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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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-.......

Representative Results

A brief overview of the entire protocol is depicted in Figure 1, which represents the critical steps involved in the growth, purification, and titration of oHSV. CPE in Vero cells can be detected as early as 4 h post-HSV infection19. Figure 2 demonstrates CPE in Vero cells at three different time points following oHSV infection. The level of the CPE is increased over time. In this protocol, 90-100% CPE is usually observed within 48 h of l.......

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.......

Acknowledgements

Research in the Saha lab was supported in part by funds from the DOD (W81XWH-20-1-0702) and Dodge Jones Foundation-Abilene. Samuel D. Rabkin and Melissa R.M. Humphrey were partially supported by NIH (R01 CA160762).

....

Materials

NameCompanyCatalog NumberComments
1.7 mL centrifuge tubesSigmaCLS3620
15 mL polypropylene centrifuge tubesFalcon352097
5 mL polypropylene tubesFalcon352063
50 mL polypropylene centrifuge tubesFalcon352098
6-well cell culture platesFalcon353046
Benzonase NucleaseSigmaE8263-25KU
Cell scraperFisher Scientific179693
Dimethyl sulfoxideSigmaD2650-100ML
Dulbecco’s Modified Eagle MediumCorningMT-10-013-CV
Dulbecco’s Phosphate Buffered SalineCorningMT-21-031-CV
Fetal Bovine SerumHycloneSH3007003
Giemsa StainSigmaG3032
GlutaraldehydeFisher Scientific50-262-23
GlycerolSigmaG5516
Hank's Balanced Salt Solution (HBSS)CorningMT-21-021-CV
High-Glucose Dulbecco’s Phosphate-buffered SalineSigmaD4031
Human immune globulinGamastanNDC 13533-335-12
Magnesium chlorideFisher ChemicalM33-500
Media Sterilization filter, 250 mLNalgene, Fisher Scientific09-740-25E
Media Sterilization filter, 500 mLNalgene, Fisher Scientific09-740-25C
Neutral Red solutionSigmaN4638
ParaformaldehydeFisher scientific 15710S
Plate rockerFisher88861043
Potassium FerricyanideSigmaP8131
Potassium FerrocyanideSigmaP9387
Sodium chlorideFisher ChemicalS271-3
Sorvall ST 16R CentrifugeThermoFisher Scientific75004381
Sorvall ST 21R CentrifugeThermoFisher Scientific75002446
Sterile Microcentrifuge Tubes with Screw CapsFisher Scientific02-681-371
SucroseFisher ScientificBP220-1
Syringe Filter, 0.45 PVDFMilliporeSigmaSLHV033RS
Syringe Filter, 0.8 MCEMilliporeSigmaSLAA033SS
Syringe filter, 5 µm PVDFMilliporeSigmaSLSV025LS
T150 culture flaskFalcon355001
Tris-HClMP Biomedicals LLC816116
Ultrasonic water bathBransonCPX-952-116R
X-galCorning46-101-RF

References

  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.

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