Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains March 26, 2013 Prepared by: Charles Bourne Strictly Private and Confidential

Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine StrainsMarch 26, 2013

Prepared by: Charles Bourne

Strictly Private and Confidential

Introduction

• Despite its limitations for preventing pulmonary TB, BCG continues to provide a platform for the construction of live, recombinant vaccines.

• The success of these vaccines relies heavily on their genetic stability throughout development, manufacturing and evaluation.

Outline

• Requirements of a vaccine

• Basic designs

• Early testing and observations

• Strategies for improvement

Requirements

• Flexibility for multiple gene evaluations

• Capacity for increased expression

• Absence of selection markers

• Stable gene maintenance

Design

• Phasmids

• Marked extrachromosomal plasmids

• Site-specific integration

Original Integration SystemMycobacteriophage L5

• Gene constructs reside on

plasmids carrying

mycobacteriophage

recombinase gene (integrase)

with the corresponding phage

attachment site (attP).

• Integrase catalyzes site-

specific recombination at the

bacterial attachment site

(attB) and plasmid is inserted

into genome.

Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains.

March 26, 2013

6

Genome

attL Integrase Gene Backbone(oriE)

AntibioticSelection

attR

Genome

Genome attB

Integration Vector

Backbone(oriE)

AntibioticSelection

attP

Integrase

Gene

Integration

X

Lee, M.H., et. al. (1991) Site-specific integration of mycobacteriophage L5: Integration-proficient vectors for Mycobacterium

smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 3111-3115.

Original L5 Integration SystemUnmarking with γδ Resolvase

• Resolvase is expressed from a subsequently introduced plasmid and recombines

sequences in the binding sites.

• Intervening sequence is excised and a single resolvase binding site remains.


March 26, 2013.

7

Genome

attL Integrase Gene AntibioticSelection

attR

Genome

TT TT resBackbone(oriE) res

Resolvase

GeneTT

Backbone(oriE) res attR

GenomeGenome

attL IntegraseTT

Stover, C. K., et. al. (1991) Nature 351, 456-460. Hatfull, G. F., et. al. (1988) Cambridge University Press.Reed. R. R. (1981) Proc Natl Acad Sci USA 78, 3428-3432. Berg, C. M., et. al. (1992) Gene 113, 9-16.

Observations

• Flexibility for multiple gene evaluations

• Capacity for increased expression

• Genetic stability in absence of antibiotic selection?

PCR Confirmation of Unmarked Integrated Construct


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9

• Stably integrated plasmid allows detection of integration

junctions (attL, attR) as well as all inserted genes.

Left Junction(attL)

Right Junction(attR)

Antigen Cassette/ Vector Backbone

GeneTT

Backbone(oriE) res attR

GenomeGenome

attL Integrase TT

Detection of attL During Manufacturing• Left integration junction PCR product weakens following unmarking.

• Disappearance of product following protein-free scale-up in manufacturing.

Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March

26, 2013.

10

1000 bp

Mar

ked

Contro

l 1

Paren

t 1Par

ent 2

Mar

ked

Contro

l 2

Unmar

ked

R&D Sto

ck 2

Acces

sion

Ban

k 2

Proce

ss D

evel

opm

ent 2

Mas

ter C

ell B

ank

2

Wat

er

850 bp

Expected Product928 bp

Detection of Antigen Cassette During Unmarking• Internal PCR shows individual colonies demonstrate varying degrees of

antigen loss during earliest stages of unmarking.


March 26, 2013.

11Pa

rent

(-)

Plas

mid

(+)

Wat

er

Expected Product1050 bp

1650 bp

1000 bp850 bp

Col

ony

20

Col

ony

19

Col

ony

18Colonies 1-17

Bla

nk

PCR Detection of Plasmid Excision


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12

• Excised plasmid with regenerated

attP site may eventually be lost

from culture entirely.

• PCR detection of regenerated “wild-

type” bacterial attachment site

(attB) indicates plasmid loss from

genome.

Wild-type Junction(attB) PCR

attBGenome

IntegrationVector

attP

Detection of attB After Culture PassageConfirmation of plasmid loss• PCR of isolated colonies passed in liquid culture (without selection) generates strong

products.

• Sequencing confirms “wild-type” attB site is re-generated after plasmid excision.


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13

Parent

Water

Expected ProductattB = 1213 bp

1650 bp

1000 bp

Strain 1

Final Pass

Strain 2

Final Pass

Original Integration SystemMycobacteriophage L5

• Site-specific recombination is

reversible in the presence of

integrase.

• Plasmid and chromosome

exist in an equilibrium

between integrated and

excised states.


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14

Genome

attL Integrase Gene Backbone(oriE)

AntibioticSelection

attR

Genome

Genome attB

Integration Vector

Backbone(OriE)

AntibioticSelection

attP

Integrase

Gene

ExcisionIntegration

X

Springer B, et. al. (2001) Instability and site-specific excision of integration-proficient mycobacteriophageL5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol. 290(8):669-75.

Strategies

• Integrase in trans

• Removable Integrase

Integrase in trans“Dual Plasmid System”

• Integrase gene is relocated to a

separate suicide plasmid.

• Both plasmids introduced into

parent strain as a co-

transformation.

• Stable construct is unmarked

with recombinase.

Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26,

2013.

16

IntegrationVector

TT

Backbone(oriE)

res

AntibioticSelection

resattP

Integrase

TT

Gene

SuicidePlasmid

AntibioticSelection(A)

oriE

AntibioticSelection(B)

sacB

CisIntegration

Vector

Backbone(oriE)

res

AntibioticSelection

res

attPTT

Gene

TT

TransIntegrasePlasmid

sacB

AntibioticSelection(A)

oriE

AntibioticSelection(B)

Integrase

Springer B, et. al. (2001) Instability and site-specific excision of integration-proficient mycobacteriophageL5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol. 290(8):669-75.

Removable Integrase“pRIAR System”

17

IntegrationVector

TT

Backbone(oriE)

res

AntibioticSelection

resattP

Integrase

TT

Gene

pRIAR

res

Integrase

aphR

resattPTT

Gene

TT Backbone(oriE)

• Integrase gene is relocated to another position on the same

integration vector between the resolvase binding sites.

Jason Huff et. al. (2010) Taking phage integration to the next level as a genetic tool for mycobacteria. Gene 468:8-19.


March 26, 2013.

Removable Integrase SystemUnmarking with γδ Resolvase

• Resolvase recombination removes antibiotic selection marker and the integrase gene.

• Stability achieved through unmarking.


March 26, 2013.

18

Genome

attL TT Gene TT Backbone(oriE)

res attR

Genome

Genome

attL TT Gene TTBackbone(oriE)

res Integrase AntibioticSelection

res attR

Genome

Resolvase

Jason Huff et. al. (2010) Taking phage integration to the next level as a genetic tool for mycobacteria. Gene 468:8-19.

Enhanced Stability Following Integrase RemovalConfirmatory PCR Testing (pRIAR)• Left Junction (attL) PCR product detected after unmarking.

• Right Junction (attR) PCR product detected after unmarking. Smaller size reflects omission

of marker/Int.

• Wild-type junction (attB) PCR product disappears following successful unmarking.


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1 2 3 4 5

Right Integration Junction (attR)

1 2 3 4 5

Wild-type Junction (attB)

1 2 3 4 5

Left Integration Junction (attL)

Enhanced Stability in Absence of IntegraseConfirmatory PCR Testing (Dual-plasmid)• Wild-type Junction (attB) PCR remains negative (below detection) throughout

manufacturing process.


March 26, 2013.

20

Wat

er

R&

D C

ultu

re

Pare

nt S

trai

n

Acc

essi

on B

ank

Mas

ter C

ell B

ank

Form

ulat

e B

ulk

(20L

)

Lyop

hiliz

ed T

OX

(10L

)

300 bp400 bp


Sensitivity of Wild-type Junction (attB) Detection PCRTesting of Serial Dilutions• Genomic DNA from parental strain serially diluted in genomic DNA of stable strain.

• Equal DNA template used in PCR and equal product loaded on gel.


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21


400 bp300 bp

Stable

Stra

in

Wat

er

1:10

,000

1:50

,000

1:10

0,00

0

1:50

0,00

0

1:1,

000,

000

1:1,

000

1:5,

000

1:10

01:

500

Conclusions

• Mycobacteriophage integration systems can be used for chromosomal expression of antigens in BCG.

• Elimination of integrase allows for stable maintenance of gene inserts in absence of selection.

• Stability of constructs can be effectively monitored by PCR.

• Genetic stability provides an opportunity for more accurate and relevant evaluation of new recombinant BCG-based vaccine candidates.

Advancing Tuberculosis Vaccines for the World

Aeras gratefully acknowledges the support of our funders and partners including the Bill & Melinda Gates Foundation, UK Department for International Development, Netherlands Ministry of Foreign Affairs, National Institutes of Health, Food and Drug Administration, European & Developing Countries Clinical Trials Partnerships, and a number of other donors and partners.

Acknowledgements

Aeras Vaccine Discovery Group

• Syed Zeyad • Kamalakannan Velmurugan• Joanna Manoranjan • Nathalie Cadieux• Megan Fitzpatrick • Arin Teymouri• Moisés Hernandez • Rosemary Chang• Ellen Wu • John Fulkerson• Barry Walker

Aeras Manufacturing Group CEO: Thomas Evans

Thank you.

Documents

Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains March 26, 2013 Prepared by: Charles Bourne Strictly Private and Confidential