Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose 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, 33, 34 (UNM/ASU), 35,
41, 42, 43, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 40, 48,
Inactive milestones: 6-10, 11, 14, 15, 17, 18, 20, 22, 23, 24, 29, 30, 31, 36-38, 44, 45,
47, 52-54, Working Group
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. LBERI and True Foundation have signed a subcontract for project management
associated with the LVS vaccinations, which will be provided by USAMRIID.
b. Five way CRDA between USAMRIID, USAMMDA, True Foundation, UNM and LBERI
was initially accepted by all parties including UNM Legal. UNM HSC Grants and
Contracts requested additional language to clarify the financial roles for UNM and LBERI;
this has delayed the signing of the CRDA. We hope that the CRDA can be re-reviewed
and signed within 2 weeks.
c. Nicole Banks (LBERI), Terri Nakamura (True) and Barbara Griffith (UNM) are developing:
i. Timeline in MS Project for the vaccination process- Nicole drafted and Barbara
returned edits
ii. Database for tracking vaccinee documents submitted to USAMRIID- Barbara
drafted data types, Terry draft database, Barbara edited the database
d. UNM EOHS has acquired current documents:
i. Normal values for prehealth screening tests to be performed in NM at TriCore
Reference Laboratory
ii. CAP certificate for TriCore Reference Laboratory
iii. CLIA certificate for TriCore Reference Laboratory
iv. Medical Director’s signed CV, from TriCore Reference Laboratory
v. Medical Director’s license, from TriCore Reference Laboratory
vi. University Hospital’s JCAHO accreditation – covers Radiology’s certification
e. UNM EOHS pending
i. Radiology Director’s CV and license- have requested current
ii. Final pricing for pre health and post health screenings
f. UNM and LBERI have prioritized the 46 scientists and staff who will be offered the LVS
vaccinations
1 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
g. USAMRIID Correspondence: Bev Fogtman, Dr. John Aldis, Jeannine Haller, and Cindy
Barrick have responded to 3 sets of questions and been extremely helpful.
i. USAMRIID is requiring that UNM EOHS nurses be GCP trained prior to arriving
at USAMRIID for LVS vaccination site training
ii. UNM has access to a course and Dr. Boudreau will review the course content
4. Significant decisions made or pending
a. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
b. 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
c. UNM and LBERI will offer the LVS vaccinations to 46 scientists; USAMRIID will be
providing the LVS vaccinations over the next 8 months, approximately.
d. Dr. Lyons will request IRB approval to allow blood draws on the vaccinated LBERI and
UNM scientists after their LVS vaccinations. UNM will share the IRB proposal with
USAMRIID.
5. Problems or concerns and strategies to address
a. UNM may need an external source of human cells from LVS vaccinated individuals, in
order to develop the immunoassays in humans. Within approximately 4 months, UNM
may have access to the blood of UNM and LBERI scientists who have been vaccinated
with LVS at USAMRIID.
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; Work stop has occurred on the SCHU S4 aerosols in primates, until the
LBERI scientists and staff receive the LVS vaccinations.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
18%
9. Work plan for the next month
a. Complete the 5 way CRDA between USAMRIID, True Foundation, UNM and LBERI
b. Formally, start the relationship with USAMRIID
i. provide Laboratory and Radiology documents to USAMRIID
ii. Begin Hazard Analysis Risk assessments
iii. Begin informed consent process and HIV
iv. iv Hold SIP Informed consent teleconference with UNM/LBERI/USAMRIID
c. EOHS nurses start GCP training, if USAMRIID approves the CITI course content
d. Obtain final pricing from UNM EOHS for prehealth and post health screenings
e. Maintain excellent communications with USAMRIID to understand the SIP protocol
requirements
10. Anticipated travel
Travel to USAMRIID could occur in summer 2007 to fall 2007
11. Upcoming Contract Authorization (COA) for subcontractors
a. 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.
b. UNM will request a COA to authorize expending funds on the pre-health screenings and
travel for the vaccinations, for 46 scientists and staff.
2 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
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
LVS focused on Micropump generator and 4 days of bioaerosol testing were conducted
i. 3 days of testing using frozen LVS stock (Figures 1 and 2)
1. 15 total sprays
2. 3 target concentrations: 1x105, 1x106, and 1x107 cfu/mL
3. Actual vs. Target cfu/mL values were accurate
4. Calculated spray factors were not consistent and were overall poor
a. Values at the lower tested concentrations (1x105 and 1x106
cfu/mL) were comparable to those observed using the Collison
nebulizer
b. Values decreased (i.e., efficiency decreased) as concentration
increased, a consistent observation seen with other aerosol
generators tested to date, including the Collison nebulizer
5. Data filed in the following folders:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump\1May07
b. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump\4May07
c. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump\10May07
Micropump: Target vs. Actual CFU/mL (Frozen LVS)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
1-May-07
5.50
4-May-07
5.00
10-May-07
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)
Figure 1. Target vs. Actual CFU/mL at three concentrations of frozen LVS using the Micropump generator on three separate
bioaerosol dates
3 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Micropump: Actual CFU/ml vs. Spray Factor (Frozen LVS)
Spray Factor (Log10)
-6.00
0.00
-6.50
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
-7.00
-7.50
1-May-07
-8.00
4-May-07
10-May-07
-8.50
-9.00
-9.50
-10.00
Actual CFU/mL (Log 10)
Figure 2. Actual CFU/mL vs. Spray Factor at three concentrations of frozen LVS using the Micropump generator on three separate
bioaerosol dates
ii. 2 days of testing using fresh LVS stock (Figures 3 and 4)
1. 12 total sprays
2. 3 target concentrations: 1x105, 1x106, and 1x107 cfu/mL
3. Actual vs. Target cfu/mL values were accurate
4. As with the frozen stocks, calculated spray factors were not consistent
and were overall poor
a. Values at the lower tested concentrations (1x105 and 1x106
cfu/mL) were comparable to those observed using the Collison
nebulizer
b. Values decreased (i.e., efficiency decreased) as concentration
increased, a consistent observation seen with other aerosol
generators tested to date
5. Data filed in the following folders:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump\10May07
b. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump\17May07
4 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Micropump: Target vs. Actual CFU/mL (Fresh LVS)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
10-May-07
5.50
17-May-07
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)
Figure 3. Target vs. Actual CFU/mL at three concentrations of fresh LVS using the Micropump generator on two separate
bioaerosol dates
Micropump: Actual CFU/ml vs. Spray Factor (Fresh LVS)
Spray Factor (Log10)
-6.00
0.00
-6.50
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
-7.00
-7.50
-8.00
10-May-07
-8.50
17-May-07
-9.00
-9.50
-10.00
-10.50
Actual CFU/mL (Log 10)
Figure 4. Actual CFU/mL vs. Spray Factor at three concentrations of fresh LVS using the Micropump generator on two separate
bioaerosol date

Outside of the ABSL-3, work was conducted on two separate generators (the Aeromist
and Aeroeclipse II) using Bacillus globigii (BG) spores, which can be used in a BSL2
environment. The aeromist and aeroeclipse are different air jet nebulizers from the
Collison and micropump which use lower air pressures to generate aerosols and thus
may be gentler on bioagents.
i. 2 days of testing using freshly prepared BG spores in solution (Figures 5-8)
1. 9 total sprays
a. 6 using the Aeromist (3 of which were conducted 1 June 2007,
but are included in this May 2007 report)
5 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
b. 3 using the Aeroeclipse II
2. 2 target concentrations: 1x104 and 1x105 CFU/mL
3. Aeromist data (Figures 5 and 6):
a. Actual vs. Target CFU/mL values were within 1 log10 of the
target values
b. Calculated sprays factor were very consistent and more efficient
than those observed with other generators tested to date
4. Aeroeclipse II data (Figures 7 and 8)
a. Actual vs. Target CFU/mL values were within 0.5 log10 of the
target values
b. Calculated spray factors were consistent, but comparable to the
Collison nebulizer; for this reason, no further testing was/is
planned for this device.
5. Data filed in the following folder:
a. \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\BSL-2 (BG) testing
Aeromist: Target vs. Actual CFU/mL (BG spores)
7.00
Actual CFU/ml (Log10)
6.50
6.00
5.50
17-May-07
5.00
1-Jun-07
4.50
4.00
3.50
3.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
Target CFU/ml (Log10)
Figure 5. Target vs. Actual CFU/mL at two concentrations of BG spores using the Aeromist generator on two separate bioaerosol
dates
6 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Aeromist: Actual CFU/ml vs. Spray Factor (BG spores)
Spray Factor (Log10)
-5.00
-5.200.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
-5.40
-5.60
-5.80
17-May-07
-6.00
1-Jun-07
-6.20
-6.40
-6.60
-6.80
-7.00
Actual CFU/mL (Log 10)
Figure 6. Actual CFU/mL vs. Spray Factor at two concentrations of BG spores using the Aeromist generator on two separate
bioaerosol dates
Aeroeclipse II: Target vs. Actual CFU/mL (BG spores)
7.00
Actual CFU/ml (Log10)
6.50
6.00
5.50
5.00
17-May-07
4.50
4.00
3.50
3.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
Target CFU/ml (Log10)
Figure 7. Target vs. Actual CFU/mL at 1x104 BG spores/mL using the Aeroeclipse generator on one bioaerosol date
7 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Aeroeclipse II: Actual CFU/ml vs. Spray Factor (BG spores)
Spray Factor (Log10)
-5.00
-5.200.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
-5.40
-5.60
-5.80
17-May-07
-6.00
-6.20
-6.40
-6.60
-6.80
-7.00
Actual CFU/mL (Log 10)
Figure 8. Actual CFU/mL vs. Spray Factor at 1x104 BG spores/mL using the Aeroeclipse generator on one bioaerosol date
4. Significant decisions made or pending
a. Will continue to perform Micropump testing using LVS
b. The Aeromist generator is a promising device that will be incorporated into further testing
in June 2007. It will be tested with frozen and/or fresh LVS in June.
c. The Aeroeclipse II will not be tested further due to its indifference from the Collison
nebulizer. The Aeroeclipse does not perform better than the Collison nebulizer.
5. Problems or concerns and strategies to address
The annual ABSL-3 Facility shutdown will delay further LVS testing by approximately 2 weeks;
BG testing will continue in June. The ABSL-3 facility will be available for testing sprays again on
June 8th.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
61%
9. Work plan for upcoming month




Perform additional bioaerosol experiments on BG spores using the Aeromist nebulizer
Perform bioaerosol experiments on fresh and frozen LVS with the Aeromist nebulizer
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue using frozen and fresh LVS, not lyophilized for sprays.
Begin initial testing of ultrasonic generator using BG spores
Continue to investigate other possible generators to use for LVS bioaerosols
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
8 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
No new experimental work on this milestone was completed in the last month.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
12.5%
9. Work plan for upcoming month
None. These NHPs will continue to be bled as a source of cells for Milestone 12/13; however, no
work is anticipated on these NHPs until they are challenged with aerosol SCHU S4 sometime
after November 2007.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Fischer 344 rats
a. Experiment Ftc32 study 1 (Notebook 104, pages 1-5, 24-25, 36, 40)
i. The purpose of this experiment was 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. Fischer 344 rats tolerated s.c., i.d. and i.t. vaccination with LVS at 4 x
107CFU/rat extremely well. However, only 14 of 24 rats survived i.t.
vaccination with 50 SCHU S4, suggesting that SCHU S4 vaccination is not
an acceptable method for vaccinating rats
iii. The LVS and SCHU S4 vaccines were cleared from the lungs, liver, and
spleen 42 days after vaccination.
9 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
iv. 60 days after vaccination, control naïve rats and vaccinated rats were
challenged i.t. with SCHU S4
v. We found that a calculated dose of as few as 57 SCHU S4 killed 5 of 6 naïve
rats (Figure 1), confirming our previous results that Fischer 344 rats are
extremely sensitive to pulmonary SCHU S4 challenge.
vi. All of the vaccinated rats were protected against i.t. challenge with 8.7 x 103
SCHU S4. Most of the vaccinated rats also survived higher challenge doses
of 2.8 x 105 and 2.6 x 106 SCHU S4. Although more of the i.t. LVS
vaccinated rats died from SCHU S4 challenge, there was no statistically
significant difference between the four vaccination groups (One-way ANOVA
with Bonferroni’s Mulitple Comparison Test)
vii. The rats were observed for 21 days. On day 23, half of the survivors from
each group were killed to determine whether they had cleared the SCHU S4
challenge and, if not, the bacterial burden was determined in the lungs,
spleens and liver. We found < 5,000 CFU SCHU S4 in the lungs of a third of
the rats examined. There was no correlation between the number of SCHU
S4 and the vaccination route or the SCHU S4 challenge dose.
viii. We will check for clearance again in 3 weeks
10 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
11 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 1. Comparison of the immunity generated by various vaccination routes and strains against
i.t. SCHU S4 challenge. Vaccinated Fischer 344 rats (n = 6/group) were challenged i.t. with the
indicated doses of SCHU S4 60 days after vaccination. Survival was monitored for 21 days.
b. Experiment Ftc37 study 2 (Notebook 104, pages 6-8)
i. The purpose was to determine the histological appearance of the lungs, liver
and spleen from naïve Fischer 344 rats infected i.t. with a lethal dose of
SCHU S4. These results will be compared with those from vaccinated rats
challenged i.t. with SCHU S4 (Ftc40 study 2)
ii. Naïve rats were infected i.t. with 400 SCHU S4. Three rats were killed on
days 0, 3, 6, 9 days to collect the lungs, liver, and spleen
iii. The tissues are currently being processed at LRRI and, upon return, will be
examined by Dr. Julie Hutt at UNM
c.
Experiment Ftc40 study 1 (Notebook 104, pages 15-17, 37-38, 42)
i. The purpose was to determine the kinetics of SCHU S4 proliferation,
dissemination and clearance in s.c. LVS-vaccinated rats
ii. Fischer 344 rats were vaccinated s.c. with 2.7 x 10 7 LVS
iii. 55 days after vaccination, the rats were challenged i.t. with 1.5 x 10 4 SCHU S4
Three days after SCHU S4 challenge, we observed 1.6 log10 SCHU S4
expansion in the lung and dissemination to the liver and spleen (Table
2). On day 9, the numbers of SCHU S4 were lower than those on day 3,
suggesting that the vaccinated rats were controlling SCHU S4
proliferation. In contrast, we showed previously that naïve rats failed to
control SCHU S4 proliferation, even though they were challenged with 2
log10 fewer SCHU S4 (Ftc37; Table 3) Values are expressed as log10 in
Tables 2 and 3.
iv.
Table 2. Kinetics of SCHU S4 proliferation in
s.c. LVS-vaccinated Fischer 344 rats*
CFU/organ (log10)
Day
Lung
Liver
Spleen
0
4.18  0.20
3
5.80  0.22 5.22  0.62 4.96  0.52
9
5.50  0.35 3.36  0.19 4.34  0.74
* n = 3 rats/group
Table 3. Kinetics of SCHU S4 proliferation in
naïve Fischer 344 rats*
Day
0
3
6
9#
#
Lung
2.17  0.36
8.04  0.12
8.42  0.09
8.81
CFU/organ (log10)
Liver
Spleen
6.46  0.32
7.42  0.24
8.66
6.06  0.51
7.44  0.34
7.49
*
n = 3 rats/group
2 of 3 rats died by day 9
d. Experiment Ftc40 study 2 (Notebook 104 pages 31-32, 34)
12 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
i. The purpose of this experiment was to determine histological appearance of
the lungs, liver, and spleens from s.c. vaccinated rats after i.t. SCHU S4
challenge. These results will be compared with those generated from naïve
rats challenged i.t with SCHU S4 (Ftc37 study 2)
ii. 43 days after s.c. LVS vaccination, rats were challenged i.t. with 320 SCHU
S4
iii. 3 rats were killed on days 0, 3, 6, 9 to collect lungs, liver, and spleens
iv. The tissues are currently being processed at LRRI and, upon return, will be
examined by Dr. Julie Hutt at UNM
e. Experiment Ftc46 (Notebook 104 pages 47-49)
i. We have noticed that when we use our non-surgical method of intratracheal
infection (using an i.v. catheter inserted into the trachea to deliver inoculum),
we would occasionally deliver the inoculum down the esophagus instead of
the trachea. Since this would induce a gastrointestinal disease instead of a
pulmonary disease and affect our interpretation of the results, we have been
looking for ways to track pulmonary delivery in rats.
ii. QD655-luc8 is a bioluminescent quantum dot conjugate that, in the presence
of its substrate coelentrazine, emits light at 655 nm without the need for
external excitation. It has been shown to emit enough light to successfully
allow in vivo imaging in mice using the Xenogen IVIS 200 imager (Nat
Biotechnol. 2006 Mar;24(3):339-43]. Thus, it may be possible to use QD655luc8 as a molecular tracker for pulmonary infection in rats
iii. The purpose of this experiment was to determine whether we can use
QD655-luc8 and coelentrazine to track pulmonary infection of rats inoculated
using the non-surgical i.t. delivery method
iv. QD655-luc8 and coelentrazine are both commercially available from Zymera
v. We prepared an inoculum containing 5 pmol of QD655-luc8 and 10 g
coelenterazine and delivered 100l of it down the trachea using the nonsurgical i.t. delivery method. We also intentionally delivered the inoculum
down the esophagus to determine whether we can discriminate between the
two infection routes
vi. As show in Figure 2, it is very easy to distinguish pulmonary delivery from
gastrointestinal delivery. With pulmonary delivery, light emitted from either
the left or right lobes, but not both. In contrast, with gastrointestinal delivery,
light emitted from the throat. The lack of light emission farther down the
gastrointestinal tract may be due to inactivation of QD655-luc8 at low pH but
this has yet to be proven
vii. Extended anesthesia was not required because in vivo imaging took less
than 1 min
viii. Emission was detectable more than 30 min after inoculation
ix. We will perform additional experiment to make sure that QD655-luc and
coelentrazine do not affect the Francisella virulence. Once proven, we will
include this tracker in all future infections.
13 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 2. In vivo imaging of rats after inoculation of QD655 and coelentrazine down the
trachea or esophagus.
Hartley Guinea Pigs
a. Experiment Ftc41 (Notebook 104, pages 18-21)
i. The purpose was to determine whether i.n. LVS vaccination protects Hartley
guinea pigs from i.n. SCHU S4 challenge. This is a repeat of Experiment
Ftc28 (Notebook 94, pages 152-156)
ii. Naïve guinea pigs (n = 6 to 10) were vaccinated i.n. with 103, 1.3 x 105, and
6.7 x 106 CFU LVS
iii. 49 days after vaccination, we collected sera from all of the vaccinated guinea
pigs to confirm sero-conversion
iv. 55 days after vaccination, we challenge the i.n. vaccinated guinea pigs with
5.5 x 104 SCHU S4 i.n. We selected this challenge dose because even
when it is diluted with the large buffer volume required to homogenize guinea
pigs lungs, the bacteria concentration will still be high enough to give us a
reliable lung deposition
14 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
v. LVS vaccination, regardless of the vaccination dose, had very little, if any,
effect on the sensitivity of guinea pigs to i.n. SCHU S4 challenge (Table 4),
confirming our previous results that vaccination did not increase the
resistance of guinea pigs to SCHU S4 challenge
i.n. LVS vaccination
Dose
(CFU/guinea pig)
None
~ 103
1.3 x 105
6.7 x 106
Dose
(CFU/guinea pig)
5 x 104
5 x 104
5 x 104
5 x 104
i.n. SCHU S4 challenge
Survival ratio
(No. alive/total)
0/6
0/6
0/6
0/6
Mean-time-to
death (days)
4.0
4.5
5.3
4.8
Table 4. Resistance of i.n. LVS-vaccinated guinea pigs to intranasal SCHU S4 challenge
b. Experiment Ftc42 (Notebook 104, pages 22-23)
i. The purpose of this experiment was to determine whether s.c. LVS
vaccination protects Harley guinea pigs from i.n. SCHU S4 challenge. This is
a repeat of Experiment Ftc28 (Notebook 94, pages 152-156)
ii. Naïve guinea pigs (n = 6/group) were vaccinated s.c. with 103, 105, and 107
CFU LVS
iii. All of the guinea pigs survived vaccination and cleared the LVS vaccine from
lungs, liver and spleen
iv. We collected sera from all of the vaccinated guinea pigs to test seroconversion
v. We are waiting to challenge the vaccinated guinea pigs i.n. with SCHU S4
c.
Experiment Ftc 39 (Notebook 103, pages 25-27)
i. The purpose of this experiment was to develop an ELISA to test guinea pig
sero-conversion after LVS vaccination
ii. We titrated sera from naïve and LVS-vaccinated guinea pigs from 1:200 to
1:102,400 dilution on plates coated with heat killed LVS.
iii. Naïve guinea pig sera produced extremely high background and had to be
diluted 1:12,800 to 1:25,600 to eliminate background (Figure 3)
iv. It was very clear that LVS-vaccinated guinea pigs sero-converted following
LVS vaccination
v. We will use this assay to test the guinea pigs in Ftc41 and Ftc42 for seroconversion
15 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 3. Titration of sera from naïve and LVS-vaccinated guinea pigs in a
LVS-specific ELISA. Diluted sera were tested in a sandwich ELISA using
plates coated with heat-killed LVS and HRP-conjugated goat anti-guinea pig
IgG for detection.
4. Significant decisions made or pending
We will use surgical instead of non-surgical i.t. delivery methods for pivotal studies in rats to
avoid the unintentional gastrointestinal infection.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
50%
9. Work plan for upcoming month
Rats
a. Determine the SCHU S4 burden in the s.c. LVS-vaccinated rats 2 months after i.t.
SCHU S4 challenge. It is possible that SCHU S4 persists in vaccinated rats as well
as vaccinated mice.
b. Repeat the s.c. LVS vaccination/i.n. SCHU S4 challenge experiment
c. Characterization of the Fischer 344 rat model
i. Repeat the experiments measuring the kinetics of SCHU S4 proliferation and
dissemination in lungs, spleens, and livers of naïve and LVS vaccinated rats.
16 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
The proposed experiments will fill the gaps in the previous experiments,
focusing on days 1-6 of infection
ii. Repeat the experiments examining the histology of lungs, spleens and livers
from naïve and LVS-vaccinated rats infected i.t. with SCHU S4
iii. Determine the effects of T cell depletion on the protective immunity induced
by LVS vaccination
iv. Determine whether passive immunization with convalescent sera will protect
naïve Fischer 344 rats from i.t. SCHU S4 challenge
d. Optimize the use of QD655-luc8 to track pulmonary inoculation
i. Determine the minimum amount of QD655-luc8 and coelenterazine required
to produce detectable signal
ii. Determine whether co-administration of QD655-luc8 affects the virulence of
LVS or SCHU S4 in naïve Fischer 344 rats
Guinea Pig
a. Challenge the s.c. LVS-vaccinated guinea pigs i.n. with SCHU S4 (Ftc42)
b. Test sero-conversion in guinea pigs used in Ftc41 and Ftc42.
c. We will make a decision regarding the usefulness of the guinea pig model after the
two action items above have been completed. Additional experiments will be
performed if deemed necessary.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 12/13-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
a. Update on NHP PBMC Freezing protocols
1. Issue: Testing 3 different protocols (CTL: 90% human A/B serum/10%
DMSO/10 x 106/ml; CERUS: 80% FBS/20% DMSO/5 x 106/ml; and Lyons:
Frozen in Gibco Recovery Cell Culture Freezing Media (contains optimal ratio of
fetal bovine serum:bovine serum and 10% DMSO)/5 – 10 x 106/ml/thawed in
presence of DNAse and left in 37o incubator for 30 – 60 minutes before use) with
the aim to choose the protocol that spares the most viable cells that remain
functional after thawing
2. Results thus far suggest that CERUS protocol spares about 50% of Con A
proliferative capacity and 30% of antigen specific proliferation; CTL protocol,
however, did not spare antigen-specific proliferation (However, antigen-specific
proliferation has only been tested once with both protocols)
3. Results below in Table 1 are from the second time we tried freezing and
recovering antigen-specific proliferation (TUL 11; frozen on 3/26/07 and thawed
on 5/14/07)
17 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Table 1
Protocol
Cerus
CTL
Lyons
# Cells recovered
2.7 x 106
1.9 x 106
0.98 x 106
% Cells Recovered
67.5%
47.5%
24.5%
% Viable
95.1%
98.7%
95.1%
Proliferation of Cells from Two NHPs on day 117 post s.c. LVS Vaccination
A00659
A00868
A
1.20E6
1.00E6
8.00E5
6.00E5
4.00E5
LVS ff Hi
LVS hk Hi
Media
0
PHA
2.00E5
Con A
Cell Mean for RLU small
1.40E6
7.00E5
6.00E5
B
Media
LVS hk Hi
LVS ff Hi
5.00E5
4.00E5
3.00E5
2.00E5
Day 117, A00868
Day 117, A00659
Day 28, A00868
Day 28, A00659
Day 21, A00868
Day 21, A00659
Day 14, A00868
Day 14, A00659
Day 7, A00868
Day 7, A00659
0
Day 0, A00868
1.00E5
Day 0, A00659
Cell Mean for RLU small
8.00E5
Figure 1: Proliferation of FRESH cells plated at 1 x 10 6/ml with the indicated stimuli. PBMCs were
purified from two separate NHPs on day 117 post. s.c. vaccination (A) and compared to other days postimmunization (B).
Data interpretation: Proliferation to LVS is not optimum at day 117 but is still detectable.
18 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Data regarding this particular experiment (TUL 11, 3/26/07, day 117 post s.c. vaccination) is stored on Dr.
Wilder’s desktop computer in C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
contract. The relevant files are: TUL 11 protocol.doc; prep for 033007mtg.doc; 0407 seminannual and
March report.doc. These files are backed up on N:\My Documents\Tularemia Contract. Data on the
collection of fresh cells and freezing protocol is also located in the TVDC 1 Binder under the tab TUL 11.
The data used to create the graphs shown above is stored in PBMC assays 053107.svd which can be
located on \\Saturn\Group\Wilder Lab\TVDC. Any subsequent date appended to the PBMC assays.svd
file will also contain the data as the date indicates the day of update and no data is deleted once it is
entered. The raw proliferation data for TUL 11 is located on \\Saturn\Group\Wilder Lab\TVDC\BRDU in
the following files: TUL 11 040307.xls and TUL 11 proliferation assay.xls.
Proliferation of Cells from a Single NHP which had been Frozen and Thawed
A00868, Fresh, None
A00868, Frozen, Cerus
A00868, Frozen, CTL
1.20E6
1.00E6
8.00E5
6.00E5
4.00E5
PHA
LVS ff Hi
LVS hk Hi
0
Con A
2.00E5
Media
Cell Mean for RLU small
1.40E6
Figure 2: Proliferation of FROZEN/THAWED cells plated at 1 x 106/ml with the indicated stimuli. PBMCs
were purified from NHP A00868 on day 117 post. s.c. LVS vaccination, frozen down using the Cerus or
CTL protocol and thawed 7 weeks later; comparison to values obtained from FRESH cells is shown.
Data interpretation: Although LVS responsiveness is not optimum, it is preserved after thawing; neither
protocol looks superior to the other.
Data regarding the protocol used to thaw the cells and set them up in the proliferation assay is located in
TVDC Bound Notebook 1, pages 24-27. . Data regarding the proliferation of thawed cells is stored on
\\Saturn\Group\Wilder Lab\TVDC\freezing_thawing test in the following files: TUL11 thawing 05-18-07.xls
and TUL11 thawing.xls. The data used to create the graph shown above (Figure 2) is stored in PBMC
assays 053107.svd which can be located on \\Saturn\Group\Wilder Lab\TVDC. Any subsequent date
appended to the PBMC assays.svd file will also contain the data as the date indicates the day of update
and no data is deleted once it is entered. The data was summarized and presented in a UNM/LBERI
Internal Tech Meeting and also at the June UNM:TVDC LBERI Tech Call with the NIAID representatives
and the related files are stored in C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia
Contract\ prep for 060107 mtg.doc and LBERI Tech Call.6.5.2007.final.ppt; and these files are backed up
on N:\My Documents\Tularemia Contract\ prep for 060107 mtg.doc and LBERI Tech
Call.6.5.2007.final.ppt.
19 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
b. Update on IgG anti-LVS ELISA
1. Performed optimization of LVS coating concentration
Comparison of Heat-Killed and Formalin-Fixed LVS Preparations as IgG anti-LVS ELISA Capture
Antigens
HK LVS ELISA Optimization
A
2.500
1/200
2.000
OD405
1/1000
1.500
1/5000
1.000
1/25000
1/125000
0.500
1/625000
0.000
0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5
5
10
Ag Concentration (x106/ml)
FF LVS ELISA Optimization
B
OD405
1.400
1.200
1/200
1.000
1/1000
0.800
1/5000
0.600
1/25000
0.400
1/125000
0.200
1/625000
0.000
0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5
5
10
Ag Concentration (x 106/ml)
Figure 3: LVS, either heat-killed (A) or formalin-fixed (B) was used at varying concentrations to coat a 96
well plate. Sera was pooled from 6 NHPs vaccinated 21 days previously with LVS and diluted as
indicated. Bound IgG was detected with goat anti-monkey IgG-HRP.
20 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Data interpretation: The optimum coating concentration for HK LVS is 2.5 x 10 6/ml, whereas the optimum
coating concentration for FF LVS is greater than 10 x 106/ml. However, as using more than 10 x 106 FF
LVS/ml is impractical, we will pursue this antigen no further in this assay.
Serum IgG anti-LVS in LVS-vaccinated NHPs
A
10000
1000
Day 28
Day 21
Day 14
B
A00659
A00868
A00896
A00902
A00908
A00937
10000
Day 28
Day 21
100
Day 14
1000
Day 7
IgG anti-LVS Titer
100000
Day 0
100
Day 7
ID
SC
Day 0
IgG anti-LVS Titer
100000
Figure 4: HK-LVS (2.5 x 106/ml) was used to coat ELISA plates. Sera from NHPs vaccinated via either
the s.c. or i.d. route were diluted 1/200 – 1/625,000 using 5-fold serial dilutions and plated in duplicate.
Bound IgG was detected with goat anti-monkey IgG-HRP. Titers were determined as the highest dilution
producing an average OD405 value above background. Panel A shows the data expressed as an average
of the 3 NHPs similarly vaccinated while Panel B shows each individual NHP expressed. Open symbols
show NHPs vaccinated via the s.c. route and closed symbols show those vaccinated via the i.d. route.
Data interpretation: There is no apparent difference in the titer of IgG anti-LVS mounted by the i.d.
21 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
vaccinated group as compared to the s.c. vaccinated group. The titers appear to be maximal by day 14
or 21 in all animals.
2. Update on IFN detection
i.
I am in contact with the ViruSpot detection system representative who is
currently looking at the data showing faint spots in wells in which cells
are unstimulated which are being detected as readily as much darker
spots in stimulated wells; she (Jaya Ghosh) will work with me on
adjusting the setting to exclude these faint spots from being counted
ii.
We attempted one assay to detect intracellular IFN staining by flow
cytometry, but it was unsuccessful due to a loss of CD4+ staining after
stimulation overnight and failure to detect increased production of IFN;
we are currently looking up some manuscripts which will guide us on this
issue
3. All ELISA data is stored in binder TVDC 1 in the Wilder laboratory as well as in
summary form on C:\Documents and Settings\jwilder.LOBOS\My
Documents\Tularemia Contract\ prep for 060107 mtg.doc and LBERI Tech
Call.6.5.2007.final.ppt backed up on N:\My Documents\Tularemia Contract\prep for
060107 mtg.doc and LBERI Tech Call.6.5.2007.final.ppt; all protocols and
procedures for setting up the ELISA can be found in the TVDC bound notebook,
pages 22–23; the raw data is also stored in \\Saturn\Group\wilder lab\TVDC\LVS
ELISA data: summarized in LVS ELISA 5-11-07.xls and raw data for plates in
051107 10’ (or 20’ or 30’).mpl.
4.
Significant decisions made or pending
Heat-killed LVS will be used to coat ELISA plates at 2.5 x 106/ml in order to detect NHP IgG
anti-LVS.
5.
Problems or concerns and strategies to address
6.
Deliverables completed
7.
Quality of performance
8.
Percentage completed
None
None
Good
75% of scientific work has been completed (Note that the goals and anticipated scientific
work in this milestone has changed in the past 6 months; thus the percentage completed is
now lower)
9.
Work plan for upcoming month
1. Continue to freeze down PBMCs using the 3 different protocols
2. Contact the ELISPOT kit and reader representatives to discuss issues in optimization
3. Optimize the coating concentrations of HK- and FF-LVS for the IgA anti-LVS ELISA
4. Test LPS responsiveness of whole blood and PBMC preparations to determine
whether B cells are being lost in the preparation
5. Attempt intracellular IFN gamma staining again with established published protocols
10.
Anticipated travel
Julie Wilder is attending the CMI course from 6/3 to 6/7/07 in Washington DC, which is
sponsored by NIAID. Per Andrew Cherry and Dr. Vicki Pierson, no COA was needed for this
NIAID sponsored course
11.
Upcoming Contract Authorization (COA) for subcontractors
None
22 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new experimental work done; however, we consulted with Dr. Karen Elkins and
received advice to improve the assays under development
b. We will apply this assay to the rat because there is accumulating data suggesting
that Fischer 344 rats may be a better model than the mouse.
c. UNM is also providing supplies to LBERI on this milestone
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
a. We had previously indicated that we have optimized the T cell proliferation assay for
the mouse: 5 x 104/well nylon wool-enriched T cells and 106/well formalin-fixed LVS
produced the best balance of background, specificity and sensitivity. However,
during a troubleshooting session, Karen Elkins suggested that we should be able to
use many more cells in our proliferations assays without increasing the background.
She suggested that our problem with non-specific proliferation at high cell numbers
may be related to the quality of fetal calf serum (FCS) that we have been using.
Thus, she sent us an aliquot from her FCS stock and we obtained an aliquot from a
reserve lot at Hyclone. We will test these two FCS stocks in parallel with our current
FCS stock in the proliferation assay and macrophage killing assay to find one that
produces minimal background
b. When we have determined the effects of FCS on T cell proliferation, we will apply this
assay to identify peptides from F. tularensis proteins that would stimulate T cells from
LVS-vaccinated BALB/c mice and potentially other vaccinated small animals models
(milestone 27)
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month
a. Test three lots of FCS (one from Karen Elkins, one from a reserve lot at Hyclone, and
one from current Hyclone lot at UNM) for background in T cell proliferation assays.
Our goal is to increase the number of cells that can be added to the assay without
increasing the background proliferation. If we can achieve this, then we further
optimize the assay in the mouse model
b. We will develop the T cell proliferation assay for the Fischer 344 rat
i. Develop procedures for isolating T cells from whole blood, spleen, lymph
node
ii. Develop procedures for stimulating T cells with Con A and killed LVS and
SCHU S4
iii. Optimize the T cell proliferation assay
23 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
10. Anticipated travel
Alexandra Scrymgeour traveled to Washington DC (June 3 to June 7) to attend the NIAID
sponsored CMI course and also planned to visit Dr. Karen Elkins’ laboratory for more advice
the T cell stimulation assay
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
No new experimental work done because no human cells were available during this period
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
We have contacted Ms Tereassa Archibeque, the respiratory nurse, to clarify how frequently
we may anticipate securing a donor.
6. Deliverables completed
NA
7. Quality of performance
Limited progress due to unavailability of human alveolar macrophages
8. Percentage completed
3%
9. Work plan for upcoming month and next 6 months
a. Determine the optimal MOI for infecting human alveolar macrophages. Since we
observed cytopathogic effects at MOI = 1, we will titrate MOI down to 0.1, 0.5, and 1
b. Determine macrophage viability by lactate dehydrogenase (LDH) release and trypan
blue exclusion after infection
c. Determine kinetics of bacterial proliferation after infection
d. Measure cytokine (e.g. TNF, IL-1, and IL-6) production by macrophages infected with
SCHU S4 or LVS
e. Determine whether recombinant IFN would inhibit SCHU S4 and LVS intracellular
growth
f. Determine whether PBMC from vaccinated human volunteers can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
24 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
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
No new experimental work done
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
We noticed in several experiments of this series that uninfected macrophages did not
survive 3 days in culture, the length of a typical experiment. During the same trouble
shooting session described earlier in Milestone 12, Karen Elkins suggested that L929
cells from different sources behave very differently and may produce different levels of MCSF, thus affecting the quality of the bone marrow-derived macrophages; her lab uses a
L929 line that is especially sensitive to TNF. She also suggested that the quality of
macrophages can be affected by the quality of FCS, just like T cells. Thus, She sent us
an aliquot of L929-conditioned medium to compare with the ones we have generated and
to measure the amount of M-CSF contained therein with a M-CSF ELISA. As indicated
earlier, she also sent us an aliquot of FCS.
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
10%
9. Work plan for upcoming month and next 6 months
a. Determine the concentration of M-CSF in the L929-conditioned medium from Karen
Elkins
b. Compare three lots of FCS (one from Karen Elkins, one from a reserve lot at
Hyclone, and one from current Hyclone lot at UNM) for ability to support robust
macrophage differentiation and maintain macrophage viability
c. Determine the optimal MOI for LVS and SCHU S4 infection of macrophages
d. Determine whether vaccinated splenocytes can induce BMM (bone marrow
macrophages) to kill intracellular LVS
e. Determine whether vaccinated splenocytes can induce BMM to kill intracellular
SCHU S4
f. 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
None
25 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. As discussed in our last conference call, we have reconstructed the IVT template so
as to remove the sequences accommodating biotin purification (the TEV and BAP
sites), and we have added another 6x His sequence so that a tag will be encoded at
the C terminus, as shown in Figure 1.
2. If the unpurified IVT lysates are sufficient for specific T cell stimulation then these
template modifications will not be necessary. However the His tags will be
maintained, since they are very short and may be useful at some downstream step.
Figure 1
26 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
B. Select and test IVT Protocols
1. We have repeated 35S methionine-labeled IVT reactions with 4 FTU ORFs,
calmodulin, and GFP expression templates, to confirm optimal protocol with respect
to both yield and cost efficiencies. In this experiment we used plasmid templates.
However, we have repeatedly shown equivalent product yields with either plasmid or
linear expression templates. Up to now we have been concerned with getting as
much product as possible. Now we are looking for a practical balance between
product needs and relative costs.
2. First we confirmed that addition of fresh substrate mix, or “feed” at later time-points
increases yields, while lower doses of template reduce polypeptide yields, as seen in
Table 1. The substrate mix includes components such as amino acids and ATP in
appropriate reaction buffer. This is referred to as the “feed”. Reactions were
supplemented one, two, three, four, or five times with feed, at given time-points; extra
template additions were not made. Aliquots were removed immediately before each
feed addition and counted. Counts are expressed as million cpm/met. This is
calculated by determining the total measured Cpms/# of met in the polypeptide/106.
(met= methionine)
Table 1: Testing starting template doses, and effect of feed additions on counts
incorporated into newly synthesized polypeptides.
Time of feed additions after start of rxn
Template
FTU 901 1ug
CalM 1ug
GFP LEE 1.0 ug
GFP LEE 0.5 ug
# met
0.5h
1
10
7
7
1h
0.005
0.067
0.006
-0.001
2h
0.010
0.302
0.048
0.048
6h
-0.001
0.446
0.093
0.071
o/n
0.052
0.506
0.186
0.180
0.046
0.818
0.221
0.230
GFP LEE 0.25 ug
7
-0.005
0.022
0.045
0.100
0.102
GFP LEE 0.10 ug
7
-0.003
0.008
0.004
0.018
0.032
No Template
0.000
0.000
0.000
0.001
-0.001
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data.
File name: Invitrogen IVT test with FTU template 5-21-07
3. Consideration of feed system costs led us to isolate the impact of both frequency of
feeds and amount of template. We confirmed supplementing versus not
supplementing with only kit-supplied buffer at 0.5h and not adding extra template.
We conclude that adding feed is advantageous, however multiple feed supplements
is not necessary. In table 2 below, feed was added only at 0.5h, then aliquots were
removed for analyses at 1h, 2h 3h, and overnight.
Table 2: Impact of only one early timepoint feed on polypeptide yields.
CPM/met/10^6
No Template
GFP plasmid No Feed
GFP Plasmid With Feed
0
0.000
0.005
0.007
1
0.000
0.206
0.173
2
0.000
0.162
0.323
3
0.000
0.211
0.270
o/n
0.287
0.316
0.766
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data.
27 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
File name: Invitrogen IVT test with FTU template 5-21-07
4. In prior experiments we had tested adding template at 1, 2, 4, 6 and 24h after
reaction start. The increases in product peaked at the 6 h time-point. While not
reduced at the 24 h time-point, no further increases were observed. To discern the
impact of minimally adding template, we tested adding DNA at only the 6 h timepoint. The results in Table 3 show that this is advantageous.
Table 3: Effect of only one template supplement
Protocol
Template
Feed @ 30min, no template supplement
FTY 901 LEE
Feed @ 30min, 0.5ug template supplement at 6h
FTU 901 LEE
CPM/met/10^6
.211
.557
5. We tested a similar protocol on 4 FTU samples. Normalized cpm calculations show
that yields from FTU and control samples are comparable, as shown in Table 1. In all
reactions, 1 ug of linear template was used and the reactions were supplemented
once with feed at 1hr. The feed reagent was supplied in the standard kit as merely
extra buffer (at no additional cost). One template supplemented was made at 4 h.
Table 4: Assessing yields of different FTU polypeptides
Template
Total CPM
CPM/met/10^6
FTU 1419
482,867
0.489
FTU 1602
332,867
0.229
FTU 1695
457,167
0.420
FTU 1712
300,000
0.213
CalM3
639,733
0.755
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data.
File name: Invitrogen IVT test with FTU template 5-21-07, which contains worksheets for
different experiments
6. We conclude that we can substantially reduce the cost of performing these reactions by
reducing the number of feed supplements to one, and adding more template only once. This
can be accomplished because we have determined when the reaction spends out these
components. The additional substrate and ATP in the “feed” are optimally added between
0.5 and 1h after reaction initiation. The DNA expression template is optimally added between
4 and 6 h after reaction start.
C. Select and test protocols for protein purification
1. Using the products from the double-tagged His templates, we are currently testing
the efficiency of product binding to nickel beads in the presence of less urea (2M,
4M). We are also using small columns for these steps rather than free beads to
reduce sample loss.
2. The flow chart of activity for the pilot purification trial has been updated to reflect our
results (figure 2). We currently are using a His-based purification approach, with
sandwiched tags to improve tag exposure
3. To assess the need for purification relative to the endpoint use of the polypeptides,
we are preparing several pilot samples. Unpurified samples alongside His purified
sample will be delivered to UNM as soon as possible for use in their T cell assays.
These samples are described in table 5 below.
28 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 2
Table 5: Test samples to be delivered to UNM
TEMPLATE
LYSATE
ACETONE->PBS
None
unpurified
buffer exchange
GFP
unpurified
buffer exchange
FTU 1419
unpurified
buffer exchange
FTU 1712
unpurified
buffer exchange
NI+ PURIFIED
purified
purified
purified
purified
4. Significant decisions made or pending
a. The new IVT protocol is very attractive. This cost and technically optimized protocol will
not require purchase of additional feed reagent. The standard kit format supplies extra
buffer which constitutes additional substrate. This is the material that we used in the
above experiments. We observe that this is sufficient to provide high yield reactions.
b. His/Nickel-based purification may require double tagged polypeptides and partial folding
for optimal binding and release, respectively.
c. If raw or buffer-exchanged lysates work in T cell assays no purification will be necessary.
5. Problems or concerns and strategies to address
We are working to address the elution problem in our purification steps for the His tag/nickel bead
strategy and for the biotin/avidin strategy. We are testing the utility of including a His tag at both
ends of the molecule, so as to enable the use of reduced urea concentrations without reduced tag
exposure. Partial folding will prevent exposure of very hydrophobic, and thereby sticky, regions.
This should improve elution efficiency.
6. Deliverables completed
None
29 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
7. Quality of performance
Very good
8. Percentage completed
96%
9. Work plan for upcoming month
a. We will be sending UNM 2 FTU and 1 non-FTU polypeptides prepared 3 different ways
by July 15th or sooner.
b. We look forward to working with them on selecting a final protocol for proteome
preparation.
c. Our goals for the next month or two are to complete Milestone 26 and confer with UNM
on their work on Milestone 27.
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
No new experimental work done
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
a. One of the impediments we have encountered in this milestone is the lack of bona
fide positive control proteins or peptides that we can use to develop the peptide
screening assay. We have recently obtained from Dr. Dan Clemens purified GroEL,
KatG, and Bfr proteins, which he had shown to consistently stimulate antigen-specific
T cell proliferation. ASU is also synthesizing long peptides from F. tularensis
proteins, such as Tul4, that are known or hypothesized to stimulate antigen-specific T
cell proliferation. We will now use these proteins and peptides to develop our peptide
screening assay.
6. Deliverables completed
NA
7. Quality of performance
No progress
8. Percentage completed
10%
9. Work plan for upcoming month
a. Test three different lots of FCS for the one that will allow for the highest number of T
cells we can include in the assay. This may increase the sensitivity of our assay
b. Develop the peptide screening assay using the purified GroEL, KatG, and Bfr as
positive controls
c. Determine whether increasing the number of T cells and/or APC would be better for
this peptide screen
30 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
d. Determine whether IFN ELISpot assay would be better than T cell proliferation
assay for this peptide screen
e. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
f. 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)
Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1. We have been finding that the chip-synthesized “oligomixes” from LC Sciences are highly
variable in quality and yield. In particular we found that the concentrations of the oligomixes
varied by almost 3 fold, whereas they should all be very similar. Also we used the oligomixes
in synthesis reactions to assess quality. We found a 100% correlation between the yield of
the mix and their successful use in gene building. Control oligos were used in side by side
reactions. This month we identified an alternative source: Agilent. While they are not
commercially selling these parallel-synthesized oligos we have been able to work out an
arrangement to test 4 chip libraries. Their technology is more advanced and oligo quality is
initially assured by their QC’ ing of DNA concentration before mailing out. This is very
encouraging.
2. We have competed the design and predictions of all gene building oligos. Two chips are on
order from LC science and we are placing an order for 4 chips with Agilent. This will allow us
to directly compare results.
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
The protocol for synthesis of recoded ORFs is likely to take some development, but no major
impediments are expected.
6. Deliverables completed
None
7. Quality of performance
31 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Very Good
8. Percentage completed
14%
9. Work plan for upcoming month
Chip synthesized oligos will be received and sets of initial synthetic ORFs will be assembled, and
tested in IVT reactions.
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

Reconstruction RNA samples are prepared with purified RNA from SCHU S4 bacteria diluted
into normal mouse lung RNA. These are then amplified via LAPT with genome-directed
primers (GDPs). We found that lung RNA prepared by Tri-Reagent RNA isolation was
incompatible with the LAPT system in that these samples did not amplify as expected.
However, if the RNA was subsequently cleaned with Qiagen Rneasy columns, amplification
yields increased 3-5 fold (Table 1).
Sample
Experiment
RNAeasy Cleanup
NML + 1.0 g SCHU S4
NML + 0.1 g SCHU S4
NML + 0.01 g SCHU S4
NML + 0.001 g SCHU S4
Total g Yield Post Amplification
LAPT-5
LAPT-6
LAPT-7
No
Yes
Yes
15
38
87
15
56
49
8
86
44
16
*
58
Table 1. Total microgram yields of amplified samples showing that using RNA after clean-up
increases amplification efficiency. NML= normal mouse lung. *Indicates lost sample as a
result of tube failure during centrifugation.

RNA samples from LAPT 7 have been processed for labeling and hybridizations are
scheduled for this next work period.

Notebook/File locations …
a. LAPT-6 Notebook 405, pages 21-22;
b. LAPT-7 Notebook 405, pages 23-24;
4. Significant decisions made or pending.
All reconstitution RNA will be cleaned through Qiagen RNAeasy columns before amplification.
32 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
5. Problems or concerns and strategies to address
We need to continue these studies to determine the lower detection limit of the LAPT
amplification process.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
90%
9. Work plan for upcoming month



Perform hybridizations with samples from LAPT-6 and 7
Repeat reconstitution experiments with SCHU S4-spiked normal lung RNA. (LAPT-8)
Perform hybridizations of total SCHU S4 and LVS RNAs and amplified reconstituted RNA on
in-house and TIGR slides.
10. Anticipated travel
None
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

Sample

Samples reported at last teleconference from LAPT-5 revealed that the RNA from UNM had
not been processed for cleanup. Despite the problems noted for reconstruction samples in
LAPT-4, Milestone 33, it appeared that there were minimal problems with the amplification.
However, problems were noted on the hybridizations with very low signal intensities. We
determined that the UNM samples needed to be processed for clean-up and repeated for
LAPT amplification. Two experiments were performed LAPT-6 and LAPT-7 revealed that
RNA after cleaning resulted in a 2-3 fold increase in amplification efficiency (Table 2)
Experiment
RNAeasy Cleanup
MS2
MS3
MS5
Total g Yield Post Amplification
LAPT-5
LAPT-6
LAPT-7
No
Yes
Yes
78
73
102
53
73
91
76
*
118
Table 2. Total microgram yields of amplified samples showing that using RNA after clean-up increases
amplification efficiency and consistency. *Indicates lost sample as a result of tube failure during
centrifugation.
33 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam


RNA from LAPT-6 and LAPT-7 were processed for labeling and equal amounts of labeled
cDNA were hybridized to test substrates Poly-L-Lysine (PLL) as compared to Corning
UltraGap microarray slides. These slides hybridized in the ArrayIt hybridization chambers
which are a standard static, slide-coverslip process. These chambers have been used for all
of the early hybridizations using purified RNA described in previous reports.
As shown in Figure 1, samples from each of the three mice had an overall increase in, and
broader range of, signal intensities on PLL slides as compared to Corning Ultragaps. The
broader range of signal intensities may be useful to analyze differences of expression from
signal intensities in the lower range. Normalized signal intensities are presented for
comparison in Figure 2. We interpret that PLL substrate slides are suitable for detecting
gene expression of amplified F. tularensis RNA from mouse tissues.
Figure 1. Raw signal intensities of amplified samples
from Mouse 2, 3, and 5 (MS2, MS3, and MS5,
respectively) on either Corning Ultragap (C) or inhouse poly-L-lysine (PLL) substrates.

Figure 2. Normalized signal intensities of amplified
samples from Mouse 2, 3, and 5 (MS2, MS3, and
MS5, respectively) on either Corning Ultragap (C) or
in-house poly-L-lysine (PLL) substrates.
We next compared whether a mixing hybridization system would give superior detection of
signal, relative to the static hybridization system. Again, equal amounts of labeled cDNA
were hybridized in the various hybridization systems. In LAPT-6, we hybridized 5
micrograms of each labeled RNA in both the MAUI and ArrayIt chambers. The Maui system
did provide enhanced signal detection as evidenced by the marked increase in raw signal
intensities for MS 2 sample. However, when comparing 5 micrograms of purified labeled
cDNA, this difference was not observed (data not shown). The increase in raw signal
acquisition was not evidenced in LAPT-7. Normalized signal intensities are presented in
Figure 4 for comparison.
34 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 3. Raw signal intensities of amplified
samples from Mouse 2, or 3, (MS2, MS3) using
either the Maui mixing hybridization station (M) or
the ArrayIt static chamber (A)
Figure 4. Normalized signal intensities of amplified
samples from Mouse 2, or 3, (MS2, MS3) using either
the Maui mixing hybridization station (M) or the ArrayIt
static chamber (A)

We next compared the normalized gene expression profiles by Spearman rank correlations
using GeneSpring GX to determine if there were differences in ranking resulting from the
difference in signal intensities. As shown in Figure 5, the Spearman correlation coefficient (
we utilize a value of >50 as a good coefficient) shows that between hybridizations and
experiments we obtained adequate correlations for all but one sample (LAPT-7 Maui v LAPT7 ArrayIt), however, this value is close to our cutoff of acceptability. The reason for the
differences in the Maui v ArrayIt comparison between LAPT-6 and 7 is not readily evident; we
obtained roughly equivalent labeling efficiency between LAPT-6 and LAPT-7 and used
equivalent amounts of labeled cDNA for each slide. Our initial interpretation, based on LAPT7 and the spearman correlation results is that, we can get equivalent data interpretation
independent of mixing the sample during hybridization.

We next combined data from multiple hybridizations into a single analysis of to average all of
the gene expression profiles of all SCHU S4 from in vivo mouse samples from all substrates
and hybridization conditions and compare the expression levels to all previous RNA samples
from in vitro grown SCHU S4. This stringent comparison allowed us to utilize a T test
analysis to identify genes equivalently expressed between the replicates which were
significantly different as a result of the in vivo vs. in vitro growth condition. The T test
analysis performed in GeneSpring identified 208 genes significantly different between the in
vivo and in vitro conditions Figure 6. These genes were further processed to identify only
those with signal intensities greater or less than 1.5 fold difference between the in vivo and in
vitro conditions. The results are shown in Table 3. Two genes are highlighted in the
increased In Vivo group. FTT0199 was the only gene identified in the previous comparison
that was also identified in the current analysis. Since, as noted in section 3, there were lower
hybridization signals in that first comparison, the data were preliminary. We have a higher
level of confidence in the current two experiments because of the reproducibility between
amplifications and the stringency of requiring similar expression profiles between experiments
and hybridization conditions. FTT0956c has been recently identified as immunogenic in
BALB/c mice after immunization with killed LVS cells by Eyles et al (EPublication ahead of
print in Proteomics 2007, Immunodominant Francisella tularensis antigens identified using a
35 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
proteome microarray). This list will be further refined with repeated experiments that will be
done in the next month.
Figure 5. Spearman correlation coefficients
analyzed by GeneSpring GX between various
samples between experiments and hybridization
systems
Figure 6. Heatmap of the 208 significantly different
genes over multiple hybridizations between SCHU
S4 RNA isolated from mouse lungs (In Vivo) to RNA
isolated from in vitro grown SCHU S4.
Table 3. Ranked genes differentially expressed, greater than 1.5 fold, identified by T-Test analysis when
comparing in vivo lung samples to in vitro SCHU S4 transcripts. The systematic gene and common gene
names are presented. The Log Ratio of the normalized expression values of In Vitro to In Vivo are
36 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
presented negative values would indicate increased expression In Vivo and positive values are
decreased In Vivo relative to the In Vitro results.

Notebook/File locations …For the data in figures 1-6 and tables 1-3 of Milestone 34
LAPT-6.
Notebook 405, pages 21-22;
Electronic location… R:\GeneVac\FTU\Contract\Microarray\Milestones\34\LAPT-6
LAPT-7
Notebook 405, pages 23-24
Electronic location… R:\GeneVac\FTU\Contract\Microarray\Milestones\34\LAPT-7.
Directory structure for LAPT 6
Directory structure for LAPT-7
4. Significant decisions made or pending.



All in vivo RNA samples must be cleaned on Qiagen (or equivalent) RNAeasy columns
before performing amplifications
PLL slides as substrates for printing provide equivalent performance for in vivo amplified RNA
samples as compared to Corning Ultragaps.
Using standard ArrayIt static hybridization chambers can provide equivalent data to Maui
mixing hybridization system.
37 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
50%
9. Work plan for upcoming month


Perform additional reproducibility studies to amplify the three UNM mouse lung samples and
test the amplified, labeled RNA on both in house and TIGR arrays.
We will perform at least one additional Maui v ArrayIt comparisons.
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
No new experimental work done on this milestone
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
10%
9. Work plan for upcoming month and next 6 months
UNM will isolate RNAs from LVS, SCHU S4,and infected mouse organs, as needed by ASU.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
38 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
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
This milestone has just begun.
4. Significant decisions made or pending.
None at this time
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
0%
9. Work plan for upcoming month
UNM will perform log dose titration of mice with varying numbers of F. tularensis SCHU S4 from
100 to 106 organisms per mouse intranasally and harvest tissues within several hours. RNAs will
be isolated and sent to ASU to determine lower level of detection of LAPT process.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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: 4/30/07
3. Work performed and progress including data and preliminary conclusions
Summary: NER deficient strains of Ft novicida (uvrB and uvrA single and the uvrA uvrB
double mutant strains grow at the same rate as the wild-type U112 strain in Chamberlain’s
defined medium (CDM), in J774 macrophages, and in lungs, livers and spleens of Balb/c mice
following intravenous (IV) injection. NER deficient strains were all highly virulent when
administered IP or IV, but when delivered SC, all Ft novicida NER mutants were approximately 1
log reduced in virulence compared to U112. This correlates with a decrease in the ability of the
uvrB mutant to disseminate to the lung following SC administration.
4. Significant decisions made or pending
The scientific work for this milestone is complete. Ft novicida NER mutants are not significantly
attenuated for virulence in mice. All of the Ft novicida NER mutants had indistinguishable
phenotypes, suggesting that there is no advantage to using the uvrA uvrB double mutant.
These observations have led us to make the decision to go forward with MS 43, in which we
proposed to screen a panel of attenuated NER-deficient double mutants of Ft novicida. For these
39 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
experiments we have made the decision to use uvrB as the NER mutation in combination with
pdpD, iglA, iglB, iglC, iglD mutations.
5. Problems or concerns and strategies to address
Abrogation of the NER pathway does not result in a dramatic loss in virulence, thus we will screen
for a secondary attenuating mutation that can be used in SchuS4–based vaccine to ensure safety
of this vaccine.
6. Deliverables completed
Growth rates of Ft novicida wild type and Ft novicida uvrA, uvrB, and uvrAuvrB mutants
determined in broth and in macrophages. LD50 comparison between strains administered by the
IP, IV, and SC routes have been completed. In vivo growth rates have been determined following
IV and SC administration.
7. Quality of performance
Excellent progress
8. Percentage completed
100% scientific work completed. Milestone completion report is pending.
9. Work plan for upcoming month
Work for this milestone is complete, and we expect to finish the milestone completion report by
the end of June.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: We have determined that all the NER-deficient strains of Ft. novicida are slightly
more sensitive to photochemical inactivation than wild type. 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. We have demonstrated that KBMA Ft. novicida are highly
attenuated for virulence. We are in the process of testing the stability of a frozen KBMA lot of
uvrB Ft. novicida at –80oC and have demonstrated that metabolic activity is retained for 3
months.
1) To address the concern that the NER-deficient strains of Ft. novicida are not more significantly
sensitive to photochemical inactivation, we have proposed a series of additional experiments to
compare the sensitivity of the uvrB to U112 to alternative DNA damaging agents, and measure
uvrB transcriptional response. We have ordered and obtained 4 alternative DNA damaging
agents to including mitomycin C, doxorubicin, benzo[a]pyrene and 4 nitroquinoline-N-oxide. We
have designed, ordered, and received 2 pairs of oligonucleotides for amplification of the uvrB
gene for quantitative real time PCR (qPCR). We have confirmed that both pairs of
oligonucleotides amplify a PCR product from U112 colonies but do not amplify a product from
40 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
uvrB strain. These primers will be used to measure the amount of uvrB RNA by reverse
transcriptase (rt)qPCR. (NB 980-039)
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: One possible
explanation for this is that there is a redundant DNA repair mechanism functioning in Ft novicida;
however, the high degree of metabolic activity retained by the mutant and wild-type strains after
photochemical inactivation suggests that the wild type may be highly sensitive to photochemical
inactivation under these conditions and that the KBMA strategy is still viable. We will measure the
sensitivity of NER mutants to a panel of DNA damaging agents and compare them to wild type.
We will investigate whether the uvrB gene is induced in response to photochemical inactivation
with S-59 and UVA light or in response to other DNA damaging agents. These experiments
should help us understand why the NER mutants are only slightly more sensitive to
photochemical inactivation compared to wild-type.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
Good progress
8. Percentage completed
75% of scientific work completed on the milestone
9. Work plan for upcoming month
We will compare the sensitivity of uvrB and U112 to 4 DNA-damaging agents including
mitomycin C, doxorubicin, benzo[a]pyrene and 4 nitroquinoline-N-oxide. If there is a difference in
the sensitivity of uvrB and U112 to these agents, we will determine whether the uvrB gene is
induced in the wild type after DNA damage by rtqPCR. We will then compare the uvrB gene
induction to treatment with S-59 and UVA.
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.
41 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
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 (AS7-017). 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
(AS07-045) in which only the serum from CD8 depleted animals provided any protection
against a lethal U112 challenge.
1) This month we performed a “whole bug” ELISA analysis on the serum that was used for
the adoptive transfer study to measure the amount of anti-Ft antibody that was transferred in
AS07-045. KBMA Ft novicida uvrB were used to coat wells of microtiter plates at a
concentration of 5.12 x106 bacteria per well. Serum from naïve mice or surviving mice
vaccinated twice with KBMA Ft novicida uvrB, depleted of T cell populations or mock
depleted and subsequently challenged with a 100x LD50 dose of U112 were adsorbed to the
killed bacteria and detected with an goat anti-mouse HRP conjugated antibody. As shown
below, all vaccinated animals had significant anti Ft antibodies, and the animals that were
depleted of CD8+ T cells had the highest anti-Ft titer. Transfer of this high-titer serum
correlated with increased protection in the adoptive transfer study (AS07-045), and suggests
that the protection we see after vaccination with KBMA Ft novicida uvrB correlates with
humoral immune responses.
Anti-FT ELISA
7000
6000
Titer
5000
4000
3000
2000
1000
0
Naive
Mock
depleted
CD4
depleted
CD8
CD4+CD8
depleted depleted
NB: 980-031
4. Significant decisions made or pending
The KBMA uvrB Ft. novicida vaccine was 100% protective only after a single administration at
very high doses, so we have chosen to pursue a repeat dosing regimen that appears to provide
100% protection at sub-toxic levels.
5. Problems or concerns and strategies to address
We have recently demonstrated that protection against lethal Ft novicida challenge is T-cell
independent and that adoptive transfer of high-titer anti-Ft serum correlates with survival benefit.
These data suggest that humoral immunity plays a significant role in protection of mice against a
42 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
lethal Ft novicida challenge and make it difficult to rank KBMA vaccine candidates that elicit a
potent T cell response. 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
Good progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We will wait for delivery of the ova-tagged strain of uvrB from Karl Klose to determine whether
KBMA Ft novicida can induce a potent CD8 T cell response
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
To inactivate uvrA in LVS, we were in the process of constructing a Targetron vector for targeting and
inactivating the uvrA gene. The goals of this milestone have changed somewhat to minimize work on
uvrA and to incorporate creating a T-cell epitope tagged protein. The work described below is
finishing up the uvrA work in progress; the work on the T-cell tagged protein is described afterwards.
uvrA Mutant in LVS:
3.1
The plasmid pKEK1140 was modified as an intron expression vector and will serve as the
backbone for TargeTron re-targeted for Ft. A 350bp PCR product will be cloned into pKEK1140
to mutate (re-target) intron RNA. Then the mutated pKEK1140 will be transformed into LVS and
express RNA-protein complex (RNP). Re-targeted RNP will locate the LVS genomic target and
insert RNA, reverse transcribe cDNA, and LVS enzymes will repair to create an UvrA mutant
LVS.
3.1.1 Made 4-primer master mixes for PCR with IBS, EBS1d, EBS2, and EBS Universal
primers which were selected from TargetTron Design Web site and ordered.
3.1.2 Performed PCR to get 350bp product with 4-primer mixes. Set up PCR as follows;






23 ul ddH2O
1.0 ul 4-primer mix
1.0 ul Intron PCR template
25.0 ul JumpStart RED taq Ready mix
AT 94C 30sec, 94C 15sec/55C 30sec/72C 30sec//30 cycles, then 72C 2min .
Gel picture:Figure 1. The largest band is about 350 bp.
43 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Data recorded on UTSA TVDC notebook #2, page 103.
3.1.3
3.1.4
3.1.5
3.1.6
3.1.7
3.1.8
3.1.9
Performed gel purification for 350bp PCR product with QIAquick Gel Extraction Kit.
Double digested gel purified 350bp PCR product and pKEK1140 with restriction enzyme XhoI and
BsrGI to generate the correct fragment ends to allow insertion of the digested PCR product into
the pKEK1140 vector.
Performed gel purification for both double digested products with QIAquick Gel Extraction Kid.
Ligated digested 350bp PCR product into digested pKEK1140 with T4 DNA ligase.
Transformed pKEK1140 inserted with 350bp PCR product into DH5 E.Coli. competent cells
using electroporation technique.
Spread transformed bacteria onto LB/Xgal/Kanamycin(50ug/ml) plate, and incubated at 37C for
overnight.
Screen white colonies for insertion of 350bp PCR product into the pKEK1140 using BglII
digestion with pKEK1140 for control.
Gel picture: Figure 2
Figure 2
3.1.10 The difference between the parent pKEK1140 and the mutant pKEK1140 is that the largest band
is a little bit more than 4.0kb in parent plasmid and a little less than 4.0kp in mutant plasmid after
being digested with BglII. As the gel picture shows, the colonies above (except for colony6) are
correct with 350bp PCR product being inserted into pKEK1140.
3.1.11 Since UvrB mutant LVS has been made, there is no need to make UvrA mutant LVS. We decided
to stop at this stage.
Data recorded on UTSA TVDC notebook #2, page 75-78 for both the figures above.
T-cell epitope tagged protein.
3.2
A new focus of this milestone is to create a T-cell epitope tagged protein that is expressed by F.
tularensis within host cells. Ideally, the protein should be secreted into the host cell. The only
well-characterized secreted protein is PepO, and the Tcell tag is SIINFEKL. In consultation with
44 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Justin Skoble, we are creating a plasmid to express PepO-SIINFEKL to transform into F.
tularensis, then send to Cerus. SIINFEKL is derived from a model MHC class I-restricted antigen,
OVA, and there are Tcell clones that specifically recognize SIINFEKL bound to MHC-I. Thus if F.
tularensis expresses SIINFEKL, then it can be determined how well MHC-I presentation occurs in
F. tularensis-infected cells/animals. We already have a plasmid, pKEK1145, which is a pBAD24
derivative that expresses PepO- Flag. A pair of complimentary oligonucleotides encoding
SIINFEKL will be used to replace the FLAG tag fragment in pKEK1145.
3.2.1 Performed annealing PCR for SIINFEKL. Two primers for PCR are shown below:
 TCell tag for: 5’-TCG AGT CAA TAA TAA ATT TCG AAA AGC TTT AGC TGC A-3’
 TCell tag rev: 5’-GCT AAA GCT TTT CGA AAT TTA TTA TTG AC-3’
Set up PCR reaction:
1ul TCell tag for (100pmol/ul)
1ul TCell tag rev (100pmol/ul)
18ul DNA Bind Buffer
At 96ºC 1min, then 0.1ºC/s to 4.0ºC
Gel picture: Figure 3.The expected bands should be about 37bp which are visible on the gel.
Figure 3
3.2.2
3.2.3
3.2.4
3.2.5
3.2.6
3.2.7
Performed gel purification for PCR product using QIAquick Gel Extraction Kit.
At the same time, digested the plasmid pKEK1145 with XhoI and PstI individually, then purified
gel for digested plasmid using the same kit as above.
Ligated gel purified SIINFEKL PCR product with digested pKEK1145 at 16ºC for overnight.
Transformed pKEK1145 with SIINFEKL insertion into DH5α E.Coli., then spread transformed
DH5α onto LB/Amp(100ug/ml) plate. Incubated at 37ºC for overnight.
Performed mini prep for 10 colonies from LB/Amp plate after transformation with QIAprep Spin
Miniprep Kit. Run gel to select correct colonies for PCR later with pKEK1145 for control.
Gel Picture: Figure 4
Figure 4
The mutant pKEK1145 should be about the same size as the parent plasmid, so colony 7 and
colony 9 are not correct
3.2.8
Performed PCR for SIINFEKL insertion for the mini preps (except for colony 7 and 9) with
pKEK1145 for control. The positive PCR product should be about 300bp. Set up following PCR
reaction:
45 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
32.6ul ddH2O
5.0ul 10xBuffer#1 for KOD
5.0ul KOD dNTPs
2.0ul MgCl2
1.0ul mini prep DNA
2.0ul PepO For primer
2.0ul SIINFEKL Rev primer
0.4ul KOD HiFi DNA polymerase
At 98ºC 1min, 98ºC 15sec/57ºC15sec/72ºC 1min//30cycles
Gel picture: Figure 5
Figure 5
3.2.9
The gel picture showed that there was SIINFEKL insertion in the plasmid pKEK1145, but the new
construct needs to be confirmed by west blotting for SIINFEKL protein detection and also
sequencing for SIINFEKL genes.
Data recorded on UTSA TVDC notebook #2, page 103-106 for figures 3, 4 and 5 above.
4. Significant decisions made or pending
It was suggested that it was unnecessary to make UvrA mutant LVS since UvrB mutant LVS had
been made, so we made decision to stop making UvrA mutant LVS, and start working on the project
about T-Cell tagged protein.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed.
Approximate 30% of scientific work completed on the milestone for T-Cell tagged protein project. Prior
month for uvrA/uvrB mutant effort was about 70%. 50% of the original milestone was completed with
the creation of the uvrB mutant in LVS
9. Work plan for upcoming month
i. Send sequence to confirm SIINFEKL insertion.
ii. Perform west blotting with SIINFEKL antibody to detect SIINFEKL protein. We will receive the
antibody to SINFEKL this month.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
46 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
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 replicates 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. 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. We have demonstrated that KBMA LVS (Wt) IV LD50 is 6.8x108,
which represents a 4-5 log attenuation compared with live LVS. We have demonstrated that
doses of KBMA 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.
Our prediction is that the KBMA LVS may be more potent than HK in a SchuS4 challenge
model. We also anticipate that a uvrB mutant may be slightly more potent.
1) Vials of KBMA LVS (WT) were sent to Terry Wu at UNMHSC for SchuS4 protection
studies in mice.
2) In order to measure the T-cell response to vaccination with KBMA LVS, we attempted to
directly measure the frequency of Ft specific T-cells after vaccination. At the International
Tularemia meeting in Woods Hole, Dr. Jeff Frelinger's group presented a poster in which they
isolated a C57Bl/6 mouse CD4+ T cell clone that reacted to the Ft Tul4 protein. They performed
mapping experiments by deletion analysis and defined the immunoreactive amino acid sequence
to be RLQWQAPEGSKCHDTS. We had this peptide synthesized and attempted to measure the
number of T-Cells that respond to this peptide by intracellular interferon-gamma (IFN-) cytokine
staining (ICS) or ELISpot assay. 5 mice per group were vaccinated with HBSS, 1x108 KBMA
LVS or 1x103 live LVS. 7 days after vaccination, spleens were harvested and single cell
suspensions were analyzed for IFN--producing cells by ICS and ELISpot (IM07-058).
For ICS staining, isolated spleen cells were stimulated with 1 uM of the peptide for 5 hours in the
presence of brefeldin A, surface stained with anti-CD4 and anti-CD8, then fixed, permeabilized
and stained for intracellular IFN-. Data was acquired on a FACSCanto flow cytometer. By ICS
staining there were no measurable IFN--secreting CD4+ T cells responding to the Tul4 peptide
after vaccination with live or KBMA LVS compared to vehicle control animals.
47 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
IM07-058 ICS Data
1.0
1.0
0.8
0.8
%IFNg+CD4+
%IFNg+CD4+
Unstim CD4
0.6
0.4
0.2
Ft peptide CD4
0.6
0.4
0.2
0.0
0.0
HBSS
LVS
KBMA
HBSS
Immunization
Unstim CD8
KBMA
Ft peptide CD8
1.0
1.0
0.8
0.8
%IFNg+CD8+
%IFNg+CD8+
LVS
Immunization
0.6
0.4
0.2
0.6
0.4
0.2
0.0
HBSS
LVS
KBMA
0.0
HBSS
LVS
Immunization
KBMA
Immunization
For ELISpot analysis of IFN- production, 2x106 cells were placed onto anti- IFN- coated filter
plates with 1uM of the peptide. 14 hours later, cells were washed away and bound IFN- detected
with anti-IFN--biotin and finally streptavidin-AlkPhos. Plates were developed using AlkPhos
substrate kit and read on an automated ELISpot reader. By ELISpot analysis there was no
significant tul-4 peptide-specific increase in IFN-producing cells, although LVS immunization
appears to non-specifically increase the number of IFN-producing cells.
IM07-058 ELISpot Data
Unstim
Ft peptide
100
SFU per 2e5 cells
SFU per 2e5 cells
100
80
60
40
20
80
60
40
20
0
0
HBSS
LVS
Immunization
KBMA
HBSS
LVS
KBMA
Immunization
48 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
3) We have performed stability studies on KBMA LVS lot 968-040 Arm-1 that demonstrate
that the metabolic activity of the lot is stable for 3 months. We will continue to perform MTS
assays at 6 and 12 months.
Nominal 1e8 particle/mL (KBMA) F. tularensis holarctica LVS
1.0
0.9
OD (490nm)
0.8
0.7
0.6
0.5
0.4
T=0 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=1 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=2 698-040 Arm-1 (10uM S-59, 6J/cm2 UVA) "
T=3 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
0.3
0.2
0.1
NB968-105
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
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. We will
compare the photochemical inactivation profile of a uvrB mutant LVS when it arrives from UTSA.
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS appears to be independent of whether the vaccine
has metabolic activity. This suggests that comparison of various routes, regimens, or
formulations will be difficult to optimize by protective efficacy. A SchuS4 challenge model may be
more appropriate.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
45% of scientific work completed on the milestone
9. Work plan for upcoming months
We expect to receive a uvrB mutant LVS from UTSA and we will measure its sensitivity to
photochemical inactivation and degree of metabolic activity and compare the NER-deficient
strain with wild-type LVS.
We will measure the mouse cytokine response after vaccination with LVS in order to
characterize the innate immune responses that correlate with a protective vaccination.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
49 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose 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. The IgLD 30 PCR fragment and the IgLD 255 PCR fragment which were digested
(XhoI/BsrGI) and gel isolated were used in ligation reaction with the KEK1140 prepared
plasmid as described in last month’s report (i.e. this was also digested with the same
enzymes and gel isolated).
b. These ligations were chloroform:phenol extracted followed by ethanol precipitation and
the final pellets were reconstituted with 10 ul sterile water and 3 ul of these purified
ligations were used to transform DH5α cells.
c. This resulted in only one colony per transformation reaction; that is, the KEK1140+ IgLD
30 XhoI/BsrGI and the KEK1140+ IgLD 255 XhoI/BsrGI, respectively. In addition, the
religation (plasmid without insert ligation) resulted in no colonies, as expected for this
negative control.
d. Plasmid was isolated from these the KEK1140+ IgLD 30 XhoI/BsrGI and the KEK1140+
IgLD 255 XhoI/BsrGI, colonies via Qiagen’s mini plasmid kit and PCR was performed to
check for insertion of the intron fragments (i.e. IgLD 30 and 255, respectively). The result
was negative; therefore, we prepared more IgLD 30 and IgLD 255 to use in another
ligation reaction. That is, these “intron fragments” were re-amplified and as described in
earlier report and digested with appropriate enzymes then gel isolated with Qiagen kit to
purify for ligation reaction. Data located in TVD UTSA notebook 5, page 25.
e. Attempted again to amplify the KEK1090 plasmid described earlier to remove the
chloramphenicol gene and replace with ampicillin gene. Two attempts did not yield the
≈5 Kb fragment expected from this amplification based on sequence data. We decided to
remake this construct by re-amplifying the parent plasmid pDS132 to use to clone in the
GroEL promoter and the ampicillin gene. We used the oligos pDS132R and pDS132F
described in milestone 39 earlier report. These oligos will replace the multi-cloning sites
(MCS) and the SacB promoter with a new MCS and will allow for the introduction of the
GroEL promoter.
f. Amplification of the pDS132 was successful (Fig 3) so I digested with Pst I enzyme and
after gel isolation I did a ligation reaction and transformed into SM10λpir cells. Will
analyze the results on next months’ report. Data located in TVD UTSA notebook 5, page
24.
50 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 3
This figure 3 represents the PCR products generated from using the pDS132 plasmid as
a template and oligos pDS132R and pDS132F with deletions made and addition of the
new MCS we expect the PCR product to be 5.2 Kb. Lanes 2 thru 5 are the same reaction
loaded for band isolation. The lower band is the correct product.
g. We currently still have to make deletions in SCHU S4 and in this milestone all the
required genes are duplicated in SCHU S4. The organization of these genes are such
that the genes pdpA and pdpD are positioned at the extreme 5’ and 3’ end on this gene
cluster (i.e. the FPI pathogenicity island). Therefore, we will attempt to create insertions
in pdpD and pdpA that contain a recognition site for the FLP recombinase (“FRT
recognition site”). Insertion of FRT sites in pdpA and pdpD, followed by the expression of
FLP recombinase in this strain will result in the excision of the entire FPI gene cluster
(≈31.5 Kb fragment). This will facilitate strain construction in SchuS4, because then only
one FPI gene will have to be mutated instead of two. The idea is based on the Wanner,
B. L paper in The Proceeding of the National Academy of Science (PNAS 2000;97;66406645).
We have on hand a pdpA deletion in Francisella .novicida, KKF82
pdpA::KanFRT, which is the pdpA deletion with a Kanamycin selection gene and the FRT
recognition site.
h. Chromosomal DNA was isolated from the KKF82 strain and used as a template to
amplify the entire pdpA deletion with designed oligos that will allow for cloning this into
KEK962. This plasmid is a pUC118 construct which contains the Francisella promoter
driving the expression of an erythromycin gene (FnErmC). The cloning sites used were
KpnI and EcoRI for the plasmid and the oligos were designed with a Mfe I site which
creates a compatible cohesive end for EcoRI. The oligos (the underline is the restriction
site) are as follows: Before pdpA Forward Mfe I : 5’-cgcgcgcaattgtatcctagaaactttagctcc3’ and
After pdpA Reverse KpnI: 5’ –ggacgcgcgggtaccttcattatttgcaaatacgagagatg- 3’
i. The PCR product resulting with these oligos was digested subsequently with Kpn I then
Mfe I and the KEK962 was digested with Kpn I then EcoRI. After gel isolations to purify
these DNAs, a ligation was performed and then we transformed DH5α cells.
j. Many colonies resulted and three constructs resulted with a BamHI digestion screen
which should generate approximately ≈6100 bp (mostly vector) and ≈2500 bp (3’ end of
pdpA and the FnKan fragment). See Figure 1.
51 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
Figure 1:
This figure represents some of the pKEK962+pdpA constructs that resulted from transformation
of DH5α cells. Lanes 2 and 8 are uncut plasmid profiles. Lane 3 is the parent plasmid KEK962
which when cut with BamHI is linearized. Lanes 4, 5, 6, and 7 are various clone plasmids
digested with BamHI. The expected bands are 6.1 Kb and 2.5 Kb for the correct construct profile.
Lane 4, clone 1 may be correct. Data located in TVD UTSA Notebook 5 page 27.
k.
Prepared a large plasmid preparation of pKEK962+pdpA Clone 1 and sent for
sequencing also will transform SCHU S4 with this construct (See below for more
information). Data located in TVD UTSA Notebook 5, page 26 and 27.
II. Experiments to generate deletions in Schu4:
a. The resulting colonies from the transformation of KEK906 into KKT1 SCHU S4 strain
were analyzed and found to be only the wild type genotype. Chromosomal DNA was
isolated from 8 isolated ampicillin resistant colonies. These did grow very poorly under
ampicillin however, they did grow. Probably the fact that the ampicillin gene in this
plasmid (KEK906) is not driven by the Fn promoter makes selecting for this resistance
difficult. These genomic preparations were used as templates to perform PCR with
oligos ΔiglB Down and ΔiglD up described earlier. Results are in figure 2.
Figure 2:
This figure represents PCR products generated with oligos ΔiglB Down and ΔiglD up with various
genomic template and the plasmid KEK906 positive control. Lane 3 is the KEK906 product which should
52 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
be smaller than the wild type profile, lane 2. Lanes 4-11 are the various 906 clones resulting for the
SCHU S4 transformation experiment. They all show the wild type genotype profile. Yes. Data located in
TVD UTSA Notebook 5, page 25.
b. Performed a transformation experiment with the KEK962+pdpA construct from above with
KKT1 SCHU S4 strain, as described earlier. We first grew an overnight culture of KKT1
then prepared a log phase culture of KKT1. The log culture was harvested and cells were
washed with 0.5M Sucrose (twice). The final pellet was resuspended in 200 ul 0.5 M
Sucrose and split in half. One suspension was electroporated with 2 ug of KEK962+pdpA
and the other suspension was electroporated with no DNA as a negative control. The
transformations were place in TSA +++ media and grown for 5 hours under no pressure at
37°C then the cells were harvested and plated onto TSA +++ with 70 ug/ml Kanamycin
selection. These were incubated for 6 days and only 9 colonies (parent clones) resulted.
c. These were streaked onto another TSA+++ Kanamycin plates to generate single colonies.
The resulting single colonies (52 total) from each parent clone were patched onto various
TSA+++ plates: 70 ug/ml Kanamycin; 100 ug/ml Erythomycin and a non-select plate.
d. All colonies grew on each plate. Took 4 representative clones and did liquid cycling in
Chamberlains media (1 ml) these were grown at 30°C for several days passaging every
day. In addition, the plated colonies were passaged on nonselect TSA+++ plates for 5
cycles. That is, patched each colony from c. above onto a new plate everyday and grown
at 37°C.
e. The KEK962+pdpA construct contains a erythromycin gene in the plasmid portion of the
construct and a kanamycin gene in the pdpA deletion portion of the construct. Therefore,
our expectation is that integration of the construct will initially result is kanamycin and
erythromycin resistance. Our hope is that we will be able to get clones that have the
plasmid removed yielding kanamycin resistant clones that are erythromycin sensitive.
f. Once this cycling was complete, we prepared dilutions of all 4 liquid cycles and plated on
TSA+++Kanamycin plates to generate single colonies. From the plated cycling only two
colonies were taken to prepare a dilution to plate on the Kanamycin plates.
g. Many colonies (hundreds) resulted and approximately 22 colonies from each liquid cycle
were patched onto the three sets of TSA+++ plates described in II. c. The total number of
patched colonies among all groups was 104 and of these 19 appeared to be Erythromycin
sensitive. So these 19 colonies are Kan resistant and Erythromycin sensitive; the
combination should indicate the presence of a pdpA deletion in SCHU S4 and loss of the
plasmid
h. These will be evaluated on next month’s report. Data located in TVD UTSA Notebook 5,
page 28-29. (Is this for the data in figure 2 for this milestone,) No, this is for the SChU S4
transformation with the construct used from Figure 1, lane 4. Figure 2 is the results from
the attempt to get the igLC deletion using the KEK906 plasmid which did not work and we
will not continue with this construct.
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. Will continue with the iglD cloning with the KEK1140 plasmid
53 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
b. Will continue with the re-modification of pDS132 to create a ampicillin resistant plasmid to
use with KKT1 SCHU S4 strain
c. Will continue to screen for pdpA deletions in SCHU S4
d. Begin another igLC transformation experiment using KEK1160 construct developed in the
lab which contains a “intron igLC fragment” that may work in SCHU S4 strain to create
the igLC deletion. This was created in the same way as the iglD is being cloned; only
with different oligos (directed to the igLC gene). This is the targetron approach to
introduce this deletion into SCHU S4.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 05/01/2006
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 uvrBiglC mutant as a vaccine
candidate (Note book #4, page 98-99). Groups of BALB/c mice (female, 4-6 weeks) were
intranasally (i.n.) immunized with 105, 106 or 107 CFU of uvrBiglC. Mice treated with PBS
were used as a mock-control. The immunized mice were challenged with 1000 CFU of F.
novicida (~100 LD50) by the i.n. route after 30 days of vaccination. As shown in Fig. 1
(below) , uvrBiglC -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 6.
54 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
b. Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglC mutant
after vaccination (Note book #4, page 96-97). Blood was collected from the PBS- and
ΔuvrBiglC - immunized mice (as described above in) at day 14 and day 28 after priming.
Specific anti-ΔuvrBiglC total antibody titer as well as IgG1 and IgG2a isotypes were
determined by ELISA. Antigens, either UV-irradiated ΔuvrBiglC (106/well) or HEL (Hen
Egg Lysozyme, 50ng/well, an unrelated antigen as control), were coated onto 96-well
microplates and reacted with serial dilutions of sera. Goat anti mouse Ig(H+L), IgG1 and
IgG2a antibody conjugated with peroxidase were used as the secondary antibody to
determine serum antibody isotypes and titers. As shown in Fig. 2, mice immunized with
ΔuvrBiglC produced significant amounts of specific serum total 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 indicated both Th1 (IgG2a) and Th2
(IgG1)- type antibodies were produced in mice after the ΔuvrBiglC immunization. No
ΔuvrBiglC - 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.
10
8
8
Total Ab
Day 14
Titer (x1000)
6
6
Day 28
4
4
2
2
0
0
5
5
IgG1
4
4
3
3
2
2
1
1
0
0
PBS
105
106
107
0
IgG2a
PBS
105
ΔuvrBiglC
106
107
ΔuvrBiglC
Fig. 2. Humoral response to ΔuvrBiglC immunization. BALB/c mice were
intranasally immunized with 105, 106 or 107 CFU of the ΔuvrBiglC mutant or PBS
alone as mock vaccination. Sera were collected 2 weeks and 4 weeks after
immunization and used to determine titers of anti- ΔuvrBiglC 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
38 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Determine the LD50 of Ft subsp. novicida uvrBiglD double mutant.
b. Monitor Ft subsp. novicida ΔuvrBiglD replication and dissemination in mice.
10. Anticipated Travel
None
55 of 56
Tularemia Vaccine Development Contract: Technical Report
Period: 5/01/2007 to 5/31/2007
Due Date: 6/18/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn Sykes,
Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose and Bernard
Arulanandam
11. 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
a. Chromosomal DNA was purified from the F. novicida iglB mutant KKF235. 10 ug of this
DNA was cryotransformed into a F. novicida uvrB mutant KKF110 in hopes of generating
a uvrB + iglB double mutant. Cryotransformants were plated on TSA++ Erm for initial
selection. ~300 colonies were generated from this crytransformation. Colony PCR with
primers specific for iglB and ermC will be used to screen for the correct mutant.
b. Chromosomal DNA from the uvrB mutant KKF110 will be isolated and cryotransformed
into the iglB mutant as a backup to the above strategy. These cryotransformants will be
plated on Kan plates and screened by PCR with UvrBup and UvrBdn1 primers and then
cut with Bgl2.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The uvrB mutant was annotated incorrectly in the previous milestone reports as KKF71. The
correct annotation is KKF110.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
90%
9. Work plan for upcoming month
Screen potential double mutants by colony PCR and restriction digest.
10. Anticipated travel
None.
11. Upcoming Contract Authorization
None
56 of 56