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

2. 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.

3. Outline Requirements of a vaccine Basic designs Early testing and observations Strategies for improvement

4. Requirements Flexibility for multiple gene evaluations Capacity for increased expression Absence of selection markers Stable gene maintenance

5. Design Phasmids Marked extrachromosomal plasmids Site-specific integration

6. Original Integration System Mycobacteriophage 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, 20136 Genome attL IntegraseGene Backbone (oriE) Antibiotic Selection attR Genome attB Integration Vector Backbone (oriE) Antibiotic Selection 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.

7. Original L5 Integration System Unmarking 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. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.7 Genome attLIntegrase Gene Antibiotic Selection attR Genome TT res Backbone (oriE) res Resolvase Gene TT Backbone (oriE) res attR Genome attLIntegrase TT 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.

8. Observations Flexibility for multiple gene evaluations  Capacity for increased expression  Genetic stability in absence of antibiotic selection?

9. PCR Confirmation of Unmarked Integrated Construct Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.9 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 Gene TT Backbone (oriE) res attR Genome attLIntegrase TT

10. 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 Marked Control 1 Parent 1Parent 2 Marked Control 2 Unmarked R&D Stock 2 Accession Bank 2Process Development 2 Master Cell Bank 2 Water 850 bp Expected Product 928 bp

11. Detection of Antigen Cassette During Unmarking Internal PCR shows individual colonies demonstrate varying degrees of antigen loss during earliest stages of unmarking. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.11 Parent (-) Plasmid (+) Water Expected Product 1050 bp 1650 bp 1000 bp 850 bp Colony 20 Colony 19Colony 18 Colonies 1-17 Blank

12. PCR Detection of Plasmid Excision Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.12 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 Integration Vector attP

13. Detection of attB After Culture Passage Confirmation 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. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.13 Parent Water Expected Product attB = 1213 bp 1650 bp 1000 bp Strain 1 Final Pass Strain 2 Final Pass

14. Original Integration System Mycobacteriophage L5 Site-specific recombination is reversible in the presence of integrase. Plasmid and chromosome exist in an equilibrium between integrated and excised states. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 201314 Genome attL IntegraseGene Backbone (oriE) Antibiotic Selection attR Genome attB Integration Vector Backbone (OriE) Antibiotic Selection attP Integrase Gene ExcisionIntegration X Springer B, et. al. (2001) Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol. 290(8):669-75.

15. Strategies Integrase in trans Removable Integrase

16. 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 Integration Vector TT Backbone (oriE) res Antibiotic Selection res attP Integrase TT Gene Suicide Plasmid Antibiotic Selection(A) oriE Antibiotic Selection(B) sacB Cis Integration Vector Backbone (oriE) res Antibiotic Selection res attP TT Gene TT Trans Integrase Plasmid sacB Antibiotic Selection(A) oriE Antibiotic Selection(B) Integrase Springer B, et. al. (2001) Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol. 290(8):669-75.

17. Removable Integrase “pRIAR System” 17 Integration Vector TT Backbone (oriE) res Antibiotic Selection res attP Integrase TT Gene pRIAR res Integrase aphR res attP TT 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. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.

18. Removable Integrase System Unmarking with γδ Resolvase Resolvase recombination removes antibiotic selection marker and the integrase gene. Stability achieved through unmarking. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.18 Genome attLTT Gene TT Backbone (oriE) resattR Genome attLTTGeneTT Backbone (oriE) res Integrase Antibiotic Selection 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.

19. Enhanced Stability Following Integrase Removal Confirmatory 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. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.19 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)

20. Enhanced Stability in Absence of Integrase Confirmatory PCR Testing (Dual-plasmid) Wild-type Junction (attB) PCR remains negative (below detection) throughout manufacturing process. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.20 Water R&D Culture Parent Strain Accession Bank Master Cell Bank Formulate Bulk (20L) Lyophilized TOX (10L) 300 bp 400 bp Expected Product attB = 382 bp

21. Sensitivity of Wild-type Junction (attB) Detection PCR Testing 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. Methods in the Development of Stable, Unmarked Recombinant BCG Vaccine Strains. March 26, 2013.21 Expected Product attB = 382 bp 400 bp 300 bp Stable Strain Water 1:10,000 1:50,000 1:100,000 1:500,000 1:1,000,000 1:1,000 1:5,000 1:100 1:500

22. 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.

23. 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.