Summary

单纯疱疹病毒的生长、净化和滴定

Published: May 13, 2021
doi:

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

Referências

  1. Harrington, K., Freeman, D. J., Kelly, B., Harper, J., Soria, J. -. C. Optimizing oncolytic virotherapy in cancer treatment. Nature Reviews Drug Discovery. 18 (9), 689-706 (2019).
  2. Zhang, S., Rabkin, S. D. The discovery and development of oncolytic viruses: are they the future of cancer immunotherapy. Expert Opinion on Drug Discovery. 16 (4), 391-410 (2021).
  3. Bommareddy, P. K., Peters, C., Saha, D., Rabkin, S. D., Kaufman, H. L. Oncolytic herpes simplex viruses as a paradigm for the treatment of cancer. Annual Review of Cancer Biology. 2 (1), 155-173 (2018).
  4. Peters, C., Rabkin, S. D. Designing herpes viruses as oncolytics. Molecular Therapy-Oncolytics. 2, 15010 (2015).
  5. Nguyen, H. -. M., Saha, D. The current state of oncolytic herpes simplex virus for glioblastoma treatment. Oncolytic Virotherapy. 10, 1-27 (2021).
  6. Koch, M. S., Lawler, S. E., Chiocca, E. A. HSV-1 oncolytic viruses from bench to bedside: an overview of current clinical trials. Cancers. 12 (12), 3514 (2020).
  7. Menotti, L., Avitabile, E. Herpes simplex virus oncolytic immunovirotherapy: the blossoming branch of multimodal therapy. International Journal of Molecular Sciences. 21 (21), 8310 (2020).
  8. Nguyen, H. M., Guz-Montgomery, K., Saha, D. Oncolytic virus encoding a master pro-inflammatory cytokine interleukin 12 in cancer immunotherapy. Cells. 9 (2), 400 (2020).
  9. Agarwalla, P. K., Aghi, M. K. Oncolytic herpes simplex virus engineering and preparation. Methods in Molecular Biology. 797, 1-19 (2012).
  10. Grosche, L., et al. Herpes simplex virus type 1 propagation, titration and single-step growth curves. Bio-protocol. 9 (23), 3441 (2019).
  11. Sutter, S. O., Marconi, P., Meier, A. F. Herpes simplex virus growth, preparation, and assay. Methods in Molecular Biology. 2060, 57-72 (2020).
  12. Froechlich, G., et al. Integrity of the antiviral STING-mediated DNA sensing in tumor cells is required to sustain the immunotherapeutic efficacy of herpes simplex oncolytic virus. Cancers. 12 (11), 3407 (2020).
  13. Froechlich, G., et al. Generation of a novel mesothelin-targeted oncolytic herpes virus and implemented strategies for manufacturing. International Journal of Molecular Sciences. 22 (2), 477 (2021).
  14. Mosca, J. D., Pitha, P. M. Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis. Molecular and Cellular Biology. 6 (6), 2279-2283 (1986).
  15. Gousseinoz, E., Kools, W., Pattnaik, P. Nucleic acid impurity reduction in viral vaccine manufacturing. BioProcess International. 12 (2), 59-68 (2014).
  16. Diefenbach, R. J., Fraefel, C. Herpes simplex virus: methods and protocols. Methods in Molecular Biology. , (2014).
  17. Hadi, A. M., et al. An experimental trial to prepared γ1 34.5 herpes simplex virus 1 immunogene by cloning technique. Systematic Review Pharmacy. 11 (5), 140-149 (2020).
  18. Kuroda, T., Martuza, R. L., Todo, T., Rabkin, S. D. Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases. BMC Biotechnology. 6, 40 (2006).
  19. Motamedifar, M., Noorafshan, A. Cytopathic effect of the herpes simplex virus type 1 appears stereologically as early as 4 h after infection of Vero cells. Micron. 39 (8), 1331-1334 (2008).
  20. Blaho, J. A., Morton, E. R., Yedowitz, J. C. Herpes simplex virus: propagation, quantification, and storage. Current Protocols in Microbiology. , 1 (2005).
  21. Malenovska, H. The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage. Journal of Applied Microbiology. 117 (6), 1810-1819 (2014).
  22. Vahlne, A. G., Blomberg, J. Purification of herpes simplex virus. Journal of General Virology. 22 (2), 297-302 (1974).
  23. Sathananthan, B., Rodahl, E., Flatmark, T., Langeland, N., Haarr, L. Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion. APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 105 (3), 238-246 (1997).
  24. Mundle, S. T., et al. High-purity preparation of HSV-2 vaccine candidate ACAM529 is immunogenic and efficacious in vivo. PLoS One. 8 (2), 57224 (2013).
  25. Jiang, C., et al. Immobilized cobalt affinity chromatography provides a novel, efficient method for herpes simplex virus type 1 gene vector purification. Journal of Virology. 78 (17), 8994-9006 (2004).
  26. Grosche, L., et al. Herpes simplex virus type 1 propagation, titration and single-step growth curves. Bio-protocol. 9 (23), 3441 (2019).
  27. Svennerholm, B., et al. Separation of herpes simplex virus virions and nucleocapsids on Percoll gradients. Journal of Virological Methods. 1 (6), 303-309 (1980).
  28. Baer, A., Kehn-Hall, K. Viral concentration determination through plaque assays: using traditional and novel overlay systems. Journal of Visualized Experiments: JoVE. (93), e52065 (2014).
  29. Miyatake, S., Iyer, A., Martuza, R. L., Rabkin, S. D. Transcriptional targeting of herpes simplex virus for cell-specific replication. Journal of Virology. 71 (7), 5124-5132 (1997).
  30. Fabiani, M., Limongi, D., Palamara, A. T., De Chiara, G., Marcocci, M. E. A novel method to titrate herpes simplex virus-1 (HSV-1) using laser-based scanning of near-infrared fluorophores conjugated antibodies. Frontiers in Microbiology. 8, 1085 (2017).

Play Video

Citar este artigo
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).

View Video