Engineering Conferences International
ECI Digital Archives
Vaccine Technology IV
Proceedings
Spring 5-24-2012
Viral sensitizer technology improves yield of
modified vaccinia ankara from available cell
substrates
Facrice Le Boeuf
Ottawa Hospital Research Institute, Center for Innovative Cancer Therapeutics, Canada
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Recommended Citation
Facrice Le Boeuf, "Viral sensitizer technology improves yield of modified vaccinia ankara from available cell substrates" in "Vaccine
Technology 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/47
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Viral 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 Diallo
The Problem
• There is a rising global demand for virusbased vaccines and therapeutics
• Viruses need to be propagated in cells
• Innate cellular antiviral responses are a
primary hurdle for efficient viral replication
V
V
V
V
= Antiviral defense pathway
V
= Virus particle
V
V
2
The 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-fold
Virus output using VSe1
Red = Viral Growth
$$ = over 1000-fold increase
Diallo JS et al. Mol Ther, 2010
Viral sensitizer compounds, through a variety of mechanisms,
affect the innate cellular antiviral response in order to
promote more efficient growth of attenuated viruses
V
V
V
Viral Sensitizer
V
V
V
V
V
= Antiviral defense pathway
V
V
V
V
V
= Virus particle
4
Viral sensitizers repress cellular antiviral response
ISRE-Luciferase activity
VSV∆
∆51 titers
Diallo JS et al. Mol Ther, 2010
VST represses cellular antiviral response
Diallo JS et al. Mol Ther, 2010
CELL-BASED VACCINE MANUFACTURING
50+ year old method
New method
•
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 VST
VST 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
8
VST INCREASES YIELD OF MVA IN BHK21
MVA Titer (Log10 pfu/ml)
** P-Value:0.039
**
CTRL
CTRL
VST
VSe7
>10-fold increase in viral titers
** Average of four separate experiments in sextuplicate
VST SUBSTANTIALLY IMPROVES MVA YIELDS COMPARED
TO STANDARD METHODS AT ROLLER BOTTLE SCALE
MVA yields
1.0E+09
10 X
45X
PFU / ml
8.0E+08
6.0E+08
4.0E+08
4.5 X
2.0E+08
0.0E+00
CEF
CEF + VST
BHK21
BHK21 + VST
The AMES mutagenicity test
VST IS NOT DNA REACTIVE IN SALMONELLA
TA100 strain (base subs tu on) revertants/plate
2000
1800
1600
1400
1200
1000
800
600
400
200
0
MMS (+)
2AA (+)
VST 20 ug
VST 10 ug
VST 5 ug
Neg CTRL
VST IS NOT DNA REACTIVE IN SALMONELLA
TA98 strain (frameshi dele on) revertants/plate
1600
1400
1200
1000
800
600
400
200
0
2AA (+)
2NF (+)
VST 20 ug
VST 10 ug
VST 5 ug
Neg CTRL
VST does not increase frameshift deletion rate
MRM method for detection of
VST in purified virus cultures
MRM is a sensitive mass spectrometry-based method that
allows for specific detection of peptides or molecules even
in complex mixtures
MRM method for detection of
VST in purified virus cultures
We can easily detect MRM transition of purified VST (1ug)
MRM method for detection of
VST in purified virus cultures
MVA preparation spiked with VST =>
Compound is easily detected
VST-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 cultures
Acknowledgements
OHRI
Dr. Jean-Simon Diallo
Dr. John Bell
Dr. Rozanne
Arulanandam
Dr. Kelley Parato
Andrew Chen
McMaster University
Br. Brian Lichty
Dr. Eric Brown
Jan Blanchard
Jenny Wang
Ryan Brown
Carleton University
Dr. Jeff Smith
Karl Wasslen
University of Ottawa
Dr. Chris Boddy
Mark Dornan
Christina Moi
NRC-HHT
Dr. Amine Kamen
Danielle Jacob
Emma Petiot
Funding agencies
CIHR
NSERC
Grand Challenges
Canada
OICR