@article{d3d94eeefa0e468ba76784eb3e07e3bd,
title = "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.",
keywords = "cancer therapy, gradient ultracentrifugation, OrfV, Parapoxvirus ovis, poxvirus, tangential flow filtration, vaccinology, virus purification",
author = "{van Vloten}, {Jacob P.} and Minott, {Jessica A.} and McAusland, {Thomas M.} and Ingrao, {Joelle C.} and Santry, {Lisa A.} and Grant McFadden and Petrik, {James J.} and Bridle, {Byram W.} and Wootton, {Sarah K.}",
note = "Funding Information: This research was supported by a National Sciences and Engineering Research Council of Canada (NSERC) Discovery and Cancer Research Society grants to S.K.W. and B.W.B. Stipend funding was from: Canadian Graduate Scholarship-Doctoral Award (NSERC), Ontario Graduate Scholarship and Ontario Veterinary College Graduate Scholarship (for J.P.v.V.); Ontario Graduate Scholarship and Nora Cebotarev & Ellen Nilsen Memorial Scholarship (for J.A.M.). We thank Campus Animal Facilities, University of Guelph, for animal care services. Conception and design: J.P.v.V. J.C.I. G.M. J.J.P. S.K.W. and B.W.B.; development of methodology: J.P.v.V. J.A.M. T.M.M. L.A.S. and S.K.W.; acquisition of data: J.P.v.V. T.M.M. and J.C.I.; analysis and interpretation of data: J.P.v.V. J.A.M. T.M.M. J.C.I. G.M. J.J.P. B.W.B. and S.K.W.; writing, review, and/or revision of the manuscript: J.P.v.V. J.A.M. J.C.I. T.M.M. L.A.S. G.M. J.J.P. B.W.B. and S.K.W. The authors declare no competing interests. Funding Information: This research was supported by a National Sciences and Engineering Research Council of Canada ( NSERC ) Discovery and Cancer Research Society grants to S.K.W. and B.W.B. Stipend funding was from: Canadian Graduate Scholarship-Doctoral Award (NSERC), Ontario Graduate Scholarship and Ontario Veterinary College Graduate Scholarship (for J.P.v.V.); Ontario Graduate Scholarship and Nora Cebotarev & Ellen Nilsen Memorial Scholarship (for J.A.M.). We thank Campus Animal Facilities, University of Guelph, for animal care services. Publisher Copyright: {\textcopyright} 2021 The Authors",
year = "2021",
month = dec,
day = "10",
doi = "10.1016/j.omtm.2021.08.004",
language = "English (US)",
volume = "23",
pages = "434--447",
journal = "Molecular Therapy - Methods and Clinical Development",
issn = "2329-0501",
publisher = "Nature Publishing Group",
}
