Presented by:
Stephen Johnston and Phillip Stafford, and Kathryn Sykes
Slide 1

• As a complement to the development of improved live vaccine strains for tularemia, the
TVDC pursues 3 projects directed at identifying
F. tularensis antigens to serve as components of a protective subunit vaccine.
• ASU conducts one based on surveying all T cell responses elicited by infection and another based on measuring and categorizing changes in pathogen gene expression during host infection.
2

• The Proteomic project goal is to assess each protein as a cellular response immunogen- a feature believed critical for a successful tularemia vaccine.
• Completed Milestones have been to:
– Design a complete SCHU S4 library of highly expressed coding sequences
– Develop protocols for a high-throughput ORF and polypeptide production system
– Produce and purify 2,229 FTT polypeptides, comprising the complete proteome.
– Conduct (UNM) and Analyze ELISpot assays from 319 pools of 7 polypeptides each.
• 3 pools scored as positive were selected for individual polypeptide assays.
• Results indicate that 9 of the 21 tested polypeptides are immunogenic
Slide 3

• The Transcriptome project goal is to detect transcripts that follow specific patterns of expression during infection. These provide a list of potential proteins that are displayed to the immune system during infection.
Slide 4

• Stephen Johnston
• Kathryn Sykes
• Phillip Stafford
• Lori Phillips
• Andrey Loskutov
• Felicia Craciunescu
Slide 5

• 2 nd proteome synthesis and plans for 2 nd
ELISpot screen of NHP and human immune cells for tularemia immunogens
Slide 6

Positively scored polypeptides in individual ELISpot assays
FTT1079 conserved hypothetical protein
• Pooled antigens tested against NHP spleen cells
• Individual antigens tested against frozen lymph node cells
FTT0323 fusA elongation factor G (EF-G) peptide #171: SGQTIISGM
FTT1130 cphA cyanophycin synthetase
FTT0955 gor
Pyruvate/2-oxoglutarate dehydrogenase complex,dihydrolipoamide dehydrogenase
FTT0323 fusA elongation factor G (EF-G) peptide #171: SGQTIISGM
FTT1531 fadA
3-ketoacyl-CoA thiolase
FTT1377 fabH
3-oxoacyl-[acyl-carrier-protein] synthase II peptide #131: SGIGGIETL
FTT1269 Chaperone protein dnaK (heat shock protein family 70 protein)
FTT0087 acnA aconitate hydratase
Slide 7

• Objective:
– Remake the FTU SCHUS4 proteome as individual ~400 amino acid polypeptides (mostly full length proteins) in order to repeat the T cell assay (ELISpot) screen in NHPs.
– Fresh cells from spleen and lymph tissues will be used.
• Status:
– Stored templates have been QC’ed and quantitated.
• High quality ones have been re-amplified
• Degraded ones are being re-assembled
– PCR primers have been QC’ed
– New quotes for lysate, antibody, and E-gel reagents have been obtained from NEB and Invitrogen
Slide 8

Quality control of 2008 LEE template
Slide 9

Re-evaluation of the concentration of 2008 LEE templates
G
H
E
F
C
D
A
B
6
7
4
5
8
Plate 384-well plate Average-2010-concentration(ng/ul) Average-2008-concentration(ng/ul)
1 BAG plate 1 15.91
22.50
2 Short1_ Plate 1
3 Short 2 _Plate 1
14.90
14.79
14.90
17.10
Long 1_Plate3
Long 2_Plate 3
Long 3_Plate 3
Long 4_Plate 1
Long 5_Plate 2
Average
22.55
14.79
22.55
15.92
11.05
16.56
26.80
33.00
13.97
10.46
18.40
19.64
SHORT 1 2008 Target template amount - 350 ng ng/ul Vol. ul ng/ul Vol. ul ng/ul Vol. ul ng/ul Vol. ul
1 2 3 4
23.21
19.22
22.51
20.19
15.08
18.21
15.55
17.33
27.25
16.58
3.48
16.82
12.84
21.10
100.58
20.81
22.52
19.09
23.36
21.61
15.54
18.33
14.99
16.20
23.21
19.22
22.51
20.19
15.08
18.21
15.55
17.33
20.69
19.02
18.66
16.92
18.40
18.76
18.69
17.33
26.65
18.73
20.20
13.13
23.82
20.64
23.86
14.69
16.95
14.67
20.69
19.02
18.66
16.92
18.40
18.76
Slide 10

Summary of LEE volume estimation for re-building the library
BAG
SHORT
1
SHORT
2
IVT
1 Yes
2 Yes
3 Yes
IVT
1 Yes
2 Yes
3 Yes
4 Yes
IVT
1 Yes
2 Yes
3 Yes
QC
Yes
Yes
Yes
LONG 1 IVT QC
1 No No
2 Need to check DNA contration_volume is greater than 40uL
3 Yes
4 Yes
Yes
Yes
LONG 2 IVT
1 Yes
2 4 out 7rows (need A, B, and F)
3 Yes
4 Yes
LONG 3 IVT
1 3 out of 7 ( need A, B, and C only)
2 5 out of 7 ( need A and B)
3 None
4 3 out of 7
LONG 4 IVT
1 None
2 4 out of 7
3 none
4 None
LONG 5 IVT
1 None
2 Yes finished re-amplification and purification of LEE finished re-amplification and purification of LEE
QC
Yes
Yes
Yes
Yes
QC
Yes
Yes
Yes
Excepti on
A1
None
Excepti on row A for QC row E for QC and F8 for IVT, need 20ul but only 15ul left row D for QC
Excepti on
A1 only has 15uL
None
None
Excepti on
QC
None
None
None
None
QC
None
Yes
QC
Yes
3 out of 7 rows
Yes
Yes
QC
None
None
None
None
Excepti on row A for both IVT and QC
D9 and D12, E5 and E9 are empty
None
None
Excepti on
D1,E1,F1, G1
F1
B1,E12,F8, and F9
Excepti on row A got enough for both IVT and QC
Exception row 3
########
########
84
36
36
24
84
12
Slide 11

• New IVTT capture beads are being tested
– IglC was selected as a test antigen to evaluate the merits of using smaller diameter
(1 vs. 2.8 micron) magnetic capture beads.
– IglC has been made synthetically as a full length protein and purified on both small and large beads. These preps are ready for
ELISpot testing.
Slide 12

• New efficiencies have been identified
– Due to using the smaller diameter beads, we have retested ratios of anti-Trx antibody, lysate, and beads.
beads concentration optimal Ab binding/mg of beads
M-280 Dynabeads standard control
30ug/ul
20ug/mg sample 1
100ug/ul
40ug/mg
My ONE beads sample 2
100ug/ul
40ug/mg sample 3
100ug/ul
40ug/mg
Beads/ AB set up beads volume
Ab volume (1ug/ul) binding buffer ug Ab /mg beads standard control
25ul
15ul
15ul
20ug/mg sample 1
8ul
32ul
32ul
40ug/mg sample 2
8ul
15ul
15ul
18.75ug/mg sample 1
Price of beads Price of Ab MyOne
MyOne vs M280 (std) vs M280 (std) same higher sample 2 sample 3 same less same same sample 3
3.75ul
15ul
15ul
40ug/mg
Slide 13

kDa
115
82
64
49
37
26
Smaller diameter beads change optima .
• A given amount of lysate can be captured with less beads (by weight), presumably due to greater surface area.
• No additional Trx antibody is required.
Std 1 2 3 C Std 1 2 3 C
Coomassie Phosphor image
Slide 14

IVTT of IglC 1 on both small and large beads
2 1 0.5 0.2
BSA, ug
2 1 0.5 0.2
BSA, ug
Slide 15

• Objective:
Recombinantly produce and purify a set of FTU proteins in ~mg quantities for testing in NHP challenge-protection assays at UNM.
Status:
• IglC is first to be put on the list.
• Construct is completed.
• IVT worked
• Any difficulties with in vivo expression will be addressed with molecular manipulations
Slide 16

Long2
Plate 1
LEE 2008(384 wells plate)
E+
G+
I+
K+
P1
AX
C+
M+
O
1 3
35
5
25
7 9 11 13
15
15 17 19
15
IVT 2010
21 23 P1
A
B
E
F
C
D
G
H
1 2
35
3
25
4
8.8
8.9
8.1
8.7
8.0
5
7.0
9.4
6 7 8
15
15
9 10 11 12
Plate 2 old
LEE 2008(384 wells plate)
2 4 6 8 10 x
12 14 16 x
25 x
25
18 20 22 x
P2
24
AX
P2
A
CX
E+
B
C
G+
D
I-I
KX
E
F
M+
O
G
H
Plate 2 new A
B
C
LEE 2010 (96 well plate- mix plate 2/23/2010 ) mix 1 2 3 4 to row A on IVT plate 2 to row B on IVT plate 2 to row F on IVT plate 2
5 6 7 8 9 10 11 12
1 2 3 4 5
16.4
12.8
7.0
6.4
5.2
10.1
6.1
6 7 8 9 10 11 12 x
25 x
25 x
Plate 3
LEE 2008(384 wells plate)
L+
N+
P
P3
B+
D+
F+
H+
J+
1
15
3 5 7 9 11 13
15
15 17 19 21 23 P3
A
B
C
D
E
F
G
H
Plate 4
LEE 2008(384 wells plate)
2 4 6 8 10 12 14 16
25
18 20 22
P4
24
B+
P4
A
D+
B
F+
C
H+
D
J+
E
L+
F
N+
G
P
H
1 2 3
15
4 5
9.1
10.8
10.6
10.8
9.0
12.5
12.5
6 7 8 9 10 11 12
15
1 2 3 4 5
18.5
23.1
19.7
15.3
12.6
12.9
12.1
6 7 8 9 10 11 12
25
Slide 17

• Morning break
Slide 18

Potential vaccine candidates
Slide 19

Multidataset Candidate Comparison
Summary of datatypes
Expression: array and RT-PCR
Western Blot
Felgner
Slide 20

Multidataset Candidate Comparison
Multi-type data are combined through trinary transformation
Weights are used in computing positive or negative votes
0 is “no-call” and is assigned a 10% weight, and is dilutive
1 is a “positive call” and is assigned 33% weight and is non-dilutive
1 is a “negative call” and is assigned a 25% weight and is non-dilutive
Each component is given a vote in the final tally based on weight
Each biological question is asked by the electoral college, with cumulative weights and calls being averaged for the final vote.
Slide 21

• Proteome:
– We have newly opened milestones, so no problems have been encountered.
– Anticipated challenges are center on data analysis strategies and possible use of additional immune analysis prior to selecting antigens for vaccine testing.
Slide 22

• Transcriptome:
Amplification (LAPT) – caused by reagents
Slide efficiency – caused by spotter
Sensitivity – rectified by modifying number of infecting organisms
Model differences – mouse and rat show different responses. Rectified by keeping models separate.
Slide 23

• Proteome:
– Milestone 25,26,28 MSCR: Finalized with
NIAID
– Milestone 28: re-opened due to re-synthesis
– Milestone 37/38: merged and selection of antigens for recombinant production is under discussion.
Slide 24

• Transcriptome:
– MS 32 MSCR: Finalized with NIAID
– MS 33 MSCR: Under NIAID review (3/2/10)
– MS 34,35,36 MSCR: Pending at ASU
Completed gene selection process for rat and mouse
GEO (Gene Expression Omnibus) has accession number for our arrays. We are assembling the SOFT-format upload
Extra slides are available for researchers, and more can be printed (good supply of probes). Note that ASU slides outperformed TIGR slides
Slide 25

• We have a working draft of a manuscript describing the high throughput IVTT production and purification protocol used for the FTT proteome.
• We are assembling a transcriptome-based timecourse study
• We are proposing a statistics-based multidatatype analysis using available data
(Hypothesis based)
Slide 26

•
• Rick: Andrew Hahn’s data is interesting regarding vaccinated human cellular responses to control Ft bacterial growth in vitro (vaccinated human cells respond to control the tularemia bacteria growth in culture). Nonvaccinated people can control in an interferon independent way. Vaccinated people are extraordinarily able to control in an interferon dependent way. It is interesting that humans sometimes have mild disease and sometimes have severe disease.
• Kathy: those that control on their own FT growth, don’t need the FT vaccine as much as those humans that need vaccination and interferon stimulation to survive.Kathy: is interferon the gate keeper?Rick: UNM is getting more data that interferon is a highly likely correlate that we should screen for. We are doing the best screen. Rick:
Terry has data that CD8 T cells are incredibly important for controlling the infection. CD 8 cells make interferon too. Can measure a different cytokine too. Perhaps measure interferon release (CD4 response) and then also measure CD8 cells also include other cells that otherwise might be missing in peripheral blood. Question, is how much antigen can be spared? Kathy: Have 8 pools of 7 (56 peptides/antigens), so now also do cytotoxicity assays as well as Elispot assays? Rick: yes Kathy: do interferon gamma positives (ELIspot) first and then do cytoxicity.
Rick: reproducibility between wells was not good in past and hopefully this new smaller bead strategy will increase the reproducibility because the smaller beads stay in suspension and don’t fall out, like the larger beads did in the past. Rick: UNM is working with Cerus and IglC Lm is working to protect in rat. Kat G didn’t work well to protect in rat. Is why Rick wanted to make IglC protein first.Kathy: with Lm IglC, what does the Elispot look like?Rick: UNM is looking at it. UNM is excited about the 75% protection with vaccination with one IglC protein vaccination.
Kathy: Maybe due a DNA prime and protein boost next, to increase protection?Rick: Wants to discuss with Cerus, as Cerus pushes Lm as a platform. There are storage issues and for cancer clinical trials the route is IV. Cerus is testing different vaccination routes. Cerus hopes to deliver vaccine candidates into humans in live Lm platform in the future.
(CONTINUED IN SLIDE NOTES)
Slide 27

• Malu: no new topics.
• Rick: Felgner arrays (~260 UNM TVDC samples were run on the arrays) are part of the multi data set, with samples from human, NHP, mouse and rat species.
• Rick: will use IglC synthesized at ASU to optimize the Elispot assay with vaccinated human and
NHP cells. Then will repeat the NHP vaccination cells with Elispot, with NHP and human cells in the Elispot
• Kathy: ASU may have enough IglC antigen for both NHP and human, after the initial screen.
• Rick: Cross validation with multiple species may enhance the confidence level for the selected proteins/gene
• (CONTINUED IN SLIDE NOTES)
Slide 28

TVDC Annual Meeting 2010
Slide 29

• ASU will continue using silver staining rather than radioactivity, for synthesis controls
• ASU will continue using the smaller beads for synthesis
•
ASU will make IglC protein w/o tags in future to use as a vaccine candidate
• Priorities for the next peptide screening in the ELIspot are fresh spleen and lymph node cells and with optimal concentrations of peptide, then if enough peptide remains could also use the frozen cells.
• Mitch: will submit MS 34 MSCR to Barbara on 3/31/10 Wednesday Mitch is working on MS 35 and 36 MSCRs to give to UNM.
• ASU will put the Ft probe sequences in GEO, so others can synthesize their own probes and so ASU can publish methods.
• The Transcriptome time course manuscript is being written and will shoot for publication by mid year 2010. Multiple data set papers are rare and statistics are hard to get. ASU should try to publish this data set and it will be valuable to the literature.
• Phil will set up mutiple data set analysis over course of a week and then can add more data and questions, as they develop.
Slide 30