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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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This 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 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

Share

Identify

Share

People die while they wait for the new

Influenza in a Connected World

Share

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