Exploiting Synthetic Genomics to Create Influenza Vaccines

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Exploiting Synthetic Genomics to Create Influenza
Vaccines
David E. Wentworth J. Craig Venter Institute,
Rockville, Maryland
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Outline
Influenza Virus Genome Sequencing (NIH/NIAID)
rg-Influenza virus
Synthetic Genomics Preparedness (NIH/NIAID)
Rapid Response (BARDA/Novartis/SGVI)
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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 -gt NCBI, IRD
• Mitigate the impact influenza epidemics/pandemics

http//www.niaid.nih.gov/LabsAndResources/resource
s/dmid/gsc/Influenza/Pages/overview.aspx
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Influenza Genome Sequencing Project Collaborators
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Influenza Virus Sequencing Pipeline
http//gsc.jcvi.org/projects/msc/influenza/
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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. 8310309-10313.
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JCVI Influenza Virus Sequencing Pipeline
Data merged
Roche 454 GS FLX Illumina GAII,
HiSeq Invitrogen Ion torrent
http//gsc.jcvi.org/projects/msc/influenza/
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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/
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(No Transcript)
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Reading and Writing DNA
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Synthetic Genomics Tools
Gibson Assembly
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Synfluenza Project Details

• NIAID project to create 1000 HAs and NAs
• 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
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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)
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5
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Assembly
H5.1
H5.2
H5.3
Assembled HA Molecules
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HAs and NAs Constructed Via Automated DNA
Synthesis and Assembly
HA, NA Genes
Designed Sequence
Colony picking
Template production
E. coli transformation
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
µFill
µFill
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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

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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
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Virus was rescued from synthetic HA and NA made
by rapid assembly RG virus was harvested 4 days
after initiation of oligo synthesis
  1 2 3 4 5
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
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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
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In MiceTS2-LAIV Is
Attenuated
Protective
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Summary

• 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 - 62 vaccine seeds
  (TIV, LAIV)
• Pre-existing stocks ?
• Engineered complete genomes as LAIVs?

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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

• 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

• 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

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• 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)

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Synfluenza Project Breakdown
Host Subtype Segment Molecules Unique Cassettes Unique Oligos Intial 1000Molecules Intial 1000Unique Cassettes Intial 1000Unique Oligos
AVIAN H5N1 HA 992 2982 5913 289 1629 4318
AVIAN H5N1 NA 874 1848 3729 322 1287 3111
AVIAN H7N3 HA 84 232 815 16 108 586
AVIAN H7N3 NA 36 101 408 11 53 286
AVIAN H7N7 HA 28 128 564 14 95 492
AVIAN H7N7 NA 31 103 478 12 60 349
AVIAN H9N2 HA 273 1167 3822 148 906 3427
AVIAN H9N2 NA 160 568 2446 101 470 2297
HUMAN FLUB HA 363 659 1158 13 85 348
HUMAN FLUB NA 487 602 1030 64 240 567
HUMAN H1N1 HA 829 1528 2220 92 441 947
HUMAN H1N1 NA 849 1065 1546 63 238 549
HUMAN H1N1PDM HA 3103 2149 2636 171 519 977
HUMAN H1N1PDM NA 2860 1259 1557 121 297 514
HUMAN H3N2 HA 1058 1660 2322 142 609 1181
HUMAN H3N2 NA 1050 1330 1762 187 576 1043
PORCINE H1N1 HA 88 378 1685 42 282 1493
PORCINE H1N1 NA 81 255 1082 40 180 929
PORCINE H1N2 HA 67 290 1452 36 241 1380
PORCINE H1N2 NA 72 226 1071 37 181 1009
PORCINE H3N1 HA 3 14 111 2 14 111
PORCINE H3N1 NA 2 10 80 2 10 80
PORCINE H3N2 HA 69 319 1233 41 260 1139
PORCINE H3N2 NA 63 216 907 36 169 796