Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 5, 12 (UNM & LBERI), 25, 26, 32, 33, 39, 40,
41, 43, 46, 49, 50
Completed milestones: 1, 16
Inactive milestones: 4, 6-11, 13-24, 27-31, 34-38, 42, 44-45, 47-48, 51-54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. NIAID is working on the IAA with USAMRIID and a legal and financial liability review is
pending.
b. UNM received a copy of the current CAP certificate for TriCore Reference Laboratories.
The CAP certificate expires after April 23, 2008.
4. Significant decisions made or pending
a. UNM and NIAID continue to wait for a change in the status of the IAA between NIAID
and USAMRIID. Kristin DeBord conveyed that the IAA may take a year to achieve.
b. UNM and LBERI will use their biobubbles as additional physical protective equipment
c. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
d. UNM EOHS has obtained many of the laboratory documents
i. Documents pending
1. Radiology Facility Accreditation Certificate
5. Problems or concerns and strategies to address
UNM will need an external source of human cells from LVS vaccinated individuals, in order to
develop the immunoassays in humans.
6. Deliverables completed
None
7. None Quality of performance
Good
8. Percentage completed
16%
Page 1 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
9. Work plan for upcoming month
Ross Kelley will continue to monitor the progress of whether Martin Crumrine's IAA between
NIAID and USAMRIID will inform UNM when and whether the TVD Contractors can be
vaccinated under this IAA.
10. Anticipated travel
Travel could occur in September 2006 to September 2007, depending on the completion of the
IAA.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM may request a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for training on
LVS site vaccination evaluations. The timing of the COA request depends on the achievement of
the IAA.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions

Performed additional bioaerosol sprays
 Sprayed approx. 5x107 CFU/mL for 10 min
 Data shown on page 2. Spray factors were comparable between AGI and
Biosampler. The AGI and Biosampler are being compared to determine if the
Biosampler collection efficiencies are better than the standard method (AGI).
\\Saturn\ABSL3\ABSL3 Study Data\Tularemia\Tularemia Milestone 3 (Bioaerosol
Development)
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
16%
9. Work plan for upcoming month

Continue preliminary bioaerosol experiments with reconstituted LVS and Collison generator
 Plan to quantitate LVS on CHAB
 Will test bioaerosol w/ and w/o antifoam. Antifoam is often added to generation
suspensions because of complications caused by foaming in generating high
numbers of aerosolized particles. These experiments will determine if antifoam is
necessary.
Page 2 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble


Will test different LVS concentrations to determine spray factors. The spray factor is
a value used by aerobiologists. It is a ratio of viable bacteria obtained in the sampler
as compared to the number of bacteria per liter of air in the aerosol. The spray factor
is used as an indicator of the quality of the spray with spray factors of 10 -6 considered
better than 10-7 or 10-8.
Perform bioaerosol experiments on vegetative LVS with Collison generator. These
experiments will help determine if fresh vegetative cells react in the same manner to
aerosolization as do reconstituted cells.
 Repeat of studies performed on reconstituted LVS
 Plan to grow LVS in CB
 Plan to quantitate LVS on CHAB
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Page 3 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr, Mitch Magee, Kathryn Sykes,
Stephen Johnston, Karl Klose, Justin Skoble
Project No.
UNM
Spray Day
Friday
Spray Date
Microbe
14-Jul-06
F.
tularensis
LVS
Date plates counted
17-Jul-06
Counted by:
RW
Microbial growth medium:
CHAB
Inoculation Date:
14-Jul-06
Harvested Date:
14-Jul-06
Initial
Vol (ml)
Suspension
Final
Vol
(ml)
4
0
Volume
Spray
(ml)
Target
CFU/mL
Avg
Dilution
Actual
CFU/mL
Approx.
CFU
Sprayed
Sample
Time
(min)
Flow
rate
(L/min)
CFU
CFU/L
SPRAY
FACTOR
1.00E+07
Spray #
1
10
8.4
1.6
0.00E+00
52
1.00E+06
5.17E+07
8.27E+07
10
16.0
8.27E+07
5.17E+05
Spray #
2
10
8.3
1.7
2.00E+05
52
1.00E+06
5.17E+07
8.78E+07
10
16.0
8.78E+07
5.49E+05
Spray #
3
10
8.3
1.7
2.00E+05
46
1.00E+06
4.57E+07
7.76E+07
10
16.0
7.76E+07
4.85E+05
Spray #
4
10
8.5
1.5
2.00E+05
33
1.00E+06
3.30E+07
4.95E+07
10
16.0
4.95E+07
3.09E+05
Spray #
5
10
5.3
4.7
2.00E+05
30
1.00E+06
3.00E+07
1.41E+08
10
16.0
1.41E+08
8.81E+05
Spray #
6
10
9.6
0.4
2.00E+05
56
1.00E+06
5.60E+07
2.24E+07
10
16.0
2.24E+07
1.40E+05
AGI #
1
20
18.8
15
1.00E+04
1.50E+05
10
5.0
2.82E+06
1.88E+05
3.64E-06
AGI #
2
20
18.7
25
1.00E+04
2.53E+05
10
5.0
4.74E+06
9.47E+04
1.83E-06
AGI #
3
20
18.7
13
1.00E+03
1.30E+04
10
5.0
2.43E+05
4.86E+03
1.06E-07
Biosampler #
4
10
8.2
53
1.00E+03
5.33E+04
10
5.0
4.37E+05
8.75E+03
2.65E-07
Biosampler #
5
10
8.6
59
1.00E+03
5.93E+04
10
5.0
5.10E+05
1.02E+04
3.40E-07
Biosampler #
6
10
8.7
38
1.00E+03
3.80E+04
10
5.0
3.31E+05
6.61E+03
1.18E-07
Page 4 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Working Group
Milestone description: Determine appropriate solid and liquid media for growth of tularemia for
project team
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
(Data found in folder on Saturn server entitled ABSL3/Study Data/Tularemia)
a. Repeated 48 hr growth curve
Chamberlain's
Chamberlain's
Chamberlain's
1:1
1:2
1:4
1:8
1:16
1:1
1:2
1:4
1:8
1:16
1:1
1:2
1:4
1:8
1:16
Flask
1
2
3
Timepoint
(Hrs)
48
48
48
Ct#1
105
63
30
Ct#2
304
40
25
Ct#3
300
33
81
Avg
2.36E+02
4.53E+01
4.53E+01
Flask
Timepoint
(Hrs)
CFU/mL
Log10
OD(600)
1
1
1
1
1
2
2
2
1
1
3
3
3
1
1
48
48
48
48
48
48
48
48
48
48
48
48
48
48
48
2.36E+07
1.18E+07
5.91E+06
2.95E+06
1.48E+06
4.53E+07
2.27E+07
1.13E+07
5.67E+06
2.83E+06
4.53E+07
2.27E+06
1.13E+06
5.67E+05
2.83E+05
7.37
7.07
6.77
6.47
6.17
7.66
7.36
7.05
6.75
6.45
7.66
6.36
6.05
5.75
5.45
0.305
0.155
0.058
0.025
-0.002
0.423
0.181
0.084
0.03
0.025
0.408
0.185
0.076
0.032
0.007
Dilution
1.00E+05
1.00E+06
1.00E+06
CFU/mL
2.36E+07
4.53E+07
4.53E+07
Avg
CFU/mL
3.81E+07
Avg
CFU/mL
Avg
Log10
Avg
OD(600)
3.81E+07
1.23E+07
6.13E+06
3.06E+06
1.53E+06
7.56
6.93
6.63
6.33
6.02
0.379
0.174
0.073
0.029
0.010
Page 5 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
48 Hr Curve
8.00
Log10 CFU/mL
7.50
7.00
6.50
6.00
5.50
5.00
0.379
0.174
0.073
0.029
0.010
OD(600)
b. \\Saturn\ABSL3\ABSL3 Study Data\Tularemia\Tularemia Milestone 3 (Bioaerosol
Development)
4. Significant decisions made or pending
A meeting of the working group examined blue/grey colony morphology data produced by DSTL
and determined that Chamberlain’s broth and CHAB agar outperformed the other media tested
and recommended that these media be used in growth of LVS stocks.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
80% (though LBERI is 100% done; UNM’s virulence studies remain to be performed)
9. Work plan for upcoming month
UNM will perform comparative virulence studies in mice, with lyophilized LVS as the baseline and
with LVS grown in Chamberlain’s broth. UNM will complete the virulence testing by 8/30/2006.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractor
None anticipated
Page 6 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Milestone 5
Milestone description: Species tested for sensitivity to LVS & generation of immunity against a
pulmonary challenge of Schu4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc12, study 6 (notebook 85, pages 16-17) and study 7 (notebook 85,
pages 18-19)
i. The purpose was to determine whether 0.1 mg/ml DNase I will reduce the
viscosity of rat lung and spleen homogenates without affecting the viability of
LVS and SCHU S4
ii. Lungs and spleens homogenized in PBS with 0.1 mg/ml DNase I with
Beadbeater remained fluid and easy to pipette 80 minutes after
homogenization.
iii. LVS and SCHU S4 remained viable after incubation in PBS with 0.1 mg/ml
DNase I at for 1 hour at room temperature (Figure 1)
iv. Conclusion: 0.1 mg/ml DNase I eliminated the viscosity problem with the
tissues homogenates without affecting the viability of LVS or SCHU S4
b. Feasibility study on PennCentury Microsprayer Aerosolizer (no notebook entry)
i. The purpose was to determine whether the microspray aerosolizer is a
feasible method for pulmonary infection of Fischer 344 rats
ii. UNM has no experience using the microsprayer aerosolizer on rats but has
used a microsprayer for mouse infections.
iii. In training sessions using the techniques and equipment developed for mice,
it was difficult to visualize the trachea in Fischer 344 rats and insert the
aerosolizer tip of the microsprayer.
iv. To improve the visualization of the rat trachea, UNM purchased a small
animal laryngoscope with an attached fiber optic light and blade for
depressing the tongue. This enabled us to visualize the trachea.
v. Additional training is planned to gain expertise locating the trachea, inserting
the aerosolizer tip, and delivering the inoculum
Page 7 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
4. Significant decisions made or pending
a.
We will generate a LVS working stock from DVC’s lot 16 using the exact conditions
established by the TVDC working group. This stock will then be used for all future
TVDC experiments
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
12%
9. Work plan for upcoming month
a. Microspray aerosolizer in rat model
i. Continue training on locating the trachea, inserting the aerosolizer tip, and
delivering the inoculum
ii. When the microspray aerosolizer has been fully evaluated and deemed
suitable for infecting rats, we will use it to determine the pulmonary LD50 of
LVS and SCHU S5
b. Vaccinate Fischer 344 rats with LVS s.c. and i.d. so they will be ready when the
appropriate method of pulmonary infection (microspray aerosolizer or i.t.) has been
determined
c. Determine the intranasal LD50 of SCHU S4 in vaccinated BALB/c and NIH-Swiss
mice
i. We vaccinated BALB/c and NIH-Swiss mice with LVS approximately two
months ago by 3 routes of infection, s.c., i.n. and i.d.
ii. We will confirm LVS clearance from these mice and infect them with various
doses of SCHU S4 to determine the i.n. LD50 of SCHU S4 in LVS-vaccinated
mice.
d. Prepare working LVS stocks from DVC’s LVS lot 16 and determine blue/gray ratio
10. Anticipated travel
Terry Wu, Rick Lyons and Barbara Griffith plan to attend the Second DVC/UNM/NIAID TVDC
meeting (10/30-10/31/06) and the Fifth International Tularemia Conference (11/1-11/4/06)
both in Woods Hole, MA.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM has received COA #11 to allow 7 TVDC scientists to attend the Second
DVC/UNM/NIAID TVDC meeting (10/30-10/31/06) and the Fifth International Tularemia
Conference (11/1-11/4/06) both in Woods Hole, MA.
Milestone 12 -UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Page 8 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
a. Correlate of protection: Control of F. tularensis growth in murine macrophages
i. We would like to use LVS-luciferase from Dr. Karl Klose (UTSA) as a marker
for killing of intracellular LVS. To that end, we are determining whether the
amount of luciferase activity correlates with the number of viable intracellular
LVS
ii. Our preliminary studies indicate (Notebook 85, pages 25-30)
1. LVS-luciferase is functional, but the activity is low
2. The luciferase plasmid is unstable and requires continuous 10 l/ml
Kanamycin selection
3. Intact LVS can take up and process exogenously-added luciferin
substrate but the luciferase activity is lower than that of lysed LVS,
suggesting that there is a barrier against substrate uptake.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
1% for UNM; we reported 20% for LBERI on 7/15/06
9. Work plan for upcoming month
a. Develop correlate of protection: Control of F. tularensis growth in murine
macrophages
i. Optimize the assay system by increase the amount of luciferin substrate
ii. Determine the minimum detectable number of LVS-luciferase
iii. Determine whether luciferase activity is an indicator of bacterial viability?
iv. Determine whether intracellular LVS-luciferase detectable?
b. Develop correlate of protection: Antigen-specific T cell proliferation and cytokine
production
i. Determine the best antigen (heat-killed, formaldehyde-fixed, or UVinactivated F. tularensis) for stimulating proliferation and cytokine production
by antigen-specific T cells from vaccinated mice
ii. UNM will include the antigen killing procedure provided by Vicki Pierson of
NIAID, in the comparison for the best antigen.
Page 9 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
10. Anticipated travel
Alexandra Scrymgeour and Tara Hendry-Hofer, who are UNM Research Associates, plan to
attend the Cell-Mediated Immunity Assay Development Course in Rockville, MD on 9/20 and
9/21/2006. COA # 10 has been provided for this travel.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM has received COA #10 to allow 2 UNM Research Associates to attend the CellMediated Immunity Assay Development Course in Rockville, MD on 9/20 and 9/21/2006. This
course is sponsored by the NIAID/DMID/ Office of Regulatory Affairs.
Milestone 12-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. Purpose: Comparison of 3 different protocols to purify PBMCs from cynomologous
macaques
i. Goal is to determine which protocol gives the highest yield of cells which are not highly
contaminated by RBCs, and that respond vigorously and specifically to immune
stimulation (mitogenic initially and later, antigenic)
ii. Raw data: TVDC Binder 1 (Wilder Lab), TUL-1 (Purdue Cytometry test, 7/12/06) and
TUL-2 (Williams’ Lab and NHPRR tests, 7/24/06)
iii. Summary data: C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\cyno blood table.doc, Tul 1 and 2 summary tables 073106.doc and Tech Call
080106.ppt
b. Methods
i. Brief outline of each protocol is presented in Cyno Blood Table.doc
ii. Full protocols can be found in TVDC Binder 1 (Wilder Lab)
Page 10 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Cyno Blood Table.doc
Separation medium
Protocol
Expected yield
NHPRR TN2005-03
Purdue Cytometry
90% Ficoll Hypaque
Lymphoprep
1. Dilute blood
1. Dilute blood
1:1 with HBSS
1:3 with PBS
2. Layer 1 part
2. Layer 1 part
90% Ficoll
LP/2 parts
Paque Plus/1.3
blood
parts diluted
3. Spin 30 min
blood
RT, no brake,
3. Spin 30
1800 RPM
minutes RT,
4. Harvest
no brake, 1600
interface
RPM
5. Wash 2x and
4. Harvest
lyse RBCs
interface
5. Add HBSS
and spin at
1000 rpm
6. Lyse RBCs
25 x 106/5ml blood
10 x 106/5 ml blood
Williams’ Lab
Ficoll Paque Plus
1. Spin blood in
vacutainer
3000 rpm, 10
min, no brake
2. Harvest buffy
coat and add 5
ml HBSS
3. Layer over 5
ml FPP
4. Spin 3000
rpm, 30 min,
RT, no brake
5. Harvest
interface
6. Spin 1000
rpm, 10 min to
remove
platelets
20-60 x 106/5 ml
blood
c. Results
i. Summary of results shown below in Tul 1 and 2 summary tables 073106
ii. Tables show that the Purdue CML protocol proved superior in resultant yield, RBC
contamination and % of lymphocytes
1. one concern with the Purdue CML protocol is that the resultant cells did not
proliferate in response to Con A when cultured at 0.5 x 106/ml
2. evaporation of up to one-half of the culture volume was the probable cause
of failure of the cells to proliferate
iii. Williams’ Lab protocol also resulted in cells that could be cultured
1. many RBCs however (actual data will be supplied in next tech report
as it is with my technician who is currently on vacation)
2. fewer lymphocytes and more macrophages upon differential counting
3. great proliferative capacity to both Con A and PHA at three different cell
concentrations (0.5 x 106/ml, 1 x 106/ml and 1.5 x 106/ml)
4. Cells were cultured in quadruplicate with Con A (10 g/ml) and PHA (2.5
g/ml) for 4 days before addition of BRDU; BRDU was left in for 18 hours
before development of the anti-BRDU ELISA as per kit instructions (Cell
Proliferation ELISA, BrdU (chemiluminescent), Roche Applied Science, Cat. #
11 669 915 001)
5. Proliferation data shown below and also in TVDC Binder 1, Tul 2
Page 11 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
a. Raw data: C:\Documents and Settings\jwilder.LOBOS\My
Documents\Tularemia Contract\TUL2 072806.xls
b. Summary data: C:\Documents and Settings\jwilder.LOBOS\My
Documents\Tularemia Contract\TUL2 summary.xls
iv. NHPRR protocol failed to produce any mononuclear cell layer above the RBC pellet
after spinning, thus, no mononuclear cells could be harvested
1. Possible problem was our use of Ficoll Paque Plus rather than Ficoll Hypaque
Tul 1 and 2 summary tables 073106.doc
Yield and Purity of PBMCs
Date
7/12/06
Protocol
Purdue
CML
Animal
F2519
7/24/06
Williams’
Lab
2146C
7/24/06
NHPRR
55DIE
Differential Counts of PBMCs
Protocol
% Lymphs
Purdue CML
76.0
Williams’ Lab
59.6
Yield
19.4 x
106/4.2 ml
blood
10.4 x
106/4.7 ml
blood
0/4.8 ml
blood
% Macs
12.9
35.9
% Viable
74%
% RBCs
10.7%
> 80%
Approx.
70.0%
n/a
n/a
% Eos
10.9
4.4
% PMNs
0.17
0.15
Page 12 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
TUL2 Summary.xls
Cyno PBMC Proliferation
1600000
1400000
1200000
RLU
1000000
0.5 x 106/ml
1 x 106/ml
1.5 x 106/ml
800000
600000
400000
200000
0
958
21954
Nothing
Media only
Cells only
Cells + ConA
Cells + PHA
Key to Graph:
-Nothing: No cells in wells; no addition of BRDU or anti-BRDU or substrate to those wells
-Media only: Media in wells for 4 days; addition of BRDU, anti-BRDU and substrate
-Cells only: Unstimulated cells in wells; addition of all reagents to detect proliferation
d. Summary
i. Purdue cytometry mailing list protocol is superior for yield, low RBC contamination, %
lymphocytes and simplicity
a. lack of cell proliferation likely due to fluid evaporation in incubator
b. evaporation can be avoided by adding sterile water to surrounding wells
ii. NHPRR protocol failed
iii.Williams Lab protocol is moderately simple but results in high RBC contamination
a. cells proliferated well to both Con A and PHA
iv. Cell proliferation ELISA BRDU kit is simple and sensitive
4. Significant decisions made or pending
Pending: Use of Purdue Cytometry Mailing List (PCML) protocol employing Nycomed
Lymphoprep separation media IF cell purification is reproducible and results in cells that will
proliferate in response to Con A and PHA
5. Problems or concerns and strategies to address
Concern that PCML protocol results in cells that fail to proliferate; will address by repeating
purification with a new sample on 8/14/06 and assuring that fluid evaporation does not occur by
filling surrounding wells with sterile water
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30% of scientific work has been completed
9. Work plan for upcoming month
Page 13 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble

LBERI: We will prepare another batch of cynomologous macaque PBMCs using the
PCML protocol and stimulate them in culture with Con A and PHA. We will also use
these or a separate set of similarly prepared PBMCs to begin working out the conditions
of the IFNgamma ELISPOT assay
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None for this milestone.
Milestone 25
Milestone description: Design protein-fragment library based on SCHU S4 sequence
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
The computer program for analyzing and predicting the SCHU4 genome has been rewritten to
include modules for mass production and linking to IVT constructs and other key aspects of the
gene analysis, amplification, and gene-building algorithms for use in this project. The previously
designed primers are being confirmed with the new algorithm for completing the purchase order.
The list of 500 synthetic peptides covering MHC Class I and Class II epitopes have been
reviewed by Drs. Sykes, Johnston, Lyons, and Breen. An order has been placed for their
delivery.
Files are stored at R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_output.xls
4. Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
75%
9. Work plan for upcoming month
Confirm the oligonucleotide design to amplify all of the coding region fragments for the SCHU4
proteome and order the primer set. Receive, dilute, and create the pools of peptides for sending
to UNM for in vitro testing.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 14 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Milestone 26
Milestone description: Confirmation of gene expression (Design HTP SOPs, Test HTP SOPs,
ORF library production, confirm gene expression)
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
We have designed and received primers for amplifying 8 targeted F. tularensis genes (Table 1).
We have successfully amplified genes 1-6 and are undergoing PCR optimization studies to
amplify gene segments for proteins 7 and 8 (see below). The amplification product will be ligated
to mass produced promoter and terminator regions of varying construction (Figure 1). The key
aspects are that we will be testing promoters with and without the ubiquitin (UB) leader sequence
and with and without the T7 terminator. The ubiquitin leader sequence may be a factor in
promoting stable protein folding. However, since this sequence adds over 250 base pairs to the
construct, it may reduce translation efficiency. Since our constructs will be linear elements, they
may not need a terminator sequence to end the reaction.
Table 1 Selected F. tularensis genes for IVT production
1.
FTT0208c
ABC transporter
2.
FTT1695
groES chaperone
3.
FTT1712c
23 kDa protein
4.
FTT0901
TUL4
5.
FTT1419
hypothetical lipoprotein (p11)
6.
FTT1602
hypothetical lipoprotein (p12)
7.
FTT0613c
hypothetical membrane protein (p15)
8.
FTT1060c
50s ribosomal protein
Figure 1. IVT promoter and terminator construct variations. Acronyms; T7 = T 7 promoter region; SD =
Shine-Delgarno ribosome binding site; ;6xHis six histidine coding region, BAP, Biotinylation acceptor site,
U-F is the universal forward linker sequence; Insert = gene fragment of interest; U-R is the universal
reverse linker sequence; HA, hemagglutinin antigen tag coding region; T7 term, the sequence of the T7
terminator sequence
.
Page 15 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Files are stored at R:\GeneVac\FTU\Contract\Proteome\Milestones\26\
4. Significant decisions made or pending
To determine the optimum promoter/terminator combination using the 8 F. tularensis genes. All 8
genes will be produced in all four constructs using overlap PCR. This will lead to the testing of 32
total representative fragments for IVT production comparison
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed
60%
9. Work plan for upcoming month
Finish the construction of the 8 protein fragments and perform the IVT reaction and send 20-50
micrograms of IVT produced protein to UNM.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 32
Milestone description: Oligos selected for microarray production; Oligos list refined, 70mer
oligos procured, GDP oligos defined, Based on SCHU S4 sequence.
Institution: ASU-Johnston
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
The 70mer oligo set has been designed to cover the SCHU S4 genome; the set has been
ordered and received. The GDP primers were redesigned and a set of 183 7mers are now on
order to complete the set for amplification of 100% of the F. tularensis coding regions.
File located at…
R:\genevac\ftu\contract\microarray\milestones\32\FTT_GDP_7-TTTT.xls
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Oligonucleotide probes and controls (1,824, 70 mers) were arrayed into 5, 384 master and one
working spotting plates for creating spotted microarrays to assess the gene expression profile of F.
tularensis.
7. Quality of performance
Good
8. Percentage completed
95%
9. Work plan for upcoming month
Upon receipt of the GDPs, this milestone will be completed.
Page 16 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 33
Milestone description: Microarrays constructed and confirmed; First printing of arrays, Testing
with DNA from Ft, Arrays GDPs validated at ASU
Institution: ASU-Johnston
1. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
We received 19, 96 well plates containing a minimum of 65 microliters of an 80 µM stock solution
of each of the 1,824, 70mer oligonucleotides and controls. These have been re-arrayed into
master and working 384 well plates for printing.
4. Significant decisions made or pending
Prepare a test plate of 70mer probes at the final concentration in 3XSSC vs. Array-It
Hybridization buffer. The Array-It buffer contains surfactants and proprietary additives that
provide superior spot morphology as compared to SSC alone. However, we need to confirm
hybridization efficiency with genomic DNA in both spotting buffer conditions before final dilution of
working plates to 40 µM.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
20%
9. Work plan for upcoming month
Perform hybridization tests using the two spotting buffer conditions with a limited set of probes.
With this result, complete the dilution of the first working set and print test slides on both Corning
Ultragaps and in-house produced poly-L-lysine-coated slides.
Perform initial hybridizations with RNA and DNA obtained from UNM.
Obtain F. tularensis LVS isolate from UNM to test GDPs by performing RNA isolations on various
numbers of bacteria to determine the minimum detectable bacterial limit. Repeat these findings
when bacteria are diluted in mouse macrophage cell line lysate.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 17 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Milestone 39
Milestone description: Create uvrA or uvrB mutant F. tularensis subsp. novicida
Institution: UTSA
1. Date started: 4/1/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
3.1 pKEK952 is the uvrA:ermC deletion plasmid that will be cryotransformed into Ft. subsp
novicida
3.2 After increasing the concentration of the pKEK952 during the cryotransformation, we
got colonies grown on TSA/Erythromycin plate. Chromosomal DNAs were extracted
from the colonies, and PCR were performed on DNA from the individual colony The
resultant PCR fragments were cut with BamHI, and band pattern is correct for some
colonies, supporting that the uvrA;ermC construct was being integrated into the
chromosomal DNA.
3.3 The PCR fragment was sent out for sequencing ，and waiting for sequencing result.
All data are recorded in page 14-15, TVD UTSA notebook #2.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
No problems so far.
6. Deliverables completed
pKEK951(uvrB::ermC deletion plasmid), KKF71( uvrB::ermC mutant in U112),
pKEK952(uvrA::ermC deletion plasmid).
7. Quality of performance
Excellent progress.
8. Percentage completed
Approximate 85% of scientific work completed on the milestone
9. Work plan for upcoming month
If the DNA sequencing result confirms the uvrA deletion and the ErmC insertion, the correct
uvrA:ermC strain will be frozen down and given a KKF number.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 40
Milestone description: Phenotyping of Ft novicida mutants; Measure degree of attenuation of
uvr mutants in macrophages and in mice
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Phenotyping of the F. tularensis ssp. novicida U112 uvrB strain from Dr. Karl Klose at UTSA is
ongoing. We previously demonstrated that the uvrB mutant has no detectable growth defect in
Chamberlain’s medium.
Page 18 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
CFU/ml
1) The intracellular growth rate of U112 or uvrB within J774 murine macrophages was repeated
without and with gentamicin added to the medium to prevent extracellular bacterial growth. Both
the U112 strain and the uvrB strain were able to replicate at the same rate (with a doubling time
of approximately 2h) in presence or absence of gentamicin. The data confirm our earlier finding
that the deletion of uvrB does not render the bacteria more sensitive to macrophage-mediated
killing.
Intracellular Growth of Ft novicida in J774 Macrophages
in the Presence or Absence of Gentamicin
1.E+10
1.E+09
uvrB - Gent
1.E+08
U112 - Gent
1.E+07
1.E+06
uvrB + Gent
1.E+05
U112 + Gent
1.E+04
1.E+03
0.0
10.0
20.0
30.0
40.0
Cerus_Ft_Protocol_007
hours post infection
Notebook 920-078
2) We received the Ft novicida uvrA mutant from Dr. Klose at UTSA on July 28. We have
prepared frozen stocks and working cell banks using DMSO as a cryopreservation agent.
4. Significant decisions made or pending
We have selected Chamberlain’s Defined Medium (CDM) and Cystine Heart Agar with
Hemoglobin (CHAH) as liquid and plate medias for cultivation and enumeration of Ft novicida.
5. Problems or concerns and strategies to address
Mutation of uvrB gene does not appear to result in a macrophage growth defect. If this trend
persists in animals, this suggests that a secondary attenuating mutation would be required if
the SchuS4 strain were to be used as the vaccine background (since LVS is already
attenuated in humans it does not require a secondary attenuating mutation). We will be
screening attenuated Ft novicida mutants that also have uvr mutations for immunogenicity in
milestone 43, with the ultimate goal of selecting an attenuating mutation to construct in
SchuS4.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
15%
9. Work plan for upcoming month
We will assess the phenotype of the Ft novicida uvrA mutant. The growth rate in CDM will be
evaluated and the growth rate in J774 murine macrophages will be compared with the wild type
U112 strain.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 19 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Milestone 41
Milestone description: Optimization of psoralen treatment and characterization of KBMA F.
novicida; Determine the amount of S-59 and UVA required to inactivate uvr mutants, determine
extent of metabolic activity of uvr mutants after S-59 and UVA inactivation, determine the level
of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
We previously identified the minimum concentration of S-59 required to inactivate wild type Ft
novicida U112 was 20M and the minimum concentration required to inactivate Ftn uvrB
was 5M when exposed to 6.5 J/cm 2 UVA. We also found that the uvrB mutant maintained
metabolic activity for at least 6 hours but that the level of metabolic activity was reduced upon
exposure to UVA light. In order to minimize the exposure to UV light we performed
photochemical inactivation of U112 and uvrB using the minimum concentrations of S-59
required for inactivation defined earlier, and then exposed the cultures to 6.5J/cm 2, 4J/cm2,
2J/cm2, or 0J/cm2 UVA. The number of colony forming units was assessed by plating the
cultures on CHAH plates and then the level of metabolic activity was assessed using the Cell
Titer 96 assay (Cerus_Ft_protocol_006).
1) UVA dose titration of U112. UVA dose titration of the wild-type Ftn strain U112 was
performed twice using 20uM S-59. There were 0 cfu on all plates that received 2, 4,or 6.5
J/cm2 UVA on plates with 100ul of culture thus our limit of detection was 10cfu/ml. There
were colonies corresponding to 6.5x109 or 1.5x1010 in cultures that received 0 UVA (NB933050, and 933-063). The metabolic activity of the cultures was determined using Cell Titer 96
assay using a series of 3-fold dilutions of particles ranging from 1x 109 to 3x107. Below are
representative data from the nominal 1e8 group. In this experiment the groups that received
no UVA (and were therefore live) demonstrated a much higher degree of metabolic activity
than the groups that were killed with S-59 and UVA. There appeared to be a negative impact
of the S-59 psoralen alone in the absence of UVA, which may be due to some exposure of
the culture to ambient light. All three photochemically inactivated cultures demonstrated a
reduced degree of metabolic activity that was indistinguishable from each other.
UVA Dose Effect on Metabolic Activity of Ftn wt Strain U112
1.64e8 particles/mL
1.8
1.6
Ftn wt -S59, 0J UVA
1.4
Ftn wt +S59, 0J UVA
OD490
1.2
1.0
Ftn wt +S59, 2J UVA
0.8
Ftn wt +S59, 4J UVA
0.6
Ftn wt +S59, 6.5J UVA
0.4
0.2
0.0
0
2
4
6
8
10
12
NB 993-063
Time (h)
Page 20 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
2) UVA dose titration of the uvrB mutant. UVA dose titration of the uvrB mutant was
performed twice using 5uM S-59. There were 0 cfu on all plates that received 4,or 6.5 J/cm 2
UVA. There were colonies corresponding to 6.3x103 and 2.3x103 cfu/ml in cultures that
received 2J/cm2 and 9.1x109 or 2.3x1010 in cultures that received 0 J UVA (NB933-050, and
933-063). The metabolic activity of the cultures was determined using a series of 3-fold
dilutions of particles ranging from 1x 109 to 3x107. Below are representative data from the
nominal 1e8 group. In this experiment, the groups that received no UVA (and were therefore
live) had a much higher degree of metabolic activity than the groups that were killed with S59 and UVA. In this case, there appeared to be no negative impact of the S-59 psoralen
alone in the absence of UVA. All three photochemically treated cultures were
indistinguishable from each other but demonstrated a reduced degree of metabolic activity
compared to live cultures. Since there were some residual viable bacteria capable of colony
formation remaining in cultures exposed to 2J/cm 2 UVA we have selected 4 J/Cm 2 as our
minimum UVA dose for future inactivation experiments.
UVA Dose Effect on Metabolic Activity of Ftn uvrB Strain
2.58e8 particles/mL
Ftn uvrB -S59, 0J UVA
Ftn uvrB +S59, 0J UVA
1.2
1
0.8
0.6
Ftn uvrB +S59, 2J UVA
Ftn uvrB +S59, 4J UVA
Ftn uvrB +S59, 6.5J UVA
0.4
0.2
0
0
2
4
6
8
10
12
NB 993-063
Time
3) Comparison of metabolic activity of WT and uvrB mutant after photochemical inactivation.
The metabolic activity of the WT and uvrB Ft novicida strains inactivated with 20 or 5 uM S59 (respectively) and 4J/cm2 UVA (+S59, 4JUVA) were compared to the relevant live
controls: no treatment (-S-59 0J UVA), S59 without UVA treatment (+S-59, OJ UVA) or UVA
treatment without S-59 (– S-59, 4J UVA). All live cultures had similar degrees of metabolic
activity while both killed cultures had reduced metabolic activity that appeared nearly
identical. While this level of metabolic activity is reduced compared to live, the continued
metabolic activity over a 12 hour period is encouraging.
Comparison of Metabolic Activity of WT and uvrB Ft novicida
~2e8 cfu/mL
Ftn wt -S59, 0J UVA
1.8
Ftn uvrB -S59, 0J UVA
1.6
Ftn wt +S59, 0J UVA
Ftn uvrB +S59, 0J UVA
1.4
Ftn wt -S59, 4J UVA
1.2
OD490
OD490
1.8
1.6
1.4
Ftn uvrB -S59, 4J UVA
1.0
Ftn uvrB +S59, 4J UVA
Ftn wt +S59, 4J UVA
0.8
0.6
0.4
Page 21 of 29
0.2
NB 993-063
0.0
0
2
4
6
8
Time
10
12
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Conclusions: The uvrB mutant is slightly more sensitive to photochemical inactivation than
the WT strain, but this does not translate into a dramatic increase in the level of metabolic
activity observed after photochemical inactivation. However, photochemically inactivated Ft
novicida demonstrate significant metabolic activity for greater than 12 hours. This might be
enough metabolic activity to maintain immunogenicity.
4. Significant decisions made or pending
The minimum S-59 dose required for inactivation of U112 is 20 uM, the minimum S-59
concentration required for inactivation of Ftn uvrB is 5 uM. The minimum UVA dose required to
achieve complete inactivation is 4 J/Cm 2.
5. Problems or concerns and strategies to address
We have determined the concentration of S-59 required to inactivate Ftn uvrB is only one
fourth the concentration required to inactive the wild-type U112 strain. We have determined
that the level of metabolic activity between WT and uvrB mutant strains is indistinguishable.
This difference is less than we have observed for other organisms. It is still possible that the
uvrA or the uvrA+uvrB strain will be more sensitive to photochemical inactivation, and
hence my display a higher degree of metabolic activity. The uvrA mutant will be evaluated in
the upcoming month. Since live strains can replicate during the MTS assay, growth of nonKBMA strains in the MTS assay will be evaluated. Samples will be plated to determine if
replication is contributing to the difference in metabolic activity between KBMA and nonKBMA samples.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
20% of scientific work completed on the milestone
9. Work plan for upcoming month
We have determined the minimum concentration of S-59 required for complete inactivation of
U112 and uvrB strains (20M and 5M respectively). We have determined that the lowest dose
of UVA that results in complete killing (4 J/cm2). We have determined that the two strains have
comparable metabolic activity when inactivated. During the upcoming month, we will compare
the uvrA mutant to the uvrB and WT strains. We will determine the lowest concentration of S59 required to achieve complete inactivation of uvrA strain and determine the level of metabolic
activity after photochemical inactivation using the MTS assay.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 43
Milestone description: Create uvrA or uvrB mutants in LVS
Institution: UTSA
1. Date started: 5/01/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
As overlapping PCR did not work for long PCR fragments, each individual fragment of UvrB was
amplified and cloned into pUC 19.
Page 22 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
The primers used are :
UvrBLVSUpEcoRI GGA gaattc GCA GCA GAT GAT ATT GCA CGC ACA
UvrBSchu4LVSDnBamHI ggaggatcc TTG TAT TGC TTG AGG CTG ATC GCC
UvrBSchu4LVSUp1BamHI ggaggatcc GCT ACG AAG GTT ATC AAA GCT CTC G
UvrBLVSDn1PstI GGA ctgcag TTG CAC CAA TCC CGG CAA GTA A
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
UvrBLVS Upstream Fragment(UvrBLVSUpSeq)
Set up PCR reaction as follows:
10 X KODXL DNA polymerase buffer
5.0 ul
dNTP 2 mM
5.0 ul
UvrBLVSUpEcoRI 2uM
5.0 ul
UvrBLVSDnBamHI 2uM
5.0 ul
LVS Chromosomal DNA 10 ng/ul
5.0 ul
KOD XL DNA polymerase 2.5 u/ul
0.5 ul
dH2O
24.5 ul
at 94C 2’, then 94C 15”, 55C 15’, 72C 2’ for 35 cycles with final extension for 10 minutes at
72C.
The UvrBLVSUpSeq was cut with EcoRI and BamHI, and ligated to pUC19 treated with same
enzymes.
Plasmids were prepared, and identified with restriction enzyme digestion with EcoRI and
BamHI.
Appropriate plasmids from step 3.3 were sent out for sequencing, and one correct plasmid was
frozen down in E. coli, and designated as pKEK1028.
UvrBLVS Downstream Fragment(UvrBLVSDnSeq)
Set up PCR reaction as follows:
10 X KODXL DNA polymerase buffer
5.0 ul
dNTP 2 mM
5.0 ul
UvrBLVSUp1BamHI 2uM
5.0 ul
UvrBLVSDn1PstI 2uM
5.0 ul
LVS Chromosomal DNA 10 ng/ul
5.0 ul
KOD XL DNA polymerase 2.5 u/ul
0.5 ul
dH2O
24.5 ul
at 94C 2’, then 94C 15”, 55C 15’, 72C 2’ for 35 cycles with final extension for 10 minutes at
72C.
The UvrBLVSDnSeq was cut with PstI and BamHI, and ligated to pUC19 treated with same
enzymes.
Plasmids were prepared and identified with restriction enzyme digestion with PstI and BamHI.
Appropriate plasmids from step 3.7 were sent out for sequencing, and one correct plasmid was
frozen down in E. coli, and designated as pKEK1029.
All the data were documented in page 54-57, TVD UTSA notebook #2.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
No problems so far. .
6. Deliverables completed
pKEK1028(pUC19UvrBLVSUpSeq), and pKEK1029(pUC19UvrBLVSDnSeq).
7. Quality of performance
Good
8. Percentage completed
Approximate 25% of scientific work completed on the milestone
Page 23 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
9.Work plan for upcoming month
3.1 Put UvrBLVSUpSeq from pKEK1028 into pKEK1029
3.2 Put FpKan into the new plasmid from step 8.1
3.3 If plasmid from step 8.2 is correct, start cryotransformation of LVS with new plasmid。
10.Anticipated travel
None.
11.Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale
Francisella tularensis culture conditions, Establish 3L culture scale purification conditions,
Optimize 3L scale photochemical inactivation process, Verify protective immunogenicity of
vaccine candidates produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
We have demonstrated previously that LVS grows robustly in Chamberlain’s defined medium
(CDM) and that WT Ft novicida and Cerus’ strains of LVS are inactivated with similar
concentrations of S-59.
1) We have received our APHIS permit from USDA for import of LVS and Ft novicida (permit
# 54834). We have requested vials of DVC LVS to be shipped to Cerus from UNM and
expect arrival by Aug 8, 2006.
2) Large scale fermentation of LVS. We have repeated our attempt to propagate Cerus LVS
in the fermentor following Cerus_FT_protocol_008. Once again, the bacteria failed to
replicate appreciably in CDM (Notebook 935-076). In order to understand the reasons for this
we are in the process of determining whether the bacteria are not growing because of the
number and duration of passages from the frozen stock, or whether there are fermentorspecific conditions that are preventing the growth of LVS.
4. Significant decisions made or pending
We have selected Chamberlain’s Defined Medium (CDM) and Cystine Heart Agar with
Hemoglobin (CHAH) as liquid and plate medias for cultivation and enumeration of LVS. We have
switched from glycerol to DMSO for cryopreservation of stocks.
5. Problems or concerns and strategies to address
We do not know why LVS failed to grow in CDM in the fermentor but grows well in CDM in shaker
flasks. We have followed the propagation procedure for large-scale fermentation of LVS but kept
the bacteria in shaker flasks. We determined that the series of amplification steps is sub optimal
and results in a reduced growth rate (with a doubling time of 4h). Therefore, we will modify the
initial propagation procedure so that the inoculum to go into the fermentor is in early stationary
phase rather than late stationary phase. After the medium in the fermentor is inoculated, we will
then remove a sample and propagate it in shaker flasks. This should distinguish whether there is
bactericidal residue in the fermentor or whether the agitation and aeration conditions are suboptimal in the fermentor.
Page 24 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
6. Deliverables completed
None
7. Quality of performance
fair progress
8. Percentage completed
10% of scientific work completed on the milestone
9. Work plan for upcoming month
We will perform a S-59 dose-titration for photochemical inactivation of DVC LVS using the
Cerus_Ft_protocol_005 to determine the minimum concentration required for complete
inactivation of DVC LVS (focusing on concentrations between 5 and 20 uM). We will modify
the large-scale propagation procedure so that the inoculum going into the fermentor is in
early stationary phase rather than late stationary phase and attempt 3L-scale propagation.
During the cultivation we will monitor the pH, dissolved oxygen concentration, and optical
density of the bacteria and determine the number of colony forming units at various time
points. We will compare the growth characteristics of LVS in Chamberlain’s medium and the
DVC medium.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHU S4)
(iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis
subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis
subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. Designed oligos for cloning IglC deletion into pUC118 (KEK964) using KEK906
as the PCR template. (Note: last month this plasmid was identified as KEK999—
it actually is KEK964)
i. iglC schu4 deletion down BamHI
5’- cgc gga tcc atc tac aga agt tga tag tgt ac – 3’
ii. iglC schu4 deletion up SphI
5’- gcg gcg gca tgc atc tac aga agt tga tag tgt ac – 3’
b. Using the above oligos and template PCR was done generating
approximately 3000 base pair product which was digested with BamHI enzyme.
The final restriction digestion product was phenol:chloroform extracted then
ethanol precipitated and rinsed DNA pellet with 70% ethanol, to purify and
concentrate the digested PCR product.
Page 25 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
c. This digested PCR (IglC deletion) product was then digested with SphI enzyme
and run on a 1% agarose gel where it was purified from the gel using the Qiagen
QIAquick Gel Extraction Kit (Cat No. 28706).
d. The pUC118 (KEK964) plasmid containing the a Francisella promoter driving the
expression of a Kanamycin gene was also digested with the same two enzymes
as in b and c above and treated similarly.
e. The isolated PCR product in c. and the final purified digested plasmid in d. were
used in a ligation reaction. This reaction was cleaned up by phenol:chloroform
extraction and ethanol precipitation and used to transform DH5α cells.
f. The resulting colonies from the transformation in e. was screened with NdeI and
EcoRI double digestion which should results in three bands for the correct
construct; of which, only two will be easily visible in a 1% agarose gel since the
smallest band is 189 BP. Thirteen colonies were screen along with the KEK964
as negative control and found two colonies that could be correct.
g. Prepared clean plasmid preparation with these two possible clones containing
the IglC deletion and Kan insertion into the pUC118 plasmid KEK964 and sent
for sequencing.
h. The sequence results were inconclusive therefore, we will continue to screen
more of the transformed colonies from e.
i. Data located in TVD UTSA Notebook 3, page 18-21.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
14%
9. Work plan for upcoming month
a. Prepare more mini plasmid preparations from the transformation resulting from
the ligation reaction of the IglC deletion PCR product and the pUC118 KEK964
construct. Will run diagnostic digestions with other enzymes including NdeI and
EcoRI to check for correct construct before sending next candidate for
sequencing.
b. Once we verify the correct construct, I will try cryotransformation and
electrotransformation to attempt to generated the IglC deletion in Schu4.
c. Will be working in ABSL 3 lab at this point to create the Schu4 mutation.
d. Order more supplies as needed
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 26 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
a. Respiratory cellular responses: Mice (BALB/C 6-week-old females) were challenged
intranasally with 10 LD50 of Ft LVS (20,000 CFU) or Ft. subspecies novicida (100 CFU).
Lungs were collected from Ft-infected mice 24h-, 48h-, and 72h-post challenge (3
mice/time point/group), single cells were made and subjected to flow cytometric analysis
to determine the infiltrating populations of macrophages, dendritic cells, CD8 + T cells,
CD4+ T cells, B-cells, NK cells, and neutrophils (Note Book #4 page:21-22, 28-30). Naive
and PBS-treated mice were used as a base line control. Flow cytometry analyses
revealed that at 24 hr post challenge, there was minimal induction of all cell types with
both organisms. At 48 hr, an increase in numbers of macrophages, dendritic cells and
neutrophils were observed upon infection with both organisms. At 72 hr, while all other
cell types examined were nominal, the increases in numbers of macrophages, dendritic
cells and neutrophils were more clearly evident. A similar phenotype analyses on spleen
cells from these infected animals did not show any remarkable differences (Table1, File
name:0706 infiltrate.doc). It needs to be noted that CD11c is commonly used as a pan
marker for dendritic cells, but may also be expressed on other cell types. CD11b, a
macrophage marker, is also expressed on granulocytes. More stringent double staining
of cells may provide a clear assessment of these cell types upon pulmonary challenge.
Table 1. Cellular infiltration in lungs of mice upon pulmonary Francisella infection
Lungs
Isotype
Naive
CD11b
1
CD11c
1
CD8
1
4.6
15.6
3.3
1.1
0.8
1.2
8.4
CD4
1
CD19
1
CD59
1
Ly6
TOTAL
0.1
35
24 hr
LVS
F nov
3.5
4.2
5.2
12.3
15
10.8
7
3.3
3.6
3.4
PBS
4.6
11.9
Naive
CD11b
1
CD11c
1
CD8
2
CD4
4
CD19
3.9
CD59
1
Ly6
TOTAL
0.7
3.6
3.7
10.9
16.5
20.5
2.2
5.3
62.7
48 hr
LVS
F nov
7.1
8.8
16.2
16.7
4.4
2.1
72 hr
LVS
F nov
4.7
11.9
10.5
12.9
26.4
24
3.6
2.6
2.2
PBS
2
1.5
1.4
1.5
2
1.4
2
1.5
1.2
1.2
0.9
1
0.7
1.5
1.1
1
1.8
0.9
1
0.6
1
1.2
0.4
0.2
1.2
0.3
0.3
7.1
34.8
5.9
28
6.2
32.4
8.4
31.2
11.7
43.3
15.8
46.1
7.3
32.7
26.4
70.9
32.9
72
24 hr
LVS
F nov
PBS
48 hr
LVS
F nov
PBS
72 hr
LVS
F nov
1.1
2.6
3.1
3.5
3.4
2.7
2.9
2.7
4
9.2
4.2
4.2
4.8
4.5
4
4.6
5.7
7.7
13.9
15.5
14.2
17
16.2
14.9
18.3
18.8
20.8
23.4
21
23.1
27.2
24.4
23.5
23
22.7
18.2
22.4
21.5
24.3
27.2
22.7
21
22.8
23.6
2.2
2.6
2.7
2.2
2.1
2.9
1.7
2
5.8
76.1
5.4
72
6.4
80.8
6.4
87.9
7.9
80
6.1
75.9
9.4
83.6
17.1
95.9
Spleen
Isotype
PBS
PBS
2
5.6
70.8
16
Page 27 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
Mice (BALB/C 6-week-old females) were challenged intranasally with 10 LD50 of Ft LVS (20,000 CFU) or
Ft. subspecies novicida (100CFU). Lungs were collected from Ft-infected mice 24h-, 48h-, and 72hpostinfection (3 mice/time point/group), single cells were made and subjected to flow cytometry analysis
to determine the population of inflammatory cells. CD11b, CD11c, CD8, CD4, CD19, CD59, and Ly6, are
commonly used markers for macrophages, dendritic cells, CD8 + T cells, CD4+ T cells, B-cells, NK cells,
and neutrophils, respectively.
b. Cytokine (TNF-α, IL-5, IL-12, IFN-γ) measurement in the lungs after LVS and F. novicida
intranasal challenges were also performed: Mice (BALB/C 6-week-old females) were
challenged intranasally with 10 LD50 of Ft LVS (20,000 CFU) or Ft. subspecies novicida
(100 CFU). Lungs were collected from Ft-infected mice 24h-, 48h-, and 72h-postinfection
(3 mice/time point/group), and individually homogenized in the presence of Hank’s
solution with a protease inhibitor cocktail (Roche Diagnostic) (Note Book #4 page:25-27).
Protein concentration of each sample was determined using a Bio-Rad protein assay kit,
and 20μg of sample was used for cytokine assays. Pro-inflammatory cytokines (TNF-α,
IL-12, and IFN-γ) were induced in mice infected with either LVS or F. novicida but not
with mock (PBS) infection (Fig.1. File name: 0706 cytokine.ppt). The levels of cytokines
increased with time. However, Th2- type cytokines, IL-5 and IL-4 (not shown) were
minimally produced in all samples. These results may suggest a Th1-bias cytokine
induction after pulmonary exposure to Ft.
Pulmonary cytokine induction following F. novicida / LVS challenge
TNF-
ng/ml
IL-12
IL-5
ng/ml
ng/ml
F. novicida
LVS
Mock (PBS)
ng/ml
IFN-
Page 28 of 29
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2006 to 7/31/2006
Due Date: 8/15/2006
Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie Wilder, Ed Barr,
Mitch Magee, Kathryn Sykes, Stephen Johnston, Karl Klose, Justin Skoble
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
12 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Acute inflammatory cell infiltration to site of infection is common among bacterial
infections. Our results indicated a lack of cellular infiltrate in the lung at 24h post-Ft.infection. Does Francisella suppress/inhibit the recruitment of inflammatory cells during
the first (two?) day of infection? We would like to set up experiments using heat-killed
Francisella (LVS and F.novicida) and klebsiella pneumoniae as controls in our
established flow cytometry assays to examine this question.
10. Anticipated travel
Describe request
11. Upcoming Contract Authorization (COA) for subcontractors
Describe request
Page 29 of 29