Elsevier

Vaccine

Volume 34, Issue 29, 17 June 2016, Pages 3371-3380
Vaccine

Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective influenza vaccine candidate

https://doi.org/10.1016/j.vaccine.2016.04.089Get rights and content

Highlights

  • Development of stable cell line expressing hemagglutinin and neuraminidase (293HA-NA).

  • Gag HIV-1 protein is more efficient than M1 to induce VLPs production in 293HA-NA cells.

  • Influenza VLPs production using Gag as scaffold was evaluated in a 3-L bioreactor and purified.

  • Characterization of VLPs showed typical size and morphology of HIV-1 immature particles.

  • The immunogenicity and protective efficacy of the VLPs was demonstrated in mice.

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 (150–200 nm) 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.

Introduction

Influenza is an illness that causes high morbidity and mortality to human population worldwide [1]. The antigenic “drift” and “shift” phenomena are the origin of the appearance of new strains that cannot be recognized by the host immune system causing severe infections [2], [3]. Thus, every year, seasonal influenza vaccines are produced in embryonated chicken eggs depending on the circulating strains [4]. The production in hen's eggs carries major drawbacks that have been described elsewhere [5], [6]. Additionally, the egg produced seasonal vaccines do not provide full protection in all ages groups [7], [8]. These facts are driving the scientific community to urgently develop a new generation of influenza vaccines that is supported by a robust production platform taking advantages of recent progress in the fields of immunology, molecular and cellular biology, and bioprocessing sciences.

Mammalian cells possess several attractive attributes as a robust production platform candidate due to their ability to perform complex post-translational modifications and the high cell densities reached in suspension cultures in bioreactors. This fact has allowed to increase the platform yields and produce bioproducts of very high quality [6]. Additionally, the required-time to develop stable cell lines has decreased considerably in the past decade. Inducible promotors have been developed in mammalian cells as well, mainly to deal with the overexpression of toxic proteins [9]. The use of inducible promotors also allows to separate the growth phase from the production phase, thereby reducing the metabolic burden during biomass growth [10], [11].

From the perspective of new generation influenza vaccines, different approaches have been investigated to overcome the disadvantages associated to egg produced vaccines [12], [13], [14], [15], [16], [17], [18], [19]. Virus-like particle (VLPs) constitute a promising alternative to safely elicit an effective immune response since they mimic native virus [20]. Influenza VLPs have been mostly produced in insect cells. However, this expression platform has the inconvenience of baculovirus contamination in final samples [21]. With the aim of avoiding these drawbacks and exploit the advantages of a superior platform, previous works have explored the production of influenza VLPs in mammalian cells. Most of these studies have been focused in elucidating the virus budding mechanisms [22], [23], [24], [25] or testing the protective immunogenicity of VLPs in animals by directly using the sucrose cushion preparations [26], [27]. From a bioprocessing perspective, influenza VLPs have been produced in human embryonic kidney cells (HEK-293) cells but the production levels were significantly lower in comparison with insect cells, and contamination with extracellular vesicles was observed in final samples [28]. In this work, we have developed and characterized an efficient procedure to produce influenza VLPs from a cell clone stably expressing the hemagglutinin (HA) and neuraminidase (NA) of influenza subtype H1N1 (293HA-NA cells). It is demonstrated that HA and NA containing VLPs can be efficiently produced following transfection of the 293HA-NA cells with a plasmid encoding the gag gene of human immunodeficiency virus (HIV) whose product acted as scaffold. The extracellular vesicles were efficiently removed from final preparation by tangential flow filtration (TFF). The Gag-made nanoparticles assembled from 293HA-NA cells showed the typical morphology and expected size for immature HIV-1 particles. An extensive characterization and quantification of the influenza VLPs produced was performed by using different analytical techniques. The immunogenicity and protective efficacy of the VLPs was demonstrated in mice.

Section snippets

Cells, plasmids and antibodies

The cells were cultured in Hyclone SFM4Transfx-293 supplemented with 4–6 mM of glutamine in a humidified incubator at 37 °C with 5% CO2, at an agitation rate of 100–110 rpm. The gene for M1 (CY033578.1) was codon optimized to human cells and the restriction sites for Hind III were added at both ends of the gene (GenScript) for subsequent cloning in pKCR5 plasmid. The plasmids pUC-HA and pUC-NA H1N1 A/Puerto Rico/8/1934 described in [28] were used as template for PCR to introduce the Kozak sequence

Development of 293HA-NA stable cells

To generate the 293HA-NA cells the HA, NA and Blasticidin containing plasmids were transfected into the parental cell line 293CymR-rcTA (Fig. 1A). At 48 h post-transfection (hpt) cells were maintained under blasticidin selection. Several cell clones were isolated, induced with cumate and the cell lysates were analyzed by Western Blot (data not shown). The clone exhibiting the highest expression levels of HA and NA was selected as our stable 293HA-NA cells. The expression of HA and NA proteins at

Acknowledgements

The authors would like to thank Viktoria Lytvyn for the precious help with the confocal images and Melanie Leclerc for helping out with the maintenance and freezing of the clones. Authors would like to acknowledge Daniel Jacob for the bioreactor operation and Aziza Manceur for the valuable expert aid with the quantification techniques. We are also grateful of Amalia Ponce for the technical assistance with the immunogenicity and animal study. This work was partially funded through Natural

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