Production du virus influenza dans des cellules HEK-293 cultivees en suspension.

摘要

HEK-293 cell lines have been widely used for over 30 years by the scientific community. Currently HEK-293 cells are the most efficient system for the production of r-proteins and viral vectors by large-scale transfection of suspension-growing cells in serum-free medium. It is the most established cell line for the production of adeno- and adeno-associated viruses, retro-and lentiviruses for gene therapy applications. Numerous viral vectors produced in HEK-293 cells are currently involved in phase II and phase III clinical trials. Noteworthy, a protein manufactured by Ely Lily using an HEK-293 clone has been approved for commercialization.This study was designed primarily to evaluate if the HEK-293 cell line could efficiently replicate the influenza virus. Experiments were performed in serum-free suspension cultures. Preliminary studies consisted in investigating the presence of Sia2-3Gal and Sia2-6Gal receptors on the cell surface to evaluate the potential of HEK-293 cells to be infected. First infections, done on both MDCK and HEK-293 cells, confirmed this hypothesis by leading to interesting viral titers (640 HA/mL, 106 IVP/mL), even though lower than those obtained with the MDCK reference cell line (2560 HA/mL, 1010 IVP/mL). Optimization of infection parameters to reach best yields and adaptation of the virus to the cell line were the next points investigated. The effect of trypsin on cells during infection was first studied. Best production was obtained at a 1mug/mL concentration, which allowed virus activation without inhibiting effect on the cells. Low multiplicity of infection (moi=10 -3) at infection efficiently amplified the virus strain without leading non-infected cells to apoptosis. The infection conditions (1x106 cell/mL, 1mug/mL trypsin, moi=10-3) allowed increased HA titers by 4, reaching HA titers equivalent to MDCK, and increased infectious titers by 2 logs to reach 108 IVP/mL. HEK-293 cells were also capable of producing high titers of infectious influenza viruses for different subtypes and variants, including A/H1, A/H3 and B strains, to up to 10 10 IVP/mL. The study of cell density effects lead to the observation that a density of 4x106 cell/mL at infection allowed to multiply by 4-fold the HA titer (10,240 HA/mL) and increase by one log the infectious titer (109 IVP/mL). This study also showed that SFM4Transfx-293 (HyQ) medium could support both cell growth to high density, followed by infection. These results were validated at small and large scale (3-L bioreactor), without medium exchange prior to infection. First purification steps isolated the virus, and its characterization indicated that pleomorphic influenza particles produced in HEK-293 cells presented well all the virus characteristics needed to infect cells and replicate. Therefore, it is concluded that the HEK-293 expression platform may be a suitable system for industrial manufacturing of influenza vaccines.Influenza A and B viruses cause annual epidemics in the human population worldwide. Influenza vaccines are still produced using embryonated hen eggs despite important drawbacks. Considering the high probability of new influenza pandemics, like the recent swine flu H1N1, there is a need to find new alternative production methods for influenza vaccines. Mammalian cells such as MDCK or VERO have been considered for the production of influenza viral strains. However these strains present significant disadvantages of being cultivated in adherence and in serum containing medium. The human cell line PER.C6 is currently employed by Sanofi-Pasteur, but no data has been released to date. In many laboratories, HEK-293 clones have been used in combination with MDCK cells to rescue the influenza strains.