Exploiting Synthetic Genomics to Create Influenza Vaccines

1. Exploiting Synthetic Genomics to Create Influenza Vaccines David E. Wentworth J. Craig Venter Institute, Rockville, Maryland

2. Outline Influenza Virus Genome Sequencing (NIH/NIAID) rg-Influenza virus Synthetic Genomics: Preparedness (NIH/NIAID) & Rapid Response (BARDA/Novartis/SGVI)

3. NIAID Collaborative Influenza Genome Sequencing Project Goals • Increase genome knowledge base • Improve understanding • Evolution, spread, and disease • Aid in the development of: • Vaccines, Therapies, Diagnostics • Data generated is publicly available • GenBank • Analysis tools -> NCBI, IRD • Mitigate the impact influenza epidemics/pandemics http://www.niaid.nih.gov/LabsAndResources/resources/dmid/gsc/Influenza/Pages/overview.aspx

4. Influenza Genome Sequencing Project Collaborators

5. Influenza Virus Sequencing Pipeline http://gsc.jcvi.org/projects/msc/influenza/

6. Sequence M-RTPCR Amplicons NP/OP Swabs Controls L + - 6 5 4 3 2 1 HA primer PB1, PB2 PA HA NP NA • Genetic/Molecular Analysis • Phylogeny • Virulence Determinants • Used in NIAID/JCVI influenza sequencing pipeline M NS Real time (CT) 23 23 29 30 30 ND Genomic Amplification Directly From Clinical Specimens Zhou, B., M. E. Donnelly, D. T. Scholes, K. St.George, M. Hatta, Y. Kawaoka, and D. E. Wentworth. 2009. J.Virol. 83:10309-10313.

7. JCVI Influenza Virus Sequencing Pipeline Closure Data merged Roche: 454 GS FLX Illumina : GAII, HiSeq Invitrogen: Ion torrent http://gsc.jcvi.org/projects/msc/influenza/

8. JCVI Influenza Virus Sequencing Pipeline Emergency Production Capacity 3730: up to 60 virus genomes/week 454: up to 300 virus genomes/week (60X coverage) Drug Resistance Detection: up to 1000 isolates/week Closure Data merged Roche: 454 GS FLX Illumina : GAII, HiSeq Invitrogen: Ion torrent http://gsc.jcvi.org/projects/msc/influenza/

10. Reading and Writing DNA

11. Synthetic Genomics Tools Gibson Assembly

12. Synfluenza Project Details • NIAID project to create ~1000 HA’s and NA’s • 12 host subtype combinations • Span sequence diversity (past 5 years) • Human – H1N1pdm, H1N1, H3N2, Influenza B • Avian – H5N1, H7N3, H7N7, H9N2 • Swine – H1N1, H1N2, H3N1, H3N2 • Algorithms to maximize reuse of oligos/cassettes and minimize costs • Each molecule made from 7 (HA) or 5 (NA) cassettes (~350bp) • Each cassette is made from 8 oligos (~65 bp) • Designs based on GenBank sequences with consensus UTRs Oligonucleotides Assemble &, clone Cloned Cassettes Sequence &, assemble Gene Segment Clones

13. Synfluenza Gene Cassette/Molecule Design • 1 copy of each unique oligo/cassette is made for each unique position • Many non-unique cassettes can be reused Non-unique, duplicate cassettes HA Cassettes (~350 bp) 1 2 3 4 5 6 7 Assembly H5.1 H5.2 H5.3 Assembled HA Molecules

14. HA’s and NA’s Constructed Via Automated DNA Synthesis and Assembly HA, NA Genes Designed Sequence E. coli transformation Colony picking Template production Sequencing reaction Sequencing Select clones pass Order/Synthesize Oligonucleotides QPix Biomek FX ABI3730 Biomek FX Biomek FX µFill ABI9700 Thermal Cycler µFill Assembly reaction Cloning ~13 kb per 384-well oligo plate Iterative assembly and amplification Culturing or PCR Biomek FX Hamilton μStar µFill µFill

15. Synfluenza Summary Purpose: Develop a technical capability to generate and stockpile synthetic DNA encoding influenza gene segment, which could be used to produce virus seeds stocks. Deliverable Library of ~1000 sequence verified HA & NA genes Available through the Biodefense and Emerging Infections Research Resource Program (BEI) Synthetic gene segment generation Gibson in-vitro assembly Assembly uses automated robotic systems Enables construction of an extensive library of influenza genes Potential to use cassettes in the future for new viruses Library of clones Vaccine seeds Diagnostics Basic Research

16. Speeding vaccine seed generation A BARDA-funded collaboration between Novartis, Synthetic Genomics Vaccines Inc. (SGVI)/J. Craig Venter Institute (JCVI) • Rapidly synthesize flu gene segments (HA and NA) directly from sequence information using synthetic oligos. • Combine newly synthesized genes with regulatory elements needed for virus rescue. • Introduce nucleic acids into cells and rescue viruses with optimized flu backbone genes. Milestone 1 (Sept. 2011): Demonstrate virus rescue within 7 days of receiving HA and NA sequence information Status – Milestone surpassed We were able to confirm rescue of an H7N9 virus within 5 days of initiating the process Slide Provided by Peter Mason, Novartis

17. Virus was rescued from synthetic HA and NA made by rapid assembly RG virus was harvested 4 days after initiation of oligo synthesis • Virus recovery has been demonstrated using several different synthetic HA and NA gene segments. • Recovery is efficient in 293T/MDCK co-cultures • Next steps include transitioning to rescue in vaccine-approved MDCK cells, in which virus rescue is less efficient. Slide Provided by Peter Mason

18. Is it Possible to Create Live Attenuated Vaccines From Emerging Viruses? • Engineer temperature sensitive mutations into H1N1pdm virus • Could be used as live attenuated vaccine • Likely to have better efficacy • Cross-protection H1N1pdm

19. In MiceTS2-LAIV Is: Attenuated Protective

20. Summary rg-Influenza Virus DNA synthesis Transfection MDCK cell RG influenza genome • High throughput genomic surveillance- circulatingsubtypes, drift variants, pandemic threats completely sequenced • Synthetic genomics - create gene segments (BARDA/Novartis) or pre-existing gene segments could be used (synfluenza) • Rescue vaccine pre-seeds - 6:2 vaccine seeds (TIV, LAIV) • Pre-existing stocks ? • Engineered complete genomes as LAIVs?

21. Thanks to all: • J. Craig Venter Institute • Craig Venter • Karen Nelson • Bill Nierman • John Glass • Dan Gibson • Mikkel Algire • Jayshree Zaveri • ZheniaDenisova • Admasu Melake • Tim Stockwell • Danny Katzel • Brian Bishop • Shiliang Wang • Brian Blanton • David Wentworth • Vivien Dugan • Suman Das • Xudong Lin • Bin Zhou • Rebecca Halpin • Elodie Ghedin • IndreshSingh • Ishwar Chandramouliswaran • Tony Yee • NCBI • David Lipman • Tatiana Tatusova • YimingBao • Novartis Vaccines and Diagnostics • Phil Dormitzer • Christian Mandl • RinoRappuoli • Peter Mason • Pirada Suphaphiphat • Melissa Sackal • Terika Spencer • Ivna de Souza • Stewart Craig • Gene Palmer • Wadsworth Center, NYSDOH • Jill Taylor • Deborah Blog • NIH/NIAID • Maria Giovanni • David Spiro • Valentina Di Francesca • Collaborators • Jill Taylor • Kirsten St George • Peter Palese • Adolfo Garcia-Sastre • Rob Webster • Gavin Smith • Lance Jennings • Nancy Cox • Robert Couch • Dick Slemons • Jonathan Yewdell • Jack Bennink • Ilaria Capua • Giovanni Cattoli • Laurel Edelman • David Boyle • Kim Halpin • Ted Leighton • John Pasick • Doris Bucher • Eva Harris • Aubree Gordon • Earl Brown • Carol Cardona • Ron Fouchier • Mona Aly • Shin Ru Shih • Hon Ip • Jonathan Runstadler

22. These projects have been funded with federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services through the Genomic Sequencing Centers for Infectious Diseases and by the Biomedical Advanced Research and Development Authority (BARDA)

23. Synfluenza Project Breakdown