禽流感病毒的悬浮培养.doc

VaccineNew avian suspension cell lines provide production of influenza virus and MVA in serum-free media: Studies on growth, metabolism and virus propagation.新的禽流感悬浮细胞系提供生产的流感病毒和MVA无血清媒体:研究生长、新陈代谢和病毒传播。1.IntroductionSeveral vaccine viruses ,including most of the human influenza vaccines,are still produced in the allantoic cavity of embryonated hens eggs or in primary chickenbroblasts(CEF). Main draw-backs of this method are limited scalability of the process, possible shortage of material in case of an avian pandemic and occasional difculties in amplication of certain strains with required seasonal antigenicity1. Continuous cell lines to replace primary material for robust industrial production especially of inuenza A and B are therefore highly desirable. MDCK and Vero cells can be used for production of vaccines. However,due to inherent genetic instability of Vero cells a production processis usually limited to only 20pas-sages2. Scale-up is limited by their requirement for an attachment surface and cultivation typically is performed in mediaContaining serum. Serum even of pharmaceutical grade suffers from lot-to-lot variations and is a potential source for contamination with adventitious agents.Contrary to inuenza vaccines, modied vaccinia Ankara viruses(MVA,recombinant or wild type)are strongly host restricted so that most mammalian cells(including MDCK and Veroc ells) are not fully permissive.For this reason production of MVA requires CEF cultures, again limiting the scalability and robustness of the process.MVA vectors against complex infectious diseases will be required at high titres so that efcient production processes are highly desirable.In order to overcome the problems in inuenza and MVA production new cell lines were developed recently35.Development and documentation of these cell lines was performed to meet current regulatory requirements. The cell lines were designed and optimized for a particular production process and typically proliferate with an indenite life span in suspension in serum-free medium with zero or low protein content.The best known example of adesigner cell is the human PER.C6 line which was derived from a primary culture of human fetal retinoblasts immortalized by transfection with the E1expression cassette of adenovirus type 55.Cell densities of up to 1.0107 cells/mL and inuenza virus yields up to 2100HA units/mL are described 6.However, the human PER.C6 cells cannot be productively inf ected by MVA.On the other hand, the avian EB14 cells are permissive not only for inuenza but also forMVA7. Derived from spontaneous immortalization of embryonic chicken cells,they grow to cell densities of up to 1.02.0107 cells/mL and provide MVA virus titres up to 1.0108pfu/mL4. However, the detection of endogenous retroviral particles in chicken embryo-derived yellow fever vaccines affects the safety of this cell line for vaccine manufacturing8. As ana lternative,the avian cell line AGE1.CR(CR)was created by immortalizing cells from duck retina with adenovirus type 5E1 genes3.Ducks as opposed to chickens carry signicantly fewer endogenous retroviral inserts and noactive elements have been described9. The CR line was further modied for constitutive expression of the adenoviral pIX gene(to obtain CR.pIX) that encodes for a structural protein involved in capsid stabilization of cognate adenovirus.This protein was stably inserted because pIX is also implicated in biochemical processes that may confer new properties to thecell line10.CR and CR.pIX cell lines proliferate ins uspension in serum-free media. Exceptional genetic stability was demonstrated for more than 90 passages. The cells can be productively infected with viruses of diverse families including different inuenza strains and MVA.Here, we present data for the cultivation of CR and CR.pIX cells in T-asks,roller bottles and small scale bioreactors(stirred tank and wave bioreactor) and infection with inuenza virus and MVA.In vestigations of cell growth and metabolism in non-infected cells and infection experiments were done.Various inuenza virus subtypes were tested and the impact of the pIX gene on MVA yield was evaluated.Titres obtained with either inuenza A or MVA demonstrate that these suspension cell lines are promising candid ates for large-scale manufacturing of vaccines in serum-free media.翻译：一些疫苗病毒主要分心的方法是有限的可伸缩性的过程,可能缺少材料,以防禽流感大流行和偶尔的困难与需要放大某些菌株的季节性抗原性。连续细胞系取代的主要材料为强劲的工业生产尤其是流感A和B。病毒与流感疫苗、修改牛痘安卡拉(MVA,重组或野生型)等过程中由于实验对象绝大多数是哺乳动物，因此在试验中具有局限性。为了克服这些问题对流感和MVA生产新细胞系培养的阻碍，只能开发和文档执行这些细胞系来满足当前监管要求。细胞系是特定生产过程设计和优化,通常增殖与无限期的寿命在无血清悬浮零或低蛋白质含量。有大量实验证明感染病毒的细胞可以是各种各样的集团,包括不同的流感毒株和MVA。在这里,我们目前的数据在T-flasks CR和CR.pIX细胞的培养指明可用小型生物反应器(搅拌釜和微生物反应器)和感染流感病毒及MVA在未受感染的环境中完成细胞生长和新陈代谢。根据对各种流感病毒亚型进行的测试和焦油的影响可证明这些悬浮细胞系可以进行无血清疫苗媒体的大规模生产。作 者Lohr, V.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；Rath, A.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；Genzel, Y.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；Jordan, I.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；Sandig, V.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；Reichl, U.(Max Planck Inst Dynam Complex Tech Syst, Sandtorstr 1, D-39106 Magdeburg, Germany.)；刊 名Vaccine2009年27卷36期4975-4982页关键词Inf