exploiting synthetic genomics to create influenza vaccines

WHO 12/08/11, D. Wentworth

Exploiting Synthetic Genomics to Create Influenza Vaccines

David E. WentworthDavid E. Wentworth J. Craig Venter Institute, J. Craig Venter Institute,

Rockville, MarylandRockville, Maryland


OutlineOutline

Influenza Virus Genome Sequencing (NIH/NIAID)

Synthetic Genomics: Preparedness (NIH/NIAID) & Rapid Response (BARDA/Novartis/SGVI)

rg-Influenza virus


NIAID Collaborative Influenza Genome NIAID Collaborative Influenza Genome Sequencing Project GoalsSequencing 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


Influenza Genome Sequencing Project Influenza Genome Sequencing Project CollaboratorsCollaborators


Influenza Virus Sequencing PipelineInfluenza Virus Sequencing Pipeline

http://gsc.jcvi.org/projects/msc/influenza/


Genomic Amplification Directly From Genomic Amplification Directly From Clinical SpecimensClinical Specimens

NP/OP Swabs Controls

1 2 3 4 5 6 - + L

M

PB1, PB2

NS

NPNA

HA

PA

Real time (CT) 23 23 29 30 ND30

G G A T T G A A T G G A T G G G A T G T T C C T T A G T

G G A T T G A A T G G A T G G G A T G T T C C T T A G T48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75

1369

0

FragmentUSSR-PCR-HA-1206

R

Sequence M-RTPCR Amplicons

Genetic/Molecular Analysis• Phylogeny• Virulence Determinants•Used in NIAID/JCVI influenza sequencing pipeline

HA primer

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.


JCVI Influenza Virus Sequencing JCVI Influenza Virus Sequencing PipelinePipeline


Roche: 454 GS FLXIllumina : GAII, HiSeqInvitrogen: Ion torrent

Closure

Data merged


JCVI Influenza Virus Sequencing JCVI Influenza Virus Sequencing PipelinePipeline


Roche: 454 GS FLXIllumina : GAII, HiSeqInvitrogen: Ion torrent

Closure

Data merged

Emergency Production Capacity3730: up to 60 virus genomes/week

454: up to 300 virus genomes/week (60X coverage)

Drug Resistance Detection: up to 1000 isolates/week


Reading and Writing DNA Reading and Writing DNA


Synthetic Genomics ToolsSynthetic Genomics ToolsGibson Assembly


Synfluenza Project DetailsSynfluenza 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

Cloned Cassettes

Sequence &, assemble

Gene Segment Clones

Assemble &, clone


•1 copy of each unique oligo/cassette is made for each unique position•Many non-unique cassettes can be reused

1 2 3 4 5 6 7

Non-unique, duplicate cassettes

Synfluenza Gene Cassette/Molecule Synfluenza Gene Cassette/Molecule DesignDesign

HA Cassettes (~350 bp)

Assembled HA Molecules

H5.1

H5.2

H5.3

Assembly

HA’s and NA’s Constructed Via Automated DNA Synthesis and Assembly

pass Colony picking

Template production

QPix

µFill

Biomek FX

E. coli transformation

Sequencing reaction

Biomek FX

ABI9700ThermalCycler

Sequencing

ABI3730

Select clones

Biomek FX

Culturing or PCR

µFill

Iterative assembly and amplification

Cloning Assembly reaction

µFill

Designed Sequence

HA, NA Genes

Order/Synthesize Oligonucleotides

Biomek FX

µFill

~13 kb per 384-well oligo plate Hamilton

μStar


Synfluenza SummarySynfluenza 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

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.

Speeding vaccine seed generation A BARDA-funded collaboration between Novartis, Synthetic Genomics Vaccines Inc. (SGVI)/J. Craig Venter Institute (JCVI)

Milestone 1 (Sept. 2011): Demonstrate virus rescue within 7 days of receiving HA and NA sequence information

Status – Milestone surpassedWe were able to confirm rescue of an H7N9 virus within5 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

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.

1 2 3 4 5

HA synthetic synthetic synthetic PR8X none

NA synthetic synthetic synthetic N9 none

backbone PR8x #19 #21 PR8x PR8X

Slide Provided by Peter Mason


Is it Possible to Create Live Attenuated Is it Possible to Create Live Attenuated Vaccines From Emerging Viruses?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


In MiceTS2-In MiceTS2-LAIV Is:LAIV Is:

Attenuated Protective


SummarySummary

• 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?

DNA synthesis Transfection

MDCK cell

rg-InfluenzaVirus

RG influenza genome


Thanks to all: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


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)


Synfluenza Project BreakdownSynfluenza Project Breakdown

Host Subtype Segment MoleculesUnique Cassettes

Unique Oligos

Intial 1000Molecules

Intial 1000Unique Cassettes

Intial 1000Unique Oligos

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

exploiting synthetic genomics to create influenza vaccines

Documents

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rescue viruses

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