Max Planck Institute for Dynamics of Complex Technical Systems, Bioprocess Engineering, Sandtorstrasse 1, 39106 Magdeburg, Germany.
Vaccine. 2012 Jul 27;30(35):5253-61. doi: 10.1016/j.vaccine.2012.05.065. Epub 2012 Jun 12.
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.
在基于细胞培养的流感疫苗生产中，人们致力于对病毒种子进行优化，以获得最大产量。通常，通过从野生型株系中生成含有例如流感 A/PR/8 的六个基因片段的骨干的高生长重配体（HGR）。然而，获得的 HA 和 TCID₅₀ 滴度通常不符合预期，需要进一步优化措施。流式细胞术是一种非常宝贵的工具，可以帮助我们更好地理解与感染进展、病毒诱导的细胞凋亡和细胞特异性产率相关的机制。在这项研究中，我们用两种流感 A/PR/8 变体（来自 NIBSC 和 RKI）和两种基于 A/PR/8 的 HGR（Wisconsin-like 和 Uruguay-like）进行了感染，以研究不同感染复数（MOI 0.0001、0.1、3）下的病毒复制、凋亡和病毒滴度。流式细胞术分析表明，在 MOI 0.0001 下，所有四种测试株系的感染和凋亡细胞群体的时间进程中显示出相似的动态。有趣的是，较高的 MOI 导致感染和凋亡细胞群体更早增加，并显示出株系特异性差异。与 A/PR/8 RKI 相比，用 A/PR/8 NIBSC 感染导致两个细胞群体的增加更早。Uruguay-like 重配体在所有测试 MOI 下，表现出最早增加感染细胞的浓度，并在晚期诱导凋亡。相比之下，Wisconsin-like 重配体在高 MOI 下显示出强烈的细胞凋亡诱导作用，导致与低 MOI 相比，病毒滴度降低。两种 A/PR/8 和 Uruguay-like 重配体的 HA 最大滴度不受 MOI 变化的影响。然而，所有菌株的最大 TCID₅₀ 滴度随着 MOI 的增加而降低。总体而言，在非常低的 MOI（0.0001）下进行感染，不仅在感染进展和诱导凋亡方面具有相似的动态，而且在最大病毒产量方面也具有相似的动态。对于将感染快速进展与晚期细胞凋亡相结合的病毒种子株系，获得了最高的 HA 滴度。因此，在建立稳健的流感疫苗生产工艺时，应该考虑这两个因素。
