Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 4, 5, 12 (UNM &LBERI), 13, 26, 27, 33, 40, 41,
43, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 48
Inactive milestones: 6-11, 14,15, 17-24, 28-31, 34-38, 42, 44, 45, 47, 52-54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. NIAID is working on the IAA with USAMRIID and a legal liability review is pending.
b. As of 12/6/06, Dr. Lyons submitted a request for programmatic support to Dr. Ed Nuzum,
Chief of the Product Development Section in the Office of Biodefense Research Affairs at
NIAID
4. Significant decisions made or pending
a. UNM and NIAID continue to wait for a change in the status of the IAA between NIAID
and USAMRIID.
b. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
c. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
d. UNM EOHS has obtained many of the laboratory documents
i. Documents pending
1. Radiology Facility Accreditation Certificate
5. Problems or concerns and strategies to address
a. UNM will need an external source of human cells from LVS vaccinated individuals, in order to
develop the immunoassays in humans.
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; Work stop has occurred on the SCHU S4 aerosols in primates, until the LBERI
scientists and staff receive the LVS vaccinations.
6. Deliverables completed
None
7. Quality of performance
1 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Good
8. Percentage completed
16%- no change relative to 11/15/06 and 12/15/06 reports
9. Work plan for upcoming 6 months
NIAID Contract Officer will continue to monitor the progress of the IAA between NIAID and
USAMRIID and will inform UNM when and whether the TVD Contractors can be vaccinated under
this IAA.
10. Anticipated travel
Travel could occur in Spring 2007 to Fall 2007, depending on the completion of the IAA.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM may request a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for training on
LVS site vaccination evaluations. The timing of the COA request depends on the achievement of
the IAA.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
No sprays were conducted in December
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
May need to add sonication step to eliminate possible clumping in sprays to reduce variability in
CFU detected.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30%
9. Work plan for upcoming month


Perform additional bioaerosol experiments on vegetative LVS with Collison generator
i. Repeat of studies performed on frozen LVS, but now with fresh LVS
ii. Plan to grow LVS in CB
iii. Plan to quantitate LVS on CHAB
Perform bioaerosol experiments on frozen LVS with sparging generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing frozen and fresh, not lyophilized.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
2 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Working Group
Milestone description: Determine appropriate solid and liquid media for growth of tularemia for
project team
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
None
4. Significant decisions made or pending
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Determined liquid and solid media for LVS growth
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming months
Finalize the protocol for the growth of LVS in liquid and on solid media.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 4
Milestone description: Confirmation of aerosol in vivo in primates
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:



Vaccinated 3 NHPs with 1 x 107 CFU LVS by intradermal route on 11/20/06 = TUL 8
Vaccinated 3 NHPs with 3 x 106 CFU LVS by subcutaneous route on 11/29/06 = TUL 9
Blood collection for PBMC preparation proceeded as indicated in the Table below
(several blood draws occurred in the month of December 2006)
3 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Experiment
Animal IDs
Tul-8
Tul-9
Date of PreDate of
vaccination Bleed Vaccination
Dose of
LVS
Route of
Vaccination
Dates of
Bleeds
A00896 (male); 11/16/06 (Day -4) 11/20/06 (Day 0)
A00908 (male);
A00937 (female)
1 x 107
Intradermal
11/27 (D7),
12/4 (D14),
12/11 (D21),
12/18 (D28)
A00868 (male); 11/29/06 (Day 0)
A00902 (male);
A00659 (female)
3 x 106
Subcutaneous
12/6 (D7),
12/13 (D14),
12/20 (D21),
12/27 (D28)
11/29/06 (Day 0)
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address


LVS titer from previously frozen vials was shown in late November to be variable and not
as predicted, resulting in different doses given to NHPs in TUL8 and TUL9
Trevor Brasel titered 3 LVS vials to obtain an average value of remaining frozen vials;
titers calculated to be 2.2 x 108/ml, 4.4 x 108/ml and 2.9 x 108/ml for an average of 3.2 x
108/ml; this is approximately double the titer originally calculated (1.5 x 108/ml)
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
10%
9. Work plan for upcoming month
Write an IACUC protocol to allowed continued blood collection from vaccinated NHPs (see
Milestone 13 for assays to be conducted with these samples)
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Species tested for sensitivity to LVS & generation of immunity against a
pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Rats Fischer 344
a. Experiment Ftc23 (notebook 94, in progress)
i. The purpose was to compare the protection induced by:
1. Vaccination with LVS s.c., i.d., or i.t.
2. Vaccination with LVS or SCHU S4 i.t.
4 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
ii. Rats were vaccinated s.c., i.d., or i.t. with LVS, i.t. with SCHU S4, or left
unvaccinated (table 1)
iii. Nearly all of the rats survived the vaccination by LVS and SCHU S4
iv. 4 weeks after vaccination, two rats from each group were killed and shown to
be free of the vaccine bacteria in the lungs, spleen, and liver.
v. 7 weeks after vaccination, the vaccinated rats were challenged i.t. with
SCHU S4 to measure the level of protection induced by the vaccine
vi. The challenge doses were chosen to include LD50 based on preliminary
experiments with naïve and vaccinated rats listed below
1. s.c. and i.d. vaccinated rats (Ftc20 notebook 94 pages 5-9)
2. i.t. vaccinated rats (Ftc22 study 1 notebook 94 pages 26-27 and
Ftc22 study 2 notebook 94 pages 28-30)
3. naïve rats (Ftc16 study 1 notebook 85 pages 55-56; Ftc16 study 2
notebook 85 pages 90-93; Ftc16 notebook 85 study 2 pages 75-77)
vii. We will monitor the infected rats for 21 days and should have final results by
1/9/07.
Table 1. Experimental design to determine the effect of bacteria strain and vaccination
route on the level of protection against i.t. SCHU S4 challenge
Vaccination
Bacterial
strain
None
i.t. SCHU S4 challenge
Dose
(CFU/rat)
103 104 105
Route
Dose
(CFU/rat)
LVS
i.d.
s.c.
i.t.
5 x 107
5 x 107
5 x 107
104
104
104
105
105
105
106
106
106
107
107
107
SCHU S4
i.t.
1 x 103
104
105
106
107
Guinea Pigs Hartley Strain
a. Experiment Ftc24 study 3 (Notebook 94, pages 23-25)
i. The purpose was to determine whether increasing the i.n. inoculation volume
from 400 to 500 l will increase the deposition of bacteria into the lungs
ii. 400 l is the maximum volume that can be delivered i.n. without any of the
inoculum being expelled by the guinea pig (Table 2)
iii. An average of 42% of the inoculum was deposited in the lungs.
iv. This is similar to the lung deposition achieved in mice following i.n.
inoculation
v. We will continue to optimize the delivery procedure to improve consistency of
deposition
Table 2. Lung deposition associated with intranasal inoculation of guinea pigs
Animal
1
2
3
Target dose
5 x 104
5 x 104
7 x 104
Actual
lung deposition
1.13 x 104
2.13 x 104
4.12 x 104
%
lung deposition
22.6
42.7
60.5
5 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
LVS and SCHU S4 expansion
a. Experiments Ftc25 (Notebook 94, pages 31-35)
i. The purpose was to compare the virulence of DVC’s SCHU S4 before and
after 48 h expansion in Chamberlain’s broth by intranasal infection of BALB/c
mice
ii. 48 hr expansion culture increased virulence (Figure 1)
1. Reduced mean-time-to-death from 7 days to 5 days
2. Reduced i.n. LD50 from 480 CFU to less than 146 CFU
Figure 1. BALB/c mice (n = 5) were infected i.n. with the indicated doses of SCHU S4
before and after 48 h culture in Chamberlain’s medium. Survival was monitored daily for
14 days.
b. Experiment Ftc26 (Notebook 94, pages 40-43)
i. Justin Skoble from Cerus Corporation indicated that the i.p. LD50 of DVC’s lot
16 LVS before and after 48 h culture in Chamberlains’ broth was 104-105
CFU instead of 1 CFU as published in the literature
The purpose was to determine the i.p. LD50 of DVC’s LVS before and after
48 h culture in Chamberlain’s broth
ii. LD50 is 7.1 x104 CFU before culture and 7.2 x 103 CFU after culture in
Chamberlain’s broth (Figure 2)
iii. Our results is similar to those from Cerus
6 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Figure 2. BALB/c mice (n = 5) were infected i.p. with the indicated doses of LVS before
and after 48 h culture in Chamberlain’s medium. Survival was monitored daily for 15
days.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
37%
9. Work plan for upcoming month
a. Rats
i. Monitor survival of vaccinated rats challenged i.t. with SCHU S4
ii. Repeat experiment to measure the protection induced by vaccination
b. Guinea Pigs
i. Determine the i.d., s.c. and i.n. LD50 for LVS
ii. Determine the i.n. LD50 for SCHU S4
iii. Vaccinate guinea pigs with LVS i.n., s.c., or i.d. routes
c. Hamster – training
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 12-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: UNM
Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1.
a. Experiment Ftc18 study 6 (Notebook 94, pages 16-22-25)
i. The purpose was to determine the minimum number of splenocytes and
formalin-fixed/heat-killed LVS required for detectable proliferation of antigen
specific T cells from vaccinated BALB/c
ii. Titrated splenocytes from 5 x 103 to 105 cells/well and LVS from 5 x 103 to 5 x
105 CFU/well.
iii. Combination of 105 splenocytes and 5 x 105 LVS/well produced the best
signal (Figure 2)
iv. Lower numbers of LVS still stimulated measurable proliferation, but with
diminishing signal
v. Formalin-fixed bacteria appear to be more stable over time than heat-killed
bacteria since both were prepared on 10/19/06 but only
7 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
vi. Anders Sjostedt also reported that formalin-fixed bacteria is more stable over
time than heat-killed bacteria (Woods Hole meeting)
Figure 2. Titration of LVS and splenocytes for optimal T cell proliferation. Splenocytes from
vaccinated BALB/c mice were cultured with heat-killed or formalin-fixed LVS for 5 days. T cell
proliferation was measured by BrdU incorporation and chemiluminescent anti-BrdU ELISA. The
bars show mean value from three wells
4. Significant decisions made or pending
a. Formalin-fixed bacteria will be used for future proliferation assays because they are
more stable over time
b. Proliferation assays will include splenoyctes from unvaccinated BALB/c mice to
ensure that any measured signal is due to antigen-specific proliferation and not nonspecific response to bacterial components
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
15%
9. Work plan for upcoming month
a. Optimize SOP for measuring T cell proliferation stimulated by formalin-fixed bacteria
b. Develop an SOP for generating bone-marrow derived macrophages using the
protocol described by Cowley SC and Elkins KL (2003) J. Exp. Med 198:379-89.
c. Develop an SOP for measuring T cell-stimulated macrophage killing of intracellular F.
tularensis once we are able to reproducibly generate BMDM
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
8 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,




Froze extra PBMCs from 12/18/06 (TUL 8 Day 28) and 12/27 (TUL9 D28) samples using
the CTL and Cerus protocols, respectively
- Cells are frozen at 10 x 106/ml or 5 x 106/ml respectively in 0.5 ml aliquots
- Cells are stored at LRRI, in Cryofreezer in Room 313 North Building 14A, Tower C,
Box 1
- Details on procedure can be found in the binder TVDC 1 under Tab for TUL 8, Day
28 and TUL 9 Day 28
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
87% of scientific work has been completed
9. Work plan for upcoming month



Test whether NHP PBMCs stain positively for anti-human CD19 and anti-human IgM.looking for B cells
Set up a proliferation assay and IFNγ ELISPOT assay with frozen PBMCs and compare
to values obtained with fresh cells. Thaw at a pre-determined timepoint in January (1/11
would be 8 weeks for the cells frozen down on 11/16/06) and set up in the same assays
to see what fraction of function can be recovered
Test intracellular staining of IFNg in whole blood and PBMC preparations
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None for this milestone.
Milestone 13-LBERI
Milestone description: Compare assays in animal models (sensitivity)
Institution: LBERI
1. Date started: 11/16/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions






Processed blood from NHPs before and after LVS vaccination according to schedule
shown in Table above under Milestone 4
PBMCs are enumerated and an aliquot stained on a cytospin for differential cell typing;
Data is shown below in Table 1.
Vaccination by either route appears to increase the numbers of neutrophils and activated
monocytes in the blood
Plasma is being saved for future anti-LVS immunoglobulin ELISA
PBMCs are set up at various cell concentrations (1, 0.25 and 0.0625 x 10 6/ml) and
stimulated to proliferate with Con A (positive control), heat-killed and formalin-fixed and
formalin-fixed LVS (at 1, 0.25 and 0.0625 x 105/ml)
PBMCs (if extra are available) are set up at 20,000 cells/well and stimulated to secrete
IFN with Con A (positive control), heat-killed and formalin-fixed LVS (at 1, 0.25 and
9 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,




0.0625 x 105/ml)
Data on proliferation is shown in Figures 1-6
- S.C. vaccination appears to induce an earlier and superior response to LVS by
PBMCs as compared to I.D. vaccination (Figure 1)
- PBMCs from I.D. vaccinated NHPs do proliferate in response to LVS at later time
points (Days 21 and 28), especially evident when 1 x 106 cells/ml are plated (Figure
2)
- Curiously, Con A responsiveness, even on Day 0, is increased in PBMCs from S.C.
vaccinated NHPs (Figure 3)
- Proliferation in response to LVS increases as more cells are plated (1 x 106/ml vs.
0.25 x 106/ml; Figure 4)
- Both LVS preparations (heat-killed and fixed) induce proliferation from S.C.
vaccinated NHPs at all concentrations tested (Figure 5)
- Yield of PBMCs is variable but improved after animal caretakers were instructed to
remove the needle from the syringe before expelling the blood into the collection tube
to prevent cell shearing (TUL 8 d28 and TUL9 D21 and D28 bleeds were NOT
expelled through a needle; Figure 6)
Data on IFN ELISPOT is pending analysis
Monkeys are healthy, showing normal activity
All data is stored in binder TVDC 1 in the Wilder laboratory as well as in C:\Documents
and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\ TUL 8 or TUL 9;
10 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Table 1. Effect of LVS Vaccination on Cellular Yields and Cell Types
Experiment Animal
ID
Tul 8 (ID)
A00896
A00908
A00937
Day Cell yield % Mononuclear
post- (x 106)
Cells
LVS
(Lymphs/Monos)
-4
30.8
81.0
-4
44.1
90.3
-4
29.0
86.8
% Activated
monocytes
%
PMNs
16.7
9.5
5.3
1.7
0.25
2.5
A00896
A00908
A00937
7
7
7
1.63
3.08
7.50
13.2
36.3
34.0
60.4
52.4
54.1
26.1
11.1
11.5
A00896
A00908
A00937
14
14
14
3.68
2.78
0.6
25.5
40.25
51.0
53.5
33.75
42.25
21
26
7.0
A00896
A00908
A00937
21
21
21
4.05
4.11
1.75
43
51.8
47.5
49
47
48
9
1.25
5.25
A00896
A00908
A00937
28
28
28
24.8
17.8
8.8
69.3
82.3
60.8
26.8
15.6
34
4.25
2.2
5
11 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
RLU (Mean +/- SEM)
140000
ID, Media
120000
ID, LVS hk Hi
ID, LVS ff Hi
100000
SC, Media
80000
SC, LVS hk Hi
60000
SC, LVS ff Hi
40000
20000
0
Day 0
Day 7
Day 14
Day 21
Day 28
Figure 1: S.C. Vaccination induces higher and earlier proliferative responses to LVS
preparations (either heat-killed or formalin fixed) than does I.D. vaccination. An average of 3
NHPs is shown for each time point and stimuli shown. Only responses to LVS plated at 1 x
105/ml and PBMCs plated at 0.25 x 106/ml are shown. RLU = relative light units as measured
on the plate-reading luminometer.
RLU (Mean +/- SEM)
9.00E5
6.00E5
ID, Media
ID, LVS hk Hi
ID, LVS ff Hi
SC, Media
SC, LVS hk Hi
SC, LVS ff Hi
3.00E5
0
Day 0
Day 7
Day 14
Day 21
Day 28
Figure 2: Proliferative response to LVS preparations are more apparent in the I.D.
vaccinated group when higher numbers of PBMCs are plated (1 x 106/ml). An average of 3
NHPs is shown for each time point and stimuli shown. Only responses to LVS plated at 1 x
105/ml and PBMCs plated at 0.25 x 106/ml are shown. . RLU = relative light units as
measured on the plate-reading luminometer.
12 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
RLU (Mean +/- SEM)
1400000
1200000
ID, Con A
SC, Con A
1000000
800000
600000
400000
200000
0
Day 0
Day 7
Day 14
Day 21
Day 28
Figure 3: Proliferation to Con A is increased in the S.C. vaccinated NHPs even before LVS
vaccination (Day 0), as compared to the I.D. vaccinated NHPs. Only responses with PBMCs
plated at 0.25 x 106/ml are shown. RLU = relative light units as measured on the platereading luminometer.
RLU (Mean +/- SEM)
1.40E6
1.20E6
1
.25
1.00E6
8.00E5
6.00E5
4.00E5
Day 28, LVS ff Hi
Day 28, LVS hk Hi
Day 28, Con A
Day 7, LVS ff Hi
Day 7, LVS hk Hi
0
Day 7, Con A
2.00E5
Figure 4: Proliferation in response to LVS is most evident when cells are plated at 1 x10 6/ml
as compared to 0.25 x 106/ml. An average of 3 NHPs is shown for each time point and
stimuli shown. Only responses to LVS plated at 1 x 10 5/ml and PBMCs from S.C. vaccination
group are shown. RLU = relative light units as measured on the plate-reading luminometer.
13 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
RLU (Mean +/- SEM)
9.00E5
6.00E5
LVS
LVS
LVS
LVS
LVS
LVS
hk Hi
hk Mid
hk Lo
ff Hi
ff Mid
ff Lo
3.00E5
0
Day 7
Day 28
Figure 5: Proliferation to LVS in PBMCs from S.C. vaccinated NHPs is equivalent when
various doses of LVS are tested (Hi = 1 x 105/ml, Mid = 0. 25 x 105/ml and Lo = 0.0625 x
105/ml). Responsiveness to formalin-fixed (ff) and heat-killed (hk) preparation are also
equivalent. An average of 3 NHPs is shown for each time point and stimuli shown. Only
responses from PBMCs plated at 0.25 x 106/ml are shown. RLU = relative light units as
measured on the plate-reading luminometer.
PBMC Yield (Mean +/- SEM)
45
40
ID
SC
35
30
25
20
15
10
5
0
Day 0
Day 7 Day 14 Day 21 Day 28
Figure 6: Yield of PBMCs from I.D. vs. S.C. vaccinated NHPs on various days pre- and postLVS vaccination is shown in millions. We can provide no explanation for the very high yield
of cells on Day 0 for the I.D. group;however, on days 21 and 28 from the S.C. group and on
day 28 for the I.D. group, blood was collected and expelled into a collection tube AFTER
removal of the needle from the syringe to prevent cell shearing.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have a concern that the response to Con A, a T cell mitogen, was increased in the S.C.
vaccinated group of NHPs above the I.D. group, even at day 0, thus unrelated to LVS
14 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
vaccination. We will address this concern by phenotyping the blood from these NHPs in the
coming month to determine if there is a higher number of T cells for some reason in the S.C.
group compared to the I.D. group. We have already confirmed that no treatment of these
NHPs in the previous PK study in which they were used can explain this phenomenon.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
10% of scientific work has been completed
9. Work plan for upcoming month





Analyze all IFN ELISPOT data to determine if NHPs are producing IFN in response to
LVS
Develop anti-LVS immunoglobulin ELISA for testing plasma (compare formalin-fixed and
heat-killed LVS as antigen)
Write an IACUC amendment to allow us to continue bleeding the vaccinated NHPs
Once IACUC amendment is approved, phenotype the blood cells and PBMCs from the 6
vaccinated NHPs to determine if differences exist in T cell numbers in the S.C. vs. I.D.
group
Once IACUC amendment is approved, test whether more PBMCs/well will give a better
IFNg ELISPOT response to LVS
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None for this milestone.
Milestone 26
Milestone description: Design of ORF expression templates and testing by evaluating subprotein production (Design HTP SOPs, Test HTP SOPs for making sub-proteins from ORFs)
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Prior to testing calmodulin and GFP in our IVT system (data below), we had tested 6 F.
tularensis (FTU) ORFs as IVT templates. However these were done without specific
labeling, rendering detection difficult and polypeptide assignment uncertain. As a means
of distinguishing newly synthesized proteins from the pre-existing proteins of the lysate
without radioisotope, we supplemented the lysate with a charged E. coli lysine tRNA
labeled with the fluorophore BODIPY-FL, as presented in the December 20 Technical
call. Since this worked well for the 2 test proteins (GFP and calmodulin), we have
placed the 6 FTU ORFs in our linear templates and will be using them to generate
proteins. We are also placing the ORFs in a plasmid IVT expression vector as a positive
control throughout manipulations and for future quality control assays.
We have also conducted preliminary IVTs in the presence of the BirA enzyme,
which biotinylates the nascent polypeptides. These reactions will be optimized and
strepavidin purifications will be conducted next.
15 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Figure 1. Evaluation of Templates for in vitro ORF expression
GFP
Calmodulin
1)
2)
3)
4)
5)
6)
7)
molecular weight ladder,
GFPexpressed with plasmid template,
calmodulin expressed with plasmid template,
calmodulin expressed with covalent LEE template,
calmodulin expressed with linked LEE template,
calmodulin expressed with linked LEE template, short promoter,
calmodulin expressed with linked LEE template, short promoter and biotin attachment
site,
8) calmodulin expressed with plasmid template, short promoter,
9) calmodulin expressed plasmid template, short promoter and biotin attachment site.
These results show that similar amounts of product are generated under all conditions
tested in fig 1 above. Therefore, we conclude that our recombinant LEE attachment
protocol works, that we can include a biotin binding site to use for high efficiency
purification, and a streamlined T7 promoter can be used.
The results are located on CIM’s internal R server:
peptide\Research\CIM\Mol_Biol_Tech\IVT\Hetal's IVTs
4. Significant decisions made or pending
No decisions have been made this month. In the upcoming month we plan to have
purification results to permit us to select the best scheme.
16 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
5. Problems or concerns and strategies to address
Now that we have seen 2 proteins successfully made, of 2 different lengths, we do not
anticipate any difficulty expressing the FTU sub-proteins in vitro. While protein
production can be highly problematic when using in vivo systems such as E. coli, the far
less complex and non-biological nature of the in vitro lysates renders production far less
protein dependent.
Identifying the best purification method will be our next challenge. If strepavidin does not
prove successful for the purification of the newly synthesized proteins, we have 2 other
tags in our template with which to work.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
70%
9. Work plan for upcoming month
Determine the yield of the 6 other FTU sub-proteins in the total lysates and begin
optimizing purification strategies.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Vaccine candidates
Institution: UNM
Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
1.
a. Ftc27 study 1 (notebook 94, in progress)
i. We received approximately 500 overlapping peptides derived from known F.
tularensis proteins that are predicted to bind H-2d
ii. The purpose was to develop a standard operating procedure for identifying
peptides that stimulate proliferation of antigen-specific T cells from
vaccinated BALB/c mice
iii. 5 x 105 splenocytes/well were cultured with 94 separate peptides (final 20
nM) for 5 days.
iv. T cells proliferation was measured by BrdU incorporation and anti-BrdU
chemiluminscent ELISA
v. Approximately half of the peptides stimulated ~106 RLU and the remainder
104 RLU, clearly identifying peptide with and without stimulatory activity
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
17 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
NA
8. Percentage completed
2%
9. Work plan for upcoming month
a. Continue to develop and optimize a SOP for measuring peptide-induced T cell
proliferation
b. Test all 500 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
i. Splenocytes from vaccinated and unvaccinated BALB/c mice will be used to
demonstrate antigen-specific response
ii. Formalin-fixed bacteria will be used as positive control
c. Assemble a list of stimulatory peptides for ASU to analyze for common stimulatory
motifs
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 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


Completed the comparison of PLL and Corning UltraGAPs slides as substrates for printing
microarrays
Data is stored at our server location
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\FTU_Substrate_Test
18 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Corning
UltraGAPs
PLL

•
•
•
Arrays printed on PLL-coated slides perform comparably to Corning
Ultragaps using median normalized signal intensities
Comparable signal to noise ratios
Comparable background signal intensities
• PLL spot sizes are ~50% larger than the Corning
ultragaps slides
• So visually, the differences appear greater than
when you analyze the median intensity value across
the slide. PLL is a little greater median intensity
than Corning Ultra GAPS (see chart below)
19 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
5
4.5
Median Normalized Intensities
4
3.5
3
2.5
2
1.5
1
0.5
0
Corning



PLL
Full-deck prints have been delayed because of printer problems (See #5. below).
Initial predictions are completed for designing oligonucleotides to detect LVS missing genes
or those with >3 mis-matched nucleotides to current probe sets. Verification of predictions is
ongoing.
Submitted PFGRC request for TIGR tularemia microarrays.
4. Significant decisions made or pending
Have chosen to print on PLL slides rather than Corning UltraGAPs
5. Problems or concerns and strategies to address

Both microarray printers are exhibiting mechanical problems and are undergoing service calls
(1/8/2007 for the Nanoprint 60 and 1/9/2007 for PE Spottarray 72).
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month




Perform full deck (57 slide) print runs on PLL slides
Will start testing with GDPs with purified RNA
Perform test comparisons between TIGR and ASU arrays
Finish design and order 70mer oligonucleotides to use as probes to detect extra LVS genes,
not detectable with the SCHU S4 probe set..
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
20 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Milestone 40
Milestone description: Phenotyping of Ft novicida nucleotide excision repair mutants; Measure
degree of attenuation of uvr mutants in macrophages and in mice
Institution: Cerus
1. Date started: 3/2/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
We previously demonstrated that the uvrB and uvrA single and the uvrA uvrB double mutant
Ft novicida strains have no growth defect in Chamberlain’s defined medium (CDM) or in J774
macrophages and that all of the mutants are fully virulent in BALB/c mice when administered by
the intraperitoneal (IP) route of administration. All Ft novicida strains were still virulent by the IV
route, but were previously unable to determine the LD 50 because the LD50 were below 100 cfu.
1) We repeated our attempt to compare the virulence of U112, uvrA, uvrB, and uvrA uvrB Ft
novicida strains when administered by the IV route. Each strain was diluted to an approximate
concentration of 1000 cfu/ml and a series of four 5-fold dilutions were then made and 100 ul of
each was injected into the tail veins of Balb/c mice (hence a range of 100cfu-<1cfu was
administered IV). 100ul samples of the initial dilution tube was plated on CHAH plates in triplicate
to determine the actual number of cfu administered, and these numbers were used in the
calculation of LD50. The LD50 values ranged from 1 to 38 cfu indicating that all the strains were
highly virulent when administered IV. These data, combined with our earlier study of IP virulence,
demonstrate that wild-type Ft novicida are equally virulent when administered via the IP or the IV
route, although when administered IV the animals died between 4 and 7 days after infection,
which is longer survival than seen with comparable doses administered IP. While both the uvrA,
and uvrA uvrB mutants appear to be more than 20 fold attenuated compared to the wild type
strain when administered IV it is not clear whether these differences are meaningful, because the
numbers of cfu are so low. We will next determine whether any of the Ftn strains is attenuated
for virulence when administered by the subcutaneous (SC) route because this route is supposed
to require the highest dose to initiate a lethal infection and may accentuate minor differences in
virulence.
Calculated IV LD50 (cfu)
Calculated IP LD50 (cfu)
U112
0.95
0.57
uvrA
27
0.82
uvrB
8.1
0.2
uvrAuvrB Study #
38
AS06-112
2.72
AS06-090
4. Significant decisions made or pending
We have selected CDM and cystine heart agar with hemoglobin (CHAH) as liquid and plate
medias for cultivation and enumeration of Ft novicida. Ft novicida nucleotide excision repair
mutants are not attenuated in vitro and do not appear to be dramatically attenuated for virulence
in mice.
5. Problems or concerns and strategies to address
Abrogation of the nucleotide excision repair pathway through uvrA and/or uvrB deletions
does not result in a significant loss in virulence. This suggests that a secondary attenuating
mutation will be required if the SCHU S4 strain were to be used as the vaccine background.
Since LVS is already attenuated in humans, it may not require a secondary attenuating
mutation. We will be screening attenuated Ft novicida mutants that also have uvr mutations
for immunogenicity in milestone 43, with the ultimate goal of selecting an attenuating
21 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
mutation to construct in SCHU S4. Our results suggest that the virulence of Ft novicida is not
as route dependent as LVS.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
70%
9. Work plan for upcoming month
SC LD50 will be initiated with all Ft novicida strains using doses ranging between 1x107 and 1 cfu.
SC is the route by which Francisella are the least virulent and hence may accentuate minor
differences in virulence. We will also monitor the growth of Ft novicida in lungs livers and
spleens after IV and IP infection with 100 cfu (~100 x LD50) in order to determine the growth
kinetics in organs after systemic infection. Once we have established the growth kinetics of wildtype Ft novicida we will compare the growth rates of the nucleotide excision repair mutants and
we will be able to measure the effect of vaccination on organ burden.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA F. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Using a small-scale (3.5mL) procedure we previously identified the minimum dose of UVA
required to achieve complete photochemical inactivation was 4 J/cm 2. We found that
minimum concentration of S-59 required to inactivate wild type Ft novicida U112 was 40M,
which was only slightly higher than the 20M concentration required to inactivate Ftn uvrB,
Ftn uvrA, and uvrA uvrB double mutant strains. Our first attempt to scale up to a 400ml
scale failed to achieve complete inactivation and was not dependent on S-59 dose,
suggesting that 4 J/cm 2 was an insufficient UV dose at this scale.
1) This month we completed the direct comparison of the metabolic activity of each Ftn strain
after photochemical inactivation at the 3.5 mL scale using 2x the minimum concentration of
S-59 required for complete inactivation and 4J/cm 2 UVA. Each KBMA strain had less
metabolic activity over 12 hours than live, which is to be expected because the KBMA
bacteria cannot replicate. All strains had measurable increases in metabolic activity for at
least 12 hours. The KBMA wild-type which was inactivated with 80uM S-59 had slightly less
activity than the nucleotide excision repair mutants that were inactivated with 40uM S-59.
The KBMA uvrB strain had slightly more metabolic activity, than uvrA, and uvrA uvrB.
These data demonstrate that there is no advantage to having both genes knocked out and
confirm that it is acceptable to select the uvrB strain for further evaluation.
Metabolic Activity of KBMA vs. Live Ft novicida Strains
2.0
1.8
1.6
90
1.4
1.2
22 of 38
Ftn wt Live
Ftn uvrA Live
Ftn uvrB Live
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
2) We attempted to optimize the scaled-up inactivation process by titrating the UVA dose
required to achieve complete inactivation. Ftn uvrB was grown in CDM in shaker flasks to
early stationary phase in the presence of 40 or 120M S-59. 400mL of culture was
transferred to a UV-transparent container and illuminated with 2, 4, 6, 8 J/cm 2 of UVA. After
each UVA exposure 1mL of bacterial suspension was plated for cfu on CHAH plates.
Complete inactivation was achieved after illumination with 6 J/cm 2 UVA. This UVA dose is
comparable to what is used for making lots of KBMA L. monocytogenes and B. anthracis.
400ml scale photochemical inactivation results
notebook
[S-59]
UVA
Particles/mL
cfu/mL
location
948-155 Arm-1 40M
2J/CM2
1 x109
TMTC
2
9
948-155 Arm-1 40M
4J/CM
1 x10
18
948-155 Arm-1 40M
6J/CM2
1 x109
0
948-155 Arm-1 40M
8J/CM2
1 x109
0
948-155 Arm-2 120M
2J/CM2
1 x109
TMTC
948-155 Arm-2 120M
4J/CM2
1 x109
1
2
9
948-155 Arm-2 120M
6J/CM
1 x10
0
948-155 Arm-2 120M
8J/CM2
1 x109
0
These results demonstrate that complete inactivation of the uvrB Ft novicida strain can be
achieved with 40M S-59 and 6 J/cm2 at the 400ml scale. We are now poised to produce a
400ml lot of KBMA Ft novicida. Once this lot is completed, metabolic activity will be
measured by MTS assay, degree of attenuation will be determined by infection of Balb/c
mice, stability of metabolic activity will be monitored at -80o C monthly.
4. Significant decisions made or pending
All nucleotide excision repair mutants (uvrA, uvrB, and uvrA uvrB) were equally sensitive to
S-59 and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 6J/cm 2 as the
conditions for making 400ml-scale KBMA lots.
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms;however,
the high degree of metabolic activity retained by all strains suggests that the wild-type is
highly sensitive to photochemical inactivation under these conditions and that the KBMA
strategy is still viable.
23 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
45% of scientific work completed on the milestone
9. Work plan for upcoming month
We will produce a 400mL lot of KBMA urvB Ft novicida, measure the metabolic activity of
the lot by MTS assay, determine the degree of attenuation by IP and IV infection of Balb/c
mice, and monitor the stability of metabolic activity after storage at -80o C.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 43
Milestone description: Create uvrA or uvrB mutants in LVS
Institution: UTSA
1 Date started: 5/01/2006
2 Date completed: In progress
3 Work performed and progress including data and preliminary conclusions
. The plasmid pDS132 is being modified for use in F. tularesis. This is a conditionally-replicative plasmid
that has a counterselectable marker (sac B), we have adapted for use in F.t. by first altering the multiple
cloning site and then inserting a Ft promoter (gro ELp) to facilitate expression of sacB and the antibiotic
resistance marker (cat). The mating plasmid pKEK1090, which has the GroEL promoter properly inserted
to facilitate sacB/cat expression, will be used as a vector to clone UvrBUpFpKanDn. The plasmid will be
mated into the LVS strain, as this is the most efficient way to make the mutant, and the selective markers
in the plasmid such as SacB will help to eliminate the plasmid backbone.
3.1 pKEK1006 was cut with NotI to release UvrBFpKan fragment, and pKEK1090 was cut
with NotI. The both fragments were ligated together, and electroporated into
Dapa- e. coli cells.
3.2 The electroporated E. coli was grown onto LB/Kanamycin/Dapa plate, and one positive
plasmid was designated as pKEK1114.
3.3 The positive colony pKEK1114 in dapa cell was grown up onto LB/Kanamyin/Dapa
plate to mid-log phase(about 3-4 hrs onto fresh plate from overnight plate); and
LVS strain was grown up onto TSA/+++ plate to mid-log phase( about 6-8 hrs).
3.4 The bacteria were scraped off from the corresponding plates, and measured OD600, to
let the OD600 ratio of e.coli to LVS be 1:10. The equal volume of bacterial
suspension of e.coli and LVS were put onto sterile filter membrane, and put
onto TSA/+++/Dapa plate for overnight mating.
3.5 The mating mixture was scraped off the filter, and making serial 1:10 fold dilution in
TSA/+++/Chloramphenicol (10 ug/ml). The diluted solution was spread onto
TSA/+++/Agar/Chloramphenicol (10 ug / ml), and incubated at 37C for at least
4-6 days.
3.6 The colonies from TSA/+++/Agar/Chloramphenicol(10ug/ml) were patched onto
TSA/+++/Agar/Kanamycin(15ug/ml)/Sucrose(5%), and incubated at 37C for 48
hrs.
24 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
3.7 The colonies resistant to kanamycin and sucrose were grown into
TSA/+++/Kanamycin(15 ug/ml) liquid for 24hrs.
3.8 The 24 hrs cultured liquid was grown up onto TSA/+++/Agar /Kanamycin (15
ug/ml)/Sucrose(5%) to get a single colony.
3.9. The single colony from step 3.8 was grown up and frozen away. Simultaneously colony
PCR was performed to identify the colony with different primer combinations.
LVSUvrBUp+LVSUvrBDn1, LVSUvrBUp+KanIdentifR, LVSUvrBDn1 + KanIdentifF,
CatIdentifF+CatIdentifR to identify the probable mutants.
1
2
3
4
5
6
7
8
Lane 1-4, LVSUvrBFpKan mutant
Lane 5-8, LVS strain
Lane 1, 5 CatIdentifF+CatIdentifR (these 2 lanes differ in pattern. These should be different as wild type
LVS does not have chloramphenicol resistance gene and the wild type is used as a control. This
indicates the plasmid backbone is still in there as only plasmid backbone has chloramphenicol resistance
gene )
Lane 2,6 LVSUvrBDn1 + KanIdentifF ( these 2 lanes appear the same, because the plasmid backbone is
still in there, so with this pair of primers will not give amplified product if the mutant still keeps the plasmid
backbone, )
Lane 3,7 LVSUvrBUp + KanidentifR (these 2 lanes differ in pattern. Lane 7 is a control. The different
pattern in these two lanes is a clear indication the pKEK1114 was inserted into UvrB site of LVS strain . )
Lane 4,8 LVSUvrBUp+ LVSUvrBDn1 (these 2 lanes appear the same. This primer will give an amplified
product at least more than 4.5 kbs in control lane 8( which sometimes cannot be amplified due to larger
25 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
size), and will not give an amplified product in the lane 4 as the plasmid backbone of pKEK1114 is still in
there. All these patterns, particularly Lane 3,7, and Lane 1,5 indicate the UvrBUpFpKanDn was inserted
into the UvrB site with the plasmid backbone)
3.10 Based on the PCR patterns, the mutant is definitely correct , i.e., the UvrB is mutated
in LVS strain. The only problem is that plasmid backbone is still in there. Based
on PCR pattern on lane 1, which clearly indicates the mutant still has the
chloramphenicol resistance gene, and only place the chloramphenicol
resistance gene kept is plasmid backbone; PCR pattern on lane 3 indicates that
the UvrB gene is mutated, as the pattern would be the same in comparison with
the wild type (lane 7) if the Uvrb is not mutated; Lane 2 should give an amplified
product with similar size as lane 3 if the plasmid backbone is lost. As there is no
amplified band in lane 2, which means that the backbone is still in there. All
these PCR patterns indicate that the UvrB is mutated, but the plasmid backbone
is still in there.
All the data were recorded on UTSA TVDC notebook #2, page 57-59.
4
Significant decisions made or pending
The UvrB mutant appears to be created in LVS, but we need to remove the plasmidbackbone.To
get rid of plasmid backbone in the probable mutant.
5
Problems or concerns and strategies to address
The TSA media is pre-made mixture and contains sodium chloride, which might negatively
impact the effect of sucrose to remove the plasmid backbone. To remove the potential
complications of the sodium chloride, we will order the individual ingredients for the TSA media,
and prepare the TSA agar plate without sodium chloride and with 5% sucrose added.
6
Deliverables completed
PKEK1006 (contains theUvrBUp FpKanDn fragment), and pKEK1114(containing
UvrBUpFpKanDn in pKEK1090)
7
Quality of performance
8
Percentage completed
Good
Approximate 48% of scientific work completed on the milestone
9
Work plan for upcoming month
Repeat the sucrose selection process to get rid of the plasmid backbone in the LVS
strain. Fresh TSA media with 5% sucrose and without sodium chloride will be used.
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
26 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
3. Work performed and progress including data and preliminary conclusions
We have demonstrated that LVS grows robustly in CDM and have prepared expanded DVC
lot 16 LVS cultures grown in CDM for 36 hours, and stored at -80oC. We have determined
that the minimum concentration of S-59 required for complete inactivation of DVC LVS is 5µM
and that photochemically inactivated LVS maintain metabolic activity for at least 12 hours.
We were recently able to achieve growth of LVS at a 3L scale in our fermentor using .001%
Sigma antifoam A in CDM and have been monitoring the stability of the stored product in 2
cryopreservation medias. We have found that the LVS provided by DVC is greatly attenuated
for virulence in mice when administered IP compared to literature reports.
1) The stability of fermentor-grown LVS culture at -80o C in 2 different freezing solutions is
being monitored on a monthly basis. The titer of each cryopreserved stock was determined
by making duplicate dilution series and plating 100 ul of each of the 10-7 dilution on 5 CHAH
plates, hence each bar in the graph represents the mean of 10 plate counts and error bars
represent the standard deviation. After one month at -80o C both samples had titers greater
than or equal to the pre-freeze titers, suggesting that both cryopreservation solutions appear
to be performing well. We will continue to monitor the viability of these samples by plating for
cfu, we will also compare the metabolic activity by MTS assay, and we will measure the
virulence of each in mice. Since 10% sucrose is less toxic to animals than 8% DMSO we will
switch to this as a cryopreservation agent in future preparations.
Fermentor-Grown LVS Viability (cfu/mL) Pre-Freeze, T=0, and T=1 Month Stability
HBSS + 1% sucrose + 8% DMSO
1.E+10
2.7E+09
2.9E+09
4.9E+09
HBSS + 10% Sucrose
4.5E+09
2.1E+09
4.5E+09
cfu/mL
Pre-Freeze
1.E+09
T=0
T=1 Month
1.E+08
1.E+07
Lot: 948-119 Arm-1 (Freeze Buffer)
Lot: 948-119 Arm-2 (10% Sucrose)
2) We previously demonstrated that our frozen stocks of expanded lot 16 LVS were greater
than 10x more virulent than the lyophilized DVC lot 16 LVS when administered via the IP
route, but that both were greatly attenuated compared to literature reports of the virulence of
LVS. We were concerned that the HBSS buffer used for the serial dilutions may have been
responsible for the attenuation and thus compared the virulence of LVS diluted in PBS or
HBSS administered by the IP route. The IP LD50 of LVS diluted in either PBS or HBSS buffer
was approximately 1000 cfu. This is approximately 10-fold lower than our first LD50 value
using HBSS but still significantly higher than literature reports of LVS virulence when
administered by this route. We will determine the IP LD50 of Cerus expanded LVS for one
more time using 5-fold serial dilutions starting at 1x105 for better resolution.
3) We determined the virulence of Cerus expanded LVS when administered IV to Balb/c
mice. The LD50 was determined twice and the range was 3.16x103 to 7.1x104 for IV
administration which is similar to the value of 2.2 x104 IV reported for NDBR Lot 4 LVS in
Green et. al (2005). These data suggest that the LVS provided by DVC is not globally
attenuated, but may have an IP-specific phenotype.
27 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Route
LD50 Expanded LVS lot 16
LD50 DVC LVS Lot16
Study Number
IP
>100
ND
AS06-090
LVS LD50 Values
IP
IP
3.4x104
1.7x103
(?HBSS?)
(HBSS)
>4.5x105
ND
AS06-106
AS06-109
IP
1.1x103
(PBS)
ND
AS06-109
IV
3.16 103
IV
7.1x104
ND
AS06-108
ND
AS06-112
4. Significant decisions made or pending
We have selected CDM and CHAH as liquid and plate medias for cultivation and enumeration of
LVS. We have determined the minimum concentration of S-59 psoralen required for complete
inactivation of DVC LVS is 5µM. We have switched to using Sigma Antifoam A concentrate as
our antifoam agent for large-scale propagation of LVS. We have selected 10% sucrose as our
cryopreservation agent.
5. Problems or concerns and strategies to address
The LVS provided by DVC appears to be highly attenuated when administered IP, the degree of
attenuation is less when LVS is expanded in CDM. The attenuation is not evident when the
expanded LVS is administered IV.
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
The stability of LVS stored at -80oC in various cryopreservation agents will continue to be
evaluated by MTS assay and plating monthly on CHAH plates. We will compare the
virulence of LVS stored in the various cryopreservation agents by injecting IP into BALB/c
mice.
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
28 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generated mutants in SCHU S4 we need to develop tools to generate successful
deletions. Our first desired deletion for the project is igLC; therefore, we are trying to clone this
gene into a modified vector of pDS132 described in milestone 43 in November’s report. This
plasmid is essentially a suicide vector than can be used in mating into SCHU S4 to initiate a
cross-over into the chromosome and generate the deletion. Secondly, we have a strategy of a
construct for which experiments have indicated that it can be used to generate the insertion of the
deletion; however, we had trouble removing the plasmid part of the construct from the
chromosome. Thus, we are working on getting the Sac B gene cloned into this construct
(KEK964) which should help resolve the retention of the plasmid in the chromosome.
I. Cloning of igLC:
a. Isolated plasmid from KEK1090 and KEK906 bacteria stocks to use in cloning the
igLC deletion.
b. KEK1090 was digested with XhoI then subsequently with Not I; the DNA was gel
purified using the Qiagen gel extraction kit described earlier.
c. KEK906 plasmid was digested with Sal I then subsequently with Not I. This digestion
resulted in two fragments of DNA; the smallest of about 3 Kb is the igLC deletion
fragment which was gel purified and used to ligate into the digested plasmid KEK1090
(b. above).
d. The ligations were then used to transform into DH5 λpir and SM10λpir cells,
respectively.
e. The transformations were placed onto 20 ug/ml Chloramphenicol selection plates and
place at 37°C for growth.
f. Will evaluate results on next report. Data located in TVD UTSA Notebook 3, page 7072
II.
Experiments to generate deletions in Schu4:
a. Prepared a new PCR product with oligos mentioned earlier: SacB XbaI BHI UP NEW
5’ -gcg ctc tag agg atc ctt att tgt taa ctg tta att g- 3’ and
SacBDownBgl2new (note this was mislabeled as up instead of down on earlier
report)
5’ –gcg cag atc tcc atc ttc aaa cag gag ggc tgg aag aag c -3’
b. The template used to generate the SacB product was pCVD422 (mentioned earlier).
This product was used to prepare for two different ligations.
c. The 1498 base pair (bp) SacB PCR product was digested with Bgl II then
subsequently with BamHI. This PCR product was used to ligate into the KEK964 and
KEK1023 plasmids, respectively. Both plasmids were digested with BamHI (BHI)
and alkaline phosphatase (CIP) treated before setting up the ligation reactions.
d. A separate SacB PCR product was digested with Xba I then subsequently with Bgl II
and used to ligate into KEK964 plasmid digested with Xba I and BamHI.
e. The ligation reactions in c. and d. (above) were used in transformation reactions into
DH5α cells.
f. Prepared dilutions of each of the transformations and spread onto selective
Kanamycin (70 ug/ml) plates.
g. KEK964 BHI/CIP + SacB Bgl II/BHI and KEK964 Xba I/BHI + SacB Xba I/Bgl II
transformations gave many colonies. Prepared pool suspensions of 10 colonies
each into 100 ul sterile water; this was boiled for 5 minutes then spun at 13 k rpms for
2 minutes. Used 2 ul of each pool suspension and set up a PCR with oligos Fn Bgl2
For and SacB XbaI BHI UP NEW the resulting product will be 2485 bp. This product
29 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
will indicate that the Sac B gene is in the plasmid and also that this gene will be in the
5’ to 3’ orientation relative to the upstream Francisella promoter already in the
KEK964 plasmid.
h. Three colony pools from each KEK964 +SacB contructs was used in the first set of
PCR mentioned in g. (above). The KEK964 BHI/CIP+SacB Bgl II/BHI only one pool
(pool 2) of three yielded a product of 2485 bp; and two pools yielded the correct
product size in the KEK964 Xba I/BHI + SacB Xba I/ Bgl II transformants.
i. Prepared plasmid preparations from clones 11 thru 20 from the KEK964 BHI/CIP +
SacB Bgl II/BHI transformants from pool 2 screened in g. (above)
j. Prepared plasmid preparations of clones 21-30 (pool 3) from the KEK964 XbaI/BHI +
SacB Xba I/Bgl II transformants.
k. All the plasmid preparations were then used in a PCR with the oligos mentioned in g.
(above); those plasmid giving a 2485 bp product were used in restriction digests
analysis.
l. Used the following enzymes to identify the correct construct based on sequence of
Sac B gene: EcoRI – correct construct should yield 4398 bp fragment (mostly vector)
and SacB fragment 1287 bp; Kpn I correct construct should yield 3553 bp (mostly
vector) fragment and 2132 bp (containing SacB) fragment; EcoRi/Kpn I double
digestion correct construct should yield 3539 bp (vector fragment), 1287 bp and 859
bp (containing SacB) fragments.
m. Found Clone 13 for the KEK964 BHI/CIP + Sac B Bgl II/BHI transformant to be
correct by restriction analysis and C26 for the KEK964 Xba I/ BHI + SacB Xba I / Bgl
II to be correct by restriction analysis (See Figure 1). Will send for sequencing
confirmation.
This figure represents part of the restriction analysis done on the
KEK964 + SacB contructs. C13 and C14 represent clones from the
KEK964 BHI/CIP+SacB Bgl II/BHI transformation; and C26 and C27
represent clones from the KEK964 Xba I/BHI + SacB Xba I/ Bgl II
transformation. Lanes 2, 4, and 9 are all uncut plasmids and lanes
3, 5, 6, 7 and 8 are plasmid DNA cut with EcoRI and Kpn I (E/K -double digestion). The constructs containing the SacB gene when
cut with these enzymes should yield 3539 bp (vector); 1287 bp and
859 bp fragments in the profile. Lane 6 is the cloning vector
KEK964 digested with the mentioned enzymes (yielded only 1
band). Clones 13 and 26 look correct, showing bands indicative of
the SacB insert.
30 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
n. KEK1023 BHI/CIP + SacB Bgl II/ BHI gave only 8 colonies with the undiluted 100 ul
plating; and the religation control transformation yielded 4 colonies.
o. Prepared plasmid from the colonies resulting from the KEK1023+SacB transformants
from n. (above) none were found to be potential candidates. Will perform another
transformation with DH5α competent cells with the same ligation reaction used in this
previous one and will use undiluted cells to plate to generate more colonies to
screen.
p. Data located in TVD UTSA Notebook 3, page 50-53.
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
34%
9. Work plan for upcoming month
a. Will need to prepare more competent (DH5α and DH5αλpir) cells for future
transformations.
b. Will evaluate any transformants resulting from the ligation with KEK1090 and the igLC
deletion fragment from KEK906.
c. Will send the two clones from the KEK964 constructs with SacB to be sequenced.
d. Will set up another transformation reaction with the 1023 BHI/CIP + SacB Bgl II/BHI
ligation to generate more colonies to screen.
e. Also, the KEK964+SacB contructs, once confirmed by sequencing, will be used to clone
in the MglA deletion to test this construct in Schu S4 strain.
f. The confirmation of the correct constructs will be done by restriction endonuclease
digestions and sequencing.
g. Order more supplies as needed
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
31 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
a. Isotyping of anti-Δiglc antibodies in sera of mice immunized with Ft novicida Δiglc + LVS
LPS (Note book #4, page 55-56). Groups of BALB/c mice (6 mice per group) were
vaccinated with Ft novicida Δiglc (106 CFU per mouse via intranasal (i.n.) route) and
purified LVS LPS (50μg per mouse via intraperitoneal injection). These mice received an
additional boost with Ft novicida Δiglc (106 CFU, i.n.) + LVS LPS (25µg, i.p.) two weeks
after the first immunization. Mice were bled after vaccination and sera were analyzed by
isotype-specific ELISAs using UV-inactivated Δiglc-coated microtiter plates. As shown in
Fig. 1, mice primed with Δiglc+ LVS LPS exhibit the induction of specific total, IgG1 and
IgG2a antibodies. No binding of the immune sera was observed with the unrelated
antigen hen egg lysozyme (HEL).
.
Figure 1. S
b. Determine the kinetic growth and clearance of the Ft novicida Δiglc mutant in target
organs after i.n. vaccination (Note book #4, page 58-62). BALB/c mice were vaccinated
with Ft novicida Δiglc mutant (106 CFU) intranasally. Lungs, liver, spleen, and lymph
nodes were collected from the vaccinated mice at a three day interval (3 mice per time
point). Numbers of bacteria in each organ were determined by dilution plating. As shown
in Fig. 2, there was heightened replication of the organism in the lungs within the first 12
days, with reduction noted at day 15. There were lower levels of replication within the
liver and spleen, with minimal detection of replicating organism by day 15. There were
organisms recovered from the draining lymph nodes, but at much lower levels than that
seen with the other target organs. Although Ft novicida Δiglc is highly attenuated in mice,
there is detectable replication of the organism up to 15 days. This replication pattern may
account for the robust priming of the immune system, with this attenuated vaccine
candidate, Ft novicida Δiglc.
32 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Fig. 2 Kinetic growth and clearance of the Ft novicida Δiglc mutant in target organs after i.n. vaccination.
Bacterial burdens were determined from lungs, liver and spleen of individual mouse and from pooled lymph
nodes at each time point (3 mice per time point). Numbers ( *) of mice without detectable bacterial burden are
indicated.
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
25 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Determine the LD50 of Ft subsp. novicida iglB mutant. The route of infection will be
intranasal and the dose range will be 103 to 106 CFU.
b. Monitor Ft subsp. novicida iglB mutant replication and dissemination in mice
10. AnticipatedTravel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
33 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
Milestone 51
Milestone description: Construction and delivery of Ft subsp. Novicida uvrA or uvrB plus
pdpD, iglA, iglB, iglC or IglD double mutant
Institution: UTSA
1 Date started: 11/01/2006
2 Date completed: In progress
3 Work performed and progress including data and preliminary conclusions
Creation of Ft novicida mutants:
We intend to create all ten permutations of mutants: uvrA + pdpD, iglA, iglB, iglC, and iglD, and
uvrB + pdpD, iglA, iglB, iglC, and iglD. Within the Klose laboratory, we already have the
constructs to make pdpD, iglA, iglC, and iglD mutants, which will be combined with the two
constructs to make uvrA and uvrB mutants. However, we do not yet have the construct to make
the iglB mutation, so our first goal of this milestone is to construct this particular mutation. Once
we have this mutation, we will then make the entire set of double mutant strains listed.
3.1 Amplify upstream and downstream fragments of IglB:
Set up:
10X KOD XL Buffer
5.0ul
dNTP 2mM
5.0ul
IglBUp 2uM
5.0ul
IglBDn 2uM
5.0ul
U112 chromosomal DNA 10ng/ul 5.0ul
KOD XL DNA Polymerase 2.5u/ul 0.4ul
DH2O
24.6ul
AND
10X KOD XL Buffer
5.0ul
dNTP 2mM
5.0ul
IglBUp1 2uM
5.0ul
IglBDn1 2uM
5.0ul
U112 chromosomal DNA 10ng/ul 5.0ul
KOD XL DNA Polymerase 2.5u/ul 0.4ul
DH2O
24.6ul
At 94C 2’, then at 94C 15”, 55C 15”, 72C 30” for 35 cycles.
34 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
3.2. Recover the IglB up and down fragments from gel with Qiagene gel recovery kit.The upstream
fragment is 575bp and downstream fragment is 624bp, and as we mention in the paragraph 3.5, the
plasmid was sequenced, and has correct sequence)
1
2
3
4
5
6
7
8
Lane 1-4 IglBUpstream fragment; Lane 5-8 IglBDownstream fragment.
3.3 Overlapping PCR to get IglBUpFpErmCDn fragment:
10X KOD XL Buffer
5.0ul
dNTP 2mM
5.0ul
IglBUp 2uM
5.0ul
IglBDn1 2uM
5.0ul
IglBupstream fragment
3.0ul
FpErmC fragment
3.0 ul
IglBDownstream fragment
3.0ul
KOD XL DNA Polymerase 2.5u/ul 0.4ul
DH2O
20.6ul
At 94C 2’, then at 94C 15”, 55C 15”, 72C 3’ for 35 cycles.
3.4 The overlapping PCR IglBUpFpErmCDn fragment and pGEMT were cut with Sac2, and
corresponding to fragments were recovered from gel with Qiagen kit. The size of the fragment is
around 2.6kb, and can be seen on the gel very easily
35 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
1
2
3
4
5
6
Lane 1-3, pGEMT; Lane 4-6, IglBUpFpErmCDn fragment
3.5 The two fragments were ligated together and electroporated into E. coli, and one colony with
Erythromycin resistance was sent out for sequencing, and has the correct sequence. The
plasmid was designated as pKEK1118.
3.6 The pKEK1118 was cryotransformed into U112, and colony PCR was performed with colonies
grown on TSA/+++/Agar/Erythromycin plate with IglBUp and IglBDn1 primers. Each PCR
fragment was cut with Bgl2. The universal primers pUCori XhoI and pUCoriEcoRI were used to
check for loss of plasmid backbone.
36 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Lane 1: U112 control; Lane 2: pKEK1118 control; Lane 3-10 IglB mutant cut with BglII.
Lane11: Negative control; Lane 12: pUC ori positive control; Lane 13-20, IglB mutant for pUC ori. As the
primers used for checking pUC ori are always used in the lab for all mutants, there is some pcr product
contamination for negative control but based on band intensity we know which ones definitely lose the
plasmid backbone. We always load same volume of PCR reaction product for electrophoresis and the
band intensity of lane 13-20, except lane 16, is at the same level of negative control. We will repeat the
checking of pUCori experiment in next experiment. When we do not use the primer pair in the lab for
about one week, the negative control will become really negative( without any band), indicating that the
contamination level is decreased. Also we may design a pair of new primers in another region of the
plasmid backbone, so we can alternatively use the new primer pairs to check for the presence of the
plasmid backbone and to reduce the chance of pUC ori pcr product contamination .
37 of 38
Tularemia Vaccine Development Contract: Technical Report
Period: 12/01/2006 to 12/31/2006
Due Date: 1/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Bob
Sherwood, Julie Wilder, Justin Skoble, Kathryn Sykes, Mitch Magee, Stephen Johnston, Karl
Klose, and Bernard Arulanandam,
3.7 One of the positives was frozen away, and designated as KKF 235 representing the IglB mutant.
All data were recorded in UTSA TVDC notebook #2, page 101-102.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
pKEK1118 (IglBUpFpErmCDn), KKF235 (IglB mutant)
7. Quality of performance
Good
8. Percentage completed
Approximate 10% of scientific work completed on the milestone
9. Work plan for upcoming month
Double mutants will be made for each mutant IglA, IglB, IglC, pdpD for UvrA and UvrB, respectively.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None.
38 of 38