Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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, 21, 26,
27, 28, 33, 34 (UNM/ASU), 40, 41, 42, 43, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 48
Inactive milestones: 6-11, 14, 15, 17, 18, 20, 22-24, 29-31, 35-38, 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. Nicole Banks (LBERI) is in contact with Kristin DeBord regarding a subcontract as of
3/7/07)
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.
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.
1 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
16%- no change since the 11/15/06 report
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 Summer 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 February (1 frozen, 2 fresh LVS in 48 hr. Chamberlain’s broth)
Both sprays with fresh LVS had problems with contamination
i. Performing QC to assess and mitigate future occurrences
Performed 9 sprays with frozen material
i. 3 sprays at target concentration of 1x105 cfu/mL
ii. 3 sprays at target concentration of 1x106 cfu/mL
iii. 3 sprays at target concentration of 1x107 cfu/mL
iv. Data for 2/23/007 show excellent predictability for spray concentration
2 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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
2/23/2007
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
v. Spray factor data for 2/23/007 show lower than expected values
1. Possible indication of stability effects on glycerol stocks? We will use
both the glycerol and sucrose stocks in upcoming sprays with the
sparging generator to determine if stability is an issue.
Spray Factor (Log10)
Actual CFU/ml vs. Spray Factor (Frozen)
0.00
0.00
-2.00
2.00
4.00
6.00
8.00
10.00
7/14/2006
8/8/2006
8/15/2006
2/23/2007
-4.00
-6.00
-8.00
-10.00
CFU/mL (Log 10)
Data found at Saturn/ABSL3/Agent and Study Specific Data/TUL-03
3 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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







Vaccination status may affect the amount of delay
If vaccination not required, plan to complete by 4/15/07
Both fresh LVS sprays were contaminated
First spray had contaminated plates, so now that the autoclave has been repaired, longer
autoclave times are necessary. The autoclave time was doubled and all subsequent runs
have been sterile.
Second spray had contaminated growth broth, so Chamberlain’s broth will now be made
one week before the aerosol challenge and QCed prior to spray. The draft SOP for this
procedure was modified to include review of all media QC data prior to initiating sprays
so that non-sterile media is not a factor in future studies.
QC records of all sterile materials will be reviewed by study personnel prior to each spray
to reduce problems with contamination.
Getting signatures to finalize LVS Growth SOP
6. Deliverables completed
None
7. Quality of performance
Good for the frozen spray; Poor for the fresh LVS sprays
8. Percentage completed
30%
9. Work plan for upcoming month



Perform additional bioaerosol experiments on vegetative LVS with Collison generator
i. Finish fresh LVS sprays (2 total)
ii. Finalize LVS growth SOP
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 frozen and fresh, not lyophilized.
Perform bioaerosol experiments on frozen LVS with micropump generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing frozen and fresh, not lyophilized.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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 twice at Barbara Griffith’s request
4 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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
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 by incorporating changes suggested by Marlene Hammer for
quality
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 4
Milestone description: Development of infectious model 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:
IACUC protocol has been approved to allow continued blood collection from vaccinated NHPs
(see Milestone 13 for assays to be conducted with these samples)
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
Vaccinated primates are a source of blood for the development of immunoassays
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
5 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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 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 Ftc31 study 1 (Notebook 94, pages 110-117)
i. The purpose was to determine the i.t. LD50 of SCHU S4 expanded in
Chamberlain’s broth.
ii. Experiment Ftc23 study 2 suggested that the i.t. LD50 is less than 3,000 CFU.
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.
iii. The discrepancy may be explained by the fact that 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
iv. 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
v. The results showed that 300 SCHU S4 killed 80% (4/5) of rats and 30 SCHU
S4 killed 20% (1/5) rats (Figure 1)
vi. The calculated i.t. LD50 for the expanded SCHU S4 is 180 CFU, considerably
lower than reported in the literature
Figure 1. Intratracheal challenge of naive Fischer 344 rats
with SCHU S4 expanded in Chamberlain’s broth. Naive
Fischer 344 rats (n = 5) were challenged i.t. with SCHU S4
and monitored for survival. The challenge doses indicated in
the figure legend reflects actual lung deposition measured 1
h after challenge
b. Clinical signs of infection
i. Based on the clinical signs that develop in infected rats in the experiments so
far, we have developed the following criteria for scoring disease severity
(-)
Active, bright, alert, and responsive to cage movement
(+/-) Active, alert, and responsive to cage movement, slight piloerection,
slight porphyrin secretion around eyes
6 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
(+)
Slightly decreased activity, alertness and responsiveness to cage
movement, clear piloerection, eyes open but with slight porphryin
secretion
(++) Definite decreased activity, moves slowly to cage movement, very
ruffled coat, rapid and shallow breathing, hunched over, huddled in
group, eyes half closed with large amount of porphyrin secretion
(+++) Inactive and unresponsive to cage movement, very ruffled coat,
weight loss, wobbly, difficult waling, labored breathing, eyes
closed, isolated from group
c.
Experiment Ftc32 study 1 (in progress)
i. The purpose of this experiment is to repeat the vaccination/challenge
experiment (Ftc23 study 2) comparing different vaccination routes and
strains in their ability to protect Fischer 344 rats against i.t. SCHU S4
challenge
ii. Rats have been vaccinated according to Table 1
Table 1. Experimental design to compare the ability of different vaccination routes and
to protect Fischer 344 rats against i.t. SCHU S4 challenge
i.t. SCHU S4 challenge
Vaccination
(number of rats/dose)
Bacterial
Dose
strain
Route
CFU/rat
101 102 103 104 105 106 107 108
None
6
6
6
LVS
i.d.
s.c.
i.t.
5 x 107
5 x 107
5 x 107
6
6
6
6
6
6
6
6
6
SCHU S4
i.t.
5 x 101
6
6
6
Guinea Pigs Hartley Strain
a. Experiment Ftc28 study 2 (Notebook 94, in progress)
i. The purpose was to use the guinea pigs that survived i.n. or s.c. LVS
infection in Ftc28 study 1 in a pilot experiment to determine whether LVS
vaccination protects guinea pigs against pulmonary SCHU S4 challenge
ii. 33 days after LVS vaccination, the number of LVS in the lungs, spleens, and
livers was below our limit of detection in two guinea pigs vaccinated s.c. with
4.4 x 107 LVS and in two guineas pigs vaccinated i.n. with 4.7 x 106 LVS
iii. 47 days after LVS vaccination, guinea pigs (n = 2-4/group) were challenged
i.n. with an estimated dose of 500 SCHU S4. At such a low dose, the large
buffer volume used to homogenize infected lungs makes it difficult to
accurately determine the actual lung deposition by plating lung homogenates
onto cystine heart agar plates
iv. The experiment is in progress, however, the preliminary results suggest that
i.n. vaccination protected guinea pigs better than s.c. vaccination and that the
level of protection is directly related to number of LVS used for vaccination
(Table 2)
v. These results are consistent with those reported in HT Eigelsbach et al
(1961) Proc. Soc. Exp. Biol. Med 108:732-4
7 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Table 2. Survival of vaccinated guinea pigs challenged i.n. with SCHU S4
Vaccination
LVS dose
Animal ID
Disease severity1
Time to death
Route
(CFU/animal)
(days)
i.n.
1.5 x 103
9949
dead
6
9950
1
9948
1.5
7.8 x 104
9947
2
9946
0
9945
1
9944
1
4.7 x 106
9943
0
9942
0
4.4 x 103
s.c.
4.4 x 105
4.4x 107
1Disease
9938
9937
9936
9932
9933
9935
9934
9930
9931
dead
dead
dead
0
0
dead
dead
dead
2
6
6
6
6
6
6
-
severity: 0 = no sign of illness to 3 = moribund
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
39%
9. Work plan for upcoming month
a. Rats -- Characterization of the Fischer 344 rat model
i. Kinetics of SCHU S4 proliferation in lungs, spleens, and livers of naïve and
vaccinated rats
ii. Histology of lungs, spleens and livers of naïve and vaccinated rats infected
with SCHU S4
b. Guinea Pigs
i. Vaccinate guinea pigs with LVS i.n., s.c., or i.d. to repeat vaccination and
challenge experiment
c. SCHU S4 expansion (TVDC working group)
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
8 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
11. Upcoming Contract Authorization (COA) for subcontractors
NA
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
Experiment Ftc27 study 6 (Notebook 94, pages 96-101) and study 7 (Notebook 94,
pages 102-109)
i. The purpose was to identify the conditions that will allow antigen-specific T
cell proliferation to be measured
ii. Total splenocytes and nylon-wool splenocytes from naïve and vaccinated
mice were titrated from 5 x 105 to 5 x 103/well and stimulated with 105/well or
106/well LVS or Con A for 5 days
iii. 5 x 105 total splenocytes/well produced unacceptably high background with
medium alone (Figure 2). The background was improved considerably by
reducing the cell number to 5 x 104 or less per well. However, reducing the
cell numbers also decreased the assay sensitivity
iv. We detected antigen-specific responses with total splenocytes (comparing
the responses of naïve and vaccinated splenocytes to formalin-fixed LVS).
However, naïve splenocytes proliferated to a higher level with formalin-fixed
LVS than culture medium, suggesting there is some non-antigen specific
response.
v. The non-antigen specific response by naïve splenocytes was eliminated
when the splenocytes were passed through nylon wool columns
vi. We concluded from this experiment that 5 x 104/well nylon wool-enriched T
cells and 106/well formalin-fixed LVS produced the best balance of
background, specificity and sensitivity
9 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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 2. T cell proliferative response to formalin-fixed LVS and Con A. Splenocytes from naïve and vaccinated BALB/c
mice were cultured with formalin-fixed 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
10 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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
NA
7. Quality of performance
Good
8. Percentage completed
16%
9. Work plan for upcoming month
Repeat T cell proliferation assay using the experimental protocol described in Ftc27 study 7
until we can achieve consistent and reproducible results
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
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 2/12/07, thawed PBMCs which were frozen down on 12/18/06 using the CTL
protocol and tested them for proliferation to Con A and LVS to compare to the freshly
isolated cells (12/18/06); also, on 2/21/07, thawed PBMCs which were frozen down on
12/27/06 using the Cerus protocol and tested their proliferative response to Con A and
LVS to compare to freshly isolated cells
 Details on procedure and data resulting from this test can be found in the binder TVDC
1 under Tab for TUL 8, Day 28 (2/12/07) and TUL 9, Day 28 (2/21/07)
Summary of Data from cells frozen down with CTL protocol:
1. Cells were originally frozen using the CTL protocol (90% human A/B
serum/10% DMSO at 10 x 106/ml)
2. On 2/12/07, thawed 2 vials from each of 2 animals; these cells were frozen on
12/18/06, TUL 8, d28 in 0.5 ml aliquots
Viable Cell Recovery/Percent viability from CTL freezing procedure (each vial
contained 5 x 106 cells):
A00896 Vial 1: 0.74 x 106= 21.1% recovered/86.4% viable
A00896 Vial 2: 2.84 x 106= 56.8% recovered/91.9% viable
A00908 Vial 1: 5.81 x 106= 102.8% recovered/77.5% viable
A00908 Vial 2: 5 x 106= 100% recovered/70.1% viable
11 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Conclusion: Viability and cell recovery were quite varied with CTL protocol
Proliferation: Cells were resuspended at 1 x 106/ml and 0.25 x 106/ml and stimulated
with Con A (5 g/ml) or LVS at Hi (1 x 105/ml) and Mid (0.25 x 105/ml) doses for 4 days
using the standard protocol in the BRDU proliferation assay
Proliferative Response of CTL Frozen/Thawed Cells (Top panel) Vs. Fresh Cells (Bottom Panel)
1
.25
2.50E6
2.00E6
1.50E6
1.00E6
LVS ff Mid
LVS ff Hi
LVS hk Mid
LVS hk Hi
0
Con A
5.00E5
Media
Cell Mean for RLU normal
Relative Light Units
(Mean +/- SEM)
3.00E6
12 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Cell Mean for RLU small
Relative Light Units
(Mean +/- SEM)
3.50E5
1
.25
3.00E5
2.50E5
2.00E5
1.50E5
1.00E5
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Lo
LVS hk Mid
LVS hk Hi
Con A
0
Media
5.00E4
Figure 1: Proliferation of frozen/thawed cells (top panel) compared to fresh cells (bottom panel). Cells
are plated at 1 x 106/ml (open bars) or 0.25 x 106/ml and stimulated with Con A (5 g/ml) or LVS (Hi = 1 x
105/ml and Mid = 0.25 x 105/ml; Lo = 0. 0625 x 105/ml). Note deficiency in the ability of the frozen/thawed
cells to proliferate in response to LVS in comparison to the fresh cells.
A potential caveat is that the proliferative response of the fresh cells was measured at the Victor at UNM,
whereas the frozen/thawed cell response was measured at LRRI using the Victor Light. The latter
machine at the time was not set to limit the light reaching the wells and thus the scale is 10 fold higher.
This situation has been rectified by a visit from the Perkin Elmer representative on 3/5/07. In the future,
we will be able to directly compare Relative Light Unit values using the same settings on both machines.
In the meantime, we show the data below in the manner of a stimulation index which = proliferation to
stimulus/proliferation to media.
13 of 50
Tularemia Vaccine Development Contract: Technical Report
10
9
8
LVS ff Hi
LVS hk Mid
2
1
0
LVS hk Hi
7
6
5
4
3
LVS ff Mid
Fresh, 1
Fresh, .25
Frozen, 1
Frozen, .25
Con A
Cell Mean for Stimulation Index
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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 2: Proliferation of Fresh and Frozen (thawed/frozen) PBMCs comparing the stimulation index.
Cells were plated at 1 x 106/ml or 0.25 x 106/ml.
Conclusion: Freeze/thaw protocol from CTL resulted in good proliferative response to
Con A but no response to LVS. This could be a problem as the primary goal of the assay
is to detect an LVS antigen specific proliferative response
Summary of Data from cells frozen down with the Cerus Protocol
(80% FBS, 20% DMSO, final concentration of 5 x 106/ml); TUL 9, Day 28, thawed on
2/21/07:
On 2/21/07, thawed 2 vials from 1 animal (A00659) and 1 vial from 1 animal (A00868);
these cells were frozen on 12/27/07, TUL 9, d28, in 1 ml aliquots
Viable Cell Recovery/Percent viability from Cerus freezing procedure
A00659 Vial 1: 3.94x10^6 = 78.8% recovered/94% viable
A00659 Vial 2: 2.69x10^6 = 53.8% recovered /95.5 % viable
A00868 Vial :3.31x10^6 =66.2%recovered /91.3% viable
Conclusion: Cell recover and viability were good for the Cerus protocol similar to last
time cells were thawed using this protocol (1/11/07)
Proliferation: Cells were resuspended at 1 x 106/ml and 0.25 x 106/ml and stimulated
14 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
with Con A (5 g/ml) or LVS at Hi dose (1 x 105/ml) for 4 days using the standard
protocol in the BRDU proliferation assay
Average proliferation as measured by RLU:
Relative Light Units
(Mean +/- SEM)
1.40E6
Fresh, 1
Fresh, .25
Frozen, 1
Frozen, .25
1.20E6
1.00E6
8.00E5
6.00E5
4.00E5
2.00E5
0
Media
Con A LVS hk Hi LVS ff Hi
Figure 3: Proliferation of Cerus frozen/thawed cells compared to fresh cells. Cells are plated
at 1 x 106/ml or 0.25 x 106/ml and stimulated with Con A (5 g/ml) or LVS (Hi = 1 x 105/ml).
Conclusion:
Using the Cerus protocol, we can recover at least 50% of the cells and a portion of the
proliferative response to both Con A and LVS. Responsiveness to LVS is better when 1 x 106
cells/ml are plated regardless of whether the cells are fresh or frozen. Thus, due to some
retention of antigen-specific responsiveness as measured by proliferation, the Cerus protocol
seems superior to the CTL protocol for freezing and thawing cells.
Data Location: C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract and backed up on N:\My Documents\Tularemia Contract





Update on possible B cell staining artifact:
Issue: CD20+ cells were abundant in peripheral blood of NHPs by FACS staining, but did not
appear in PBMC preparation (as if B cells were being excluded by the Percoll gradient while
T cells were being enriched – didn’t make sense)
Possible artifact is being tested by staining B cells with other antibodies (specific for CD19
and surface IgM)
Blood drawn on 2/15/07 from 3 NHPs (1 from LVS ID group (TUL 8) and 2 from LVS SC
group (TUL 9))
Preliminary results suggest that anti-human CD19 does not bind NHP B cells in blood or
PBMC preparation; anti-IgM binds a small proportion of cells in the whole blood and positive
cells are enriched in PBMCs; anti-CD20 binds a large proportion of cells in whole blood, but
not in PBMCs (these cells do look lymphocytic/monocytic)
Full details will be shown at next tech call; however, at the 3/6/07 LBERI Technical call, Freyja Lynn conveyed that
antibodies developed for human assays can lead to unusual results when primate cells are assayed.
15 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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
Concern that freezing cells down and thawing them using the CTL protocol may not preserve
the proliferative response to LVS antigen; we will test this again to be sure before making this
conclusion.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
90% of scientific work has been completed
9. Work plan for upcoming month



We will fully analyze the B cell staining from blood and PBMCs stained on 2/15/07
We will also analyze the T cell phenotype of the NHPs re-bled on 2/15/07 to determine
whether S.C. (TUL 9) groups of NHPs have more T cells in their blood (suspected due to
their superior response to Con A as compared to TUL 8 NHPs)
We will test intracellular staining of IFN in whole blood and PBMC preparations (from LVSvaccinated NHPs; Milestone 4)
10. Anticipated travel
None
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 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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
IFN ELISPOT:
 Data on IFN ELISPOT assays performed in November and December indicated that
no response to LVS was observed in the LVS vaccinated NHPs (all below background)
 Suspected that not enough cells were originally plated/well (20,000 cells/well) to detect
LVS-specific IFN secretion
 Preliminary data on blood drawn on 2/15/07 suggest that if 100,000 or 500,000
cells/well are plated), IFN spots can be seen in wells stimulated with LVS; however,
the background in media only wells is quite high (spots are about half that seen in LVS
stimulated wells); the spots in the unstimulated wells are of a different character (more
pale, less discreet); Julie will contact the representative to discuss options for changing
the settings on the ELISPOT reader to exclude spots; also, capture and detection
antibody concentrations may need to be adjusted
 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;
Development of anti-LVS IgG ELISA:
 Plasma was stored from vaccinated NHPs (TUL 8 and TUL 9)
 ELISA plates were coated with either heat-killed (HK) LVS or formalin-fixed (FF) LVS at
three different concentrations (1 x 106/ml, 0.5 x 106/ml and 0.25 x 106/ml), diluted in PBS,
0.1 ml/well, incubated overnight at 4 degrees.
 Plates were blocked with 5% w/v nonfat milk in PBS for 2 hours at RT.
 D0 and D21 sera from vaccinated NHPs were diluted 1/100, 1/500, 1/2500 and 1/12500
in 5% nonfat milk and plated at 0.1 ml/well in duplicate; incubated overnight at 4
degrees.
 Antibody was detected with Goat anti-monkey IgG-HRP for 2 hours at RT, followed by
substrate (ABTS) addition.
 Data are expressed as arbitrary units: lowest OD405 above background (no sera added to
well) x dilution factor (i.e. 0.1 OD x 12500 = 1250 units).
17 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Comparison of HK-LVS coating concentration:
Units of anti-HK-LVS
3500
Arbitrary units
3000
2500
2000
1500
1000
500
0
896
908
937
659
868
902
TUL 8
TUL 8
TUL 8
TUL 9
TUL 9
TUL 9
1 x 106 HK-LVS Day 0
0.5 x 106 HK-LVS Day 0
0.25 x106 HK-LVS Day 0
1 x 106 HK-LVS Day 21
0.5 x 106 HK-LVS Day 21
0.25 x106 HK-LVS Day 21
Figure 1: ELISA plates were coated with various dilutions of HK-LVS and both Day 0 and Day 21 sera
from vaccinated NHPs was tested for IgG anti-LVS.
Conclusion: Day 0 sera gives very low background with the exception of NHP A00896 binding to plates
coated with 0.25 x 106 HK-LVS/ml. IgG anti-LVS can be detected in all day 21 sera regardless of HK-LVS
concentration used to coat the plates. These data suggest that we may be able to use less antigen to
coat the plates.
18 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Comparison of HK- and FF-LVS used to coat the ELISA plates:
Units of anti-HK vs FF-LVS
3500
Arbitrary Units
3000
2500
2000
1500
1000
500
0
896
908
937
659
868
902
TUL 8 TUL 8 TUL 8 TUL 9 TUL 9 TUL 9
Units on HK-LVS 1 x 106 HK-LVS Day 0
Units on HK-LVS 1 x 106 HK-LVS Day 21
Titer on FF-LVS 1 x 106 Day 0
Titer on FF-LVS 1 x 106 Day 21
Figure 2: ELISA plates were coated with either HK-LVS or FF-LVS at 1 x 106/ml. Reactivity of Day 0 and
Day 21 sera are shown as arbitrary units (Note: titer should read Units).
Conclusions: In all cases, day 21 sera is more reactive to HK-LVS than to FF-LVS, particularly in NHPs
A00896 and A00908 (TUL 8). In addition, not shown is the fact that peak OD405 values were 3 – 4 times
higher when tested on HK-LVS coated plates as opposed to FF-LVS coated plates.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have a concern that the ELISPOT reader settings need to be fine tuned to be able to exclude
faint spots which result when large numbers of cells are plated/well (required to detect LVSinduced IFN secretion). Julie will talk to the service representative about this issue.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
15% of scientific work has been completed
9. Work plan for upcoming month

Analyze the phenotype of the blood cells and PBMCs from the 3 vaccinated NHPs
19 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,




which were re-bled on 2/12/07 to determine if differences exist in T cell numbers in the
S.C. vs. I.D. group that may explain why the Con A proliferative response of the S.C.
group is higher than the I.D. group
Further test whether a cell number between 100,000 and 500,000 PBMCs/well will give
detectable IFN ELISPOT results without a high background
Run the rest of the vaccinated NHP sera (Day 7, 14 and 28) on HK-LVS coated ELISA
plates to determine arbitrary units of IgG anti-LVS; also fine tune the ELISA protocol
Optimize coating the ELISA plates, per Freyja Lynn’s recommendations of 3/6/07
Schedule LVS-DTH test (injection of 100,000 LVS into vaccinated NHPs by the i.d.
route and monitor whether inflammation develops as a sign of vaccination)
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F. tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc36 study 2 (notebook 94, in progress)
i. The purpose was to develop an assay that will allow us to measure the
growth of F. tularensis in human alveolar macrophages and how it is affected
by cytokines and T cells from vaccinated individuals
ii. 2.5 x 104 human alveolar macrophages were infected with SCHU S4 at MOI
of 100, 10 and 1 for 2 h at 37oC. Infected macrophages were treated with 50
g/ml gentamcin for 1 h to eliminate extracellular bacteria and then
maintained with 2 g/ml gentamicin to kill any bacteria that escapes from the
infected macrophages
iii. On day 0, 2, and 4, the infected macrophages were lysed and the cell lysates
were serially diluted 10-fold and plated onto cystine heart agar plates to
quantify the number of intracellular bacteria in the macrophages.
iv. SCHU S4 clearly proliferated in human alveolar macrophages (Figure 3)
v. The assay needs to be optimized because 1) there is some variability among
replicate samples and 2) the dilutions did not proportionally reduce the
number of colonies
20 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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. Growth of SCHU S4 in human alveolar macrophages. Human alveolar macrophages were
infected with SCHU S4 at MOI of 100, 10, or 1. On the d0 and 2 of infection, the infected
macrophages were lysed and the cell lysates were serially diluted 10-fold and plated onto cystine
heart agar plates. Each plate contains undiluted cell lysates and 3 (d0) or 7 (d2) 10-fold dilutions
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
There is some variability in the number of colonies from replicate samples and the dilutions
did not proportionally reduce the number of colonies. We will try to improve consistency and
reproducibility of this assay by increasing the number of macrophages/well (thus the number
of bacteria) and increasing the volume of cell lysate plated.
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
1%
9. Work plan for upcoming month
a. Determine whether F. tularensis would grow more robustly and thereby create a
larger assay window in the absence of gentamicin. Currently, 2 g/ml gentamicin is
added to the culture medium to minimize extracellular growth. However, there is a
possibility that gentamicin may penetrate into the cells and kill intracellular bacteria
as well. Since LVS did not proliferate in the absence of murine bone marrow-derived
macrophages (milestone 21, Ftc30 study 3), we may be able to leave out gentamicin
for this assay.
b. Determine whether recombinant IFN would inhibit SCHU S4 and LVS intracellular
growth
c. Measure cytokine (e.g. TNF, IL-1, and IL-6) production by macrophages infected with
SCHU S4 or LVS
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
21 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30 study 3 (notebook 94, pages 93-95)
i. The purpose is to develop an assay that will allow us to measure the growth
of F. tularensis in murine macrophages and how growth is affected by
cytokines and T cells from vaccinated mice
ii. BALB/c bone marrow cells were cultured in DMEM with 10% fetal calf serum
and 10% conditioned L929 supernatant as a source of M-CSF
iii. After 7 days, the cells became adherent and had macrophage morphology
i. The macrophages were infected with LVS at MOI of 10:1, 20:1 or 40:1
Infected macrophages were treated with 50 g/ml gentamcin for 1 h to
eliminate extracellular bacteria and then cultured in the absence antibiotics
ii. On day 0 and 3, the infected macrophages were lysed with sterile water.
The cell lysates were serially diluted 10-fold and plated onto cystine heart
agar plates to quantify the number of intracellular bacteria in the
macrophages
iv. LVS cultured without macrophages did not proliferate. Similar numbers of
LVS were recovered from the macrophages on d0, suggesting that within the
range used, the MOI did not determine the number of bacteria that entered
the macrophage (Table 2)
v. After 3 days in culture, LVS expanded by > 3 logs
MOI
(bacteria:cell)
10:1
20:1
40:1
Number of bacteria recovered
(log10 CFU/well)
Day 0
Day 3
2.47  0.11
6.07  0.56
2.20  0.06
6.50  0.14
2.55
5.91  0.22
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
5%
9. Work plan for upcoming month
a. Determine whether infected macrophages can be induced by cytokines such as IFN
or vaccinated splenocytes to kill intracellular bacteria
22 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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. Titrate the stimuli (cytokine or vaccinated T cells) to induce optimal macrophage
killing of intracellular bacterial
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone title: 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
A. Select and test ORF expression constructs
B. Select and test IVT Protocols
C. 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
We have previously demonstrated that our constructs work to efficiently express inserted
genes or ORFs (sub-gene fragments). We now are making alterations in the design of the
basic expression constructs only so as to accommodate optimizations of the IVT and
purification steps.
Current data: We asked whether the resolution and sensitivity of our IVT readouts could be
improved by switching from a fluorescent tRNA-based to a 35S-met -based detection method.
1. Observations:
a. The modular components of the expression construct used in the experiments are
illustrated in Fig. 1.
b. For comparison, IVT products labeled with the fluorescent tRNA reagent are shown in
Fig. 2. Note lanes in red box.
i. Lysate alone, without IVT template, displays a number of endogenous
fluorescent proteins, which are background in the detection system.
ii. IVT reactions yield detectable polypeptide products, but the intensity of the
specific band is almost as high as the background bands.
c. 35S-radiolabeled IVT reaction is shown in Fig. 3.
i. Lane 2: lysate without template. No background signal is visible.
ii. Lane 1: lysate with GFP template. A specific full-length GFP polypeptide is
produced with a minor component of GFP degraded products. Detection is clear
and band intensity is greater. No background.
2. Interpretation: 35S-methione labeling of in vitro translated products provides greater
specificity of detection. This protocol will be used in future.
23 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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:
Figure 2:
24 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Fig. 3.
Files are stored at: R:\\peptide\Research\CIM\GeneVac\FTU
B. Select and test IVT protocols
•
•
Protein yields obtained from test reactions appear to be generally lower than control templates.
Using label intensity as a relative measure, we roughly estimated our yields to be ~50% of kit
control plasmid templates. Kit information indicates yields can range widely, but typically yielding
from 5 to 10ug before purification. We are aiming for 10-15ug of purified product.
Rather than attempt protein purification from samples, with small quantities of protein, yield
improvement was pursued.
Current Data: We tested an IVT lysate feeding system so as to generate more polypeptide
product in a single IVT reaction. The Roche system, based on a wheat germ lysate is illustrated
in fig. 4.
25 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Fig. 4.
1. Observation #1: We used both supercoiled (SC) plasmid and linear templates in the
feeding system, with reagent. The linear template is advertised as working poorly in this
system. We hypothesized that this may be due to exonucleases in the reactions, which
degrade template. Therefore we also tested the effect of adding fresh DNA template at 3
hour time points. We chose 3 hours because reaction kinetic analyses have shown that
product yields plateau at this time. Indeed comparison of lanes 2 and 5 demonstrate that
the linear template yielded less product than was generated by the plasmid template in
the same system. However, comparison of lanes 2 and 8 show that addition of template
at later time-points raised levels of linear-template generated product such they appear
similar to that generated by the SC plasmid.
26 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Fig. 5.
2. Conclusions:
a. Use of linear templates in extended IVT reactions is improved by addition of fresh
template at later timepoints.
b. Polypeptide yields from the IVT reactions with sequential additions of linear template
are similar to that from a supercoiled circular template.
Files are stored at: R:\\peptide\Research\CIM\GeneVac\FTU
3. Observation #2: We further tested the feeding system using one of the relevant tularemia
ORFs in addition to the test ORF, GFP. We compared the wheat germ lysate (Roche) to
the E. coli lysates (Roche and Invitrogen) in the feeding system (Roche). This required
mixing the Roche and Invitrogen components. We also tested the effect of adding
additional T7 polymerase enzyme at later timepoints. We did a relative quantitation of
yields by TCA precipitating reactants and comparing counts. The absolute number of
precipitable counts per minute allowed us to calculate an estimate of total product yields
using the known specific activity of the 35S on a known date.
27 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Fig. 6.
4. Conclusions
a. Comparison of lanes 5 and 6, and lanes 8 and 9, and lanes 14 and 15 confirm that more
product is generated with feeding supplements. Using TCA to precipitate radiolabeled
products, we have determined the increase to be 2 to 4 fold. In the Roche feeding
system, we calculate that ~30ug of GFP and 8ug of FTU1712 is generated.
b. The wheat germ lysate was less efficient than the E. coli lysates from either vendor.
c. Comparison of lanes 10 and 17 indicate that the Invitrogen lysate in the Roche feeding
buffer is most efficient.
d. Comparison of linear to plasmid templates confirms that yield is similar when additional
template is added into the feeding system.
e. Comparison of lanes 15 and 16 indicate that the template and substrate supplemented
reactions may be RNA polymerase limited.
f. Optimal IVT plan to date has these features:
i. E. coli lysate and buffer from Invitrogen.
ii. Dialysis feeding system from Roche
iii. Linear expression template, with feeding and template supplements.
5. Observation #3: We have identified a BirA ligase site that is ~10 fold more efficient than the
consensus site. We have redesigned our template to encode this optimal site.
28 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Fig. 7.
4. Significant decisions made or pending
IVT reaction supplementation is worthwhile.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
85%
9. Work plan for upcoming month





Yields will be further optimized by testing T7 pol supplementation, and also shorter timepoints of addition, such as 30 min and 1h instead of 3h.
High efficiency biotin attachment peptide will be tested within template in cotranslation/biotinylation reactions.
Strepavidin purifications of protein-fragments will be tested.
Nickel purification will be conducted in parallel to allow comparison of scheme efficiencies
and purities.
Yield and solubility of pilot set of the SCHU S4 protein-fragments will be assessed by
measuring purified, antibody-bound protein on Luminex.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
29 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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 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.
No progress this month
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
No progress
8. Percentage completed
2%- this is the same amount of work reported as in the 1/15/07 report due to no progress 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, under Milestone 12.
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
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of peptide libraries (Optimize IVT protein-fragment
production, Develop IVT protocol for high-throughput production, Validate immunogenecity
of protein-fragments, Full scale production of protein-fragment library, Purification of proteinfragment library, Array protein-fragment into overlapping pools, Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
30 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Our electronic management system, GEMS, has designed and then instructed our robot to
normalize concentration of all oligos, pool forward and reverse gene specific primers, then identify
pairs designated for PCR-amplifying natural sequence ORFs from SCHU S4 genomic DNA. The
remaining primers will be used as part of the gene building protocol.
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
5%
9. Work plan for upcoming month
Production of wild type SCHU S4 ORFs will begin.
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


One full-deck print has been completed and we now have 32 Poly-L-Lysine slides and 15
Corning Ultragaps of the completed 1804 Schu S4 probe set and ribosomal controls. Both
microarray printers are functional. The Perkin Elmer Spotarray was sent to the factory for
refurbishing and returned within 21 days. Motion QC testing occurred over 4 straight days
with mock printing conditions. The Nanoprint had a redesigned sonicator system installed
and new water sensor and system verification testing is underway.
RNA from both LVS and SCHU S4 strains have been received from UNM and additional
hybridizations performed. These results have been combined with the previous data and
initial differential gene lists identified showing 61/1804 down-regulated comparing SCHU S4
to LVS and 26/1804 up-regulated comparing SCHU S4 to LVS grown in vitro.
31 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Up in SCHU
S4
Down in SCHU S4
Figure 1. Combined data (all substrates) looking for genes up or down regulated between LVS and
SCHU S4 > 3 fold and significant between replicate slides.


R:\GeneVac\FTU\Contract\Microarray\Milestones\33
Submitted PFGRC request for TIGR tularemia microarrays has been approved. Material
transfer agreements are in processing before slide delivery.
4. Significant decisions made or pending.
We will perform comparisons of hybridizations of serially diluted microbial RNA to determine the
amplification efficiency of the genome directed primers using the linear amplification of
prokaryotic transcript protocol.
5. Problems or concerns and strategies to address
Printer malfunctions addressed
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
70%
9. Work plan for upcoming month
a. Will start testing with GDPs with purified Schu S4 RNA in log serial dilutions to test the
amplification efficiency.
b. We will compare data from in-house produced slides to TIGR produced slides.
c. Order 70mer oligonucleotides to detect extra LVS gene probes and create array plate for
this gene set.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
32 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
None
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
a. Initial hybridization studies have been performed using standard slide-coverslip
hybridizations.
b. RNA has been isolated from normal mouse lung for dilutions studies of Schu S4 RNA
and GDP testing.
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
a. We will perform comparisons of hybridizations of microbial RNA diluted into RNA from
normal mouse lung on both Poly-L-Lysine and Corning Ultragaps before final substrate
decision.
b. We will be comparing automated hybridization chamber systems including our in house
Genomics Solutions GeneTac Hybridization system and an evaluation test of the
BioMicro Maui Hybridization system.
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
Date started: 03/01/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1.
a. Experiment Ftc34 study 2 (Notebook 94, in progress)
i. The purpose was to determine the RNA yield from freshly cultured LVS and
SCHU S4
ii. In Ftc34 study 1, the yield from both SCHU S4 and LVS was approximately
50 g/1010 bacteria. This surprisingly low yield may be attributed to fact that
33 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
we were using bacteria stored in RNAlater rather than freshly harvested
bacteria.
iii. For Ftc 34 study 2, LVS and SCHU S4 were grown on cystine heart agar
plates for 48 h before they were scraped from the plate and resuspended in
PBS to for RNA isolation.
iv. The concentration of bacteria in the suspension was roughly estimated by
extrapolating the OD600 to previously established growth curves. Based on
this estimation, a volume equivalent to 1010 bacteria was used for RNA
isolation. The suspension was also plated on cystine heart agar plates to
obtain a more accurate concentration
v. RNA was isolated using Qiagen RNeasy Midi kit
vi. The yield from the fresh culture is similar to that from bacteria stored in
RNAlater
b. Experiment Ftc35 (Notebook 94, in progress)
i. The purpose was to isolate RNA from the lungs of BALB/c mice infected with
SCHU S4. ASU will use this mixture of prokaryotic and eukaryotic RNAs,to
optimize their procedure to detect SCHU S4 gene expression in the presence
of a relative excess of mouse organ RNA
ii. BALB/c mice were infected i.n. with 500 SCHU S4
iii. 3 days after infection, 2 infected mice were killed to determine the bacterial
burden in the lungs and 5 infected mice were killed to harvest lungs for RNA
isolation
iv. There were 7 x 107 CFU SCHU S4 in the lungs 3 days after infection
v. Each lung was homogenized in 5 ml Tri-reagent (Molecular Research
Center) and RNA isolated immediately following manufacturers protocol
4. Significant decisions made or pending
a. ASU currently has enough mass of pure SCHU S4 and LVS RNA . More SCHU S4
and LVS RNA will be isolated for ASU, as needed in the future.
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
10%
9. Work plan for upcoming month
a. Analyze the quality of RNA isolated from infected mouse lung before shipping the
RNAs to ASU
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
34 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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.
The subcutaneous (SC) route of infection is the route by which Francisella strains are the least
virulent (suggesting that dissemination from this site may represent a significant barrier). Our
initial observation was that the uvrB mutant was attenuated by 2 logs when compared to U112.
1) In order to confirm that the uvrB mutant is attenuated and to evaluate whether this phenotype
is the same for all the nucleotide excision repair mutants, we repeated the SC LD50 study using
10-fold serial dilutions (ranging from ~1x107 to ~1x101 cfu) of U112, uvrA, uvrB, uvrA uvrB.
3 Balb/c mice per group were injected. In this study, we determined that the median lethal dose
of U112 was 2.1 x 105 cfu. This level of virulence is dramatically less than our first observation
(1.17 x 103). The second SC LD50 determination with U112 was confounded by the fact that
deaths of 1 animal per group were observed over a 4 log range making the calculated LD50
determination highly suspect. The median lethal dose of uvrB was 1.17 x 105 which is very
similar to the original finding of 1.28 x 105. The other nucleotide excision repair mutants had
similar virulence to uvrB: uvrA 2.43 x 105 and uvrA uvrB 3.48 x 104. This data suggests that
the uvrA, uvrB, uvrA uvrB mutants all have similar levels of virulence by the SC route, but it
is not clear whether they are attenuated when compared to the wild-type strain, because the
virulence of U112 was so highly variable. We will repeat the SC LD 50 of each strain to determine
whether the mutants are in fact attenuated when administered by this route.
IV LD50 (cfu)
IP LD50 (cfu)
SC LD50 (cfu)
SC LD50 (cfu)
U112
0.95
0.57
1.17 x 103
2.10 x 105
uvrA
27
0.82
ND
2.43 x 105
uvrB
8.1
0.2
1.28 x 105
1.17 x 105
uvrAuvrB
38
2.72
ND
3.48 x 104
Study #
AS06-112
AS06-090
AS07-014
AS07-025
2) In order to determine whether animals vaccinated with live Ft novicida were protected against
a lethal infection with wild-type Ft novicida, we challenged all surviving mice from the SC LD 50
experiment (AS07-014) with 100 x IP LD50 dose of U112 (~100cfu) 4 weeks after the initial
vaccination. All control (HBSS vaccinated) animals died within 3 days of lethal challenge. All
animals vaccinated with 10 or more cfu of U112 or uvrB survived. Deaths in vaccinated animals
were only observed in the lowest dose cohorts: 66% of the animals that were vaccinated with
~2.08 cfu of U112 died and 33% of the animals that were vaccinated with ~1.28 cfu of uvrB
died. At doses this low, it is likely that the reason the animals were not protected was that they
were not infected during the initial vaccination. These data demonstrate that SC vaccination with
live Ft novicida can protect against lethal IP Ft novicida challenge one month after vaccination.
35 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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) In order to determine whether the nucleotide excision repair (NER) mutants were attenuated
for growth in vivo, we performed a study to evaluate the growth rates of the mutant strains
compared to wild-type Ft novicida U112 in spleen, liver, and lungs of Balb/c mice infected IV.
Mice were injected IV with approximately 100 cfu (which represents approximately 10-100 x
LD50). Cohorts of 3 mice were sacrificed at 4, 24, 48, and 72 hours post infection. At each time
point, spleen, liver, and lungs were harvested and homogenized in HBSS. 100ul of each
homogenate were plated directly on CHAH plates and a series of tenfold dilutions were plated to
calculate the number of cfu per organ. Each symbol represents the mean of three organs and the
bars represent the standard error. While there were some significant differences in the number of
cfu at day 3, all Ft novicida NER mutant strains replicated at the same rate or more rapidly than
the wild-type strain. These data suggest that the NER machinery is not required for growth in
lungs livers or spleens.
AS07-034
Spleen
1.0×10
8
F.t.n.
F.t.n.
F.t.n.
F.t.n.
CFU/Organ
1.0×10 7
1.0×10 6
1.0×10 5
U112
uvrA
uvrB
uvrAuvrB
1.0×10 4
1.0×10 3
1.0×10 2
LOD
1.0×10 1
1.0×10
AS07-034
0
0 (4hr)
1
2
3
Day
Lungs
Liver
1.0×10
1.0×10
7
1.0×10
6
F.t.n.
F.t.n.
F.t.n.
F.t.n.
1.0×10 5
U112
uvrA
uvrB
uvrAuvrB
1.0×10 4
1.0×10 3
1.0×10 2
LOD
1.0×10 1
1.0×10 5
CFU/Organ
CFU/Organ
1.0×10 8
6
1.0×10 4
1.0×10 3
1.0×10 2
LO
1.0×10 1
1.0×10 0
1.0×10 0
0 (4hr)
1
2
3
Day
0 (4hr)
1
2
Day
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.
36 of 50
3
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
5. Problems or concerns and strategies to address
Abrogation of the NER pathway may result in a loss in virulence when administered by the
SC route, however this observation has yet to be reproduced. If this attenuation is
reproducible, it 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
90%
9. Work plan for upcoming month


SC LD50 with all Ft novicida strains will be repeated
If there is an attenuation by the SC route, we will monitor the growth of Ft novicida NER
mutants in lungs, livers, and spleens after SC infection 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
We have demonstrated that NER mutants of Ft novicida are two times more sensitive to
photochemical inactivation that wild type. We have optimized conditions for a 400 mL scale
inactivation process for the Ftn uvrB strain using 40M S-59 and 7J/cm2 UVA that resulted
in a vaccine that was completely inactivated and maintained metabolic activity for 12 hours.
We have demonstrated that KBMA Ftn uvrB are attenuated for virulence by up to 8 logs.
1) We have been monitoring the stability of various lots of KBMA Ft novicida uvrB by
measuring the degree of metabolic activity using the MTS assay after storage at –80o C in
10% sucrose. Lot 948-164 is the oldest lot of KBMA vaccine that was produced thus we
have the most data on stability: the metabolic activity after 1 month of storage was equal to or
greater than the initial metabolic activity, but there was only a minor decrease in metabolic
activity after 2 months of storage. This data suggests that 10% sucrose is an appropriate
cryopreservation agent for KBMA Ft novicida vaccines for at least 2 months, and we will
continue to monitor the metabolic activity of this lot on a monthly basis.
.
37 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
Lot #
[S-59]
UVA dose
Particles/ mL
cfu/mL
948-164
948-202 arm-1
948-202 arm-2
40M
40M
40M
6J/cm2
6J/cm2
7J/cm2
3.9 x 1010
5.4 x 1010
5.12 x 1010
1
91
0
Lot 948-164 (Nominal 1.5e8 particles/mL)
1.40
Pre-Freeze
t=0
t=1 Month
t= 2 Month
OD (490nm)
1.20
1.00
0.80
0.60
0.40
0.20
0.00
0
1
2
3
4
5
6
7
Time (hours)
8
9
10
11
12
NB968-050
The metabolic activity of the sterile lot (948-202 arm-2) was determined and compared to lot
948-164 (which was produced with 6 J/cm 2 UVA instead of 7 J/cm 2 and as a result had 1 cfu
per 3.9 x 1010 particles). These lots had similar degrees of metabolic activity.
400mL KBMA F. tularensis novicida delta uvrB (Nominal 1.5e8 particle/mL)
Lot 948-164 (40uM S-59, 6J/cm2 UVA) vs. Lot 948-202 Arm-2 (40uM S-59, 7J/cm2 UVA).
1.40
Lot 948-164, T=2 Month, Run date: 02-23-07
OD (490nm)
1.20
Lot 948-202 Arm-2, T=1 Month, Run date: 02-23-07"
1.00
0.80
0.60
0.40
0.20
0.00
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
NB968-050
4. Significant decisions made or pending
All NER 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/cm2 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 wild type strains suggests
that the wild-type is highly sensitive to photochemical inactivation under these conditions and
that the KBMA strategy is still viable.
38 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
60% of scientific work completed on the milestone
9. Work plan for upcoming month


Stability at -80o C of lot 948-202 arm-2 KBMA urvB Ft novicida will be monitored by
metabolic activity assays on a monthly basis and compared to lot 948-164.
The degree of attenuation via the IV route will be established using KBMA urvB Ft
novicida lot 948-202 arm-2.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
In Milestone 41 (study # ASO7-009) we demonstrated that KBMA F.t. novicida uvrB is
dramatically attenuated for virulence when administered by the SC, IP, or IV routes. One
month after a single vaccination, animals from this study were challenged with 100 x LD 50 of
wild type F.t. novicida to determine whether a single immunization with the KBMA vaccine is
protective.
AS07-009
HBSS
HK Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
KBMA Ftn uvrB
Vaccination
dose (particles)
1.00E+09
1.00E+09
1.00E+08
1.00E+09
1.00E+08
1.00E+09
1.00E+08
Route
IV
IP
IP
IP
IV
IV
SC
SC
Vaccination
survivors
5
5
2
5
0
2
5
5
100 x IP LD50
challenge survivors
0
5
2
3
0
2
3
0
Protection
0%
100%
100%
60%
100%
60%
0%
Mean time
to death
3d
NA
NA
4d
NA
NA
4.5d
4d
These results demonstrate that mice can be protected from a 100 x IP LD50 challenge of
Wild-type Ft novicida using a KBMA Ft novicida vaccine. The doses of KBMA vaccine that
were 100% protective were at or near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV).
Also to note, the heat-killed control administered at 1 x 109 IP was also 100% protective
(despite being avirulent). These data suggest that, by these routes of administration, an
unacceptably high dose of KBMA vaccine needs to be administered to confer protection after
a single vaccination. We will next try to increase the potency of the KBMA vaccine by
administering the vaccine 2 times.
4. Significant decisions made or pending
39 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
None
5. Problems or concerns and strategies to address
The KBMA vaccine was only 100% protective at very high doses. At these dose levels heat
killed bacteria were also protective. We will attempt to protect mice from a lethal challenge
using lower doses administered 2 times.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
5% of scientific work completed on the milestone
9. Work plan for upcoming month


We will vaccinate mice with a 0.1 x LD50 dose of KBMA urvB Ft novicida IV
administered 2 times, and challenge mice 1 month post-boost with 100 x IP LD50 dose of
live wild-type Ft novicida.
We will also determine whether CD4+, CD8+, or CD4+ and CD8+ T cells contribute to
this protection by doing antibody depletion studies.
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 Prepared TSA plates with individual components containing no salt and added 5% sucrose. In
addition, the plates contained Sodium Pyruvate, Sodium Metabisulfite, Ferrous Sulfate, Casamino
Acid and L- Cysteine at concentrations indicated in earlier report. LVS was inoculated onto these
plates, but did not grow.
3.2 I inoculated the Schu S4 strain on the LB+++ with 5% Sucrose, previously mentioned; the
result showed growth of the Schu S4 strain.
3.3 Since the LVS doesn’t seem to grow on TSA+++ without salt, we decided to prepare TSA+++
containing Salt and Kanamycin (final concentration 30 ug/ml), and added Sucrose to 10% final
40 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
concentration. We are hopeful that the increased sucrose concentration will compensate for the
small amount of salt contained in the TSA plates.
3.4 LVS with UvrB insertion was inoculated on these plates with 10% sucrose, and we did see
growth.
3.5 Data recorded on UTSA TVDC notebook #2, page 61 and 62.
4
Significant decisions made or pending
5
Problems or concerns and strategies to address
None.
Preparing a media that will support bacterial growth, but also support SacB removal of the plasmid
back bone in the presence of sucrose.
6
Deliverables completed
None.
7
Quality of performance
Good
8
Percentage completed
Approximate 51% of scientific work completed on the milestone
9
Work plan for upcoming month
i.
ii.
iii.
Patch the resulting colonies from 10% Sucrose plates (3.4) onto TSA+++ with
chloramphenicol and TSA+++ with Kanamycin to see if the plasmid backbone has been
lost.
We will order some Thayer-Martin, modified( MTMII) agar plates and Mueller Hinton
Chocolate agar plates from BD Biosciences. These plates will allow for growth of LVS and
we hope that if we add 10% liquid sucrose to these plates as a spread, we may be able to
get sucrose selection by this means.
Continue work with UvrA plasmid construction.
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
41 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
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 demonstrated that
LVS is nearly avirulent when administered by the SC route.
1) 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 three months at -80o C, both samples showed a
decrease in titer that was 2.5-3 fold less than the t=0 time point. It is not clear why both
samples appeared to increase in titer over the first to months and then drop after 3 months,
but this suggests that there is variability in the assay and that both cryopreservation solutions
appear to be performing equally well. If viability decreases significantly at the next time point,
then additional stabilizers will be explored.
8.40E+08
5.85E+09
4.47E+09
2.13E+09
4.47E+09
9.28E+08
4.89E+09
1.00E+09
Pre-Freeze
T=0
T=1 Month
T=2 Months
T=3 Months
9.43E+09
CFU/mL
1.00E+10
2.88E+09
1.00E+11
2.73E+09
Viability Count (CFU/mL) for Pre-Freeze, T=0, T=1, T=2 and T=3 Month
Stability Time Point
1.00E+08
1.00E+07
Lot: 948-119 Arm-1 (Freeze Buffer)
Lot: 948-119 Arm-2 (10% Sucrose)
NB968-029
DVC F. tualrensis holarctica LVS
The metabolic activity of the cryopreserved LVS was also evaluated after 3 months using the
MTS assay. There was no significant decrease in metabolic activity of either lot after 3
months. We will continue to monitor the stability of lot 948-119 LVS samples by plating for
cfu and by MTS assay.
Nominal 2.22e7 cfu/mL
1.00
T=0 Arm-1 (Freeze Buffer)
T=0 Arm-2 (10% Sucrose)
T=1 Month Arm-1 (Freeze Buffer)
T=1 Month Arm-2 (10% Sucrose)
T=2 Month Arm-1 (Freeze Buffer)
T=2 Month Arm-2 (10% Sucrose)
T=3 Month Arm-1 (Freeze Buffer)
T=3 Month Arm-2 (10% Sucrose)
0.90
0.80
OD (490nm)
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
0
2
4
6
Time (hours)
8
10
12
NB968-019
2) Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher
than NER mutants and we do not yet have a NER deficient LVS strain, we have decided to
look at whether a KBMA LVS vaccine can be protective. We have produced two 400mL lots
of photochemically treated LVS treated with either 10 or 20uM S-59 and 6 J/cm 2 UVA
(representing 2x or 4x the minimum concentration required for complete inactivation at a
42 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
small scale). There were no residual cfu in either lot. The metabolic activity of each lot was
compared by MTS assay and each was identical. These data demonstrate that we can
produce a KBMA LVS vaccine in the absence of a nucleotide excision repair mutation. We
will use these lots to vaccinate animals to determine whether they protected against a lethal
LVS challenge.
Lot#
968-040 Arm-1
968-040 Arm-2
[S-59]
10uM S-59
20uM S-59
UVA
6 J/cm2
6 J/cm2
Particles/mL
2.64 x 1010
2.60 x 1010
cfu
0
0
Nominal 1e8 particle/mL (KBMA) F. tularensis holarctica LVS
OD (490nm)
1.0
0.9
T=0 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
0.8
T=0 968-040 Arm-2 (20uM S-59, 6J/cm2 UVA)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0
1
2
3
4
5
6
7
Time (hours)
8
9
10
11
12
NB968-050
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.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
30% of scientific work completed on the milestone
9. Work plan for upcoming month

The stability of live LVS stored at -80oC in various cryopreservation agents will be
evaluated monthly by MTS assay and plating on CHAH plates.
43 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,




We will compare the virulence of live LVS stored in the various cryopreservation agents
by injecting IV into BALB/c mice.
We will measure the stability of KBMA LVS stored at -80oC in 10% sucrose
We will measure the reduction in virulence of KBMA LVS administered IV or IP compared
to Live LVS
We will use these lots (KBMA LVS) to vaccinate animals to determine whether they
protected against a lethal LVS challenge.
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
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. We are
utilizing a construct to delete the mglA gene as our model deletion construct, for two reasons:
first, there is a single copy of the mglA gene in the chromosome (unlike iglC, of which there are
two copies), thus the manipulations necessary to obtain a mutant are minimized; second, there is
a phenotypic difference of mglA mutant strains that can be determined on indicator media
containing X-P, because MglA controls an acid phosphatase, which makes screening potential
mutants easier.
I. Cloning of igLC:
a. Prepared the pools of 10 each from the KEK1090+iglC deletion fragment colonies that
resulted from the transformed DH5αλpir cells. Had only three total pools and 3 microliters from
each was used in a PCR to screen for the iglC deletion fragment. The results indicated all
were the plasmid with no iglC deletion inserts.
b. Transformed more DH5αλpir cells with the ligation reaction of KEK1090+iglC deletion
fragment to generate more colonies to screen for the iglC deletion fragment.
44 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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. Will evaluate results on next report. Data located in TVD UTSA Notebook 3, page 75-76.
II. Experiments to generate deletions in Schu4:
a. The plasmid KEK1133+MglA clones C7 and C8 were sent for sequencing and found to
be correct.
b. These constructs were used in a transformation reaction with Schu S4 strain to determine
whether the SacB gene will allow the plasmid backbone to be removed from the
chromosome once the deletion is inserted.
c. Schu S4 (40 ml) was grown to O.D. 600 of 0.625 in TSB +++ (described in earlier report),
these cells were washed twice with 0.5M Sucrose then resuspended final pellet with 400
ul 0.5 M Sucrose and split into two sterile microcentrifuge tubes.
d. Added 2.5 ug of plasmid DNA (C7 and C8, respectively) to the Sucrose treated Schu S4
cells and electroporated in 0.2 cm gap cuvette with setting of 2.5 KV at 400 Ohms.
e. Add cells to a TSA+++ plate and incubate at 37° C for 6 hours. Transfer these cells from
the non-selective plate onto TSA+++ containing 70 ug/ml Kanamycin final concentration.
f. Placed these kanamycin plates at 37° C for up to 6 days checking for grown on plates
everyday.
g. This experiment resulted in twelve initial colonies that were kanamycin resistant
indicating that the SCHU S4 was potentially successfully transformed with the MglA
construct (C7/C8). We isolated genomic DNA from each of these colonies and used
MglA specific oligos to screen for the integration of the plasmid with deletion. Eight out
of the 12 colonies clearly showed that the MglA plasmid (s) integrated into the
chromosome (See Figure 1 and 2).
Figure 1.
This figure shows the results of a PCR using oligos Mgl AB H3 and Mgl AB BHI (described in
an earlier report) which are specific to the Mgl A gene. Lane 3 is Schu S4 wild type (wt),
Lane 4 is KKF34 which is a U112 Mgl A deletion control which contains an erythromycin
insertion making this larger than the Wt profile, Lanes 5 and 6 are the plasmids used in the
experiment C7 and C8 pUCFnKanSacBΔMglA, respectively. Lanes 7-14 represents various
clones isolated from MglA transformation experiment (K1-K8). Four out of 8 isolates ( K1, K4,
K6 , K8 in lanes 7,10, 12, 14, respectively) clearly showed the deletion insertion as compared
45 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
to the vector control lanes 5,6 and the Wt control. The wt banding pattern remains because
the vector is still in the chromosome. Lane 2 is the PCR product resulting from iglC oligos
used on KEK906 which was Sal I digested then isolated from and agarose gel using the
Qiagen gel extraction kit described earlier.
Figure 2.
This figure represents results from a PCR using MglA gene specific oligos mentioned in
figure 1. Lane 2 is the Wt control, Lane 3 is KKF34, Lane 4 is C8 pUCFnKanSacBΔMglA
(KEK1133+ΔMglA C8). Lanes 5-8 represents various clones generated from the MglA
transformation experiment. Lane 9 and 10 are TSA+++ plate cycled clones Cyc3 K7 and
Cyc3 K9, respectively. Comparing the wt profile (ln2) along with the plasmid profile (ln 4)
Lanes 6-8 shows that the deletion plasmid integrated into the Schu S4 chromosome and
lanes 9 and 10 show that simply passaging on non-selective plates will not force the plasmid
out of the chromosome (not a surprise).
h. Also, these colonies were cycled onto nonselective plates for six passages at 37° C.
Prepared only two genomic isolates of K7 and K9 (just randomly picked) from the cycled
plated cells.
i. In addition, these 12 clones were passaged in liquid media (Chamberlains) at 30°C for
four passages then dilutions were made and plated onto TSA +++ with no salt containing
5% Sucrose. This resulted in more than 500 colonies per clone.
j. Some of these sucrose colonies were patched onto TSA +++ and TSA+++ containing
kanamycin. Kanamycin sensitive colonies (which presumably lost the plasmid) were
screened for retention of the MglA deletion in the chromosome. So far, of 100 colonies
total patched we have only 12 that are still Kanamycin resistant.
k. I will prepare MglA PCR screen for at least 7 picks from the sucrose Kanamycin sensitive
colonies resulting from each original clone (ie 7 from K2, 7 from K3, etc). Results will be
reported next month.
l. KEK1133 (C13) and KEK1134 (C26) which are the pUC constructs reported in previous
report that contain the SacB gene were cut with Sal I restriction endonuclease and then
alkaline phosphate (CIP) treated; this was isolated from an agarose gel using the Qiagen
extraction kit described earlier.
m. In addition, KEK906 plasmid containing the iglC deletion fragment was used as the
template to generate this iglC fragment to contain Sal I restriction sites at both the 5´ and
3´-ends. I used the Sal I oligos mentioned in last month’s report. The resulting PCR
product was phenol/chloroform extracted, ethanol precipitated and used in a Sal I
46 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
n.
o.
p.
q.
restriction endonuclease digestion reaction. The completed reaction was run on an
agarose gel and isolated using the Qiagen extraction kit. The isolated iglC Sal I deletion
PCR fragment was eluted with 40 ul Tris pH 8.5 and 2 ul of this was run on a agarose gel
to check integrity (See figure 1).
The plasmids KEK1133 and KEK1134 Sal I/CIP isolates from l. was used with the iglC
Sal I PCR isolate from m. to set up a ligation reaction at 16°C. Once the reaction was
complete this was chloroform/phenol extracted and ethanol precipitated then
reconstituted to 10 microliters with sterile water.
Transformation of DH5α cells was done with various ligation reactions from n. and this
resulted in about 40 colonies per vector construct (KEK1133 SalI/CIP + iglCSalI and
KEK1134 SalI/CIP + iglC SalI , respectively).
I will report the results of these colonies next month, we are looking for a clone containing
the iglC deletion construct in this mutagenesis vector
Data located in TVD UTSA Notebook 3, pages 58-63
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
40%
9. Work plan for upcoming month
a. Prepare the pool suspensions of the KEK1090 + iglC clones from new transformations
and run PCR with iglC oligos to find the correct iglC deletion construct.
b. Will evaluate any potential candidates from the PCR screen for the iglC constructs by
preparing plasmid from these clones and doing restriction analysis.
c. Will set up PCR reactions to screen for complete deletion of the MglA gene in Schu S4
from the transformation experiment (II. c-k. above) using the MglA specific oligos as well
as other oligo sets specific to the plasmid.
d. Will do mini plasmid preparations on the resulting 10 colonies from each of the
transformations with KEK1133 and KEK1134 containing the iglC deletion fragment.
e. Will screen for the correct iglC deletion construct by restriction endonuclease digestion
and PCR using iglC specific oligos.
f. Order more supplies as needed
10.Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
47 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
a. Evaluate the protective efficacy of the Ft subsp. novicida iglB mutant as a vaccine
candidate (Note book #4, page 77-78). Groups of BALB/c mice (female, 4-6 weeks) were
intranasally (i.n.) immunized with 104, 105, 106 or 107 CFU of the ΔiglB mutant. Mice treated
with PBS were used as a mock-control. The immunized mice were rested for 30 days and
challenged with 1000 CFU of F. novicida (~100 LD50) by the i.n. route. As shown in Fig. 1,
iglB-vaccinated 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 4.
100
104
105
106
107
PBS
% Survival
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14
% 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 ΔiglB immunization against F. novicida infection. BALB/c
mice were immunized intra-nasally with 4 doses (104, 105, 106, and 107 CFU) of ΔiglB
or PBS and i.n. challenged with lethal dose of F. novicida (1000CFU). Mice were
monitored for survival rate and weight change.
b. Analyze the antibody profiles of mice immunized with the Ft novicida iglB mutant after
vaccination (Note book #4, page 72-76). Blood was collected from the PBS- and ΔiglBimmunized mice (as described above in a) at day 14 and day 28 after priming. Specific antiΔiglB total antibody titer as well as IgG1 and IgG2a isotypes were determined by ELISA.
Antigens, either UV-irradiated ΔiglB (105/well) or HEL (Han 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, mice immunized with ΔiglB produced significant amounts of
specific serum antibody at day 14 after priming for all vaccination doses. The titers were
increased at day 28 after priming (2 days before bacterial challenge). Isotyping analyses
48 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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,
indicated both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were produced in mice after
the ΔiglB immunization. No ΔiglB- 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.
5
4
Total Ab
Day 14
Titer (x1000)
3
Day 28
2
1
0
0
5
5
IgG1
4
4
3
3
2
2
1
1
0
0
0
0
PBS 104
105
106
107
IgG2a
PBS 104
ΔiglB
105
106
107
ΔiglB
Fig. 2. Humoral response to ΔiglB immunization. BALB/c mice were intranasally
immunized with 104, 105, 106 or 107 CFU of the ΔiglB mutant or PBS alone as mock
vaccination. Sera were collected 2 weeks and 4 weeks after immunization and used
to determine titers of anti-ΔiglB specific antibody.
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
31% of scientific work completed on the milestone
9. Work plan for upcoming month
a. Analyze the cell mediated antigen-specific cytokine response of mice immunized with the
Ft novicida iglB mutant.
b. H&E analyses of lungs and spleens from ΔiglB immunized/ F. novicida challenged mice
(day 30).
10. Upcoming Contract Authorization (COA) for subcontractors
None
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
49 of 50
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2007 to 2/28/2007
Due Date: 3/15/2007 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. Chromosomal DNA was purified from the F. novicida uvrB mutant KKF71. 10 ug of this
DNA was cryotransformed into a F. novicida iglD mutant KKF37 in hopes of generating a
uvrB + iglD double mutant. Cryotransformants were plated on TSA++ Kan for initial
selection and then 6 colonies were further screened by colony PCR with the primers
UvrBUP and UvrBDn1. The resulting 3.5 kilobase PCR fragments were digested with
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 ,7 and 8 show two fragments
and thus are correct double mutants. Colony 1 (lane 3) was frozen away as KKF225.
Data described in Notebook 1, page 13. The strain KKF225 is a uvrB + iglD double
mutant of Ft. subsp. novicida.
Figure 1.
1.
2.
3.
4.
5.
6.
7.
8.
Ladder
Ft. subsp novicida
colony 1
colony 2
colony 3
colony 4
colony 5
colony 6
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
40%
9. Work plan for upcoming month
Create a uvrB + iglA double mutant
10. Anticipated travel
None.
11. Upcoming Contract Authorization
None
50 of 50