Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Contract No. HHSN266200500040-C
ADB Contract No. N01-AI-50040
Section I: Purpose and Scope of Effort
The Tularemia Vaccine Development Contract will lead to vaccine candidates, two
animal models and cellular assays vital for testing vaccine efficacy.
Sections II and III:Progress and Planning Presented by Milestone
Active milestones: 2, 3, 4, 5, 7, 8, 9, 11, 12/13(UNM/LBERI), 14, 17, 19,
21(UNM/LBERI), 26, 27, 28, 35(ASU/UNM), 49, 50, 52, 55, 56, 57
Completed milestones: 1, 25, 32, 33, 34 (UNM/ASU), 16, 39, 40, 43 (UTSA), 48, 51
Inactive milestones: 6, 10, 15, 18, 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 11 annual health screenings since 8/26/08 for the LVS
vaccinees originally vaccinated on in September and October 2007.
b. One UNM and possibly 2-3 LBERI scientists will request vaccinations in the
January 2009.
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 and will offer
LVS vaccinations in January 2009.
c. UNM (4) and LBERI (33) are vaccinated; UNM and LBERI could offer the LVS
vaccinations up 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. One UNM and one LBERI scientist are medically pending. One UNM scientist
may be rescheduled for LVS vaccination. USAMRIID is now offering the LVS
vaccinations as of October 7, 2008 but the UNM scientists will be attending the
Page 1 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
UNM TVDC annual meeting on that date. USAMRIID will offer UNM and LBERI a
date in January 2009.
6. Deliverables completed
A total of 37 participants (33 LBERI and 4 UNM participants) have received the LVS
vaccination since 9/11/07.
7. Quality of performance
Excellent
8. Percentage completed
70% 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 1 UNM and 2-3 LBERI scientists for the pre-vaccination
health screenings required for vaccinations in January 2009.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 4
Milestone description: Confirmation of aerosol in vivo in NHP
Institution: LBERI
1. Date started: 11/1/06
2. Date completed: In progress
3. Work performed and progress including data and preliminary conclusions:
a. Continued writing the Milestone Completion report. Completion of the draft
report is dependent on the completion of the pathology report.
b. The LBERI Veterinary pathology has read the histopathology slides from Cohort
2 and will write the histopathology report next 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
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 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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 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 using different bacteria strains, dose
and methods of pulmonary infection, we would like to repeat the
comparison only with 1000 SCHU S4 delivered intratracheally.
iii. We decided to take advantage a program project grant study headed
by Julie Hutt and Amanda Dubois (PFt5) to examine the
histopathology of NHP infected with SCHU S4 by bronchoscopy at 1,
4, and 7 days post challenge.
iv. We started a parallel experiment at UNM on the TVDC in mice and
rats using intratracheal infection with 103 SCHU S4. The tissues for
days 1- 4 post infection (p.i.) have already been collected for the
mice and rats; the next time point will be day 7 p.i.
v. The tissues will be processed at LBERI and analyzed by Julie Hutt.
b. Experiment Ftc74 (Notebook 116 pages 28-35)
i. The purpose of this experiment was to determine whether inclusion
of quantum dots (QD) in the SCHU S4 inoculum would affect the
virulence of SCHU S4 following i.t. infection of Fischer 344 rat model
ii. We showed previously that addition of QD had no effect on the
virulence of SCHU S4 in naïve BALB/c and Fischer 344 rats.
However, these results have to be interpreted carefully because both
mice and rats are very sensitive to pulmonary SCHU S4 infection
and would only survive if SCHU S4 was extremely attenuated or the
immune response was very effective. Therefore, we repeated this
experiment in LVS vaccinated rats which have much higher
resistance to SCHU S4.
iii. 30 rats were vaccinated s.c. with 107 LVS and rested for 1 month for
the vaccine to clear.
Page 3 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
iv. The vaccinated rats were then divided into 5 groups of 6 rats each
and challenged with SCHU S4 in the presence or absence of
quantum dots
v. As shown in Fig 1, QD had little impact on the virulence of SCHU S4
when the challenge dose was in the range of 3-4 x 103 CFU.
However, at a 10-fold higher dose, rats infected with SCHU S4 in the
presence QD lost less weight and had better overall survival than
rats infected with SCHU S4 alone.
vi. This experiment will have to be repeated with more animals for us to
make a decision whether to include QD or not in future experiments
100
80
60
3.7 x 10 3 cfu no QD
3.1 x 10 3 cfu w/ QD
40
20
Percent survival
Percent survival
100
0
80
60
4.7 x 10 4 cfu no QD
4.1 x 10 4 cfu w/ QD
40
20
0
0
5
10
15
0
5
Days
15
105
100
95
3
3.7 x 10 no QD
3.1 x 10 3 w/ QD
90
85
Percent starting weight
105
Percent starting weight
10
Days
80
100
4.7 x 10 4 no QD
4.1 x 10 4 w/ QD
95
90
85
80
0
5
10
15
0
5
Days
10
15
Days
Figure 1. Effect of QD on the survival and weight loss of LVS vaccinated Fischer 344
rats infected i.t. with SCHU S4. LVS vaccinated rats (107 dose) (n = 6) were challenged
with SCHU S4 in the presence or absence of quantum dots and substrate. Weight loss
and survival were monitored daily
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
a. Mouse model completed
b. Guinea pig model completed
c. Rat model completed
7. Quality of performance
NA
8. Percentage completed
86%
9. Work plan for upcoming month
a. Complete Ftc75 as described above
b. Repeat Ftc74 as described above with more animals
Page 4 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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. Wave 1 high dose challenges were performed with target doses of approximately
5000, 50,000, and 250,000 CFU. Wave 2 low dose challenges were performed
on October 17, 2008. There were 2 groups with 4 animals each. The animals
were presented with 1-2 CFU (target dose 25 CFU) and 19-90 CFU (target dose
250 CFU). Below is the respiratory and temperature clinical data collected to
date.
ID#
28615
28624
28581
28588
28618
28463
28571
28499
Sex
F
F
M
M
F
F
M
M
Study Day/Respiration Rates (breaths per minute)
-11 to -1
-11 to -1
-11 to -1
1400
2000
0800 Baseline ± SD
± SD
±
Baseline
Baseline
±
8
65
± 12
63
±
62
62
± 13
68
± 10
65
±
69
± 11
61
±
8
65
±
56
± 14
55
±
9
56
±
58
±
9
57
±
7
58
±
64
±
8
61
±
7
63
±
61
±
6
62
± 10
62
±
63
±
9
66
±
8
65
±
SD
8
10
9
10
6
6
6
7
Table 1. Average Baseline Respiration Rate (breaths per minute) for Study Days
-11 thru -1. Respiratory rates were captured daily at 0800 and 1400 prior to
animal exposure to establish a baseline for use in post-exposure comparisons.
Due to variability in animal activity and to provide a more accurate basis for
comparison, the baselines for each time point were calculated as separate
entities. The 2000 observations were not performed until after exposure, so the
2000 baseline was calculated using each animal’s average daily respiration rate.
Time acronyms used: 0800 is 8am, 1400 is 2pm and 2000 is 8pm. Electronic file
located on http://wss/sites/alst/Shared%20Documents/Forms/AllItems.aspx.
Page 5 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
A
0800 Respiratory Rates
0
1
2
3
4
5
6
7
8
9
48
40
48
44
48
56
60
56
68
56
58
44
44
68
60
72
52
44
64
44
44
52
52
56
52
48
56
48
48
56
56
56
60
48
64
44
52
64
54
52
60
60
72
52
72
60
68
64
40
52
64
52
56
52
68
60
68
68
76
56
64
52
76
68
60
44
48
60
64
64
52
84
60
76
64
40
68
48
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
28615
28624
28581
28588
28618
56
52
52
48
44
72
80
56
52
48
52
60
64
52
52
80
68
60
56
60
56
52
56
56
64
56
56
56
52
64
60
48
68
52
60
64
52
72
68
56
52
52
32
56
68
60
68
50
72
76
60
68
64
72
54
56
56
64
68
80
60
64
64
28463
28571
28499
56
68
60
68
68
76
64
60
64
72
72
80
56
60
56
56
64
60
56
76
64
40
80
68
44
76
48
40
68
56
28615
28624
28581
28588
28618
28463
28571
28499
10
11
12
13
14
56
56
60
72
80
68
68
58
60
68
56
68
76
72
64
60
60
48
Baseline
62
62
69
56
58
64
61
63
±30%
81
81
89
73
76
83
80
82
43
43
48
39
41
45
43
44
±50%
93
93
103
85
88
96
92
95
31
31
34
28
29
32
31
32
B
1400 Respiratory Rates
42
52
52
Baseline
±30%
±50%
65
68
61
55
57
84
89
79
71
74
45
48
43
38
40
97
103
92
82
85
32
34
31
27
28
61
62
66
79
81
86
43
44
46
92
94
99
31
31
33
C
2000 Respiratory Rates
28615
28624
28581
28588
28618
28463
28571
28499
0
1
2
3
4
5
6
7
81
9
10
11
12
13
14
64
68
60
44
44
56
76
60
60
48
56
56
56
68
76
60
68
60
48
52
52
56
72
68
64
60
76
48
56
68
72
80
56
52
60
48
56
60
52
56
60
48
56
56
56
60
64
60
64
52
100
60
52
48
72
52
68
48
76
80
60
52
84
52
52
52
52
60
56
80
76
72
72
48
44
60
64
60
60
76
72
60
72
72
60
68
48
52
1Respiratory
60
48
68
48
Baseline
63
65
65
56
58
63
62
65
±30%
82
85
84
72
75
81
81
84
44
46
45
39
40
44
43
45
±50%
95
98
97
83
86
94
93
97
32
33
32
28
29
31
31
32
rate not recorded on animal #29463 at 2000 obs on study day 8.
Table 2. Respiratory Rates (breaths per minute) for Individual Animals on Study Days 1
thru 14. A) Respiratory rates at 0800. B) Respiratory rates at 1400 C) Respiratory rates
at 2000. Pink data points indicate a 30% increase in respiration rate when compared to
baseline; red data points indicate a 50% increase in rate. Light blue data points indicate
a 30% decrease in respiration rate when compared to baseline. Electronic file located
on http://wss/sites/alst/Shared%20Documents/ Forms/AllItem .aspx.
Page 6 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Respiratory Rate (breaths per
minute)
ED50 Wave 2 Respiratory Rates
100
28615
90
80
28624
70
28581
60
28588
50
28618
40
28463
30
28571
-168-144-120-96-72-48-24 0 24 48 72 96 120144168192216240264288312336
28499
Study Day (Hours)
Figure 1. ED50 Wave 2 Respiratory Rates. Each data series represents
an individual animal, with the identifiers on the right side of the table
depicting each animal’s study ID number. No dramatic changes in
respiration rate were observed with the exception of animal #28581 at
150 hours post exposure. Electronic file located on http://wss/sites/alst/
Shared%20Documents/Forms/AllItems.aspx.
ID#
28615
28624
28581
28588
28618
28463
28571
28499
Sex
F
F
M
M
F
F
M
M
-11 to -1
0800 Base
Avg
±
99.4
±
98.7
±
101.0
±
97.9
±
96.3
±
97.4
±
100.5
±
100.0
±
SD
1.00
1.72
1.05
2.37
1.87
1.24
0.72
1.69
-11 to -1
1400 Base
Avg
±
100.2
±
99.9
±
101.4
±
99.9
±
97.2
±
99.4
±
101.5
±
101.0
±
SD
0.54
1.12
0.98
1.07
1.09
0.40
0.55
0.90
-11 to -1
2000 Base
Avg
±
99.8
±
99.3
±
101.2
±
98.9
±
96.8
±
98.3
±
100.9
±
100.5
±
SD
0.64
1.28
0.80
1.55
1.09
0.70
0.54
0.90
Table 3. Average Baseline Temperature for Study Days -11 thru -1.
Body
temperatures were captured daily at 0800 and 1400 prior to animal exposure to
establish a baseline for use in post-exposure comparisons. Due to variability in
animal activity and to provide a more accurate basis for comparison, the
baselines for each timepoint were calculated as separate entities. The 2000
observations were not performed until after exposure, so the 2000 baseline was
calculated using each animal’s average daily body temperature. Electronic file
located on http://wss/sites/alst/Shared%20Documents/Forms/AllItems.aspx.
Page 7 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
A.
28615
28624
28581
28588
28618
28463
28571
28499
0
98.7
97.6
100.9
98.4
96.5
97.6
99.4
100.2
1
99.6
99.1
100.8
91.6
96.9
97.4
100.8
100.4
2
100.3
97.4
101.2
97.0
95.6
97.7
99.7
99.2
3
100.7
100.0
102.9
96.5
97.9
99.3
102.1
100.1
4
100.2
96.7
102.0
99.5
97.6
99.6
99.6
99.6
5
98.9
99.6
102.2
97.7
99.0
99.7
100.1
99.4
6
99.5
97.7
100.6
99.3
98.9
97.5
97.7
99.2
7
98.2
98.1
100.3
98.2
99.1
95.2
95.6
100.4
0800 Temps
8
9
97.0
88.6
98.7
98.7
98.9
97.8
95.7
93.7
100.5
98.2
99.1
91.0
86.9
98.3
10
11
12
13
14
99.4
100.0
98.9
97.2
99.0
99.9
98.1
98.5
98.2
98.8
100.9
97.1
97.7
98.7
99.3
99.1
95.6
95.7
11
12
13
14
Baseline
99.4
98.7
101.0
97.9
96.3
97.4
100.5
100.0
Febrile
101.4
102.1
103.1
102.6
100.0
99.8
102.0
103.3
Hypo
97.4
95.2
98.9
93.1
92.5
94.9
99.1
96.6
Baseline
Febrile
Hypo
100.2
101.3
99.2
B.
1400 Temps
0
1
2
3
4
5
6
7
8
9
28615
99.3
100.0
100.6
102.3
101.3
98.5
99.5
99.2
94.9
75.1
10
28624
96.5
99.9
100.0
100.0
100.7
99.8
99.2
99.7
99.7
98.8
28581
99.9
100.9
101.7
104.9
104.0
101.9
101.4
98.1
28588
98.6
96.9
99.4
99.4
103.0
100.2
99.5
100.2
92.0
100.3
100.7
100.1
98.1
99.2
28618
96.3
95.7
98.0
100.9
99.6
99.6
99.2
98.1
98.9
98.0
99.6
100.3
100.9
98.9
28463
97.4
99.4
99.8
102.2
100.5
100.0
97.9
94.6
94.4
90.3
28571
101.3
101.9
102.2
101.5
97.4
99.1
96.7
95.5
97.5
81.6
28499
96.9
101.5
101.7
101.6
100.5
100.4
98.9
99.9
99.2
98.5
97.8
98.1
97.4
97.8
97.8
98.4
98.1
99.9
102.1
97.6
101.4
103.4
99.4
98.3
99.9
102.1
97.8
100.0
97.2
99.4
95.1
99.4
100.2
98.6
101.5
102.6
100.4
101.0
102.8
99.2
95.2
C.
2000 Temps
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
28615
99.8
99.9
99.6
102.0
100.7
100.3
100.1
100.1
94.6
28624
100.5
99.5
99.7
98.6
99.8
99.1
98.7
99.6
98.3
98.4
100.7
100.5
100.3
99.9
100.6
28581
100.0
102.0
100.2
102.7
102.0
101.7
101.5
97.1
28588
94.8
98.6
97.9
97.5
101.8
99.5
97.5
98.1
99.4
99.8
100.2
99.8
99.8
100.6
100.1
28618
98.1
97.4
99.0
99.0
99.0
99.5
100.3
100.1
99.2
99.6
101.3
98.7
98.7
100.4
102.0
28463
98.9
100.5
99.6
100.4
99.6
28571
103.7
102.3
102.2
97.7
100.6
98.5
97.8
96.0
93.3
89.3
101.3
97.5
97.2
92.4
28499
100.1
101.4
103.4
100.1
99.3
99.7
100.4
101.2
100.8
100.3
98.6
97.4
Baseline
Febrile
Hypo
99.8
101.1
98.5
99.3
101.8
96.7
101.2
102.8
99.6
98.9
102.0
95.8
96.8
98.9
94.6
98.3
99.7
96.9
100.9
102.0
99.9
100.5
102.3
98.7
Table 4. Temperatures for Individual Animals on Study Days 1 thru 14. A) Temperatures at
0800. B) Temperatures at 1400 C) Temperatures at 2000. Red data points indicate a fever
(defined as a temperature greater than the baseline plus 2 standard deviations). Blue data points
indicate hypothermia (defined as a temperature less than the baseline plus 2 standard
deviations).Electronic file located on http://wss/sites/alst/Shared%20Documents /Forms/AllItems
.aspx.
Page 8 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
ED50 Wave 2 Body Temperatures
Body Temperature (°F)
105.0
100.0
28615
28624
95.0
28581
90.0
28588
85.0
28618
28463
80.0
28571
75.0
-168-144-120-96-72-48-24 0 24 48 72 96 120144168192216240264288312336
28499
Study Day (Hours)
Figure 2. ED50 Wave 2 Body Temperatures. Each data series represents an individual
animal, with the identifiers on the right side of the table depicting each animal’s study ID
number. No dramatic changes were observed until approximately 18 hours prior to
sacrifice, when body temperatures dropped significantly. Electronic file located on
http://wss/sites/alst/Shared%20Documents/Forms/AllItems.aspx.
Death
ID#
Sex
Presented
Dose
Manner
of
Death1
Timepoint
Study
Day
28615
28624
28581
28588
28618
28463
28571
28499
F
F
M
M
F
F
M
M
1-2 cfu
2-3 cfu
1-2 cfu
1-2 cfu
19.3 cfu
30.3 cfu
89.6 cfu
60.7 cfu
E
NA
FD
E
E
E
E
E
1400
NA
0800
1400
2000
2000
1400
1400
9
NA
8
20
17
9
9
12
1E
= euthanized, FD = found dead
Table 5. ED50 Wave 2 Mortality Data. All animals were euthanized with the exception of
#28581, which was found dead approximately 186 hours after exposure. Animals dosed
with 1-2 cfu succumbed between 174 and 480 hours post exposure; animals dosed with
19-90 cfu succumbed between 216 and 414 hours post exposure. Electronic file located
on http://wss/sites/alst/Shared%20Documents/Forms/AllItems.aspx.
Page 9 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
b. The batch of Chamberlains media that had been used in previous studies was
depleted. A new batch was purchased from Teknova. Upon using a new batch
of media from Teknova, LBERI was unable to successfully grow the bacteria and
there appeared to be an issue with the media. The decision was made to
prepare the Chamberlain’s defined media in-house at LBERI instead of
outsourcing it to Teknova as a commercial vendor. Using the media prepared inhouse, multiple growth curves have been performed to redefine the curve to
ascertain where mid log growth occurs. These data are shown below in Figures
3 and 4.
Time vs Normalized OD600: Individual Flasks
2.50
Normalized OD600
2.00
1.50
1.00
0.50
0.00
15
20
25
30
35
40
45
50
Time (h)
Flask 1: 27OCT08
Flask 1: 29OCT08
Flask 1: 3NOV08
Flask 2: 27OCT08
Flask 2: 29OCT08
Flask 2: 3NOV08
Flask 3: 27OCT08
Flask 3: 29OCT08
Flask 3: 3NOV08
Figure 3. Francisella tularensis SCHU S4 18-24h growth curve using in-house prepared
Chamberlain’s broth: Time vs. Normalized OD600. These data represent nine separate
flasks analyzed on three separate days. Data are located in the following folder:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY08\FY08-074 (TUL-07)\Growth curves
Page 10 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Time vs CFU(log10)/mL: Individual Flasks
Normalized OD600
12.00
11.50
11.00
10.50
10.00
9.50
9.00
8.50
8.00
7.50
7.00
0
5
10
15
20
25
30
35
40
45
50
Time (h)
Flask 1: 27OCT08
Flask 3: 27OCT08
Flask 2: 29OCT08
Flask 1: 3NOV08
Flask 2: 27OCT08
Flask 1: 29OCT08
Flask 3: 29OCT08
Flask 2: 3NOV08
Figure 4. Francisella tularensis SCHU S4 18-24h growth curve using in-house prepared
Chamberlain’s broth: Time vs. CFU(log10)/mL. These data represent nine separate
flasks analyzed on three separate days. Data are located in the following folder:
\\Saturn\absl3\Agent and Study Specific Data and Miscellaneous Documents\STUDY
SPECIFIC DATA\FY08\FY08-074 (TUL-07)\Growth curves
Figures 3 and 4 demonstrate that log phase initiates at approximately 23h postinoculation and is short-lived (4-5h based on the beginning of stationary phase at 2728h). This is similar to what was observed with past batches of Chamberlain’s medium
from Teknova. Though not shown here, the initial concentration of the 100 mL volume
immediately following SCHU S4 inoculation was consistently 2.0 x 10 6 CFU/mL; this
indicates a 3-4 log10 increase by 24h. Interestingly, viable bacterial concentrations did
not decrease sharply as expected at 48h as was seen previously with Teknova-produced
Chamberlain’s medium. At certain time points, bacterial concentration outliers are
present. This may be attributed to the presence of clumps in the bacterial suspension, a
phenomenon that we have observed with numerous types of microbes. The 27OCT08
OD600 data demonstrated significantly lower OD600 values, though the associated
bacterial concentrations were as expected (1 x 109 CFU/mL). The reasons behind the
OD600 discrepancy are currently unknown. Hypothetically, an incorrect blank (perhaps
from an older, darker Chamberlain’s lot) was used, though this cannot be confirmed. To
rule this out, future spectrophotometer readings will incorporate the blank as an actual
sample (i.e the OD600 value obtained from the blank will be subtracted from the
measured test sample values). Taken together, the growth curve data demonstrate that
the optimal bacterial harvest time for bioaerosol challenge material preparation is 24-25h;
this is the same range that has been targeted for all recent NHP exposures using SCHU
S4 grown in Chamberlain’s media purchased from Teknova .
4. Significant decisions made or pending
a. Wave 3 presented target doses will be 250 CFUs and 500 CFUs. These doses
were chosen by NIAID to represent an intermediate dose between the low dose
Page 11 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
presented in Wave 2 (90 CFU) and the high dose presented in Wave 1 (5000
CFU).
b. Challenge date for Wave 3 has not yet been determined due to re-evaluation of
bacterial growth curve.
5. Problems or concerns and strategies to address
The batch of Chamberlains media that had been used in previous studies was depleted.
A new batch was purchased from Teknova. Upon using a new batch of media from
Teknova, LBERI was unable to successfully grow the bacteria and there appeared to be
an issue with the media. The decision was made to prepare the Chamberlain’s defined
media in-house at LBERI instead of outsourcing it to Teknova as a commercial vendor.
Using the media prepared in-house, multiple growth curves have been performed to
redefine the curve to ascertain where mid log growth occurs.
6. Deliverables completed
None
7. Quality of performance
Good.
8. Percentage completed
45% of the scientific work is complete.
9. Work plan for next month
a. Day 35 Terminal sacrifices for Wave 2 survivors are scheduled for November 21,
2008.
b. Wave 3 challenges at 250 and 500 CFU presented doses will be performed once
the re-evaluation of the bacterial growth curve is complete..
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated.
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. On October 16, 2008, 5 animals were vaccinated with LVS. Three of the animals
were vaccinated via scarification and the other two via subcutaneous injection;
the comparison of s.c. and scarification is being conducted to prepare for a future
study in which we will compare our original lot of LVS with a new lot of LVS; by
direction from USAMMDA, the entity providing us with the new LVS IND 157 lot,
we will perform this comparison using scarification as the route of inoculation;
thus, it is important to establish that we can scarify the monkeys with LVS and
that they develop an immune response as a result of the scarification; S.C. is
being used as a positive control as we have shown previously that this route of
LVS inoculation induces humoral and cellular immunity in NHPs
b. Target dose was 60 x 106 LVS organisms/inoculum. This mimics the dose given
to humans in the clinical trial of the USAMMDA LVS IND 157 protocol.
Page 12 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
c.
Blood draws were performed on Day 0, 7 and 14. Plasma was collected and
stored and will be analyzed in an ELISA to determine the level of serum IgG antiLVS produced in the vaccinated animals.
d. The ability of PBMCs to respond to LVS and SCHU S4 antigens by proliferation
is shown in Figure 1.
300000
250000
200000
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
150000
100000
Scarification, Day 7
Scarification, Day 0
0
SC, Day 7
50000
SC, Day 0
RLU (Mean+/- SEM)
350000
Figure 1: Blood was collected from NHPs vaccinated with 60 x 106 LVS organisms either
by subcutaneous inoculation (SC, n = 2) or by scarification (n=3). PBMCs were purified
from blood collected either immediately prior to vaccination (Day 0) or on Day 7 postvaccination. All PBMCs were plated at 1 x 106 cells/ml and incubated with the indicated
stimuli (1 x 105/ml) for 5 days before addition of BRDU and measurement of luciferase
activity by anti-BRDU antibodies. Presented data represents the averages for 2 NHP
vaccinated by the subcutaneous route and for 3 NHP vaccinated by the scarification
method.
a. The ability of PBMCs to respond to LVS and SCHU S4 antigens by IFNγ
secretion as measured by ELISPOT is shown in Figure 2.
Page 13 of 62
Tularemia Vaccine Development Contract: Technical Report
450
400
350
300
250
200
150
TNTC
100
450
400
Media
LVS hk Hi
350
LVS ff Hi
300
SCHUS4 hk Hi
250
SCHUS4 ff Hi
A06199, Day 15
A06199, Day 7
A06199, Day 0
A04169, Day 15
A04169, Day 7
A04169, Day 0
28656, Day 15
28656, Day 7
28656, Day 0
50
0
B
200
150
100
A05403, Day 15
A05403, Day 7
A05403, Day 0
0
28461, Day 15
50
28461, Day 7
IFNg Spots (Mean +/- SEM)
A
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
28461, Day 0
IFNg Spots (Mean +/- SEM)
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 2: Blood was collected from NHPs vaccinated with LVS organisms either by
scarification (n=3, panel A) or by subcutaneous inoculation (SC, n = 2, panel B). PBMCs
were purified from blood collected either immediately prior to vaccination (Day 0) or on
Day 7 or 15 post-vaccination. All PBMCs were plated at 1.33 x 106 cells/ml and
incubated with the indicated stimuli (1 x 105/ml) for 20 hours. LVS hk hi and ff hi
stimulation of PBMCs from A04169 was conducted but too many spots developed in the
well (TNTC) making the count inaccurate. Data are graphed for individual NHPs tested
and annotated by NHP identification numbers (28461 and A05403)
a. Preliminary conclusions
a. LVS vaccination by either scarification or s.c. inoculation appears to
stimulate PBMCs to proliferate (A) and secrete IFNγ (B). This is
particularly evident when examining the response to LVS hk Hi.
Page 14 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC assay
11072008.svd; TVDC (5) bound notebook (9247), pp. 7 -18; 23 -39
4. Significant decisions made or pending
a. Based on the serum IgG anti-LVS ELISA data a decision will need to be made
regarding whether these 5 vaccinated animals will be challenged with Schu S4.
5. Problems or concerns and strategies to address
a. Although we targeted a dose of 60 x 106 LVS organisms/inoculation, when
aliquots of the inoculum were plated, nothing grew on the plates.
b. We reconstituted another vial of lyophilized LVS in the exact manner as we had
previously and plated that on 10/21/08. The results showed that the
concentration in the reconstituted vial was 8.7 x 106/ml rather than 1 x 109/ml as
expected. Therefore, the 5 animals vaccinated on October 16, 2008 were
dosed with 4.8 x 105 CFU rather than the proposed 60 x 106 CFU.
6. Deliverables completed
None
7. Quality of performance
Good.
8. Percentage completed
12% of the scientific work is complete.
9. Work plan for upcoming month
a. Blood draws will be performed on Day 21, 28, and 35 post-vaccination.
b. We will continue to measure the proliferative response of PBMCs to LVS and
SCHU S4 antigens in vitro, as well as the ability of the PBMCs to respond to
these antigens by IFNγ secretion as measured by ELISPOT assay.
c. We will determine IgG anti-LVS titers from all the plasma collected from each of
the 5 NHPs(d0 – d35)
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
a. The draft SOP “LBERI 3.1 Operation of the Aeromist, Hospitak, Collison 3-jet,
Aeroneb® Micropump, and Sparging Generators During Francisella tularensis
Bioaerosol Studies” was sent to UNM.
b. Preparing the validation plan and the list of studies needed for the aerosol
procedure.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Page 15 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
None
7. Quality of performance
Good.
8. Percentage completed
15% of the scientific work is complete.
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
Page 16 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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
a. No new work done this period
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
Mouse proliferation assay, IFN and IL-2 Elispot, anti-Ft antibody titration
Rat IFN Elispot, anti-Ft antibody titration
Guinea pig anti-Ft antibody titration 
7. Quality of performance
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 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
a. We have previously observed that some non-LVS vaccinated animals respond to
LVS antigens by IFNγ secretion; we were unsure whether this pattern would hold
if animals were bled on separate days or whether the responsiveness was
inconsistent from day to day
b. We have also occasionally observed high backgrounds in the IFNγ ELISPOT
assay (i.e. large numbers of spots in unstimulated wells); we have determined
that often this is due to high RBC content (contamination) but sometimes also
occurs in PBMC preparations that contain less than 2% RBCs; we also reasoned
that the background responses may be coming from platelets; we did not know
whether this response would be consistent if the same NHP was tested on
different days
Page 17 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
We repeated the IFNγ ELISPOT assay on PBMCs isolated from non-LVS
vaccinated animals to determine whether the pattern of responsiveness (nonspecific responses and high background) seen in previous analysis of former
PBMC preparations were consistent when comparing independent PBMC
purifications.
350
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
300
250
200
150
100
28587, TUL57
28587, TUL53
28587, TUL52
28587, TUL49
28571, TUL57
28571, TUL53
28571, TUL52
28571, TUL49
28463, TUL57
28463, TUL53
28463, TUL52
28463, TUL49
28461, TUL57
28461, TUL53
0
28461, TUL52
50
28461, TUL49
IFNg Spots (Mean +/- SEM)
c.
Figure 3. PBMCs were prepared from non-LVS vaccinated NHPs, plated
at 1.33 x 106 cells/ml and incubated with the indicated stimuli (1 x 105/ml)
for 20 hours.
Data interpretation: In all cases, the pattern of non-specific
responsiveness to LVS and SCHU S4 antigens was similar in each of the
two instances in which each PBMC preparation was tested. In two of the
four cases, 28461 and 28587, the second PBMC preparation
demonstrated less background (response to media) than the first.
Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay
statview\PBMC assay 11072008.svd; TVDC (5) bound notebook (9247),
pp. 7 -18; 23 -39
d. We have been testing the ability of previously frozen and thawed PBMCs
to produce IFNγ as measured by the IFNγ ELISPOT assay; two different
protocols have been tested (Cerus and CTL); Figure 4 shows the result.
Page 18 of 62
Tularemia Vaccine Development Contract: Technical Report
400
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
350
300
250
150
100
350
300
250
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
28664, None
28664, CTL
28664, Cerus
28656, None
28656, CTL
28656, Cerus
28627, None
28627, Cerus
400
28627, CTL
50
0
B
200
150
100
28651, None
28651, Cerus
28549, None
0
28549, Cerus
50
28503, None
IFNg Spots (Mean +/- SEM)
A
200
28503, Cerus
IFNg Spots (Mean +/- SEM)
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 4. Effects of freeze/thaw on the ability of PBMCs to react in the
IFNγ ELISPOT assay. PBMCs were prepared from non-LVS
vaccinated NHPs, plated at 1.33 x 106 cells/ml and incubated with the
indicated stimuli (1 x 105/ml) for 20 hours. Panel A shows a pattern that
we have observed in 7/14 of the recent non-LVS vaccinated NHPs we
have tested: The freeze thaw process reduces the overall response of
the PBMCs and responsiveness to FF LVS hi is not evident. Panel B
shows a pattern that we observed in the other 7/14 of the recent nonLVS vaccinated NHPs we have tested: responses were low both before
and after the freeze/thaw process.
Data Interpretation: It appears as though the freeze/thaw process can
reduce the background responsiveness, and sometimes the non-specific
responsiveness of PBMCs if it is initially apparent in the fresh
Page 19 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
preparation. There also appears to be no difference between the two
freeze/thaw protocols. It also appears that if high non-specific
responsiveness is not apparent, the freeze/thaw process does not
decrease that responsiveness any further.
Data storage:
Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC
assay 11082008.svd; TVDC (3) bound notebook (9225), pp. 58 – 63; 68
– 92; TVDC (4) notebook (9235) pp. 1-2; 51 – 55; 78 -81.
a. We have observed that some non-LVS vaccinated NHPs respond to
formalin fixed and heat-killed LVS and SCHU S4 antigens in the IFNγ
ELISPOT assay; we obtained mutant LVS and SCHU S4 organisms from
Anders Sjostedt’s lab that are lacking the 0-antigen (LPS) and tested hk
and ff preparations of these mutants; Figure 5 shows the results
350
A
Media
LVS hk Hi
300
LVS ff Hi
250
SCHUS4 hk Hi
200
SCHUS4 ff Hi
LVS hk Mutant Hi
150
LVS ff Mutant Hi
100
50
0
28656
A04169
SCHUS4 hk Mutant Hi
A06199
B
300
Media
LVS hk Hi
LVS ff Hi
250
SCHUS4 hk Hi
200
SCHUS4 ff Hi
150
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
350
100
TNTC
IFNg Spots (Mean +/- SEM)
400
SCHUS4 ff Mutant Hi
TNTC
IFNg Spots (Mean +/- SEM)
400
50
0
28461
A05403
Figure 5: Blood was collected from NHPs vaccinated with LVS organisms either by
scarification (n=3, panel A) or by subcutaneous inoculation (SC, n = 2, panel B). PBMCs
were purified from blood collected on Day 15 post-vaccination. All PBMCs were plated at
Page 20 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
1.33 x 106 cells/ml and incubated with the indicated stimuli (1 x 105/ml) for 20 hours. LVS
hk hi and ff hi stimulation of PBMCs from A04169 was conducted but too many spots
developed in the well (TNTC) making the count inaccurate. Patterns of responsiveness
were similar when analyzing PBMCs from day 7 post-LVS inoculation (data not shown).
Data Interpretation: Several points are suggested by this pattern of responsiveness, 1)
the response of PBMCs to LVS hk hi is abrogated by loss of the 0 antigen (no response
to LVS hk mutant hi); 2) the response to LVS ff hi is only partially reduced by loss of the
0 antigen (compare to LVS ff mutant hi); 3) LVS vaccinated NHPs make no response to
SCHU S4 hk or ff antigens UNLESS the organisms have been mutagenized to remove
their O antigen (with the exception of 28656 which does not respond to SCHU S4
antigens in any form).
Data storage: Raw Data \\Saturn\Group\Wilder Lab\TVDC\PBMC assay statview\PBMC
assay 11072008.svd; TVDC (5) bound notebook (9247), pp. 32 -39.
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% of the scientific work is complete.
9. Work plan for upcoming month
a. Process PBMCs from the LVS-vaccinated NHPs from Milestone 8 blood draws
Day 28 and 35.
b. Continue to test mutant LVS and SCHU S4 antigens (O antigen negative strains)
in the proliferation and IFNγ ELISPOT assays with both LVS-vaccinated and nonvaccinated NHPs..
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None anticipated
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
Received convalescent PBMC and sera from tularemia patients on Martha’s Vineyard on
9/30/2008.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
Page 21 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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
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 Ptran10 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Ptran\Ptran10)
i. The purpose of this experiment was to confirm that 0.25 ml of
immune rat serum is sufficient to protect naïve rats against an i.t.
challenge of ~200 SCHU S4. This is a repeat of experiment Ptran 9.
ii. Naïve Fischer 344 rats were passively immunized i.p. with 0.25 ml of
heat inactivated normal rat serum, immune rat serum or PBS (naïve)
and challenged i.t. 1 day later with 217 SCHU S4 (actual lung
deposition).
iii. Fig. 3 shows that 0.25 ml of immune rat serum but not normal serum
or PBS very effectively protected naïve rats against SCHU
challenge. This reproduced the results from Ptran9.
Page 22 of 62
Tularemia Vaccine Development Contract: Technical Report
Percent survival
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
100
80
60
NAIVE
NRS
IRS
40
20
0
0
10
20
30
Days P.I.
Fig. 3. Passive immunization of Fischer 344 rats. Fischer 344 rats
(n = 6) were treated with 0.25 ml normal rat serum (NRS), immune
rat serum (IRS) or PBS (Naïve) and challenged i.t. 1 day with 217
SCHU S4. Survival was monitored daily.
b. Experiment Ptran11 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Gopi's experiments\Ptran\ptran-11)
i. The purpose of this experiment was to determine whether passively
immunized rats that survived an i.t. SCHU S4 challenge would
develop resistance to subsequent challenges with larger SCHU S4
doses.
ii. It is possible that immune rat serum provides only temporary and
limited protection against SCHU S4 challenge but does not lead to
long lasting, specific, cell-mediated immunity against F. tularensis.
This may occur if immune serum caused SCHU S4 to be cleared
from the infected rats before cell-mediated immunity could develop.
iii. To address this possibility, passively immunized rats that had
survived a primary i.t. challenge with 200 SCHU S4 were challenged
again with 2 x 104 SCHU S4. The interval between the primary and
the secondary challenge was 1 month; previous studies had
indicated that this may not be sufficient time for the rats to clear the
primary challenge. With this caveat in mind, Fig. 4 shows that
passive immunization enabled rats not only to survive a primary
SCHU S4 challenge but also develop specific immunity against
subsequent SCHU S4 exposure.
iv. In future iterations of this experiment, we will have to treat infected
rats with antibiotics after the primary infection to completely eliminate
SCHU S4 before the secondary challenge.
Page 23 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Percent survival
100
80
60
Naive
LVS
LVS/SCHU S4
IRS/SCHU S4
40
20
0
0
10
20
30
40
Days P.I.
Fig. 5. Passively immunized rats develop enhanced immunity after
recovering from a primary i.t. SCHU S4 infection. Rats that had been
vaccinated with LVS or with immune rat serum and then recovered from a
primary i.t. SCHU S4 challenge (LVS/SCHU S6 and IRS/SCHU S4,
respectively; n = 6) were challenged again with 2 x 104 SCHU S4 1 month
after the primary challenge. The survival curves of naïve rats and LVS
immune (LVS) rats that had not received that primary SCHU S4 challenge
were included as controls.
c.
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
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. 6). 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.
Page 24 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Lungs
Liver
Spleen
10
10
8
8
Naive
LVS vaccinated
NRS
IRS
5
3
CFU (log)
7
CFU (log)
CFU (log)
9
6
4
2
1
5
10
15
4
2
0
0
6
0
0
Days post infection
5
10
15
Days post infection
0
5
10
15
Days post infection
Fig. 6. 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
Very Good
8. Percentage completed
15%
9. Work plan for upcoming month
a. Titer the amount of Ft-specific antibodies in the immune sera
b. Continue to reduce the volume of IRS used for passive immunization to titer
out the protection.
c. Repeat SCHU S4 growth kinetics experiment in actively and passively
immunized rats
d. Confirm that passively immunized rats that recovered from a primary SCHU
S4 challenge develop resistance to subsequent SCHU S4 challenge
e. Perform histological analysis on the tissues from passively immunized rats
challenged with SCHU S4
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 25 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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
4. Significant decisions made or pending
NA
5. Problems or concerns and strategies to address
NA
6. Deliverables completed
NA
7. Quality of performance
Good
8. Percentage completed
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
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
NA
6. Deliverables completed
NA
Page 26 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
7. Quality of performance
Good
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-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 AHft1 (L:\Lyonslab\Tularemia\Tularemia Contract
Folder\Experiments and Results\Andrew's experiments\AHft1)
i. The purpose of this experiment was to determine whether human
monocytes can be infected with SCHU S4 and used as the target cell
instead of macrophages in the killing assay.
ii. We are attempting this approach because the 6-day culture with MCSF required to generate macrophages from monocytes or bone
marrow may be impractical in the clinical setting. We could eliminate
this 6-day culture by working directly with monocytes, which have
been shown to internalize F. tularensis novicida and produce IL-1.
However, we do not know whether infected monocytes have the
effector mechanism capable of controlling intracellular growth upon
activation by immune T cells.
iii. Monocytes and lymphocytes were isolated from human buffy coat
(unvaccinated) and infected with SCHU S4 at MOI = 0.1. After 72 h,
SCHU S4 was detected in the cultures containing monocytes with
and without lymphocytes but not in the lymphocyte cultures (Fig 2).
The actual bacterial burden could not be determined because the
samples were diluted too far for plating. Nevertheless, these results
demonstrated that monocytes can be infected and support the
growth of SCHU S4 in our hands.
iv. This experiment will be repeated to gain consistency before we
determine whether they can be activated to control intracellular
bacterial growth.
Page 27 of 62
Tularemia Vaccine Development Contract: Technical Report
total CFU per well
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
3.010 7
Monocyte-enriched
Lymphocyte-enriched
Monos + Lymphs
Ft + media only (approximate)
2.010 7
1.010 7
0
0
24
48
72
hours post-infection
Fig. 2. Infection of human monocytes with SCHU S4. Monocytes ± lymphocytes
were infected with SCHU S4 at MOI = 0.1. The intracellular bacterial burden was
determined on the indicated days.
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
48 %
9. Work plan for upcoming month
a. Optimize the rat macrophage SCHU S4 killing assay with naïve and
vaccinated splenocytes. We will first titrate the MOI for infecting rat
macrophages with SCHU S4
b. Repeat SCHU S4 infection of human monocytes to gain consistency and
reproducibility
c. 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
Page 28 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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 done on this Milestone 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
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 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 (active)
 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
Page 29 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
3. Work performed and progress including data and preliminary conclusions
B.
Build ORF expression library corresponding to proteome
a. 2,065 linear expression elements (LEE) were successfully assembled and
are now ready for IVT production of FTU polypeptides
Figure 1: 384-well FTU LEE amplification. Lanes 13 and 26 are molecular weight markers. These
are samples of 25x 96-well plates of amplification reactions run in order to generate the 2065
ORFs. These were next used to assemble LEE templates.
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU proteomic library\E-gel\Long ORF 2
b. These templates were quality controlled by gel electrophoresis, quantitated
by radioactive label, dried, and finally stored at -20oC.
c. Initially 128 of the 2065 ORFs were not sufficiently amplified. We suspected
that this was caused by FTU primer degradation during storage of the diluted
primers
d. We selected12 ORFs from this subset and reordered respective primers.
These were tested in amplification reactions.
e. The results in figure 2 confirmed that the failure of ORF amplification was
due to degraded primers and the problem was resolved by amplifying with
newly purchased primers.
Page 30 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Kb
1.5
1
2 3
4 5
6 7
8 9 10 11 12
1.0
0.5
Figure 2: LEE re-amplification of 12 FTU ORFs using new primers
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT DNA gels\FTU Wild
Type PCR 10-24-08 crop

Amplification of these 128 ORFS using fresh primers is in progress, and the
complete LEE assembling of all FTU ORFs from SchuS4 will be ready for the
construction of the polypeptide library by next week
C.
Generate polypeptide library

The 25 plates of 2,065 fully assembled LEE expression templates have
been transferred to the appropriate 96-well plates and readied for
performing the FTU IVT reactions.
We also arrayed 8 LEE template plates to be used for QC of the IVT
reactions. The design of the QC plates was based on the arrangements
of the primers plates and ORF amplification plates, as follows The PCR
primers were initially arrayed in 8x 384-well plates, and then re-arrayed
into 25x 96-well plates for PCR. A random sampling of 4x 12 ORFs
corresponding to 1 row of each PCR plate and the 4 plates
corresponding to each primer plate was duplicated in a QC plate. Only 4
rows are filled.
The first well of the 5th row will contain a green fluorescence protein
(GFP) template as a positive control.
35S-methionine will be included only in the QC plate IVT reaction mix
We tested a large batch ,,of the NEB IVT pure system, sufficient to
translate the 2065 ORFs, to determine the quality of the lysate batch
(without purification). See figure 3 for results showing that 12
polypeptides were successfully synthesized.




Page 31 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
100
75
50
37
25
Figure 3: NEB IVT of row A of FTU LEE amplification PCR plate 2
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F
tularensis proteomic library\NEB IVT kit testing 10-8-02 crop 2

The new large batches of beads and mAb were tested in the IVT
polypeptide purification protocol for these 12 successful IVTs. Twelve
IVT templates from FTU LEE amplification of FTU Long ORF 2 PCR
plate 2 were chosen for the test
Figure 4: Testing anti-thioredoxin antibody and beads from the bulk order
Odd number: IVT proteins eluted from beads (captured sample)
Even number: 1/5 IVT reaction supernatant from beads (uncaptured sample)
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S gels\F
tularensis proteomic library\Testing large batch of beads and Ab 10-23-08 crop


Figure 4 showed that most IVT proteins were captured efficiently via antithioredoxin tags leaving very little amount of proteins in the supernatant
The storage ability of bead-bound protein was also tested:
Page 32 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Ova
FTU 721A
Kda
37
Beads
Sup.
Beads
Sup.
25
15
10
1
2
3 1 2 3
1
2
3
Figure 5: Testing storability of bead-bound proteins at different temperatures. 1:
Immediately after IVT reaction was completed; 2: Storage at 4oC after 5 days; 3 :
Storage at -80oC after 5 days. Arrow points at the correct size protein
R:\GeneVac\FTU\Contract\Proteome\FTU IVT Data\FTU gels\FTU HTP IVT 35S
gels\Testing bead storage temperature 2




D.

We are currently constructing the polypeptide library of F. tularensis
Four 96-well and one QC 48-well IVT will be run at each time
After the reaction is completed, 96-well IVT plates will be split into two wells
and then stored at -20oC .
The QC control plate (48-well) is washed with PBS and resuspend with SDS
loading buffer for TCA assay and gel electrophoresis This control plate
assesses whether sufficient mass and appropriately sized products are
synthesized
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.
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
75%
9. Work plan for upcoming month
Complete the last of the LEE assembling of 116 ORFs
Generate the polypeptide library from all 2065 F. tularensis ORFs
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 33 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Milestone 27-UNM
Milestone description: Optimization of T cell assays and endpoints in mice. UNM will
use ASU’s protein fragments in lymph node proliferation assays to define vaccine
candidates
Institution: UNM
1. Date started: 12/15/06
2. Date completed: Pending
3. Work performed and progress including data and preliminary
conclusions
a. Ftc77 (No data entry has been made at UNM yet because the work was
vaccination was performed at LBERI)
i. The purpose of this experiment will be to screen the library of 2065
linear expression element constructs of F. tularensis for potential
vaccine candidates.
ii. No new work was performed at UNM
iii. Two NHP have been vaccinated with LVS at LBERI and are waiting
to be boosted, prior to harvesting LN and spleens for screening the
ASU polypeptide library
4. Significant decisions made or pending
None
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
a.
Hold teleconference with ASU to discuss the complexity of the peptide pools
to be tested
10. Anticipated travel
NA
11. Upcoming Contract Authorization (COA) for subcontractors
NA
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
a. Ftc64 study 5 (L:\Lyonslab\Tularemia\Tularemia Contract Folder\Experiments
and Results\Ftc experiments\Ftc64\Ftc64 study 5)
Page 34 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
i. The purpose of this experiment was isolate RNA from SCHU S4
infected rat lungs 1, 3, 5, 7, and 24 hours after i.t. challenge.
ii. Table 1 shows the bacterial burden in the lungs at the time when the
lungs were collected
iii. The RNAs have been isolated but their quality could not be
determined because our Agilent analyzer was not working. ASU has
indicated that they will accept these samples from UNM without this
information
Table 1. Lung bacterial burden at early after infection
Time post infection (h)
Bacteria load (CFU/lung)
1
1.21 x 105
3
2.01 x 105
5
2.86 x 105
7
1.01 x 106
24
5.24 x 107
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 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 have previously reported on the first round of LAPT
amplifications from the first time course challenge experiment. We identified genes
by varying expression pattern changes over time. We have also been optimizing
Page 35 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
parameters for the qPCR verification of microarray selected genes using
reconstitution samples of SCHU S4 RNA diluted into normal mouse lung RNA

The results presented in Figure 1 shows qPCR relative quantification of iglC gene
expression relative to MutS. The MutS gene was used as the normalization control
to calculate the relative gene expression values. The samples under analysis were
from the initial dose response experiment where mice were challenged with varying
doses of SCHU S4 bacteria and the lungs taken at 4 hours post infection for LAPT.
The figure below represents the amplification plot of the Sybr green incorporation
during PCR analyses. The samples in orange hues represent the iglC gene traces
and those in the green hues are MutS and each grouping represents the sample from
the highest to lowest bacterial challenge. The major findings are that the iglC traces
were reasonable for all of the samples and detectable cycle threshold calculations
could be made for each of the doses. This was not observed with MutS. The curves
of the MutS amplifications were biphasic indicating poor specificity of amplification.
Importantly, only the samples from mice challenged with 104 organisms or more had
reliably detectable signals.
Figure 1. Amplification plot of the qPCR reactions from dose response experiments for the IglC
gene (orange curves) and for the Mut S (green curves) gene (control) Bacterial Doses ranged
from 1 x 107 to 1 x 101
Notebook/File locations …, ASU: Notebook 804, Relative Quantification, page 134.
 Melting curve analysis of the qPCR reactions confirmed that the amplifications of
MutS (Green curves) had a major shift in melting temperature. This is a further
indication of a lack of high level specificity in the RNA amplification in the low-dose
challenge experiments.
Page 36 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 2. Melting curve analysis of qPCR results for the IglC (orange) and Mut S (green)
products.
Notebook/File locations …, ASU: Notebook 804, Relative Quantification, page 135.

Using the data from the interpretable curves, we were able to establish relative
quantification changes of iglC between several of the dose-response challenge
doses in the two experiments to date. The key data column is the expression level
(right column). We see that the expression level increased by a factor of 2-2.5 fold
increase as the bacterial dose increased by 1 log. Additional analyses are need to
validate these expression level changes as a function of bacterial load.
Figure 3. Relative quantification values for iglC to MutS for the two dose response experiments
(1712c is the IglC gene)
Page 37 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Notebook/File locations …, ASU: Notebook 804, Relative Quantification, page 136 and
137.
4. Significant decisions made or pending
We need to re-evaluate the genes used for relative quantification as the Mut S gene
transcripts did not amplify consistently from RNA preparations generated from mouse
lungs exposed to the full range of SCHU S4 doses. Bioinformatic searched for additional
genes is warranted. We will redesign and test new primers for the 16S ribosomal genes.
5. Problems or concerns and strategies to address
Several LAPT attempts have failed during the last month. We have performed extensive
QC analyses on all the components of the amplification kits. All of the enzymes
performed with test control samples. We have concluded that the problem resides in the
template switch primer. We have re-ordered the primer and will re-establish the LAPT.
6. Deliverables completed
None
7. Quality of performance
Good
8. Percentage completed
62.5%
9. Work plan for upcoming month


Procure new template switch primer and re-establish the LAPT procedure.
Acquire new primers for 16S Ribosomal gene for relative quantification and seek
another gene to test as a control.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Milestone 49
Milestone description: Construct single mutants in F. tularensis subsp. tularensis
(SCHU S4) (iglC, pdpD, iglD, iglA, iglB)
49.1: Construct iglC F. tularensis subsp. tularensis (SCHU S4)
49.2: Construct pdpD F. tularensis subsp. tularensis (SCHU S4), Construct iglD F.
tularensis subsp. tularensis (SCHU S4)
49.3: Construct iglA F. tularensis subsp. tularensis (SCHU S4), Construct iglB F.
tularensis subsp. tularensis (SCHU S4)
Institution: UTSA
1. Date started: April 1, 2006
2. Date completed: in progress
3. Work performed and progress including data and preliminary conclusions
In order to generate mutants in SCHU S4 we need to develop tools to generate successful
deletions. Therefore, our focus is two fold, one is cloning experiments to get our target
deletions into vectors that we can use in creating these deletions and experiments with
SCHU S4 itself using constructs that we believe will allow us to make deletions into SCHU
S4.
In response to discussion at the annual TVDC meeting and in discussions with Dr. Lyons,
milestone 49 was altered to replace the SCHU S4 iglA mutant with a SCHU S4 nadM mutant.
The rationale is based upon experiments performed in the Klose laboratory with a F.
Page 38 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
tularensis subsp. Novicida nadM mutant, which was attenuated for virulence in mice and
demonstrated protective efficacy against subsequent homologous challenge. The nadM
gene is involved In NAD biosynthesis and is thus considered an important type of attenuating
mutation, since this is predicted to affect growth in the host but not necessarily any specific
step in virulence, thus allowing for a more robust immune
response to the attenuated organism.
I.
Cloning:
a. UTSA began a strategy to create a metabolic gene mutant, Nad+M. Based on gene
analysis of Sigma’s algorithm software scores were assigned to various oligo sets to use
in creating a tulatron plasmid construct with Nad+M as the target gene. We decided to
try the gene location for intron insertion at 602/603. The oligos were ordered and
designated names and sequences are as follows:
i. NadM 602/603s IBS:
5’-AAAAAAGCTTATAATTATCCTTATCCATCTAAGCCGTGCGCCCAGATAGGGTG-3’
ii. NadM 602/603s EBS1d:
5’-CAGATTGTACAAATGTGGTGATAACAGATAAGTCTAAGCCAATAACTTACCTTTCTTTGT-3’
iii. NadM 602/603s EBS2:
5’-TGAACGCAAGTTTCTAATTTCGGTTATGGATCGATAGAGGAAAGTGTCT-3’
These oligos were ordered to use with the tulatron vector KEK1140, already on hand, to
create the Nad+M construct which will be used with SchuS4 once the Nad+M construct is
completed. Once again, as in other “tulatron” constructs, we will use the restriction
endonuclease sites of Bgl II and Xho I to clone in the resulting Nad+M gene PCR product
that will be generated when using the oligo set described above. Data located in UTSA
TVD Notebook 7, page 44.
II.
Experiments to generate mutants in Schu4:
a. From the previous report, VgrG mutant clones V17T4E, V17T4F and V17Tef.1 were
used as templates with oligos specific to the VgrG gene (FTT1346 fwd NdeI and VgrG
rev EcoRI) generating a 1600 bp product seen on September’s report figures 4 and 5,
respectively. These products were subsequently purified from the 1% agarose gel and
sent for sequencing using the oligos which generated these products, in addition to the
EBS universal primer, to confirm that this mutant is correct. The resulting sequencing
showed that these mutants were correct based on gene alignments with sequence data
and verification of intron insertion at correct gene position. Data located in UTSA TVD
Notebook 7, page 35.
b. Therefore, the VgrG mutant was assigned KKT13 number for identification and the
V17Tef.1 clone was used to freeze for permanent frozen stock of the VgrG mutant of
SCHU S4. In addition, this clone was grown up in liquid culture and used in a mouse
experiment to determine whether this mutation affects the virulence of SCHU S4. The
intranasal inputs (colony forming units {CFUs}) where discussed in October’s TVD
meeting in Arizona. In summary, 30 mice were used with 5 mice per group; the highest
CFU input was 1.988E6 and all VgrG groups survived after 38 days post infection. The
wild type group KKT1, positive control, (76 CFUs) died at day five. The VgrG mutant of
SCHU S4 is attenuated in Balb/c mice (see graph below).
Page 39 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
VgrG Intranasal Inoculation in Balb/c Mice
120
Percentage Survival of Mice
100
PBS
VgrG
1.988E6
VgrG
1.988E5
VgrG
1.988E4
VgrG
1.988E3
WT (KKT1CFU 76)
80
60
40
20
0
D1
D2
D3
D4
D5
D6
D7
D8
D10
D11
D30
Day after Inoculation
c. A challenge experiment was performed on the VgrG intranasally inoculated mice
along with the PBS group. The SchuS4 wild type strain, KKT1, was used for the
challenge experiment which was done intranasally at a dose of 81 CFUs, empirically
calculated. Lung depositions were not measured. There were no survivors after day 5;
that is, every group in the experiment did not survive the wild type challenge (Table 1).
Table 1: Challenge with wild type SchuS4 of Mice inoculated with VgrG mutant (KKT13)
Group of Mice
PBS
VgrG 1E6
VgrG 1E5
VgrG 1E4
VgrG 1E3
Inoculation
Route
I.n.
I.n.
I.n.
I.n.
I.n.
Dose
(CFU)
81
81
81
81
81
D1
5/5
5/5
5/5
5/5
5/5
D2
5/5
5/5
5/5
5/5
5/5
Survival Rate
D3
5/5
5/5
5/5
5/5
5/5
D4
5/5
5/5
5/5
5/5
5/5
D5
0/5
0/5
0/5
0/5
0/5
Table 1 is the results of the wild type challenge on mice inoculated first with the
attenuated VgrG mutant of SCHU S4 (KKT13). There was no difference between the
naïve mice (PBS) and the VgrG mice groups from time to death after challenge;
indicating that this VgrG mutant was not protective to a 81 CFU SCHU S4 dose
challenge, intranasally. Data located in UTSA TVD Notebook 7, page 48.
d. In addition, we isolated genomic DNA from the KKT13, vgrG mutant using the Easy
DNA kit from Invitrogen following the protocol with some modifications, to include two
phenol chloroform extractions following the initial chloroform extraction after the lysis
step. The final preparation was quantitated by O.D 260 using 50 ug/ml per 1 O.D. 260 as
my standard in calculating this genomic isolation. This experiment is just to further
Page 40 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
confirm that the VgrG intron insertion is at the correct location; although previous tulatron
mutants have not had a discrepancy between the sequence verification of the “gene
intron” and the results seen from a Southern blot. Our goal is to use 12 ug total genomic
DNA in our restriction endonuclease digestions; which will then be electrophoresed on a
0.85 % agarose gel and transferred to nitrocellulose paper for subsequent labeling with
the “intron sequence” as probe, to locate the gene insertions in each candidate that will
be run on this blot. Set up reactions with EcoRI, EcoRV and Hind III, respectively, these
are generally very good cutting enzymes and based on sequence analysis we will be able
to differentiate the “intron” band which will result from the Blac2 insertion and the other
band which will be a result of the desired gene insertion (for example, the VgrG gene).
We will use the KKT1 (wild type, Blac2 insertion) and KKT10 (IgLC mutant) as controls
for this experiment. I will screen a small amount (1.5 ul/35 ul reaction) of these
digestions to check for complete cutting before actually running the large gel which will be
used in the Southern blot experiment.
UTSA routinely screens and verifies mutants with at least two methods such as DNA
sequencing, Southern blotting/restriction mapping, protein production detected by
antibodies, vaccination and protection in animal models as examples, to prove the mutant
gene has been created.
Data located in UTSA TVD Notebook 7, page 39, 41.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
This milestone involves many mutants: the following have been completed; IgLC, igLD
and VgrG
7. Quality of performance
Good
8. Percentage completed
79%
9. Work plan for upcoming month
a. Need to run the Southern blot to verify expected VgrG intron location on the genome.
(Although sequence analysis showed the VgrG mutant is correct.)
b. The Biosafety level 3 laboratory will be closed for re-certifications and repairs of
various equipment in this lab. The prediction for the lab closure is from November 10
to November 21, 2008 given there are no unforeseen complications during repair and
re-certifications of equipment. Therefore, Dr. Klose’s team will be part of a rotation to
escort repair personnel into the lab during this shut down period.
c. Will start the cloning of the Nad+M gene into the KEK1140, “tulatron” vector
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 41 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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) Evaluate the protective efficacy of KKT10 (ΔiglD of SCHU S4) vaccination
against intradermal SCHU S4 challenge. (Note book #9, page 8-10). Mice were given
intradermally (BALB/c) or intragastrically (C57BL/6) a single dose of KKT10 (10 3
CFU) and challenged i.d. with either 20 or 100 CFU of SCHU S4 three weeks after
the immunization. Control mice were mock vaccinated with PBS. Vaccination with
KKT10 (ΔiglD of SCHU S4) either by the i.d. or i.g. route did not protect mice against
i.d. SCHU S4 challenge as shown in Fig.1 and 2. The evaluation of KKT10 i.g.
vaccination against i.n. SCHU S4 challenge is currently underway.
% Survival
100
80
60
Mock 20 CFU
Mock 100 CFU
KKT10 20 CFU
KKT10 100 CFU
40
20
0
0
1
2
3
4
5
Days after challenge
6
7
8
Fig. 1. Protective efficacy of KKT10 (ΔiglD of SCHU S4) immunization against SCHU
S4 infection. BALB/c mice (5 per group) were immunized intradermally (i.d.) with 10 3
CFU of KKT10 or PBS and i.d. challenged with lethal dose of F. tularensis SCHU S4
strain (20 or 100 CFU). Mice were monitored for survival rate.
Page 42 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
100
% Survival
80
Mock 20 CFU
Mock 100 CFU
KKT10 20 CFU
KKT10 100 CFU
60
40
20
0
0
1
2
3
4
5
Days after challenge
6
7
Fig. 2. Protective efficacy of KKT10 (ΔiglD of SCHU S4) immunization against SCHU
S4 infection. BALB/c mice (8 per group) were immunized intragastrically with 10 3 CFU
of KKT10 or PBS and intradermally challenged with lethal dose of F. tularensis SCHU
S4 strain (20 or 100 CFU). Mice were monitored for survival rate.
50A-b: Measure humoral responses after KKT10 (ΔiglD of SCHU S4) oral
immunization and evaluation of protective efficacy of KKT10 oral immunization
against SCHU S4 intranasal challenge. Mice have been vaccinated orally with KKT10
and are resting. We will collect sera and fecal pellets from these mice to assess
antigen-specific antibody production. We will also challenge these mice intranasally
with SCHU S4 and monitor the survival rate.
50B-a: Analyze the cellular responses to mice vaccinated orally with F. holarctica
LVS at 8 and 12 weeks after vaccination. (Note book #8, pages 15, 49-50, 66-67,
and 135-37) Mice were vaccinated I.G. with 103 CFU of LVS or mock vaccinated with
PBS alone. At either 8 or 12 weeks after immunization, spleens were collected, single
cells were made and incubated in the presence of increasing amounts of UVinactivated LVS (103-105 CFU) for 72 hours. Splenocytes were also cultured in the
presence of the unrelated antigen HEL or media alone as controls. At the end of the
culture period, supernatants were collected and analyzed for IFN-production using
the BD OptEIA Mouse IFN- ELISA Set (BD Biosciences) per manufacturer’s
instructions. As seen in figure 3A, cells from mice collected at 8 weeks after LVS
vaccination produced significant amounts of IFN-when cultured with higher doses of
LVS when compared to mock vaccinated mice or to cells cultured with HEL. As seen
in Figure 3B, IFN- was also produced by cells collected at 12 weeks similarly to the
8 week timepoint. Collectively the levels of antigen-specific IFN-wane from week 2
till week 12.
Page 43 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Mo ck (PBS) I.G.
LVS I.G.
B 4
a
ed
i
EL
10 4
M
S
LV
S
a
ed
i
M
H
S
10 5
10 4
LV
S
LV
S
LV
H
<31.250
10 5
<31.250
S
1
LV
1
10 3
2
EL
2
10 3
3
IFN-  (ng/mL )
3
12 Weeks
LV
8 Weeks
A 4
Fig.3. Cellular responses to LVS IG vaccination. Groups of mice (3 mice/group) were inoculated
IG with 103 CFU OF LVS. At either 8 weeks (A) 12 weeks (B) after vaccination, spleens were
collected, single cells were prepared and incubated in the presence of LVS, and supernatants
were analyzed for IFN- production.
50B-b: Survival after LVS I.G. vaccination and CD8 + T cell depletion/F. t. subsp.
tularensis SCHU S4 challenge. (Notebook #8, pages 133-34, 139-41, 142-47)
Groups of Balb/c mice (8 mice/group) were vaccinated IG with 10 3 CFU of LVS or
mock vaccinated (PBS) and rested for three weeks. One group of mice were treated
IP with 200g of neutralizing anti-CD8 antibody, prepared from the hybridoma cell
line TIB-210 (ATCC), at days -2, -1, 0 and every subsequent third day after intranasal
challenge with 140 CFU of F.t. SCHU S4. One group of mice received IP injections of
rat IgG and another group received no treatment as positive controls. Some mice,
which were given parallel IP treatments, were sacrificed at day 4 after challenge in
order to measure the level of CD8+ T-cell depletion. Single cell suspensions of
splenocytes were incubated with PE-CY7 labeled CD8 antibody and measured for
fluorescence by flow cytometry. As shown in Fig 4, splenocytes from mice which
received the rat IgG control contained 10.2% CD8+ T-cells. Splenocytes from mice
which received the anti-CD8 antibody contained only 0.2% CD8+ T-cells which was
comparable to the fluorescence of the PE-CY7 isotype control at 0.1%. These results
show that the anti-CD8 antibody treatment adequately depleted the splenic
compartments of CD8+ T-cells as desired. Mice which received the F.t. SCHU S4
challenge were then monitored daily for survival and weight loss. As shown in Fig. 5,
mice which received either rat IgG or no treatment exhibited a median level of
survival (50%). Mice which received the anti-CD8 antibody showed early symptoms
of disease and complete mortality by day 11 post challenge. All mock vaccinated
mice succumbed to infection by day 6 after challenge. These results indicate that
CD8+ T-cells play a role in clearance of infection following oral vaccination with LVS.
Page 44 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Unstained
Isotype Control
0.0%
Rat Ig Treated CD8
0.1%
Anti-CD8 Ab Treated CD8
10.2%
0.2%
Fig. 4. in vivo depletion of CD8+ T-cells. BALB/c mice were given IP injections of 200g of
either anti-CD8 antibody or rat IgG as a control at D -2, -1, 0 and 3. On day 4, single cell
suspensions of splenocytes were stained with PE-CY7 labeled CD8 antibody and
fluorescence was measured by flow cytometry.
Page 45 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
LVS Vac c
100
LVS Vac c /Rat Ig
LVS Vac c /anti-CD8 Ab
Moc k Vac c
% Survival
80
60
40
20
0
0
5
10
15
20
25
30
Day s After Challenge
Fig.5. Protective efficacy of LVS intragastric immunization followed by anti-CD8 antibody treatment and
F. tularensis SCHU S4 challenge. Groups of BALB/c mice (8 mice per group) were immunized IG with
103 CFU of LVS and rested for three weeks. Mice were then treated IP with 200g of either anti-CD8
antibody or rat IgG as a control (day -2, -1, 0 and every subsequent third day), or given no treatment. On
day 0, all mice were challenged with 140 CFU of SCHU S4 and monitored daily 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
86% of scientific work completed on milestone 50A (original plans)
95% of scientific work completed on milestone 50B (intragastric plan)
Average % completed across the milestone is 90.5%
9. Work plan for upcoming month
50A: (1) Measure humoral responses after KKT10 (iglD mutant of SCHU S4) oral
immunization and evaluate protective efficacy of KKT10 oral immunization against
intranasal SCHU S4 challenge.
(2) Measure intramacrophage growth of SCHU S4 vgrG mutant.
50B: Evaluation of protective efficacy of LVS I.G. vaccination against SCHU S4
challenge at 8 week post vaccination.
10. Anticipated Travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 46 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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
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 a 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
Since NR5330 and NR7241 were not constructed by UTSA, we need to confirm the
presence of the transposon insertion in the targeted gene of the mutant by PCR and
sequencing. The primers for PCR are:
FTN0720 Rev: 5’- CGA TGC TGT GGT GCA AGT ATC G -3’
FTN0757 For: 5’- GGC AAG TGG TGG TCG TCA TCG -3’
Kan2- 125: 5’- AAC GCA GAC CGT TCC GTG GC -3’
3.2
PCR was carried out with the primers “FTN0720 Rev” and “Kan2-125” for NR5330 and the
primers “FTN0757 For” and “Kan2-125” for NR7241 to confirm the transposon insertion in
the target gene. PCR reaction was set up as follows:
5XGreen GoTaq Buffer (Promega)
dNTP mix, 10mM each (Promega)
Kan2-125
FTN0720 Rev or FTN0757 For
GoTaq DNA polymerase (Promega)
DNA
H2O
4.0ul
0.4ul
1.0ul
1.0ul
0.1ul
1.0ul
12.5ul
At 95ºC 2min, 95ºC 30sec/53ºC 30sec/72ºC 1min//30cycles, 72ºC 5min
Page 47 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 1: Gel picture for PCR to verify the insertion in NR5330 and NR7241
Figure 1 legend and data location: Lane1 and 3 were the negative controls --wild type U112 which
had no insertion in either FTN0720 or FTN0757. Lane2 was the transposon mutant NR5330 and
lane4 was NR7241. Both mutants generated PCR products of approximately 500bp in size.
Data recorded on UTSA TVDC notebook #6, page47 for Figure above.
Kan2-125 was the transposon specific primer for both mutants. “FTN0720 Rev” was the
gene specific primer of FTN0720, whereas “FTN0757” was the FTN0757 gene specific
primer. PCR production for NR5330 (FTN0720) should be about 570bp, and NR7241
(FTN0757) should produce 550bp PCR production. The PCR amplifications both
generated the correctly sized product and proved the presence of the transposon insertion
in both mutated strains.
3.3
The PCR productions from Step3.2 were gel purified separately using QIAquick Gel
Extraction Kit and sent for sequencing with the primers FTN0720 Rev and Kan2-125 for
NR5330, FTN0757 For and Kan2-125 for NR7241. The sequencing data confirmed the
insertion was at 193bp in FTN0720 of NR5330, and 1339bp in FTN0757 of NR7241.
The sequencing data recorded on UTSA TVDC notebook #6, page49-52.
3.4
To evaluate the attenuation of the transposon mutants NR5330 and NR7241, the Balb/C
mice were vaccinated with NR5330 or NR7241 intranasally on Oct. 24 th. The dose of
inoculum was 200CFU for NR5330 group and 534CFU for NR7241 group. On the third day
after inoculation, 3 mice from NR5330 group and 2 mice from NR7241 group were
observed sick, but they recovered from sickness two to three days latter except for one
from NR5330 group. It died on day 8. The following table showed the data of this
experiment. We planned to use the dose of 1000CFU for each mutant, but the actual dose
was lower than expected, which was still acceptable. It appeared that the mutants were
attenuated at the dose used after 11 days of the inoculation, but we’ll know for sure if they
are attenuated or not in one month post vaccination, which will be reported in next monthly
technical report.
Page 48 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Table 1: Evaluation for the Attenuation of NR5330 and NR7241 in Balb/C mice
Group of
Mice
NR5330
NR7241
PBS
Dose of
Inoculum
(CFU)
200
534
Route of
Inoculation
i.n.
i.n.
i.n.
Survival Rate
D1
6/6
5/5
5/5
D2
6/6
5/5
5/5
D3
6/6
5/5
5/5
D8
5/6
5/5
5/5
D11
5/6
5/5
5/5
Data recorded on UTSA TVDC notebook #6, page53 for Table 1 above.
4. Significant decisions made or pending
None
5.
Problems or concerns and strategies to address
None
6. Deliverables completed
pKEK1186 (the tulatron vector for retarget site at 720/721bp of recA)
pKEK1187 (the tulatron vector for retarget site at 840/841bp of recA)
pKEK343 (recA mutant LVS)
KKT11 (recA mutant Schu S4)
pKEK1193 (pWSK30/lux operon without the Ft promoter)
pKEK1194 (pKEK843/lux operon with the Ft promoter)
KKF337 (LVS carrying Lux operon under the Ft promoter)
KKF341 (U112 carrying lux operon under the Ft promoter)
7. Quality of performance
Good
8. Percentage completed.
About 40% of scientific work completed.
9. Work plan for upcoming month
i. Check the survival of mice vaccinated with NR5330 and NR7241 on Oct 24 th daily until 30
days after the vaccination.
ii. Challenge the surviving mice vaccinated with NR5330 and NR7241 on Oct 24 th with
wildtype F. novicida
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 KBMA Listeria monocytogenes
(Lm) vaccines expressing 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
Page 49 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
ovalbumin epitope SIINFEKL (SL8) and these will be compared to Ft vaccines expressing pepOSL8 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.
We previously demonstrated that iglC-SL8 fusion proteins are expressed to a higher level than
katG-SL8 in the cytosol of macrophages. Live-attenuated vaccines expressing either antigen
were able to 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.
Incorporation of a constitutively active prfA allele into the chromosome of the live-attenuated LMIglC-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.
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. All epitope-tagged expression cassettes have been sequenced
verified.
Page 50 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Table 1
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
Cloning of bivalent Lm strains expressing both KatG-SL8 and IglC-B8R epitope-tagged strains
was completed previously. This month, we have compared the levels of expression and the
immunogenicity of bivalent strains with the monovalent vaccine strains expressing KatG-SL8 or
IglC-B8R. Comparisons were done in both the actAinlB and the actAinlBuvrABprfAG155S
genetic backgrounds.
1) Intracellular antigen expression analysis. DC2.4 mouse dendritic cells were infected with
each strain for 6 hours in the presence of gentamycin to prevent extracellular bacterial growth.
Infected cell monolayers were lysed with detergent and run on 4-12% gradient SDS-PAGE gels,
transferred to nitrocellulose. KatG and IglC fusion proteins were detected by Western blot using
rabbit polyclonal anti-ActA antibodies and fluorescent secondary antibodies. The fluorescence
intensity was quantified using a Li-Cor Odyssey Infrared Imaging System. As previously
described for ActAN100-IglC-SL8, ActAN100-IglC-B8R fusions were expressed to a much greater
extent than ActAN100-katG-SL8 fusions. In order to quantify the amount of each fusion protein,
the blot was also probed with a monoclonal antibody against p60 (a constitutively-expressed Lm
protein involved in cell wall remodeling) and an anti-mouse secondary antibody with a different
fluorophore. This anti-p60 signal was used to normalize to the number of bacteria in each well by
dividing the intensity of ActA signal by the intensity of the p60 signal. By ratiometric analysis, IglC
is expressed 20-200 times more than KatG (with IglC/p60 ratios ranging from 3.0-8,6 vs.
KatG/p60 ratios ranging from 0.04-0.14). Both bivalent strains produced KatG and IglC, but there
appears to be a slight decrease in the amount of IglC secreted from the bivalent strains. In the
prfA* background the difference was less than 2-fold (with IglC/p60 ratios of 5.63 vs. 7.5). In the
CRS-100 background the difference was greater (with IglC/p60 ratios of 3.01 vs. 8.55). KatG
expression was not decreased in the prfA* strain (both with KatG/p60 ratios of.04), but was
decreased by less than 2-fold (with KatG/p60 ratios of 0.14 vs. 0.08) in the CRS-100 background.
This is the first attempt at using the Li-Cor Odyssey Infrared Imaging System to quantify antigen
expression and to normalize to a constitutively expressed protein using a multiplex Western blot
approach. Thus, we will continue to characterize the p60 expression to ensure that it correlates
linearly with cfu.
Page 51 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Insert
Strain
iglc
katG
BH2292
+
+
BH2182
+
BH2172
BH2316
+
BH2180
+
BH2282
BH1029
iglC*
p60*
iglC/p60 katG/p60
0.26
33.68
5.98
5.63
0.04
0
30.06
4.01
7.50
0.00
+
0.19
0
4.76
0.00
0.04
+
0.31
11.02
3.66
3.01
0.08
0.05
30.87
3.61
8.55
0.01
0.41
-0.03
3.01
-0.01
0.14
+
NS5b
katG*
52.97
3.12
16.98
Figure 1. Multiplex Western blot for ratiometric analysis of intracellular KatG and IglC expression from
ANZ207
Meso
0.36
3.13
0.12
bivalent and monovalent Lm strains. Cell lysates from LM-infected DC2.4 cells were separated on 4-12%
SDS-PAGE gels and blotted with rabbit anti-ActA and mouse anti-P60. IR-Fluorescent secondary
antibodies were detected using an Odyssey Infrared Imaging System. The IglC and KatG antigens are
pseudo colored red, and p60 is pseudo colored green. The fluorescence intensities were quantified,
background was subtracted (from same area of gel but in a lane that did not have expected band),
expression was normalized within each sample to p60 (a constitutively expressed Lm housekeeping gene).
Data in red are from strains with wild-type prfA and data in blue are strains with prfAG155S constitutively
active allele. Data are located in NB2006-053.
2) Immunogenicity of live-attenuated Lm strains expressing epitope-tagged KatG and IglC.
C57BL/6 mice were vaccinated IV with 1e6 cfu of strains in the Lm677 (prfA* ) background or 5e6
wild-type prfA background (CRS-100; aka prfAwt). In C57BL/6 mice, 5e6 cfu of prfA* strains
appears to be less well tolerated (data not shown). 1 week after vaccination, splenocytes were
harvested and incubated with peptides corresponding to the epitopes B8R, SL8, or LLO190-201 as
a positive control in the presence of brefeldin A. ICS analysis was used to quantify the T- cell
responses. All strains expressing IglC-B8R were able to stimulate a response against the B8R
epitope (Figure 2, left panel). Similarly, all strains expressing KatG-SL8 were able to stimulate a
response against the SL8 epitope (Figure 2, middle panel). The monovalent strains expressing
either IglC or KatG in both the actAinlB (CRS-100; aka prfAwt). and the
actAinlBuvrABprfAG155S (prfA*) backgrounds elicited a significantly stronger response when
compared with coinjection of ½ the dose of both monovalent strains (equivalent total dose of Lm).
The bivalent vaccines induced comparable B8R- and SL8-specific responses as the monovalent
strains, except for the bivalent strain on the actAinlB background (BH2184) which induced
lower B8R-specific responses than BH2180. Overall, differences seen between strains appeared
to be greater in the actAinlB background(prfAwt), than in the prfA* background.
Page 52 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
*
*
***
**
*
Figure 2. ICS analysis 7 days after a single IV vaccination with epitope-tagged monovalent and bivalent liveattenuated Lm strains. Lm strain genotypes are listed in Table 1, but are in the following order for each
background: KatG-SL8 monovalent, IglC-B8R monovalent, katG and iglC bivalent, ½ dose of both
monovalent strains. Blue circles represent mice immunized with expression cassettes in
actAinlBuvrABprfAG155S background (aka prfA*) and red circles are mice immunized with actAinlB
background (aka prfAwt). strains. Unpaired T tests were used to determine statistical significance *
p<.05, **p<.005, ***p<.005. IM08-086, Notebook #1002; pp 174, 178-181.
3) Evaluation of primary T cells responses after vaccination with KBMA Lm vaccine.
C57BL/6 Mice were immunized IV with 5e6 cfu live attenuated LM-strains BH1222 (actAinlBIglC-SL8) and BH1228 (actAinlBuvrAB-IglC-SL8) or 1e8 particles of KBMA BH1228
(actAinlBuvrAB-IglC-SL8)). 1 week after vaccination, splenocytes were harvested and
analyzed by ICS (figure 3). After vaccination with either live strain, 18% of the CD8+ T cells
induced IFN in response to the SL8 peptide. After a single vaccination with KBMA BH1228, the
SL8-specific response was 2.8%, an 85% reduction in potency compared to live BH1228. A 70%
reduction in CD4+ T cell responses to LLO190-201 was also seen. The reduction in potency of
KBMA compared to live Lm strain is consistent with our previous work with other antigens;
however, potency of KBMA can often be improved with a boost vaccination.
Figure 3. ICS analysis of primary T cell response induced by live and KBMA Lm. 1 week after vaccination
with 5e6 live or 1e8 KBMA Lm vaccines, splenocytes were harvested and pulsed with SL8 peptide or
LLO190 peptide for ICS analysis by flow cytometry. IM08-087 Notebook #2000, pp 28-30, 34.
Page 53 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
4) Analysis of Tcell responses after homologus and heterologus prime-boost. We
previously reported that LVS-PepO-SL8 did not induce measurable T cell responses against IglC
peptide pools or against the SL8 peptide after a single ID vaccination with 1x10 4. We then
attempted to determine whether LVS-PepO-SL8 could prime an immune response that could be
boosted by a second administration of LVS or Lm-IglC-SL8 or if LVS could boost a response
primed by LVS or Lm (Figure 4). C57BL6 mice were administered 1x106 BH1222
(LmactAinlBuvrAB-IglC-SL8) IV or 1x104 LVS pepO-SL8 ID, Animals received a boost
vaccination 3 weeks after the prime, and 6 days after boost vaccination spleens were harvested
and analyzed by ELISpot (figure 4). LVS prime followed by LVS boost did not induce T cells
against the encoded SL8 antigen, nor did it induce a response against the endogenous IglC
antigen. Lm-prime followed by Lm-boost elicited high numbers of T cells responsive to IglC
peptides or SL8. Lm prime followed by LVS boost responses were low. This is consistent with
LVS being unable to stimulate a secondary response and reflects the contraction of Lm primary
responses in the 4 weeks after the primary vaccination. In the animals that received LVS as a
prime and LM as a boost the responses were similar to those reported with an LM primary
response alone. Together, these data suggest the LVS is a poor stimulator of T-cell immunity
against these antigens. While it is possible that LVS-Lm combination may provide protective
immunity the combination does not appear to provide a synergistic T-cell response (as we have
seen with other vaccine modalities e.g. vaccinia virus). This type of analysis will need to be
repeated with higher doses of LVS (equivalent to Lm) in order to determine whether this lack of
an immune response was due to insufficient numbers of LVS cfu.
Figure 4. ELISpot analysis of splenic T-cell responses 6 days after boost vaccination. Animals were primed
or boosted with 1x106 BH1222 (LmactAinlBuvrAB-IglC-SL8) IV or 1x104 LVS pepO-SL8 ID as indicated.
IM08-090 Notebook #2000, pp 22-27, 29-34.
5) Quality control of 400mL-scale LVS lot 16 vaccine lot. A new lot of DVC lot 16 LVS was
amplified according to the TVDC SOP (NB# 2001-012); this lot number is LVS lot 2002-022.
Initial titers were performed using in-house prepared CHAH plates but yielded cfu counts that
were unexpectedly low (0.1-1E9 cfu/mL). Based on conversations with Trevor Brasel from
LBERI, we ordered chocolate agar plates from Hardy Diagnostics and found that the titer was
much higher and the colonies appeared more rapidly and with more consistent size. The final
titer of this lot is 3.34E9 cfu/mL and is consistent with expected cfu titer based on OD 600
measurements. Chocolate agar will be used for all future cfu analysis of LVS.
Page 54 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
4. Significant decisions made or pending


Because the vaccinia virus quadrotope tag significantly decreased the
immunogenicity of the iglC vaccine, this tag will not be used for further
immunogenicity studies.
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
45%
9. Work plan for upcoming month


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
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-KatG-SL8 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).
Previously, 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 asays 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
Page 55 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
responses were identified in Balb/c, C3H/HeJ, and FVB/NJ mice. We mapped CD8+ T cell
epitopes using 9 mers overlapping by one amino acid, identifying IglC 34-142 (LFIDSLTIA) in Balb/c
mice and IglC137-144 (IMIDLSNL) in C57BL/6.
1) Comparison of IglC responses after vaccination with Live and KBMA Lm vaccines. As
described in MS55 section 3-3, C57BL/6 Mice were immunized IV with 5e6 cfu live attenuated
Lm-strains BH1222 (actAinlB-IglC-SL8) and BH1228 (actAinlBuvrAB-IglC-SL8) or 1e8
particles KBMA BH1228. 1 week after vaccination, splenocytes were harvested and analyzed by
ELISpot analysis (figure 5) for responses to the IglC peptide pool #2, or individual peptide 33-19
(IglC137-144, IMIDLSNL). IglC-specific responses induced by KBMA BH1228 were significantly
lower compared to the responses induced by the live strain. As we have seen with other vaccine
strains, use of a prfA* KBMA strain will likely increase the primary immune responses, and use of
a boost KBMA vaccination is likely to increase T cell responses to levels similar to live attenuated
.
Figure 5. ELISpot analysis of live and KBMA primary T cell response. 1 week after vaccination with 5e6 live
or 1e8 KBMA Lm vaccines, splenocytes were harvested and immune responses to IglC pool 2 peptide
library, 8mer individual iglC peptide 33-19, or SL8 peptide were measured by ELISpot analysis. IM08-087
Notebook #2000, pp 28-30, 34.
2) Protection against lethal LVS challenge after Live-Lm vaccination. We performed an LVS
protection study to determine whether Lm-based vaccines can protect against a lethal LVS
challenge as a preliminary step prior to SchuS4 challenge. Balb/c mice were IV vaccinated twice
separated by 4 weeks with HBSS (buffer), 1e3 cfu LVS-pepO-SL8, 5e6 cfu Lm strain CRS-100,
BH1228 (Lm expressing iglC), BH1398 (expressing katG), or 2.5e6 cfu of both BH1228 and
BH1398 for a total of 5e6 per injection. 30 days after boost vaccination the animals were
challenged IV with a 10x LD50 dose of DVC lot 16 LVS. The actual number of cfu administered is
uncertain due to complications from the use of CHAH plates to determine cfu after vaccination.
However, after performing an IV LD50 study for the newly produced lot of DVC Lot 16 LVS, a dose
that was ten times LD50 was administered for in the challenge study. As expected, all the HBSS
and CRS-100 vaccinated negative control animals died (figure 6). 100% of the mice vaccinated
with LVS, Lm-IglC or Lm-IglC and LM-KatG survived the lethal challenge whereas only 40% of
the mice vaccinated with only Lm-katG survived. These data demonstrate that Lm vaccines
expressing Ft antigens can provide protective immunity against lethal LVS challenge and confirm
data generated by the Horwitz lab at UCLA.
Page 56 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
IM08-056: 10XLD50 LVS Challenge (IV)
% survival
100
50
8+
1
39
8
8
13
9
H
B
H
12
2
B
B
H
12
2
8
0
R
S10
LV
S
C
H
B
SS
0
Figure 6 Protection against lethal IV LVS challenge. Animals received two IV vaccinations of 1x10 3 cfu LVSPepO-SL8, or 5x106 cfu live attenuated Lm expressing IglC, KatG, or ½ the dose of both. 30 days after the
boost vaccination animals received a 10x IV LD50 dose of DCV lot16 LVS. Survival was monitored for 2
weeks. Lack of a bar indicates that all animals in the group died.
3) Comparison of IglC responses after vaccination with monovalent and bivalent Lm
vaccine candidates. As reported in MS55 section 3-2, monovalent and bivalent strains of Lm
were analyzed for their ability to stimulate an IglC-specific T cell response in C57BL/6 mice. For
this study, the 9mer peptide 33-10 (IglC136-144, GIMIDLSNL) was used, since this study was
initiated prior to mapping of the optimal 8mer 33-19 (IglC137-144, IMIDLSNL). By both ICS and
ELISpot analysis, there was a high degree of background induced by vaccination with the strains
expressing only KatG (BH2172 and BH2282, figure 7). This suggests that either Lm alone is
inducing a response against this peptide or KatG is. Nothing similar to the 33-10 (GIMIDLSNL)
amino acid sequence is present in KatG, but a BLAST search performed against the Lm
database revealed that a hypothetical protein lmo0368 contains a sequence that is 100%
identical for the first 7 amino acids of the 33-10 peptide (GIMIDLS). This suggests that Lm alone
is inducing a partial response to the 33-10 IglC peptide. Of note, all the IglC-expressing strains
induced stronger 33-10 IglC responses than induced by KatG-expressing strain. However, these
data suggest that there might be cross-reactivity between IglC and a Listeria protein, so 33-10
might not be the best epitope to assess immune responses induced by Lm strains in C57BL/6
mice.
Page 57 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 7, ICS and ELISpot analysis against IglC peptide 33-10 after a single vaccination
in C57BL/6 mice. IM08-086, Notebook #1002; pp 174, 178-181.
4. Significant decisions made or pending
We will not continue to measure 33-10 responses from C57BL/6 mice for quantification of
immune responses because of the high background presumably induced by Lm alone.
5. Problems or concerns and strategies to address
UNM and Anza continue to negotiate the MTA language to allow sharing of information
and reagents from UCLA. Without this MTA we cannot share our LM vaccine strains
expressing UCLA antigens with UNM for Schu4 Challenge studies.
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
25%
9. Work plan for upcoming month



Anza will vaccinate mice with various live and KBMA Lm vaccines to determine
whether IglC, KatG, or both protect against lethal LVS infection
Entire IglC peptide library will be tested with Lm expressing an irrelevant antigen
to determine if there is cross-reactivity between Lm and IglC in Balb/c mice as
well.
Once MTA is approved, live and KBMA Lm lots will be sent to UNM for
evaluation in SchuS4 challenge model.
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 58 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
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 inlA gain of
function mutants to our traditional platform strain. Routes will be ranked by ability to induce a
cellular immune response using ELISpot, ICS, and in vivo cytotoxicity. We will optimize
dosing regimen of most potent and tolerable route. Lm expressing IglC and/or KatG will be
used to evaluate immunogenicity. Optimized route and regimen will be confirmed by SchuS4
protection studies at UNM.
We have previously constructed vaccine candidates that contain the inlA gain of function
mutations (Table 2). 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-iglC-SL8 expression cassette was inserted using the integration vector
pINT. We performed initial evaluation of immunogenicity in C57BL/6 mice after IV and Oral
immunization and found little difference between the strains when splenic immune responses
were measured.
Table 2
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
1) Cellular invasion assays. In order to determine whether the inlAM gain of function mediates
enhanced entry into the CaCo2 cell line (as reported in Wollert et al.), we performed cellular
invasion assays as described in their manuscript. Monolayers of CaCo2 cells were infected with
Lm strains, washed with PBS, and then treated with gentamycin to kill extracellular bacteria.
Monolayers were then lysed by hypotonic shock and plated for CFU. Invasion of the CaCo2 cells
was dependent on inlA, as a inlA strain was nearly completely unable to invade, but after 4
independent assays, we were not able to demonstrate that the inlAM allele increased invasion
(figure 8). We were also unable to demonstrate an increase in invasion when prfA* strains were
compared (figure 8, right panel). While this result is different from what was published, it is
Page 59 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
important to note that CaCo2 cells are of human origin and thus there is a possibility that murine
epithelial cells could be better infected by the InlAM-expressing bacteria (which was not reported
by Wollert et al.).
CaCo2 Invasion NB2006-024
10 6
CFU/mL
10 5
10 4
10 3
10 2
10 1
68
B
H
21
66
B
H
21
94
H
21
B
B
H
21
64
10 0
Figure 8. CaCo2 cellular invasion assay. CaCo2 cells were infected with Lm strains for 1 hour,
washed 3x with PBS and treated with gentamycin. Wells were lysed by hypotonic shock and
diluted and plated for CFU. Data are located in NB2006 p.049, p.024.
2) Comparison of oral and IV immunogenicity in spleens and intestinal epithelium. Live
BH1228 (LmactAinlBuvrAB-iglC-SL8) was administered to C57BL/6 mice. 5x106 cfu were
administered IV or 1x 109 cfu were administered by oral gavage. Splenic and intra-epithelial
lymphocytic (IEL) responses from small intestine were measured by ICS (figure 9) and ELISpot
(not shown). In spleens, SL8 and IglC responses were 2-3 times lower after oral immunization
than with IV administration. Responses as a percentage of CD8 positive cells was lower in IELs
than in spleens, but the responses in IELs after oral and IV immunization were similar. Thus, if
protection correlates with IEL responses, the oral route may be equivalent to IV, but if protection
correlates with splenic responses, then IV would be expected to be a superior route of
immunization.
Page 60 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 9. ICS analysis of immune responses against ovalbumin SL8 peptide or IglC 33-10
peptide after a single oral or IV vaccination in C57BL/6 mice. Mice were vaccinated with 5x10 6
cfu IV, or 1x 109 cfu orally. 7 days after immunization, spleens and intestines were harvested and
splenocytes or intraepithelial lymphocytes (IEL’s) were pulsed with peptides for ICS analysis by flow
cytometry. Data are located in notebook #1002, pp 173, 182, 183..
3) Comparison of immunogenicity after oral administration of strains with various inlA
alleles. C57BL/6 mice were vaccinated orally with 1x109 BH2164 (LmactAinlBinlAWT-IglCSL8) and BH2194 (LmactAinlBinlAM-IglC-SL8) and BH1228 (LmactAinlBuvrAB-IglC-SL8)
which expresses the endogenous inlA allele of our host strain to determine whether the inlAM gain
of function contributes to immunogenicity. Immune responses were measured in splenocytes and
IEL’s from the small intestine (figure 10). In spleens (figure 10, top row and bottom row left and
center), IglC, SL8, and LLO190-201 responses were measurable, and BH2194 induced responses
that were marginally higher than BH2164, but not consistently higher than BH1228 (which does
not have a gain of function inlA allele). In IEL’s the responses were approximately 10-fold lower
than in spleen (figure 10, bottom row, right panel). The only peptide that stimulated a measurable
response was SL8, but interestingly BH2194 induced responses that were 3-4 times higher than
BH2164 and BH1228, this may indicate some function of the inlAM gain of function allele in mice
after oral vaccination. Because the responses in IEL’s are so low and the differences between
strains are so small these analyses will need to be repeated to determine whether the differences
are reproducible and significant.
Page 61 of 62
Tularemia Vaccine Development Contract: Technical Report
Period: 10/01/2008 to 10/30/2008
Due Date: 11/7/2008 and Prepared by: Rick Lyons, Barbara Griffith,Terry Wu, Justin
Skoble, Bob Sherwood, Trevor Brasel, Michelle Valderas, Julie Wilder, Stephen
Johnston, Kathryn Sykes, Mitch Magee, Karl Klose, Bernard Arulanandam
Figure 10. ELISpot responses after oral administration of Lm vaccine strains with various alleles of inlA
expressing IglC-SL8. Top panel, IglC responses in splenocytes were measured one week after oral
immunization. Bottom panel, SL8 splenic responses (left), LLO190 splenic responses (center), and SL8
intra-epithelial lymphocytic (IEL) responses (right). Data are located in notebook #1002, pp 184-186.
4. Significant decisions made or pending
None
5. Problems or concerns and strategies to address
None
6. Deliverables completed
None
7. Quality of performance
Excellent
8. Percentage completed
10%
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.
An intranasal LD50 will be performed with DVC lot16 LVS as this route of infection
may be more relevant for investigation of tularemia vaccines.
A murine epithelial cell line will be purchased for invasion assays
10. Anticipated travel
None
11. Upcoming Contract Authorization (COA) for subcontractors
None
Page 62 of 62