Name: growth, purification, and titration of oncolytic herpes simplex virus
Uploaded: 2021-05-13T14:30:00
Description: Watch this Scientific Journal Video about Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus at JoVE.com

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).

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.
<ol>
	<li>On day -1, seed low-passage Vero cells in 20 T-...

<ol>
	<li>Harrington, K., Freeman, D. J., Kelly, B., Harper, J., Soria, J. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+oncolytic+virotherapy+in+cancer+treatment.">Optimizing oncolytic virotherapy in cancer treatment.</a> Nature Reviews Drug Discovery. 18 (9), 689-706 (2019).</li><li>Zhang, S., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+and+development+of+oncolytic+viruses:+are+they+the+future+of+cancer+immunotherapy.">The discovery and development of oncolytic viruses: are they the future of cancer immunotherapy.</a> Expert Opinion on Drug Discovery. 16 (4), 391-410 (2021).</li><li>Bommareddy, P. K., Peters, C., Saha, D., Rabkin, S. D., Kaufman, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+herpes+simplex+viruses+as+a+paradigm+for+the+treatment+of+cancer.">Oncolytic herpes simplex viruses as a paradigm for the treatment of cancer.</a> Annual Review of Cancer Biology. 2 (1), 155-173 (2018).</li><li>Peters, C., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+herpes+viruses+as+oncolytics.">Designing herpes viruses as oncolytics.</a> Molecular Therapy-Oncolytics. 2, 15010 (2015).</li><li>Nguyen, H. -. M., Saha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+state+of+oncolytic+herpes+simplex+virus+for+glioblastoma+treatment.">The current state of oncolytic herpes simplex virus for glioblastoma treatment.</a> Oncolytic Virotherapy. 10, 1-27 (2021).</li><li>Koch, M. S., Lawler, S. E., Chiocca, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSV-1+oncolytic+viruses+from+bench+to+bedside:+an+overview+of+current+clinical+trials.">HSV-1 oncolytic viruses from bench to bedside: an overview of current clinical trials.</a> Cancers. 12 (12), 3514 (2020).</li><li>Menotti, L., Avitabile, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+oncolytic+immunovirotherapy:+the+blossoming+branch+of+multimodal+therapy.">Herpes simplex virus oncolytic immunovirotherapy: the blossoming branch of multimodal therapy.</a> International Journal of Molecular Sciences. 21 (21), 8310 (2020).</li><li>Nguyen, H. M., Guz-Montgomery, K., Saha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virus+encoding+a+master+pro-inflammatory+cytokine+interleukin+12+in+cancer+immunotherapy.">Oncolytic virus encoding a master pro-inflammatory cytokine interleukin 12 in cancer immunotherapy.</a> Cells. 9 (2), 400 (2020).</li><li>Agarwalla, P. K., Aghi, M. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+growth,+preparation,+and+assay.">Herpes simplex virus growth, preparation, and assay.</a> Methods in Molecular Biology. 2060, 57-72 (2020).</li><li>Froechlich, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrity+of+the+antiviral+STING-mediated+DNA+sensing+in+tumor+cells+is+required+to+sustain+the+immunotherapeutic+efficacy+of+herpes+simplex+oncolytic+virus.">Integrity of the antiviral STING-mediated DNA sensing in tumor cells is required to sustain the immunotherapeutic efficacy of herpes simplex oncolytic virus.</a> Cancers. 12 (11), 3407 (2020).</li><li>Froechlich, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+novel+mesothelin-targeted+oncolytic+herpes+virus+and+implemented+strategies+for+manufacturing.">Generation of a novel mesothelin-targeted oncolytic herpes virus and implemented strategies for manufacturing.</a> International Journal of Molecular Sciences. 22 (2), 477 (2021).</li><li>Mosca, J. D., Pitha, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+and+posttranscriptional+regulation+of+exogenous+human+beta+interferon+gene+in+simian+cells+defective+in+interferon+synthesis.">Transcriptional and posttranscriptional regulation of exogenous human beta interferon gene in simian cells defective in interferon synthesis.</a> Molecular and Cellular Biology. 6 (6), 2279-2283 (1986).</li><li>Gousseinoz, E., Kools, W., Pattnaik, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleic+acid+impurity+reduction+in+viral+vaccine+manufacturing.">Nucleic acid impurity reduction in viral vaccine manufacturing.</a> BioProcess International. 12 (2), 59-68 (2014).</li><li>Diefenbach, R. J., Fraefel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+methods+and+protocols.">Herpes simplex virus: methods and protocols.</a> Methods in Molecular Biology. , (2014).</li><li>Hadi, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+trial+to+prepared+&#947;1+34.5+herpes+simplex+virus+1+immunogene+by+cloning+technique.">An experimental trial to prepared &#947;1 34.5 herpes simplex virus 1 immunogene by cloning technique.</a> Systematic Review Pharmacy. 11 (5), 140-149 (2020).</li><li>Kuroda, T., Martuza, R. L., Todo, T., Rabkin, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flip-Flop+HSV-BAC:+bacterial+artificial+chromosome+based+system+for+rapid+generation+of+recombinant+herpes+simplex+virus+vectors+using+two+independent+site-specific+recombinases.">Flip-Flop HSV-BAC: bacterial artificial chromosome based system for rapid generation of recombinant herpes simplex virus vectors using two independent site-specific recombinases.</a> BMC Biotechnology. 6, 40 (2006).</li><li>Motamedifar, M., Noorafshan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytopathic+effect+of+the+herpes+simplex+virus+type+1+appears+stereologically+as+early+as+4+h+after+infection+of+Vero+cells.">Cytopathic effect of the herpes simplex virus type 1 appears stereologically as early as 4 h after infection of Vero cells.</a> Micron. 39 (8), 1331-1334 (2008).</li><li>Blaho, J. A., Morton, E. R., Yedowitz, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus:+propagation,+quantification,+and+storage.">Herpes simplex virus: propagation, quantification, and storage.</a> Current Protocols in Microbiology. , 1 (2005).</li><li>Malenovska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+stabilizers+and+rates+of+freezing+on+preserving+of+structurally+different+animal+viruses+during+lyophilization+and+subsequent+storage.">The influence of stabilizers and rates of freezing on preserving of structurally different animal viruses during lyophilization and subsequent storage.</a> Journal of Applied Microbiology. 117 (6), 1810-1819 (2014).</li><li>Vahlne, A. G., Blomberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+herpes+simplex+virus.">Purification of herpes simplex virus.</a> Journal of General Virology. 22 (2), 297-302 (1974).</li><li>Sathananthan, B., Rodahl, E., Flatmark, T., Langeland, N., Haarr, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+herpes+simplex+virus+type+1+by+density+gradient+centrifugation+and+estimation+of+the+sedimentation+coefficient+of+the+virion.">Purification of herpes simplex virus type 1 by density gradient centrifugation and estimation of the sedimentation coefficient of the virion.</a> APMIS: Acta Pathologica, Microbiologica, et Immunologica Scandinavica. 105 (3), 238-246 (1997).</li><li>Mundle, S. 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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 &#176;C&#160;for later purification. Blaho and colleagues employed a slig...

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 cancer types3,4,5,6,7,8. The current advanced recombinant DNA biotechnology has further increased the feasibility of engineering new oHSVs coding for therapeutic transgene(s)3,5. An efficient system of oHSV propagation, purification, and titer determination is critical before any (newly developed) oHSV can be tested for in vitro and in vivo studies. This paper describes a simple step-by-step method of oHSV growth (in Vero cells), purification (by the sucrose-gradient method), and titration (by an oHSV plaque assay in Vero cells) (Figure 1). It can be easily adopted in any Biosafety Level 2 (BSL2) laboratory setting to achieve a high-quality viral stock for preclinical studies.
Vero, an African green monkey kidney cell line, is the most commonly used cell line for oHSV propagation9,10,11,12,13 as Vero cells have a defective antiviral interferon signaling pathway14. Other cell lines with inactivated stimulator of interferon genes (STING) signaling can also be used for oHSV growth12,13. This protocol utilizes Vero cells for oHSV growth and&#160;plaque assay. Following propagation, oHSV-infected cells are harvested, lysed, and subjected to purification, wherein lysed cells are first treated with benzonase nuclease to degrade host cell DNA, prevent nucleic acid-protein aggregation, and reduce the viscosity of the cell lysate. As proper activation of benzonase often requires Mg2+, 1-2 mM MgCl2 is used in this protocol15. The host cell debris from the benzonase-treated cell lysate is further eliminated by serial filtration before high-speed sucrose-gradient centrifugation. A viscous 25% sucrose solution cushion helps to ensure a slower rate of virus migration through the sucrose layer, leaving host cell-related components in the supernatant, thus improving purification and limiting virus loss in the pellet16. The purified oHSV is then titrated on Vero cells, and viral plaques are visualized by Giemsa staining17 or X-gal staining (for LacZ encoding oHSVs)18.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.7 mL centrifuge tubes</td>
			<td>Sigma</td>
			<td>CLS3620</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL polypropylene tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Falcon</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well cell culture plates</td>
			<td>Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzonase Nuclease</td>
			<td>Sigma</td>
			<td>E8263-25KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell scraper</td>
			<td>Fisher Scientific</td>
			<td>179693</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>Corning</td>
			<td>MT-10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td>Corning</td>
			<td>MT-21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH3007003</td>
			<td></td>
		</tr>
		<tr>
			<td>Giemsa Stain</td>
			<td>Sigma</td>
			<td>G3032</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Fisher Scientific</td>
			<td>50-262-23</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td>Corning</td>
			<td>MT-21-021-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Glucose Dulbecco&#8217;s Phosphate-buffered Saline</td>
			<td>Sigma</td>
			<td>D4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Human immune globulin</td>
			<td>Gamastan</td>
			<td>NDC 13533-335-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher Chemical</td>
			<td>M33-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 250 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25E</td>
			<td></td>
		</tr>
		<tr>
			<td>Media Sterilization filter, 500 mL</td>
			<td>Nalgene, Fisher Scientific</td>
			<td>09-740-25C</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral Red solution</td>
			<td>Sigma</td>
			<td>N4638</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Fisher scientific</td>
			<td>&#160;15710S</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate rocker</td>
			<td>Fisher</td>
			<td>88861043</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferricyanide</td>
			<td>Sigma</td>
			<td>P8131</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Ferrocyanide</td>
			<td>Sigma</td>
			<td>P9387</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher Chemical</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 16R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75004381</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall ST 21R Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002446</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Microcentrifuge Tubes with Screw Caps</td>
			<td>Fisher Scientific</td>
			<td>02-681-371</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>BP220-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.45 PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLHV033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter, 0.8 MCE</td>
			<td>MilliporeSigma</td>
			<td>SLAA033SS</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter, 5 &#181;m PVDF</td>
			<td>MilliporeSigma</td>
			<td>SLSV025LS</td>
			<td></td>
		</tr>
		<tr>
			<td>T150 culture flask</td>
			<td>Falcon</td>
			<td>355001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>MP Biomedicals LLC</td>
			<td>816116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic water bath</td>
			<td>Branson</td>
			<td>CPX-952-116R</td>
			<td></td>
		</tr>
		<tr>
			<td>X-gal</td>
			<td>Corning</td>
			<td>46-101-RF</td>
			<td></td>
		</tr>
	</tbody>
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growth, purification, and titration of oncolytic herpes simplex virus

Oncolytic viruses (OVs), such as&#160;the&#160;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 &#34;plaque assay&#34; 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.

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...

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Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

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

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

Research

JoVE Journal

Immunology and Infection

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