Department of Immunotherapeutics and Biotechnology, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center.
Molecular Neurosurgery Laboratory and the Brain Tumor Research Center, Department of Neurosurgery, Massachusetts General Hospital and Harvard Medical School.
J Vis Exp. 2021 May 13(171). doi: 10.3791/62677.
Oncolytic viruses (OVs), such as the oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a "plaque assay" in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.
溶瘤病毒（OVs），如溶瘤单纯疱疹病毒（oHSV），是癌症免疫治疗领域中一种快速发展的治疗策略。OVs，包括 oHSV，选择性地在癌细胞中复制并杀死癌细胞（不伤害健康/正常细胞），同时诱导抗肿瘤免疫。由于这些独特的特性，基于 oHSV 的治疗策略越来越多地用于癌症的治疗，包括临床前和临床，包括 FDA 批准的 talimogene laherparevec（T-Vec）。在进行实验研究之前，任何 OVs，包括 oHSVs，都需要进行生长、纯化和滴定这三种基本的实验室技术。本文描述了一种在 Vero 细胞中扩增 oHSV 的简单逐步方法。随着 oHSV 的繁殖，它们会在 Vero 细胞中产生细胞病变效应（CPE）。一旦 90-100%的感染细胞出现 CPE，就可以轻轻收获细胞，用溶菌酶和氯化镁（MgCl2）处理，过滤，然后用蔗糖梯度法进行纯化。纯化后，通过 Vero 细胞中的“空斑测定”确定具有感染力的 oHSV（称为噬菌斑形成单位或 PFUs）的数量。本文所述的方案可用于制备高滴度的 oHSV 用于细胞培养中的体外研究和动物实验体内实验。
