Cellular signaling pathways modulated by influenza infection in HEK293 cells
© Manceur et al. 2015
Published: 14 December 2015
Cell-culture based vaccines are a valuable alternative to egg-produced vaccines. The equivalent of 1500 influenza vaccine doses can be produced in a 1L bioreactor within 48 hours with HEK293 cells. The kinetics of production of H1N1 A/Puerto Rico/8/68 in HEK293 cells has been previously characterized by our group; higher viral production is achieved when cells are infected at a multiplicity of infection (MOI) of 0.01 compared to an MOI of 1. Also, two cycles of infection take place: a first round of virus is produced at 8 hours post-infection (hpi) which then infect cells again and lead to a second viral exit at 16 hpi.
Infection triggers a cascade of intracellular responses which contributes to viral replication, but eventually leads to cell death. Most studies focus on one pathway at a time, and aim at limiting the extent of the infection through the use of inhibitors. Our goal however is to increase viral yield and quality. The main objective is therefore to understand and manipulate the molecular events taking place at the cellular level upon influenza infection and replication, with particular attention to key time points corresponding to viral production and exit. Signaling pathways examined include the Akt, mTOR and ERK pathway, and their activation levels were determined by measuring their phosphorylation states.
Materials and methods
HEK293 cells were grown in suspension in serum free media (SFM4Transfx-293, HyClone). Cells at a density of 2E06 cells/ml were infected at an MOI of 0.01, in the presence of 1 μg/ml trypsin-TPCK, with H1N1 A/Puerto Rico/8 (H1N1 PR/8) or H3N2 A/Aichi/8/68 (H3N2 Aichi). Non-infected cells, but treated with trypsin-TPCK were used as a negative control. Samples were collected at 0.5 to 42 hpi, fixed with 2% paraformaldehyde, permeabilized and stored in methanol at -80oC until analysis. Phospho-specific antibodies (Cell Signaling) were used to measure phospho-Akt (S473), phospho-mTOR (S2448) and phospho-Erk (Thr202-Tyr204) using flow cytometry. The presence of influenza virus was simultaneously assessed by staining hemagglutinin (HA), the main surface protein of influenza. Cell viability was monitored throughout the infection period with an automated cell counter (Cedex HiRes, Roche). Viral production was quantified by measuring HA concentration using the dot-blot technique and an anti-HA antibody developed in house. The infectious titer was determined by TCID50 in MDCK cells.
In parallel, the phosphorylation of mTOR was increased during the first 24hpi but remained stable, and was similar for the two strains of influenza examined (Figure 1B). Finally, ERK was initially activated by infection with H3N2 Aichi but not with H1N1 PR/8, and decreased at 24hpi with both strains (Figure 1C).The profile of activation of each kinase reflects different cellular events caused by viral infection: mTOR is solicited during protein synthesis while Erk is involved in export of viral ribonucleoproteins from the nucleus to the cytoplasm [7–9].
Effect of small molecules used to modulate Akt activity at different time points after infection with H1N1 PR/8
Akt activator (T.O.I)
Akt activator (17hpi)
Akt inhibitor (T.O.I.)
Akt inhibitor (17hpi)
HA concentration (μg/ml)
18.4 ± 0.8
2.9 ± 0.6
35.1 ± 5.2
31.2 ± 3.9
21.0 ± 4.4
We have systematically investigated multiple signaling pathways using flow cytometry, with particular attention to time points corresponding to viral entry and exit. An understanding of the timing of these events will facilitate the selection of small molecules to control signaling pathways and we have first evidence that this will lead to an improved feeding strategy. As a proof of principle, we show that modulation at Akt at strategic time points significantly increases viral yield. Other important signaling molecules that will be examined include PKC, NFkB and P53. Based on the data thus obtained, a cocktail of small molecules can be prepared and added to the culture at the most appropriate time in order to improve the yield and the quality of the virus generated for vaccine production.
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