped with VSV.G glycoprotein which may also contribute to the strength of the IDLV-induced immune responses by increasing the tropism of the vector.…”

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza

“…For new strains, a recombinant protein can be used to generate the standard curve even though the binding affinity might vary depending on the nature of the sample (S4 Fig). Alternatively, a calibrating antigen can be produced and quantified using a method based on SDS-page as recently described [28]. HA concentrations obtained by dot blot were compared to values obtained in the hemagglutination assay and the SRID assay.…”