Le Boeuf, Facrice

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Engineering Conferences InternationalECI Digital ArchivesVaccine Technology IV ProceedingsSpring 5-24-2012Viral sensitizer technology improves yield ofmodified vaccinia ankara from available cellsubstratesFacrice Le BoeufOttawa Hospital Research Institute, Center for Innovative Cancer Therapeutics, CanadaFollow this and additional works at: http://dc.engconfintl.org/vaccine_ivPart of the Biomedical Engineering and Bioengineering CommonsThis Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion inVaccine Technology IV by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.Recommended CitationFacrice Le Boeuf, "Viral sensitizer technology improves yield of modified vaccinia ankara from available cell substrates" in "VaccineTechnology IV", B. Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves , ITQB/IBET; K. Jansen,Wyeth Vaccine Research Eds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/47Viral Sensitizer Technology Improves Yield of Modified Vaccinia Ankara from Available Cell Substrates*Dr. Fabrice Le Boeuf, Dr. Rozanne Arulanandam, Dr. Paul White, Julie Cox, Dr. Jeff Smith, Dr. Chris Boddy, Dr. John C. Bell, Dr. Jean-Simon DialloThe Problem• There is a rising global demand for virus-based vaccines and therapeutics• Viruses need to be propagated in cells• Innate cellular antiviral responses are a primary hurdle for efficient viral replicationV = Antiviral defense pathwayVVVVVV= Virus particle2The solution: Viral Sensitizer Technology (VST)Viral sensitizer technology (VST) encompasses a collection of small molecules identified by high-throughput screening that enhance viral growth in some cases up to over 1000-foldVirus output using VSe1Red  = Viral Growth $$ = over 1000-fold increaseDiallo JS et al. Mol Ther, 2010Viral sensitizer compounds, through a variety of mechanisms, affect the innate cellular antiviral response in order to promote more efficient growth of attenuated viruses V = Antiviral defense pathwayVVVVVVVVVVVV= Virus particleViral Sensitizer4ISRE-Luciferase activity VSV∆51 titersViral sensitizers repress cellular antiviral responseDiallo JS et al. Mol Ther, 2010VST represses cellular antiviral responseDiallo JS et al. Mol Ther, 201050+ year old method New methodCELL-BASED VACCINE MANUFACTURING• Most vaccines are virus-based and many are poorly growing attenuated virus strains• Production of several vaccines (eg. Influenza, MVA) is dominated by egg-based methods which have a number of issues • Producers are starting to move towards continuous cell-line based productions and chemically defined media• Few cell lines are approved for vaccine manufacturing• The innate antiviral response can limit the production of virus from cells, providing an opportunity for VSTVST APPLICATION: MODIFIED VACCINIA ANKARA (MVA)• Modified Vaccinia Ankara (MVA) is a multi-application vaccine platform approved for smallpox vaccination and in Phase II/III evaluation for several indications including Malaria, HIV, Influenza, and Cancer  • MVA is currently produced in eggs or egg-derived CEF cells.• BHK21 cell line is the only continuous mammalian cell line that supports MVA production MVA 8VST INCREASES YIELD OF MVA IN BHK21(Log10 pfu/ml)** P-Value:0.039**>10-fold increase in viral titers** Average of four separate experiments in sextuplicateCTRL VSe7MVA TiterCTRL VSTMVA yields8.0E+081.0E+0910 X45XVST SUBSTANTIALLY IMPROVES MVA YIELDS COMPARED TO STANDARD METHODS AT ROLLER BOTTLE SCALE0.0E+002.0E+084.0E+086.0E+08CEF CEF + VST BHK21 BHK21 + VSTPFU / ml4.5 XThe AMES mutagenicity test1200 1400 1600 1800 2000 TA100 strain (base subs tu on) revertants/plate VST IS NOT DNA REACTIVE IN SALMONELLA0 200 400 600 800 1000 MMS (+) 2AA (+) VST 20 ug  VST 10 ug  VST 5 ug Neg CTRL 1000 1200 1400 1600 TA98 strain (frameshi  dele on) revertants/plate VST IS NOT DNA REACTIVE IN SALMONELLA0 200 400 600 800 2AA (+) 2NF (+) VST 20 ug VST 10 ug VST 5 ug Neg CTRL VST does not increase frameshift deletion rateMRM method for detection of VST in purified virus culturesMRM is a sensitive mass spectrometry-based method that allows for specific detection of peptides or molecules even in complex mixturesMRM method for detection of VST in purified virus culturesWe can easily detect MRM transition of purified VST (1ug)  MRM method for detection of VST in purified virus culturesMVA preparation spiked with VST => Compound is easily detectedVST-assisted MVA preparation flowthrough => No compound-associated MRM transition signal detected (background)Conclusions• VST can increase yields of MVA by 4.5X in CEF and > 10-fold in BHK21 cells• VST activity is maintained at the roller bottle scale• VST is not DNA reactive based on AMES tests• MRM methodologies can be used to detect VST, which is absent from purified VST-assisted MVA culturesAcknowledgementsOHRIDr. Jean-Simon DialloDr. John BellDr. RozanneArulanandamDr. Kelley ParatoCarleton UniversityDr. Jeff Smith Karl WasslenUniversity of OttawaDr. Chris BoddyFunding agenciesCIHRNSERCGrand Challenges CanadaAndrew ChenMcMaster UniversityBr. Brian LichtyDr. Eric BrownJan BlanchardJenny WangRyan BrownMark DornanChristina MoiNRC-HHTDr. Amine KamenDanielle JacobEmma PetiotOICR

Viral sensitizer technology improves yield of modified vaccinia ankara from available cell substrates

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Viral sensitizer technology improves yield of modified vaccinia ankara from available cell substrates

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