Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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, 44, 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, 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. UNM/USAMRIID/USAMMDA/LBERI/True Foundation held LVS Vaccination “kick off”
teleconference on 7/16/07. First groups of participants were tentatively scheduled for
9/11/07, 10/2/07 and possibly 9/25/07
b. All laboratory, radiology, medical director and nurse certification documents were
submitted to USAMRIID
c. Requested TVDC COA to cover travel and health screenings for participants and travel
for UNM EOHS nurses
d. True Foundation completed and implemented web database to track documents and
samples being shipped to USAMRIID
e. Risk assessment forms were submitted on 16 LBERI participants and 14 attended the
first SIP Informed Consent teleconference with USAMRIID on 8/2/07
f. LVS vaccine web database is being populated by UNM and True Foundation to track
Risk Assessment form submission and acceptance, Informed Consent submission and
acceptance, Health screening appointments, and planned dates for receipt of LVS
vaccinations
g. UNM HIPAA authorization form was prepared by 8/6/07
h. Health Screenings began on 8/7/07
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
1 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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.
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
20%
9. Work plan for the next month
a. Submit Health screening test results to USAMRIID and obtain USAMRIID medical review
b. Make travel arrangements for medically eligible participants to enter LVS Vaccination
program at USAMRIID on 9/11/07 and 10/2/07. Participants will be at USAMRIID for 2
days following the vaccinations
c. Start Risk Assessments, SIP informed consent teleconference, and health screenings for
the next group of participants.
d. Maintain excellent communications with UNM EOHS, LBERI and USAMRIID
10. Anticipated travel
Travel to USAMRIID will begin on 9/9/07 when UNM EOHS nurses travel to USAMRIID for
training and on 9/10/07 when the first group of participants travel to USAMRIID for the LVS
vaccinations.
11. Upcoming Contract Authorization (COA) for subcontractors
a. UNM requested a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for
training on LVS site vaccination evaluations. (Electronic email of 8/7/07 authorized the
expenses and signed COA letter will follow)
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. (Electronic email of 8/7/07
authorized the expenses and signed COA letter will follow)
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

July 2007 LVS work focused on the Aeromist Nebulizer testing:
i. One day of bioaerosol testing was completed incorporating dilutions of fresh LVS
stock (48h culture) in the generator (Figures 1 and 2); for comparison, 3
additional sprays were conducted using the Collison nebulizer following the
completion of the Aeromist testing.
2 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
1. 12 total sprays with fresh LVS
2. 3 target concentrations for the Aeromist (1x105, 1x106, and 1x107 cfu/mL)
and 1 target concentration for the Collison (1x107 cfu/mL).
3. Actual vs. Target cfu/mL values were 0.5-1.0 log10 lower than desired
for the Aeromist nebulizer, whereas they were within 0.5 log10 for the
Collison generator. One hypothesized reason for this is that the
Aeromist nebulizer induced increased stress on the bacteria relative to
the Collison. The Aeromist nebulizer may cause greater shear forces
than the Collison. Further testing will be required to confirm or disprove
this.
4. Calculated spray factors were consistent at the three tested
concentrations
a. Values were better than those observed using the Collison
nebulizer in this series of bioaerosols and those of previous
dates. Data indicate that the Aeromist nebulizer is a promising
alternative to the Collison generator, though further testing is
required.
b. Values decreased (i.e., efficiency decreased) as concentration
increased, a consistent observation seen previously.
5. Aeromist nebulizers are low cost at $15 each and have the advantage
that the spray solution can be prepared in the Aeromist and then
transported to the testing location.
6. 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\Aeromist nebulizer\19July07
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
Figure 1. Target vs. Actual CFU/mL at three target concentrations of fresh LVS using the Aeromist and Collison generators
3 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Aeromist: Actual CFU/mL vs. Spray Factor (Fresh)
-4.50
0.00
-4.70
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-4.90
-5.10
-5.30
-5.50
-5.70
-5.90
-6.10
-6.30
-6.50
Actual CFU/mL (Log10)
7/19/2007 (Aeromist)
7/19/2007 (Collison)
Figure 2. Actual CFU/mL vs. Spray Factor at three target concentrations of fresh LVS using the Aeromist and Collison generators
4. Significant decisions made or pending
The Aeromist nebulizer continues to show promise as an alternative to the Collison nebulizer. A
significant attempt will be made to complete the Aeromist bioaerosol testing with LVS by the end
of August 2007.
5. Problems or concerns and strategies to address
Only one day of bioaerosols was conducted during the month of July. This was primarily due to
hardware issues with the exposure line. See attached memorandum from the Aerosol group for
details.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
72%
9. Work plan for upcoming month



Continue and complete bioaerosol experiments on fresh LVS with the Aeromist nebulizer
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on SBCGA
iii. Will continue doing frozen and fresh, not lyophilized
Perform bioaerosols using new frozen stock of LVS
i. Aeromist and Collison; same setup as bioaerosols performed on 19 July 2007
using fresh LVS
ii. Verify consistency with old frozen LVS stock
Initiate final decision on bioaerosol generator to be used on animal studies
10. Anticipated travel
None
4 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
No 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
a. 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
Schu4 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 Ftc40 study 2 (Notebook 104 pages 31-32, 34)
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 s.c., 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
5 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
iv. The tissues are currently being processed at LRRI and, upon return, will be
examined by Dr. Julie Hutt at UNM
b. Experiment Ftc47 (Notebook 104, pages 59-62, 66, 89-94)
i. The purpose of this experiment was to compare the kinetics of SCHU S4
proliferation and dissemination in naïve and s.c. LVS-vaccinated rats after i.t.
challenge. This is a repeat of Ftc37 study 1 and Ftc40 study 1
ii. 28 rats were left unvaccinated and 40 rats were vaccinated s.c. with 5 x 10 7
LVS
iii. 33 days after vaccination, the naïve and vaccinated rats were challenged i.t.
with 1.7 x 104 SCHU S4. This dose was similar to the one used to challenge
vaccinated rats in Ftc40 study 1 but was 100-fold higher than the one used to
challenge naïve rats in Ftc 37 study 1
iv. The numbers of SCHU S4 in the naïve lungs on days 1 and 2 were higher
than we expected and exceeded our dilution series; therefore, we do not
have reliable data for the naïve lungs at these time points. Similarly, we do
not have reliable data for the vaccinated lungs on day 1 and the vaccinated
spleens on days 2-4 because our dilution series were too narrow.
Nevertheless, table 1 shows the mean bacterial load when there were
reliable data from at least 3 rats/group
v. In naïve rats, SCHU S4 disseminated from the lungs to the spleens and liver
within the first day (culture plates contained too many bacteria to count) and
the bacterial burden reached 108 – 109 CFU by the time the rats died 4 to 5
days after infection.
vi. In vaccinated rats, SCHU S4 also disseminated from the lungs to the spleen
and liver within the first day (culture plates contained too many bacteria to
plate). However, the numbers of SCHU S4 were lower at every time point
compared with those found in naïve rats, suggesting that the vaccinated rats
were more effective at controlling SCHU S4 proliferation.
vii. We will continue to measure the bacterial load in the vaccinated rats to
determine if and when they clear the SCHU S4 challenge
Table 1. Kinetics of SCHU S4 proliferation and dissemination in naïve and s.c. LVS
vaccinated ratsa
Log10 mean bacterial load ± SD
Naive
s.c. LVS vaccinated
Day
Lungs
Liver
Spleen
Lungs
Liver
Spleen
0
4.2 ± 0.1
4.2 ± 0.1
1
NDb
2.5 ± 0.5
2.5 ± 0.4
ND
2.2 ± 0.3
0
2
ND
5.7 ± 0.2
5.0 ± 0.4
7.4 ± 0.1
4.5 ± 0.3
ND
3
8.5 ± 0.3
7.2 ± 0.2
7.1 ± 0.4
7.4 ± 0.1
4.5 ± 1.0
ND
4
8.9 ± 0.3
7.9 ± 0.3
8.4 ± 0.3
7.7 ± 0.7
5.1 ± 0.3
ND
5
Died
6.8 ± 0.4
5.0 ± 0.2
4.6 ± 0.3
6
Died
6.0 ± 0.8
3.9 ± 0.7
4.1 ± 0.8
a n = 3-5/group
b ND = No quantitative data as bacterial counts were too high in the dilutions performed
c.
Experiment Ftc48 (Notebook 104 pages 80-88)
i. The purpose of the experiment was to determine the kinetics of LVS
proliferation, dissemination, and clearance after s.c. vaccination in rats. This
is a repeat of Experiment Ftc38.
ii. 54 rats were vaccinated s.c. with 3.7 x 107 LVS
6 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
iii. 6 rats were killed every day for the first 6 days and weekly thereafter to
determine the bacterial burden in the lungs, livers, and spleen as well as the
clearance kinetics
iv. As indicated in Table 2, LVS disseminated from the inoculation site in the
skin to the lungs, livers, and spleen within the first day. However the total
number of LVS in the lungs, spleen and liver was 3-4 log10 lower than the
inoculum. This suggests that LVS was either trapped or eliminated in the
skin or localized in other sites that we did not sample.
v. Bacterial numbers peaked on day 2 in the spleen and liver and day 3 in the
lungs and declined thereafter
vi. On day 6, LVS was cleared from the liver in all six rats and from the lungs in
5 of 6 rats. LVS was completely cleared by day 14.
Table 2. Kinetics of LVS proliferation and dissemination after s.c.
vaccinationa
Log10 mean bacterial load ± SD
(No. infected tissue/total)
Day p.i.
Spleen
Liver
Lungs
1
4.2 ± 0.7 (6/6)
3.7 ± 0.2 (5/6) 2.5 ± 0.2 (3/6)
2
4.5 ± 0.2 (6/6)
4.3 ± 0.4 (6/6) 3.1 ± 0.4 (5/6)
3
4.0 ± 0.6 (5/6)
4.0 ± 0.6 (6/6) 3.5 ± 0.6 (4/6)
4
4.1 ± 0.1 (6/6)
3.5 ± 0.3 (6/6) 2.5 ± 0.6 (2/6)
5
3.8 ± 0.3 (6/6)
2.7 ± 0.3 (3/6) 2.8 ± 0.4 (4/6)
6
3.5 ± 0.2 (6/6)
0 (0/6)
2.1 (1/6)
14
0 (0/6)
0 (0/6)
0 (0/6)
21
0 (0/6)
0 (0/6)
a inoculum = 3.7 x 107CFU/rat
d. Experiment Ftc46 study 2 (Notebook 106 pages 3-6)
i. The purpose of the experiment was to determine the stability of QD655-luc8
and coelentrazine and the minimum amount of each component required for
in vivo detection after i.t. delivery
ii. QD655-luc8 was titrated from 0.05 to 5 g/rat and coelentrazine from 0.1 to
10 g/rat and delivered i.t.
iii. As shown in figure 1, at least 0.5 g QD 655-luc8 and 1 g coelentrazine are
required for detection. Therefore, we decided to use 5 g QD655-luc8 and 1
g Coelentrazine for future experiments
iv. QD 655-luc8 and coeletrazine appear to be stable for at least 1 month when
stored at 4oC and –20oC respectively. This conclusion is based on the fact
that the same reagents were used in Ftc46 study 1 performed on 6/4/07
7 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 1. Titration of QD655-luc8 and coelentrazine in Fischer 344 rats
BALB/c mice
a. Experiment Ftc 44 (Notebook 104, pages 43-46, 53, 67)
i. The purpose of this experiment was to determine whether KBMA-LVS
vaccination protects BALB/c mice from i.n. SCHU S4 challenge. In
experiments performed at Cerus, KBMA-LVS was found to be no better than
heat-killed LVS at generating protection against a lethal LVS challenge.
ii. The experimental design is shown in Table 3.
iii. As shown in Figure 2, i.n. vaccination with live LVS protected BALB/c mice
against i.n challenge with 50 and 500 SCHU S4.
iv. In contrast, KBMA LVS vaccination, with or without boost, may have only
extended survival after i.n challenge with 50 SCHU S4 by one day
v. We are continuing to monitor the survival of the mice vaccinated with live
LVS to make sure that the SCHU S4 challenge does not eventually kill them
as well
8 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Table 3. Experimental design to determine the effectiveness of the KBMA-LVS vaccine
Vaccination or boost
Challenge
Vaccination
(3 wk after primary)
(1 mo after boost)
Group
Dose
Dose
Dose
(6/grp)
vaccine route (CFU/ms)
vaccine route (CFU/ms)
strain
route (CFU/ms)
1
None
None
SCHU S4 i.n.
50
2
500
3
5000
4
5
6
Live LVS
i.n.
103
7
8
9
None
-
-
10
11
12
13
14
15
16
17
18
KBMA LVS i.v.
None
-
KBMA LVS i.n.
-
50
500
5000
KBMA LVS i.v.
108
50
500
5000
108
KBMA LVS i.v.
108
50
500
5000
-
KBMA LVS i.n.
108
50
500
5000
KBMA LVS
108
50
500
5000
108
None
-
in
9 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
10 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 2. Protection generated by vaccination (and boost) with KBMA-LVS. BALB/c mice (n = 5/group)
vaccinated and boosted, when indicated, with live LVS or KMBA-LVS were challenged with the indicated
doses of SCHU S4.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
7. Quality of performance
Good
8. Percentage completed
62%
9. Work plan for upcoming month
Rats
a. Repeat the s.c. LVS vaccination/i.n. SCHU S4 challenge experiment
b. Characterization of the Fischer 344 rat model
i. Repeat Ftc47 to determine the kinetics of SCHU S4 proliferation and
dissemination in lungs, spleens, and livers of naïve and LVS vaccinated rats.
We will add more dilutions to make sure we obtain reliable data
ii. Complete the experiment examining the histology of lungs, spleens and
livers from 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
c. Optimize the use of QD655-luc8 to track pulmonary inoculation in rats
i. Determine whether co-administration of QD655-luc8 affects the virulence of
SCHU S4 in naïve Fischer 344 rats
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
11 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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. Previous results suggested that both the CERUS and CTL protocols spared
some antigen specific (LVS) proliferation and resulted in greater than 50% cell
recovery from thawing; the Lyons protocol resulted in less than 26% cell
recovery in each of two experiments in which it was tested, but did spare an
equivalent amount of LVS-specific proliferation
3. PBMCs were prepared and frozen using the 3 different protocols on 6/11/07
(TUL 14) and 6/12/07 (TUL 15); they will be thawed on 8/6/07 and 8/7/07 and
tested for their proliferative capacity to LVS and mitogens
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 073107.svd; and in the
TVDC 1 bound notebook pages 30 – 39 and 49 (TUL 14) and pages 40 – 48 and 50 (TUL 15)
b. Update on IFN detection
i.
We have been attempting to titrate the number of cells/well in relation to
the number of LVS-induced IFN ELISPOT spots detected
a. Figure 1 shows the results of 3 experiments combined (TUL 14, 15
and 17)
ii.
We have also begun to titrate the amount of antibody used to coat the
wells of the ELISPOT plate (monoclonal mouse anti-human IFNg; G7-4,
MABtech); the manufacturer recommends 15 μg/ml; we have now tested
7.5 and 30 μg/ml
a. Figure 2 shows the results a single experiment in which we tested
PBMC from 3 NHPs on 3 different coating concentrations
PBMCs from LVS-vaccinated NHPs respond to FF LVS by secreting IFN as detected by ELISPOT
IFNgamma Spots/well
(Mean +/- SEM)
60
50
40
*
.33
.67
1
1.33
*
*
*
30
20
*
10
*
0
Media
LVS hk Hi
LVS ff Hi
Figure 1: PBMCs (from 6 vaccinated NHPs, TUL 14, 15 and 17) were plated at varying concentrations (x
106 cells/ml) and stimulated with either HK or FF LVS (1 x 105 cells/ml); * = significantly different than
media at that concentration. 1.33 x 106/ml corresponds to 200,000 cells/well.
12 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
IFNgamma spots/well
(Mean +/- SEM)
Data interpretation: Both HK and FF LVS stimulate IFN production detectable by ELISPOT when
100,000 – 200,000 cells/well are plated.
100
90
80
70
60
50
40
30
.67
1
1.33
*
IFNgamma spots/well
(Mean +/- SEM)
.67
1
1.33
LVS hk Hi
Media
70
60
50
40
30
*
*
.67
1
1.33
*
LVS hk Hi
LVS ff Hi
30 g/ml
*
*
Media
LVS hk Hi
C
*
*
20
10
0
B
*
*
20
10
0
LVS ff Hi
15 g/ml
70
60
50
40
30
100
90
80
*
*
Media
IFNgamma spots/well
(Mean +/- SEM)
A
20
10
0
100
90
80
*
7.5 g/ml
*
LVS ff Hi
13 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 2: PBMCs from 2 vaccinated NHPs, TUL 17) were plated at varying concentrations (x 10 6 cells/ml)
on IFN ELISPOT plates that were coated with varying concentrations of anti-IFN antibody (A: 7.5
g/ml; B: 15 g/ml and C: 30 g/ml) and stimulated with either HK or FF LVS (1 x 105 cells/ml); * =
significantly different than media at that concentration. 1.33 x 106/ml corresponds to 200,000 cells/well.
Data Interpretation: Differing coating antibodies concentrations didn’t make a large difference in the
ability to detect IFN spots; however, 30 g/ml looks suboptimal compared to either 7.5 or 15 g/ml in that
fewer spots are detected at the highest cell concentration tested (200,000/well). Coating with higher than
7.5 g/ml allows significant detection of spots at the lowest cell concentration tested (100,000/well; 0.67 x
106/ml). 15ug/ml appears to be the best coating concentration for the antibody to IFN
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 073107.svd;
Summary data: N:My Documents\Tularemia Contract\Internal Tech Mtg\agenda prep for 080307 and
\\Saturn\Group\Wilder Lab\TVDC\Summary reports\ agenda prep for 080307
Notebook data: TVDC 1 bound notebook pages 30 – 39 and 49 (TUL 14); pages 40 – 48 and 50 (TUL
15) and pages 63 – 70 (TUL 17).
c.
Update on LVS-stimulated proliferation
i.
Terrry Wu prepared new batches of formalin fixed (FF) and heat-killed (HK)
LVS which we tested in our proliferation assay
ii.
Figure 3 shows the results and direct comparison to the current lot of FF LVS
and HK LVS (undesignated as compared to New FF LVS and New HK LVS)
800000
ID
SC
*
600000
400000
*
x
x
New LVS ff Hi
New LVS hk Hi
LVS ff Hi
0
LVS hk Hi
200000
Media
Relative Light Units (small)
(Mean +/- SEM)
LVS-stimulated Proliferation of PBMCs from LVS-vaccinated NHPs
Figure 3: PBMCs from 6 NHPs (TUL 16 and 17) were stimulated at the indicated cell concentration with
LVS preparations at 1 x 105/ml. Two way ANOVA analysis shows that the ID group differs from the SC
group overall; * indicates different than all other stimuli within vaccination route; x indicates different than
14 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
all other stimuli within vacation route except each other (i.e. LVS ff Hi = LVS hk Hi in the SC group).
Data interpretation: The new preparations of hk and ff LVS are less able to stimulate PBMC proliferation
at the same concentration as the original preparations. The NHPs vaccinated with LVS via the ID route
appear to preferentially proliferate to the ff LVS preparation.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 073107.svd;
Summary data: N:My Documents\Tularemia Contract\Internal Tech Mtg\agenda prep for 080307 and
\\Saturn\Group\Wilder Lab\TVDC\Summary reports\ agenda prep for 080307
Notebook data: TVDC 1 bound notebook pages 63 - 70 (TUL 16 and 17).
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Will titrate the new lots of HK LVS and FF LVS to try to improve the PBMC responses. Will ask UNM
to prepare another lot of HK LVS and FF LVS.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
83% of scientific work has been completed
9. Work plan for upcoming month
1. Thaw PBMCs frozen down in experiments TUL 14 and 15 and test in proliferation and IFN
ELISPOT assays if enough cells permit
2. Begin to optimize the conditions of the IFNg ELISPOT assay by testing different detection
antibody concentrations; we’ll keep the coating concentration constant at 15 g/ml
3. Optimize the IgA anti-LVS ELISA and begin testing banked sera from TUL 8 and TUL 9
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc27.14 (Notebook 101, pages 51-53) and Experiment Ftc27.14b
(Notebook 101, pages 57-60)
i. The purpose of these experiments was to screen fetal bovine serum (FBS)
for use in T cell proliferation assays. Our goal is to identify a batch of FBS
15 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
ii.
iii.
iv.
v.
vi.
with low mitogenic activity so that we can increase our assay sensitivity by
increasing the number of splenocytes from 5 x 104/well.
We tested 4 lots of FBS: HyClone lot APA20504 (from Karen Elkins),
HyClone lot FSC28994 (reserved at HyClone), Hyclone lot FQK25099
(currently used in Lyons lab), and a batch of FBS without any identifier that
we have been using for the T cell proliferation and macrophage killing assays
We used total splenocytes rather than nylon-enriched splenocytes
As shown in Figure 3, there was no obvious difference between the four
batches of FBS when used in the T cell proliferation
5 x 104 and 1 x 105 splenocytes/well showed antigen-specific proliferation
with very little background. This result suggests that we can use twice as
many splenocytes (1 x 105) per well as before and that we do not have to
enrich for T cells in nylon wool columns. However, given that the results with
all 4 lots of FBS were similar, it is not clear why 1 x 10 5 cell/well had such
high background before
5 x 105 and 1 x 106 cell/well proliferated non-specifically in the absence
antigen.
Figure 3. Screening FBS for use in T cell proliferation assays. T cells were incubated with 5 x 10 5 formalin-fixed
LVS in the presence of various FBS. After 5 days, T cell proliferation was measured indirectly by the
incorporation on BrdU
b. Experiment Ftc27.15 (Notebook 101, pages 67-69)
i. The purpose of this experiment was to determine whether we can use more
than 1 x 105 splenocytes/well in the T cell proliferation assay
ii. Total splenocytes were titrated from 1 x 105 to 4 x 105/well
iii. As shown in Figure 4, we can use up to 2 x 105 cell/well with very little
background
16 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 4. Screening FBS for use in T cell proliferation assays. T cells were incubated with 5 x 10 5 formalin-fixed
LVS in the presence of various FBS. After 5 days, T cell proliferation was measured indirectly by the
incorporation on BrdU
4. Significant decisions made or pending
Will use 2 x105 cells/well in proliferation assays and have ordered 12 bottles of a single lot of
tested FBS for assay usage.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
42%
9. Work plan for upcoming month and next 6 months
a. Optimize T cell proliferation assay using positive control proteins GroEL, KatG, Bfr
b. Optimize T cell proliferation assay using in vitro translated peptides from ASU
(GroEL, KatG, Bfr, Tul4)
c. Develop ELISpot assay
d. We will develop the T cell proliferation assay for the Fischer 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
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
17 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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 work done because no human cells were available during this period. Two donors are
scheduled in August 2007
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
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
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
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30.10 (Notebook 101, pages 45-47), Experiment Ftc30 study 11
(Notebook 101, pages 48-50) and Ftc30.11b (Notebook 101, pages 54-56)
i. Karen Elkins suggested that our problems with the macrophage culture may
be attributed to 1) the amount of M-CSF and/or 2) the quality of FBS. We
ruled out the possibility that we did not have enough or had too much M-CSF
18 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
ii.
iii.
iv.
v.
by showing that the amount of M-CSF in the L-929 conditioned media from
the Lyons lab and the Elkins lab was very similar (Ftc30.8b)
We tested 4 lots of FBS: HyClone lot APA20504 (from Karen Elkins),
HyClone lot FSC28994 (reserved at HyClone), Hyclone lot FQK25099
(currently used in Lyons lab), and a batch of FBS without any label that we
have been using for the macrophage culture
As shown in Table 4, the unlabeled FBS that we have been using for the
macrophage culture only supported about 50% differentiation and 50%
confluency. The 3 remaining FBS all supported > 80% differentiation and
confluency
These results suggested that we were using a batch of FBS that was not
suitable for culturing macrophages
We have purchased 12 x 500 ml bottle of the HyClone lot FSC28894 for work
related to the TVDC
Table 4. Screening FBS for ability to support macrophage
differentiation and growth
FBS lot
% differentiated
% confluency
HyClone FQK25099
> 80
> 80
(Lyons Lab)
HyClone APA20504
> 80
> 80
(Elkins lab)
HyClone FSC28994
> 80
> 80
(Reserved at HyClone)
Unlabeled
~ 50
~ 50
(Lyons lab)
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Purchased large volume of FBS lot that better supported macrophage differentiation and
growth
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
15%
9. Work plan for upcoming month
a. Determine the optimal MOI for LVS and SCHU S4 infection of murine macrophages
b. Determine whether vaccinated mouse splenocytes can induce BMM to kill
intracellular LVS
c. Determine whether vaccinated mouse splenocytes can induce BMM to kill
intracellular SCHU S4
d. 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
19 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. We have prepared a set of modular IVT templates, which differ by the arrangement of
the His tag. These are diagrammed in Figure 1.
2. An available choice of single tagged, double-tagged, and tagless constructs will
increase the possibility for successful affinity purification of the encoded polypeptide.
3. This will give us flexibility in potentially finding a tagged polypeptide that can be used
for purification of all samples.
4. The double HIS tag presented a PCR design challenge, given that the same
sequence will be at both ends. This was addressed by designing alternative
sequence to encode His.
Figure 1. Modular component tools for assembling alternative IVT templates.
20 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
T7
RBS ATG
T7
RBS ATG
T7
RBS ATG
T7
RBS ATG
His
ORF
His
Term
N-term tag
ORF
His
Term
C-term tag
ORF
His
Term
double-tag
Term
w/out-tag
ORF
5. We tested the translation efficiency of the ORFs as provided to the IVT reaction in either
a sequence-verified, cloned plasmid construct or provided as an unverified, mix of linear
constructs. The latter design is planned for our ORF library.
6. Cpm determination of our radio labeled translation product yields is very encouraging.
The results shown in Table 1 indicate that there is no appreciable difference in yields
generated from the crude linear templates relative to a cloned, perfect-sequence
template.
Table 1: Transcription/translation efficiency of crude vs. perfect PCR products
MW
#
ul total ul
CPM
CPM
Met
sample counted measured Total
CPM/
met
ug prot
(yield)
FTU 728a_a Clone
21,703
4
85
5
11274
191,658
47,915
20.74
FTU 728a_b Clone
24308
4
85
5
12653
215,101
53,775
26.07
FTU 1434c_a
Clone
FTU 728a_a mix
19007
5
85
5
20006
340,102
68,020
25.79
21,703
4
85
5
3725
63,325
15,831
6.85
FTU 728a_b mix
24308
4
85
5
13990
237,830
59,458
28.83
FTU 1434c_a mix
19007
5
85
5
23193
394,281
78,856
29.90
Data located at: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT\IVT data: FTU
Short Plate 1 Row B 7-26-07
7. We conclude that the protocols for production of both wild type (PCR) and recoded (gene
assembly) ORFs, and for their expression in the modular linear construct are ready.
B. Select and test IVT Protocols
1. We tested the adaptability of out IVT reactions to the demands of a high-throughput
protocol.
2. The results in Table 2 show excellent yields for all of 12 randomly chosen polypeptides.
21 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
3. We are getting at least 20 ug of protein produced.
Table 2. Test of IVT protein production in a HTP-formatted protocol.
MW w/
#
ul sample
ul for CPM
CPM Total
tag
Met
CPM
CPM
#met
ug prot
1
2
3
4
5
6
7
8
9
10
11
12
13
169,546
80,699
67,405
62,546
79,172
66,423
128,962
67,314
164,849
207,037
61,462
98,611
109,877
63.88
20.70
27.16
17.60
28.67
29.02
28.84
29.94
49.27
42.78
29.33
26.45
25.38
CALM3
FTU 582 B
FTU 887 A
FTU 329 A
FTU 319 A
FTU 1550 A
FTU 1284 A
FTU 1368 B
FTU 784 A
FTU 741 A
FTU 196 A
FTU 1472 A
FTU 150 A
19,500
13,277
20,851
14,567
18,743
22,611
11,573
23,019
15,470
10,693
24,698
13,882
11,956
10
2
3
6
5
9
4
6
3
3
5
3
3
85
85
85
85
85
85
85
85
85
85
85
85
85
5
5
5
5
5
5
5
5
5
5
5
5
5
99733
9494
11895
22075
23286
35165
30344
23758
29091
36536
18077
17402
19390
1,695,461
161,398
202,215
375,275
395,862
597,805
515,848
403,886
494,547
621,112
307,309
295,834
329,630
Data located at: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT\IVT data: FTU
Chip BAG 7-30-07
4. These reactions were performed without a template spike. Even though this omits a
step, we know it is advantageous relative to final yields. At this time, we do plan to
include a template spiking step in the protocol. Other methods for further increasing
yield, which are HTP adaptable if needed, are longer reaction times and performing
multiple reactions.
5. Figure 2 displays the non-reducing SDS-PAGE analysis of 11 of the 12 FTU IVT
reactions quantitated in Table 2. Each of these FTU polypeptides has an N-terminal His
tag. S-35 labeling enables visualization of all products, whether full length, prematurely
terminated, or degraded.
6. We conclude that all 11 products are migrating to positions similar to their predicted
monomer or multimer molecular weights, and they being made with reasonable specificity
and integrity.
22 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 2. Visualization of polypeptides generated by IVT.
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Contract\Proteome\Tien's data\E page: IVT FTU
Shrt Plt 01 Row B Chip BAG 7-31-07 02
C. Select and test protocols for protein purification
1. To assess the level of purity needed to successfully apply the polypeptides to the T
cell proliferation assays, we prepared a set of pilot samples. Unpurified and buffer
exchanged, and affinity purified samples were delivered to UNM as acetone
precipitates for use in development of their T cell assays. These samples are
reviewed in Table 3.
Table 3: Test samples delivered to UNM
TEMPLATE
LYSATE
ACETONE->PBS
None
unpurified
buffer exchange
GFP
unpurified
buffer exchange
FTU 1419
unpurified
buffer exchange
FTU 1695
unpurified
buffer exchange
FTU 901
unpurified
buffer exchange
FTU 1602
buffer exchange
FTU 1712
buffer exchange
NI+ PURIFIED
purified
purified
purified
purified
purified
23 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
2. More recently we sent a second batch of IVT polypeptides to UNM for testing in T cell
proliferation assays.
Table 4: Additional IVT samples for evaluation of protein purity demands in T-cell
stimulation assays
FTU 1696, 1602, 1712for Ship
7/16/2007
#
ul total ul
CPM
CPM/
MW
Met sample counted CPM
Total
met
ug prot
1
2
3
4
5
6
7
FTU 1696 Aa
FTU 1696 Ab
FTU 1696 Ba
FTU 1696 Bb
FTU 1602
FTU 1712
FTU 901
21,612
26,139
23,795
22,733
15,393
26,032
7
5
6
12
2
7
85
95
85
85
285
285
5
5
5
5
5
5
48273
11928
29716
46055
23646
24851
820,641
226,632
505,172
782,935
1,347,822
1,416,507
117,234
45,326
84,195
65,245
673,911
202,358
uCi
43.80
21.14
34.63
25.64
179.33
91.06
69.98
Total uCi
0.385
0.106
0.237
0.368
0.633
0.665
2.394
Data located at: R:\GeneVac\FTU\Contract\Proteome\Hetal's data\Hetal IVT\IVT data: IVT FTU 1696
1602 1712 for shipment 7-16-07
3. We have defined the purification process as:
i. acetone precipitation of the IVT reaction products
ii. pellet solubilization in presence of urea
iii. Affinity binding of the His fusion products to Ni+-bead column
iv. washing bead column in presence of urea
v. specific elution of the bound products with imidazole
vi. precipitation of the affinity purified polypeptides with acetone
4. We have found that purification process is associated with significant loses of the IVT
synthesized products. Namely, ~ 50% during acetone precipitation/urea
solubilization, and ≥50% during Ni-His purification. Overall loses between 75% and
100%
5. To improve recoveries from purification, we carried out an experiment suggested in
our technical call: we added urea directly to the IVT reaction without prior acetone
precipitation. We also tried using guanidinium-HCl instead of urea at this step. The
rationale for trying this- the elimination of the acetone precipitation- is that we are not
resolubilizing the full sample. If we do not precipitate we would not need to
resuspend. The idea was that this might improve yields. The results are shown in
Table 5. Each set of lysate-samples are labeled A. through E.
6. Comparison of A. to B. demonstrates that direct addition of urea to the lysate does
not increase the purified product yields (elution). Likewise, Comparison of C. to D.
demonstrates that direct addition of guanidinium-HCl to the lysate does not increase
the purified product yields (elution).
7. In sample E. we compared the acetone-pellet’s solubilization efficiency when using
urea versus Gu-HCl.
8. Unfortunately, we conclude that there is no measurable improvement in yields with
either protocol modification. We are purifying about the same amount of sample
even though we are not suffering the 50% loss at the resolubilization step. This
24 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
suggests that there is some other downstream advantage to clearing the lysate by
performing the acetone precipitation prior to bead binding,
9. However, we do note that we may choose to omit the acetone precipitation step just
for simplicity.
Table 5. Attempts to improve solubilization efficiency
FTU 1695 Urea GuHCl Comparison
#
Met
ul
sample
14185
14185
14185
5
5
5
100
350
160
14185
14185
14185
5
5
5
14185
14185
14185
CPM
CPM
Total
5
5
5
24985
373
1195
499,700
26,110
38,240
100
200
180
5
5
5
28976
1393
1816
5
5
5
100
270
170
5
5
5
14185
14185
14185
5
5
5
100
250
175
14185
14185
14185
5
5
5
25
25
25
MW
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
A. Rxn Mix +Urea
IVT
Flow thru urea wash
Elution
B. Acetone ppt->Urea
IVT
Flow thru urea wash
Elution
C. Rxn Mix + GuHCl
IVT
Flow thru urea wash
Elution
D. Acetone Ppt -> GuHCl
IVT
Flow thru urea wash
Elution
E. Act Ppt ->solubilize
IVT
Act/Urea rspsd
Act/Gu-HCl rspsd
8/3/2007
ul
for
CPM
CPM
/met
ug
prot
uCI
99,940
5,222
7,648
29.19
1.53
2.23
0.235
0.012
0.018
579,520
55,720
65,376
115,904
11,144
13,075
33.85
3.25
3.82
0.272
0.026
0.031
29387
273
1457
587,740
14,742
49,538
117,548
2,948
9,908
34.33
0.86
2.89
0.276
0.007
0.023
5
5
5
42508
620
1528
850,160
31,000
53,480
170,032
6,200
10,696
49.66
1.81
3.12
0.399
0.015
0.025
5
5
5
43560
23455
23404
217,800
117,275
117,020
43,560
23,455
23,404
12.72
6.85
6.84
0.102
0.055
0.055
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Contract\Proteome\Tien's data: IVT FTU 1696
Urea GuHCl Comparison 8-3-07
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
By our current protocol, purification of IVT products costs significant losses. In case
purification is to be needed, we plan to initiate a set of solubility and elution titration
experiments on multiple template arrangements. These will identify the most commonly
successful (most robust) parameters for retrieving the largest portion of the sample.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
98%
25 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
9. Work plan for upcoming month
We will initiate a series of experiments to titrate down the amount of nickel beads used in the
binding steps. This will limit the amount non-specific surface area available to the
polypeptide. We will also titrate the reaction time for the binding reaction. The kinetics of the
specific His-Ni interaction is likely to be faster than the non-specific and hydrophobic
interactions. We will identify the optimal time for complete specific binding without
encouraging non-specific interactions.
In addition we will be testing our alternative tagged constructs in the purification protocols.
These are intended to enable us to lower the urea concentrations, and thereby discourage
exposure of hydrophobic “sticky” surfaces of the polypeptides.
10. Anticipated travel
We’ve made our reservations for the October 2007 Annual TVDC meeting in Santa Fe!
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiments performed under milestone 12/13 to troubleshoot the T cell proliferation
assay can be considered under this milestone as well. We now know that we can
increase the number of splenocytes from 5 x 104 to 2 x 105/well and that we have a
batch of FBS that produces negligible background
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
No progress
8. Percentage completed
12%
9. Work plan for upcoming month
a. Determine whether the draining lymph nodes would be a better source of T cells for
the proliferation assay than the spleen. Draining lymph nodes are frequently used in
various T cell assays to demonstrate the existence of antigen specific responses
because they contain more antigen-specific T cells than the spleen. In addition, more
than 106 cells/well can be used in these assays and thereby increasing the assay
sensitivity. We will determine whether the hilar lymph nodes draining the lung after
i.n. LVS vaccination will be a better source of T cells for screening peptides than
splenocytes
26 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
b. Develop the peptide screening assay using the purified GroEL, KatG, and Bfr as
positive controls
c. Determine whether IFN ELISpot assay would be better than T cell proliferation
assay for this peptide screen
d. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
e. 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 performed PCR’s on all wild type designed ORFs. Amplification protocols are in
place. All have been run and we know how many rxns will get us the desired amount of
product. However, ORFs will be freshly amplified when we are ready to assemble and run
the full scale IVT reactions We will await final assembly of LEE templates until final decision
on purification methods and requirements are determined.
2. Templates and primers for promoter and terminator amplification have been tested and
readily available.
3. Database management and tracking system is in place.
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. In particular, we will be testing the new Agilent chips in our protocols.
We are still waiting on these chips.
6. Deliverables completed
None
7. Quality of performance
27 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Very Good
8. Percentage completed
20%
9. Work plan for upcoming month
Chip synthesized oligos will be hopefully received. As soon as they are in hand, sets of initial
recoded, synthetic ORFs will be assembled, and tested in IVT reactions.
10. Anticipated travel
We’ve made our reservations for the October 2007 Annual TVDC meeting in Santa Fe!
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

The initial studies have been performed with reconstitution samples prepared by diluting
purified SCHU S4 RNA in normal mouse lung RNA. Previous results show that we could
amplify and detect over 100 genes with as little as 0.001 g of SCHU S4 RNA..

We next prepared a set of reconstitution samples that included dilutions to 0.0001 g SCHU
S4 RNA. Comparing the 0.01 samples between LAPT 8 and 9 and the 0.001 identified 59
genes reproducibly detected (Figure 1).
28 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Figure 1. Venn diagram showing overlap of genes from the 0.01 g samples in LAPT 8 and 9
and the 0.001 g LAPT 9 sample.

Comparing the 0.01 samples between LAPT 8 and 9 and 0.0001 identified 45 genes
reproducibly detected (Figure 2).
Figure 2. Venn diagram showing overlap of genes from the 0.01 g samples in LAPT 8 and 9
and the 0.0001 g LAPT 9 sample.
29 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee

Comparing the 0.001 samples between LAPT 8 and 9 and 0.0001 identified 40 genes
reproducibly detected (Figure 2).
Figure 3. Venn diagram showing overlap of genes from the 0.0001 g samples in LAPT 8 and 9
and the 0.0001 g LAPT 9 sample.
The graphs above were designed to test the sensitivity and specificity of the LAPT process across
two experiments and through a dose response curve. The analysis shows that we are able to
maintain high specificity with no false positive genes being detected even at 0.0001 g (Figure 1, 2,
and 3, dark blue = 0). With the current analyses we have established a high level of specificity
leading to the consistent detection of 40 genes across experiments through a dose response.
However, with high specificity we have sacrificed some sensitivity (the ability to detect a gene
expression above background level). It still needs to be determined if 40-60 genes will be enough to
monitor gene pathways as the organism grows in tissues, but we will be able to relax the specificity to
increase the number of detectable genes and will have to rely on replication of experimental
conditions to minimize the detection false positive genes.


Notebook/File locations … LAPT-9 Notebook 514, pages 33-36;
Electronic file locations… R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-9

ASU has been using the Clontech kit for the LAPT reactions. After noting the changes in
Clontech kit design for the first strand synthesis (See problems to address), we have
performed one amplification comparing Superscript II (SSII) to Superscript III (SSIII) reverse
transcriptase. As shown in Table I., the first strand reactions were performed in either the
supplied Invitrogen buffers or in the Clontech buffer system. The optimum amplification
occurred with the SSII enzyme in the Clontech buffer. The level of amplification is
acceptable, although on the low side of expected yields.
Table 1. Amplified yield of RNA (in micrograms expected yield of 30-50 g) of SCHU S4 RNA amplified
by LAPT. Comparison of Superscript II to Superscript III enzymes and buffer compositions on
amplification yield.
30 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
g SCHU S4 amplified
1.0
0.1
0.0

SSII-SSII Buffer
4
9.3
13.8
SSIII-SSIII Buffer
6.2
6.3
Not done
SSII-Clontech Buffer
24.2
31.3
15.7
Notebook/File locations … SSII vs. SSIII, Notebook 514, page 37.
4. Significant decisions made or pending.
Find suitable replacement enzyme for first strand cDNA synthesis, since the components of the
Clontech kit have changed.
5. Problems or concerns and strategies to address
Changes in kit components have caused a problem. For the LAPT process, the original protocol
utilized two kits from Clontech; the Super SMART PCR cDNA synthesis kit for the first strand
cDNA synthesis and the Advantage 2 PCR kit for amplification. One of the key activities for the
first strand synthesis relies on the ability of the reverse transcriptase to add a few nontemplate
deoxynucleotides (cytosines) to the 3’ end of the nascent cDNA. This allows for the template
switch primer to anneal via a guanine and add the T7 promoter for the downstream in vitro
transcription. The Clontech kit provided an enzyme termed “Powerscript” that had the tailing
activity. There was a lawsuit between Clontech and Invitrogen resulting in the inability of
Clontech to continue to provide the “Powerscript” reverse transcriptase. We received new kits,
without enzyme, and were instructed to utilize any MMLV reverse transcriptase and were directed
to Invitrogen’s Superscript product. We performed a series of LAPT amplifications utilizing
Superscript III from Invitrogen and all of the amplifications failed. We subsequently obtained
information from Invitrogen that the migration of Superscript from version II to version III resulted
in the loss of tailing activity. To address this, we will be preparing an “in house” version of the kit
for performing the first strand synthesis that will include the c tailing activity.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
95%
9. Work plan for upcoming month
Identify and compare several MMLV reverse transcriptase enzymes with tailing activity for
preparing the in house first strand synthesis reaction components.
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 lab work done
31 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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
12%
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
Milestone 34-ASU
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

We were performing amplifications for continuing the comparison between TIGR and ASU
arrays, but amplifications failed due to changes in Clonetech cDNA synthesis kit.

Notebook/File locations … Notebook 514, pages 17-30;
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
See MS 33 above for details on microarray target synthesis troubleshooting
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
65%
9. Work plan for upcoming month


Perform comparisons of known genes and their ability to hybridize to both the ASU and TIGR
arrays.
Perform additional hybridizations between ASU and TIGR arrays with unamplified and
amplified SCHU S4 RNA.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
32 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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


From samples obtained from UNM, RNA was purified over Qiagen RNAeasy columns.
Pooled samples were prepared and processed for LAPT but all amplifications failed due to
changes in Clontech kit.
Notebook/File locations … Notebook 514, pages 17-30;
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
See troubleshooting plans for target amplifications reported under Milestone 33
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
2.5%
9. Work plan for upcoming month
LAPT process will start on the 15 new RNA samples from UNM. During clean-up we will create a
pool of each challenge dose for initial testing. The samples will be subsequently prepared for the
LAPT process for hybridization to the ASU array.
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 Ft. novicida. We have optimized
photochemical inactivation conditions at a 3.5 mL scale and a 400mL scale and produced a
lot of KBMA uvrB Ft. novicida for potency testing in MS42. We have demonstrated that
KBMA Ft. novicida are highly attenuated for virulence. Frozen KBMA uvrB Ft. novicida
maintain metabolic activity at –80oC for at least 3 months.
33 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Because the inactivated NER-deficient strains have a similar degree of metabolic
activity as the wild-type Ft. novicida strain (which is different than has been seen with L.
monocytogenes or B. anthracis) we have initiated a series of experiments to determine the
cause of this observation. There are 2 obvious and distinct possibilities 1) is that the NER
genes are not turned on during photochemical inactivation with S-59 and UVA light or 2)
there may be a redundant mechanism for repair of DNA damage that prevents inactivation of
the uvr mutants at low S-59 concentrations. We previously evaluated the sensitivity of the
uvrB mutant and U112 to 6 alternative DNA damaging agents: S-303 (a nitrogen mustard
crosslinking agent that is not activated with UV-light), mitomycin C, cisplatin, doxorubicin
hydrochloride, benzo[a]pyrene, and 4 nitroquinoline-N-oxide using a 96-well format minimum
inhibitory concentration (MIC) assay. 4 of the DNA damaging agents inhibited growth of the
bacteria. Of the 4 agents that inhibited growth of Ft novicida, only 2 inhibited growth of the
uvrB mutant at lower concentrations (S-303 and 4 nitroquinoline-N-oxide).
1) This month we repeated the MIC assays using a two-fold dilution series over a smaller range
of concentrations of mitomycin, cisplatin, and S-303. The growth of U112 and the uvrB mutant
Ftn strains in the presence of cisplatin or mitomycin were indistinguishable. The minor difference
in sensitivity to S-303 was reproduced in this second experiment and was repeated a third time.
NB920-189
Ftn sensitivity to DNA damage by S-303
Ftn sensitivity to cisplatin
0.7
uvrB T16h
0.4
U112 T0
0.3
0.2
uvrB T0
0.7
0.6
U112 T16h
0.5
0.4
uvrB T16h
0.3
0.2
uvrB T0
U112 T0
2.
3
4.
6
9.
3
0.
9
1.
7
3.
4
6.
9
S-303 uM
11
85
.0
59
2.
5
29
6.
3
14
8.
1
74
.1
37
.0
18
.5
0.1
0.1
44
0.
0
22
0.
0
11
0.
0
55
.0
27
.5
13
.8
OD600 nm
0.5
OD600 nm
U112 T16h
0.6
Cisplatin ug/ml
The fact that there were either no or only minor differences in sensitivity to DNA damage between
the uvrB mutant and wild type Ft novicida demonstrates that this phenomenon is not unique to
photochemical inactivation with S-59 and UVA and suggests that there is a redundant mechanism
for repairing or preventing DNA damage in Ft novicida. However, to rule out the possibility that
the uvrB gene is not induced by DNA damage in Ft novicida, samples of U112 treated with
various concentrations of S-303 were taken and frozen for processing of RNA to measure uvrB
transcriptional response after DNA damage. We have previously designed, and tested the
specificity of 2 pairs of oligonucleotide primers for amplification of the uvrB gene for reverse
transcriptase quantitative real time PCR (rt)qPCR (qPCR). Over the next month we will perform
rtqPCR to determine whether the uvrB gene is induced after DNA damage.
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.
34 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms. This
appears to hold true for other methods of induced DNA damage. One possible explanation
for this is that there is a redundant DNA repair mechanism functioning in Ft novicida that may
limit the sensitivity of the NER-deficient mutants to DNA damage and thereby limit the
metabolic activity and potency of KBMA Ft novicida. If there is a redundant repair
mechanism, we may not be able to produce a highly potent KBMA vaccine utilizing
Francisella species as a platform.
6. Deliverables completed
400mL-sacle photochemical inactivation process defined
7. Quality of performance
Good progress
8. Percentage completed
85% of scientific work completed on the milestone
9. Work plan for upcoming month
We will determine whether the uvrB gene is induced in U112 by rtqPCR after DNA damage at
a concentration that inhibits growth of the uvrB mutant more than U112. We will also
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.
KBMA Ft novicida uvrB were 100% protective when a single dose was administered at or
near the LD50 of the KBMA vaccine (1 x 109 IP, 1 x 108 IV). 100% protection was also
achieved by administration of 1 x 107 KBMA particles IV when the vaccine was given twice
separated by 3 weeks. Depletion of CD4+ T cells prior to the challenge decreased the
survival rate to 80%, depletion of C8+ T cells had no effect, and depletion of both cell
populations resulted in 90% survival. Together, these data demonstrated that CD4 T cells
contribute to a protective immune response in a non-CD8 T cell-dependent manner. These
data suggest that the CD4 T cells may be boosting humoral immunity by stimulating B cells.
This interpretation was supported by an adoptive transfer experiment in which only the hightiter serum from CD8-depleted animals provided any protection against a lethal U112
challenge. Together these data demonstrate that the protection we see after vaccination with
KBMA Ft novicida uvrB correlates with humoral immune responses and explains why the
KBMA vaccine does not perform better than heat killed vaccine. This also makes it nearly
impossible to rank attenuated Ft novicida mutants by their ability to protect mice against a
lethal challenge. We instead plan to evaluate the ability of KBMA vaccines to induce a potent
CD8 T-cell response to an introduced ovalbumin epitope tag and are awaiting the
construction of this strain from UTSA.
35 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
4. Significant decisions made or pending
We have decided to evaluate the potency of the KBMA Ft novicida vaccine by measuring the
CD8 T cell response to an ovalbumin epitope tag.
5. Problems or concerns and strategies to address
Because humoral immunity plays a significant role in protection of mice against a lethal Ft
novicida challenge it is essentially impossible to rank KBMA vaccine candidates that elicit a
potent T cell response using survival after a lethal Ft novicida challenge in MS 43. We have
requested that Karl Klose construct an ovalbumin epitope-fusion protein to facilitate screening
strains of Ft novicida for their ability to elicit a T cell response to this well-defined epitope.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
25% of scientific work completed on the milestone
9. Work plan for upcoming month
We have put this milestone on hold while we wait for delivery of the ova-tagged strain of
uvrB from UTSA to determine whether KBMA Ft novicida-OVA epitope tagged strains 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 have constructed 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.
We have reported in the technical report for May that a Targetron vector, which was the
plasmid pKEK1140 inserted with a 350bp PCR product and named pKEK1167, had been
created. We tried to transform the plasmid pKEK1167 into wild type LVS afterwards, and got
the mutant UvrA LVS. The insertion should happen in UvrA(1312C) at bp 72/73.
3.1.1 To make competent LVS for transformation, mid-log phase LVS was washed in 0.5M
Sucrose for 3 times. The final LVS pellet was resuspended in 50ul 0.5M Sucrose for
transformation.
36 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
3.1.2 Transformed the plasmid pKEK1167 into the competent LVS using electroporation, and
spread the transformed LVS onto TSA+++/ Kanamycin(50ug/ul) plate to select single
colonies after being incubated at 30C for 3-4 days.
3.1.3 Patched the single colonies from TSA+++/ Kanamycin(50ug/ul) plate onto the same fresh
plate, and incubated it at 30C to get colonies for colony PCR.
3.1.4 Performed colony PCR to confirm insertion in LVS with the primers UvrASchu4Up(from LVS
genomic DNA) and EBS Universal (from the insertion DNA), UvrASchu4LVSDn
(from LVS genomic DNA) and EBS Universal. UvrASchu4Up was in the upstream of UvrA,
whereas UvrASchu4LVSDn was located in the downstream of UvrA.
Set up PCR reaction:
32.6ul ddH2O
5.0ul 10XBuffer #1 for KOD
5.0ul dNTPs
2.0ul MgCl2
1.0ul DNA
2.0ul UvrAShcu4Up
2.0ul EBS Universal
0.4ul KOD HiFi polymerase
At 98C 1min, 98C 15sec/ 55C 15sec/ 72C 1min// 30 cycles
34.0ul ddH2O
5.0ul 10XKOD XL Buffer
5.0ul dNTPs
1.0ul DNA
2.0ul UvrASchu4LVSDn
2.0ul EBS Universal
1.0ul KOD XL DNA polymerase
At 94C 1min, 94C 30sec/ 55C 10sec/ 72C 2min// 30 cycles, 72C 10min
Gel picture: Figure1
1
1000bp
500bp
2
3
4
5
6
7
8
9 10 11 12
1.1kb ladder
2.Colony 1
3.Colony 2
4.Colony 3
5.Colony 4
6.Wild type LVS
7.Colony 1
8.Colony 2
9.Colony 3
10.Colony 4
11.Wild type LVS
12.1kb ladder
Lane 2-6 PCR with UvrASchu4Up and EBS Universal primers
Lane 7-11 PCR with UvrASchu4LVSDn and EBS Universal primers
3.1.5 The gel picture showed that the insertion was in UvrA LVS. PCR product with UvrASchu4Up
and EBS Universal primer was about 698bp, which meant the insertion had been inserted in
UvrA(FTT1312C) at 72/73 bp and EBS Universal primer was in the reverse direction
comparing to UvrA sequence. There was not specific PCR product for primers
UvrASchu4LVSDn and EBS Universal, which meant the two primers were in the same
orientation.
3.1.6 Colony PCR with UvrASchu4Up and UvrASchu4LVSDn primers to further confirm the
insertion.
37 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
32.6ul ddH2O
5.0ul 10XBuffer #1 for KOD
5.0ul dNTPs
2.0ul MgCl2
1.0ul DNA
2.0ul UvrAShcu4Up
2.0ul UvrASchu4LVSDn
0.4ul KOD HiFi polymerase
At 98C 1min, 98C 15sec/ 55C 15sec/ 72C 1min30sec// 30 cycles
Gel picture: Figure 2
1
2000bp
1500bp
1000bp
517bp
2
3
4
5
6
1.1kb ladder
2.Colony1
3.Colony2
4.Colony3
5.Colony4
6.Wild type LVS
3.1.7 The gel picture confirmed the insertion. With the insertion in UvrA LVS, PCR product should
be bigger than wild type LVS with which we got about 600bp PCR product. There were two
bands for colony 1,2 and 3. The smaller band is the same size (600bp) as one of the wild
type LVS bands, which meant that the colony had LVS with the plasmid but no insertion
happening. The larger band (1300bp) is a different size from the wild type LVS bands,
which meant that the colony also contained LVS with the insertion in the UvrA gene. As for
colony 4(lane 5), there was only one larger band (about 1300bp), which meant that this
colony possibly contained only LVS with the insertion in the UvrA gene, and no LVS with the
plasmid but no insertion happening. So colony4 is probably pure UvrA mutant LVS. For
completely pure UvrA mutant LVS, we proceeded with step 3.1.9 to 3.1.11.
3.1.8 Sent the gel purified DNA from PCR product with UvrASchu4Up and EBS Universal primers
for sequencing with the same primers, and the sequencing result confirmed that the insertion
was in UvrA(1312C) at 72/73bp in LVS.
3.1.9 In order to separate LVS bacteria with the insertion and LVS bacteria without insertion, we
streaked colony4 onto TSA+++/ Kanamycin(50ug/ul) plate and incubated at 30C to get
single colonies. Then colony PCR was done with the same primers as 3.1.4 and 3.1.6 to
screen the colonies until we got the pure right colony without being mixed with the colony
without insertion. All incubation was done at 30C.
3.1.10
Even if the insertion had been inserted into UvrA , the plasmid had to be removed from
the UvrA mutant LVS. Since the plasmid was temperature sensitive and resistant to
Kanamycin, the UvrA mutant LVS with plasmid was streaked onto TSA+++/
Ampicillin(100ug/ul) plate and incubated at 37C to get single colonies. Then the single
colonies were patched onto TSA+++/ Ampicillin( (100ug/ul) and TSA+++/
Kanamycin(50ug/ul) plates, and incubated at 37C. The same procedure was repeated until
we got Kan sensitive and Amp resistance colony. All incubation was done at 37C.
3.1.11
Performed the colony PCR with UvrASchu4Up and EBS Universal, UvrASchu4Up and
UvrASchu4LVSDn primers for the Kanamycin sensitive colony with the same settings as
3.1.4 and 3.1.6.
38 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Gel picture: Figure3
1 2 3 4
5 6 7 8 9 10
1.0kb
0.5kb
1.5kb
1.0kb
0.5kb
1.1kb ladder
2.Positive cotrol
3.Wild type LVS
4.Colony2
5.Colony 3
6.Colony 13
7.Colony 21
8.Colony 24
9.Colony 27
10.1kb ladder
11.1kb ladder
12.Positive control
13.Wild type LVS
14.Colony 2
15.Colony 3
16.Colony 13
17.Colony 21
18.Colony 24
19.Colony 27
20.1kb ladder
11 12 13 14 15 16 17 18 19 20
Lane 2-9: Colony PCR with UvrASchu4Up and EBS Universal primers (approximately 600bp)
Lane 12-19: Colony PCR with UvrASchu4Up and UvrASchu4LVSDn primers(approximately
1300bp)
3.1.12
The gel picture (Fig 3) showed that we had got the UvrA mutant LVS with the insertion in
UvrA(1312C) at 72/73 bp and the plasmid being removed. This UvrA mutant LVS was
Named KKV317.
Data recorded on UTSA TVDC notebook #2, page 79-83 for Figure1,2,3.
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 T cell tag is SIINFEKL. In
consultation with 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 T cell 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 PepOFlag. A pair of complimentary oligonucleotides encoding SIINFEKL will be used to replace the
FLAG tag fragment in pKEK1145.
3.2.1 In the last technical report, we have reported that the Flag from pKEK1149 had been
replaced with SIINFEKL and we named this plasmid pKEK1168. Both of the plasmid
pKEK1149 and pKEK1168 have the Francisella promoter “Fn-promoter” to transcribe the
cloned gene. We did western blotting with anti-SIINFEKL, but didn’t detect protein expression
in DH5 E.Coli.. So we ordered OVA peptide as the positive control for later western blotting.
The backbone of the plasmid pKEK1169, which was pKEK1145 replaced FLAG with
SIINFEKL, and pKEK1145 is the PBAD24 plasmid, which has the ARA-promoter to transcribe
the cloned gene. The plasmid pKEK1169 was checked to see if there was SIINFEKL
expression in DH5 E.Coli..
3.2.2 The whole cells lysates were made to check the expression of SIINFEKL. For pKEK1168,
overnight culture was used, and as for pKEK1169, 1.5 Arabinose was added to overnight
culture and 4 hours culture to induce the protein expression since pKEK1169 was a PBAD
39 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
plasmid which has Arabinose promoter (ARA-Promoter) and all the genes cloned into the
PBAD plasmid can be induced transcription and thus protein expression by adding certain
amount Arabinose.. In the Western blot, the primary antibody was anti-SIINFEKL from cells
culture and the second antibody was anti-mouse antibody. PepO-SIINFEKL protein is about
78KD.
Figure1:
1
2
3
4
5
1.Pkek1169 overnight culture
2.pKEK1168 overnight culture
3.OVA
4.Pkek1169 4hrs culture
5.DH5 E.coli.
75KD
50KD
3.2.3 From the picture above (Fig 1), we still didn’t see the SIINFEKL expression in pKEK1168, but
we did see the corresponding band to the protein sized about 75KD from the PBAD plasmid
Pkek1169 (PepO-SIINFEKL protein is about 78KD). No positive signal in OVA is possibly
because the anti-SIINFEKL didn’t recognize the antigen site in OVA. From this experiment, at
least we confirmed that the antibody to SIINFEKL worked.
3.2.4 One more western blotting was done for Pkek1169 in DH5 E.Coli. using the same
procedure to confirm the SIINFEKL expression, and the time course was set up. 1.5
glucose was added in overnight culture to inhibit the protein expression(lane1), and 1.5
Arabinose was added in cultures at time point 1hr, 2hrs, 3hrs, 4hrs and overnight(lane2-6) to
induce protein expression.
Figure2:
1
2
3
4
5
6
7
75KD
50kd
1.Overnight culture for pkek1169
2.1hr culture for pkek1169
3.2hrs culture for pkek1169
4.3hrs culture for pkek1169
5.4hrs culture for pkek1169
6.Overnight culture for pkek1169
7.DH5  E.coli.
Lane1: with1.5 glucose
Lane2-6: with 1.5 Arabinose
3.2.5 The result from 3.2.4 did show that there was SIINFEKL expression for pKEK1169 (PBAD
plasmid) in DH5 E.Coli. since the protein expression was increasing as the time went on.
3.2.6 For some reason, we didn’t detect SIINFEKL expression from pKEK1168, which had
Francisella promoter, in DH5 E.Coli. So we tried to transform pKEK1168 into wild type LVS
to see if the expression would happen in LVS where the Francisella promoter should work
better , but we have not gotten the right colony yet.
Data recorded on UTSA TVDC notebook #2, page 109-111 for Figure1,2.
4
Significant decisions made or pending
We will send LVS transformed with the Pkek 1168 to Cerus Lab to let them identify SIINFEKL
expression if we cannot detect SIINFEKL expression using western blotting after transforming
pKEK1168 to LVS.
5
Problems or concerns and strategies to address
SIINFEKL didn’t express in Pkek1168 with Francisella promoter in E.coli., so we need to check if
it will express in LVS after Pkek1168 has been transformed into LVS.
6
Deliverables completed
pKEK1167 (Pkek1140 inserted with a 350bp PCR product, for constructing UvrA mutant LVS.)
KKV317 (UvrA mutant LVS)
Pkek1168 ( pKEK1149 with FLAG being replaced with SIINFEKL, but under a Francisella
promoter)
40 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
Pkek1169 ( Pkek1145 with FLAG being replaced with SIINFEKL but under an ARA-promoter from
the backbone PBAD24 plasmid)
7
Quality of performance
8
Percentage completed
9
Work plan for upcoming month
Good
Approximate 70% of scientific work completed on the milestone
i.
ii.
iii.
iv.
Transform Pkek1168 into LVS.
Perform western blotting for Pkek1168/LVS
Ligate PepO-SIINFEKL cut from pKEK1169 into Pkek894 with Francisella promoter to
construct a new plasmid to see if it will express SIINFEKL in E.coli. and LVS.
Send the strain to Cerus Lab if ii and iii don’t work.
10 Anticipated travel
None.
11 Upcoming Contract Authorization (COA) for subcontractors
None.
Milestone 44
Milestone description: Formulation and evaluation of KBMA LVS: establish photochemical
inactivation regimen of selected uvr mutant of LVS and measure metabolic activity and virulence
of KBMA LVS.
Institution: Cerus
1. Date started: 6/18/2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1) using a small-scale inactivation procedure we have determined that the S-59 psoralen
concentration required to inactivate uvrB LVS is 5uM. This is the same concentration at which
we have been able to inactivate WT LVS. We will next perform a parallel inactivation profile
directly comparing Wt LVS and uvrB mutant and compare the metabolic activity of the
inactivated bacteria.
Photochemical Inactivation of LVS uvrB
NB 963-081
1.E+10
1.E+08
CFU
LVS uvrB
1.E+06
1.E+04
1.E+02
1.E+00
0
1
2
3
4
5
6
7
8
9
10
[S-59] M
2) In order to determine whether the uvrB mutant LVS is more sensitive to other DNA damaging
agents than WT we performed MIC assays using the DNA damaging agents S-303 and cisplatin.
Surprisingly, growth of the WT LVS strain was inhibited at lower concentrations of both S-303 and
cisplatin than either of the 2 uvrB clones we received from UTSA.
41 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
(NB920-184)
LVS and uvrB sensitivity to S-303
OD600 nm
0.45
0.4
0.35
uvrB 1-A T16h
uvrB 1-B T16h
0.3
0.25
LVS 16 h
0.2
0.15
uvrB 1-A T0
LVS T0
uvrB 1-B T0
0.
9
1.
7
3.
4
6.
9
13
.8
27
.5
55
.0
44
0.
0
22
0.
0
11
0.
0
0.1
uM S-303
uvrB 1-A T16h
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
uvrB 1-B T16h
LVS 16h
LVS T0
uvrB 1-A T0
2.
3
4.
6
9.
3
uvrB 1-B T0
11
85
.0
59
2.
5
29
6.
3
14
8.
1
74
.1
37
.0
18
.5
OD600 nm
LVS and uvrB sensitivity to Cisplatin
ug/ml cisplatin
The MIC assay using S-303 was repeated and the WT LVS appeared again appeared be slightly more
sensitive to DNA damage induced by S-303. One possible explanation for the apparent increase in
sensitivity to the DNA damaging agents after deletion of the uvrB gene is that the mutants may grow at a
slightly higher rate than the wild type after serial passage.
S303
NB 920-189
0.35
uvrB 16h
0.2
LVS 16h
0.15
uvrB 1h
0.1
LVS 1h
9
4
7
9
6.
3.
1.
0.
.8
13
.5
27
.0
55
0.
0
11
0.
0
0.05
22
OD600
0.3
0.25
nM
42 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
4. Significant decisions made or pending
none
5. Problems or concerns and strategies to address
The uvrB mutant of LVS does not appear to be more sensitive to DNA damage induced by
photochemical inactivation with S-59 and UVA or by other chemical means. This suggests that
the potency of a KBMA uvrB LVS vaccine is likely to be the same as KBMA Wt LVS which failed
to protect mice against lethal a schuS4 challenge (see MS46). These results suggest that we
reevaluate the KBMA tularemia vaccine strategy and we suggest comparing the efficacy of a
KBMA LVS vaccine to a KBMA Listeria monocytogenes vaccine that expresses Ft antigens.
6. Deliverables completed
none
7. Quality of performance
Good
8. Percentage completed
5%
9. Work plan for upcoming month
The inactivation profile of wt LVS will be directly compared with the uvrB mutant. The metabolic
activity of the inactivated strains will also be compared. Our prediction is that there not be a
significant difference between the two strains.
10. Anticipated travel
none
11. Upcoming Contract Authorization (COA) for subcontractors
none
Milestone 46
Milestone description: Scale up of KBMA LVS vaccine production; Optimize large–scale LVS
culture conditions, Establish 3L culture scale purification conditions, Optimize 3L scale
photochemical inactivation process, Verify protective immunogenicity of vaccine candidates
produced by optimized large-scale process
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: we have demonstrated that LVS grows robustly in Chamberlains Defined Media
(CDM) and have prepared expanded DVC lot 16 LVS cultures grown in CDM for 36 hours,
and stored at -80oC. We have determined that the minimum concentration of S-59 required
for complete inactivation of DVC lot 16 LVS is 5µM and that photochemically inactivated LVS
maintain metabolic activity for at least 12 hours. We produced a 3L lot of LVS in our
fermentor using .001% Sigma antifoam A in CDM and have demonstrated stability for 4
months at -80o in 2 cryopreservation medias. We have found that the LVS provided by DVC
is greatly attenuated for virulence in mice when administered IP compared to literature
reports. We have demonstrated that LVS replicate rapidly in livers and spleens of mice
immediately following IV injection; however, it appears that there is a lag that specifically
affects growth in the lungs. We have also demonstrated that LVS is nearly avirulent when
administered by the SC route.
We have produced a 400mL lot of KBMA wild-type LVS using 10 uM S-59 and 6 J/cm 2 UVA
for initial proof of concept studies, and for later comparison with NER-deficient uvrB LVS
and we have demonstrated that the metabolic activity of this lot is stable for 3 months. We
have demonstrated that KBMA WT LVS IV LD50 is 6.8x108, which represents a 4-5 log
attenuation compared with live LVS. We have demonstrated that doses of KBMA WT LVS as
low as 1 x107 provide protection against 100 x IP LD50 challenge of live LVS. However, none
43 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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. We recently attempted to
measure the T-cell response to a CD4 Tul4 epitope in mice vaccinated with live or KBMA
LVS by intracellular interferon-gamma (IFN-) cytokine staining (ICS) or ELISpot assay, but
were unable to detect an induced response to this epitope. This may be because this epitope
does not bind the MHC molecule with high affinity, or the T cell response elicited by LVS may
actively suppress T cell responses. We recently demonstrated that LVS does not induce IL-6
or MCP-1which are critical hallmarks of a protective inflammatory response. Furthermore,
co-vaccination with LVS decreased the innate inflammatory response to Lm. Administration
of LVS decreased the ability of the elicited T cells to produce the cytokine IL-2.
1) Terry Wu at UNM has completed a protection study with KBMA WT LVS. The data below
demonstrate that neither a (IV or IN) prime nor a prime and boost (separated by 3 weeks)
vaccination regimen with KBMA WT LVS protected against a lethal SchuS4 challenge in
mice.
.
Terry also demonstrated that in this study that live LVS administered IN protected against 50
and 500 cfu challenge of SchuS4. Together these data demonstrate the KBMA WT LVS
vaccine is less potent than live attenuated LVS. Next steps for this type of study include
regimen optimization including increasing the IN dose, increasing the number of vaccinations
(e.g. 3 daily administrations as a prime followed by daily administrations as a boost) and a
comparison with the efficacy of the KBMA uvrB vaccine.
44 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been investigating the efficacy of a wild-type KBMA LVS vaccine. Now
that we have received the uvrB mutant we will focus on producing a lot of KBMA uvrB LVS
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS against a lethal LVS challenge is independent of
metabolic activity. This suggests that comparison of various routes, regimens, or formulations will
be difficult to optimize by protective efficacy. The SchuS4 challenge model in mice is more
stringent, but KBMA LVS failed to protect after two doses. It is possible that the rat model may
allow a higher degree of sensitivity. The suppression of the innate inflammatory response and
the suppression of CD4 T cell cytokine production may potentially indicate that LVS is not a
potent inducer of protective T cell responses. We would like to screen for T-cell responses using
the peptides generated by ASU as an alternative method for optimization of vaccine potency.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
53% of scientific work completed on the milestone
9. Work plan for upcoming months
We will compare the photochemical inactivation profile of a uvrB mutant of LVS and their
metabolic activity in MS44. We will prepare a 400mL scale lot of KBMA uvrB LVS.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis (SCHU S4)
(iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F. tularensis
subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F. tularensis
subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful deletions.
Therefore, our focus is two fold, one is cloning experiments to get our target deletions into vectors
that we can use in creating these deletions and the other is experiments with SCHU S4 itself using
constructs that we believe will allow us to make deletions into SCHU S4.
I. Cloning:
a.
Used an “on hand” plasmid pKEK1042 to finish a pdpD construct that will be needed to
create a deletion of an entire pathogenicity island in Schu S4. This vector is pwsk30 based
already containing a 1 Kb 3’-pdpD fragment at Pst I and Sal I cloning sites; in addition, an
erythromycin gene containing a FRT (flip recombinase transferase) site at the 5’-end is at
EcoRI and Pst I cloning site.
b.
Primers were designed to generate 5’ end pdpD fragment of 1000 bp size:
i. pdpD 1 Kb NotI S4 Forward: 5’-gcgcaaaaaagcggccgctatcgtaagagcctataagg-3’
45 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
c.
ii. pdpD 1 Kb BamHI S4 Reverse: 5’-gcgggatcctaatgccgatgaagctttaccac-3’
Used Schu S4 chromosomal DNA as template and amplified the pdpD 1 Kb fragment using
the above oligos. This product was digested with Not I and BamHI enzymes along with the
plasmid pKEK1042 (Figure 1).
Figure 1.
This represents the 1 Kb 5’-end pdpD PCR product and the pKEK1042 plasmid both digested with Not I
and BamHI and run on a gel for band isolation using the Qiagen Gel Extraction Kit. Lane 1 is the 1 Kb
pdpD fragment; Lane 5 and 6 is digested pKEK1042; Lane 8 is uncut pKEK1042 and Lane 3 is the 1 Kb
ladder.
d.
The isolated DNA from figure 1 was used in ligation reaction and then subsequently used to
transform DH5α cells. This only resulted in 3 colonies; these where grown in liquid cultures
and plasmid was isolated. Will continue with restriction analysis on next report. Data located
in TVD UTSA Notebook 5. What page number? UTSA did not provide before report was due;
Barbara will obtain and insert into this document, after 8/15/07
II. Experiments to generate deletions in Schu4:
a.
Continued with PCR analysis with the pdpA Schu S4 clones generated from last month.
Used kanamycin gene specific oligo (Kan identi Up) paired with a gene downstream of the
pdpA gene to determine in which pathogenicity island (FPI-I, II) this deletion is located. The
goal is to isolate one clone for each of these pathogenicity islands (FPI-I and FPI-II,
respectively) pdpA deletions.
b.
The FPI-I diagnostic gene based on sequence analysis will be FTT1343. This oligo paired
with the kanamycin oligo should generate approximately a 7 Kb PCR product. FTT1343 5’
down: 5’-gccaaagtacaaatagcctgttgctgatcattcacg-3’
c.
The FPI-II diagnostic gene based on sequencing analysis will be FTT1696. This oligo paired
with the kanamycin oligo should generate approximately a 9.6 Kb PCR product. FTT1696
(groL) 3’ UP: 5’-cggtggcatgggcggtatgcctggcatg-3’
d.
Five of the pdpA clones generated last month were used in this long range PCR screen to try
and identify a deletion in each of the pathogenicity islands. Figure 2 below presents two
possible candidates.
Figure 2.
46 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
This figures represents four out of five analyzed using long range PCR to determine in which
pathogenicity island, the pdpA deletion is located in Schu S4. The FTT1343 oligo is used as the FPI-I
specific gene target for both panels. Lanes identified as 2 are the Schu S4 control; lane 3 is the
plasmid used to generate the pdpA deletion; lane 4 is clone 1B pdpA; lane 5 is clone 2C pdpA; lane 6
is clone 3D pdpA and lane 7 is clone 4B pdpA. Based on profile clone 1B pdpA seems to be a
deletion in FPI-I and clone 4B pdpA may be a deletion in FPI-II since a product of the correct size was
generated in the kan specific oligo set. In addition, the PCR reaction run with the FTT1696 (groL)
specific oligo with the Kan oligo did generate a product. (data not shown). Lane 8 is a PCR product
resulting from the iglC specific gene and mod.methelase directed oligo (not part of this discussion).
e.
The PCR products were isolated using the Qiagen gel extraction kit and sent for sequencing.
Some of these product did not generate any sequence with the oligos used to generate the
product. The problem according the company was low DNA concentration of template.
Therefore, I will generate other PCR products with the same oligos and others to generated
confirmation data on each deletion by sequencing. Data located in TVD UTSA Notebook
5.What page number? UTSA did not provide before report was due; Barbara will obtain and
insert into this document, after 8/15/07
f.
Long range PCR was performed to determine if the Schu S4 igLC candidates generated in
last month’s work contains this “intron” inserted in both copies to the pathogenicity islands.
Initially used a representative from each group of experiments performed. That is, 2A and 2B
are from the cryotransformation experiments which was done in duplicate (A and B,
respectively); and the electroporation experiment which generated less number of clones, 3E
is the designated clone used in screening.
g.
The FPI-I diagnostic gene based on sequence analysis will be Trehalase (FTL1151) this oligo
paired with iglC specific oligo (iglC NdeI rev or EBS2 427iglC) will generate approximately 10
Kb gene product. FTL_1152 Rev (LVS): 5’-act tta gat cct ata gta ccc cct aag gct gaa-3’
h.
The FPI-II diagnostic gene based on sequencing analysis will be FTT1719 (modification
methylase) this oligo paired with iglC specific oligo will generate a 10 Kb gene product.
Mod.Methylase Forward 3’ end: 5’-cgt ttt ctt cgt tgg tat ccg taa aga ttt agc-3’
Figure 3.
This represents PCR profiles generated when using chromosomal templates of various iglC clones of
SCHU S4 and Schu S4 as a negative control. In both A and B panels the mod.methylase oligo (FTI-II)
was used. In panel A the intron iglC oligo EBS2 427iglC was used and the expectation is that only the
clones containing the iglC intron insertion will generate a product. In panel A, lanes 3,4, and 5 show
10kb products indicative of the iglC intron insertion. In panel A, Lane 2 is the Schu S4 profile and
does not show a product. In panel B the iglC Nde I rev gene specific oligo was used and all should
generate a product since both are directed to Schu S4 DNA independent of the iglC intron insertion.
i.
Other PCR experiments were done on various iglC clones and some were sent for
sequencing but, the isolated PCR products sent did not generate enough sequence to
confirm the deletion. Therefore, we are preparing more PCR products and will islolate and
purify a more concentrated sample to send for sequencing.
47 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
j.
In addition, one of the igLC clones generated was grown in Chamberlains medium and
prepared for SDS-PAGE. Subsequent western analysis was performed using a goat antiigLC serum to check for the absence of the igLC protein. The results indicated that there is
not wild-type igLC detected in the igLC clone tested. This indicated that the intron insertion
occurred into both pathogenicity islands for this tested igLC clone. We still need sequence
confirmation. Data located in TVD UTSA Notebook 5. What page number? UTSA did not
provide before report was due; Barbara will obtain and insert into this document, after 8/15/07
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
41% (In the 7/15/07 report, the % completed should have been reported as 39% and not as
65%)
9. Work plan for upcoming month
a. Will continue with the 5’-end pdpD cloning into pKEK1042.
b. Will continue with the analysis of the pdpA deletions by sending PCR products for sequencing.
c. Will continue to verify by PCR and sequencing for the correct igLC deletion in Schu S4.
d. Based on immunoblot results we may have the iglC clone therefore, will perform an animal
experiment to see if this clone is attenuated.
e. Order supplies as required.
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 uvrBiglD mutant as a vaccine
candidate (Note book #4, page 106-108). Groups of BALB/c mice (female, 4-6 weeks) were
intranasally (i.n.) immunized with 105, 106 or 107 CFU of ΔuvrBiglD. 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 21 days of vaccination. As shown in Fig. 1,
ΔuvrBiglD-vaccinated mice were highly protected against subsequent pulmonary challenge
48 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
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.
100
105 CFU
106 CFU
107 CFU
PBS
% Survival
80
60
40
20
0
0
6
12
18
24
30
% Body weight
110
105
100
95
90
85
80
0
2
4
6
8
10
12
14
Days after challenge
Fig. 1. Protective efficacy of ΔuvrBiglD immunization against F. novicida infection.
BALB/c mice were immunized intra-nasally with 3 doses (105, 106, and 107 CFU) of
ΔuvrBiglD or PBS and i.n. challenged with lethal dose of F. novicida (1000CFU). Mice
were monitored for survival rate and weight change.
b.
Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglD mutant after
vaccination (Note book #4, page 109-111). Blood was collected from the PBS- and
ΔuvrBiglD- immunized mice (as described above in) at day 21 after priming. Specific antiΔuvrBiglD total antibody titer as well as IgG1 and IgG2a isotypes were determined by ELISA.
Antigens, either UV-irradiated ΔuvrBiglD (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 ΔuvrBiglD produced significant amounts of
specific serum total antibody. Isotyping analyses indicated both Th1 (IgG2a) and Th2 (IgG1)type antibodies were produced in mice after the ΔuvrBiglD immunization. No ΔuvrBiglDspecific antibody was detected in mice mock-vaccinated with PBS at day 21 after
immunization. All tested serum samples showed no reactivity to the unrelated HEL protein.
49 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
3000
PBS
uvrBiglD 10 5
uvrBiglD 10 6
uvrBiglD 10 7
Ab Titer
2500
2000
1500
1000
0500
500
00
Total Ab
IgG1
IgG2a
Fig. 2. Humoral response to ΔuvrBiglD immunization. BALB/c mice were
intranasally immunized with 105, 106 or 107 CFU of the ΔuvrBiglD mutant or PBS
alone as mock vaccination. Sera were collected 3 weeks after immunization and
used to determine titers of anti- ΔuvrBiglD 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
42 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Determine the LD50 of Ft subsp. novicida uvrBiglA double mutant.
b. Monitor Ft subsp. novicida ΔuvrBiglA replication and dissemination in mice.
10. Anticipated Travel
None
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.
The potential uvrB + iglB double mutants are still in the process of being sequenced to
confirm the correct mutations. Originally, several clones were PCR amplified with primers
specific for uvrB. Clones that contained the correct size PCR product had the PCR product
gel purified and sent for sequencing to confirm the mutation. However, the sequencing
50 of 51
Tularemia Vaccine Development Contract: Technical Report
Period: 7/01/2007 to 7/31/2007
Due Date: 8/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob Sherwood, Julie
Wilder, Kathryn Sykes, Stephen Johnston, Justin Skoble, Karl Klose, Bernard Arulanandam, Mitch Magee
b.
center was unable to retrieve any sequence from the clones that I sent. To get around this,
different primers will be used in hopes of getting better sequence conformation. Each clone
grows on both Erythromycin and Kanamycin which is indicative of having the uvrB and iglB
mutation.
Each of the uvrB double mutants were screened for their ability to grow and survive in
macrophages compared to wildtype (WT). Single mutants of the Francisella Pathogenicity
Island have been shown to be important for intramacrophage growth, with the exception of
pdpD. Therefore it was predicted that the uvrB + FPI gene double mutants would be
attenuated for intramacrophage growth as well. Indeed, KKF224 (uvrB + iglC), KKF225 (uvrB
+ iglD), KKF226 (uvrB + iglA) were all attenuated in macrophage growth by ~3 logs (Figure
1). Whereas KKF227 (uvrB + pdpD) retained its ability to grow and survive in macrophages
to approximately wildtype levels (Figure1). Therefore the double mutants behave identically
to the FPI single mutants in their ability to grow and survive in macrophages. Data recorded
in notebook #1 pages 17-18.
Figure 1.
.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
98%
9. Work plan for upcoming month
Further sequencing analysis of the uvrB + iglB clones to confirm the mutations are correct. Send
completed F novicida double mutants [KKF224 (uvrB + iglC), KKF225 (uvrB + iglD), KKF226
(uvrB + iglA) KKF227 (uvrB + pdpD) ] to Cerus group.
10. Anticipated travel
None.
11. Upcoming Contract Authorization
None
51 of 51