Production and purification of high-titer OrfV for preclinical studies in vaccinology and cancer therapy
2021; Cell Press; Volume: 23; Linguagem: Inglês
10.1016/j.omtm.2021.08.004
ISSN2329-0501
AutoresJacob P. van Vloten, Jessica A. Minott, Thomas M. McAusland, Joelle C. Ingrao, Lisa A. Santry, Grant McFadden, Jim Petrik, Byram W. Bridle, Sarah K. Wootton,
Tópico(s)Animal Virus Infections Studies
ResumoPoxviruses have been used extensively as vaccine vectors for human and veterinary medicine and have recently entered the clinical realm as immunotherapies for cancer. We present a comprehensive method for producing high-quality lots of the poxvirus Parapoxvirus ovis (OrfV) for use in preclinical models of vaccinology and cancer therapy. OrfV is produced using a permissive sheep skin-derived cell line and is released from infected cells by repeated freeze-thaw combined with sonication. We present two methods for isolation and purification of bulk virus. Isolated virus is concentrated to high titer using polyethylene glycol to produce the final in vivo-grade product. We also describe methods for quantifying OrfV infectious virions and determining genomic copy number to evaluate virus stocks. The methods herein will provide researchers with the ability to produce high-quality, high-titer OrfV for use in preclinical studies, and support the translation of OrfV-derived technologies into the clinic. Poxviruses have been used extensively as vaccine vectors for human and veterinary medicine and have recently entered the clinical realm as immunotherapies for cancer. We present a comprehensive method for producing high-quality lots of the poxvirus Parapoxvirus ovis (OrfV) for use in preclinical models of vaccinology and cancer therapy. OrfV is produced using a permissive sheep skin-derived cell line and is released from infected cells by repeated freeze-thaw combined with sonication. We present two methods for isolation and purification of bulk virus. Isolated virus is concentrated to high titer using polyethylene glycol to produce the final in vivo-grade product. We also describe methods for quantifying OrfV infectious virions and determining genomic copy number to evaluate virus stocks. The methods herein will provide researchers with the ability to produce high-quality, high-titer OrfV for use in preclinical studies, and support the translation of OrfV-derived technologies into the clinic. The application of poxviruses in human and animal health has expanded dramatically since Edward Jenner first used cowpox as a vaccine against smallpox. The advent of recombinant DNA technology enabled the expansion of poxvirus vaccines to target diverse human and veterinary pathogens, with vaccinia virus strains being the most tested platform.1Garcia-Arriaza J. Esteban M. Enhancing poxvirus vectors vaccine immunogenicity.Hum. Vaccin. Immunother. 2014; 10: 2235-2244Google Scholar Recently, a new application for poxviruses as oncolytic viruses (OV) has emerged, with Parapoxvirus ovis (OrfV) among them.2Rintoul J.L. Lemay C.G. Tai L.-H. Stanford M.M. Falls T.J. de Souza C.T. Bridle B.W. Daneshmand M. Ohashi P.S. Wan Y. et al.OrfV: a novel oncolytic and immune stimulating parapoxvirus therapeutic.Mol. Ther. 2012; 20: 1148-1157Google Scholar,3Chan W.M. McFadden G. Oncolytic poxviruses.Annu. Rev. Virol. 2014; 1: 119-141Google Scholar OVs are multi-mechanistic immunotherapy tools that fight cancer by selectively targeting and killing tumor cells and by activating a host immune response against the tumor.4Twumasi-Boateng K. Pettigrew J.L. Kwok Y.Y.E. Bell J.C. Nelson B.H. Oncolytic viruses as engineering platforms for combination immunotherapy.Nat. Rev. Cancer. 2018; 18: 419-432Google Scholar Since the arrival of T-Vec, the first OV to be Food and Drug Administration approved for treating solid tumors,5Pol J. Kroemer G. Galluzzi L. First oncolytic virus approved for melanoma immunotherapy.Oncoimmunology. 2016; 5: e1115641Google Scholar research has expanded to take advantage of the unique biology of different viral backbones and develop improved immunotherapies. OrfV is a highly immunogenic poxvirus that targets ungulates as a primary host.6de la Concha-Bermejillo A. Guo J. Zhang Z. Waldron D. Severe persistent orf in young goats.J. Vet. Diagn. Invest. 2003; 15: 423-431Google Scholar Similar to other poxviruses, it has a large double-stranded DNA genome with a central region of conserved genes required for viral genome replication and morphogenesis, which are flanked by regions that encode accessory virulence and immune-modulatory genes.7Mercer A.A. Lyttle D.J. Whelan E.M. Fleming S.B. Sullivan J.T. The establishment of a genetic map of orf virus reveals a pattern of genomic organization that is highly conserved among divergent poxviruses.Virology. 1995; 212: 698-704Google Scholar These flanking regions are of considerable interest, as they provide a location for targeted insertion of transgenes for vaccine and cancer therapy and serve as targets for basic knockout studies to modulate the immunogenicity of the viral backbone. OrfV is a known OV capable of infecting multiple types of human and murine cancer cells both in vitro and in vivo, leading to drastic reductions in tumor burden in preclinical murine models of metastatic melanoma and colon carcinoma.2Rintoul J.L. Lemay C.G. Tai L.-H. Stanford M.M. Falls T.J. de Souza C.T. Bridle B.W. Daneshmand M. Ohashi P.S. Wan Y. et al.OrfV: a novel oncolytic and immune stimulating parapoxvirus therapeutic.Mol. Ther. 2012; 20: 1148-1157Google Scholar The OrfV genetic system is amenable to the generation of transgenic viruses, akin to other poxviruses. Recombinant viruses can be generated through homologous recombination between a transfer plasmid and the parental replicating virus in permissive cells.8Tai L.H. Tanese de Souza C. Bélanger S. Ly L. Alkayyal A.A. Zhang J. Rintoul J.L. Ananth A.A. Lam T. Breitbach C.J. et al.Preventing postoperative metastatic disease by inhibiting surgery-induced dysfunction in natural killer cells.Cancer Res. 2013; 73: 97-107Google Scholar The large genome size and inclusion of non-essential genes provides multiple targets for insertion of therapeutic transgenes and the potential to incorporate multiple transgenes for multivalent vaccine strategies.9Rziha H.J. Rohde J. Amann R. Generation and selection of orf virus (OrfV) recombinants.Methods Mol. Biol. 2016; 1349: 177-200Google Scholar Recombinant OrfV can and has been used as a vaccine vector targeting a number of pathogens, including rabies, influenza, and herpesvirus,10Rziha H.-J. Henkel M. Cottone R. Bauer B. Auge U. Gӧtz F. Pfaff E. Rӧttgen M. Dehio C. Büttner M. Generation of recombinant parapoxvirus—non essential genes for insertion.J. Biotechnol. 2000; 83: 137-145Google Scholar, 11Amann R. Rohde J. Wulle U. Conlee D. Raue R. Martinon O. Rziha H.-J. A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.J. Virol. 2013; 87: 1618-1630Google Scholar, 12Rohde J. Amann R. Rziha H.J. New Orf virus (Parapoxvirus) recombinant expressing H5 hemagglutinin protects mice against H5N1 and H1N1 influenza A virus.PLoS One. 2013; 8: e83802Google Scholar demonstrating the value of OrfV as a viral vector system. Further expanding OrfV-based vaccine and OV technologies will require accessible and reliable methods for producing high-titer virus. OrfV can be grown in cell culture, but the production of high-titer ultrapure virus has been historically challenging and has restrained in vivo preclinical testing. The purity of OrfV propagations is of significant importance because contaminants can alter immunological outcomes, which can make rational design of OVs as immunotherapies challenging. Additionally, both low-titer and low-purity virus prevents systemic administration of OVs, which is critical for targeting metastatic disease.13Fischer T. Planz O. Stitz L. Rziha H.J. Novel recombinant parapoxvirus vectors induce protective humoral and cellular immunity against lethal herpesvirus challenge infection in mice.J. Virol. 2003; 77: 9312-9323Google Scholar Unfortunately, the literature at the time of this writing is sparse with respect to OrfV production. Therefore, we present a comprehensive method for producing, purifying, quality testing, and titrating OrfV for use in preclinical murine models of vaccination and cancer therapy. This information will endow researchers with the ability to translate OrfV-based technologies from the bench to the bedside. Cell culture:•Sheep skin fibroblasts14Breitbach C.J. Lichty B.D. Bell J.C. Oncolytic viruses: therapeutics with an identity crisis.EBioMedicine. 2016; 9: 31-36Google Scholar—the authors can provide these cells upon request•OA3.T (ATCC CRL-6546)•Complete Dulbecco's modified Eagle's medium (DMEM) (Fisher Scientific, Cat. #SH30022.01)○10% fetal bovine serum (VWR, PA, USA, Cat. #97068-085)○Penicillin-streptomycin cocktail (Fisher Scientific, Cat. #SV30010)○1× non-essential amino acids (Fisher Scientific, Cat. #11140050)•0.25% trypsin-ethylene-diamine-tetra-acetic acid (EDTA) (Corning, NY, USA, Cat. #25-052-CI)•Phosphate-buffered saline (Fisher Scientific, Cat. #SH30256.01)•MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Basel Switzerland, Cat. # LT07-703)•Cell culture plates including 150-mm 6-well and 96-well flat-bottomed plates (Corning, NY, USA) Virus harvest and purification:•Disposable cell scraper (Fisher Scientific, Cat. #179693PK)•50-mL conical tubes (Fisher Scientific, Cat. # 14-432-22)•0.3 M NaOH•1 M NaOH•Ultrapure H2O for buffer preparation•36% sucrose-PBS•5% sucrose-PBS•Iodixanol OptiPrep Density Gradient Medium (Sigma-Aldrich, Cat. # D1556)•38-mL ultracentrifuge tubes (Beckman Coulter, Cat. #355631 or 344058)•250-mL centrifuge bottles, polypropylene (Beckman Coulter, Cat. # 355627), for Type 19 fixed-angle aluminum rotor•Ultra-clear ultracentrifuge tubes, 13 mL (Beckman Coulter, Cat. #344059)•Polyethylene glycol (PEG), molecular weight 20,000 g/mol (Sigma-Aldrich, Cat. #81300)•3-mL and 5-mL syringes•18-gauge sharp Luer-Lok needle (Fisher Scientific, Cat. #14-826-5G)•18-gauge blunt-tip Luer-Lok needle (Becton Dickinson, Cat. # 305181)•Pierce Universal Nuclease for Cell Lysis (Fisher, Cat. #PI88702) Virus titration•Multi-channel 10–50-μL and 30–300-μL pipettes•UltraPure low-melting-point agarose (Fisher Scientific, Cat. #16520050)•2× MEM (Temin's modification), no phenol red (Fisher Scientific, Cat. #11935046)•Proteinase K (Ambion, Cat. #AM2546)•Phenol/chloroform/isoamyl alcohol (Fisher Scientific, Cat. #BP17521-400) •Biological safety cabinet—all steps which involve virus manipulation must be conducted in the biological safety cabinet•Clinical and high-speed centrifuges (must accommodate 50-mL conical tubes)•Ultracentrifuge with swinging bucket SW 41 Ti rotor (methods 1 and 2), SW32 Ti and Type 19 fixed-angle aluminum rotor (method 2 only)•Probe sonicator (Fisher Scientific, Model FB120)•Supracap 50 Depth Filter Pall V100P (Pall Laboratory, Cat. #SC050V100)•Omega Membrane LV Centramate Cassette, 300 kDa (Pall Laboratory, Cat. #OS300T12)•Slide-A-Lyzer dialysis cassette, 10,000 kDa molecular weight cutoff (Fisher Scientific, Cat. #66380)•Centramate Cassette Holder (Pall Laboratory, Cat. #CM018V)•Tubing screw clamp (Pall Laboratory, Cat. #88216)•Utility pressure gauge (×2) (Cole-Palmer, Cat. #68355-06)•Male and female Luer-Lok with 1/8 in national pipe thread (NPT; Cole-Palmer, Cat. #41507-44 and -46)•Female threaded tee fittings, nylon, 1/8 in NPT(F) (Cole-Palmer, Cat. #06349-50)•Masterflex C-Flex ULTRA tubing, L/S 16, 25 ft (Cole-Palmer, Cat. #06434-16)•Masterflex L/S Easy-Load Head for Precision Tubing, PARA, SS Rotor (Cole-Palmer, Cat. #UZ-07514-10)•Masterflex L/S variable-speed drive with remote I/O; 100 rpm (Cole-Palmer, Cat. #UZ-07528-30) or equivalent•Thermocycler•Microscope capable of brightfield and fluorescence (optional) •For method overview, refer to Figure 1. 1.Thaw sheep skin fibroblasts (SSFs) from liquid nitrogen, remove dimethyl sulfoxide, and recover in complete DMEM (cDMEM) in a plate or flask. Incubate at 37°C, 5% CO2, and 21% O2 until cells reach 85%–95% confluency.•This usually requires 3–5 days.•We recommend using cells at a low passage number, as cell quality decreases as passage number increases.•All cells in these experiments were confirmed mycoplasma free using the MycoAlert PLUS Mycoplasma Detection Kit.2.Seed SSF cells from the recovery plate into larger plates. In this protocol we expand into 3 × 150-mm plates by seeding 1.5 × 106 cells per plate, but flasks can be used. Incubate until cells reach 85%–95% confluency, which typically takes 3–5 days.•We recommend seeding SSFs in 20 mL of medium when using 150-mm plates to ensure even coverage of cells and to reduce risk of evaporation.3.Continue to amplify SSFs to desired propagation size. For a typical preclinical batch, we recommend 50 × 150-mm plates. To accomplish this, expand the three plates from step 4 to six plates, and then to 15 and finally up to 50 plates, waiting for 85%–95% confluency before moving to the next amplification. SSFs should not be seeded at lower than 1.5 × 106 for 150-mm plates, as lower initial densities reduce growth time.•Seed an additional three 150-mm plates for each batch produced. One plate is used to count cell number to calculate the amount of infectious virus needed, and the other two are further passaged for virus quantification (see below). 1.Begin virus infection when plates reach 90%–95% confluency.2.Detach cells from only one of the extra plates by removing medium, washing with PBS, and adding 4 mL of trypsin-EDTA.a.Neutralize trypsin-EDTA with 6 mL of complete medium for a total of 10 mL.b.Count cells using a hemocytometer and calculate the total number of cells per 150-mm plate.c.Multiply the total number of cells to be seeded per plate (1 × 107 cells) by the total number of plates, to obtain the total number of cells for infection.d.Example: 1 × 107 cells/plate × 50 plates = 5.0 × 108 cells total.e.Use a titrated virus stock to calculate the total volume of virus required to infect cells with a multiplicity of infection (MOI) of 0.05. For example:(Total no. of cells × MOI)/Stock virus conc.= (5.0 × 108 cells × 0.05)/ 1 × 109 PFU/mL= 2.5 × 106/1 × 109= 0.0025 mL or 2.5 μL total for propagation3.Add required virus to basal DMEM, for a total volume of 4 mL per plate (e.g., 200 mL for a 50-plate propagation) in 50-mL conical tubes.4.Remove medium from 150-mm plates of SSF cells and replace with 4 mL of basal DMEM containing virus.5.Incubate plates at room temperature for 30 min on a rocker to distribute the virus.•Alternatively, incubate at 37°C, 5% CO2, and 21% O2 for 30 min, rocking plates every 10 min by hand.6.Add an additional 12 mL of cDMEM to each plate, for a total of 16 mL per plate.7.Incubate for 4–6 days, or until 90%–100% of SSF cells are showing signs of cytopathic effect (CPE; Figure 2A).•The duration of infection will depend on multiple factors including the health of the SSF cells and the accuracy of the titer of the virus used for the infection. We recommend checking the progress of infection daily. 1.This process enables recovery of virus from both the supernatant and the cell pellet to maximize propagation yields.2.Once CPE is observed in 90%–100% of SSF cells, detach cells using a disposable cell scraper.3.Collect cells and medium in 50-mL conical tubes and incubate on ice.4.Centrifuge tubes at 1,500 × g for 15 min at 4°C to pellet cell debris.5.Decant supernatant into a sterile 1-L vessel, seal with parafilm, and store at 4°C.•A significant amount of virus will be found secreted in the supernatant, which can be purified using the methods described herein.•OrfV can be stored for up to a month at 4°C without significant loss of titer (data not shown).6.Resuspend cell pellets in 5 mL of clarified virus containing supernatant and pool in 50-mL conical tubes. Fill tubes to a maximum of 45 mL to accommodate expansion during freezing. Store tubes at −80°C.a.Tubes can be stored long-term at this step.7.Freeze-thaw of cell pellet suspension at −80°C should be performed three times to disrupt cell membranes and release intracellular virus.8.After three freeze-thaw cycles, sonicate the sample for six cycles of 10 s on at an amplitude of 50% followed by 10 s off.a.Conical tubes should be placed on ice during sonication to prevent excessive heating of samples.9.Centrifuge the sonicated cell pellet suspension at 6,000 × g for 30 min at 4°C to separate the virus from cell debris.a.This is a critical step, as slower centrifugation speeds fail to remove cell debris and result in clogging of depth filter and tangential flow filtration membranes, resulting in extremely long purification times. Additionally, cell debris not removed by high-speed centrifugation can be carried through to iodixanol gradient separation and can retain virus particles in the wrong bands.10.Decant supernatant (containing virus) into a sterile vessel. Take care not to dislodge the cell pellet. Discard remaining cell pellets.11.Add 25 U of nuclease/mL of supernatant (e.g., add 1,250 U of nuclease [Fisher, Cat. #PI88702]) to 50 mL. Incubate at room temperature for 30 min and place on ice.12.Proceed to method 1 or method 2 for virus concentration. All buffers should be autoclaved or passed through a 0.22-μm filter prior to use. All filtration steps should be performed in a biological safety cabinet.1.Set up the peristaltic pump and tubing in a biological safety cabinet as per Figure 3. Before attaching the depth filter, sterilize the tubing by running 50 mL of 1 M NaOH-PBS followed by 200 mL of Milli-Q H2O.2.Attach the depth filter and remove the vent cap. Be sure to include a pressure gauge after the pump outflow but before the depth filter to measure the pressure being applied to the virus going through the depth filter cassette. Slowly pump another 100 mL of PBS through and watch for the release of all air from the depth filter. Once all the air is removed and PBS flows through the top, cap the vent and allow the PBS to continue to flow through.•The depth filter can be angled to position the air release at the top to ensure removal of all air from the filter.•Stop pump when 5 mL of PBS remains; introducing minor air bubbles is acceptable. Avoid introducing excessive amounts of air or venting will have to be repeated.3.Before running virus through, place a new sterile bottle at the flow-through to collect the virus as it exits the depth filter. Mix virus supernatants from virus harvest steps 5 and 10 and begin pumping the virus supernatant through the depth filter.•Use the pressure gauge to monitor the pressure as virus supernatant moves through the filter. We have not seen loss of virus at pressures up to 15 psi, but the flow rate can dramatically decline as the filter fills with debris. We recommend replacing the filter halfway through to maintain pressures lower than 15 psi and a reasonable flow rate.4.Chase the final volumes of virus supernatant with 50 mL of PBS without allowing air bubbles to enter the system. This will flush any remaining virus out of the depth filter. Keep virus on ice while setting up the tangential flow filtration equipment. Pause point: If tangential flow filtration cannot be initiated immediately, store filtered virus at 4°C overnight. Do not freeze filtered virus.5.Discard the depth filter. Run 50 mL of 1 M NaOH through the tubing to sterilize. Disassemble the tubing and store in 0.5 M NaOH. As above, all steps should be performed in a biological safety cabinet.1.Remove the 300 kDa Centramate cassette from the 0.5 M NaOH storage solution and rinse with Milli-Q H2O. Sandwiching the cassette between two plastic gaskets, set up the Centramate cassette holder following the diagram in Figure 4. Using a torque wrench set to 6 newton-meters, tighten the nut half a rotation and move to the opposite nut—this ensures equal distribution of weight—and then to the nut beside and its opposite. Repeat until all bolts have been tightened fully until the torque wrench clicks, identifying that each bolt is tightened to the torque and that the seal around the cassette is even.2.Connect the tubing to the peristaltic pump, cassette, and reservoir as shown in Figure 4. Ensure that the cassette is installed so that the filtrate and retentate ports are properly oriented.3.Install a pressure gauge between the peristaltic pump output and the cassette to monitor the pressure going into the cassette.4.Sterilize the tubing and the cassette by adding 300 mL of 0.3 M NaOH into the reservoir. Position the tubing running from the filtrate and the retentate so that it goes directly into the waste. Run this for the first 100 mL and then switch tubing so that the filtrate and retentate circulates back into the reservoir for 20 min, then remove the filtrate lines from the reservoir and pump the rest of the 0.3 M NaOH into the waste. This removes debris that may have accumulated in the cassette during storage.5.Remove any residual 0.3 M NaOH from the system by running 200 mL of ultrapure H2O from the reservoir, through the cassette, and into the waste. Make sure to properly rinse the entirety of the reservoir, as any remaining NaOH can neutralize virus particles. Repeat this process twice.6.Stop the pump when 5 mL of the last wash with ultrapure H2O remains in the reservoir. Add 50 mL of sterile PBS and pump through the system until the reservoir contains only 5 mL of PBS. Remove the waste container and replace with a new sterile waste container. 1.Add virus to be purified into the reservoir, making sure retentate lines go back to the reservoir and filtrate lines into a new sterile waste container to avoid inactivation of any virus that might end up in the waste. Begin pump and carefully monitor the pressure gauge, and adjust the flow rate so that pressure is always below 10 psi. At this step, virus will be retained in the system.•As virus is concentrated, the pressure of the cassette increases. It is important to regularly check the pressure gauge and adjust the flow rate to keep the pressure below 10 psi.•We recommend sampling the waste during virus concentration to ensure that no virus is being lost. Ensure that sampled waste does not contain NaOH-PBS, as this can neutralize the virus.2.Run virus through the system until only 5–10 mL remains in the reservoir. 3.Exchange the buffer from concentrated virus supernatant to 5% sucrose-PBS for subsequent virus elution. Elution in 5% sucrose-PBS limits the aggregation of virus as it leaves the membrane.4.Add 50 mL of 5% sucrose-PBS to the reservoir and run until only 5 mL remains in the reservoir.5.Stop the pump and close the filtrate lines with caps to prevent the flow of liquid into the waste. Detach the retentate line circulating from the cassette back into the reservoir, from the reservoir itself and place in a 50-mL conical tube.6.Turn on the pump and collect elution 1 in the 50-mL conical tube. Stop the pump when elution 1 is finished.7.Repeat the elution process another two times by adding 5 mL of 5% sucrose-PBS to the reservoir to produce elution 2 and elution 3. The approximate volume of elutions 1, 2, and 3 are 10 mL, 5 mL, and 5 mL, respectively.8.Store elutions and waste (for future testing) at 4°C or proceed directly to gradient purification and final concentration. 1.Move the retentate and filtrate lines to a new waste container. Add 250 mL of 0.3 M NaOH-PBS to the reservoir, and pump 100 mL to remove any debris in the system.2.Once 100 mL has entered the waste, attach the retentate and filtrate lines to the reservoir to make a closed system and continue to pump for at least 1 h to clean the tubing and cassette. Pause point: The cleaning process can be extended overnight if the cassette has been used multiple times. Keep in mind that long exposure to high NaOH (>0.5 M) can degrade the filter.3.Remove the TFF cassette from the Centramate and store the cassette submersed in 0.3 M NaOH at 4°C. Wipe the Centramate down with Milli-Q H2O to remove excess NaOH and prevent corroding the metal. 1.Distribute the virus containing supernatant that was harvested from the virus-infected cells in step 5 of the virus harvest procedure into 250-mL centrifuge bottles, ensuring each bottle is balanced to 0.01 g.2.Centrifuge at 20,000 × g for 1.5 h at 4°C to pellet OrfV. We use an ultracentrifuge with a fixed-angle Type 19 rotor, but any high-speed centrifuge capable of achieving 20,000 × g can be used.•If a high-speed centrifuge is unavailable, this step can be skipped and the virus containing supernatant that was harvested from the virus-infected cells can instead be used to balance the sucrose cushion in step 5 below, and discarded.3.Decant the supernatant into a vessel and resuspend the virus pellet in a total of 25 mL of the supernatant. Discard the remaining supernatant.4.Place 10 mL of 36% sucrose-PBS into autoclaved 38-mL ultracentrifuge tubes.5.Carefully overlay 25 mL of the cleared lysate from step 11 of the virus harvest on top of the sucrose cushion. Repeat this step until the entire volume of cleared lysate has been loaded on top of a sucrose cushion.6.Repeat steps 4 and 5 for the resuspended virus pellet obtained in step 3.7.Centrifuge all sucrose cushion tubes at 55,000 × g for 1.5 h at 4°C in a swinging bucket SW 32 Ti rotor.8.Decant the supernatant and resuspend and pool viral pellets in a total volume of 7 mL of 5% sucrose-PBS. Store at 4°C. Gradient purification removes additional impurities from virus eluted following either two-step filtration or sucrose cushioning and is required to achieve the purity required for in vivo experiments.1.Dilute 60% (w/v) iodixanol (which is the concentration of iodixanol in Opti-Prep) in PBS to generate 15%, 25%, and 35% (v/v) iodixanol-PBS.2.Create the iodixanol gradient as per Figures 5B and 5C in 13-mL ultra-clear ultracentrifuge tubes. Initially, layer 2 mL of the 60% at the bottom of tube. Then add 1 mL of the 35% gently above the 60% layer. Next, gently layer on 1 mL of the 25% followed by 1 mL of the 15%.•Care should be taken to prevent mixing layers of the gradient.•For the TFF virus product, we typically only subject elutions 1 and 2 to gradient centrifugation, as they contain the majority of virus. Pause point: Iodixanol gradients can be stored at 4°C overnight.3.Gently pipette elutions 1 and 2 from method 1 or the resuspended virus pellet from method 2 on top of the 15% iodixanol gradient. Fill tubes to within 1 cm of the top to avoid collapse during centrifugation; if needed, supplement with 5% sucrose-PBS.4.Carefully balance each tube inside its ultracentrifuge canister to within 0.01 g.5.Centrifuge gradients in the SW 41 Ti rotor at 80,000 × g for 3 h at 4°C.6.OrfV will accumulate as three distinct bands in the 15%, 25%, and 35% iodixanol-PBS bands (Figures 5B and 5C).7.Extract virus using a P1000 pipette. Remove as much liquid on top of the 15% virus band as possible. Proceed to pipette out the virus band into a new collection tube. Repeat this with the remaining bands. Pool the collected virus.8.Repeat this step for all gradients poured, and pool the collected virus. Sonicate the pooled virus using a probe sonicator.•Conical tubes should be placed on ice during sonication to prevent excessive heating of samples.•Sonicator parameters should follow two cycles of 10 s on with 10-s rests in between at an amplitude of 20%.9.Centrifuge sonicated cell pellet suspension at 1,500 × g for 5 min at 4°C to remove any remaining cell debris from virus, and proceed to dialysis with the decanted supernatant. 1.Pour 1 L of sterile PBS into a 1-L beaker and add a sterile magnetic stir bar.2.Rehydrate a 3-mL Slide-A-Lyzer dialysis cassette (10,000 kDa cutoff) in PBS for 30 s.3.Load a 10-mL syringe with virus collected from the gradient after sonication and clarification, and attach an 18-gauge blunt-tip needle.4.Carefully insert the needle into one of the injection ports of the dialysis cassette, inject the virus, and remove any air before removing the needle. Mark the port used with a sharpie.•Avoid going back into the same port if needing to access the cassette again for any reason.5.Place the dialysis cassette in the 1 L of sterile PBS and stir slowly on a magnetic stir plate at room temperature for 2 h.6.After 2 h, replace the PBS with fresh PBS and transfer the dialysis setup to 4°C, stirring gently for at least 4 h to overnight. Pause point: After refreshing the PBS, the dialysis can be continued overnight. 1.Dialysis can result in volumetric expansion of the virus propagation.15Dirks C. Duh F.M. Rai S.K. Lerman M.I. Miller A.D. Mechanism of cell entry and transformation by enzootic nasal tumor virus.J. Virol. 2002; 76: 2141-2149Google Scholar For preclinical applications, it is desirable to concentrate virus into the smallest possible volume. To accomplish this, proceed with PEG concentration.2.Load a resealable plastic bag with 25 mL of 40% (w/v) PEG 20,000 in PBS. Place the dialysis cassette in the bag and seal so that all the air is removed and the entirety of the cassette is covered with PEG 20,000.3.Incubate at room temperature until volume is reduced to a desired level.•The duration required to reduce volume is variable, and depends on the amount of starting volume and the desired final volume. We typically concentrate 10 mL into 3 mL in 4.5 h. We recommend checking the cassette for contraction after the first 2 h and then every hour afterward.4.Following PEG concentration, gently rinse the cassette in PBS to remove external PEG.5.Specifically rinse the injection port (different from the one used to inject the virus) to be used with 5 mL of sterile PBS to remove any remaining PEG.6.Remove the virus by first injecting 5 mL of air into the dialysis cassette using a 10-mL syringe with an 18-gauge blunt-tip needle. Extract the virus, then reload the syringe with 350 μL of sucrose-PBS diluted for a final concentration of 5% sucrose-PBS. Inject sucrose-PBS back into the dialysis cassette. Gently palpate the cassette to remove any residual virus, and once again fill with 2 mL of air and collect all remaining liquid.7.Aliquot virus (typically 200–500 μL) including one 20-μL aliquot for titration. Store virus at −80°C. 1.The TCID50 assay for
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