Hemagglutinin and neuraminidase containing virus-like particles produced in HEK-293 suspension culture: An effective 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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2019, VaccineCitation Excerpt :The MFGE8 plays a role in the secretion of lipids. In a previous study we have shown the feasibility of producing influenza VLPs in HEK-293SF cells and the VLP efficacy as vaccine in mice [17]. In the present work, we have characterized the composition of those influenza Gag-VLPs generated using an inducible HEK-293 producing cell line [17] and extracellular vesicles (EVs) produced from non-transfomed HEK-293SF.
Influenza A and B virus-like particles produced in mammalian cells are highly immunogenic and induce functional antibodies
2019, VaccineCitation Excerpt :In the literature, influenza VLPs are composed of one or different combinations of the viral surface glycoproteins, HA, NA, and the structural influenza protein matrix 1 (M1), with or without the matrix-2 (M2) ion channel [11,12]. Influenza VLPs have been produced in various expression systems; mammalian, insect and plant cells [13–15] and in Eri silkworm pupae [16]. In this study, we chose to produce influenza VLPs in mammalian cell lines using transient transfection [17].