Transcript Synthetic Genomics - JCVI - J. Craig Venter Institute

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)
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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 -> 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
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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
NP/OP Swabs Controls
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2
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5
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-
+
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
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64 65 66
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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/
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Reading and Writing DNA
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Synthetic Genomics Tools
Gibson Assembly
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Synfluenza Project Details
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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
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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
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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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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
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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
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Synthetic gene segment generation
• Gibson in-vitro assembly
• Assembly uses automated robotic systems
• Enables construction of an extensive library of influenza genes
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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
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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
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
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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
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In MiceTS2LAIV Is:
Attenuated
Protective
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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?
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Thanks to all:
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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
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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
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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
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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
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
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