Getting the Most Out of Baculovirus · Enterovirus-like Particles as Vaccines • Virus-like...
Transcript of Getting the Most Out of Baculovirus · Enterovirus-like Particles as Vaccines • Virus-like...
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Getting the Most Out of Baculovirus Linda Lua
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Enabling World Class Research
• Recombinant Protein Production ▫ Discovery, Translational, Preclinical ▫ Drug discovery, vaccinology, diagnostics, functional
materials, targeting and delivery, therapeutics, bioprocess development, agricultural science
• Research Training ▫ Up-skill professional development ▫ Tailored workshops and symposium
• Consultation
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Design & HTP Molecular Cloning
HTP Expression Screening
Scale-up Expression
Protein Purification & Analysis
Verified Construct
“A focus of expertise in protein research and a research emphasis on platform technologies”
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Which Way Should I Go?
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Types of Recombinant Protein
• Enzymes • Glycoproteins • Cytokines/interleukins • Kinases • Transcription factors • Structural proteins • Membrane proteins • Receptors • Viral proteins • Recombinant peptides
http://www.rsc.org/ej/NP/2010/c0np00005a/c0np00005a-f4.gif http://nfs.unipv.it/nfs/minf/dispense/immunology/lectures/files/structure_abs_tcr.html
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Army Malaria Institute
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International Academic & Industry
Australia
• Brazil • Germany • New Caledonia • New Zealand • North America • Malaysia • Singapore • United Kingdom
• 16 Australian universities • 11 Government research
organizations • 13 Companies
www.uq.edu.au/pef
• >1000s expression constructs
• >100s proteins • Simple biomolecules to
complex multi-protein assemblies
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Baculovirus Technology 1980s • Occasional production of
‘difficult to express’ proteins
• Rescue projects
2014 • A major expression tool in
most pharma/biotech labs • 50-60% proteins expressed
using baculo • Vaccines and gene delivery
system
Technology enhancement Automation
High-throughput
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COST%
SPEED%
TYPICAL%YIELD%
POST%–%TRANSLATION%MODIFICATION%
FDA%APPROVAL%
LOW% HIGH%
BACULO8INSECT%CELL% CELL8FREE%BACTERIA%MAMMALIAN% YEAST%
BACULO8INSECT%CELL%CELL8FREE%BACTERIA% MAMMALIAN%YEAST%
BACULO8INSECT%CELL%CELL8FREE% BACTERIA% MAMMALIAN%YEAST%
BACULO8INSECT%CELL%CELL8FREE% BACTERIA%MAMMALIAN% YEAST%
BACULO8INSECT%CELL%CELL8FREE% BACTERIA% MAMMALIAN%YEAST%
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rBaculovirus
H1, H5, H10 wild-type & mutants
Influenza VLP
HPV VLP
chimeric VLP
• Dengue viral proteins • Malaria proteins • TB proteins • Hendra viral proteins • Receptors and ligands • Ion channels • Kinases • Glycoproteins • Enzymes • Interleukins
Virus-like particles Hemagglutinin
100s recombinant proteins produced in this system
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HTP Baculovirus/Insect Cell Platform Recombination
24-well Virus amplification
24-deep well
Expression screening 24-deep well
Day 0 Day 7
Day 10
Expression analysis
Day 13
Cloning 96-well
1 week
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Common Challenges for Secreted Proteins
Low expression/secretion
Large volume of supernatant
Poor target protein recovery and purity
Contaminating host proteins
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Media: Sf900II P3 virus stock MOI 5 TOI hi5-1.5x106 cells/ml TOI Sf9-3x106 cells/ml
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27oC
21oC
FlashBACGOLD™ Bac to Bac™
HBM HBM gp67 gp67
High Five™ gp67 Bac to Bac™ - 21oC FlashBACGOLD™ Δv-cath ΔchiA- 27oC
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Ni Sepharose™ Excel Bind-Elute
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Stability of Virus Stock
P3 P4 P5 P6 P7 P8
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Virus-Like Particle (VLP)
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Simple to Complex Virus-Like Particles
• Single layer • Multiple layers • Non-enveloped • Enveloped
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Expert Rev. Vaccines 9(10), 1149–1176 (2010)
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Enteroviruses
• Hand Foot and Mouth Disease (HFMD) • Poliomyelitis
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Enterovirus Genome and Viral Structure
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Enterovirus-like Particles as Vaccines • Virus-like particle (VLP) resemble virus • Non-infectious as without genetic
material • Produce using insect cells • Co-expression of polyprotein (P1) and
protease (for processing of polyprotein into structural proteins)
• Assembly of structural proteins in VLPs in cells
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Flow fractionation (AF4)
Membrane Crossflow
MALS, UV & RI
size
%
Size Distribution Velocity
1. Sample enters chamber.
Process:
2. Crossflow drags particles to membrane 3. Particles diffuse back into flow (rate based on size).
4. Smaller particles flow faster than larger particles due to laminar flow.
5. UV, MALS & RI used to determine size distribution
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Analysis by field-flow fractionation
VLPs
Aggregates
Misformed
VLPs
VLPs
Misformed VLPs
Aggregates
Chuan et al., Biotech Bioeng 2008
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Biophysical Characterization
DLS analysis: Diameter - 32 nm TEM analysis: Diameter – 25-30 nm
AF4 analysis: Diameter - 30 nm
0"
10"
20"
30"
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50"
60"
70"
80"
90"
100"
110"
0"
0.2"
0.4"
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0.8"
1"
1.2"
0" 10" 20" 30" 40" 50" 60"
Radius'(n
m)'
UV'absorba
nce'(rela4
ve'scale)'
Time'(min)'
Soluble""Proteins"
VLPs"
Aggregates"
Misformed""VLPs"
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Modular VLP Vaccine Design
Virus-like particle
VP1 Modular VP1 Modular capsomere (5 VP1)
Modular VLP (72 capsomeres)
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Modular VLP Vaccine Design
Modular VP1
1. Correct epitope presentation is crucial to induce immune response against target pathogen
2. Module insertion must not disrupt capsomere structure
Computational tools to design and predict modular vaccine
candidates, test using insect cells
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Modular VLP Vaccine Design
Virus-like particle
VP1 Modular VP1 Modular capsomere (5 VP1)
Modular VLP (72 capsomeres)
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The Future…
FLU
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“A focus of expertise in protein research and
a research emphasis on platform technologies”
Our track record Our expertise Our capabilities Our platform
Chancellor’s Award for Team Excellence 2013
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THANK YOU