Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
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, 4, 5, 12/13(UNM/LBERI), 19, 21, 26, 27, 28, 33, 34 (UNM/ASU), 35,
41, 42, 43, 44, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 40, 48,
Inactive milestones: 6-10, 11, 14, 15, 17, 18, 20, 22, 23, 24, 29, 30, 31, 36-38, 45, 47,
52-54, Working Group
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. Five way CRDA between USAMRIID, USAMMDA, True Foundation, UNM and LBERI
has been fully executed as of approximately 7/2/2007.
b. Nicole Banks (LBERI), Terri Nakamura (True) and Barbara Griffith (UNM) are developing:
i. Timeline in MS Project for the vaccination process- Nicole updated dates based
on CRDA and Barbara needs to edit
ii. Database for tracking vaccinee documents submitted to USAMRIID is being
developed by True.
c. UNM EOHS has acquired current documents:
i. Normal values for prehealth screening tests to be performed in NM at TriCore
Reference Laboratory
ii. CAP certificate for TriCore Reference Laboratory
iii. CLIA certificate for TriCore Reference Laboratory
iv. Medical Director’s signed CV, from TriCore Reference Laboratory
v. Medical Director’s license, from TriCore Reference Laboratory
vi. University Hospital’s JCAHO accreditation – covers Radiology’s certification
vii. Radiology Director’s CV and licenseviii. Final pricing for pre health and post health screenings
d. UNM EOHS pending
i. Nurses need to take GCP training course before being trained at USAMRIID
ii. Barbara needs to modify HIPPA consent to include knowledge that dates of
sample and document shipping will be shared with True Foundation.
e. UNM and LBERI have prioritized the 46 scientists and staff who will be offered the LVS
vaccinations
f. USAMRIID Correspondence: Bev Fogtman, Dr. John Aldis, Jeannine Haller, and Cindy
Barrick have responded to 2 more sets of questions and been extremely helpful.
1 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
i. Dr. Boudreau liked the content of the GCP training course which is readily
available to the UNM EOHS nurses.
ii. Barbara will be contacting USAMRIID, True, USAMMDA, LBERI, and UNM
EOHS to set up a “kick off” teleconference for tentatively 7/18 to begin work on
the first set of 8 vaccinees.
4. Significant decisions made or pending
a. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
b. 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
c. UNM and LBERI will offer the LVS vaccinations to 46 scientists; USAMRIID will be
providing the LVS vaccinations over the next 8 months, approximately.
d. Dr. Lyons will request IRB approval to allow blood draws on the vaccinated LBERI and
UNM scientists after their LVS vaccinations. UNM will share the IRB proposal with
USAMRIID.
5. Problems or concerns and strategies to address
a. UNM may need an external source of human cells from LVS vaccinated individuals, in
order to develop the immunoassays in humans. Within approximately 4 months, UNM
may have access to the blood of UNM and LBERI scientists who have been vaccinated
with LVS at USAMRIID.
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; Work stop has occurred on the SCHU S4 aerosols in primates, until the
LBERI scientists and staff receive the LVS vaccinations.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
19%
9. Work plan for the next month
a. Complete the 5 way CRDA between USAMRIID, True Foundation, UNM and LBERI
b. Formally, start the relationship with USAMRIID
i. provide Laboratory and Radiology documents to USAMRIID
ii. Begin Hazard Analysis Risk assessments
iii. Begin informed consent process and HIV
iv. iv Hold SIP Informed consent teleconference with UNM/LBERI/USAMRIID
c. EOHS nurses start GCP training, if USAMRIID approves the CITI course content
d. UNM will request a COA for travel and for UNM EOHS for prehealth and post health
screenings for 46 vacinees.
e. UNM will modify the HIPPA consent to include knowledge that True Foundation will be
aware of the dates that documents and samples are sent to USAMRIID
f. Maintain excellent communications with USAMRIID to understand the SIP protocol
requirements
10. Anticipated travel
Travel to USAMRIID could occur in summer 2007 to fall 2007
11. Upcoming Contract Authorization (COA) for subcontractors
a. 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.
2 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
b. UNM will request a COA to authorize expending funds on the pre-health screenings and
travel for the vaccinations, for 46 scientists and staff.
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

June 2007 LVS work focused on the Aeromist Nebulizer testing;
i. Both June dates incorporated dilutions of frozen LVS stock in the generator
(Figures 1 and 2)
1. 18 total sprays performed to date
2. 3 target concentrations: 1x105, 1x106, and 1x107 cfu/mL
3. Actual vs. Target cfu/mL values were inaccurate for the 13 April and 21
June set of sprays due to a drop in titer of the frozen stock. This
phenomenon was observed in the previous frozen stock as well;
together, these data indicate the need to create a new frozen stock every
4-6 months. Values were accurate for the second set due to a correction
in titer calculations
4. Calculated spray factors were consistent at equal concentrations
a. Values were comparable to those observed using the Collison
nebulizer, and in numerous cases better.
b. Values decreased (i.e., efficiency decreased) as concentration
increased, a consistent observation seen with other aerosol
generators tested to date, including the Collison
5. Aeromist nebulizer continues to be a promising alternative to the Collison
generator and will be further tested.
6. Data filed in the following folders:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Aeromist nebulizer\21Jun07
b. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Aeromist nebulizer\26Jun07
3 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Aeromist: Target vs. Actual CFU/mL (Frozen)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
4/13/2007
6.00
6/21/2007
6/26/2007
5.50
5.00
4.50
4.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
Figure 1. Target vs. Actual CFU/mL at three target concentrations of frozen LVS using the Aeromist generator on three separate
bioaerosol dates
Aeromist: Actual CFU/ml vs. Spray Factor (Frozen)
-5.80
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-6.00
-6.20
4/13/2007
6/21/2007
-6.40
6/26/2007
-6.60
-6.80
-7.00
Actual CFU/mL (Log 10)
Figure 2. Actual CFU/mL vs. Spray Factor at three target concentrations of frozen LVS using the Aeromist generator on three
separate bioaerosol dates
4 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam

Outside of the ABSL-3, work was conducted using ultrasonic generation technology
(generator modified in-house) using BG spores
i. 3 days of testing using freshly prepared BG spores in solution (Figures 3 and 4)
1. 9 total sprays
2. 1 target concentration: 1x105 CFU/mL
3. Results
a. Actual vs. Target CFU/mL values were within 0.5 log10 of the
target values, which is excellent
b. Calculated sprays factor were inconsistent and much less
efficient than those observed with other generators tested to date
4. Data filed in the following folder:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\BSL-2 (BG) testing
Ultrasonic Generator: Target vs. Actual CFU/mL (BG spores)
7.00
Actual CFU/ml (Log10)
6.50
6.00
5.50
6/11/2007
5.00
6/13/2007
6/15/2007
4.50
4.00
3.50
3.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
Target CFU/ml (Log10)
Figure 3. Target vs. Actual CFU/mL at one target concentration of BG spores using an ultrasonic generator on three separate
bioaerosol dates
5 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Ultrasonic Generator: Actual CFU/ml vs. Spray Factor (BG spores)
Spray Factor (Log10)
-5.00
5.30
-5.50
5.35
5.40
5.45
5.50
5.55
5.60
5.65
-6.00
-6.50
6/11/2007
-7.00
6/13/2007
6/15/2007
-7.50
-8.00
-8.50
-9.00
Actual CFU/mL (Log 10)
Figure 4. Actual CFU/mL vs. Spray Factor at one target concentration of BG spores using an ultrasonic generator on three
separate bioaerosol date

Because of repeated culture problems encountered with the CHAB (i.e., inconsistencies
with titers, colony morphology, absence of growth, etc.), a second media type was
chosen to determine if an alternative to CHAB could be used for future LVS testing:
i. Supplemented Blood Cysteine Glucose Agar (SBCGA; originally tested upon
initiation of this study; determined to be “second best” growth medium)
ii. Results:
Figure 5. Frozen LVS stock titer after 48h of growth at 37°C. Left, CHAB. Right, same culture on
SBCGA.
iii. After 48h of growth on SBCGA, LVS growth was consistent with that reported in
the literature (i.e., colonies 3-4mm in diameter, gray-white to slightly blue and
opaque, non-hemolytic, raised, convex and demonstrating a wet, shiny surface)
and with what we observed in our previous media comparison study. This
growth pattern was not apparent on the CHAB until 96h post-inoculation. In
addition, growth was not consistent between the CHAB plate replicates as it was
on the SBCGA; some plates presented absence of growth, a phenomenon seen
in previous bioaerosol runs. It can be concluded that the CHAB media is not
reliable and future experiments should incorporate SBCGA.
1. Data filed in the following folder:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
6 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\BSL-2 (BG) testing
iv. Images (Pics) filed in the following folder:
1. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\Francisella tularensis\CHAB vs. BCGA pics
v. Titer comparisons calculated in the following file:
1. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Aeromist nebulizer\26Jun07\LVS spray data sheet 26Jun07
4. Significant decisions made or pending



Will continue to perform Aeromist testing using LVS
i. A new frozen LVS stock will be created and tested; because of the 1 log10 drop
in titer, remaining prior stocks of LVS will be destroyed following confirmation of
purity and titer of the new stock.
ii. Fresh culture will be tested
Ultrasonic generators will not be tested further due to their demonstrated loss in
efficiency versus the Collison nebulizer. Furthermore, this type of generator requires
increased suspension volume and is more difficult to setup.
Because of inconsistencies with culture titers seen using CHAB, the decision was made
to use SBCGA on all future LVS bioaerosols.
5. Problems or concerns and strategies to address

Following the annual ABSL-3 Facility shutdown, hardware issues were observed with the
exposure line used for LVS testing (Class III Biosafety Cabinet located in Room 1028).
These issues primarily included signal degradation to generator and humidity control
components and were thought to be due to faulty wiring and cabling. To address this, the
ABSL-3 Aerosol group is currently replacing wires and leads throughout the system and
restoring integrity. This procedure is approximately 80% complete and is demonstrating
restoration of critical functions such as generator flow and humidity control.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
70%
9. Work plan for upcoming month



Continue bioaerosol experiments on fresh and frozen LVS with the Aeromist nebulizer
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on SBCGA
iii. Will continue doing frozen and fresh, not lyophilized
Continue to investigate other possible generators to use for LVS bioaerosols
Initiate final decision on bioaerosol generator to be used on animal studies
10. Anticipated travel

Travel to Aerobiology Meeting and presentation of LVS bioaerosol data (July 9-11, 2007)
11. Upcoming Contract Authorization (COA) for subcontractors

None anticipated
7 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
No work on this milestone was completed in the last month.
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
12.5%
9. Work plan for upcoming month
a. None. These NHPs will continue to be bled as a source of cells for Milestone 12/13;
however, no work is anticipated on these NHPs until they are challenged with aerosol
Schu4 sometime after November 2007.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Fischer 344 rats
a. Experiment Ftc37 study 2 (Notebook 104, pages 6-8) and electronic files of the
histology images are stored at L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Experiments\Ftc37\Ftc37 study 2
i. The purpose was to determine the histological appearance of the lungs, liver
and spleen from naïve Fischer 344 rats infected i.t. with a lethal dose of
SCHU S4. These results will be compared with those from vaccinated rats
challenged i.t. with SCHU S4 (Ftc40 study 2)
ii. Naïve rats were infected i.t. with 400 SCHU S4. Three rats were killed on
days 0, 3, 6, 9 days to collect the lungs, liver, and spleen
8 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
iii. On day 3, the lungs of 2/3 rats exhibited mild to moderate, multifocal,
pyogranulomatous bronchopneumonia with small scattered foci of necrosis
(Figure 1). Each of these rats also had minimal, multical areas of
pyogranulomatous hepatitis, and mild to moderate pyogranulomatous
splenitis (Figure 2). Bacteria were not detectable (H and E stained sections)
in either of the rats. The third rat had no lesions in the lungs, liver or spleen,
and was presumed to have not been infected.
iv. On day 6, all 3 rats had moderate to severe, multifocal to coalescing
pyogranulomatous bronchopneumonia with mild hyperplasia of bronchial
associated lymphoid tissue. Each of the rats also had moderate to severe,
multifocal pyogranulomatous hepatitis, with minimal lymphocytic and
histiocytic infiltrates in the portal triads. There was multifocal, moderate to
severe, pyogranulomatous splenitis, a slight reduction in the size of the
periarteriolar lymphoid sheaths (without active necrosis/apoptosis), and a
slight increase in the number of splenic plasma cells in all animals. Bacteria
were not detected (H and E stained sections) in any of the sections. Note
that the pyogranulomatous splenic infiltrates consisted of more macrophages
than neutrophils on day 6 compared to day 3.
v. On day 9, all 3 rats had severe, multifocal to coalescing pyogranulomatous
bronchopneumonia with mild hyperplasia of bronchial associated lymphoid
tissue. Each of the rats also had mild to moderate, multifocal
pyogranulomatous hepatitis, with mild lymphocytic and histiocytic infiltrates in
the portal triads. There was multifocal, moderate to severe,
pyogranulomatous splenitis, a slight reduction in the size of the periarteriolar
lymphoid sheaths (without active necrosis/apoptosis), and a mild increase in
the number of splenic plasma cells (compared to day 6) in all animals.
Bacteria were not detected (H and E stained sections) in any of the sections.
Note again that the pyogranulomatous splenic infiltrates consisted of more
macrophages than neutrophils on day 9 compared to day 3. There was also
less active necrosis and fewer neutrophils, and more macrophages in the
livers on day 9 then on days 3 and 6.
9 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 1. Histological appearance of the lungs from naïve Fischer 344 rats infected with SCHU S4.
10 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 2. Histological appearance of the liver and spleen from naïve Fischer 344 rats infected
with SCHU S4.
b. Experiment Ftc40 study 1 (Notebook 104, pages 15-17, 37-38, 42, 52, 63, 77)
i. The purpose was to determine the kinetics of SCHU S4 proliferation,
dissemination and clearance in s.c. LVS-vaccinated rats
ii. Fischer 344 rats were vaccinated s.c. with 2.7 x 10 7 LVS
iii. 55 days after vaccination, the rats were challenged i.t. with 1.5 x 10 4 SCHU S4
iv. Three days after challenge, the number of SCHU S4 in lungs had increased
1.6 log10 and dissemination to the liver and spleen was evident (Table 1, right
side). The numbers of SCHU S4 in the lungs, liver and spleen on day 9 were
all lower than those on day 3, suggesting that the vaccinated rats were
controlling SCHU S4 proliferation. The SCHU S4 burden continued to
11 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
decreased thereafter, but had not been cleared by day 28. This was
consistent with our result from Experiment Ftc32 that clearance of SCHU S4
in LVS vaccinated rats occurred between 31 and 56 days after challenge.
The kinetics of SCHU S4 proliferation and dissemination in naïve rats (Table
1, left side; Ftc37) is included for reference. Note that the challenge dose
was 2 log10 lower than used for the s.c. vaccinated rats
Table 1. Kinetics of SCHU S4 proliferation in naïve and s.c. LVS-vaccinated Fischer 344 rats*
Log10 CFU/organ  SD#
Naive
s.c. LVS vaccinated
Day
Lung
Liver
Spleen
Lung
Liver
Spleen
0
2.2  0.4
4.2  0.2
3
8.0  0.1
6.5  0.3
6.1  0.5
5.8  0.2
5.2  0.6
5.0  0.5
6
8.4  0.1
7.4  0.2
7.4  0.3
9†
8.8
8.7
7.5
5.5  0.4
3.4  0.2
4.3  0.7
14
4.5  0.2
2.6  0.4
3.0  0.5
21
0
0
3.3  0.8
28‡
0
2.6
2.0
*n = 3/group
†
2 of 3 rats died by day 8
‡
only 1 rat in this group
c.
Experiment Ftc40 study 2 (Notebook 104 pages 31-32, 34)
i. The purpose of this experiment was to determine histological appearance of
the lungs, liver, and spleens from s.c. vaccinated rats after i.t. SCHU S4
challenge. These results will be compared with those generated from naïve
rats challenged i.t with SCHU S4 (Ftc37 study 2)
ii. 43 days after s.c. LVS vaccination s.c., rats were challenged i.t. with 320
SCHU S4
iii. 3 rats were killed on days 0, 3, 6, 9 to collect lungs, liver, and spleens
iv. The tissues are currently being processed at LRRI and, upon return, will be
examined by Dr. Julie Hutt at UNM
d. Experiment Ftc47 (Notebook 104, pages 59-62, 66)
i. The purpose of this experiment is to compare the kinetics of SCHU S4
proliferation and dissemination in naïve and s.c. LVS vaccinated rats after i.t.
challenge. This is a repeat of Ftc37 study 1 and Ftc40 study 1 and the
experimental design is shown in Table 2
ii. We vaccinated 40 rats with 5 x 107 LVS and are waiting for LVS to clear
Table 2: Experimental design comparing kinetics of SCHU S4
proliferation and dissemination in naïve and s.c. LVS vaccinated
rats after i.t. challenge
No. of rats per group-day
Day
0 1 2 3 4 5 6 21 49 Total
Naive
3 5 5 5 5 5
28
Vaccinated - 5 5 5 5 5 5
5
5
40
12 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
e. Experiment Ftc48 (Notebook 104)
i. The purpose of the experiment is to determine the kinetics of LVS
proliferation, dissemination, and clearance after s.c. vaccination in rats. This
is a repeat of Experiment Ftc38 and the experimental design is shown in
Table 3.
ii. We vaccinated 54 rats wit 5 x 107 LVS and have started data collection
Table 3. Experimental design to determine the kinetics of LVS
proliferation, dissemination, and clearance after s.c. vaccination
Day
No. per group-day
1
6
2
6
3
6
4
6
5
6
6
6
14
6
21
6
28
6
Total
54
Hartley Guinea Pigs
a. Experiment Ftc41 (Notebook 104, pages 18-21, 39, 41, 79; Notebook 103, pages 7
-9, 20)
i. The purpose was to determine whether i.n. LVS vaccination protects Harley
guinea pigs from i.n. SCHU S4 challenge. This is a repeat of Experiment
Ftc28 (Notebook 94, pages 152-156)
ii. Naïve guinea pigs (n = 6 to 10) were vaccinated i.n. with 103 (theoretical
dose), 1.3 x 105, and 6.7 x 106 CFU LVS
iii. All of the guinea pigs survived vaccination with minimal signs of illness
iv. 28 days after vaccination, one guinea pig vaccinated with 1.3 x 10 5 LVS and
one with 6.7 x 106 LVS were killed and found to be free of LVS in the lungs,
livers and spleens. We confirmed LVS clearance on day 55 using one
additional guinea pig from each LVS vaccination dose group
v. 49 days after vaccination, we tested the sera from all of the vaccinated
guinea pigs for sero-conversion. As shown in Figure 3, all of the vaccinated
guinea pigs sero-converted regardless of the LVS vaccination dose.
However, we did not test for a correlation between the LVS vaccination dose
and the antibody titer
vi. 55 days after vaccination, the i.n. vaccinated guinea pigs were challenged
i.n. with 5.0 x 104 SCHU S4. We selected this challenge dose so that we will
be able to reliably determine the lung deposition even in the large buffer
volume required to homogenize guinea pig lungs.
vii. LVS vaccination increased survival 1-1.5 days but ultimately did not prevent
the guinea pigs from dying of i.n. SCHU S4 infection (Table 4).
13 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 3. Sero-conversion in intranasal LVS vaccinated guinea pigs. Sera collected from
i.n. LVS vaccinated guinea pigs were diluted 1:25,600 and tested by ELISA using heatkilled LVS. Each bar represents a single animal. The naïve serum was pooled from
several naïve guinea pigs.
Table 4. Resistance of i.n. LVS vaccinated guinea pigs to i.n. SCHU S4 challenge
i.n. LVS vaccination
Dose
(CFU/guinea pig)
None
~ 103
1.3 x 105
6.7 x 106
Dose
(CFU/guinea pig)
5 x 104
i.n. SCHU S4 challenge
Survival ratio
(No. alive/total)
0/6
0/6
0/6
0/6
Mean-time-to
death (days)
4.0
4.5
4.3
4.0
b. Experiment Ftc42 (Notebook 104, pages 22-23; Notebook 103, pages 7-20)
i. The purpose of this experiment was to determine whether s.c. LVS
vaccination protects Harley guinea pigs from i.n. SCHU S4 challenge. This is
a repeat of Experiment Ftc28 (Notebook 94, pages 152-156)
ii. Naïve guinea pigs were vaccinated s.c. with 10 3, 105, and 107 LVS
iii. All of the guinea pigs survived LVS vaccination with minimal signs of illness
iv. 56 days after vaccination, 2 guinea pigs from each dose group were killed
and found to be free of LVS in the lungs, spleens and livers.
v. We also tested the sera from all of the vaccinated guinea pigs for seroconversion. As shown in Figure 4, all of the vaccinated guinea pigs seroconverted regardless of the LVS vaccination dose. However, we did not test
for a correlation between the LVS vaccination dose and the antibody titer
vi. 60 days after vaccination, the i.n. vaccinated guinea pigs were challenged
i.n. with 2.2 x 103 SCHU S4. This challenge dose is approximately 20-fold
less than the dose used to challenge the i.n. LVS vaccinated guinea pigs in
Experiment Ftc41
14 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
vii. LVS vaccination increased survival in a vaccination dose-specific manner,
but did not ultimately prevent the guinea pigs from dying of i.n. SCHU S4
infection (Table 5)
Figure 4. Sero-conversion in subcutaneous LVS vaccinated guinea pigs. Sera collected from i.n. LVS
vaccinated guinea pigs were diluted 1:25,600 and tested by ELISA using heat-killed LVS. Each bar
represents a single animal; the gaps in the 103 and 107 LVS groups represent guinea pigs that were not
analyzed. The naïve serum was pooled from several naïve guinea pigs.
Table 5. Resistance of i.n. LVS vaccinated guinea pigs to i.n. SCHU S4 challenge
s.c. LVS vaccination
Dose
(CFU/guinea pig)
None
103
105
107
Dose
(CFU/guinea pig)
2.2 x 103
i.n. SCHU S4 challenge
Survival ratio
(No. alive/total)
0/6
0/6
0/6
0/6
Mean-time-to
death (days)
5.0
6.3
6.5
7.5
BALB/c mice
a. Experiment Ftc 44 (Notebook 104, pages 43-46, 53, 67)
i. In experiments performed at Cerus, KBMA-LVS was no better than heatkilled LVS at generating protection against a lethal LVS challenge. Thus, the
purpose of this experiment determine whether KBMA-LVS vaccination
protects BALB/c mice from i.n. SCHU S4 challenge.
ii. The experimental design is shown in Table 6
iii. We have completed the vaccination and the boost regime and are currently
waiting 1 month to challenge the mice with SCHU S4
15 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Table 6:
Vaccination or boost
(3 wk after primary)
Dose
vaccine route (CFU/ms)
None
-
Vaccination
Group
(6/grp)
1
2
3
vaccine
None
Dose
route (CFU/ms)
-
4
5
6
Live LVS
i.n.
103
7
8
9
None
-
-
10
11
12
13
14
15
16
17
18
KBMA LVS i.v.
None
-
KBMA LVS i.n.
-
strain
route
SCHU S4 i.n.
Dose
(CFU/ms)
50
500
5000
-
50
500
5000
KBMA LVS i.v.
108
50
500
5000
108
KBMA LVS i.v.
108
50
500
5000
-
KBMA LVS i.n.
108
50
500
5000
KBMA LVS
108
50
500
5000
108
None
Challenge
in
4. Significant decisions made or pending
a. We will characterize the Fischer 344 rats in detail since they are currently the best
model we have found
b. We will only use the s.c. vaccination route because s.c., i.t. and i.d. LVS vaccination
of Fischer 344 rats produced similar levels of protection against i.t. SCHU S4
challenge and this vaccination route is most applicable for human vaccination
c. We have decided, with Vicki’s approval, to cease further model development using
the guinea pigs because LVS vaccination did not protect them against SCHU S4
challenge
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
7. Quality of performance
Good
16 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
8. Percentage completed
60%
9. Work plan for upcoming month
Rats
a. Repeat the s.c. LVS vaccination/i.n. SCHU S4 challenge experiment
b. Characterization of the Fischer 344 rat model
i. Complete the experiment measuring the kinetics of LVS proliferation,
dissemination, and clearance after s.c. vaccination
ii. Complete the experiment measuring the kinetics of SCHU S4 proliferation
and dissemination in lungs, spleens, and livers of naïve and LVS vaccinated
rats.
iii. Complete the experiment examining the histology of lungs, spleens and
livers from LVS-vaccinated rats infected i.t. with SCHU S4
iv. Determine the effects of T cell depletion on the protective immunity induced
by LVS vaccination
v. Determine whether passive immunization with convalescent sera will protect
naïve Fischer 344 rats from i.t. SCHU S4 challenge
c. Optimize the use of QD655-luc8 to track pulmonary inoculation in rats
i. Determine the minimum amount of QD655-luc8 and coelenterazine required
to produce detectable signal
ii. Determine whether co-administration of QD655-luc8 affects the virulence of
SCHU S4 in naïve Fischer 344 rats
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 12/13-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. Update on NHP PBMC Freezing protocols
1. Issue: Testing 3 different protocols (CTL: 90% human A/B serum/10%
DMSO/10 x 106/ml; CERUS: 80% FBS/20% DMSO/5 x 106/ml; and Lyons:
Frozen in Gibco Recovery Cell Culture Freezing Media (contains optimal ratio of
fetal bovine serum:bovine serum and 10% DMSO)/5 – 10 x 106/ml/thawed in
presence of DNAse and left in 37o incubator for 30 – 60 minutes before use) with
the aim to choose the protocol that spares the most viable cells that remain
functional after thawing
2. Previous results suggested that the CERUS protocol (tested twice) spared at
least 30% of antigen specific proliferation (LVS); CTL protocol (tested twice)
spared between 0 and >100% of antigen-specific proliferation and the Lyons
protocol had not yet been tested due to a low recovery of cells after thawing
17 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
3. Results below reflect the continuation of these investigations
Table 1
Protocol
Cerus
CTL
Lyons
# Cells recovered
NT
1.8 x 106
0.9 x 106
% Cells Recovered
NT
54.1%
25.9%
% Viable
NT
96%
96%
1.60E6
1.40E6
1.20E6
B
CTL
Lyons
None
1.00E6
8.00E5
6.00E5
Frozen, LVS ff Hi
Frozen, LVS hk Hi NT
Frozen, Con A NT
Frozen, Media
NT
Fresh, LVS ff Hi
0
NT
Fresh, LVS hk Hi
2.00E5
Fresh, Con A
4.00E5
Fresh, Media
Relative Light Units (Mean +/- SEM)
Proliferation of Fresh and Frozen/Thawed NHP PBMCs
Figure 1: Proliferation of Fresh and Frozen cells plated at 1 x 106/ml with the indicated stimuli. PBMCs
were purified from one NHP (A00896) on day 140 post. i.d. vaccination; NT = not tested due to lack of
cells
Data interpretation: Proliferation to LVS is spared by either freeze/thaw protocol; but Lyons protocol
results in fewer total cells recovered; this is the 2 nd time that the Lyons protocol has resulted in less than
26% cell recovery.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 070207.svd;
\\Saturn\Group\Wilder Lab\TVDC\BRDU\TUL12; \\Saturn\Group\Wilder Lab\TVDC\freezing_thawing
test\TUL 12 thawing 6-04-07 and in the TVDC 1 bound notebook pages 28 -29 and the TVDC 1 binder
under the TUL 12 tab
Summary data: \\Saturn\Group\Wilder Lab\TVDC\Summary reports\062907 UNM TVD Internal Tech
Minutes Action items jw rev 070507.doc and \\Saturn\Group\Wilder Lab\TVDC\Summary reports\SubNIAID Tech call.7.3.2007 RS&JW.ppt.
18 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
4. We had been concerned, based on data presented last month (day 117)(TUL 11), that the
response to LVS may be waning over time. The data shown in Figure 2 (comparing days 21 day 195) suggests that this is not true.
Proliferation of Cells from a Single NHP which had been Frozen and Thawed
Cell Mean for RLU small
1.60E6
Media
Con A
LVS hk Hi
LVS ff Hi
1.40E6
1.20E6
1.00E6
8.00E5
6.00E5
4.00E5
2.00E5
0
Day 21
Day 28
Day 117
Day 195
Figure 2: Proliferation of fresh cells from NHP A00659 plated at 1 x 106/ml with the indicated stimuli.
PBMCs were purified from at various days post. s.c. LVS vaccination.
Data interpretation: Although LVS responsiveness was not optimum at Day 117, this trend did not hold
for cells purified on Day 195 post s.c. LVS vaccination. The reasons why the day 117 cells did not
proliferate well to LVS is unclear but seem to be related to the proliferation assay on that particular day
rather than the waning of the LVS response.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 070207.svd;
Summary data: \\Saturn\Group\Wilder Lab\TVDC\Summary reports\062907 UNM TVD Internal Tech
Minutes Action items jw rev 070507.doc and \\Saturn\Group\Wilder Lab\TVDC\Summary reports\SubNIAID Tech call.7.3.2007 RS&JW.ppt.
b. Update on IFN detection
i.
We contacted with the ViruSpot detection system representative who
looked at the data showing faint spots in wells in which cells are
unstimulated which are being detected as readily as much darker spots
in stimulated wells; she (Jaya Ghosh) suggested some new detection
settings, and will send us the factory suggested settings. Upon rereading the plate again with the new settings, the faint spots were still
being detected. We continue to work with Jaya on adjusting the settings
to exclude these faint spots from being counted
ii.
Dr. Wilder attended the DMID Cell Mediated Immunity Assays:
Development, Qualification and an Overview of Validation course in
Gaithersburg, MD (June 4 – 7, 2007). There, she spoke with other
experts in ELISPOT assays. One suggestion was that the faint spots
may have been caused by platelet contamination. RBC contamination
from those PBMC preps that gave faint spots were 9.3 – 18.6% (TUL
19 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
iii.
iv.
Experiment
NHP
TUL 10
TUL 10
TUL 11
TUL 11
TUL 12
TUL 14
TUL 14
TUL 15
10), 5.5 – 13.8% (TUL 11) and 7.4% (TUL 12).
Recently (TUL 14 and TUL 15), attempted to titer the number of PBMCs
that would show spots when stimulated with LVS. The % RBCs in these
PBMC preps were 0.4 – 0.8%; less background staining was observed.
Data from TUL 10 – 12, TUL 14 and 15 are summarized in Table 2.
Figure 3 shows the cell titration data from TUL 14 and 15.
Table 2: Apparent Effect of RBC Contamination on IFN ELISPOT Spot
Detection
% RBC
Media
A00868
A00902
A00659
A00902
Cells
plated/well
500,000
500,000
200,000
200,000
167,159,140
144,135,107
39,34
207,184,174
LVS FF (1 x
105/ml)
509,528,424
307,330,278
NT
244,161,144
LVS HK (1 x
105/ml)
323,331,349
320,283,244
63,70,25
172,160,155
18.6%
9.3%
5.5%
13.8%
A00896
A00908
A00868
200,000
200,000
200,000
0.4%
0.8%
0.4%
0,5,0
2,3,3
16,14,13
10,14,5
15,18,4
27,22,39
4,3,4
4,3,1
20,13,16
PBMCs from LVS-vaccinated NHPs respond to FF LVS by secreting IFN as detected by ELISPOT
*
Cell Mean for IFNg Spots
25
20
15
1
1.33
.67
.33
.1
*
10
5
*
0
Media
LVS hk Hi
LVS ff Hi
Figure 3: PBMCs (from 3 vaccinated NHPs, TUL 14 and 15) were plated at varying concentrations (x 106
cells/ml) and stimulated with either HK or FF LVS (1 x 105 cells/ml); * = significantly different than media
at that concentration. 1.33 x 106/ml corresponds to 200,000 cells/well.
Data interpretation: High RBC contamination appears to contribute to high backgrounds in the ELISPOT
assay. All the background spots look paler when compared to the spots made in the wells stimulated with
LVS, but are nevertheless being counted with the current settings on the ELISPOT reader. When only
data from TUL 14 and 15 are analyzed (low RBC contamination), the background levels are low and it
becomes clear that FF LVS stimulates a low level of IFN production.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 070207.svd;
20 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Summary data: \\Saturn\Group\Wilder Lab\TVDC\Summary reports\062907 UNM TVD Internal Tech
Minutes Action items jw rev 070507.doc and \\Saturn\Group\Wilder Lab\TVDC\Summary reports\SubNIAID Tech call.7.3.2007 RS&JW.ppt.
Notebook data: TVDC 1 bound notebook pages 30 – 45 (TUL 14 and 15)
c.
Update on B cell artifact
1. NHP whole blood contains a population that stains positively with anti-CD20
antibody and thus looks to be B cells; however, upon purification of PBMCs, this
population disappears or is greatly decreased; we are attempting to find out whether
this is a real loss that we need to be concerned about
2. CD20+ cells are lost in the PBMC population to varying degrees. In the
blood, they do not express surface IgM; in the PBMC preparation, they do
co-express IgM.
3. Tested whether B cells are functional in the PBMC preps by stimulating the
cells with LPS to test their proliferative response; compared to lysed whole
blood stimulated with LPS similarly (TUL 14 and 15). The results are shown
in Figure 4.
LPS responsiveness of NHP Whole Blood vs. PBMCs
Cell Mean for RLU small
1.40E6
1.20E6
Fresh
Whole Blood
1.00E6
8.00E5
6.00E5
*
4.00E5
2.00E5
0
Media
LPS
Figure 4: Lysed whole blood cells or PBMCs (Fresh) were cultured at 1 x 106/ml with LPS (1 g/ml).
Proliferation was measured after 5 days in culture. * p = .04 compared to media stimulation of the PBMC
preparation.
Data interpretation: Despite the loss of CD20+ cells in the PBMC preparation, there is not a signficant
loss of B cell responsiveness to LPS. There is a high background of proliferation when using whole blood
that possibly contributes to the non-significant (p = .06) effect of adding LPS to the culture.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 070207.svd;
Summary data: \\Saturn\Group\Wilder Lab\TVDC\Summary reports\062907 UNM TVD Internal Tech
Minutes Action items jw rev 070507.doc and \\Saturn\Group\Wilder Lab\TVDC\Summary reports\SubNIAID Tech call.7.3.2007 RS&JW.ppt.
Notebook data: TVDC 1 bound notebook pages 30 – 45 (TUL 14 and 15)
21 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
d. Stimulation of NHP PBMCs with recombinant proteins from LVS
1. Recombinant proteins from LVS were provided to us from Dan Clemens;
these proteins (Bfr, KatG, GroEL) were originally selected as stimulatory in
that they stimulated a proliferative response from spleen cells in an LVS
vaccinated mouse (Lee, B-Y., Horwitz, M.A. and D.L. Clemens, Infection and
Immunity 74:4002- 13, 2006)
2. Although the suggested stimulatory conditions were published to be 20 x 106
splenocytes/ml and 10 g/ml protein, we could not spare that many cells;
instead we cultured the cells at 1 or 2.5 x 106 cells/ml and used 10 or 5 g/ml
of each protein
3. Data are shown in Figure 5.
Proliferation of PBMCs from LVS-vaccinated NHPs to LVS recombinant proteins
Cell Mean for RLU small
8.00E5
10 g/ml,
Day
203, 11 x 106/ml
10 g/ml,
Day
203, 2.5
2.5 x 106/ml
Day
195,11x 106/ml
5 g/ml,
7.00E5
6.00E5
5.00E5
4.00E5
3.00E5
*
*
2.00E5
*
1.00E5
0
Media
Bfr
GroEL
KatG
Figure 5: PBMCs (TUL 14 and 15) were stimulated at the indicated cell concentration with LVS
recombinant proteins at the indicated concentration. * p <.05 when compared to Media within the same
stimulation conditions.
Date interpretation: A proliferative response to LVS recombinant proteins was observed and reached
significance when a low number of cells were plated and a low antigen concentration used. However, the
total proliferation increases if either more antigen is used or more cells are plated.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 070207.svd;
Summary data: \\Saturn\Group\Wilder Lab\TVDC\Summary reports\062907 UNM TVD Internal Tech
Minutes Action items jw rev 070507.doc and \\Saturn\Group\Wilder Lab\TVDC\Summary reports\SubNIAID Tech call.7.3.2007 RS&JW.ppt.
Notebook data: TVDC 1 bound notebook pages 30 – 45 (TUL 14 and 15)
4.
Significant decisions made or pending
None
5.
Problems or concerns and strategies to address
None
6.
Deliverables completed
22 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
None
7.
Quality of performance
8.
Percentage completed
Good
80% of scientific work has been completed
9.
Work plan for upcoming month
1. Thaw PBMCs frozen down in experiments TUL 14 and 15 and test in proliferation and
IFN ELISPOT assays if enough cells permit
2. Begin to optimize the conditions of the IFNg ELISPOT assay by testing different cell
numbers plated (between 200,000 and 500,000/well), antibody coating concentrations
and detection antibody concentrations
3. Optimize the IgA anti-LVS ELISA and begin testing banked sera from TUL 8 and TUL
9 provided that the Goat anti-monkey IgA-HRP reagent arrives (it has been on backorder
the last 2 months)
10.
Anticipated travel
11.
Upcoming Contract Authorization (COA) for subcontractors
None
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new lab work done
b. We had previously indicated that we have optimized the T cell proliferation assay for
the mouse: 5 x 104/well nylon wool-enriched T cells and 106/well formalin-fixed LVS
produced the best balance of background, specificity and sensitivity. However,
during a troubleshooting session, Karen Elkins suggested that we should be able to
use many more cells in our proliferation assays without increasing the background.
She suggested that our problem with non-specific proliferation at high cell numbers
may be related to the quality of fetal calf serum (FCS) that we have been using.
Thus, she sent us an aliquot from her FCS stock and we obtained an aliquot from a
reserve lot at Hyclone. We will test these two FCS stocks in parallel with our current
FCS stock in the proliferation assay and macrophage killing assay to find one that
produces minimal background
c. When we have determined the effects of FCS on T cell proliferation, we will apply this
assay to identify peptides from F. tularensis proteins that would stimulate T cells from
LVS-vaccinated BALB/c mice and potentially other vaccinated small animals models
(milestone 27)
d. We will apply this assay to the rat because there is accumulating data suggesting
that Fischer 344 rats may be a better model than the mouse
e. UNM is providing supplies to LBERI
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
23 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month and next 6 months
a. Test three lots of FCS (one from Karen Elkins, one from a reserve lot at Hyclone, and
one from current Hyclone lot at UNM) for background in T cell proliferation assays.
Our goal is to increase the number of cells that can be added to the assay without
increasing the background proliferation. If we can achieve this, then we further
optimize the assay in the mouse model
b. We will develop the T cell proliferation assay for the Fischer rat
i. Develop procedures for isolating T cells from whole blood, spleen, lymph
node
ii. Develop procedures for stimulating T cells with Con A and killed LVS and
SCHU S4
iii. Optimize the T cell proliferation assay
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No work done because no human cells were available during this period
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
Our Bronchoscopy nurse is likely to accrue a donor in July 2007
6. Deliverables completed
NA
7. Quality of performance
Limited progress due to unavailability of human alveolar macrophages
8. Percentage completed
3%
9. Work plan for upcoming month
a. Determine the optimal MOI for infecting human alveolar macrophages. Since we
observed cytopathogic effects at MOI = 1, we will titrate MOI down to 0.1, 0.5, and 1
b. Determine macrophage viability by lactate dehydrogenase (LDH) release and trypan
blue exclusion after infection
c. Determine kinetics of bacterial proliferation after infection
d. Measure cytokine (e.g. TNF, IL-1, and IL-6) production by macrophages infected with
SCHU S4 or LVS
e. Determine whether recombinant IFN would inhibit SCHU S4 and LVS intracellular
growth
24 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
f.
Determine whether PBMC from vaccinated human volunteers can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30.9c (Notebook 101, pages 34-38)
i. The purpose of the experiment was to determine whether mouse
macrophages differentiated with 100 ng/ml recombinant M-CSF instead of L929 conditioned medium can be used to measure T cell induced macrophage
killing of intracellular LVS
ii. Macrophages were infected with LVS at MOI of 1:40 (LVS:macrophages)
and, after 3 days, the number of macrophage-associated LVS and free LVS
in the media were determined
iii. Addition of 5 x 105 nylon wool enriched naïve splenocytes had little effect on
the total (macrophage-associated + medium associated) LVS in the culture.
However, addition of the same number of vaccinated splenocytes
significantly reduced the total LVS in the culture (P < 0.05 by one-way
ANOVA with Bonferoni posttest; Figure 5). This is attributed to a reduction of
LVS in both fractions.
iv. Addition of 5 x 104 naïve or vaccinated splenocytes had no significant effect
on the total LVS in the culture (P > 0.05)
v. Although the results are encouraging, the large number of free LVS in the
media (> 104 CFU) is disconcerting. Karen Elkins also finds extracellular
LVS, but the number is usually negligible compared with intracellular LVS.
She suggested that our macrophages may not have been healthy and
allowed LVS to escape prematurely into the medium.
25 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 5. Inhibition of LVS by vaccinated mouse splenocytes. LVS-infected bone marrow
derived macrophages were incubated with nylon wool enriched naïve or vaccinated
splenocytes. After 3 days, the number of LVS in the media and inside the macrophages was
determined by plating the medium and the lysed macrophages, respectively.
b. Experiment Ftc30.8b (Notebook 101, pages 39-44)
i. The purpose of this experiment was to compare the amount of M-CSF in the
L-9292 conditioned media from the Elkins and the Lyons lab. This will help
us determine whether our inability to generate quality macrophages is due to
the amount of M-CSF. It will also give us the optimal concentration of
recombination M-CSF that should be used to differentiate macrophages
ii. We used the Quantikine Mouse M-CSF ELISA kit from R&D Systems
iii. Our results indicated that the L-929 conditioned media from the Lyons lab
and the Elkins lab contained 5.4 ng/ml and 1.7 ng/ml M-CSF, respectively.
Thus, the amount of M-CSF does not appear to be the cause of our problem.
iv. This result also suggest that we have been using too much recombinant MCSF to induce macrophage differentiation (61.6 ng/ml vs. 0.17 ng/ml after
diluting the L-929 conditioned medium 1:10 into the working medium).
v. We are titrating the amount of recombinant M-CSF to determine the optimal
concentration for differentiating macrophages.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
The quality of macrophages has to be improved.
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
12%
26 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
9. Work plan for upcoming month
a. Compare three lots of FCS (one from Karen Elkins, one from a reserve lot at
Hyclone, and one from current Hyclone lot at UNM) for ability to support robust
macrophage differentiation and maintain macrophage viability
a. Determine the optimal MOI for LVS and SCHU S4 infection of macrophages
b. Determine whether vaccinated splenocytes can induce BMM to kill intracellular LVS
c. Determine whether vaccinated splenocytes can induce BMM to kill intracellular
SCHU S4
d. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. We are reconstructing the IVT template to both streamline and double tag with 6x
His. The streamlining rearrangement is complete. For the double His tagging,, 2
single tagged His templates are being made; one with an N-terminal and the other
with a C terminal tag. This will permit directed amplifications.
2. We have generated 1712 and 1602 with the C-terminal constructs. We have good
yields but did not purify. Since these were not able to bind the Nickel beads, we will
try the N-terminal constructs on these polypeptides for purification.
B. Select and test IVT Protocols
27 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
1. As discussed in the last conference call, we tested our protocol on 4 FTU samples.
Normalized cpm calculations show that yields from FTU and control samples are comparable.
In all reactions, 1 ug of linear template was used and the reactions were supplemented once
with feed at 1hr. The feed reagent was supplied in the standard kit as merely extra buffer (at
no additional cost). One template supplemented was made at 4 h.
Table 1: Assessing yields of different FTU polypeptides
Template
Total CPM
CPM/met/10^6
FTU 1419 (p11)
482,867
0.489
FTU 1602 (p12)
332,867
0.229
FTU 1695 (GroES)
457,167
0.420
FTU 1712 (IglC2)
300,000
0.213
CalM3
639,733
0.755
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data.
File name: Invitrogen IVT test with FTU template 5-21-07
2. We concluded that we can substantially reduce the cost of performing these reactions by
reducing the number of feed supplements to one, and adding template only once. This can
be accomplished because we have determined when the reaction spends out these
components. The additional substrate and ATP in the “feed” are optimally added between
0.5 and 1h after reaction initiation. The DNA expression template is optimally added between
4 and 6 h after reaction start.
C. Select and test protocols for protein purification
1. To assess the need for purification relative to the endpoint-use of the polypeptides,
we are preparing several pilot samples, independent from the ones shown above.
Unpurified and buffer exchanged, and affinity purified samples will be delivered to
UNM as acetone precipitates for use in development of their T cell assays. These
samples are described in table 2 below.
Table 2: Test samples to be delivered to UNM
TEMPLATE
LYSATE
ACETONE->PBS
None
unpurified
buffer exchange
GFP
unpurified
buffer exchange
FTU 1419
unpurified
buffer exchange
FTU 1695
unpurified
buffer exchange
FTU 901
unpurified
buffer exchange
FTU 1602
buffer exchange
FTU 1712
buffer exchange
NI+ PURIFIED
purified
purified
purified
purified
purified
2. We have analyzed the 1419 (p11), 1695 (GroES) and GFP samples by SDS-PAGE.
These are shown in Figure 1. Samples were loaded by volume, not cpms. We
conclude that the products show no signs of degradation, and are predominantly fulllength, migrating at the expected molecular weights. Faint, lower molecular weight
bands in the unpurified lanes are likely to correspond to specific, early termination
products, since they are regular in size and are removed by C-terminal His
purification. Positions of non-radioactive, colorimetric markers were dotted on sides
of gel. Sizes are indicated.
28 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 1.
\\peptide\Research\CIM\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal's IVT gels\IVT 6-2107br200
3. In preparation for mailing these samples to UNM, we have determined the curies in each
sample, and calculated the amount of product in each. These are presented in Table 3
below.
4. The purified protein yields range from ~7ug to 16ug from the 3 originally selected samples
(1419, 1694, GFP).
5. We include TUL4 (901), which had a marginal purified sample yield of 2.3ug, because of its
interest as a T cell antigen. Unpurified sample yields range from 34ug to 140ug.
6. We will also include 2 additional samples, 1619 (4.1ug) and 1702 (2.3ug), that were acetone
precipitated from the total lysate reaction, but not purified.
7. Urea-exposed preps #2 and #3 have been acetone precipitated, preps #1 are unmanipulated
lysates and will be shipped frozen.
8. Preps #2 and #3 will be resuspended in DMSO at different concentrations for T cell testing.
9. Hetal has contacted Ralph Becker at the UNM Safety Health and Environmental Affairs Office
regarding mailing and receipt of radioactive samples.
29 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Table 3. Amount of radioactivity and calculated amount of protein in samples for UNM.
Sample
ug Protein
Total uCi
FTU 901 (TUL4)
1
2
3
IVT lysate (frozen)
Acetone Precipitated
Purified and ppt
69.36
60.98
2.27
4..0
0.114
0.004
1
2
3
IVT lysate (frozen)
Acetone Precipitated
Purified and ppt
39.60
140.64
7.38
4.386
0.912
0.048
1
2
3
IVT lysate (frozen)
Acetone Precipitated
Purified and ppt
101.19
1066.33
12.08
3.246
12.752
0.144
1
2
3
IVT lysate (frozen)
Acetone Precipitated
Purified
34.00
88.53
16.26
1.216
0.809
0.148
FTU 1419 (p11)
FTU 1695 (IglC2)
GFP
1602
Acetone Precipitated
4.12
0.018
Acetone Precipitated
2.284
0.021
1712
No Template
1 IVT lysate (frozen)
4.723
2 Acetone Precipitated
1.285
\\peptide\Research\CIM\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT\FTU IVT
samples for ship 7-06-07
4. Significant decisions made or pending
a. His/Nickel-based purification may require double tagged polypeptides and partial folding
for optimal binding and release, respectively.
b. If raw or buffer-exchanged lysates work in T cell assays at UNM, then no purification will
be necessary.
5. Problems or concerns and strategies to address
We believe we have addressed the elution problem in our purification steps for the His tag/nickel
bead strategy, by maintaining constant urea concentrations through binding and elution steps.
We are testing the utility of including a His tag at both ends of the molecule, so as to enable the
use of reduced urea concentrations without reduced tag exposure. Partial folding will prevent
exposure of very hydrophobic, and thereby sticky, regions. This should improve elution
efficiency.
6. Deliverables completed
None
7. Quality of performance
Very good
30 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
8. Percentage completed
97%
9. Work plan for upcoming month
a. We are sending UNM 3 FTU and 1 non-FTU polypeptides prepared 3 different ways as
soon as paperwork is in place.
b. We will be using the products from the double-tagged His templates to testing the
efficiency of product binding to nickel beads in the presence of less urea (2M, 4M). We
are also using small columns for these steps rather than free beads to reduce sample
loss.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new lab work done
b. One of the impediments we have encountered in this milestone is the lack of bona
fide positive control proteins or peptides that we can use to develop the peptide
screening assay. We have recently obtained purified GroEL, KatG, and Bfr proteins
from Dr. Dan Clemens, who reproducibly showed that these proteins stimulated
antigen-specific T cell proliferation. Dr. Julie Wilder also found that these proteins
stimulated PBMCs from vaccinated monkeys. We will soon use these proteins to test
splenocytes from vaccinated mice and rats.
c. ASU is also synthesizing long peptides from F. tularensis proteins, such as Tul4, that
are known or hypothesized to stimulate antigen-specific T cell proliferation.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
No progress
8. Percentage completed
10%
9. Work plan for upcoming month
a. Test three different lots of FCS for the one that will allow for the highest number of T
cells we can include in the assay. This may increase the sensitivity of our assay
b. Develop the peptide screening assay using the purified GroEL, KatG, and Bfr as
positive controls
c. Determine whether increasing the number of T cells and/or APC would be better for
this peptide screen
31 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
d. Determine whether IFN ELISpot assay would be better than T cell proliferation
assay for this peptide screen
e. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
f. Assemble a list of stimulatory peptides for ASU to analyze for common stimulatory
motifs
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of peptide libraries (Optimize IVT protein-fragment
production, Develop IVT protocol for high-throughput production, Validate immunogenecity
of protein-fragments, Full scale production of protein-fragment library, Purification of proteinfragment library, Array protein-fragment into overlapping pools, Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)

Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1. We have almost completed a “first pass” of the natural sequence ORFs. These are the ORFs
that our algorithms indicate should be efficiently and specifically amplified. Very
encouragingly, no amplification hurdles were encountered among these PCR-fragments,.
Based on calculations of the total amount of DNA estimated to be needed (5ug), 75% are
complete. The remaining yields will be increased by performing a “second pass” set of
reactions.
2. PCR products were evaluated in a digitized gel system that enables fast, accurate
visualization and analysis of 96 samples simultaneously.
3. This enables electronic quality control.
4. All results are automatically entered into electronic management.
5. Raw data of duplicate samples run on an agarose e-gel are shown in Figure 2.
32 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 2
\\peptide\Research\CIM\GeneVac\FTU\chemidoc\ FTU WT test83FINAL E-gel
6. The raw results are scanned and electronically “cut” so as to be arranged by microtiter plate
locations. An example of this rearranging is shown in Figure 3.
33 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 3
\\peptide\Research\CIM\GeneVac\FTU\chemidoc\ FTU WT test83FINAL E-gel
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
The protocol for synthesis of recoded ORFs is likely to take some development, but no major
impediments are expected. In particular, we will be testing the new Agilent chips in our protocols.
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
16%
9. Work plan for upcoming month
Chip synthesized oligos will be received and sets of initial recoded, synthetic ORFs will be
assembled, and tested in IVT reactions.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
34 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
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

Reconstruction RNA samples are prepared with purified RNA from SCHU S4 bacteria diluted
into normal mouse lung RNA. These are then amplified via LAPT with genome-directed
primers (GDPs).

We had some initial problems if the mouse-lung RNAs were isolated by Tri-Reagent RNA
extraction only. All RNA needs to be subsequently purified over RNAeasy columns.

Previous samples (LAPT-7) were hybridized to arrays and used to test in the Maui
hybridization system (used in QC for next milestone).

RNA samples from LAPT 8 have been processed for labeling and hybridizations. We
performed some additional dilutions in an attempt to better define the lower level of detection.


Notebook/File locations … LAPT-8 Notebook 514, pages 7-17;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-8
35 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 1. Un-normalized raw signal intensities of amplified SCHU S4 RNA after LAPT amplification
The raw signal intensities of reconstituted samples show a lowering of overall signal intensities after
dilution to 0.1 g. However, the overall expression pattern was maintained down to 0.001 g. Spearman
correlations between the amplified 1.0 and other samples indicate a very strong correlation on through to
0.001 g. Utilizing a 1000 raw intensity cut-off, the number of detectable genes dropped from a high of
1,400 in the 1.0 g sample to 335 in the 0.001 g sample. However, the average background on these
slides is routinely very low (200-400) and we should be able to use a lower level cutoff to increase the
number of detectable genes. This result indicates that we will be able to amplify and detect as little as
0.001 g of SCHU S4 RNA in infected lung samples
Table 1. Spearman correlations and number of genes > 1000 raw intensity values after LAPT.
# genes > 1000 raw signal
g SCHU S4 amplified
Spearman Correlation to 1.0 g sample
0.5
.797
994
0.1
.648
359
0.05
.667
609
0.01
.722
494
0.001
.744
335
36 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
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
92%
9. Work plan for upcoming month
Perform one additional dose response of reconstitution samples to determine if samples less than
0.001 g can be detected. Dose responses will include from 0.01, 0.005, 0.001, 0.0005, and
0.0001,.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34-UNM
Milestone description: Pilot Studies for the optimization of RNA isolation and hybridization
conditions
Institution: UNM
1. Date started: 03/01/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc45 (Notebook 104, pages 54-55, 64, 68-74)
i. The purpose of this experiment was to isolate RNA from SCHU S4-infected
mouse lung for ASU to measure the sensitivity of their F.tularensis
microarray
ii. BALB/c mice (n = 5) were infected i.n. with 3 x 102, 2 x 103, 5 x 104, 5 x 105,
and 4 x 106 SCHU S4
iii. 4 h after infection, 2 mice were killed to determine the lung burden and 3
mice were killed for RNA isolation using Tri Reagent (Ambion)
iv. The RNAs have been transferred to ASU for usage on MS35
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
12%
9. Work plan for upcoming month and next 6 months
UNM will isolate RNAs from LVS, SCHU S4,and infected mouse organs, as needed by ASU.
37 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 34-ASU
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions





We utilized samples from LAPT-7 and compared a reconstituted sample (0.1 µg SCHU S4
RNA in 10 µg Mouse lung RNA) on both the Maui and ArrayIt platforms. In this last
comparison, there was no significant increase by raw signal intensity by using the Maui
mixing system. (Figure 1)
Figure 1 (below)
We performed a comparison of the ASU and TIGR Francisella arrays. We labeled 10
micrograms of unamplified SCHU S4 RNA and hybridized equal amounts to either the inhouse Poly-L-Lysine or TIGR arrays. Comparison of the raw image file is shown in Figure 2.
Notebook/File locations … ASU v TIGR, Notebook 405, page 25;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\34\ASU_TIGR_1.
38 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 2. Acquired image of 5 g SCHU S4 RNA hybridized via static ArrayIt chamber to either in-house
ASU PLL microarray slide or TIGR array on Corning slide

The acquired images were processed in GeneSpring and histograms of both raw and
normalized data are shown in Figure 3. There was a broader range of signal intensities with
the ASU PLL slides. This result is similar to the comparison of the ASU array when
processed onto Corning Ultragap slides. The TIGR arrays are provided on Corning Ultragap
slides. The normalized signal intensities also had a broader range of signals. However, as
shown by the color map in the normalized image, the correlation between the ASU and TIGR
arrays is very poor. If correlations were high the TIGR side of the image would be colored
similarly to the ASU side of the array with red signals at the top of the histogram, yellow in the
middle, and blue on the bottom.
39 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 3. Histogram plots of raw and normalized signal intensities comparing ASU and TIGR arrays

The differences between the TIGR and ASU arrays was explored further to look at the
extremes of the genes that were > 8 fold different between the ASU and TIGR arrays. Thirty
eight genes were identified as being detected as oppositely regulated solely by the source of
the array (Figure 4).
40 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 4. Greater than 8-fold difference plot between ASU and TIGR arrays and histogram plot showing
the placing of these 38 genes at polar opposites between ASU and TIGR arrays.
4. Significant decisions made or pending.
The Maui Hybridization system does not consistently provide enhanced signal detection.
5. Problems or concerns and strategies to address
Differences exist between the genes identified by the TIGR as compared to the ASU array.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
60%
9. Work plan for upcoming month

We are planning on limited studies to begin to assess some of the differences by either
quantitative PCR or by acquiring a custom Agilent arrays to increase the number of probes
per gene to identify key differences between platforms.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
41 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent Schu 4
infection & RT PCR confirmation of candidates.
Institution: UNM/ASU-Johnston
1. Date started: 06-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

We have received 15 RNA samples from UNM of a dose-response SCHU S4 ?challenge
with mouse lung harvest 4 hours post challenge. PLEASE ADD THE UNM EXPERIMENT
Identifying number here so that the source of the RNA can be tracked. Each lung was
processed for total RNA isolation at UNM using TriReagent. At ASU, the RNA samples have
started being processed over RNAeasy columns for cleanup.
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%
9. Work plan for upcoming month
LAPT process will start on the 15 new RNA samples from UNM. During clean-up we will create a
pool of each challenge dose for initial testing. The samples will be subsequently process for the
LAPT process for hybridization to the ASU array.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. 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
Summary: We have determined that all the NER-deficient strains of Ft. novicida are slightly
more sensitive to photochemical inactivation than wild type Ft. novicida. We have optimized
photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and produced a
lot of KBMA uvrB Ft. novicida for potency testing in MS42. We have demonstrated that
KBMA Ft. novicida are highly attenuated for virulence. We are in the process of testing the
42 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
stability of a frozen KBMA lot of uvrB Ft. novicida at –80oC and have demonstrated that
metabolic activity is retained for 3 months.
Because the inactivated NER-deficient strains have a similar degree of metabolic
activity as the wild-type Ft. novicida strain (which is different than has been seen with L.
monocytogenes or B. anthracis) we have initiated a series of experiments to determine the
cause of this observation. There are 2 obvious and distinct possibilities 1) is that the NER
genes are not turned on during photochemical inactivation with S-59 and UVA light or 2)
there may be a redundant mechanism for repair of DNA damage that prevents inactivation of
the uvr mutants at low S-59 concentrations.
1) We have evaluated the sensitivity of the uvrB mutant and U112 to 6 alternative DNA
damaging agents: S-303 (another synthetic psoralen that is not activated with UV-light),
mitomycin C, cisplatin, doxorubicin hydrochloride, benzo[a]pyrene, and 4 nitroquinoline-N-oxide
using a 96-well format minimum inhibitory concentration (MIC) assay. Briefly: U112 and uvrB
mutant Ft. novicida were incubated overnight in 15 ml CDM. In the morning, cultures were diluted
1:100 in fresh CDM. Serial 1:4 dilutions of chemicals were made in 96 well plates and 50 ul/well
were transferred to duplicate 96 well plates to which 100 ul/well of U112 or uvrB were added.
The OD600 nm was read at Time 0 and again after incubation at 37oC for 16h without shaking.
Due to dramatic differences in solubility, the dose ranges of each agent varied significantly, but in
total, 4 of the DNA damaging agents were added at concentrations that inhibited growth of the
bacteria. Of the 4 agents that inhibited growth of Ft novicida, only 2 inhibited growth of the uvrB
mutant at lower concentrations (S-303 and 4 nitroquinoline-N-oxide shown below). NB 920-180
OD600 nm
um
1/1
no
yes
yes
no
0
50
.2
20
00
4 NitroquinolineDifference
Growth
inhibition
yes
no
no
yes
yes
yes
between
U112 and uvrB
83
.3
52 13
0.
8
13 28
0.
20
32 7
.5
52
8.
13
8
2.
03
4
0.
50
9
0.
12
7
U112 uvrB T 16h
33
3.
33
13
30
U112 uvrB T 0
H2O
hot acetone
0.
0
33 0
25
.0
0
83
1.
25
20
7.
81
51
.9
5
12
.9
9
3.
25
0.
81
0.
20
0.
05
0.
01
0.83 ug/ml
50000 ug/ml
3333 ug/ml
33333 ug/ml
0.3
0.2
media/ethanol
0.1
DMSO 0
83
OD600
Mitomycin
4 nitroquinoline-N-oxide
S303
Highest
Solubility
0.8
0.7
concentration
Solvent
in media
13300 uM
media0.6
yes
0.5
U112 T 0
33.3 ug/ml dichloromethane
no
0.4
U112 T 16h
33
DNA damaging
agent
0.9
0.8
0.7
S-303
0.6
0.5 pyrene
Benzo
0.4
Doxorubicin
0.3
0.2
hydrocholoride
0.1
Cisplatin
0
minor
no
no
U112 T 0
U112 T 16h
uvrB T 0
uvrB T 16h
no
no
minor
ug/ml
The fact that there were either no or only minor differences in sensitivity to DNA damage between
the uvrB mutant and wild type Ft novicida demonstrates that this phenomenon is not unique to
photochemical inactivation with S-59 and UVA and may suggest that there is a redundant
mechanism for repairing or preventing DNA damage in Ft novicida. As a next step, the MIC
assay will be repeated with a smaller dose range using 2-fold serial dilutions. We will then
measure uvrB transcriptional response after DNA damage. We have previously designed, and
tested the specificity of 2 pairs of oligonucleotide primers for amplification of the uvrB gene for
reverse transcriptase quantitative real time PCR (rt)qPCR (qPCR).
43 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
4. Significant decisions made or pending
All NER mutants (uvrA, uvrB, and uvrA uvrB) of Ft. novicida were equally sensitive to S-59
and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 7J/cm 2 as the
conditions for making 400ml-scale KBMA lots, and have produced a lot of KBMA uvrB Ft.
novicida vaccine that is sterile for further characterization. We have decided to open MS 42 in
order to determine whether KBMA Ft novicida can protect against a lethal wild-type Ft novicida
challenge.
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms. This
appears to hold true for other methods of induced DNA damage. One possible explanation
for this is that there is a redundant DNA repair mechanism functioning in Ft novicida that may
limit the sensitivity of the NER-deficient mutants to DNA damage and thereby limit the
metabolic activity and potency of KBMA Ft novicida.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
Good progress
8. Percentage completed
80% of scientific work completed on the milestone
9. Work plan for upcoming month
We will repeat the MIC assay comparing the sensitivity of uvrB and U112 to S-303, cisplatin
and mitomycin C using 1:2 serial dilutions. We will also compare the sensitivities of the
uvrB mutant and WT LVS to Ft novicida to determine whether this is unique to the novicida
subspecies. We will determine whether the uvrB gene is induced in the wild type by rtqPCR
after DNA damage at a concentration that inhibits growth of the uvrB mutant but not U112.
We will also compare the uvrB gene induction to treatment with S-59 and UVA.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: KBMA Ft novicida uvrB vaccine stocks produced in MS41 have been tested in mice
for virulence and protection against a 100 x IP LD50 challenge of Wild-type Ft novicida. KBMA Ft
novicida uvrB were 100% protective when a single dose was administered at or near the LD 50 of
the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also achieved by administration
of 1 x 107 KBMA particles IV when the vaccine was given twice separated by 3 weeks. Depletion
of CD4+ T cells prior to the challenge decreased the survival rate to 80%, depletion of C8+ T cells
had no effect, and depletion of both cell populations resulted in 90% survival. Together, these
data demonstrated that CD4 T cells contribute to a protective immune response in a non-CD8 T
cell-dependent manner. These data suggest that the CD4 T cells may be boosting humoral
44 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
immunity by stimulating B cells. This interpretation was supported by an adoptive transfer
experiment in which only the high-titer serum from CD8-depleted animals provided any protection
against a lethal U112 challenge. Together these data demonstrate that the protection we see
after vaccination with KBMA Ft novicida uvrB correlates with humoral immune responses and
explains why the KBMA vaccine does not perform better than heat killed vaccine. This also
makes it nearly impossible to rank attenuated Ft novicida mutants by their ability to protect mice
against a lethal challenge. We instead plan to evaluate the ability of KBMA vaccines to induce a
potent CD8 T-cell response to an introduced ovalbumin epitope tag and are awaiting the
construction of this strain from UTSA.
4. Significant decisions made or pending
We have decided to evaluate the potency of the KBMA Ft novicida vaccine by measuring the
CD8 T cell response to an ovalbumin epitope tag.
5. Problems or concerns and strategies to address
Because humoral immunity plays a significant role in protection of mice against a lethal Ft
novicida challenge it will make it difficult to rank KBMA vaccine candidates that elicit a potent
T cell response by survival after a lethal challenge in MS 43. We have requested that Karl
Klose construct an ovalbumin epitope-fusion protein to facilitate screening strains of Ft
novicida for their ability to elicit a T cell response to this well-defined epitope.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We will wait for delivery of the ova-tagged strain of uvrB from Karl Klose to determine
whether KBMA Ft novicida can induce a potent CD8 T cell response
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
T-cell epitope tagged protein.
A new focus of this milestone is to create a T-cell epitope tagged protein that is expressed by F.
tularensis within host cells. Ideally, the protein should be secreted into the host cell. The only wellcharacterized secreted protein is PepO, and the Tcell tag is SIINFEKL. In consultation with Justin
Skoble, we are creating a plasmid to express PepO-SIINFEKL to transform into F. tularensis, then send to
Cerus. SIINFEKL is derived from a model MHC class I-restricted antigen, OVA, and there are Tcell
clones that specifically recognize SIINFEKL bound to MHC-I. Thus if F. tularensis expresses SIINFEKL,
then it can be determined how well MHC-I presentation occurs in F. tularensis-infected cells/animals. We
already have a plasmid, pKEK1145, which is a pBAD24 derivative, that expresses PepO- Flag. A pair of
complimentary oligonucleotides encoding SIINFEKL will be used to replace the FLAG tag fragment in
pKEK1145.
45 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
3.1 We have another plasmid pKEK1149, which has been inserted with Francisella
promoter and expresses PepO-Flag. The plasmid pKEK1149 with FLAG being
replaced with SIINFEKL can be transformed into F. tularensis directly, whereas
the plasmid pKEK1145 with SIINFEKL insertion needs to be digested to get PepOSIINFEKL which will be inserted into another plasmid with Fransicella promoter.
3.2 The same procedure described in last technical report was performed. The plasmid
pKEK1149 was double digested with XhoI and PstI restriction enzymes. PCR
product SIINFEKL was ligated into the digested plasmid pKEK1149, then the mutant
plasmid pKEK1149 was transformed into DH5 E.coli. competent cells. The
transformed cells were spread onto LB/Tetracycline(10ug/ml) plate and incubated at
37C for overnight.
3.3 Only one colony grew on LB/Tetraycline plate. Performed mini prep for this colony with QIAprep Spin
Miniprep Kit.
3.4 Screened this colony with PCR for SIINFEKL insertion. The positive PCR product should be about
300bp. Set up pKEK1149 for negative control and one of the pKEK1145 inserted with SIINFEKL for
positive control.
32.6ul ddH2O
5.0ul 10xBuffer#1 for KOD
5.0ul KOD dNTPs
2.0ul MgCl2
1.0ul mini prep DNA
2.0ul PepO For primer
2.0ul SIINFEKL Rev primer
0.4ul KOD HiFi DNA polymerase
At 98ºC 1min, 98ºC 15sec/57ºC15sec/72ºC 1min//30cycles
Gel picture: Figure1
1
1kb
2
3
4
1 1kb ladder
2 pKEK1149(SIINFEKL) mini prep
3 pKEK1145(SIINFEKL) mini prep
4 pKEK1149
0.5kb
Data recorded on UTSA TVDC notebook #2, page 107-108 for the figure above.
3.5 The gel picture showed that there was SIINFEKL insertion in the plasmid pKEK1149. In order to
confirm SIINFEKL insertion in the plasmid pKEK1149 and pKEK1145, pKEK1149(SIINFEKL) mini
prep DNA and pKEK1145(SIINFEKL) mini prep #1 DNA were sent out for sequencing with “PepO
For” primer.
3.6 The sequence results confirmed that SIINFEKL genes had been inserted into the plasmid pKEK1149
and pKEK1145.
3.7 Western blot has been done once for pKEK1149(SIINFEKL) with anti-SIINFEKL antibody from cells
culture supernatant sent by University of Massachusetts Medical School to see if SIINFEKL protein
46 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
was expressed, but it didn’t work well. OVA will be purchased and used as the positive control for
further western blot.
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
Approximate 40% of scientific work completed on the milestone for T-Cell Tagged protein project.
Up from 30% on this T cell tagged project reported last month. uvrB mutant
completion was 50% of the original milestone.
9
Work plan for upcoming month
i.
ii.
Continue doing western blot with the positive control OVA to detect the expression of
SIINFEKL protein.
Once we know that the SIINFEKL protein is expressed, the plasmid inserted with SIINFEKL
will be transformed into F. tularensis.
10 Anticipated travel
None.
11 Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
2 stabs of uvrB LVS mutant were received from UTSA. Both were streaked out on CHAH agar
for single colony isolation and then single colonies were inoculated into CDM and grown
overnight and suspended in freezing medium and stored at -80oC for frozen stocks.
4. Significant decisions made or pending
none
5. Problems or concerns and strategies to address
none
6. Deliverables completed
none
7. Quality of performance
none
8. Percentage completed
none
9. Work plan for upcoming month
The sensitivity of the LVS uvrB mutant to photochemical inactivation with S-59 and UVA light will
be determined and compared with the wild type LVS strain using the 3.5 mL inactivation process.
47 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
The MIC assay with alternative DNA damaging agents will also be performed with LVS and the
uvrB mutant.
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 LVS
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
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route. We have produced a 400mL lot of KBMA wild-type LVS using
10 uM S-59 and 6 J/cm 2 UVA for initial proof of concept studies, and for later comparison with
NER-deficient uvrB LVS and we have demonstrated that the metabolic activity of this lot is
stable for 3 months. We have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which
represents a 4-5 log attenuation compared with live LVS. We have demonstrated that doses
of KBMA WT LVS as low as 1 x107 provide protection against 100 x IP LD50 challenge of live
LVS. However, none of the mice vaccinated with the equivalent doses of HK LVS died either.
This is consistent with protection against an LVS challenge being largely humoral. Our
prediction is that the KBMA LVS may be more potent than HK in a SchuS4 challenge model
that was initiated at UNMHSC. We also anticipate that a uvrB mutant may more potent than
WT LVS.
We recently attempted to measure the T-cell response to a CD4 Tul4 epitope in mice vaccinated
with live or KBMA LVS by intracellular interferon-gamma (IFN-) cytokine staining (ICS) or
ELISpot assay, but were unable to detect an induced response to this epitope. This may be
because this epitope does not bind the MHC molecule with high affinity, or the T cell response
elicited by LVS may actively suppress T cell responses.
1) Protective immunity to intracellular pathogens requires the lytic activity of antigen-specific
CD8+ T cells. The priming of a protective CD8+ T cell population following vaccination requires
the combination of antigen presentation, cytokines, and CD4+ T cell help. Recent data from our
lab using another intracellular pathogen, Listeria monocytogenes (Lm), has shown that
attenuated strains of this bacterium which do not elicit protective CD8+ T cell responses are not
simply non-immunogenic, but actively suppress the CD8+ T cell response. The attenuation of
these Listeria strains limits their escape into the host cell cytosol. Because LVS demonstrates a
48 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
delayed egress from the phagocytic vacuole relative to other intracellular pathogens, (40 minutes
versus <1 minute for Listeria), we questioned if LVS would have a similar negative impact on
inflammation and CD8+ T cell priming.
C57BL/6 Mice were vaccinated IV with 1x105 Lm expressing the ovalbumin epitope SIINFEKL,
1x103 live LVS, or both. 4 and 24hours later, serum sample were obtained for cytokine analysis
and 7 days later spleens were harvested for ICS and ELIspot analysis. At these doses, LVS does
not induce IL-6 or MCP-1which are critical hallmarks of a protective inflammatory response.
Furthermore, co-vaccination with LVS decreased the innate inflammatory response to Lm.
IM07-067
24H MCP-1
24H IL-6
H
LV
S
LV
S
ac
tA
ac
tA
+L
VS
0
1e
5
H
B
SS
0
1000
ac
tA
ac
tA
+L
VS
50
2000
1e
5
100
3000
B
SS
MCP-1 @ 24 HPI (pg/mL)
IL-6 @ 24 HPI (pg/mL)
150
ac
tA
+L
VS
0
6
4
2
0
ac
tA
+L
VS
5
8
ac
tA
10
%LLO190-201 specific CD4+ T cells
15
ac
tA
%OVA257-264 specific CD8+ T cells
After 7 days post-vaccination, the magnitude of the Lm-induced T cell response to either an
endogenous CD-4 Lm epitope from listeriolysin-O (LLO) or to the CD-8 Ova epitope were not
decreased by co-infection of LVS with LmOva. However, administration of LVS appeared to
decrease the ability of the elicited T cells to produce the cytokine IL-2.
60
50
40
30
ac
tA
+L
VS
20
ac
tA
IM07-067
%IL-2+ of LLO190-201 -specific
IFN-+CD4+ T cells
IL-2 production within LLO190 specific CD4s
49 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been to investigating the efficacy of a wild-type KBMA LVS vaccine.
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS appears to be independent of whether the vaccine
has metabolic activity. This suggests that comparison of various routes, regimens, or
formulations will be difficult to optimize by protective efficacy. Thus, a SchuS4 challenge model
may be more appropriate. The suppression of the innate inflammatory response and the
suppression of CD4 T cell cytokine production may potentially indicate that LVS is not a potent
inducer of protective T cell responses.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
50% of scientific work completed on the milestone
9. Work plan for upcoming months
We will compare the photochemical inactivation profile of a uvrB mutant LVS and their
metabolic activity in MS44. We will measure the mouse cytokine response after vaccination
with LVS in order to characterize the innate immune responses that correlate with a
protective vaccination.
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
In order to generate mutants in SCHU S4 we need to develop tools to generate successful
deletions. Therefore, our focus is two fold, one is cloning experiments to get our target deletions
into vectors that we can use in creating these deletions and experiments with SCHU S4 itself
using constructs that we believe will allow us to make deletions into SCHU S4.
I. Cloning
a. The new transformation performed to generate the igLD construct was not successful.
This resulted in only one colony per igLD transformation and these were not correct.
Data located in TVD UTSA notebook 5, page 33.
50 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
b. The transformation with the re-amplified pDS132 creating a new multiple cloning sight
(MCS) generated hundreds of colonies. Isolated plasmids from 20 colonies using the
Qiagen Mini plasmid isolation kit.
c. Digested these preparations along with the parent vector pDS132 with various
enzymes including EcoRV, Not I and Bgl II. The EcoRV will cut the plasmids in two
locations creating a profile that will differ between the parent and the desired construct.
In addition, the other two enzymes are actually restriction sites that have been introduced
into the new construct which should cut the correct plasmid and not the parent plasmid.
d. Found one transformant that gave the correct profiles (C6 pDS132+new MCS); refer
to figure 1 for further description.
Figure 1.
This figure represents three sets of restriction endonuclease digestion profiles resulting when using the
plasmid pDS132 and a desired plasmid containing a new MCS (C6 pDS132+nMCS) with EcoRV
(lanes 3 and 4); Not I (lanes 5 and 6) and Bgl II (lanes 8 and 9). Lanes 2 and 10 are uncut pDS132
and lane 7 is uncut C6 pDS132+nMCS. The correct clone will yield 3298 bp and 1564 bp fragments in
the EcoRV profile compared with original plasmid yielding 1988 bp fragment size for the second band.
In addition, the Not I and Bgl II digestions should linearize the correct plasmid which contains a new
MCS whereas; the original plasmid will not be cut by these enzymes. Lanes 3, 5, and 9 are pDS132
profiles and lanes 4, 6 and 8 are C6 pDS132+nMCS profiles.
e. The sequencing of the pdpA construct into KEK962 verified the correct construct (C1962+pdpA::Kan). This was used in a transformation experiment into Schu S4 KKT1
strain mentioned in earlier report. (see next section for more description).
II. Experiments to generate deletions in Schu4:
a. The erythromycin sensitive pdpA transformants mentioned in last report were
analyzed by PCR using the oligos Before pdpA Forward Mfe I and After pdpA Reverse
KpnI (described in last report). The correct DNA fragment should yield a larger size
compared with the parent. Of the 19 colonies screened sixteen of them appeared to be
correct. Figure 2 below represents 9 of these 19 colonies screen by PCR.
51 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 2.
This figure represents the resulting PCR products when using pdpA specific primers to help screen
for integration of the pdpA::Kan (FRT) deletion fragment into Schu S4 KKT1 strains clones resulting
in a transformation with the pdpA deletion plasmid (lane 3). Lane 4 is the negative control KEK962
plasmid and lanes 5 through 13 represents various pdpA::Kan KKT1 clones isolated from the
transformation experiments. Lanes 5, 6, 11, 12, and 13 seems to be correct compared to the wild
type product in lane 2.
b. These pdpA clones will be further evaluated on next month’s report. This pdpA
construct appears to be integrated into the chromosome of Schu S4. We need to
determine which pathogenicity island the integration has occurred (FPI or FPII). Thus,
we will perform additional PCR experiments and send some of these products for
sequencing to make this determination. Data located in TVD UTSA Notebook 5, page
30, 32, 33.
c. Began transformation experiments with a construct created in our lab using the Sigma
tulatron concepts; this construct is KEK1160. The target sight of this intron is at position
427/428 of the igLC Schu S4 chromosome sequence.
d. Performed cryotransformation and electroporation experiments using 5 ug of this
KEK1160 vector for each experiment using the Schu S4 strain KKT1 (beta lac 2 deletion).
The selection plates used in these experiments were TSA+++ with 70 ugl/ml kanamycin
(Kan). These plates were placed at 30 C for 4 days and checked for growth everyday.
e. These experiments resulted in thousands of colonies for the cryotransformations (this
experiment was done in duplicate) and seven colonies for the electroporation experiment.
Some of the resulting colonies were patched on TSA+++ with 70 ug/ml Kan. Ten from
each cryotransformation group was used to prepare genomic preparations to use for
PCR screening for the disruption of igLC.
f. Used iglC specific oligos located at the 5’ and 3’ end from the integration site of the
igLC gene. The resulting PCR profile should result with approximately 800 bp difference
from the wild type fragment size. One from each group was used initially to screen for an
insertion. (Figure 3).
52 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 3:
This represents PCR products resulting from mostly genomic templates used with igLC
Nde I rev and igLC Nco I forward oligos. Lane 2 is the wild type profile at ≈ 600 bp, lane
3 is the plasmid negative control and lanes 4, 5 and 6 are possible igLC insertion clones
which are yielding ≈1400 bp DNA fragments. The clones 2A, 2B and 3E all look correct.
g. The KEK1160 vector’s intron seems to have integrated into the KKT 1 chromosome we now will
determine which igLC gene this “intron” has disrupted. We expect that both copies have been
disrupted but we need to verify by using long range PCR using oligos specific to other genes
which can be used to identified each pathogenicity’s island gene. Resulting PCR products will
be sent for sequencing to determine the location of the igLC integrations. This should result in
the deletion of the igLC gene. Data located in TVD UTSA Notebook 5, page 34-36.
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
65%
9. Work plan for upcoming month
a. Will continue with the iglD cloning with the KEK1140 plasmid
b. Will continue with the re-modification of pDS132 to create a ampicillin resistant plasmid to
use with KKT1 SCHU S4 strain
c. Will continue to screen for pdpA deletions in SCHU S4
d. Will continue to verify by PCR and sequencing for the correct igLC deletion in Schu S4. Also,
will prepare protein preparations of the igLC candidate and run a SDS-PAGE to do a western
assay to check for the protein deletion of igLC.
e. Status of attempts to delete the whole FPI: The pdpA has been integrated into the Schu S4
chromosome. We will freeze a clone for each FPI island integration location and these will be
used for the second transformation experiment where the pdpD deletion will be made. We
currently need to create this plasmid construct to use in this experiment (Cloning required).
This section only completes a portion of a multi-step process to achieve this goal.
53 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
f. Order supplies as required.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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. Determine the LD50 of Ft subsp. novicida uvrBiglD double mutant. (Note book #4 page
100-101): Groups of BALB/c mice (female, 4-6 weeks) were intranasally challenged with
105, 106 or 107 CFU of ΔuvrBiglD. As shown in Fig. 1, there is no mortality observed at
any given challenge dose, indicating the high degree of attenuation with this organism.
No significant weight loss of infected mice was also observed. The LD 50 of ΔuvrBiglD in
the intranasal infection model (BALB/c mice) was greater than 107 CFU.
% Survival
100
80
10 5
10 6
10 7
60
40
20
% Body weight
0
0
2
4
2
4
6
8
10 12 14 16 18 20
6
8
10
110
105
100
95
90
85
80
0
12
14
Days post-challenge
Fig.1. Survival of mice infected with Ft subsp. uvrBiglD double mutant. Groups
of BALB/c mice (female, 6-week old) were challenged intra-nasally with 3
doses (105, 106, and 107 CFU) of ΔuvrBiglD to determine LD50 of this strain.
b. Monitor Ft subsp. novicida ΔuvrBiglD replication and dissemination in mice after
intranasal challenge (Note book #4, page 102-105). BALB/c mice were challenged with
ΔuvrBiglD mutant (106 CFU) intranasally. Lungs, liver, spleen, and lymph nodes were
collected from the infected mice at day 3, 7 and 14 after challenge (3 mice per time
point). Numbers of bacteria in each organ were determined by dilution plating. As shown
in Fig. 2, there was heightened replication of the organism in the lungs within the first 7
54 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
days post-challenge, with slight reduction noted at day 14. Numbers of bacteria in the
liver, spleen and lymph node are lower than that in the lungs and are consistent
throughout the observed period. The growth kinetics of ΔuvrBiglD showed some
differences to the uvrBiglC mutant. In the first week of infection, there are fewer bacteria
(2 logs) in the ΔuvrBiglD-challenged spleens than that seen with ΔuvrBiglC (see April
2007 report). However, at day 14, while there were still significant numbers of ΔuvrBiglD
in the lungs and spleen, the bacterial load within the same tissues in the ΔuvrBiglCchallenged animals was almost not detectable (see April 2007 report).
10
8
6
Lungs
Liver
Log10 CFU/organ
4
**
2
1
10
8
6
Lymph node
Spleen
4
2
1
3
7
3
7
14
Days post-challenge
14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrBiglD in target
organs after i.n. vaccination. Bacterial burdens were determined from lungs,
liver and spleen of individual mouse and from pooled lymph nodes at each
time point (3 mice per time point). Numbers (*) of mice without detectable
bacterial burden are indicated.
4. Significant decisions made or pending
The Ft subsp. novicida uvrBiglD double mutant is highly attenuated in mice infected intranasally,
though the growth of the uvrBiglD double mutant in lungs, liver and spleen is evident.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
40 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Evaluate the protective efficacy of the Ft subsp. novicida uvrBiglD mutant as a vaccine
candidate. Groups of vaccinated mice will be challenged i.n. with Ft subsp. novicida.
Animals will be monitored for survival and weight loss.
b. Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglD mutant at
day 14 and 28 after vaccination.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
55 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
Milestone 51
Milestone description: Construction and delivery of Ft subsp. novicida uvrA or uvrB plus pdpD,
iglA, iglB, iglC or iglD double mutants.
Institution: UTSA
1. Date started: 11/01/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. The initial strategy to generate a iglB + uvrB double mutant was to purify chromosomal DNA
from the iglB mutant KKF235 and cryotransform that DNA into the uvrB mutant KKF110. This
generated several transformants however many of these were spontaneous Erythromycin
mutants.
b. In order to get around this problem, chromosomal DNA from the uvrB mutant KKF110 was
purified and 10 ug of DNA was cryotransformed into the iglB mutant KKF235 and plated on
TSA ++ Kanamycin. Around 40 colonies were obtained from this transformation and eight of
them were chosen for chromosomal preparation. PCR using the primers uvrBindentF and
uvrBidentR was performed on the chromosomal DNA to screen for the presence of the uvrB
deletion and Kan insertion (Fig. 1). In a wild type strain the uvrB gene is 2007 bp in length.
The primers used in this screen sit ~200 bp down stream of the start site and ~200 bp
upstream of the stop codon giving a ~1600 bp amplified product in a wild type strain.
However, in a uvrB::kan strain 500 bp of DNA has been deleted and a 900 bp kan gene has
been inserted into the gene. Therefore, a 2000 bp fragment is to be expected if the mutant is
correct. Clones 1-3 and 6-8 look correct and further screening will be done to confirm the
clone is correct.
Figure 1.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
2000 bp
1600 bp
1000 bp
500 bp
5. Problems or concerns and strategies to address
None
1. F. novicida wild type
2. empty
3. clone 1
4. clone 2
5. clone 3
6. clone 4
7. clone 5
8. clone 6
9. clone 7
10. clone 8
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
95%
9. Work plan for upcoming month
Screen potential double mutants (clones 1-3 and 6-8) by further PCR and restriction digest.
Characterize double mutants (uvrB + iglA/iglB/iglC/iglD/pdp
D)for their ability to grow and survive in macrophages.
10. Anticipated travel
None.
56 of 57
Tularemia Vaccine Development Contract: Technical Report
Period: 6/01/2007 to 6/30/2007
Due Date: 7/13/2007 and Prepared by: C.Rick Lyons, Barbara Griffith,Terry Wu, Kathryn Sykes, Stephen
Johnston, Mitch Magee, Bob Sherwood, Julie Wilder, Justin Skoble, Karl Klose, Bernard Arulanandam
11. Upcoming Contract Authorization
None
57 of 57