Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, 4, 5, 12/13 (UNM/LBERI), 19, 21, 26, 27, 28, 34 (UNM/ASU), 35, 41,
42, 44, 46, 49, 50, 52
Completed milestones: 1, 16, 25, 32, 33, 39, 40, 43 (UTSA), 48, 51
Inactive milestones: 6, 7, 8, 9,10, 11, 14, 15, 17, 18, 20, 22,23, 24, 29, 30, 36, 37, 38,
43 (Cerus), 45, 47, 53, 54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. The Fourth group of 6 LBERI participants were vaccinated on 11/13/07.
b. To date, 28 LBERI participants have been vaccinated.
c. Fifth group of 6 LBERI participants being scheduled to travel to Washington DC on 1/7/08
to be vaccinated on 1/8/08.
d. The UNM group of 6-7 participants are tentatively scheduled for vaccination on 3/18/08
e. UNM and USAMRIID are actively using the LVS vaccine web database to track Risk
Assessment form submission and acceptance, Informed Consent submission and
acceptance, Health screening appointments, planned dates for receipt of LVS
vaccinations, dates of medical clearance, and travel arrangements
4. Significant decisions made or pending
a. UNM and LBERI will use their biobubbles as additional physical protective equipment
b. The LBERI work stoppage has been lifted for SCHU S4 aerosols as of 11/7/07.
c. In approximately 1 month, UNM may have access to a local source of human cells from
LVS vaccinated individuals.
d. Dr. Lyons is requesting UNM IRB approval to allow blood draws on the vaccinated LBERI
and UNM scientists after their LVS vaccinations. The LBERI and UNM scientists and
staff will be offered the opportunity to volunteer to donate bloods for the development of
immunoassays, approximately 2 months after receiving the LVS vaccination.
e. UNM and LBERI are offering the LVS vaccinations to 24 more scientists to total 46;
USAMRIID will continue to provide the LVS vaccinations over the next 5-6 months.
5. Problems or concerns and strategies to address
a. Within approximately 1 month, UNM may have access to the blood of UNM and LBERI
scientists who have been vaccinated with LVS at USAMRIID. This is dependent on
UNM’s IRB approval
Page 1 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
6. Deliverables completed
22 LBERI scientists and staff have received the LVS vaccination between 9/11/07 and 10/23/07.
7. Quality of performance
Excellent
8. Percentage completed
40%
9. Work plan for the next month
a. Submit Health screening test results to USAMRIID and obtain USAMRIID medical review
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 1/8/08 (5th group) .Participants will be at USAMRIID for 2 days
following the vaccinations
c. Start Risk Assessments, SIP informed consent teleconference, and health screenings for
the UNM group of approximately 7 participants.
d. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
LVS vaccination participants will be traveling to USAMRIID on 1/7/08 and 3/17/08.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM received a signed COA letter for COA 15 on 9/11/07.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions

November 2007 bioaerosol optimization work focused on Aeromist nebulizer testing
using S4 Schu4 and confirmation of pathogenicity via aerosol delivery in mice:
i. One day of bioaerosol testing was completed incorporating dilutions of fresh (48h
culture) S4 Schu4 stock in the generator. One main objective was to determine if
fresh Schu4 behaved similarly to fresh LVS under similar bioaerosol testing
conditions.
1. 9 total sprays with fresh Schu4
a. 16NOV: 9 sprays conducted at 3 target concentrations (1x105,
1x106, and 1x107 cfu/mL) and a delivered pressure of 10 psi
2. Results (Figures 1 and 2)
a. 16NOV:

Actual vs. Target cfu/mL values were accurate prior to
the bioaerosol (i.e., pre-spray values were as
targeted). Post-spray values demonstrated an
approximate 0.5 log10 drop; this was an expected
result and was seen previously when testing LVS.
Page 2 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam

As with LVS, spray factor values indicated increased
efficiency when using post-spray versus pre-spray
generator suspension concentrations. This is due to
the decrease in viability observed in the bacterial
suspension following the bioaerosol run. Spray factor
values were lower (i.e., less efficient) as compared to
similar testing using frozen LVS. The reason for this is
unknown, but is discussed further below.
ii. One day of bioaerosol testing was completed incorporating dilutions of frozen S4
Stock stock using the Aeromist and Collison generators. Here, the Collison was
tested for comparison purposes.
1. 9 total sprays with frozen S4 Schu4
a. 7NOV:


6 sprays conducted using the Aeromist at 1 target
concentrations (1x106 cfu/mL) and two delivered
pressures (7.5 and 10 psi)
3 sprays conducted using the Collison at 1 target
concentration (1x106 cfu/mL) and a delivered pressure
of 25 psi.
2. Results (Figures 3 and 4)
a. 7NOV:

Target cfu/mL values were approximately 0.5log10
lower than desired in both generators. It was
hypothesized that this was due to a titer drop observed
in the frozen stock (i.e., the target was lower than
expected). Titers dropped slightly following the
bioaerosol, an observation consistent with LVS and
fresh Schu4, and no significant difference was seen
between the three setups (Aeromist at 7.5 and 10psi
and Collison at 25psi).

Spray factor values indicated that the Aeromist was
inefficient at a delivered pressure of 7.5psi and just as
efficient as the Collison at 10psi. However, similar to
the frozen stock testing, overall spray factors were low.
Though further testing is needed, our preliminary
conclusion is that S4 Schu4 is more fragile as a
bioaerosol when compared to LVS grown and tested
under similar conditions.
Page 3 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
3. Data filed in the following folders:
a. 7NOV: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06078_TUL-03\TUL-03\Aeromist nebulizer\7NOV07
b. 16NOV: \\Saturn\absl3\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY06078_TUL-03\TUL-03\Aeromist nebulizer\16NOV07
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Post)
11/16/2007 (Post)
11/16/2007 (Post)
Figure 1. Target vs. Actual CFU/mL at three target concentrations of fresh Schu4 using the Aeromist generator
Page 4 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Aeromist: Actual CFU/mL vs. Spray Factor (Fresh)
-6.00
4.00
-6.20
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-6.40
-6.60
-6.80
-7.00
-7.20
-7.40
-7.60
-7.80
-8.00
Actual CFU/mL (Log10)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Post)
11/16/2007 (Post)
11/16/2007 (Post)
Figure 2. Actual CFU/mL vs. Spray Factor at three target concentrations of fresh Schu4 using the Aeromist generator
Page 5 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Aeromist: Target vs. Actual CFU/mL (Frozen)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
4.00
4.50
5.00
5.50
6.00
6.50
Target CFU/ml (Log10)
11/7/2007 (Pre, 7.5psi)
11/7/2007 (Pre, 10psi)
11/7/2007 (Pre, Collison, 25psi)
11/7/2007 (Post, 7.5psi)
11/7/2007 (Post, 10psi)
11/7/2007 (Post, Collison, 25psi)
Figure 3. Target vs. Actual CFU/mL at one target concentration of frozen S4 Schu4 using the Aeromist generator
Page 6 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Aeromist: Actual CFU/mL vs. Spray Factor (Frozen)
-5.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Spray Factor (Log10)
-5.50
-6.00
-6.50
-7.00
-7.50
-8.00
Actual CFU/ml (Log10)
11/7/2007 (Pre, 7.5 psi)
11/7/2007 (Pre, 10psi)
11/7/2007 (Pre, Collison, 25psi)
11/7/2007 (Post, 7.5psi)
11/7/2007 (Post, 10psi)
11/7/2007 (Post, Collison, 25psi)
Figure 4. Target vs. Actual CFU/mL at one target concentration of frozen S4 Schu4 using the Aeromist generator
iii. Mouse exposures were conducted on 15NOV to assess and confirm the
infectivity, pathogenicity and lethality of LVS and S4 Schu4 in mice. Data were
summarized and presented to TVDC on 30NOV in report format. The conduct of
the study and results are summarized below:
1. Swiss Webster mice were exposed to either LVS or S4 Schu4 using the
nose-only exposure system in the ABSL-3 Facility
a. 48h fresh culture of LVS in Chamberlain’s broth was used to
prepare the challenge material for this organism.
b. Frozen Schu4 stock was diluted and used for challenge material
because of poor growth in Chamberlain’s broth at 48h. The
reason for the poor growth is unknown. It should be noted that
good growth was observed at 72h; it can be hypothesized that
Schu4 requires a longer incubation time than LVS.
2. Mice were exposed in two groups of 20 and observed for up to 10 days
post-challenge for morbidity and mortality.
3. LVS-exposed mice received a challenge dose of 10,200 CFU; Schu4exposed mice received a challenge dose of 1,580 CFU.
Page 7 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
4. Results demonstrated a higher pathogenicity/lethality with Schu4:
Effect of Aerosol Tularemia on Mouse Mortality
% Survival
95
75
LVS - M
55
LVS - F
Schu-4 M
35
Schu-4 F
15
-5
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
Study Day
Figure 5. Effect of aerosolized F. tularensis on mouse mortality.
a. 100% mortality was observed in the Schu4-challenged mice by
Study Day 5
b. Survivors were noted in the LVS group and survival time was
longer when compared to Schu4.
c.
All mice exhibited significant clinical signs of infection including
unkempt presentation, lethargy, extreme piloerection, and
hunched posture. Those animals challenged with LVS also
demonstrated rapid breathing and closed eyes.
d. Weight loss was significant in all exposed animals:
Page 8 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Effect of Aerosol Tularemia Challenge on Percent
Body Weight Change in Mice
% BW Change
0.0
-5.0 Day 3 - 0 Day 4 - 0 Day 6 - 0 Day 8 - 0 Day 10 0
-10.0
LVS M
-15.0
LVS F
-20.0
Schu-4 M
-25.0
Schu-4 F
-30.0
-35.0
Figure 6. Effect of aerosol F. tularensis challenge on percent body weight change in mice
5. It was concluded that the strains of LVS and S4 Schu4 maintained at
LBERI were pathogenic and lethal in a mouse aerosol model. This
indicates that NHP exposures may proceed once animal acclimation and
scheduling is complete. The procedures are currently underway.
6. Data filed in the following folder:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07-089 (TUL04)\15NOV07 Mouse exposure
4. Significant decisions made or pending
Bioaerosol data suggest that F. tularensis S4 Schu4 is not as stable (as an aerosol) as LVS
regardless of fresh broth or frozen culture use. Further refining is needed for Schu4 though we
are still confident in our choices of test generators (i.e., the Collison and Aeromist Nebulizers).
Our strains of LVS and Schu4 were lethal in a mouse aerosol challenge model; steps are being
taken to move forward with NHP exposures.
5. Problems or concerns and strategies to address
S4 Schu4 growth is unpredictable in Chamberlain’s broth; though it grows well, we cannot be
certain at this time whether it will grow at 48 or 72h. We are proceeding to perform bioaerosols
using material harvested from solid BCGA media. This stems from our experience with Y. pestis
in that we observed very stable cultures harvested from solid media.
6. Deliverables completed
None
7. Quality of performance
Good
Page 9 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
8. Percentage completed
95%
9. Work plan for upcoming month


4 or 5DEC: Schu4 bioaerosols using fresh Schu4 grown in Chamberlain’s and the
Collison
7DEC: Schu4 bioaerosols using culture harvested from solid BCGA and the Aeromist
nebulizer
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress, (2/1/08 target date of completion)
3. Work performed and progress including data and preliminary conclusions:
Planning and scheduling are currently underway to expose 2 naïve NHPs to S4 Schu4. Dates
are pending.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Discussions need to be conducted concerning the desired endpoints, tissue analyses, challenge
dose and other points.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
12.5%
9. Work plan for upcoming month
a. Planning on challenging 2 naïve NHPs in December (date TBD).
b. LVS-vaccinated NHPs will continue to be bled as a source of cells for Milestone 12/13;
however, no work is anticipated on these NHPs until they are challenged with aerosol
Schu4 sometime after January 2008.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
Page 10 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ptran2 (Notebook 112 pages 3-5)
i. The purpose of this experiment is to produce antibodies that can be used to
deplete CD4 and CD8 T cells from naïve and LVS-vaccinated rats.
ii. The hybridoma lines W3/23 (mouse anti rat-CD4) and OX-8 (mouse anti-rat
CD8) were obtained from European Collection of Cell Cultures (ECACC) and
TS2/18.1.1 (isotype control; mouse anti-human LFA-2) was obtained from
American Type Culture Collection (ATCC)
iii. Culture supernatants from all three hybridomas were tested for the presence
of antibodies by FACS analysis. Culture supernatants were incubated with
rat peripheral blood cells and antibody binding was detected with PEconjugated anti-mouse IgG1 antibodies
iv. We detected binding of W3/25 but not OX-8. It is possible that the
concentration OX-8 antibodies in the supernatant was below the level of
detection or that it no longer produces any antibody. Therefore, we will
culture the OX-8 hybridoma longer without refreshing the culture medium and
use a Centricon column to increase the antibody concentration.
v. We will also use an ELISA to determine whether the TS2/18.1.1 is producing
antibodies.
vi. When we have confirmed the hybridomas are producing antibodies, we will
ship them to Taconic Farms for ascites production
b. Experiment Ftc62 (Notebook 106, page 95)
i. The purpose was to prepared heat killed and formalin fixed SCHU S4 for use
in proliferation, Elispot, and Elisa
ii. SCHU S4 was cultured for 48 hours in Chamberlain’s broth and then either
heated at 70oC for 2 h or fixed with 3% neutral-buffered formalin
iii. The heat-killed SCHU S4 is at 3.1 x 109/ml. The exact number of formalin
fixed SCHU S4 was not determined
iv. To confirm that all bacterial were killed, 100 l from a total of 50 ml were
injected s.c. into 2 BALB/c mice. No mouse died after 7 days, suggesting
that all bacteria were killed by our procedure
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model almost completed – additional characterization underway
7. Quality of performance
Good
8. Percentage completed
68%
9. Work plan for upcoming month
a. Produce anti-CD4, anti-CD8 and isotype control antibodies
i. Confirm antibody production by OX-8 and TS2/18.1.1 by ELISA and FACS
analysis
ii. Ship hybridomas to Taconic for ascites production
iii. Vaccinate rats with LVS so they can be used to determine the role of CD4
and CD8 T cells in vaccine-induced protection when the ascites are available
Page 11 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
b. Determine whether passive immunization with convalescent sera will protect naïve
Fischer 344 rats from i.t. SCHU S4 challenge
i. Vaccinate rats for use as serum donors and as controls in challenge
experiments
c. Determine whether QD655-luc8 can be used to track pulmonary deliver
i. Determine whether QD655-luc8 and its substrate are toxic to SCHU S4
ii. Determine whether QD655-luc8 signal is indicative of the SCHU S4 location
iii. Determine whether co-administration of QD655-luc8 affects the virulence of
SCHU S4 in naïve Fischer 344 rats
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new work done because assays for the mouse and rat have been optimized and
we are applying these assays to measure response to in vitro translated proteins and
peptides provided by ASU in milestone 27
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
Good
8. Percentage completed
55%
9. Work plan for upcoming month
none
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
Page 12 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Milestone 12/13-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a.
Update on IFN detection by ELISPOT analysis
1.
2.
We have been attempting to optimize the ELISPOT assay that detects
IFN secretion; initial assays used only PBMCs from NHPs that had
been previously vaccinated with LVS;
More recent assays have included PBMCs from non-LVS vaccinated
NHPs; these assays have revealed that:
i. PBMCs from non-LVS vaccinated NHPs respond to FF LVS more
than HK LVS
ii. The response to FF LVS appears to dilute out in a non-linear
fashion when less cells are used suggesting possibly that a small
percentage of the cells are producing the majority of the IFN (see
Figure 1)
iii. Using less FF LVS also reduces the amount of IFN secreted by
PBMCs from non-LVS vaccinated NHPs but it still induces enough
to be significantly different than unstimulated PBMCs (see Figure
2)
iv. The amount of IFN secreted by PBMCs from non-vaccinated
NHPs is variable (see Figure 3)
Effect of Cell Concentration on IFN secretion by PBMCs
Cell Mean for IFNg Spots
180
160
140
120
Media
LVS hk Hi
LVS ff Hi
A
*
100
80
60
40
20
0
1
1.33
Concentration of Cells Plated (million/ml)
Page 13 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Cell Mean for IFNg Spots
40
35
30
*
25
20
15
*
B
Media
LVS hk Hi
LVS ff Hi
Day 195 post S.C.
LVS Vaccination
*
*
10
5
0
.33
.67
1
1.33
Cell Mean for IFNg Spots
Concentration of Cells Plated (million/ml)
100
90
80
70
60
50
40
30
20
10
0
Media
LVS hk Hi
LVS ff Hi
Day 237 post S.C.
LVS Vaccination
*
C
*
*
*
* *
.67
1
1.33
Concentration of Cells Plated (million/ml)
Figure 1: PBMCs from non-LVS vaccinated NHPs (A), or S.C. LVS vaccinated NHPs 195 (B) or 237 (C)
days post vaccination were plated at varying cell concentrations and stimulated with either HK or FF LVS
(1 x 105 cells/ml). * Significantly different than media at the same cell concentration. N = 2 – 4
NHPs/group.
Page 14 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Effect of LVS Concentration on IFN secretion by PBMCs
Cell Mean for IFNg Spots
350
*
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
300
250
200
*
150
100
*
*
*
50
0
1
1.33
Concentration of Cells Plated (Million/ml)
Figure 2: PBMCs from non-LVS vaccinated NHPs were plated at varying cell concentrations and
stimulated with either HK or FF LVS (Hi = 1 x 105 cells/ml; Mid = 0.25 x 105/ml). * Significantly different
than media at the same cell concentration. N = 3 NHPs/group.
IFN Secretion by Individual Non-LVS Vaccinated NHPs
Media
LVS hk Hi
LVS ff Hi
400
350
300
250
200
150
100
A05477
A04367
A04344
A04339
A04274
A04168
A03033
0
A03016
50
A02386
Cell Mean for IFNg Spots
450
Figure 3: PBMCs from non-LVS vaccinated NHPs were plated at 1.33 x 106 cells/ml and stimulated with
either HK or FF LVS (1 x 105 cells/ml).
3.
Data interpretation
i. Whereas no significant IFN secretion is detected from non-LVS
vaccinated NHPs if the PBMCs are plated below 1.33 x 106
cells/ml, significant LVS-specific IFN secretion is noted in s.c.
Page 15 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
vaccinated NHPs when PBMCs are plated as low as 0.67 – 1 x
106/ml. Note: PBMCs from I.D. vaccinated NHPs are not shown
due to their poor production of IFN
ii. Reducing the concentration of FF LVS used to stimulate PBMCs
reduces the amount of IFN detected, however not to the same
level as unstimulated PBMCs from non-vaccinated NHPs
iii. Non-vaccinated NHPs secrete IFN to varying degrees after HK or
FF LVS stimulation, but uniformly more after FF LVS stimulation
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 120707.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 120707.svd and TVDC 1 bound notebook:
TUL 15 (pps. 41 – 48, 50, 78 – 80, 119 – 20), TUL 17 (pps. 63 – 70, 112 – 116), TUL 19 (pps. 99 – 108),
TUL 22 (pps. 141 – 145) and TUL 23 (pps. 146 – 151).
b. Update on proliferative response of PBMCs
Cell Mean for RLU normal
1. We established the conditions such that PBMCs from LVS-vaccinated NHPs
showed a specific proliferative response to LVS above media
2. Recently, we have been screening more non-LVS vaccinated NHPs in the
proliferation assay
3. Summary results (Figure 4) show the response of 15 non-LVS vaccinated NHPs
(Day 0) compared to the 6 LVS-vaccinated NHPs
4. The response of individual non-vaccinated NHPs is shown in Figure 5
3.50E6
Media
3.00E6
LVS hk Hi
2.50E6
2.00E6
1.50E6
LVS hk Mid
LVS hk Lo
LVS ff Hi
LVS ff Mid
LVS ff Lo
1.00E6
5.00E5
0
Day 0
Day 7
Day 14
Day 21
Day 28
Figure 4: PBMCs from NHPs were plated at 1x 106/ml and stimulated with either HK or FF LVS at various
doses (Hi = 1 x 105 cells/ml; Mid = 0.25 x 105 cells/ml and Lo = 0.0625 x 105 cells/ml). Day 0 represents
non-vaccinated NHPs (n = 3 – 15) and days 7 – 28 represent both ID and SC-vaccinated NHPs (n = 6).
Page 16 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
3.00E6
Media
LVS hk Hi
LVS ff Hi
2.50E6
2.00E6
1.50E6
1.00E6
A05477
A04367
A04344
A04339
A04274
A04168
A03033
A03016
A02386
A00937
A00908
0
A00896
5.00E5
Figure 5: PBMCs from non-LVS vaccinated NHPs were plated at 1x 106/ml and stimulated with either HK
or FF LVS at 1 x 105 cells/ml.
5. Data Interpretation: Although our initial studies initiated in November 2006 with 6
naïve NHPs suggested that they had little proliferative response to LVS (only 3 shown in Figure 3,
A00896, A00908 and A00937), the response of the other 9 NHPs tested has been more varied
and responsive to LVS.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 120507.svd; N:My
Documents\Tularemia Contract\Statview Data\PBMC assay 120507.svd; TVDC 1 bound notebook: TUL
18 (pps. 81 – 91, 124 – 125); TUL 19 (pps. 99 – 108); TUL 21 (pps. 135 – 140) and TUL 22 (pps. 141 –
145); and TVDC binder 1(TUL 8 and TUL 9).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We need to decide which non-LVS vaccinated NHPs will be used to test Schu 4 pathogenicity
(scheduled for December) and which will be used to pair with LVS-vaccinated NHPs as nonvaccinated controls (late January or early February).
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
92% of scientific work has been completed
9. Work plan for upcoming month
1. Test whether lower concentrations of HK and FF LVS will reduce background LVS-stimulated
proliferation and IFNγ in PBMCs from non-vaccinated NHPs.
2. Test the sera of non-vaccinated NHPs for the presence of IgG anti-LVS activity.
3. Test PBMCs from ID and SC vaccinated NHPs on the same day to determine if there are
actual differences in IFNγ secretion using both HK and FF LVS as antigens.
Page 17 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
11. Anticipated travel
None
12. 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 5 (Notebook 106, pages 91-94, 96 and Notebook 115, pages
9-12)
i. The purpose of this experiment was to determine whether IFN inhibits F.
tularensis growth in human alveolar macrophages.
ii. Human alveolar macrophages were infected with LVS, SCHU S4 and a
second Biovar A strain, NMFTA1 at MOI = 1and 0.1
iii. Cells were treated with 15 ng/ml (10ED50) recombinant human IFN or left
untreated
iv. Bacterial burden were determined 1, 3, and 5 days after infection
v. MOI of 1 was too low for LVS but ideal for SCHU S4 and NMFTA1
vi. SCHU S4: On day 1, the bacterial load was clearly lower with IFN than
without. However, unlike previous experiments, there was almost no SCHU
S4 on day 3, even in the absence of IFN. The difference between this and
past experiments may be associated with donor variability or different media
used (DCMM in previous experiments and RPMI in this experiment). DCMM
is a defined medium for culturing dendritic cells
vii. NMFTA1: the number of NMFTA1 was lower with IFN than without in two of
three replicates on day 1. In the absence of IFN, the bacterial load more
than doubled by day 3 and extracellular bacteria was detected. IFN
completely eliminated both intracellular and extracellular NMFTA1
viii. These results suggest that IFN can suppress NMFTA1 and SCHU S4
growth in human alveolar macrophage cultures. We would like to repeat this
experiment to confirm the differences between SCHU S4 and NMFTA1 as
well as the differences in SCHU S4 growth in this and previous experiments
Page 18 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
9%
9. Work plan for upcoming month
a. Determine kinetics of bacterial proliferation in human alveolar macrophages after F
tularensis infection with and without recombinant IFN
b. Determine kinetics of bacterial proliferation in human monocyte derived macrophages
after F. tularensis infection with and without recombinant IFN
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30.15e (Notebook 101, pages 44-47) and Experiment Ftc30.15f
(Notebook 101, pages 48-51)
i. The purpose of these experiments was to repeat Ftc30.15b and Ftc30.15d
which showed that T cells from LVS vaccinated but not naïve mice induced
macrophage killing of SCHU S4.
Page 19 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
ii. In contrast to the earlier experiments, naïve and vaccinated splenocytes
reduced the SCHU S4 load to similar extent.
iii. These results suggest that the procedure developed for this assay is not
robust enough and will require troubleshooting.
iv. We do not have an explanation yet for these problems, but we are reviewing
the notebook to find inconsistencies in the way the experiments were
performed
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
a. Troubleshoot mouse macrophage killing assay
i. Review the notebook to find inconsistencies in the way the experiments were
performed
b. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
Page 20 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
1. Together with UNM we are exploring wheat germ and rabbit reticulocyte lysates as
alternative systems to the currently used E. coli IVT system. For this purpose we
redesigned and assembled ten linear expression cassettes for eukaryotic expression.
Each LEE carries 1 full-length or sub-gene ORF of ~500bp. Together the 10 ORFs
correspond to five complete FTU genes (groES, groEL, IglC, katG, Tul4) and two
irrelevant antigens (OVA and Calm), which currently are being amplified to generate
sufficient amounts for planned reactions. Templates will be shipped next week to UNM
for expression in wheat germ and rabbit reticulocyte IVT systems.
Figure 1. Assembled LEEs for eukaryotic expression of tularemia and control ORFs.
1 – OVA; 2 – FTU0721a (KatG); 3 – FTU0721b (KatG);; 4 – FTU0721c (KatG);; 5 –
FTU1696a (GroEL); 6 – FTU1696b (GroEL); 7 – Calm; 8 – FTU0901 (TUL4); 9 – FTU1712
(IglC2), 10 – FTU1695 (GroES)
Data location: R:\GeneVac\FTU\Contract\Proteome\Derek's data\DNA Gels\Wheat Germ
Amp final 12-4-07.jpg
B. Select and test IVT Protocols
1. As previously reported all protocols for HTP protein production in the E. coli based IVT
system have been tested and optimized. We anticipate that most of these protocols will
also be useful in the eukaryotic expression systems; however, this will be empirically
tested if a decision to switch to a eukaryotic system is made.
C. Select and test protocols for protein purification
1. We are continuing to test various protocols for removing an unknown component(s) from
E. coli extracts which cross-reacts with LVS. A set of IVT samples made without
templates have been filtered through membranes with 100kDa, 50kDa, 30kDa, 10kDa
and 3kDa cutoffs., these have been generated and shipped to UNM for evaluation. The
sample was an IVT reaction mixture without a template, which was filtered through
membranes with various pore sizes. These are the pore size characteristics provided by
the manufacturer. They define the size of dextrans stopped by the filters. Calculated
sizes for our IVT products are <45kDa. A 100kDa filter should completely stop whole
ribosomes and their subunits, but not smaller complexes or our polypeptides.
2. To investigate the possibility that E. coli chaperones are the source of cross-reactivity, we
sent UNM a protocol for removing chaperones from Ni-beads bound proteins using an
ATP wash step. Terry will try the protocol on the IVT proteins shipped to him earlier.
3. To eliminate the possibility of IVT template and LVS DNA cross-reacting, Terry will also
test effect of i) spiking irrelevant DNA and ii) DNAse treatment of the above samples on
cross-reactivity.
4. Significant decisions made or pending
We are waiting for results of the T-cell stimulation experiments to decide on expression
cassette format, IVT system and needs for depletion/purification.
5. Problems or concerns and strategies to address
Purification of IVT products is associated with significant losses and results in variable purity
of polypeptide samples.
Page 21 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Switching to a eukaryotic IVT system will increase cost. Therefore, resolving the crossreactivity problem in E. coli via removal/depletion steps is preferable. Feasibility evaluation is
in progress.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
99%
9. Work plan for upcoming month
Depending on the results of the ongoing depletion experiment (UNM) we will test a polyclonal
E. coli IgG-based depletion step, and follow through on other assay validation experiments.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc59 study 6 (Notebook 115, pages 1-8)
i. The purpose of these experiments was to determine whether the
crossreactive material in the E. coli lysate can be removed by 1) washing or
2) size exclusion.
ii. To remove the crossreactive materials by washing, a mixture of ivt proteins
were bound via their His tag to BcMag.His beads from BioClone, washed 3
times with 1%FBS/PBS, and then the bead-bound protein were used to
stimulate T cells in IL-2 and IFN Elispot assays
iii. To remove the crossreactive materials by size exclusion, the E. coli lysate
mix without DNA template was fractionated with an 80kD cutoff filter. The
retained fraction and the eluate were both used to stimulate T cells in the
IFN and IL-2 Elispot assays
iv. As shown in Fig 2, the bead-bound proteins stimulated IL-2 and IFN
secretion, but we were unable to remove the crossreactive material by
washing. In this experiment, the “ivt protein mix” is a mixture of 4 different ivt
proteins in E coli lysate before they were denatured, bound to the beads and
washed. This would show the crossreactivity before any manipulation. The
“ivt protein mix – bead bound” is mixture after denaturation, binding and
washing. UNM was expecting that the washing would remove most if not all
of the crossreactive material.
v.
Page 22 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Figure 2. Failure to remove crossreactive materials in E. coli lysate by washing. A
mixture of His-tagged ivt proteins were bound to His beads, washed with 1%FBS/PBS
and then tested for stimulation of IL-2 and IFN secretion
Figure 3. Failure to eliminate crossreactive material in E. coli lysate by size exclusion. E.
coli lysate was fractioned with a 100 kD cutoff filter and the fractions used to stimulate IL2 and IFN production. If the crossreactive material is a ribosome, we expected no IL-2 or
IFN response in the “EC filtrate” 
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
a. LVS vaccinated T cells cross react with proteins in the E. coli ivt reactions.
Page 23 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
i. ASU will fractionate the E. coli lysate with smaller cutoffs to remove the
crossreactive proteins
ii. We also hypothesized that the crossreactive material is a chaperone
protein(s), which are very immunogenic. We will determine whether stripping
the chaperone with ATP would reduce the background
iii. Another possibility is that the crossreactivity against the DNA used as
template in the ivt reaction. We will spike in irrelevant DNA into the ivt
reaction buffer to determine if it increases crossreactivity. If so, we will treat
the ivt reactions with DNase before using them in the Elispot assays 
iv. We also do not know the sensitivity of the Elispot assay. With this
information, we can determine whether alternative ivt systems such as wheat
germ and rabbit reticulocyte lysate systems, which are less efficient than the
E. coli lysate, may be used. Therefore, we will immunize mice with DNA
encoding F. tularensis proteins rather than whole bacteria to avoid
immunizing the mice with proteins that might be crossreactive to the E. coli
lysate and to have vaccinated mice with known specificities to F. tularensis
proteins.
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
13%; no change because we have not resolved the problem with crossreactivity
9. Work plan for upcoming month
See discussion in the section “Problems or concerns and strategies to address”
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of peptide libraries (Optimize IVT protein-fragment
production, Develop IVT protocol for high-throughput production, Validate immunogenecity
of protein-fragments, Full scale production of protein-fragment library, Purification of proteinfragment library, Array protein-fragment into overlapping pools, Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions


All activities related to the ORF library production are on hold. We are waiting for the final
decision on the system, yield, format, pooling, and purification.
To assist UNM with optimization immuno detection protocols we assembled nine genetic
Page 24 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam

immunization constructs for mouse immunizations against five FTU proteins, groES, groEL,
IglC, katG, Tul4 and chicken ovalbumin (OVA). Animals were primed on Nov 28 and will be
boosted on Dec 19.
The corresponding set of genes was also cloned into a prokaryotic expression vector. To
date all clones have been confirmed. Protein production for a protein boost is in progress.
We are planning to ship the purified proteins to UNM during the first week of January.
4. Significant decisions made or pending.
Decision on the design of the expression cassette, type of IVT system, and needs for depletion or
purification are pending. A number of alternatives have been prepared. These will be instituted as
soon as that T cell assay is defined.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
28%
9. Work plan for upcoming month
Make and ship to UNM bullets for the boost. Continue to work on protein production and
purification for the protein boost of genetically immunized mice.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34-UNM
Milestone description: Pilot Studies for the optimization of RNA isolation and hybridization
conditions
Institution: UNM
1. Date started: 03/01/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
None
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
14%
9. Work plan for upcoming month
UNM will infect BALB/c mice i.n. with 101 to 107 SCHU and isolate RNA 4 hours after
infection. ASU will use these RNA to measure the sensitivity of their microarray analyses
10. Anticipated travel
None
Page 25 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Table 1 was previously shown in the last progress report and represents the hybridization of
select PCR-amplified targets to its designed probe on either the ASU or TIGR array. 10
random subgene fragments were amplified labeled and hybridized to each array. On the
ASU array, two of the probes did not selectively bind to their cognate probe (713B and 897A).

FTU0242A
FTU0457A
FTU0580A
FTU0713B
FTU0890A
FTU0897A
FTU0988A
FTU1295B
FTU1365B
FTU1658A
Exp 1
ASU Array
Y
Y
Y
N
Y
N
Y
Y
Y
Y
Exp 2
ASU Array
TIGR
Y
N
Y
N
Y
Y
N
N
Table 1. Hybridization of indirectly labeled PCR products of known genes to ASU and TIGR arrays.
We performed bioinformatic alignment analyses to determine if the probes should detect the
fragment. For both the 713B and 897A fragment, the probe did not align to the amplified fragment. The
713 fragment aligned with the 713A fragment and not the 713B fragment. The 897 probe did not align
with the 897A fragment but did align with the 897B fragment. Thus, these probes should not have been
hybridized by the fragment that was amplified. This observation serves as negative control to show that
the probes do not nonspecifically crosshybridize.


Notebook/File locations …, Notebook 404, page 117-120.
Electronic file locations…
R:\GeneVac\FTU\Contract\Microarray\Milestones\34\Testing Arrays
4. Significant decisions made or pending.
No additional studies will be done to compare the TIGR arrays
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
Page 26 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
7. Quality of performance
Good
8. Percentage completed
92%
9. Work plan for upcoming month


Perform QPCR verification of to establish the QPRC assay for verification of microarray
results.
Continue studies to establish the optimum conditions for genomic normalization of
microarrays using genomic SCHU S4 RNA
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent Schu 4
infection & RT PCR confirmation of candidates.
Institution: UNM/ASU-Johnston
1. Date started: 06-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Using the data from the initial studies of mouse lungs harvested after a dose response
challenge of SCHU S4 (103-107 organisms), we performed a bioinformatic comparison of
biologically pooled data versus averaged data from individual samples. The results are
shown as heat-map comparisons of the biologically pooled samples to the individually run
samples (Figure 1). The color coding of the heatmap was performed in two ways. On the
right side of the figure the colormap was derived from the individual dataset and the left panel
was derived from the biologically pooled dataset. In both instances the derived color map
was then projected on the other dataset. The results show that both heatmap process project
from one dataset to the other with a high degree of similarity indicating that biological pooling
is acceptable. Biological pooling will become advantageous when the quantity of Ft RNA is
low in the animal organs and biological pooling will be advantageous for assay sensitivity.
This was further dissected by performing spearman correlations between the biologically
pooled and bioinformatically pooled data (Figure 2). The two extremes of the data
comparisons show that with the lowest challenge dose (left panel, 10 3 organisms) had a
reasonable correlation of 0.58. This correlation increased to a maximum of 0.84 in the
animals challenged with 107 organisms (right panel).
Page 27 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Figure 1. Heatmap of biologically pooled and bioinformatically pooled data of Francisella tularensis
SCHU S4 RNA amplified from mouse lungs.
Page 28 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Figure 2. Spearman correlation graphs of data from biologically pooled data to bioinformatically averaged
to biologically pooled data from the dose response challenge.


Notebook/File locations … Notebook 514, pages 67-81;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\35\LAPT-11LAPT-12
4. Significant decisions made or pending.
There may be increased power, stability and sensitivity utilizing biologically pooled samples of
RNA over processing of individual RNAs.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
7.5%
9. Work plan for upcoming month
Initial studies will be performed to repeat the early dose response studies and include a lower
dose down to 10 organisms per animal to validate the dose response detection limit. Work with
UNM to establish a continuous sample stream for this milestone.
10. Anticipated travel
None
Page 29 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: We have determined that all the NER-deficient strains of Ft. novicida are only
slightly more sensitive to photochemical inactivation than wild type Ft. novicida. We have
optimized photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and
produced a lot of KBMA uvrB Ft. novicida for potency testing in MS42. We have
demonstrated that KBMA Ft. novicida are highly attenuated for virulence. Frozen KBMA
uvrB Ft. novicida maintain metabolic activity at –80oC for at least 3 months. Inactivated
NER-deficient strains have a similar degree of metabolic activity as the wild-type Ft. novicida
strain (which is different than has been seen with L. monocytogenes or B. anthracis), and we
have demonstrated that this lack of sensitivity to DNA damage is universal to numerous DNA
damaging agents.
1) This month, the vaccines research program was “spun out” of Cerus Corporation. All of the
personnel, intellectual property and other assets related to immunotherapy were transferred
from Cerus to Anza Therapeutics Inc. We had a conference call with NIAID and UNM to
discuss possible mechanisms for continuing with the TVDC contract going forward. A
number of possibilities were discussed, but the most expedient mechanism that was
recommended by NIAID was for Anza to work with cerus as a subcontractor on the TVDC.
There was then a meeting with Cerus' financial and legal teams who conveyed willingness to
maintain the NIAID subcontract with UNM while farming out the work to Anza as another
subcontract. This would theoretically maintain Cerus established indirect rate.
4. Significant decisions made or pending
All NER mutants (uvrA, uvrB, and uvrA uvrB) of Ft. novicida were equally sensitive to S-59
and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 7J/cm 2 as the
conditions for making 400ml-scale KBMA lots, and have produced a lot of KBMA uvrB Ft
novicida vaccine that is sterile for further characterization. We have decided to open MS 42 in
order to determine whether KBMA Ft novicida can protect against a lethal wild-type Ft novicida
challenge.
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms. This
appears to hold true for other methods of induced DNA damage. One possible explanation
for this is that there is a redundant DNA repair mechanism functioning in Ft novicida that may
limit the sensitivity of the NER-deficient mutants to DNA damage and thereby limit the
metabolic activity and potency of KBMA Ft novicida. If there is a redundant repair
mechanism, we may not be able to produce a highly potent KBMA vaccine utilizing
Page 30 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Francisella species as a platform. A new concern is that Cerus may no longer have enough
human resources to complete this milestone in a timely manner.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
fair progress
8. Percentage completed
85% of scientific work completed on the milestone
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: KBMA Ft novicida uvrB vaccine stocks produced in MS41 have been tested in
mice for virulence and protection against a 100 x IP LD50 challenge of Wild-type Ft novicida.
KBMA Ft novicida uvrB were 100% protective when a single dose was administered at or
near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also
achieved by administration of 1 x 107 KBMA particles IV when the vaccine was given twice
separated by 3 weeks. Depletion of CD4+ T cells prior to the challenge decreased the
survival rate to 80%, depletion of C8+ T cells had no effect, and depletion of both cell
populations resulted in 90% survival. Together, these data demonstrated that CD4 T cells
contribute to a protective immune response in a non-CD8 T cell-dependent manner. These
data suggest that the CD4 T cells may be boosting humoral immunity by stimulating B cells.
This interpretation was supported by an adoptive transfer experiment in which only the hightiter serum from CD8-depleted animals provided any protection against a lethal U112
challenge. Together these data demonstrate that the protection we see after vaccination with
KBMA Ft novicida uvrB correlates with humoral immune responses and explains why the
KBMA vaccine does not perform better than heat killed vaccine. This also makes it nearly
impossible to rank attenuated Ft novicida mutants by their ability to protect mice against a
lethal challenge. We instead plan to evaluate the ability of KBMA vaccines to induce a potent
CD8 T-cell response to an introduced ovablumin epitope tag and are awaiting the
construction of this strain from UTSA.
1) This month, no new progress was achieved on this milestone as we work towards modification
of the milestones. However, the vaccines research program was “spun out” of Cerus
Corporation. All of the personnel, intellectual property and other assets related to immunotherapy
were transferred from Cerus to Anza Therapeutics Inc. We had a conference call with NIAID and
UNM to discuss possible mechanisms for continuing with the TVDC contract going forward. A
number of possibilities were discussed, but the most expedient mechanism that was
Page 31 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
recommended by NIAID was for Anza to work with cerus as a subcontractor on the TVDC. There
was then a meeting with Cerus' financial and legal teams who conveyed willingness to maintain
the NIAID subcontract with UNM while farming out the work to Anza as another subcontract. This
would theoretically maintain Cerus established indirect rate.
4. Significant decisions made or pending
We have decided to evaluate the potency of the KBMA Ft novicida vaccine by measuring the
CD8 T cell response to an ovalbumin epitope tag.
5. Problems or concerns and strategies to address
Because humoral immunity plays a significant role in protection of mice against a lethal Ft
novicida challenge it is essentially impossible to rank KBMA vaccine candidates that elicit a
potent T cell response using survival after a lethal Ft novicida challenge in MS 43. We have
requested that Karl Klose construct an ovalbumin epitope-fusion protein to facilitate screening
strains of Ft novicida for their ability to elicit a T cell response to this well-defined epitope.
6. Deliverables completed
None
7. Quality of performance
fair progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary: using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. The uvrB LVS was also not more sensitive to DNA
damaging agents compared to WT. This suggests that there may be redundant DNA repair
mechanisms in LVS that may be functioning to repair photochemically induced crosslinks.
1) This month, no new progress was achieved on this milestone as we work towards modification
of the milestones.
4. Significant decisions made or pending
none
5. Problems or concerns and strategies to address
The uvrB mutant of LVS does not appear to be more sensitive to DNA damage induced by
photochemical inactivation with S-59 and UVA or by other chemical means. This suggests that
the potency of a KBMA uvrB LVS vaccine is likely to be the same as KBMA Wt LVS which failed
to protect mice against lethal a schuS4 challenge (see MS46). These results suggest that we
Page 32 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
reevaluate the KBMA tularemia vaccine strategy and we suggest comparing the efficacy of a
KBMA LVS vaccine to a KBMA Listeria monocytogenes vaccine that expresses Ft antigens.
6. Deliverables completed
none
7. Quality of performance
fair
8. Percentage completed
5%
9. Work plan for upcoming month
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
none
11. Upcoming Contract Authorization (COA) for subcontractors
none
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA
for initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS
and we have demonstrated that the metabolic activity of this lot is stable for 3 months. We
have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log
attenuation compared with live LVS. We have demonstrated that doses of KBMA WT LVS as
low as 1 x107 provide protection against 100 x IP LD50 challenge of live LVS. However, none
of the mice vaccinated with the equivalent doses of HK LVS died either. This is consistent
with protection against an LVS challenge being largely humoral. b We recently attempted to
measure the T-cell response to a CD4 Tul4 epitope in mice vaccinated with live or KBMA
LVS by intracellular interferon-gamma (IFN-) cytokine staining (ICS) or ELISpot assay, but
were unable to detect an induced response to this epitope. This may be because this epitope
does not bind the MHC molecule with high affinity, or the T cell response elicited by LVS may
actively suppress T cell responses. We recently demonstrated that LVS does not induce IL-6
Page 33 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
or MCP-1which are critical hallmarks of a protective inflammatory response. Furthermore,
co-vaccination with LVS decreased the innate inflammatory response to Lm. Administration
of LVS decreased the ability of the elicited T cells to produce the cytokine IL-2. Terry Wu at
UNM completed a protection study with KBMA WT LVS in which neither a (IV or IN) prime
nor a prime and boost (separated by 3 weeks) vaccination regimen with KBMA WT LVS
protected against a lethal SchuS4 challenge in mice. KBMA WT LVS vaccine appears to be
less potent than live attenuated LVS.
1) This month, no new progress was achieved on this milestone as we work towards
modification of the milestones. However, the vaccines research program was “spun out” of
Cerus Corporation. All of the personnel, intellectual property and other assets related to
immunotherapy were transferred from Cerus to Anza Therapeutics Inc. We had a conference
call with NIAID and UNM to discuss possible mechanisms for continuing with the TVDC
contract going forward. A number of possibilities were discussed, but the most expedient
mechanism that was recommended by NIAID was for Anza to work with cerus as a
subcontractor on the TVDC. There was then a meeting with Cerus' financial and legal teams
who conveyed willingness to maintain the NIAID subcontract with UNM while farming out the
work to Anza as another subcontract. This would theoretically maintain Cerus established
indirect rate.
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been investigating the efficacy of a wild-type KBMA LVS vaccine. Now
that we have received the uvrB mutant we will focus on producing a lot of KBMA uvrB LVS
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS against a lethal LVS challenge is independent of
metabolic activity. This suggests that comparison of various routes, regimens, or formulations will
be difficult to optimize by protective efficacy. The SchuS4 challenge model in mice is more
stringent, but KBMA LVS failed to protect after two doses. It is possible that the rat model may
allow a higher degree of sensitivity. The suppression of the innate inflammatory response and
the suppression of CD4 T cell cytokine production may potentially indicate that LVS is not a
potent inducer of protective T cell responses. We would like to screen for T-cell responses using
the peptides generated by ASU as an alternative method for optimization of vaccine potency or
construct an overlapping peptide library for IglC.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
53% of scientific work completed on the milestone
9. Work plan for upcoming months
We will work to generate a modified set of milestones that are scientifically appropriate and
achievable.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 34 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHU S4)
(iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis
subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis
subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful
deletions. Therefore, our focus is two fold, one is cloning experiments to get our target deletions
into vectors that we can use in creating these deletions and experiments with SCHU S4 itself
using constructs that we believe will allow us to make deletions into SCHU S4.
I. Cloning:
a. Using the newly developed Tulatron system, work has been performed to develop a vgrG mutant
in the highly virulent Francisella tularensis subsp. tularensis strain Schu4. Continued cloning was
performed to achieve a plasmid that contains an intron targeted to vgrG. As described in
previous month’s report, two potential sites(30/31 and 81/82) within vgrG have been chosen in
hopes of generating this mutant. Both products amplified and were gel purified. These two
retargeted intron products were cut with XhoI and BsrGI and ligated into pKEK1140 cut with the
same enzymes. These cut DNA products were ligated and transformed into E.coli DH5.
Transformants were plated on LB + Kanamycin. ~80 colonies were obtained by this
transformation and minipreps were made from each set of potential constructs.
b. To screen for potential clones, the restriction enzyme Bgl2 was chosen. The parent plasmid,
pKEK1140, when cut with Bgl2 will give 3 bands. Clones that are cut with Bgl2 containing the
retargeted intron to vgrG, will yield 3 bands, with the highest band shifting to a smaller size than
the parent. 5 clones of each potential insertion site (30/31 and 81/82) were screened for this shift
in size (figure 1). All 10 clones appeared identical, clone’s 1 of both 30/31 and 81/82 were rerun
for longer time on a gel to better visualize the shift in size of the upper band (figure 2). Both
potential clones show a shift in size compared to the parent. The vgrG 30/31 clone was frozen
away as pKEK1161 and the vgrG 81/82 clone was frozen away as pKEK1162. Future work will
be done to transform these plasmids into Schu4, in hopes of generating a vgrG mutant. Data
stored in TVDC UTSA Notebook #1, pages 23-24.
Page 35 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Figure 1.
1
2
3
4
5
Lane 1. ladder
Lane 2. pKEK1140
Lane 3. clone 1 (30/31)
Lane 4. clone 2 (30/31)
Lane 5. clone 3 (30/31)
Lane 6. clone 4 (30/31)
Lane 7. clone 5 (30/31)
6
7
8
9
10 11 12 13
Lane 8. pKEK1140
Lane 9. clone 1 (81/82)
Lane 10. clone 2 (81/82)
Lane 11. clone 3 (81/82)
Lane 12. clone 4 (81/82)
Lane 13. clone 5 (81/82)
Figure 2.
1
2
3
4
5
6
Lane 1. ladder
Lane 2. pKEK1140
Lane 3. clone 1 (30/31)
Lane 4. ladder
Lane 5. pKEK1140
Lane 6. clone 1 (81/82)
c.
The igLD cloning into KEK1140 did not yield any correct clones with both the 30/31a or 255/256a
“intron products” from the ligation described in previous report. Each component in the cloning
procedure will be created new and will try again. That is, will prepare a new purified plasmid
preparation of the vector KEK1140, will prepare a new oligo preparation mixture of each of the
oligo sets (30/31a and 255/256a, respectively) and then do the polymerase chain reactions
following appropriate digestions to do this cloning. Results will be reported on next report. Data
located in TVD UTSA Notebook 5, page 86 and 90.
d. In order to delete an entire pathogenicity island (FPI) from Schu 4 we need a construct of pdpD
containing a flip recombinase transferase. This cloning was last reported in July. The first three
clones isolated from the first ligation reaction were not correct. However, another preparation of
the vector and 5’-end pdpD PCR were made and these were digested, purified and ligated as
described before. This ligation resulted in hundreds of transformants and the re-ligation control
(vector only) had about 60 clones. Ten clones from the pKEK1042 + 5’ pdpD PCR ligation were
screened by digesting with BamHI and Not I restriction endonucleases. Most of the clones
looked correct. Took 4 representative clones and prepared larger plasmid preparations and
digested with the same enzymes mentioned and again the plasmid seem to have the correct size
insert. (1000 bp -- see figure 3.) Data located in TVD UTSA Notebook 5, page 87-89.
Page 36 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
Figure 3.
Legend:
BamHI / Not I Digested
1. 1 Kb Ladder (Invitrogen)
1 Kb
1
2
3
4
5
6
7
8
2. Uncut pKEK1042
3. pKEK1042
9.0
1.0
4. C1 pKEK1042+5’pdpD
5. C2 pKEK1042+5’pdpD
6. C4 pKEK1042+5’pdpD
7. C9 pKEK1042+5’pdpD
8. Uncut C2 pKEK1042+5’pdpD
This represents mostly pKEK1042 + 5’ end pdpD plasmids digested with BamHI and Not I restriction
endonucleases (a double digestion). These were the enzymes used to clone this pdpD PCR product
into the vector. The correct construct should yield a 1000 bp fragment as well as the pKEK1042
portion of the plasmid. Lanes 3 thru 7 are digested plasmids. Lanes 2 and 8 are uncut plasmid
profiles of plasmid and C2 pKEK1042+5’pdpD, respectively. Lane 3 is the negative control plasmid
only. C1, C2 and C4 look correct compared with the parent plasmid. Data located in TVD UTSA
Notebook 5, page 91.
II. Experiments to generate deletions in Schu4:
a. We had no new constructs to perform any work with Schu4 from this past month.
b. We are awaiting all data from the sequencing reactions to be returned to complete analysis of the
pdpA and the igLC clones made earlier.
c.
Various events regarding the BSL3 lab were addressed this past month.
i.
The repaired -85 C was returned from the contracted company and seems to be
keeping temperature will be moving this into BSL3 this week.
ii.
Had to participate in a mandated drill involving the BSL3 lab and emergency
procedures which also included our local fire department emergency response
team.
d.
Did some ordering for enzymes and general supplies for ongoing experiments.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
54%
9. Work plan for upcoming month
a.
b.
c.
d.
Will continue analysis of sequence data received from PCR igLC and pdpA DNA samples
Will sequence the vgrG clone and start transformation experiments with Schu S4.
Will continue with the igLD cloning into vector KEK1140
Will continue verification of the 5’end pdpD construct by restriction analysis and send clone
for sequencing
e. Order supplies as required.
10.Anticipated travel
None
Page 37 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
a. Determine the LD50 of Ft subsp. novicida uvrBpdpD double mutant. (Note book #4 page
136-137): Groups of BALB/c mice (female, 4-6 weeks) were intranasally (i.n.) challenged
with 105, 106 or 107 CFU of ΔuvrBpdpD. As shown in Fig. 1, mice infected with 105
CFU of the bacteria exhibited 80% survival, while animals infected with 106 CFU and 107
CFU maintained a 40% survival rate respectively.
The LD50 of ΔuvrBpdpD in the intranasal infection model (BALB/c mice) was determined
to be between 105 and 106 CFU. Significant weight loss was observed in groups of mice
infected with higher doses of ΔuvrBpdpD (106 and 107 CFU). Mice infected with 105
CFU lost an average of 10% body weight.
% Survival
100
80
60
40
20
% Body weight
0
110
105
100
95
90
85
80
0
4
8
12
16
20
105 CFU
106 CFU
107 CFU
0
2
4
6
8
10
12
14
Days post-challenge
Fig.1. Survival of mice infected with Ft subsp. uvrBpdpD double mutant.
Groups of BALB/c mice (female, 6-week old) were challenged intra-nasally with
3 doses (105, 106, and 107 CFU) of ΔuvrBpdpD to determine LD50 of this strain.
b. Monitor Ft subsp. novicida ΔuvrBpdpD replication and dissemination in mice after
intranasal challenge (Note book #4, page 138-141). BALB/c mice were challenged with
ΔuvrBpdpD mutant (105 CFU) intranasally. Lungs, liver, spleen, and lymph nodes were
collected from the infected mice at day 3, 7 and 14 after challenge (3 mice per time
point). Numbers of bacteria in each organ were determined by dilution plating. As shown
in Fig. 2, there was heightened replication of the organism in the lungs within the first 7
Page 38 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
days post-challenge, with slightly reduced viable bacteria recovered from two mice at day
14. The bacteria were rapidly disseminated to lymph nodes, liver and spleen as early as
3 days after intranasal infection, even at day 7, greater than 10 5 viable bacteria were still
detectable in all examined organs. At day 14, significant numbers of ΔuvrBpdpD can
still be found in the lungs, liver, spleen and lymph nodes from at least two of the
three infected mice. Dissemination of ΔuvrBpdpD from lungs to lymph nodes and liver
was faster than our previously characterized ΔuvrBiglA (see August 2007report), and
ΔuvrBiglC (April 2007 report). Bacterial loads in the target organs of ΔuvrBpdpD-infected
mice are also higher than uvrBigl mutants (ΔuvrBiglA,B,C,D). In summary, ΔuvrBpdpD is
moderately attenuated but more virulent than uvrBigl mutants.
9
7
5
Lung
Spleen
Liver
Spleen
*
*
Log 10 CFU / Organ
3
1
9
7
S pleen
Spleen
Spleen
Lymph nodes
5
*
3
1
3
7
14
3
Days after Challenge
7
14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrpdpD in target
organs after i.n. vaccination. Bacterial burdens were determined from lungs,
liver and spleen of individual mouse and from pooled lymph nodes at each
time point (3 mice per time point). Numbers (*) of mice without detectable
bacterial burden are indicated.
c.
Monitor Ft subsp. holarctica LVS replication and dissemination in mice after intragastric
challenge (Note book #8, page 1-4). BALB/c mice were challenged with LVS (1600 CFU)
intragastrically. Lungs, liver, spleen, and lymph nodes were collected from the infected
mice at day 1, 2, 3, 5, 7 and 14 after challenge (3 mice per time point). Numbers of
bacteria in each organ were determined by dilution plating. As shown in Fig. 3, there
was heightened replication of the organism in the lungs within the first 7 days postinoculation, with decreased numbers of viable bacteria recovered at day 14. Elevated
numbers of bacteria were not present in the liver and spleen until day 5 after inoculation,
after which bacterial numbers in the spleen decline until day 14 while they persist in the
livers. Detectable numbers of LVS were only present in lymph nodes at day 7 after
inoculation. Also planned was to look at bacterial loads at day 21 after inoculation, but 1
mouse died before the time point. This time point will therefore have to be repeated and
an update will be given upon obtaining the results.
Page 39 of 42
Tularemia Vaccine Development Contract: Technical Report
CFU Per Organ
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
10
10
10
10
10
10
10
10
10
10
10
10
8
Lungs
7
6
5
4
3
8
Spleen
7
6
5
4
3
1
2
3
5
10
10
10
10
10
10
8
10
10
10
10
10
10
8
Liv er
7
6
5
4
3
Ly mph N odes
7
6
5
4
3
1
2
14
7
Days After Inoculation
3
5
7
14
Fig. 3 Kinetic growth and clearance of Ft LVS in target organs after i.g.
vaccination. Bacterial burdens were determined from lungs, liver, spleen and
lymph nodes of individual mouse (3 mice per time point).
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
52% of scientific work completed on milestone 50a (original plans)
08% of scientific work completed on milestone 50b (intragastric plan)
9. Work plan for upcoming month
a. (50A) Evaluate the protective efficacy of the Ft subsp. novicida uvrBpdpD mutant. Groups
of vaccinated mice will be challenged i.n. with Ft subsp. novicida. Animals will be monitored
for survival and weight loss.
b. Analyze the antibody profiles of mice immunized with the Ft novicida uvrBpdpD mutant at
day 14 and 28 after vaccination.
c. (50B) Evaluate the protective efficacy of intragastric LVS vaccination. Groups of
vaccinated mice will be challenged i.n. with two doses of Ft subsp. tularensis SCHU S4 at
three weeks after inoculation. Animals will be monitored for survival and weight loss.
d. Analyze the serum, fecal and respiratory antibodies of mice vaccinated intragastrically with
LVS at 21 days after immunization.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
Page 40 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
3. Work performed and progress including data and preliminary conclusions
To inactivate RecA in Schu S4, we were in the process of constructing a Targetron vector for
targeting and inactivating the RecA gene. The Targetron vector was designed to be constructed
with the intron expression vector pKEK1140 for the backbone, and a 350bp PCR product to
retarget the intron RNA for insertion mutagenesis of Rec A.
3.1 In last technical report, we reported that the backbone pKEK1140 and 350bp PCR products had
been double digested with XhoI and BsrGI restriction enzymes. After digestion, the insertion DNA—
350bp PCR was ligated into the backbone of the plasmid pKEK1140.
350bp PCR cut with XhoI and BsrGI
5.0ul
(at 720/721s or 840/841s RecA retarget site)
10X T4 DNA Ligase Buffer
1.5ul
T4 DNA Ligase
1.0ul
ddH2O
6.0ul
pKEK1140 cut with XhoI and BsrGI
1.5ul
Incubated the ligation solution at 16C for 16 hours. Purified the ligation DNA using Phenol and
Chloroform before transformation.
3.2 Transformed the ligation DNA into the host cells-- E.Coli. DH5 competent cells using
electroporation. After transformation, the transformed cells were spread onto LB/Xgal/Kanamycin(50ug/ml) plate, and incubated at 37C for overnight.
3.3 Screened the transformants by digesting the miniprep DNA from the white colonies on the plate
using BglII restriction enzyme.
Figure for the digestion as followings:
Data recorded on UTSA TVDC notebook #6, page 4-6 for the figure above.
3.4 The figure for the digestion indicated that all the 8 colonies (lane3-6 and lane 10-13) were
correct. Lane8 was the parent plasmid digested with BglII. It wasn’t digested completely because
there were some undigested DNA at the same size as lane7 (uncut pKEK1140). The difference
between the mutant plasmid and the parent plasmid is that after digestion with BglII, the largest
fragment among the three fragments was slightly bigger than 4000bp for the parent plasmid (lane8)
and a little bit smaller than 400bp for the mutant plasmid(lane3-6 and lane10-13).
3.5 We sent “pKEK1140/350bp PCR at 720/721s recA retarget site” miniprep DNA #2 and #4,
“pKEK1140/350bp PCR at 840/841s recA retargeted site” miniprep DNA #1 and #2 for sequencing
with “groELp Down BglII XhoI” for the primer. The sequencing results confirmed that the four
Page 41 of 42
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2007 to 11/30/2007
Due Date: 12/7/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Trevor Brasel, Mitch Magee, Kathryn Sykes, Stephen Johnston, Justin
Skoble, Karl Klose, and Bernard Arulanandam
mutants were correct. “pKEK1140/350bp PCR at 720/721s recA retarget site” #2 was named as
pKEK1186 and ” pKEK1140 /350bp PCR at 840/841s recA retargeted site” #1 was named as
pKEK1187.
4 Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
pKEK1186 (pKEK1140 with 350bp PCR insertion at 720/721s recA retarget site)
pKEK1187 (pKEK1140 with 350bp PCR insertion at 840/841s recA retarget site)
7. Quality of performance
Good
8. Percentage completed.
About 10% of scientific work completed.
9. Work plan for upcoming month
Transform pKEK1186 or pKEK1187 into F.novicida U112 or LVS to make sure that RecA can be
mutated in them before working on F.tularensis Schu S4.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 42 of 42