Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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, 34 (UNM/ASU), 35, 41,
42, 44, 46, 49, 50, 52
Completed milestones: 1, 16, 25, 32, 33, 39, 40, 43 (UTSA), 48, 51
Inactive milestones: 6, 7, 8, 9, 10, 11, 14, 15, 17, 18, 20, 22, 23, 24, 29, 30, 36, 37, 38,
43 (Cerus), 45, 47, 53, 54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. First group of 7 LBERI participants received the LVS vaccination on 9/11/07and their Day
7 to 28 day follow-up completed.
b. Second group of 8 LBERI participants received LVS vaccination on 10/2/2007 and their
day 7 to 28 day follow-up should be completed.
c. Third group of 7 LBERI participants received LVS vaccination on 10/23/07 and their day
7-28 day follow-up is completed.
d. Fourth group of 6 LBERI participants received the LVS vaccination on 11/13/07 and their
day 7 to 28 day follow-up is completed.
e. Fifth group of 4 LBERI participants received the LVS vaccination on 1/8/08 and their day
7-28 day follow up is pending
f. LBERI has 32 staff and scientists vaccinated as of 1/9/2008.
g. First group of 7 UNM participants are being scheduled to receive the LVS vaccination on
3/18/2008
h. 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
b. The LBERI work stoppage has been lifted for SCHU S4 aerosols as of 11/7/07.
c. In approximately 1 month, UNM may have access to a local source of human cells from
LVS vaccinated individuals.
d. Dr. Lyons has requested UNM IRB approval to allow blood draws on the vaccinated
LBERI and UNM scientists after their LVS vaccinations. The LBERI and UNM scientists
Page 1 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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.
e. UNM (7) and LBERI (32) are offering the LVS vaccinations up to 7 more scientists to total
46; USAMRIID will continue to provide the LVS vaccinations over the next 3-4 months.
5. Problems or concerns and strategies to address
a. Within approximately 1 month, UNM may have access to the blood of UNM and LBERI
scientists who have been vaccinated with LVS at USAMRIID. This is dependent on
UNM’s IRB approval
6. Deliverables completed
32 LBERI scientists and staff have received the LVS vaccination between 9/11/07 and 1/8/08.
7. Quality of performance
Excellent
8. Percentage completed
42%
9. Work plan for the next month
a. Start Risk Assessments, SIP informed consent teleconference, and health screenings for
the next group of 7 UNM participants.
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 3/18/07 – first group of UNM participants. Participants will be
at USAMRIID for 2 days following the vaccinations.
c. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
LVS vaccination participants will be traveling to USAMRIID on 3/17/08
11. Upcoming Contract Authorization (COA) for subcontractors
UNM received a signed COA letter for COA 15 on 9/11/07.
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

December 2007 bioaerosol optimization work focused on bioaerosol testing using SCHU
S4 and confirmation of SCHU S4 pathogenicity via aerosol delivery in primates:
i. Two days of bioaerosol testing were completed incorporating dilutions of fresh
(48h Chamberlain’s broth and 72h BCGA cultures) SCHU S4 stock in the
Collison and Aeromist generators, respectively. November studies indicated that
SCHU S4 was less stable as a bioaerosol versus LVS under similar spray testing
conditions. Decreased SCHU S4 viability post spray is the measure for lower
stability. Therefore, one main objective for December testing was to determine
whether SCHU S4 bioaerosol stability could be increased via growth on a solid
nutrient medium, BCGA:
1. 15 total sprays with fresh SCHU S4
a. 4DEC: 9 sprays conducted at 3 target concentrations (1x105,
Page 2 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
1x106, and 1x107 cfu/mL) using a 48h broth culture of SCHU S4
and the Collison nebulizer at a delivered pressure of 25psi
b. 7DEC: 6 sprays conducted at 3 target concentrations (1x105,
1x106, and 1x107 cfu/mL) using a 72h BCGA plate culture of
SCHU S4 and the Aeromist nebulizer at a delivered pressure of
10psi
2. Results (Figures 1 and 2)
a. 4DEC:

Actual vs. Target cfu/mL values were slightly higher
prior to the bioaerosol (i.e., pre-spray values were
higher than targeted). This was likely due to the lack
of sufficient SCHU S4 growth curve data. Only one
set of data was available so there were too few data
points and the growth curve improves as each new
study is added to the curve. Post-spray values
demonstrated a low < 0.5 log10 drop indicating good
stability in the Collison nebulizer during bacterial
bioaerosol generation.

As with LVS, SCHU S4 spray factor values indicated
increased efficiency when using post-spray versus
pre-spray generator suspension concentrations. This
is due to the decrease in viability observed in the
bacterial suspension following the bioaerosol run.
Spray factor values were lower (i.e., less efficient) as
compared to similar testing using fresh LVS. This
same result was observed when using the Aeromist
nebulizer under similar testing conditions in November
2007.
b. 7DEC:

Actual vs. Target cfu/mL values were slightly lower
prior to the bioaerosol (i.e., pre-spray values were
lower than targeted). This was likely due to the lack of
sufficient SCHU S4 growth curve data. Post-spray
values demonstrated <= 0.5 log10 drop indicating
good stability in the Aeromist nebulizer during bacterial
generation. This slight drop was better than expected
with the Aeromist; prior studies demonstrated upwards
of a 1log10 drop in concentration during the bioaerosol
run. This is indicative of increased stability/viability
when using SCHU S4 harvested from a 72h BCGA
culture.

As with LVS, spray factor values indicated increased
efficiency when using post-spray versus pre-spray
generator suspension concentrations. This is due to
the decrease in viability observed in the bacterial
Page 3 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
suspension following the bioaerosol run. Spray factor
values were similar to those observed when testing
fresh LVS and were higher than those seen with 48h
SCHU S4 Chamberlain’s broth cultures. The exact
reasons for this are unknown, but are discussed
further below.
3. Data filed in the following folders:
a. 4DEC: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06078_TUL-03\TUL-03\Collison Generator\Fresh Schu4
b. 7DEC: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06078_TUL-03\TUL-03\Aeromist nebulizer\7DEC07
Aeromist and Collison: Target vs. Actual CFU/mL (Fresh Chamberlain's and BCGA)
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
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
11/16/2007 (Pre, CB, Aeromist)
11/16/2007 (Post, CB, Aeromist)
12/7/2007 (Pre, BCGA, Aeromist)
12/7/2007 (Post, BCGA, Aeromist)
12/4/2007 (Pre, CB, Collison)
12/4/2007 (Post, CB, Collison)
Figure 1. Target vs. Actual CFU/mL at three target concentrations of fresh Chamberlain’s broth and BCGA SCHU S4 using the
Collison and Aeromist generators, CB: Chamberlains liquid broth culture
BCGA: culture on solid medium BCGA plates
Figure 1 demonstrates that target versus actual CFU/mL were accurate and that post-bioaerosol
concentrations were less than or equal to 0.5 log10 lower than pre-bioaerosol values (an expected result
based on previous experiments conducted similarly).
Page 4 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Aeromist and Collison: Actual CFU/mL vs. Spray Factor (Fresh Chamberlain's and BCGA)
-5.50
4.00
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-6.00
-6.50
-7.00
-7.50
-8.00
Actual CFU/mL (Log10)
11/16/2007 (Pre, CB, Aeromist)
11/16/2007 (Post, CB, Aeromist)
12/7/2007 (Pre, BCGA, Aeromist)
12/7/2007 (Post, BCGA, Aeromist)
12/4/2007 (Pre, CB, Collison)
12/4/2007 (Post, CB, Collison)
Figure 2. Actual CFU/mL vs. Spray Factor at three target concentrations of fresh Chamberlain’s broth and BCGA SCHU S4 using
the Collison and Aeromist generators
CB: Chamberlains liquid broth culture
BCGA: culture on solid medium BCGA plates
Figure 2 demonstrates that F. tularensis SCHU S4 is less stable as a bioaerosol (based upon calculated
spray factors) when grown on and harvested from a 48h Chamberlains liquid broth culture versus a 72h
BCGA culture. Here, the data indicate that bioaerosol stability is more dependent upon the bacterial
culture method than the bioaerosol generation technique.
4. Significant decisions made or pending
Bioaerosol data suggest that F. tularensis SCHU S4 is more stable as a bioaerosol when cultured
from a solid nutrient medium, BCGA. The impact on virulence, however, was significant and is
discussed below under Milestone 4.
5. Problems or concerns and strategies to address
Data continue to indicate that the Collison and Aeromist nebulizers are the optimal choices for
SCHU S4 bioaerosol generation. Based solely on cost effectiveness and ease of use, we are
strongly considering the Aeromist nebulizer for all future F. tularensis bioaerosols .The primary
question now is what culture approach should be used for NHP challenges. This remains to be
discussed.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
96%
Page 5 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
9. Work plan for upcoming month
Initiate Milestone completion report and finalize additional bioaerosol testing including SCHU4 S4
bioaerosols using the Aeromist while focusing on bacterial culture methods (e.g., fresh
Chamberlain’s vs. fresh BCGA vs. frozen stock prepared from Chamberlain’s broth)
10. Anticipated travel
ASM Biodefense Meeting Feb 24-27 in Baltimore, MD. Abstract entitled, “Optimization of
Bioaerosol Generation Techniques for Francisella tularensis” accepted for poster presentation.
LBERI will use non-TVDC funds for this travel per Dr. Sherwood.
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:
Two naïve cynomolgus macaques were exposed to a high dose of SCHU S4 via aerosol
challenge in order to confirm pathogenicity and define endpoints in a primate model. It was
hypothesized that the NHPs would rapidly succumb to infection based on our initial mouse
pathogenicity studies. The challenge material was prepared fresh from a 72h BCGA culture.
Results are summarized below. Electronic data are located in the following folder:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY07-083 and -089 (TUL-04)\14DEC07 NHP exposure
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis SCHU S4 Bioaerosol Challenge Data
Tissue Culturec
Animal ID
Challenge
Date
Inhaled
Dose
(CFU)a
Nx Dateb
A04339
14-Dec-07
2670
A04344
14-Dec-07
5030
Bloodd
Spleen
Liverd
TBLN
Lung
27-Dec-07
1.70E+06
1.20E+06
3.50E+04
7.20E+06
5.70E+04
28-Dec-07
BLD
2.60E+02
2.20E+02
6.70E+02
8.20E+04
a
Dose is based on viable bacteria collected into an all-glass impinger following achievement of 3.5L inhaled
b
Animal A04339 succumbed to infection prior to euthanasia
c
Blood data presented as CFU/mL; tissue data presented as CFU/g
d
Bacteria cultured from the blood of A04339 and the liver of A04344 were inconsistent with F. tularensis
Data indicate that each NHP was challenged with a high dose (2000 to 5000 CFU) of SCHU S4.
Animal A04339 succumbed to infection 13 days post-exposure, while A04344 was euthanized on
Study Day 14 as per the LBERI IACUC protocol. Primary clinical signs over the duration of the
infection included general lethargy, lack of eating, hunched posture, and coughing. The absence
of F. tularensis in the blood and the low numbers present in the spleen, liver, and TBLN of
A04344 are suggestive of a primarily pneumonic disease though further testing is required to
confirm this. The high tissue concentrations of SCHU S4 observed in A04339 indicate that
septicemia probably did not occur until end of life. Bacterial contaminants were observed in the
blood of A04339 and the liver of A04344. It was hypothesized that the blood contaminant was
Page 6 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
due to introduction of normal gut flora during the aortic draw. Similarly, the liver contaminant was
observed only on the undiluted sample suggesting inadvertent introduction of normal flora.
4. Significant decisions made or pending
SCHU S4 harvested from a 72h BCGA culture is stable as a bioaerosol (see MS3 data), but
virulence is decreased (relative to LBERI’s initial mouse bioaerosol studies using frozen stock as
the challenge material)
5. Problems or concerns and strategies to address
Future NHP aerosol challenges will require further investigation into the decreased virulence seen
with the two naïve NHPs discussed herein. Additional mouse bioaerosol exposures using various
SCHU S4 culture approaches will likely address this.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
20%
9. Work plan for upcoming month
a. Decide on exposure approaches for next set of NHPs. Focus will primarily be on the
culture method used for the challenge material (i.e., fresh Chamberlain’s versus fresh
BCGA versus frozen stock). This will ultimately depend on the additional mouse
pathogenicity as described in Item 5.
b. LVS-vaccinated 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 January 2008.
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
a. Experiment Ptran2 (Notebook 112 pages 3-5)
i. The purpose of this experiment is to produce antibodies that can be used to
deplete CD4 and CD8 T cells from naïve and LVS-vaccinated rats.
ii. The hybridoma lines W3/23 (anti-CD4) and OX-8 (anti-CD8) were obtained
from European Collection of Cell Cultures (ECACC) and TS2/18.1.1 (isotype
control; anti-human LFA-2) was obtained from American Type Culture
Collection (ATCC)
iii. Culture supernatants from all three hybridomas were tested for the presence
of antibodies by FACS analysis. Culture supernatants were incubated with
rat peripheral blood cells and antibody binding was detected with PEconjugated anti-mouse IgG1 antibodies
Page 7 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
iv. We detected binding of W3/25 but not OX-8. It is possible that 1) the
concentration OX-8 antibodies in the supernatant was too low; 2) the
hybridoma did not produce any antibody; or 3) the number of OX-8-positive
cells in the blood was too low to detect antibody binding. Therefore, we
cultured the hybridoma cells longer without refreshing the culture medium to
increase the antibody concentration and then tested the culture supernatant
again, this time on rat splenocytes.
v. The supernatants from OX-8 was titrated from neat to 1:1000 dilution and
TS2/18.1.1 and W3/25 from neat to 1:100 on two different rat spleen
preparations. OX-8 antibody purchased from BD Biosciences was used as a
positive control for the OX-8 culture supernatant generated at UNM.
vi. We found that both W3/25 and OX-8 hybridoma lines produced antibodies;
W3/25 bound 30-50% of rat splenocytes and OX-8 bound 20-40% of rat
splenocytes. (See data in Table 1) We showed separately by ELISA that
TS2/18.1.1 also produced antibodies (data not shown) but did not bind rat
cells here because it is specific for human LFA-2
vii. The hybridomas have been shipped to Taconic Farms for ascites production
to generate a large stock of antibodies for long term usage.
Table 1. Titration of hybridoma supernatants on rat splenocytes
b. Experiment Ftc46 study 4 (Notebook 103, page 74-75, 81-83, 94)
i. In Ftc46 study 3, we found that the delivered location of the quantum dots did
not always correlate with the recovered location of SCHU S4. Specifically,
the Xenogen images indicated that 3 of 4 rats had received SCHU S4 down
the esophagus when in fact they all had SCHU S4 in their lungs (the
esophagus and gut were not sampled). However, the number of SCHU S4
recovered in the lungs was only 20 to 50% of the inoculum. It possible,
therefore, that most of the inoculum had been in fact been delivered down
the esophagus but some of the inoculum ended up in the lungs.
ii. This Ftc46 study 4 experiment had two purposes: 1) to determine whether
SCHU S4 can be found in the esophagus after intratracheal inoculation and
2) to determine the best position to image the rats
Page 8 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
iii. Rats were inoculated i.t. with 100 l of a mixture containing 5 pmol/ml
QD655-luc8, 14.5 g/ml coelentrazine, and 9.1 x 105 cfu/ml SCHU S4. The
rats were imaged in supine, prone and both right and left lateral positions and
then the lungs and esophagus were collected to quantify the number of
SCHU S4 in these tissues
iv. The Xenogen images taken of one rat from the four perspectives are shown
in Figure 1. The quantum dots are clearly localized in the lungs
v. The distribution of SCHU S4 in the lungs and esophagus are shown in Table
2. While the luminescence emitted primarily from the lungs, the number of
SCHU S4 recovered from the lungs is only 10% of the inoculum; this is
consistent with the average actual lung deposition of 20% found in earlier
experiments. We have never been able to account for the remaining 80-90%
of the inoculum.
vi. Few or no bacteria were detected in the esophagus. We may have to sample
the gut because there may be bacteria in the gut but the quantum dot
emission may be inactivated in the acidic environment. Another potential
loss may be in the syringe and this can be easily tested.
Figure 1. Xenogen images taken of a single rat from 4 different perspectives
after intratracheal inoculation with a mixture of SCHU S4 and quantum dots
Table 2. Distribution of SCHU S4 after intratracheal inoculation
Rat
1
2
3
No. bacteria recovered (cfu)
Lungs
Esophagus
1.1 x 104
ND
3
9.5 x 10
ND
1.5 x 104
200
ND = below detection level
c.
Experiment Ftc46 study 5 (Notebook 103, page 76-77, 84-86, 95)
i. Another way to do the experiment described in Experiment Ftc46 study 4 is
to show that when the inoculum is deliberately delivered down the
Page 9 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
esophagus and the Xenogen images reflects such, no bacteria will be found
in the lungs.
ii. Rats were inoculated esophageally with 100 l of a mixture containing 5
pmol/ml QD655-luc8, 14.5 g/ml coelentrazine, and 9.1 x 105 cfu/ml SCHU S4.
iii. The Xenogen images of three rats are shown in Figure 2. The emission
patterns suggested that SCHU S4 was in the lungs for rat 1 and in the gut for
rat 2. Rat 3 had some initial luminescence the lungs which disappeared in
subsequent imaging attempts; thus it was expected to have some bacteria in
the lungs
iv. The distribution of SCHU S4 in the lungs and esophagus are shown in Table
3. Optimistically, these data suggest that whenever there is a quantum dot
signal in the lungs, it is a good indication that the bacteria is also there.
However, they also suggest that even when we intentionally deliver the
inoculum esophageally, some bacteria ends up in the lungs. Moreover, as
discussed in the previous experiment, approximately 80% of the inoculum
remains unaccounted and some or all of them may be in the gut
Figure 2. Xenogen images taken of three rats’ esophageal inoculation with a mixture
of SCHU S4 and quantum dot
Table 3. Distribution of SCHU S4 after esophageal
inoculation
Rat
1
2
3
No. bacteria recovered (cfu)
Lungs
Esophagus
3.8 x 104
1.0 x 103
ND
2.8 x 103
3
1.2 x 10
6.5 x 102
ND = below detection level
4. Significant decisions made or pending
None
Page 10 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed – additional characterization underway
7. Quality of performance
Good
8. Percentage completed
69%
9. Work plan for upcoming month
a. Role of CD4 and CD8 T cells in the ability of rats to resist tularemia
i. Wait for ascites to be produced at Taconic and, in the mean time, vaccinate
rats with LVS so they can be used to determine the role of CD4 and CD8 T
cells in vaccine-induced protection when the ascites are available
b. Determine whether passive immunization with convalescent sera will protect naïve
Fischer 344 rats from i.t. SCHU S4 challenge
i. Vaccinate rats for use as serum donors and as controls in challenge
experiments
ii. Collect and passively transfer serum from naïve and LVS vaccinated rats into
naïve rats to determine the ability of antibodies to protect against tularemia
c. Sample the gut for SCHU S4 after i.t. inoculation.
10. Anticipated travel
Travel to FDA tularemia vaccine workshop and NIAID/UNM/DVC joint TVDC team meetings
in Washington DC from March 11 to March 13, 2008.
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 IFN detection by ELISPOT analysis
1.
We have been screening non-LVS vaccinated NHPs to test their
responsiveness to LVS before they were exposed to SCHU S4 or
consideration for future vaccination with LVS; Figure 1 shows the
results
i. PBMCs from non-LVS vaccinated NHPs respond to FF LVS more
than to HK LVS
ii. The amount of IFN secreted by PBMCs from non-vaccinated
NHPs is variable
Page 11 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
IFN Secretion by Individual Non-LVS Vaccinated NHPs
Media
LVS hk Hi
LVS ff Hi
400
350
300
250
200
150
100
A05477
A04367
A04344
A04339
A04274
A04168
A03033
0
A03016
50
A02386
Cell Mean for IFNg Spots
450
Figure 1: PBMCs from non-LVS vaccinated NHPs were plated at 1.33 x 106 cells/ml and stimulated with
either HK or FF LVS (1 x 105 cells/ml).
2.
Data interpretation
i. Non-vaccinated NHPs secrete IFN to varying degrees after HK or
FF LVS stimulation, but uniformly more after FF LVS stimulation
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay010408.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 010408.svd and TVDC 1 bound notebook:
TUL 15 (pps. 41 – 48, 50, 78 – 80, 119 – 20), TUL 17 (pps. 63 – 70, 112 – 116), TUL 19 (pps. 99 – 108),
TUL 22 (pps. 141 – 145) and TUL 23 (pps. 146 – 151).
b. Update on proliferative response of PBMCs
1. We have been screening more non-LVS vaccinated NHPs in the proliferation
assay
2. The response of individual non-vaccinated NHPs is shown in Figure 2
Page 12 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Proliferation by PBMCs from Individual Non-LVS Vaccinated NHPs
3.00E6
Media
LVS hk Hi
LVS ff Hi
2.50E6
2.00E6
1.50E6
1.00E6
A05477
A04367
A04344
A04339
A04274
A04168
A03033
A03016
A02386
A00937
A00896
0
A00908
5.00E5
Figure 2: PBMCs from non-LVS vaccinated NHPs were plated at 1x 106/ml and stimulated with either HK
or FF LVS at 1 x 105 cells/ml.
3. Data Interpretation:
i. Although our initial studies initiated in November 2006 with 6 naïve NHPs
suggested that they had little proliferative response to LVS (only 3 shown
in Figure 2, A00896, A00908 and A00937), the response of the other 9
NHPs tested has been more varied and responsive to LVS.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay010408.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 010408.svd and TVDC 1 bound notebook:
TUL 15 (pps. 41 – 48, 50, 78 – 80, 119 – 20), TUL 17 (pps. 63 – 70, 112 – 116), TUL 19 (pps. 99 – 108),
TUL 22 (pps. 141 – 145) and TUL 23 (pps. 146 – 151); and TVDC binder 1(TUL 8)..
c.
Update on IgG anti-LVS ELISA
1. We screened the plasma of several non-LVS vaccinated NHPs in order to insure
that they did not have antibodies to LVS before we exposed two of them to
aerosolized SCHU S4 (Figure 3)
Page 13 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
IgG Anti-LVS in Non-LVS Vaccinated NHPs
IgG anti-LVS Titer
6000
5000
4000
3000
2000
A05477
A04344
A04339
A04168
A03033
A03016
A02386
A00937
A00908
A00902
A00896
0
A00868
1000
Figure 3: Plasma from non-LVS vaccinated NHPs was tested for heat-killed LVS reactivity by ELISA.
Titers were determined as the highest dilution tested which had a greater OD405 than the background
value. Dilutions tested were 1/200, 1/1000, 1/5000, 1/25000 and 1/125,000.
2.
Data Interpretation
i. Most of the non-LVS vaccinated NHPs had titers of 200 (the lowest dilution
tested); i.e. they had no reactivity above background
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 010408.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 010408.svd; TVDC 1 bound notebook: TUL
19 (pps. 99 – 108); TUL 21 (pps. 135 – 140) and TUL 22 (pps. 141 – 145); and TVDC binder 1(TUL 8 and
TUL 9).
d. Choice of two non-LVS vaccinated NHPs for exposure to aerosolized SCHU S4
1. We needed to choose two non-LVS vaccinated NHPs to expose to aerosolized
SCHU S4
i.
We compared the response of all non-LVS vaccinated NHPs to LVS by
ELISA (IgG), proliferation and IFNγ production
ii. A04339 and A04344 were the two NHPs which had the lowest responses to
LVS in the majority of the assays; None of the tested NHPs had low
responses to LVS in all assays (Figure 4A shows proliferation; 4B shows
IFNγ production)
Page 14 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Response of A04339 and A04344 to LVS
Cell Mean for RLU small
225000
200000
175000
Media
LVS hk Hi
LVS ff Hi
A
150000
125000
100000
75000
50000
25000
0
A04339
Cell Mean for IFNg Spots
180
A04344
Media
LVS hk Hi
LVS ff Hi
160
140
B
120
100
80
60
40
20
0
A04339
A04344
Figure 4: PBMCs from non-LVS vaccinated NHPs were plated at 1x 106/ml (A; proliferation assay) or
1.33 x 106/ml (B;IFNγ production) and stimulated with either HK or FF LVS at 1 x 105 cells/ml.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 010408.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 010408.svd; TVDC 1 bound notebook: TUL
19 (pps. 99 – 108); and TUL 22 (pps. 141 – 145).
4. Significant decisions made or pending
LBERI has decided that all NHPs will be pre-screened for reactivity to LVS before use in any
experimentation involving LVS vaccination and/or SCHU S4 challenge
5. Problems or concerns and strategies to address
None
Page 15 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
93% of scientific work has been completed
9. Work plan for upcoming month
1. Continue to screen non-LVS vaccinated NHPs for their reactivity to LVS by proliferation and
IFNγ secretion
2. Test PBMCs from ID and SC vaccinated NHPs on the same day to determine if there are
actual differences in IFNγ secretion using both HK and FF LVS as antigens.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc60.7 (Notebook 103, page 78-80, 84-86, 95)
i. The limited sensitivity of our antigen response assays may become
problematic when we use them to screen vaccinees and vaccine candidates.
In small animal models, the assay sensitivity may be increased by boosting
the vaccinated animals with inactivated or live LVS or SCHU S4. The
purpose of this experiment was to determine whether boosting LVS
vaccinated mice with live or heat killed LVS would increase the number of
IFN producing cells in the spleen.
ii. LVS vaccinated mice were boosted i.n. with 5 x 104 live or heat killed LVS 48
days after initial vaccination. One week after the boost, splenocytes from
boosted mice were collected and used in an IFN Elispot. By mistake, mice
that had been vaccinated 36 days earlier than the ones that were boosted
were used as source of control, “vaccinated only” splenocytes.
iii. The IFN Elispot assay results shown in Figure 3 suggest that boosting
vaccinated mice with HK LVS increased the number of LVS-specific, IFN
producing cells and that boosting with live LVS increased the number further.
However we have to interpret these data carefully given the caveat described
in (ii) and the fact that the duplicate sample for the mice boosted with live
LVS did not work (Figure 3).
Page 16 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Figure 3. Increasing the number of antigen-specific IFN
producing cells by boosting vaccinated BALB/c mice with live
or heat-killed LVS.
b. Experiment Ftc60.8 (Notebook 103, page 87-93)
i. To confirm and improve the experiment just described (Ftc60.7), we repeated
the experiment with three changes: 1) all mice were vaccinated on the same
day and later boosted on the same day; 2) the number of splenocytes per
well were titrated; and 3) naïve mice were included as a negative control
ii. The results from the IFN Elispot assay shown in Figure 4 suggest that
boosting LVS vaccinated mice did not increase the frequency of IFNproducing antigen-specific splenocytes.
Figure 4. Effect of boosting on the frequency of antigen-specific IFN producing cells
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
Good
Page 17 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
8. Percentage completed
56%
9. Work plan for upcoming month
a. Repeat the experiment described above to increase assay sensitivity
b. Determine whether the IFN response would be better if splenocytes are harvested
earlier after boost
c. Determine whether boosting with SCHU S4 would be better than with LVS because it
persists longer in mice.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new lab work performed; Theresa Archibeque is working to accrue more donors
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
9%
9. Work plan for upcoming month
a. Determine kinetics of bacterial proliferation in human alveolar macrophages after F
tularensis infection with and without recombinant IFN
b. Determine kinetics of bacterial proliferation in human monocyte derived macrophages
after F. tularensis infection with and without recombinant IFN
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
Page 18 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
3. Work performed and progress including data and preliminary conclusions
No new work performed
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
25%
9. Work plan for upcoming month
a. Troubleshoot mouse macrophage killing assay with SCHU S4
b. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. Ten eukaryotic in vitro expression cassettes for five complete FTU (Francisella tularensis)
genes (groES, groEL, IglC, katG, Tul4) and two non-FTU antigens antigens (OVA and
Calm) were shipped to UNM. These template samples will be used for expression in
wheat germ and rabbit reticulocyte IVT systems, if necessary.
Figure 1. Quality control of the IVT expression cassettes.
Page 19 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
1 – OVA; 2 – FTU0721a (KatG); 3 – FTU0721b (KatG);; 4 –
FTU0721c (KatG);; 5 – FTU1696a (GroEL); 6 – FTU1696b (GroEL);
7 – Calm; 8 – FTU0901 (TUL4); 9 – FTU1712 (IglC2), 10 – FTU1695
(GroES)
Data location: R:\GeneVac\FTU\Contract\Proteome\Derek's data\DNA Gels\WG Amp final
optimized 12-11-07.jpg
B. Select and test IVT Protocols
1. As previously reported all protocols for HTP protein production in the E. coli based IVT
system have been developed and optimized. We are anticipating that developed
protocols will be applicable to the eukaryotic expression systems, but will test them if
decision to switch is made.
C. Select and test protocols for protein purification
1. We have found that the FTU T cell assays respond nonspecifically to some factor in the
E. coli IVT lysates. Therefore, a set of IVT lysates without added template were passed
through several different size exclusion filters with different molecular weight cutoffs.
These samples were prepared, shipped to UNM and tested by Dr. Wu in his T cell assay
for cross-reactivity. The cross-reactivity in the interferon ELISpot was eliminated in all
filtered samples.
2. The stability of the macro-complexes and the corresponding efficiency of macro-complex
elimination were evaluated by PAGE. Fresh, frozen/thawed and 37 oC shaken IVT
samples were tested. Filtration of fresh IVT through a 100kDa filter removes most E. coli
proteins as visualized by Coomassie stain (fig 2, lines 1, 2). Three cycles of freezing and
thawing resulted in significant degradation of the macro-complexes as visualized by
reduced removal efficiency. Noticeable amounts of E. coli proteins were detected in the
100kDa-filtered and 50kDa-filtered, but not the 30kDa filtered samples (fig 2, lines 3-8).
Therefore, freezing/thawing of IVT lysates and reactions should be avoided. However,
Fig 3 (next figure) shows that shaking at 37oC increases reaction yield without disrupting
the complexes (fig 3, lines 1, 2).
Fig. 2. Effect of freezing/thawing on the macro-complex stability.
Page 20 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
1 – fresh IVT; 2 – fresh IVT, 100kDa filtered;
3 – fr/thwed IVT; 4 – fr/thwed IVT, 100kDa filtrated;
5 – fr/thwed IVT; 6 – fr/thwed IVT, 50kDa filtrated;
7 – fr/thwed IVT; 8 – fr/thwed IVT, 30kDa filtrated.
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\Presentation\Testing IVT
protein filtration using Microcon.ppt
3. Next, IVT template was added to the reactions and these samples with radiolabeled de
novo polypeptides were filtered. Calmodulin was selected as the test template. The
efficiency of separating the in vitro synthesized Calmodulin polypeptide from the E. coli
proteins was evaluated by PAGE (fig 3A). The gel was stained to visualize total proteins
and then autoradiographed to visualize de novo polypeptide (fig 3B). Calmodulin was
detected in the 100kDa and 50kDa (lines 4-7), but not in the 30kDa filtrates (lines 8, 9).
The reduced yield of de novo synthesized protein in the filtered samples relative to the
original IVT reaction indicates its possible association with either E coli macro-complexes
or attachment to the filtration membrane.
Fig 3. Separation of the de novo synthesized protein from E.
coli macro-complexes by filtration.
Page 21 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
A – Coomassie stained PAGE
B – Autoradiograph of the lower part of the gel in A
1 – IVT lysate w/out template, shaken 4hrs at 37oC
2 – Sample #1 passed through 100kDa filter
3 – IVT with template, w/out shaking at 37oC
4 – IVT with template, shaken 4hrs at 37oC
5 – Sample #4 passed through 100kDa filter
6- IVT with template, shaken 4hrs at 37oC
7 – Sample #6 passed through 50kDa filter
8 – IVT with template, shaken 4hrs at 37oC
9 – Sample #8 passed through 30kDa filter
Data location: R:\GeneVac\FTU\Contract\Proteome\Tien's data\Presentation\Testing IVT
protein filtration using Microcon.ppt
4. A set of IVT reactions which have been frozen/thawed, incubated at 37oC with and w/out
template (OVA) samples and their 100kDa and 50kDa filtrates have been prepared and
are being shipped to UNM for evaluation in the T-cell assay
4. Significant decisions made or pending
None at this time, but decisions on a delivery format for the polypeptide samples is pending.
5. Problems or concerns and strategies to address
To minimize loss of polypeptide sample during filtration and maximize separation from most
E. coli proteins in lysate, we will test effects of the diluting the reaction mix and adding low
concentrations of mild detergents.
6. Deliverables completed
None
Page 22 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
7. Quality of performance
Very good
8. Percentage completed
99%
9. Work plan for upcoming month
Generate and test effect of IVT dilution and use of mild detergents on the de novo protein
recovery efficiency and T-cell stimulation specificity.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc59 study 9 (Notebook 115, pages 13-15, 20) and Experiment Ftc59
study 11 (Notebook 115, pages 21-25)
i. The purpose of these experiments was to determine whether the
crossreactive material in the E. coli lysate can be removed by size exclusion.
ii. Two independent sets of ivt E. coli lysates (without template) were filtered
through Centricon filters with MW cutoff of 3, 10, 30, 50 and 100kDa. We
hoped that the crossreactive material would be retained above the filter
leaving the flow through free of the crossreactive material.
iii. The results from both experiments showed that removal of fractions above
100kDa from ivt lysate dramatically reduced cross reactive stimulation of
IFN release
Figure 5. Elimination of crossreactive stimulation by fractionating E. coli ivt mixture
through 100 kDa cutoff filter.
Page 23 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
15%
9. Work plan for upcoming month
a. Test the size exclusion strategy with ivt samples. In the experiments described thus
far, we tested only the E. coli lysates without any DNA template and therefore there
were no ivt proteins. We would like to show that the size exclusion strategy also
works when DNA templates are included. Specifically, we would like to show that the
ivt proteins flow through the filter and stimulate IFN production while the
crossreactive material is retained above the filter.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenecity of protein-fragments, Full scale production of protein-fragment library,
Purification of protein-fragment library, Array protein-fragment into overlapping pools, Ship
to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (active)
 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
A. Build ORF expression library corresponding to proteome
1. In anticipation that decisions in milestone 26 are near, we are pooling PCR primers ORF library
production. We will proceed following decisions on expression system, yield needs, delivery
format, pooling capacity, and purification requirements.
Page 24 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
2. Genetic immunization of mice with groES, groEL, IglC, katG, Tul4 and OVA has been completed.
Animals were boosted with genetic immunization expression plasmids on Dec 20 and are ready
for protein boost.
3. Proteins for the final boost have been partially purified. Samples are being sent to UNM on Jan
7. These immunized mice will be used as a source of immune splenocytes for the T cell assay
development and the animals will also be bled for immune sera, as reagent.
Fig. 3 Coomassie stained PAGE of partially purified recombinant antigens.
1 – OVA; 2 – KatG A; 3 – GroEL B; 4 – IglC2; 5 – KatG B; 6 – KatG C; 7 – TUL4; 8 - GroES
Data location: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT data\FTU gels\FTU
HTP IVT Coomassie gels. Files: FTU proteins for immunization boost IB washed 12-13-07
01.jpg; FTU proteins for immunization boost Final 12-13 and 14 batch.jpg; FTU proteins for
immunization boost 12-14-07 batch.jpg
4. Significant decisions made or pending.
Following results of T-cell stimulation experiments, we will initiate FTU IVT template library
production.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
29%
9. Work plan for upcoming month
We are planning to prepare 2 96-well plates of FTU ORFs and then prepare these as LEE IVT
template constructs. This exercise will help us identify any further protocol adaptations that may
be needed and teach us the feasibility of different cascading schedules so as to optimize the
upcoming scaled production schemes.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 25 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Milestone 34-ASU
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

We performed studies to optimize labeled cDNA and genomic DNA for the genomic
normalization process. In the experiment below, we labeled fragmented genomic SCHU S4
DNA with Alexaflour 488 and SCHU S4 cDNA with Alexafluor 555 and performed a doseresponse titration. Holding the cDNA constant at 200 pmoles labeled, we performed
hybridizations after mixing in defined ratios of labeled genomic DNA. The ratios were 200
pmoles cDNA:0 pmoles genomic DNA, 200 pmoles cDNA:50 pmoles genomic DNA; 200
pmoles cDNA:100 pmoles genomic DNA; and 200 pmoles cDNA:200 pmoles genomic DNA.
The cDNA and genomic DNA were mixed in hybridization buffer and hybridized to the ASU
array by standard techniques. In Figure 1, the raw signal intensity histograms of the labeled
genomic DNA show equal low-level signal intensities across the competition range. Figure 2
shows the cDNA raw signal intensity histograms. Using the 200:0 as a no-competition
control, we observed no significant inhibition of cDNA signal at either 200:50 or 200:100
genomic competition. However, at the 200:200 competition ratios there was a decrease in
overall signal intensity and the Spearman correlation to the non competition control showed a
marked reduction in correlation. The Spearman correlations of the 200:50 and 200:100
competitions to controls were very close and show minimal disruption to hybridization signals.
Figure 1. Raw signal intensity of the genomic DNA
channel

Figure 2. Raw signal intensity of SCHU S4 cDNA
channel.
We next performed a correlation analysis of the raw signal intensities and genomically
normalized data (Figure 3). Raw signal intensities between 200:50 and 200:100 competition
samples were quite high. Similar high levels of correlations were observed with genomically
normalized data. High correlations were not found when using the 200:200 genomically
normalized data (data not shown).
Page 26 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Figure 3. Correlation analyses of raw signal data and genomically normalized signal data
 Notebook/File locations …, Notebook 514, page 115-122.
 Electronic file locations…
R:\GeneVac\FTU\Contract\Microarray\Milestones\34\LAPT-16 (Genomic Normalization)
4. Significant decisions made or pending.
When using 200 labeled pmoles of cDNA, competition with 50 pmoles genomic DNA does not
lead to significant changes in hybridization kinetics and provides consistent base values for
genomic normalization of the data.
5. Problems or concerns and strategies to address
Two series of amplifications recently failed. The reason for one failure was a technical problem
with one step being performed at the wrong incubation temperature (47 ○C instead of 37 ○C).
The second failure was attributed to an enzyme that was warm on receipt (improper shipping).
Standard Operating Procedures (SOPs) are being reviewed and re-written for all the steps of
amplification process to ensure proper steps are followed. If enzymes are received without dry
ice, the shipment will be rejected. The company is re-supplying enzymes that were shipped
improperly.
 Notebook/File locations …, Notebook 514, LAPT 17, page 125-128.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
98%
9. Work plan for upcoming month


Perform QPCR verification of to establish the QPRC assay for verification of microarray
results.
Continue studies to establish the optimum conditions for genomic normalization of
microarrays using genomic SCHU S4 RNA
Page 27 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34-UNM
Milestone description: Pilot Studies for the optimization of RNA isolation and hybridization
conditions
Institution: UNM
1. Date started: 03/01/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc45 study 2 (Notebook 115, pages 26-33)
i. The purpose of this experiment was to determine the sensitivity of the F.
tularensis expression microarray developed at ASU.
ii. We are providing ASU with lung RNA from mice inoculated i.n. with 101 to 107
CFU SCHU S4. We have isolated the RNA and will ship them to ASU after
we verify sterility and RNA integrity.
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
16%
9. Work plan for upcoming month
a. In the experiment just described, we are testing the sensitivity of the microarray
analysis by infecting mice with various doses of SCHU S4 and isolating RNA at 4 h
post infection. We would also like to test the array sensitivity by infecting mice with a
single, minimal lethal dose of SCHU S4 and isolate RNA at various time points
thereafter. Essentially, these experiments are measuring the sensitivity of the
microarray to detect genes expressed at a single and then multiple time points.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 28 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Milestone 35-ASU
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

A new dose response experiment has been performed at UNM by infecting mice with log
gradient doses of SCHU S4 bacilli ranging from 10 to 107. The lungs were removed at 4
hours post challenge and total RNA isolated. The RNA is scheduled to be shipped to ASU
the week of January 7-11th.
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
10%
9. Work plan for upcoming month
Perform RNAeasy purification of received RNA and perform LAPT amplification and microarray
hybridization of samples.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: We have determined that all the NER-deficient strains of Ft. novicida are only
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 maintain metabolic activity at –80oC for at least 3 months. Inactivated
NER-deficient strains have a similar degree of metabolic activity as the wild-type Ft. novicida
Page 29 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
strain (which is different than has been seen with L. monocytogenes or B. anthracis), and we
have demonstrated that this lack of sensitivity to DNA damage is universal to numerous DNA
damaging agents.
1) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
Nov 16, the vaccines research program was “spun out” of Cerus Corporation. All of the
personnel, intellectual property and other assets related to immunotherapy were transferred
from Cerus to Anza Therapeutics Inc. Cerus and Anza are working to establish a
professional service agreement that will allow the work on the TVDC to proceed using Anza
personnel and that is acceptable to UNM and NIAID. Numerous discussions have been held
between Cerus and Anza representatives and with UNM to discuss possible mechanisms for
continuing with the TVDC contract going forward. Cerus, Anza, and UNM have agreed in
principle to establish a service agreement between Cerus and Anza. However, until this
relationship is approved by NIAID there will be no direct costs charged by Anza to Cerus for
the TVDC. Anza is currently in the process of getting an IACUC established in order to
obtain our OLAW assurance and Anza has submitted APHIS documents to the USDA for
transport of Francisella tularensis strains (application # 07361052).
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. 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 to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 30 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
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 work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
None
Page 31 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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
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) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
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 schuS4 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
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
none
11. Upcoming Contract Authorization (COA) for subcontractors
none
Page 32 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA
for initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS
and we have demonstrated that the metabolic activity of this lot is stable for 3 months. We
have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log
attenuation compared with live LVS. We have demonstrated that doses of KBMA WT LVS as
low as 1 x107 provide protection against 100 x IP LD50 challenge of live LVS. However, none
of the mice vaccinated with the equivalent doses of HK LVS died either. This is consistent
with protection against an LVS challenge being largely humoral. 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) This month, no new progress was achieved toward this milestone as we work towards
modification of the milestones and establish a service agreement between Cerus and Anza.
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
Page 33 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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 work to generate a modified set of milestones that are scientifically appropriate and
achievable.
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. All igLD components needed for cloning looked very good; the concentration of DNA yielded for
each component was the highest (especially for the igLD 30a and 255a products) compared to
the previous preparations (figure 1). A ligation was done with these preparations and
transformation of DH5 cells have been completed; however, UTSA did not get a chance to
screen resulting clones. Will isolate plasmid from the resulting clones and results will be reported
on next report. Data located in TVD UTSA Notebook 5, page 92.
Page 34 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Figure 1.
This represents the igLD cloning components used in the new ligation and transformation. The
enzymes used for cloning were Xho I and BsrGI. Lanes 2 and 4 are independently digested and
gel purified KEK1140 plasmid; lanes 3 and 5 are igLD 30/31a and igLD 255/256a oligo set PCR
products which were digested and gel purified, respectively. Lane 1 is Low Mass Ladder from
Invitrogen which gives an idea of nanogram (ng) amounts of DNA loaded on this gel (e.g. 2.0 Kb
band represents 100 ng). The yield is sufficient for at least 3 independent ligations for 30a and
255a, respectively.
Lane 6 is the 1 Kb ladder from Invitrogen. The expected sizes for each
component is correct; 8.0 Kb for the KEK1140 and 350 bp for the respective iglD PCR products.
b. In order to delete an entire pathogenicity island (FPI) from Schu 4 we need a construct of pdpD
containing a flip recombinase transferase. We’ve confirmed the first pdpD construct to contain
this gene sequence (this plasmid is named KEK1188). Plasmid from potential clones were used in
a polymerase chain reaction (PCR) using oligos directed to the 5’ and 3’ ends of pdpD which also
corresponded to the cloned region of pdpD. The resulting PCR product should be approximately
3.4 Kb in size which includes the cloned 5’ and 3’ ends of pdpD; the Flip recombinase recognition
sites; and an erythromycin gene (figure 2). In addition, plasmid (KEK1188) was sent for
sequencing; results indicated that cloned regions are pdpD. We now need to move this deletion
construct into a pUC vector; since this background plasmid has been shown to integrate and
resolve from the Schu S4 chromosome with better results (that is, retaining the desired deletion).
The present vector is pwsK30 and has shown not to work well with SchuS4.
Figure 2.
1 Kb
1
9.0
3.0
1.5
2
3
4
Legend:
1, 1 Kb Ladder (Invitrogen)
2. C9 1042+pdpD
3. KEK1188 (C1-pdpD)
4. Uncut KEK1188
This PCR screen used two potential pdpD constructs believed to contain both 5’ and 3’ ends of this
gene; in addition; to the already cloned flip recombinase recognition sites and the erythromycin
gene. The oligos used for this reaction were pdpD 1 Kb Not I S4 Forward (described in earlier
Page 35 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
report) and pdpD up Sal I (5’-gcacgcgtcgacctaagccagtaaaataagcatac-3’). Lane 2 is clone 9 (C9)
that did not yield a correct product; lane 3 is clone 1 (C1) of the 1042+pdpD plasmids initially
screen by digestion (figure1 from November 2007 report) which yielded the expect 3.4 Kb product
and was designated KEK1188. Lane 4 is a sample of uncut plasmid KEK1188; and Lane 1 is 1Kb
ladder. Data located in TVD UTSA Notebook 5, page 93.
II. Experiments to generate deletions in Schu4:
a. In hopes of generating a vgrG mutant in Schu4, two plasmids were created using the tulatron
method (pKEK1161 and pKEK1162). pKEK1161 targets base pairs 30 and 31 for insertion;
pKEK1162 targets base pairs 81 and 82 of vgrG. These plasmids were Midi prepped and
cryotransformed into Schu4. Transformants were plated on TSA++ with Kanamycin for selection
at 30ºC. Both plasmids yielded ~200 colonies on their respective plates. 20 colonies from each
plate were patched to a fresh TSA++ Kan plate and incubated at 30ºC. These clones will be
further screened by PCR. Primers to be used will include primers specific to vgrG, this will ensure
that there is a shift in size of vgrG due to the intron insertion. A second set of primers will be
utilized including a primer specific to the intron itself and a primer specific to vgrG. This will insure
that the shift in size of vgrG is due to the insertion of the intron. Chromosomal preps have been
made from 10 clones from each plate and are ready for PCR analysis. Data located in TVD UTSA
Notebook 1, page 25.
b. Analysis of the sequencing data for pdpA and the igLC clones made earlier are not complete;
most sequencing were fine confirming gene deletions are correct; but the igLC need additional
sequencing to confirm that both copies are deleted. Need to design other oligos to verify both
locations. However, based on the immunoblot done earlier we believe both genes must be
deleted we just want clean confirmation sequence to show this fact.
c. Did some ordering for enzymes and general supplies for ongoing experiments.
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
55.5%
9. Work plan for upcoming month
a. Will continue analysis of sequence data received from PCR igLC and pdpA DNA samples.
b. Will sequence the vgrG clone and continue with screening of the transformation colonies
obtained from on going experiment by PCR.
c. Will continue with the igLD cloning into vector KEK1140.
d. Will continue to create the pUC pdpD-Flp construct; will design strategy for this construct.
10.Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
Page 36 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
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: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A. Evaluate the protective efficacy of the Ft subsp. novicida uvrBpdpD mutant as a vaccine
candidate (Note book #4, page 144-145). Groups of BALB/c mice (female, 4-6 weeks) were
intranasally (i.n.) immunized with 105 CFU of ΔuvrBpdpD. Mice treated with PBS were used
as a mock-control. The immunized mice were challenged with 1000 CFU of F. novicida (~100
LD50) by the i.n. route after 30 days of vaccination. As shown in Fig. 1, ΔuvrBpdpDvaccinated mice were highly protected against subsequent pulmonary challenge with F.
novicida. No significant loss of body weight was also observed in the protected animals. As
expected PBS-treated mock-vaccinated mice succumbed by day 5.
100
% Survival
80
uv rBpdpD
PBS
60
40
20
0
0
4
8
12
16
20
% Body weight
110
105
100
95
90
85
80
0
2
4
6
8
10
12
14
Days after challenge
Fig. 1. Protective efficacy of ΔuvrBpdpD immunization against F. novicida infection.
BALB/c mice were immunized intra-nasally with 105 CFU of ΔuvrBpdpD or PBS and
i.n. challenged with lethal dose of F. novicida (1000 CFU). Mice were monitored for
survival rate and weight change.
Analyze the antibody profiles of mice immunized with the Ft novicida uvrBpdpD mutant after
vaccination (Note book #4, page 142-143). Blood was collected from the PBS- and
ΔuvrBpdpD (105 CFU)- immunized mice (as described above in a) at day 14 and day 28 after
priming. Specific anti-ΔuvrBpdpD total antibody titer as well as IgG1 and IgG2a isotypes were
determined by ELISA. Antigens, either UV-irradiated ΔuvrBpdpD (106/well) or HEL (Hen Egg
Lysozyme, 50ng/well, an unrelated antigen as control), were coated onto 96-well microplates
and reacted with serial dilutions of sera. Goat anti mouse Ig(H+L), IgG1 and IgG2a antibody
conjugated with peroxidase were used as the secondary antibody to determine serum
antibody isotypes and titers. As shown in Fig. 2, specific serum total antibody in mice
immunized with ΔuvrBpdpD was detectable at day 14 and increased by day 28 after priming
Page 37 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
(2 days before bacterial challenge). Isotyping analyses indicated both Th1 (IgG2a) and Th2
(IgG1)- type antibodies were produced in mice after the ΔuvrBipdpD immunization. However,
the presence of dominant IgG2a isotype implied strong Th1 immune responses in the
ΔuvrBipdpD-immunized mice. The calculated IgG2a/IgG1 ratio (4:1) is much higher than all
previously characterized uvrBigl mutants. No ΔuvrBpdpD-specific antibody was detected in
mice mock-vaccinated with PBS at day 28 after immunization. All tested serum samples
showed no reactivity to the unrelated HEL protein. In summary, the ΔuvrBpdpD is less
attenuated than ΔuvrBigl mutants (A,C,D).
4
Total Ab
IgG1
Day 14
Day 28
IgG2a
Titer (x1000)
3
2
1
0
PBS
uvrBpdpD
PBS
uvrBpdpD
PBS
uvrBpdpD
Fig. 2. Humoral response to ΔuvrBpdpD immunization. BALB/c mice were
intranasally immunized with 105 CFU of the ΔuvrpDpD mutant or PBS alone as
mock vaccination. Sera were collected 2 weeks and 4 weeks after immunization
and used to determine titers of anti- ΔuvrBpdpD specific antibody.
50B. Evaluate the protective efficacy of intragastric LVS vaccination against F. tularensis
SCHU S4 challenge. (Note book #8, page 6, 10). Groups of BALB/c mice (female, 4-6 weeks)
were intragastrically (i.g.) immunized with 1600 CFU of LVS. Mice treated with PBS were
used as a mock-control. The immunized mice were challenged 3 weeks later with either 100
or 500 CFU of SCHU (100 or 500LD50) by the i.n. route. As shown in Fig. 3, LVS-vaccinated
mice were highly protected against subsequent pulmonary challenge with SCHU S4 and
exhibited no appreciable weight loss. As expected, PBS-treated mock-vaccinated mice
succumbed to the infection by day 6 after challenge.
Page 38 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
% Weight Loss
% Survival
100
80
60
40
20
0
110
105
100
95
90
85
80
1600 CFU LVS I . G. / 100 CFU SCHU S4
1600 CFU LVS I . G. / 500 CFU SCHU S4
Mock (PBS) I . G. / 100 CFU SCHU S4
0
0
5
2
10
15
20
25
4
6
8 10 12
Days After Challenge
30
14
Fig. 3. Protective efficacy of LVS intragastric immunization followed by F.
tularensis SCHU S4 challenge. Groups of BABL/c mice were immunized I.G. with
1600 CFU LVS or PBS and rested for 3 weeks. Mice were then challenged i.n.
with SCHU S4 (100 or 500 CFU) and monitored daily for survival and weight loss.
Analyze the antibody profiles of mice intragastrically immunized with F. LVS. (Note book #8,
pages 6-7, 9-10, 16-17). Blood, feces and bronchalveolar lavage fluid (BAL) was collected
from the PBS- and LVS (1600 CFU)- immunized mice (as described above in a) at day 21
after vaccination. Specific anti-LVS total antibody titer, as well as IgG1, IgG2a, and IgA
isotypes for serum, IgA and IgM isotypes for fecal samples, and IgG1, IgG2a, IgA and IgM
isotypes for BAL, were determined by ELISA. Antigens, either UV-irradiated LVS (106/well) or
HEL (Hen Egg Lysozyme, 100ng/well, an unrelated antigen as control), were coated onto 96well microplates and reacted with serial dilutions of sera or undiluted fecal and BAL samples.
Goat anti mouse Ig(H+L), IgG1, IgG2a, IgA and IgM antibody conjugated with horseradish
peroxidase were used as the secondary antibodies to determine antibody isotypes and titers.
As shown in Fig. 5, mice immunized i.g. with LVS produced high titers of LVS-specific total,
IgG1 and IgG2a antibodies, but did not produce any measurable IgA. The response of the
IgG2a isotype was 2:1 over IgG1 implying a dominant Th1 immune response. No LVSspecific antibody was detected in mice mock-vaccinated with PBS. All tested serum samples
showed no reactivity to the unrelated HEL protein. As shown in Fig. 6, intragastrically
immunized mice produce a high amount of LVS-specific IgA in the G.I. tract, while only
producing a small amount of total antibody and almost no IgM. Little to no LVS-specific
antibody was detected in mice mock-vaccinated with PBS. All tested serum samples showed
no reactivity to the unrelated HEL protein. As shown in Fig. 7, mice vaccinated i.g with LVS
produce a wide array of specific antibodies in the BAL including high numbers of total
antibody as well as IgA, IgM, and IgG2a. There was also some IgG1 produced, but again, the
high ratio of IgG2a to IgG1, (more than 3:1) indicate a predominant Th1 response. Little to no
LVS-specific antibody was detected in mice mock-vaccinated with PBS. All tested serum
samples showed no reactivity to the unrelated HEL protein except for IgM which showed a
low level response. In summary, intragastric immunization with LVS induces significant LVSspecific systemic, secretory and respiratory antibody responses.
Page 39 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
20000
10000
LVS
Mock (PBS)
50% Binding Titer
LVS/ HEL
Mock (PBS)/ HEL
1000
100
Total Ab
IgG1
IgA
IgG2a
Fig. 4. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 CFU of LVS or PBS as a
control. Sera were collected 3 weeks later and analyzed to determine titers
for anti-LVS specific antibodies.
LVS
Moc k (PBS)
LVS/HEL
Moc k (PBS)/HEL
O.D. (630 nm)
0.80
Ig (H+L)
2.00
IgA
0.20
0.60
1.50
0.15
0.40
1.00
0.10
0.20
0.50
0.05
0.00
0.00
0.00
IgM
Fig. 5. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 CFU of LVS or PBS as a
control. Fecal samples were collected 3 weeks later and analyzed to
determine titers for anti-LVS specific antibodies.
Page 40 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
2. 00
Tot al Ab
1. 50
O .D.
1. 50
O .D.
2. 00
I gA
1. 00
1. 00
1. 00
0. 50
0. 50
0. 50
0. 00
0. 00
0. 00
2. 00
2. 00
I gG1
1. 50
O .D.
O .D.
1. 50
1. 00
I gM
1. 50
O .D.
2. 00
I gG2a
LVS
Mock (PBS)
LVS/ HEL
Mock (PBS)/ HEL
1. 00
0. 50
0. 50
0. 00
0. 00
Fig. 6. Humoral responses to intragastric LVS immunization. Groups of
BALB/c mice were vaccinated I.G. with 1600 CFU of LVS or PBS as a
control. Bronchoalveolar lavage fluid was collected 3 weeks later and
analyzed to determine titers for anti-LVS specific antibodies.
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
53% of scientific work completed on milestone 50A (original plans)
11% of scientific work completed on milestone 50B (intragastric plan)
9. Work plan for upcoming month
50A: Measure intramacrophage (J774) replication of Ft subsp. tularensis (SCHU S4) iglC
and mglA mutants
Evaluate the protective efficacy of intragastric F. novicida iglB vaccination against Francisella
type A (SCHU S4) and type B (OR96-0246) challenge. Analyze the serum, fecal and
respiratory antibodies of mice vaccinated intragastrically with iglB at 21 days after
immunization. Groups of vaccinated mice will be challenged i.n. with two doses of SCHU S4
or OR96-0246 at three weeks after inoculation. Animals will be monitored for survival and
weight loss.
50B: Measure bacterial dissemination in various tissues at early time points following
intragastic LVS immunization, including in the BAL, trachea and mesenteric lymph nodes, to
determine the cause of previously seen high bacterial numbers present in the lungs.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 41 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
To inactivate RecA in Schu S4, we constructed a Targetron vector for targeting and inactivating the
RecA gene. The Targetron vector was constructed with the intron expression vector pKEK1140 for
the backbone, and a 350bp PCR product to retarget the intron RNA for insertion mutagenesis of
RecA.
3.1 We have constructed the Targetron vector pKEK1186 (720/721s) and pKEK1187 (840/841s). Before
making RecA mutants in SchuS4, we want to know if it works in LVS with the same techniques since
RecA sequence in LVS is homologous with it in Schu S4.
3.2 To make competent host cells, wild type LVS was cultured in liquid medium TSB++/ Amp (100ug/ml)
at 37C until it reached its mid-log phase. Washed LVS with 0.5M Sucrose at 6000rpm, 5minutes for 3
times, then resuspended the final pellet in 0.5M sucrose for transformation.
3.3 The plasmid pKEK1187 was transformed into LVS prepared in step3.2 using electroporation. The
transformed cells were incubated in liquid medium TSB++ at 30C with shaking for 1 hour, then plated
onto TSA++/Kanamycin (30ug/ml) plate and incubated at 30C for about 3-4 days to get single colonies.
3.4 Performed miniprep for the transformants using QIAprep miniprep kit.
Figure:
Data recorded on UTSA TVDC notebook #6, page6-7.
From the figure above, it was sure that the plasmid pKEK1187 had been transformed into LVS because
the size of plasmid purified from the colony #7--#10 (lane3-lane6) was similar to pKEK1187 (lane2), but
we don’t know if the insertion has happened. To screen the Kanamycin resistant colonies for gene
disruption, the primers for colony PCR were ordered, but we haven’t received it yet.
4 Significant decisions made or pending
None.
Page 42 of 43
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2007 to 12/31/2007
Due Date: 1/14/2008 and Prepared by: Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Trevor Brasel,
Bernard Arulanandam, and Karl Klose
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed.
About 12% of scientific work completed.
9. Work plan for upcoming month
Screen the Kanamycin resistant colonies by colony PCR. Remove the plasmid from recA mutant LVS.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
Page 43 of 43