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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • 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 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 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.

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-150 cm2 flasks at a density of 7-8 × 106 cells/flask in regular Vero cell medium.
    NOTE: Vero medium is prepared by supplementing Dulbecco's modified eagle medium (DMEM) with 10% heat-inactivated fetal calf serum (IFCS).
  2. On Day 0 (cells are 80-90% confluent), add oHSV inoculum on the Vero cells.
    1. Preparation of virus inoculum
      1. Use a multiplicity of infection (MOI) of 0.01 (MOI can vary from 0.01 to 0.1 depending on the replication capacity of virus) for virus amplification. Use formula (1) to calculate the amount of virus (mL) for 20 flasks.
        ​Amount of virus (mL) for 20 flasks = amount of virus needed (pfu)/titer of virus stock (pfu/mL)(1)
      2. Use high-glucose Dulbecco's phosphate-buffered saline (DPBS) supplemented with 1% IFCS to prepare the virus inoculum (7 mL per T-150 cm2 flask, i.e., ~140 mL for 20 flasks). Add the required amount of oHSV to 140 mL of high-glucose DPBS/1% IFCS solution, vortex for 1 min, and keep the mixture ready for addition to Vero cells.
    2. Wash the T-150 cm2 flasks 2x with high-glucose DPBS supplemented with 1% IFCS (10 mL/wash). Aspirate the DPBS/1% IFCS and add 7 mL of virus inoculum/T-150 cm2 flask.
    3. Gently rock the flasks for 5 min using a flask rocker for proper distribution of the inoculum over the Vero monolayer, and then incubate the flasks at 37 °C for 1.5-2 h. Make sure the incubator shelf is level.
    4. Remove the inoculum and add DMEM supplemented with 1% IFCS (25 mL/flask). Incubate the flasks for 2-4 days.
  3. Check the flasks daily for 90-100% CPE (see Figure 2).
  4. Harvest the oHSV-infected Vero cells.
    1. Collect the culture supernatant (~20 mL) from each flask (leave ~5 mL in each flask) in 50 mL conical centrifuge tubes or media container.
    2. Use a cell scraper to scrape the cells from the bottom of the flasks gently.
      NOTE: The cells should quickly come off the flasks.
    3. Add ~15 mL of the culture supernatant (collected in step 1.4.1) to each flask (which brings the volume of ~20 mL in each flask, i.e., 400 mL for 20 flasks) and gently wash the bottom of the flasks a few times using a 10 mL sterile serological pipet.
      NOTE: Do not pipet vigorously. Aim to keep all cells intact.
    4. Collect the cells (+ medium) into 50 mL conical centrifuge tubes on ice (use 8 tubes to hold 400 mL of harvested cells from 20 flasks).
    5. Spin the cells at 300 g for 10 min at 4 °C and aspirate the supernatant.
    6. Add 1.25 mL (50%) of Virus Buffer (VB) and 1.25 mL (50%) of culture supernatant (collected in step 1.4.1) to each centrifuge tube and re-suspend each pellet thoroughly. Transfer the re-suspended cells from eight 50 mL centrifuge tubes to one 50 mL conical centrifuge tube.
      NOTE: Refer to the preparation of VB solution in Table 1. Filter-sterilize the VB solution using a media sterilization filter. Use 0.5 mL of VB + 0.5 mL of supernatant per T-150 cm2 flask for re-suspension, i.e., for 20 flasks (20 mL), use 10 mL of VB and 10 mL of culture supernatant.
    7. Snap-freeze the re-suspended cells using dry ice/100% ethanol and store at -80 °C.

2) oHSV purification

  1. Snap-freeze (in dry ice and 100% ethanol)/thaw (in a 37 °C warm water bath) the cells followed by water bath-sonication for 1 min for a total of 3 cycles to ensure proper lysis of the cells to release virus in the supernatant.
    NOTE: Perform sonication for 1 min using 40 kHz, 120 V power. If a tunable sonicator is not available, use an ultrasonic water bath sonicator. Take a 50 μL aliquot for titration in section 3, which will help to identify which of the following step(s) is responsible for potential virus loss during the purification procedure.
  2. Treat the cell lysate with Benzonase Nuclease (175 units/mL) + 2 mM MgCl2 (1 M stock = 2 µL/mL), vortex, and incubate for 30 min at 37 °C. Place the tube on ice and perform the following steps at 4 °C.
  3. Pellet the cell debris by low-speed centrifugation.
    1. Spin the cell lysate at 300 g for 10 min.
    2. Collect the supernatant in a new 50 mL conical centrifuge tube (re-suspend the cell pellet in 0.5 mL of VB, designate it as pellet-1, and store at 4 °C for use in step 2.3.5; see Figure 1).
    3. Spin the supernatant (obtained in step 2.3.2) again at 500 g for 10 min.
    4. Collect the supernatant in a new 50 mL conical centrifuge tube (re-suspend the cell pellet in 0.5 mL of VB, designate it as pellet-2, and store at 4 °C for use in step 2.3.5; see Figure 1).
    5. Combine the re-suspended pellet-1 (from 2.3.2) and pellet-2 (from 2.3.4) into a new 1.7 mL centrifuge tube, vortex/sonicate (water bath) 2x, and spin at 400 g for 10 min. Collect the supernatant and combine it with the supernatant obtained in step 2.3.4; see Figure 1).
      NOTE: Perform sonication for 1 min using 40 kHz, 120 V power to prevent viral aggregation before the filtration procedure in step 2.4. Take a 50 μL aliquot of the combined supernatant for titration in section 3.
  4. Filter the combined supernatant (~21 mL, i.e., 20 mL from 1.4.6, 0.5 mL from 2.3.2, and 0.5 mL from 2.3.4) using the following 3-step filtration method.
    1. Draw 21 mL of the supernatant using a 10 mL syringe (5-7 mL each time for easy passage through the filter) and pass it through a sterile 5 μm polyvinylidene difluoride (PVDF) membrane filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 1).
      1. Add 1 mL of VB to a 50 mL conical centrifuge tube emptied in 2.4.1 (to collect the remaining trace amount of virus supernatant), vortex, and pass through same 5 μm PVDF filter placed on TUBE 1 (bringing the total to 22 mL of filtrate). Proceed to step 2.4.2.
    2. Draw 22 mL of the filtrate from TUBE 1 (as in 2.4.1) and pass it through a sterile 0.8 μm mixed cellulose ester (MCE) membrane filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 2).
      1. Add 1 mL of VB to TUBE 1 emptied in step 2.4.2, vortex, and pass it through the same 0.8 μm MCE filter placed on TUBE 2 (bringing the total to 23 mL of filtrate). Proceed to step 2.4.3.
    3. Draw 23 mL of filtrate from TUBE 2 (as in 2.4.1) and pass it through a sterile 0.45 µm PVDF filter placed on a new 50 mL conical centrifuge tube (labeled as TUBE 3).
      1. Add 1 mL of VB to TUBE 2 emptied in step 2.4.3, vortex, and pass it through the same 0.45 μm PVDF filter placed on TUBE 3 (bringing the total to 24 mL of filtrate).
        NOTE: Take a 50 μL aliquot of the filtrate for titration in section 3.
  5. High-speed centrifugation using the sucrose-gradient method
    NOTE: A fixed angle F13-14x50cy rotor was used in this protocol. Both the rotor and the centrifuge must be at 4 °C before proceeding with the following steps.
    1. Add 10 mL of an ice-cold, sterile-filtered 25% sucrose solution (prepared by dissolving 25 g of sucrose powder in 100 mL of Hank's Balanced Salt Solution) in a new 50 mL conical centrifuge tube.
    2. Slowly (3 mL/min) add 24 mL of the virus filtrate (obtained from step 2.4.3.1) on top of the sucrose layer. Take care to maintain separate layers of the virus filtrate and the sucrose solution.
      NOTE: Up to 30 mL of the virus layer can be added over 10 mL of the sucrose solution.
    3. Centrifuge the tube for 90 min at 22,620 g at 4 °C.
    4. Remove the supernatant and the sucrose layer from the 50 mL conical centrifuge tube.
      NOTE: The virus pellet should be whitish. Take a 50 μL aliquot of the supernatant and a 50 μL aliquot of the sucrose layer for titration in section 3.
  6. Re-suspend the pellet in 10% glycerol/PBS solution.
    1. Add 1 mL of sterile 10% glycerol (diluted in PBS) to the 50 mL conical centrifuge tube to cover the pellet.
      NOTE: The amount of the re-suspended mixture can vary (such as 0.8-1.2 mL) depending on the pellet size. A smaller volume would give a higher concentration of virus.
    2. Place the 50 mL conical centrifuge tube on ice for 2-4 h. During this period, sonicate/vortex the pellet every 15 min for 30 s to help dislodge/re-suspend the pellet.
    3. Collect the re-suspended pellet (1 mL of 10% glycerol/PBS + the pellet size will bring the total volume to ~1.3 mL) in a 2 mL microcentrifuge tube. [Optional: Add another 0.3 mL of 10% glycerol/PBS to the same 50 mL conical centrifuge tube to collect the remaining trace amount of the pellet, pipet up and down, and combine with the re-suspended pellet in step 2.6.3 to bring the total volume to ~1.6 mL.]
      1. If the suspension in step 2.6.3 is turbid or cloudy (due to cell debris), centrifuge the tube at 500 g for 10 min, transfer the supernatant to a new 2 mL microcentrifuge tube, and proceed to step 2.6.4.
    4. Take a 50 μL aliquot for oHSV titration in section 3. Aliquot the rest of the solution (250 μL/aliquot) into sterile microcentrifuge tubes with screw caps (sealed well for long-term storage), snap-freeze, and store at -80 °C until use (ready for experimental studies once the oHSV titer is determined).
      ​NOTE: All materials that come in contact with the virus must be bleached or treated with ultraviolet radiation before removal from the hood or disposal.

3. oHSV titration and plaque assay

  1. Seed 1.7-1.8 x 105 Vero cells/well in a 6-well cell culture plate in Vero cell medium (DMEM with 10% IFCS; see step 1.1).
    NOTE: Make sure that the cells are homogeneously distributed throughout the well; do not swirl the plate, which can cause accumulation of cells in the middle of the wells. To prevent swirling, slowly rock the plate by hand vertically, then horizontally, and then gently place the plate in the incubator.
  2. The next day (when the cells reach 70-80% confluency), aspirate the culture medium, and add 1 mL/well of high-glucose PBS supplemented with 1% IFCS. Leave the plate in the cell culture hood until step 3.3 is complete.
  3. Serially dilute the virus in 5 mL polypropylene tubes using PBS/1% IFCS (Figure 3).
    NOTE: Use the 50 μL aliquot collected in step 2.6.4 for serial dilution. In the 1st tube (10-3 dilution), add 2 μL of the virus in 1998 μL of PBS/1%IFCS, vortex. In the 2nd tube (10-5 dilution), take 10 μL from the 1st tube in 990 μL of PBS/1% IFCS, vortex. In the 3rd tube (10-6 dilution), take 100 μL from the 2nd tube in 900 μL of PBS/1% IFCS, vortex; continue this 10-fold serial dilution until 10-9 dilution. See the details in Figure 3.
  4. Aspirate PBS/1% IFCS, and add 0.7 mL/well of the serially diluted virus (starting from 10-5 to 10-9) to Vero cells.
    NOTE: Do not let the cells dry.
  5. Gently rock the plate on a rocker for 5 min at room temperature (to ensure homogeneous distribution of the virus inoculum).
  6. Incubate the plate for 1.5 h at 37 °C.
    NOTE: During this incubation period, prepare 1:1000 dilution of human immunoglobulin G (IgG) in DMEM supplemented with 1% IFCS. For a 6-well plate, prepare 12.5 mL so that 2 mL/well can be used in step 3.7. Adjust the dilution to account for lot variations in the human IgG.
  7. Remove the virus inoculum from the wells, and add 2 mL/well of 0.1% human IgG (to neutralize the oHSV in the culture medium and prevent the formation of secondary plaques) in DMEM supplemented with 1% IFCS. Incubate the plate at 37 °C for 3-4 days.
    NOTE: Clear plaques usually form in 3 days.
  8. Fixing and staining plates
    1. Remove the supernatant, and fix the cells in pure methanol (1 mL/well) for 5 min. Remove the methanol, and allow the plates to air-dry.
    2. Dilute Giemsa stain (1:5) with deionized water, and add 1 mL of the diluted Giemsa stain per well. Incubate the plate at room temperature for 10-15 min.
    3. Remove the stain, rinse with tap water, and allow the plates to air-dry.
    4. Count the plaques using a dissecting microscope.
    5. Optional for oHSVs with lacZ expression:
      1. Remove the supernatant, and fix the cells with cold 0.2% glutaraldehyde/2% paraformaldehyde for 5-10 min at room temperature.
      2. Remove the fixative solution, and wash the cells 3x with PBS.
      3. Add X-gal solution to the cells (1 mL/well), and incubate the plate at 37 °C for 2 h.
        NOTE: The X-gal stain should be prepared freshly on the day of staining; long-term storage might lead to fading of color. See the preparation of X-gal solution in Table 1.
      4. Remove the X-gal stain, and wash the plate with tap water for 1 min.
      5. Counter-stain with Neutral Red solution (1 mL/well) for 2 min at room temperature.
        NOTE: See preparation of Neutral Red solution in Table 1.
      6. Wash the plate with tap water for 1 min; allow the plates to air-dry.
      7. Count blue plaques using a dissecting microscope (Figure 4).
  9. Calculate the titer by using formula (2)
    Titer in pfu/mL (plaque-forming units) = Number of plaques/0.7 mL × dilution factor (2)
    NOTE: For example, if 25 plaques are found in the 10-9 dilution well, the titer is 25/0.7 × 109 = 35.7 × 109 = 3.57 × 1010 pfu/mL. This is the final oHSV titer of aliquots prepared in step 2.6.4.

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...

Disclosures

SDR is a co-inventor on patents relating to oncolytic herpes simplex viruses, owned and managed by Georgetown University and Massachusetts General Hospital, which have received royalties from Amgen and ActiVec Inc. and is on the Scientific Advisory Board of EG 427. The other authors have nothing to disclose.

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

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