Abstract
Background 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 [1]; 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.
Highlights
Cell-culture based vaccines are a valuable alternative to egg-produced vaccines
The kinetics of production of H1N1 A/Puerto Rico/8/68 in HEK293 cells has been previously characterized by our group [1]; higher viral production is achieved when cells are infected at a multiplicity of infection (MOI) of 0.01 compared to an MOI of 1
H3N2 Aichi activated Akt but to a lesser extent with a 6-fold increase in phosphorylation at 24hpi (Figure 1A). These results suggest that signaling pathways are differentially modulated depending on the strain of influenza virus
Summary
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 [1]; higher viral production is achieved when cells are infected at a multiplicity of infection (MOI) of 0.01 compared to an MOI of 1. Two cycles of infection take place: a first round of virus is produced at 8 hours post-infection (hpi) which 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. The main objective is 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
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