Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 4, 5, 12 (UNM/LBERI), 13(UNM/LBERI), 19, (26, 27,
33, 34, 40, 41, 43, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 48
Inactive milestones: 6-11, 14, 15, 17, 18, 20-24, 28-30, 35-38, 42, 44, 45, 47, 52-54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. NIAID is working on the IAA with USAMRIID and a legal liability review is pending.
b. As of 12/6/06, Dr. Lyons submitted a request for programmatic support to Dr. Ed Nuzum,
Chief of the Product Development Section in the Office of Biodefense Research Affairs at
NIAID,
c. As of 1/25/07, Kristin DeBord returned a response to UNM and LBERI regarding possible
programmatic support for the LVS vaccinations at USAMRIID.
d. Barbara Griffith of UNM and Nicole Banks of LBERI have been exchanging reviews of
Kristin DeBord’s response (1/26, 2/4 and 2/9/07)
4. Significant decisions made or pending
a. UNM and NIAID continue to wait for a change in the status of the IAA between NIAID
and USAMRIID.
b. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
c. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
d. UNM EOHS has obtained many of the laboratory documents
i. Documents pending
1. Radiology Facility Accreditation Certificate
5. Problems or concerns and strategies to address
a. UNM will need an external source of human cells from LVS vaccinated individuals, in order to
develop the immunoassays in humans.
1 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; Work stop has occurred on the SCHU S4 aerosols in primates, until the LBERI
scientists and staff receive the LVS vaccinations.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
16%- no change relative to 11/15/06 and 12/15/06 reports
9. Work plan for upcoming 6 months
NIAID Contract Officer will continue to monitor the progress of the IAA between NIAID and
USAMRIID and will inform UNM when and whether the TVD Contractors can be vaccinated under
this IAA.
10. Anticipated travel
Travel could occur in Spring 2007 to Fall 2007, depending on the completion of the IAA.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM may request a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for training on
LVS site vaccination evaluations. The timing of the COA request depends on the achievement of
the IAA.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions


3 sprays performed in January (1 frozen, 2 fresh LVS in 48 hr. Chamberlain’s broth)
Able to accurately predict actual LVS titer from frozen stock. Accurate prediction is critical
later for giving the correct dose to the primate animals, as the confirmation of the dose
delivered is determined after the primate is exposed. For each spray, the target cfu/ml is
selected and then following the spray, the sample is serially diluted and plated on CHAB
to determine the actual titer. The figure demonstrates the results to date which indicate
that with more experience LBERI is getting better at predicting the actual titer. 1/16/07
spray was on the curve and thus the prediction worked well.
2 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Actual vs. Target CFU/mL (Frozen)
Actual CFU/ml (Log10)
9.00
8.00
6/9/2006
7/14/2006
8/8/2006
8/15/2006
1/16/2007
7.00
6.00
5.00
4.00
3.00
3.00
4.00
5.00
6.00
7.00
8.00
Target CFU/ml (Log10)
Data found at Saturn/ABSL3/Agent and Study Specific Data/TUL-03

Able to accurately predict actual LVS titer from fresh, 48 hour stock. The following figure
demonstrates the ability to accurately predict the actual LVS titer from fresh 48 hr culture

Target vs. Actual CFU/mL (Fresh)
Actual CFU/mL (Log10)
9.00
8.00
7.00
1/17/2007
6.00
1/24/2007
10/27/2006
5.00
4.00
3.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Target CFU/mL (Log10)
Data found at Saturn/ABSL3/Agent and Study Specific Data/TUL-03

Spray factor excellent for frozen LVS. The figure below demonstrates that frozen LVS
has very good spray factors at 10-6 to 10-7 up to 8 logs of LVS ( over the log 4 to log 8
range)
3 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Spray Factor (Log10)
Actual CFU/ml vs. Spray Factor (Frozen)
0.00
-1.000.00
-2.00
-3.00
2.00
4.00
6.00
8.00
10.00
7/14/2006
8/8/2006
8/15/2006
-4.00
-5.00
-6.00
-7.00
-8.00
CFU/mL (Log 10)
Data found at Saturn/ABSL3/Agent and Study Specific Data/TUL-03
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address




Delay due to limited access to bioaerosol suite – repeat a few sprays in Feb. because of
contamination issues and concern to replicate observed spray factors from January data.
LVS Vaccination status for scientific and animal care personnel may affect the amount of
delay
If LVS vaccination is not required for the scientific personnel, plan to complete by
4/15/07. We feel that LVS vaccination may not be needed for the planned work because
all of the work is done in BSC with no animal work.
In the January report, LBERI stated that a sonication step may need to be added to
eliminate possible clumping in sprays to reduce variability in CFU detected. We are still
evaluating the data to determine if this may be an issue. We have not yet generated
additional data to answer this question.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30%- Though LBERI has performed sprays this month, they have chosen to report no increase in
the % work completed, relative to the prior month.
9. Work plan for upcoming month
4 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,


Perform additional bioaerosol experiments on vegetative LVS with Collison generator
i. Finish frozen and fresh LVS sprays (3 total)
ii. Titer LVS stock grown in 10% sucrose
Perform bioaerosol experiments on frozen LVS with sparging generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing aerosols with frozen and fresh LVS, not with lyophilized LVS.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Working Group
Milestone description: Determine appropriate solid and liquid media for growth of tularemia for
project team
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
Bob Sherwood has revised the LVS Growth SOP to add sections which NIAID requested in a
quality SOP.
Barbara Griffith reviewed Bob’s revisions and requested additional edits on 2/6/07 to meet
NIAID’s quality SOP standards.
4. Significant decisions made or pending
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Determined liquid and solid media for LVS growth
7. Quality of performance
Good
8. Percentage completed
100%- except that the final formatted SOP is 95% complete
9. Work plan for upcoming months
a. Finalize LVS growth SOP
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in primates
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
5 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,


Wrote an IACUC protocol to continue to bleed the vaccinated NHPs; blood is required for
perfecting immunoassays such as IFN ELISPOT; IFNg intracellular staining and
phenotyping of cells in the blood and PBMC preparations (work to be completed under
Milestone 12 and 13); IACUC protocol was approved 1/23/07
LVS-vaccinated NHPs received their semi-annual TB test on 1/22/07 – the results were
all negative
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
11%
9. Work plan for upcoming month
Continue blood collection from vaccinated NHPs (see Milestone 12 and 13 for assays to be
conducted with these samples)
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
Rats Fischer 344
a. Experiment Ftc23 study 1 (notebook 94, pages 46-49) and Ftc23 study 2 (notebook
94, pages 59-65
i. The purpose was to measure the resistance of LVS (s.c., i.d. or i.t.) or SCHU
S4 (i.t.) vaccinated rats to i.t. SCHU S4 challenge.
ii. The vaccination strain, route, and dose are shown in Table 1
iii. All of the rats survived s.c. and i.d. LVS vaccination and 93% (28 of 30)
survived i.t. LVS or SCHU S4 vaccination
iv. 4 weeks after vaccination, two rats from each group were killed and shown to
be free of the vaccine bacteria in the lungs, spleen, and liver.
v. 7 weeks after vaccination, the vaccinated rats were challenged i.t. with
SCHU S4 to measure the level of protection generated by vaccination. The
challenge dose and number of animals per group are shown in Table 1
vi. Naïve rats were killed by as few as 3,000 SCHU S4 (Figure 1).
6 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
1. This was surprisingly low compared to our previous results and
Jemski’s results (Infect. Immun 1981 Dec;34(3):766-72) indicating an
i.n. LD50 between 104 and 105 SCHU S4
2. Since we used a SCHU S4 stock that was expanded in
Chamberlain’s broth in this experiment and DVC’s stock without
expansion in earlier experiments, the increased virulence may have
resulted from the 48 h culture in Chamberlain’s broth
vii. Vaccination protected 50% of rats from 3 x 107 SCHU S4 and at least 60%
from lower challenge doses (Figure 1)
1. Regardless of the LVS vaccination route (i.d., s.c. or i.t.), similar
protection was generated against i.t. SCHU S4 challenge. This is in
line with Jemski’s results
2. We did not reach an LD99 because we were limited by the
concentration of our SCHU S4 stock (9 x 108/ml). To reach LD99, we
will have to concentrate our stock by centrifugation.
viii. SCHU S4 vaccination does not offer significant advantage over LVS
vaccination (Figure 2)
b. Experiment Ftc31 study 1 (Notebook 94, in progress)
i. The purpose is to determine the i.t. LD50 of SCHU S4 expanded in
Chamberlain’s broth. Results from Ftc23 study 2 suggested that the i.t. LD 50
is lower than 3,000 CFU.
ii. Fischer 344 rats (n = 5) were challenged i.t. with 30 (2 logs lower than the
lowest dose used in Ftc23 study 2) to 3 x 105 SCHU S4
iii. The rats are being monitored daily and final results will be ready for the next
report.
Table 1. Experimental design to determine the effect of bacteria strain and
vaccination route on the level of protection against i.t. SCHU S4 challenge
i.t. SCHU S4 challenge
(#/dose)
Vaccination
Strain
None
Route
Dose
(CFU)
Clearance
at 4 wk
Survival at
7 wk (%)
LVS
s.c.
i.d.
i.t.
4.6 x 107
4.6 x 107
2.2 x 107
Yes
Yes
Yes
SCHU S4
i.t.
7.2 x 102
Yes
104
6
105
6
106
6
107
108
100
100
93
6
6
6
6
6
6
6
6
6
6
6
6
93
6
6
6
6
7 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Figure 1. Resistance of LVS-vaccinated Fischer 344 rats to i.t. SCHU S4 challenge.
Vaccinated Fischer 344rats (n = 6) were challenged i.t. with SCHU S4 and monitored
for 2 weeks. The challenge doses indicated in the figure legend reflects actual lung
deposition measured 1 h after challenge
Figure 2. A comparison of the protection generated by i.t. LVS or SCHU S4
vaccination. Vaccinated Fischer 344rats (n = 6) were challenged i.t. with SCHU S4
and monitored for 2 weeks. The challenge doses indicated in the figure legend
reflects actual lung deposition measured 1 h after challenge
Guinea Pigs Hartley Strain
a. Experiment Ftc28 study 1 (Notebook 94, pages 70-73)
i. The purpose was to determine the i.n. and s.c. LD50 for LVS in guinea pigs
ii. Guinea pigs were challenged i.n. or s.c. with target doses of 103, 105, and
107 LVS. These doses were selected according to HT Eigelsbach et al
(1961) Proc. Soc. Exp. Biol. Med 108:732-4
8 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
iii. Consistent with Eigelsbach’s results, our results showed that guinea pigs
very resistant to LVS, able to withstand i.n. challenge with 4.73 x 106 LVS
and s.c. challenge with 8.8 x 106 LVS. There were four animals in each group
and all four survived.
b. Experiment Ftc29 study 1 (notebook 94, pages 67-69)
i. The purpose was to determine the i.n. LD50 for SCHU S4 in guinea pigs
ii. Guinea pigs (n = 4) were challenged i.n. with target doses of 1, 10, and 100
SCHU S4
iii. We were unable to determine actual lung deposition because the large buffer
volume required to homogenize the lungs diluted the small number of SCHU
S4 used for infection to below detectable levels. However, it is our
experience that the actual lung deposition is always lower than the target
dose. Thus, the target dose is an overestimation of the lung deposition
iv. As few as 10 SCHU S4 (target dose) killed guinea pigs. The lethal dose may
be even lower since 1 guinea pig died from 1 SCHU S4 (target dose)
v. The mean-time-to death varied inversely with the i.n. SCHU S4 challenge
dose
vi. We terminated the experiment 2 wk after challenge because the surviving
guinea pigs (target dose of 100) never showed any sign of infection during
the two weeks of observation. We concluded that because the inoculum was
so low, they were essentially not challenged
Table 3. 14 d survival of guinea pigs challenged
intranasally with SCHU S4
Survival ratio d14
Target dose
(# alive/total)
MTD1
2
10
0/4
5.8
101
0/4
6.2
100
3/4
7.0
1 MTD = mean-time-to-death
LVS and SCHU S4 expansion
a. Experiments Ftc25 study 4 (Notebook 94, pages 50-53)
i. The purpose was to determine the growth kinetics of SCHU S4 in
Chamberlain’s broth over 3 days
ii. 2 separate vials of DVC’s SCHU S4 each was inoculated into 30 ml
Chamberlain’s broth and cultured with shaking at 37oC. Aliquots were
removed every 12 h to determine OD600nm and bacterial concentration
iii. SCHU S4 grew exponentially up to 36 h and then plateaus
iv. Optical density is less sensitive than plating for measuring bacterial growth
9 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Figure 3. SCHU S4 growth kinetics in Chamberlain’s broth. Two independent
cultures were prepared in Chamberlain’s broth and samples were taken every 12
h to measure OD600nm (left) and bacterial concentration by plating on cystine heart
agar plates (right).
4. Significant decisions made or pending
a. We will continue to prepare SCHU S4 working stocks from 48 h cultures in
Chamberlain’s broth.
b. We will wait until the rat and guinea pig models are completed before proceeding to
hamsters and rabbits
5. Problems or concerns and strategies to address
a.
For small infection doses, the large volume required to homogenize guinea pig lungs
makes it very difficult to accurately determine the lung deposition. Therefore, we will
plate as much of the lung homogenates as possible and report whether the lungs
have bacteria or not
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
38%
9. Work plan for upcoming month
a. Rats
i. Determine the i.t. LD50 for SCHU S4 expanded in Chamberlains.
ii. Vaccinate rats with LVS i.d., s.c., and i.t., and with SCHU S4 i.t. The
vaccinated rats will be challenge i.t. with SCHU S4 seven weeks after
vaccination.
b. Guinea Pigs
i. Check guineas pig in Ftc28 study 1 for LVS clearance and then challenge
them i.n. with SCHU S4 in a preliminary, dose-finding experiment
ii. Determine the i.d., s.c. and i.n. LD50 for LVS
iii. Determine the i.n. LD50 for SCHU S4
iv. Vaccinate guinea pigs with LVS i.n., s.c., or i.d. routes
c. SCHU S4 expansion
i. Compare the virulence of SCHU S4 stock generated with and without
passage on agar before expansion in Chamberlain’s broth culture
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
10 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Milestone 12-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc27 study 2 (Notebook 94, pages 54-58)
i. The purpose was to make sure that killed Francisella and Francisella-derived
peptides stimulated antigen-specific and not mitogenic T cell proliferation
ii. Antigen-specific responses can be distinguished from mitogenic responses
by assaying both naïve and vaccinated splenocytes
1. If the response is mitogenic, then both naïve and vaccinated
splenocytes will proliferate
2. If the response is antigen-specific, then only the vaccinated
splenocytes will proliferate
iii. Naïve and vaccinated splenocytes were stimulated with formalin-fixed LVS,
Con A or not stimulated.
iv. Formalin-fixed LVS stimulated mitogenic and not antigen-specific T cell
proliferation (Figure 4), as the naïve splenocytes proliferated very similarly to
the splenoctyes derived from LVS vaccinated mice.
v. The background increased with more splenocytes, such that there was as
much proliferation without any stimulus as with formalin-fixed LVS or Con A
Figure 4. T cell proliferative response to formalin-fixed LVS and Con A.
Splenocytes from naïve and vaccinated BALB/c mice were cultured with formalinfixed LVS for 5 days. T cell proliferation was measured by BrdU incorporation
and chemiluminescent anti-BrdU ELISA. The bars show mean value from three
wells
11 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
b. Experiment Ftc30 study 3 (Notebook 94, in progress)
i. The purpose is to develop the SOP for generating bone marrow-derived
macrophages (BMDM) that will be used in the macrophage killing assay
developed by Karen Elkin’s lab [Cowley SC and Elkins KL (2003) J. Exp.
Med 198:379-89]
ii. We generated BMDM from BALB/c mice following the protocol from Elkin’s
lab. The cells look like macrophages when viewed under a microscope and
adhere very tightly to the tissue culture plate.
iii. We are now infecting the macrophages with LVS and SCHU S4 to begin
developing a macrophage killing assay
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The two problems with the T cell proliferation assay are the high background and the non-antigen
specific proliferation. It is known that some batches of fetal calf serum can have high mitogenic
activity; therefore, we will test different batches of fetal calf serum for one that has very low
mitogenic activity. In addition, we had demonstrated in the past antigenic-specific response using
naïve and vaccinated T cells enriched over Thy1 MACS columns . Since it is possible that the
non-specific proliferation was caused by non-T cells, we will repeat the assay using a T cell
enrichment protocol.
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
15%- we are reporting no increase in work performed because we are troubleshooting basic
parameters of the T cell proliferation assay
9. Work plan for upcoming month
a. T cell proliferation
i. Compare batches of fetal calf serum for low mitogenic activity
ii. Determine whether T cell enrichment will increase antigen-specific responses
iii. When the high background and antigen-specificity problems have been
corrected, we will again titer the amount of antigen required to optimal
proliferation
b. Macrophage killing assay
i. Develop method to infect BMDM with LVS (are you testing parameters like ratio
of bacteria to macrophages, or exposure time or what?)
ii. Develop method to quantify the bacterial load inside infected macrophage.
1. Treat the macrophage culture with 10 g/ml gentamicin 1 h after infection
to eliminate all extracellular bacteria
2. Lyse infected macrophages with sterile water at various times after
infection
3. Plate macrophage lysate onto cystine heart agar plates to determine the
bacterial load
iii. Determine whether mouse BMDM can be activated with cytokine or vaccinated T
cells to kill intracellular LVS
iv. Optimize SOP for the macrophage killing assay
1. Titrate the MOI and time of infection so that the macrophages remain
capable of carrying out their effector functions.
2. Titrate the stimuli (cytokine or vaccinated T cells) to induce optimal
macrophage killing of intracellular bacterial
12 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Choosing a PBMC Freezing Method
 On 1/11/07, thawed PBMCs which were frozen down on 11/16/06 using the Cerus
protocol and tested them for proliferation to Con A to compare to the freshly isolated cells
(11/16/06)
 Details on procedure and data resulting from this test can be found in the binder TVDC 1
under Tab for TUL 8, Day 0
 Cells were originally frozen using the Cerus protocol (80% FBS, 20% DMSO, final
concentration of 5 x 106/ml)
 Thawed 2 vials from each animal (A00896, A00908, A00937); these cells were frozen on
11/16/06, the day of the TUL 8 pre-bleed in 1 ml aliquots
 Cells were thawed in a 37 degree water bath, transferred from the cryovial to a 15 ml
conical tube and RPMI media containing 10% FBS and antibiotics
(penicillin/streptomycin) was then added in dropwise fashion until total volume was 4 ml;
added 10% RPMI medium until 15 ml conical was full; washed 3 times in 10% RPMI,
resuspended in 0.5 ml 10% RPMI and counted
Viable Cell Recovery/Percent viability from freezing procedure (each vial contained 5 x
106 cells):
A00896 Vial 1: 3.125 x 106= 62.5% recovered/87.4% viable
A00896 Vial 2: 3.25 x 106= 65% recovered/93.8% viable
A00908 Vial 1: 3.24 x 106= 64.8% recovered/96.3% viable
A00908 Vial 2: 2.85 x 106= 57% recovered/97.4% viable
A00937 Vial 1: 2.49 x 106= 49.8% recovered/93.3% viable
A00937 Vial 2: 2.4 x 106= 48% recovered/91.4% viable
Conclusion: Viability and cell recovery were quite acceptable
Proliferation: Cells were resuspended at 1 x 106/ml and 0.25 x 106/ml and stimulated with
Con A (
Average proliferation as measured by RLU:
13 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Relative Light Units
(Mean +/- SEM)
2.50E6
2.00E6
1.50E6
1, Media
1, Con A
.25, Media
.25, Con A
1.00E6
5.00E5
0
Fresh
Frozen
Proliferation shown with each individual animal plotted:
Relative Light Units
(Mean +/- SEM)
4.00E6
3.50E6
1, Media
1, Con A
3.00E6
.25, Media
2.50E6
.25, Con A
2.00E6
1.50E6
1.00E6
Frozen, A00937
Frozen, A00908
Frozen, A00896
Fresh, A00937
Fresh, A00908
0
Fresh, A00896
5.00E5
Caveat: Frozen/thawed cell proliferation was read on the Victor Light luminometer at LRRI
while the fresh cell proliferation was read at UNM on the Victor; the Victor allows one to
adjust the aperture of the light source and thus we read at both “small” and “normal” aperture
for 0.1 seconds whereas the Victor Light does not; we are presumably comparing “normal” to
“normal” RLU/second. I am checking with the Perkin-Elmer rep on this issue.
14 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Regardless, we should be able to compare the Stimulation Index:
Descriptive Statistics
Split By: Cell Num ber Plated, Treatm ent, Cell Source
Inclusion criteria: Day 0 tul 8 not .06 con a/m edia from PBMC assays 010807.svd
Mean
Std. Dev.
Std. Error Count
RLU normal, Total
813638.729 903552.312
Maximum
# Missing
20490.000 5005305.000
0
29330.000
73530.000
0
18 117337.000
304285.000
0
87349.699
107
9
RLU normal, 1, Media, Fresh
46092.222
13673.555
4557.852
RLU normal, 1, Media, Frozen
207047.778
58033.149
13678.544
Minimum
RLU normal, 1, Con A, Fresh
1077417.778 346188.607 115396.202
9 666820.000 1644670.000
0
RLU normal, 1, Con A, Frozen
2187327.765 972047.910 235756.247
17 759713.000 5005305.000
0
RLU normal, .25, Media, Fresh
43161.111
31640.810
10546.937
9
20490.000
115330.000
0
RLU normal, .25, Media, Frozen
161639.111
71451.693
16841.326
18
76698.000
301773.000
0
RLU normal, .25, Con A, Fresh
664572.222 239278.509
79759.503
9 312750.000
978210.000
0
18 540791.000 2206851.000
0
RLU normal, .25, Con A, Frozen
1486512.111 477470.425 112540.858
Ratio of Con A:Media = Stimulation Index: used averaged RLU numbers, not individual
NHPs or vials
1 x 106/ml plated Fresh: 1.08 x 106/0.046 x 106 = 23.5
1 x 106/ml plated Frozen: 2.19 x 106/0.21 x 106 = 10.4
0.25 x 106/ml plated Fresh: 0.66 x 106/0.043 x 106 = 15.3
0.25 x 106/ml plated Frozen: 1.49 x 106/0.16 x 106 = 9.31
Conclusion:
Using the Cerus protocol, we can recover at least 50% of the cells and approximately 45% of
the activity.
Data Location: C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract and backed up on N:\My Documents\Tularemia Contract
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Contacting the Victor Technical Service to determine whether the setting differences between the
Victor (UNM) and Victor Light (LBERI) may explain the higher RLU detected with the frozen cells
assayed for proliferation at LBERI
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
88% of scientific work has been completed
9. Work plan for upcoming month
Choosing PBMC freezing protocol
 Monday, 2/12: Will thaw cells from TUL 8, Day 28 (12/18/06), at 8 weeks post freezing
using the CTL protocol; and Wednesday, 2/21 will thaw cells from TUL 9, Day 28 at 8
weeks post freezing using the CERUS protocol
15 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,

if cell recovery numbers permit, we can test their proliferative capacity to both Con A and
LVS and also by ELISPOT (Con A only, as we have still seen no response to LVS by
ELISPOT using the cell numbers we originally tested)
Developing immunoassays
 Test whether NHP PBMCs stain positively for anti-human CD19 and anti-human IgM.looking for B cells in the PBMC population as compared to whole blood
 Test intracellular staining of IFN in whole blood and PBMC preparations (from LVSvaccinated NHPs; Milestone 4)
 Begin development of anti-LVS ELISA using serum from LVS-vaccinated NHPs
(Milestone 4 and 13)
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 13-UNM
Milestone description: Compare assays in animal models (sensitivity)
Institution: UNM
Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1.
UNM has not started working on this milestone yet but have been providing supplies to
LBERI
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
NA
9. Work plan for upcoming month
NA
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
16 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Milestone 13-LBERI
Milestone description: Compare assays in animal models (sensitivity)
Institution: LBERI
1. Date started: 11/16/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Comparing assays in NHP vaccinated with LVS by SC or ID routes
 Data on IFN ELISPOT is pending final analysis but preliminary data indicate that no
response to LVS was observed in the LVS vaccinated NHPs (all below background)

Suspect that not enough cells were originally plated/well to detect LVS-specific IFN
secretion
 Monkeys are healthy, showing normal activity
 All data is stored in binder TVDC 1 in the Wilder laboratory as well as in C:\Documents
and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\ TUL 8 or TUL 9;
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have a concern that the response to Con A, a T cell mitogen, was increased in the S.C.
vaccinated group of NHPs above the I.D. group, even at day 0, thus unrelated to LVS
vaccination. We will address this concern by phenotyping the blood from these NHPs in the
coming month to determine if there is a higher number of T cells for some reason in the S.C.
group compared to the I.D. group. We have already confirmed that no treatment of these
NHPs in the previous pharmacokinetic study in which they were used can explain this
phenomenon. We had planned on starting this analysis in January, however, we were
delayed by the need to write and submit an IACUC protocol amendment to continue to bleed
the NHPs, as well as their semi-annual TB testing.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
11% of scientific work has been completed
9. Work plan for upcoming month



Develop anti-LVS immunoglobulin ELISA for testing plasma (compare formalin-fixed and
heat-killed LVS as antigen)
Phenotype the blood cells and PBMCs from the 6 vaccinated NHPs to determine if
differences exist in T cell numbers in the S.C. vs. I.D. group
Test whether more PBMCs/well will give a better IFN ELISPOT response to LVS
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None
17 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Milestone 19
Milestone description: Direct cytokine effects on growth of F. tularensis in human
macrophages determined
Institution: UNM/LBERI
1. Date started: 1/1/2007
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
UNM: Respiratory nurse is recruiting normal human donors for collection of alveolar
macrophages. Two donors are scheduled in February.
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
0.5%
9. Work plan for upcoming month
Collect human alveolar macrophages and begin developing in vitro assays
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
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
Designs for an expression template for in vitro translation (IVT) of the FTU ORFs have been
constructed and evaluated.
a) The first feature evaluated was aimed at testing the effect on polypeptide synthesis levels of
i) optimizing the spacing between transcription and translation start sites, and ii) including the
T7 g10 enhancer originally described by Studier. E. coli transcription/translation lysates
(Invitrogen, Inc.) were used for all reactions. Identical design features were implemented in
both plasmid and linear expression element (LEE) constructs. Reactions were carried out in
the presence of fluorescently labeled tRNA lysine, and then fractionated by SDS-PAGE.
Translated products were visualized by their fluorescence. The fluorescent sensitivity is fairly
comparable to the radioactive method, but the disadvantage is that some E. coli normal
18 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
proteins are fluorescent. These fluorescent E.coli bands give rise to a background that is
visible on the gels, including an 18kD band. Currently, ASU is moving to S 35 labeling to allow
visualization of nascent IVT proteins in the size range of the fluorescent background of the
auto fluorescing bacterial proteins.
Observations: (see Figure 1)
Lanes 2&3: Slight reduction in CALM (calmodulin) synthesis is observed in the construct
without the G10 enhancer and spacer.
Lanes 7&8: Slight reduction in GFP (green-fluorescence protein) synthesis is observed in the
construct without the G10 enhancer and spacer.
Lanes 2 &9: LEE and plasmid expression levels are similar.
Conclusion:
The spacer and g10 spacer are worth including in the constructs so as to optimize polypeptide
yields.
Files are stored at: R:\\peptide\Research\CIM\GeneVac\FTU
Figure 1:
b) The second design feature that we tested was aimed at determining whether or not the
presence of a sequence encoding a biotinylation site (BAP) affected polypeptide synthesis?
19 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
In vitro translation samples are presented below (Figure 2) which were fractionated on two
different gels but electrophoresed simultaneously in the same electrophoresis apparatus. The
gel shown on the right displays generally higher fluorescence than the left gel; this is apparent if
one compares the CALM LEE sample run on both gels. The templates used for the samples in
the left gel did not carry the BAP sequence; templates used for samples fractionated in the right
gel did have BAP, as indicated.
Observations:
The BAP sequence did not cause expression problems for any of the 8 different ORF-encoded
polypeptides. The Tobacco Etch Virus (TEV) cleavage site is marked on the cartooned
template below (in pink in Figure 2). It is positioned such that all fused amino acids encoded by
the template can be efficiently removed with exposure to the TEV protease. Currently this
includes the His and BAP tags, and may potentially include ubiquitin, if it proves advantageous.
Adding the BAP site enables sensitive protein detection and also a high affinity tag for the
protein purification.
Conclusion: The BAP sequence provides a potentially useful tag without compromising yield or
specificity.
Figure 2
Files are stored at: R:\\peptide\Research\CIM\GeneVac\FTU
20 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
c) The next point we considered relative to optimizing a template design was its robustness. To
address this we evaluated the ability of the template to generate an assortment of FTU
proteins. We synthetically built 6 “improved” FTU ORFs, and these were placed in the linear
templates described above. Namely, the LEE templates carried a promoter spacer,
g10enhancer, and BAP site. For comparison to supercoiled templates, plasmids versions
were constructed by cloning the same ORFs and design features into pEXP5-NT. These two
sets of 6 FTU constructs in addition to the GFP and CALM control gene constructs were
added to IVT lysates.
Observations (Figure 3):
The SDS-PAGE analysis of the products is shown, with the calculated molecular weights
provided below. The linear constructs are presented on the left and plasmid constructs on the
right.
Conclusions:
The template design in vitro synthesis protocol is sufficiently robust to generate 6 out of 6 FTU
proteins without individual ORF reaction optimizations. Linear and plasmid reaction yields are
comparable. Since LEE construction enables us far greater speed and flexibility, LEEs are
preferable over plasmid.
Occasional multimers appear to have formed, consistent with properly folded products.
21 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Figure 3
ORF
Calculated MW
FTT208c
FTT0901
FTT1419
FTT1602
FTT1695
FTT1712
25.7kDa
15.9
11.7
12.2
10.4
22.5
CALML3
GFP
19.5
26.5
Files are stored at: R:\\peptide\Research\CIM\GeneVac\FTU
d) Purification of in vitro translated and biotinylated products via strepavidin bead binding to is
being evaluated. Current templates also encode a His tag. Nickel bead purification will be
investigated, in parallel. We will be looking at the yields and solubility of the newly
synthesized proteins. We will add ubiquitin to the template to potentially improve translation
22 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
levels and to facilitate refolding. We anticipate that this approach will avoid many possible
solubility problems.
The inclusion of ubiquitin in the template design depends on the downstream impact. If
ubiquitin solves a solubility problem but decreases yields, then ASU will only use ubiquitin for
problematic polypeptides on a second pass (redo). If the ubiquitin fusion does not negatively
impact yield, then ASU may use ubiquitin generally.
4. Significant decisions made or pending
No significant decisions have been made this month, although we are progressing our way to a
template decision. In the upcoming month we plan to have a first set of data from purification
protocols to evaluate.
5. Problems or concerns and strategies to address
Identifying the best purification method is our current challenge. The parameters of success will
by optimizing yield, purity, and solubility. If biotin/strepavidin and His/Ni+ do not prove successful
we have a number of other tags ready to include in our template. These include HA, myc, and
FLAG sequences.
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
80%
9. Work plan for upcoming month
a. We will scale-up production of the FTU sub-proteins in the IVT reactions so as to have
sufficient material for testing several purification schemes. IVT’s have been performed in the
presence of the BirA enzyme, which biotinylates the nascent polypeptides while translation is
occurring. These reactions will be further optimized.
b. Strepavidin purifications of protein-fragments will be conducted and protocols evaluated to
optimize both yield and purity. A His/ nickel bead purification approach will simultaneously be
pursued. While biotin/avidin is a very high affinity interaction, enzymatically-driven biotinylation
may not be complete. By comparison, His/Ni+ is a lower affinity interaction, however 100% of the
polypeptides will carry the His tag. Finally, if neither of these is deemed optimal, redesign is
simple. The in vitro assembled LEEs will enable facile exchange with an alternative tag.
c. Yield of the SCHU S4 protein-fragments will be determined and adequacy assessed.
d. Improved yield strategies, such as feeder systems, will be evaluated
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
AS’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1.
a. Experiment Ftc27 study 2 (Notebook 94, in progress)
i. The purpose was to identify peptides that would specifically stimulate T cells
derived from LVS vaccinated BALB/c mice
23 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
ii. This experiment is also reported under milestone 12
iii. Vaccinated and naïve splenocytes were incubated with 20 nM peptides for 5
days and T cell proliferation was measured by BrdU incorporation
iv. Our results suggest that peptides or the amount of DMSO (used to
resuspend the peptides) in the culture may have suppressed proliferation
since the amount of proliferation was lower with peptide than without (Figure
5)
Figure 5. Peptide-stimulated T cell proliferation. Naïve and vaccinated
splenocytes (5 x 104/well) were cultured with 20 nM peptides for 5 days. T cell
proliferation was measured by BrdU incorporation followed by anti-BrdU ELISA.
Each well contains a single peptide and each peptide was assayed in triplicate.
The error bar shows SD.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
In addition to the high background and non-antigen specific proliferation described in the T
cell proliferation assay in Milestone 12, it also appears that the peptides and/or the amount of
DMSO in the culture may be suppressive. Once we have resolved the first two problems, we
will titrate the amount of peptides used in the assay so that we will maximize antigen-specific
proliferation and minimize the suppressive effects of the peptides.
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
2%- this is the same amount of work reported as in the 1/15/07 report due to the basic
troubleshooting being performed this month
9. Work plan for upcoming month
a. Continue to develop and optimize a SOP for measuring peptide-induced T cell
proliferation, after the high background and non-antigen specific proliferation
problems are resolved in general.
24 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
b. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
i. Splenocytes from vaccinated and unvaccinated BALB/c mice will be used to
demonstrate antigen-specific response
ii. Formalin-fixed bacteria will be used as positive control and media alone will
be used as a negative control
c. Assemble a list of stimulatory peptides for ASU to analyze for common stimulatory
motifs
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 33
Milestone description: Microarrays constructed and confirmed; First printing of arrays,
Testing with DNA from Ft, Arrays GDPs validated at ASU.
Institution: ASU-Johnston
1. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions




Full-deck prints have been delayed because of printer problems (See #5. below) but are now
in the schedule for running on 2/8/2007 for 25 poly-L-Lysine and 25 Corning Ultragaps
substrate slides.
RNA from both LVS and Schu4 strains have been received from UNM
Initial predictions are completed for designing oligonucleotides to detect LVS missing genes
or those with >3 mis-matched nucleotides to current probe sets. Verification of predictions is
ongoing.
Submitted PFGRC request for TIGR tularemia microarrays and we are awaiting review. This
should be completed by 2/16/2007.
4. Significant decisions made or pending.
We will perform comparisons of hybridizations of microbial RNA diluted into RNA from normal
mouse lung on both Poly-L-Lysine to Corning Ultragaps before final substrate decision
5. Problems or concerns and strategies to address
Both microarray printers are exhibiting mechanical problems and are undergoing service calls
(1/8/2007 for the Nanoprint 60 and 1/9/2007 for PE Spotarray 72). The PE Spotarray 72 has been
shipped back to the manufacturer for complete review and repair of all systems. We have been
promised a 21 day turn around for repairs and return of the instrument. The Nanoprint 60 was
repaired, but sonicator water sensor problems are still an issue. However, we developed a workaround and the system is functioning. We have also contacted a colleague on ASU campus who
runs a BioRad VersArray ChipWriter Pro and he has agreed to help by allowing us to run a print
run should dual machine problems arise again.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
50%
9. Work plan for upcoming month
25 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,





Perform full deck print runs on PLL slides and Corning UltraGaps.
Will start testing with GDPs with purified SCHU S4 and LVS RNA first alone and then in the
presence of normal mouse RNA.
Perform test comparisons between TIGR and ASU arrays
Finish design and order 70mer oligonucleotides to detect extra LVS gene probes.
We will perform comparisons of hybridizations of microbial RNA diluted into RNA from normal
mouse lung on both Poly-L-Lysine to Corning Ultragaps before final substrate decision
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 Ftc34 study 1 (Notebook 94, pages 72-75)
i. The purpose was to isolate total RNA from LVS and SCHU S4 for ASU
ii. 150 g of total RNA was isolated from LVS and SCHU S4 stored in RNAlater
at 4oC
iii. Estimated yield approximately 50 g/1010 bacteria
iv. The RNA integrity was excellent based on an Agilent profile
v. Ambion RiboPure and Qiagen RNeasy Midi kits produced similar yield
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The RNA yield of 50 g/1010 bacteria was extremely low. Qiagen tech support suggested that
RNAlater may interfere with RNA isolation, which contradicts advice from Ambion tech support.
We will do a direct comparison using Francisella stored in RNAlater or freshly lysed without
RNAlater addition.
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
5% ( previously reported at 0.5 % on 04/15/06 Technical Report)
9. Work plan for upcoming month
a. Compare RNA yield from freshly-cultured and RNAlater-archived Francisella
b. Isolate additional 350 g l LVS and SCHU S4 total RNA for ASU
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
26 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Milestone 40
Milestone description: Phenotyping of Ft novicida nucleotide excision repair mutants; Measure
degree of attenuation of uvr mutants in macrophages and in mice
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
We previously demonstrated that the uvrB and uvrA single and the uvrA uvrB double mutant
Ft novicida strains have no growth defect in Chamberlain’s defined medium (CDM) or in J774
macrophages and that all of the mutants are fully virulent in BALB/c mice when administered by
the intraperitoneal (IP) route of administration. All Ft novicida strains were virulent by the IV
route, but LD50 values ranged from 1 for wild-type to 38 cfu for the uvrA uvrB double mutant
suggesting that the nucleotide excision repair mutants may be slightly attenuated by this route.
1) The subcutaneous (SC) route of infection is the route by which LVS is the least virulent (Green
et. al, 2005); thus, this is the route by which subtle differences in virulence may best be
evaluated. Because we were unable to find published information on the virulence of Ft novicida
by the SC route, 10-fold serial dilutions ranging from 1x108 to 1x100 were administered. Because
such a broad dose range was used, we limited our evaluation of Ft novicida strains to U112 and
uvrB, and used only 3 mice per group. We determined that the median lethal dose of U112 was
1.17 x 103 cfu while the median lethal dose of uvrB was 1.28 x 105. This data suggests that the
uvrB mutant is attenuated by approximately 2 logs when administered by the SC route. Next
month, we will confirm the SC LD50 of U112 and uvrB and compare these values with the uvrA
single and the uvrA uvrB double mutant.
Calculated IV LD50 (cfu)
Calculated IP LD50 (cfu)
Calculated SC LD50 (cfu)
U112
0.95
0.57
1.17 x 103
uvrA
27
0.82
ND
uvrB
8.1
0.2
1.28 x 105
uvrAuvrB
38
2.72
ND
Study #
AS06-112
AS06-090
AS07-014
2) We initiated preliminary studies to evaluate the growth rate of wild-type Ft novicida U112 and
Cerus expanded lot 16 LVS in spleen, liver, and lungs of Balb/c mice infected IV. This
preliminary experiment was performed so that we could determine the biodistribution and the
range of dilutions that would be required to eventually compare the growth rate of various
mutants following IV administration of a lethal dose of bacteria. Mice were injected IV with
approximately 100 cfu (which represents approximately 100 x LD 50 for U112 but 0.01 LD50 for
LVS). Cohorts of 3 mice were sacrificed at 4, 24, 48, 72 and 96 hours post infection (for the fifth
cohort of U112 that was to be sacrificed at 96 hours, all the animals died prior to the 96 hour time
point). At each time point spleen, liver, and lungs were harvested and homogenized in HBSS.
100 microliters of each homogenate were plated directly on CHAH plates and a series of 8 tenfold
dilutions were also plated to calculate the number of cfu per organ. Each symbol represents the
mean of three organs and the bars represent the standard error. Both Ft novicida and LVS
replicate rapidly in livers and spleens immediately following IV injection, however it appears that
there is a lag that specifically affects growth in the lungs. It is interesting to note that the LVS
(given at a sublethal dose IV) is capable of replicating in all the organs, but appears to stop
replicating by day 4.
27 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
cfu/organ
Spleen
1.0×10 10
1.0×10 09
1.0×10 08
1.0×10 07
1.0×10 06
1.0×10 05
1.0×10 04
1.0×10 03
1.0×10 02
1.0×10 01
F.t. novicida U112
F.t. holarctica LVS
AS07-002
0 (4hr)
1
2
3
4
Day
* All animals injected w ith F.t. novicida died prior to day 4.
Lungs
Liver
1.0×10
1.0×10
8
1.0×10 6
1.0×10
7
1.0×10 5
1.0×10
6
cfu/organ
cfu/organ
1.0×10 9
7
1.0×10 5
1.0×10
4
1.0×10 3
1.0×10 4
1.0×10 3
1.0×10 2
1.0×10 1
1.0×10 2
1.0×10 0
1.0×10 1
0 (4hr)
1
2
3
4
Day
* All animals injected w ith F.t. novicida died prior to day 4.
0 (4hr)
1
2
3
4
Day
* All animals injected w ith F.t. novacida died prior to day 4.
4. Significant decisions made or pending
We have selected CDM and cystine heart agar with hemoglobin (CHAH) as liquid and plate
medias for cultivation and enumeration of Ft novicida. Ft novicida nucleotide excision repair
mutants are not attenuated in vitro, but may have route-specific attenuation of virulence in mice.
5. Problems or concerns and strategies to address
Abrogation of the nucleotide excision repair pathway may result in a loss in virulence
depending on the route of administration. If the SC route of administration is used for
administration of a KBMA vaccine, this level of attenuation may provide an added degree of
safety to the vaccine in the event that an organism is not killed by photochemical inactivation.
However, since the attenuation is modest and only measured when the bacteria are
administered via the SC route, finding a secondary attenuation mutation that can be used in
SchuS4 –based vaccine would still be preferred.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
75%
9. Work plan for upcoming month
28 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,


SC LD50 with the remaining Ft novicida strains (uvrA and uvrAuvrB) will be evaluated
and compared with uvrB and U112. Doses will be administered ranging between 1x107
and 100 cfu.
We will also monitor the growth of Ft novicida nucleotide excision repair mutants in
lungs, livers, and spleens after IV infection with 100 cfu in order to determine whether the
nucleotide excision repair machinery is required for growth or dissemination to specific
organs.
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
Using a small-scale (3.5mL) photochemical treatment regimen using 4 J/cm 2 UVA, we found that
the minimum concentration of S-59 required to inactivate wild type Ft novicida U112 was 40M,
which was only slightly higher than the 20M concentration required to inactivate Ftn uvrB, Ftn
uvrA, and uvrA uvrB double mutant strains. We have been optimizing conditions for 400 mL
scale inactivation process. We have found that the UVA dose needed to be increased to 6 J/cm 2
to achieve 9 logs of inactivation.
1) This month we produced three 400mL lots of KBMA Ft novicida uvrB to determine whether
this process resulted in a vaccine that was completely inactivated and maintained metabolic
activity. Lots that pass these specifications will be used for stability studies, immunogenicity
studies, and virulence determination. Ft novicida uvrB was grown in CDM in the presence of
40M S-59 until early stationary phase, illuminated with UVA, washed 2x, and suspended in
HBSS containing 10% sucrose at a concentration of approximately 5 x 10 10cfu/ml. 1 ml of final
product was plated to determine whether there were any residual cfu. The first two attempts to
produce lots of KBMA vaccine using 6 J/cm 2 failed due to residual cfu; however, when process
was performed with 7 J/cm 2 a sterile lot was produced. The metabolic activity of lot 948-164 was
determined prior to (pre-freeze) and after freezing at –80o C (t=0) and is comparable. The
metabolic activity of the sterile lot (948-202) will be determined and tested for stability on a
monthly basis.
Lot #
948-164
948-202 arm-1
948-202 arm-2
[S-59]
40M
40M
40M
UVA dose
6J/cm2
6J/cm2
7J/cm2
Particles/ mL
3.9 x 1010
5.4 x 1010
5.12 x 1010
cfu/mL
1
91
0
29 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Metabolic activity of KBMA uvrB Lot 948-164 (Nominal 1.5e8 particles/mL)
1.40
OD (490nm)
1.20
Pre-Freeze
1.00
t=0
0.80
0.60
0.40
NB948-169
0.20
0.00
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
2) This month we demonstrated that KBMA Ft novicida uvrB is attenuated for virulence. Doses
of 1x109 and 1x108 particles of KBMA Ft novicida uvrB lot 948-164 were administered IP, IV,
and SC to BALB/c mice and compared to heat-killed Ft novicida uvrB administered IP. The
KBMA vaccine was avirulent at both doses when administered SC. The KBMA vaccine was more
virulent than heat killed Ft novicida when administered IP in that at the 1x109 dose only 40% of
the mice survived compared to 100% with heat killed bacteria. The KBMA vaccine was most
virulent when administered IV: all the mice in the 1e9 group died and only 40% of the 1x10 8group
survived. Despite the fact that lot 948-164 had 1 cfu per 3.9 x1010, we believe that the toxicity of
the KBMA vaccine in these studies is most likely due to the input toxicity of the KBMA organisms
rather than an infection caused by a rare residual live organism because all of the animal deaths
occurred within the first 24 hours after infection and the time to death following live Ft novicida
infection is usually after 3 days. To determine whether vaccination with KBMA Ft novicida uvrB
can protect against a lethal wild type Ft novicida infection, the surviving animals from this study
will be challenged with 100 x LD50 of U112 (100 cfu) via the IP route one month after the
vaccination. All further characterization of the KBMA Ft novicida will be performed with lot 948202 arm-2 which had no detectable cfu in 1mL of final product.
Route
Calculated LD50
KBMA uvrB
SC
>1 x 109
KBMA uvrB
IV
<1 x 108
KBMA uvrB
IP
6.8 x 108
HK uvrB
IP
>1 x 109
Study #
AS07-009
These results demonstrate that complete inactivation of greater than 1010 uvrB Ft novicida can
be achieved with 40M S-59 and 7 J/cm2 at the 400ml scale, and that KBMA Ft novicida are
attenuated for virulence by as much as 8 logs.
4. Significant decisions made or pending
All nucleotide excision repair mutants (uvrA, uvrB, and uvrA uvrB) 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 vaccine
that is sterile for further characterization.
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; however, the
high degree of metabolic activity retained by the mutant and wildtype strains suggests that the
wild-type is highly sensitive to photochemical inactivation under these conditions and that the
KBMA strategy is still viable.
6. Deliverables completed
None
30 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
7. Quality of performance
Good progress
8. Percentage completed
50% of scientific work completed on the milestone
9. Work plan for upcoming month




Stability at -80o C of lot 948-202 arm-2 of KBMA urvB Ft novicida will be monitored by
metabolic activity assays on a monthly basis.
The degree of attenuation via the IV route will be established using KBMA urvB Ft
novicida lot 948-202 arm-2.
The survivors from the KBMA LD50 study will be challenged IP with 100 cfu of U112 to
determine the degree of protection afforded by vaccination via the SC, IP or IV routes.
If vaccination with KBMA urvB Ft novicida provides a significant level of protection
against a wild-type Ft novicida challenge, then we will vaccinate mice with a 0.1 x LD50
dose of KBMA urvB Ft novicida and deplete CD4+, CD8+, or CD4+ and CD8+ T cells to
determine the contribution of T cells to protection.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 43
Milestone description: Create uvrA or uvrB mutants in LVS
Institution: UTSA
1. Date started: 5/01/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
The plasmid pDS132 was modified for use in F. tularensis. This is a conditionally-replicative
plasmid that has a counterselectable marker (sac B). We have adapted pDS132 for use in F.t. by
first altering the multiple cloning site and then inserting a Ft promoter (gro ELp) to facilitate
expression of sacB and the antibiotic resistance marker (cat). The mating plasmid pKEK1090,
which has the GroEL promoter properly inserted to facilitate sacB/cat expression, was used as a
vector to clone UvrBUpFpKanDn (KEK1114). The plasmid was mated into the LVS strain and the
SacB is expected to help to eliminate the plasmid backbone.
3.1 Ordered individual components for the TSA media. One of the components for this media
was on backorder; therefore, I made media using LB supplemented with the various
additional chemicals used in the TSA and used this media to grow up Schu S4
strain. The supplements were 250 ug/ml final of sodium pyruvate, sodium
metabisulfite, and ferrous Sulfate; also 1% final of casamino acids and L-cysteine
hydrochloride. LVS did not grow on this media. I should receive the necessary
components for TSA media by February 9th. I will prepare this TSA media with 5%
Sucrose and no sodium chloride and repeat sucrose selection process to rid the
plasmid backbone from LVS containing uvrB insertion; described in previous report.
Data recorded on UTSA TVDC notebook #2, page 60.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
31 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
After the components are received, UTSA will be making TSA media w/o NaCl to facilitate
removal of the plasmid backbone from LVS containing the urvB insertion.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
Approximate 49% of scientific work completed on the milestone
9. Work plan for upcoming month
i.
ii.
Repeat the sucrose selection process to get rid of the plasmid backbone in the LVS strain.
Fresh TSA media with 5% sucrose and without sodium chloride will be used. This should
complete the uvrB LVS mutant.
Although the LB was supplemented with the various TSA +++ chemicals described in 3.1—
did not work for LVS—I will see if the Schu S4 will grow on these plates.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
We have demonstrated that LVS grows robustly in 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 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 been monitoring
the stability of the stored product 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.
1) We have performed our first biodistribution study using Cerus expanded lot 16 LVS injected IV
into BALB/c mice. The data are presented in milestone 40 because LVS and Ft novicida were
compared.
2) The stability of fermentor-grown LVS culture at -80o C in 2 different freezing solutions is being
monitored on a monthly basis. The titer of each cryopreserved stock was determined by making
duplicate dilution series and plating 100 ul of each of the 10-7 dilution on 5 CHAH plates, hence
each bar in the graph represents the mean of 10 plate counts and error bars
represent the standard deviation. After two months at -80o C both samples had titers greater than
or equal to the pre-freeze titers, suggesting that both cryopreservation solutions appear to be
performing well.
32 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Fermentor-Grown LVS Viability (cfu/mL) Pre-Freeze, T=0, and T=1 Month T=2 Month
HBSS + 1% sucrose + 8% DMSO
HBSS + 10% Sucrose
5.85E+09
4.47E+09
2.13E+09
4.47E+09
9.43E+09
4.89E+09
2.88E+09
2.73E+09
1.00E+10
Pre-Freeze
T=0
T=1 Month
T=2 Months
1.00E+09
Lot: 948-119 Arm-1 (Freeze Buffer)
Lot: 948-119 Arm-2 (10% Sucrose)
NB948-178
The metabolic activity of the cryopreserved LVS was also evaluated after 2.5 months using the
MTS assay and was compared to DVC lyophilized lot 16 LVS. The degree of metabolic activity of
DVC lot 16 was significantly lower than Cerus frozen LVS when the vaccine stocks were
normalized to OD600. This suggests that the DVC lyophilized product contains many dead
bacterial particles. However, when the data were normalized to cfu the DVC lot 16 had much
higher degree of metabolic activity. Together these data suggest that the non-viable particles in
the DVC vaccine may actually retain some metabolic activity but are not capable of forming a
colony on a plate. We will continue to monitor the stability of lot 948-119 LVS samples by plating
for cfu, we will also compare the metabolic activity by MTS assay, and we will measure the
virulence of each in mice.
Metabolic Activity of Cerus Lot 948-119 vs. DVC Lot: 703-0303-016
OD (600nm) = 0.05
0.80
Cerus lot:948-119 Arm-1
Cerus lot: 948-119 Arm-2
DVC lot: 703-0303-016
OD (490nm)
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0
2
4
6
8
10
12
Time
(hours)vs. DVC Lot: 703-0303-016
Metabolic Activity of Cerus Lot
948-119
Nominal 1e7 cfu/mL
1.20
DVC lot: 703-0303-016
Cerus lot: 948-119 Arm-1
Cerus lot: 948-119 Arm-2
1.00
OD (490nm)
CFU/mL
1.00E+11
0.80
0.60
0.40
NB668-001
0.20
0.00
0
2
4
6
Time (hours)
8
10
12
33 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
3) We have attempted to determine the LD50 of Cerus expanded lot 16 LVS via the SC route. 10fold serial dilutions ranging from 1.26 x108 to 1.26 x 102 were administered. At the highest dose
only 33.3% lethality was achieved. This confirms the observation of Green et. al, 2005, that LVS
is nearly avirulent when administered via the SC route.
4. Significant decisions made or pending
We have selected CDM and CHAH as liquid and plate medias for cultivation and enumeration of
LVS. We have determined the minimum concentration of S-59 psoralen required for complete
inactivation of DVC LVS is 5µM. We have switched to using Sigma Antifoam A concentrate as
our antifoam agent for large-scale propagation of LVS. We have selected 10% sucrose as our
cryopreservation agent.
5. Problems or concerns and strategies to address
The LVS provided by DVC appears to be highly attenuated when administered IP, the degree of
attenuation is less pronounced when LVS is expanded in CDM. The attenuation is less evident
when the expanded LVS is administered IV.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
28% of scientific work completed on the milestone
9. Work plan for upcoming month



The stability of LVS stored at -80oC in various cryopreservation agents will be evaluated
monthly by MTS assay and plating on CHAH plates.
We will compare the virulence of LVS stored in the various cryopreservation agents by
injecting IV into BALB/c mice.
We will attempt to produce a sterile 400mL lot of KBMA LVS using 10, 20, and 40uM S59 and 6J/cm2 UVA.
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. Our first desired deletion for the project is igLC therefore, we are trying to clone this
gene into a modified vector of pDS132 described in milestone 43 in November’s report. This
plasmid is essentially a suicide vector than can be used in mating into SCHU S4 to initiate a
cross-over into the chromosome and generate the deletion. Secondly, we have a strategy of a
34 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
construct for which experiments have indicated that it can be used to generate the insertion of the
deletion; however, we had trouble removing the plasmid part of the construct from the
chromosome. Thus, we are working on getting the Sac B gene cloned into this construct
(KEK964) which should help resolve the retention of the plasmid in the chromosome.
I. Cloning of igLC:
a. Prepare more DH5αλpir competent cells to use with KEK1090+iglC deletion fragment
from KEK906; described in earlier report.
b. The transformation of KEK1090 + iglC deletion fragment from KEK906 yielded 48
colonies. Prepared mini plasmid preparations of 15 colonies and digested with EcoRI
enzyme. The results indicated that these were all vector with no insert. Will therefore,
make colony pools of 10 each and screen by PCR first to find the possible correct clone
in these transformants.
c. Will evaluate results on next report. Data located in TVD UTSA Notebook 3, page 7374.
II. Experiments to generate deletions in Schu4:
a. The two clones were sequenced to confirm SacB cloned into KEK964 and the
sequences were correct. Construct C13 KEK964 BHI/CIP+SacB Bgl II/BHI was
frozen as KEK1133 and the Construct C26 KEK964 XbaI/BHI + SacB XbaI/ Bgl II
was frozen and KEK1134.
b. Digested the KEK1133 with BamHI enzyme the subsequently with Sph I enzyme and
this vector was gel isolated with the Qiagen gel purification kit described earlier.
c. The MglA deletion fragment was generated from KEK1023 by using oligos MglA new
Down BHI and MglA new Up SphI. This product was digested as done with the
vector KEK1133. This MglA fragment was also gel isolated with the Qiagen gel
extraction kit. The KEK1133 BHI/SphI was ligated with the MglA BHI/SphI
fragment.
d. Did a transformation with DH5α cells using the ligation reaction above and this
resulted in hundreds of colonies, potentially containing the construct to make the
MglA deletion mutation for SCHU S4.
e. Did mini plasmid preparation on 10 colonies and screened with Hind III digestion and
ran the cloning vector KEK1133 and a control to compare profile.
f. Based on the figure 1 below clone 8 may be correct as compared to the vector and
expected fragment sizes.
Figure 1.
35 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
This represents some KEK1133+MglA clones generated from transformation into DH5λ
cells. The various plasmids were digested with Hind III enzyme and the correct construct
should yield 4 fragments ≈3800 bp (vector mostly); ≈2100 bp (MglA fragment);1385 bp
(Kan and SacB fragment) and ≈ 150 bp (part of MglA and vector). The control vector will
only show three fragments. Lane 1 is the 1 Kb ladder (Invitrogen); Lanes 2, and 4-7 are
Hind III digested plasmids and Lanes 3 and 8 are uncut plasmids. Lane 7 is Clone 8 of
the KEK1133 + MglA DH5α transformant’s plasmid and it appears to be correct.
Although the 150 bp band doesn’t show up well in this photo, which may be due to the
amount of DNA loaded on the gel.
g. I performed a PCR reaction using MglA oligo set; mentioned in II.c. above; that would
generate ≈ 2200 bp fragment if the MglA deletion is present in the KEK1133. I used
KEK1023 as the positive control as the template for PCR. In addition, I ran 9 of the
10 plasmids prepared from the resulting DH5α clones. Based on that result Clones
7 and 8 looks the most promising candidates (see Figure 2).
Figure 2.
This represents the results of a polymerase chain reaction using various plasmid
templates (as indicated in legend) and the oligo PCR primer set MglA new Down
BHI and MglA new Up SphI. The positive control is the KEK1023 pUC construct
containing the MglA deletion fragment, lane 2. Lanes 3-5 and 7-12 are results
from transformants of KEK1133 + MglA. Lane 6 is KEK1090 + iglC (which
should not have been loaded in this gel). Lanes 10 and 11 look like they may be
correct, these are C7 and C8 KEK1133+MglA constructs. (note: The extra band in
lane 10 is an non-specific band resulting from the plasmid used in cloning. Since plasmid
will be removed from chromosome this will not present any problems in the next steps)
h. Will prepare a larger plasmid preparation from Clones 7 and 8 do a few more
restriction digests for confirmation. Then we will send these two clones for
sequencing if all looks good.
i. Meanwhile, I have designed an oligo to amplify the iglC deletion fragment from
KEK906 to allow me to clone this into the KEK1133 vector via a Sal I site.
IgLC schu4 up Sal I: gcg gcg gtc cac gaa gaa tct cca cca gaa gc and the
primer pair has already been described earlier IglC schu4 down Sal I.
36 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
j.
All data from figure 1 and 2 is located in TVD UTSA Notebook 3, page 54-57.
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
37%
9. Work plan for upcoming month
a. Prepare the pool suspensions of the KEK1090 + iglC clones and run PCR with iglC oligos
to find the correct iglC deletion construct.
b. Will evaluate any potential candidates (C7 and C8 KEK1133+MglA constructs) from the
PCR screen above by preparing plasmid from these clones and doing restriction analysis
c. Will prepare a large plasmid preparation of the KEK1133 + MglA clone and send for
sequencing once the restriction analysis is complete.
d. Will order the Sal I oligo to generate the iglC deletion fragment to clone into KEK1133
plasmid.
e. Digest both the KEK1133 and the iglC deletion PCR fragment with Sal I restriction
endonuclease and then will isolate the band to prepare for ligation.
f. Perform plasmid preparations and PCR of constructs resulting from ligation above (in e.).
Will do restriction endonuclease digest analysis to screen for the correct construct.
g. Order more supplies as needed
10.Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: In Progress
3. Work performed and progress including data and preliminary conclusions
37 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
a. Determine the LD50 of Ft subsp. novicida iglB mutant. (Note book #4 page 63-65):
Groups of BALB/c mice (female, 4-6 weeks) were intranasally challenged with 104, 105,
106 or 107 CFU of the ΔiglB mutant. As shown in Fig. 1, there is no mortality observed
at any dose, indicating the high degree of attenuation with this ΔiglB mutant organism.
No significant weight loss of infected mice was also observed. The LD 50 of ΔiglB in the
intranasal infection model (BALB/c mice) is greater than 107 CFU.
b. Monitor Ft subsp. novicida iglB mutant replication and dissemination in mice after
intranasal challenge (Note book #4, page 66-71). BALB/c mice were challenged with
ΔiglB mutant (106 CFU) intranasally. Lungs, liver, spleen, and lymph nodes were
collected from the infected mice at a three day interval (3 mice per time point). Numbers
of bacteria in each organ were determined by dilution plating. As shown in Fig. 2, there
was heightened replication of the organism in the lungs within the first 12 days, with
reduction noted at day 15. There were lower levels of replication within the liver and
spleen. Numbers of bacteria in the spleen are consistent through out the observed
period. In the liver, the bacterial burden was decreased by day 15. There were
organisms recovered from the draining lymph nodes, but at much lower levels than that
seen with the other target organs. The growth kinetics of ΔiglB in the animal organs is
comparable to ΔiglC as reported previously.
38 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
29%
9. Work plan for upcoming month
a. Evaluate the protective efficacy of the Ft subsp. novicida iglB mutant as a vaccine
candidate. Groups of vaccinated mice will be challenged i.n. with Ft subsp. novicida.
Animals will be monitored for survival and weight loss.
b. Analyze the antibody profiles of mice immunized with the Ft novicida iglB mutant at day 30
after vaccination.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
39 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
Milestone 51
Milestone description: Construction and delivery of Ft subsp. novicida uvrA or uvrB plus pdpD,
iglA, iglB, iglC or iglD double mutants.
Institution: UTSA
1. Date started: 11/01/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
Chromosomal DNA was purified from the uvrB F.novicida mutant KKF71. 10 ug of this
chromosomal DNA was cryotransformed into an iglC F. novicida mutant KKF24 in hopes of
generating a uvrB + iglC double mutant. Cryotransformants were plated on TSA/++/Kanamycin
for the initial selection and 4 colonies were screened by colony PCR with the primers UvrBUP
and UvrBDn1. The resulting 3.5 Kb PCR fragments were subjected to restriction digest using the
enzyme Bgl2 and run on a DNA agarose gel. If the mutant is correct, you would expect to see
two fragments on the gel due to the presence of a Bgl2 site within the Kan marker that is not
present in the wild type copy of uvrB (Figure 1). Lanes 3, 5 and 6 show the correct restriction
digest pattern (two bands). Colony 1 (lane 3) was frozen away as KKF224 (Data described in
Notebook 1, pages 12-13). This strain KKF224 is a uvrB + iglC double mutant in Ft subsp.
Novicida. (Note: Sequencing is not necessary due to the ability of the new mutant to grow on both
Erythromycin and Kanamycin. Recombination can only occur at the site of uvrB. This, along with the
PCR/restriction digest data is sufficient proof that this mutant is correct.)
a.
Figure 1.
1.
2.
3.
4.
5.
6.
1Kb ladder
U112 wild type control
colony 1
colony 2
colony 3
colony 4
4. Significant decisions made or pending
The decision was made to focus making all the double mutants in Ft novicida in a uvrB
background per the 1/25/07 conference call with Dr. Klose, Skoble and Lyons
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
20%
40 of 41
Tularemia Vaccine Development Contract: Technical Report
Period: 1/01/2007 to 1/31/2007
Due Date: 2/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam,
9. Work plan for upcoming month
Create a uvrB + iglD double mutant
10. Anticipated travel
None.
11. Upcoming Contract Authorization
None
41 of 41