syntura: a new ribonuclease resistant viral rna control material ralf schönbrunner, ph.d. sogat xxi...
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SynTura: A New Ribonuclease Resistant
Viral RNA Control Material
Ralf Schönbrunner, Ph.D.SoGAT XXI
Brussels, BelgiumMay 28, 2009
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SynTura Project
• The SynTura project was created to provide RNA Internal Controls and Quantification Standards which behave like the target they intend to control
• Synthetic & Natural = SynTura
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SynTura Project Objectives
• Develop technology platform which can provide control material close to real analyte
• HCV was used as model system• Also functional as QS and IC
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The Best Theoretical Alternative for Real HCV
• Similar virus to HCV
• Easy to culture
• Grows to high titers
• Non infectious to humans
• Detectable by HCV assays
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CONFIDENTIAL
Viruses Related to HCV
Flaviviridae
Flavivirus
Yellow fever virus,
West Nile virus
Dengue virus
St. Louis encephalitis virus
Tick-borne encephalitis virus
Japanese encephalitis virus
Pestivirus
• Classical swine fever virus (CSFV) • Border disease virus (BDV)• Bovine Viral Diarrhea Virus 1 & 2
(BVDV)
Hepacivirus HCV
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CONFIDENTIAL
BVDV, HCV and MS2
BVDV HCV MS2
Mammalian virus Mammalian virus E. Coli Bacteriophage
Flaviviridae Flaviviridae Leviviridae
Genome 12 kb Genome 9.5 kb Genome 3.57 kB
Enveloped (lipid bilayer) Enveloped (lipid bilayer) Protein coat
Detergent sensitive Detergent sensitive Detergent resistant
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HCV & BVDV Genome Comparison
BVDV genome is similar to HCV but can be easily cultured and manipulated
Target Region of NAT HCV assays
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Flaviviridae TranslationHCV & BVDV
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5’UTR Secondary Structure
• Critical 5’UTR secondary structure– Transcripts often do not have correct
secondary structure– Might give misleading results for certain
HCV genotypes
• Is the Flaviviridae 5’-3’ Ring formation important?
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5’ & 3’ NTR Strategies
SynTura
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Position of HCV 5’NTR in BVDV
5’HCV NTRNFAR
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Generation of a BVDV-Hybrid
BVDV Plasmid(BVDV in blue)
Insert desired Sequence (red)
Make RNA Transcripts
Transfect Cell Line
Grow transfected Cell in culture
Viral Particles
Infected Cells produce Virus
DNA RNA
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Development Timeline
Milestones Time Receive and confirm synthetic DNA Sequence for cloning 2 - 4 weeks
Cloning into 3’NTR (DNA into 20 kb Plasmid) 4 – 5 weeks or longer Transfection 1 week
Grow first passage – initial titer (used as seed stock for subsequent lots)
2 weeks
Grow material for first lot (100 mL – 300 mL) 3 weeks Inactivation and confirmation 2 weeks
Total time: 4 – 6 month
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SynTura HCV Growth Curve
growth curve
0
1
2
3
4
5
6
7
8
9
10
10 20 30 40 50 60 70
time (h)
titr
e/ m
L (
log
10)
NCP7
B5UH
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Inactivation of BVDV
• Inactivated with ß-Propiolactone
• Protein-modifying agent – reacts with amides of Lys or Arg
• Commonly used for BVDV vaccines
• Alternative: Heat – 90% loss
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Initial Titer Determination
Samples Ct QS Ct Dilution Factor log IU/ml Final Conc IU/ml
HCV-BVDV K8 20.3 31.4 10 7.46 2.9.E+08
HCV-BVDV K4 20.2 31.4 10 7.49 3.1.E+08
HCV control 26.6 31.3 1 5.30 2.0.E+05
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Effect of Inactivation on TMA
SAMPLE Invalid/valid (IC)
Interpretation Avg S/CO
(n=5) Active 1E2 Valid reactive 5.65 (n=5) Inactive 1E2 Valid reactive 5.83 (n=5) Active 1E3 valid reactive 9.07 (n=5) Inactive 1E3 valid reactive 8.73
Results with ProCleix Ultrio TMA Assay
• No impact of ß-PL inactivation on TMA
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Sequence Stability
• 3 passages 5 clones partially sequenced– No mutations found in 5 clones
• 7 passages 5 clones partially sequenced – 1 clone no mutation – 2 clones had 1 point mutation in BVDV sequence– 2 clones had 1 point mutation in HCV insert
• Rate of mutation in insert similar to virus
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SynTura Thermal Stability
Thermal Stability of β-Propiolactone inactivated SynTura HCV
0
20
40
60
80
100
0 20 40 60 80 100
Temp. (ºC)
% R
eco
very
BVDV β-PL
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37ºC Accelerated Stability
37ºC
-6
-4
-2
0
2
4
6
0 5 10 15 20 25
Days
Ref C
t-Sa
mpl
e Ct
BVDV
HCV
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SynTura HCV as Calibrator
HCV vs.BVDV-HCV as Calibrators
BVDVy = -3.8043x + 43.98
R2 = 0.9772
HCVy = -3.8196x + 44.319
R2 = 0.9911
15
20
25
30
35
40
45
0.00 2.00 4.00 6.00 8.00
Log conc (IU/mL)
Ct
HCV
BVDV
Linear (BVDV)
Linear (HCV)
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OptiQuant-S HCV RNA Quantification Panel
• Based on SynTura Technology
• 100, 500, 5e3, 5e4, 5e5, 5e6 & 2.5e7 IU/mL
• Plasma Matrix
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Multicenter Study
0.00
2.00
4.00
6.00
8.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Expected Titer (log10 IU/ml)
Ob
serv
ed
Tite
r (l
og
10 IU
/ml)
Tricore
NYPH
CCPHL
ACL
BayCare
Covance
Overall Results from all Testing Sites
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Roche CAP/CTM HCV IVD
BayCare Laboratories
y = 0.9462x + 0.3496
R2 = 0.9984
0.00
2.00
4.00
6.00
8.00
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
Expected Titer (log10 IU/ml)
Ob
serv
ed
Tite
r (l
og
10 IU
/ml)
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Comparison of two Abbott realTime HCV ASR Assays
OptiQuant-S HCV Quantification Panel inAbbott Systems
Abbott Lab 2y = 1.0004xR2 = 0.9977 Abbott Lab 1
y = 0.9892xR2 = 0.9992
1
2
3
4
5
6
7
8
1 2 3 4 5 6 7 8
Expected Titer log10 (IU/ml)
Ob
serv
ed T
iter
log
1
0
(IU
/ml)
Abbott Lab 1
Abbott Lab 2
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Key Results
• SynTura HCV titer 2.0E+08 IU/mL
• Concentrated to 1.5eE+10 IU/mL
• Stable over 7 passages– Insert remains intact– No mutation after 3 passages – Only 1 point mutation after 7 passages
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Summary
• SynTura has properties very similar to HCV and probably other enveloped mammalian viruses
• SynTura technology allows integration of defined RNA sequences
• SynTura technology can be used for RNA assays as:– QS and IC– Positive Control– Calibrator
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Thank You
• Martin Luther Universität Halle/Saale– Sven Behrens – Martina Behrens
• Acrometrix– Mona Shahbazian– Jerry Boonyaratanakornkit– Reina Karunaratne
• Steve Young, Jessie Kilgore –Tricore
• Hanna Rennert, John Sipley, - NYPH, Cornell Medical Center
• Melody Hung-Fan, Kara Lee, -Contra Costa Public Health Lab
• Linda Sabatini, Lech Mazur, -ACL Central Lab
• Maura Pieretti, Carolyn Dowell, -BayCare Lab
• Ted Schutzbank- Covance