单纯疱疹病毒的生长、净化和滴定
Summary
在本手稿中，我们描述了一个简单的方法的生长，纯化和滴定肿瘤疱疹简单病毒的药物前使用。
Abstract
肿瘤病毒（OVs），如肿瘤疱疹单纯病毒（oHSV），是癌症免疫治疗领域快速增长的治疗策略。OVs，包括oHSV，有选择地复制和杀死癌细胞（拯救健康/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，临床前和临床上，包括 FDA 批准的塔利莫金·拉赫帕雷韦克 （T-Vec）。生长、纯化和滴定是任何 OEV（包括 oHSV）的三种基本实验室技术，在它们可用于实验研究之前。本文描述了一个简单的分步方法来放大维罗细胞中的 oHSV。当 oHSV 成倍增加时，它们在 Vero 细胞中产生细胞病变效应 （CPE）。一旦90-100%的受感染细胞显示CPE，它们就会被轻轻收获，用苯甲酸酯和氯化镁（MgCl2）进行处理，过滤后，使用蔗糖梯度法进行净化。净化后，传染性 oHSV（指定为斑块形成单位或 PPF）的数量由 Vero 细胞中的”斑块检测”确定。此处描述的协议可用于为细胞培养和体内动物实验的体外研究准备高滴度 oHSV 库存。
Introduction
肿瘤病毒（OVs）是癌症免疫治疗的一种新兴和独特的形式。OVs有选择地复制和解毒肿瘤细胞（备用正常/健康细胞）1，同时诱导抗肿瘤免疫2。单纯疱疹病毒（oHSV）是所有OV中研究最广泛的病毒之一。这是最远的沿诊所， 与塔利莫金拉赫帕雷普韦克 （T-VEC） 是第一个也是唯一的 OV 获得 FDA 批准在美国治疗晚期黑色素瘤3.除了T-VEC，许多其他基因工程的oHSV正在临床和临床上测试不同的癌症类型3，4，5，6，7，8。目前先进的重组DNA生物技术进一步增加了工程新oHSV编码治疗转基因的可行性。在任何（新开发的）oHSV能够进行体外和体内研究测试之前，有效的 oHSV 传播、纯化和滴定测定系统至关重要。本文描述了一个简单的分步方法（在维罗细胞中）、纯化（通过蔗糖梯度法）和滴定（通过在维罗细胞中的 oHSV 斑块检测）（图 1）。它可以很容易地在任何生物安全级别 2 （BSL2） 实验室设置中采用，以实现高品质的病毒库存，用于前科研究。
维罗，非洲绿猴肾细胞系，是最常见的细胞系为oHSV传播9，10，11，12，13，因为维罗细胞有一个有缺陷的抗病毒干扰素信号通路14。其他具有干扰素基因（STING）信号的灭活刺激器的细胞系也可用于oHSV增长12，13。此协议利用 Vero 细胞进行 oHSV 生长和斑块检测。繁殖后，oHSV感染的细胞被收获、解毒并接受净化，其中被浸渍细胞首先用苯甲酶核酶处理，以降解宿主细胞DNA，防止核酸蛋白聚集，降低细胞的粘度。由于适当激活苯甲酶通常需要毫克2+，1-2 mm MgCl2用于此协议15。在高速蔗糖梯度离心之前，通过连续过滤进一步消除苯甲酸酯处理细胞裂解物中的宿主细胞碎片。粘性25%蔗糖溶液垫有助于确保通过蔗糖层的病毒迁移速度更慢，将宿主细胞相关成分留在超自然体内，从而改善净化和限制颗粒16中的病毒损失。净化的 oHSV 然后滴定在 Vero 细胞上，病毒斑块通过 Giemsa 染色17或 X gal 染色（用于 LacZ 编码 oHSV）18来可视化。
Protocol
1） 奥赫斯夫增长 注：在与 oHSV 合作之前，确保机构生物安全委员会的批准。这项研究是根据经核准的IBC第18007号议定书进行的。保持BSL2预防措施：漂白所有与病毒接触的管道、尖端、管子和其他材料。在手离开BSL2细胞培养罩之前，用70%异丙醇喷洒手套。使用病毒后，始终用肥皂水彻底洗手。 在第 -1 天，种子低通道 Vero 细胞在 20 T-150 厘米2 烧瓶密度为 7-8 × 1…
Representative Results
图 1中描述了整个协议的简要概述，该图代表了 oHSV 的生长、净化和认证的关键步骤。Vero细胞中的CPE最早可检测到4小时后HSV感染19。图2在 oHSV 感染后的三个不同时间点演示 Vero 细胞中的 CPE。随着时间的推移，CPE 的水平会提高。在此协议中，90-100% CPE 通常在低 MOI oHSV 接种的 48 小时内观察到（这是收获细胞进行净化的最佳时机）。但?…
Discussion
该协议从低通道维罗细胞中的 oHSV 生长开始。在病毒接种时，Vero细胞单层的对流应为+80%，因为过度生长的细胞可以发展出紧密的纤维结构，减少OHSV进入Vero细胞20。观察到 90-100% CPE 后，培养超自然体被去除，细胞被收获，在 VB/超自然剂中再悬浮（参见步骤 1.4.6），快速冷冻，并储存在 -80 °C 以供以后净化。Blaho和他的同事采用了一种略有不同的方法来采集和储存受感染的维罗细…
Declarações
The authors have nothing to disclose.
Acknowledgements
萨哈实验室的研究部分得到了美国劳工部（W81XWH-20-1-0702）和道奇琼斯基金会-阿比林的资金支持。塞缪尔·拉布金和梅丽莎·汉弗莱.M部分得到国家卫生研究院（R01 CA160762）的支持。
Materials
| 1.7 mL centrifuge tubes | Sigma | CLS3620 | |
| 15 mL polypropylene centrifuge tubes | Falcon | 352097 | |
| 5 mL polypropylene tubes | Falcon | 352063 | |
| 50 mL polypropylene centrifuge tubes | Falcon | 352098 | |
| 6-well cell culture plates | Falcon | 353046 | |
| Benzonase Nuclease | Sigma | E8263-25KU | |
| Cell scraper | Fisher Scientific | 179693 | |
| Dimethyl sulfoxide | Sigma | D2650-100ML | |
| Dulbecco’s Modified Eagle Medium | Corning | MT-10-013-CV | |
| Dulbecco’s Phosphate Buffered Saline | Corning | MT-21-031-CV | |
| Fetal Bovine Serum | Hyclone | SH3007003 | |
| Giemsa Stain | Sigma | G3032 | |
| Glutaraldehyde | Fisher Scientific | 50-262-23 | |
| Glycerol | Sigma | G5516 | |
| Hank's Balanced Salt Solution (HBSS) | Corning | MT-21-021-CV | |
| High-Glucose Dulbecco’s Phosphate-buffered Saline | Sigma | D4031 | |
| Human immune globulin | Gamastan | NDC 13533-335-12 | |
| Magnesium chloride | Fisher Chemical | M33-500 | |
| Media Sterilization filter, 250 mL | Nalgene, Fisher Scientific | 09-740-25E | |
| Media Sterilization filter, 500 mL | Nalgene, Fisher Scientific | 09-740-25C | |
| Neutral Red solution | Sigma | N4638 | |
| Paraformaldehyde | Fisher scientific | 15710S | |
| Plate rocker | Fisher | 88861043 | |
| Potassium Ferricyanide | Sigma | P8131 | |
| Potassium Ferrocyanide | Sigma | P9387 | |
| Sodium chloride | Fisher Chemical | S271-3 | |
| Sorvall ST 16R Centrifuge | ThermoFisher Scientific | 75004381 | |
| Sorvall ST 21R Centrifuge | ThermoFisher Scientific | 75002446 | |
| Sterile Microcentrifuge Tubes with Screw Caps | Fisher Scientific | 02-681-371 | |
| Sucrose | Fisher Scientific | BP220-1 | |
| Syringe Filter, 0.45 PVDF | MilliporeSigma | SLHV033RS | |
| Syringe Filter, 0.8 MCE | MilliporeSigma | SLAA033SS | |
| Syringe filter, 5 µm PVDF | MilliporeSigma | SLSV025LS | |
| T150 culture flask | Falcon | 355001 | |
| Tris-HCl | MP Biomedicals LLC | 816116 | |
| Ultrasonic water bath | Branson | CPX-952-116R | |
| X-gal | Corning | 46-101-RF |
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Tags
Oncolytic Herpes Simplex VirusCancer ImmunotherapyVirus PropagationVirus PurificationVirus TitrationBiosafety Level TwoVirus StockAnticancer Immune ResponseVirus-based Cancer ImmunotherapyWild-type Herpes Simplex VirusGenetically Engineered Herpes Simplex VirusT-150 Square Centimeter FlasksDPBSIFCSDMEMCell ScraperCentrifugationVirus BufferBenzonase NucleaseVirus Titer
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Nguyen, H., Sah, N., Humphrey, M. R. M., Rabkin, S. D., Saha, D. Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus. J. Vis. Exp. (171), e62677, doi:10.3791/62677 (2021).