Production and purification of high-titer OrfV for preclinical studies in vaccinology and cancer therapy

Abstract

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. 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 which virus be in the and conical with and and Type 19 fixed-angle aluminum (Fisher Scientific, Cat. Cat. molecular weight (Fisher Scientific, Cat. Cat. Cat. Cat. and Luer-Lok with in Cat. and in Cat. Cat. for Cat. with Cat. or capable of and method to sheep skin from and in in a or at and cells using cells at a as cell quality as number cells in these using the PLUS Mycoplasma Detection cells from the into this we into 150-mm plates by cells but can be cells which in mL of medium using 150-mm plates to of cells and to of to to a preclinical we 150-mm the plates from to and to and to for to the be at for 150-mm as an 150-mm plates for is used to cell number to the of infectious virus and the other two are for virus virus infection plates cells from of the plates by with and mL of with mL of medium for a of cells using a and the number of cells 150-mm the number of cells to be by the number of to the number of cells for plates cells a virus to the of virus required to cells with a of infection of of cells virus cells mL or for required virus to for a of mL mL for a in conical medium from 150-mm plates of cells and with mL of plates at for on a to the at and for plates by an mL of to for a of mL for or of cells are of of infection will on multiple including the health of the cells and the of the titer of the virus used for the We the of infection of virus from both the and the cell to is in of cells using a cell cells and medium in conical tubes and on tubes at for at to cell into a with and at significant of virus will be in the which can be using the methods can be for to a at significant of titer cell in mL of virus and in conical tubes to a of mL to expansion tubes at can be at this of cell at be to cell and freeze-thaw the for of on at an of by tubes be on to of the cell at for at to the virus from cell is a critical as to cell and in of and in purification Additionally, cell by can be through to and can virus in the into a to the cell cell of of of Cat. to at for and on to method or method for virus be or through a to be in a the and in a as the the by mL of M by mL of the and the to a the but the to the being to the virus through the mL of through and for the of from the the is and through the the and the to to can be to the at the to of from the mL of is of or will have to be virus a new at the to the virus as it the virus from virus harvest and and the virus through the the to the as virus through the We have of virus at to but the can dramatically as the with We the through to and a the final of virus with mL of to the system. This will virus of the virus on the be virus at the mL of M through the to the and in M be in a the from the M and with the between two the the in a to the a and to the of to the and have been the that is to the and that the the is the to the and as in that the is that the and are a between the and the to the into the the and the by mL of M into the the from the and the that it into the this for the first mL and that the and into the for the from the and the of the M into the This that have in the M from the system by mL of ultrapure H2O from the through the and into the to the of the as can virus this the mL of the with ultrapure H2O in the mL of and through the system the mL of the and with a new virus to be into the to the and into a new to of virus that in the and the and the that is this virus will be in the virus is the of the is to the and the to the the virus to that no virus is being that as this can the virus through the system mL in the the buffer from concentrated virus to for virus in the of virus as it the mL of to the and mL in the the and the with to the of into the the line from the into the from the and in a conical on the and in the conical the is the two by mL of to the to produce and The of and are and and at or to purification and final the and to a new mL of M to the and mL to in the mL has entered the the and to the to make a system and to for at to the and The can be the has been used multiple in that to high can the the from the and the in M at the with H2O to and the the virus that from the cells in of the virus harvest into centrifuge bottles, is to at 20,000 for at to We use an ultracentrifuge with a fixed-angle Type 19 but centrifuge capable of 20,000 can be a centrifuge is this can be and the virus that from the cells can be used to the in and the into a and the virus in a of mL of the the mL of into ultracentrifuge mL of the from of the virus harvest on of the this the of has been on of a and for the virus in tubes at for at in a the and and viral in a of mL of at Gradient purification from virus or and is required to the purity required for in vivo is the of in in to and the as and in ultracentrifuge mL of the at the of mL of the the on mL of the by mL of the be to of the the virus we and to as they the of virus. can be at and from method or the virus from method on of the tubes to of the to with ultracentrifuge to in the at for at will as in the and and virus using a as on of the virus as to the virus into a new this with the the this for and the virus. the virus using a tubes be on to of two of on with in between at an of cell at for at to cell from and to with the of into a and a a in for a with virus from the and and an blunt-tip the needle into of the of the the and the the used with a into the to the for the in the of and on a at for the with and transfer the to for at to the the can be can in expansion of the virus C. of cell and by tumor Virol. Scholar preclinical it is to virus into the with a with mL of 20,000 in the in the and that the is and the of the is with at is to a required to is and on the of and the final We mL into mL in We the for the first and the in to the from the used to the to be used with mL of to the virus by first mL of into the using a with an blunt-tip the the with of for a final of into the the to and with mL of and virus including for virus at for OrfV is using which are permissive to OrfV and at a time for Additionally, cells have an to the to for of cells infected with OrfV or cells at cells in flat-bottomed 96-well cell culture plates in of that cell is at by of virus in to with for two to for potential a virus we to is to a virus that has been as this can infectious virus of cells with the in at and for the of infection by in a that as and the using the L. Bridle B.W. and purification of high-titer disease virus for use in preclinical models of Ther. 2018; 9: Scholar can be to by by a of C. of cell and by tumor Virol. Scholar to of OrfV by is on to the cells that and of cells for OrfV to as in cells at cells in mL of in a 6-well and or cells of virus in in to with a virus we to medium from cells and with of virus. that is infected with medium virus for at the to of mL of low-melting-point agarose in H2O and in a agarose in a by in a mL of no phenol with fetal bovine serum and cocktail and to of infectious and with mL of agarose and MEM at a and mL to that no as these will of by agarose to plates into at and for or are by at a with and the by the of can be with red or and with to ability to and OVs in and is for preclinical of virus genome copy number by provides and and for these types of We describe herein a method to and OrfV virus by using and using DNA from virus as an virus DNA from virus we with at virus with to genomic cellular of of with to of virus and at for the by of and at for viral DNA using a Scientific, Cat. or by the method virus with of K and of to to a of to for in a alcohol and by at for at viral DNA the which and phenol viral DNA by of and of M at for to and at for by or viral genomic DNA can be by using the in and the plasmid target in and The plasmid the target for for genome copy number by size in a new on virus DNA with from for OrfV We on a of virus DNA virus genome copy the of the plasmid is by and the copy number is using the is generated by the determining at and the line of The of the virus are and on the line of to the virus genome copy number cells to using method 1: using method for infectious genome copy Cell contaminants through the purification and can of virus and the of in vitro and in vivo cellular and DNA can of cell used for that can This is in the because the of virus infection is Cell from contaminants can in high which in and vitro with virus are killing can be cell contaminants are lethal to cells or the infectious is the The use of in preclinical models for vaccination or cancer can and immune response The critical in the OrfV and purification method that of cellular contaminants in virus and for The infectious of OrfV in the of virus being produced as virions that from the infected cell but with of titer using a J. Virol. 2016; 5: Scholar virus a and from infected the but this is produced at a the virus and Scholar The of virus be released from which by freeze-thaw combined with which a high of cell This the of cellular which can be by of the virus can to of the it is to high and on through can of virus from cellular We have large being to the of the which large of the is or in or Additionally, we using virus from for purification through an contaminants with which can be to both in vitro and in vivo the and with the an can be to this with by using a the an be in as and other can to cell that are of as infected the quality of virus we titrating a known and virus in with virus be in to multiple We have a of to of infectious a freeze-thaw of virus but have of infectious freeze-thaw new of OrfV be tested for in vivo, OrfV is to be through the purification and can to with of OrfV require from to virus have a combination of including and but to animal We have systemic administration of at as high as The we have to mice is produced using method but a for therapy is preclinical cancer we this quality in the tumor at the disease as is This that virus is in mice that have immune to cancer and in from replication of the virus. an cell line to produce we on C.J. Lichty B.D. Bell J.C. Oncolytic viruses: therapeutics with an identity crisis.EBioMedicine. 2016; 9: 31-36Google Scholar sheep cells primary fetal cells and human cells for cell line in at using the virus for and and virus at and infection the cell line for producing we the the for cell the and produced the of with no significant between the two cell and cells produced virus and but from We production of OrfV on virus are to culture and have replication cells we have that the quality of cells in cell culture, which is the with the of OrfV using the purification we produced and by The virus in a the virus a OrfV can at Additionally, for to be in a the virus of to mL of in which is an for the using the purification we produced and The virus and different from that of the method be into that OrfV produced using the purification method have in vivo at high with produced by no in purity or between both at that and are to we from multiple propagations and The virus to virus the we from the and tested virus in the The of virus in the first with of virus the and in the final this we to purification with the first and OrfV genome copy number in virus and OrfV in we targeting genes for the virus the and a We these genes because they are of the of poxvirus genes and are to be in immune-modulatory or virulence tested by for from OrfV genomic DNA target regions and into a plasmid which used to a for and of OrfV in quantifying genome copy number by that and produced a that of they are the for of genome copy number for this of the methods provide researchers with the tools to produce OrfV of high and purity for use in preclinical models of vaccination and cancer therapy. will also methods for and quantifying OrfV in virus and We that these methods will to the of OrfV as a vaccine and oncolytic vector This research by a and of and to and and and We of for animal and and of and of and and of and of the and The authors no

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