Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
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, 8, 9, 10, 11(UNM &LBERI), 12/13 (UNM &LBERI), 14, 17, 18, 19, 21(UNM
29 (UNM &LBERI), 35 (UNM/ASU), 36, 49, 52, 53
Completed milestones: 1, 3, 4, 5, 6, 7, 16, 25, 26, 27, 28, 32, 33, 34, 39, 40, 43, 48, 50, 51
Inactive milestones: 15, 20, 22, 23, 24, 30, 37, 38, 54
&LBERI),
Milestones terminated after initiation: 41, 42, 44, 46, 55, 56, and 57 (MSCR will be written)
Milestones terminated before initiation: 43 (Cerus) 45, 47, 58, and 59 (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.Three UNM and possibly 6 LBERI scientists will receive LVS vaccinations in 2009 and 2010.
b.USAMRIID (United States Army Medical Research Institute for Infectious Disease) tentative
vaccination dates are November 18, 2009 and January 26, 2010.
c.UNM submitted risk assessment forms for 9 potential vaccinees to USAMRIID in preparation for the
LVS vaccinations.
4. Significant decisions made or pending
a.USAMRIID tentatively will resume offering vaccinations to UNM and LBERI in 2009/2010.
b.UNM (4) and LBERI (33) are vaccinated; UNM and LBERI will offer the LVS vaccinations to 9 more
scientists to total up to 46.
c.Dr. Lyons received UNM IRB re-approval to allow blood draws on the vaccinated LBERI and UNM
scientists after their LVS vaccinations
d.Dr. Lyons received UNM HRRC approval to allow 150ml blood draws rather than 100ml blood
draws, to increase the number of cells available for correlates of protection assays. UNM HRRC
also approved blood draws 1 month before LVS vaccination as well as up to 6 blood draws in the
12 months post vaccination. A volunteer donor may donate blood no more than once per month.
e.UNM, True, LBERI, USAMMDA and USAMRIID fully executed the CRDA modification #1 to extend
the termination date from 6/29/09 to September 29, 2010.
5. Problems or concerns and strategies to address
a.Nine scientists could be vaccinated in 2009 and 2010.
b.USAMRIID may restart LVS vaccinations on November 18, 2009 and in January 2010.
6. Deliverables completed
Page 1 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
a.A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS vaccination
since 9/11/07.
b.37 participants have terminated from the USAMRIID SIP, after completing the one year health
follow-up at UNM EOH (Employee Occupational Health).
7. Quality of performance
Excellent
8. Percentage completed
77% of the scientific work is complete
9. Work plan for upcoming month
Schedule LVS vaccinations for 9 remaining LBERI and UNM scientists on November 18, 2009 and
January 26, 2010.
Milestone 8
Milestone description: LVS vaccination protection of aerosol Schu S4 confirmed 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. LBERI repeated an LVS vaccination/SCHU S4 challenge study in NHP to determine
whether that DVC Lot 16 LVS vaccine protects against a lethal SCHU S4 aerosol
challenge.
b. NHPs were LVS vaccinated via subcutaneous inoculation or scarification in May 2009.
Group1, Group 2, and Group 3 were vaccinated with 1.1 x 107, 1.2 x107 and 1 x 106 CFU
LVS, respectively. The vaccinated NHPs and naïve controls were challenged with
approximately 1000 CFU SCHU S4 (target presented value). NHPs that survived to Day
21 post-challenge were euthanized.
c. Complete necropsies with tissues being collected for histology and bacteriological burden
were performed on all animals euthanized or found dead. Blood was collected to
measure bacteremia on Day 3, Day 10 (if the animal was alive) and from NHPs which
were euthanized due to moribundity. The data on tissue and blood burden has now been
quality controlledand is re-presented in Tables 1 and 2 below (originally presented in the
August monthly report and no mistakes were found).
d. A scatter plot showing time to death post-SCHU S4 aerosol vs. challenge dose is shown
in Figure 1. The Figure shows all non-vaccinated and LVS vaccinees that were
vaccinated in the 5 – 7 weeks prior to aerosol SCHU S4 challenge in both the original
TUL 08 and the more recent TUL 08B experiment; (LVS vaccine/SCHU S4 aerosol
challenge experiments in February 2009 and July 2009, respectively).
e. A statistical evaluation of tissue burden of SCHU S4 post-aerosol challenge combining
data from both TUL08 and TUL08B is presented in Figure 2.
f. A subjective measure of severity of histopathological lesions from TUL 08 (February 09)
experiment is presented in Figure 3.
Page 2 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Table 1: SCHU S4 Bacterial Tissue Burden post-Aerosol Challenge
Vaccine
Group
Presented
Dose (CFU)
Day to
Death
A06694
Control
865
A06882
Control
A07427
Animal ID
CFU/g
Spleen
Liver
Mes LN
TBLN
Lung
14
2.16E+06
5.94E+03
3.92E+06
8.43E+06
1.41E+08
2140
6
3.71E+08
2.75E+06
1.33E+06
6.84E+08
9.38E+08
Control
1950
5
1.91E+09
2.37E+07
4.87E+08
1.40E+09
2.34E+09
A06873
Scarified
1240
7
3.03E+07
2.19E+06
3.86E+03
6.49E+06
7.84E+08
A07386
Scarified
1260
10
2.60E+08
1.38E+06
BLD
1.68E+07
7.72E+08
A06674
Scarified
3740
6
1.16E+04
2.28E+03
BLD
6.11E+03
2.28E+08
A07682
Scarified
1690
6
2.08E+03
2.50E+03
9.08E+02
3.34E+05
7.96E+07
A06693
Scarified
1330
7
3.00E+05
4.56E+04
BLD
7.82E+06
1.99E+09
A07686
Scarified
2080
8
1.17E+04
1.63E+04
1.17E+04
8.23E+04
1.96E+08
A07395
S.C.
1410
21*
BLD
BLD
BLD
1.38E+04
1.36E+04
A07418
S.C.
791
21*
BLD
5.95E+01
BLD
1.37E+04
1.38E+04
A06675
S.C.
2610
6
1.24E+05
1.73E+04
5.66E+02
6.65E+05
1.32E+09
A07566
S.C.
1250
6
8.94E+03
3.10E+03
BLD
3.01E+06
1.12E+08
A06702
S.C.
500
8
6.44E+05
6.06E+04
2.34E+07
1.39E+07
2.66E+09
A07610
S.C.
519
5
4.92E+03
2.38E+03
BLD
1.73E+06
1.27E+08
aBLD,
below limit of detection; * Terminal sacrifice
Table 1. The SCHU S4 bacterial burden is indicated for each NHP exposed in spleen, liver, mesenteric
lymph node (Mes LN), tracheobronchial lymph node (TBLN) and lung. Also noted are the vaccine
status, the dose of SCHU S4 presented in the aerosol upon challenge and the number of days each
NHP survived. Highlighted animals survived to day 21 and were subjected to terminal euthanasia at
that time.
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Page 3 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Table 2: SCHU S4 Bacterial Burden in the Blood post-Aerosol Challenge
Vaccine
Group
Presented
Dose (CFU)
Day to
Death
A06694
Control
865
14
BLD
BLD
TNTC
A06882
Control
2140
6
3.33E+00
N/A
No data
A07427
Control
1950
5
BLD
N/A
TNTC*
A06873
Scarified
1240
7
BLD
N/A
1.75E+02
A07386
Scarified
1260
10
BLD
N/A
No data
A06674
Scarified
3740
6
BLD
N/A
No data
A07682
Scarified
1690
6
BLD
N/A
BLD
A06693
Scarified
1330
7
BLD
N/A
BLD*
A07686
Scarified
2080
8
BLD
N/A
BLD
A07395
S.C.
1410
21*
BLD
BLD
BLD
A07418
S.C.
791
21*
BLD
BLD
BLD
A06675
S.C.
2610
6
BLD
N/A
No data
A07566
S.C.
1250
6
BLD
N/A
No data
A06702
S.C.
500
8
BLD
N/A
TNTC
A07610
S.C.
519
5
BLD
N/A
BLD
Animal ID
Day 3
Day 10
@ Necropsy
Table 2. The SCHU S4 bacterial burden is indicated for each NHP exposed, Blood bacterial burden were
assessed in every NHP on Day 3, any NHPs remaining alive on Day 10 (A06694, A07395 and A07418),
and any animal who was euthanized due to moribundity (i.e. not found dead; A07427, A06873, A07682,
A06693, A07686, A07395, A07418, A06702 and A07610). BLD = below detection. N/A = not applicable
(i.e. NHP was dead). TNTC = Too numerous to count.
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Figure 1: Relationship of SCHU S4 Challenge Dose, Vaccine Status and Time to Death
Page 4 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
SCHU S4 CFU Challenge
5000
Control
Scarification
S.C.
4000
3000
2000
1000
0
-1000
4
6
8
10
12
14
16
18
20
22
Days Survival Post-SCHU S4 Challenge
Figure 1: Relationship of SCHU S4 challenge dose and time to death in Control (non-vaccinated) NHPs
and those vaccinated with LVS by either scarfication or sub-cutaneous (S.C.) inoculation. Vaccinees
receiving LVS by scarification or sub-cutaneous inoculation in experiments conducted in February 2009
(TUL 08, n = 3 each) and July 2009 (TUL 08B, n = 6 each) are shown. Non-vaccinated NHPs from both
experiments are shown (n = 6, 3 from each experiment). Three vaccinees originally vaccinated in
November 2006 and challenged in February 2009 are excluded from this analysis.
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Plotted Data: N:\My Documents\Tularemia Contract\Summary Data\SCHU S4 TTD.svd
Page 5 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 2: SCHU S4 Bacterial Burden in Tissues post-Aerosol Challenge
Control
Scarification
S.C.
1E10
SCHU S4 CFU/gram
(Mean +/- S.D.)
1E9
1E8
1E7
* *
*
*
1E6
1E5
1E4
1E3
1E2
1E1
Spleen
Liver
TBLN
MLN
Lung
Figure 2: SCHU S4 burden in select organs following aerosol challenge. Vaccinees receiving LVS by
scarification or sub-cutaneous inoculation in experiments conducted in February 2009 (TUL 08, n = 3
each) and July 2009 (TUL 08B, n = 6 each) are shown. Non-vaccinated NHPs from both experiments are
designated Control (n = 6, 3 from each experiment). Three vaccinees originally vaccinated in November
2006 and challenged in TUL 08 are excluded from this analysis. * indicates significantly different (p < .05)
than Control when analyzed by one-way ANOVA.
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Plotted Data: N:\My Documents\Tularemia Contract\Summary Data\SCHU S4 TTD.svd
Page 6 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 3: Lesion scores are assessed for liver and spleen based on histological sections stained with
H&E. Vaccinees receiving LVS by scarification (n = 3) or sub-cutaneous (n = 3) inoculation in the
experiment conducted in February 2009 (TUL 08) are combined and designated as Vaccinated. Lesions
were scored on a scale from 0-4, based upon distribution and severity, and summed for each animal.
The data presented represent the average for each group. Non-vaccinated NHPs (n = 3) are designated
Control. Three vaccinees originally vaccinated in November 2006 are excluded from this analysis.
Data storage N:\07-083 tularemia dec 07\07-083A vaccine
g. Data on respiration rate and temperatures pre- and post-SCHU S4 aerosol exposure
were compiled and are presented in Figures 4 and 5 respectively.
Page 7 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 4: Respiration rates post-SCHU S4 infection are plotted from the TUL 08B experiment. Control
non-vaccinated NHPs are noted by arrows (A06694, A06882 and A07427).
Electronic file located at Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 (TUL-08B).
Page 8 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 5: Body temperatures post-SCHU S4 infection are plotted from the TUL 08B experiment. Control
non-vaccinated NHPs are noted by arrows (A06694, A06882 and A07427).
Data Storage: Raw Data :\\saturn\ABSL3\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY07\FY07-083 (TUL-08B)
Electronic file located at Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY07\FY07-083 (TUL-08B).
Plotted Data: N:\My Documents\Tularemia Contract\Prime Tech NIAID Calls\LBERI NIAID Tech Call July
2009 final.ppt; N:\My Documents\Tularemia Contract\Summary Data\SCHU S4 TTD.svd
Data interpretation:
i.LVS vaccination does not fully protect NHPs from death due to SCHU S4 aerosol
challenge but does lead to significantly decreased SCHU S4 bacterial burden in
draining (TBLN) and distal (MLN) lymph nodes and a trend to decreased
bacterial burden in spleen and liver (Table 1, Figures 1 and 2).
Page 9 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
ii.The reduced bacterial burden in spleen and liver of LVS-vaccinated and challenged
NHPs likely contributes to the decreased histopathology scores in those
vaccinees compared to non-vaccinated controls (Figure 3).
iii.SCHU S4 aerosol challenge of both LVS-vaccinated and non-vaccinated NHPs
results in decreased temperatures (as measured by microchip) and increased
respiration rates prior to death in the TUL 08B experiment (Figures 4 and 5).
These findings are similar to those observed in the original TUL 08 experiment.
h. The IACUC protocol and ES&H review for the next vaccine/challenge study were
reviewed and approved.
4. Significant decisions made or pending
Based on discussions between LBERI, UNM, and NIAID another study will be performed in which
NHPs will be vaccinated subcutaneously with DVC Lot 16 (n=3), DVC Lot 17 (n=3), DVC Lot 20
(n=8), and USAMMDA IND 57 Lot 4 (n=8).
5. Problems or concerns and strategies to address
LBERI’s goal is to develop an model of vaccine-induced protection against aerosol deliver you
SCHU S4. We have been testing DVC Lot 16 as the source of LVS we use to vaccinate the
NHPs. As DVC Lot 16 has not proven to show complete protection from death due to aerosol
challenge with SCHU S4, we will be testing other lots and sources of LVS in upcoming
experiments and comparing their efficacy directly to DVC Lot 16.
6. Deliverables completed
None
7. Quality of performance
Good this month
8. Percentage completed
72% of the scientific work is complete.
9. Work plan for upcoming month
a.Write study protocol for upcoming study which will include 4 different lots of LVS,
vaccination subcutaneously on the abdomen (w/o shaving and normalization to LVS CFU
based on “CFU/vial at vial creation” of USAMMDA IND 157 lot4 .
b.Perform physical exams and draw baseline blood from NHPs to assess the response of
PBMCs to formalin fixed and heat-killed LVS and SCHU S4 in immunological assays and
clinical chemistry parameters.
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 new work was performed during the month of August.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
a.LBERI encountered growth issues and is currently addressing these. Trevor will expand upon
the growth issues in the next monthly report. Next aerosols are likely in October 2009.
Page 10 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
b. The Quality Assurance reviewer most familiar with the plan was out of the country for several
weeks. The LBERI TVDC team is awaiting her return for the review of the Aerosol Qualification
Plan.
6. Deliverables completed
None
7. Quality of performance
NA since no new work was done.
8. Percentage completed
45% of the scientific work is complete.
9. Work plan for upcoming month
a.Compliance Group will complete review of the Aerosol Qualification Plan.
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
No new work was performed during the month of August.
4. Significant decisions made or pending
Animals arrived at LBERI during July. Due to the results of the vaccination/SCHU S4 challenge
study performed under Milestone 8 the animals will be used for an additional
vaccination/challenge study under Milestone 8.
5. Problems or concerns and strategies to address
Testing of new vaccine candidates is dependent on MS8 demonstrating that 4 various DVC and
USAMMDA LVS lots of vaccine can protect NHP from SCHU S4 aerosol challenge. Freyja Lynn
of NIAID requested that the 4 lots of LVS be tested under MS 8 as animal model development,
rather than under Milestone 10 as discussed with prior NIAID Project Officers.
6. Deliverables completed
None
7. Quality of performance
None
8. Percentage completed
2% of the scientific work is completed.
9. Work plan for upcoming month
No work is planned for the month of September.
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
Page 11 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
a.SCHU S4 challenges were performed on August 18th and 22nd for the telemetered portion
of this milestone. The target presented dose was 1000 CFU. Table 3 below reports
actual presented doses.
Table 3: Presented doses and Day of Death for Telemetered Arm of the Natural History
Study.
Aug 18 exposures
Presented Day of Death
Animal #
Dose (cfu) (study day)
Aug 25 (7)
A07752
2.85E+02
Aug 25 (7)
A07755
3.09E+02
A07776
1.19E+02
alive
A07801
9.62E+01
Aug 25 (7)
A07805
1.02E+02
Aug 26 (8)
A07808
1.21E+02
Aug 26 (8)
Aug 22 exposures
Presented
Animal #
Dose
A07783
4.30E+01
Day of Death
(study day)
Aug 29 (7)
A07784
1.82E+02
Aug 29 (7)
A07788
2.48E+02
Aug 28 (6)
A07811
6.27E+02
Aug 28 (6)
A07828
3.27E+02
Aug 29 (7)
Aug 28 (6)
A07841
2.73E+02
Animals were exposed to a target dose of 1000 cfu on study day 0. Exposure days were
staggered due to the number of animals. Actual presented doses are listed by animal
and study day and date are captured.
b.Clinical signs observed were rapid respiration rate, hunched posture, decreased food
intake, and coughing progressing to either respiratory distress or unresponsiveness.
Fevers were seen in the second set of exposed animals as well. The signs are
consistent with what was observed in the LD50 and non-telemetered natural history. Two
NHP died and the rest were euthanized due to moribundity, except for one survivor
described in “c” below.
c.Hematology, clinical chemistry and bacteremia from days 0-6 were assessed. Terminal
bacteremia was also determined. For the lone survivor, day 10 and day 14 blood was
analyzed for bacteremia, clinical chemistry and hematology. Large amounts of terminal
sera from each animal, with the exception of the survivor, were collected. The survivor
became very ill but has appeared to recover. Currently the animal is scheduled to be
euthanized on September 9th. Clinical chemistry, hematology are to be analyzed as well
as the amount of bacteria in the blood determined.The bacteremia, tissue burden,
hematology and clinical chemistry data will be compiled and reported next month.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Page 12 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
None
6. Deliverables completed
In-life non-telemetered arm of the SCHU S4 natural history study is completed.
7. Quality of performance
Good
8. Percentage completed
75% of the scientific work is complete.
9. Work plan for upcoming month
a.Histopathology slides for lungs, spleen, liver, kidney, nasal cavity, oropharynx with tonsils,
brain, bone with marrow (rib/femur), lymph nodes (tracheobronchial, retropharyngeal,
submandibular, mesenteric, axillary and inguinal), and representative lesions will be read
for the non-telemetered arm of the study. It is expected that LBERI will read the slides
and prepare a histopathology report by the end of September 2009 for the nontelemetered arm of the study.
b.The lone survivor of the telemetered arm will be euthanized on September 9th.
c.The bacteremia, tissue burden, hematology and clinical chemistry data will be compiled and
reported next month for the telemetered arm of the study.
d.Telemetry data will be analyzed.
e.When will histopathology phases be completed for the telemetered arm of the study? The
last animal (A07776) was euthanized on September 9, 2009. After tissues are removed
from the ABSL3, we will be able to give an accurate date as to when histopathology
slides will be complete for pathologist review.
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.This milestone examines the cellular and humoral immunity associated with effective
vaccination of Fischer 344 rats with LVS
b.Experiment PtrIgG3 (Notebook 138, pages 141-148)
i. UNM has been using passive immunization as a model system to
characterize the role of antibodies in protecting rats vaccinated with LVS
against intratracheal SCHU S4 challenge.
ii.
UNM has shown repeatedly that passive immunization of Fischer 344
rats with LVS-immune serum protected them against a low dose intratracheal
SCHU S4 challenge. To determine whether IgG was sufficient for protection,
purified IgG equivalent to twice the amount of LVS-specific IgG in 250 l of
immune serum was given before challenge with 150 CFU SCHU S4. Rats
treated with immune rat serum or IgG purified from normal serum were
included as controls. The results showed that purified immune IgG provided
a similar level of protection as immune serum (Fig. 1).
Page 13 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Percent survival
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
100
75
50
Purified Normal IgG
25
Purified Immune IgG
Immune serum
0
0
10
20
30
Days Post-infection
Figure 1. Purified immune IgG and immune serum produced similar protection in
Fischer 344 rats. IgG was purified from normal or immune rat sera and passively
transferred into naïve Fischer 344 rats (n=6) one day before i.t. challenge with
150 CFU SCHU S4.
c.Experiment Pdep-1 (Notebook 138, pages 153-155)
i.UNM has been characterizing the role of humoral immunity in the defense
against SCHU S4 infection in the Fischer 344 rat model. Historical studies in
mice suggested that cellular immunity is also important for defense against
this intracellular pathogen. One approach to study the importance of cellular
immunity is to selectively deplete T cell subsets using cell-type specific
antibodies. Previous experiments showed that weekly injections of 250 g
OX-8 antibody effectively depleted rats of CD8 T cells. Injections of W3/25
or OX-38 antibodies did not result in depletion of CD4 T cells, but UNM has
experimental evidence that the W3/25 antibody functionally inactivated CD4
T cells.
ii.To determine whether T cell immunity is essential for the survival of passively
immunized rats, Fischer 344 rats were treated with antibodies against CD4
and/or CD8 three day before passive immunization and four days before i.t.
SCHU S4 challenge.
iii.Treatment with the OX-8 ascites fluid (anti-CD8) completely eliminated the
protective effect of immune serum and all of the rats died within 10 days of
infection (Fig. 2). However, the OX-8 antibody also depletes NK cells and
therefore specific depletion of NK cells with asialo-GM antibodies may be
required to exclude the contribution of NK cells.
iv.Treatment with anti-CD4 ascite fluid reduced the survival to 50% but this was
not statistically different from treatment with isotype control ascites fluid (Fig
2). We cannot confirm that the W3/25 antibody had any effect in vivo
because it does not deplete CD4 T cells, but future experiments will try to
determine whether W3/25 treatment inactivates T cells functionally. A more
significant effect may be observed only with a higher challenge dose.
Page 14 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
NRS
IRS/Isotype Control
IRS/anti-CD4
IRS/anti-CD8
IRS/anti-CD4+anti-CD8
Percent survival
100
80
60
40
20
0
0
5
10
15
20
25
30
Days Post-challenge
Figure 2. T cell depletion abrogates protection associated with passive
immunization. Naïve Fischer 344 rats (n = 6) were treated i.p. with 1 mg/rat of
anti-CD8 (clone OX-8), anti-CD4 (clone W3/25), or isotype control (clone
TS2/18.1.1) antibodies 3 days before passive immunization and every 7 days
thereafter. One day after passive immunization, treated rats were challenged
i.t. with SCHU S4.
d.
Experiment Ptiter-1 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Gopi's experiments\Ptiter)
i. The passive immunization experiments performed so far have used immune
sera collected 4 weeks after s.c. vaccination because LVS is known to be
cleared by that time. This experiment was performed to determine the
kinetics of the LVS-specific antibody responses in the vaccinated rats. The
sera have been collected and ELISA will be performed.
e.
Experiment Ftc65.9 (Notebook 128, pages 108-109)
i. The T cell depletion strategy will be used to determine whether cellular
immunity is essential for the survival of LVS-vaccinated rats. As a
preliminary experiment, UNM used rats from a separate line of experiments
that had been infected with SCHU S4 and treated successfully with 1 mg/kg
or 10 mg/kg of the antibiotics levofloxacin. A similar study in mice showed
that mice infected with SCHU S4 and then treated therapeutically with
levofloxacin developed immunity to a subsequent SCHU S4 challenge and
therefore may be considered “vaccinated” [Klimpel GR Vaccine
(2008)26:6874]. This study also showed an inverse correlation between the
dose of antibiotics and survival upon re-challenge (90% @ 6.25 mg/kg, 80%
@ 12.5 mg/kg, 60% @ 25 mg/kg, and 50% @ 50 mg/kg)
ii. Rats from the two levofloxacin doses were each divided into 2 groups (n = 2
or 3) and then treated with the combination of anti-CD4 (clone W3/25) and
anti-CD8 (clone OX-8) antibodies or with isotype control (clone TS2/18.1.1)
antibodies one day before i.t. challenge with 5 x 104 SCHU S4 and weekly
thereafter. The dosage of antibiotics had no effect on survival of the TS2treated groups (a higher dose of levofloxacin may produce results similar to
mice experiments). However, rats treated with 10 mg/kg and depleted of
Page 15 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
CD4 and CD8 T cells were more susceptible to SCHU S4 re-challenge than
those treated with 1 mg/kg.
iii. Since the higher doses of antibiotics may have eliminated the LVS vaccine
earlier, the results from the mouse and rat studies suggest that the
persistence of the live vaccine may affect the strength of immunity and the
mechanism of protection
Percent survival
100
80
Vaccinated
TS2 (1mg/Kg Levo)
W3/25 + OX-8 (1mg/Kg Levo)
TS2 (10mg/Kg Levo)
W3/25 + OX-8 (10mg/Kg Levo)
60
40
20
0
0
10
20
30
40
Days Post-infection
Fig. 3. Variable requirement for T cells in immunity against i.t. SCHU S4
challenge.
f.
Experiment Ftc90 (Notebook 128, pages 110-111)
i. UNM is providing pre- and post-LVS vaccination sera from Fischer 344 rats
to Susan Twine at National Research Council Canada to look for
immunoreactive proteins across animal species. Blood has been collected
from 12 rats before and after LVS vaccination. UNM will send the sera to
NRC shortly.
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
62%
9.Work plan for upcoming month
a.Humoral immunity
i.Repeat experiment to determine the antibody response after LVS vaccination and
after passive immunization just before SCHU S4 challenge
ii.Repeat experiment to determine whether purified immune IgG is protective.
iii.Repeat experiment to determine the importance of T cells in passive immunization
iv.Analyze cytokine profiles associated with passive immunization
v.Determine the kinetics of T cell development in passively immunized rats
Page 16 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
b.
vi.Consult with biostatistician on the presentation of results on bacterial growth in
passively immunized rats
Cellular immunity
i.Determine the importance of CD4 and/or CD8 T cells in the protection of LVS
vaccinated rats.
ii.Develop procedures for adoptive transfer of immune T cells into naïve rats.
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and compared to those in other species.
Institution: LBERI
1. Date started: 2/23/2006
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No work was performed during the month of August.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
a. Protein assays are being tested and optimized in order to compare LVS and SCHU
S4 antigen preparation CFU to protein content. LBERI and UNM wish to standardize
the quantitation of antigen preparations. UNM has not been able to show a linear
relationship between LVS protein and CFU. The UNM continues to test optimized
procedures such as using sonication, but thus far have not found an adequate
method which produces the desired linear relationship.
b. We wish to set up the microagglutination assay as directed by NIAID, however, we
have not yet received all the necessary critical reagents from external collaborators.
In late August, an aliquot of positive control rabbit sera was received from Dr. Stzein.
We await delivery of the LVS antigen and the protocol to stain that antigen with
hematoxylin.
6. Deliverables completed
None
7. Quality of performance
None in August 2009
8. Percentage completed
91% of the scientific work is complete.
9. Work plan for upcoming month
a.Consult with UNM on proceeding with the LVS and SCHU S4 protein:CFU assay. UNM
has not been able to show a linear relationship between LVS protein and CFU.
b.Construct a pooled negative control plasma sample for use in IgG anti-LVS ELISAs; this
would be used in conjunction with the positive control sample reported on in April.
c.Initiate the development of the microagglutination assay, per the procedure provided by
Freyja Lynn at NIAID. The procedure was from Dr. Marcelo Sztein of the University of
Maryland. Currently LBERI is waiting on the positive control antigen to arrive in order to
begin the development of the assay. UNM received the positive control antigen from Dr.
Sztein’s lab on approximately 9/3/09. UNM will receive the Ft antigen lot of LVS (lot#9)
from USAMMDA and USAMMDA’s antigen preparation protocol, after joinders to the
USAMMDA MTA are signed by UNM and UNM’s four TVDC subcontractors.
Page 17 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 12/13
Milestone description: Assays for detecting relevant immune responses in animals & humans
developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc86,9 (Notebook 128, pages 112-114)
i. An important consideration for development of immunoassays is whether
antigen preparations can be standardized and accurately quantified such that
comparisons between experiments and antigens can be made. UNM’s
approach to address this issue is to correlate the protein content of antigen
preparations to bacteria number. In July, UNM performed several
experiments to optimize the conditions for extracting proteins from live LVS
using the B-PER protein extraction reagent from Pierce. The results
suggested that the B-PER reagent will not meet our needs.
ii. In August, UNM performed two experiments to determine whether physical
disruption by sonication would be a better approach. UNM tested two
different sonication settings on live LVS: 10% pulses up to 10 minutes and
continuous sonication for 5 min. UNM did not find a difference between the
protein content in sonicated and non-sonicated samples, suggesting that
sonication did not disrupt the bacteria. This will be confirmed by plating the
sonicated bacteria and checking for growth of viable bacteria.
iii. UNM will continue to test sonication settings as well as other methods of
physical disruption (such as bead-beating) and detergent lysis (such as RIPA
buffer)
4.Significant decisions made or pending
None
5.Problems or concerns and strategies to address
a.UNM has not been able to show a linear relationship between bacteria number and
protein content and suspect that this problem is due to the bacterial lysis conditions.
So far, UNM has tried unsuccessfully with the B-PER reagent from Pierce and
sonication in a cup horn. UNM will attempt to improve protein extraction efficiency by
extending sonication period and duty cycle or using a more powerful sonication
probe, bead beating, and RIPA detergent lysis.
6.Deliverables completed
None
7.Quality of performance
NA
8.Percentage completed
80%
9.Work plan for upcoming month
a.Determine whether sonication would be better for protein extraction than achieved with the
B-PER protein extraction reagent. UNM will attempt to improve protein extraction
efficiency by extending sonication period and duty cycle and using a more powerful
sonication probe
b.Establish the microagglutination assay to measure humoral immune responses, per Freyja
Lynn’s request
Page 18 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
c.Test the robustness of the IFN ELISpot assay and ELISA for human samples when they
are available. UNM’s priority is to develop the correlate of protection assay described
under milestone 21. UNM is aiming to test plasma/sera from approximately 20
unvaccinated humans and 20 LVS vaccinated humans.
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 work was done
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
a.All work is contingent on the availability of assays and reagents described in MS21
b.Determine the titer of anti-Ft antibodies in sera from convalescent tularemia patients on
Martha’s Vineyard. This is dependent on the work being performed under MS12/13 to
quantify protein content in antigen preparations
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 work was done during this month.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
No work was done during this month because UNM is developing human and rat assays to
selectively deplete effector subsets in vitro, under Milestone 11.
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
0%
Page 19 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
9.Work plan for upcoming month
No work planned
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 work was done
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
UNM cannot start this milestone until a human assay is developed in MS21.
6. Deliverables completed
None
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
No work was done
4. Significant decisions made or pending
UNM will prepare a manuscript for publication based on these findings. No additional experiments
with human alveolar macrophages will be performed unless needed for manuscript.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
NA
8. Percentage completed
27%
9. Work plan for upcoming month
No lab work planned. Continue work on manuscript preparation
Page 20 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 21
Milestone description: Correlates of protection: in vitro assay or other readout of effector
function of Ft developed for multiple species.
Institution: LBERI
1. Date started: 4/8/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a.The goal of this milestone is to develop a correlate of protection assay that differentiates
unvaccinated from vaccinated individuals in multiple species. LBERI used flow cytometry
to detect cell surface markers or secretion of intracellular cytokines from PBMC from LVS
vaccinated NHPs. Surface staining was for CD3, CD4 and CD8 and intracellular staining
was for IFNγ, TNFα and IL2. LBERI will develop the flow cytometry assay to enhance
sensitivity for the intracellular staining with the goal of distinguishing vaccinated from
unvaccinated individuals by expression of a set of cytokines.
b.Another attempt at detecting intracellular cytokine expression in vaccinated NHP PBMCs by
flow cytometry was made.
i.Cryopreserved PBMCs from NHP A06873 that were collected and frozen Day 8 postLVS vaccination (1.1 X 107 via scarification), were thawed and stimulated at 4 X
105 cells per well for a total of 6 hours (4 hours with Golgi plug). Cells were
stimulated with either media only; 8 X 103 hkLVS or ffLVS per well; 4 X 104
hkLVS or ffLVS per well; 2 X 105 hkLVS or ffLVS per well; 1 µg PHA per well; or
10 ng PMA/100 ng ionomycin per well. As an additional control for cytokine
staining HiCK-1 cells were permeabilized and stained along with the NHP
samples
ii.For automatic compensation with FACSDiva (software) anti-mouse Ig  Compbeads
were single stained with mAb-conjugates: IFN--FITC, TNF--PE, CD4-PECy7,
IL-2-APC, CD8-APCCy7. For the CD3-PerCP conjugate, PBMC had to be used
for compensation as the conjugate consists of a mouse Ig antibody and the
CompBeads only bind  light chain antibodies. This discovery solved the
compensation problems we have been working on. The staining amount per
sample has also been increased for the IL-2-APC antibody, from the previously
used 1 ul per sample to 20 ul per sample, following a titration with CompBeads.
Non-stained PBMC were used for voltage gain PMT settings.
iii.
a.The samples were run at the FACS Canto and this time the compensation
setting worked very well and no spectral overlaps were too large to be
well compensated for.
b.The control HiCK-1 cells showed both CD4 T cells and CD8 T cells
producing all three cytokines (IFN-g, IL-2 and TNF-a).
Figure 6: Human HiCK-1 T cells producing intracellular IFN-, IL-2 and
TNF-
Page 21 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
a)
c)
b)
d)
Figure 6. HiCK-1 cells gated for CD4 (panels a and b) or CD8 T cells (panels c and d). The dotplot in
panel b shows only the IL-2+IFN-+ double positive events gated in panel a, and approximately half of
the IFN-+IL-2+ double positive also express TNF- (triple positive). The gating of the double and triple
positive CD8 T cells was done in a similar fashion in panel c and panel d.
Data located at “sirius\users”(Z:) crietz\BD Canto Experiments\September 2009\Experiment 090109.
A significant proportion of both CD4 T cells and CD8 T cells from HiCK-1 cells simultaneously express
IFN-, IL-2 and TNF- which can be visualized with a 6 color surface- and intracellular staining in the
FACS Canto.
c.The PMA/ionomycin control for the NHP A06873 worked well and 6 h stimulation induced
all three cytokines from both CD4 T cells and CD8 T cells. This is promising as it shows
that the anti-human cytokine conjugates (Quality Controlled for NHP by manufacturer)
work well also for NHP cells and our procedure for intracellular staining is adequate.
Page 22 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 7: CD4 and CD8 T cells from a LVS vaccinated NHP producing intracellular IFN-g, IL-2 and TNF-a
in response to PMA/ionomycin activation.
a)
c)
b)
c)
c)
d)
Figure 7. PBMC from NHP A06873 (day 8 post LVS) stimulated for 6 h with PMA/ionomycin and
gated for CD4 T cells (panels a and b) and CD8 T cells (panels c and d).
Data located at “sirius\users”(Z:) crietz\BD Canto Experiments\September 2009\Experiment
090109.
Our selected reagents and developed staining procedure detects the three cytokines also in NHP
PBMC stimulated by PMA/ionomycin.
d)
The hkLVS or ffLVS stimulated PBMCs from NHP A06873 (days 8 post LVS) did not produce
any amount of the three cytokines above media control background. 6 h of stimulation with whole
bacteria antigen may not be enough for antigen processing, presentation and activation of T cells
to produce cytokines. Even the PHA control (not shown) did not show any cytokines above media
background at the 6 h time point.
Page 23 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 8: CD4 or CD8 T cells from NHP A06873 stimulated for 6 h with ffLVS or hkLVS did not produce
IFN-g, IL-2 or TNF-a above background control
a)
c)
b)
d)
Figure 8. PBMC from NHP A06873 stimulated for 6 h with 0.5 hkLVS/PBMC (panels a and b) or
0.5 ffLVS/PBMC (panels c and d). 0.5 LVS organisms per PBMC is the same as 2x10 5 organisms
per well with 4x105 PBMC. Panel a) and panel c) show gated CD4 T cells, panel b) and panel d)
show gated CD8 T cells.
Data located at “sirius\users”(Z:) crietz\BD Canto Experiments\September 2009\Experiment 090109.
The hkLVS or ffLVS stimulated T cells from NHP A06873 (day 8 post LVS) did not produce any amount
of IFN-, IL-2 or TNF- above media control background. 6 h of stimulation with whole bacteria antigen
may not be enough for antigen processing, presentation and activation of T cells to produce cytokines.
Later time points of stimulation with these antigens will be tested as well as later time points post LVS
vaccination.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
LBERI will troubleshoot the ICS assay, per the work plan outlined below.
6. Deliverables completed
None
7. Quality of performance
Fair
8. Percentage completed
9% of the scientific work is complete
9. Work plan for upcoming month
a. Repeat ICS assay for later time points of stimulation (24-48 h suitable for human PBMC
from vaccinated donors) as well as later time points post LVS vaccination.
Page 24 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
b.As an additional control to set the lower fluorescence limits of detection for the cytokines,
include FMO (fluorescence minus-one) controls. These are positive control cells (e.g
stimulated with PMA/ionomycin) stained with all conjugates except the one where it
controls for background fluorescence. E.g, a sample stained for CD3, CD4, CD8, IL-2
and TNF- (but not IFN--FITC) will control for background fluorescence in the IFN-
fluorescence detector, and guide subsequent gating.
Milestone 21
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
a.The goal of this milestone is to develop correlates of protection for vaccinated humans.
UNM appears to have developed an assay that distinguishes vaccinated and
unvaccinated individuals based the ability of vaccinated human PBMC to produce IFN
and control SCHU S4 growth after pre-stimulation with inactivated LVS antigen
preparations. The numbers of samples are quite low at this point and additional donors
(both LVS-vaccinated and unvaccinated) will be needed to increase the significance of
these findings.
b.Experiment FT-AH-27 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments and
Results\Andrew's experiments\FT-AH-27)
i.
UNM tested PBMC from an additional 2 unvaccinated donors and the results
were consistent with previously observed patterns for unvaccinated donors. HKLVS pretreatment did not significantly affect SCHU S4 bacterial growth at 72h in
either PBMC sample. FF-LVS pretreatment decreased CFU in PBMC #32 by
approximately 6-fold and approximately 2-fold in PBMC #43 relative to the
bacterial growth detected with no pretreatment. The effect of FF-LVS
pretreatment was not as strong as seen in previous vaccinated human PBMC
preps. For example, the magnitude of decrease was 250-fold and 20-fold in
independent vaccinated human PBMC preps in experiment FT-AH-26.
Page 25 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
PBMC #32
PBMC #43
1.010 8
1.010 8
HK-LVS
3530-FOLD INCREASE
1.010
7
FF-LVS
1600-FOLD INCREASE
1.010 5
1.010 4
NO PRETREATMENT
4590-FOLD INCREASE
FF-LVS
2300-FOLD INCREASE
1.010 6
total CFU / well
total CFU / well
1.010 6
HK-LVS
3150-FOLD INCREASE
1.010 7
NO PREATMENT
10,300-FOLD INCREASE
1.010 5
1.010 4
NO PRETREATMENT
FF-LVS
HK-LVS
1.010 3
1.010 2
NO PRETREATMENT
FF-LVS
HK-LVS
1.010 3
1.010 2
0
24
48
72
HOURS POST-INFECTION
0
24
48
72
HOURS POST-INFECTION
Figure 4. Bacterial burden in unvaccinated PBMC stimulated with inactivated LVS. 2.5 x 10 6
Ficoll-purified human PBMCs per well from unvaccinated human donors were incubated for 48
hours with formalin fixed LVS or heat-killed LVS. 48 h after pre-stimulation, F. tularensis strain
SCHU S4 was added to wells at MOI=1 and incubated for 2 hours, followed by addition of
gentamicin to 50ug/mL for 45 minutes, followed by PBS wash and resuspension in gentamicinfree cDMEM. Bacterial burden was determined 72 h after infection. Data points indicate
average +/- SD of triplicate infected wells, and duplicate uninfected wells
4.Significant decisions made or pending
None
5.Problems or concerns and strategies to address
NA
6.Deliverables completed
None
7.Quality of performance
Good
8.Percentage completed
71 %
9.Work plan for upcoming month
a.Gather more data on IFN secretion and CFU reduction from both vaccinated and
unvaccinated donors
b.Determine whether addition of anti-IFN antibody prevents LVS vaccinated PBMC control of
SCHU S4 growth.
c.Identify cell types associated with IFN production by intracellular cytokine staining
Page 26 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 29
Milestone description: Analysis of T cells from lymph nodes & T cell epitopes
Institution: LBERI
1. Date started: 8/7/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No new work was performed during the month of August.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
None
8. Percentage completed
16% of the scientific work is complete
9. Work plan for upcoming month
a.LBERI is waiting for the UNM team to request the tissues from this animal. The NHP will be
exposed to LVS by bronchoscopy when UNM is ready to test the lymph node tissues in
the peptide array assay again.
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
No new work done this period
4.Significant decisions made or pending
Perform repeat screen of polypeptide library with 6.0 x 105 frozen lymph node cells per well
5.Problems or concerns and strategies to address
NA
6.Deliverables completed
None
7.Quality of performance
Good
8.Percentage completed
25%
9.Work plan for upcoming month
a.Meet with the ASU group during the TVDC annual meeting in San Antonio to discuss next
step for testing/confirming stimulatory polypeptides
Page 27 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
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
a. Experiment Ftc64,9 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc experiments\Ftc64)
i. ASU requested additional lung RNA from SCHU S4 infected rats 1, 3, 5, 7,
and 24 h after infection. The failure to amplify RNA from the previous batch
of RNA (Ftc64, 8) appears to be caused by a dilution error in the inoculum.
ii. Lung RNAs have been isolated and UNM is currently check sample sterility
before shipping them to ASU.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
UNM missed a calculation/dilution error. UNM will double check calculations and institute a check-off
system into the protocol
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
35%
9. Work plan for upcoming month
Ship the Rat lung RNAs to ASU during the week of 9/14/09
Milestone 35
Milestone description: Array hybridizations with mouse RNAs from virulent Schu 4 infection &
RT PCR confirmation of candidates.
Institution: UNM/ ASU-Johnston
1. Date started: 04-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Current status: The early time course kinetic experimental design includes two mouse challenge level
groups (low-dose (~200-500 CFU/lung at 1 hour post challenge) and high dose (~4-5,000 CFU/lung
at 1 hour post challenge) and a rat challenge (~2,000 CFU/lung at 1 hour post challenge). Each
challenge dose time course has been performed twice at UNM. Time course samples were taken at
1, 3, 5, 7, and 24 hours post challenge. There are multiple animals per sample time, a pool of RNA
was prepared for each time point and the pool of RNA was purified over RNAeasy columns. After
purification, the RNA pools are processed using the Linear Amplification of Procaryotic Transpcript
(LAPT) protocol. The amplified RNA is processed into cDNA, fluorescently labeled, and allowed to
hybridize to the Francisella tularensis 70-mer oligonucleotide microarray printed on slides.
ASU will create two LAPT samples per time point RNA and perform two independent labelings per
LAPT. ASU has completed the hybridizations of the mouse sample sets and one rat sample set. A
Page 28 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
second rat experiment failed during multiple attempts at the LAPT amplifications. A repeat rat
experiment has been performed at UNM and samples are expected within the next 2 weeks at ASU.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
The last rat experiment samples failed to amplify in the LAPT process in 3 separate experiments.
The reason for this failure is unclear. ASU verified that the kits and reagents were working by using a
mouse RNA sample that had previously amplified as a positive control and this sample was included
in the second run at the same time as the new rat samples. A second RNAeasy purification was
performed with the rat RNA to ensure that the samples were adequately clean. Bioanlayzer analysis
revealed that the RNA was of good quality and not degraded. Yet, in a third attempt the LAPT
process did not amplify the samples. ASU requested a new set of infected rat lung RNAs from UNM
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
95%
9. Work plan for upcoming month
Printed and QC validated microarray slides are ready for hybridizations. ASU will finish independent
LAPT amplifications and the labeling and hybridizations of the second rat experiment. Initial QPCR
testing is underway of the 15 selected genes identified in Milestone 36. Final gene selection will
occur as described in the Milestone 36.
Milestone 36
Milestone description: Final integration of expression data and informatics analysis
Institution: UNM/ ASU-Johnston
1.Date started: 03-01-2009
2.Date completed: Pending
3.Work performed and progress including data and preliminary conclusions
The strategy for gene selection utilizes pattern mapping algorithms to
determine any one of three basic patterns of gene expression (Up, Down,
and Flat) over the time course of infection. ASU assigns the pattern in the
GeneSpring analysis software so that any signals will be low in the
uninfected lung (T0) and then progress across the time course with the
defined patterns.
The following data are derived from the two high dose mouse experiments and
one rat experiment. The data were used to create restricted patterns of
expression using genomically normalized (GN) two color and single color
(SC) data sets. In addition, ASU utilized the data from two species (rat and
mouse) experiments. Venn diagram comparisons were utilized to create
individual data sets containing genes identified across the patterns of
microarray gene expression and across the various data sets. These
combined data sets were hand curated to select genes with smooth
transitions across the time course and for high level expression within the
subgroup. These final microarray gene expression data sets were
Page 29 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
visualized and are shown in the following graphs. Figure 1 represents the
gene lists that were determined to match the increase in expression over
time. The identified genes are shown to the right of the graph and the order
of the genes was determined by the level of expression at the 24 hour time
point. The data sets used to select the genes are also shown. For the
increasing in expression over time gene list, two were derived from
comparing GN and SC data and three were derived from the mouse and rat
experiments. The selected genes for the decreasing over time pattern are
shown in Figure 2. For the hand curated set, one gene was identified at the
intersection of all data sets and two each for the GN and SC data sets and
mouse and rat data sets. Similarly, the flat over time expression set is
shown in Figure 3. Two genes were identified using the GN and SC data
sets and three were from the mouse to rat comparisons. The Kegg
information for these 15 selected genes is shown in Figure 4. The genes
highlighted in red were also identified in the publication by Weiss et al
investigating the identification of potential virulence genes utilizing signature
tagged mutagenesis.
Figure 1. Expression patterns of curated
gene sets of 5 selected genes increasing in
expression over the infection time course.
The identified genes are shown to the right
along with the experiments used in the Venn
diagram for the gene selection.
Figure 2. Expression patterns of curated
gene sets of 5 selected genes decreasing in
expression over the infection time course.
The identified genes are shown to the right
along with the experiments used in the Venn
diagram for the gene selection.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milesto
nes\36\08-2009\IOT_8-25-09.JPG
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milesto
nes\36\08-2009\DOT_8-25-09.JPG
Page 30 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 3. Expression patterns of curated
gene sets of 5 selected genes flat in
expression over the infection time course.
The identified genes are shown to the right
along with the experiments used in the Venn
diagram for the gene selection.
Figure 4. Kegg information of the 15 selected
gene sets.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Mileston
es\36\08-2009\Kegg_8-25-09.JPG
File locations …
R:\GeneVac\FTU\Contract\Microarray\Mileston
es\36\08-2009\FOT_8-25-09.JPG
From the primate ELISpot data, 10 proteins were identified with the highest interferon
gamma stimulation activity. We utilized these gene lists selected for the top 5 and
second 5 in activity and graphed their expression patterns on the mouse expression
data. The resulting graphs are shown below. The top 5 genes are shown in Figure
5. Interestingly, two of the top 5 protein fragments were from the same gene,
FTT0323 and thus there are only 4 genes listed in the top 5 category. These genes
would not have been identified in our microarray gene expression analyses because
the high level of T0 expression levels were very high supporting that there may be
some non-specific priming during the amplification. However, there were some
changes in expression over time but not in a consistent smooth transition. The
second set of 5 proteins was mapped onto the mouse data set and is shown in
Figure 6. This second set had a lower T0 level of detection and for the most part
would have been considered in the flat over time category
Page 31 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 5. Expression patterns of the top 5 proteins
selected by the primate ELISpot data analyses.
Figure 6. Expression patterns of the second top 5
proteins selected by the primate ELISpot data analyses.
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milestones\36\082009\PS_Top-5_8-31-09.JPG
File locations …
R:\GeneVac\FTU\Contract\Microarray\Milestones\36\082009\PS_2nd-5_8-31-09.JPG
4.Significant decisions made or pending
After the next rat samples are received and processed, the combined datasets need to be utilized for
final gene list selections. Once genes are selected by expression pattern analysis, the results will be
confirmed by qPCR analyses. From those genes that are confirmed by qPCR, 10 will be selected for
evaluation for vaccine efficacy in Milestone 37.
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
Combine the last rat experiment data sets into the analyses. We plan to establish a realtime data selection session utilizing a WebEx session so that participants can ask questions
of the data and receive real-time results. Initially the real time WebEx session will be
between ASU and UNM before inviting additional participants.
Page 32 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
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 twofold, 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.
Currently, UTSA worked on determining if the FTT0748 mutant is attenuated by performing a
mouse experiment. In addition, UTSA tried to create the last SCHU S4 mutant of this milestone,
NadM.
UTSA used the KKT1 SCHU S4 strain for experiments where electroporation was
performed to get the plasmid constructs containing the desired gene targets into the bacteria.
Once the plasmid is in the bacteria; the entire plasmid will be incorporated into the genome of
SCHU S4 (as for pKEK1305 pJC84 T20+NadM). The potential correct clone is initially
determined by the antibiotic selection used in the transformation experiment. Typically the gene
sequence that is specific to the target gene will have a sequence cassette, such as kanamycin,
which allows for selection of the clones which integrated at the correct location in their genome.
The plasmid which carries this target gene sequence will have a different “marker” (antibiotic
resistance) which will allow for counter selection to verify the removal of the plasmid from the
bacteria. In order to verify potential correct clones we perform polymerase chain reactions
(PCRs) using genomic DNA isolated from a potential clone and various oligo sets. The oligo sets
are typically directed to an antibiotic resistant sequence cassette, gene targets at the 5’ or 3’ end
of the desired gene and origin of replication specific oligos to check for the presence of the
plasmid in the bacteria. Once the correct mutant is identified by PCR results, some PCR
products are sent for sequencing for verification. UTSA then will remove the plasmid from the
bacteria and the end result is the final mutant which will be used in subsequent experiments that
will help determine if this mutant is a good vaccine candidate. UTSA did not generate the NadM
SchU S4 mutant this month.
•Mouse Experiment with the FTT0748 SCHU S4 mutant.
•Result: The FTT0748 SCHU S4 mutant was found not to be attenuated in mice.
•1st A working culture stock of FTT0748 was grown at 37°C then harvested and frozen to be used
in mouse experiment. Various titrations were done on stock and the working stock
concentration was determined.
•Result: Based on three independent vials used the average titration concentration was
determined as 6.0 X 108 CFU per ml.
nd
•2 Balb/c mice were intranasally inoculated with various dilutions of FTT0748 mutant.
Subsequently, the stock vial used was plated at various dilutions to determine empirically the
various inoculation doses used. Our target doses were 10, 100, 1000, 1x10 4 and 1x105
CFU/ml, respectively. A PBS negative control group was done and each test group had 5
mice.
Page 33 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
•Results: The empirically measured intranasal doses were 5, 50, 177, 1974 and 1.97 x10 4
CFU/ml, respectively.
•3rd Mice were observed everyday following the inoculations.
•Results: Only one mouse in the test group receiving 5 CFUs survived after day 8. There is
no significant difference in time of death compared to the Schu S4 strain.
Table 1.
Group of Mice
Intranasal
inoculations
Dose (CFUs)
FTT0748
mutant
5
50
177
1974
1.97E4
PBS
Survival Rate
D1
D2
D3
D4
D5
D6
D7
D8
D22
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
5/5
3/5
5/5
5/5
5/5
4/5
0/5
0/5
5/5
4/5
0/5
0/5
2/5
1/5
1/5
−
−
−
−
−
−
−
−
−
−
−
−
−
−
5/5
5/5
5/5
5/5
Table 1 represents
the results of a mouse experiment where mice were intranasally inoculated with the FTT0748 Schu S4
mutant. By day 5 all mice in the 1x103 and 1x104 test groups died. By day 6 all the mice in test group 1x10 2
and 50 CFU test groups died. Finally, by day 7 all but one mouse in the test group receiving 5 CFUs died.
The LD50 for the Schu S4 strain is 10 CFUs and typically all mice die by day 5. Data located in TVD UTSA
Notebook 7, page 125-127. The FTT0748 SCHU S4 strain is not attenuated in mice.
•Creation of the NadM SCHU S4 mutant.
•Result the NadM mutant of SCHU S4 was not isolated.
•1st Clones 1 and 5 from previous screen were streaked to generate single clones on fresh
kanamycin plates.
•Result: Two clones were generated in the C1 plate and 4 clones were generated in the C5
plate.
•2nd UTSA patched all of the resulting colonies from these groups onto fresh kanamycin plates,
allowed these to grow.
•Result: The patched C1 singles did not grow on the subsequent patch plate and two of the four
single clones grew (slowly). UTSA has not isolated the genomic DNA from these two clones
to determine whether UTSA has a pure clone of NadM.
Data located in TVD UTSA Notebook 7, page 123 and 128.
•Cloning of the mobRP4 gene into the pKEK1305 (pJC84_T20 NadM) plasmid. UTSA pursued
increasing the number of transformants with this construct (pKEK1305) by introducing the
mobRP4 gene. This mobRP4 gene facilitates movement of genes from plasmids into bacterial
genomic targets.
•Result: Completed digestions, ligations and generated transformants to screen for plasmid
containing the MobRP4 gene.
•1st UTSA used the MobRP4 pcr product, the pJC84 and pKEK1305 plasmid and digested with
Sal I
•Result: The restriction enzyme digestions were successful.
•2nd UTSA used the digested pJC84 and pKEK1305 then alkaline phosphatase (CIP) treated
these plasmids to reduce the background of re-ligation frequency of these plasmids during
the followed ligation reactions.
•Result: These manipulations were successful (figure 1).
Page 34 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Figure 1.
Figure 1 represents various isolated DNA components used in the cloning of the MobRP4 gene into pKE1305
(lane4 and 5) and pJC84 (lane3), respectively. Lane 2 is the MobRP4 gene pcr product Sal I digested; lanes 3
and 4 are the plasmids which were Sal I digested then alkaline phosphatase treated (CIP) and all were gel
purified using a kit. These profiles represent only 2 ul of 28 ul purified DNAs, respectively. Lane 5 is uncut
pKEK1305 profile which should run slightly lower than the cut plasmid (lane4). The integrity of the DNA looked
good (no degradation visible and sizes are correct) and was used in a ligation reaction. Data located in TVD
UTSA Notebook 7, page 124.
•3rd UTSA used the DNA components in figure 1 in various ligation reactions and transformed DH5α
cells to try and generate the pKEK1305 (T20 NadM construct) and the pJC84 parent plasmid
containing the MobRP4 gene.
•Result: Generated several hundred transformants in each plasmid ligations, pJC84 +
MobRP4 and pKEK1305+ MobRP4, respectively. Will screen these transformants for
correct plasmid construct(s) next month.
4. Significant decisions made or pending
UTSA has been working for several months to try and generate the SCHU S4 NadM mutant
without success. UTSA tried the “tulatron technique” and the last attempt was to use a different
transposon and delivery plasmid pJC84 containing T20 NadM (pKEK1305) recovered from a
F.novicida strain in hopes that the change in chromosomal structure around NadM may allow the
deletion to be successful. However, two attempts did not yield an isolated NadM; and there
appears still to be substantial growth defect so the “health” of this mutant is still in question.
UTSA believe perhaps the role NadM plays in the virulent Francisella strain, SCHU S4 is
significantly more linked to its survival and therefore we’ve chose to discontinue pursuing this
mutant.
5. Problems or concerns and strategies to address
UTSA was using the pJC84+T20 NadM construct with the goal to generate this NadM mutant in
SCHU S4 by this method. The clones processed from last month did not generate very many
single colonies and those that did arise, grew slowly. UTSA chose to introduce the MobRP4
gene back into this mating plasmid with the goal to the increase the number of transformants
by mating this plasmid into the SCHU S4 bacteria instead of using electroporation. Although
UTSA generated transformants for this MobRP4 containing plasmid UTSA, has run out of time for
this milestone and UTSA does not have an isolated NadM Schu S4 mutant.
6. Deliverables completed
KKT5: igLC1 IgLC2 Schuh4: KKT10: iglD1 igLD2 Schuh4; KKT13: VgrG1 VgrG2; and KKT26:
FTT0748 mutants are completed Schuh4 strains to date.
7. Quality of performance
Good
8. Percentage completed
100%
9. Work plan for upcoming month
a. UTSA will begin milestone 54 gene deletion constructs in SCHU S4, after discussions with
UNM to finalize the genes in the next couple of weeks. MS 54 is dependent on the genes
Page 35 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
identified by the proteome (MS30) and transcriptome (MS 37) approaches. The genes will be
selected due to their potential to attenuate Schu S4. They may lead to a vaccine candidate
based on work done in F.novicida work in other labs and based on proteome and transcriptome
work performed at ASU and UNM. During discussions in the yearly UNM meeting, as well as, in
the last site visit in San Antonio, there were discussions to pursue the gene atpC (UTSA 2009 site
visit slide for Milestones in the next 18 months) and 1pxF.
b. Will begin work with FTT0065 gene identified as atpC gene in F.tularensis. UTSA will begin
with analysis of gene to generate oligos to use in cloning into the “tulatron plasmid” pKEK1140.
c. Will order appropriate supplies as required.
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
UTSA created FTN1487 gene mutants in Fn.U112 (KKF378) and FTT1579 gene mutants in Ft.Schu
S4 (KKT19). UTSA is in the process of constructing the tulatron vector for insertion mutagenesis of
FTT0523, which is another restriction barrier in Schu S4, in both wild type Schu S4 and in KKT19
(FTT1579).UTSA will create the single FTT0523 mutant in Schu S4 and a double FTT0523 and
FTT1579 mutants in Schu S4. This method allows retargeting the restriction enzyme and inactivating
a specific gene to facilitate introduction of foreign plasmid DNA into Schu S4 strain.
. Verification of KKT19 frozen stock
. Result: KKT19 (FTT1579 restriction enzyme barrier mutant in Schu S4) was verified by
sequencing and PCR.
.
1st PCR amplification of KKT19 using both sets of primers
.
Result: PCR confirmed that the insertion was in FTT1579 gene of KKT19.
Figure1: Gel picture of PCR
Figure1 legend, results and data location: lane2-6 was PCR amplified using FTT1579 gene primers,
whereas the insertion primer was used for lane7-11. KKT19 (lane2 and 7) produced the same PCR
product as the positive control KKF378 (lane5 and10) compared to the negative control KKT1 (lane6
and 11), which indicated the existence of intron insertion in FTT1579 of KKT19.
Data recorded on UTSA TVDC notebook #6, page97 for Figure1.
.
2nd The gel purified PCR DNA amplified with the insertion primer in Figure1 (lane7) was
sequenced using the FTT1579 gene primer “FTN1487 Rev” and the insertion primer “EBS
Universal”.
Page 36 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
. Result: the intron insertion at 849/850bp of FTT1579 gene in KKT19 was confirmed
by sequencing
.
Data location: UTSA TVDC notebook #6, page98 and 99.
.
Construction of the tulatron vector for insertion mutagenesis of FTT0523 in wild type
Schu S4 and in KKT19
.
Result: The primers for amplifying 350bp PCR were designed and ordered. 350bp PCR
product was produced.
.
1st Designed the primers for amplifying 350bp PCR
.
.
Result: 2 sets of primers for insertion of mutated intron at 390/391bp or 364/365bp of
FTT0523 were designed and ordered.
2nd Generation of 350bp PCR product
.
Result: 350bp PCR product for insertion at 390/391bp of FTT0523 was amplified.
Figure2: Gel picture of 350bp PCR
Figure2 legend, results and data location: the band below 0.5kb on lane2-4 was the expected 350bp
PCR product. Data recorded on UTSA TVDC notebook #6, page101 for Figure2.
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 71% of scientific work completed.
9. Work plan for upcoming month
i. Digest 350bp PCR and the parent plasmid pKEK1140 with XhoI and BsrGI to produce cohesive
ends for ligation of 350bp PCR fragment into pKEK1140.
ii. Ligate the digested 350bp PCR into the plasmid pKEK1140 digested with the same
enzymes.
iii Transform the ligation reaction from above into DH5 competent cells.
iv Screen the potential transformants.
Page 37 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Milestone 53A
Milestone description Phenotyping and vaccine efficacy demonstration of recA, other subsp.
tularensis mutants.
53.1: phenotyping and immunologic characterization of Ft subsp. tularensis recA and recAiglC
strains, as well as strains generated in milestone #54.
53.2: phenotyping and immunologic characterization of Ft subsp. tularensis recA plus best
attenuating mutation(s) strains (generated in milestone #52), as well as strains generated in
milestone #54.
Institution: UTSA
1. Date started: 04/01/2009
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
53A: Intramacrophage growth of KKT19, a SCHU restriction enzyme (FTT1579) mutant
(Note book # 9, page 44-46). Murine macrophage cell line (J774) were seeded in a 96-well
plate (2x105/200 μl/well) overnight and infected with the KKT19 mutant or its parental strain
(SCHU S4) using an infection dose of 10 or 100 MOI. Numbers of viable bacteria in
macrophages were measured at 3 hr and 24 hr post-infection. As expected, the wild type
SCHU S4 replicated rapidly inside macrophages within a day using MOI of 10 or 100.
Intramacrophage replication of KKT19 was comparable to the wild type SCHU S4 strain at
the end of 24 hr incubation with both infection doses. This implies that KKT19 may not be
attenuated. A murine intranasal infection model has been set up to study the virulence of this
KKT19 mutant in vivo.
Fig. 1. Intramacrophage growth of SCHU S4 FTT1579 mutant (KKT19).
Murine macrophage cell line (J774) were infected with KKT19 or its parental
strain (SCHU S4) using an inoculum of 10 or 100 MOI. Numbers of viable
bacteria in macrophages were measured at 3hrs and 24 hrs post-infection.
4. Significant decisions made or pending
None
Page 38 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25% of scientific work completed on milestone 53A.
9. Work plan for upcoming month
1.Determination of LD50 of KKT19 using an intranasal challenge model.
2.Evaluation of the cellular response of mice immunized with F. novicida iglB (KKF235) by the
intranasal and oral routes.
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: Bacterial dissemination profile in F344 rats following F. novicida intratracheal
challenge. (Notebook # 10, pages 90, 91) Groups of F344 rats (3 rats per group) were
challenged intratracheally (IT) with 105 CFU of F. novicida U112. Rats were sacrificed at one
and three days after challenge and lungs, livers, spleens and cervical lymph nodes were
removed aseptically in to DMEM supplemented with 10% Fetal Bovine Serum (D10). Tissues
were then homogenized and 10-fold serial dilutions were plated on tryptic soy agar to
determine bacterial numbers. As seen in Figure 1, by 24 hours after challenge, bacteria have
replicated 2-3 logs in the lungs, 4 logs of bacteria are already present in both the liver and
spleen, and a small number of bacteria can be found in the cervical lymph nodes. By 3 days
after challenge, the numbers of bacteria present in the lungs has stayed steady, whereas
bacteria have replicated an additional 2-3 logs in the liver, spleen and cervical lymph nodes.
This data indicates that upon IT challenge, F. novicida is able to replicate to a high degree
and disseminate very quickly to systemic organs.
Page 39 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Fig. 1. Bacterial dissemination in target organs following F. novicida U112 intratracheal
challenge. Groups of rats (3 rats per group) were challenged IT with 10 5 CFU of U112. At
days 1 and 3 after challenge, lungs, livers, spleens and cervical lymph nodes were
homogenized and serial dilutions were plated to determine bacterial numbers.
53B-b: Cellular responses in F344 rats following F. novicida U112 intratracheal challenge
were determined (notebook # 10, pages 86, 93, 96). Groups of Fisher 344 rats (3 rats per
group) were challenged IT with 105 CFU of F. novicida U112 in PBS, or mock challenged with
PBS alone, and rested for 14 days. Rats were then sacrificed, spleens and cervical lymph
nodes were removed in groups, and single cell suspensions were made. Cells were then
incubated in the presence of UV-inactivated U112 (103, 104, 105 CFU), or the unrelated
antigen hen egg lysozyme (HEL), for 72 hours. Culture supernatants were then collected and
analyzed for the presence of IFN- by cytokine ELISA. As seen in Figure 2, F. novicida
intratracheal challenge induces high levels of antigen-specific IFN- in both the spleens and
cervical lymph nodes. There was little IFN- produced by incubation with the unrelated
antigen HEL, or from mock challenged rats.
Page 40 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Fig. 2. Cellular response of Fisher 344 rat intratracheal F. novicida challenge. Groups of F344
rats (3 rats per group) were challenged IT with 105 CFU of F. novicida and rested for 14 days.
Single cell suspensions of spleens and cervical lymph nodes were incubated with increasing
concentrations of UV-inactivated F. novicida ,or the unrelated antigen HEL, for 72 hours.
Culture supernatants were collected and analyzed for antigen specific IFN- by ELISA.
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
53B. Bacterial dissemination in Fisher 344 rats following LVS vaccination and SCHU S4
challenge.
Page 41 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
Acronym List Updated 9/10/09
ABSL3: Animal Biosafety Level 3 lab
AM: alveolar macrophages
ASU: Arizona State University
BLD: below limit of detection
BMDM: bone marrow derived macrophages
BP: base pair
BSL2: Biosafety Level 2 lab
BSL3: Biosafety Level 3 lab
C: centrigrade
cDNA: complimentary deoxyribonucleic acid
CDA: confidentiality and disclosure agreement
CDM: Chamberlains defined media
CFU: colony forming units
COA: contract officers authorization
CRDA: Cooperative research and development agreement
C-term: Carboxy terminus
DNA: deoxyribonucleic acid
DVC: Dynport Vaccine Company
ED50: Effective Dose, 50 % dead
ELISA: enzyme-linked immunosorbent assay
ELISpot: enzyme-linked immunospot assay
EOH: Employee Occupational Health
FDA: Federal Drug Administration
FF: formalin fixed
FSC: forward scatter, equivalent to cell size FT or Ft or F. tularensis: Francisella tularensis
GDP: genome directed primers
GI: gastrointestinal
GLP: good laboratory practice
KBMA: killed but metabolically active
HK: heat killed
HTP: high through put
IAA: interagency agreement
IACUC: Institutional animal care and use committee
ICS: Intracellular staining
ID: intradermal
IFNγ: interferon gamma
Page 42 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
IL2: interleukin 2
IN: intranasal
IND: investigational new drug
IP: intraperitoneal
IRB: institutional review board
IRS: immune rat sera
IT: intratracheal
IVT: in vitro translation/transcription
KB: kilobase
L: liter
LAPT: Linear Amplification of Procaryotic Transpcript
LBERI: Lovelace Biomedical and Environmental Research Institute
LD50: Lethal dose 50% dead
LD99: lethal dose, 99% dead
LN: lymph node
LPS: lipopolysaccharide
LRRI: Lovelace Respiratory Research Institute
LVS: Live vaccine strain (tularemia)
NHP: nonhuman primate
MDM: monocyte derived macrophages
mL: milliliter
MLN: mesenteric lymph node
mM: Milli molar
MOI: multiplicity of infection
MS: Milestone
MSCR: Milestone Completion Report
MTA: material transfer agreement
MTS reagent: (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2Htetrazolium, inner salt
N: number
NIAID: National Institute of Allergy and Infectious Disease
NRS: normal rat sera
N-term: amino- terminus
OD: optical density
ORF: open reading frame
PBMC: peripheral blood mononuclear cells
PBS: phosphate buffered saline
PFA: paraformaldehyde
Page 43 of 44
Tularemia Vaccine Development Contract: Monthly Technical Report
Period: 8/01/2009 to 8/31/2009
Due Date: 9/25/2009
Prepared by: C. Rick Lyons, Terry Wu, Barbara Griffith, Karl Klose, Bernard Arulanandam, Stephen Johnston, Mitch
Magee, Kathryn Sykes, Bob Sherwood, Michelle Valderas, Dana Pohlman, Julie Wilder, Julie Hutt, and Trevor Brasel
pM: pico Molar
pmol: picomole
PCR: polymerase chain reaction
QA: quality assurance
QC: quality control
qPCR: quantitative polymerase chain reaction
RNA: ribonucleic acid
SC: subcutaneous
SDS: sodium dodecyl sulfate
SCHU S4: virulent strain of Francisella tularensis
SIP: Special Immunizations Program at USAMRIID
SOP: standard operating procedure
SSC: side scatter, equivalent to cell density or granularity
TBLN: tracheobronchial lymph node
Tn: transposon
TNFα: tumor necrosis factor alpha
TVDC: Tularemia vaccine development contract
uL: microliter
UNM: University of New Mexico
USAMMDA: United States Army Medical Materiel Development Activity
USAMRIID: United States Army Medical Research Institute for Infectious Disease
UTSA: University of Texas at San Antonio
WT: wild type
Page 44 of 44