Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Exploiting Synthetic Genomics to Create Influenza Vaccines David E. Wentworth J. Craig Venter Institute, Rockville, Maryland WHO 12/08/11, D. Wentworth Outline Influenza Virus Genome Sequencing (NIH/NIAID) rg-Influenza virus Synthetic Genomics: Preparedness (NIH/NIAID) & Rapid Response (BARDA/Novartis/SGVI) WHO 12/08/11, D. Wentworth 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 WHO 12/08/11, D. Wentworth Influenza Genome Sequencing Project Collaborators WHO 12/08/11, D. Wentworth Influenza Virus Sequencing Pipeline http://gsc.jcvi.org/projects/msc/influenza/ WHO 12/08/11, D. Wentworth Genomic Amplification Directly From Clinical Specimens NP/OP Swabs Controls 1 2 3 4 5 6 - + Sequence M-RTPCR Amplicons L G G A T T G A A T G G A T G G G A T G T T T C C C C T T A G T T T A G T 71 72 73 74 75 1369 Fragment USSR-PCR -HA-1206 R HA primer PB1, PB2 PA 0 HA NP NA M G G A T T G A A T G G A T G G G A G T T 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 Genetic/Molecular Analysis • Phylogeny • Virulence Determinants •Used in NIAID/JCVI influenza sequencing pipeline NS Real time (CT) 23 23 29 30 30 ND 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. WHO 12/08/11, D. Wentworth JCVI Influenza Virus Sequencing Pipeline Data merged Roche: 454 GS FLX Illumina : GAII, HiSeq Invitrogen: Ion torrent http://gsc.jcvi.org/projects/msc/influenza/ WHO 12/08/11, D. Wentworth 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 Data merged Roche: 454 GS FLX Illumina : GAII, HiSeq Invitrogen: Ion torrent http://gsc.jcvi.org/projects/msc/influenza/ WHO 12/08/11, D. Wentworth WHO 12/08/11, D. Wentworth Reading and Writing DNA WHO 12/08/11, D. Wentworth Synthetic Genomics Tools Gibson Assembly WHO 12/08/11, D. Wentworth 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) Oligonucleotides Assemble &, clone Cloned Cassettes Sequence &, assemble Gene Segment Clones – Designs based on GenBank sequences with consensus UTRs WHO 12/08/11, D. Wentworth 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 WHO 12/08/11, D. Wentworth HA’s and NA’s Constructed Via Automated DNA Synthesis and Assembly HA, NA Genes Designed Sequence E. coli transformation Order/Synthesize Oligonucleotides Assembly reaction Biomek FX Hamilton μStar Colony pass picking QPix Cloning Template production Sequencing reaction Sequencing Biomek FX Biomek FX ABI3730 µFill ABI9700 Thermal Cycler µFill Iterative assembly andor Culturing PCR amplification µFill µFill Select clones Biomek FX ~13 kb per 384-well oligo plate 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 • • Synthetic gene segment generation • Gibson in-vitro assembly • Assembly uses automated robotic systems • Enables construction of an extensive library of influenza genes • • Available through the Biodefense and Emerging Infections Research Resource Program (BEI) Potential to use cassettes in the future for new viruses Library of clones • Vaccine seeds • Diagnostics • Basic Research WHO 12/08/11, D. Wentworth 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 Virus was rescued from synthetic HA and NA made by rapid assembly RG virus was harvested 4 days after initiation of oligo synthesis Rescue of H7N9 virus 1.0E+08 virus titer (IU/ml) 1.0E+07 1.0E+06 1.0E+05 1.0E+04 48h rescue 1.0E+03 72h rescue 1.0E+02 1.0E+01 1.0E+00 11 22 33 44 55 HA synthetic synthetic synthetic PR8X none NA synthetic synthetic synthetic N9 none backbone PR8x #19 #21 PR8x PR8X 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 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 WHO 12/08/11, D. Wentworth In MiceTS2LAIV Is: Attenuated Protective WHO 12/08/11, D. Wentworth Summary rg-Influenza Virus DNA synthesis Transfection MDCK cell RG influenza genome • High throughput genomic surveillance- circulating subtypes, 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? WHO 12/08/11, D. Wentworth Thanks to all: – – – – – – – – – – J. Craig Venter Institute Craig Venter Karen Nelson Bill Nierman John Glass – Dan Gibson – Mikkel Algire – Jayshree Zaveri – Zhenia Denisova – 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 Indresh Singh Ishwar Chandramouliswaran Tony Yee – – – – – – – – – – – NCBI David Lipman Tatiana Tatusova Yiming Bao Novartis Vaccines and Diagnostics Phil Dormitzer Christian Mandl Rino Rappuoli 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 WHO 12/08/11, D. Wentworth 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) WHO 12/08/11, D. Wentworth Synfluenza Project Breakdown Host AVIAN AVIAN AVIAN AVIAN AVIAN AVIAN AVIAN AVIAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN HUMAN PORCINE PORCINE PORCINE PORCINE PORCINE PORCINE PORCINE PORCINE Subtype H5N1 H5N1 H7N3 H7N3 H7N7 H7N7 H9N2 H9N2 FLUB FLUB H1N1 H1N1 H1N1PDM H1N1PDM H3N2 H3N2 H1N1 H1N1 H1N2 H1N2 H3N1 H3N1 H3N2 H3N2 Segment HA NA HA NA HA NA HA NA HA NA HA NA HA NA HA NA HA NA HA NA HA NA HA NA Intial Intial 1000 Intial 1000 Unique Unique 1000 Unique Unique Molecules Cassettes Oligos Molecules Cassettes Oligos 992 2982 5913 289 1629 4318 874 1848 3729 322 1287 3111 84 232 815 16 108 586 36 101 408 11 53 286 28 128 564 14 95 492 31 103 478 12 60 349 273 1167 3822 148 906 3427 160 568 2446 101 470 2297 363 659 1158 13 85 348 487 602 1030 64 240 567 829 1528 2220 92 441 947 849 1065 1546 63 238 549 3103 2149 2636 171 519 977 2860 1259 1557 121 297 514 1058 1660 2322 142 609 1181 1050 1330 1762 187 576 1043 88 378 1685 42 282 1493 81 255 1082 40 180 929 67 290 1452 36 241 1380 72 226 1071 37 181 1009 3 14 111 2 14 111 2 10 80 2 10 80 69 319 1233 41 260 1139 63 216 907 36 169 796 WHO 12/08/11, D. Wentworth