Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Summary

In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use.

Abstract

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.

Introduction

Oncolytic viruses (OVs) are an emerging and unique form of cancer immunotherapy. OVs selectively replicate in and lyse tumor cells (sparing normal/healthy cells)¹ while inducing anti-tumor immunity². Oncolytic herpes simplex virus (oHSV) is one of the most extensively studied viruses among all OVs. It is furthest along in the clinic, with Talimogene laherparepvec (T-VEC) being the first and only OV to receive FDA approval in the USA for the treatment of advanced melanoma³. In addition to T-VEC, many other genetically engineered oHSVs are being tested preclinically and clinically in different cance....

Protocol

1) oHSV growth

NOTE: Ensure institutional biosafety committee approval before working with oHSV. This study was conducted under approved IBC Protocol no. 18007. Maintain BSL2 precautions: bleach all pipets, tips, tubes, and other materials that come into contact with the virus. Spray gloves with 70% isopropyl alcohol before hands leave the BSL2 cell culture hood. Always thoroughly wash hands with soap water after working with a virus.

1. On day -1, seed low-passage Vero cells in 20 T-.......

Discussion

The protocol starts with the growth of oHSV in low-passage Vero cells. The confluency of the Vero cell monolayer should be ~80% at the time of virus inoculation as overgrown cells can develop tight fibrous structures that can reduce oHSV entry into Vero cells²⁰. Once 90-100% CPE is observed, the culture supernatant is removed, cells are harvested, resuspended in VB/supernatant (see step 1.4.6), snap-frozen, and stored at -80 °C for later purification. Blaho and colleagues employed a slig.......

Materials

Name	Company	Catalog Number	Comments
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