Microarray assay for evaluation of the genetic stability of modified vaccinia virus ankara B5R gene
Microarray assay for evaluation of the genetic stability of modified vaccinia virus ankara B5R gene
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DOI：
10.1002/jmv.20889
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发表时间：
2007-06-01
影响因子：
12.7
通讯作者：
Chumakov, Konstantin
中科院分区：
文献类型：
--
作者：
Laassri, Majid;Meseda, Clement A.;Chumakov, Konstantin
与使用活天花疫苗相关的不良事件导致了新一代减毒天花疫苗的开发，这些疫苗是在培养细胞中制备的，作为替代品。无法对其在人体上的疗效进行直接临床评估，这要求许可必须基于动物研究和对其体外特性的详尽评估。活病毒疫苗最重要的特征之一是它们的遗传稳定性，包括疫苗毒株回复到更强的毒力形式，与其他病毒序列重组产生潜在的致病病毒，以及可能导致免疫原性和效力降低的遗传漂移。为了研究新一代天花疫苗中免疫必需的痘苗病毒基因的遗传稳定性，开发了一种先进的寡核苷酸芯片，用于检测B5R基因可能出现的突变。B5R基因是一种痘苗病毒基因，编码一种含有非常重要的中和表位的蛋白质。这个微阵列包含重叠的寡核苷酸，覆盖修饰的痘苗病毒安卡拉(MVA)的13513个基因，MVA是一种研究得很好的候选天花疫苗。微阵列分析被证明能够检测到甚至是一个单点突变，并能够区分牛痘病毒株。同时，它还可以检测到牛痘病毒株克隆中新出现的突变。在本文描述的工作中，MVA B5R基因在Vero和MRC-5细胞中传代34次后是稳定的，这两种细胞被建议用作疫苗生产的细胞底物。潜在地，建议的方法可以用作身份测试，并可以扩展到整个病毒基因组，并用于监测疫苗生产的一致性。J.Med.维罗尔。79：791-802,2007。(C)2007年Wiley-Liss，Inc.
Adverse events associated with the use of live smallpox vaccines have led to the development of a new generation of attenuated smallpox vaccines that are prepared in cultured cells as alternatives. The inability to conduct direct clinical evaluation of their efficacy in humans demands that licensure be based on animal studies and exhaustive evaluation of their in vitro properties. One of the most important characteristics of live viral vaccines is their genetic stability, including reversion of the vaccine strain to more virulent forms, recombination with other viral sequences to produce potentially pathogenic viruses, and genetic drift that can result in decrease of immunogenicity and efficacy. To study genetic stability of an immunoessential vaccinia virus gene in a new generation smallpox vaccine, an advanced oligonucleotide microchip was developed and used to assay for mutations that could emerge in B5R gene, a vaccinia virus gene encoding for a protein that contains very important neutralizing epitopes. This microarray contained overlapping oligonucleoticles covering the 13513 gene of modified vaccinia virus Ankara (MVA), a well-studied candidate smallpox vaccine. The microarray assay was shown to be able to detect even a single point mutation, and to differentiate between vaccinia strains. At the same time, it could detect newly emerged mutations in clones of vaccinia strains. In the work described here, it was shown that MVA B5R gene was stable after 34 passages in Vero and MRC-5 cells that were proposed for use as cell substrates for vaccine manufacture. Potentially, the proposed method could be used as an identity test and could be extended for the entire viral genome and used to monitor consistency of vaccine production. J. Med. Virol. 79:791-802, 2007. (C) 2007 Wiley-Liss, Inc.