Progress toward identification of
Fransicella tularensis subunit vaccine
candidates using the transcriptome approach
Stephen A. Johnston
Arizona State University
Transcriptome Analysis of Francisella tularensis
• Is there a relation between expression of genes
in vivo and antigenicity?
• Gene expression profiling of F. tularensis in host
tissues will allow us to compare transcriptional
activity to in vitro grown bacteria
• Compare bacterial transcripts in immune and
non-immune animals
• Identify genes or gene islands regulated by host
factors
Slide 2
LAPT Process
LAPT Process
Slide 3
Project Milestones
• 32
• 33
• 34
Oligos list refined, 70mer oligos procured, GDP oligo defined
Printing and Testing and GDP confirmed
Pilot studies for optimization of RNA isolation & hybridization
conditions done
• 35
Array hybridations with mouse RNAs from virulent Schu 4
infection & RT PCR confirmation of Candidates
• 36
• 37
Final integration of expression data and informatics analysis
Production of protein candidates and testing in mice & candidate
selection done
Milestones 32-34 are completed, Milestone 35 is active
Slide 4
Slide 5
Differentially Expressed Genes
SCHU S4 to LVS
(3 Fold up and down and significant)
Up in SCHU S4
Down in SCHU S4
Slide 6
Raw Signal Intensity
Reconstitution Experiments
1 mg
0.5 mg
0.1 mg
0.05 mg
0.01 mg
0.001 mg
0.0001 mg 0.00001 mg
Slide 7
Spearman Analysis of the Rank Order Between Samples
MS2 MS3 MS5
MS-2 to MS-3
MS-2 to MS-5
MS-3 to MS-5
Coefficent
0.742
0.715
0.624
Slide 8
In Vivo Experiments
• Dose Response Experiments
–
–
–
–
Challenged mice with varying doses of SCHU S4
Harvested lungs at 4 hours
Performed LAPT
Determined that a dose of at least 103 was needed
• Time Course Challenge
– Challenge with 103 SCHU S4
– Harvest lungs at 1, 3, 5, 7, and 24 hours
– Assessed patterns of increasing expression, decreasing expression, or
no change
Slide 9
Targeted genes for qPCR
FTT Designation
Gene symbol
Gene Name
FTT0901
lpnA (Tul4)
conserved hypothetical lipoprotein
FTT0721c
katG
Peroxidase/catalase
FTT1712c
iglC2
intracellular growth locus, subunit C
FTT0548
dnaQ
DNA polymerase III, epsilon subunit
FTT0058
atpB
ATP synthase subunit
FTT0256c
Lipopolysaccharide protein
Slide 10
Reconstitution Samples Assessed by qPCR for FTT0721
Melting Curve
Amplification Plot
Genomic DNA Dark Blue
cDNA Light Blue
Standard Curve
0.01
0.1
Slide 11
Mean Cycle Threshold (Ct) Values for
qPCR Analysis of Reconstitution cDNA Samples
Input RNA
ng/ml
FTT0548 FTT0901 FTT0721
FTT0058
FTT1712c
1000
19.64
18.59
17.86
15.98
11.37
100
24.12
19.96
19.55
19.46
13.09
10
26.34
24.3
23.48
22.97
16.9
1
30.61
28.29
26.7
26.05
20
0.1
ND
30.07
30.01
29.76
23.58
0.01
ND
34.93
32.66
31.68
26.89
0.001
ND
ND
ND
ND
30.09
0
ND
ND
ND
ND
ND
ND = not detected
Slide 12
Next 6 Months
• MS 35
– Complete second time course experiment LAPT analyses
– Second animal model will be tested
– Perform qPCR verification of microarray data
• MS 36
– Compilation of array and qPCR analyses
– Candidate selection
• MS 37
– Create expression constructs of candidates
– Protein production
Slide 13
Acknowledgements
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Stephen Johnston
Phillip Stafford
Cristina McCarville
Lori Phillips
Nikki Hackett
John Lainson
John Lawson
Buddy Day
Elizabeth Lambert
Mara Gardner
Slide 14