New Technology inVaccine Engineering

Anton MiddelbergAustralian Institute for Bioengineering and Nanotechnology

The University of Queensland, Australia

Viruses in MayKatoomba, August, 2012

Introduction

Vaccination enormously successfulSmallpox eradicated, polio close

More to do15 M people still die annually, half children <5 yrsEmergent and re-emergent disease

Technological gap in approachesPasteur’s “Isolate-Inactivate-Inject” dominatesOpportunity to engineer better systems

3

Modular Vaccine Design

with sites for insertion of optional modules

BASEhttp://www.indiamart.com/ajantaautoacc/front-guard.html#ajanta-grill-guard-for-tavera

MODULE MODULAR DESIGN+

http://www.ssa.ford.com/servlet/ContentServer?cid=1178863133817&pagename=FSSA%2FDFYPage%2FFord-Default&c=DFYPage&site=FSSA

Front guard

Viral antigen

Murine Polyomavirus

X 72

CapsomereVirus-like Particle

(VLP or Capsid)VP1 Protein

Antigen Insertion Sites on VLP Surface

Bioprocess Engineering

Best available expressionin literature: 1 mg/L.OD

After factorial optimisation,Host selection and redesign:15-20 mg/L.OD

Confirmed 2-4 g/L in fed-batchE. coli fermentation.

J. Biotechnol. (2008), 134(1-2): 64-71

J. Biotechnol. (2010), 150(2): 224-231

7

A practical model

Biotechnol. Bioeng. (2010), 107:550

VP1 self-assembly in vitro

9

Speedsame process for different virusestime from DNA to purified antigen < 1 weekprocessing can be automated

ScaleMakes protein using industrial biotechnology tools100M doses per kL of bacterial culture in 24 h

Safetywe make protein, not viruswe can sterile filter before virus assembly

The UQ Microbial Vaccine Platform (MVP)

PurificationAnd Assembly

(48 h) >100,000 dosesper litre

1000L pilot= 100M doses

In 24 h

WaterGlucoseSaltsBacteria

Within 24 h2 g/l Soluble Antigen

Purification and Assembly

The UQ Microbial Vaccine Platform (MVP)

“Dose Excess” regime.Everyone can cultivate bacteria.

11

Application of the Platform: Influenza

12

Influenza

www.cdc.gov

http://microbiology2009.wikispaces.com/Influenza

Global ChallengeWhen the virus changes, existing vaccine does not work.

13

H1N1 (2009): April 27th

14

H1N1 (2009): May 27th

15

H1N1 (2009): September 27th

16

Influenza in a Connected World

Identify

ShareControl

People die while they wait for the new vaccine

17

Vaccine available for in vivo testing

0 2 4 5 7

Laboratory Timeline (Days):

Rapid response for emergent virus

Microbial culture

Modular capsomere

Modular VLP

18

Target Epitopes

Strain-specificHA1 – receptor binding regionsOther HA1 epitopes

Broadly cross-protectingM2e of matrix protein 2HA stalk regions

AssumeViruses

are Static

AssumeVirusesChange

19

Target Epitopes

Strain-specificHA1 – receptor binding regionsOther HA1 epitopes

Broadly cross-protectingM2e of matrix protein 2HA stalk regions

AssumeVirusesChange

20

Haemagglutinin Helix 190 Influenza A virus

http://viromag.files.wordpress.com/2009/02/influenza-virus-diagram.jpg

Receptor binding site.

Biology 101 – blocking the receptor binding site will block viral entry.

Glycosylation?Structure?

21

Viralprotein

VP1

Target epitope

VLP presenting target epitope

Modularize into VLP format

x5

x72

Capsomere presenting target epitope

22

Structural analysis of helix 190 peptide

MD simulationGromacsIn PBS solution

Peptide B1 Peptide B2

Helix 190in native HA

20 ns

23

Module Structure MattersPeptide Recombinant HA

***

***

ns = not significant

***ns

24

Initial Target Epitopes

Strain-specificHA1 – receptor binding regionsOther HA1 epitopesBiology 101

Broadly cross-protectingM2e of matrix protein 2HA stalk regions

AssumeViruses

are Static

25

ImmunogenicBroad cross protectionComplementary mechanism

Matrix Protein M2eInfluenza A virus

http://viromag.files.wordpress.com/2009/02/influenza-virus-diagram.jpg

VP1

x5

Modularize into capsomere format

26

Screening of modularized capsomere

Expression levelSolubility levelDownstream bioprocessing yield

1011 and 2022

27

Modular capsomere

28

Capsomere format improves immunogenicity

Alhydrogel

PBS

p = 0.0005

Endp

oint

titr

e(A

nti-M

2e s

peci

fic Ig

G)

Alhydrogel

1011

29

AlhydrogelAlhydrogel

Modular capsomere 1011 vs 2022

Adjuvant necessary for capsomere format

Excellent epitopetolerance

1011 2022 2022Wild-type capsomere

Little sensitivity to more modules

Excellent IgG titres

Endp

oint

titr

e (A

nti-M

2e s

peci

fic Ig

G)

ConclusionsVLP and capsomere platform developed

Remarkable productivity, protein not virus basedExcellent developability and manufacturabilityExcellent end point titres

Moving to protection studiesMultitude of insertions successfully handled

Flexibility afforded by VLP and Capsomere formats

30

31

Influenza in a Connected World

Identify

ShareControl

32

Acknowledgements

Dr Linda Lua & UQ Protein Expression Facility