Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Oncolytic herpes simplex virus is an emergent form of cancer immunotherapy. An efficient system of virus propagation, purification, and titeric determination is critical for the use in experimental studies. This method is very simple and can be easily adopted in any biosafety level two laboratory setting to receive a high quality viral stock for preclinical studies.

Production of a high quality, pure viral stock is crucial since it is used to study anticancer immune response and to develop a novel form of virus based cancer immunotherapy. This protocol can be used to purify any wild-type or genetically engineered herpes simplex virus. This protocol should be easy to read and easy to follow for an individual who have never performed this technique before.

The listed materials and reagents should be in place before trying this technique for the first time. Begin by washing the T-150 square centimeter flasks with two times high glucose DPBS supplemented with 1%IFCS. Aspirate the DPBS and add seven milliliters of virus inoculum per flask.

Gently rock the flasks for five minutes and incubate at 37 degrees Celsius for 1.5 to 2 hours. Then, remove the inoculum and add 25 milliliters of DMEM supplemented with 1%IFCS to each flask. After incubation at 37 degrees Celsius and 5%carbon dioxide for two to four days, collect 20 milliliters of the culture supernatant from each flask into 50 milliliter centrifuge tubes.

Using a cell scraper, scrape the cells from the bottom of the flasks. Add approximately 15 milliliters of the previously collected culture supernatant to each flask to bring the volume up to 20 milliliters, then use a 10 milliliter sterile serological pipette to gently wash the bottom of the flasks a few times. Collect the cells with medium into 50 milliliter conical centrifuge tubes placed on ice.

Centrifuge the tubes at 300 times G for 10 minutes at four degrees Celsius. Resuspend each pellet thoroughly in 1.25 milliliters of virus buffer, or VB, and 1.25 milliliters of previously collected culture supernatant. Mix all resuspended pellets into one 50 milliliter conical centrifuge tube.

Snap freeze the resuspended cells using dry ice and 100%ethanol and store at minus 80 degrees Celsius. Thaw the previously snapped frozen cells in a warm water bath at 37 degrees Celsius and sonicate for one minute for three cycles in a water bath. Add 175 units of Benzonase Nuclease and two microliters of two millimolar magnesium chloride per milliliter of the cell lysate and vortex.

After an incubation of 30 minutes and warm water bath at 37 degrees Celsius, place the tube on ice. Spin the cell lysate at 300 times G for 10 minutes. Collect the supernatant in a new 50 milliliter conical centrifuge tube and resuspend the cell pellet in 500 microliters of VB.Designate it as pellet one.

Spin the collected supernatant again at 500 times G for 10 minutes and store the supernatant separately in a fresh 50 milliliter conical centrifuge tube to use later. Resuspend the cell pellet in 500 microliters of VB, designating it as pellet two. Combine the resuspended pellet one and pellet two in a new 1.75 milliliter centrifuge tube and vortex and sonicate twice in a water bath before spinning the tubes at 400 times G for 10 minutes.

Collect the supernatant from 1.7 milliliter centrifuge tube and combine it with the supernatant previously collected 50 milliliter conical centrifuge tube. Filter the supernatant through a sterile five micrometer PVDF membrane filter into a new 50 milliliter centrifuge tube called tube one. Add one milliliter of VB to the 50 milliliter centrifuge tube, emptied after filtering the supernatant from the previous step, vortex, and pass it through the same PVDF membrane filter placed on tube one.

Pass the filtrate from tube one through a sterile 0.8 micrometer MCE membrane filter placed on a new 50 milliliter centrifuge tube called tube two. Add one milliliter of VB to the emptied tube one, vortex, and pass it through the same MCE filter placed on tube two. Pass the filtrate from tube two through a sterile 0.45 micrometer PVDF filter placed on a new 50 milliliter centrifuge tube labeled tube three.

As described before, repeat the washing of tube two with one milliliter of VB to add filtrate to tube three. In this representative analysis, the cytopathic effect could be observed in Vero cells at 36, 48, and 72 hours post oHSV infection with the rounding of the affected cells. The increasing level of CPE over time is evident as visualized by mCherry expression.

At 72 hours post infection, viral plaques were stained with X-gal to visualize oHSVs with lacZ expression. Gently scrubbing off virus infected cells and gently washing the bottom on the flask is critical to keep all HSV infected cell intact so that a high titer viral stock can be obtained.