Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two
animal models and cellular assays vital for testing vaccine efficacy.
Sections II and III: Progress and Planning Presented by
Milestone
Active milestones: 2, 4, 5, 7, 8, 9, 10, 11(UNM/LBERI), 12/13(UNM/LBERI), 14, 17, 18,
19, 21(UNM/LBERI), 29(UNM/LBERI), 35(ASU/UNM), 49, 50, 52, 53, 55, 56, 57
Completed milestones: 1, 3, 6, 16, 25, 26, 27, 28, 32, 33, 34 (UNM/ASU), 39, 40, 43
(UTSA), 48, 51
Inactive milestones: 15, 20, 22, 23, 24, 30, 36, 37, 38, 54, 58, 59
Milestones terminated after initiation: 41, 42, 44, 46, (MSCR will be written)
Milestones terminated before initiated: 43 (Cerus), 45, 47 (MSCR will not be written)
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/2005
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. UNM EOH has performed 29 annual health screenings since 8/26/08 for the LVS
vaccines originally vaccinated through December 2007.
b. Three UNM and possibly 6 LBERI scientists will request vaccinations in 2009.
c. USAMRIID tentative vaccination date is June 2009, pending FDA approval
4. Significant decisions made or pending
a. Dr. Lyons received UNM IRB approval to allow blood draws on the vaccinated LBERI
and UNM scientists after their LVS vaccinations. The LVS vaccinated LBERI and
UNM scientists and staff have been offered the opportunity to volunteer to donate
bloods for the development of immunoassays, approximately 2 months after
receiving the LVS vaccination.
b. USAMRIID tentatively will resume offering vaccinations to UNM and LBERI in June
2009 if FDA approval is given.
c. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI will offer the LVS
vaccinations to 9 more scientists to total up to 46. The CRDA with USAMRIID is valid
for 2 years, ending June 29 2009.
5. Problems or concerns and strategies to address
a. Nine scientists could be vaccinated in 2009 if USAMRIID receives FDA approval for
the new Tularemia vaccination protocol.
b. USAMRIID may restart LVS vaccinations in June 2009 pending FDA approval
Page 1 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
c. UNM will contact USAMRIID regarding extension of the CRDA from ending on
6/29/09 to ending on September 29, 2010.
6. Deliverables completed
A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS
vaccination since 9/11/07.
7. Quality of performance
Excellent
8. Percentage completed
74% of the scientific work is complete
9. Work plan for the next month
a. Continue annual health screenings required by USAMRIID and being performed at
UNM for the LBERI and UNM LVS vaccines.
b. UNM will be obtaining blood donations from LVS vaccinees for immunoassay
development and reimbursing participants $40/ donation.
c. UNM will work with 3 UNM and 6 LBERI scientists for the pre-vaccination health
screenings required for vaccinations once USAMRIID has FDA approval to offer the
LVS vaccinations again.
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
No work was performed during this reporting period
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
98% of the scientific work is complete
9. Work plan for next month
a. Liz Zinter and Michelle Valderas will write the Milestone Completion Report by April
1, 2009
Page 2 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
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
a. Experiment Ftc71.2 (Notebook 130 pages 31, 34-35)
i. The purpose of this experiment was to determine the effect of LVS
vaccination dose on the resistance of vaccinated rats to i.t. SCHU S4
challenge. This is a repeat of experiment Ftc71.1 which showed that a s.c.
vaccination dose as small as 103 protected 5 of 6 Fischer 344 rats against an
i.t. challenge of 3.3. x 103 SCHU S4.
ii. In this repeat experiment, Fischer 344 rats (n = 6) were either left
unvaccinated as a negative control or vaccinated s.c. with 6.8 x 101, 6.8 x
102, or 6.8 x 104 cfu/rat based on the inoculum plating results. The rats are
scheduled to be challenged on March 10, 2009
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
7. Quality of performance
NA
8. Percentage completed
94%
9. Work plan for upcoming month
a. Complete experiment Ftc71.2 by challenging the LVS vaccinated rats i.t. with SCHU
S4
b. Complete the histopathological analyses of tissues from mice, rats, and NHPs
infected with SCHU S4
c. Complete milestone completion reports for the mouse, rat, and guinea pigs
Milestone 7
Milestone description: SCHU S4 ED50 in primates determined from selection of
challenge dosing
Institution: LBERI
1.
2.
3.
4.
Date started: 2/25/08
Date completed: In progress.
Work performed and progress including data and preliminary conclusions:
Significant decisions made or pending
No work was performed during this reporting period.
5. Problems or concerns and strategies to address
None
Page 3 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
90% of the scientific work is complete.
9. Work plan for next month
a. Respiratory rates and temperatures previously reported will be modified as per the
format provided by Kristin DeBord.
b. Histopathology will continue to be analyzed. Slide preparation will be completed midMarch. Based on this date, a draft pathology report will be prepared by the end of
April with a final pathology report at the end of May.
c. Liz Zinter and Michelle Valderas will write the Milestone Completion report by June
15, 2009.
Milestone 8
Milestone description: LVS vaccination protection of aerosol Schu S4 validated in
primates
Institution: LBERI
1. Date started: 8/15/2008
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions
a. On 2/12/09 and 2/13/09, 12 NHPs (3 controls; 6 vaccinated with LVS in 1/09; 3
vaccinated with LVS 11/06) were challenged with Schu S4 by aerosol.
b. LVS inoculum size and route of vaccination varied and is detailed in Table 1. Briefly,
i. LVS vaccination was delivered by the intradermal (i.d.) route on 11/20/06 to 3
NHPs
1. The target dose was 1 x 105, however, the dose delivered was 1.5 x
107;
2. The dose was prepared by thawing a previously frozen stock of LVS;
the target dose was supposed to have been calculated based on the
known titer of the LVS when frozen
3. However, a 10-fold dilution error was made and the titer of the vial
was approximately 10-fold higher than expected as well, resulting in
a delivered dose of 1.5 x 107 rather than 1 x 105
4. One NHP (A00896) had to be used for a non-ABSL3 study in July
2008 due to his aggressive behavior (he was challenged with LVS by
bronchoscopy)
ii. LVS was delivered by the subcutaneous (s.c.) route on 11/29/06 to 3 NHPs
1. In an attempt to match the i.d. delivered dose of 11/20/06, the target
dose for this vaccination was 1.5 x 107
2. A frozen vial of LVS was thawed and diluted based on the
anticipated higher titer observed on 11/20/06
3. The resultant dose was actually 2.7 x 106, as determined by plating
the remaining inoculums post-vaccination
4. One NHP (A00902) was mistakenly used in another study in August
2007
Page 4 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
5. One NHP (A00868) developed behavioral problems and had to be
excluded from the ABSL3 portion of this study (he was challenged
with LVS by bronchoscopy in January 2009
iii. LVS was delivered either by the s.c. route or by scarification to 3 NHPs each
on 1/8/09
1. These vaccinations were done in an attempt to mimic the vaccination
protocol performed at USAMRIID
2. As such, a lyophilized vial of DVC Lot 16 LVS was resuspended
directly in 1.4 ml of sterile water
3. In 2005, DVC indicated that LVS Lot#16 vials each contained 1.4 x
109 LVS. This dilution could have resulted in 1.3 to 1.3 x 109 LVS/ml;
however, Baylor University had titered the vials at 1 to 2 x 108 in
2005 and UNM had titered the vials at 5 to 7x107 in 2006.
4. 3 NHPs were scarified with 60 µl LVS which should have resulted in
a dose of 60 x 106 organisms based on DVC’s reported 1.3 to 1.4x
109/vial.
5. A 1:1 dilution of the inoculums was prepared and 120 µl was injected
s.c. for a calculated dose of 60 x 106 organisms
6. The actual inoculum dose delivered was 31,200 organisms as
determined by plating the inoculums and counting the resultant
colonies
c. The animals were challenged with 27-1780 CFU (target dose 500 CFU).
d. Table 1 reports the route of vaccine administration, presented dose (CFU) exposure
date, and date of death or euthanasia date for each animal. Low dosing of animals
on 2/12/09 flask 1 were due to a microbiological miscalculation. The Study Specific
Protocol for preparation of bioaerosol material is being changed to have a calculation
verification by a second scientist so that a miscalculation cannot occur again.
Page 5 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Table 1: Summary of LVS Vaccination/SCHU S4 Challenge Study
Animal
Number
Vaccine Group
Presented
Dose
(CFU)
Exposure Date
Date of Death (Study
Day)
A03152
Control
50
2/12/09 – Flask 1
Euthanized 3/5 (21)
28643
Jan. 8, 2009 scarified
(31,200 LVS)
43
2/12/09 – Flask 1
Euthanized 3/5 (21)
28671
Jan. 8, 2009 scarified
(31,200 LVS)
34
2/12/09 – Flask 1
2/21/09 (9)
A04994
Jan. 8, 2009 scarified
(31,200 LVS)
27
2/12/09 – Flask 1
2/21/09 (9)
A05895
Control
89
2/12/09 – Flask 2
2/18/09 (6)
28627
Jan. 8, 2009
subcutaneous
(31, 200 LVS)
117
2/12/09 – Flask 2
2/28/09 (16)
28587
Jan. 8, 2009
subcutaneous
(31, 200 LVS)
293
2/12/09 – Flask 2
2/24/09 (12)
A06587
Jan. 8, 2009
subcutaneous
(31, 200 LVS)
1690
2/12/09 – Flask 2
2/22/09 (10)
A06626
Control
684
2/13/09
2/18/09 (5)
A00937
Oct 2006 Intradermal
(1.5 x 107 LVS)
754
2/13/09
Euthanized 3/5 (21)
A00908
Oct 2006 Intradermal
(1.5 x 107 LVS)
1780
2/13/09
2/17/09 (4)
A00659
Oct 2006 Subcutaneous
(2.7 x 106 LVS)
1270
2/13/09
2/21/09 (8)
Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\MicroBioaerosol
Summary Report
e. Table 2 shows the SCHU S4 tissue burden at necropsy from those NHPs
succumbing to disease prior to Day 16.
f. Bacteria were culturable from all tissues except for the liver from animal A00908
which was below the detection limit. In general, lung bacterial burden was not
reduced in vaccinated animals. Bacterial burden in other tissues was more variable,
as we have previously observed and did not appear to be impacted by vaccination
status.
Page 6 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Table 2. SCHU S4 Tissue Bacterial Burden.
a
CFU/g
Vaccine
Group
Presented
Dose (CFU)
A05895
Control
43
18-Feb-09 1.56E+07 4.34E+05 1.54E+08 7.00E+04 6.39E+08
25671
8JAN09
Scar
27
21-Feb-09 4.60E+05 4.60E+04 1.19E+07 1.12E+06 3.83E+08
A04994
8JAN09
Scar
89
21-Feb-09 1.20E+06 1.20E+05 4.83E+06 9.80E+02 8.18E+08
28587
8JAN09
Sub
293
24-Feb-09 4.34E+05 9.00E+03 2.73E+06 4.76E+05 7.67E+08
A06587
8JAN09
Sub
1686
22-Feb-09 7.92E+05 9.46E+04 9.80E+05 4.90E+07 2.12E+08
A06626
Control
684
18-Feb-09 7.41E+07 1.92E+06 1.61E+09 2.17E+06 4.09E+08
A00908
OCT06 ID
1777
17-Feb-09 4.34E+04
A00659
OCT06
Sub
1274
21-Feb-09 3.07E+04 3.07E+04 6.30E+06 4.76E+03 3.32E+08
Animal ID
Nx Date
Spleen
Liver
BLD
Mes LN
TBLN
Lung
4.62E+08 9.80E+05 1.46E+08
BLD, below limit of detection
Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\12Feb09
Bioaerosols\Group 1 tissue forms and on Z:\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\ 13Feb09
Bioaerosols\Group 2 tissue forms
g. Bacteremia post SCHU-S4 aerosol exposure is reported for each NHP in Table 3.
Bacteremias were not detected on every animal in this study, unlike what we
observed in the ED50 study, though many progressed to death in this
vaccination/challenge study. It is possible that vaccination resulted in decreased
bacteremia, or that bacteremia resulted later than the planned blood draws on days
0 to 6 due to prior LVS vaccination. Another interpretation is that, because the
aerosol dose was a log lower than targeted and the blood draws were timed for
animals with primary pulmonic disease, that LBERI also missed the window to
observe bacteremia.
Page 7 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Table 3. SCHU S4 Bacteremia Post-Aerosol Exposure
b
Animal Vaccine
ID
Group
Study Day
Presented
Nx
Dose
Date
(CFU)
0
1
2
3
4
5
6
BLD
BLD
BLD
Term
A03152 Control
50
n/a
BLD BLD
BLD
BLD
A05895 Control
43
18Feb- BLD BLD
09
BLD
BLD
28643
8JAN09
Scar
34
n/a
BLD BLD
BLD
BLD
BLD
BLD
BLD
25671
8JAN09
Scar
27
BLD BLD
BLD
BLD
BLD
BLD
BLD
n/a
A04994
8JAN09
Scar
89
BLD BLD
BLD
BLD
1.00E+01
BLD
3.33E+00
BLD
28627
8JAN09
Sub
117
BLD BLD
BLD
BLD
3.33E+00
BLD
BLD
n/a
28587
8JAN09
Sub
293
BLD BLD
BLD
BLD
6.67E+00
BLD
BLD
BLD
A06587
8JAN09
Sub
1686
BLD BLD
BLD
BLD
BLD
BLD
BLD
BLD
A06626 Control
684
A00937
OCT06
ID
754
A00908
OCT06
ID
1777
A00659
OCT06
Sub
1274
21Feb09
21Feb09
28Feb09
24Feb09
22Feb09
18Feb09
n/a
BLD BLD 3.33E+00
BLD
BLD BLD
BLD
17Feb- BLD BLD
09
21Feb- BLD BLD
09
BLD
3.33E+00 1.30E+02 4.87E+02 4.88E+02
6.33E+01 1.53E+03
BLD
BLD
3.33E+00 3.33E+00
BLD
1.27E+02
BLD
BLD
3.33E+00
BLD
2.50E+02
BLD
. The data is reported for each individual animal as CFU/mL. Boxes are grey where animals
have died before the six days of blood draws were complete. BLD indicates below level of
detection; n/a indicates that blood could not be drawn from a moribund or dead animal. Blank
cells in the table indicate that the NHP survived until termination (3/5/09) and the bacterial counts
are not yet back at the time of this report..
Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08)\12Feb09 Bioaerosols\Group 1
blood forms and on Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 (TUL-08)\ 13Feb09 Bioaerosols\Group 2 blood forms
g.
IFN-γ production by PBMCs stimulated with heat killed and formalin-fixed LVS
was analyzed for all animals shortly before SCHU S4 aerosol challenge.
Page 8 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
i. The response of the PBMCs from non-LVS vaccinated controls is shown
in Figure 1.
ii. The response of PBMCs from the NHPs vaccinated in 11/06 is shown in
Figure 2.
iii. The response of PBMCs from the NHPs vaccinated via scarification
(Figure 3) and subcutaneous inoculation (Figure 4) in 1/2009 is shown
both prior to (Day 0) LVS vaccination and post-LVS vaccination (Day 25).
IFNg Spots (Mean +/- S.D.)
350
89 CFU
Day 6
300
250
50 CFU
200
Media
LVS hk Hi
LVS ff Hi
664 CFU
Day 5
150
100
50
0
A03152
A05895
A06626
Figure 1. Antigen-stimulated IFN-γ production by non-LVS vaccinated controls. All cells
were plated at 1.33 x 106/ml; All IgG anti-LVS titers were 1/20000. “Day x” indicates the day
that the NHP succumbed to SCHU S4 infection. A03152 was euthanized on Day 21, the last
day of the study.
Page 9 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
IFNg Spots (Mean +/- S.D.)
350
300
250
1270 CFU
Day 8
Media
LVS hk Hi
LVS ff Hi
754 CFU
200
1740 CFU
Day 4
150
100
50
0
A00659
A00908
A00937
Figure 2. Antigen-stimulated IFN-γ production by LVS vaccinated NHPs. The animals were
vaccinated in 11/06. All cells plated at 1.33 x 106/ml; Day post-LVS vaccination = 786
(A00659, 2.7 x 106 CFU LVS by subcutaneous inoculation) – 795 (A00908 and A00937, 15 x
106 CFU LVS by intradermal vaccination); “Day x” above the bars indicates the day that the
NHP succumbed to SCHU S4 infection. A00937 was euthanized on Day 21, the last day of
the study.
Page 10 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
250
43 CFU
500K
200
34 CFU
Day 9
Media
LVS hk Hi
LVS ff Hi
20K
500K
150
4K
100
27 CFU
Day 9
0.8K
A04994, Day 0
28671, Day 25
28671, Day 0
0
28643, Day 25
50
100K
A04994, Day 25
300
28643, Day 0
IFNg Spots (Mean +/- S.D.)
350
Day Post-LVS Vaccination
Figure 3. Antigen-stimulated IFN-γ production by NHPs vaccinated with LVS by scarification.
The animals were vaccinated in 1/2009 with 31,200 CFU LVS.. All cells plated at 1.33 x
106/ml; IgG anti-LVS titers are shown above the bars (ex. 4K); “Day x” above the bars
indicates the day that the NHP succumbed to SCHU S4 infection; 28643 was euthanized on
Day 21, the last day of the study.
Page 11 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
400
350
300
250
283 CFU
Day 12
4K
100K
Day 16
117 CFU
1690 CFU
Day 10
100K
100K
4K
20K
200
150
100
A06587, Day 25
A06587, Day 0
28627, Day 25
28627, Day 0
0
28587, Day 25
50
28587, Day 0
IFNg Spots (Mean +/- S.D.)
450
Media
LVS hk Hi
LVS ff Hi
Day Post-LVS Vaccination
Figure 4. Antigen-stimulated IFN-γ production by NHPs vaccinated with LVS by
subcutaneous inoculation. The animals were vaccinated in 1/2009 with 31,200 CFU LVS
although the target vaccination dose was 60 x 106. All cells plated at 1.33 x 106/ml; IgG antiLVS titers are shown above the bars (ex. 4K); “Day x” above the bar indicates the day that
the NHP succumbed to SCHU S4 infection.
Data Interpretation:
 Delivery of SCHU S4 by aerosol was complicated by the fact that a dilution error was
made with Flask 1 resulting in a delivered target dose of 50 CFU rather than the desired
500 CFU
 Survival post-SCHU S4 aerosol challenge does not correlate with IgG anti-LVS titers as
all the vaccinated NHPs had high titers post-vaccination yet succumbed to disease at
different rates
 Presence of high IgG anti-LVS titers induced by LVS vaccination did not protect NHPs
from SCHU S4-induced lethal disease
 Sensitivity of recently vaccinated NHPs (1/09) to SCHU S4 may be related to the
relatively weak IFNγ production by their PBMCs post-vaccination
– The lack of responsiveness to HK LVS has not been observed previously in any
LVS-vaccinated NHP (10/10 vaccinated and tested for responsiveness) and may
be due in this case to the low LVS vaccination inoculum (31,200 CFU) used.
– Previous vaccinees (10/08) received 2.6 x 105 CFU LVS and all 5 produced more
IFNγ upon HK LVS stimulation after LVS vaccination as compared to before
vaccination (see Figure 5 for historical data)
Page 12 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
700
600
500
Media
LVS hk Hi
LVS ff Hi
400
300
200
A06199, Day 0
A06199, Day 7
A06199, Day 15
A06199, Day 21
A06199, Day 28
A06199, Day 35
A05403, Day 0
A05403, Day 7
A05403, Day 15
A05403, Day 21
A05403, Day 28
A05403, Day 35
A04169, Day 0
A04169, Day 7
A04169, Day 15
A04169, Day 21
A04169, Day 28
A04169, Day 35
0
28656, Day 0
28656, Day 7
28656, Day 15
28656, Day 21
28656, Day 28
28656, Day 35
100
28461, Day 0
28461, Day 7
28461, Day 15
28461, Day 21
28461, Day 28
28461, Day 35
IFNg Spots (Mean +/- S.D.)
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Figure 5. IFN-γ production by LVS-vaccinated NHPs. The animals were vaccinated in
10/2008. All cells plated at 1.33 x 106/ml; an arbitrary value of 600 was assigned to wells
which were TNTC; LVS vaccination dose = 2.6 x 105.
Data Storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay
02028009.svd; TVDC bound notebook (5) 9247, pp. 7 – 46, 54 – 69. TVDC bound notebook (6)
9616, pp. 4 – 12, 47 - 57.
4. Significant decisions made or pending
A new experiment has been proposed to test four different LVS vaccination doses (102, 104,
106, and 108) delivered by subcutaneous inoculation followed by aerosol challenge with 500
CFU SCHU S4.
5. Problems or concerns and strategies to address
LVS vaccinated animals from this study unexpectedly succumbed to SCHU S4 challenge
potentially indicating that the LVS vaccination dose was insufficient to provide protection. A
new LVS vaccination dose range experiment using freshly grown, viable LVS has been
proposed to determine whether the concentration of the LVS vaccine can affect the
susceptibility to SCHU S4 aerosol challenge.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
30% of the scientific work is complete.
9. Work plan for upcoming month
a. Survivors from the study were euthanized on day 21post-challenge (3/5/09) and
tissues were taken for pathology, microbiology, and immunologic assessment.
b. We will consult with the NIAID project officer and specialists to review and refine the
design of the next LVS vaccination and challenge experiment.
Page 13 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Milestone 9
Milestone description: Aerosol SOP developed for GLP transition
Institution: LBERI
1. Date started: 8/13/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No work was performed during the reporting period.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Practice bioaerosols in conjunction with actual NHP exposures have demonstrated the
difficultly in consistently achieving a presented dose of 500 CFU. We need to determine what
will be considered an acceptable range (keeping in mind that pulmonary disease is
established at or above 89 CFU.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
40% of the scientific work is complete.
9. Work plan for upcoming month
a. Establish a broader acceptable challenge dose range (with input from the TVDC
team) and conduct an additional day of mock-qualification runs.
b. Revise and complete qualification plan for the aerosol and send out for TVDC review
c. Conduct an additional day of mock-qualification runs with the revised Qualification
Plan. Focus will be on achieving a more broadly defined SCHU S4 aerosol
concentration.
Milestone 10
Milestone description: Efficacy testing of vaccine candidates (LBERI) and
Characterization of selected small animal model (UNM)
Institution: LBERI /UNM
1. Date started: 1/1/2009
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
19 NHPs were ordered for the testing of the USAMMDA IND 157 LVS vaccine.
4. Significant decisions made or pending
The testing of the USAMMDA IND 157 vaccine may be delayed until LBERI further tests the
vaccine efficacy of the DVC LVS lot#16, used on the TVDC to date.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
2%
Page 14 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
9. Work plan for upcoming month
We will discuss plans for this experiment with the NIAID project officers. It will likely be
delayed until we can find a dose or dosing regimen of DVC LVS lot #16 that will reliably
protect NHPs from aerosol SCHU S4 challenge.
Milestone 11
Milestone description: In vivo GLP NHP model efficacy SOP and efficacy testing of
vaccine candidates
Institution: LBERI
1. Date started: 1/16/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. We completed immunological screening of the NHPs ordered for the natural history
study (measured IgG anti-LVS titers and the performance of PBMCs in the IFNγ and
proliferation assays).
4. Significant decisions made or pending
Approval of Natural History Study Protocol using a targeted 500 CFU SCHU S4 on
unvaccinated NHP
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
15% of the scientific work is complete.
9. Work plan for upcoming month
a. Animals will receive physical examination, pole/collar/chair training, and will be
moved into the ABSL3 pending approval of the Natural History Study Protocol by Dr.
Lyons and NIAID. Exposure dates are tentatively set for 4/1-2/09.
b. BERI plans to submit the study protocol to Dr Lyons and to NIAID on approximately
3/10/09.
Milestone 11
Milestone description: In vivo GLP model efficacy SOPS developed in one small
species and primate and efficacy testing of vaccine candidates
Institution: UNM
1. Date started: 1/16/2008
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment CDep3 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Gopi's experiments\cdep\Cdep3)
i. The long term goal is to determine the role of CD4 and CD8 T cells in the
immune response for protecting vaccinated animals from SCHU S4 challenge.
The purpose of this experiment was to test the effectiveness of the OX-38
Page 15 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
ii.
iii.
iv.
v.
vi.
ascites fluid for depleting CD4 T cells in rats and to optimize the conditions for
depletion.
We had previously obtained the W3/25 ascites fluid for in vivo depletion of
CD4 T cells, but found out afterwards that it is an inactivating antibody rather
than a depleting antibody and there is no way for us to confirm depletion in
vivo. OX-38 has been reported to be a depleting antibody for CD 4 T cells
Fischer 344 rats were injected i.p. with OX-38 as follows:
1. 100 g once a week or
2. 100 g twice a week or
3. 250 g once a week or
4. 500 g once a week
Spleens of normal rats contain about 10% CD8 T cells, 25% CD4 T cells, 45%
B cells and 20% of neutrophils, NK cells and monocytes
7 days after the first injection, splenocytes were collected and the percent of
CD4 T cells was determined by FACS analyses. As shown in Figure 1,
treatment with OX-38 ascites fluid (100 ug 2/wk, 250 ug 1/wk and 500 ug
1/wk) reduced CD4 T cells from 15% to 5% of total splenocytes. One
treatment per week with 250 g of ascites fluid appears to be as effective as a
higher dose (500 g) once a week and a lower dose (100 g) twice a week.
There are a couple of obvious problems: 1) The percent of CD4+ T cells in
untreated or isotype control treated rats was 10% less than the expected 25%
of total splenocyte population. This may be problem in the gating strategy
used in the analysis and we are reanalyzing this data. 2) The depletion was
not complete, i.e. ~5% CD4+ T cells are left. We will modify the treatment
schedule to obtain better depletion efficiency
Percent of CD4 T cells
20
15
10
5
ty
pe
10
0u
g
1/
w
10
k
0u
g
2/
w
25
k
0u
g
1/
w
50
k
0u
g
1/
w
k
is
o
P
B
S
0
OX-38
Figure 1. Fischer 344 rats (1 per treatment schedule) were injected i.p. with the OX38 ascites fluid following the indicated dose and treatment schedule. One week after
the first treatment, the splenocytes were collected and analyzed for the presence of
CD4+ T cells by flow cytometry
b. Experiment Ptran 12B (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Ptran\Ptran-12B)
i. The purpose of this experiment was to determine the kinetics of SCHU S4
proliferation and dissemination in passively immunized rats.
ii. The pattern of bacterial growth and dissemination in this experiment was very
similar to that observed in experiment Ptran12. The results from the earlier
Page 16 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
experiment suggested that the SCHU S4 burden in the passively immunized rats
reached a peak 5 days after infection and was then maintained at a plateau for
several days it declined. However, in this experiment we observed a second
peak on day 10 post infection.
iii. This suggests that the control mediated by the passively transferred immune rat
serum loses strength by day 7 and 10. However, shortly thereafter, the host
regains control when the cell-mediated immunity is induced and the infection is
eventually cleared.
Liver
Spleen
10
8
8
8
6
4
6
4
2
2
0
0
0
3
6
9
12
15
18
Days Post-challenge
21
CFU (log10)
10
CFU (log10)
CFU (log10)
Lung
10
Naive (0.25ml PBS)
NRS (0.25ml)
Vaccinated
IRS (0.25ml)
6
4
2
0
0
3
6
9
12
15
18
Days Post-challenge
21
0
3
6
9
12
15
18
21
Days Post-challenge
Figure 2. Fischer 344 rats (n = 4) were either vaccinated s.c. with LVS or passively
immunized with 0.25 ml immune rat serum. One month after LVS vaccination or 1 day
after passive immunization, Fischer rats were challenged i.t. with SCHU S4. AT the
indicated time point, the bacterial burden in the lungs, liver and spleen were determined.
The curves show mean bacterial burden ± SD.
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
30%
9. Work plan for upcoming month
a. Continue efforts to purify IgG from immune and normal rat serum and to
demonstrate that the protection is mediated solely by antibodies and not other serum
components
b. Complete histopathological analyses of tissues from actively and passively
immunized rats after i.t. SCHU S4 challenge
c. Optimize CD4+ T cell depletion with the OX-38 ascites fluid
d. Determine whether CD4 and/or CD8 T cells are required for LVS-mediated protection
e. Determine whether immune mouse serum (Experiment Pmouse1) and human
convalescent sera from Martha’s Vineyard protects rats against i.t. SCHU S4
challenge
f. Continue draft of qualifying plan
Page 17 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals &
humans developed and compared to those in other species.
Institution: LBERI/UNM
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No work performed on this MS this month.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We have yet to standardize the LVS and Schu S4 antigens (protein content versus CFU/mL).
The plan is to construct a standard curve correlating CFU/ml and protein content using LVS
and SCHU S4; aliquots will be plated and lysed; lysates will be measured for protein content.
Preparations of heat-killed and formalin-fixed LVS will also be lysed and measured for protein
content; the standard curve will allow correlation to CFU/mL.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
88% of the scientific work is complete.
9. Work plan for upcoming month
a. Determine the relationship between LVS protein content and CFU/mL.
b. Begin to re-titrate the WT and mutant LVS antigens based on protein content.
c. Prepare a positive and negative control plasma reference for use in future IgG antiLVS assays and test in IgG anti-LVS ELISA
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals &
humans developed and Compare assays in animal models (sensitivity)
Institution: UNM/LBERI
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc80 (Notebook 128 pages 34-36)
i. The purpose of this experiment was to test the ability of a human IFN
ELISpot kit to detect Ft-specific IFN secreting T cells in PBMCs from LVS
vaccinated individuals and tularemia patients. In this experiment, we used
frozen PBMC from a patient on Martha’s Vineyard who had recovered from
tularemia.
ii. We did not detect IFN secreting cells after stimulation with formalin-fixed
LVS as an antigen. This may reflect a low number of Ft-specific T cell
precursors in this tularemia patient.
iii. We will test sensitivity of the IFN ELIspot using PBMC from volunteers LVS
vaccinated under this contract. It is possible that we may have to restimulate
Page 18 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
the PBMC in vitro to expand the Ft-specific precursor frequency and work
within the assay sensitivity.
b. Experiment Ftc86 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc experiments\Ftc86)
i. The purpose of this experiment is to establish a standard curve correlating
the total protein in an antigen preparation to the number of bacteria. This will
allow us to compare different antigen preparations and experimental results.
We are designing the experiment and coordinating with LBERI to perform
parallel experiments at both sites. We have purchased the Pierce reducing
agent compatible with the BCA protein assay kit for this analysis.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
Good
8. Percentage completed
71%
9. Work plan for upcoming month
a. Test human IFN ELISpot with PBMC from LVS vaccinated volunteers
b. Establish a standard curve correlating the total protein in an antigen preparation to the
number of bacteria
c. Micro-agglutination titer for vaccinated individuals with protocol Freyja provided.
Milestone 14
Milestone description: Assays in vaccinated humans validated (sensitivity)
Institution: UNM/LBERI
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
No new work done this period.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
We had planned to test serum and PBMC from convalescent tularemia patients on Martha’s
Vineyard. However, due to limited samples, we decided to postpone testing the serum
samples until we can normalize the heat killed and formalin-fixed F. tularensis preparations
(Milestone 12/13) and test for reactivities to LVS, SCHU S4 and the respective O-antigen
mutants. We also decided to postpone testing the Martha’s Vineyard PBMC samples until we
can increase the sensitivity of the IFN ELISpot assay because our results from experiment
Ftc80 indicated that it is unlikely for us to detect tuli-specific T cells in Martha’s Vineyard
patient PBMC with additional stimulation/manipulations.
Page 19 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
None
Milestone 17
Milestone description: In vitro assay for analysis of cellular and humoral elements of
the immune response in vaccinated human and animal’s response to F. tularensis
established
Institution: UNM
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
No new work done this period. We are waiting for a human and rat assay to be developed so
that we can selectively deplete effector subsets in vitro
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Needs improvement, but progress depends on other milestones
8. Percentage completed
25%
9. Work plan for upcoming month
Milestone 18
Milestone description: Role of specific  T cells in protection
Institution: UNM/LBERI
1. Date started: 7/1/08
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
No new work done this period
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
Page 20 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
No work planned
Milestone 19
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
a. Experiment FT-AH-6 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Andrew's experiments\FT-AH-6)
i. The purpose of this experiment was to repeat FT-AH-5 in determining the
effect of cytokines TNF and IFN on the growth of SCHU S4 in naïve human
alveolar macrophages (AM)
ii. 106 AMs were cultured in 14mL polypropylene Falcon culture tubes
iii. AMs were infected with SCHU S4 at MOI = 10 and treated with 500 units/ml
TNF, IFN or both. The amount of cytokine used was 5 times higher than
that used in FT-AH-5. Bacterial burden and cell recovery from the cultures
were determined on days 0, 2, and 3 post infection.
iv. We observed a sharp decline in the cell recovery over the 3 day course (Fig.
3) that was associated with a 10-fold increase in the bacterial burden in the
culture after 48 h. In FT-AH-5, the cell recovery was slightly higher and the
bacterial burden increased by almost 100-fold.
v. Cytokine treatment had modest if any impact on the bacterial burden at 72 h.
These results together with those from FT-AH-5 suggest that human naïve
alveolar macrophages cannot control SCHU S4 growth.
vi. As in previous experiments, the limited number of AMs does not allow for
enough replicates and time points and large variability
Page 21 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
6
control
+IFNg
+TNFa
+IFNg +TNFa
75
50
25
0
total CFU / tube (log 10)
%input recovered
100
5
control
+IFNg
+TNFa
+IFNg +TNFa
4
3
2
0
24
48
72
Time post-infection (hours)
3
48
72
Hours post-infection
Figure 3. Effect of IFN and TNF on SCHU S4 growth in human alveolar macrophages.
Human alveolar macrophages were infected with SCHU S4 at MOI = 10 and incubated with
500 U/ml IFN and/or TNF. At the indicated time points, infected macrophages were lysed
to determine the total bacterial load
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
UNM obtains a limited quantity of AMs from human donors, relative to the variety of in vitro
AM conditions to be tested. We will determine whether number of human alveolar
macrophage cells per well can be reduced for each assay so that the number of replicate
wells can be increased to improve reproducibility.
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
20%
9. Work plan for upcoming month
a. Search published literature and review previous studies in the lab with human AMs
for more consistent and reproducible techniques, e.g. comparing culture in tubes and
on plates.
b. Reduce the number of AMs per well required for assay to increase the number of
replicates.
c. Repeat experiment to examine the effect of cytokines on bacterial growth in human
alveolar macrophages
Milestone 21
Milestone description: Correlates of protection: in vitro assay or other readout of
effector function of Ft developed for multiple species.
Institution: LBERI/UNM
1. Date started: 4/8/2008
2. Date completed: In progress
Page 22 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
3. Work performed and progress including data and preliminary conclusions
No work was performed during this reporting period.
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
2% of the scientific work is complete
9. Work plan for upcoming month
Repeat the ICCS assay and include a positive mitogen control (Con A). PBMCs from the
NHPs vaccinated in October 2008 will be used in the assay.
Milestone 21
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM/LBERI
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment FT-AH-8 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Andrew's experiments\FT-AH-8)
i. The purpose of this experiment was to determine whether pretreatment with
IFN and/or TNF for 48 h would activate human PBMC sufficiently to control
LVS and SCHU S4 growth in vitro
ii. In previous experiments, PBMC treated with cytokines at the time of bacterial
infection did not have any inhibitory effect on SCHU S4 growth. This is
possible if there was not enough time to activate the PBMC before they were
overwhelmed by the growing SCHU S4 culture.
iii. In this experiment, PBMC were pretreated with 100 U/ml TNF and/or IFNg 48
h before LVS/SCHU S4 infection at MOI = 1. The pretreatment was
postulated to activate the PBMC to respond better to the SCHU S4 infection.
iv. As shown in Fig. 4 (A, B, and C), LVS and SCHU S4 infection reduced the
PBMC cell recovery at 48 and 72 h after infection to ≤ 5% of the starting cell
number. Cell recovery was considerably better from all of the cytokine pretreated PBMC cultures and the “protective” effect was stronger in LVS
cultures than with SCHU S4 cultures (note the scale on the Y axes).
v. The above observation correlated inversely with the number of bacteria
recovered from these cultures (Fig. 4 D and E). The cytokine pre-treated
cultures, especially those pre-treated with the combination of IFN and TNF
had the lowest bacterial burden. However, the results have to be interpreted
with caution because there were large variability among duplicate samples
(note the large error bars reflecting the standard deviation on some samples)
Page 23 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
vi. These results suggest that 48 h pretreatment of PBMC with TNF and/or IFN
may be required for any PBMC inhibitory effect on the bacterial growth to be
observed. These results also suggest that further development of a PBMCbased assay is warranted.
PBMC
+ IFNg
+ TNFa
+ IFNg/TNFa
60
40
20
LVS
C
50
40
PBMC
+ IFNg
+ TNFa
+ IFNg/TNFa
30
20
10
0
0
0
20
40
60
SCHU S4
25
20
10
5
72
48
Time post infection (h)
Time (h)
total CFU / well (log
LVS
E
5
4
3
PBMC
+ IFNg
+ TNF
+ IFNg/TNFa
2
1
0
3
48
72
Time post-infection (hours)
72
Time post infection (h)
10)
10)
D
PBMC
+ IFNg
+ TNFa
+ IFNg/TNFa
15
0
48
80
total CFU / well (Log
% of starting cell No.
80
% of starting cell No.
B
100
% of starting cell No.
Uninfected
A
SCHU S4
6
5
4
PBMC
+ IFNg
+ TNFa
+ IFNg/TNFa
3
2
1
0
3
48
72
Time post-infection (hours)
Figure 4. Effect of IFN and/or TNF pretreatment on PBMC cell recovery and bacterial growth. Human PBMC were
pretreated with 100 U/ml TNF and/or IFN 48 h before LVS or SCHU S4 infection and fresh cytokines were added daily.
The PBMCs were infected with LVS or SCHU S4 at MOI = 1. (A, B, and C) PBMC cell recovery and (D and E) bacterial
burden were determined at the indicated time points. Each bar represents the mean of two wells ± SD
a. Experiment FT-AH-7 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Andrew's experiments\FT-AH-7)
i. The purpose of this experiment was to determine whether PBMC from
unvaccinated and LVS vaccinated individuals can be differentiated.
ii. 2.5 x 105 PBMC from unvaccinated and LVS vaccinated individuals were
infected with SCHU S4 at MOI = 10. After 48 and 72 h, the cell recovery and
bacterial burden was determined
iii. Surprisingly, at 72 h, the naïve PBMC cultures had slightly better recovery
and lower bacterial burden than the vaccinated PBMC cultures (Fig. 6). We
will perform statistical analysis on these results to determine whether these
differences are significant.
Page 24 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
5
100
naive PBMC
4
total CFU/well
%input recovered
vaccinated PBMC
75
50
25
3
2
naive PBMC
vaccinated PBMC
1
0
0
0
24
48
72
Time post-infection (hours)
0
24
48
72
Time post-infection (hours)
Figure 6. Comparison of PBMC from LVS vaccinated and unvaccinated volunteers. 2.5 x 105
PBMC from unvaccinated and LVS vaccinated individuals were infected with SCHU S4 at MOI =
10. After 48 and 72 h, the cell recovery and bacterial burden was determined. The data
represent the mean of two wells ± SD
4. Significant decisions made or pending
Decide whether PBMC or monocytes can be used for the macrophage killing assay
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
55 %
9. Work plan for upcoming month
a. Measure the maximum potential of human PBMC/monocyte to be activated to inhibit
F. tularensis growth by testing parameters including reactivation with IFNγ, lower
MOI, and cytokine combination
b. Addition of vaccinated human T cells to PBMC/monocytes, if they are useful
c. Develop rat PBMC/monocyte assay similar to humans
d. Evaluate the usefulness of LVS/lux operon in RAW 264 murine macrophage-like cell
line
Milestone 29
Milestone description: Analysis of T cells from lymph nodes & T cell epitopes
Institution: LBERI/UNM
1. Date started: 8/7/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
Page 25 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
a. LVS bronchoscopy of A05403 was performed on 2/11/09 to boost. A05403 was
vaccinated via the LVS route on October 16, 2008.
b. Necropsy of A05403 occurred on 2/23/09 (day 12 post-LVS boost)
c. PBMC and spleen responses to HK and FF LVS are currently being analyzed.
d. Lymph nodes and spleen cells were transferred to UNM on 2/23/09 for use in the ASU
Ft polypeptide library cellular screening assay.
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
15% of the scientific work is complete
9. Work plan for upcoming month
Analysis of the PBMC and spleen responses to HK and FF LVS will be completed.
Milestone 29
Milestone description: Analysis of T cells from NHP lymph nodes and T cell epitopes
Institution: UNM
1. Date started: 10/1/08
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc82 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and
Results\Ftc82)
b. The purpose of this experiment was to screen the F. tularensis polypeptide library from
ASU with splenocytes and tracheobronchial lymph node cells from a LVS
vaccinated/boosted cynomolgus macaque using IFN ELISpot assay.
c. The entire set of polypeptides (in pools of 7peptides/well) was screened in duplicate.
There were clear plate to plate variations in the background which required us to
analyze the plates individually. The number spots per well was counted manually and
entered into an excel spreadsheet. We used the mean of all the samples on each plate
except the positive and negative controls plus 2 standard deviations as the minimum
cutoff for a positive. Based on this analysis we found several positive samples.
However, for most them, the positive results were not confirmed by the duplicate
sample or by the second tissue.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
UNM and ASU are developing a plan to confirm the positives detected. The remaining mass
of each polypeptide is the limiting reagent.
6. Deliverables completed
NA
7. Quality of performance
Good
Page 26 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
8. Percentage completed
7%
9. Work plan for upcoming month
a.
Develop and execute plan with ASU to confirm the results from the polypeptide
screening experiment
Milestone 35
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM/ASU Johnston
1. Date started: 8/1/2006
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
No new work performed since ASU made no request for RNA samples
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
None
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent
Schu 4 infection & RT PCR confirmation of candidates.
Institution: ASU-Johnston/ UNM
1. Date started: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Previous Results: We had noticed a drop in signal intensities on the most recent
print run of arrays. After an extensive evaluation, it was noted that there had been a
change in the hybridization temperature chamber from 60 to 65 C. As part of the
trouble shooting we had heat-treated the probe oligos used for printing (See Fig 2,
02/06/2009 Technical report). This figure showed that we could replicate the
previous hybridization signals with unamplified labeled SCHU S4 RNA, but that the
heat-treated oligos were slightly less efficient than non-heat-treated oligo probes
(average signal of 1,792 vs 860 respectively). Because the efficiency seemed
Page 27 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
reasonable and microarray printer problems which would delay analyses, we
proceeded with the hybridization using slides made from heat-treated oligos.

We have successful LAPT amplifications from both of the time course mouse and
single time course rat experiments. These LAPT-amplified RNA’s were labeled and
hybridized to the most recent print-run from heat-treated oligo’s. Data were median
normalized between experiments from at least two sets of hybridizations per time
point and the time points. For the two mouse time course and one rat time course
samples, the data were bioinformatically averaged within species. The expression
patterns across time were use to select the top two hundred genes that were either
up-regulated or down-regulated across the time course within a species response.
The data shown in Figure 1 are a heatmap of the mouse time-course samples for
both the up-regulated and down-regulated gene lists.
Figure 1. Heatmap of the average of the two mouse time courses top two hundred genes that
follow an up-regulation or down-regulation of expression across the time course of the infection.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\Lapt_Comparisons\LAPT29, 30, 31 tc1 tc2
tc3 comparisons\heatmap.png
Page 28 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam

The exact same analyses were carried out for the data from the rat time course and
using the gene lists for both mouse and rat data sets. The results of the Venn
diagram for the up-regulated genes are shown in Figure 2. Between the two hundred
independent gene lists, 42 upregulated genes cross-mapped between the infected
mice and rats. Interestingly, for the down-regulated gene sets, there was also an
intersection of 42 genes. The gene identifiers for these sets and the names, if
known, are presented in Table 1. This initial set of genes is a starting point for final
selections. We still have multiple samples of each time course to process for data
acquisition. The plan is to utilize multiple LAPT samples from each time course with
at least two labeling of each time course before the final gene sets are selected for
confirmation. The strategy will be to compare the gene lists obtained with mouse
studies and rat studies, and primate studies if available. These gene lists will be
compared to those from the literature regarding vaccine and virulence-related genes
to identify a set of genes for cloning and testing as potential vaccine candidates.
Page 29 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Figure 2. Venn-diagram analysis of the top 200 genes up-regulated in mice and rats.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milestones\35\Lapt_Comparisons\LAPT29, 30, 31
tc1 tc2 tc3 comparisons\Venn.png
FTT1008c
FTT0582
FTT0138
FTT1219c
FTT0115
FTT1353
FTT0526
FTT1399
FTT1442c
FTT0144
FTT0061
FTT1380
FTT1195c
FTT0503c
FTT0121
FTT0509c
FTT0150
FTT0562
FTT0778
FTT1184c
FTT1412
FTT0364c
FTT0969c
FTT1667
FTT0043
FTT0370c
FTT1369c
FTT0800
FTT1385c
FTT0844
FTT0130
FTT1375
FTT1034c
FTT0576
FTT0079
FTT1709
FTT0935c
FTT1336
FTT0982
FTT0445
FTT0988
FTT1405c
Up-regulated in both mice and rats
hypothetical protein
Ferredoxin
preprotein translocase, subunit E, membrane protein
conserved hypothetical protein
nucleoside permease NUP family protein
conserved hypothetical protein
hypothetical protein
conservered hypothetical membrane protein
DNA-directed RNA polymerase, alpha subunit
DNA-directed RNA polymerase beta chain
ATP synthase delta chain
conserved hypothetical membrane protein,pseudogene
conserved hypothetical protein, pseudogene
Succinyl-CoA synthetase, alpha subunit
DNA helicase II
conserved hypothetical protein
30S ribosomal protein S16
polyamine transporter, ABC transporter,ATP-binding protein
hypothetical protein
conserved hypothetical protein
Prolyl-tRNA synthetase
hypothetical protein
potassium uptake protein TrkA
hypothetical protein
NADH dehydrogenase I, M subunit
Nucleotide-binding protein, yjeE
Transketolase
haloacid dehalogenase-like hydrolase family protein
hypothetical protein
rossman fold oxidoreductase, pseudogene
glycerol kinase
3-oxoacyl-(acyl-carrier-protein) reductase
NADH dehydrogenase
conserved hypothetical protein
phosphoglucosamine mutase
conserved hypothetical protein
biotin synthesis protein BioC
ABC transporter, ATP-binding and membrane protein
hypothetical membrane protein
ABC transporter, ATP-binding, pseudogene
hypothetical protein
hydroxyacylglutathione hydrolase
FTT1534c
FTT0741c
FTT0564
FTT0260
FTT0990
FTT0247
FTT0656
FTT1476
FTT0169
FTT1443c
FTT0255c
FTT0286c
FTT1426c
FTT0068
FTT0311c
FTT0827c
FTT0802
FTT1120c
FTT0680c
FTT0475
FTT1435c
FTT0484
FTT0688c
FTT0610
FTT0269
FTT0327
FTT1452c
FTT0012
FTT1679
FTT1401
FTT1526c
FTT0174
FTT0857c
FTT1112c
FTT0862c
FTT0712c
FTT1675
FTT1754
FTT0536
FTT0535c
FTT0687c
FTT1475
Down-regulated in both mice and rats
conserved hypothetical protein
hypothetical protein
polyamine transporter, subunit I, ABC transporter, membrane protein
CrcB family protein
Leucyl-tRNA synthetase
Transposase
holliday junction endodeoxyribonuclease
Galactokinase
hemK protein homolog
ATPase, AAA family
hypothetical protein
UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase
conservered hypothetical membrane protein
superoxide dismutase [Fe]
conserved hypothetical protein
Hypothetical protein yieG
Cyanophycinase
queuine tRNA-ribosyltransferase.
Peptidyl-tRNA hydrolase
mechanosensitive ion channel protein
ABC transporter, ATP-binding protein
hypothetical protein
ATP-dependent protease, proteasome-related peptidase subunit
DNA/RNA endonuclease family protein
carbonic anhydrase, family 3
50S ribosomal protein L23
Glycosyltransferase
conserved hypothetical protein, pseudogene
30S ribosomal protein S20
prophage repressor protein
isocitrate dehydrogenase
YggT family protein
conserved hypothetical protein
RNA polymerase sigma-32 factor
heat shock protein HtpX
serine transporter
peptide deformylase
phosphate acetyltransferase
hypothetical membrane protein
lactate dehydrogenase
ATP-dependent protease, ATP-binding subunit
Galactose-1-phosphate uridylyltransferase
Table 1. List of genes that overlap between mice and rats for both the up- and down-regulated
mapped expression patterns.
File locations R:\GeneVac\FTU\Contract\Microarray\Milestones\35\Lapt_Comparisons\ LAPT29,
30, 31 tc1 tc2 tc3.xls
Page 30 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Perform a new microarray print from non-heat-treated oligo probes. We have made a new
set of non-heat-treated oligo master plates for dispensing, but a minor printer problem has
resulted in delays of printing. The printhead needs to be treated by electroplating to
resurface the head. The current head surface has deteriorated resulting in pins sticking in
the up position leading to drop-outs of the deposited oligos. A new surfacing will completed
by 3/13/2009 and a new set of printed slides will be available by 3/18/2009.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
76%
9. Work plan for upcoming month
Continue processing the hybridizations to finalize the data comparisons.
Identify gene lists for work-up by qPCR
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis
(SCHU S4) (iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F.
tularensis subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F.
tularensis subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful
deletions. Therefore, our focus is two fold, one is cloning experiments to get our target
deletions into vectors that we can use in creating these deletions and experiments with
SCHU S4 itself using constructs that we believe will allow us to make deletions into SCHU
S4.
I.
Cloning:
a. UTSA is continuing with a cloning strategy where we will be using a mating
vector pJC84 to facilitate the integration of NadM construct sequence into the
SchuS4 genome which will disrupt the NadM gene. The initial plasmid pGem-T
is only a cloning plasmid it does not have the required components which will
allow for this NadM T20 sequence to integrate into the genome of SchuS4. The
new NadM plasmid (pGemNadMT20) construction was verified to be correct by
sequencing. However, the first ligation between the pJC84 Sal I plasmid and the
Sal I NadM T20 isolated from the pGemNadM T20 plasmid did not yield the
correct construct; UTSA only found re-ligation profiles in the first ten screened
from the resulting DH5α transformants (Figure 1). So, UTSA decided to use two
Page 31 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
different restriction endonucleases to clone these components together. Instead
of using just one restriction site, we will use two restriction sites to directional
ligate the NadM T20 into pJC84. This will reduce the re-ligation frequency since
the two chosen enzymes do not have compatible ends for ligation. The pJC84
has a Sac I and a Sma I site in its multi cloning site and the pGemNadM T10
construct has Sac I and Nco I sites. The Sma I creates a blunt end. The Nco I
digested fragment can be treated with Klenow to generate a blunt end w for
ligation to the Sma I portion of the pJC84 plasmid. This is currently in progress.
Data located in TVD UTSA Notebook 7, page 73 and 75.
Figure1.
1 Kb
4.0
1.0
Sal I digested
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Legend:
1. 1 Kb Ladder
8. C5pJC84+NadMT20
2. Uncut pJC84
9. C6pJC84+NadMT20
3. pJC84
10. C7pJC84+NadMT20
4. C1pJC84+NadMT20 11. C8pJC84+NadMT20
5. C2pJC84+NadMT20 12. C9pJC84+NadMT20
6. C3pJC84+NadMT20 13. C10pJC84+NadMT20
7. C4pJC84+NadMT20 14. Uncut C10pJC84+NadMT20
Figure 1 represents ten plasmids resulting from the pJC84 Sal I +NadM T20 Sal I ligation transformants.
Lane 2 (uncut) and 3 (Sal 1 digested) are the pJC84 plasmid; lanes 4-14 are various pJC84+NadM T20
transformant’s plasmids (Sal1 digested except lane 14 which is undigested). The correct combined
pJC84 plus NadM plasmid should have yielded a ≈3600 bp band in addition to the ≈3800 bp plasmid
(pJC84) upon Sal 1 digestion. The transformants resulted in self re-ligation plasmid as evidenced by
the single 3800 bp band only rather than ligating the two different DNA components (the plasmid
pJC84 and the isolated NadM T20 fragment) together and giving rise to two Sal 1 restriction fragments
at 3600 and 3800.. Data located in TVD UTSA Notebook 7, page 75.
b. We proceeded with the cloning of the final mutant construct FTT0748 and based
on a algorithm analysis the following oligos were used to generate the “intron”
FTT0748::Ll:LtrB to clone into the pKEK1140. This represents the transposon
approach to cloning the NadM gene into SCHUS4.
FTT0748-561/562S-IBS
FTT0748-561/562S-EBS1d
FTT0748-561/562S-EBS2
The result of this polymerase chain reaction (PCR) is shown in figure 2. The
resulting 380 bp product was cleaned up by using the Qiagen gel extraction kit.
Page 32 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Figure 2.
1 Kb
1
2
Legend:
1. 1 Kb Ladder
2. FTT0748::Ll:LtrB
3. FTT0748::Ll:LtrB
3
1.5
0.5
Figure 2 represents the PCR products resulting when the FTT0748 561/562s oligo set and EBS
universal primer was used with Sigma’s intron template (Ll:LtrB). The correct product is the ≈380
bp product just below the 500 bp fragment in the 1 Kb ladder marker. Lane 2 and 3 are
independent polymerase chain reactions. Data located in TVD UTSA Notebook 7, page 79.
c.
The pKEK1140 plasmid along with the PCR products from figure 2 were digested
with Xho I and BsrGI restriction endunucleases simultaneously since they both
have 100% activity in the same buffer. These digestions were at 37ºC and
allowed to go overnight. The resulting digestions were run on a 1% agarose gel
(figure 3) and subsequently purified with the Qiagen gel extraction kit.
Figure 3.
1 Kb
1
12.5
0.5
2
3
4
Legend:
1. 1 Kb Ladder
2. FTT0748::Ll:LtrB
3. FTT0748::Ll:LtrB
4. KEK1140
Figure 3 represents the FTT048::Lt:LtrB PCR products (lanes 2 and 3) and the pKEK1140
plasmid (lane 4) digested with Xho I and BsrG1 restriction endonucleases. In lane 4 the
pKEK1140 with removal of the lacZ gene being results in the 900 bp fragment. We use
the remaining pKEK1140 vector portion (≈8.2 kb). Data located in TVD UTSA Notebook
7, page 80.
d. The purified cloning components mentioned in I.c. will be used in a ligation
reaction that will be run in the upcoming month. This will allow for the new
tulatron construct which we hope with generate the FTT0748 genetic tool to
create the FTT0748 Schu S4 mutant.
Page 33 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
II.
Experiments to generate mutants in Schu4:
a. The last report indicated various potential NadM mutants which were selected for
further passaging to generate more isolated clones. The original clones N2, N7
and N3 were used to passage. From the last report the candidates to continue
with passaging were N2F, N7-6 and N3-4B. The single colonies generated at
each passage were selected randomly from plate and given a name as indicated
on figures 4 and 5; also genomic isolations were made from some of each group
and used in a polymerase chain reaction using the earlier mentioned oligos:
nadM-NcoI: 5’- cgcgcgccatgggcatgtatgatatttcagtttttataggaagatttcag -3’
nadM-EcoR1: 5’- cggaattcttatagtttcttaccacattcctctaataaaatc -3”
These oligos will yield the desired mutant profile which is one band at ≈1900 bp
for the complete correct NadM mutant (Figures 4 and 5).
Figure 4.
1 Kb
1
2 3 4 5 6 7 8 9 10 11 12 13
2.0
1.2
Legend:
1. 1 Kb Ladder 13. N3-4 cont
2. KKT1
14. N3-4B,4-33A
3. N7-6,4-23A
15. N3-4B,4-33B
4. N7-6,4-23B
16. N3-4B,4-33C
5. N7-6,4-23C
17. N3-4B,4-33D
6. N7-6,4-23D
18. N3-4B,4-33E
7. N7-6,4-23E
19. N3-4B,4-33F
8. N7-6,5-3A
20. N3-4B,4-33G
9. N7-6,5-3B
21. N3-4B,4-33H
10. N7-6,5-3C
22. N3-4B,4-33I
11. N7-6,5-3D
23. N3-4B,4-33J
12. N7-6,5-3E
3.0
1.0
1 2 14 15 16 17 18 19 20 21 22 23 13
Figure 5.
1 Kb
1 2 3 4 5 6 7 8 9 10 11 12 13
2.0
0.9
Legend:
1. 1 Kb ladder
2. KKT1
3. N2F,3-1A
4. N2F,3-1B
5. N2F,3-1C
6. N2F,3-1D
7. N2F,3-1E
8. N7 orig
9. N2F,3-4A
10. N2F,3-4B
11. N2F,3-4C
12. N2F,3-4D
13. N2F,3-4E
Figures 4 and 5 represent PCRs using various cycled NadM mutants using oligos nadMNcoI and nadM-EcoR1 which target the 5’ and 3’ ends of the NadM gene, respectively.
Most of the screened clones are from cycle 5 except for the N3-4B,4-33A-J (figure 4,
lanes 14-23). These are from cycle 6. The correct NadM mutant should yield a ≈1900 bp
(“mutant band”) product only if the passaging has successfully selected for the mutant and
has eliminated the wild type. The KKT1 is used as the wild-type control yielding ≈1100 bp
fragment with this oligo set (lane 2 in both figures). In figure 5, lanes 9-13 reactions had
very little of the reaction left in their tubes after removing from thermocycler so, these are
Page 34 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
not accurate profiles for these clones (will need to redo these reactions, fyi). In figure 4
lane 13 (N3-4) and in figure 5 lane 8 (N7 orig) are profiles from original NadM clones used
in the passaging and are controls to following progression of passaging results. The
mutant band is very light on some of these original profiles but it is there at 1900 bp.
Comparatively, some of the various passaged NadM mutants seems to be yielding a
darker mutant band than their parents (original clones). In figure 4 lanes 3-12 and in
figure 5 lanes 3-7 seems to have at least equal the intensity of the correct mutant band
(≈1900 bp) compared to the parent. Some of these cycled clones appear to be yielding a
stronger mutant band which may indicate potential success in generating a clone
containing only the mutant band with additional passaging. Data located in TVD UTSA
Notebook 7, pages 76 and 77.
b. The genomic templates of the NadM clones N2F,3-1E, N7-6, 4-23A, N7-6, 5-3D,
N3-4B, 4-33g and N3-4, 4-33h in figures 4 and 5 were used for further screening
to insure that those clones yielding a ≈1900 bp band do in fact still have the
NadM intron inserted at the correct location in the chromosome. Therefore, the
oligo set EBS Universal and the forward NadM directed primer, nadM-Nco I
(described earlier) were used to check for presence of this NadM intron in the
chromosome (Figure 6). All of these candidates did have the correct size
product so these were chosen to continue with passaging. Data located in TVD
UTSA Notebook 7, page 77.
Figure 6.
1 Kb
1 2 3 4 5 6 7 8 9
1.5
0.9
Legend:
1. 1 Kb Ladder
2. KKT1
3. N3-4 control
4. N2F,3-1E
5. N7-6,4-23A
6. N7-6,5-3D
7. N3-4B,4-33g
8. N3-4B,4-33h
9. pKEK1261
Figure 6 represent the PCR products resulting when using oligos EBS Universal
and the forward NadM directed primers with various potential NadM Schu S4
mutants’ genomic preparations as templates. The expected size product for the
correct location of the NadM intron insertion in the Schu S4 chromosome should
be ≈900 bp. Lane 2 is the KKT1 which is considered the wild type profile where
there should be no product. Lane 9 is the tulatron plasmid construct of NadM
which was used to generate possible NadM mutants; this should result in no
product. Lanes 3 thru 8 are cycled NadM mutants which generated a correct
mutant band product during the first PCR screen (figure 4 & 5) using forward and
reverse primers to NadM gene. The selected mutant set here all have the intron
still intact at the correct gene location.
Page 35 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
c.
Therefore, this coming month will continued to passage some select clones by
streaking for single colonies on a TSA+++ 60ug/ml kanamycin plates which will
be grown and 30°C. Isolated clones from this passage will be screen by PCR to
search for the correct ≈1900 bp band without any wild type band present (1100
bp).
4. Significant decisions made or pending
The Milestone 49 is extended 6 months from original completion date and will end on
8/31/09 rather than 2/27/09.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
KKF5: igLC1 IgLC2 Schuh4: KKF10: iglD1 igLD2 Schuh4; and KKF13: VgrG1 VgrG2 Schuh4
mutants are completed Schuh4 strains to date.
7. Quality of performance
Good
8. Percentage completed
89%
9. Work plan for upcoming month
a. Will continue with the screening of the NadM mutant which will require cycling of various
clones to facilitate the effective insertion of the NadM intron into the SchuS4
chromosome.
b. Will continue with second strategy for generating NadM Schu S4 mutant via the pJC84
mating vector from Dr. Celli lab at the Rocky mountain labs. Once NadM T20 is cloned
into pJC84 will do restriction digestions and send for sequencing for confirmation.
c. Will continue with the FTT0748 construct cloning by performing a ligation of the prepared
components mentioned in report followed by transformation of DH5α cells. Resulting
transformants will be screen for the correct plasmid construct by both by restriction
analysis and sequencing.
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB;
LVS uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
pdpD, iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double
mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
iglA, iglB strains
Institution: UTSA
1. Date started: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A: Analyze the antibody profiles of mice orally immunized with KKF235 (Ft subsp.
novicida iglB) (Note book #9, pages 22-26). Blood, feces and bronchalveolar
lavage fluid (BAL) was collected from the PBS- and KKF235 (2350 CFU)- immunized
mice at day 21 after vaccination. Specific anti- KKF235 total antibody titer, as well as
IgG1, IgG2a, and IgA isotypes for serum, IgA and IgM isotypes for fecal samples,
Page 36 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
and total antibody, IgG1, IgG2a, IgA and IgM isotypes for BAL, were determined by
ELISA. Antigens, either UV-irradiated KKF235 (106/well) or HEL (Hen Egg Lysozyme,
1µg/well, an unrelated antigen as control), were coated onto 96-well microplates and
reacted with serial dilutions of sera or undiluted fecal and BAL samples. Goat anti
mouse Ig(H+L), IgG1, IgG2a, IgA and IgM antibody conjugated with horseradish
peroxidase were used as the secondary antibodies to determine antibody isotypes
and titers.
As shown in Fig. 1, mice immunized orally with KKF235 produced high serum titers
of KKF235-specific total, IgG1 and IgG2a antibodies. The response of the IgG2a
isotype was equally expressed compared to IgG1 implying dominants of both Th1
and Th2 immune response. KKF235-specific antibody was not detected in mice
mock-vaccinated with PBS. All tested serum samples showed no reactivity to the
unrelated HEL protein (data not shown). As shown in Fig. 2A, orally immunized mice
produce a low level of KKF235-specific IgA in the G.I. tract, while almost no IgM.
However, mice vaccinated orally with KKF235 produce a wide array of specific
antibodies in the BAL including high level of total antibody as well as IgG1, IgG2a,
IgA, and small amount of IgM as shown in Fig. 2B,. Again, almost same amount of
IgG1 and IgG2a were produced indicating a mixed Th1 and Th2 response. Little to
no KKF235-specific antibody was detected in mice mock-vaccinated with PBS. All
tested BAL samples showed no reactivity to the unrelated HEL protein (data not
shown). In summary, oral immunization with KKF235 (Ft subsp. novicida ΔiglB)
induces significant KKF235-specific systemic and respiratory antibody production.
10000
KKF235
Ab Titer
Mock (PBS)
1000
100
Total Ab
IgG1
IgG2a
Fig 1. Humoral responses to oral KKF235 immunization. Groups of mice (6 mice
/group) were vaccinated orally with 2350 CFU of KKF235 or PBS as a control. Sera
were collected 3 weeks later and analyzed to determine titers for anti-KKF235
specific antibodies.
Page 37 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
A
Intestinal Antibodies
O.D.
1. 00
IgA
0. 80
0. 60
0. 60
0. 40
0. 40
0. 20
0. 20
0. 00
0. 00
IgM
KKF235
Mock (PBS)
Respiratory Antibodies
B
1. 00
Total
Ab
1. 00
IgG1
1. 00
IgG2a
1. 00
IgA
1. 00
0. 80
0. 80
0. 80
0. 60
0. 60
0. 60
0. 60
0. 60
0. 40
0. 40
0. 40
0. 40
0. 40
0. 20
0. 20
0. 20
0. 20
0. 20
0. 00
0. 00
0. 00
0. 00
0. 00
0. 80
O.D.
1. 00
0. 80
0. 80
IgM
Fig 2. Humoral responses to oral KKF235 immunization in Fecal and BAL.
Groups of mice (6 mice /group) were vaccinated orally with 2350 CFU of
KKF235 or PBS as a control. Fecal (intestinal) and BAL (respiratory)
samples were collected 3 weeks later and analyzed to determine titers for
anti-KKF235 specific antibodies.
Oral LVS vaccination was used as a control, the serum and fecal antibodies were
detected at D21 after vaccination (2130 CFU). As shown in Fig. 3, anti-LVS antibody
was induced in LVS-vaccinated mice. Similar amount of IgG1 and IgG2a were
produced suggesting mixed Th1 and Th2 response. Moreover, significant amount of
anti-LVS specific IgA was detected in fecal samples of the LVS-vaccinated but not
PBS mock vaccinated mice (Fig. 4). All tested sera and fecal samples showed no
reactivity to the unrelated HEL protein (data not shown).
Collectively, oral
immunization with LVS induces significant LVS-specific systemic and secretory
antibody responses.
Page 38 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
10000
LVS
Ab Titer
Mock (PBS)
1000
100
Total Ab
IgG1
IgG2a
Fig 3 Humoral responses to oral LVS immunization. Groups of mice (5 mice
/group) were vaccinated orally with 2130 CFU of LVS or PBS as a control.
Sera were collected 3 weeks later and analyzed to determine titers for antiLVS specific antibodies.
Intestinal Antibodies
O.D.
1.00
IgA
1.00
0.80
0.80
0.60
0.60
0.40
0.40
0.20
0.20
0.00
0.00
IgM
LVS
Mock (PBS)
Fig 4 Humoral responses to oral LVS immunization in Fecal . Groups of mice (5 mice
/group) were vaccinated orally with 2130 CFU of LVS or PBS as a control. Fecal samples
were collected 3 weeks later and analyzed to determine titers for anti-LVS specific
antibodies.
Page 39 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
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
96% of scientific work completed on milestone 50A (original plans)
9. Work plan for upcoming month
50A. Evaluation of protective efficacy of oral KKF235 (iglB of U112) vaccination
against Francisella Type B (OR96-0246 virulent strain) challenge.
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu
S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
3.1 Creation of RecA and IglC double mutant in F. tularensis tularensis (SCHU S4).
This part of Milestone 52 is to create recA and IglC double mutant in F. tularensis tularensis.
Inactivating the recA gene will stabilize the strain and prevent the strain from any additional
genetic changes. We already have the IglC mutant of Schu S4, and the tulatron vector
pKEK1186 for disturbing and inactivating the recA gene in Francisella tularensis.
3.1.1 In last monthly report, it was reported that colony PCR was performed using recA gene
primers to screen 10 colonies obtained from cryotransformation, and all of 10 colonies had the
same result as the positive control, which indicated that the intron insertion was in the gene of
recA. Another colony PCR was amplified using the primer from the insertion and the other
primer from recA gene to further confirm the intron insertion in recA gene (see Step3.1.2 in
this monthly report for details).
3.1.2 Colony PCR for the same 10 colonies was performed using the recA gene primer “recA
Schu4 for” and the insertion specific primer “EBS Universal” to verify the insertion in recA
gene of KKT5. PCR reaction was set up as follows and the reagents for PCR were purchased
from Promega Inc:
5XGreen GoTaq Buffer
dNTPs mix, 10mM each
recA Schu4 for (25pmol/ul)
EBS Universal (25pmol/ul)
GoTaq DNA polymerase
DNA
DNAse,RNAse free water
4.0ul
0.4ul
1.0ul
1.0ul
0.1ul
1.0ul
12.5ul
At 95°C 2 min, 95°C 30 sec/55°C 30 sec/72°C 1 min//30 cycles, 72°C 5 min
Page 40 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Figure1: Gel picture of colony PCR using the insertion specific primer.
Figure1 legend, results and data location: Lane7 was KKF348 (recA mutant U112) as the positive control,
which had the intron insertion in recA gene of U112 and the PCR product generated was about 0.5kb. Lane8
was the parent strain KKT5 (IglC mutant Schu S4) as the negative control without PCR product generated
since one of the primers was from the insertion. Lane2-lane6 was colony1-5 and lane9-lane13 was colony610. All of the colonies produced the same result as the positive control (lane7), which meant that the
mutated intron was inserted into recA gene to generate PCR product using the primer in the insertion. The
result confirmed that the mutated intron was in recA gene of the screened colonies.
Data recorded on UTSA TVDC notebook #6, page62 for Figure1.
3.1.3 To remove the remaining tulatron vector from the strain, colony8 was streaked onto
TSA++ agar plate and incubated at 37°C to kill the plasmid in the strain. The tulatron vector is
a temperature sensitive plasmid that is removed from the strain as the temperature changed
from 30°C to 37°C. After 3-4 days incubation at 37°C, 52 single colonies were obtained and
patched onto both TSA++/Kanamycin(50ug/ml) and TSA++ plates, and incubated at 37°C for
2-3 days to select Kanamycin sensitive colonies. About 27 colonies did not grow on
TSA++/Kanamycin agar media. Of 27 colonies, colony8-1, 8-2, 8-3, 8-6, 8-10, 8-26, 8-29 and
8-33 were patched onto fresh TSA++/Kanamycin(50ug/ml) agar plate to make sure no growth
on Kanamycin media. After 3-4 days incubation at 37°C, colony8-1, 8-2, 8-3, 8-26 and 8-29
were sensitive to Kanamycin definitely, which indicated that the plasmid from these 5 colonies
were removed.
Data recorded on UTSA TVDC notebook #6, page62-63.
3.2 Creation of FTT1579 and FTT0523 gene mutants in Schu S4. FTT1579 functions as Type
III restriction enzyme, and FTT0523 as the hypothetical protein that is similar to Q89Z57 Type
I restriction enzyme EcoAI specificity protein. Both of FTT1579 and FTT0523 genes limit the
entrance of the plasmid DNA into Schu S4 strain. This goal is to break down the restriction
barriers (FTT1579 and FTT0523) of Schu S4. The method allows us to retarget these two
restriction enzymes and inactivate certain gene(s) to facilitate introduction of the plasmid DNA
into Schu S4 strain.
3.2.1 Since FTN1487 and FTT1579 are the same genes but in different strains (U112 and
Schu S4) respectively, we decided to knockout the gene in U112 before working on Schu S4.
Page 41 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
FTN1487 (same as FTT1579) and FTT0523 gene sequences were obtained from Francisella
Tularensis web site at www.francisella.org and pasted into the algorithm on the Targetron
Design Site at www.sigma-aldrich.com/targetron. We searched for potential insertion sites.
849/850bp was chosen for FTN1487 or FTT1579 insertion site and 390/391bp for FTT0523
gene insertion site.
Data recorded on UTSA TVDC notebook #6, page 69-70.
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.
About 52% of scientific work completed
9. Work plan for upcoming month
i Screen the Kanamycin sensitive colonies obtained on Step3.1.3 using colony PCR.
ii Sequence the PCR product from above (i) to determine the insertion in RecA of
KKT5.
iii Design the primers to make the tulatron vectors for FTN1487 (FTT1579) and FTT0523
gene disturbance FTN1487 and FTT1579 have the same gene sequences. FTN1487
functions as restriction endonuclease in U112. Similarly FTT1579 functions as Type III
restriction enzyme, and FTT0523 as the hypothetical protein that is similar to Q89Z57 Type I
restriction enzyme EcoAI specificity protein.
Milestone 53B
Milestone description: Examining the protective efficacy of LVS and two attenuated
SCHU S4 mutant strains via oral vs. intradermal inoculations in the rat model;
50.1: replication of LVS, Schuh4, iglC Schuh4, and one additional attenuated Schuh4
mutant derived in milestone 49 in rat macrophages .
50.2: protective efficacy of LVS, iglC Schuh4, and one additional attenuated Schuh4
mutant derived in milestone 49 against Schuh4 intratracheal challenge (oral vs.
intradermal vaccinations in rats)
50.3: antigen specific cellular and humoral responses of rats following vaccination with
LVS, iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49
50.4: bacterial dissemination and lung pathology of rats following vaccination with LVS,
iglC Schuh4, and one additional attenuated Schuh4 mutant derived in milestone 49
Institution: UTSA
1. Date started: 12/01/2008
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
53B-a: (1) Replication of F. novicida U112 and F. holarctica within rat bone marrow
derived macrophages. (Note book # 10, pages 29, and 31-32). Bone marrow derived
Page 42 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
macrophages were derived from Fisher 344 rats, seeded in 96-well culture plates at a
density of 2 X 105 cells per well and allowed to adhere over night. Cells were infected
with either F. novicida or F. holarctica at 10 and 100 MOI for 2 hours. Cells were then
pulsed with Gentamicin for 1 hour to kill any remaining extracellular bacteria, after
which they were incubated at 37 degrees C. Cells were lysed at 3, 24, 48, or 72
hours after infection and serial dilutions of lysate were plated on TSA plates to
enumerate intracellular bacteria. As shown in Figure 1, there was an initial uptake of
103-104 CFU of F. novicida at 3 hours followed by 1-2 logs of replication by 24 hours
post-infection. From 48 to 72 hours after infection, the numbers of viable F. novicida
slowly decreased. In contrast, only 101 – 102 CFU of F. holarctica were taken up by
the macrophages initially. However, these bacteria continued to slowly replicate
throughout the time course and reached numbers which were similar to that of F.
novicida. This data indicates that while F. novicida is able to only replicate early in the
infection process, F. holarctica is able to continually replicate throughout a longer
period of time.
F. nov ic ida
F. holarc tic a
10 MOI
10
7
10 6
10
6
10 5
10
5
10 4
10
4
10 3
10
3
10 2
10
2
10
1
CFU
10 7
10 1
3
24
48
72
100 MOI
3
24
48
72
Hours After Inoc ulation
Fig. 1. Intramacrophage growth of F. novicida and F. holarctica in rat BMDM.
Primary bone marrow derived macrophages derived from Fisher 344 rats were
infected with F. novicida U112 or F. holarctica at either 10 or 100 MOI. Cells were
lysed and viable bacteria were counted at 3, 24, 48 and 72 hours after infection.
(2) Replication of F. novicida U112 and F. tularensis SCHU S4 within rat bone
marrow derived macrophages. The bone marrow derived macrophages are being
generated from rats and this assay is under development progress.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
12%
Page 43 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
9. Work plan for upcoming month
53B. (1) Perform phagocytosis assay of F. tularensis SCHU S4 with F344 rat bone
marrow derived macrophages.
Milestone 55
Milestone description: Compare cellular Immunogenicity of Francisella and ListeriaBased vaccine platforms. Measure cellular immunogenicity of live-attenuated vaccine
platforms. Compare immunogenicity of KBMA tularemia vaccine platforms
Institution: Cerus/Anza
1. Date started: 4/1/2008
2. Date completed: Pending;
3. Work performed and progress including data and preliminary conclusions
Anza terminated scientific support for Cerus as of 2/15/09; no new work reported
for February
Summary of objectives: We will construct and prepare live and Killed But Metabolically
Active (KBMA) Listeria monocytogenes (Lm) vaccines expressing Francisella tularensis
(Ft) antigens. To directly compare the cellular immunogenicity of Lm and Ft-based
vaccines, each Lm vaccine candidate will express an antigen fused to a model ovalbumin
epitope SIINFEKL (SL8) and these will be compared to Ft vaccines expressing pepO-SL8
fusions (provided by UTSA). We will measure the ability of each vaccine to stimulate a
CD8 T cell response in vitro using a B3Z assay. We will measure the cytokine responses
elicited by vaccination with each platform in mice, compare the CD8 T cell response to
SL8 after prime and boost vaccinations in mice using intracellular cytokine staining (ICS)
and ELISpot assays and measure the potency of the T cells elicited by use of an in vivo
cytotoxicity assay.
Summary of key achievements: We have demonstrated that IglC-SL8 fusion proteins are
expressed to a much higher level than KatG-SL8 in the cytosol of macrophages and
dendritic cells (DCs). Live-attenuated vaccines expressing either fusion protein were
able to secrete antigen within DCs and stimulate the B3Z T cell line that responds to the
SL8 peptide. The IglC-SL8 fusion protein induced a stronger immune response in mice
than KatG-SL8 by ICS and ELISpot analysis. Incorporation of a constitutively active prfA
allele (G155S) into the chromosome of the live-attenuated Lm-IglC-SL8 vaccine
increased immunogenicity by 2-fold. Inclusion of a much larger tag (containing an
additional 4 epitopes from vaccinia virus) decreased the immunogenicity of the Lm
vaccine. We also cloned bivalent vaccine strains (in both native prfA and prfAG155S
backgrounds) that express both KatG-SL8 and IglC-fused to a single strong vaccinia
virus epitope (B8R). The amount of intracellular antigen expression was measured using
a semi-quantitative Western blot and was found to be similar to each of the monovalent
strains but there appears to be a slight decrease in the amount of IglC secreted from the
bivalent strains. In the prfAG155S background the difference was less than 2-fold. The
bivalent vaccine strains also induced immune responses in C57BL/6 mice against the
epitope tags that were similar in magnitude to an equivalent dose of monovalent strains
expressing either KatG-SL8 or IglC-B8R; however the bivalent strain with the native prfA
background induced significantly lower B8R-specific responses. Overall, differences
seen between bivalent and monovalent strains appeared to be greater in the native prfA
than in the prfAG155S background. We also compared the primary immune response
after a single vaccination with Live and KBMA Lm-IglC-SL8 and found that KBMA Lm
induced T cell responses that were approximately one fifth the magnitude of live-
Page 44 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
attenuated. This reduction in potency of KBMA compared to live Lm immunogenicity is
consistent with our previous work with other antigens and it is likely that the potency of
the KBMA vaccine will be improved with a boost vaccination and by the use of the
prfAG155S allele. An initial comparison of Lm and Ft vaccines was performed and
suggested that LVS-pepO-SL8 did not induce a primary T-cell response against SL8 nor
did it boost a response induced by Lm-IglC-SL8.
1) Cloning and characterization of live attenuated bivalent Listeria monocytogenes
(Lm) tularemia vaccine strains. A new expression technology has been employed by
Anza. By using the carboxy-terminal region of ActA (including the transmembrane
domain) we have been able to target antigen expression to the surface of the bacteria
rather than have it secreted. This has two potential advantages: 1) any antigen preexpressed by the bacteria in culture remains associated with the bacteria rather than
being washed away when the vaccines are formulated for injection stocks; 2) the
proximity to the bacterial membrane may increase expression of hydrophobic antigens
that are poorly secreted. In order to determine whether this surface anchoring helps to
increase antigen expression or immunogenicity we have constructed a KatG-surface
anchored expression construct and have put it in the Lm11 and Lm677 background. We
have also cloned bivalent strains that co-express surface anchored-KatG and secreted
IglC to determine the impact this alternative targeting has on the secretion of IglC.
Intracellular antigen expression will be determined using the multiplex Western blot
described previously. A summary of vaccine candidates that have been constructed is
presented in table I below for reference; the new strains are highlighted in red.
Table I
Strain
Lm11
Genetic Background
actAinlB
Antigen Cassette
none
Status
Sequence verified
Notebook, page
Lm583
actAinlBuvrABprfAG155S
none
Sequence verified
Lm677
actAinlBuvrABprfAG155S
none
Sequence verified
BH137
actAinlB
ActAN100-Ova
Sequence verified
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
NB977, p52
BH2282
actAinlB
ActAN100-KatG-SL8
Sequence verified
NB736, p137
BH1228
actAinlBuvrAB
ActAN100-IglC-SL8
Sequence verified
NB977, p52
BH1398
actAinlBuvrAB
ActAN100-KatG-SL8
Sequence verified
NB977, p152
BH2094
actAinlBuvrABprfAG155S
ActAN100-IglC-SL8
Sequence verified
NB899, p11
BH2172
actAinlBuvrABprfAG155S
ActAN100-KatG-SL8
Sequence verified
NB899, p49
BH2098
actAinlB
ActAN100-IglC-VacQuad-SL8
Sequence verified
NB899, p13
BH2100
actAinlBuvrABprfAG155S
ActAN100-IglC-VacQuad-SL8
Sequence verified
NB899, p13
BH2180
actAinlB
ActAN100-IglC-B8R (@ comK)
Sequence verified
NB899, p51
BH2182
actAinlBuvrABprfAG155S
ActAN100-IglC-B8R (@ comK)
Sequence verified
NB899, p51
BH2316
actAinlB
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
NB899, p56
BH2292
actAinlBuvrABprfAG155S
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8 (@tRNAarg)
Remade and verified
(BH2184 had point
mutation in KatG)
Sequence verified
BH2562
actAinlB
ActAN100-KatG-Anchored
Not sequenced
NB2008, p62
BH2568
actAinlBuvrABprfAG155S
ActAN100-KatG-Anchored
Not sequenced
NB2008, p62
BH2564
actAinlB
Not sequenced
NB2008, p62
BH2566
actAinlBuvrABprfAG155S
ActAN100-KatG-Anchored
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-Anchored
ActAN100-IglC-B8R (@ comK)
Not sequenced
NB2008, p62
NB736, p138
Page 45 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
2) Lots of Live attenuated and KBMA vaccines produced. In order to facilitate
testing of the monovalent and bivalent strains of Lm at UNM and at Anza, we previously
Strain
Antigen cassette
type
Titer (CFU/mL)
Lot#
location
BH2172
Genetic
Background
Lm677
KatG-SL8
Live
CH-FR80-015
Lm677
IglC-B8R
Live
2.41 x 1010
1.96 x 1010
837-15-A
BH2182
837-15-B
CH-FR80-002
BH2292
Lm677
KatG-SL8/IglC-B8R
Live
CH-FR80-002
Lm11
KatG-SL8/IglC-B8R
Live
2.20 x 1010
1.74 x 1010
837-15-C
BH2316
837-15-D
CH-FR80-002
BH2100
Lm677
IglC-VacQuad
KBMA
963-104a
CH-FR80-002
BH2182
Lm677
IglC-B8R
KBMA
9.9 x 109 P/mL
0 cfu/mL
9.7 x 109 P/mL
2002-060A
CH-FR80-002
0 cfu/mL
9.6 x 109 P /mL
2002-060B
CH-FR80-002
2002-070
CH-FR80-042
BH2292
Lm677
KatG-SL8/IglC-B8R
KBMA
BH2172
Lm677
KatG-SL8
KBMA
0 cfu/mL
8.9 x 109 P /mL
0 cfu/mL
produced 100mL scale lots of live attenuated BH2172, BH2182, BH2292, and BH2316
and 400mL scale lots of KBMA
vaccines. A summary of the available lots is presented in Table II.
Table II. Lm vaccine lots produced and available for distribution to TVDC team members.
4. Significant decisions made or pending

All work at Anza has ceased. Cerus will respond to UNM’s request for a letter of
determination.
5. Problems or concerns and strategies to address



Due to Anza’s termination, all scientific and management personnel left Anza
UNM requested a letter of determination from Cerus
UNM can test Cerus/Anza generated bacterial strains at UNM
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
65%: Anza terminated scientific support for Cerus as of 2/15/09; no new work
reported for February
9. Work plan for upcoming month





Transfer bacterial strains to UNM.
Inventory equipment at Cerus for transfer to UNM
Complete Milestone Completion Reports for MS41,42,44,46,55,56,57
Hire Justin Skoble as a consultant with Cerus to write the TVDC MSCRs
MTA is fully executed by UNM/Cerus/LBERI/UCLA. Live and KBMA Lm lots will be
sent to UNM for evaluation in SchuS4 challenge model.
Page 46 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Milestone 56
Milestone description: Characterize the cellular immune response that correlates
with protection against an LVS Challenge and demonstrate that Cerus strains of
live and KBMA Lm-IglC and Lm-KatG protect against a SchuS4 challenge
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Anza terminated scientific support for Cerus as of 2/15/09; no new work reported
for February
Summary of objectives: We will measure the T cell response to IglC induced by live and
KBMA Lm expressing IglC compared with those elicited by Ftn or LVS vaccination. We
will produce an IglC overlapping peptide library (15aa overlapping by 11aa) to identify
IglC epitopes that are recognized by mouse T cells. We will use the IglC peptide library
for ELISpot and ICS assays to measure the IglC-specific T cell responses induced after
vaccination with live and KBMA Lm-IglC and to compare responses induced by live and
KBMA Ftn and LVS vaccination. We will demonstrate that the mechanism of protection
induced by Lm vaccines is cellular, by depletion of T cell populations and passive transfer
studies. We will demonstrate that strains of live and KBMA Lm-IglC-SL8 and Lm-KatGSL8 protect against a SchuS4 challenge and we will produce lots of KBMA vaccine and
send to UNM for testing in animal models (mice and rats).
Summary of key achievements: We determined that Lm strains expressing IglC can
induce IglC-specific immune responses in five different strains of mice (Balb/c, C57BL/6,
FVB/NJ, C3H/HeJ, and SJL/J). Immune responses were primarily observed to peptides
in IglC pool2 (peptides 26-51). By performing ELISpot assays using individual peptides,
we were able to map the responses to specific regions of the IglC protein. Using ICS and
flow cytometry, we were able to determine which responses were mediated by CD4+ or
CD8+ positive T cells. IglC-specific CD4+ T cell responses were identified in Balb/c,
C3H/HeJ, and FVB/NJ mice. We mapped CD8+ T cell epitopes using 9 mers
overlapping by one amino acid, identifying IglC34-142 (LFIDSLTIA) in Balb/c mice and
IglC137-144 (33-19, IMIDLSNL) in C57BL/6. We demonstrated that Lm vaccines
expressing IglC can provide 100% protective immunity against a 10 LD 50 LVS challenge
and Lm expressing KatG provided 40% protection (confirming data generated by the
Horwitz lab at UCLA). A single vaccination with KBMA-IglC induced an IglC response
that was barely distinguishable from background.
4. Significant decisions made or pending
All work at Anza has ceased. Cerus will respond to UNM’s request for a letter of
determination.
5. Problems or concerns and strategies to address



Due to Anza’s termination, all scientific and management personnel left Anza
UNM requested a letter of determination from Cerus
UNM can test Cerus/Anza generated bacterial strains at UNM
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
Page 47 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
45%- Anza terminated scientific support for Cerus as of 2/15/09; no new work
reported for February
9. Work plan for upcoming month





Transfer bacterial strains to UNM.
Inventory equipment at Cerus for transfer to UNM
Complete Milestone Completion Reports for MS41,42,44,46,55,56,57
Hire Justin Skoble as a consultant with Cerus to write the TVDC MSCRs
MTA is fully executed by UNM/Cerus/LBERI/UCLA. Live and KBMA Lm lots will be
sent to UNM for evaluation in SchuS4 challenge model.
Milestone 57
Milestone description: Optimization of KBMA Lm Vaccination Route and Regimen.
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Anza terminated scientific support for Cerus as of 2/15/09; no new work reported
for February
Summary of objectives: We will compare various routes of administration including IV, IM,
IN, ID and oral. For oral, IN, and ID administration in mice, we will first mutate the inlA
gene of Lm to allow for binding of murine E-cadherin in order to mimic the human
interaction (as described in Wollert et al., Cell, 2007). We will compare the potency of the
M
inlA gain of function mutants to our traditional platform strain. Routes will be ranked by
ability to induce a cellular immune response using ELISpot, ICS, and in vivo cytotoxicity.
We will optimize dosing regimen of most potent and tolerable route. Lm expressing IglC
and/or KatG will be used to evaluate immunogenicity. Optimized route and regimen will
be confirmed by SchuS4 protection studies at UNM.
Summary of Key achievements: We have constructed vaccine candidates that contain
M
the inlA gain of function mutations (Table III). The sequence of the wild-type EGDe inlA
gene (from the Lm strain used in the Wollert manuscript) was synthesized and the inlA
WT
gene in our platform strain was replaced (inlA ) in our live-attenuated and KBMA
platform strains as there are a number of differences in the sequence between the native
sequences between these strains. Two point mutations, S192N and Y369S, were
M
incorporated into the EGDe inlA sequence (inlA ) and inserted into the chromosome of
our live-attenuated and KBMA platform strains. Into these 4 strains the ActAN100-iglCSL8 expression cassette was inserted using the integration vector pINT. Cellular
invasion assays were performed: invasion of CaCo2 cells was dependent on inlA, as a 
M
inlA strain was unable to invade, but we were not able to demonstrate that the inlA gain
wt
of function allele increased invasion compared to inlA (as published by Wollert et.al).
Oral and IV routes of administration were compared: In spleens, SL8 and IglC responses
were 2-3 times lower after oral immunization than with IV administration, but mucosal
responses from intra-epithelial lymphocytes (IELs) were similar after immunization by
M
either route. Mice that were vaccinated orally with the inlA strain had marginally higher
splenic T cell responses and IEL responses that were 3-4 times higher than the isogenic
wt
strain expressing inlA . This preliminary result suggests that there may be a slight
M
increase in immunogenicity when the inlA vaccine strain is administered orally.
Page 48 of 49
Tularemia Vaccine Development Contract: Technical Report
Period: 2/01/2009 to 2/28/2009
Due Date: 3/12/2009 and Prepared by: Rick Lyons, Barbara Griffith ,Terry Wu, Bob Sherwood,
Michelle Valderas, Julie Wilder, Julie Hutt, Trevor Brasel, Dana Pohlman, Mitch Magee, Kathryn
Sykes, Stephen Johnston, Karl Klose, and Bernard Arulanandam
Table III: Strains constructed for murine route of administration studies
Strain
Genetic Background
Antigen Cassette
Status
Notebook,
page
CRS-100
actAinlB
none
Sequence verified
WT
none
Sequence verified
WT
ActAN100-IglC-SL8
Sequence verified
none
Sequence verified
ActAN100-IglC-SL8
Sequence verified
none
Sequence verified
NB899, p. 44
NB899, p. 48
NB899, p.49
NB899, p. 52
NB899, p. 44
NB899, p.48
ActAN100-iglC-SL8
Sequence verified
NB899, p. 44
M
none
Sequence verified
M
ActAN100-iglC-SL8
Sequence verified
NB899, p.48
NB899, p.44
BH2130
actAinlBinlA
BH2164
actAinlBinlA
BH2170
actAinlBinlA
M
BH2194
M
BH2132
actAinlBinlA
actAinlB
BH2166
uvrABprfAG155SinlA
actAinlB
WT
WT
uvrABprfAG155SinlA
BH2134
BH2168
actAinlBuvrABprfAG155SinlA
actAinlBuvrABprfAG155SinlA
4. Significant decisions made or pending
All work at Anza has ceased. Cerus will respond to UNM’s request for a letter of
determination.
5. Problems or concerns and strategies to address



Due to Anza’s termination, all scientific and management personnel left Anza
UNM requested a letter of determination from Cerus
UNM can test Cerus/Anza generated bacterial strains at UNM
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
25%- Anza terminated scientific support for Cerus as of 2/15/09; no new work
reported for February
9. Work plan for upcoming month




Transfer bacterial strains to UNM.
Inventory equipment at Cerus for transfer to UNM
Complete Milestone Completion Reports for MS41,42,44,46,55,56,57
Hire Justin Skoble as a consultant with Cerus to write the TVDC MSCRs
.
Page 49 of 49