禽流感病毒的悬浮培养

VaccineNew avian suspension cell lines provide production of influenza virus and MV A 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 hen’s eggs or in primary chickenfibroblasts(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 difficulties in amplification of certain strains with required seasonal antigenicity[1]. Continuous cell lines to replace primary material for robust industrial production especially of influenza 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-sages[2]. 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 influenza vaccines, modified vaccinia Ankara viruses(MV A,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 MV A requires CEF cultures, again limiting the scalability and robustness of the process.MV A vectors against complex infectious diseases will be required at high titres so that efficient production processes are highly desirable.In order to overcome the problems in influenza and MV A production new cell lines were developed recently[3–5].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 indefinite 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 5[5].Cell densities ofup to 1.0×107 cells/mL and influenza 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 influenza but also forMV A[7]. Derived from spontaneous immortalization of embryonic chicken cells,they grow to cell densities of up to 1.0–2.0×107 cells/mL and provide MV A virus titres up to 1.0×108pfu/mL[4]. However, the detection of endogenous retroviral particles in chicken embryo-derived yellow fever vaccines affects the safety of this cell line for vaccine manufacturing[8]. As ana lternative,the avian cell line AGE1.CR(CR)was created by immortalizing cells from duck retina with adenovirus type 5E1 genes[3].Ducks as opposed to chickens carry significantly fewer endogenous retroviral inserts and noactive elements have been described[9]. The CR line was further modified 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 line[10].CR and CR.pIX cell lines proliferate ins uspension in serum-free media. Exceptional genetic stability wasdemonstrated for more than 90 passages. The cells can be productively infected with viruses of diverse families including different influenza strains and MV A.Here, we present data for the cultivation of CR and CR.pIX cells in T-flasks,roller bottles and small scale bioreactors(stirred tank and wave bioreactor) and infection with influenza virus and MV A.In vestigations of cell growth and metabolism in non-infected cells and infection experiments were done.Various influenza virus subtypes were tested and the impact of the pIX gene on MV A yield was evaluated.Titres obtained with either influenza A or MV A 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。