Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells
Growth Determinants for H5N1 Influenza Vaccine Seed Viruses in MDCK Cells
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DOI：
10.1128/jvi.00970-08
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发表时间：
2008-11-01
影响因子：
5.4
通讯作者：
Kawaoka, Yoshihiro
中科院分区：
文献类型：
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作者：
Murakami, Shin;Horimoto, Taisuke;Kawaoka, Yoshihiro
H5 N1甲型流感病毒正在造成越来越多的人类死亡，迄今已有240多例死亡病例。如果这些病毒引起流感大流行，作为人类灭活疫苗生产的批准基质的鸡胚可能会供应短缺，因为鸡将被这些病毒杀死或被扑杀以限制感染的全球传播。Madin-Darby犬肾（MDCK）细胞系是一种有前途的替代候选底物，因为与其他细胞系相比，它支持流感病毒的有效生长。在这里，我们解决了在MDCK细胞中的H5 N1疫苗种子病毒的生长的分子决定因素，揭示了PB 2的位置360处的Tyr残基的关键责任，血凝素（HA）和神经氨酸酶（NA）之间的功能平衡的相当大的要求，和NS 1的位置55处的Glu残基的部分责任。基于这些发现，我们产生了针对该细胞系优化的PR 8/H5 N1抑制剂，其除了NS基因之外，其内部蛋白的所有基因都来自PR 8（UW）菌株，NS基因来自PR 8（UW）菌株。（剑桥）毒株;其N1 NA基因，该基因具有长柄并源自早期H5 N1毒株;以及其HA基因，其具有亲和型切割位点序列并且来源于循环的H5 N1病毒。我们的研究结果证明了基于细胞培养的方法生产灭活H5 N1疫苗的种子病毒的重要性和可行性，该方法可以以及时，具有成本效益的方式稳健生长，作为基于鸡蛋的疫苗生产的替代方案。
H5N1 influenza A viruses are exacting a growing human toll, with more than 240 fatal cases to date. In the event of an influenza pandemic caused by these viruses, embryonated chicken eggs, which are the approved substrate for human inactivated-vaccine production, will likely be in short supply because chickens will be killed by these viruses or culled to limit the worldwide spread of the infection. The Madin-Darby canine kidney (MDCK) cell line is a promising alternative candidate substrate because it supports efficient growth of influenza viruses compared to other cell lines. Here, we addressed the molecular determinants for growth of an H5N1 vaccine seed virus in MDCK cells, revealing the critical responsibility of the Tyr residue at position 360 of PB2, the considerable requirement for functional balance between hemagglutinin ( HA) and neuraminidase (NA), and the partial responsibility of the Glu residue at position 55 of NS1. Based on these findings, we produced a PR8/H5N1 reassortant, optimized for this cell line, that derives all of its genes for its internal proteins from the PR8(UW) strain except for the NS gene, which derives from the PR8( Cambridge) strain; its N1 NA gene, which has a long stalk and derives from an early H5N1 strain; and its HA gene, which has an avirulent-type cleavage site sequence and is derived from a circulating H5N1 virus. Our findings demonstrate the importance and feasibility of a cell culture-based approach to producing seed viruses for inactivated H5N1 vaccines that grow robustly and in a timely, cost-efficient manner as an alternative to egg-based vaccine production.