Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two
animal models and cellular assays vital for testing vaccine efficacy.
Sections II and III:Progress and Planning Presented by Milestone
Active milestones: 2, 4, 5, 7, 8, 9, 11, 12/13(UNM/LBERI), 14, 17, 18, 19,
21(UNM/LBERI), 27, 28, 35(ASU/UNM), 49, 50, 52, 55, 56, 57
Completed milestones: 1, 3, 25, 26, 32, 33, 34 (UNM/ASU), 16, 39, 40, 43 (UTSA),
48, 51
Inactive milestones: 6, 10, 15, 20, 22, 23, 24, 29, 30, 36, 37, 38, 53, 54, 58, 59
Milestones terminated after initiation: 41, 42, 44, 46, (MSCR will be written)
Milestones terminated before initiated: 43 (Cerus), 45, 47 (MSCR will not be written)
Milestone 2
Milestone description: Vaccinations performed on relevant personnel
Institution: UNM/LRRI
1. Date started: 11/01/2005
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
a. UNM EOH has performed 18 annual health screenings since 8/26/08 for the LVS
vaccinees originally vaccinated on in September and October 2007.
b. Three UNM and possibly 6 LBERI scientists will request vaccinations in 2009.
c. USAMRIID canceled the 1/27/09 vaccination date.
4. Significant decisions made or pending
a. Dr. Lyons received UNM IRB approval to allow blood draws on the vaccinated
LBERI and UNM scientists after their LVS vaccinations. The LVS vaccinated
LBERI and UNM scientists and staff have been offered the opportunity to
volunteer to donate bloods for the development of immunoassays, approximately
2 months after receiving the LVS vaccination.
b. USAMRIID resumed offering the LVS vaccine as of October 7, 2008 but will not
offer vaccinations to UNM and LBERI until FDA approval is given.
c. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI will offer the LVS
vaccinations to 9 more scientists to total up to 46. The CRDA with USAMRIID is
valid for 2 years, ending June 2009.
5. Problems or concerns and strategies to address
a. Nine scientists could be vaccinated in 2009 if USAMRIID receives FDA approval
for the new Tularemia vaccination protocol.
6. Deliverables completed
Page 1 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS
vaccination since 9/11/07.
7. Quality of performance
Excellent
8. Percentage completed
71% of the scientific work is complete
9. Work plan for the next month
a. Continue annual health screenings required by USAMRIID and being performed
at UNM for the LBERI and UNM LVS vaccinees.
b. UNM will be obtaining blood donations from LVS vaccinees for immunoassay
development and reimbursing participants $40/ donation.
c. UNM will work with 3 UNM and 6 LBERI scientists for the pre-vaccination health
screenings required for vaccinations in January, February and March 2009
10. Anticipated travel
Four LVS vaccinees to USAMRIID from 1/26/09 to 1/29/09
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
No work was performed during this reporting period.
4. Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed
95% of the scientific work is complete
9. Work plan for next month
a. Continue working on the Milestone Completion Report.
b. Continue reading the histopathology slides from Cohort 3 as they become
available.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Page 2 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Milestone 5 - UNM
Milestone description: Small species tested for sensitivity to LVS & generation of
immunity against a pulmonary challenge of SCHU S4
Institution: UNM
1. Date started: 12/12/2005
2. Date completed: pending
3. Work performed and progress including data and preliminary conclusions
a. Experiment Ftc75 (Notebook 115, pages 178-179)
i. The purpose of this experiment is to compare the histopathology of
BALB/c mice, Fischer 344 rats, and Cynomolgus monkeys with
pulmonary SCHU S4 infection
ii. In preparing the manuscript describing the Fischer 344 rat model,
Julie Hutt noticed that rats infected intratracheally (i.t.) with SCHU S4
developed a different pulmonary disease than mice infected
intranasally with NMFTA1 (biovar A) or by aerosol with strain 33
(biovar A); infected rats developed bronchopneumonia whereas
infected mice developed vasculitis. She further indicated that the rat
disease is similar to the NHP and human diseases that had been
described in the literature. Since these interpretations were based
on histological data generated in separate experiments using
different bacteria strains, dose and methods of pulmonary infection,
we performed a side-by-side comparison of mice, rats, and NHPs
challenged with 1000 SCHU S4 by surgical i.t., non-surgical i.t. and
bronchoscopy, respectively.
iii. Tissues were collected on the following schedule
1. NHP -- 1, 4, and 7 days post challenge
2. Mice -- 1, 2, 3, and 4 days (infected mice did not survive to
day 7)
3. Rats -- 1, 2, 3, 4 and 7 days.
iv. The tissues are being processed at LBERI
b. Experiment Ftc71.1 (Notebook 130 pages 18-21)
i. The purpose of this experiment is to determine the effect of LVS
vaccination dose on the resistance of vaccinated rats to i.t. SCHU S4
challenge
ii. Fischer 344 rats (n = 6) were either left unvaccinated as a negative
control or vaccinated s.c. with 103, 105, or 107 LVS
iii. In early January 2009, slightly more than 1 month after LVS
vaccination, the vaccinated rats and control rats will be challenged
i.t. with 104 SCHU
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
Page 3 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
7. Quality of performance
NA
8. Percentage completed
90%
9. Work plan for upcoming month
a. Complete the histopathological analyses of tissues from mice, rats, and
NHPs infected with SCHU S4
b. Complete the experiment to determine whether the effect of vaccination is
dose dependent
c. Complete and submit manuscript describing the Fischer 344 rat model
d. Complete milestone completion reports for the mouse, rat, and guinea pigs
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 7
Milestone description: SCHU S4 ED50 in primates determined from selection of
challenge dosing
Institution: LBERI
1. Date started: 2/25/08
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions:
a. Overview of presented dose by animal versus time to death is depicted in Figure
1. Animals were given between 1 cfu and 1,260,000 cfu in waves 1, 2 and 3.
The data presented below (presented dose versus time to death graph) was
compiled to act as a guide to the data that follows in this report. It summarizes
the dose each animal received and the trend is such that the higher the dose an
animal receives, the shorter the time to death. Similarly, the lower the dose, the
longer the time to death. The lone survivor, animal 28624 (2.85 cfu presented
dose) was not included on this graph because it was alive as of Nov. 30. This
same data will be used to help calculate the LD50.
Page 4 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 1. Presented dose vs. time to death for all animals in all waves of ED50. Electronic file
located on Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 1.
b. Wave 1 high dose challenges were performed in October 2008 with target doses
of approximately 1000, 10,000, and 100,000 CFU. Hematology data (Figures 2
thru 4) demonstrates an increase in white blood cells by Day 2 followed by a
decrease before death or euthanasia; an increase in % neutrophils by Day 2
followed by a decrease before death or euthanasia; and a decrease in %
lymphocytes by Day 2 followed by an increase before death or euthanasia.
Leukocytosis (a high white blood cell count) can be a reaction to various infectious
diseases. As such we collected blood for hematology from infected animals.
Leukocytosis may be caused by an increase in neutrophils, lymphocytes, and other
cell types. Data from wave 1 indicates that leukocytosis did occur in these animals 2
days after challenge and that phenomenon was due to an increase in the number of
neutrophils despite a decrease in the number of lymphocytes. Leukocytosis occurred
on day 2 and was followed by either death or leucopenia prior to death. Leucopenia
was due to a sharp drop in the number of neutrophils and in spite of an increase in
lymphocytes. The significance of this data will become clear after all waves are
compared and trends observed.
Page 5 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 2. Schu S4 Wave 1 Total White Blood Cells Counts. The data is reported for each
individual animal starting at Day -14 through time of death or euthanasia. Normal hematology
range was 6.9 to 19 X 103 per µL. Electronic file located on Z:\Agent and Study Specific Data
and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave
1.
Page 6 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 3. Schu S4 Wave 1 Percentage of Neutrophils in the blood of infected animals. The data
is reported for each individual animal starting at Day -14 through time of death or euthanasia.
Normal % neutrophil range was 38 to 80.8 . Electronic file located on Z:\Agent and Study
Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL07)\ED50 Wave 1.
Figure 4. Schu S4 Wave 1 Percentage of Lymphocytes in the blood of infected animals. The
data is reported for each individual animal starting at Day -14 through time of death or
euthanasia. Normal % lymphocyte range was 27.9 to 80.8 . Electronic file located on Z:\Agent
and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08074 (TUL-07)\ED50 Wave 1.
Bacteremia data (Figure 5 and Table 1) showed that many animals died or were
euthanized before there were culturable bacteria in the blood or that there was a
small amount of bacteria that was cultured on Day 4. No trend was observed
and many animals died very quickly and in spite of having no detectable bacteria
within the blood. The dose may simply be too high to observe bacteremia in
every animal.
Page 7 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 5. Schu S4 Wave 1 Bacteremia. The data is reported for each individual animal starting at
Day 0 through time of death or euthanasia. Electronic file located on Z:\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50
Wave 1.
Animal #
28511
28438
28565
28496
28559
28569
28395
28447
28525
28549
28570
28617
Day 0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
4
0
10
0
16.7
8.67
5667
440
233
6
0
0
233
Table 1. Schu S4 Wave 1 Bacteremia. The data is reported for each individual animal as
CFU/ml. Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 1.
To summarize, data from wave 1 indicates that leukocytosis did occur in these
animals 2 days after challenge and that phenomenon was due to an increase in the
number of neutrophils despite a decrease in the number of lymphocytes.
Leukocytosis occurred on day 2 and was followed by either death or leucopenia prior
to death. Leucopenia was due to a sharp drop in the number of neutrophils and in
spite of an increase in lymphocytes. The significance of this data will become clear
after all waves are compared and trends observed. many animals died or were
euthanized before there were culturable bacteria in the blood or that there was a
Page 8 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
small amount of bacteria that was cultured on Day 4. No trend was observed
and many animals died very quickly and in spite of having no detectable bacteria
within the blood. The dose may simply be too high to observe bacteremia in
every animal.
c.
Wave 2 challenges were performed in October 2008. The animals were
presented with 1-2 CFU (target dose 25 CFU) and 19-90 CFU (target dose 250
CFU). Again, because leukocytosis is a common response to infectious agents,
hematology was performed on study days 0, 2, 4, 6, 10, 14, 20, and 26 in
November. Hematology data (Figures 6 thru 8) demonstrates a slight decrease
in white blood cells on Day 2 followed an increase in white blood cells on Day 4
followed by a decrease and, in some cases, a slight increase before death or
euthanasia; a decrease in % neutrophils on Day 2 followed by an increase on
Day 4 followed by a decrease and, in some cases, a slight increase before death
or euthanasia; and an increase in % lymphocytes on Day 2 followed by a
decrease on Day 4 followed by an increase and in some cases a slight decrease
before death or euthanasia. This data is quite different from the data observed in
wave 1. The wave 2 animals did not present with symptoms indicative of primary
pulmonic disease, and this hematology data may distinguish between the more
protracted non-pulmonic disease and primary pulmonic disease. The significance
of this data will become clear after all waves are compared and trends observed.
Figure 6. Schu S4 Wave 2 Total White Blood Cells Counts. The data is reported for each
individual animal starting at Day -14 through time of death or euthanasia. Normal hematology
range was 6.9 to 19 X 103 per µL. Electronic file located on Z:\Agent and Study Specific Data
and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave
2.
Page 9 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 7. Schu S4 Wave 2 Percentage of Neutrophils in the blood of infected animals. The data
is reported for each individual animal starting at Day -14 through time of death or euthanasia.
Normal % neutrophil range was 38 to 80.8. Electronic file located on Z:\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50
Wave 2.
Figure 8. Schu S4 Wave 2 Percentage of Lymphocytes in the blood of infected animals. The
data is reported for each individual animal starting at Day -14 through time of death or
euthanasia. Normal % lymphocyte range was 27.9 to 80.8 Electronic file located on Z:\Agent
and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08074 (TUL-07)\ED50 Wave 2.
Bacteremia data (Figure 9 and Table 2) showed that most animals had culturable
bacteria in the blood by Day 4 or Day 6. Two animals, 28624 and 28588, didn’t
have a positive bacteremia until Day 26 and Day 14, respectively. 28588 died on
day 20, whereas 28624 survived until scheduled euthanasia (day 46).
Page 10 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 9. Schu S4 Wave 2 Bacteremia. The data is reported for each individual animal starting at
Day 0 through time of death or euthanasia. Electronic file located on Z:\Agent and Study Specific
Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50
Wave 2.
Animal #
28463
28571
28615
28618
28499
28624
28581
28588
Day 0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
4
10
0
6.7
0
13.3
0
3.3
0
6
190
36.7
153.3
3.3
46.7
0
243.3
0
10
14
20
26
32
36
0
0
0
0
0
0
3.3
226.7
0
0
30
0
Table 2. Schu S4 Wave 2 Bacteremia. The data is reported for each individual animal as
CFU/ml. Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 2.
Temperatures were taken for each animal beginning on Day 15 through day 35.
Each day the temperature was taken at 0800, 1400, and 2000. Temperatures for
Day 0 through Day 14 were reported in the October 2008 monthly report. Table
3 reports the temperature for Days 15 through Day 35 for those animals that
were still alive.
Animal 28588 became hypothermic preceding death while
animal 28618 became febrile preceding death. Animal 28624, the lone survivor,
had variable hypothermic episodes throughout the in-life phase. Challenge
doses were low enough that we were able to capture bacteremias on every
animal.
Page 11 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Table 3. Schu S4 Wave 2 Temperatures for Day 15 thru Day 35.. The data is reported for each
individual animal. Blue indicates a decrease in temperature by 2 standard deviations from the
baseline. Red indicates a increase in temperature by 2 standard deviations from the baseline.
Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 2.
Respiratory rates were taken for each animal beginning on Day 15 through day
35. Each day the respiratory rates were taken at 0800, 1400, and 2000.
Respiratory rates for Day 0 through Day 14 were reported in the October 2008
monthly. Table 4 reports the respiratory rates for Days 15 through Day 35 for
those animals that were still alive. As shown by the data outlined in Table 4, the
dramatic increase in respiratory rates (> 30% over baseline) seen in wave 1 did
not occur in wave 2.
Page 12 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Table 4. Schu S4 Wave 2 Respiratory Rates. The data is reported for each individual animal.
Pink indicates a 30% increase in rate. Red indicates a 50% increase in rate. Electronic file
located on Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 2.
In summary, wave 2 hematology was markedly different from that observed in
wave 1. Perhaps this will provide a way to distinguish primary pulmonic disease
from the more protracted disease seen with very low challenge doses of Schu
S4. Respiratory rates and temperatures were unremarkable except prior to
death. Challenge doses were low enough that we were able to capture
bacteremias on every animal.
d. Wave 3 challenges were performed in November 2008. The animals were
presented with 237-444 CFU (target dose 250 CFU) and 621-1150 CFU (target
dose 500 CFU). Mortality data (Table 5) shows that all animals were euthanized
between Day 4 through Day 8 post challenge once moribundity criteria were met.
Page 13 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
ID#
Gender
Presented
Manner of
Timepoint at
Study Day at
Dose
Death
Death
Death
(CFU/ml)
28467
F
363
Euthanized
8 am
6
28484
F
444
Euthanized
8 am
8
28601
M
237
Euthanized
8 pm
6
28585
M
417
Euthanized
8 am
6
28479
F
675
Euthanized
8 am
5
28664
F
1150
Euthanized
2 pm
4
28512
M
884
Euthanized
8 pm
6
28572
M
621
Euthanized
8 am
8
Table 5. Schu S4 Wave 3 Mortality Data. The data is reported for each individual animal.
Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 3.
Tissue bacterial burden at time of death shows consistently high levels of Schu
S4 in lung and TBLN, with more variation by animal in the other organs (Table 6).
ID#
Spleen
Liver (cfu/g)
TBLN (cfu/g)
MLN (cfu/g)
Lung
(cfu/g)
(cfu/g)
28467
2.6e8
5.9e6
5.3e7
2.4e6
8.2e8
28484
1.4e7
1.5e5
1.3e8
1.5e5
9.7e8
28601
3.7e8
1.1e7
1.8e7
3.0e8
8.8e8
28585
1e6
1e5
1.1e8
0
1.3e9
28479
5.8e7
9.4e4
5.0e8
2.3e5
1.1e9
28664
1e8
3.0e6
2.5e8
6.3e3
2.8e8
28512
1.8e5
4.0e4
8.3e7
1.2e6
8.3e7
28572
3.6e8
7.0e6
1.5e8
3.3e7
1.0e9
Table 6. Schu S4 Wave 3 Tissue Bacterial Burden. The data is reported for each individual
animal. Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 3.
Temperatures were taken for each animal beginning on Day -7 through time of
death. Day -7 to day -1 are averaged and give the baseline shown to the right of
the figure. Each day the temperature was taken at 0800, 1400, and 2000. As
shown by the data outlined in Table 7 some animals became febrile within one to
four days post-challenge but all animals became hypothermic one to four days
preceding death. Severe hypothermia (less than 90 degrees) appears to be an
apt indicator of impending death.
Page 14 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Table 7. Schu S4 Wave 3 Temperatures. The data is reported for each individual animal. Blue
indicates a decrease in temperature by 2 standard deviations from the baseline. Red indicates a
increase in temperature by 2 standard deviations from the baseline. Electronic file located on
Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 3.
Respiratory rates were taken for each animal beginning on Day -7 through time
of death. Day -7 to day -1 are averaged and give the baseline shown to the right
of the figure. Each day the respiratory rates were taken at 0800, 1400, and
2000. As shown by the data outlined in Table 8, all animals had a 30% to 50%
increase in respiratory rates anywhere from one day to seven days preceding
death. Respiratory rates appear to be an indication of primary pulmonic disease;
similar respiratory rates were seen in wave 1 but not wave 2.
Page 15 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Table 8. Schu S4 Wave 3 Respiratory Rates. The data is reported for each individual animal.
Pink indicates a 30% increase in rate. Red indicates a 50% increase in rate. Electronic file
located on Z:\Agent and Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 3.
Bacteremia data (Figure 10 and Table 9) showed that all animals had culturable
bacteria in the blood by Day 4 or Day 6. Challenge doses were low enough that
we were able to capture bacteremias on every animal.
Page 16 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 10. Schu S4 Wave 3 Bacteremia. The data is reported for each individual animal starting
at Day 0 through time of death or euthanasia. Electronic file located on Z:\Agent and Study
Specific Data and Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL07)\ED50 Wave 3.
Animal #
28479
28664
28601
28512
28572
28484
28467
28585
Day 0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
4
6.67
570
33.3
0
10
0
0
0
6
6000
13.3
520
10
7666.7
180
8
0
Table 9. Schu S4 Wave 3 Bacteremia. The data is reported for each individual animal as
CFU/ml. Electronic file located on Z:\Agent and Study Specific Data and Miscellaneous
Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 3.
e. Clinical observations demonstrated ocular tularemia in two animals, 28588 and
28618, from Wave 2. Both animals had flocculent white material from which F.
tularensis was cultured. Figure 11 is representative pictures of the eyes
demonstrating the ocular tularemia. In Waves 2 and 3 at least 6 animals, 28588,
28615, 28571, 28467, 28512, 28601, had red skin lesions which resemble
petechiae. Figure 12 is a representative picture of the skin lesions. In all waves
but not limited to all animals there was coughing, nasal discharge, and anorexia.
Waves 1 and 3 had greatly increased respiratory rates, gasping, labored
breathing, and mouth breathing.
Page 17 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 11. Schu S4 Wave 2 Ocular Tularemia. Eyes from animals 28588 and 28618
demonstrating ocular tularemia by the flocculent white spots, and confirmed by microbiology
(plating of ground ocular tissue). Electronic file located on Z:\Agent and Study Specific Data and
Miscellaneous Documents\STUDY SPECIFIC DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 2.
Figure 12 Schu S4 Wave 2/3 Red Skin Lesions. Animals with red skin
lesions that may be petechiae. Electronic file located on Z:\Agent and
Study Specific Data and Miscellaneous Documents\STUDY SPECIFIC
DATA\FY08\FY08-074 (TUL-07)\ED50 Wave 2 (or 3).
4. Significant decisions made or pending
The challenge dose that will be used in the Natural History Study is dependent on this
ED50 study.
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None.
7. Quality of performance
Good.
8. Percentage completed
85% of the scientific work is complete.
9. Work plan for next month
Animal 28624, the lone survivor to date from Wave 2 will be euthanized on December 2,
2008.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Page 18 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Milestone 8
Milestone description: LVS vaccination protection of aerosol Schu4 validated in
primates
Institution: LBERI
1. Date started: 8/15/2008
2. Date completed: In progress.
3. Work performed and progress including data and preliminary conclusions
a. Update on Scarification vs. S.C. LVS vaccination routes; The two vaccination
routes are being compared, in preparation for USAMMDA’s requested test of the
IND 157 LVS by the scarification route.
i. 5 NHPs were vaccinated with LVS on 10/16/08; 2 by subcutaneous
and 3 by scarification. During this reporting period the animals were bled
on days 21, 28 and 35 to compare the relative immune response
resulting from the two immunization routes.
ii. Figure 12 shows the IgG anti-LVS results; both routes of vaccination
are effective at inducing antibody to LVS.
iii. Figure 13 shows the proliferative capacity of PBMCs post-LVS
vaccination; by either route of vaccination, peak proliferative response
occurs at day 28.
iv. Figure 14 shows the ability of PBMCs to secrete IFNg post-LVS
vaccination; responses to LVS are apparent by day 15 and peak on day
28; responses to SCHU S4 antigens are less than those to LVS
antigens.
Page 19 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Attachment A
Gantt Chart
1000000
A
10000
Day 35
Day 35
Day 21
Day 28
1000000
Day 15
100
Day 7
1000
Day 28
SC
None
Scarification
Day 0
B
100000
28461
28656
A04169
A05403
A06199
10000
Day 15
Day 7
100
Day 0
1000
Day 21
Plasma IgG anti-LVS Titer (Mean +/- SEM)
100000
Figure 12. IgG anti-LVS titers. Panel A shows group average; Panel B shows individual NHPs.
Green = Scarification; Blue = S.C. vaccination with LVS. Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 12042008.svd;
TVDC (5) bound notebook (9247), pp. 52 – 53; 68 – 69.
Page 20 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
8.00E5
6.00E5
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
4.00E5
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
0
Day 7, Scarification
2.00E5
Day 7, SC
RLU (Mean +/- SEM)
1.00E6
Figure 13. Proliferation of PBMCs from LVS-vaccinated NHPs to LVS or SCHU S4 antigens (1 x
105/ml). All PBMCs plated at 1 x 106/ml. Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 12042008.svd;
TVDC (5) bound notebook (9247), pp. 23 – 46; 54 – 67.
Page 21 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
500
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
400
300
200
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 7, SC
0
Day 15, SC
100
Day 7, Scarification
IFNgamma Spots
(Mean +/- SEM)
A
400
B
Media
LVS hk Hi
LVS ff Hi
300
200
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
0
Day 7, Scarification
100
Day 7, SC
IFNgamma Spots
(Mean +/- SEM)
500
Figure 14. IFN secretion by PBMCs from LVS-vaccinated NHPs in response to LVS or SCHU
S4 antigens (1 x 105/ml). In panel A, all PBMCs are plated at 1.33 x 106/ml, however some NHPs
were TNTC (too numerous to count) in response to LVS antigens by day 15, 21 and 28. In Panel
B, the IFN secretion pattern in shown when PBMCs are plated at 1.0 (day 21) or 0.67 x 10 6/ml
(day 28 and 35). Only those stimuli shown were tested. Data storage:
Page 22 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay 12042008.svd;
TVDC (5) bound notebook (9247), pp. 23 – 46; 54 – 67.
Preliminary Data Interpretation: Both the s.c. and scarification routes of vaccination are effective
at inducing IgG antibody to LVS. When analyzing the proliferative response of PBMCs from
vaccinated NHPs to LVS and SCHU S4 antigen, peak proliferative response occurs at day 28
when using either route of vaccination. When examining the ability of PBMCs to secrete IFN in
response to LVS antigens, responses are apparent by day 15 and peak on day 28. Although
responses to SCHU S4 antigens appear to be less than those to LVS antigens, in the IFN
ELISPOT assay, we need to insure that the same amount of fixed or heat killed antigen is being
delivered to each well by performing a BCA protein assay
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
15% of the scientific work is complete.
9. Work plan for upcoming month
Schedule vaccination of 6 NHPs (3 scarification vs. 3 subcutaneous route) based on
optimum timing of challenge and ABSL3 schedule. This experiment will be to test
whether either scarification or s.c. vaccination with LVS will protect NHPs from an aerosol
challenge with SCHU S4.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Milestone 9
Milestone description: Aerosol SOP developed for GLP transition
Institution: LBERI
1. Date started: 8/13/2008
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
No work was performed during this reporting period.
4. Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None.
7. Quality of performance
Good.
8. Percentage completed
15% of the scientific work is complete.
Page 23 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
9. Work plan for upcoming month
a. Continue to work on the validation plan for the aerosol procedure.
b. Address any comments that are received from UNM on the aerosol SOP.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Milestone 11 - UNM
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. No new work done this month
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
None
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
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
Page 24 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Update on response of PBMCs to O-mutant antigens
a. LBERI is testing whether some of the background responsiveness seen to FF
LVS and SCHU S4 may be due to the LPS moieties on the surface of the whole
bacteria as antigens by comparing the response to O-antigen mutants lacking
LPS.
b. Figure 15 shows the proliferative response of LVS-vaccinated PBMCs; there
is little difference between Wild Type (WT) whole bacterial antigen preparations
and mutant O minus antigens.
c. Figure 16 shows the ability of PBMCs from non-LVS vaccinated NHPs to
secrete IFN in response to the mutant and WT antigens; in general, the
response to FF LVS is decreased when 0 antigen is missing, but the response to
other antigens is either unchanged or increased (in one NHP, A06199 to HK
SCHU S4).
d. Figure 17 shows the ability of PBMCs from LVS-vaccinated NHPs to secrete
IFN in response to the mutant and WT antigens; in general, the response to HK
and FF LVS is often decreased when 0 antigen is missing but is also sometimes
the same, the response to HK SCHU S4 is often increased when LPS is mutated;
the response to FF SCHU S4 antigen is varied when comparing WT and mutant
antigens
Page 25 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
RLU (Mean +/- SEM)
1.00E6
8.00E5
A
6.00E5
4.00E5
2.00E5
0
Day 7
Day 15
Day 21
Day 28
Day 35
Media
LVS hk Hi
LVS ff Hi
RLU (Mean +/- SEM)
1.00E6
8.00E5
B
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
6.00E5
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
4.00E5
SCHUS4 hk Mutant Hi
2.00E5
0
Day 7
Day 15
Day 21
Day 28
Day 35
Figure 15. Proliferation of PBMCs from LVS-vaccinated NHPs to LVS or SCHU S4 WT and Omutant antigens (1 x 105/ml). All PBMCs plated at 1 x 106/ml. A: 2 S.C. vaccinated NHPs; B: 3
scarified NHPs.
Page 26 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
IFNg Spots (Mean +/- SEM)
160
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
140
120
100
80
60
40
20
0
28461
28656
A04169
A05403
A06199
Figure 16. IFN production by non-LVS vaccinated NHPs in response to LVS and SCHU S4 WT
and 0-mutant antigens (1 x 105/ml).
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
IFNgamma Spots
(Mean +/- SEM)
500
400
A
300
200
100
0
Day 7
Day 15
Day 21
Day 28
Day 35
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
IFNgamma Spots
(Mean +/- SEM)
500
400
B
300
200
100
0
Day 7
Day 15
Day 21
Day 28
Day 35
Figure 17. IFN production by LVS vaccinated NHPs (A: 2 S.C.; B: 3 scarified) in response to
LVS and SCHU S4 WT and 0-mutant antigens (1 x 105/ml). All PBMCs are plated at 1.33 x
Page 27 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
106/ml. Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay
12042008.svd; TVDC (5) bound notebook (9247), pp. 23 – 46; 54 – 67.
Preliminary Data Interpretation: When examining the proliferative response of PBMCs from LVS
vaccinated NHPs to LVS or SCHU S4 antigens, there is little difference between WT and mutant
antigens (Figure 15). When examining the response of PBMCs from non-LVS vaccinated NHPs
to secrete IFN in response to LVS or SCHU S4 antigens, Figure 16 shows that in general, the
response to FF LVS is decreased when 0-antigen is missing, but the response to other antigens
is either unchanged or increased (note the response of A06199 to HK SCHU S4). Finally, Figure
17 shows that when examining the ability of PBMCs from LVS-vaccinated NHPs to secrete IFN
in response to LVS and SCHU S4 antigens, the response to HK and FF LVS is often decreased
when 0-antigen is missing but is also sometimes the same; the response to HK SCHU S4 is often
increased when LPS is mutated; and the response to FF SCHU S4 antigen is varied when
comparing WT and mutant antigens, thus, there is no clear pattern. Once again, we must confirm
that the same amount of antigen is being delivered per well when comparing WT and mutant
organisms and will do so by setting up a BCA assay to measure total protein.
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
84% of the scientific work is complete.
9. Work plan for upcoming month
Continue to test freeze/thaw protocol; specifically concentrate on cells from the newly
vaccinated NHPs.
10. Anticipated travel
None.
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
Milestone 12/13-UNM
Milestone description: Assays for detecting relevant immune responses in animals &
humans developed and Compare assays in animal models (sensitivity)
Institution: UNM
1. Date started: 7/15/06 (MS12) and 12/06 (MS13)
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
None
5. Problems or concerns and strategies to address
None
Page 28 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
NA
8. Percentage completed
63%
9. Work plan for upcoming month
Start work on milestone completion report
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 14
Milestone description: Assays in vaccinated humans validated (sensitivity)
Institution: UNM/LBERI
1. Date started: 2/29/2008
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
No new work done this period
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
5%
9. Work plan for upcoming month
a. Test the Martha’s Vineyard PBMC samples for F. tularensis specific proliferation
and IFN production
b. Test PBMC from human LVS vaccinees at UNM for F. tularensis specific
proliferation and IFN
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 29 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
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
a. Experiment ELISA-2 L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\ELISA 2
i. The purpose of this experiment was to determine the titer of F.
tularensis-specific antibodies in the immune rat serum used in the
passive immunization experiments. An accurate antibody titer would
allow us to standardize different batches of serum preparation and to
compare the effects of serum with purified immunoglobulin.
ii. The antibody titer in the immune and non-immune rat sera was
determined by ELISA using heat killed LVS as the capture antigen.
iii. The titration curves are shown in Fig 1. The antibody titer is the
highest dilution with a detectable signal. The antibody titer is
1:16,000 for the immune serum and 1:2,500 for the normal serum.
0.3
OD405 nm
Immune Rat Serum
Naive Rat Serum
0.2
0.1
1:50000
1:25000
1:16000
1:8000
1:5000
1:2500
1:1600
1:800
0.0
Figure 1. Titer of F. tularensis-specific antibody in immune and normal rat
serum measured by ELISA using heat killed LVS as capture antigen
b. Experiment Pdose-1 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Pdose 1)
i. The purpose of this experiment was to determine the serum volume
required to protect against a i.t. SCHU S4 challenge dose of 200 cfu
Page 30 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
ii. We showed in a previous experiment that 250 l of immune rat
serum was able to protect naïve rats against i.t. challenge with 217
SCHU S4.
iii. In this experiment, the challenge dose was kept the same as the
previous experiment but the volume of immune serum was reduced
from 250 l to 125 l and 50 l. We found that even 50 l of immune
serum protected rats against an i.t. challenge with 130 SCHU S4
(Fig. 2).
iv. We will continue to reduce the serum volume until no protection is
observed with a similar SCHU S4 challenge dose
Figure 2. Titration of serum volume required to protect rats against
i.t. SCHU S4 challenge. Rats (n = 6) were injected i.p. with the
indicated volumes of normal (NRS) or immune (IRS) rat serum and
one day later challenged i.t. with 130 SCHU S4. Survival was
monitored daily
c.
Experiment Pdose-1 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Pdose 1)
i. The purpose of this experiment was to determine the limit of
protection generated by passive immunization with 250 l immune
rat serum.
ii. Rats were passively immunized with 250 l immune or normal rat
serum and 1 day later challenged with 102, 103 or 104 SCHUS4.
iii. As shown in Fig. 3, the highest challenge dose protected by passive
immunization with 250 l was 103 SCHU S4.
Page 31 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure 3. Limit of protection conferred by 250 l immune rat serum.
Rats (n = 6) were injected i.p. with 250 l immune rat serum and
one day later challenged i.t. with the indicated dose of SCHU S4.
The numbers in parenthesis indicate the actual lung deposition
measured one hour after challenge

d. Experiment Phist-1 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\phist-1)
i. The purpose of this experiment was to compare the histopathology
of tissues from passively immunized rats and s.c. LVS vaccinated
rats after i.t. SCHU S4 challenge
ii. Rats were either vaccinated s.c. with LVS one month before SCHU
S4 challenge or injected i.p. with 150 l PBS (Naïve), normal rat
serum (NRS) or immune rat serum (IRS) one day before SCHU S4
challenge.
iii. On the indicated days post i.t. SCHU S4 challenge (Table 1), 3 rats
from each group were euthanized to collect the lungs, lung draining
lymph node, liver and spleen.
iv. The tissues are being processed for analyses
Table 1. Experimental design to determine the histopathology of
passively immunized rats after i.t. SCHU S4 challenge
Group
Treatment
Time Points
No.
(Days post challenge)
1
2
3
4
Naïve
NRS
IRS
Vaccinated
1, 3, 5, 7
1, 3, 5, 7
1, 3, 5, 7, 10, 14, 21
1, 3, 5, 7, 10, 14, 21
e. Experiment Ptran12 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Ptran\ptran-12)
i. The purpose of this experiment was to determine the kinetics of
SCHU S4 proliferation and dissemination in passively immunized
rats
Page 32 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
ii. Naïve rats, LVS immune rats and rats passively immunized with 0.25
ml of normal or immune rat serum were challenged i.t. with 242
SCHU S4 (n = 3 for all groups). Bacterial burden in the lungs, liver,
and spleen were determined on days 2, 5, 7, 14, and 21.
iii. As shown previously, LVS vaccination did not prevent SCHU S4
infection or systemic dissemination but allowed the rats to gain
control over bacterial proliferation by day 2 p.i. and to eventually
clear the SCHU S4 infection (Fig. 4). In contrast, naïve rats and rats
that had received normal rat serum (NRS) were never able to control
bacterial proliferation and died. Passively immunized rats (IRS)
demonstrated an intermediate phenotype; their bacterial burden in all
three tissues was higher than those in LVS vaccinated rats but not
as high as those in naïve or the NRS rats. It also appears that after
reaching its peak, the bacterial burden remained stable for a few
days and was cleared with much slower kinetics than observed in
vaccinated rats. This plateau may represent a transition from an
antibody to a cell mediated mechanism of protection.
iv. As of day 21 post challenge, the passively immunized rats had not
yet cleared the SCHU S4 infection
Spleen
Lungs
Liver
10
10
10
Naive (0.25ml PBS)
NRS (0.25ml)
Vaccinated
IRS (0.25ml)
8
8
8
6
6
4
4
2
2
6
4
0
0
0
5
10
15
20
25
2
0
5
10
15
20
25
0
5
10
15
20
25
Fig. 4. Kinetics of SCHU S4 proliferation and dissemination in passively immunized rats. Fischer
344 rats (n = 3) were either vaccinated with LVS or treated with 0.25 ml normal rat serum (NRS)
or immune rat serum (IRS) and challenge i.t. 1 day later with 242 SCHU S4. On the indicated
days, bacterial burden in the lungs, liver and spleen were measured.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
NA
8. Percentage completed
20%
9. Work plan for upcoming month
a. Continue to reduce the volume of IRS used for passive immunization until no
protection is observed against i.t. challenge with 200 SCHU S4
Page 33 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
b. Repeat SCHU S4 growth kinetics experiment in actively and passively
immunized rats
c. Complete histopathological analyses of tissues from actively and passively
immunized rats after i.t. SCHU S4 challenge
d. Purify IgG from immune and normal rat serum in order to demonstrate that
the protection is mediated by antibodies and not other serum components
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 18-UNM
Milestone description: Role of specific  T cells in protection
Institution: UNM
1. Date started: 7/1/08
2. Date completed: Pending
3. Work performed and progress including data and preliminary
conclusions
a. No new work done because we are waiting for the ascites fluid for depleting
CD4 T cells to arrive the week of 12/15/08
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
The receipt of the ascites has caused a delay, which should end during the week of
12/15/08
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
5%
9. Work plan for upcoming month
a. Determine the role of CD4 and CD8 T cells in LVS vaccinated rats. We
already have the ascites fluid for depleting CD8 T cells in the lab but we are
waiting for the ascites fluid for depleting CD4 T cells. When we have both
sets of ascites fluids, then we will vaccinate and treat Fischer 344 rats to
determine the importance of these T cell subsets in protection.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 34 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Milestone 19-UNM
Milestone description: Interaction between human alveolar macrophages and F.
tularensis
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary
conclusions
We received only one human alveolar macrophage sample and it was contaminated
during the experiment. No new results to report for this period.
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
Many of the human alveolar macrophage samples we have received have been
contaminated. We are going to add 100 units/ml polymyxin B, 100 units/ml penicillin
G, and amphotericin B to try to control the contamination problem . UNM must use
antibiotics that are not toxic to tularemia.
6. Deliverables completed
NA
7. Quality of performance
Needs improvement
8. Percentage completed
17%
9. Work plan for upcoming month
a. Analyze cytokine production by human alveolar macrophages cultured in
non-tissue culture treated tubes and on tissue culture treated plates.
b. Determine the effect of recombinant IFN on intracellular growth of SCHU S4
and LVS.
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
None
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. No work was performed during this reporting period..
4. Significant decisions made or pending
Page 35 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
None
5. Problems or concerns and strategies to address
None.
6. Deliverables completed
None.
7. Quality of performance
Good
8. Percentage completed
2% of the scientific work is complete
9. Work plan for upcoming month
Repeat the ICCS assay and include a positive mitogen control (Con A). PBMCs from
the newly LVS-vaccinated NHPs will be used in the assay.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
Milestone 21-UNM
Milestone description: T cell-induced macrophage killing of intracellular bacteria
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary
conclusions
a. Experiment Ft-AH2 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Andrew's experiments\Ft-AH2)
i. The overarching goal for this series of experiments is to determine
whether human monocytes can be infected with SCHU S4 and used
as the effector cell in the killing assay instead of macrophages. The
procedure used to generate human macrophages from peripheral
blood monocytes is very time consuming and limits the number of
experiments we can do. Since F. tularensis have been shown to
infect monocyte, we may be able to shorten the experiments
considerable by using them as the effector cells instead of
macrophages. In this experiment, we wanted to determine the
optimal multiplicity of infection (MOI) for monocytes and the serum
requirement for infection of monocytes
ii. Monocytes were isolated from human buffy coat and infected with
SCHU S4 at MOI = 0.1 and 1.0 in the presence or absence of serum.
The bacterial burden was measured daily for each of the four culture
conditions.
iii. The results shown in Fig 5 suggest:
1. Serum enhances bacterial uptake (time 0) but has no effect
on subsequent growth because the slope of the growth
curves with and without serum is similar
Page 36 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
2. Infection at MOI = 1.0 is preferred over MOI = 0.1 because
there is no initial decline in the bacterial load at 24 h. The
reason for this decline is unknown
3. A combination of serum and MOI = 1.0 can only support
growth for 48 h since the slope of the growth curve
decreased from 48 to 72 h.
iv. This experiment will be repeated to gain consistency before we
determine whether they can be activated with IFN to control
intracellular bacterial growth.
1.010 6
CFU / well
1.010 5
1.010 4
1.010 3
MOI=0.1 (-) serum
MOI=0.1 (+) serum
MOI=1.0 (-) serum
MOI=1.0 (+) serum
1.010 2
1.010 1
1.010 0
0
24
48
72
hours post-infection
Fig. 5. Optimization of the conditions for infecting human monocytes. Human
monocytes in DMEM were plated at a concentration of 2.5 x 106 cells/well in 48well flat-bottom plates and infected at the indicated MOI. The bacterial burden
was measured over a 72 h period by plating the cultures on cystine heart agar
plates
b. Experiment Ftc61 study 8d (Notebook 128 pages 12-15)
i. The purpose of this experiment was to determine the optimal MOI for
infecting rat bone marrow derived macrophages (BMM).
ii. Rat BMM were infected with SCHU S4 at MOI of 1:50, 1:100, 1:200
and 1:1000 (SCHU S4:BMM). 48 h later, the bacterial burden at
each MOI was determined.
iii. As shown in Table 2, there is a direct correlation between MOI and
the bacterial load recovered on day 2. At MOI 1:50, we observed
some cytopathic effects.
iv. We will repeat this experiment again to gain consistency before we
determine whether they can be activated to control intracellular
bacterial growth.
Table 2. MOI Optimization for infecting rat bone marrow
derived macrophages
Page 37 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
MOI
(SCHU S4: macrophage)
1:50
1:100
1:200
1:1000
No. of bacteria
recovered on day 2
6
5.26 x 10
6
3.88 x 10
5
6.13 x 10
5
2.33 x 10
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
50 %
9. Work plan for upcoming month
a. Repeat MOI optimization for infecting rat BMM with SCHU S4
b. Determine whether rat the cytotoxic activity in BMM can be activated with
recombinant IFN
c. Determine whether rat monocytes can be infected with SCHU S4
d. Repeat SCHU S4 infection of human monocytes
e. Determine whether infected human monocytes can be activated to control
intracellular bacterial growth with recombinant IFN and immune T cells
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will
use ASU’s protein fragments in lymph node proliferation assays to define vaccine
candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: 10/15/2008
3. Work performed and progress including data and preliminary
conclusions
No new work done this period.
4. Significant decisions made or pending
None
Page 38 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
5. Problems or concerns and strategies to address
None
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
40%
9. Work plan for upcoming month
Boost the LVS vaccinated NHP before harvesting the organs. Boost will occur after
the ASU peptides are received at UNM.
Test peptides from ASU with lymph node cells and spleens from LVS vaccinated
NHP, on Milestone 29
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
Milestone 28
Milestone description: Generation of polypeptide libraries (Optimize IVT proteinfragment production, Develop IVT protocol for high-throughput production, Validate
immunogenicity of protein-fragments, Full scale production of protein-fragment library,
Purification of protein-fragment library, Array protein-fragment into overlapping pools,
Ship to UNM)
Milestone description: Build SCHU4 proteome
 Build ORF expression library corresponding to proteome (complete)
 Generate complete protein-fragment library (active)
 Array protein-fragments into measurable pools for T cell stimulation
(inactive)
Institution: ASU-Sykes
1. Date started: 03-01-2007
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
B. Build ORF expression library corresponding to proteome
a. All ORFs that were not amplified previously due to degradation of stored FTU
primers have been successfully amplified using new primers
b. Concentration of these templates and amounts needed for IVT reactions
were determined
c. Appropriate volumes were transferred to IVT plates, dried, and stored at 20oC
d. 2,229 linear expression elements were successfully assembled and now
ready for IVT production of FTU polypeptides
Page 39 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Figure1: Re-amplification of 86 Linear Expression Elements using the new primers.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA
gels\HTP Egel AMP\Long ORF 5\Long ORF 5 Plate 2 and short 2 with new primers 11-708_Eeditor
 Figure 1 shows the 128 ORFs that could not be amplified last month with old
oligo primers, which have now been amplified and assembled into the LEE
cassettes for IVT.
C.
Generate polypeptide library
C. Initial large-scale batch of protein production
 The first batch of IVT high throughput protein production containing
336 polypeptides from 4 of the 96-well plates

For the QC plate, we in vitro translated 12 ORF constructs from
each of these 4x96 well plates that give a total of 48 ORFs. In
addition, 1 GFP template was included in this QC plate for positive
control. Radiolabel (35S-Methionine) was used only in this QC plate
for visualization and analysis
Page 40 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
1 2
3
4 5 6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 GFP
Figure 2: Autoradiograph of QC plate containing 24 IVT samples from 2
of 96-well plate. The last lane is GFP
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP
IVT 35S gels\F tularensis proteomic library\Long ORF 2\Long 2 and GFP
IVT QC row A and B

Figure 2 shows that only the GFP template, which was added
directly into QC plate without lyophilization, was translated. Since
GFP template was not dried before IVT reaction, it is possible
that this step may also have interfered with the IVT reactions for
the 336 FTU library samples. Figures 3 through Figure 5 show
the results from the troubleshooting steps taken. These explain
the absent IVT products in Figure 2 and despite the QC plate
results, the following experiments confirm the integrity of the
library samples.
1
2
3
4
5
6
7
8
Figure 3: DNA agarose gel of 8 IVT templates which were used
for IVT reaction shown in figure 2 ( lane 1, 3, 5, 7, 9, 11, 13, and
15).
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU
gels\FTU HTP IVT DNA gels\Testing Lee Storage 11-12-08 Long
ORF2 PCR plate 2 Row B1-B8 2

Figure 3 shows that LEE IVT templates are not degraded and
should be sufficient for IVT reactions.
Page 41 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
1 2 3 4 5 66
Figure 4: IVT of 4 FTU LEE that were not translated in QC plates
(lane 1, 3, 5, and 7). These FTU IVT templates (lanes 1 to 4) were
not dried before performing the IVT reactions. Lane 5 and 6 are IVT
reactions from the same GFP template dried in 2 different speed
vacs.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU
HTP IVT 35S gels\F tularensis proteomic library\Long ORF 2\
Long ORF 2 Plate 2 Row B1-B4 and GFP 11-17-08 crop

Figure 4 lanes 1-4 shows that the FTU templates are sufficient
for in vitro translation and that normally drying does not interfere
with the translational process (lanes 5 and 6). Since the 48 QC
templates and 336 FTU templates used for the construction of
the library were prepared at different times and in different
speed-vac machines, the library plates containing the
preparative quantities of polypeptide were directly assayed
following the final purification step.
Figure 5: silver stain of 4 bead samples picked from the library plates
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU
HTP IVT silver stain\LOng ORF2 B1 to B4 11-12-08 crop
Page 42 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee

Figure 5 shows that the unlike the samples in the QC plates, the
FTU polypeptides in the library plates were successfully
translated and efficiently bound on beads as purified products.
This again suggests that the smaller batch of QC samples, which
were lyophilized in a borrowed chemistry speed vac, were
exposed to a substance that inhibited the subsequent IVT
reactions, and that this one time event did not harm the larger
batch of library samples, which were dried in the standard
equipment.
II. Second large-scale batch of IVT protein production

This batch contains 144 ORFs from 2x 96-well PCR plates (Long
ORF 5 )
1 2 3
4 5
6 7
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 GFP
Figure 6: Autoradiograph of 24 IVT polypeptides from QC Long ORF 5
plate. GFP is used as positive control
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP
IVT 35S gels\F tularensis proteomic library\Long ORF 5\Long ORF 5QC
11-21-08 2


Figure 6 shows that 92% of FTU proteins were translated
successfully in vitro
The average amount of proteins bound on beads is 10.2 ug per
reaction
Page 43 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008
to 11/30/2008
150
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
III. Current status of library protein production
250
150
75
50
37
25
20
15
10
1 2
3 4
5
6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Figure 7: Coomassie blue stain of BAG (Build a gene) QC plate
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT
Coomassie gels\F tularensis Library\BAG QC plate 1 and 2 11-25-08 crop
250
150
75
50
25
20
15
10
Figure 8: Autoradiograph of BAG QC plate
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S
gels\F tularensis proteomic library\BAG\QC PCR plate 1 and 2
 We have successfully synthesized 1,222 FTU proteins in vitro
 Proteins bound on bead are purified and sufficient for T-cell assay
 The remainder of the 1,007 FTU proteins will be translated in the
next weeks and the library will be completed by the end of this
month
D. Array polypeptide library

We will use 1 of the half-sets of IVT reactions to initially array. The current
plan is to array these reactions into pools comprised of 7 polypeptides.
Each pool will be split into 4 wells for delivery to UNM.
Page 44 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
4. Significant decisions made or pending.
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Very Good
8. Percentage completed
95%
9. Work plan for upcoming month

Complete the F. tularensis polypeptide library production, arraying and pooling. We
anticipate that Milestone 28 will be completed in December 2008.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 35 - UNM
Milestone description: Array hybridization with mouse RNA from virulent SCHU S4
infection and RT PCR confirmation of candidates
Institution: UNM
1. Date started:
2. Date completed: pending
3. Work performed and progress including data and preliminary
conclusions
Isolated RNAs from SCHU S4 infected rat lungs 1, 3, 5, 7, and 24 hours after i.t.
challenge and shipped them to ASU
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
25%
9. Work plan for upcoming month
None
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 45 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
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: 08-01-2006
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions

Previous Results: We reported on the problems with consistent amplifications for
the LAPT process. QC check analyses indicated that the failure of the process most
likely resided in problems with the template switch primer. The problems resulted
from personnel turnover leading to miscommunication around the handover of
samples and reagents. We also reported on potential problems with the relative
quantification gene, MutS, for the qPCR analyses. We noted that there was a
problem with the sensitivity in the ability for this gene to detect the number of bacteria
in the samples.

We had a new lot of template switch primer synthesized and have re-diluted and
aliquoted this primer and ensured accurate labeling. An initial LAPT study was
performed to verify dose response range of that the concentration of the primer was
consistent with previous experiments. With the concentration confirmed, we
performed a LAPT analysis with a reconstitution sample. Purified SCHU S4 was
serially-10use lung RNA. The results,
shown in Table 1, are consistent with all previous amplifications and a total of 45- 60
μg of RNA obtained. Thus, the LAPT problem has been resolved this month, using
the new lot of template switch primer.
g SCHU-S4 Microgram Yield
1
58.56
0.1
45.96
0.01
50.52
0.001
50.22
0.0001
51.66
0
49.26
Table 1.
and the LAPT process performed.
Notebook/File locations …, ASU: Notebook 804, LAPT-29, page 186

There is an estimated 5 femtograms of total RNA in one bacterium. Based on this
estimate, it would take over 2,000 bacteria to be detected in our current qPCR assay.
Page 46 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
The current detection limit of 10 picograms was determined by a reconstitution
sample analysis using purified SCHU S4 RNA and 10 picograms represents more
than 2000 bacteria. The current time course samples have been collected after
infection was initiated with a 103 SCHU S4 infection dose. With this dose, the
numbers of bacteria were quantified during the time course and the results are shown
in Table 2 (data from UNM). Based on the detection limits, before 24 hours there
would not be adequate numbers of bacteria to be detected by the qPCR assay.
Harvest Time Bacterial Load
1h
144
3 h
256
5 h
330
7 h
608
24 h
83,000
Table 1. Number of bacteria in the lungs of mice after intranasal infection with
103 SCHU S4 bacteria (Data provided by Terry Wu).
4. Significant decisions made or pending
Depending upon the limit of detection, additional infections may need to be performed to
acquire samples within the detection range.
5. Problems or concerns and strategies to address

Using the 24h sample listed above representing more than 80,000 bacteria, we will
be performing a serial dilution qPCR analysis to determine a more quantifiable,
empirically determined detection limit.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
65%
9. Work plan for upcoming month



Now that the LAPT process has worked in several consecutive experiments, we will
be amplifying the second time course mouse experiment.
We have received the RNA samples from the rat infection model from UNM. These
samples will be purified by RNAeasy purification and processed for LAPT analysis.
We will perform a dilution series with the 24 hour sample, containing over 80K
bacteria for empirical determination of the detection limits in the qPCR using the
MutS and iglC genes.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 47 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis
(SCHU S4) (iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F.
tularensis subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F.
tularensis subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate
successful deletions. Therefore, our focus is two fold, one is cloning experiments to get
our target deletions into vectors that we can use in creating these deletions and
experiments with SCHU S4 itself using constructs that we believe will allow us to make
deletions into SCHU S4.
I.
Cloning:
a. The NadM PCR product was generated successfully by using the SchuS4 DNA as
template and the three NadM oligos as PCR primers (NadM 602/603s IBS, NadM
602/603s EBS1d and NadM 602/603s EBS2). See figure 1. Data located in UTSA TVD
Notebook 7, page 49.
Figure 1.
1 Kb
1
3.0
2
3
4
5
Legend:
1. 1 Kb Ladder
2. PCR NadM
3. PCR NadM
4. PCR NadM
5. PCR NadM
0.5
Figure 1 legend, results, data location: represents the PCR profile generated with the
NadM oligos mentioned and SchuS4 genomic DNA as template. Lanes 2 thru 5 are
aliquot loads from the same PCR NadM reaction vial. The desired band is ≈350 bp
which is seen in each lane as the darkest band. Lane 1 is 1 Kb ladder from Invitrogen.
Data located in UTSA TVD Notebook 7, page 49.
b. The ≈350 bp NadM PCR product was isolated from the gel in Figure 1 via the Qiagen
gel extraction kit then subsequently digested with Bgl II and Xho I to prepare this
Page 48 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
DNA to ligate into the pKEK1140 which was also treated similarly. Once the ligation
reaction was completed this was chloroform: phenol extracted to clean the reaction
and then ethanol precipitated. This ligation preparation was then used to transform
competent DH5α cells. As a standard protocol, we did a ligation reaction with the
vector KEK1140 BsrG1 II/Xho I treated without “insert” (i.e. NadM PCR product) this
is run as a control to check how well the various digestions worked. Therefore, both
the KEK1140 re-ligation (positive control) and the KEK1140+NadM ligations were
used respectively, in a transformation experiment with DH5α cells.
c. The transformed DH5α cells were plated onto TSA +++ 60ug/ml Kanamycin plates to
select for correct NadM construct. This resulted in hundreds of clones for the
KEK1140+NadM (1140+NadM) transformation whereas, the re-ligation (KEK1140
only) resulted in 15 clones.
d. Selected six clones from the KEK1140+NadM transformed DH5α cells to prepare
plasmid from cells and screen with a Bgl II digestion. This is our standard initial
screen when using this cloning vector (KEK1140) since it typically will give a banding
pattern which is different from the parent vector by itself and is indicative of DNA
being cloned into this vector (Figure 2).
Figure 2.
1 Kb
1
4.0
1.5
1.0
2
3 4 5
6 7 8 9 10
Legend
1. 1 Kb ladder
2. Uncut C1 1140+NadM
3. KEK1140
4. C1 1140+NadM
5. C2 1140+NadM
6. C3 1140+NadM
7. C4 1140+NadM
8. C5 1140+NadM
9. C6 1140+NadM
10. Uncut KEK1140
Figure 2 legend, results, data location: represents a Bgl II digestion profile of six
plasmids generated from clones resulting from the transformation experiment with
KEK1140+NadM ligation into DH5α cells. The parent plasmid KEK1140 (lanes 3 BglII cut
and 10 uncut) were run as controls on this gel for comparison. The correct clone should
generate three bands when digested with Bgl II enzyme; these are ≈3800 bp, ≈2700 bp
and 1500 bp. This profile differs from the parent only by the highest molecular weight
band; the cloning vector by itself will yield a ≈4000 bp band whereas the NadM clone will
be at ≈3800 bp. Lanes 4 thru 9 represent various NadM plasmid constructs and of these
lanes 4, 6 and 8 look to be correct. Data located in UTSA TVD Notebook 7, page 56.
e. From figure 2 results we decided to prepare a large plasmid preparation of clone 1
(C1 1140+NadM) and send for sequencing using an oligo mentioned earlier, GroEL
Down Bgl II Xho I. This oligo should allow for the entire cloned NadM (insert) to be
Page 49 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
sequenced since this primer will anneal at the promoter of the construct which is just 5’ of
the cloned insert. We should have results on sequence by next report date.
II.
Experiments to generate mutants in Schu4:
a. The genomic isolates from the controls (KKT1 and KKT10) did not have enough DNA
for the various digestions we hope to profile on the southern blot. Since the BSL3 lab
was closed for two weeks these genomic isolations were prepared at the end of the
month. Data located in UTSA TVD Notebook 7, page 57. We will do various digestions
of these preparations in this coming month to try and finish the VgrG (KKT13) Southern
blot mentioned in last month’s report.
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
82%
9. Work plan for upcoming month


Need to run the Southern blot to verify expected VgrG intron location on the genome.
(Although sequence analysis showed the VgrG mutant is correct.)
Will continue verification of the new KEK1140+NadM construct via digestions and
sequencing of plasmid.
10. Anticipated travel
None
11.Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 50
Milestone description: Phenotyping and confirmation of single gene mutants;
50.1: phenotyping and immunologic characterization of Ft subsp. novicida uvrA or uvrB;
LVS uvrA or uvrB, and Ft subsp. tularensis (SCHU S4) iglC strains,
50.2: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
pdpD, iglD strains, Ft subsp. novicida uvrA or uvrB plus pdpD/iglA/iglB/iglC/iglD double
mutant strains,
50.3: phenotyping and immunologic characterization of Ft subsp. tularensis (SCHU S4)
iglA, iglB strains
Institution: UTSA
1. Date started: 05/01/2006
2. Date completed: provide date when milestone is completed
3. Work performed and progress including data and preliminary conclusions
50A-a: (1) Measure humoral responses after KKT10 (iglD mutant of SCHU S4) oral
immunization. (Note book # 9, page 10-12). BALB/c mice were orally immunized with
Page 50 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
KKT10 (103 CFU) or PBS (mock control). Sera and fecal pellets were collected at day
21 after immunization and assayed for anti-KKT1 specific antibody titers by ELISA.
Antigens, either UV-irradiated KKT10 (106/well) or HEL (Hen Egg Lysozyme,
1g/well, an unrelated antigen as control), were coated onto 96-well microplates and
reacted with fecal samples or serial dilutions of sera. Mice received single
immunization of KKT10 induced significant amount of antigen-specific total serum
antibody (Ig(H+L)) as shown in Fig. 1A. Further IgG isotyping analyses of the sera
indicated oral immunization of KKT10 resulted in producing high titer of IgG1 but
negligible amount of IgG2a. This dominant IgG1 isotype switching also showed in
intranasally KKT10-immunized mice (our Oct. 2008 report). Oral immunization also
induced low but measurable anti-KKT10 specific secretory IgA in the prepared fecal
pellet samples (Fig. 1B.). No KKT10- specific antibody was detected in mice mockvaccinated with PBS at day 21 after immunization. All tested samples showed no
reactivity to the unrelated HEL protein (data not shown).
A
.
5000
K K T10
B
.
0.30
K K T10
Mock/P B S
IgA
IgA
1000
A405
T iter
0.20
0.10
100
Ig(H+L) IgG1 IgG2a
0.00
IgM
IgM
Fig.1. Mucosal immune responses induced by KKT10 (iglD of SCH S4) oral
immunization. Mice were immunized with 103 CFU of KKT10 or mock vaccinated
with PBS. Sera (A) and fecal pellets (B) were collected 3 weeks after immunization,
and assayed for anti-KKT10 specific antibody.
50A-b Evaluate the protective efficacy of oral KKT10 vaccination against SCHU S4
intranasal challenge. (Note book #9, page 13-14). BALB/c mice were given orally a
single dose of KKT10 (103 CFU), rested for 32 days, and challenged intranasally with
either 80 or 400 CFU of SCHU S4. All KKT10- and PBS/mock- vaccinated mice
succumbed to SCHU S4 challenge by day 9. However, between the groups of mice
challenged with 400 CFU of SCHU S4, the KKT10 immunized mice has a prolonged
median-time-to-death of 6.5 days compared to 4 day for the PBS mock immunized
mice. This difference is significant (p < 0.005) using the Kaplan-Meier survival
analysis. There is no difference in survival between KKT10- and PBS-immunized
Page 51 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
mice when challenged with low dose (80CFU) of SCHU S4. In summary, oral
immunization with KKT10 induced Th-2 type immune response and did not protect
mice from lethal SCHU S4 challenge. However, KKT10 immunization did increase
median survival time.
100
80
Moc k /PBS 80 CFU
% survival
Moc k /PBS 400 CFU
KKT10 80 CFU
60
KKT10 400 CFU
40
20
0
0
2
4
6
8
10
Day s after c hallenge
Fig. 2. Protective efficacy of KKT10 immunization against F. tularensis
infection. BALB/c mice were orally immunized with 103 CFU of KKT10 or
PBS and i.n. challenged with lethal dose of F. tularensis SCHU S4 strain
(80 or 400 CFU). Mice were monitored for survival rate.
50B: Determine long-term protective efficacy of oral LVS vaccination against Ft
subsp. tularensis SCHU S4 challenge. (Notebook #8, pages 130, 138).Several
previous studies have shown that protection conferred by LVS vaccination against Ft
subsp. tularensis is short lived. (Chen et al., Vaccine 2003, Conlan et al., Vaccine
2005) We have previously conducted a study which showed that administration of a
primary dose of LVS orally followed by a secondary boost vaccination, was able to
extend the length of protection against 100 and 500 CFU of SCHU S4. (Ray et al., In
Review) In this experiment, we wished to increase the challenge dose to test the
limits of this extended protection. BALB/c mice were vaccinated orally with LVS (103
CFU) or mock (PBS) vaccinated. Mice were rested for eight weeks. Some mice were
given a second oral dose of LVS (103 CFU) and rested for an additional four weeks.
All mice were challenged intranasally with 1200 CFU of F.t. SCHU S4. Mice were
monitored daily for morbidity and mortality. As shown in Fig. 3, all of the mice that
only received one vaccination dose died by day 7 after challenge. However, mice that
received a second vaccination dose, exhibited 34% survival. All of the mock (PBS)
vaccinated mice died by day 5 after challenge as expected. These results suggest
that while a single vaccination dose was only able to confer a slight increase in time
to death at this higher challenge dose, administration of an additional vaccination
Page 52 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
dose was able to confer some protection. This provides further evidence that giving
vaccination boosts might be advantageous in protecting against Ft. subs. tularensis.
LVS 8 wk/ SCHU S4 challenge
100
LVS 8 wk/ boost / SCHU S4 challenge
Mock (PBS)/ SCHU S4 challenge
% Surv iv al
80
60
40
20
0
0
5
10
15
20
25
30
Day s After Challenge
Fig. 3. Long term protective efficacy of oral LVS vaccination followed by SCHU S4 challenge.
BALB/c mice were vaccinated orally with 103 CFU of LVS or mock (PBS) vaccinated. Mice were
rested for 8 weeks. Some mice were given a second vaccination (103 CFU) and rested for an
additional 4 weeks. Mice were challenged with 1200 CFU SCHU S4 and monitored for survival
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
87% of scientific work completed on milestone 50A (original plans)
100% of scientific work completed on milestone 50B (intragastric plan)
93.5% average work completed
9. Work plan for upcoming month
50A. (1) Intramacrophage growth of SCHU S4 vgrG mutant (KKT13).
(2) Evaluate the protective efficacy of oral KKT13 SCHU S4 vgrG mutant
vaccination against SCHU S4 intranasal challenge.
50B: Milestone 50B is complete. UTSA will initiate a MS#50 MSCR draft.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 53 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Milestone 52
Milestone description: Create RecA mutants in F. tularensis subsp. tularensis(Schu
S4)
Institution: UTSA
1. Date started: 9/15/2007
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions
3.1 Evaluation of Attenuation and Protective Efficiency of Transposon Mutants NR5330 and
NR7241
NR5330 (FTN0720) and NR7241 (FTN0757) are the F.novicida transposon mutants provided
by the University of Washington. The gene FTN0720 functions as transcriptional regulator,
and FTN0757 is known as the “membrane protein of unknown function”. NR5330 was
mutated by insertion of the transposon “<Kan-2>” at 193bp in FTN0720, whereas NR7241
was created by insertion of the transposon “T20” at 1339bp in FTN0757. Our goal in this
study is to evaluate the attenuation of those mutants in Balb/C mice and subsequently
protective efficiency of the mutants against wild type F.novicida challenge. Then we can
decide the valuation of making and studying the mutation in the same gene in Schu S4
background.
3.1.1 In the last monthly report, it was reported that the mice immunized with
transposon mutants NR5330 and NR7241 survived except one (NR5330) which died 11
days after inoculation. We continue to observe the vaccinated mice until one month
post-inoculation. None of them looked sick or died, which convinced us that both of
NR5330 and NR7241 were attenuated in BalbC mice. Below is the graph of mice
survival over time .
Graph 1: Mice survival rate 33 days after vaccination.
% Survival Rate
100
75
N
R5330
D
ose 200 C
FU I . N
50
N
R7241
D
ose 534 C
FU I . N
25
N=6 or
5
PBS
I.N
0
1
2
3
4
5
6
7
8
14 20 26 33
Da y a fte r I.N. i n fe c ti o n
Page 54 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Graph 1 legend, results and data location Data recorded on UTSA TVDC notebook #6,
page56 for Graph 1 above. NR5330 was attenuated at the dose 200CFU in BalbC
mice, and NR7241 was attenuated at the dose of 534CFU.
3.1.2 On day33 (Nov26), the survived mice immunized with NR5330 and NR7241, and
the mice inoculated with PBS were challenged with wild type U112 at dose of 242 CFU
intranasally to evaluate the protective efficiency of the mutants against wild type strain
in BalbC mice. PBS control group died on day4 and day5, whereas both mutant groups
were fine after 6 days of inoculation. The table below described the data of this
experiment.
Table 1: Survival rate after challenge with wt U112
Group
Dose of
Route of
of Mice Inoculum(CFU) Inoculation
NR5330
242
i.n.
NR7241
242
i.n.
PBS
242
i.n.
D1
5/5
5/5
5/5
D2
5/5
5/5
5/5
Survival Rate
D3
D4
5/5
5/5
5/5
5/5
5/5
2/5
D5
5/5
5/5
0/5
D6
5/5
5/5
Table I legend, results and data location: It appeared that both mutants protected the mice
against wt U112 challenge, but we need to check the mice until one month post- challenge, which
will be reported in next monthly report. Data recorded on UTSA TVDC notebook #6, page57 for
Table 1 above.
3.2 Create recA and IglC double mutant in F. tularensis tularensis (SCHU S4).
This part of Milestone 52 is to create recA and IglC double mutant in F. tularensis tularensis.
Inactivating the recA gene will stabilize the strain and prevent the strain from any additional
genetic changes. We already have the IglC mutant of Schu S4, and the tulatron vector pKEK1186
for disturbing and inactivating the recA gene in Francisella tularensis.
3.2.1 The tulatron vector pKEK1186 (at 720/721 retarget site) was purified from its host
cells E.Coli.DH5 using QIAfilter plasmid Midi and Maxi kits.
3.2.2 The host cell KKT5 (IglC Schu S4) was inoculated onto TSA++ agar medium from
the frozen stock, and incubated at 37C for 2—3 days for cryotransformation.
3.2.3 Our BSL3 lab was shut down for maintenance for two weeks in November, so
very limited time was provided for us to work in BSL3 lab. I didn’t get the chance to
perform the procedure of crytransformation of pKEK1186 into KKT5.
Data recorded on UTSA TVDC notebook #6, page58.
4. Significant decisions made or pending
None
5.
Problems or concerns and strategies to address
None
6. Deliverables completed
Page 55 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
None
7. Quality of performance
Good
8. Percentage completed.
About 43% of scientific work completed.
9. Work plan for upcoming month



Daily check the survival of mice challenged with wt U112 on Nov 26 th until 30 days after
the challenge.
Transform tulatron vector pKEK1186 into KKT5 (IglC Schu S4).
Screen the transformants.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) subcontractors
None
Milestone 55
Milestone description: Compare Cellular Immunogenicity of Francisella and ListeriaBased Vaccine Platforms. Measure cellular immunogenicity of live-attenuated vaccine
platforms. Compare immunogenicity of KBMA tularemia vaccine platforms
Institution: Cerus/Anza
1. Date started: 4/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will construct and prepare live and Killed But Metabolically
Active (KBMA) Listeria monocytogenes (Lm) vaccines expressing Francisella tularensis
(Ft) antigens. To directly compare the cellular immunogenicity of Lm and Ft-based
vaccines, each Lm vaccine candidate will express an antigen fused to a model ovalbumin
epitope SIINFEKL (SL8) and these will be compared to Ft vaccines expressing pepO-SL8
fusions (provided by UTSA). We will measure the ability of each vaccine to stimulate a
CD8 T cell response in vitro using a B3Z assay. We will measure the cytokine responses
elicited by vaccination with each platform in mice, compare the CD8 T cell response to
SL8 after prime and boost vaccinations in mice using intracellular cytokine staining (ICS)
and ELISpot assays and measure the potency of the T cells elicited by use of an in vivo
cytotoxicity assay.
Summary of key achievements: We have demonstrated that iglC-SL8 fusion proteins are
expressed to a much higher level than katG-SL8 in the cytosol of macrophages and
dendritic cells (DCs). Live-attenuated vaccines expressing either fusion protein were
able to secrete antigen within DCs and stimulate the B3Z T cell line that responds to the
SL8 peptide. The IglC-SL8 fusion protein induced a stronger immune response in mice
than KatG-SL8 by ICS and ELISpot analysis. Incorporation of a constitutively active prfA
allele (G155S) into the chromosome of the live-attenuated Lm-IglC-SL8 vaccine
increased immunogenicity by 2-fold. Inclusion of a much larger tag (containing an
additional 4 epitopes from vaccinia virus) decreased the immunogenicity of the Lm
vaccine. We recently cloned bivalent vaccine strains (in both native prfA and prfAG155S
backgrounds) that express both KatG-SL8 and IglC-fused to a single strong vaccinia
virus epitope (B8R). The amount of intracellular antigen expression was measured using
a semi-quantitative Western blot and was found to be similar to each of the monovalent
Page 56 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
strains but there appears to be a slight decrease in the amount of IglC secreted from the
bivalent strains. In the prfAG155S background the difference was less than 2-fold. The
bivalent vaccine strains also induced immune responses in C57BL/6 mice against the
epitope tags that were similar in magnitude to an equivalent dose of monovalent strains
expressing either KatG-SL8 or Iglc-B8R, however the bivalent strain with the native prfA
background induced significantly lower B8R-specific responses. Overall, differences
seen between bivalent and monovalent strains appeared to be greater in the native prfA
than in the prfAG155S background. We also compared the primary immune response
after a single vaccination with Live and KBMA Lm-IglC-SL8 and found that KBMA Lm
induced T cell responses that were approximately one fifth the magnitude of liveattenuated. This reduction in potency of KBMA compared to live Lm immunogenicity is
consistent with our previous work with other antigens and it is likely that the potency of
the KBMA vaccine will be improved with a boost vaccination and by the use of the
prfAG155S allele. An initial comparison of Lm and Ft vaccines was performed and
suggested that LVS-pepO-SL8 did not induce a primary T-cell response against SL8 nor
did it boost a response induced by Lm-iglC-SL8.
1) Cloning and characterization of live attenuated bivalent Listeria monocytogenes (Lm)
tularemia vaccine strains. A summary of vaccine candidates that have been constructed is
presented in table #1 below for reference. All epitope-tagged expression cassettes have been
sequenced verified.
Table I
Strain
CRS-100
Genetic Background
actAinlB
Antigen Cassette
none
Status
Sequence verified
Notebook, page
BH137
actAinlB
ActAN100-Ova
Sequence verified
BH1222
actAinlB
ActAN100-IglC-SL8
Sequence verified
NB977, p52
BH2282
actAinlB
ActAN100-KatG-SL8
Sequence verified
NB736, p137
BH1228
actAinlBuvrAB
ActAN100-IglC-SL8
Sequence verified
NB977,p52
BH1398
actAinlBuvrAB
ActAN100-KatG-SL8
Sequence verified
NB977, p152
BH2094
actAinlBuvrABprfAG155S
ActAN100-IglC-SL8
Sequence verified
NB899, p11
BH2172
actAinlBuvrABprfAG155S
ActAN100-KatG-SL8
Sequence verified
NB899,p49
BH2098
actAinlB
ActAN100-IglC-VacQuad-SL8
Sequence verified
NB899,p13
BH2100
actAinlBuvrABprfAG155S
ActAN100-IglC-VacQuad-SL8
Sequence verified
NB899, p13
BH2180
actAinlB
ActAN100-IglC-B8R (@ comK)
Sequence verified
NB899, p51
BH2182
actAinlBuvrABprfAG155S
ActAN100-IglC-B8R (@ comK)
Sequence verified
NB899, p51
BH2316
actAinlB
actAinlBuvrABprfAG155S
Remade and verified
(BH2184 had point
mutation in KatG)
Sequence verified
NB899, p56
BH2292
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8
(@tRNAarg)
ActAN100-IglC-B8R (@ comK)
ActAN100-KatG-SL8
(@tRNAarg)
NB736, p138
2) The effects of increasing dose of live attenuated Lm strains was evaluated comparing
doses ranging from 1 x103 cfu to 1x106 cfu administered IV. For this experiment the strain
BH2098 was used because the VacQuad epitope tag (which contains 4 epitopes from vaccinia
virus of known antigenic strength with B8R as the strongest and decreasing with A42R, C4L, and
K3L). SL8, IglC-33-19, and LLO immunogenicity were also evaluated (figure 1). While the SL8
epitope tag immunogenicity increased with increasing dose, the IglC (33-19) immunogenicity
peaked at the 1x105 dose and decreased at the 1x106 dose. The response to the Lm protein LLO
Page 57 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
reached a plateau at the 1x105 dose. When the vaccinia virus epitopes were evaluated (figure 2)
the strongest epitopes B8R and A42R induced strongest responses at the highest doses, while
the weaker epitopes C4L and K3L either reached a plateau at the 1x105 dose or peaked at the
1x105 dose. Together these data demonstrate that there is a different optimal dose for strong
and weaker epitopes. The reason for this difference is unclear, but could be related to antigenic
competition or due to negative regulation caused by increased inflammation at the higher doses
which may have a greater effect on weaker antigens. It will be interesting to perform this type of
dose-response analysis with vaccine strains that contain the prfAG155S allele to see whether the
dose response curves are similar.
BH2098 (QuadVac-IglC in Lm11)
BH2098 (QuadVac-IglC in Lm11)
200
100
1e3
1e4
0
1e5
0
300
1e6
IFN- SFC/2e5 splenocytes
50
1e3
1e3
1e4
1e5
0
100
1e4
200
150
1e5
400
200
1e6
IFN- SFC/2e5 splenocytes
600
1e6
IFN- SFC/2e5 splenocytes
LLO190-201 responses
33-19 responses
SL8 responses
BH2098 (QuadVac-IglC in Lm11)
Figure 1. ELISpot analysis of live primary T cell response as a function of dose. 1 week after vaccination
with varying doses of live Lm vaccine strain BH2094 (Lm actAinlB IglC-QuadVac) splenocytes were
harvested and immune responses to SL8, IglC 33-19, or LLO190 peptide were measured by ELISpot
analysis.
IM08-098 Notebook #2000, pp35-38.
Page 58 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
C4L responses
200
200
100
BH2098 (QuadVac-IglC in Lm11)
1e3
100
50
1e3
0
1e4
1e3
1e4
1e5
0
150
1e5
100
1e6
IFN- SFC/2e5 splenocytes
A42R responses
200
1e4
BH2098
(QuadVac-IglC
K3L
responsesin Lm11)
BH2098 (QuadVac-IglC in Lm11)
300
1e5
1e6
0
1e3
1e4
1e5
0
300
1e6
200
IFN- SFC/2e5 splenocytes
400
400
IFN- SFC/2e5 splenocytes
600
1e6
IFN- SFC/2e5 splenocytes
B8R responses
800
BH2098 (QuadVac-IglC in Lm11)
Figure 2. ELISpot analysis of live primary T cell response to vaccinia virus epitopes as a function of dose. 1
week after vaccination with varying doses of live Lm vaccine strain BH2094 (Lm actAinlB IglC-QuadVac)
splenocytes were harvested and immune responses to B8R, C4L, A42R, K3L peptides were measured by
ELISpot analysis.
IM08-098 Notebook #2000, pp35-38.
2) 100mL-scale Lots of Live attenuated Lm vaccines produced. In order to facilitate testing of
the monovalent and bivalent strains of Lm at UNM and at Anza, 100mL scale lots of BH2172,
BH2182, BH2292, and BH2316 were produced and stored in 0.3mL aliquots at -80oC (NB837,
p.15-16). The post-thaw titer of each lot was determined and is approximately 2 x 1010 cfu/mL
(table II), thus each aliquot contains ~6x109 cfu or enough material to vaccinate ~1000 mice.
There are 50 aliquots remaining which should provide enough material to ship to UNM for
SchuS4 protection studies and for upcoming Anza studies.
Strain
BH2172
BH2182
BH2292
BH2316
Table II. Live-attenuated Lm lots produced
Genotype
Type Titer (CFU/mL)
Lm677:KatG-SL8
Live
2.41 x 1010
LM677:IglC-B8R
Live
1.96 x 1010
Lm677:KatG-SL8/IglC-B8R
Live
2.20 x 1010
LM11: KatG-SL8/IglC-B8R
Live
1.74 x 1010
Lot#
837-15-A
837-15-B
837-15-C
837-15-D
Location
CH-FR80-015
CH-FR80-015
CH-FR80-015
CH-FR80-015
4. Significant decisions made or pending
 Because the vaccinia virus quadrotope tag significantly decreased the
immunogenicity of the Lm-IglC vaccine, strains with this tag will not be used as
vaccine candidates, but may be used further immunogenicity studies.
Page 59 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee

Chocolate Agar plates from Hardy Diagnostics will be used for cfu titers of LVS
strains.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
50%
9. Work plan for upcoming month




We will produce KBMA lots of prfAG155S vaccine candidates
We will evaluate the immunogenicity of KBMA strains after a prime and boost
vaccination
We will confirm that p60 expression correlates with cfu by performing an MOI
dose response and perform western blot and cfu analysis in parallel
We will repeat dose-response study using prfAG155S platform
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 56
Milestone description: Characterize the Cellular Immune Response that Correlates
with Protection Against an LVS Challenge and demonstrate that Cerus Strains of
Live and KBMA Lm-IglC and Lm-KatG Protect Against a SchuS4 Challenge
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will measure the T-cell response to IglC induced by live and
KBMA Lm expressing IglC compared with those elicited by Ftn or LVS vaccination. We
will produce an IglC overlapping peptide library (15aa overlapping by 11aa) to identify
IglC epitopes that are recognized by mouse T cells. We will use the IglC peptide library
for ELISpot and ICS assays to measure the IglC-specific T cell responses induced after
vaccination with live and KBMA Lm-IglC and to compare responses induced by live and
KBMA Ftn and LVS vaccination. We will demonstrate that the mechanism of protection
induced by Lm vaccines is cellular, by depletion of T cell populations and passive transfer
studies. We will demonstrate that strains of live and KBMA Lm-IglC-SL8 and Lm-KatGSL8 protect against a SchuS4 challenge and we will produce lots of KBMA vaccine and
send to UNM for testing in animal models (mice and rats).
Summary of key achievements: We determined that Lm strains expressing IglC can
induce IglC-specific immune responses in five different strains of mice (Balb/c, C57BL/6,
FVB/NJ, C3H/HeJ, and SJL/J). Immune responses were primarily observed to peptides
in IglC pool2 (peptides 26-51). By performing ELISpot assays using individual peptides,
we were able to map the responses to specific regions of the IglC protein. Using ICS and
flow cytometry, we were able to determine which responses were mediated by CD4+ or
CD8+ positive T cells. IglC-specific CD4+ T cell responses were identified in Balb/c,
Page 60 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
C3H/HeJ, and FVB/NJ mice. We mapped CD8+ T cell epitopes using 9 mers
overlapping by one amino acid, identifying IglC34-142 (LFIDSLTIA) in Balb/c mice and
IglC137-144 (IMIDLSNL) in C57BL/6. We demonstrated that Lm vaccines expressing IglC
can provide 100% protective immunity against a 10 LD 50 LVS challenge and Lm
expressing KatG provided 40% protection (confirming data generated by the Horwitz lab
at UCLA). A single vaccination with KBMA-IglC induced an IglC response that was
barely distinguishable from background. We found that in C57BL/6 mice vaccinated with
LM-KatG that there was a low T cell response induced against the IglC-33-10 peptide
suggesting that there may be cross reactivity to a listeria antigen or KatG.
1) Testing whether Lm alone induces responses to IglC peptide library: Previously we
found that C57BL/6 mice vaccinated with Lm-KatG-SL8 induced responses against the IglC 3310 peptide. By BLAST analysis a Lm protein was found to have homology with the 33-10
peptide, so it was assumed that Lm alone may induce a cross-reactive response to this iglC
peptide. To determine whether Lm-alone induces IglC responses, C57Bl/6 mice and Balb/c mice
were vaccinated IV with 5x106 Lm actAinlB expressing non-ft antigens and splenocytes were
analyzed to see if they were responsive to any IglC peptides in the IglC library. Lm-alone did not
induce significant responses to the peptides in the IglC peptide library (Figure 3). These data
suggest that either KatG induces cross reactive responses against IglC in C57BL/6 mice or that
the data reported last month from study IM08-086 may be spurious (e.g could be result of peptide
contamination).
100
IglC library in Balb/c mice:
P002-08-003, Notebook #2005, pp 54-56
90
IFNg SFC/2e5 cells
80
70
60
50
40
30
20
10
41
42
43
44
45
46
47
48
49
50
51
33-19
43
44
45
46
47
48
49
50
51
33-10
40
42
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
9
8
10
7
6
5
4
3
2
1
IglC library in C57BL/6 mice
P002-08-001 Notebook #2005, pp 57-59
90
80
IFNg SFC/2e5 cells
41
100
unstim
0
70
60
50
40
30
20
10
40
39
38
37
36
35
34
33
32
31
30
29
28
27
26
25
24
23
22
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
unstim
0
Figure 3. ELISpot analysis of live primary T cell response to IglC peptides from Lm expressing an irrelevant
antigen. 1 week after vaccination with 5e6 Lm actAinlB expressing irrelevant antigens, splenocytes were
harvested and immune responses to individual IglC peptides were measured by ELISpot analysis.
4. Significant decisions made or pending
None.
5. Problems or concerns and strategies to address
UNM, Anza, UCLA and LBERI have negotiated MTA language to allow sharing of
information and reagents from UCLA, but this need to be approved by NIAID. Without
Page 61 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
this MTA we cannot share our LM vaccine strains expressing UCLA antigens with UNM
for Schu4 Challenge studies. A final draft MTA is under review at NIAID as of 12/10/08.
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
30%
9. Work plan for upcoming month



Balb/c mice will be vaccinated (2x) with Lm-IglC, then prior to lethal LVS
challenge antibodies will be injected to deplete T cell populations: -CD4, -CD8,
both, or irrelevant Ig.
Once MTA is approved by NIAID and signed by UNM/Cerus/Anza/LBERI/UCLA,
live Lm lots will be sent to UNM for evaluation in SchuS4 challenge model.
Anza will vaccinate mice with live Lm vaccine candidates to determine whether
IglC, KatG, or both protect against lethal LVS infection using increased
stringency of LVS challenge.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 57
Milestone description: Optimization of KBMA Lm Vaccination Route and Regimen.
Institution: Cerus/Anza
1. Date started: 6/1/2008
2. Date completed: Pending
3. Work performed and progress including data and preliminary conclusions
Summary of objectives: We will compare various routes of administration including IV, IM,
IN, ID and oral. For oral, IN, and ID administration in mice, we will first mutate the inlA
gene of Lm to allow for binding of murine E-cadherin in order to mimic the human
interaction (as described in Wollert et al., Cell, 2007). We will compare the potency of the
inlAM gain of function mutants to our traditional platform strain. Routes will be ranked by
ability to induce a cellular immune response using ELISpot, ICS, and in vivo cytotoxicity.
We will optimize dosing regimen of most potent and tolerable route. Lm expressing IglC
and/or KatG will be used to evaluate immunogenicity. Optimized route and regimen will
be confirmed by SchuS4 protection studies at UNM.
Summary of Key achievements: We have constructed vaccine candidates that contain
the inlAM gain of function mutations (Table III). The sequence of the wild-type EGDe inlA
gene (from the Lm strain used in the Wollert manuscript) was synthesized and the inlA
gene in our platform strain was replaced (inlAWT) in our live-attenuated and KBMA
platform strains as there are a number of differences in the sequence between the native
sequences between these strains. Two point mutations, S192N and Y369S, were
incorporated into the EGDe inlA sequence (inlAM) and inserted into the chromosome of
our live-attenuated and KBMA platform strains. Into these 4 strains the ActAN100-iglCSL8 expression cassette was inserted using the integration vector pINT. Cellular
Page 62 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
invasion assays were performed: invasion of CaCo2 cells was dependent on inlA, as a
inlA strain was unable to invade, but we were not able to demonstrate that the inlAM
gain of function allele increased invasion compared to inlAwt (as published by Wollert
et.al). Oral and IV routes of administration were compared: In spleens, SL8 and IglC
responses were 2-3 times lower after oral immunization than with IV administration, but
mucosal responses from intra-epithelial lymphocytes (IELs) were similar after
immunization by either route. Mice that were vaccinated orally with the inlAM strain had
marginally higher splenic T cell responses and IEL responses that were 3-4 times higher
than the isogenic strain expressing inlAwt. This preliminary result suggests that there may
be a slight increase in immunogenicity when the inlAM vaccine strain is administered
orally.
Table III
Strain
Genetic Background
Antigen Cassette
Status
Notebook,
page
CRS-100
actAinlB
none
Sequence verified
BH2130
actAinlBinlAWT
none
Sequence verified
BH2164
actAinlBinlAWT
ActAN100-IglC-SL8
Sequence verified
BH2170
actAinlBinlAM
none
Sequence verified
BH2194
actAinlBinlAM
ActAN100-IglC-SL8
Sequence verified
BH2132
actAinlBuvrABprfAG155SinlAWT
none
Sequence verified
BH2166
actAinlBuvrABprfAG155SinlAWT
ActAN100-iglC-SL8
Sequence verified
BH2134
actAinlBuvrABprfAG155SinlAM
none
Sequence verified
BH2168
actAinlBuvrABprfAG155SinlAM
ActAN100-iglC-SL8
Sequence verified
NB899, p. 44
NB899, p. 48
NB899, p.49
NB899, p. 52
NB899, p. 44
NB899, p.48
NB899, p. 44
NB899, p.48
NB899, p.44
In order to evaluate which route of administration is the most effective we intend to perform
protection studies in which mice are vaccinated with Lm strains by various routes and then
challenged with a lethal dose of LVS. Because tularemia is a pathogen that is of most
concern when it is aerosolized, we have decided to do these pivotal protection studies with
an intranasal (IN) LVS challenge. In order to prepare for these studies we initiated an IN LVS
LD50 in Balb/c mice. Doses of amplified DVC lot 16 LVS were administered IN to
anesthetized mice ranging from 500 cfu to 100,000 cfu. Unfortunately, only one mouse in the
highest dose group died (Table IV). This is much higher than the LD100 dose reported by the
Horwitz lab. We were later informed by Dr. Horwitz that use of Isofluorane as an inhaled
anesthetic can increase the IN LD50 by orders of magnitude and he strongly recommended
the use of injected ketamine/xylazine as an anesthetic. The Lyons lab uses isofluorane
anesthesia prior to IN administration of LVS, but cautions that the animals must be deeply
anesthetized and breathing deeply, which was not the case during this experiment. Thus this
LD50 analysis will need to be repeated with an alternate method of anesthesia.
Table IV. P001-08-001: LVS intranasal LD50 data. Notebook #2000, p45
Page 63 of 64
Tularemia Vaccine Development Contract: Technical Report
Period: 11/01/2008 to 11/30/2008
Due Date: 12/18/2008 and Prepared by: C. Rick Lyons, Barbara Griffith, Terry Wu, Julie
Wilder, Trevor Brasel, Julie Hutt, Dana Pohlman, Michelle Valderas, Bob Sherwood, Karl
Klose, Bernard Arulanadam, Justin Skoble, Stephen Johnston, Kathryn Sykes, Mitch
Magee
Grp #
Mice
Strain
Dose Route
Vx date
Survial
1
4
Ft-LVS
1e5
2
4
Ft-LVS
5e4
IN
11/5
3
IN
11/5
4
3
4
Ft-LVS
1e4
IN
11/5
4
4
4
Ft-LVS
5e3
IN
11/5
4
5
6
4
Ft-LVS
1e3
IN
11/5
4
4
Ft-LVS
5e2
IN
11/5
4
4. Significant decisions made or pending
Isofluorane will not be used as method of anesthesia for IN studies.
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
12%
9. Work plan for upcoming month



Mucosal immunity will be evaluated again after oral immunization to determine
whether the >2fold increase in mucosal immunity seen with the inlAM strain is
reproducible.
The intranasal LD50 will be repeated with DVC lot16 LVS using alternate anesthesia
methodology
Murine epithelial cell line CT-26 will be used for invasion assays
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 64 of 64