Title
Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells
Conference Dates
June 12-17, 2016
Abstract
Virus like particles (VLPs) can be formulated into promising vaccines to prevent influenza infection. In addition of having a structure and composition that mimic the wild type virus, VLPs are safe since they are devoid of viral genes and consequently are not infectious. One approach to scale up the manufacturing of VLPs is to produce them in a serum-free suspension culture using a stable mammalian cell line. Importantly, with VLPs synthetized by mammalian cells, the post-translational modifications of the surface antigens should be similar to the wild type virus, and therefore should trigger a potent and specific immune response for the pathogen. As a proof of concept, we first established a cell line that was stably expressing hemagglutinin (HA) and neuraminidase (NA) proteins of influenza (subtype H1N1) using our patented cGMP human embryonic kidney (HEK293) cell line (clone 293SF-3F6). Transcription of the genes for these two glycoproteins was regulated by the inducible cumate transcription gene-switch. Next, to establish our capability to produce VLPs, we compared the formation of VLPs using these cells after forced expression of two scaffold proteins: Gag from the human immunodeficiency virus and M1 protein from influenza A (H1N1). In addition, monitoring of the VLPs was facilitated by fusing the Gag protein to the green fluorescent protein (GFP). VLP production was therefore initiated by transient transfection of plasmid encoding Gag or M1 and by addition of cumate to the culture medium. The VLPs secreted in the culture medium were recovered by ultracentrifugation on a sucrose cushion. The presence of HA an NA within the VLP fraction was demonstrated by western blot and quantified by dot blot. Interestingly, VLPs were produced more efficiently in the presence of Gag, indicating that Gag is a better scaffolding protein than M1 in this context. Under the electron microscope, the Gag-VLPs appeared as vesicles of 100 to 150 nm of diameter, containing a denser internal proteinous ring, which is a typical morphology for VLPs produced through Gag expression. The production of Gag-VLPs was also validated in a 3-L stirred tank bioreactor in serum-free medium. The immunogenicity of the VLPs is currently under investigation in a murine model for influenza. In conclusion, VLPs containing HA and NA can be manufactured in serum free suspension culture of HEK293 cells through forced expression of Gag. The efficacy of these VLPs for vaccination remains to be demonstrated.
Recommended Citation
Amine Kamen, Alina Venereo-Sanchez, Rénald Gilbert, Aziza Manceur, Parminder Chahal, Sven Ansorge, and Wangxue Chen, "Characterization of HA and NA-containing VLPs produced in suspension cultures of HEK 293 cells" in "Vaccine Technology VI", Laura Palomares, UNAM, Mexico Manon Cox, Protein Sciences Corporation, USA Tarit Mukhopadhyay, University College London, UK Nathalie Garçon, BIOASTER Technology Research Institute, FR Eds, ECI Symposium Series, (2016). https://dc.engconfintl.org/vaccine_vi/55