Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 4, 5, 12/13(UNM/LBERI), 19, 21, 26 27, 28, 33, 34
(UNM/ASU), 40, 41, 42, 43, 46, 49, 50, 51
Completed milestones: 1, 16, 25, 32, 39, 48
Inactive milestones: 6-10, 11, 14, 15, 17, 18, 20, 22, 23, 24, 29, 30, 31, 35-38, 44, 45,
47, 52-54
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/1005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. 4/2/2007: Nicole Banks (LBERI) submitted a draft subcontract with True Foundation and
a draft budget for the LVS vaccinations to Brian Jamieson, the NIH Contract Officer
affiliated with the IDIQ with LBERI.
b. 4/24/07: LBERI received funding from NIAID for IDIQ C13 to support the receipt of the
LVS vaccinations from USAMRIID and to support the subcontract with the TRUE
Foundation which will provide project management for the LVS vaccinations and
administer the payments to USAMRIID.
c. Four way CRDA between USAMRIID, True Foundation, UNM and LBERI is under
development
d. Subcontract between LBERI and True Foundation is under development
e. UNM EOHS is acquiring current certifications, CV’s for Radiology Facility and TriCore
Laboratories which will provide the local pre-health screenings for the LBERI and UNM
scientists
f. USAMRIID (Bev Fogtman, Dr. John Aldis, Marilynn Lee, and Cindy Barrick) have
provided the current Tularemia SIP Protocol, Informed Consent, Instructions to External
Participants, Risk Assessment Form, and Fee Schedule
g. During a 5/7/07 conference call between USAMRIID, UNM, and LBERI, the USAMRIID
team answered questions regarding the logistics of the LVS vaccinations and the prehealth screenings. Minutes of the meeting are available and have been reviewed by the
USAMRIID team. Once the CRDA and subcontract are completed, it will take at least 6
months to vaccinate 46 total vaccinees, in monthly groups of 8 vaccinees. USAMRIID will
allow two UNM EOHS nurses to be trained to read the LVS vaccination sites for days
7,14, 28 etc.
1 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
4. Significant decisions made or pending
a. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
b. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
c. Dr. Lyons has arranged to obtain the JCAHO certificate from University Hospital as
demonstration that the UH Radiology Facility is accredited; USAMRIID will accept this
certification.
d. UNM and LBERI will offer the LVS vaccinations to 46 scientists; USAMRIID will be
providing the LVS vaccinations over the next 8 months, approximately.
e. 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.
c. LBERI is prioritizing the LBERI scientists and staff who will be offered the LVS vaccinations
through USAMRIID over the next 2-8 months.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
17%
9. Work plan for the next month
a. Complete the 4 way CRDA between USAMRIID, True Foundation, UNM and LBERI
b. Complete the subcontract between True Foundation and LBERI
c. Prioritize the LBERI and UNM scientists who will be offered the LVS vaccination
d. Maintain excellent communications with USAMRIID to understand the SIP protocol
requirements
10. Anticipated travel
Travel to USAMRIID could occur in summer 2007 to fall 2007
11. Upcoming Contract Authorization (COA) for subcontractors
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 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
SUMMARY through 4/30/07
a. Standardized growth conditions for LVS have been determined which allow reproducible
growth and minimize batch to batch media variability
i. LVS is grown 48 hrs in Chamberlain’s broth with shaking at 37ºC
ii. LVS is stored at -80ºC in 10% sucrose in Chamberlain’s broth
b. Effects on LVS aerosols have been determined for the Collison generator
i. LVS reconstituted from lyophilized media is killed by aerosolization in the Collison
generator.
ii. Frozen LVS provides stable bioaerosols between the concentrations tested (10 3 –
108 CFU/mL) with spray factors ranging from approximately 4x10-7 to 1x10-6.
iii. Fresh LVS also provides stable bioaerosols between the concentrations tested (10 4 –
108 CFU/mL) with spray factors ranging from approximately 2x10-7 to 1x10-6.
b. Studies were carried out on the effects on LVS aerosols using the sparging generator.
Upon completion of numerous bioaerosol runs, it was determined that the system
involved a complex setup, demonstrated irreproducibility between runs, and poorly
aerosolization LVS. Because of these observations, it was decided to halt testing of the
sparging generator.
c. Preliminary studies were initiated on the effects on LVS aerosols using a disposable,
plastic nebulizer (Aeromist).
d. Studies have begun on the effects on LVS aerosols using the micro-pump generator.
Monthly: April 2007:
a. A final seven sprays were performed with frozen LVS at a target spray concentration of
107 cfu/mL using the sparging generator
i. Target concentrations were accurate and consistent with previous tests as shown in
Figure 1.
ii. Spray factors were consistently low with those seen in previous studies (see Figure
2).
iii. Data located in the following folder: \\Saturn\absl3\Agent and Study Specific Data
and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078 (TUL03)\TUL-03\Sparging Generator. Specific day-to-day data files are assigned their
own folders are as follows:
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Sparging
Generator\Frozen LVS\20Mar2007
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Sparging
Generator\Frozen LVS\23Mar2007
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Sparging
Generator\Frozen LVS\28Mar2007
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Sparging
Generator\Frozen LVS\6Apr2007
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Sparging
Generator\Frozen LVS\13Apr2007
3 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Sparging: Actual vs. Target CFU/mL (Frozen)
1.80E+07
Actual CFU/ml (Log10)
1.60E+07
1.40E+07
3/28/2007
1.20E+07
3/20/2007
1.00E+07
3/23/2007
8.00E+06
4/6/2007
6.00E+06
4/13/2007
4.00E+06
2.00E+06
3.00E+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)
Figure 3-1. Actual vs. Target Spray Concentration for Frozen LVS using the Sparging Generator.
Note: Microsoft Excel was used to create the graph.
Sparging: Actual CFU/ml vs. Spray Factor (Frozen)
Spray Factor (Log10)
0.00
-1.006.85
6.90
6.95
7.00
7.05
7.10
7.15
7.20
7.25
-2.00
-3.00
3/20/2007
-4.00
3/23/2007
-5.00
3/28/2007
-6.00
4/6/2007
-7.00
4/13/2007
-8.00
-9.00
-10.00
Actual CFU/mL (Log 10)
Figure 3-2. Spray Concentration vs. Spray Factor for Frozen LVS using the Sparging Generator.
Note: Microsoft Excel was used to create the graph.
b. Three sprays were performed with frozen LVS at a target spray concentrations of 10 7
cfu/mL using an Aeromist disposable, plastic nebulizer
4 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
i.
Target concentrations were lower than those observed with other generators (Figure
3).
ii. Spray factors were better than those seen using the sparging generator, but
consistent with those seen using the Collison nebulizer (see Figure 4).
iii. Data located in the following folder: \\Saturn\absl3\Agent and Study Specific Data
and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078 (TUL03)\TUL-03\Plastic Nebulizer. Specific day-to-day data files are assigned their
own folders are as follows:
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Aeromist
nebulizer\13Apr07
Aeromist: Actual vs. Target CFU/mL (Frozen)
4.00E+06
Actual CFU/ml (Log10)
3.50E+06
3.00E+06
2.50E+06
2.00E+06
4/13/2007
1.50E+06
1.00E+06
5.00E+05
3.00E+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)
Figure 3-3. Actual vs. Target Spray Concentration for Frozen LVS using the Aeromist Plastic
Nebulizer. Note: Microsoft Excel was used to create the graph.
5 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Aeromist: Actual CFU/ml vs. Spray Factor (Frozen)
Spray Factor (Log10)
-5.00
-5.205.60
5.70
5.80
5.90
6.00
6.10
6.20
6.30
6.40
6.50
6.60
-5.40
-5.60
-5.80
4/13/2007
-6.00
-6.20
-6.40
-6.60
-6.80
-7.00
Actual CFU/mL (Log 10)
Figure 3-4. Spray Concentration vs. Spray Factor for Frozen LVS using the Aeromist Plastic
Nebulizer. Note: Microsoft Excel was used to create the graph.
c.
Three sprays were performed with freshly reconstituted Bacillus globigii (BG) spores
(provided by Dugway) at target spray concentration of 104 105and 106 cfu/mL using the
micropump generator. This was an initial test to determine if the micropump was capable
of effectively generating biological aerosols before beginning LVS aerosols. BG spores
were chosen because they can safely be handled in a BSL-2 environment. Though they
do not mimic LVS, they were used to provide an initial evaluation of the micropump
generator.
i. Target concentrations were lower than those observed with other generators (Figure
5), but this was likely due to titer calculations. LBERI was provided a dry powder
form of the spores with an estimated titer. Based on our observations, the actual
concentration value was approximately 1 log10 lower than the value provided to us.
ii. Spray factors were better than those seen using any generator to date (see Figure 6).
The spray factor is a unitless measurement and is defined as the ratio of the aerosol
concentration to the starting concentration. The bioaerosol is defined as being more
efficient as this value approaches 1.
iii. Data located in the following folder: \\Saturn\absl3\Agent and Study Specific Data
and Miscellaneous Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL03\Micro Pump. Specific day-to-day data files are assigned their own folders are as
follows:
 \\Saturn\absl3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY06-078 (TUL-03)\TUL-03\Micro
Pump\25Apr07
6 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Micropump: Actual vs. Target CFU/mL (BG spores)
Actual CFU/ml (Log10)
2.52E+05
2.02E+05
1.52E+05
4/25/2007
1.02E+05
5.20E+04
2.00E+03
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
Target CFU/ml (Log10)
Figure 3-5. Actual vs. Target Spray Concentration for BG Spores using the Micropump Generator.
Note: Microsoft Excel was used to create the graph.
Micropump: Actual CFU/ml vs. Spray Factor (BG spores)
Spray Factor (Log10)
-5.00
-5.200.00
1.00
2.00
3.00
4.00
5.00
6.00
-5.40
-5.60
-5.80
4/25/2007
-6.00
-6.20
-6.40
-6.60
-6.80
-7.00
Actual CFU/mL (Log 10)
Figure 3-6. Spray Concentration vs. Spray Factor for BG Spores using the Micropump Generator.
Note: Microsoft Excel was used to create the graph.
7 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
4. Significant decisions made or pending
Testing of the sparging generator has halting due to numerous encountered problems.
Data consistent with those observed using the Collison nebulizer were seen with the Aeromist
nebulizer experiments presented herein. Best spray factors seen to date with the micropump
generator.
5. Problems or concerns and strategies to address
Not applicable
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
59% - Milestone deadline was extended 4-6 weeks to allow completion of Micropump and one
other (TBD) generator (from 6/1/07 to 8/31/07)
9. Work plan for upcoming month



Perform bioaerosol experiments on frozen and fresh LVS with Micropump generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
Perform bioaerosol experiments on frozen and fresh LVS with another generator
(probably an ultrasonic generator)
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing frozen and fresh, not lyophilized.
iv. Generator choice yet to be determined. One possibility is the Sonik ultrasonic
aerosol generator.
Select optimized method for LVS bioaerosol generation and determine if SCHU-4
bioaerosols also behave similarly using that method.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Working Group
Milestone description: Determine appropriate solid and liquid media for growth of tularemia for
project team
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
Bob Sherwood completed the SOP for LVS Growth in Chamberlains Medium
4. Significant decisions made
Standardized the SOP for the growth of LVS in Chamberlain’s medium
Distributed the SOP to NIAID, DVC and UNM teams
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Determined liquid and solid media for LVS growth
Expanded LVS shows no reduction in virulence in mice
8 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Expanded SCHU S4 in Chamberlains or on agar shows no difference in virulence in mice
LVS Growth in Chamberlain’s Medium SOP is finalized
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month and next 6 months
No new work is planned
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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:
a. 3 non-human primates originally vaccinated by the i.d. route (A00896, A00906 and
A00937) were re-injected with 150,000 formalin fixed LVS via the i.d. route on the upper back
and they were observed for 3 days for a delayed type hypersensitivity (DTH) response
b. The results are presented below in table format (Table 1). The results obtained from an
identical skin test performed on 3 NHPs originally vaccinated via the s.c. route are presented
for comparison (A00659, A00868 and A00902).
Table 4-1
A00896
A00906
A00937
A00659
3/26/07
i.d. 150K LVS
i.d. 150K LVS
i.d. 150K LVS
i.d. 150K LVS
3/27/07
No reaction
No reaction
No reaction
No reaction
A00868
i.d. 150K LVS
No reaction
A00902
i.d. 150K LVS
No reaction
3/28/07
No reaction
No reaction
No reaction
5 mm, slightly
red; raised bump
4 mm, slightly
raised and red
bump; diffuse red
rash of 35 mm
No reaction
3/29/07
No reaction
No reaction
No reaction
8 mm, slightly
red, raised bump
4 mm, slightly
raised and red
bump; diffuse red
rash of 35 mm
No reaction
b. Data interpretation:
i. None of the NHPs that were originally vaccinated via the i.d. route had a response to an
i.d. injection of formalin fixed LVS. In contrast, two of the three NHPs originally
vaccinated via the s.c. route had a reaction to the formalin fixed LVS that resembled a
DTH reaction.
iii. The reactions mounted by A00659 and A00868 suggest that s.c. vaccination of these
NHPs 16.5 weeks previously resulted in activation of the cellular immune response
9 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
i.

It is unclear why A00902 had no response to the formalin fixed LVS as its
proliferative response to LVS was not that different than the other two NHPs
when tested in vitro; it is also unclear as to why none of the i.d. vaccinated NHPs
responded to the i.d. formalin fixed LVS; however, in general we have observed
that the i.d. vaccinated NHPs proliferated less well in vitro to LVS as compared to
the s.c. vaccinated NHPs.
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\ prep for 041307
mtg.doc and N:\My Documents\Tularemia Contract\prep for 041307 mtg.doc
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
No work is planned on this milestone in the next month as we are waiting to challenge these
NHPs with Schu4 once the aerosolization protocol is developed. We plan to do this challenge in
November 2007 when these NHPs will have been vaccinated one year previously.
10. Anticipated travel
None anticipated at the present time
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 5
Milestone description: Small species tested for sensitivity to LVS & generation of immunity
against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Fischer 344 rats
a. Experiment Ftc32 study 1 (Notebook 104, pages 1-5)
i. The purpose of this experiment is to repeat the vaccination/challenge
experiment (Ftc23 study 2) comparing different vaccination routes and
strains in their ability to protect Fischer 344 rats against i.t.. SCHU S4
challenge
ii. All of the LVS-vaccinated rats recovered completely from vaccination;
however, only 14 of 24 rats survived i.t. SCHU S4 vaccination
10 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
iii. After confirming clearance of the LVS and SCHU S4 vaccine, the vaccinated
rats were challenged i.t. with SCHU S4 (Table 1)
iv. We are monitoring the challenged rats for clinical signs of illness and survival
and will report the results in the next report
Table 1. Experimental design to compare the ability of different vaccination routes and to protect Fischer 344 rats
against i.t. SCHU S4 challenge
Vaccination
Bacterial
strain
None
No. of vaccinated rats infected i.t. with indicated SCHU S4 dose
Route
-
Dose
CFU/rat
-
LVS
i.d.
s.c.
i.t.
4.0 x 107
4.0 x 107
4.4 x 107
SCHU S4
i.t.
4.4 x 101
5.7 100
6
5.7 x 101
6
6.0 x 102
6
8.7 x 104
2.8 x 105
2.6 x 106
6
6
6
6
6
6
6
6
6
6
6
b. Experiment Ftc37 study 2 (Notebook 104, pages 6-8)
i. The purpose is to determine the histological appearance of the lungs, liver
and spleen of naïve Fischer 344 rats infected i.t. with a lethal dose of SCHU
S4
ii. Naïve rats were infected i.t. with 400 SCHU S4. Three rats were killed on the
day of infection and every 3 days thereafter to collect the lungs, liver, and
spleen
iii. The tissues are currently being processed at LRRI and, upon return, will be
examined by Dr. Julie Hutt at UNM
c. Experiment Ftc38 study 1 (Notebook 104, pages 9-14)
i. The purpose is to determine the kinetics of LVS proliferation, dissemination
and clearance after s.c. vaccination
ii. We decided to vaccinate the rats by s.c. route because previous results
(Experiment Ftc23) showed that s.c. vaccination was as good as other
vaccination routes in protecting rats against respiratory SCHU S4 challenge
and because it is the preferred route to vaccinate humans
iii. Naïve rats were vaccinated with 2.7 x 107 LVS s.c.
iv. Systemic dissemination was observed on day 3 post vaccination
v. The bacterial burden was also highest on day 3 post vaccination and
declined thereafter
vi. The number of LVS isolated from the lungs, spleen and liver varied among
the three rats at all time points.
vii. The rats cleared LVS 21 days after vaccination
11 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Table 2. Proliferation and dissemination of LVS following s.c. vaccination of naïve Fischer 344 rats
CFU/organ
Rats- set 1
Rats set 2
Rats-set 3
Day p.i.
lung
Liver
spleen
Lung
Liver
spleen
lung
liver
spleen
3
2,160 324,000 550,000
192 132,000
600,000
96
4,800
165,000
6
120
480
1,300 53,400
6,000
5,500
720
2,880
3,700
9
1,200
0
100
120
0
4,500
0
0
4,200
21
0
0
0
0
0
0
0
0
0
d. Experiment Ftc40 (Notebook 104, pages 15-17)
i. The purpose is to determine the kinetics of SCHU S4 proliferation,
dissemination and clearance in s.c. LVS vaccinated rats
ii. Naïve rats were vaccinated s.c. with LVS
iii. We are waiting 6 weeks for the rats to clear the LVS vaccine before
challenging them i.t. with SCHU S4
iv. The challenge dose will be approximately 50 SCHU S4 to allow us to
compare kinetics of SCHU S4 proliferation and dissemination before
(reported previously from Experiment Ftc37 study1) and after LVS
vaccination
e. Experiment Ftc39 study 1 (Notebook 94 pages 140-143), Experiment Ftc39 study 2
(Notebook 94 pages 163-167) and Experiment Ftc39 study 3 (Notebook 184-185)
i. The purpose is to develop an ELISA assay to confirm LVS vaccination in rats
based on sero-conversion.
ii. We followed instructions from Freyja Lynn to optimize the antigen coating
concentration and sera concentration
iii. Sera were collected from naïve and LVS-vaccinated rats
iv. 96-well plates were coated with 2 x 104 to 1 x 107 CFU/ml heat-killed or
formalin-fixed LVS
v. Sera from naïve and LVS-vaccinated rats were titrated from 1:800 to
1:25,600 dilution. At 1:800 dilution, sera from naïve rats produced minimal, if
any, background signal
vi. Both formalin-fixed and heat-killed LVS could be used for coating plates;
however, the optimum coating concentration was lower using heat-killed
LVS. This is similar to Julie Wilder’s results using vaccinated monkey sera
(Figure 1)
vii. The optimal coating concentration is 1-5 x 106/ml heat-killed LVS and the
optimal serum dilution is between 1:1600 to 1:800
12 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Figure 1. Optimization of ELISA for measuring sero-conversion in LVS-vaccinated
Fischer 344 rats. Heat-killed and formalin-fixed LVS were titrated from 2 x 104 to 1 x 107
CFU/ml and the sera from LVS-vaccinated rats were titrated from 1:800 to 1:25,600
dilution.
Hartley Guinea Pigs
a. Experiment Ftc41 (Notebook 104, pages 18-21)
i. The purpose is to determine the resistance of i.n. LVS vaccinated Harley
guinea pigs to i.n. SCHU S4 challenge. This is a repeat of Experiment Ftc28
(Notebook 94, pages 152-156)
ii. Naïve guinea pigs (n = 6 to 10) were vaccinated i.n. with 103, 105, and 107
CFU LVS
iii. We killed two vaccinated guinea pigs/group to determine whether they have
cleared the LVS vaccine. We are waiting for the plating results.
iv. When we have verified that the vaccinated guinea pigs have cleared the LVS
vaccine, we will challenge them i.n. with SCHU S4
b. Experiment Ftc42 (Notebook 104, pages 22-23)
i. The purpose is to determine the resistance of s.c. LVS vaccinated Harley
guinea pigs to i.n. SCHU S4 challenge. This is a repeat of Experiment Ftc28
(Notebook 94, pages 152-156)
ii. Naïve guinea pigs (n = 6) were vaccinated s.c. with 103, 105, and 107 CFU
LVS
iii. We are waiting 4 weeks for the vaccinated guinea pigs to clear the LVS
vaccine
iv. When we have verified that the vaccinated guinea pigs have cleared the LVS
vaccine, we will challenge them i.n. with SCHU S4
4. Significant decisions made or pending
As we move from mice to rats, guinea pigs, and rabbits, we will need to use larger and larger
volumes of buffer to adequately homogenize the infected tissues to measure lung deposition
and bacterial burden. Since increasing the buffer volume reduces our level of detection, we
will now only use vaccination or challenge doses that can be measured with confidence
5. Problems or concerns and strategies to address
a. We noticed in Ftc32 (described above) that we were only able to recover SCHU S4
from the lungs of 2/3 of the rats challenged i.t. with SCHU S4. This suggested that
13 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
1/3 of the rats were infected through the esophagus instead the trachea. This is a
serious problem for interpreting any survival and protection result. Thus we are
looking for a marker that would allow us to track pulmonary infection. We are testing
the feasibility of adding self-illuminating quantum dots to the inoculum [So MK et al
Nat Protoc. 2006;1(3):1160-4] and then using the Xenogen in vivo imaging system
we have in the lab to verify pulmonary delivery
b. We noticed that the Bead-Beater tubes that we have been using to homogenize
mouse and rat tissues are not suitable for homogenizing infected rat lungs (because
the rat lungs become rubbery 3-6 days after SCHU S4 infection) and guinea pig
tissues (because of their large size). Thus we are evaluating a disposable, sealed
homogenizer system offered by Omni International (Marietta, GA).
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
47%
9. Work plan for upcoming month
Rats
a. Measure the resistance of vaccinated Fischer 344 rats to i.t. SCHU S4 challenge
b. Characterization of the Fischer 344 rat model
i. Kinetics of SCHU S4 proliferation and dissemination in lungs, spleens, and
livers of naïve and vaccinated rats
ii. Histology of lungs, spleens and livers of naïve and vaccinated rats infected
with SCHU S4
Guinea Pigs
a. Check for LVS clearance in lungs, liver, and spleen 4 weeks after vaccination
b. Check for sero-conversion using ELISA similar to the one we developed for mice and
rats
c. Challenge the vaccinated guinea pigs intranasally with SCHU S4
d. Decide whether to pursue or abandon guinea pigs as a model
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
14 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Milestone 12/13-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared in animal models
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
i. Issue: Testing 3 different protocols (CTL, CERUS and Lyons) to choose
the protocol with spares the most viable cells that remain functional after
thawing
ii. On 4/9/07, we bled the 3 NHPs that were inoculated with LVS via the i.d.
route and attempted to freeze down cells from the same NHP using each
of the 3 different protocols
1. One animal’s blood clotted and we couldn’t use it
2. A second animal had a low yield of cells (1.9x 106); set up for
IFN ELISPOT
3. The third animal yielded 8.25 x 106 cells: Set up in a
proliferation assay and froze down cells using the CTL and
Lyons protocols; these will be thawed on 6/4 and re-tested for
proliferation
b.
Update on IFN detection (ELISPOT assay and intracellular IFN staining)
i. Issue: Although we are readily able to detect IFN by ELISPOT when PBMCs
are stimulated with Con A, we were unable to detect LVS-driven IFN production
by ELISPOT
ii. Experimental test: Try to use more PBMCs/well; 20,000 is sufficient for Con A,
but may not be for antigen-specific induction of IFN secretion
iii. Using cells from TUL 11 (3/26/07), we set up 200,000 PBMC/well and observed
that we are still having problems with detection of faint spots in the unstimulated
wells (Table 2)
TABLE 12/13-1: IFN spots induced using 200,000 cells/well (TUL 11)
Animal
Stimuli
Spots
A00902 (S.C.)
Media
207, 184, 174 (faint)
HK LVS
172, 160, 155
FF LVS
244, 161, 140
A00659 (S.C.)
Media
39, 34
HK LVS
63, 70, 25
iv. As an alternative to the IFN ELISPOT we attempted to detect IFNγ production
by intracellular staining technology
1. Using cells from TUL 13 (blood drawn on 4/30/07), we stimulated
PBMCs overnight with Con A, PHA or LVS;
2. On day 2, we re-stimulated with PMA + ionomycin in the presence of
Brefeldin A for 6 hours in order to capture any IFN induced by the
stimulus inside the cell;
15 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
3. The cells were then stained with antibodies specific for CD3, CD4, CD8
and anti-IFN
4. Preliminary results suggest that IFN was not induced under these
conditions and that the cells lost CD3 during the stimulation process; a
full description of the data will be provided in the June report of May
activities.
v. Data interpretation: LVS-induced IFN ELISPOT detection requires more
PBMCs/well which also results in higher background; currently in discussion with
the ELISPOT reader representative about this; in addition, we need to research
the appropriate methods to allow detection of intracellular IFN staining in NHPs
c.
Update on LVS ELISA
i.
Performed the optimization protocol for the IgG anti-LVS ELISA suggested
by Freyja Lynn
a. Heat killed and formalin fixed LVS were resuspended to various
concentrations and used to coat wells of an ELISA plate
b. A pooled serum sample from day 21 post-LVS vaccinated NHPs was
used as a positive control and was diluted to various degrees
c. Results are shown in Figures 8 and 9
HK LVS ELISA Optimization
2.500
1/200
2.000
OD405
1/1000
1.500
1/5000
1.000
1/25000
1/125000
0.500
1/625000
0.000
0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5
5
10
Ag Concentration (x106/ml)
Figure 12/13-1: Plates were coated with increasing concentrations of HK LVS. Pooled serum from LVS-
vaccinated NHPs was diluted from 1/200 to 1/625,000. The optimum coating concentration is the lowest
that still provides maximal color development. In this assay, the optimal coating concentration appears to
be 2.5 x 106/ml.
16 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
FF LVS ELISA Optimization
OD405
1.400
1.200
1/200
1.000
1/1000
0.800
1/5000
0.600
1/25000
0.400
1/125000
0.200
1/625000
0.000
0.02 0.04 0.08 0.16 0.31 0.63 1.25 2.5
5
10
Ag Concentration (x 106/ml)
Figure 12/13-2: Plates were coated with increasing concentrations of FF LVS. Pooled serum from LVSvaccinated NHPs was diluted from 1/200 to 1/625,000. The optimum coating concentration is the lowest
that still provides maximal color development. In this assay, the optimal coating concentration has not yet
been determined.
ii.
iii.

Preliminary Data interpretations:
a. The optimum coating concentration for the NHP IgG anti-LVS ELISA is
2.5 x 106 HK LVS/ml.
b. Although we have not yet determined the optimal coating concentration
of FF LVS in this assay, it is impractical to be using more than 10 x 106
cells/ml; therefore, we do not propose to continue testing this antigen
preparation for the NHP IgG anti-LVS ELISA.
Future experiments
a. Test all banked serum from LVS-vaccinated NHPs (TUL 8 and TUL 9;
days 0, 7, 14, 21 and 28) for IgG anti-LVS reactivity using ELISA plates
coated with 2.5 x 106 HK LVS cells/ml
b. Optimize the IgA anti-LVS ELISA using the same methodology as
described for the IgG anti-LVS ELISA
All data for TUL 11 is stored in binder TVDC 1 in the Wilder laboratory as well as in
C:\Documents and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\ prep
for 042707.doc; and N:\My Documents\Tularemia Contract\prep for 042707.doc; All
data for TUL 13 is in bound notebook TVDC 1 pgs.12-21; the raw data is also stored in
\\Saturn\Group\wilder lab\TVDC\PBMC assays032907.svd; all data for the LVS ELISA
is stored in binder TVDC1 in the Wilder laboratory as well as in C:\ Documents and
Settings\jwilder.LOBOS\My Documents\Tularemia Contract\ prep for 042707.doc and
LVS ELISA Ag optimization.xls; and N:\My Documents\Tularemia Contract\prep for
042707.doc and LVS ELISA Ag optimization.xls;
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
17 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
i.
ii.
Concern that IFN ELISPOT assay is not optimized for LVS-specific induction
of IFN secretion, will talk to the kit and ELISPOT reader representatives about
this concern
Concern that intracellular cytokine staining method is not working; we will
research this in the literature to find a published working protocol; possibly
contact Dr. Louis Picker about this (former colleague and NHP PBMC expert)
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
72% of scientific work has been completed
9. Work plan for upcoming month
a. Continue to freeze down and thaw PBMCs using the 3 different protocols
b. Contact the ELISPOT kit and reader representatives to discuss issues in optimization
c. Develop the IFN intracellular staining assay in whole blood and PBMC preparations
(from LVS-vaccinated NHPs; Milestone 4) using flow cytometry
d. Develop the IgA anti-LVS ELISA
10. Anticipated travel
Attend the June 4 – 7 meeting in Gaithersburg, MD on optimization and validation of
immunoassays
11. Upcoming Contract Authorization (COA) for subcontractors
Per Andrew Cherry, no COA is needed for invited attendance at the NIAID sponsored CMI
course in Gaithersburg, MD.
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. No new work done.
b. We previously have optimized the T cell proliferation assay for the mouse; 5 x
104/well nylon wool-enriched T cells and 106/well formalin-fixed LVS produced the
best balance of background, specificity and sensitivity. We are now designing
experiments to optimize this assay for the rat because our preliminary results suggest
that Fischer 344 rats may be a better model than the mouse.
c. We are now applying this assay to identify peptides from F. tularensis proteins that
would stimulate T cells from LVS-vaccinated BALB/c mice and potentially other
vaccinated small animals models (milestone 27)
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
18 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Good
8. Percentage completed
40%
9. Work plan for upcoming month
We will start developing the T cell proliferation assay for the Fischer rat
a. Verify that Fischer 344 rat is a good model of human respiratory tularemia
b. Develop procedures for isolating T cells from whole blood, spleen, lymph node
c. Develop procedures for stimulating T cells with Con A and killed LVS and SCHU S4
d. Optimize the number of T cells and amount of antigen to use in the assay
10. Anticipated travel
Alexandra Scrymgeour will be attending the NIAID sponsored CMI course in Gaithersburg,
MD from June4 to 6, 2007.
11. Upcoming Contract Authorization (COA) for subcontractors
Per Andrew Cherry, no COA is required for Alexandra Scrymgeour’s travel to the NIAID
sponsored CMI course.
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 donor alveolar cells were available during this period
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
In Ftc36 study 3, we noticed that F. tularensis caused cytopathic effects by day 2 of culture
and perhaps earlier. This will not give us enough time to follow the interactions between F.
tularensis and the human alveolar macrophages. Thus, we will reduce the MOI until the
macrophages survive for at 4-5 days.
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
3%
9. Work plan for upcoming month
a. Determine the optimal MOI for infecting human alveolar macrophages. Since we
observed cytopathic 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 macrophage infection with SCHU S4
and LVS
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
19 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
f.
Determine whether PBMC from LVS 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 (mouse and
rat models)
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc30 study 8 (notebook 101, pages 16-18)
i. We noticed in several experiments of this series that uninfected murine
macrophages did not survive 3 days in culture, the length of a typical
experiment. It is possible that the amount of M-CSF in the culture medium
was not enough to generate quality macrophages. In fact, we do not know
how much M-CSF is in the L929 conditioned medium we use as a source of
M-CSF. Thus, the purpose of this experiment was to measure the amount of
M-CSF in the L929 conditioned medium
ii. We measured the M-CSF content in the L929 conditioned medium using the
mouse M-CSF ELISA kit from R&D systems
iii. The L929 conditioned medium contains 205 pg/ml M-CSF
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Fair
8. Percentage completed
10%
9. Work plan for upcoming month
a. Determine the concentration of M-CSF required to generate bone marrow derived
murine macrophages. We will use recombinant M-CSF at a range between 1-1000
pg/ml
b. Determine the optimal MOI for LVS and SCHU S4 infection of murine macrophages
c. Determine whether vaccinated mouse splenocytes can induce BMM to kill
intracellular LVS
d. Determine whether vaccinated mouse splenocytes can induce BMM to kill
intracellular SCHU S4
e. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
20 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
v. Determine whether T cells from vaccinated rats can induce infected
macrophages to kill intracellular bacteria
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 26
Milestone description: Confirmation of gene expression (design HTP SOPs, test HTP SOP,
ORF library production and confirm gene expression)
Description: Prepare a high-throughput protein production system
 Select and test ORF expression constructs
 Select and test IVT Protocols
 Select and test protocols for protein purification
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions:
A. Select and test ORF expression constructs
1. The results of the preliminary purification experiments indicate that the 6x His
sequence will be a more effective affinity tag than biotinylation for product isolation.
We currently favor a template design that sandwiches the ORF between both an N
and C terminal His tag, to maximize opportunities for tag exposure. This is likely to
improve resin binding, and therefore purification yields. An alternative to His
purification is work to improve biotinylation efficiency. One possibility is to test a
template design in which a spacer is placed between the His, BAP and TEV protease
cleavage sites. See parenthetic area shown in Figure 1 below.
2. However, if the unpurified IVT lysates are sufficient for specific T cell stimulation then
these template modifications will not be necessary. The His tags will be maintained,
since they are very short and may be useful at some downstream step.
21 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Figure 1
B. Select and test IVT Protocols
1. We have repeated 35S-labeled IVT reactions with 5 FTU ORF templates, using the
combined Invitrogen/Roche feed system.
2. One batch of IVT’s was performed in the presence of the BirA enzyme, which
biotinylates the nascent polypeptide as translation is occurs.
3. A test scaled up reaction with FTU 901 was performed using same system. Scale up
was not successful, but neither was kit control. New kit arrived today. Scale up test
will be repeated this month, so as to have plenty of material for purification trials.
C. Select and test protocols for protein purification
1. The batch of the 5 FTU polypeptides noted above were used to further test nickel
bead elution schemes. We have previously found that, in general, imidazole elution
of His tagged proteins from nickel beads is not efficient. Therefore, we started this
project using EDTA to elute samples. The summary table below shows that EDTA
elution was also not efficient. Next, acid elution was investigated. We found that the
pH change successfully eluted product from some samples but not all. Namely,
TUL4 and p11 were efficiently eluted (77% and 57%, respectively); while p12,
GroES, and IglC2 were not (15%, 2%, 6%, respectively). The variability is likely to be
a result of different folding structures assumed by the polypeptides.
22 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Table 1: Summary of IVT purification yields
F. tularensis protein
FTU 901 (TUL4)
Purification
method
His-tag Biotin-tag
Soluble ppt in 8M Urea
Resin Bound
EDTA/Protease Elution
53%
58%
3%
pH elution
Beads post pH Elution
77%
14%
71%
12%
3%
FTU 1419 (p11)
Soluble ppt in 8M Urea
Resin Bound
EDTA/Protease Elution
100%
77%
2%
pH elution
Beads post pH Elution
57%*
99%*
100%
5%
2%
* there was a problem with
cpm’s in this sample
FTU 1602 (p12)
Soluble ppt in 8M Urea
Resin Bound
EDTA/Protease Elution
84%
88%
3%
pH elution
Beads post pH Elution
15%
100%
100%
15%
4%
FTU 1695 (Gro ES)
Soluble ppt in 8M Urea
Resin Bound
EDTA/Protease Elution
91%
ND
1%
pH elution
Beads post pH Elution
2%
59%
59%
18%
9%
FTU 1712 (IglC2)
Soluble ppt in 8M Urea
Resin Bound
EDTA/Protease Elution
79%
36%
2%
pH elution
Beads post pH Elution
6%
44%
79%
14%
8%
23 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Data located at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\ FTU 5 Ni
and Biotin Purification 4-4-07
2. The products eluted from the nickel beads were analyzed alongside an aliquot from the
unpurified IVT reaction by SDS-PAGE. These are shown in the autograph of Figure 2.
Lanes 1, 3, 5, 7, and 9 were loaded with the total lysate sample. Lanes 2, 4, 6, 8, and 10
contain the corresponding sample that has been purified via the His tag and pH elution.
The expected molecular weight for full length products are indicated with an arrow. It is
noted that Tul 4 and p11 preparations, which showed higher acid-elution efficiency, also
appear to contain significant amounts of partial polypeptides. By contrast, the 3
polypeptides showing lower acid-elution efficiencies are full length products. Perhaps
smaller products are most efficiently eluted.
Name Legend
0901
TUL4
1419
p11 lipoprotein
1602
p12 lipoprotein
1695
GroES
1712
IglC2
Figure 2
24 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Data at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT\ IVT FTU 5
Purification Dialysis Normalized 4-18-07 02
3. The flow chart of activity for the pilot purification trial has been updated to reflect our
results (figure 3). We currently favor a His-based purification approach, with
sandwiched tags to improve tag exposure. We also favor the pH-based elution
protocol. However, further optimization of elution will be necessary to make this
method more robust and less variable across multiple proteins.
4. We have these samples ready to be transferred to UNM.
5. Technically the most attractive scheme is to use unpurified sample since product
yield will be the greatest. To determine feasibility, we would like to propose testing
unpurified sample alongside His purified sample in T cell assays as soon as possible.
Figure 3
4. Significant decisions made or pending
a. In the His tag-nickel bead purification method, elution efficiency is better by using pH
rather than imidazole or EDTA.
b. Our final selection of a purification method is pending.
c. No purification is an option if lysates work in T cell assays.
25 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
5. Problems or concerns and strategies to address
We are working to address the elution problem in our purification steps for the His tag/nickel bead
strategy and for the biotin/avidin strategy. We will be testing the utility of including a His tag at
both ends of the molecule, as a means to reduce urea concentration necessary for binding. This
may also improve elution efficiency.
6. Deliverables completed
None
7. Quality of performance
Very good
8. Percentage completed
95%
9. Work plan for upcoming month
a. We will be sending UNM between 5 and 6 FTU purified polypeptide preps within 1 to 2
months.
b. We look forward to working with them on selecting a final protocol for proteome
preparation.
c. Our goals for the next month are to complete Milestone 26 and confer with UNM on their
work on Milestone 27.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will use
ASU’s protein fragments in lymph node proliferation assays to define vaccine candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc27 study 12 (Notebook 94, pages 186-192 and Notebook 101 pages14)
i. The purpose was to identify peptides that are recognized specifically by
splenocytes from vaccinated BALB/c mice and that stimulate proliferation
ii. We tested 288 peptides for ability to stimulate specific proliferation by
vaccinated splenocytes
iii. 5 x 104 nylon-wool splenocytes from naïve and LVS-vaccinated mice were
incubated with 5 g/ml peptide for 5 days. Proliferation was measured by the
incorporation of BrdU into actively dividing T cells. Specific proliferation is
indicated by proliferation of vaccinated but not naïve T cells
iv. We did not find any peptide that had specific stimulatory activity
v. Two of the peptides tested were from GroEL and KatG. In contrast to our
results, B.Y. Lee et al. showed that splenocytes from i.d. vaccinated BALB/c
mice responded specifically to whole GroEL and KatG protein [Infect Immun.
2006 Jul;74(7):4002-13.]. There are three notable differences between the
two studies
26 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
1. We used peptides whereas Lee et al used whole proteins. It is
possible that the peptides we used are not the stimulatory peptide
from the GroEL or KatG proteins
2. We used i.n. vaccinated mice whereas Lee et al used i.d. vaccinated
mice. Jeff Frelinger’s group presented an abstract at the Woods
Hole meeting that i.n. vaccination induced fewer IFN-positive cells in
the spleen than i.d. vaccination. It is therefore possible that
splenocytes from i.d. vaccinated mice would produce a stronger
response
3. We used 5 x 104 splenocytes whereas Lee et al used 2 x 106
splenocytes. We found that 5 x 104 splenocytes was optimal for
measuring stimulation by whole bacteria because a higher number of
splenocytes was associated with higher background. It is possible
that the non-specific proliferation observed previously to whole
bacteria would not apply to peptides.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
We do not have a known positive control peptide that would help us with this assay.
Therefore, we asked ASU to synthesize large peptides that cover the entire length of GroEL
and six other proteins for us to use as positive controls
6. Deliverables completed
NA
7. Quality of performance
No progress
8. Percentage completed
10%
9. Work plan for upcoming month
a. Determine whether increasing the number of T cells and/or APC would be better for
this peptide screen
b. Determine whether IFN ELISpot assay would be better than T cell proliferation
assay for this peptide screen
c. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice, after a positive control peptide is used to validate the
approach
d. 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
27 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Milestone 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. Our electronic management system, GEMS, has designed and then instructed our robot to
normalize concentration of all oligos, pool forward and reverse gene specific primers, then
identify pairs designated for PCR-amplifying natural sequence ORFs from SCHU S4 genomic
DNA. The remaining primers will be used as part of the gene building protocol.
2. A description of the oligo and block design script is located at:
\\Peptide\shared\CIM\users\Kevin\ Running the Block Design script
3. We have begun amplifying ORFs that were identified as having adequate natural sequence
for production by IVT. In the first run, we achieved 94% successful amplification. This is
defined as visualization of a specific band at the predicted molecular weight, with sufficient
yield to prepare inocula.
4. A new batch of deoxuracil primers were needed in order to have sufficient material on hand
to run the whole library with a single batch. These have been received and are currently
being QC’ed by PCR and functional LEE linking reactions. Large scale amplification will
continue this month.
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
We will proceed with the remaining (wildtype) WT PCRs. When this first run is complete all
“redos” will be compiled and rerun. In the past a large portion of the initial failures can be
captured in this second run.
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
10%
9. Work plan for upcoming month and next 6 months
In the next month, several more plates will be done.
In the next several months the first run production of wild type SCHU S4 ORFs should be
completed and we will be organizing the second run. Protocols for the synthetic ORF synthesis
will be in place.
10. Anticipated travel
None
28 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
11. Upcoming Contract Authorization (COA) for subcontractors
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

Amplifications have been done with purified SCHU S4 RNA serially ten-fold diluted which
showed we lost RNA without a carrier. Experiments were reported during the site visit of
SCHU S4 RNA diluted into normal mouse lung RNA which showed adequate amplification
down to 10 ng of SCHU S4 RNA in 10 micrograms of normal mouse lung RNA. This
experiment was named LAPT-3 (Notebook 405, pages 12-13 hard copies; electronic files at
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-3. Spearman analysis of the rank
order of genes detected with unamplified SCHU S4 RNA revealed good correlations.
Bioanalyzer QC analysis of these amplified RNA is shown in Figure 1 and reveals expected
traces of an appropriate size range of amplification products.
Figure 1. Bioanalyzer results of amplified SCHU S4 RNA diluted in normal mouse lung RNA

This experiment was repeated twice…
a. LAPT-4 (Notebook 405, pages 14-16; Electronic location
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-4
b. LAPT-5 (Notebook 405, pages 17-21; Electronic location
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-5).

Low amplified RNA yields were obtained with spiked samples with only about 20 micrograms
of RNA obtained after amplification. After verification of low yields in LAPT-5, we think that
this preparation of lung RNA has an inhibitor of the amplification step. We observed
adequate amplification of the RNA in the UNM lung samples from infected animals which
indicates there was no technical problem with the amplification reactions themselves. The
RNA obtained by LAPT from the spiked samples were concentrated by speedvac, labeled,
29 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
and an array hybridization was performed. Arrays were of very low intensity and were not
analyzed further.
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address


Low amplification yields of the RNA from the last normal lung RNA preparation indicates an
inhibitor of the amplification may be present. We need to prepare a new normal lung RNA
preparation for repeat testing of RNA spiked with SCHU S4 RNA for correlation testing
We obtained sequences of the TIGR probes for the Francisella array. A blast analysis of the
probe sequences against a database of the unique SCHU S4 and LVS genes from the most
recent annotation reveal that the TIGR arrays would not detect 91 SCHU S4 and 261 LVS
genes. We performed a similar reverse blast analysis of our probe set and found 3 missing
genes (1 SCHU S4 and 2 LVS). These will be redesigned and added to our current array.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
85%
9. Work plan for upcoming month


The MTA is approved to obtain TIGR slides and perform array comparisons between ASU
and TIGR arrays. The order for TIGR arrays has been placed. Receive TIGR arrays and run
test comparisons with ASU arrays
Repeat reconstitution experiments with SCHU S4-spiked normal lung RNA.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 34
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done.
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

RNA samples from infected mouse lungs obtained from UNM were named MS-2, MS-3, and
MS-5 had approximately 7.4 X 107 CFU SCHU S4 bacteria per lung after 3 days of infection.
Infected mouse lung samples were processed in parallel with spiked RNA into normal mouse
lung RNA in experiment LAPT-5 (Notebook 405, pages 17-21; LAPT-5 Electronic location
R:\GeneVac\FTU\Contract\Microarray\Milestones\34\LAPT-5). 10 micrograms of total lung
RNA was processed by LAPT and the adequate yields were obtained (Table 1). The yields
from infected mouse lung RNA were similar to those shown previously in LAPT-3. The fact
that these yields were 5 times that observed in the spiked samples indicate that the normal
mouse lung RNA used for spiking may have an inhibitor.
30 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Sample
MS-2
MS-3
MS-5
LAPT RNA Yield
982g
1160g
951 g
Table 1. Amplification yields of LAPT processing of lung RNA from SCHU S4 infected mice.
These amplified samples have been labeled and hybridizations are under way on the ASU array
on both poly-L-Lysine and Corning Ultragap slides.
4. Significant decisions made or pending.
Good
5. Problems or concerns and strategies to address
We will be comparing two isolations methods to include Tri-Reagent and RNAeasy protocol to
compare isolation methods.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month



Isolate more normal mouse lung RNA or purify the normal mouse lung RNA
We will be comparing automated hybridization chamber systems including our in house
Genomics Solutions GeneTac Hybridization system and an evaluation test of the BioMicro
Maui Hybridization system.
Continue the comparisons of the LAPT on UNM provided RNA from mice infected with
SCHU S4
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
a. No new work done on this milestone; ASU has not requested more RNA
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
31 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Good
8. Percentage completed
10%
9. Work plan for upcoming month and next 6 months
UNM will isolate RNAs from LVS, SCHU S4,and infected mouse organs, as needed by ASU.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
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
Summary: NER deficient strains of Ft novicida (uvrB and uvrA single and the uvrA uvrB
double mutant strains grow at the same rate as the wild-type U112 strain in Chamberlain’s
defined medium (CDM), in J774 macrophages, and in lungs, livers and spleens of Balb/c mice
following intravenous (IV) injection. NER deficient strains were all highly virulent when
administered IP or IV, but when delivered via subcutaneous (SC) route of infection (the route by
which Francisella strains are the least virulent)
all Ft novicida NER mutants were approximately 1
AS07-045
log reduced in virulence compared to U112.
1) This month we monitored the growth of Ft novicida uvrB and U112 in lungs, livers, and
spleens following SC infection in order to determine whether the nucleotide excision repair
machinery is required for growth or dissemination to specific organs.
AS07-045
Liver
10000000
F.t. n U112
F.t.n. urvB
CFU/organ
1000000
100000
10000
1000
100
limit of dection
10
1
0 (4hr)
1
2
3
Day
* One animal from each group w as found dead on Day 3.
Lungs
Spleen
1000000
10000000
F.t. n U112
F.t.n. urvB
100000
10000
1000
100
limit of dection
F.t.n. U112
F.t.n. uvrB
100000
CFU/organ
CFU/organ
1000000
10000
1000
100
limit of dection
10
10
1
1
0 (4hr)
1
2
3
0 (4hr)
1
2
3
Day
Day
* One animal from each group w as found dead on Day 3.
* One animal from each group w as found dead on Day 3.
32 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
5x105 CFU of U112 and uvrB were administered SC (representing ~100x LD50 of U112 and
~10xLD50 of uvrB. Cohorts of 3 animals were sacrificed at 4, 24, 48 and 72 hours post-infection,
and lungs, livers and spleens were harvested, homogenized, and serial dilutions were plated onto
CHAH plates for enumeration of CFU. Both U112 and the uvrB mutant were able to
disseminate from the SC injection site to the liver within 4 hours and then replicate logarithmically
for 48 hours. Surprisingly, the bacteria were cleared to below the limit of detection in livers and
spleens by 72 hours. There appears to be a significant difference in the ability of the uvrB
mutant to disseminate to the lung: no cfu were detectable at 24hours post-infection, and the peak
CFU at 48 hours post-infections was approximately one log lower than with U112. These data
suggest that perhaps the reason for the decrease in virulence of the NER mutants is due to a
decreased capacity to disseminate to the lungs following SC injection.
4. Significant decisions made or pending
The scientific work for this milestone is complete. Ft novicida NER mutants are not significantly
attenuated for virulence in mice. All of the Ft novicida NER mutants had indistinguishable
phenotypes, suggesting that there is no advantage to using the uvrA uvrB double mutant.
These observations have led us to make the decision to go forward with MS 43, in which we
proposed to screen a panel of attenuated NER-deficient double mutants of Ft novicida. For these
experiments we have made the decision to use uvrB as the NER mutation in combination with
pdpD, iglA, iglB, iglC, iglD mutations.
5. Problems or concerns and strategies to address
Abrogation of the NER pathway does not result in a dramatic loss in virulence, thus we will
screen for a secondary attenuating mutation that can be used in SchuS4–based vaccine to
ensure safety of this vaccine.
6. Deliverables completed
Growth rates of Ft novicida wild type and Ft novicida uvrA, uvrB, and uvrAuvrB mutants
determined in broth and in macrophages. LD50 comparison between strains administered by the
IP, IV, and SC routes have been completed. In vivo growth rates have been determined following
IV and SC administration.
7. Quality of performance
Excellent progress
8. Percentage completed
100% scientific work completed. Milestone completion report is pending.
9. Work plan for upcoming month
Work for this milestone is complete, and we expect to finish the milestone completion report by
the end of May for submission in June.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
33 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Milestone 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 mutants of Ft. novicida are slightly more
sensitive to photochemical inactivation than wild type. We have optimized photochemical
inactivation conditions at a 3.5 mL scale and a 400mL scale and produced a lot of KBMA
uvrB Ft. novicida. We have demonstrated that KBMA Ft. novicida are highly attenuated for
virulence. We are in the process of testing the stability of a frozen KBMA lot of uvrB Ft.
novicida at –80oC.
1) We have been monitoring the stability of KBMA Ft novicida uvrB lot#948-202 arm-2 by
measuring the degree of metabolic activity using the MTS assay after storage at –80o C in
10% sucrose. The metabolic activity after 3 months of storage was essentially equivalent to
the initial metabolic activity immediately after storage. These data indicate that KBMA Ft
novicida vaccine stocks can be stably stored without loss in metabolic activity.
Lot 948-202 Arm-2 (Nominal 1.93e8 particles/mL)
1.8
T=2 weeks
T=1 Month
T=2 Months
T=3 Months
1.6
OD (490nm)
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
NB968-082
4. Significant decisions made or pending
All NER mutants (uvrA, uvrB, and uvrA uvrB) of Ft. novicida were equally sensitive to S-59
and had comparable metabolic activity after inactivation. We have chosen to use the uvrB
single mutant for further experimentation. We have selected 40M S-59 and 7J/cm 2 as the
conditions for making 400ml-scale KBMA lots, and have produced a lot of KBMA uvrB Ft.
novicida vaccine that is sterile for further characterization. We have decided to open MS 42 in
order to determine whether KBMA Ft novicida can protect against a lethal wild-type Ft novicida
challenge.
5. Problems or concerns and strategies to address
The 2-fold difference in the concentration of S-59 required for complete inactivation of the
mutants compared to wild type is less than we have observed for other organisms: One
possible explanation for this is that there is a redundant DNA repair mechanism functioning in
Ft novicida; however, the high degree of metabolic activity retained by the mutant and wild-
34 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
type strains after photochemical inactivation suggests that the wild type may be highly
sensitive to photochemical inactivation under these conditions and that the KBMA strategy is
still viable. We will measure the sensitivity of NER mutants to a panel of DNA damaging
agents and compare them to wild type. We will investigate whether the uvrB gene is induced
in response to photochemical inactivation with S-59 and UVA light or in response to other
DNA damaging agents. These experiments should help us understand why the NER mutants
are only slightly more sensitive to photochemical inactivation compared to wild-type.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
70% of scientific work completed on the milestone
9. Work plan for upcoming month
We will compare the sensitivity of uvrB and U112 to 4 DNA-damaging agents including
mitomycin C, doxorubicin, benzo[a]pyrene and 4 nitroquinoline-N-oxide. If there is a
difference in the sensitivity of uvrB and U112 to these agents, we will determine whether the
uvrB gene is induced in the wild type after DNA damage by rt PCR. We will then compare
the uvrB gene induction to treatment with S-59 and UVA.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 42
Milestone description: Determine whether KBMA F.t. novicida vaccine protects against wildtype F.t. novicida challenge in mice: Vaccination route and regimen optimization, measure
durability of protection
Institution: Cerus
1. Date started: 2/1/07
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Summary: KBMA Ft novicida uvrB vaccine stocks produced in MS41, have been tested in
mice for virulence and protection against a 100 x IP LD50 challenge of Wild-type Ft novicida,
one month after a single vaccination with KBMA Ft novicida vaccine. The doses of KBMA
vaccine that were 100% protective were at or near the LD50 of the KBMA vaccine (1 x 109 IP,
1 x 108 IV).
We reported last month that we can achieve 100% protection from a 100 x IP LD 50 challenge
by administration of 1 x 107 KBMA particles IV if the vaccine was given twice separated by 3
weeks and that depletion of T cells had no effect. In fact this observation was made
prematurely and after 6 to 7 days following 100x LD50 challenge 2 mice in the CD4 depleted
group died and 1 mouse in the CD4 and CD8 depleted group died. CD8 depletion had no
effect by itself, and did not decrease the survival in the double depletion group. These data
suggest that CD4+ T cells contribute to protection during the challenge. The mechanism of
this contribution is unclear, and may simply provide help to the humoral response.
35 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Survival of mice vaccinated twice with 1e7 KBMA IV and challenged with 100 x IP LD 50 U112
Vaccination
No Depletion
CD4
CD8
CD4 + CD8
HBSS
0% (MTD 3d)
100%
80% (MTD 6.5d)
100%
90% (MTD 7d)
KBMA uvrB
AS07-017
To test directly whether the mechanism of protection provided by KBMA Ft novicida
vaccination is humoral, we collected serum from each of the surviving animals and adoptively
transferred 200 ul of serum IV 1 day before and 100ul of serum 4 hours before a 100x IP
LD50 challenge and compared the survival to animals that received serum from naïve mice.
While adoptive transfer of serum from KBMA vaccinated mice protected only 1 mouse of 20,
in each of the groups that received serum from KBMA vaccinated mice, there was a
significant delay in the time to death compared with mice that received naïve mouse serum.
It is likely that if we were able to transfer more serum, more mice would have survived (as
reported previously by Bernard Arulanandam’s group at UTSA). We have recently
demonstrated that protection was also observed following vaccination with heat-killed Ft
novicida. Together, these data suggest that humoral immunity plays a significant role in
protection of mice against a lethal Ft novicida challenge and make it difficult to rank KBMA
vaccine candidates.
AS07-045 Survival After Adoptive transfer of Serum
Percent survival
100
Naive serum
KBMA uvrB immunized
KBMA imunized -CD4
KBMA imunized -CD8
KBMA imunized -CD4/-CD8
80
60
40
20
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Day
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The KBMA uvrB Ft. novicida vaccine was 100% protective only after a single administration
at very high doses, so we have chosen to pursue a repeat dosing regimen which appears to
provide 100% protection at sub-toxic levels. We are concerned that the mechanism of
protection elicited by Ft novicida is humoral, which could make screening difficult for vaccine
candidates that elicit a potent T cell response. We have requested that Karl Klose construct
an ovalbumin epitope-fusion protein to facilitate screening strains of Ft novicida for their
ability to elicit a T cell response to this well-defined epitope.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
15% of scientific work completed on the milestone
9. Work plan for upcoming month
We will wait for delivery of the ova-tagged strain of uvrB from Karl Klose to determine
whether KBMA Ft novicida can induce a potent CD8 T cell response
36 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 43
Milestone description: Create uvrA or uvrB mutants in LVS
Institution: UTSA
1 Date started: 5/01/2006
2 Date completed: In progress
3 Work performed and progress including data and preliminary conclusions
The plasmid pDS132 was modified for use in F. tularensis. This is a conditionally-replicative plasmid that
has a counterselectable marker (sac B), we have adapted for use in F.t. by first altering the multiple
cloning site and then inserting a Ft promoter (gro ELp) to facilitate expression of sacB and the antibiotic
resistance marker (cat). The mating plasmid pKEK1090, which has the GroEL promoter properly inserted
to facilitate sacB/cat expression, was used as a vector to clone UvrBUpFpKanDn (KEK1114). The
plasmid was mated into the LVS strain and the SacB is expected to help to eliminate the plasmid
backbone.
3.1 The sequence result has confirmed that UvrB mutant LVS is correct. Next we will make UvrA mutant
LVS.
3.2 To mate UvrAUpFpKanDn(KEK1120) into LVS, we made TSA/+++/Amp(100ug/ml) plates,
LB/DAPA(80ug/ml)/Chloramphenicol(10ug/ml) plates, TSA/+++/DAPA(80ug/ml) plates and
TSA/+++/Chloramphenicol(10ug/ml) plates.
3.3 Scraped overnight cultured LVS and E.Coli./DAPA-/Pkek1120 from TSA/+++/Amp and
LB/DAPA/Chloramphenicol plates onto corresponding plates and let them grow until mid-log phase.
About 7-8 hrs for LVS strain, and 3-4hrs for E.Coli./DAPA- strain.
3.4 The bacteria were scraped off from their corresponding plates, and measured OD600 value.
3.5 Made mixture of LVS and E.Coli with ratio at LVS:E.Coli.=1:10. Then spread mixed bacteria onto
TSA/+++/DAPA plates for overnight mating.
3.6 The mating cells were scraped off from TSA+++/DAPA plate, and serial 1:10 fold dilution in
TSA/+++/Chloramphenicol liquid medium were made. The diluted suspension was spread onto
TSA/+++/Chloramphenicol(10ug/ml) plate, and incubated at 37C for 4-10 days.
3.7 The colonies from TSA/+++/Chloramphenicol plate were patched onto TSA/+++/Kanamycin(15ug/ml)
plate and incubated at 37C for 24-48hrs.
3.8 Performed colony PCR with KanFNdel and KanRBamH1 primers to make sure that the colonies
resistant to Kanamycin contain Kanamycin resistance gene.
PCR as following:
dd water
10xBuffer#1 for KOD
32.6ul
5.0ul
37 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
MgCl2
dNTPs
KanFNdel
KanRBamH1
KOD Hifi polymerase
DNA
2.0ul
5.0ul
2.0ul
2.0ul
0.4ul
1.0ul
At 98C 1min, then 98C 15 sec/55C 15sec/721min//30cycles
Gel picture:
1
2 3 4
1 1kb Marker
2 UvrBKan U112
3 Colony1
4 Colony2
PCR result confirmed that colony1 and 2 contained Kanamycin resistance genes, and plasmid KEK1120
(urvA gene) were in LVS.
3.9 Grew colony1 and 2 in TSA/+++/Kanamycin(15ug/ml) liquid medium. In the mean time, patched these
two colonies onto TSA/+++/Kanamycin(15ug/ml) plate to get bigger colonies.
4.0 Colony1, 2 and other colonies didn’t grow. Actually they were dead after two days incubation on the
original TSA/+++/Kanamycin(15ug/ml) plate.
4.1 It is very difficult to grow UvrA mutant LVS on TSA/+++/Kanamycin plates and they didn’t survive
after mating pKEK1120 to LVS. Another technical method will be applied to make UvrA mutant LVS in the
following month.
Data recorded on UTSA TVDC notebook #2, page70-74
38 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
4
Significant decisions made or pending
One possible reason for UvrA mutant LVS not surviving is that the UvrA gene is very important
to LVS. LVS won’t be survival without UvrA. Another possible reason is that UvrA mutant LVS
may easily loose the mutant UvrA gene. We decided to try to make UvrA mutant LVS using
intron re-targeting technique.
5. Problems or concerns and strategies to address
LVS with PKEK1120(UvrAFpKan) could not continuing being grown on low concentration
Kanamycin medium, thus other technique will be used.
6. Deliverables completed
KKF303 (UvrB mutant LVS).
7. Quality of performance
Good
8. Percentage completed
Approximate 57% of scientific work completed on the milestone
9. Work plan for upcoming month
Plasmid KEK1140 will be used as backbone to create UvrA mutant LVS with
intron re-targeting technique.
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 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/cm2 UVA for initial proof of concept studies, and for later comparison with
NER-deficient uvrB LVS.
39 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
We have performed stability studies on KBMA LVS lot 968-040 Arm-1 that demonstrate that
the metabolic activity of the lot is stable for 2 months. We will continue to perform MTS
assays at 3, 6 and 12 months.
Nominalthat
1e8 particle/mL (KBMA) F. tularensis holarctica LVS
s demonstrating
1.2
T=0 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
1.0
T=1 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
OD (490nm)
T=2 698-040 Arm-1 (10uM S-59, 6J/cm2 UVA) "
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
6
7
Time (hours)
8
9
10
11
12
NB968-082
In order to demonstrate that KBMA LVS is attenuated for virulence we performed an LD 50
study comparing KBMA and Heat killed (HK) LVS administered IV at doses of 1 x109, 1 x108,
1 x107 doses (AS07-044). 3 of 5 animals in 1e9 KBMA group died, no other animals in
KBMA or HK groups died. Thus the calculated IV LD50 of KBMA LVS is 6.8x108, which
represents a 4-5 log attenuation compared with live Cerus expanded LVS. We then
subjected all the survivors from the LD50 study to a 100 x IP LD50 LVS challenge. All of the
Buffer vaccinated mice died within 5 days, and none of the KBMA vaccinated mice died.
However, none of the mice vaccinated with the equivalent doses of HK LVS died either.
These data demonstrate that doses of KBMA LVS as low as 1 x107 provide protection, but
that this protection is not dependent on metabolic activity. This is consistent with protection
against an LVS challenge being largely humoral. Our prediction is that the KBMA LVS may
be more potent than HK in a SchuS4 challenge model. We also anticipate that a uvrB
mutant may be slightly more potent
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
uvrB mutant we have been investigating the efficacy of a wild-type KBMA LVS vaccine. We
will compare the photochemical inactivation profile of a uvrB mutant LVS when it arrives from
UTSA.
5. Problems or concerns and strategies to address
The protection seen with the KBMA WT LVS appears to be independent of whether the vaccine
has metabolic activity. This suggests that comparison of various routes, regimens, or
formulations will be difficult to optimize by protective efficacy. A SchuS4 challenge model may
be more appropriate.
6. Deliverables completed
None
7. Quality of performance
40 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Good progress
8. Percentage completed
40% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
We will ship KBMA LVS to Terry Wu at UNM for testing in a SchuS4 challenge model.
We will compare the virulence of live LVS stored in the various cryopreservation agents by
injecting IV into BALB/c mice
We expect to receive a uvrB mutant LVS from UTSA and we will measure its sensitivity to
photochemical inactivation and degree of metabolic activity and compare the NER-deficient
strain with wild-type LVS.
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 experiments with SCHU S4 itself using constructs
that we believe will allow us to make deletions into SCHU S4.
I. Cloning
a. Started cloning for igLD gene using a vector designed in our lab designated KEK1140. This
plasmid was designed with sequences from a Sigma vector pACD4K-C. This contains a
transposon like sequence that is designed such that one can direct an insertion into a
chromosome using gene specific sequences placed into this intron region. In addition, the gene
specific oligos are generated with the help of a special program that has been designed by
Sigma. The KEK1140 contains also a kanamycin gene, a repA gene (which makes plasmid
temperature sensitive); a sac B gene (which makes the plasmid sucrose sensitive); a reverse
transcriptase gene; and a groEL promoter. We had success with this vector when making the
ampicillin deletion mutant in SCHU S4 (KKT1).
b. The igLD gene sequence was sent to Sigma and they provided oligo sequences that can be
used to generate the desired PCR product (insert) to use for cloning into the intron-retargeting
sight of our plasmid (ie where the transposon-like sequence is located). Two sets 3 oligos each
were picked for our use in these sets of experiments. These are as follows:
FTT1356_30/31a-IBS:
5’-aaaactcgagataattatccttagctgccgtcatggtgcgcccagatagggtg-3’
41 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
FTT1356_30/31a-EBS1d:
5’-cagattgtacaaatgtggtgataacagataagtcgtgatgattaacttacctttctttgt-3’
FTT1356_30/31a-EBS2:
5’-tgaacgcaagtttctaatttcgattgcagctcgatagaggaaagtgtct-3’
FTT1356_255/256s-IBS:
5’-aaaactcgagataattatccttaagtttccagacagtgcgcccagataggggtg-3’
FTT1356_255/256s-EBS1d:
5’-cagattgtacaaatgtggtgataacagataagtccagacaggtaacttacctttctttgt-3’
FTT1356_255/256s-EBS2:
5’-tgaacgcaagtttctaatttcgattaaacttcgatagaggaaagtgtct-3’
EBS Universal Primer: This one is directed to vector intron sequence (will always use this with
each primer set in a PCR reaction):
5’-cgaatttagaaacttgcgttcagtaaac-3’
The 30/31 set of oligos will direct the insertion between site 30 and 31 of the iglD gene and the
255/256 oligo set will direct insertion between 255 and 256 site of the iglD gene. I will refer to the
PCR product inserts as iglD 30 and iglD 255, respectively. Two different targets are chosen in
iglD; in UTSA’s experience, a minimum of two targets need to be chosen in order that one will
work. UTSA;s success rate is approximately 50%.
c. The PCR reactions were set up as follows:
1ul Intron PCR Template (this is the intron sequence that will be changed in our specific
gene sequence)
1ul Four primer mix (contains 2ul of each primer 3-set (above) and 2ul Universal primer—
each at 25 pmole/ul stocks)
23 ul RNAse/DNAse free water
25 ul JumpStart RED Taq Ready Mix (Sigma).
50 ul is total reaction volume.
The parameters for the PCR were 94 C for 30 seconds for initial denaturation; 94 C for 15
seconds denaturation; 55 C for 30 seconds for annealing; 72 C for 30 seconds for extension
for 30 cycles; then 72 C for 2 minutes final extension step. (see figure 1). The correct size
fragments for both the iglD products were successfully amplified.
d. The final PCR products from above and the plasmid KEK1140 were both cleaned up and
digested with BsrGI and Xho I restriction endonucleases at 37 C overnight.
e. These digested DNA were run on a 1% agarose gel and band isolated using the Qiagen gel
extraction kit. (see figure1).
42 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Figure 1.
Legend:
•
1 Kb
1
2
3
4
5
6
•
12.5
1.5
0.5
•
•
•
•
1. 1 Kb
Ladder
(Invitrogen)
2. KEK1140 a
BsrGI/Xho I
isolate
3. KEK1140 a
BsrGI/Xho I
isolate
4. igLD 30
PCR product
5. igLD 255
PCR product
6. Low Mass
Ladder
This figure represents two separate KEK1140 band gel-isolated plasmids which had been
cut with BsrGI/XhoI enzymes (lanes 2 and 3) and the PCR products resulting when using
the oligo sets mentioned in I.b.; IgLD 30 set is lane 4 and IgLD 255 set is lane 5. Lanes 1
and 6 are molecular weight markers used on gel.All the fragments, including the PCR
product and the cut vector , are the expected size.
f.
The isolated DNA from e. above were used in a ligation reaction that was done at 16 C
overnight.
g. Data located in TVD UTSA Notebook 5, pages 17-20.
h. Started a re-modification of KEK1090 plasmid to make it ampicillin resistant instead of
chloramphenicol resistant. We now have a ampicillin sensitive SCHU S4 mutant and we will
be able to use a ampicillin selection when creating a mutant. This is useful so we can use
counter-selection with gene markers inside the desired gene deletion. This makes screening
for a deletion much faster.
i. Analyzed sequences and ordered oligos to re-amplify the KEK1090 plasmid minus the
chloramphenicol gene and also order oligos to amplify the ampicillin gene from the pwsk30
plasmid so we can use this to clone into the KEK1090.
j. The oligos were designed so that we can use Nco I and Spe I restriction sites for cloning.
The oligos are as follows:
pKEK1090 NcoI: 5’-gcgcccatggtagcttccttagctcctgccc-3’
pKEK1090 SpeI: 5’-gcgcactagtttcccgggtcatggctgcgc-3’
bla NcoI: 5’-gcgcccatgggtattcaacatttccgtgtcgcc-3’
bla SpeI: 5’-gcgcactagtttaccaatgcttaatcagtgagg-3’
k. Set up the PCR reaction following the HiFi KOD DNA Polymerase parameters to generate
the ampicillin gene from pwsk30. And use the XL KOD DNA Polymerase parameters to
amplify the pKEK1090 plasmid.
l. The amplification of the ampicillin gene was successful; however, the pKEK1090 did not
generate the desired product. Will retry this amplification with lower annealing temperatures.
In the meantime, started the first digestion of the ampicillin gene with Spe I restriction
endonuclease from Biolabs. Data located in TVD UTSA notebook 5, page 21 and 22.
43 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
II.
Experiments to generate deletions in Schu4:
a. The previous screen of the MglA SCHU S4 transformation experiment indicated that we
might have the MglA deletion. I had re-streaked the potential clone for singles again on a
TSA+++ plate and selected 20 colonies from this plate to screen for the correct clone.
b. The results indicated that all were wildtype genotype. There are more singles to screen;
however, we feel that without a selection marker inside this gene and its slight growth defect
it may take quite a few colonies in order to find this deletion. We therefore will drop this
experiment and focus on our target genes (iglC, pdpD, iglD, iglA, iglB). Data located in TVD
UTSA Notebook 5, pages 8,9,11 and 15.
c. Started the transformation experiment using our existing KEK906 iglC deletion plasmid and
the new ampicillin sensitive SCHU S4 deletion (KKT1). We used 2.5 ug of KEK906 and
electroporated into 0.5M Sucrose washed KKT1. These cells were placed into Tryptic Soy
Broth +++ for 6 hours without selection at 37 C. Then cells were harvested and resuspended
in 200 ul of sterile PBS and plated onto two 1 mg/ml Ampicillin TSA+++ plates, respectively.
Also, KKT1 cell control were also electroporated without DNA and grown as described and
also plated onto 1 mg/ml Ampicillin TSA+++ plates (as further control plated 10 ul on TSA+++
without selection just to check viability of cells).
d. After several days about 130 colonies resulted which were patched on fresh TSA+++ 1mg/ml
Ampicillin plates and of these patched colonies only 27 grew again on the fresh ampicillin
plates. Ten of these were used to screen with the iglC specific oligos via the “colony picks”
PCR screen. Unfortunately, the first screen generated a product only in the controls and one
of the “colony picks”. This amplification was for 3000 bp product so decided to use oligos
that generate a smaller product and can still differentiate the deletion from the wild-type.
e. These oligos are named ΔiglB Down and ΔiglD Up and these were used first with only
wildtype SCHU S4 (S4) genomic DNA and the KEK906 deletion plasmid to make sure we get
the expected sizes. (Figure 2).
Figure 2.
Legend:
1 Kb
1
3
2
•
•
•
2.0
1.0
1. 1 Kb
Ladder
(Invitrogen)
2. Schu S4
Wild Type
3. KEK906
ΔigLC
plasmid
This figure represents the PCR products generated by using deletion oligos that are
located outside the iglC gene and directed to the neighboring iglB and iglD genes. The
results indicated the correct expected sizes and also we are able to differentiate the
deletion from wild-type gene size (lane 2 wildtype S4 and lane 3 igLC deletion plasmid).
f.
Data located on page 14 TVD UTSA Book 5
44 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
4. Significant decisions made or pending
Will not continue to attempt the MglA gene deletion, but will focus on the iglC, pdpD, iglD, iglA,
iglB gene deletions in SCHU S4
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8 .Percentage completed
52%
9. Work plan for upcoming month
a. Will continue with the iglD cloning with the KEK1140 plasmid
b. Will continue with the re-modification of KEK1090 to create an ampicillin resistant plasmid
to use with the KKT SCHU S4 strain.
c. Will continue to screen for any possible iglC deletion insertion resulting from the
transformation experiment with KEK906.
d. 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: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
a. Determine the LD50 of Ft subsp. novicida uvrBiglC double mutant. (Note book #4 page
84-86): Groups of BALB/c mice (female, 4-6 weeks) were intranasally challenged with
105, 106 or 107 CFU of ΔuvrBiglC. As shown in Fig. 1, there is no mortality observed at
any given challenge dose, indicating the high degree of attenuation with this organism.
No significant weight loss of infected mice was also observed. The LD 50 of ΔuvrBiglC in
the intranasal infection model (BALB/c mice) was greater than 107 CFU.
45 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
100
% Survival
80
10 5
10 6
10 7
60
40
20
0
0
2
4
6
8
10
12
14
2
4
6
8
10
12
14
16
18
% Body weight
110
105
100
95
90
85
80
0
Days post-challenge
Fig.1. Survival of mice infected with Ft subsp. uvrBiglC double mutant. Groups
of BALB/c mice (female, 6-week old) were challenged intra-nasally with 3
doses (105, 106, and 107 CFU) of ΔuvrBiglC to determine LD50 of this strain.
b. Monitor Ft subsp. novicida ΔuvrBiglC replication and dissemination in mice after
intranasal challenge (Note book #4, page 87-89). BALB/c mice were challenged with
ΔuvrBiglC mutant (106 CFU) intranasally. Lungs, liver, spleen, and lymph nodes were
collected from the infected mice at day 3, 7 and 14 after challenge (3 mice per time
point). Numbers of bacteria in each organ were determined by dilution plating. As shown
in Fig. 2, there was heightened replication of the organism in the lungs within the first 7
days post-challenge, with dramatic reduction noted at day 14. There were lower levels of
replication within the liver and spleen. Numbers of bacteria in the spleen are consistent at
day 7 and 14. In the liver, the bacterial burden was decreased by day 14. There were
also organisms recovered from the draining lymph nodes at all three time points, with
greater replication noted on days 7 and 14. The growth kinetics of ΔuvrBiglC in the
animal is comparable to the single ΔiglC strain as reported previously.
Spleen
Spleen
10
9
8
7
6
5
Lungs
Liver
44
Log10 CFU/organ
33
2
11
Spleen
10
9
8
7
6
5
D3
D7
D14
Lymph node
Spleen
44
33
2
11
3
7
3
7
14
Days post-challenge
14
Fig. 2 Kinetic growth and clearance of Ft novicida ΔuvrBiglC 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).
46 of 48
Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
4. Significant decisions made or pending
The Ft subsp. novicida uvrB iglC double mutant is highly attenuated in mice infected intranasally,
though the growth of the Ft subsp. novicida uvrB iglC double mutant in lungs, liver and spleen is
evident.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
36 % of scientific work completed on the milestone
9. Work plan for upcoming month
a. Evaluate the protective efficacy of the Ft subsp. novicida uvrBiglC mutant as a vaccine
candidate. Groups of vaccinated mice will be challenged i.n. with Ft subsp. novicida.
Animals will be monitored for survival and weight loss.
b. Analyze the antibody profiles of mice immunized with the Ft novicida uvrBiglC mutant at
day 14 and 28 after vaccination.
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. Chromosomal DNA was purified from the F. novicida uvrB mutant KKF71. 10 ug of this
DNA was cryotransformed into a F. novicida pdpD mutant KKF37 in hopes of generating
a uvrB + pdpD double mutant. Cryotransformants were plated on TSA++ Kan for initial
selection and then 6 colonies were further screened by colony PCR with the primers
UvrBUP and UvrBDn1. The resulting 3.5 kilobase PCR fragments were digested with
Bgl2 and run on a DNA agarose gel. If the mutant is correct, you would expect to see two
fragments on the gel due to the presence of a Bgl2 site within the Kan marker that is not
present in the wild type copy of uvrB (Figure 1). Lanes 3 and 5 show two fragments and
thus are correct double mutants. Colony 1 (lane 3) was frozen away as KKF227. Data
described in Notebook 1, page 15. The strain KKF227 is a uvrB + pdpD double mutant of
Ft. subsp. novicida.
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Tularemia Vaccine Development Contract: Technical Report
Period: 4/1 to 4/30/2007
Due Date: 5/15/2007 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Kathryn
Sykes, Stephen Johnston, Mitch Magee, Justin Skoble, Bob Sherwood, Julie Wilder, Karl Klose,
Bernard Arulanandam
Figure 1.
1.
2.
3.
4.
5.
6.
7.
8.
Ladder
Ft. subsp novicida
colony 1 (KKF227)
colony 2
colony 3
colony 4
colony 5
colony 6
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
KKF71 (Francisella subsp. novicida mutant uvrB)
KKF72 (Francisella subsp novicida mutants , uvrA)
KKF100 Francisella subsp. novicida mutant uvrB+ uvrA double mutant)
KKF224 (Francisella subsp novicida uvrB + iglC double mutant)
KKF225 (Francisella subsp novicida uvrB + iglD double mutant)
KKF226 (Francisella subsp novicida uvrB + iglA double mutant)
KKF227 (Francisella subsp novicida uvrB + pdpD double mutant)
7. Quality of performance
Excellent
8, Percentage completed
80%
9. Work plan for upcoming month
Create iglB + uvrB double mutant
10. Anticipated travel
None.
11. Upcoming Contract Authorization
None
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