Otto-von-Guericke-University Magdeburg, Chair of Bioprocess Engineering, Magdeburg, Germany.
German Primate Center-Leibniz Institute for Primate Research, Infection Biology Unit, Göttingen, Germany.
BMC Biol. 2021 May 3;19(1):91. doi: 10.1186/s12915-021-01020-5.
Infections with influenza A virus (IAV) cause high morbidity and mortality in humans. Additional to vaccination, antiviral drugs are a treatment option. Besides FDA-approved drugs such as oseltamivir or zanamivir, virus-derived defective interfering (DI) particles (DIPs) are considered promising new agents. IAV DIPs typically contain a large internal deletion in one of their eight genomic viral RNA (vRNA) segments. Consequently, DIPs miss the genetic information necessary for replication and can usually only propagate by co-infection with infectious standard virus (STV), compensating for their defect. In such a co-infection scenario, DIPs interfere with and suppress STV replication, which constitutes their antiviral potential.
In the present study, we generated a genetically engineered MDCK suspension cell line for production of a purely clonal DIP preparation that has a large deletion in its segment 1 (DI244) and is not contaminated with infectious STV as egg-derived material. First, the impact of the multiplicity of DIP (MODIP) per cell on DI244 yield was investigated in batch cultivations in shake flasks. Here, the highest interfering efficacy was observed for material produced at a MODIP of 1E-2 using an in vitro interference assay. Results of RT-PCR suggested that DI244 material produced was hardly contaminated with other defective particles. Next, the process was successfully transferred to a stirred tank bioreactor (500 mL working volume) with a yield of 6.0E+8 PFU/mL determined in genetically modified adherent MDCK cells. The produced material was purified and concentrated about 40-fold by membrane-based steric exclusion chromatography (SXC). The DI244 yield was 92.3% with a host cell DNA clearance of 97.1% (99.95% with nuclease digestion prior to SXC) and a total protein reduction of 97.2%. Finally, the DIP material was tested in animal experiments in D2(B6).A2G-Mx1 mice. Mice infected with a lethal dose of IAV and treated with DIP material showed a reduced body weight loss and all animals survived.
In summary, experiments not only demonstrated that purely clonal influenza virus DIP preparations can be obtained with high titers from animal cell cultures but confirmed the potential of cell culture-derived DIPs as an antiviral agent.
甲型流感病毒（IAV）感染会导致人类发病率和死亡率增高。除了接种疫苗外，抗病毒药物也是一种治疗选择。除了美国食品和药物管理局（FDA）批准的药物，如奥司他韦或扎那米韦，病毒衍生的缺陷干扰（DI）颗粒（DIPs）也被认为是有前途的新型药物。IAV DIPs 通常在其 8 个基因组病毒 RNA（vRNA）片段之一中含有一个大的内部缺失。因此，DIPs 缺失了复制所需的遗传信息，通常只能通过与传染性标准病毒（STV）共感染来进行复制，从而补偿其缺陷。在这种共感染的情况下，DIPs 会干扰和抑制 STV 的复制，这构成了它们的抗病毒潜力。
在本研究中，我们通过遗传工程化的 MDCK 悬浮细胞系生产了一种纯粹的克隆 DIP 制剂，该制剂在其第 1 节（DI244）中有一个大的缺失，并且不像卵源性材料那样受到传染性 STV 的污染。首先，在摇瓶的分批培养中研究了每个细胞的 DIP 多重性（MODIP）对 DI244 产量的影响。在这里，通过体外干扰测定，在使用 1E-2 的 MODIP 时观察到最高的干扰效果。RT-PCR 结果表明，生产的 DI244 材料几乎没有受到其他缺陷颗粒的污染。接下来，该工艺成功地转移到搅拌罐生物反应器（500 毫升工作体积）中，在遗传修饰的贴壁 MDCK 细胞中确定了 6.0E+8 PFU/mL 的产量。通过基于膜的排阻色谱（SXC）对产生的物质进行了约 40 倍的纯化和浓缩。DI244 的产量为 92.3%，宿主细胞 DNA 清除率为 97.1%（在 SXC 之前用核酸酶消化时为 99.95%），总蛋白减少 97.2%。最后，在 D2（B6）。A2G-Mx1 小鼠中进行了动物实验。用致死剂量的 IAV 感染并给予 DIP 材料的小鼠体重减轻减少，所有动物均存活。
总之，实验不仅表明可以从动物细胞培养物中获得高滴度的纯克隆流感病毒 DIP 制剂，而且还证实了细胞培养衍生的 DIP 作为抗病毒药物的潜力。
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