Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, 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. First group of 8 participants traveled to Washington DC on 9/10/07 to receive the LVS
vaccination on 9/11/07. All 8 were from LBERI
b. The second group of 8 participants is being scheduled to travel to Washington DC on
10/1/07 to receive the LVS vaccination on 10/2/2007.
c. The third group tentatively will be scheduled for 10/23/07
d. UNM and USAMRIID are actively using the LVS vaccine web database to track Risk
Assessment form submission and acceptance, Informed Consent submission and
acceptance, Health screening appointments, planned dates for receipt of LVS
vaccinations, dates of medical clearance, and travel arrangements
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. The work stoppage will be rescinded approximately 1 month after the 9/11/07
vaccination date.
b. In approximately 2 months, UNM may have access to a local source of human cells from
LVS vaccinated individuals.
c. Dr. Lyons is requesting UNM IRB approval to allow blood draws on the vaccinated LBERI
and UNM scientists after their LVS vaccinations. The LBERI and UNM scientists and
staff will be offered the opportunity to volunteer to donate bloods for the development of
immunoassays, approximately 2 months after receiving the LVS vaccination.
d. UNM and LBERI are offering the LVS vaccinations to 46 scientists; USAMRIID will be
providing the LVS vaccinations over the next 8 months, approximately.
5. Problems or concerns and strategies to address
a. Within approximately 2 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
1 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
LBERI scientists and staff receive the LVS vaccinations. LBERI may rescind the work
stoppage approximately 1 month after 9/11/2007.
6. Deliverables completed
First group of 8 LBERI scientists and staff are receiving the LVS vaccination on 9/11/2007
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for the next month
a. Submit Health screening test results to USAMRIID and obtain USAMRIID medical review
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 10/2/07 (2nd group) and 10/23/07 (3rd group) Participants will
be at USAMRIID for 2 days following the vaccinations
c. Start Risk Assessments, SIP informed consent teleconference, and health screenings for
the next group of participants.
d. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
Travel to USAMRIID began on 9/9/07 when UNM EOHS nurses travel to USAMRIID for training
and on 9/10/07 when the first group of participants traveled to USAMRIID for the LVS
vaccinations. The 2nd group will be traveling on 10/1/2007.
11. Upcoming Contract Authorization (COA) for subcontractors
a. UNM requested a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for
training on LVS site vaccination evaluations. (Electronic email of 8/7/07 authorized the
expenses and signed COA letter will follow) Signed COA letter has not been received
from NIAID as of 9/10/2007
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. (Electronic email of 8/7/07
authorized the expenses and signed COA letter will follow) Signed COA letter has not
been received from NIAID as of 9/10/2007
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

August 2007 LVS work concentrated primarily on completion of the Aeromist Nebulizer
testing. Data consistently demonstrated viability issues in the generator suspension and
thus additional focus was placed on the impact of nebulization on bacteria during the
bioaerosol runs using the Aeromist and Collison generators. Specifics for the month of
August are detailed below:
i. Bioaerosol testing was conducted over five days incorporating dilutions of frozen
and fresh (48h culture) LVS stock using the Aeromist (4 days) and the Collison (1
day) generators. Figures 1 and 2 summarize the data obtained using fresh
culture (cumulative results to date) and Figures 3 and 4 summarize data for
frozen LVS in a similar manner. Additional testing with the Collison was
performed using frozen LVS to demonstrate the differences observed between
the two nebulizers regarding LVS viability and spray factors.
2 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
1. 18 total sprays were performed with frozen LVS
a. New stock used
 10% sucrose in Chamberlains
 Created on 5JUL07
 Titer = 1 x 108 CFU/mL
b. 15 using the Aeromist
c. 3 using the Collison
d. 27 total samples analyzed
 6 pre-bioaerosols with the Aeromist
1. 3 delivered pressures
a. 10 psi (n = 2; 1 x 106 CFU/mL)
b. 15 psi (n = 2; 1 x 106 CFU/mL)
c. 20 psi (n = 2; 1 x 106 CFU/mL)
 15 post-bioaerosols with the Aeromist
1. 3 delivered pressures
a. 10 psi (n = 2; 1 x 106 CFU/mL)
b. 15 psi (n = 2; 1 x 106 CFU/mL)
c. 20 psi (n = 11; 1 x 105, 6, and 7 CFU/mL)
 3 pre-bioaerosols with the Collison
1. 20 psi only (1 x 106 CFU/mL tested representing
normal operating parameters)
 3 post-bioaerosols with the Collison
1. 20 psi only (1 x 106 CFU/mL tested representing
normal operating parameters)
2. 15 total sprays were performed with fresh LVS
a. All using the Aeromist
b. 21 total samples analyzed
 6 pre-bioaerosols
1. 3 delivered pressures
a. 10 psi (n = 2; 1 x 106 CFU/mL)
b. 15 psi (n = 2; 1 x 106 CFU/mL)
c. 20 psi (n = 2; 1 x 106 CFU/mL)
 15 post-bioaerosols
1. 3 delivered pressures
a. 10 psi (n = 2; 1 x 106 CFU/mL)
b. 15 psi (n = 2; 1 x 106 CFU/mL)
c. 20 psi (n = 11; 1 x 105, 6, and 7 CFU/mL)
3. 3 target concentrations were tested (1x105, 1x106, and 1x107 cfu/mL).
The 1 x 106 CFU/mL target was used throughout the pre- and postbioaerosol testing for consistency and comparison (between the
Aeromist and Collison) purposes.
4. Francisella tularensis Schu S4 seed and working stocks were grown in
Chamberlain’s broth and frozen at -80ºC in Chamberlain’s broth plus
10% sucrose. Stock titers of both cultures are approximately 2x109
CFU/mL.
5. Results and Discussion
a. Fresh LVS (Figures 1 and 2)
 Actual vs. Target cfu/mL values were 0.5-1.0 log10
lower than desired for the Aeromist nebulizer. This is
3 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
particularly evident for the 10AUG07 bioaerosols. This
was hypothesized to be due to the shear forces on the
bacteria at 20 psi. For this reason, three different
delivered pressures to the Aeromist were tested
(17AUG07).
 At lower delivered pressures, bacterial viability
retention in the generator suspension was greater
versus the normal operating pressure of 20psi.
Interestingly, output (in mL per min) at the different
pressures was not significantly different, though output
was increased at the higher pressure. Refer to the
accessory Excel sheets for details.
 Calculated spray factors were consistent at the three
tested concentrations. Values were better than or
similar to those observed using the Collison nebulizer
in this series of bioaerosols and those of previous
dates. Data indicate that the Aeromist nebulizer is a
promising alternative to the Collison generator, though
further testing is required.
 Pre- versus post-bioaerosol spray factors were
significantly different. All values were similar for the
pre-sprays, but were different at the post-sprays. Data
demonstrate that spray factors were better at the
higher tested delivered pressure. This is logical in that
the spray factor is based on the generator suspension.
Specifically, at 20 psi, the viability in the generator
suspension decreases, thereby increasing the ratio
between the suspension and the chamber
concentration (i.e., the spray factor appears to be
more efficient).
 Spray factor values decreased (i.e., efficiency
decreased) as concentration increased, a consistent
observation seen previously.
b. Frozen LVS (Figures 3 and 4)
 Actual vs. Target cfu/mL values were 0.5-1.0 log10
lower than desired for the Aeromist nebulizer. This is
particularly evident for the 8AUG07 bioaerosols. This
was hypothesized to be due to the shear forces on the
bacteria at 20 psi. For this reason, three different
delivered pressures to the Aeromist were tested
(16AUG07). A similar titer drop was not observed in
the Collison (30AUG07).
 At lower delivered pressures to the Aeromist, bacterial
viability retention in the generator suspension was
greater versus the normal operating pressure of 20psi,
a similar result observed using fresh LVS. Again,
output at the different pressures was not significantly
differently, though output was increased at the higher
pressure. Refer to the accessory Excel sheets for
details.
 Calculated spray factors were consistent at the three
tested concentrations. Values were better than or
4 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam


similar to those observed using the Collison nebulizer
in this series of bioaerosols and those of previous
dates. Data indicate that the Aeromist nebulizer is a
promising alternative to the Collison generator, though
further testing is required.
Pre- versus post-bioaerosol spray factors were
significantly different. All values were similar for the
pre-sprays, but were different at the post-sprays. Data
demonstrate that spray factors were better at the
higher tested delivered pressure (similar to what was
observed using fresh LVS).
Spray factor values decreased (i.e., efficiency
decreased) as concentration increased, a consistent
observation seen previously.
6. Aeromist nebulizers are low cost at $15 each and have an advantage in
that the spray solution can be prepared in the Aeromist and then
transported to the testing location.
7. 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
b. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL03\Aeromist nebulizer\8Aug07
c. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL03\Aeromist nebulizer\10Aug07
d. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL03\Aeromist nebulizer\16Aug07
e. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL03\Aeromist nebulizer\17 Aug 07
f. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078_TUL-03\TUL03\Collison Generator\Frozen LVS\30Aug2007
5 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
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
7.50
Target CFU/ml (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
8/10/2007
8/17/2007 (Pre; 10psi)
8/17/2007 (Pre; 20psi)
8/17/2007 (Post; 10psi)
8/17/2007 (Post; 15psi)
8/17/2007 (Post; 20psi)
8/17/2007 (Pre; 15psi)
Figure 1. Cumulative Target vs. Actual CFU/mL data at three target concentrations of fresh LVS using the Aeromist generator.
Bioaerosols conducted on 17AUG07 were conducted at three different pressures delivered to the Aeromist (10, 15, and 20psi).
Earlier Collison data are presented as well for comparison purposes.
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
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
7.50
Target CFU/ml (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
8/20/2007 (Pre, 20psi)
8/17/2007 (Pre; 10psi)
8/17/2007 (Pre; 20psi)
8/17/2007 (Post; 10psi)
8/17/2007 (Post; 15psi)
8/17/2007 (Post; 20psi)
8/17/2007 (Pre; 15psi)
Figure 2. Cumulative Actual CFU/mL vs. Spray Factor data at three target concentrations of fresh LVS using the Aeromist
generator. Bioaerosols conducted on 17AUG07 were conducted at three different pressures delivered to the Aeromist (10, 15, and
6 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
20psi). Earlier Collison data are presented as well for comparison purposes.
Aeromist: Target vs. Actual CFU/mL (Frozen)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
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)
4/13/2007 (Pre, 20psi)
6/21/07 (Pre, 20psi)
6/26/07 (Pre, 20psi)
8/8/2007 (Pre, 20psi)
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
Figure 3. Cumulative Target vs. Actual CFU/mL data at three target concentrations of frozen LVS using the Aeromist generator.
Pre- and post-bioaerosol data for the Aeromist and Collison generators are presented as well to demonstrate the differences
observed between the two nebulizers regarding LVS viability in the generator suspension. Bioaerosols conducted on 16AUG07 were
conducted at three different pressures delivered to the Aeromist (10, 15, and 20psi).
7 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Aeromist: Actual CFU/mL vs. Spray Factor (Frozen)
-5.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Spray Factor (Log10)
-5.50
-6.00
-6.50
-7.00
-7.50
Actual CFU/ml (Log10)
4/13/2007 (Pre, 20psi)
6/21/2007 (Pre, 20psi)
6/26/2007 (Pre, 20psi)
8/8/2007 (Pre, 20 psi)
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
Figure 4. Cumulative Actual CFU/mL vs. Spray Factor data at three target concentrations of frozen LVS using the Aeromist
generator. Pre- and post-bioaerosol data for the Aeromist and Collison generators are presented as well to demonstrate the
differences observed between the two nebulizers regarding differences in spray factors when using pre- versus post-bioaerosol
generator suspension concentration values. Bioaerosols conducted on 16AUG07 were conducted at three different pressures
delivered to the Aeromist (10, 15, and 20psi).
4. Significant decisions made or pending
The Aeromist nebulizer continues to show promise as an alternative to the Collison nebulizer.
The Aeromist nebulizer provides consistent spray factors with values as good as or better than
the Collison. Based on our data, one advantage for the Collison is that it confers less shear
forces on the bacteria versus the Aeromist at the same delivered pressure. To address this, the
delivered pressure to the Aeromist must be decreased which, in turn, slightly decreases the
output. Output can be corrected by increasing the suspension concentration. A decision needs
to be made as to whether or not this is acceptable. Sprays performed with fresh LVS show better
spray factors than with frozen LVS indicating better survival of aerosolized bacteria.
5. Problems or concerns and strategies to address
See Item 4
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
88%
9. Work plan for upcoming month
8 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam




Perform bioaerosol experiments using fresh LVS with the Collison, focusing on preversus post-bioaerosol generator suspension concentrations.
Perform bioaerosol experiments using the Aeromist nebulizer at lower delivered
pressures; again, focus on pre- versus post-bioaerosol generator suspension
concentrations and aerosol output.
Plan bioaerosol experiments using F. tularensis Schu4
Initiate final decision on bioaerosol generator to be used on animal studies based upon
an evaluation of bacterial viability, reproducibility of spray factors, and reproducibility of
actual vs. target spray concentrations.
10. Anticipated travel
Attend the Annual TVDC meeting in Santa Fe, NM on October 8 and 9, 2007
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
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
9 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Fischer 344 rats
a. Experiment Ftc40 study 2 (Notebook 104 pages 31-32, 34 electronic files of the
images are stored at L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Experiments\Ftc40)
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.
ii. Rats were challenged 43 days after s.c. LVS vaccination with 320 SCHU S4 i.t.
iii. 3 rats were killed on days 0, 3, 6, 9 to collect lungs, liver, and spleens
iv. Compared with naïve rats (Ftc37 study 2), vaccinated rats challenged i.t. with
SCHU S4 had:
1. Less severe pneumonia and the lung lesions appeared to be
resolving by day 9
2. Dissemination to the liver and spleen were seen in fewer rats/group
v. These results suggest that LVS vaccination provides protection by more
effectively controlling SCHU S4 proliferation and systemic dissemination to
the liver and spleen
b. Experiment Ftc46 study 3 (Notebook 106, pages 104-106, 111)
i. The purpose of this experiment was to determine whether the route of
infection (intratracheal or esophageal) can be definitively identified based on
the location of the QD655-luc8 signal.
ii. An inoculum containing SCHU S4, QD655-luc8, and substrate was prepared
and delivered i.t. into four rats
iii. The infected rats were imaged using the Xenogen IVIS imaging system and
scored as intratracheal delivery or esophageal delivery based on the location
of the QD655-luc8 signal
iv. The infected rats were then killed to determine the number of SCHU S4
deposited in the lungs.
v. As shown in Table 1, we recovered the same number of SCHU S4 from the
lungs regardless of the location of the QD655-luc8 signal
vi. This unexpected result suggests that the location of the QD655-luc8 signal
cannot be used to identify the route of SCHU S4 infection
vii. Zymera, the manufacturer of the QD655-luc8, offered the explanation that
QD655-luc8 tend to stick to tissues and therefore may separate from the
bacteria. However, if this was the case, we would expect the QD655-luc8
signal to always be localized in the throat area and never be found in the
lungs
viii. We will repeat the experiment, this time changing 1) the inoculum
composition, 2) the insertion depth of intratracheal tube and 3) the position of
the rats in the imaging system
Table 1. Correlation between QD655-luc8 localization and infection route
Rat
Imaging results
CFU recovered
Percent recovery (%)a
from lungs
1
Esophageal
193
21
10 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
2
Esophageal
3
Intratracheal
4
Esophageal
a Target deposition = 900 CFU
c.
363
169
460
40
18
51
Experiment Ftc47 (Notebook 104, pages 59-62, 66, 89-93, 98, 112)
i. The purpose of this experiment was 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
ii. 40 rats were vaccinated s.c. with 5 x 107 LVS and 28 rats were unvaccinated
iii. 33 days after vaccination, the naïve and vaccinated rats were challenged i.t.
with 1.7 x 104 SCHU S4. This dose was similar to the one used to challenge
vaccinated rats in Ftc40 study 1 but was 100-fold higher than the one used to
challenge naïve rats in Ftc 37 study 1
iv. The numbers of SCHU S4 in the naïve lungs on days 1 and 2 were higher
than we expected and exceeded our dilution series; therefore, we do not
have reliable data for the naïve lungs at these time points. Similarly, we do
not have reliable data for the vaccinated lungs on day 1 and the vaccinated
spleens on days 2-4 because our dilution series were too narrow.
Nevertheless, table 2 shows the mean bacterial load resulting from the
reliable data from at least 3 rats/group
v. In naïve rats, SCHU S4 disseminated from the lungs to the spleens and liver
within the first day (culture plates contained too many bacteria to count) and
the bacterial burden reached 108 – 109 CFU by the time the rats died 4 to 5
days after infection.
vi. In vaccinated rats, SCHU S4 also disseminated from the lungs to the spleen
and liver within the first day (culture plates contained too many bacteria to
plate). However, the numbers of SCHU S4 were lower at every time point
compared with those found in naïve rats, suggesting that the vaccinated rats
were more effective at controlling SCHU S4 proliferation.
vii. SCHU S4 was found in the lungs, but not in the spleen or liver, of 4/5
vaccinated rats after 21 days. No detectable infection after 42 days
Table 2. Kinetics of SCHU S4 proliferation and dissemination in naïve and s.c. LVS
vaccinated ratsa
Log10 mean bacterial load ± SD
Naive
s.c. LVS vaccinated
Day
Lungs
Liver
Spleen
Lungs
Liver
Spleen
0
4.2 ± 0.1
4.2 ± 0.1
1
NDb
2.5 ± 0.5
2.5 ± 0.4
ND
2.2 ± 0.3
0
2
ND
5.7 ± 0.2
5.0 ± 0.4
7.4 ± 0.1
4.5 ± 0.3
ND
3
8.5 ± 0.3
7.2 ± 0.2
7.1 ± 0.4
7.4 ± 0.1
4.5 ± 1.0
ND
4
8.9 ± 0.3
7.9 ± 0.3
8.4 ± 0.3
7.7 ± 0.7
5.1 ± 0.3
ND
5
Died
6.8 ± 0.4
5.0 ± 0.2
4.6 ± 0.3
6
Died
6.0 ± 0.8
3.9 ± 0.7
4.1 ± 0.8
21
2.9 ± 0.3
0
0
42
0
0
0
a
b
n = 3-5/group
ND = No quantitative data as bacterial counts were too high in the dilutions performed
d. Ftc47 study 2 (Notebook 104, pages 99-103)
i. The purpose of this experiment was to fill-in missing data due to
unexpectedly high bacterial load in the naïve rats challenged i.t. with SCHU
S4 in Ftc47 study 1 (see above)
11 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
ii. Naïve rats were challenged i.t. with 1.6 x 104 SCHU S4 and groups of five
rats were killed on day 0, 1, and 2 to determine the bacterial burden in the
lungs, liver and spleen
iii. The lung deposition and the number of SCHU S4 in the liver and spleen on
day 1 and 2 in this experiment and Ftc47 study 1 were very similar
iv. Table 3 shows that SCHU S4 expanded massively by 3 log10 in the lung
within the first day of infection. Dissemination to the spleen and liver
occurred as early as day 1 (only 1 of 4 rats) but was observed in all rats on
day 2.
Table 3. Kinetics of SCHU S4 proliferation
and dissemination in naïve rats
Log10 mean bacterial load ± SD
Day p.i.
Lungs
Liver
Spleen
0
4.2 ± 0.3
1
7.3 ± 0.1
2.5 (1/4)a
1.9 (1/4)
2
8.2 ± 0.2
5.0 ± 0.6
4.7 ± 0.7
a SCHU S4 found in 1 out of 4 rats
4. Significant decisions made or pending
None
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
8. Percentage completed
65%
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. Determine the kinetics of SCHU S4 proliferation and dissemination in lungs,
spleens, and livers of LVS vaccinated rats.
ii. Determine the effects of T cell depletion on the protective immunity induced
by LVS vaccination
iii. Determine whether passive immunization with convalescent sera will protect
naïve Fischer 344 rats from i.t. SCHU S4 challenge
c. Determine whether QD655-luc8 can be used to track pulmonary deliver
i. Determine whether QD655-luc8 and its substrate are toxic to SCHU S4
ii. Determine the effect of the following on the colocalization of QD655-luc8
signal and SCHU S4 in vivo
1. The inoculum composition
2. The insertion depth of intratracheal tube
3. The position of the rats in the imaging system
iii. Determine whether co-administration of QD655-luc8 affects the virulence of
SCHU S4 in naïve Fischer 344 rats
12 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
10. Anticipated travel
Will attend the TVDC annual meeting in Santa Fe, NM on 10/8 and 10/9/2007
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 both the CERUS and CTL protocols spared
some antigen specific (LVS) proliferation and resulted in greater than 50% cell
recovery from thawing; the Lyons protocol resulted in less than 26% cell
recovery in each of two experiments in which it was tested, but did spare an
equivalent amount of LVS-specific proliferation
3. PBMCs were prepared and frozen using the 3 different protocols on 6/11/07
(TUL 14) and 6/12/07 (TUL 15) and were thawed on 8/6/07 and 8/7/07 to test
their proliferative capacity to LVS and mitogens
Table 1: Recovery of NHPs from different Freeze/Thaw Protocols
Freeze/Thaw Protocol
# Tubes thawed (TUL 14 and 15
% Recovery
combined)
Cerus
4
83%, 78%, 102%, 70%
CTL
4
75%, 122%, 74%, 72%
Lyons
5
52%, 8%, 37%, 80%, 8%
13 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Mean Proliferation of TUL 14 Freeze/Thaw Samples
Cell Mean for RLU small
9.00E5
8.00E5
7.00E5
6.00E5
5.00E5
4.00E5
Media
Con A
LVS hk Hi
LVS ff Hi
PHA
LPS
3.00E5
2.00E5
1.00E5
0
Cerus
CTL
Lyons
None
Figure 1: PBMCs, either fresh (None) or frozen and thawed using one of the three indicated protocols
(Cerus, CTL, Lyons) were plated at 1x 106 cells/ml and stimulated with either HK or FF LVS (1 x 105
cells/ml), Con A (10 μg/ml), PHA (2.5 μg/ml) or LPS (1 μg/ml); Data represent the mean of 3 LVSvaccinated NHPs, TUL 14, A00896, A00908 and A00937, day 203 post LVS vaccination.
Data Interpretation: When expressed as the mean of the 3 NHPs, it appears as though the fresh cells did
not proliferate well to either HK- or FF-LVS (3rd and 4th bars in each data set respectively), but this may be
a function of the high background proliferation to media that is not observed in the frozen/thawed cells.
Frozen/thawed cells proliferated better to FF-LVS than to HK-LVS. Frozen/thawed cells uniformly did not
proliferate well to LPS, regardless of the protocol used, suggesting that B cell function is not spared by
the freeze/thaw process. Interestingly, this is in contrast to the mitogen-stimulated T cell proliferation
which is spared by the freeze/thaw process (note good retention of proliferation to both Con A and PHA,
which we have observed previously as well).
14 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Proliferation of Individual TUL 14 Freeze/Thaw PBMC Samples
Cell Mean for RLU small
1.60E6
1.20E6
Media
Con A
LVS hk Hi
1.00E6
LVS ff Hi
8.00E5
PHA
1.40E6
6.00E5
A
LPS
4.00E5
2.00E5
0
Cerus
CTL
Lyons
None
Cell Mean for RLU small
1.20E6
1.00E6
8.00E5
6.00E5
B
Media
Con A
LVS hk Hi
LVS ff Hi
PHA
LPS
4.00E5
2.00E5
0
Cell Mean for RLU small
Cerus
1.00E6
9.00E5
8.00E5
7.00E5
6.00E5
5.00E5
4.00E5
3.00E5
CTL
Lyons
Media
Con A
LVS hk Hi
LVS ff Hi
None
C
PHA
LPS
2.00E5
1.00E5
0
Cerus
CTL
Lyons
None
Figure 2: PBMCs, either fresh (None) or frozen and thawed using one of the three indicated protocols
(Cerus, CTL, Lyons) were plated at 1x 106 cells/ml and stimulated with either HK or FF LVS (1 x 105
cells/ml), Con A (10 μg/ml), PHA (2.5 μg/ml) or LPS (1 μg/ml); Data (TUL 14) represent the response of
15 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
individual LVS-vaccinated NHPs: A00937 (A), A00908 (B) and A00896 (C), day 203 post LVS
vaccination.
Data Interpretation: When data from the 3 inidividual NHPs are plotted, it appears as though the fresh
cells from A00908 were those that did not proliferate well to either HK- or FF-LVS, perhaps being masked
by the high background proliferation to media. However, the frozen/thawed cells from A00908 also
proliferated the least well to FF-LVS. It is still apparent that frozen/thawed cells proliferated better to FFLVS than to HK-LVS and that frozen/thawed cells uniformly did not proliferate well to LPS, regardless of
the protocol used.
Proliferation of TUL 15 Freeze/Thaw PBMC Samples
Cell Mean for RLU small
1.20E6
1.00E6
8.00E5
6.00E5
Media
Con A
LVS hk Hi
LVS ff Hi
PHA
LPS
4.00E5
2.00E5
NT
0
Cerus
CTL
Lyons
None
Figure 3: PBMCs, either fresh (None) or frozen and thawed using one of the two indicated protocols
(Cerus or CTL) were plated at 1x 106 cells/ml and stimulated with either HK or FF LVS (1 x 10 5 cells/ml),
Con A (10 μg/ml), PHA (2.5 μg/ml) or LPS (1 μg/ml); Data (TUL 15) represent the response of 3 LVS
vaccinated NHPs in the case of the fresh cells (None; A00868, A00902 and A00659) and individual LVSvaccinated NHPs in the case of the frozen/thawed cells: A00868 (Cerus) and A00902 (CTL), day 195
post LVS vaccination.
Data Interpretation: Many unanticipated problems occurred during the execution of TUL 15 that affected
the quality of the data. On the day of the fresh blood draw, the tube of PBMCs from NHP A00659 was
dropped immediately after the gradient separation. We attempted to separate the cells again, and this
may have resulted in a small yield of cells. There were not enough to freeze. On the day of thawing, 2
out of 6 cryovials could not be located (A00868, CTL and A00902, Cerus). They were later located in a
different box in the liquid nitrogen tank (it is as yet unclear how they got there). Therefore, only one
sample/freezing protocol was available for testing. Neither of these thawed aliquots of cells proliferated
well to either HK- or FF LVS. As usual, the Lyons protocol resulted in poor cell recovery and there were
not enough cells to test.
Overall summary of TUL 14 and TUL 15 freeze/thaw testing: the results were less desirable than we had
anticipated due to high variability and experimental mishaps. We cannot yet make a decision regarding
whether we will choose the Cerus or CTL protocols. However, we have decided not to pursue the Lyons
protocol as the cell yield after thawing is consistently lower than either of the other two protocols. It is
also clear that B cell proliferation to LPS is not spared by any of the freeze/thaw protocols tested.
Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay
082907.svd; and in the TVDC 1 bound notebook pages 30 – 39, 49, 74 - 77 (TUL 14) and pages 40 – 48,
50, and 78 - 80 (TUL 15); summary data was presented at the Internal Tech Meeting on 8/24/07 and can
be found in N:My Documents\Tularemia Contract\Internal Tech Mtg\agenda prep for 082407
16 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
b. Update on IFN detection by ELISPOT
i.
ii.
iii.
iv.
Having already determined the optimum number of cells to plate to detect
LVS-stimulated IFN production from NHP PBMCs (100,000 – 200,000) and
the concentration of the anti-IFN capture antibody (G7-4; 15 μg/ml) used to
coat the plates, we have begun to titrate the anti-IFN detection antibody
(biotinylated 7B-6) to determine the optimal concentration at which to use it
On 8/23/07 (TUL 17), 3 identical plates were coated with 15 μg/ml G7-4 and
cells from LVS-vaccinated NHPs A00896, A00937 and an LVS-naïve NHP
A04367 were incubated on the plate
The plates were developed with 3 different concentrations of biotinylated 7B6 (0.5, 1.0 and 2.0 μg/ml; 1 μg/ml is the kit-recommended concentration)
The plates have yet to have been read, but it is apparent that the naïve NHP
responded with the most IFN spots when stimulated with either HK- or FFLVS, particularly with the latter; we need to repeat this experiment with
another naïve NHP to confirm these results
Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay
082907.svd; Notebook data: TVDC 1 bound notebook pages 81 - 91 (TUL 18);
c. Update on LVS-stimulated proliferation
i.
Terry Wu prepared new batches of formalin fixed (FF) and heat-killed (HK)
LVS which we tested in our proliferation assay
ii.
Previous results (TUL 16 and TUL 17) had suggested that it was less potent
at stimulating LVS-vaccinated PBMC proliferation than our current
preparations of FF- and HK-LVS
iii.
We re-titrated the new preparations of HK- and FF-LVS using higher
concentrations; we also included one LVS-naïve NHP (TUL 18)
17 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Proliferation of LVS-vaccinated NHP PBMCs to HK- and FF-LVS
*
A00896
A00937
120000
80000
*
*
1600000
New LVS ff 5x10^6
New LVS hk 5x10^6
New LVS ff 1x10^6
New LVS hk 1x10^6
New LVS ff 5x10^5
New LVS hk 5x10^5
New LVS ff 1x10^5
New LVS hk 1x10^5
LVS ff Hi
LVS hk Hi
40000
0
RLU (Mean +/- SEM)
*
A
Media
RLU (Mean +/- SEM)
160000
*
B
1400000
1200000
1000000
*
800000
600000
*
*
*
*
*
400000
New LVS ff 5x10^6
New LVS hk 5x10^6
New LVS ff 1x10^6
New LVS hk 1x10^6
New LVS ff 5x10^5
New LVS hk 5x10^5
New LVS ff 1x10^5
New LVS hk 1x10^5
LVS ff Hi
LVS hk Hi
0
Media
200000
Figure 4: PBMCs from 2 LVS-vaccinated NHPs (A) and one naïve NHP (B) were stimulated at 1 x 106/ml
with LVS preparations at the indicated concentrations or at 1 x 105/ml where not indicated. * indicates
different than media.
18 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Data interpretation: As initially observed, the new preparations of HK- and FF-LVS are less able to
stimulate LVS-vaccinated PBMC proliferation at the same concentration as the original preparations (1 x
105/ml). However, using a 50-fold higher concentration, the new preparations were able to stimulate
significant proliferation from one of two LVS-vaccinated NHPs (A00896 but not A00937, both day 278
post-ID LVS vaccination). However, NHP A04367, who is LVS-naïve, also exhibited significant
proliferation to the new preparations of LVS; note that the scale of the Y-axis is 10-fold higher as well,
indicating high background proliferation and higher proliferation overall.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 082907.svd; Notebook
data: TVDC 1 bound notebook pages 81 - 91 (TUL 18).
d. Update on IgA anti-LVS ELISA
i.
For the second time, the assay designed to optimize the LVS coating
concentrations necessary to detect IgA anti-LVS in LVS-vaccinated
NHP sera failed due to no color development
ii.
We have ordered a human IgA to use as a control to make sure that
our anti-IgA-HRP antibody is working correctly
4. Significant decisions made or pending
We will cease testing the Lyons protocol for freeze/thawing of PBMCs as it results in low cell
recoveries compared to either the Cerus or CTL protocols.
5. Problems or concerns and strategies to address
a. Naïve NHP A04367 appears to react to LVS both by IFN ELISPOT and proliferation
b. Freeze/thaw testing is not yet complete due to poor responses and experimental mishaps
when testing TUL 14 and TUL 15
c. IgA anti-LVS ELISA is not working; either due to no IgA in the NHP sera or a failure of the
detection antibody (anti-IgA-HRP) to detect NHP IgA; we cannot locate a preparation of
NHP IgA so we will test our detection antibody on human IgA; the manufacturer states
that their antibody also recognizes human IgA
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
86% of scientific work has been completed
9. Work plan for upcoming month
1. Thaw PBMCs frozen down in experiments TUL 16 and 17 and test in proliferation and IFN
ELISPOT assays if enough cells permit
2. Continue to optimize the conditions of the IFN ELISPOT assay by testing different detection
antibody concentrations; we’ll keep the coating concentration constant at 15 g/ml
3. Test human IgA in an ELISA as a control for the anti-IgA-HRP detection antibody
10. Anticipated travel
Attend the Annual TVDC meeting in Santa Fe, NM on October 8 and 9, 2007
11. Upcoming Contract Authorization (COA) for subcontractors
None
19 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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. Experiment Ftc27.15 (Notebook 101, pages 67-69)
i. The purpose of this experiment was to determine the maximum number of
splenocytes/well to use in the T cell proliferation assay. This experiment was
part of our objective to increase assay sensitivity
ii. Total splenocytes were titrated from 1 x 105 to 4 x 105/well
iii.
iv. Antigen specific proliferation was detected with both 1 x 105 and 2 x 105
splenocytes/well. 3 x105 and 4 x 105 splenocytes/well produced very high
background and no antigen-specific proliferation was observed (Fig. 1)
v. We decided that 2 x 105/well is the maximum number of splenocytes that can
be used in the T cell proliferation assay
Figure 1. Titration of total splenocytes in T cell proliferation assay. T cells were
incubated with 5 x 105 formalin-fixed LVS in the presence of various FBS. After 5
days, T cell proliferation was measured indirectly by the incorporation on BrdU
b. Experiment Ftc27.16 (Notebook 101, pages 76-72) and Experiment Ftc27.16b
i. The purpose of these experiments was to determine the number of nylonwool enriched splenocytes and hilar lymph node cells to use in the T cell
proliferation assay. By enriching for T cells, we may be able to increase the
assay sensitivity.
ii. Spleens and hilar lymph nodes were collected 66 days after LVS vaccination
iii. Nylon wool-enriched splenocytes
1. Cells were titrated from 5 x 104 to 1 x 106/well (Fig. 2)
2. No LVS-specific response was detected with 5 x 104 cells/well
3. LVS-specific responses were detected with 1 x 105and 2.5 x 105
cell/well. LVS-specific responses were also detected with 5 x 10 5
cells/well, but the background was extremely high
4. 1 x 106 cells/well produced very high background and no LVSspecific proliferation was observed
5. 1 x 105 nylon wool enriched splenocytes/well appear to be the
optimal cell concentration
20 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
6. This experiment will be repeated with cells from naïve mice to show
that responses are antigen specific, i.e. no proliferation by naïve cells
iv. Lung draining lymph node cells (Fig. 3)
1. No LVS-specific proliferation was detected with 5 x 104 and 1 x 105
cells/well
2. This experiment will be repeated to confirm the absence of LVSspecific T cells in the hilar lymph nodes
Figure 2. LVS-stimulated proliferation of nylon wool enriched splenocytes. Nylon wool enriched
splenocytes were incubated with 5 x 105 formalin-fixed LVS (FF LVS) and after 5 days T cell proliferation
was measured indirectly by the incorporation on BrdU
Figure 3. LVS-stimulated proliferation of hilar lymph node cells. Cells at the indicated concentration were
incubated with 5 x 105 formalin-fixed LVS (FF LVS) and after 5 days T cell proliferation was measured
indirectly by the incorporation on BrdU
c.
Experiment Ftc27.17 (Notebook 101, pages 85-87) and Ftc27.17b (Notebook 101,
pages 92-95)
i. The purpose of these experiments was to determine the conditions for
stimulating T cell proliferation with proteins in preparation for screening Ftularensis-derived peptide library from ASU
ii. The Bfr, KatG and GroEL proteins were synthesized and shown by Dr. Dan
Clemens to stimulate T cell proliferation
iii. 2 x 105 total splenocytes from naïve and LVS vaccinated BALB/c mice were
cultured with 1 or 5 g/ml Bfr, KatG or GroEL for 5 days
iv. There are a number of problems in both experiments that make data
interpretation impossible (Fig. 4).
1. All of the proteins stimulated naïve cells to proliferate to some extent
21 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
2. There was no consistent pattern in the proliferative responses
between the two experiments, such as the fact that in Ftc27.17b
vaccinated cells had higher basal proliferation than naïve cells
3. The proliferative responses in the presence of Bfr in Ftc27.17 and Bfr
and KatG inFtc27.17b were lower than those without antigen
v. Since Dan Clemens had previously shown that these proteins stimulated
antigen-specific proliferation, we should be able to reproduce his results by
troubleshooting our T cell proliferation assay. Alternatively, the IFN Elispot
assay, as discussed next, may be a better assay for detecting F. tularensis –
specific responses.
Figure 4. Stimulation of total splenocytes by recombinant F. tularensis proteins. Total splenocytes were
incubated with 1 or 5 g/ml recombinant proteins for 5 days and proliferation was measured indirectly by
the incorporation of BrdU. Bars show mean ± SD.
d. Experiment Ftc51.1 (Notebook 103, pages 21-23) and Experiment Ftc51.2 (Notebook
103, pages 24-31)
i. The purpose of these experiments was to develop the IFN Elispot assay as
an alternative to the T cell proliferation assay. The T cell proliferation assay
can at times produce extremely high background and be sensitive to
suppression (see the results from Exp Ftc27.17b)
ii. 2 x 105 total splenocytes from naïve and LVS-vaccinated BALB/c mice were
incubated with various concentrations of heat-killed or formalin-fixed LVS for
23 h and then processed for Elispot assay
iii. As shown in Figure 5, antigen specific responses against both formalin-fixed
LVS and heat-killed LVS were detected
iv. It is extremely encouraging that the experiment shown has been repeated
with similar results
v. We will determine next whether the Elispot assay can be used more reliably
for screening proteins and peptides than the T cell proliferation
22 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 5. Antigen-specific responses to formalin-fixed LVS (FF LVS) or heat-killed (HK LVS) as
measured by IFN Elispot. 2 x 105 total splenocytes from naïve and LVS-vaccinated BALB/c mice were
incubated with various concentrations of heat-killed or formalin-fixed LVS for 23 h. The results show
triplicate of each condition
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Non-specific T cell proliferation -- test IFN Elispot assay as an alternative
6. Deliverables completed
NA
7. Quality of performance
Good
23 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
8. Percentage completed
50%
9. Work plan for upcoming month
a. Evaluate and develop the IFN ELISpot assay using positive control proteins GroEL,
KatG, Bfr and ivt proteins from ASU
b. Troubleshoot T cell proliferation assay using positive control proteins GroEL, KatG,
Bfr and ivt proteins from ASU
c. Compare total splenocytes, nylon wool enriched splenocytes, and hilar lymph node
cells for best sensitivity
d. Develop the T cell proliferation assay and Elispot assay for the Fischer rat
i. Procedures for isolating T cells
ii. Procedures for stimulating T cells
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
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
a. Experiment Ftc36 study 3 (Notebook 106, pages 8-14)
i. The purpose of this experiment was to determine the MOI that would allow F.
tularensis-infected macrophages to survive 3 to 4 days with minimal
extracellular bacterial growth. The infected macrophages will be used in the
T cell-macrophage killing assay
ii. Human alveolar macrophages were infected at MOI of 1, 10, and 100 and
the cell- and medium-associated bacterial load were determined on days 0,
2, and 3
iii. The macrophage monolayer was at most 20-30% confluent after the
infection, gentamicin and washes
iv. 2 days after infection, SCHU S4 and LVS were found in the medium at all
three MOI and cytopathic effects were observed
v. These results suggest that MOI of 1, 10, and 100 may be too high to
establish an infected macrophages culture. The results may be improved by
increasing the number of macrophages in the culture or reducing the MOI
b.
Experiment Ftc36 study 4 (Notebook 106, pages 15-24, 29-31)
i. The purpose of this experiment was to determine whether reducing the MOI
to 0.1 and 0.01 would produce a viable macrophage monolayer that can be
used for the macrophage killing assay
ii. Human alveolar macrophages were infected at MOI of 0.01, 0.1, 1, 10, and
100 and the cell- and medium-associated bacterial load were determined
daily
iii. The results with MOI of 1, 10, 100 were similar to those in Ftc36 study 3
iv. An MOI of 0.01 appears close to optimal for SCHU S4 because it was found
extracellularly in only 1 of 3 wells after 2 days.
24 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
v. MOI of 1 or 0.1 appear close to optimal for LVS because it was found
extracellularly in only 1 of 3 wells infected with MOI = 1 and 0 of 3 wells
infected with MOI = 0.1 after 2 days
25 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 6. Microdot assay measuring the number of intracellular and extracelluar LVS and SCHU S4 in
culture with human alveolar macrophages. Human alveolar macrophages were infected with LVS
(above) or SCHU S4 (below) at various MOI. On the day of infection and 1 and 2 days later, the culture
supernatants and macrophage lysates were diluted and plated onto cystine heart agar plates.
c.
Experiment 021507amp (Notebook 106)
i. The purpose of this experiment was to determine the cytokine production
profile of human alveolar macrophages stimulated with SCHU S4 or LVS for
24 hours
ii. The MOI had little or no effect on the cytokine production profile (Fig. 7)
iii. LVS induced higher levels of all cytokines examined than SCHU S4
iv. SCHU S4 induced IL-1 at MOI = 1 and 10, suggesting that it is not a
completely stealth pathogen.
26 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 7. Cytokine production by human alveolar macrophages exposed to LVS and SCHU S4. Human
alveolar macrophages were infected with LVS and SCHU S4 at MOI of 1, 10, 100 for 24 hours. LPS and
heat-killed Listeria monocytogenes (HKLM) were used as positive controls. Cytokines in the culture
supernatants were subjected to Luminex multiplex analysis and the results are shown as mean ± SD
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
8%
27 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
9. Work plan for upcoming month
a. Repeat the experiment to determine the optimal MOI for infecting human alveolar
macrophages with LVS and SCHU S4
b. Determine kinetics of bacterial proliferation intracellularly after infection
c. Determine macrophage viability by lactate dehydrogenase (LDH) release and trypan
blue exclusion after infection
d. Repeat the experiment to measure cytokine production by macrophages infected with
SCHU S4 or LVS
e. Determine whether recombinant IFN would inhibit SCHU S4 and LVS intracellular
growth
f. Determine whether PBMC from vaccinated human volunteers can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
Will attend the TVDC annual meeting in Santa Fe, NM on 10/8 and 10/9/07
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 Ftc30.12 (Notebook 101, pages 73-75)
i. The purpose of this experiment was to determine whether LVS vaccinated
splenocytes can induce murine bone marrow-derived macrophages (BMM) to
kill intracellular LVS
ii. LVS-infected macrophages were incubated with 104, 105, or 106 total
splenocytes from naïve or LVS vaccinated mice. After 3 days, the number of
LVS in the macrophages and in the media was determined
iii. Total splenocytes induced some non-specific killing because the number of
LVS recovered from the cultures with naïve splenocytes decreased as the
number of splenocytes used in the assay increased from 10 4 to 106 cells (Fig
8)
iv. Nevertheless, there was specific killing with 1o6 splenocytes because the
cultures with vaccinated splenocytes had fewer LVS than cultures with naïve
splenocytes
Figure 8. Splenocyte-induced killing of intracellular LVS by bone marrow-derived macrophages (BMM).
LVS-infected BMM were incubated with 104, 105, or 106 splenocytes from naïve or vaccinated BALB/c
mice. After 3 days, the number of LVS in the BMM and in the media was determined. The bars show the
mean of triplicate samples ± SD
28 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
b. Experiment Ftc30.13 (Notebook 101, pages 82-84) and Experiment Ftc30.13b
(Notebook 101, pages 88-91)
i. The purpose of these experiments was to determine the optimal MOI for
infecting murine bone marrow-derived macrophages (BMM) with SCHU S4
to be used in the T cell-macrophage killing assay
ii. BMM were infected with 2 x 102 to 2 x 105 SCHU S4/well
iii. The infected macrophage cultures were monitored daily for changes in
morphology, intracellular proliferation, and escape into the culture medium
iv. To quantify extracellular bacteria, culture supernatants were diluted and
plated onto cystine heart agar plates; to quantify intracellular bacteria, the
macrophages were lysed with sterile water and the cell lysates plated at
various dilutes onto cystine heart agar plates
v. At 2 x 104 SCHU S4/well or more, SCHU S4 was found in the media after 1
day and expanded to 107/ml after 3 days
vi. The results with 2 x 103 SCHU S4/well or less was variable and have to be
repeated
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
20%
9. Work plan for upcoming month
a. Determine the optimal MOI for infecting murine macrophages with SCHU S4
b. Determine whether vaccinated mouse splenocytes can induce BMM to kill
intracellular SCHU S4
c. 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
29 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam


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. As previously presented, we have been further optimizing the modular construction of
IVT templates relative to polypeptide purification. These 4 proposed constructs differ
only by the location and number of His tags. While likely to be advantageous for
purification, we were unable to reliably build doubly-His tagged IVT templates by
overlapping PCR due to sequence identities. To circumvent this problem we have
now been testing a 2-step/2-component linking protocol in which the ORF is linked to
one component at a time. An experiment was set up to evaluate the assembly
efficiencies of these alternative ORF linear expression constructs, which will be the
IVT templates. We were pleased to find that the 2 step/2 component protocol is more
efficient than the 1-step/3-component protocol, Namely,, full length product assembly
is achieved with fewer cycles when performed as successive 2-component reactions
rather than a simultaneous promoter, ORF, terminator component reaction. These
results are shown by qPCR in Figure 1. The two different single tagged constructs,
and the tagless constructs were assembled in one reaction with similar efficiency,
whereas the double tagged construct was assembled in 2/ 2-component steps
Figure 1. qPCR evaluation of IVT template assembling with alternatively-positioned His
tags
Dbl His
N His
C His
No His
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\QPCR for Overlap.asy
30 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
2. Toward the aim of finding a less expensive reagent than PicoGreen for quantitating
template samples and a measurement method that conserved sample, we tested
using UV adsorption of direct samples delivered by a plate reader, and compared it to
readings obtained using picogreen fluorimetry on an aliquoted sample. The
advantage of fluorimetry is the ability to measure small quantities of material,
however, we and others recognize that fluorimetry readings can be imprecise (show
wide variability). We are now able to directly read a well sample by UV absorbance,
which is both accurate and precise without exspending it. We found the two methods
provide comparable readouts (Table 1). Therefore, we plan to switch to the OD260
readout for HTP quantification of IVT templates.
Table 1. IVT template quantification by PicoGreen and OD260
well PicoGreen
OD260
A1
1.41
1.77
A2
0.61
1.00
A3
1.44
1.64
A4
1.34
1.57
A5
1.29
1.51
A6
1.62
1.83
A7
1.18
1.37
A8
1.68
1.84
A9
1.06
1.46
A10
0.52
0.79
A11
1.55
1.64
A12
0.82
1.03
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\DNA quantification\FTU short Plate 01
PCR and Purification.xls
3. We recognize that quality control will be critical throughout the HTP protocol. To
facilitate timely identification of any “issues” we investigated the predictive value of
IVT template quantity and quality on polypeptide yields. The gels in figure 2 allowed
us to compare two templates to two IVT reactions for 3 different polypeptides, 1434a,
728a, and 728b. We note that for any one polypeptide, the IVT DNA template
amount (Presence/absence) and specificity correlated that of the subsequently
produced products(Figure 2). We are implementing an e-gel evaluation of all IVT
templates as a part of a HTP protein production protocol.
31 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 2. Template quality and quantity is predictive of IVT success.
728b Pool
1434a Pool
728a Pool
1434a Clone
728b Clone
728a Clone
728a Clone
728a Pool
728b Clone
728b Pool
1434a Clone
SDS-PAGE to visualize IVT products
6
4
7
3
5
2
1434a Pool
DNA agarose gel to visualize IVT templates
1
2
3
4
5
6
7
30 26 29 26 7 21 ug
Data location: R:\GeneVac\FTU\Contract\Proteome\KDay's data\kmday2 IVT overlap
2007-07-19 15hr 31min.jpg; R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal's IVT
gels/IVT FTU Shrt Plt 01 Row B Chip BAG 7-31-07 02.tif
4. We found that a single or double tag or lack of tag has no effect on polypeptide yields
(Table 2). This suggests that we might be able to select a double His tag IVT design
even without the results of the T-cell stimulation experiments. These samples would
be suitable for a His tag based protein purification or no purification decision later.
Both His tags have been tested by Terry already in the T cell assays.
Table 2. Effect His tag on IVT yield
Sample
FTU1419 (w/out tag)
FTU1419 (C-term tag)
FTU1419 (N-term tag)
FTU1419 (double tag)
IVT yield (ug)
26.04
36.95
38.24
33.49
Data location: R:\GeneVac\FTU\Contract\Proteome\Derek's Data\TCA of FTU with New
Prom and Term.xls.
.
B. Select and test IVT Protocols
1. To increase the throughput capacity of IVT reactions we looked for an alternative to the
currently available ProteoMaster, which accommodates only one plate at the time. We
evaluated the effect of shaking speed and radius on IVT yields (Table 3). We found that
the regular tissue cultural shaker, which cannot be used faster than 300 rpms, is
significantly less effective than the ProteoMaster which is used at 800rpms.. A high
speed four plate shaker ProteoMaster has been ordered.
32 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Table 3. Effect of shaking on IVT yield.
FTU 1419
FTU 1419
FTU 1419
FTU 1419
FTU 1419
FTU 1419
Shaking
speed
300 rpm
300 rpm
400 rpm
400 rpm
800 rpm
800 rpm
IVT yieldug protein
15.55
15.17
16.41
16.82
31.41
26.39
Data location: RR:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT\IVT data\IVT
shaking Test 8-10-07.xls
C. Select and test protocols for protein purification
1. We assessed the feasibility of using an ammonium sulfate fractionation as an alternative to
acetone precipitation of the IVT samples. The idea was that selective precipitation of the
lysate reagents might allow polypeptides to be separated without needing to precipitate
them out, and thereby avoid re-solubility problems. However, we found no useful
discrimination of IVT components vs. newly synthesized protein. In fact, the IVT proteins
are mostly precipitated instead of kept soluble by 25% ammonium sulfate, whereas most
of the lysate components remain soluble. This can be visualized by the 2 gels in figure 3.
The left panel is a Coomassie stain of all lysate components. In the absence of NH 4SO4
(0%) 5ul, 10ul, and 20ul of sample were directly loaded. Aliquots were removed at 25%,
50%, 75% and 90% NH4SO4 cuts to identify constituents in either the soluble or pelleted
fractions. On the right panel a radiograph displays the locations of the 35S-labeled IVT
products. Based on these results we decided not to pursue this approach any further
(Figure 3).
33 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 3. Ammonium sulfate fractionation of IVT reaction
Coomassie stain
0%
5 10 20
Radiograph
25% 50% 75% 90%
S
0%
P S P S P S P
5 10 20
25%
S
P
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\SDS gels\ltien 2007-0823 ammonium sulfate.jpg; R:\GeneVac\FTU\Contract\Proteome\Tien's data\E
page\ammonium 082407.tif
2. To improve solubility of the acetone precipitated IVT samples we tested several non-ionic
detergents but found none universally suitable for all samples (Table 4).
Table 4. Effect of detergents on protein solubility.
Polypeptide dH2O
20% DMF
3% CHAPS
1%Deoxycholate
FTU 1434a
8294
0
396
15466
FTU 728a
4620
352
5874
0
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\Testing for IVT protein
solubility.xls
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
By our current protocol, purification of IVT products leads to significant loss of sample. If
purification is needed, we will initiate a set of solubility and elution titration experiments on
multiple template arrangements. These will identify the most commonly successful (most
robust) parameters for retrieving the largest portion of the sample.
6. Deliverables completed
None
7. Quality of performance
Very good
34 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
8. Percentage completed
98.5%
9. Work plan for upcoming month
We will initiate a series of experiments to test alternatives to agarose nickel beads. This is
intended to reduce non-specific binding. We will try cobalt-nickel and magnetic-nickel beads
in the purification protocols.
10. Anticipated travel
We’ve made our reservations for the October 2007 Annual TVDC meeting in Santa Fe!
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. Experiment Ftc50 study 1 (Notebook 106, pages 25-28, 33), Experiment Ftc50
study2 (Notebook 106, pages 35-39), and Experiment Ftc50 study 3 (Notebook 106,
pages 40- 45)
i. The purpose of these experiments was to determine whether the in vitro
translated proteins still in the in vitro translation reaction solution can
stimulate T cell proliferation without further purification. If so, then ASU will
not have to purify their in vitro translated proteins and risk significant loss in
the process and difficulty in dissolving lyophilized samples
ii. In Ftc50 study 1, the Ftu901 protein appeared to stimulate total and nylon
wool-enriched splenocytes to proliferate. However, since naïve splenocytes
were not included in this experiment, we did not know if this response was
antigen specific.
iii. Both naïve and vaccinated splenocytes were tested in Ftc50 study 2 and the
results showed that there was no specific proliferation. This was confirmed
in Ftc50 study 3 (Figure 9).
iv. As indicated earlier, the T cell proliferation assay can sometimes have high
background which may obscure any antigen-specific response. Therefore,
we will test the ivt proteins in the IFN Elispot assay, which seemed to be
more reliable.
35 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure 9. Stimulation of naïve and vaccinated splenocytes by in vitro translated proteins. Total naïve and
LVS-vaccinated BALB/c splenocytes were cultured with ivt proteins for 5 days and proliferation was
measured by the incorporation of BrdU
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
a. Lack of antigen-specific responses
i. Test ivt proteins in IFN Elispot assay
ii. Test additional ivt proteins in case the 3 tested so far are not stimulatory
b. Solubility of lyophilized ivt proteins – ASU is testing various solvents that would work
universally to dissolve lyophilized ivt proteins
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
12%; not changed because no positive result yet
9. Work plan for upcoming month
a. Determine whether the draining lymph nodes would be a better source of T cells for
the proliferation assay than the spleen. Draining lymph nodes are frequently used in
various T cell assays to demonstrate the existence of antigen specific responses
because they contain more antigen-specific T cells than the spleen. In addition, more
than 106 cells/well can be used in these assays and thereby increasing the assay
sensitivity. We will determine whether the hilar lymph nodes draining the lung after
i.n. LVS vaccination will be a better source of T cells for screening peptides than
splenocytes
b. Develop the peptide screening assay using the purified GroEL, KatG, and Bfr as
positive controls
c. Determine whether IFN ELISpot assay would be better than T cell proliferation
assay for this peptide screen
d. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
e. 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
36 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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
We are conducting experiments with the new Agilent chips in our gene building protocols.
However results are pending
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 are using the new Agilent chips in our gene building
protocols.
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
21%
9. Work plan for upcoming month
Chip synthesized oligos will be used in building protocols.
10. Anticipated travel
We will all be at the Annual TVDC meeting in Santa Fe Oct 8th and 9th.
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 33
Milestone description: Microarrays constructed and confirmed; First printing of arrays,
Testing with DNA from Ft, Arrays GDPs validated at ASU.
Institution: ASU-Johnston
1. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Previous results, shown in Table 1, show an experiment to compare the use of Superscript II
and Superscript III reverse transcriptase in the provided buffer systems and the previous
Clonetech buffer system. We had established that the previous failed LAPT amplifications
37 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
were the result of the changes in enzyme. Superscript II is RNase H - and maintains terminal
transferase activity while Superscript III is RNAse H- but does not have terminal transferase
activity. In addition, the small but simple difference in buffer composition (Clonetech buffer
had 6 mM MgCl2 while the SSII buffer had 3 mM MgCl2) made a large difference in RNA yield
after amplification.
g SCHU S4 amplified
1.0
0.1
0.0
SSII-SSII Buffer
4
9.3
13.8
SSIII-SSIII Buffer
6.2
6.3
Not done
SSII-Clontech Buffer
24.2
31.3
15.7
Table 1. Total micrograms of RNA after LAPT amplification was compared using Superscript II (SSII) to
Superscript III (SSIII) enzymes with different buffer systems. Amplified yield of RNA (in micrograms
expected yield of 30-60 g) of SCHU S4 RNA amplified by LAPT.

Notebook/File locations … SSII vs. SSIII, Notebook 514, page 37.

The next experiment was set up to compare RNA yield with Superscript II over increasing
concentrations of MgCl2 with and without the addition of 2mM MnCl2 during the last 20
minutes of the LAPT. There is one report in the literature stating that the addition of MnCl2
may increase the efficiency of terminal transferase activity of reverse transcriptase that is
needed for effective template switching. For this assay 0.1 g SCHU S4 were diluted in 10
g normal lung RNA and processed for LAPT. The results shown in Table 2 confirm the
previous experiment in that 3mM MgCl2 is not sufficient with only 7 g yielded. However, with
3 mM MgCl2 and with MnCl2 during the last 20 minutes, a reasonable yield of 29 ug was
observed. With increasing the MgCl2 concentration to 6 or 9 mM, the effect of 2mMMnCl2
during the last 20 minutes was not as apparent with reasonable amounts of RNA obtained
with all buffer compositions.
mM MgCl2
3
6
9
No MnCl2
7
40
65
With MnCl2 (2mM)
29
54
67
Table 2. Comparison of total micrograms of RNA after amplification with the LAPT process with addition
of increasing MgCl2 with and without MnCl2. SCHU S4 RNA (0.1 g) was diluted into 10 g total normal
lung RNA.

There is a balance between amplification efficiency and specificity. The chief concern is that
while we may be increasing amplification efficiency with the increasing MgCl 2 and MnCl2
there may be a decrease in specificity. To test this potential problem, we correlated these
various samples by assessing the gene profiles after labeling and hybridization. With the
exception of the 3 mM MgCl2 alone, the samples were labeled and hybridized to the
Francisella microarray for correlation analysis. The 6 and 9 mM MgCl2 samples were labeled
at two separate times and are labeled with a -1 or -2 to show and show a high correlation
(0.872) using the whole gene set for comparison. Several other comparisons are shown in
38 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Table 3. The key observation was that at the 9mM MgCl2 concentration the addition of MnCl2
dropped the correlation from an average above 0.800 to 0.677.
Condition 1
Condition 2
Spearman Coefficient
6 mM MgCl2 alone -1
9 mM MgCl2 alone -1
0.872
6 mM MgCl2 alone -2
9 mM MgCl2 alone -2
0.838
6 mM MgCl2 alone - 1
6 mM MgCl2 alone - 2
0.793
9 mM MgCl2 alone - 1
9 mM MgCl2 alone - 2
0.801
3 mM MgCl2 + MnCl2
6 mM MgCl2 alone
0.839
3 mM MgCl2 + MnCl2
9 mM MgCl2 alone
0.807
6 mM MgCl2 + MnCl2
6 mM MgCl2 alone
0.789
9 mM MgCl2 + MnCl2
9 mM MgCl2 alone
0.677
Table 3. Spearman correlation coefficients between labelings and conditions.


Table 2 and 3. Notebook/File locations … Invitrogen buffer test Notebook 514, pages 43-48;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-9
4. Significant decisions made or pending.
We have successfully redesigned the amplification system and need to finalize the decision to
utilize 6 or 9 mM MgCl2. The addition of MnCl2 is not advantageous for efficiency (yield) or gene
product specificity at the 6 or 9 mM MgCl2. concentrations.
5. Problems or concerns and strategies to address
We have requested additional SCHU S4 RNA due to unexpected increased usage during the
amplification kit troubleshooting.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
97%
9. Work plan for upcoming month
Compare the 9mM MgCl2 to 6mM MgCl2 with a reconstitution sample of SCHU S4 RNA spiked
into normal mouse lung RNA for yield and hybridization
10. Anticipated travel
We will all be at the Annual TVDC meeting in Santa Fe Oct 8th and 9th.
11. Upcoming Contract Authorization (COA) for subcontractors
None
39 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Milestone 34
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 are purifying with RNAeasy columns the previously provided mouse samples for
amplification as soon as we determine the final MgCl 2 concentration to utilize (Milestone
33)
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
67%
9. Work plan for upcoming month


Perform comparisons of known genes and their ability to hybridize to both the ASU and TIGR
arrays.
Perform additional hybridizations between ASU and TIGR arrays with unamplified and
amplified SCHU S4 RNA.
10. Anticipated travel
We will all be at the Annual TVDC meeting in Santa Fe Oct 8th and 9th.
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. No new lab work done
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
40 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
8. Percentage completed
12%
9. Work plan for upcoming month.
ASU has requested that UNM provide more SCHU S4 DNA and RNA in September,
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
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


Using the samples provided by UNM from a dose-response challenge experiment where 3
mice per group were challenged with doses of SCHU S4 RNA ranging from 103-107
organisms. Lungs were harvested within two hours of challenge and processed for total RNA
isolation. The lung RNA samples were received at ASU and processed for RNAeasy cleanup. For the initial studies, lung RNAs from individuals within each group were pooled for a
single sample per dose and processed for LAPT amplification using Superscript II in buffer
supplemented to 6 mM MgCL2. The total micrograms of RNA yielded after amplification are
shown in the second column (Table 1). Reasonable yields were obtained with challenge
doses >104 SCHU S4 per mouse.
The RNA was labeled and processed for hybridization to the arrays. The samples from the
animals challenged with 107 CFU were analyzed and we determined that 538 genes were
represented at > 2-fold increase in intensity as compared to amplified normal lung RNA.
Using these 538 genes as a baseline we found that approximately 200 genes were still
represented throughout the rest of the dose response down to 10 3 CFU.
Infection dose
g after LAPT
# Genes >2 fold to 0
0
18
-
103
5
192
104
42
260
105
51
197
106
30
190
107
42
538
41 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam

We compared the overlap of the amplified genes across the dose response of the challenge.
Using the 538 genes as the universe of detectable genes amplified we see that between 81
and 86 genes are equally represented across the dose responses. This gene base
represents approximately 42% of the 200 genes amplified in animals challenged with doses
less than 106 CFU.
Figure 2. Venn Diagrams of the overlap of amplified genes across the dose response of the challenge.


Notebook/File locations … NM FTU Run 2, Notebook 514, pages 49-58;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-10
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
4%
9. Work plan for upcoming month
The next comparison will be to compare the pooled groups amplified with buffer supplemented to
9mM MgCl2.
10. Anticipated travel
We will all be at the Annual TVDC meeting in Santa Fe Oct 8th and 9th.
11. Upcoming Contract Authorization (COA) for subcontractors
None
42 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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. Frozen KBMA uvrB Ft. novicida are
maintain metabolic activity at –80oC for at least 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. We previously evaluated the sensitivity of the uvrB mutant and
U112 to 6 alternative DNA damaging agents: S-303 (a nitrogen mustard crosslinking agent
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. 4 of the DNA damaging agents 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).
1) This month, no new progress was achieved on this milestone due to layoffs of personnel.
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. If there is a redundant repair
mechanism, we may not be able to produce a highly potent KBMA vaccine utilizing
Francisella species as a platform.
43 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
A new concern is that Cerus may no longer have enough human resources to complete this
milestone in a timely manner.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
fair progress
8. Percentage completed
85% of scientific work completed on the milestone
9. Work plan for upcoming month
We will work with Rick Lyons to generate a modified set of milestones that are scientifically
appropriate and achievable, and we will present them at the annual TVDC conference.
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 LD50 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 immunity by stimulating B cells.
This interpretation was supported by an adoptive transfer experiment in which only the hightiter 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.
1) Ft novicida double mutant strains: KKF224 uvrBiglC, KKF225 uvrBiglD, KKF226
uvrBiglA, and KKF227 uvrBpdpD were received from UTSA on 8/28/07and frozen
stocks were prepared.
2) Ft novicida strains: KKF319 U112 expressing PepO-SIINFEKL was received from UTSA
on 9/6/07.
44 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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 is essentially impossible to rank KBMA vaccine candidates that elicit a
potent T cell response using survival after a lethal Ft novicida 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
Fair progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We will evaluate the ability of live and KBMA Ft novicida-SIINFEKL epitope tagged strains to
induce a CD8 T cell response. We will compare the magnitude of the response to Live and
KBMA SIINFEKL epitope tagged strains of Listeria monocytogenes.
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
3.1. 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
well-characterized 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.
3.2. Since pKEK1168 (the plasmid with PepO-SIINFEKL construct and Francisella promoter) did not
express SIINFEKL protein in DH5 E.coli., we decided to create a new plasmid with both PepOSIINFEKL and Francisella promoter. We already have the pBAD plasmid pKEK1169 providing
PepO-SIINFEKL and the plasmid pKEK894 that was made originally from pKK214 and provides
the Francisella promoter. Both pKEK1169 and pKEK894 were double digested with NcoI and
PstI, then PepO- SIINFEKL cut from pKEK1169 was ligated into the backbone pKEK894 and
transformed into DH5 E.coli. to get the new plasmid, which was named pKEK1177 and
contains PepO-SIINFEKL under the control of only the Francisella promoter from the backbone
pKEK894. The PCR and sequencing results confirmed it.
45 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
3.3. Western blotting was performed to detect the SIINFEKL expression from pKEK1177 in DH5
E.coli. with the whole cells lysates from overnight culture. Lane 1 was the pBAD plasmid
pKEK1169 in DH5α E.coli. and overnight culture was treated with 1.5% arabinose (see the
technical report from July for details). Lane 1 was the positive control and lane2 was the
negative control (Data shown in Figure 1). We did see the expression of SIINFEKL from lane3
and lane4, but the signals were very weak. It is because the Francisella promoter does not work
well in E.Coli. We hoped the protein expression would work better in Francisella. The primary
antibody was a mouse monoclonal antibody to SIINFEKL and the second antibody was antimouse antibody.
Figure 1:
3.4. The plasmid pKEK1177 was transformed into both wildtype LVS and U112 F. novicida using
electroporation. The positive colonies were selected on 10ug/ml Tetracycline TSA++ plate.
3.5. Screened the colonies using colony PCR with “SIINFEKL Rev “and “ pFnBglII For “ primers (
Data shown in Figure 2)
Figure 2:
The picture showed PCR products about 2.2kb from lane5 to lane10 with the positive control on
lane2 and negative controls on lane3 and lane4, which confirmed that the pKEK1177 had been
transformed into both wildtype LVS (lanes 5 to 7) and U112 (lanes 8 to 10).
3.6. Performed western blotting for LVS with pKEK1177 and U112 with pKEK1177 using the whole
cells lysates from overnight culture to detect the expression of SIINFEKL. The primary antibody
was monoclonal anti-SIINFEKL from the University of Massachusetts Medical School and the
second antibody was anti-mouse antibody purchased from GE Healthcare. PepO-SIINFEKL
protein is about 78KD. (Data shown in Figure 3)
46 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Figure3
From the picture, it was obvious that 3 colonies (lane2 to lane4) from LVS with pKEK1177 and 2
colonies(lane7 and lane8) from U112 with pKEK1177 did express SIINFEKL protein. Lane 6 had
the negative result for reasons unknown, but I will prepare the chromosomal DNA for lane6 and
perform PCR to make sure if there is PepO-SIINFEKL construct in this colony. Lane1 and lane5
were the negative controls.
All the results confirmed that we had created a T-cell epitope tagged protein-SIINFEKL that is
expressed by F. tularensis, both LVS and U112 F novicida.
3.7. We named LVS with pKEK1177 as “KKF320” and U112 with pKEK1177 as “KKF319”, and sent
both strains KKF320 and KKF319 to Cerus Lab, .
Data recorded on UTSA TVDC notebook #2, page112-114 for figure 1 and 2.
4
Significant decisions made or pending
Milestone 43 has been finished completely. We will send UvrA mutant LVS, UvrB mutant LVS,
PepO-SIINFEKL LVS and PepO-SINNFEKL U112 to UNM in a couple of weeks.
5
Problems or concerns and strategies to address
6
Deliverables completed
None
pKEK1177 (containing both PepO-SIINFEKLconstruct and Francisella promoter)
KKF320 (LVS with pKEK1177)
KKF319 (U112 with pKEK1177)
7
Quality of performance
8
Percentage completed
9
Work plan for upcoming month
Good
100% of scientific work completed on the milestone
None
10 Anticipated travel
Will attend Annual TVDC meeting in Santa Fe, New Mexico on October 8&9, 2007.
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
47 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Summary: using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. The uvrB LVS was also not more sensitive to DNA
damaging agents compared to WT. This suggests that there may be redundant DNA repair
mechanisms in LVS that may be functioning to repair photochemically induced crosslinks.
1) Ft holarctica LVS strains: KKF317 uvrA mutant, and KKF320 LVS expressing PepOSIINFEKL were received from UTSA on 9/6/07.
4. Significant decisions made or pending
none
5. Problems or concerns and strategies to address
The uvrB mutant of LVS does not appear to be more sensitive to DNA damage induced by
photochemical inactivation with S-59 and UVA or by other chemical means. This suggests that
the potency of a KBMA uvrB LVS vaccine is likely to be the same as KBMA Wt LVS which failed
to protect mice against lethal a SCHU S4 challenge (see MS46). These results suggest that we
reevaluate the KBMA tularemia vaccine strategy and we suggest comparing the efficacy of a
KBMA LVS vaccine to a KBMA Listeria monocytogenes vaccine that expresses Ft antigens.
6. Deliverables completed
none
7. Quality of performance
fair
8. Percentage completed
5%
9. Work plan for upcoming month
The inactivation profile of wt LVS will be directly compared with the uvra and the uvrB mutant.
The metabolic activity of the inactivated strains will also be compared. Our prediction is that there
will not be a significant difference between the two strains. We will evaluate the ability of live and
KBMA LVS-SIINFEKL epitope tagged strain to induce a CD8 T cell response. We will compare
the magnitude of the response to Live and KBMA SIINFEKL epitope tagged strains of Listeria
monocytogenes.
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
48 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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. b 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. We recently demonstrated that 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. Administration
of LVS decreased the ability of the elicited T cells to produce the cytokine IL-2. Terry Wu at
UNM completed a protection study with KBMA WT LVS in which neither a (IV or IN) prime
nor a prime and boost (separated by 3 weeks) vaccination regimen with KBMA WT LVS
protected against a lethal SchuS4 challenge in mice. KBMA WT LVS vaccine appears to be
less potent than live attenuated LVS.
1) No new progress on this milestone was achieved this month.
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 investigating the efficacy of a wild-type KBMA LVS vaccine. Now
that we have received the uvrB mutant we will focus on producing a lot of KBMA uvrB LVS
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS against a lethal LVS challenge is independent of
metabolic activity. This suggests that comparison of various routes, regimens, or formulations will
be difficult to optimize by protective efficacy. The SchuS4 challenge model in mice is more
stringent, but KBMA LVS failed to protect after two doses. It is possible that the rat model may
allow a higher degree of sensitivity. 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. We would like to screen for T-cell responses using
the peptides generated by ASU as an alternative method for optimization of vaccine potency or
construct an overlapping peptide library for IglC.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
53% of scientific work completed on the milestone
9. Work plan for upcoming months
We will compare the photochemical inactivation profile of a uvrB mutant of LVS and their
metabolic activity in MS44. We will prepare a 400mL scale lot of KBMA uvrB LVS. We will
49 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
work with Rick Lyons to generate a modified set of milestones that are scientifically
appropriate and achievable, and we will present them at the annual TVDC conference.
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 the other is experiments with SCHU S4 itself using
constructs that we believe will allow us to make deletions into SCHU S4.
I.
Cloning:
a
Did not get to any cloning work this month. Will continue with the cloning of the 5’end
pdpD into pKEK1042 in this coming month.
II.
Experiments to generate deletions in Schu4:
a
In order to isolate one pdpA deletion clone for each of pathogenicity islands (FPI-I and
FPI-II, respectively) we located a gene from each of the FPIs which will be diagnostic in
our PCR screen. These genes identified last month are FTT1343 (FPI-I) and FTT1696
(FPI-II). Last month, we performed PCR with these oligos paired with the Kan specific
oligo (KanIdentifUp) in order to generate a product.
b
Based on the initial screens from last month, the chromosomal preparations used to do
the above mentioned PCRs were from clones 1B (which is believed to be in the FPI-I)
and 4B clones 4B-1 and 4B-2 ( which are believed to be in the FPI-II). The resulting PCR
products were the correct size and still need to purified and then sent for sequencing
c
Polymerase chain reactions were performed with various oligo sets directed to igLC, the
cloned intron or the diagnostic FPI-I and FPI-II oligos . These PCR products were the
expected size and still need to be purified and then sent for sequencing. The oligo pairs
are listed by name below:
i. Mod.Methylase Forward 3’ :: iglC NdeI Rev
ii. Mod.Methylase Forward 3’ :: EBS2 427 igLC
iii. FTL1152 (LVS)* :: EBS2 427 igLC
iv. FTL1152 (LVS)* :: iglC NdeI Rev
v. EBS2 427 igLC :: igLC Nco I For
*This gene (FTL1152) has a homolog with the Schu S4 FTT1364. Our lab had the FTL1152 oligo on hand and it
will work for our purposes in this screen.
d
Repeated a immunoblot using goat anti-igLC serum with a potential iglC mutant T-IgLC
and with controls. We found the same results seen in the first immunoblot mentioned in
last month’s report. The immunoblot did not detect any wild-type igLC protein in the
50 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
mutant strains(Figure 1, lanes 3&6). Data located in TVD UTSA Notebook 5, page 65
and 66.
Figure 1.
This represents the SDS-PAGE gel (panel A) and resulting Immunoblot (panel B) when using
goat anti-IgLC serum on Schu S4 treated strains indicated in the legend (lanes 2 thru 5). Lanes 2,
4, and 5 should result in the wild-type band of 23 kilodaltons (kDa). The KKT3 ΔMglA mutant
(lane 5) produces less igLC protein since MgLA is the transcriptional regulator of this gene
therefore, this blot did not yield the band after 1 minute exposure (the igLC band is visible after 5
minute exposure, data not shown). The mutants strains T-IgLC single and T-igLC original (lanes 3
and 6) clearly do not illustrate the wild-type igLC.
e
Prepared working bacterial stocks (18 vials) of the potential igLC deletion (T-IgLC) to use in
experiment with mice. Our goal was to do nasal inoculations using 102, 103, 104, 105 and 106
colony forming units (CFU) to see if this mutant is attenuated. A titration (serial dilutions) was
done on the prepared bacterial stock plated onto TSA +++ plates and counted to determine
starting concentration of this stock. This initial calculation indicated stock at 6.97 X 10 9
cells/ml; thus appropriate dilutions were made to use a 20 ul inoculation dose per desired
input. The experiments results are in table 1 below:
51 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
Table 1.
Schu S4 delta-igLC nasal
Number of
Survival after 6
Survival after
inoculation CFUs
Mice
days
26 days
**7.5 X 101
5
5
5
9.0 X 102
5
5
5
3
5
5
5
9.0 X 104
5
5
5
9.0 X 105
5
5
5
PBS control
5
5
5
Schu S4 (+) 2.1 X 101
5
0
0
9.0 X 10
** This number is the 20 ul inoculum used from the 10-6 dilution made from the working stock which
was plated on TSA +++ plates and counted. The other numbers are base on the calculations made
from the titration plate counts performed on the day of experiment.
The mice infected intranasally with 7.5x101 to 9x105 CFU of the SCHU S4 iglC mutant all survived for 26
days after infection. The results of this mouse experiment indicate that the deletion is attenuated and we
hope to challenge the surviving mice this month to determine whether the SCHU S4 iglC mutant strain
might be protective to the vaccinated mice. Data located in TVD UTSA Notebook 5, page 73.
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
44%
9. Work plan for upcoming month
a
b
c
d
Will continue with the 5’-end pdpD cloning into pKEK1042.
Will isolate PCR products generated for both the pdpA and the igLC genomic templates and
send for sequencing to get confirmation of these clones at the DNA level.
Will be challenging the surviving mice from the igLC mouse experiment with our planned
dose of 100 CFUs of KKT1 SchuS4.
Order supplies as required.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
52 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
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 uvrBiglA double mutant. (Note book #4 page 122124): Groups of BALB/c mice (female, 4-6 weeks) were intranasally challenged with 105, 106
or 107 CFU of ΔuvrBiglA. 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 LD50 of ΔuvrBiglA in the intranasal
infection model (BALB/c mice) was greater than 107 CFU.
% Survival
100
10 5 CF U
10 6 CF U
10 7 CF U
80
60
40
20
0
0
2
4
0
2
4
6
8
10
12
14
6
8
10
12
14
16
18
20
% Body weight
115
110
105
100
95
90
85
80
Days post-challenge
Fig.1. Survival of mice infected with Ft subsp. uvrBiglA double mutant. Groups
of BALB/c mice (female, 6-week old) were challenged intra-nasally with 3
doses (105, 106, and 107 CFU) of ΔuvrBiglA to determine LD50 of this strain.
b. Monitor Ft subsp. novicida ΔuvrBiglA replication and dissemination in mice after intranasal
challenge (Note book #4, page 125-128). BALB/c mice were challenged with ΔuvrBiglA
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 days post-challenge, with slightly
reduced viable bacteria recovered from one single mouse at day 14. Numbers of bacteria in
53 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
the liver and spleen are lower than that in the lungs within first week but are still detectable at
day 14 in one mouse liver and in 2 of the three examined spleens. Significant number of
ΔuvrBiglA in lymph nodes only detectable at day 7 post-challenge. Dissemination of
ΔuvrBiglA from lungs to spleen was slower than the ΔuvrBiglD (see June 2007report) and
ΔuvrBiglC mutants (April 2007 report). However, at day 14, while there were still significant
numbers of ΔuvrBiglA in the lungs and spleen, the bacterial load within the same tissues in
the ΔuvrBiglC-challenged animals was almost not detectable.
Log 10 CF U / Organ
9
7
5
Lungs
Spleen
Liver
Spleen
**
3
1
9
7
5
**
Spleen
Spleen
Lymph nodes
*
3
1
Spleen
3
7
14
3
Days post-challenge
7
14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrBiglA 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 uvrBiglA double mutant is highly attenuated in mice infected intranasally,
though the growth of the uvrBiglA 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
44 % of scientific work completed on the milestone
9. Work plan for upcoming month
a
b
Evaluate the protective efficacy of the Ft subsp. novicida uvrBiglA 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.
Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglA mutant at
day 14 and 28 after vaccination.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
54 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, 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: 9/11/2007
3. Work performed and progress including data and preliminary conclusions
a. Previous work had been performed to generate a uvrB + iglB double mutant. Briefly,
genomic DNA was isolated from the uvrB::kan F. novicida mutant. This DNA was
transformed into an iglB::ermC F. novicida mutant and selected on Kanamycin plates.
Colonies were screen by PCR using primers specific for uvrB. Sequencing of this PCR
product failed to provide quality sequence.
b. This month, further PCR was performed since sequencing results were inconclusive. Five
potential uvrB + iglB double mutants’ genomic DNA were screened by PCR using Kanamycin
gene primers. This would provide a definitive confirmation that our double mutant is correct.
The primers used were KanFNdeI and KanRBamHI (Figure 1, lanes 9-14). These primers
are specific for Kanamycin and should yield a PCR product of ~750 bp in strains carrying the
Kan gene. U112 genomic DNA was used as a negative control since U112 is Kan sensitive.
Included in this PCR set were the same potential clones with primers specific for uvrB (Figure
1, lanes 3-7). This is just confirmation from previous month’s work that there is still an
insertion within the uvrB gene. The correct clone should have a uvrB::kan PCR product of
~2100 bp. The results show that clones 1-5 all contain the kanamycin insert (Figure 1,
lanes10-14). This is conclusive data that the uvrB + iglB double mutant is correct. This
mutant grows on Kanamycin (uvrB::kan) as well as Erythromicin (iglB::ermC). Clone 1 was
frozen away as KKF321. No further sequencing will be required to confirm this clone as the
PCR data is sufficient. Data and figure recorded in Notebook #1, pg 19.
Figure 1.
1
2
3
4
5
6
7
8
9
10
11
12
13 14
2000 bp
1600 bp
1000 bp
500 bp
1.
2.
3.
4.
5.
6.
7.
8.
ladder
E.coli genomic DNA (negative control)
Clone 1 (KKF321)
Clone 2
Clone 3
Clone 4
Clone 5
ladder
55 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 8/01/2007 to 8/31/2007
Due Date: 9/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Karl Klose, Bernard Arulanandam
9.
10.
11.
12.
13.
14.
U112 (negative control)
Clone 1 (KKF321)
Clone 2
Clone 3
Clone 4
Clone 5
5. Problems or concerns and strategies to address
None
6. Deliverables completed
uvrB + iglB double mutant of F novicida
7. Quality of performance
Excellent
8. Percentage completed
100%
9. Work plan for upcoming month
Send KKF321 double mutant to Cerus group.
10. Anticipated travel
Will attend the TVDC annual conference in Santa Fe, NM on 10/8 and 10/9/2007
11. Upcoming Contract Authorization
None
56 of 56