Critical assessment of influenza VLP production in Sf9 and HEK293 expression systems

Abstract

BackgroundEach year, influenza is responsible for hundreds of thousand cases of illness and deaths worldwide. Due to the virus’ fast mutation rate, the World Health Organization (WHO) is constantly on alert to rapidly respond to emerging pandemic strains. Although anti-viral therapies exist, the most proficient way to stop the spread of disease is through vaccination. The majority of influenza vaccines on the market are produced in embryonic hen’s eggs and are composed of purified viral antigens from inactivated whole virus. This manufacturing system, however, is limited in its production capacity. Cell culture produced vaccines have been proposed for their potential to overcome the problems associated with egg-based production. Virus-like particles (VLPs) of influenza virus are promising candidate vaccines under consideration by both academic and industry researchers.MethodsIn this study, VLPs were produced in HEK293 suspension cells using the Bacmam transduction system and Sf9 cells using the baculovirus infection system. The proposed systems were assessed for their ability to produce influenza VLPs composed of Hemagglutinin (HA), Neuraminidase (NA) and Matrix Protein (M1) and compared through the lens of bioprocessing by highlighting baseline production yields and bioactivity. VLPs from both systems were characterized using available influenza quantification techniques, such as single radial immunodiffusion assay (SRID), HA assay, western blot and negative staining transmission electron microscopy (NSTEM) to quantify total particles.ResultsFor the HEK293 production system, VLPs were found to be associated with the cell pellet in addition to those released in the supernatant. Sf9 cells produced 35 times more VLPs than HEK293 cells. Sf9-VLPs had higher total HA activity and were generally more homogeneous in morphology and size. However, Sf9 VLP samples contained 20 times more baculovirus than VLPs, whereas 293 VLPs were produced along with vesicles.ConclusionsThis study highlights key production hurdles that must be overcome in both expression platforms, namely the presence of contaminants and the ensuing quantification challenges, and brings up the question of what truly constitutes an influenza VLP candidate vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0152-x) contains supplementary material, which is available to authorized users.

Identification of HIV-1–Based Virus-like Particles by Multifrequency Atomic Force Microscopy

“…Additionally, the cell machinery also naturally secretes other cellular vesicles to the extracellular space such as microvesicles and exosomes (9). The presence of these cellular vesicles has been previously described in HIV-and Influenza-based VLPs produced in mammalian cells (10)(11)(12), and it has also been observed in our research laboratory in all HIV-1-based VLP preparations. The need for high-quality VLP-based vaccine production prevails, because its enduse is in its human clinical application (6).…”

“…A second population of cellular vesicles is also detected in all VLP samples. The presence of cellular debris has also been reported in Influenza-based VLP produced in mammalian cells (12). Cellular vesicles are heterogeneous in size and present a liposomelike circular structure.…”

Induction of Broad-Based Immunity and Protective Efficacy by Self-amplifying mRNA Vaccines Encoding Influenza Virus Hemagglutinin

“…Mimicking the virus structure makes VLPs highly immunogenic and able to activate both B-and T-cell responses (16,34,35), resulting in a very promising technology. However, there are still concerns related to the production capability, not only in terms of VLPs yield but also for the presence of cell-derived contaminants and vaccine purity (36).…”

Bioorthogonal Strategy for Bioprocessing of Specific-Site-Functionalized Enveloped Influenza-Virus-Like Particles

“…The size of the VLPs is different from the one presented previously (Figure 4e,f), which confirms the heterogeneity of the system. 32 It is clear that there are unwanted larger particles at this stage of the process that are not VLPs, or at least not complete ones, because of the lack of HA spikes. This result provides an indication of what to expect from both sorting populations.…”