Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Contract No.
ADB Contract No.
HHSN266200500040-C
N01-AI-50040
Contract Title:
Tularemia Vaccine Development Team
Performance Period: 10/1/06 to 3/31/07
Contractor Name:
University of New Mexico Health Science Center
Contractor Address: Controller’s Office MSC-09-5220
1 University of New Mexico
Albuquerque, New Mexico 87131-0001
Authors:
C. Rick Lyons, MD, PhD and Barbara B. Griffith, MS
Date of submission: 4/16/2007
Semi-Annual
Technical Report:
Based on Contributions from ASU, Cerus, LBERI,
UNM and UTSA
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two animal
models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by Milestone
Active milestones: 2, 3, Working Group, 4, 5, 12(UNM/LBERI), 13(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
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. NIAID is working on the IAA with USAMRIID and a legal liability review is pending.
b. As of 12/6/06, Dr. Lyons submitted a request for programmatic support to Dr. Ed Nuzum,
Chief of the Product Development Section in the Office of Biodefense Research Affairs at
NIAID,
Monthly- March 2007
a. 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 (4/2/07). LBERI proposes to subcontract with the TRUE
Foundation in order to manage the vaccination program and to administer the payments
to USAMRIID. LBERI is requesting funding from the NIH Contract Officer.
4. Significant decisions made or pending
a. UNM and NIAID continue to wait for a change in the status of the IAA between NIAID
and USAMRIID.
b. UNM and LBERI will use their biobubbles as additional physical protective equipment, but
a work stoppage has occurred for SCHU S4 aerosols until LBERI staff is vaccinated with
LVS.
c. NIAID will need to provide UNM access to human cells from other LVS vaccinated
individuals which are needed to develop in vitro immunoassays. For possibly another
year, UNM will not have access to a local source of human cells from LVS vaccinated
individuals
d. UNM EOHS has obtained many of the laboratory documents
i. Documents pending
1. Radiology Facility Accreditation Certificate
5. Problems or concerns and strategies to address
a. UNM will need an external source of human cells from LVS vaccinated individuals, in order to
develop the immunoassays in humans.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
b. LBERI does not want to begin SCHU S4 aerosols until after their staff receive the LVS
vaccinations; Work stop has occurred on the SCHU S4 aerosols in primates, until the LBERI
scientists and staff receive the LVS vaccinations.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
16%- no change since the 11/15/06 report
9. Work plan for the next month and next 6 months
a. NIAID Contract Officer will continue to monitor the progress of the IAA between NIAID and
USAMRIID and will inform UNM when and whether the TVD Contractors can be vaccinated under
this IAA.
b. LBERI is awaiting funding request response from NIH Contract Officer on the IDIQ, to support
the LVS vaccinations from USAMRIID.
10. Anticipated travel
Travel could occur in Summer 2007 to Fall 2007, depending on the completion of the IAA or
funding of the IDIQ request.
11. Upcoming Contract Authorization (COA) for subcontractors
UNM may request a COA to allow 1-2 UNM EOHS nurses to travel to USAMRIID for training on
LVS site vaccination evaluations. The timing of the COA request depends on the achievement of
the IAA.
Milestone 3
Milestone description: Bioaerosol technique selected and optimized
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/31/07 – SUMMARY/HIGHLIGHTS
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 (104 –
108 CFU/mL) with spray factors ranging from approximately 2x10-7 to 1x10-6.
b. Studies have begun on the effects on LVS aerosols for the Sparging generators.
Monthly: March 2007:
a. 18 additional sprays were performed with fresh LVS over the target spray concentrations
of 105 to 107 cfu/mL
i. Target concentrations were good and consistent with previous tests as shown in
Figure 1.
ii. Spray factors were consistent with those seen in previous studies (see Figure 2).
iii. Data located at \\Saturn\ABSL3\Agent and Study Specific Data\Study Specific
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Data\TUL-03\TUL-03\Collison Generator\Collison.data evaluation.fresh LVS.xls
Collison: Target vs. Actual CFU/mL (Fresh)
Actual CFU/mL (Log10)
9.00
8.00
1/17/2007
7.00
1/24/2007
6.00
10/27/2006
3/14/2007
5.00
3/21/2007
4.00
3.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Target CFU/mL (Log10)
Figure 1. Actual vs. Target Dose of Fresh LVS Grown in Chamberlains Media
Spray Factor (Log10)
Collison: Actual Spray Conc. vs. Spray Factor
(Fresh)
-4.000
3.00
-5.000
4.00
5.00
6.00
7.00
8.00
9.00
1/17/2007
-6.000
1/24/2007
-7.000
10/27/2006
3/14/2007
-8.000
3/21/2007
-9.000
-10.000
Actual CFU/mL (Log10)
Figure 2. Spray Concentration vs. Spray Factor for Fresh LVS Aerosolized Using a Collison
Nebulizer
b. 27 sprays were performed using the Sparging generator and frozen LVS
i. Target concentrations were good and demonstrate that the titer for current frozen
LVS stock is consistent with previous tests (Figure 3).
ii. Spray factors were inconsistent with those seen in previous studies for Collison
aerosols (see Figure 4).
a. Sprays are being varied to determine optimum media for LVS stabilization and
optimum generator settings for the Sparging generator.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
b. Media formulations used include saline, Brain heart infusion broth (BHIB), and
BHIB + 0.001% antifoam A. Various media are being investigated because the
Sparging generator uses a different mechanism to generate aerosols and
appears to be very susceptible to foaming. We are attempting to minimize
foaming and still allow aerosol generation.
c. Generator airflows have been varied from 8 liters/min to 16 liters/min
d. Spray concentration data have demonstrated that the estimated titer of the LVS
frozen stock is correct as spray concentrations have been close to predicted.
e. LVS concentrations in impinger samples have varied widely with generally low
numbers recovered (spray factors lower than 1x10 -7. The best spray (spray
factor = 3x10-6) was associated with a low flow rate through the Sparging
generator.
iii. Data located at \\Saturn\ABSL3\Agent and Study Specific Data\Study Specific Data\TUL03\TUL-03\Sparging\Sparging.data evaluation.LVS.xls
Actual CFU/ml (Log10)
Sparging: Actual vs. Target CFU/mL (Frozen)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
3.00
3/28/2007
3/20/2007
3/23/2007
4.00
5.00
6.00
7.00
8.00
Target CFU/ml (Log10)
Figure 3. Actual vs. Target Spray Concentration for Frozen LVS
Spray Factor (Log10)
Sparging: Actual CFU/ml vs. Spray Factor (Frozen)
0.00
6.80
-2.00
6.90
7.00
7.10
7.20
7.30
3/20/2007
-4.00
3/23/2007
-6.00
3/28/2007
-8.00
-10.00
CFU/mL (Log 10)
Figure 4. Spray Concentration vs. Spray Factor for Frozen LVS in the Sparging Generator
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Not applicable
6. Deliverables completed
None
7. Quality of performance
Good for both frozen spray and fresh LVS sprays with a Collison nebulizer
8. Percentage completed
38% - Milestone deadline needs to be extended 4-6 weeks to allow completion of Sparging
generator and Micropump generator testing
9. Work plan for upcoming month and next 6 months



Perform bioaerosol experiments on frozen and fresh LVS with sparging generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
Perform bioaerosol experiments on frozen LVS with micropump generator
i. Repeat of studies performed on Collison
ii. Plan to quantitate LVS on CHAB
iii. Will continue doing frozen and fresh, not lyophilized.
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:
Semi-Annual: 10/1/06 to 3/30/07- SUMMARY HIGHLIGHTS
i. Solid and liquid media for expansion have been selected
ii. Expanded LVS shows no reduction in virulence in mice
iii. Bob Sherwood has revised the LVS Growth SOP twice at Barbara Griffith’s
request and needs to make the final quality changes requested by Marlene
Hammer
Monthly- March 2007
SCHU S4 can be expanded in Chamberlains or on agar and then Chamberlains, with no
difference in virulence in mice (Figure 5)
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 5.
Chamberlains
CHAB to Chamberlain's
100
75
87
20
5
50
25
0
Percent survival
Percent survival
100
75
155
36
8
50
25
0
0
2
4
6
8
0
Days
2
4
6
8
Days
Virulence of SCHU S4 stocks expanded in Chamberlain's broth with and without agar subculture. BALB/c mice
(n = 6) were challenged i.n. with the indicated preparation and doses of SCHU S4 and monitored daily for
survival.
Materials and Methods. To prepare the stock without agar subculture, an entire vial of SCHU S4 was
inoculated into 30 ml of Chamberlain's broth and cultured at 37oC for 48 h with shaking. To prepare the stock
with agar subculture, 0.2 ml of SCHU S4 was plated onto CHAB and cultured at 37oC for 24 h. SCHU S4 was
collected from the plate with PBSand and the OD550 was adjusted to 0.1. 1 ml of the bacterial suspension was
then inoculated into 100 ml Chamberlain's broth and cultured at 37oC for 48 h with shaking. The two stocks
were aliquoted without any preservative and frozen at -80oC until use
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Determined liquid and solid media for LVS growth
7. Quality of performance
Good
8. Percentage completed
100%- except that the final formatted SOP is 95% complete
9. Work plan for upcoming month and next 6 months

Finalize LVS growth SOP by incorporating changes suggested by Marlene Hammer for
quality
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
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
1. Date started: 11/1/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions:
Semi-Annual: 10/1/06 to 3/30/07 – SUMMARY/HIGHLIGHTS ONLY
a. 6 non-human primates were vaccinated with LVS, 3 via the intradermal (i.d.) route and 3
via the subcutaneous (s.c.) route
b. Vaccinations were delivered to the skin of the upper back and were well-tolerated with no
adverse effects
c. Both i.d. and s.c. routes of vaccination induced an immune response based on the ability
of peripheral blood mononuclear cells to proliferate in response to LVS in vitro and the
presence of IgG anti-LVS in the serum as measured by ELISA
Monthly-March 2007:
a. 3 non-human primates originally vaccinated by the s.c. route (A00659, A00868 and
A00902) 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)
Table 1
A00659
3/26/07
i.d. 150K LVS
3/27/07
No reaction
A00868
i.d. 150K LVS
No reaction
A00902
i.d. 150K LVS
No reaction
3/28/07
5 mm, slightly
red; raised bump
4 mm, slightly
raised and red
bump; diffuse red
rash of 35 mm
No reaction
3/29/07
8 mm, slightly
red, raised bump
4 mm, slightly
raised and red
bump; diffuse red
rash of 35 mm
No reaction
d. Data interpretation:
i. Two of the three NHPs had a reaction to the formalin fixed LVS that resembled a DTH
reaction,
ii. The reaction mounted by A00659 most resembled a DTH reaction in that it was not
surrounded by a diffuse rash
iii. The reactions mounted by A00659 and A00868 suggest that vaccination of these
NHPs 16.5 weeks previously resulted in activation of the cellular immune response
iv. 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

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 033007
mtg.doc and N:\My Documents\Tularemia Contract\prep for 033007 mtg.doc
4. Significant decisions made or pending
a. 10/1/06 – 3/30/07: None
b. March 2007: None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. 10/1/06 – 3/30/07: None
b. March 2007: None
7. Quality of performance
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Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Good
8. Percentage completed
12%
9. Work plan for upcoming month
a. For next month: Test the skin test response of the 3 NHPs that were originally
vaccinated via the i.d. route A00937, A00896 and A00908 by administering 150,000 LVS
i.d. in the upper back and observing the reaction over the next 3 days
b. For the next 6 months: Expose the LVS vaccinated primates to Schu 4 in order to test
whether they are protected from disease
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
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. Mouse model
i. The purpose was to determine the suitability of outbred NIH-Swiss mice as
models of human respiratory tularemia
ii. Naïve Swiss mice were very sensitive to i.n. SCHU S4 challenge and the
calculated i.n. LD50 was less than 1
iii. Naïve Swiss mice were resistant to s.c. and i.d. vaccination with at least 5 x
106 LVS but no more than 1-5 x 103 LVS by the intranasal route. The
calculated i.n. LD50 is 1.1 x 104 CFU
iv. Vaccinated Swiss mice required 7 weeks to clear the LVS vaccine
v. Intranasal vaccination protected Swiss mice against intranasal SCHU S4
challenge better than s.c. or i.n. vaccination
1. SCHU S4 i.n. LD50 = 761
2. SCHU S4 s.c. LD50 = 36
3. SCHU S4 i.d. LD50 = 27
vi. We developed a scoring system for disease severity
vii. Outbred NIH-Swiss mice are no better than inbred BALB/c mice, which has
been published extensively, as a model of respiratory tularemia because
1. They were very similar to inbred BALB/c mice in all parameters
2. Vaccination induced a limited window of protection
3. Conventional vaccination routes (s.c. or i.d.) offered no protection
b. Fischer rat model
i. The purpose was to determine the suitability of Fischer 344 rats as a model
of human respiratory tularemia
ii. We developed a consistent and reproducible method for delivering F.
tularensis into rat lungs using an i.v. catheter and a lighted laryngoscope
iii. Naïve Fischer rats were more sensitive to respiratory SCHU S4 infection
than previously reported in the literature and recognized by the tularemia
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
c.
research community. They were killed by as few as 3,000 SCHU S4 and the
calculated i.t. LD50 is 180 CFU
iv. Fischer rats were very resistant to LVS vaccination and tolerated
intradermal, subcutaneous, or intratracheal vaccination with at least 5 x 10 7
SCHU S4
v. The vaccinated rats cleared LVS 4 weeks after vaccination
vi. LVS vaccination, regardless of route, protected 50% of rats from 3 x 107
SCHU S4 and at least 60% from lower challenge doses.
vii. We developed a scoring system for disease severity
viii. Fischer 344 rats appear to be better than mice as model of human
respiratory tularemia because
1. Conventional vaccination routes (s.c. or i.d.) protected against
respiratory challenge
2. The window of protection was larger than in mice
Guinea pig model
i. The purpose was to determine the suitability of Hartley guinea pigs as a
model of human respiratory tularemia
ii. Guinea pigs were very sensitive to respiratory SCHU S4 challenge and were
killed by as few as 10 SCHU. The minimum lethal dose may be even lower
because some guinea pigs were killed by a calculated dose of less than 1.
iii. Guinea pigs were very resistant to LVS vaccination and tolerated intranasal
vaccination with 4.73 x 106 LVS and s.c. vaccination with 8.8 x 106 LVS
iv. Vaccinated guinea pigs cleared LVS one month after vaccination
v. Neither s.c. nor i.n. vaccinated guinea pigs survived i.n. challenge with 1.5 x
103 SCHU S4, although i.n. vaccinated guinea pigs survived 2-3 days longer
than s.c. vaccinated guinea pigs.
vi. It appears that Hartley guinea pigs may be only as good as mice as a model
of human respiratory tularemia because LVS vaccination did not offer a big
window of protection against respiratory tularemia
Monthly: March 2007
Fischer 344 rats
a. Experiment Ftc32 study 1 (Notebook 101, in progress)
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. Rats were vaccinated according to Table 1
iii. All of the LVS-vaccinated rats have recovered from vaccination and appear
outwardly healthy. However, 10/24 of the rats vaccinated intranasally with
SCHU S4 have died. This confirms our earlier finding that Fischer 344 rats
are more sensitive to respiratory SCHU S4 challenge than previously thought
Table 1. Experimental design to compare the ability of different vaccination routes and to
protect Fischer 344 rats against i.t. SCHU S4 challenge
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Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
i.t. SCHU S4 challenge
(number of rats/dose)
Vaccination
Bacterial
strain
None
Route
-
Dose
CFU/rat
-
LVS
i.d.
s.c.
i.t.
5 x 107
5 x 107
5 x 107
6
6
6
6
6
6
6
6
6
SCHU S4
i.t.
5 x 101
6
6
6
101
6
102
6
103
6
104
105
106
107
108
b. Experiment Ftc37 study 1 (notebook 94, pages 171-175)
i. The purpose of this experiment was to determine the kinetics of SCHU S4
proliferation and dissemination in naïve Fischer 344 rats after i.t. infection
ii. Naïve Fischer 344 rats were challenged i.n. with about 30 CFU SCHU S4.
Three infected rats were killed on the day of infection and every 3 days
thereafter to determine the bacterial burden in the lungs, spleens and livers
iii. By day 3, SCHU S4 had already expanded 6 logs in the lungs, disseminated
to the spleen and liver, which contained at least 106 CFU (Table 2)
iv. The bacterial burden continued to increase and by day 6 averaged 2.7 x 108
in the lungs, 2.88 x 107 in the liver and 3.2 x 107 in the spleen
v. A similar increase tissue burden was observed in mice challenged with the
biovar A strain NMFTA1 [see Wu T et al. Infect. Immun. (2005) 73:2644]
vi. Interestingly, at this time, the lungs became very rubbery and difficult to
homogenize using beadbeater tubes
vii. Two of the challenged rats did not survive until day 9
Table 2. Kinetics of SCHU S4 proliferation and dissemination after i.t. infection of naïve Fischer 344 rats
c.
Experiment Ftc37 study 2 (Notebook 101, in progress)
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. The 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 were fixed in neutral buffered formalin for 3 days and stored in
70% ethanol until they can be processed
d. Experiment Ftc38 study 1 (in progress)
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) had showed that s.c. vaccination was as good as other
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vaccination routes in protecting rats against respiratory SCHU S4 challenge
and it is the preferred route to vaccinate human
iii. We vaccinated 27 naïve rats with 5 x 107 LVS by s.c. route and have been
measuring the bacterial load in the lungs, livers and spleen every three days.
This will continue until the infected rats clear 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 that would confirm vaccination
based on sero-conversion.
ii. We followed instructions from Freyja Lynn to optimize the antigen coating
concentration and sera concentration
iii. 96-well plates were coated with a range of heat-killed or formalin-fixed LVS
from 5 x 103 to 1 x 107/ml, in 2-fold dilutions
iv. Sera from naïve and LVS-vaccinated rats were titrated from 1:200 to 1:1,600
dilution
v. Our preliminary data suggested that heat-killed LVS was better than formalinfixed LVS for coating plates. This is similar to Julie Wilder’s results using
vaccinated monkey sera
vi. The optimal coating concentration is 5 x 105/ml heat-killed LVS and the
optimal serum concentration 1:800
f. Experiment Ftc40 study 1 (in progress)
i. The purpose is to determine the kinetics of SCHU S4 proliferation,
dissemination, and clearance in s.c. LVS vaccinated rats
ii. We have vaccinated rats s.c. with 5 x 107 LVS and will wait for the rats to
clear LVS, then challenge them i.t. with 30 and 300 SCHU S4
Hartley Guinea Pigs
a. Experiment Ftc28 study 2 (Notebook 94, pages 152-156)
i. The purpose was to use the guinea pigs that survived i.n. or s.c. LVS
infection in Ftc28 study 1 in a pilot experiment to determine whether LVS
vaccination protects guinea pigs against pulmonary SCHU S4 challenge
ii. 33 days after LVS vaccination, the number of LVS in the lungs, spleens, and
livers was below our limit of detection in two guinea pigs vaccinated s.c. with
4.4 x 107 LVS and in two guineas pigs vaccinated i.n. with 4.7 x 106 LVS
iii. 47 days after LVS vaccination, guinea pigs (n = 2-4/group) were challenged
i.n. with an estimated dose of 500 SCHU S4. At such a low dose, the large
buffer volume used to homogenize infected lungs makes it difficult to
accurately determine the actual lung deposition by plating lung homogenates
onto cystine heart agar plates
iv. Our results indicated that neither i.n. nor s.c. LVS vaccination protected
guinea pigs against a respiratory SCHU S4 challenge (Table 3), although i.n.
vaccinated guinea pigs survived 2-3 days longer than s.c. vaccinated guinea
pigs
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Table 3. Survival of vaccinated guinea pigs challenged i.n. with SCHU S4
Vaccination
LVS dose
Animal ID
Disease severity1
Time to death
Route
(CFU/animal)
(days)
i.n.
1.5 x 103
9949
Dead
6
9950
Dead
9
9948
Dead
8
7.8 x 104
9947
Dead
8
9946
Alive
9945
Dead
13
9944
Dead
9
4.7 x 106
9943
Dead
8
9942
Dead
10
s.c.
4.4 x 103
4.4 x 105
4.4x 107
9938
9937
9936
9932
9933
9935
9934
9930
9931
Dead
Dead
Dead
Dead
Dead
Dead
Dead
Dead
Dead
6
6
6
9
11
6
6
6
8
b. Experiment Ftc41 (in progress)
i. The purpose is to determine the resistance of i.n. LVS vaccinated Harley
guinea pigs to i.n. SCHU S4 challenge
ii. We vaccinated 3 groups of guinea pigs (n = 6 to 10) i.n. with 103, 105, and
107 CFU LVS
iii. We will challenge the vaccinated guinea pigs with SCHU S4 when they have
cleared the LVS vaccination
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse model completed
7. Quality of performance
Good
8. Percentage completed
45%
9. Work plan for upcoming month and next 6 months
a. Rats
i. Measure the resistance of vaccinated Fischer 344 rats to i.t. SCHU S4
challenge
ii. Characterization of the Fischer 344 rat model
1. Kinetics of SCHU S4 proliferation in lungs, spleens, and livers of
naïve and vaccinated rats
2. Histology of lungs, spleens and livers of naïve and vaccinated rats
infected with SCHU S4
b. Guinea Pigs
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Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
i. Our goal is to repeat, with statistically significant group sizes, our preliminary
experiment showing that neither i.n. nor s.c. LVS vaccination protected
guinea pigs from respiratory SCHU S4
ii. Vaccinate naïve guinea pigs i.n. and s.c. with three different LVS doses
iii. Check for LVS clearance in lungs, liver, and spleen 4 weeks after vaccination
iv. Check for sero-conversion using ELISA similar to the one we developed for
mice and rats
v. Challenge the vaccinated guinea pigs intranasally with SCHU S4
vi. Decided whether to pursue or abandon guinea pigs as a model
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12-LBERI
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 – 3/30/07 – SUMMARY/HIGHLIGHTS
a. After testing 3 different protocols for purifying PBMCs from NHP blood, we chose a
method published on the Purdue Cytometry Mailing List which uses Nycomed
LymphoprepTM; this separation medium is simple to use and results in the best recovery
of white blood cells with the least red blood cell contamination
b. We have chosen a BrdU/Chemiluminescence ELISA kit in order to measure PBMC
proliferation (Roche Applied Science, Catalog #11 669 915 001); it is easy to use, nonradioactive and gives consistent results; it is sensitive enough to detect antigen (LVS)specific and mitogen-driven cell proliferation
i.
the optimum number of cells to plate for the proliferative response is 2 x
105/well (1 x 106/ml); this concentration allows us to visualize both LVSspecific and Con A-induced proliferation
ii.
the amount of heat-killed- or formalin-fixed LVS used to stimulate PBMCs
can range from 0.0625 – 1 x 106 cells/ml and equivalent results are
obtained; HK-LVS and FF-LVS are also equivalent although the FF-LVS
induces higher background proliferation from PBMCs collected from naïve
NHPs
c.
We have set up the IFNELISPOT assay using a kit from MABTECH (Catalog #3420M2HW-Plus)
i. 20,000 cells/well plated allows detection of IFN spots when stimulated with Con A
ii. LVS-induced IFN secretion detection requires that between 100,000 and 500,000
cells/well are plated; this unfortunately results in a higher background as well
d. Antibodies to CD4, CD8, CD20, CD3 and CD56 have been selected and stain cells in
both whole NHP blood and PBMC preparations
i. CD20+ cells are much reduced in the PBMC preparation as compared to whole blood
for reasons we do not yet understand
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
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e. We have been testing 3 different, yet generally similar, methods for freezing and thawing
NHP PBMCs
i. CTL protocol (90% human A/B serum/10% DMSO at 10 x 106/ml): cells can be
recovered 8 weeks after freezing although the yield varies between 20 – 100%; viability is
good (70 – 92%); can recover about 50% of the original proliferative response to Con A
(tested twice), but see little response of the thawed cells to LVS (tested only once thus
far)
ii. Cerus protocol ((80% FBS, 20% DMSO, final concentration of 5 x 106/ml): cells can
be recovered 8 weeks after freezing with yields ranging from 66 – 79%, and viability
between 91 – 96%; can recover at least 50% of the cells after thawing and 50% of the
Con A proliferative response and 30 – 40% of the LVS-specific proliferative response;
recovery of the antigen-specific proliferative response is better when 1 x 106 cells/ml are
plated rather than 0.25 x 106 cells/ml
f. We have begun development of an ELISA to detect NHP IgG anti-LVS
i. preliminary results suggest that HK-LVS is superior to FF-LVS for use as a coating
antigen, but we have yet to formally test the optimum coating concentration
Monthly-March 2007:
a. Update on NHP PBMC Freezing protocols
i. On 3/26/07, we froze down cells from the same NHP using each of the 3 different
protocols; these will be thawed on 5/14/07 (7 weeks post-freezing) to test antigenspecific proliferation, and possibly Con A and PHA proliferation if enough cells are
available
ii. Using cells from TUL8, day 28 post i.d. LVS vaccination, we compared the IFN
ELISPOT results induced by Con A in fresh and frozen cells (Figure 6)
Partial Con A-stimulated IFN secretion can be recovered from frozen PBMCs using the CTL
protocol
Cell Mean for IFNg Spots
600
Media
Con A
500
400
300
200
100
0
Fresh
Frozen
Figure 6: Test of mitogen driven IFN ELISPOT assay using frozen vs. fresh cells; these cells are
from TUL8, day 28 and had been frozen for 8 weeks before thawing using the CTL protocol.
20,000 cells/well.
b. Update on B cell staining artifact
i. Issue: NHP whole blood contains a population that stains positively with anti-CD20
antibody and thus looks to be B cells; however, upon purification of PBMCs, this
population disappears or is greatly decreased; is this an artifact or real?
15 of 70
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Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
ii.
Suspect that this is an artifact due to the fact that T cells are enriched in the PBMC
population when compared to whole blood and B cells are approximately the same size
and density and should not be depleted during the PBMC isolations
iii. Experimental test: bled three NHPs (A00659, A00902 (day 78 post S.C. LVS
vaccination; and A00896, day 87 post I.D. LVS vaccination) and stained both blood and
PBMCs with antibodies to T cell markers (CD3, CD4, and CD8), CD20 and other B cell
markers (CD19 and IgM)
iv. Results:
Cell Mean for % Monocytic
80
70
60
A
Blood
PBMC
50
40
30
20
10
0
Cell Mean for % Granulocytic
A00659
A00896
A00902
25
Blood
PBMC
20
B
15
10
5
0
A00659
A00896
A00902
Figure 7: Demonstration that PBMC purification indeed A) enriches for monocytic cells and B)
excludes granulocytic cells as expected. Note, A00659 PBMC prep appeared to have lots of RBCs
in it, thus, the monocyte percentage was not enriched much.
16 of 70
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Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
60
Blood
PBMC
50
40
30
20
%CD3+CD8+: A00902
%CD3+CD8+: A00896
%CD3+CD8+: A00659
%CD3+CD4+: A00902
%CD3+CD4+: A00896
%CD3+CD4+: A00659
%CD3+: A00902
%CD3+: A00896
0
%CD3+: A00659
10
Figure 8: Percent of T cells in Blood and PBMCs.
16
Blood
PBMC
14
12
10
8
6
4
%CD20+IgM+: A00902
%CD20+IgM+: A00896
%CD20+IgM+: A00659
%IgM+: A00902
%IgM+: A00896
%IgM+: A00659
%CD20+: A00902
%CD20+: A00896
0
%CD20+: A00659
2
Figure 9: Percent of B cells in Blood and PBMCs
v.
Interpretation of results
i. CD20+ cells are lost in the PBMC population to varying degrees. Blood cells do not
express surface IgM
ii. IgM+ cells are enriched in the PBMC population and all of them are CD20+; thus
these look like true B cells.
17 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
iii. Not shown: Anti-human CD19 failed to stain many cells in either whole blood or
PBMCs.
iv. In summary, the flow cytometry results support the presence of low levels of B cells
in the PBMC preparations
c.
Update on IFN ELISPOT assay:
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
Higher numbers of cells plated are required to observe LVS-induced, as compared to Con Ainduced, IFN ELISPOT development
Cell Mean for IFNg Spots
700
Media
Con A
LVS hk Hi
LVS ff Hi
600
500
400
300
200
100
0
.25
.667
3.33
Concentration of cells plated in Million cells/ml
Figure 10: When more cells are plated, LVS-induced IFN secretion is detectable, however, the
background also increases. These cells came from a day 78 re-bleed of 2 NHPs, averages of the 2
NHPs are shown.
iii. Summary of Con A-induced IFN secretion as detected by ELISPOT comparing the
S.C. vs. I.D. routes of vaccination
18 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Con A-induced IFN ELISPOT results in I.D. vs. S.C. LVS vaccinated NHPs
ID, Media
ID, Con A
SC, Media
SC, Con A
1600
1400
1200
1000
800
600
400
Day 78
Day 28
Day 21
Day 0
0
Day 14
200
Day 7
Cell Mean for IFNg Spots
1800
Figure 11: IFN ELISPOT assay as induced by Con A. Average of 2 – 3 NHPs at each timepoint.
iii. Data interpretation: LVS-induced IFN ELISPOT detection requires more
PBMCs/well which also results in higher background; need to talk to the
ELISPOT reader representative about this; Con A induced IFN ELISPOT
detection is fairly equivalent between I.D. and S.C. LVS vaccinated NHPs, but
this may not be meaningful as it is not LVS-specific

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 033007
mtg.doc; and N:\My Documents\Tularemia Contract\prep for 033007 mtg.doc; the raw
data is also stored in \\Saturn\Group\wilder lab\TVDC\PBMC assays032907.svd
4. Significant decisions made or pending
a. 10/1/06 – 3/30/07: Chose a PBMC purification strategy, a proliferation kit and antibodies
that stain T and B cells in NHP whole blood and PBMCs
b. March 2007: None
5. Problems or concerns and strategies to address
a. Concern that freezing cells down and thawing them using the CTL protocol may not
preserve the proliferative response to LVS antigen; we will test this again to be sure before
making this conclusion.
b. Concern that B cells are being lost by PBMC purification, we will test the LPS
responsiveness of whole blood cells and PBMCs by proliferation
c. 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
6. Deliverables completed
a. 10/1/06 – 3/31/07: None
b. March 2007: None
7. Quality of performance
Good
8. Percentage completed
70% of scientific work has been completed (Note that the goals and anticipated scientific work
in this milestone has changed in the past 6 months; thus the percentage completed is now
lower)
19 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
9. Work plan for upcoming month and next 6 months
a. For next month:
i. Continue to freeze down PBMCs using the 3 different protocols
ii. Contact the ELISPOT kit and reader representatives to discuss issues in optimization
iii. Optimize the coating concentrations of HK- and FF-LVS for the IgG anti-LVS ELISA
b. For the next 6 months:
i. Develop the IFN intracellular staining assay in whole blood and PBMC preparations
(from LVS-vaccinated NHPs; Milestone 4) using flow cytometry
ii. Develop the IgA anti-LVS ELISA
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 12-UNM
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed
Institution: UNM
1. Date started: 7/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07
a. Detection of F. tularensis-specific T cells in LVS-vaccinated animals
i. The purpose was to develop an assay for detecting F. tularensis-specific T
cells in vaccinated animals
ii. Our assay measures incorporation of BrdU into the DNA of actively
proliferating T cells stimulated by formalin-fixed and heat killed LVS or SCHU
S4.
iii. We found that splenocytes must be enriched over a nylon wool column to
reduce non-specific proliferation
iv. We also found that a combination of 5 x 104/well nylon wool-enriched T cells
and 106/well LVS in a 96-well format produced the best balance of
background, specificity and sensitivity
v. We are currently optimizing this assay to detect T cells that recognize
peptides derived from F. tularensis proteins (milestone 27)
Monthly: March 2007
a. No work done. We found in the last reporting period that a combination of 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 have optimized this assay
as much as possible without drastically changing our approach. We are now
applying this assay to identify F. tularensis peptides that stimulate T cells from LVSvaccinated BALB/c and potentially other vaccinated small animals models (milestone
27)
4. Significant decisions made or pending
None
20 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month and next 6 months
Since Fischer 344 rats appear to be a better model than the mouse, we will start to develop
the T cell proliferation assay for the rat
a. Verify that Fischer 344 rats 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
NA
11. Upcoming Contract Authorization (COA) for subcontractors
Milestone 13-LBERI
Milestone description: Compare assays in animal models (sensitivity)
Institution: LBERI
1. Date started: 11/16/06
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 – 3/30/07 – Summary/Highlights
a. Performed a comparison of Con A and LVS-specific proliferation in NHPs vaccinated with
LVS by SC or ID routes
i. S.C. vaccinated NHPs appear to respond earlier to LVS, by day 7 post
vaccination as opposed to day 21 post vaccination in the I.D. vaccinated group;
ii. The proliferative response of the S.C. vaccinated NHPs to LVS is greater
quantitatively at all time points; however the proliferative response to Con A is
also greater in the S.C. vaccinated NHPs as compared to the I.D. vaccinated
group, even before vaccination (Day 0); this remains unexplained

All data is stored in binder TVDC 1 in the Wilder laboratory as well as in C:\Documents
and Settings\jwilder.LOBOS\My Documents\Tularemia Contract\ TUL 8 or TUL 9; and
N:\My Documents\Tularemia Contract\ TUL 8 or TUL 9;
Monthly- March 2007:
a. No data to report on this milestone
4. Significant decisions made or pending
a. 10/1/06 – 3/30/07: None
b. March 2007: None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. 10/1/06 – 3/30/07: None
b. March 2007: None
21 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
7. Quality of performance
Good
8. Percentage completed
15% of scientific work has been completed
9. Work plan for upcoming month and next 6 months
a.
b.
For next month:
i. Using blood drawn from these NHPs, further optimize the IFN ELISPOT and
serum Ig anti-LVS ELISA
For the next 6 months:
i. Run all of the vaccinated NHP sera (Days 7, 14, 21 and 28) on LVS coated
ELISA plates, after the ELISA plate coating concentration is optimized for both
IgG and IgA anti-LVS
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 13-UNM
Milestone description: Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
UNM has not started working on this milestone yet but have been providing supplies to
LBERI
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
NA
9. Work plan for upcoming month
NA
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
22 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. Measure the function of F. tularensis-specific T cells from vaccinated human
i. The purpose was to develop an assay for measuring the activity of F.
tularensis-specific T cells from vaccinated human subjects, specifically, their
ability to induce macrophages to kill intracellular F. tularensis
ii. We have been working on the procedure for infecting human alveolar
macrophages with LVS and SCHU S4
iii. Our preliminary experiments indicated that
1. MOI of 1, 10 or 100 produced a similar level of infection
2. LVS proliferated slower than SCHU S4 in human alveolar
macrophages
3. LVS and SCHU S4 caused cytopathic effects as early as 2 days after
infection and escaped from the cells into the medium
Monthly: March 2007
a. Experiment Ftc36 study 3 (Notebook 94, pages 176-181)
i. The purpose was to determine the optimal MOI for infecting human alveolar
macrophages and to determine the kinetics proliferation and escape
ii. 2.5 x 104 human alveolar macrophages were infected with LVS or SCHU S4
at MOI of 100, 10 and 1 for 2 h at 37oC. Infected macrophages were treated
with 50 g/ml gentamcin for 1 h to eliminate extracellular bacteria and then
cultured at 37oC without antibiotics
iii. On day 0, 2, and 3, a sample of the culture media was diluted and plated to
determine whether bacteria escaped from the macrophages. The infected
macrophages were then lysed and the cell lysates plated onto cystine heart
agar plates to quantify the number of intracellular bacteria
iv. Our results showed:
1. MOI of 1, 10 or 100 produced a similar level of infection for LVS and
SCHU S4 (Table 4 below)
2. LVS proliferated slower than SCHU S4
3. LVS and SCHU S4 both caused cytopathic effects by day 2 p.i., seen
as disintegration of the macrophage monolayer and changes in cell
morphology
4. LVS and SCHU S4 were found in the culture medium by day 2 p.i.
5. The number of LVS and SCHU S4 did not increase between day 2
and day 3, possibly because the macrophages were dead
23 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Table 4. Kinetics of LVS and SCHU S4 proliferation in human alveolar
macrophages
Strain
LVS
Log bacterial concentration (CFU/ml)
MOI = 1
MOI = 10
MOI = 100
Day of
infection
0
Fraction
Cell
0.4  0.4
1.0  0.1
1.2  0.1
2
Cell
Medium
total
2.9  0.1
2.4  0.5
3.1  0.2
3.2  0.3
2.0  0.6
3.2  0.3
2.5  0.2
1.6  0.1
2.6  0.2
3
Cell
Medium
total
2.8  0.4
1.9  0.2
2.9  0.4
3.4  0.4
2.3  0.4
3.4  0.4
1.9  0.5
1.0  0.3
1.9  0.5
0
Cell
0.8  0.1
1.1  0.2
0.8  0.1
2
Cell
Medium
total
5.8  0.1
4.6  0.2
5.8  0.1
5.2  0.1
4.3  0.1
5.3  0.1
5.4  0.3
4.1  0.6
5.5  0.2
3
Cell
Medium
total
5.4  0.2
4.2  0.1
5.4  0.2
5.5  0.2
4.3  0.1
5.6  0.1
5.1  0.1
4.3  0.2
5.2  0.1
SCHU S4
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 and next 6 months
b. Determine the optimal MOI for infecting human alveolar macrophages. Since we
observed cytopathogic effects at MOI = 1, we will titrate MOI down to 0.1, 0.5, and 1
c. Determine macrophage viability by lactate dehydrogenase (LDH) release and trypan
blue exclusion after infection
d. Determine kinetics of bacterial proliferation after infection
e. Measure cytokine (e.g. TNF, IL-1, and IL-6) production by macrophages infected with
SCHU S4 or LVS
f. Determine whether recombinant IFN would inhibit SCHU S4 and LVS intracellular
growth
g. Determine whether PBMC from vaccinated human volunteers can induce infected
macrophages to kill intracellular bacteria
19. Anticipated travel
24 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. Measure the function of F. tularensis-specific T cells from vaccinated animals
i. The purpose was to develop an assay for measuring the activity of F.
tularensis-specific T cells from vaccinated animals, specifically, their ability to
stimulate macrophages to kill intracellular F. tularensis
i. We are now able to generate murine bone-marrow derived macrophages and
infect them with LVS. Our preliminary results indicated that
1. MOI between 10-40 produced a similar level of infection
2. After 3-day culture, the number of LVS increased by 3.5 to 4 log 10
3. LVS proliferation is dependent macrophages
ii. Addition of splenocytes from vaccinated mice at the start of culture reduced
the number of bacteria recovered after 3 days
Monthly: March 2007
i. Experiment Ftc30 study 5 (notebook 94, pages 147-149)
ii. The purpose was to determine whether T cells from vaccinated BALB/c mice
can induce bone-marrow derived macrophages to kill intracellular LVS
iii. BALB/c bone marrow macrophages (BMM) were cultured for 7days in DMEM
with 10% fetal calf serum and 10% conditioned L929 supernatant as a
source of M-CSF
ii. The BMM macrophages were infected with LVS at MOI of 40:1 for 2 h and
then treated with 50 g/ml gentamcin for 1 h to eliminate extracellular
bacteria
iii. The infected macrophages were cultured with 5 x 106/well naïve or
vaccinated splenocytes
iv. After 3 days in culture, the infected macrophages were lysed and the cell
lysates were plated to quantify the number of intracellular bacteria in the
macrophages
v. Addition of vaccinated splenocytes completely inhibited LVS proliferation in
BMM (Fig 1)
vi. Addition of naïve splenocytes inhibited intracellular LVS proliferation by 1 log.
Since the naïve splenocytes were not enriched over a nylon wool column to
eliminate non-specific response, which we observed in milestone 12, it is
possible that non-specific IFN production caused this reduction
25 of 70
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Period: 10/01/2006 to 3/31/2007
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Figure 1. Inhibition of LVS proliferation in BMM by vaccinated BALB/c
splenocytes. LVS-infected BMM were incubated with 5 x 106 naïve or
vaccinated BALB/c splenocytes. After 3 days in culture, intracellular LVS
was quantified. Each bar represents the average of triplicate samples 
SD.
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 and next 6 months
b. Repeat experiment to determine whether vaccinated splenocytes can induce BMM to
kill intracellular LVS
c. Determine whether vaccinated splenocytes can induce BMM to kill intracellular
SCHU S4
d. Develop the macrophage killing assay using T cells from vaccinated Fischer 344 rats
i. Develop procedures for isolating and culturing macrophages from rats
ii. Develop procedures for isolating T cells from naïve and vaccinated rats
iii. Determine the optimal MOI for infecting rat macrophages
iv. Determine the kinetics of LVS and SCHU S4 proliferation in infected
macrophages
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
26 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Milestone 25
Milestone description: Design protein-fragment library based on SCHU S4 annotated
sequence. Design a genomically-complete SCHU S4 library of genetically-improved proteinfragments (polypeptides).
Institution: ASU-Sykes
1. Date started: 3/02/2006
2. Date completed: 12/01/07
3. Work performed and progress including data and preliminary conclusions
Semi-Annual 10/1/06 to 3/30/07-Summary Highlights
a. We developed a new approach to optimally redesign recoded genes.
b. We wrote oligo prediction software, called SynBuild-Htp, which is necessary to implement the
redesign approach at the genomic level.
c. We developed new gene assembly protocols that uniquely enable us to use microfluidic
(“chip”)-based oligo synthesis technologies.
i. These oligonucleotides are parallel-synthesized and therefore significantly less
expensive than standard oligos. However, production-scale and oligo quality are
much lower, and samples are delivered in a single tube as a complex mixture
called an “oligomix”.
ii. We now have working protocols for synthetically assembling genes in parallel,
using the inexpensive chip oligos.
d. The fidelity of the synthetically generated genes using our new protocols is 10 times greater
than that possible with any existing gene assembly protocols which require larger quantities
of higher quality oligos.
e. We added an addendum to this aim: To design and obtain 500 synthetic tularensis peptides
predicted to be immunogenic. These will be used in the assay development phases of UNM’s
T cell experiments, in concert with a set of initial in vitro translated tularensis polypeptides
generated at ASU. The list of peptides (20-mers) covering MHC Class I and Class II epitopes
were proposed by Dr. Sykes and reviewed by Drs. Johnston, Lyons, and Breen.
f. The peptides were ordered and then sent from ASU to UNM as lyophilized samples (~2mg) in
microtiter plates.
g. The 2 groups held a conference call to plan use, testing, and storage of the peptides.
Files are stored at R:\GeneVac\FTU\Contract\Proteome\Milestones\26\MHC\ftu_mhc_proteins.xls
Monthly – March 2007
Completed milestone
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
100%
9. Work plan for upcoming month and next 6 months
None
27 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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:
Semi-Annual 10/1/06 to 3/30/07-Summary Highlights
A. Select and test ORF expression constructs
Alternative expression template designs for in vitro translation (IVT) of the FTU ORFs were
designed, constructed and evaluated for improving ivt product yields and specificity.
1. Some spacer sequences are known to enhance expression from supercoiled plasmid
template but their impact on linear expression template, in which template length
should be minimized, is unknown.
a. The effect on polypeptide synthesis levels of i) optimizing the
spacing between transcription and translation start sites, and ii)
including the T7 g10 enhancer originally described by Studier was
evaluated.
b. We found that both spacers improve polypeptide yields.
2. We evaluated whether the presence of a sequence encoding a biotinylation site
(BAP) affected polypeptide synthesis. Engineering a BAP site into the template would
enable biotinylation of the polypeptide necessary for strepavidin-based detection
and/or affinity purification. We found that the BAP sequence did not cause
expression problems for any of the ORF-encoded polypeptides.
3. To address how robust the template design would be, we evaluated the ability of the
template to generate an assortment of FTU proteins. We synthetically built 6
recoded FTU ORFs, and these were placed in the linear templates described above.
Yields and quality of ivt products from linear templates were compared to ivt products
from supercoiled plasmid templates representing the same ORFs. We found the
template design in vitro synthesis protocol is sufficiently robust to generate 6 out of 6
FTU proteins without individual ORF reaction optimizations. Linear and plasmid
reaction yields are comparable.
Files are stored at: \\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Cheryl's data\IVT
B. Select and test IVT Protocols
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Alternative in vitro translation protocols were evaluated for improving ivt product yields and
specificity.
1. E. coli expression systems from three vendors and a wheat germ system were tested.
We found that the Invitrogen E. coli lysate performed best.
2. Two polypeptide product detection systems were compared. We found 35S-radiolabel to
be superior to a non-radioactive tRNA-lys system.
3. We evaluated a lysate reaction “feed” system as a means of improving product yields.
We found the Roche substrate-feed system to significantly improved yields when used
with the Invitrogen expression lysates.
4. We further optimized the system for use with linear template by addition of fresh template
to the feed system at later time-points.
Files are stored at: R:\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT\IVT 35S gels
C. Select and test protocols for protein purification
Polypeptide product assessment and development of purification protocols
1. The absolute number of TCA precipitable counts per minute allowed us to calculate an
estimate of total product yields using the known specific activity of the 35S on a known
date and the known number of methionines in a chain.
2. We scaled up synthesis of protein from 6 linear FTU ORF expression cassettes to test a
set of purification approaches.
3. A preliminary T cell assay suggests that lysate purification may not be critical. This needs
to be further investigated.
4. A nickel-based purification of 6 His tagged polypeptide preparations indicates quantitative
binding of polypeptides to beads in 8M urea.
5. Biotinylation by BirA ligase of polypeptides during synthesis does not compromise yields.
6. Purification of products via a biotin tag indicates partial binding to AviTag beads.
Monthly-March 2007:
A. Select and test ORF expression constructs
The results of the preliminary purification experiments indicate that His will be an effective affinity
tag to isolate products, whereas biotin may not (see data below in figure 4 & 5). Therefore we
now favor the design of sandwiching the ORF between both an N and C terminal His tags, to
maximize opportunities for tag exposure necessary for the polypeptide purifications. (Fig 1)
Figure 1
B. Select and test IVT Protocols
1. We have performed IVT reactions with FTU templates, using the combined
Invitrogen/Roche feed system.
2. Products were radiolabeled with 35S-met.
3. We tested the need for supplements of DNA template and/or polymerase during the
29 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
extended reaction times. Gel electrophoresis results and quantification are shown in
Figures 2 and 3, respectfully.
4. We conclude that intervaled supplements of linear template significantly elevated
polypeptide yields, and injection of additional enzyme has little or no real impact.
Figure 2
\\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 3
\\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT
5. IVT’s have been performed in the presence of the BirA enzyme, which biotinylates
the nascent polypeptides while translation is occurring.
C. Select and test protocols for protein purification
1. We performed a test purification of GFP via its His tag, using nickel beads. Products
were TCA precipitated and radioactive counts were measured.
2. The results in figures 4 and 5 show that a specific product of the predicted molecular
weight was obtained from the reaction (lane 1). C-terminal purification of the GFP
polypeptide yielded a full-length purified product.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 4
\\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT
Raw TCE-precipitable counts
Sample
Volume
Specific CPM
Total CPM
%
Total IVT
45
13350
600728
100
Flow through
100
1195
119450
20
Wash
1000
119
118500
20
Eluent
60
2461
147660
25
Unrecovered
(uneluted)
36
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 5
\\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT
3. We scaled-up production of the FTU sub-proteins in the IVT reactions so as to have
amounts of material sufficient for testing several purification schemes as outlined
below in Figure 6.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 6
4. We have prepared a set of lysate reactants, with and without biotinylation, and
performed an initial set of purifications with nickel and Avitag beads.
5. The results in the table below indicate that we are quantitatively resuspending our
acetone precipitated product from the IVT reaction. Affinity tag binding in 8M urea is
quantitative with the Nickel beads but not with the strepavidin beads. Neither
purification system achieved efficient elution as conducted in EDTA and enzymatic
release, respectively. Alternative elution approaches are being pursued
6.
\\peptide\Research\CIM\GeneVac\FTU\Proteome Design\Hetal's data\Hetal IVT
34 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
FTU 901
Resolubility
Resin Binding
Recovery
His-tag
Biotin
53%
58%
3%
71%
12%
3%
100%
77%
2%
100%
5%
2%
84%
88%
3%
100%
15%
4%
91%
1%
59%
18%
9%
79%
36%
2%
79%
14%
8%
FTU 1419
Resolubility
Resin Binding
Recovery
FTU 1602
Resolubility
Resin Binding
Recovery
FTU 1695
Resolubility
Resin Binding
Recovery
FTU 1712
Resolubility
Resin Binding
Recovery
4. Significant decisions made or pending
6 months:
a. We have identified an optimal working template that is linear in form and sufficient for
robust, large-scale production of polypeptides for the full tularemia proteome.
b. We have built a new set of technologies to optimize ORF production and their expression
levels.
c. We have identified an optimal set of IVT production protocols
d. We have initiated purification experiments.
e. Our final selection of purification methods is pending.
1 month:
a. Yield is noticeably improved with additional template in the IVT reactions yet additional
enzyme proves to have only a minor impact.
b. We favor 2 template additions and no enzyme additions to the feed IVT reactions
c. IVT reaction predominantly produces the intended polypeptide product.
d. Recovered product is pure. (as assessed by the gel data in figure 4 for His tagged
product recovered using nickel beads
e. Elution efficiency needs to be improved so as to retrieve the currently unrecovered
portion of the sample, for the His tag and nickel bead purification method.
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 return to using imidizole as our eluent. We will
also perform an experiment to measure the efficiency of the biotinylation reaction. We will also
test the utility of including a His tag at both ends of the molecule, as a means to reduce urea
concentration necessary for binding.
6. Deliverables completed
None
35 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
7. Quality of performance
Very good
8. Percentage completed
90%
9. Work plan for upcoming month and next 6 months
a. Within the next 2 months we will deliver 6 sets of 3 FTU polypeptide preps (18 samples)
to UNM for T cell assay evaluation. Each set will be comprised of the same polypeptide
purified by 2 different affinity methods and unpurified sample.
b. Our strategy for improving elution is to change from using EDTA for His nickel elution to
using a pH shift. We will determine which step is compromised in the biotin scheme. The
most likely losses are at the 2 enzymatic steps: BirA ligation and TEV cleavage. These
will be tested. We will conduct a set of experiments to determine which of these steps is
inefficient. If it is the biotinylation step, we will test performing the birA ligation reaction
subsequent to the IVT, so as to optimize reaction conditions. If it is the TEV cleavage
step, we will test adding a spacer sequence to the template between the BAP and TEV
sites.
c. We will be sending UNM between 5 and 6 FTU purified polypeptide preps within
1 to 2 months. We look forward to working with them on selecting a final protocol
for proteome preparation.
d. Our goals for the next 6 months are to complete Milestone 26, confer with UNM
on their work on Milestone 27, and begin generating both WT and synthetic
ORFs for the library.
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
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. Identification of stimulatory peptides derived from F. tularensis proteins
i. The purpose was to identify peptides that will stimulate proliferation of T cells
formless vaccinated BALB/c
ii. We are working with a peptide library synthesized by Alta Bioscience for ASU
that contains most of the peptides from F. tularensis that match the H-2d
MHCI and MHCII binding motifs
iii. Using a limited set of the peptides in the T cell proliferation assay we
developed for milestone 12, we found that
1. Peptides or the solvent in which they are dissolved suppressed Con
A-stimulated T cell proliferation. However, the suppressive effect
was eliminated when the peptide concentration was reduced to 50
g/well or less
2. Peptides did not have mitogenic activities and did not stimulate
proliferation of naïve T cells
iv. We have not identified a stimulatory peptide yet
36 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Monthly: March 2007
a. Experiment Ftc27 study 9 (Notebook 94 pages 158-160) and Experiment Ftc27 study
10 (Notebook 94, pages 168-170)
i. The purpose was to determine the optimal peptide concentration to use in
the T cell proliferation assay.
ii. We had observed previously that 50 g/ml peptides suppressed Con Astimulated proliferation of naïve splenocytes. Therefore, we repeated this
experiment titrating peptide concentration from 50 g/ml to 0.5 g/ml
iii. We observed peptide dose-dependent suppression of Con A-stimulated
proliferation of naïve splenocytes (Fig 2). As previously observed, 50 g/ml
peptide completely suppressed Con A-stimulated proliferation. Proliferation
was restored when the peptide concentration was reduced to 5 g/ml and the
signal intensity was reduced at 0.5 g/ml. Thus, the optimal concentration of
peptide is 5 g/ml.
Figure 2. Peptides suppress Con A-stimulated proliferation of naïve splenocytes in a
dose-dependent manner. 5 x 104 nylon wool-enriched, naive splenocytes were
stimulated with 5 g/ml Con A in the presence or absence of peptides at the indicated
concentration. The suppression caused by two representative peptides is shown.
b. Experiment Ftc27 study 11 (Notebook 94, pages 171-173)
i. The purpose is to identify peptides that are recognized specifically by
splenocytes from vaccinated BALB/c mice and that stimulate proliferation
ii. We individually tested 23 peptides using the conditions described above and
none of them stimulated splenocytes proliferation
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
37 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
6. Deliverables completed
None
7. Quality of performance
No progress
8. Percentage completed
10%
9. Work plan for upcoming month
a. Test all 600 peptides for ability to stimulate proliferation of splenocytes from
vaccinated BALB/c mice
i. Splenocytes from vaccinated and unvaccinated BALB/c mice will be used to
demonstrate antigen-specific response
ii. Dan Clemens reported that splenocytes from BALB/c mice infected i.d. with
LVS proliferated and produced IFN when stimulated with recombinant Bfr,
KatG, and GroEL. We found peptides from these proteins in the ASU
peptide library, however, we do not know if they are the stimulatory peptides
b. Assemble a list of stimulatory peptides for ASU to analyze for common stimulatory
motifs
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of peptide libraries (Optimize IVT protein-fragment
production, Develop IVT protocol for high-throughput production, Validate immunogenecity
of protein-fragments, Full scale production of protein-fragment library, Purification of proteinfragment library, Array protein-fragment into overlapping pools, Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome
 Generate complete protein-fragment library (inactive)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual 10/1/06 to 3/30/07
Inactive
Monthly-March 2007:
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
3. We have begun amplifying ORFs that were identified as having adequate natural sequence for
38 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
production by IVT.
4. 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.
Figure 7
\\peptide\Research\CIM\Mol_Biol_Tech\IVT
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
We will proceed with the remaining 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
6%
9. Work plan for upcoming month and next 6 months
In the next month, several more plates will be done.
We will also address the purification challenges as described above.
In the next 6 months, 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
11. Upcoming Contract Authorization (COA) for subcontractors
None
39 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
MILESTONE 32
Milestone description: Oligos list refined, 70mer oligos procured, GDP oligo defined. Will be
based on annotated SCHU S4 genome.
Electronic list of oligos generated from annotated F. tularensis SCHU S4 sequence that will be
used to produce microarrays for analyzing F. tularensis SCHU S4 transcriptome. Genome
Directed Primers (GDPs) were designed to amplify the gene segment that would encompass
the 70mer probe.
Institution: ASU/Johnston
1. Date started: 3/02/2006
2. Date completed: 08/28/2006
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS




1804 probes for all annotated coding sequences were designed and produced
The GDP (Genome Directed Primer) program was used to create a set of 183, 7mer
primers for amplification of the SCHU S4 genome in the context of murine tissue.
We added to this milestone the production of LVS specific probes that are not detected
by the SCHU S4 Probe set (298) new probes and GDPs for amplification.
While technically the milestone is complete. the remaining probes and GDPs need to be
received
Data files are stored for…
70mers - R:/Genevac/FTU/Contract/Microarray/Milestones/32/FTU_Microarray_Probe_Order.xls
GDPs- R:/Genevac/FTU/Contract/Microarray/Milestones/32/FTT_GDP_7-TTTT.xls
LVS 70mers- Genevac/FTU/Contract/Microarray/Milestones/32/LVS_Probes.xls
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
 A set of 1804, 70mer probes designed to cover entire transcriptome is available

GDP primers have been predicted and obtained.
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month and next 6 months
Receive LVS Probes and GDPs. With the receipt of these last probes the extended milestone will
be closed. The probes and primers will be tested and confirmed in milestone 33.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
40 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS ONLY


Array printing conditions and probe dilution conditions determined and confirmed.
We have compared Poly-L-Lysine (PLL) vs. Corning Ultragaps slides as substrate and shown
that PLL slides perform equally, if not better, than Ultragaps with purified RNA
 Initial processing of RNA from both in vitro grown SCHU S4 and LVS have been run on
multiple arrays with initial lists of differentially expressed genes identified.
 We have gotten approval to obtain Francisella arrays from TIGR and are processing the
material transfer agreement to obtain slides
Monthly -March Report
 GDP amplifications have been performed using GDP primers and purified SCHU S4 RNA
serially 10-fold diluted from 1000ng to 1 ng (results shown in Table 1). The yields from 10
and 1 ng were lower than expected. The reason for the inability of 1000 ng samples to
amplify is not clear but the reduced amplification at high levels of input RNA have been seen
in the past. There may be a problem of stoichiometry between input RNA and primer
concentration.
Input
RNA Obtained (g)
Fold Increase
1000 ng
6
6
100 ng
80.3
803
10 ng
6.8
680
1 ng
2.15
2150
Table 1. Amplification of SCHU S4 RNA using GDP Primers.

The amplified RNA was labeled with Alexa dyes and used to probe the Tularemia genome
arrays. Histogram analysis of the raw microarray intensity values were plotted to reveal
distribution of signals over the array and reveals that the amplified signals have a similar
distribution to signals of unamplified RNA (Figure 1.) This equivalent distribution between
amplified and unamplified samples indicates that there is not a bias of a subset of genes
being preferentially amplified. This provides confidence that the amplification occurs across
the complete gene set and the results should be representative of the input ratios.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Raw Intesity Values on Microarray
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
100 ng
1000 ng
LAPT Amplified
Unamplified
Figure 1. Histogram distribution of raw microarray intensities of LAPT amplified RNA (0.1 an
1.0 g as compared to unamplified SCHU S4 RNA
Files are located at
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-1
R:\GeneVac\FTU\Contract\Microarray\Milestones\33\LAPT-2
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
ng SCHU S4
1000
1000
100
100
10
10
1
1
g Mouse
Lung RNA
Spearman
0.609
10
10
10
10
0.658
0.626
0.585
0.593
nd
0.576
Table 2. Spearman correlation of normalized intensity microarray hybridization values of
diluted SCHU S4 RNA (with and without mouse lung RNA) to unamplifed SCHU S4 RNA.
Nd= not determined. Our experience with other LAPT projects of Yersina pestis and
Mycobacterium tuberculosis reveal that a Spearman correlation greater than 0.50 is the
lowest acceptable value of amplified to unamplified ratios. This is an important factor for the
validation of the GDP’s to ensure the LAPT process maintains relative gene expression levels
Significant decisions made or pending.
The initial amplifications reveal that the LAPT process is working as expected. We
will be performing additional LAPT processes and testing the hybridizations, with and
without mouse lung RNA as an experimental reconstruction, on both the poly-LLysine and Corning Ultragaps slides to ensure the selection of substrate is adequate.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Printer and speedvac malfunctions addressed and equipment in good working order.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
75%
9. Work plan for upcoming month and next 6 months



GOALS FOR THE NEXT MONTH
We will perform amplifications of serially diluted microbial RNA admixed with mouse lung
RNA using genomic DNA normalization techniques.
Finalize MTA to obtain TIGR slides and perform array comparisons between ASU and TIGR
arrays.
With the completion of these goals this milestone will close within the next 4-6 weeks.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
43 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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
Semi-Annual: 10/1/06 to 3/30/07 SUMMARY HIGHLIGHTS
a. Isolation of RNA from LVS and SCHU S4
i. We provide ASU with RNA from LVS and SCHU S4 so they can optimize
microarray hybridization conditions
1. The yield is low, approximately 50 g/1010 bacteria
2. RNA isolated using Ambion RiboPure and Qiagen RNeasy Midi
columns produced similar yield and quality
3. Fresh culture and bacteria stored in RNAlater produced similar yield
and quality
ii. We also provided ASU with RNA from the lungs of BALB/c mice infected with
SCHU S4 so they can optimize their procedure for detecting SCHU S4 gene
expression in the presence of a relative excess of mouse RNA
b.
i.
ii.
c. Experiment Ftc35 (Notebook 94, in progress)
i. The purpose was to isolate RNA from the lungs of BALB/c mice infected with
SCHU S4. ASU will use this mixture of prokaryotic and eukaryotic RNAs to
optimize their procedure to detect SCHU S4 gene expression in the presence
of a relative excess of mouse organ RNA
ii. BALB/c mice were infected i.n. with 500 SCHU S4
iii. 3 days after infection, 2 infected mice were killed to determine the bacterial
burden in the lungs and 5 infected mice were killed to harvest lungs for RNA
isolation
iv. There were 7 x 107 CFU SCHU S4 in the lungs 3 days after infection
v. Each lung was homogenized in 5 ml Tri-reagent (Molecular Research
Center) and RNA isolated immediately following manufacturers protocol
Monthly: March 2007
a. No work done on this milestone
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
10%
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
9. Work plan for upcoming month and next 6 months
UNM will isolate RNAs from LVS, SCHU S4, and infected mouse organs, as needed by ASU.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 34-ASU
Milestone description: Pilot studies for optimization of RNA isolation & hybridization
conditions done
Institution: UNM/ASU-Johnston
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS


Initial hybridization studies have been performed using standard slide-coverslip
hybridizations.
RNA has been isolated from normal mouse lung for dilutions studies of SCHU S4 RNA and
GDP testing.
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
We are waiting on the validation of the LPT and GDPs before starting on the amplification of the
samples we have received
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
11%
9. Work plan for upcoming month and next 6 months



We will perform comparisons of hybridizations of microbial RNA diluted into RNA from normal
mouse lung on both Poly-L-Lysine and Corning Ultragaps before final substrate decision.
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. We will evaluate signal to noise ratios, overall signal intensities,
and the ability to detect low-level signals as the assessment criteria
It is estimated that this milestone will be completed by 05/31/2007.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
45 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS
a. Because Francisella tularensis is highly infectious, one concern about using a KBMA vaccine is
that a single organism that escapes inactivation could potentially cause fatal disease. Thus we
would ideally prefer to use a KBMA vaccine platform strain that was attenuated for virulence. In
some organisms (e.g. Mycobacterium tuberculosis) it has been demonstrated that mutations in
nucleotide excision repair (NER) genes can result in severe attenuation of virulence. We
therefore wanted to determine whether the NER-deficient mutants of Francisella tularensis ssp.
novicida are attenuated for growth or virulence in vitro, in macrophages, and in mice.
b. Growth characterization of NER mutants
I.
uvrB and uvrA single and the uvrA uvrB double mutant Ft novicida strains
grow at the same rate in Chamberlain’s defined medium (CDM) as the wildtype U112 strain.
II.
uvrB and uvrA single and the uvrA uvrB double mutant Ft novicida strains
have no growth defect in J774 macrophages
III.
All Ft novicida NER mutant strains replicated at the same rate or more rapidly
than the wild-type strain in lungs, livers and spleens of Balb/c mice following
intravenous (IV) injection.
c. Virulence characterization of NER mutants
I.
uvrB and uvrA single and the uvrA uvrB double mutant Ft novicida strains
were fully virulent in Balb/c mice when administered by the intraperitoneal (IP)
route of administration. LD50 range was ~0.5 cfu to 3 cfu.
II.
All Ft novicida strains were highly virulent by the IV route, but LD50 values ranged
from 1 for wild type to 38 cfu for the uvrA uvrB double mutant suggesting that
the NER mutants may be slightly attenuated by this route.
III.
The subcutaneous (SC) route of infection is the route by which Francisella strains
are the least virulent (suggesting that dissemination from this site may represent
a significant barrier). All Ft novicida NER mutants were approximately 1 log
reduced in virulence compared to U112 when administered SC.
Monthly-March 2007:
The SC LD50 of U112, uvrB and uvrA single and the uvrA uvrB double mutant Ft novicida
strains was repeated (AS07-039). 10-fold serial dilutions ranging from 1x107 to 10 cfu were
administered to 3 animals per group. The LD50 of U112 was 5.26 x 103 which is similar to the first
LD50 determination 1.17 x103 and confirms that the second LD50 data were indeed spurious.
Each of the NER mutants had similar virulence ranging from 1.25 x104 to 3.25 x104. These data
suggest that the abrogation of the nucleotide excision repair machinery causes a slight virulence
attenuation when the bacteria are administered SC.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
IV LD50 (cfu)
IP LD50 (cfu)
SC LD50 (cfu)
SC LD50 (cfu)
SC LD50 (cfu)
U112
0.95
0.57
1.17 x 103
2.10 x 105
5.26 x 103
uvrA
27
0.82
ND
2.43 x 105
3.25 x 104
uvrB
8.1
0.2
1.28 x 105
1.17 x 105
1.25 x 104
uvrAuvrB
38
2.72
ND
3.48 x 104
2.46 x 104
Study #
AS06-112
AS06-090
AS07-014
AS07-025
AS07-039
4. Significant decisions made or pending
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
LD50 comparison between Ft novicida wild type and Ft novicida uvrA, uvrB, and uvrAuvrB
mutants administered by the IP, IV, and SC routes have been completed.
7. Quality of performance
Excellent progress
8. Percentage completed
95%
9. Work plan for upcoming month and next 6 months
a. During the next month, we will monitor the growth of Ft novicida NER mutants 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.
b. We expect to complete this milestone by the end of April and therefore expect file a
milestone completion report within approximately 2 months.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 41
Milestone description: Optimization of photochemical inactivation and characterization of
KBMA Ft. novicida; determine the amount of S-59 and UVA required to inactivate uvr mutants;
determine extent of metabolic activity of uvr mutants after S-59 and UVA inactivation; determine
the level of virulence attenuation of KBMA uvr strains in mice
Institution: Cerus
1. Date started: 3/2/06
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS
a. In order to select a NER-deficient background for development of KBMA tularemia
vaccines, we screened 3 mutants that should all be NER deficient: uvrB and uvrA single
and the uvrA uvrB double mutant Ft novicida strains for sensitivity to photochemical
inactivation with S-59 psoralen and UVA light. We optimized photochemical inactivation
47 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
conditions at a 3.5 mL scale, determined the level of sensitivity of NER mutants to
photochemical inactivation, and measured the degree of metabolic activity of each strain after
photochemical inactivation.
i. Determined that minimum S-59 concentration required to inactivate ~1 x 1010 cfu
of U112 was 40M while 20 M S-59 was required for inactivation of uvrA, uvrB, and
uvrAuvrB mutant strains of Ft. novicida.
ii. 4 J/cm2 was the minimum dose of UVA required to achieve consistent inactivation
iii. Metabolic activity profiles of all strains were similar
iv. Selected uvrB for further evaluation because it was equivalent to the other NER
mutants and we had the most experience with this strain as it was the first mutation
constructed
b. Optimized 400 mL-scale inactivation conditions for uvrB for stability studies and virulence
determination
i. 40M S-59 + 7 J/cm2 UVA were used to achieve inactivation 5x1010 cfu/mL
ii. Sterile lots produced that have metabolic activity
iii. MTS activity is stable at –80oC for greater than 2 months
iv. KBMA uvrB Ft. novicida are highly attenuated for virulence >8 logs IP, ~8 logs IV, ~4
logs SC
Monthly-March 2007:
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 2 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.
OD (490nm)
Lot 948-202 Arm-2 (Nominal 1.93e8 cfu/mL)
1.90
1.70
1.50
1.30
1.10
0.90
0.70
0.50
0.30
0.10
-0.10
T=2 weeks
T=1 Month
T=2 Months
0
1
2
3
4
5
6
7
8
9
10
11
12
Time (hours)
NB968-072
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
48 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Ft novicida; however, the high degree of metabolic activity retained by the mutant and wildtype 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 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
65% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
a. over the next month: stability at -80o C of lot 948-202 arm-2 KBMA urvB Ft novicida will
be monitored by MTS assay. We will compare the sensitivity of uvrB and U112 to
numerous DNA-damaging agents.
b. Over the next six months we will: investigate whether the NER machinery functions in Ft
novicida and is induced in response to DNA damage and we will attempt to devise a more
scalable inactivation process to facilitate 3-L scale production.
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
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS
a. Using KBMA Ft novicida uvrB vaccine stocks produced in MS41, we have demonstrated
that mice can be protected from 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 then demonstrated that we can achieve 100% protection from a 100 x IP LD50 challenge
by administration of 1 x 107 KBMA particles IV if the vaccine was given twice separated by 3
weeks. These data demonstrate that KBMA Ft novicida can protect against a lethal Ft
novicida challenge.
b. We have recently demonstrated that the mechanism of protection elicited by KBMA Ft
novicida is independent of CD4 and CD8 positive T cells, and that protection was also
observed with heat-killed Ft novicida raising the concern that the protection was due to a
humoral response. We will perform adoptive transfer studies of serum from vaccinated
animals to confirm this.
49 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Monthly-March 2007:
a. AS07-017: 40 Balb/c mice were vaccinated IV with 1 x 107 KBMA uvrB Ft novicida and 10
mice were vaccinated with buffer. 3 weeks later all animals were boosted with the same. 4
weeks after boost vaccination 10 animals per group were injected IP with antibodies to
deplete CD4+ cells, CD8+ cells or CD4+ and CD8+ cells. Animals were depleted of T cell
populations for 3 consecutive days prior to a 100 x IP LD50 Ft novicida challenge. All the
mice in the buffer control group died, but all 40 KBMA vaccinated animals survived the
challenge regardless of whether they had T cell populations depleted. These data suggest
that neither CD4+ nor CD8+ T cells are required for protective immunity against a lethal Ft
novicida challenge. The surviving animals from this study all received terminal heart
punctures to collect blood for adoptive transfer of serum.
Vaccination
HBSS
KBMA uvrB
Survival after 100 x IP LD50 challenge with U112
No Depletion
CD4
CD8
0%
100%
100%
100%
CD4 + CD8
100%
AS07-017
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The KBMA uvrB Ft. novicida vaccine was only 100% protective 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
10% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
a. This month we will adoptively transfer serum from KBMA-vaccinated animals that survived
a lethal challenge with Ft novicida to demonstrate that they have levels of antibodies that are
high enough to protect against a subsequent lethal Ft novicida challenge. We will also
develop an anti-Ft novicida ELISA to measure the amount of humoral immunity elicited
against Ft novicida and whether the antibody titer correlates with protection.
b. Over the next 6 months, we will investigate various vaccination routes and times between
prime and boost vaccination to establish an optimized vaccination regimen.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
50 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Milestone 43
Milestone description: Construct 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 counter-selectable 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 contains 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, through counter-selection on plates containing sucrose, and result in the generation of the
uvrB mutation in LVS.
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY HIGHLIGHTS
a. Modified mutagenesis vector pDS132 to: 1. Contain more useful restriction sites in the multiple cloning
site (MCS), 2. Contain Ft groELp promoter to drive sacB and CmR expression, this was renamed
pKEK1090
b. Introduced uvrB::Kan (previously cloned, also contains ermC) into pKEK1090, renamed pKEK1114
c. Conjugated PKEK1114 into LVS at 1: 10 ratio, isolated cointegrants by selection for CmR, PCR
indicated these were correct cointegrants.
d. Counterselection for second recombination attempted on TSA+++ 5% sucrose (+ Kan), this did not
result in loss of cointegrated plasmid
e. Several different media formulations were tested with 5% sucrose, but none allowed growth of LVS
f. Prepared TSA+++ with 10% sucrose to 10% final concentration, LVS grew on this medium.
g. LVS with cointegrated uvrB::Kan plasmid grown on TSA+++10% sucrose, single colonies isolated.
Monthly- March 2007
a. Made TSA+++/CM plates containing the same components as above, and 10ug/ml Chloramphenicol
(CM) was added.
b. Patched the colonies from TSA+++/Kan/10%Sucrose plate onto both TSA+++/Kan plates and
TSA+++/CM plates. After overnight incubation, colonies grew on TSA+++/Kan plates but no colonies
were detected on TSA+++/CM plates even if incubation was extended for one additional day. This is the
expected phenotype of strain that has lost plasmid backbone to become chloramphenical sensitive(CmS),
but has retained uvrB::Kan mutation (KanR) insertion.
c. Picked up colonies from TSA+++/Kan plate and grew in TSB+++/Kan liquid medium for overnight.
Performed chromosomal DNA purification with Invitrogen Easy-DNA kit.
d. Performed PCR with UvrBLvsPstlUp and UvrBLvsPstlDn1 primers for chromosomal DNA. Gel picture
in figure1 below.
51 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
1
2
3
4
5
6
7
8
9
1.Marker(1kb ladder)
2.UvrBFpKan U112
3.Wild type LVS
4.sample 6 (1:10 dilution)
5.sample 7 (1:10 dilution)
6.sample 8 (1:10 dilution)
7.sample 9 (1:10 dilution)
8.sample 6 (1:100 dilution)
9.Marker (1kb ladder)
Figure 1: Comparing PCR products derived from the potential uvrB LVS mutants (lanes 4-8) to LVS
parent (lane 3) with primers flanking potential Kan insertion site. The potential mutants amplified smaller
PCR products, as expected, due to the slightly smaller size of mutant insert (Kan-ermC) vs. full sized
uvrB gene in LVS parent.
Data in TVD UTSA notebook 2, page 67
Note: The samples were diluted with water because the concentration of the purified genomic DNA was
too high to perform PCR successfully. Sample 6 was diluted at 1:10 and 1:100.
e. Gel purified the PCR products from above with QIAquick gel extraction kit. Recovered gel DNA to be
digested with Bgl2 restriction enzyme and sent for sequencing to confirm the presence of the uvrB
mutation in LVS.
f. Performed PCR with SacBCDSDnBgl2 and SacBPUpBamHI primers for chromosomal DNA to make
sure that plasmid backbone has been removed. If the SacB gene marker has been removed, then the
plasmid backbone has been removed
Gel picture: Data in TVD UTSA notebook 2, page 68.
1
2
3
4
5
6
7
8
1.Marker
2.PuvD442
3.sample 6 (1:10)
4.sample 7 (1:10)
5.sample 8 (1:10)
6.sample 9 (1:10)
7.sample 6 (1:100)
8.marker
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Figure 2: This PCR indicates lack of plasmid marker (sacB) in potential mutants (lanes 3-7) vs. control
sacB plasmid (lane 2), as expected if these strains have lost plasmid backbone.
g. Digested gel purified DNA from uvrB PCR products shown above in Figure 1 with Bgl2 restriction
enzyme.
Gel picture: Data in TVD UTSA notebook 2, page 67
1 2
3 4
5
6 7
1.Marker
2.UvrBKanU112 cut
3.UvrBKanU112 uncut
4.LVS cut
5.Sample 6 cut
6.Sample 6 uncut
7.Marker
Figure 3: The Kan insertion in uvrB contains a Bgl II site introduced during construction of the mutation;
this site is absent from this region in the wildtype LVS strain. When LVS uvrB region is amplified and
digested with BglII (lane 4), this enzyme does not cut the fragment. When uvrB region from one of the
candidate uvrB mutant isolates shown above in figure 1, #6, is amplified, it produces a fragment slightly
smaller than the LVS parent strain (lane 6), but when this fragment is digested with BglII, it is cleaved into
two fragments (lane 5), as expected for the correct mutant strain. Control lanes 1&2 show that our
previously constructed uvrB mutant in Ft novicida also had a Bgl II site introduced into the chromosome.
Cumulatively these results indicate that the LVS mutant isolates have the uvrB::KanErm mutation. Thus
a uvrB LVS mutant has been generated successfully.
h. Sent gel purified DNA for sequencing, sequencing scheduled to be received on 4/2/07.
4. Significant decisions made or pending
a. We have created UvrB mutant in LVS strain and removed plasmid
backbone.
b. UvrAFpKan has been made in plasmid PKEK1120 which needs to
be mated to LVS, and get rid of the backbone.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. pKEK1190(contains GroEL promoter)
b. pKEK1006(contains UvrB FpKan fragment)
c. pKEK1114(contains UvrBFpKan in PKEK1090) resulted in the uvrB mutant in LVS
d. uvrB LVS mutant
7. Quality of performance
Good
53 of 70
Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
8. Percentage completed
Approximate 55% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
next month plan:
UvrAFpKan will be mated in LVS and the backbone must be removed.
Two months’ plan:
UvrA::Kan LVS mutant identified and verified. (this milestone is scheduled to finish 5/31/07)
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
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS
a. 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 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 been monitoring the stability of the stored product
in 2 cryopreservation medias. We have found that the LVS provided by DVC is greatly
attenuated for virulence in mice when administered IP compared to literature reports. 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.
Monthly-March 2007:
a. The stability of live fermentor-grown LVS at -80o C in 2 different freezing solutions is being
monitored on a monthly basis. The titer of each cryopreserved stock was determined by
making duplicate dilution series and plating 100 ul of each of the 10-7 dilution on 5 CHAH
plates, hence each bar in the graph represents the mean of 10 plate counts and error bars
represent the standard deviation. After 4 months at -80o C, both samples showed no
significant decrease in titer and both cryopreservation solutions appear to be performing
equally well.
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5.85E+09
3.19E+09
Pre-Freeze
8.40E+08
4.47E+09
2.13E+09
4.47E+09
9.28E+08
3.87E+09
9.43E+09
4.89E+09
2.88E+09
CFU/mL
1.00E+10
Viability Count (CFU/mL) for Pre-Freeze, T=0, T=1, T=2, T=3 and T=4 Month
2.73E+09
1.00E+11
T=0
T=1 Month
T=2 Months
T=3 Months
T=4 Months
1.00E+09
1.00E+08
Lot: 948-119 Arm-1 (Freeze Buffer)
Lot: 948-119 Arm-2 (10% Sucrose)
DVC F. tualrensis holarctica LVS
NB968-061
OD (490nm)
The metabolic activity of the cryopreserved LVS was also evaluated after 4 months using the
MTS assay. There was no significant decrease in metabolic activity of either lot after 4
months. There is a trend that Arm-1 has slightly more metabolic activity than arm-2, but this
most likely reflects the slightly higher titer of arm 1 at each timepoint, rather than a difference
resulting from the storage conditions We will continue to monitor the stability of lot 948-119
LVS samples by plating for cfu and by MTS assay.
Nominal 2.22e7 cfu/mL
1.00
0.90
0.80
0.70
0.60
0.50
0.40
0.30
0.20
0.10
0.00
T=0 Arm-1 (Freeze Buffer)
T=0 Arm-2 (10% Sucrose)
T=1 Month Arm-1 (Freeze Buffer)
T=1 Month Arm-2 (10% Sucrose)
T=2 Month Arm-1 (Freeze Buffer)
T=2 Month Arm-2 (10% Sucrose)
T=3 Month Arm-1 (Freeze Buffer)
T=3 Month Arm-2 (10% Sucrose)
T=4 Month Arm-1 (Freeze Buffer)
T=4 Month Arm-2 (10% Sucrose)
0
2
4
6
Time (hours)
8
10
12
NB968-061
b. The stability of two KBMA LVS Lots is being monitored on a monthly basis by MTS assay.
Both lots have comparable metabolic activity and are stable for 1 month at –80oC
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Nominal 1e8 particle/mL (KBMA) F. tularensis holarctica LVS
1.2
T=0 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=0 968-040 Arm-2 (20uM S-59, 6J/cm2 UVA)
T=1 968-040 Arm-1 (10uM S-59, 6J/cm2 UVA)
T=1 968-040 Arm-2 (20uM S-59, 6J/cm2 UVA)
OD (490nm)
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5 Time (hours)
6
7
8
9
10
11
12
NB968-072
4. Significant decisions made or pending
Because wt Ft novicida is inactivated with S-59 concentrations that are only slightly higher than
wild type we have decided to explore the possibility that a wild-type KBMA LVS vaccine can
provide protective immunity. We have produced 400mL lots of photochemically treated LVS that
have a high degree of metabolic activity. We will use these lots to vaccinate animals to determine
whether they are protected against a lethal LVS challenge.
5. Problems or concerns and strategies to address
The LVS provided by DVC appears to be highly attenuated when administered IP, the degree of
attenuation is less pronounced when LVS is expanded in CDM. This is not likely to be a major
problem as IP is an unlikely route of administration for vaccines.
6. Deliverables completed
None
7. Quality of performance
Good progress
8. Percentage completed
35% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
a. This month we will monitor the stability of live LVS stored at -80oC in various
cryopreservation agents by MTS assay and plating on CHAH plates. We will also measure
the stability of KBMA LVS stored at -80oC in 10% sucrose by MTS assay. We will compare
the virulence of live LVS stored in the various cryopreservation agents by injecting IV into
BALB/c mice. We will measure the reduction in virulence of KBMA LVS administered IV
compared to Live LVS.
b. Over the next six months we will continue to measure the stability of live and KBMA LVS.
We will demonstrate the degree to which KBMA LVS is attenuated and we will perform
protection experiments with KBMA wild-type LVS. We expect to receive a uvrB mutant LVS
from UTSA and we will measure its sensitivity to photochemical inactivation and degree of
metabolic activity and compare the NER-deficient strain with wild-type LVS. We will also
establish purification methods for LVS using tangential flow filtration (TFF).
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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
Semi-Annual: 10/1/06 to 3/30/07—SUMMARY HIGHLIGHTS
I. Cloning:
a. In order to generate mutants in SCHU S4, we need to develop tools to generate
successful deletions. Our first desired deletion for the project is igLC therefore, we
are trying to clone this gene into a plasmid to facilitate gene deletion into the
chromosome of SCHU S4. We started experiments to clone the iglC deletion (≈3000
bp fragment) into pDS132 plasmid which is a suicide vector derived from pCVD422.
This cloning was not successful.
b. We tried a modified version of pDS132 which was described in milestone 43
November 2006 report. The multiple cloning site was changed and the Sac B
promoter was replaced with the GroEL promoter (designated as pKEK1090). It was
used in experiments with LVS and we were able to get a uvrB deletion into the LVS
chromosome. If we are able to clone iglC into this vector, the vector probably will
work to delete the iglC gene in SCHU S4. To date we have not been able to clone
the iglC deletion in this plasmid.
c. In another approach, a construct (pKEK964 based) that was used in a SCHU S4
deletion experiment proved to work to get the iglC deletion into the chromosome;
however, we were unable to remove the plasmid from the SCHU S4 chromosome.
Therefore, we cloned SacB into pKEK964, designated pKEK1133 (Bgl II/BHI) and
pKEK1134 (BHI/XbaI). These two versions of the plasmid differ only in the way the
Sac B gene was cloned into the plasmid. In pKEK1133, we cloned in front of the
Kanamycin gene via Bgl II/ BamHI sites and in the pKEK1134, we cloned via by Bgl II
/ Xba I sites. We are currently trying to clone the iglC deletion into this plasmid
(pKEK1133).
II. Experiments to generate deletions in SCHU S4
Our first goal was to generate the IgLC deletion into SCHU S4; however, since we did not
have a mating plasmid that will allow test experiments we decided to start with the MglA
gene for proof of principle. For the MglA, we had a construct in the lab that allowed us to
test parameters that may work in SCHU S4 once we do generate the iglC deletion
construct. This MglA gene is useful as a test gene because there is only one gene copy
in the Francisella chromosome as compared to two gene copies for iglC. With only one
MglA gene present, we can more easily test the frequency of recombination and removal
of the mated or transformed plasmid from the chromosome.
a. cloned sacB into pKEK1023, the MglA deletion construct, to facilitate
counterselection necessary for mutant strain construction. Simultaneously we cloned
sacB into the parent vector pKEK964, which lacks
based containing the Francisella promoter (Fn) driving a kanamycin gene. The sacB
gene was cloned downstream of the kanamycin gene. The MglA deletion was
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subsequently cloned into pKEK964, resulting in pKEK1113. pKEK1113 was
transformed into Schuh4, And we obtained 12 colonies that were kanamycin (70
ug/ml) resistant from this experiment. These 12 clones were then grown in Tryptic
Soy Broth (4 cycles) and plated on 5% sucrose TSA ( without salt) plates to select for
colonies that had lost the plasmid due to a second recombination event (SacB
counter-selection). Out of 30 sucrose resistant colonies screened, we found one
colony that appears to have the correct
Monthly-March 2007
I. Cloning of igLC:
a. Transformation of pKEK1090+iglC deletion into DH5αλpir yielded only two colonies.
Plasmids were isolated from these colonies and screened by PCR with iglC gene specific
oligos; none of the clones contained the iglC deletion based on PCR results. However, a
DNA fragment was cloned into the plasmid, based on the restriction profile which differed
from the parent plasmid pKEK1090; however this fragment is smaller than the anticipated
size of
acquiring spontaneous deletions, as we suspect. .
b. Continued with the cloning of the iglC deletion into the plasmid(s) pKEK1133 (SacBBglI/BHI) and pKEK1134 (SacB-BHI/XbaI). The constructs are both pUCFnKan+SacB
and the difference between the two is the sites used to clone in the Sac B gene.
Prepared a total of 20 plasmid preparations from the pKEK1133+iglC transformants and
17 from the 1134+iglC transformants.
c. All of the plasmids were digested with EcoRI and compared to their parent plasmid.
Since the iglC deletion has an EcoR I site at the 5’ end at position 526 and the insertion
has two possible orientations, we expected to see the SacB gene fragment of 1265
resulting from the vector alone being digested with EcoRI to shift up by either 526 bp
(1791 bp expected size) or by 2481 bp (3736 bp expected size). Some of the screened
plasmids did show this shift; however, when I screened some of these plasmids by PCR
with iglC gene specific oligo primers, I did not generate the deletion band (Figure 1).
Figure 1: This represents PCR products (lanes2-10 and12-23) generated by using the indicated
DNA templates in legend with iglC gene specific oligo pcr primers (iglC schu4 up NEW Sal I and
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iglC schu4 down Sal I). Lanes 3 and 13 are the positive control plasmid pKEK906 generating the
≈3000 bp iglC deletion (diglC) fragment. Lanes 2 and 12 are the plasmids (pUCFnKan+SacB
BglII/BHI –1133 and pUCFnKan+SacB BHI/XbaI -1134) which we are using to clone in the diglC
fragment. Notice that there are non-specific products generated; however, it is not the diglC
fragment. Lanes 2 – 10 are possible pKEK1134 plasmid containing the diglC fragment; and lanes
14 – 23 are possible pKEK1133 plasmid containing the diglC fragment. Unfortunately, none of
the screened plasmids indicated that they contain the diglC fragment. Data located in TVD UTSA
Notebook 3, page 67.
d. I took a few of these plasmids and used only the 3′-end iglC oligos used to make the
original iglC deletion and ran a PCR screen. The controls worked fine; however, I did not
generate any products in any of the plasmids tested (Data not shown).
e. I have more transformants to screen in these test sets. I will also set up Sal I digestions
to determine the size of the inserted fragment that is resulting from this ligation/transformation
set. I will present these results on next report.
f. Data located in TVD UTSA Notebook 3, pages 64-67 and 77,
II.
Experiments to generate deletions in Schu4:
a. The
-containing plasmid pKEK1113 was previously transformed into SCHU S4
which resulted in 12 kanamycin (Kan) resistant colonies. Seven of these clearly
showed the MglA deletion present (K2, K3, K5, K7, K11, K12 and K13). Therefore,
we prepared colony picks of K2, K3, K5 and K7 groups for PCR screening from the
patched sucrose colonies reported earlier. Seven of each of these groups were used
to run PCR with the MglA gene specific oligos used in last report.
b. The basic protocol for the “colony pick” is that a sterile pipette tip is used and touched
to the colony patch of interest and then this tip is submerged in 100 ul of sterile water
and shaken well. The small amount of cells left in the water is frozen on dry ice for 5
minutes then thawed in a 37°C water bath for 5 minutes; this freeze/thaw is done
three times to lyse the bacterial cells. Then these cells are boiled for 5 minutes and
followed by centrifuging for 5 minutes. Then 90 ul of this liquid is transferred to a
sterile microcentrifuge tube and frozen until use. This provides a source of the
chromosomal DNA from the colonies, which can be used in PCR or in restriction
digests.
c. The results of the first set of sucrose resistant Kan sensitive colonies showed that the
plasmid likely resolved in 29 of the 30 colonies screened (See figure 2). We still
need to run a screen for the Kan gene or pUC origin using the gene specific oligos to
confirm that the plasmid has been removed from the chromosome. However, based
on the MglA oligo primers used, these 29 colonies lost the plasmid and reverted back
to the wild type (i.e. no mglA deletion). The one colony that still showed the MglA
deletion maintained the wild type gene profile as well. It is possible that this may be
a mixed colony containing a complete deletion colony and a reverted wild type colony
since this strain was kanamycin sensitive.
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Figure 2: This represents the first PCR screen from the sucrose singles obtained
from various lineages of the Kan resistant colonies resulting from the SCHU S4
transformation with C7- and C8-1133+dMglA, respectively. That is, K2
generated the 2A, 2B, etc sucrose colonies, K3 generated the 3A, 3B, etc
sucrose colonies and so forth. Lanes 2 and 16 represent the wild type MglA
gene size using the gene specific oligo primers MglAB BHI and MglAB H3.
Lanes 3 and 17 represents the smaller deleted MglA gene size using the
mentioned oligos. The complete deletion should only yield the lower molecular
weight band pattern and it is clear that all of the colonies, with the exception of
5D (lane 21) returned to the wild genotype since the lower molecular weight band
now is gone from the profile. Sample 5d still shows a band at the correct size for
the DmglA mutation, as well as some other mW bands, this may represent mixed
wildtype and mutant colonies. Data in TVD UTSA Notebook 3, page 78.
d. We streaked some cells from the one strain still showing the DmglA pattern, patch
(5d), onto a fresh TSA+++ plate to generate single colonies. The resulting single
colonies from this plate were patched onto TSA+++ and TSA+++ containing 70 ug/ml
Kan, respectively.
e. Twenty of these single patched colonies resulting from the parent 5d were colony
picked to prepare for PCR with the MglA oligo primers. This set was prepared in a
reaction using the MglA gene specific oligo primers to screen by PCR. The results
(Figure 3) indicated that one of the resultant colonies (lane 7 below) appears to
wildtype mglA (i.e. no deletion).
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Figure 3: This represents PCR products generated using the DNA chromosomal
templates indicated in legend and MglA gene specific oligo primers (MglAB BHI/ MglAB
H3). The majority of chromosomal DNA (SA1-SA19) were obtained from single colonies
generated from the parent 5D (figure 2 Lane 21). Lanes 12, 13 and 14 are more sucrose
singles generated from the initial parent K5. Lane 2 and 16 represent the wild type MglA
gene size using this oligo set; and lanes 3 and 17 represent the MglA deletion gene
size using the plasmids (C7- and C8-1133+dMglA, respectively) used in the SCHU S4
transformation experiment. Data in TVD UTSA Notebook 3, page 80.
f.
Data located in TVD UTSA Notebook 3, page 68-69, 78-80.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have had some difficulties obtaining the iglC deletion in the Schuh4 chromosome. However,
we have a number of alternative strategies we are pursuing concurrently to obtain this mutant,
discussed at the UTSA Site Visit (4/9/07), and we are confident we will have this mutant soon.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
49%
9. Work plan for upcoming month and next 6 months
For the next 6 months:
I.
Will begin experiments using the existing KEK906 iglC deletion construct (AmpR)
transformed into a newly created ampicillin sensitive SCHU S4 strain that has just been
constructed in our lab. We will determine if this plasmid functions to facilitate integration
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II.
a.
b.
c.
d.
e.
into the Schuh4 chromosome, and if we can select loss of plasmid and maintenance of
the DiglC mutation in the chromosome.
Will start cloning of the iglD deletion mutation.
Will design primers for the iglD gene containing 1000 bp homology on either side of this
gene. These will be amplified from SCHU S4 chromosomal DNA.
The pKEK1090 vector will be modified by replacing the chloramphenicol gene with
ampicillin gene.
The ampicillin gene will be amplified from the pwsk30 plasmid with appropriate restriction
sites.
Once the ampicillin resistant pKEK1090 construct is completed, we will begin cloning of
the iglD deletion gene fragments.
Begin experiments to create iglD mutation in SCHU S4 genome.
Upcoming month:
a. Sequence pKEK1090+iglC and pKEK1133+ iglC plasmids to determine if deletions are
occurring during/after ligation within the iglC fragment. This will allow us to determine
where the problem lies and how to proceed with this ligation.
b. Will continue to screen the remaining pKEK1133+iglC and pKEK1134+iglC transformants
that were generated from the last transformation
c. Will begin work on the iglD deletion. Will start with the pKEK1090 plasmid modifications
mentioned in forecast above.
d. Will verify that the MglA deletion is present in chromosome of SA15 mutant, shown
above.
e. Order supplies as required.
10.Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB; LVS
uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) pdpD,
iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4) iglA,
iglB strains
Institution: UTSA
1. Date started: 04/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
Semi-Annual: 10/1/06 to 3/30/07-SUMMARY/HIGHLIGHTS
The virulence and bacterial dissemination of Ft subsp. novicida uvrAuvrB double mutant and
Ft subsp. novicida iglB single mutant were evaluated in an intranasal infection model (BALB/c
mice). The protective efficacy of Ft subsp. novicida iglB mutant as a vaccine candidate was
evaluated in an intranasal Ft novicida challenge model. Additionally, we also evaluated the
protective efficacy of the Ft novicida iglC mutant plus LPS of LVS against pulmonary SCHU
S4 challenge.
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a. ΔuvrAuvrB double mutant
i. LD50 is comparable to wild type Ft novicida strain (<10 CFU).
ii. Rapid replication in the lungs and dissemination to liver and spleen after i.n.
challenge.
iii. This mutant is not attenuated in a pulmonary model of infection.
b. Δiglb mutant
i. LD50 is greater than 107 CFU.
ii. Bacteria replicated in the lungs and disseminated to the liver and spleen after i.n.
challenge, but was found to be 4-5 logs less when compared to the ΔuvrAuvrB
double mutant at day 3 after i.n. infection.
iii. Bacteria were still detectable in the lungs, liver, spleen and lymph nodes 15 days
after i.n. challenge. This replication pattern may account for the robust priming of
the immune system, with this attenuated vaccine candidate.
iv. BALB/c mice vaccinated with Δiglb produced significant amounts of antigenspecific antibody at day 14 after immunization and the titer increased by day 28.
Isotype analyses indicated both Th1 (IgG2a) and Th2 (IgG1)- type antibodies were
produced.
v. Balb/c mice intranasally immunized with Δiglb (as low as 104 CFU) were protected
from subsequent lethal pulmonary challenge with F. novicida (1000 CFU,
100LD50).
c. Δiglc mutant
i. The Ft novicida Δiglc was detectable in target organs (lungs, liver, spleen, and
lymph nodes) 15 days after i.n. immunization.
ii. Immunization with Δiglc (106 CFU i.n.) plus LVS LPS (50 μg i.p.) failed to protect
BALB/c mice against lethal pulmonary SCHU S4 challenge (300 and 3000 CFU).
Monthly-March 2007:
a. Analyze the cell mediated antigen-specific cytokine response of mice immunized with
the Ft novicida iglB mutant (Note book #4, page 79-81). BALB/c mice were immunized
with ΔiglB (106 CFU) or mock immunized with PBS. Spleens were collected from mice
at day 14 after immunization. Single cells were made and stimulated with UVinactivated ΔiglB or HEL (hen egg lysozyme) as an unrelated control. Supernatants
from cell culture were collected at 72 h and assayed for the production of cytokines. As
shown in Fig .1, ΔiglB-primed spleen cells produced antigen-specific IFNwith UV-inactivated ΔiglB (104 and 105 CFU). Minimal production of IFNin PBS/mock primed spleen cells stimulated with ΔiglB. Moreover, ΔiglB-primed spleen
cells incubated with HEL produced no measurable IFNminimal production of antigen-specific IL-4 production in the supernatants. These
results indicate that immunization with ΔiglB induced robust Th1 type cell mediated
immune responses in mice Note book #4, page 79-81.
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1500
150
1200
120
IL-4(pg/ml)
IFN-(pg/ml)
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
900
600
90
60
30
300
0
HEL
104iglB
105 iglB
PBS/mock
iglB
0
PBS/mock
iglB
Fig.1. Cytokine recall responses after immunization with ΔiglB. BALB/c
mice (3 mice/group) were immunized i.n. with ΔiglB (106 CFU) or PBS as
mock vaccination control. On day 14, spleen cells were tested for ΔiglBinduced IFN-γ and IL-4 with UV-inactivated ΔiglB (104 and 105). HEL, an
unrelated antigen, was used as a control stimulus.
b. H&E analyses of lungs and spleens from ΔiglB immunized/ F. novicida challenged mice
(Note book #4, page 82-83). Lungs were collected from ΔiglB immunized and F.
novicida challenged mice 30 days post-challenge, fixed in 10% neutral formalin and
embedded into paraffin blocks. Serial horizontal sections (4-5µm) were prepared on
silane-coated slides and stained with hematoxylin and eosin (H & E). Lung sections
prepared from naïve mice and PBS/mock immunized/wild type challenged mice at day
3 were also used for comparison. The results of histopathological analyses are shown
in Fig.2. In general few foci of inflammatory cells were observed in lungs of mice
vaccinated i.n. with 104 or 107 CFU ΔiglB and subsequently challenged with wild type,
as indicated in panel E and G (arrow heads) and the insert in panel F. In these tissue
sections, most of the airway architecture revealed open air spaces similar to naïve
animals (Panel A & B). In contrast, lungs of mice that were mock vaccinated with PBS
and then challenged with 103 CFU of the wild-type U112 strain displayed severe
consolidation and polymorphonuclear cell infiltration 3 days after challenge (Panel C &
D).
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4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
34% of scientific work completed on the milestone
9. Work plan for upcoming month and next 6 months
a. For Next one month
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i. Determine the LD50 of Ft subsp. novicida uvrBiglC double mutant.
ii. Monitor Ft subsp. novicida ΔuvrBiglC replication and dissemination in mice.
b. For Next 6 months
i Evaluate virulence of Ft subsp. novicida uvrBiglD and uvrBiglA double mutants
ii Analyze immune response to ΔuvrBiglD and ΔuvrBiglA immunization.
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
Semi-Annual: 10/01/06 to 3/30/07-SUMMARY/HIGHLIGHTS
a. The Francisella subsp novicida mutants uvrB (KKF72), uvrA (KKF71) and uvrAB
(KKF100) double mutant were tested for their ability to grow and survive in J774
macrophages in comparison to wildtype. The 3 mutants showed similar growth in the
J774 macrophages as the wildtype U112.
b. Result (in triplicate) 24 hours post infection of macrophages
i. U112
1. 3.0 x 106 cfu/ml
2. 1.0 x 106 cfu/ml
3. 3.6 x 106 cfu/ml
ii. KKF71
1. 4.6 x 106 cfu/ml
2. 4.7 x 106 cfu/ml
3. 3.4 x 106 cfu/ml
iii. KKF72
1. 3.9 x 106 cfu/ml
2. 3.3 x 106 cfu/ml
3. 2.1 x 106 cfu/ml
iv. KKF100
1. 4.7 x 106 cfu/ml
2. 8.0 x 106 cfu/ml
3. 3.3 x 106 cfu/ml
c. The Francisella subsp. novicida mutant uvrB (KKF71) was transformed with
chromosomal DNA from an iglC and an iglD mutant in order to create double mutants.
The uvrB + iglC double mutant was made and frozen away as KKF224. The uvrB + iglD
double mutant was made and frozen away as KKF225.
Monthly March 2007
d. Chromosomal DNA was purified from the F. novicida uvrB mutant KKF71. 10 ug of this
DNA was cryotransformed into a F. novicida iglA mutant KKF42 in hopes of generating a
uvrB + iglA 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
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
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 KKF226. Data
described in Notebook 1, page 14. The strain KKF226 is a uvrB + iglA double mutant of
Ft. subsp. novicida.
1.
Figure 1.
2.
3. 4.
5.
6.
7.
8.
1.
2.
3.
4.
5.
6.
7.
8.
Ladder
Ft. subsp novicida
colony 1
colony 2
colony 3
colony 4
colony 5
colony 6
4. Significant decisions made or pending
a. The decision was made to focus making all the double mutants in Ft novicida in a uvrB
background per the 1/25/07 conference call with Dr. Klose, Skoble and Lyons
b. All the uvr mutants in Francisella subsp. novicida show no defect in intramacrophage
survival at 24 hrs post infection.
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)
7. Quality of performance
Excellent
8. Percentage completed
60%
9. Work plan for upcoming month and next 6 months
Create a uvrB + pdpD double mutant in the next month
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
Sections IV: Contract Expenditures and Subcontractor Billing
1. % work completed vs. %cumulative costs incurred to date with regard to planned
effort and proposed budget
The active Milestones over the past 6 months have been: 2, 3, 4, 5, 12 (UNM &LBERI), 13 (UNM
&LBERI), 19, 21, 25, 26, 27, 28, 32, 33, 34, 40, 41, 42, 43, 46, 49, 50 and 51.
LBERI: % invoiced and work completed through Jan 2007; billed to NIAID 4/15/07
UNM: % invoiced and work completed through Mar 2007; billed to NIAID 4/15/07
ASU: % invoiced and work completed through Jan 2007; billed to NIAID 4/15/07
Cerus: % invoiced and work completed through Dec 2006; billed to NIAID 4/15/07
UTSA: % invoiced and work completed through Jan 2007; billed to NIAID 4/15/07
Data associated with most current UNM invoice submitted to NIAID ( parallels Financial Report by
Milestone submitted by UNM to NIAID 4/16/07)
Milestone
% invoiced
(same % as
reported on
4/16/07 FRM)
3- LBERI
37
% work
completed
(same % as
reported on
4/16/07 FRM)
30
Over
Milestone
budget?
(yes/no)
4-LBERI
5-UNM
9.8
52.3
11
45
No
No
12 UNM
12 LBERI
28.7
91.6
40
88
No
No
13 UNM
6
0
No
13 LBERI
27.2
11
No
19-UNM
5.9
3
No
21-UNM
2
10
No
No
Discrepancy between % invoiced and
% work completed?-briefly describe
Variance is emerging partly associated
with quality issues that will be discussed
at the 4/26 LBERI site visit (By Mar ’07,
invoicing is at 58.6% and work is at 38%)
None
UNM has performed expanded LVS and
SCHU S4 virulence testing for the
Working group; The Work completed
does not include the Working Group
efforts but the expenses of the Working
Group effort are included in this
milestone.
Work is slightly ahead of expenses
Variance is emerging partly associated
with additional goals being added to this
milestone (comparing freezing protocols,
researching B cell artifact et). This will be
discussed at the 4/26 LBERI site visit
UNM is purchasing reagents that are
used at LBERI. UNM has not started
work on this milestone, to date.
LBERI may be expensing some effort to
MS 13 that should be expensed on MS12
or another milestone. This will be
reviewed at the 4/26 LBERI site visit, as
LBERI may need to redistribute budgets
between some milestones.
Milestone start up and development of
assays requires labor in advance of
completing work for the milestone.
Future animal expenses associated with
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
25-ASU
26-ASU
100
94.4
100
80
No
No
27-UNM
28-ASU
5.3
0
10
0
No
No
32-ASU
33-ASU
100
84
100
50
No
No
34-UNM
16.9
10
No
34-ASU
0
0
No
39-UTSA
40-Cerus
96.1
82.5
100
75
No
No
41-Cerus
34.4
50
No
42-Cerus
0
0
No
43-UTSA
44.9
49
No
48-UTSA
74.4
100
No
46- Cerus
15.5
28
No
49-UTSA
50- UTSA
51-UTSA
26
20.8
14.1
37
29
20
No
No
No
this milestone will decrease the variance
None; milestone completed
Variance is expected to decrease as
work increases relative to expenses. (By
March ’07, invoicing is at 94.9% and
work is at 90%)
Work is temporarily ahead of expenses
Technical work started after Dec 06 (By
Mar ’07, invoicing is 7.5% and work is
6%)
None; milestone completed
Work is expected to catch up to
expenses ( By Mar ’07, 75% completed
March and 88% expensed)
UNM is providing RNAs to ASU for initial
assay development (microarray cDNA
synthesis and hybridization).
Technical work started after Jan 07 (By
Mar’07, invoicing at 17% and work is at
11%)
None
Work is lagging behind invoice as of
Dec’06 (By Mar 07, invoicing is 96% and
work is 95%)
Invoice lags behind “work completed” by
1 month.
Technical work started after Jan 07 (By
Mar ’07, invoicing is 6.6% and work is at
10%)
None (By Mar ’07, invoicing is 56% and
work is 55%)
Milestone is completed; vendor invoices
may continue to be received.
Invoice lags behind “work completed” by
1 month.
Invoices are expected to catch up
Invoices are expected to catch up
Invoices are expected to catch up
To date, administrative costs have been billed to NIAID that are associated with Milestones 1 and 2 and
with the management of the scientific milestones.
2. Estimates of subcontractors expenses from prior month if subcontractor did not
submit a bill. List for each subcontractor. If subcontractors were not working or did not
incur any cost in current or prior month, then include a statement to this effect.
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Tularemia Vaccine Development Contract: Semi-Annual Technical Report
Period: 10/01/2006 to 3/31/2007
Due Date: 4/7/2007 and Prepared by: Barbara Griffith and Terry Wu
LBERI, Cerus, ASU and UTSA all submitted invoices to UNM in the March 2007. The UNM invoice to
NIAID, being submitted on 4/15/07, will include subcontractor invoices from ASU, Cerus, LBERI and
UTSA.
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