bacterial expression of a vlp sub-unit for rapid and cheap
TRANSCRIPT
Engineering Conferences InternationalECI Digital Archives
Vaccine Technology IV Proceedings
Spring 5-23-2012
Bacterial expression of a VLP Sub-unit for rapid andcheap influenza vaccinationAnton MiddelbergAustralian Institute for Bioengineering and Nanotechnology The University of Queensland, Australia
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Recommended CitationAnton Middelberg, "Bacterial expression of a VLP Sub-unit for rapid and cheap influenza vaccination" in "Vaccine Technology IV", B.Buckland, University College London, UK; J. Aunins, Janis Biologics, LLC; P. Alves , ITQB/IBET; K. Jansen, Wyeth Vaccine ResearchEds, ECI Symposium Series, (2013). http://dc.engconfintl.org/vaccine_iv/31
Engineering VLPand Capsomere
Anton Middelberg
Australian Institute for Bioengineering and Nanotechnology
The University of Queensland, Australia
Engineering VLPCapsomere Vaccines
Vaccine Technology IV
May 20 – 25, 2012
Anton Middelberg
Australian Institute for Bioengineering and Nanotechnology
The University of Queensland, Australia
Introduction
� Vaccination enormously successful� Smallpox eradicated, polio close
� More to do� More to do� 15 M people still die annually, half children <5 yrs
� Emergent and re-emergent disease
� Technological gap in approaches� Pasteur’s “Isolate-Inactivate-
� Opportunity to engineer better systems
Vaccination enormously successfulSmallpox eradicated, polio close
15 M people still die annually, half children <5 yrs
emergent disease
Technological gap in approaches-Inject” dominates
Opportunity to engineer better systems
Modular Vaccine Design
http://www.ssa.ford.com/servlet/ContentServer?cid=1178863133817&pagename=FSSA%2FDFYPage%2FF
Front guard
with sites for insertion of
optional modules
BASEMODULE+
78863133817&pagename=FSSA%2FDFYPage%2FFord-Default&c=DFYPage&site=FSSA
Viral antigen
Modular Vaccine Design
Front guard
3
http://www.indiamart.com/ajantaautoacc/front-guard.html#ajanta-grill-guard-for-tavera
MODULE MODULAR DESIGN
Viral antigen
Murine Polyomavirus
CapsomereVP1 Protein
X 72
Virus-like Particle
(VLP or Capsid)
Antigen Insertion Sites on VLP SurfaceAntigen Insertion Sites on VLP Surface
Bioprocess Engineering
GST
Thrombin Cleavage Site + GG
BamHI (931)
pGEX-4T-1-V P1
6112 bp
VP1
Ampicillin Resistance Gene
XhoI (2098)
Bioprocess Engineering
Best available expression
in literature: 1 mg/L.OD
After factorial optimisation,After factorial optimisation,
Host selection and redesign:
15-20 mg/L.OD
Confirmed 2-4 g/L in fed-batch
E. coli fermentation.
J. Biotechnol. (2008), 134(1-2): 64-71
J. Biotechnol. (2010), 150(2): 224-231
VP1 self-assembly in vitroin vitro
P-1 / V-101
P-2 / HG-101
Homogenization
S-101S-102
Process Flowsheet
P-1 / V-101
Fermentation
Homogenization
P-6 / DE-101
Sterile FiltrationP-7 / V-103
Capsid Assembly
S-106
S-107
S-103
P-3 / C-101
Expanded Bed
P-4 / V-102
Tag Removal
P-5 / C-102
Capsomere Purification
S-104
P-6 / DE-101
Sterile Filtration
S-105
�SpeedSpeedSpeedSpeed� same process for different viruses
� time from DNA to purified antigen < 1 week
� processing can be automated
The UQ Microbial Vaccine Platform (MVP)
�ScaleScaleScaleScale� Makes protein using industrial biotechnology tools
� 100M doses per kL of bacterial culture in
�SafetySafetySafetySafety� we make protein, not virus
� we can sterile filter before virus assembly
same process for different viruses
time from DNA to purified antigen < 1 week
processing can be automated
Vaccine Platform (MVP)
9
Makes protein using industrial biotechnology tools
of bacterial culture in 24 h
we can sterile filter before virus assembly
The UQ Microbial Vaccine Platform (MVP)
Water
Glucose
SaltsBacteria
Within 24 h
2 g/l Soluble Antigen
Purification
Purification and Assembly
The UQ Microbial Vaccine Platform (MVP)
PurificationAnd Assembly
(48 h)>100,000 doses
per litre
1000L pilot
= 100M doses
In 24 h
“Dose Excess” regime.
Everyone can cultivate bacteria.
Pre-existing immunity against the platform?existing immunity against the platform?
11
En
dp
oin
t ti
ter
(An
ti-a
nti
gen
sp
ecific
Ig
G)
Pre-existing immunity
En
dp
oin
t ti
ter
(An
ti-a
nti
gen
sp
ecific
Ig
G)
ns = not significant
104
105
106
Day 21
Day 77
Day 21
Day 77
(An
ti-a
nti
gen
sp
ecific
Ig
G)
Response against
(Group A Streptoccocus)J8iwt VLP
ns
12
wt p
rimed
PBS p
rimed
wt p
rimed
PBS p
rimed
100
101
102
103
104
(An
ti-a
nti
gen
sp
ecific
Ig
G)
ns = not significant
Application of the Platform: InfluenzaPlatform: Influenza
13
Influenza
www.cdc.gov
Global Challenge
When the virus changes, existing vaccine does not work.
14
http://microbiology2009.wikispaces.com/Influenza
Global Challenge
When the virus changes, existing vaccine does not work.
H1N1 (2009): April 27thth
15
H1N1 (2009): May 27thth
16
H1N1 (2009): September 27H1N1 (2009): September 27th
17
Influenza in a Connected World
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Identify
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People die while they wait for the new
Influenza in a Connected World
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Control
18
Share
People die while they wait for the new vaccine
0 2 4
Laboratory Timeline (Days):
Rapid response for emergent virus
Microbial
culturecapsomere
5 7
Rapid response for emergent virus
19
Vaccine available for in vivo testing
Modular
capsomere
Modular
VLP
Initial Target Epitopes
� Completely Non-Universal
�HA1 – receptor binding regions
�Other HA1 epitopes�Other HA1 epitopes
� Somewhat Universal
�M2e of matrix protein 2
�HA stalk regions
Universal
receptor binding regions
Assume
Influenza
Changes
20
2
Assume
Influenza
“Behaves”
Haemagglutinin Helix 190
� Receptor binding site.
� Biology 101 – blocking
the receptor binding site the receptor binding site
will block viral entry.
� Glycosylation?
� Structure?
190Influenza A virus
21
http://viromag.files.wordpress.com/2009/0
2/influenza-virus-diagram.jpg
Target epitope
Modularize into VLP format
Viralprotein
VP1
x5
Modularize into VLP format
x72
Capsomere presenting target
epitope
22
VLP presenting
target epitope
Structural analysis of helix 190 peptide
MD simulation� Gromacs
� In PBS solution
Peptide B1
Helix 190in native HA
Structural analysis of helix 190 peptide
MD simulationGromacs
In PBS solution
� 20 ns
23
Peptide B1 Peptide B2
20
30
An
gstr
om
RMSDStructural deviation of designed helix 190 peptide from
C-a
lpha
Mai
n-chai
n
Side-
chai
nW
hole p
eptid
e
0
10
20
An
gstr
om
Peptide B1
Structural deviation of designed helix 190 peptide from native
24Whole
pep
tide
Peptide B2
Module Structure Matters
Peptide
***
ns
ns = not significant
Module Structure Matters
Glycosylated HA
***
***
25
***
Glycosylation Matters Less
B2 VLPs
Matters Less
26
B1 VLPs
controls
Initial Target Epitopes
� Completely Non-Universal
�HA1 – receptor binding regions
�Other HA1 epitopes�Other HA1 epitopes
�Biology 101
� Somewhat Universal
�M2e of matrix protein 2
�HA stalk regions
Universal
receptor binding regions
27
2
� Immunogenic
� Broad cross protection
� Complementary mechanism
Matrix Protein M2e
VP1
x5
Modularize into capsomere
format
Influenza A virus
28
http://viromag.files.wordpress.com/2009/02/in
fluenza-virus-diagram.jpg
Engineering VP1 for antigen modules
x5x5
� Assembly incompetent
� Improved stability
antigen modules
� Trypsin digestion site
29
� Antigen insertion:
� two surface loops
� N-terminus
� C-terminus
Modular capsomere
30
Screening of modularized capsomere
1011 and 2022
Screening of modularized capsomere
�Expression level
�Solubility level
�Downstream
31
�Downstream
bioprocessing
yield
1011 and 2022
Capsomere format improves immunogenicity
En
dp
oin
t ti
tre
M2e s
pecif
ic I
gG
)
Alhydrogel
PBS
En
dp
oin
t ti
tre
(An
ti-M
2e s
pecif
ic I
gG
)
format improves immunogenicityp = 0.0005
32
Alhydrogel Alhydrogel
1011
Modular capsomere 1011 vs 2022
M2e s
pecif
ic I
gG
)
Alhydrogel
1011 2022Wild-type capsomere
En
dp
oin
t ti
tre (
An
ti-M
2e s
pecif
ic I
gG
)
Modular capsomere 1011 vs 2022
� Little sensitivity to more
� Excellent IgG titres
33
Alhydrogel
� Adjuvant necessary
for capsomere format
� Excellent epitope
tolerance
2022
� Little sensitivity to more
modules
Conclusions
� VLP and capsomere platform developed
� Remarkable productivity, protein not virus based
� Excellent developability and manufacturability
� Excellent end point titres� Excellent end point titres
� Moving to protection studies
� Multitude of insertions successfully handled
� Flexibility afforded by VLP and
platform developed
Remarkable productivity, protein not virus based
and manufacturability
Moving to protection studies
Multitude of insertions successfully handled
Flexibility afforded by VLP and Capsomere formats
34
Influenza in a Connected World
Share
Identify
Share
Influenza in a Connected World
Share
Control
35
Share
Acknowledgements
Dr Linda Lua & UQ Protein Expression FacilityMs Melisa Anggraeni
36
Dr Linda Lua & UQ Protein Expression Facility