LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
2 December 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Milestones
#2
Active
Vaccinations of study personnel- no work
this month
#4
Active
Confirmation of aerosol in vivo in NHP
efficacy studies in primates
#7
Active
SCHU S4 LD50 in primates
#8
Active
LVS vaccination protection of aerosol Schu4
validated in primates
#9
Active
Aerosol SOP developed for GLP transition
#12/13
Active
Assays for detecting relevant immune
responses in animals and humans
#21
Active
Correlates of protection- in vitro assay or
other readout of effector function of Ft
developed for multiple species
Slide 2
MS#4 – Flow Diagram
MS 4: NHP Aerosol Confirmation
Aerosol Challenge
Approach
Naïve NHP
Challenges
Vaccinated NHP
Challenges
MS 3
Cohort 1
(n=2)
Vaccinated
NHPs available;
awaiting
completion of
naïve and LD50
challenges
Mouse
Challenges
Cohort 1
Cohort 2
Cohort 3
Cohort 2
(n=2)
Cohort 3
(n=2)
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 3
Milestone #4 – November 2008 Accomplishments

No work was performed during this month.
Slide 4
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month


Awaiting pathology reports
Milestone Completion report has been initiated but completion is
dependent on the pathology reports.
Slide 5
MS#7 – Flow Diagram
MS 7: NHP SCHU S4 ED50
Round 1 (n=4 NHP each dose)
1,000 CFU
10,000 CFU Presented
(Target) Dose
100,000 CFU
Round 2 (n=4 NHP each dose)
25 CFU
250 CFU
Round 3 (n=4 NHP each dose)
250 CFU
Red: completed
Green: in progress
Blue: steps in the milestone
500 CFU
LD50/ED50
Determination
Slide 6
MS 7 Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature and respiration monitoring, body weight records, gross
necropsy, hematology, and viable bacterial blood/tissue cultures
Slide 7
Milestone #7 November 2008 Accomplishments

Initiated Wave 3 on November 18
–

All animals deceased by November 26
Data as of 11/30/08
Slide 8
Wave 1 Hematology Data
Normal Hematology Ranges for NHP
WBC = 6.9-19 x 103 / µl
% Neutrophils = 38-80.8%
% Lymphocytes = 27.9-80.8%
Slide 9
Wave 1 - Bacteremias
Animal #
28511
28438
28565
28496
28559
28569
28395
28447
28525
28549
28570
28617
Day 0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
0
0
0
0
4
0
10
0
16.7
8.67
5667
440
233
233
6
0
0
Slide 10
Wave 2 – Temperatures, Day 15-35
8 am
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35 Baseline Febrile
28615
99.4
101.4
28624 93.6 94.5 97.9 96.3 96.4 96.7 95.9 95.5 97.5 96.0 93.8 96.0 96.9 94.1 95.6 98.6 97.5 98.2 98.6 97.1 96.0
98.7
102.1
28581
101.0
103.1
28588 90.4 95.7 95.6 92.7 89.0 81.1
97.9
102.6
28618 96.8 98.5 99.6
96.3
100.0
28463
97.4
99.8
28571
100.5
102.0
28499
100.0
103.3
Hypo
97.4
95.2
98.9
93.1
92.5
94.9
99.1
96.6
2 pm
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35 Baseline Febrile Hypo
28615
100.2
101.3
99.2
28624 98.1 95.9 97.0 98.5 97.9 98.3 97.2 95.4 96.2 98.1 96.8 97.7 97.4 96.0 98.3 98.0 98.7 98.8 100.4 98.6 97.3
99.9
102.1
97.6
101.4
103.4
99.4
28581
99.9
102.1
97.8
28588 98.1 95.9 96.5 93.0 90.0 ####
97.2
99.4
95.1
28618 100.7 100.5 99.9
99.4
100.2
98.6
28463
101.5
102.6
100.4
28571
101.0
102.8
99.2
28499
8 pm
1
15
28615
28624
28581
28588
28618
28463
28571
28499
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Baseline
99.8
96.6 98.2 98.5 99.1 96.5 98.7 98.8 99.5 98.9 100.0 100.1 99.5 99.3 101.5 101.3 100.8 98.5 101.2 98.6 98.9
99.3
101.2
97.6 96.2 93.7 91.0
98.9
100.4 102.2
96.8
98.3
100.9
100.5
Blue indicates a decrease in temperature by 2 standard deviations from the baseline
Red indicates a increase in temperature by 2 standard deviations from the baseline
#### - 2 pm temperature for 28588 was not captured on Study Day 20
33
34
35
Febrile Hypo
101.1 98.5
101.8 96.7
102.8 99.6
102.0 95.8
98.9
94.6
99.7
96.9
102.0 99.9
102.3 98.7
Slide 11
Wave 2 – Respiration Rates, Day 15-35
8 am
15
28615
28624 76
28581
28588 72
28618 64
284632
28571
28499
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
68
88
68
72
68
68
52
64
56
40
68
60
56
52
60
52
56
56
56
52
56
56
60
52
56
52
42
Baseline
62
62
69
56
58
64
61
63
±30%
81 43
81 43
89 48
73 39
76 41
83 45
80 43
82 44
±50%
93 31
93 31
103 34
85 28
88 29
96 32
92 31
95 32
Baseline
65
68
61
55
57
61
62
66
±30%
84 45
89 48
79 43
71 38
74 40
79 43
81 44
86 46
±50%
97 32
103 34
92 31
82 27
85 28
92 31
94 31
99 33
2 pm
15
28615
28624 76
28581
28588 64
28618 76
284632
28571
28499
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
72
72
68
80
88
60
60
64
80
64
64
52
52
52
60
60
56
64
52
52
60
60
64
52
60
52
0
151
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
72
88
104
72
64
68
72
60
52
56
72
52
56
54
56
60
56
60
60
64
64
60
68
56
64
56
28615
28624
28581
28588
28618
284632
28571
28499
8 pm
Baseline
63
65
65
56
58
63
62
65
±30%
82 44
85 46
84 45
72 39
75 40
81 44
81 43
84 45
±50%
95 32
98 33
97 32
83 28
86 29
94 31
93 31
97 32
1
Respiratory rates were not recorded during 2000 obs on study day 15.
Aqua Blue indicates a 30% decrease in rate
Dark Blue indicates a 50% decrease in rate
Pink indicates a 30% increase in rate
Red indicates a 50% increase in rate
Slide 12
Wave 2 Hematology Data
Normal Hematology Ranges for NHP
WBC = 6.9-19 x 103 / µl
% Neutrophils = 38-80.8%
% Lymphocytes = 27.9-80.8%
Slide 13
Wave 2 Bacteremias
Animal #
28463
28571
28615
28618
28499
28624
28581
28588
Day 0
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
4
10
0
6.7
0
13.3
0
3.3
0
6
190
36.7
153.3
3.3
46.7
0
243.3
0
10
14
20
26
32
36
0
0
0
0
0
0
3.3
226.7
0
0
30
0
Slide 14
Wave 3 – Time to Death
ID #
Sex
Presented
Dose (cfu)
Manner of
Death
Timepoint
at Death
Study Day
at Death
28467
F
363
Euthanized
8 am
6
28484
F
444
Euthanized
8 am
8
28601
M
237
Euthanized
8 pm
6
28585
M
417
Euthanized
8 am
6
28479
F
675
Euthanized
8 am
5
28664
F
1150
Euthanized
2 pm
4
28512
M
884
Euthanized
8 pm
6
28572
M
621
Euthanized
8 am
8
Slide 15
Wave 3 – Tissue Bacterial Burden
Animal #
Spleen
(cfu/g)
Liver
(cfu/g)
TBLN
(cfu/g)
MLN
(cfu/g)
Lung
(cfu/g)
28467
2.6e8
5.9e6
5.3e7
2.4e6
8.2e8
28484
1.4e7
1.5e5
1.3e8
1.5e5
9.7e8
28601
3.7e8
1.1e7
1.8e7
3.0e8
8.8e8
28585
1e6
1e5
1.1e8
0
1.3e9
28479
5.8e7
9.4e4
5.0e8
2.3e5
1.1e9
28664
1e8
3.0e6
2.5e8
6.3e3
2.8e8
28512
1.8e5
4.0e4
8.3e7
1.2e6
8.3e7
28572
3.6e8
7.0e6
1.5e8
3.3e7
1.0e9
Slide 16
Wave 3 Temperatures
28467
28484
28601
28585
28479
28664
28512
28572
28467
28484
28601
28585
28479
28664
28512
28572
28467
28484
28601
28585
28479
28664
28512
28572
0
96.9
96.6
97.5
97.0
97.1
98.8
97.9
100.6
0
95.7
95.7
95.1
94.1
88.6
95.2
96.9
97.9
0
97.3
96.9
96.7
96.3
96.5
98.9
95.5
94.9
1
95.5
95.9
98.0
98.7
96.7
96.9
95.9
100.2
1
98.0
97.5
98.3
98.1
96.9
96.5
96.8
99.3
1
98.8
97.5
97.2
97.8
97.7
99.1
96.6
98.8
2
98.7
97.5
97.5
97.1
97.1
96.6
95.5
98.1
2
99.2
98.7
98.1
98.2
98.6
99.9
96.9
100.0
2
96.9
101.2
96.9
99.0
98.7
101.1
98.6
97.9
3
95.9
99.2
94.1
97.3
96.5
98.6
95.9
97.6
3
96.2
100.0
94.1
98.6
98.8
100.2
97.2
95.8
3
98.3
98.7
96.6
98.8
97.5
97.1
98.7
96.5
0800 Temps
4
5
95.9
94.7
98.8
96.9
96.5
95.4
95.7
91.2
96.8
90.3
92.6
95.7
95.3
95.9
94.9
6
90.4
96.2
93.9
88.1
91.8
94.7
1400 Temps
4
5
97.2
94.2
97.9
96.9
97.0
96.0
95.4
91.8
97.3
86.0
90.3
97.3
95.7
95.9
95.4
4
97.1
97.3
96.8
96.9
96.5
96.7
96.1
2000 Temps
5
6
94.3
96.3
94.5
95.0
90.1
91.5
87.8
96.0
96.5
89.0
93.0
7
8
93.2
84.7
91.1
83.2
6
7
94.2
91.0
91.4
89.6
92.9
89.5
7
91.4
88.4
Baseline Febrile Hypo
96.4
98.0
94.9
96.6
97.9
95.2
96.8
99.0
94.7
96.2
98.9
93.5
96.0
99.0
92.9
97.2
100.6
93.8
96.9
98.8
95.0
98.5
102.2
94.8
Baseline
97.4
96.9
97.6
97.1
96.5
98.6
97.4
99.7
Baseline
96.9
96.7
97.2
96.5
96.1
98.0
97.1
99.3
Blue indicates a decrease in temperature by 2 standard deviations from the baseline
Red indicates a increase in temperature by 2 standard deviations from the baseline
Febrile
98.9
98.8
99.5
99.3
99.3
100.5
99.7
101.9
Febrile
98.3
98.1
99.0
98.8
98.7
100.2
99.1
101.5
Hypo
95.9
95.0
95.7
94.8
93.8
96.7
95.2
97.5
Hypo
95.5
95.2
95.4
94.2
93.4
95.8
95.2
97.2
Slide 17
Wave 3 Respiratory Rates
28467
28484
28601
28585
28479
28664
28512
28572
28467
28484
28601
28585
28479
28664
28512
28572
28467
28484
28601
28585
28479
28664
28512
28572
0
72
48
44
52
60
48
52
68
0
52
44
44
48
44
44
52
64
0
80
52
44
64
60
36
48
52
0800 Respiratory Rates
1
2
3
4
5
6
7
8
Baseline
±30%
68
76
108
164
180
184
69
90
49
44
52
60
80
120
108
92
68
46
60
32
48
44
76
68
100
84
50
65
35
56
52
76
120
140
108
53
69
37
52
60
100
96
76
55
71
38
48
44
68
68
45
59
32
52
52
48
92
90
100
51
67
36
72
104
60
80
110
132
116
76
65
85
46
1400 Respiratory Rates
1
2
3
4
5
6
7
Baseline
±30%
84
80
144
160
180
71
93
50
48
48
68
92
120
96
80
49
63
34
48
48
84
88
90
88
51
66
36
56
52
100
128
120
57
75
40
52
60
100
100
90
57
75
40
40
52
60
72
47
61
33
56
48
60
72
100
104
54
70
38
60
60
68
84
120
116
116
65
84
45
2000 Respiratory Rates
1
2
3
4
5
6
7
Baseline
±30%
88
72
140
180
180
70
91
49
48
56
80
76
112
88
88
48
62
33
48
48
92
96
92
88
50
65
35
56
44
108
128
120
55
72
39
56
72
104
108
84
56
73
39
36
56
80
46
60
32
52
56
68
80
108
96
52
68
37
60
56
80
96
112
136
108
65
85
46
±50%
104
35
70
23
74
25
79
26
82
27
68
23
77
26
98
33
±50%
107
36
73
24
76
25
86
29
86
29
71
24
81
27
97
32
±50%
106
35
71
24
75
25
83
28
84
28
69
23
79
26
98
33
Pink indicates a 30% increase in rate
Red indicates a 50% increase in rate
Slide 18
Wave 3 Bacteremias
Animal # Day 0
28479
28664
28601
28512
28572
28484
28467
28585
0
0
0
0
0
0
0
0
2
0
0
0
0
0
0
0
0
4
6.67
570
33.3
0
10
0
0
0
6
6000
13.3
520
10
7666.7
8
0
Slide 19
Summary of Clinical Observations

Ocular tularemia - wave 2,
two animals (28588, 28618),
floculent white material that returned tuli on culture of the eye

Red skin lesions (rule out petechiae), waves 2 and 3, at least 6
animals (28588, 28615, 28571, 28467, 28512, 28601)

Respiratory symptoms – greatly increased rate, gasping, labored
breathing, and mouth breathing in waves 1,3; coughing in all
waves



Nasal discharge – all waves but not all animals
Anorexia – all waves, commonplace
Hypothermia as death approaches – all waves
Slide 20

Milestone #7 – NHP SCHU S4 ED50
Plans for next month
Continue study execution
– Wave 2
2 Dec 08: Term sac
 Graph Hematology Data from Wave 3 once received from Clinical
Pathology group
 Histopathology for all 3 waves is pending
Slide 21
MS#8 – Flow Diagram
MS 8: LVS Vaccinated NHP Challenged with SCHU S4
Round 1 Vaccination Practice/Challenge (n=3 scarification; n=2
subcutaneous)
Round 2 Vaccination/Challenge (n=3 by
scarification; n=3 by subcutaneous route; n=4
previously vaccinated; 2 SC, 2 ID)
X CFU (TBD)
Round 3 Challenge (USAMMDA vaccine vs .
DVC LVS Lot 16 by scarification)
X CFU
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 22
MS 8 Objective and Endpoints


Describe the natural history of aerosol delivered Schu S4 infection in
NHPs that have been previously vaccinated with LVS. Two different
methods of vaccination will be compared (scarification and
subcutaneous).
Endpoints include histopathology and bacterial CFUs of internal organs
(lung, spleen, liver, kidneys, and lymph nodes), twice a day records of
clinical symptoms post-infection, and clinical checmisty and
hematology during infection.
Slide 23
Milestone #8 November 2008 Accomplishments


Blood draws were performed on the 5 NHPs (3 via scarification
and 2 via subcutaneous) that were vaccinated with LVS in
October 2008 on Days 21, 28, and 35 post-vaccination to assess
immunity to LVS.
Data through November 2008
Slide 24
LVS Vaccination by Either Route Elicited Increases in
IgG anti-LVS Titer
1000000
Averages shown
* p = .029
100000
Individual NHPs shown
Day 35
1000000
100000
Day 35
Day 28
Day 15
100
Day 7
1000
Day 21
28461
28656
A04169
A05403
A06199
10000
Day 0
Day 21
Day 15
100
Day 7
1000
Day 28
SC
None
Scarification
Day 0
IgG anti-LVS Titer
10000
Slide 25
LVS Vaccination by Either Route Induces PBMC
Proliferation
RLU (Mean +/- SEM)
1000000
800000
600000
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
400000
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
Day 7, Scarification
0
Day 7, SC
200000
Note: No stimulus induced more than 100,000 RLU prior to vaccination
Slide 26
LVS Vaccination by Either Route Induces PBMC
Proliferation
IFNg Spots
(Mean +/- SEM)
Media
350
LVS hk Hi
300
LVS ff Hi
250
SCHUS4 hk Hi
SCHUS4 ff Hi
200
150
100
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
Day 7, Scarification
0
Day 7, SC
50
Note: No stimulus induced more than 80 spots prior to vaccination; all NHPs
responded; One scarified NHP had too many spots to count in response to LVS
antigens on d15 and 21
Slide 27
Milestone #8 – LVS Vaccination
Plans for next month

Continue to analyze data from days 28 and 35 to determine
timing of future challenges
– IFN production looks optimal by day 15 post-vaccination
– Proliferation looks optimal by day 28
– Antibody response peaks on day 21

Schedule vaccination of 6 NHPs (3 scarification vs. 3
subcutaneous route) based on optimium timing of challenge and
ABSL3 schedule
Slide 28
MS#9 – Aerosol SOP Development
MS 9: Aerosol SOP Development
Develop Validation Plan
Perform Aerosol Validation
Prepare Aerosol SOP
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 29
MS 9 Objective

Develop a SOP compatible with GLP transition for aerosol delivery of
Schu S4.
Slide 30
Milestone #9 November 2008 Accomplishments

No work was performed during the month of November.
Slide 31


Milestone #9 – Aerosol SOP Development
Plans for next month
Continue to work on the validation plan for the aerosol
procedure.
If comments are received from UNM, address SOP comments.
Slide 32
Milestone #12/13 – Immune
Responses in Animals and Humans
Immunoassay Development and Comparisons in Animal Models
Choose PBMC
Purification Method
Choose PBMC
Freezing Method
Method chosen:
Purdue ListServ
Cerus or CTL?
Red: completed
Green: in progress
Yellow: on hold; restart if
necessary
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFN
Proliferation
assay:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 33
Update on testing Cerus Freeze/Thaw
Protocol on IFN ELISPOT Assay Results
 We have previously determined that the Cerus
Freeze thaw protocol spares the reactivity of PBMCs
in the proliferation assay



In the past months, we have thawed frozen aliquots
of cells from several experiments for testing in the
IFN ELISPOT assay
All NHPs presented are non-LVS vaccinated;
interpretation of results is limited to non-specific
responses and background responses
Responses after PBMC thawing vary but usually
Cerus and CTL protocols give equivalent results
Slide 34
Proliferative Response to LVS and SCHUS4 0-Mutants
is Generally Similar to WT Antigens
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
RLU (Mean +/- SEM)
8.00E5
6.00E5
4.00E5
2 s.c. vaccinated shown
2.00E5
0
Day 7
Day 15
Day 21
Day 28
3 scarified
shown
Media
LVS hk Hi
LVS ff Hi
1.00E6
SCHUS4 hk Hi
RLU (Mean +/- SEM)
All PBMCs plated at 1
x 106/ml; no
differences observed
using Day 0 PBMCs
8.00E5
SCHUS4 ff Hi
LVS hk Mutant Hi
6.00E5
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
4.00E5
SCHUS4 hk Mutant Hi
2.00E5
0
Day 7
Day 15
Day 21
Day 28
Slide 35
IFN Production in Response to O-mutants is Varied
in Non-LVS vaccinated NHPs
IFNg Spots (Mean +/- SEM)
160
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
LVS hk Mutant Hi
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
140
120
100
80
60
40
20
0
28461
28656
A04169
A05403
A06199
Response to FF LVS mutant is decreased;
Response to HK LVS mutant is unchanged;
Response to FF SCHU S4 mutant is usually unchanged;
Response to HK SCHU S4 mutant is usually unchanged
(except for A06199)
Slide 36
IFN Production in Response to O-mutants is Varied
in Non-LVS vaccinated NHPs
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
IFNg Spots (Mean +/- SEM)
350
300
SCHUS4 ff Hi
250
LVS hk Mutant Hi
200
LVS ff Mutant Hi
150
SCHUS4 ff Mutant Hi
SCHUS4 hk Mutant Hi
100
50
0
Day 7
Day 15
Day 21
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
Response to FF LVS mutant is
decreased;
Response to HK LVS mutant is
decreased;
Response to FF SCHU S4 mutant is
variable;
Response to HK SCHU S4 mutant is
varied but often increased
IFNg Spots (Mean +/- SEM)
350
300
250
LVS hk Mutant Hi
200
LVS ff Mutant Hi
SCHUS4 ff Mutant Hi
150
SCHUS4 hk Mutant Hi
100
50
0
Day 7
Day 15
Day 21
Slide 37
MS 12/13: Work upcoming in the next month

Continue to test freeze/thaw protocol; specifically concentrate
on cells from the newly vaccinated NHPs
Slide 38
MS #21 – Correlates of protection
Establish assays of effector function that detect correlates of
protection
Establish conditions to detect intracellular cytokines in NHP PBMCs
Confirm response in
LVS-vaccinated NHPs
Confirm low response
in non- LVS-vaccinated
NHPs
Slide 39
MS 21: Upcoming work in the next month


No work performed this month
Repeat ICCS assay and include a positive mitogen control (Con
A); use PBMCs from newly vaccinated NHPs
Slide 40
Action Items




Michelle will send ED 50 study data in xls files to Barbara and
Barbara will send to NIAID (completed 12/10/08)
Michelle will send hematology data for wave 3 to BG for transfer
to NIAID. (done 12/10/08)
Julie will send the raw data in an excel file to Barbara who will
transfer the files to Freyja and Kristin (done 12/11/08)
Barbara will request dates/ times from Julie Wilder for a call with
Freyja Lynn (BG contacted Julie on 12/10/08; waiting for J Wilder
response 1214/08. Done but Freyja requested a call in Jan 2009
rather than Dec 2008; We’ll discuss a Jan 09 date, during the
1/6/09 LBERI tech call)
Slide 41