TVDC Annual Meeting 10-2009:
Progress toward identification of
Francisella tularensis subunit vaccine
candidates using the Proteome and
Transcriptome approaches
Stephen Johnston
Kathryn Sykes
Mitch Magee
Phil Stafford
Tien Olson
Lori Phillips
Rationale for ASU approaches
• 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
Proteome Approach:
In the “Proteomic” project each
protein is assessed as a cellular
response immunogen- a feature
believed critical for a successful
tularemia vaccine.
3
Proteome Screen to identify Immunome
Generate FTU
proteome in
vitro
Generate library
of all genecoding
sequences
Screen immunome for
vaccine candidates
Identify NHP T
cell immunogens
Purify
polypeptides
4
Last Year’s Endpoint
• MS25: Design a complete SCHU S4 library of highly expressed
coding sequences
– Completed design phase
• MS26: Develop protocols for a high-throughput ORF and
polypeptide production system
– Completed new protocol development for ORF production and
expression construct assembly.
– Completed new protocol development for IVT polypeptide
production
– Completed new protocol development for protein purification
• MS28: Build SCHU S4 proteome
– 80% of ORF expression library encoding proteome was
completed.
5
Milestones Achieved in 2009
• MS25: Design a complete SCHU S4 library of highly expressed
coding sequences
– MSCR approved by NIAID
• MS26: Develop protocols for a high-throughput ORF and
polypeptide production system
– MSCR approved by NIAD
• MS28: Build SCHU S4 proteome
– Complete library of 2,229 polypeptides were produced, purified
and delivered to UNM
– MSCR submitted, reviewed and resubmitted
– ELISpot assays from 319 pools of 7 polypeptides were
analyzed
– 3 pools were selected for individual polypeptide assays.
– Analysis of these 21 assays indicates that 9 polypeptides are
immunogenic: a significant number of cells release IFNg.
6
Testing FTU Polypeptide T cell
stimulated responses
7
HTP polypeptide synthesis from linear templates
T7
RBS
ATG
TRX
ORF
His
Term
Tosyl Magnetic beads for capture
IVT proteins
kD
250
150
100
75
50
37
25
20
15
Assembling of IVT LEE cassettes
In vitro translation of proteins
8
Sub-Milestone 28.3
1. 2,229 ORFs were generated and placed into
bacterial expression constructs.
2. 2,229 FTU proteins were transcribed/
translated in vitro using reconstituted bacterial
components.
3. Polypeptides were affinity purified using the
Trx tag .
9
Sub-Milestone 28.3
4. Products were evaluated for integrity and
purity and quantitated.
5. These individual polypeptides were arrayed
into 319 pools of 7.
6. Half of each batch of purified products was
sent to UNM and used for T-cell assay.
7. The remaining half was stored at ASU for
subsequent individual polypeptide analyses.
10
Plate arrangement of FTU IVT proteins
FTU IVT
PoolPlate
(set)
Sources of FTU IVT proteins
Distribution
1
Short ORF1, Short ORF 2, and Long
ORF 5
84 pools of 7 polypeptides
84 pools of 7 polypeptides
2
Long ORF 4 and Long ORF 3
84 pools of 7 polypeptides
3
Long ORF 2 and Long ORF 1
67 pools of 7 polypeptides
4
Poor CAI ORFs
11
ELISPOT of Pool -plate1
100
Lymph node
90
Splenocytes
80
Number of spots
70
50
40
30
20
10
0
ELISPOT of pool -plate2
120
Lymph node
100
Number of spots
UNM
Elispot
results
60
Splenocytes
80
60
40
20
0
12
12
ELISPOT of Pool-plate 3
180
Lymph node
160
Splenocytes
140
Number of spots
120
100
80
60
UNM
Elispot
results
40
20
0
ELISPOT of Pool-plate 4
60
Lymph node
Splenocytes
Number of spots
50
40
30
20
10
0
13
13
Sample of data from ELISpots of pooled polypeptides
against immune NHP lymphocytes
Set , pool
lymph
lymph
spleen
spleen
1,A01
1
2
29
11
1,A02
2
2
0
3
1, A03
1
1
92
9
1, A07
3, B09
3
20
6
0
0
170
28
26
4, E04
5
6
7
7
3, E08
4
2
61
33
3, E10
6
5
35
117
3, E11
9
8
44
205
3, E12
12
11
53
293
14
Individual analysis of polypeptides from 3 selected pools
Against frozen lymph node derived lymphocytes
Media
HK LVS
1:10 1:100
Pool E08
Pool E10
Pool E11
Controls
15
Set 3 Pool Library
source
well
position
Positive
tissue, Rd #1
Gene name
Pool E10
A10
Spleen
FTT1079 conserved hypothetical protein
Pool E10
D10
Spleen
FTT0323 fusA
elongation factor G (EF-G)
peptide #171: SGQTIISGM
Pool E10
G10
Spleen
FTT1130 cphA
cyanophycin synthetase
Pool E11
B11
Spleen
FTT0955 gor
Pyruvate/2-oxoglutarate dehydrogenase
complex,dihydrolipoamide dehydrogenase
Pool E11
D11
Spleen
FTT0323 fusA
elongation factor G (EF-G)
peptide #171: SGQTIISGM
Pool E8
B8
Spleen
FTT1531 fadA
3-ketoacyl-CoA thiolase
Pool E8
C8
Spleen
Pool E8
F8
Spleen
FTT1377 fabH
3-oxoacyl-[acyl-carrier-protein] synthase II
peptide #131: SGIGGIETL
FTT1269 Chaperone protein dnaK (heat shock
protein family 70 protein)
Pool E10
E10
Spleen
FTT0087 acnA
aconitate hydratase
16
Milestone 38
Produce and purify 12 project candidates
selected from across all approaches.
•
This meetings discussions will facilitate a
selection of candidates for testing in
challenge protection assays.
17
6 month plan
• Plan out and execute the completion of the T
cell screen with the UNM team and analyze
immunogenicity results.
• Generate the subunit vaccine candidates, that
are identified by the TVDC team, for evaluation
in protection assays (Milestone 38)
18
Transcriptome Approach:
In the“Transciptome” project changes
in expression patterns of pathogen are
measured during host infection and
sorted. Categories will be tested for
correspondence with host immunity.
19
Project Milestones
• 35
Array hybridations with mouse RNAs from virulent Schu 4
infection & RT PCR confirmation of Candidates
–
–
–
–
–
• 36
Two mouse challenge doses (High Dose / Low Dose); Rat Challenge Model
Two independent LAPT amplifications per challenge dose
Two independent labelings per LAPT
Two hybridizations per labeling
One set of Rat samples left to be finished
Final integration of expression data and informatics analysis
– Identify patterns of expression over time
– Compare datasets derived from other projects and literature
• 37
Production of protein candidates and testing in mice & candidate
selection done
20
Genomically-Normalized Signal Intensity
Strategies
Up
Flat
• Using pattern maps to identify the
top ~200 genes in one of three
patterns
Down
0
1
3
121
5
23
109
11
8
33
115
7
24
– Increasing; Decreasing; Flat
• Venn the individual experiment lists
to get a restricted list of genes
• Compare the restricted sets of
genes between experiments to
identify those across experiments
yielding
21
Increasing Over Time
Mouse HD and Rat (6)
GN and SC Mouse (31)
22
Decreasing Over Time
Mouse HD and Rat (8)
All (1)
GN and SC Mouse (41)
23
Flat Over Time
Mouse HD and Rat (3)
GN and SC Mouse (18)
24
Increasing Over Time
(Hand-Curated)
FTT0594c
FTT0424
FTT1161
FTT0620
FTT1401
GN and SC Mouse
GN and SC Mouse
Mouse HD and Rat
Mouse HD and Rat
Mouse HD and Rat
25
Decreasing Over Time
(Hand-Curated)
FTT1212c
FTT0806
FTT0615c
FTT0665c
FTT0473
GN and SC Mouse
Mouse HD and Rat
Mouse HD and Rat
All
GN and SC Mouse
26
Flat Over Time
(Hand-Curated)
FTT0437c
FTT1655
FTT0014c
FTT1522c
FTT0299
GN and SC Mouse
GN and SC Mouse
Mouse HD and Rat
Mouse HD and Rat
Mouse HD and Rat
27
Flat
Decreasing
Increasing
KEGG Information
FTT0594c
hypothetical protein
FTT0424
hypothetical protein
FTT1161
adk
FTT0620
HAD superfamily protein
FTT1401
prophage repressor protein
FTT1212c
gloA
lactoylglutathione lyase (EC:4.4.1.5)
FTT0806
capC*
capsule biosynthesis protein CapC
FTT0615c
FTT0665c
FTT0473
metal ion transporter protein*
aldolase/adducin class II family protein
acetyl-CoA carboxylase, biotin carboxylase subunit (EC:6.4.1.2)
accC
FTT0437c
pyrE
FTT1655
hypothetical protein
FTT0014c
hypothetical protein
FTT1522c
hypothetical protein
FTT0299
valS
adenylate kinase (EC:2.7.4.3)
orotate phosphoribosyltransferase (EC:2.4.2.10)
valyl-tRNA synthetase (EC:6.1.1.9)
* Weiss, D. S., A. Brotcke, T. Henry, J. J. Margolis, K. Chan, and D. M. Monack. 2007. In vivo negative selection screen identifies genes
required for Francisella virulence. Proc Natl Acad Sci U S A 104:6037-6042.
28
Comparison of Microarray Profiles and qPCR
Microarray
qRT-PCR
Increasing
RAT
Mouse HD-1
Mouse HD-2
29
Problems Encountered/Overcome
• LAPT Amplification failures
– Template switch primer
• Microarray signal reduction
– Slide production problems from a bad batch
of aminosilane
30
Next 6 Months
• MS 35
– Complete last Rat LAPT analyses
– Perform qPCR verification of microarray data
• MS 36
– Final Compilation of array data
– Candidate selection
• MS 37
– Create expression constructs of candidates
– Protein production
31
10/5/09: Action Items
• Mitch will titrate the amount of rat lung
RNA in the LAPT, with the goal to titrate
out too much RNA or an inhibitor to the
LAPT processing
• Mitch will try a mixing experiment of the
positive control and the putative inhibitory
rat lung RNAs, in the LAPT process.
• Terry/Mitch- explore possibility that the
bacterial RNA is degraded or that the lysis
of the bacteria is lower than expected.
32