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Influenza vaccine Flow cytometry Multiplicity of infection Apoptosis Infection dynamics
Abstract:
In cell culture-based influenza vaccine production significant efforts are directed towards virus seed
optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of
six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and
TCID50 titres obtained do not meet expectations and further optimization measures are required.
Flow cytometry is an invaluable tool to improve our understanding of mechanism related to progress of
infection, virus-induced apoptosis, and cell-specific productivity. In this study, we performed infections
with two influenza A/PR/8 variants (from NIBSC and RKI) and two A/PR/8-based HGRs (Wisconsin-like
and Uruguay-like) to investigate virus replication, apoptosis and virus titres at different multiplicities of
infection (MOI 0.0001, 0.1, 3). Flow cytometric analyses showed similar dynamics in the time course of
infected and apoptotic cell populations for all four tested strains at MOI 0.0001. Interestingly, higher MOI
resulted in an earlier increase of the populations of infected and apoptotic cells and showed strain-specific
differences. Infections with A/PR/8 NIBSC resulted in an earlier increase in both cell populations compared
to A/PR/8 RKI. The Uruguay-like reassortant showed the earliest increase in the concentration of infected
cells and a late induction of apoptosis at all tested MOIs. In contrast, the Wisconsin-like reassortant
showed strong apoptosis induction at high MOIs resulting in reduced titres compared to lower MOI.
Maximum HA titres were unaffected by changes in the MOI for the two A/PR/8 and the Uruguay-like
reassortant. Maximum TCID50 titres, however, decreased with increasing MOI for all strains.
Overall, infections at very low MOI (0.0001) resulted not only in similar dynamics concerning progress
of infection and induction of apoptosis but also in maximum virus yields. Highest HA titres were obtained
for virus seed strains combining a fast progress in infection with a late onset of apoptosis. Therefore, both
factors should be considered for the establishment of robust influenza vaccine production processes.
Copyright © 2012 Elsevier Ltd. All rights reserved. [accessed November 2nd 2012]
