EPPIC (Efficient Purification by Parental Inducer Constraint) Platform for Rapid Generation of Recombinant Vaccinia Viruses
EPPIC (Efficient Purification by Parental Inducer Constraint) Platform for Rapid Generation of Recombinant Vaccinia Viruses
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DOI：
10.1016/j.omtm.2020.03.026
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发表时间：
2020-06-12
影响因子：
4.7
通讯作者：
Verardi, Paulo H.
中科院分区：
文献类型：
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作者：
Jasperse, Brittany;O'Connell, Caitlin M.;Verardi, Paulo H.
牛痘病毒（VACV）被成功地用作天花根除运动的疫苗。从那时起，它被广泛用于疫苗和治疗载体的开发。然而，产生和纯化重组VACV（rVACV）的方法通常是耗时的、繁琐的，并且在某些情况下需要专门的细胞系或设备。在这里，我们描述了一种新的EPPIC（通过亲本诱导物约束的有效纯化）平台，用于使用复制诱导型VACV（vIND）作为同源重组的亲本病毒快速生成rVACV。从亲本vIND纯化rVACV通过在不存在诱导物的情况下连续传代两次来实现（即，亲本诱导物“约束”），而不需要专门的设备。我们确定了同源重组和系列纯化的最佳条件，并产生了一套vIND亲本病毒，以促进平台的定制。重要的是，EPPIC平台可以通过对DNA穿梭载体的简单修饰而适于快速产生表达疫苗或治疗性抗原的复制缺陷型和复制能力型rVACV，具有或不具有筛选标记物，从而允许在几天内快速开发、更新和改进个性化或定制疫苗和治疗性载体。
Vaccinia virus (VACV) was successfully used as a vaccine in the smallpox eradication campaign. Since then, it has been widely used in the development of vaccine and therapeutic vectors. However, methods of generating and purifying recombinant VACVs (rVACVs) are often time-consuming, cumbersome, and in some cases require specialized cell lines or equipment. Here, we describe a novel EPPIC (Efficient Purification by Parental Inducer Constraint) platform for the rapid generation of rVACVs using a replication-inducible VACV (vIND) as a parental virus for homologous recombination. Purification of the rVACV from the parental vIND is achieved by two serial passages in the absence of inducer (i.e., parental inducer "constraint") in standard laboratory cell lines, without the need for specialized equipment, within 1 week. We determined the optimal conditions for homologous recombination and serial purification and generated a suite of vIND parental viruses to facilitate customization of the platform. Importantly, the EPPIC platform can be adapted to rapidly generate replication-deficient and replication-competent rVACVs expressing vaccine or therapeutic antigens, with or without screening markers, by simple modifications to a DNA shuttle vector, thus allowing the rapid development, updating, and refinement of personalized or custom vaccines and therapeutic vectors in a matter of days.