Lovelace Respiratory Research Institute
NHP Model: Vaccination / Challenge and
SCHU S4 Natural History
R. Sherwood, T. Brasel, M. Valderas, J. Wilder, J. Hutt
www.LRRI.org
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Highlights
 MS#2
37 persons have received LVS vaccination
 MS#4
Confirmed pathogenesis of aerosolized SCHU S4 in cyno
 MS#7
Determined cyno LD50 for SCHU S4 to be ~2 CFU
 MS#8
Conducted multiple LVS vaccination studies with SCHU S4
challenge in cynos
 MS#11
Telemetered natural history study in cynos was completed
 MS#12/13 Have developed ELISA and IFN-γ ELIspot assays for NHPs
 MS#21
Working on intracellular cytokine staining protocol for
PBMCs
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M. Valderas
SCHU S4 NHP NATURAL HISTORY
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Milestone 11 – Overview of the Milestone
Title: GLP model efficacy SOPs developed in 1 small species and primate & efficacy
testing of vaccine candidates
Description: GLP compatible SOP for small animal model of SCHU S4 aerosol
infection in small animals (UNM) and primates (LBERI)
Objective: to determine the natural course of disease in telemetered and nontelemetered cynomolgus macaques (Vietnamese) that have been aerosol challenged
with a dose of F. tularensis SCHU S4 that results in primary pulmonic disease.
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Milestone 11 – Key Achievements
1) Completion of in-life procedures
2) Consistent with what was seen in the LD 50 study and extends the finding
with the addition of clinical chemistry and telemetry
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Milestone 11 - Study Design
The Natural History of SCHU S4 infection in cynomolgus macaques
was split into 2 separate studies. Animals were not telemetered in the
first study, Tul11A. Serial sacrifices were performed. In the second
study, Tul11B, animals were telemetered and allowed to succumb to
the disease
Tul 11
Natural History
Tul11A
non-telemetered, serial sacrifices
Tul11B
telemetered, allowed to succumb
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Milestone 11 – Study Specific Commonalities and
Differences
Commonalities between studies:
1) Target presented dose of 1000 cfu.
2) Blood draws for clinical chemistry, hematology, CRP level, and
bacteremia on Days 0-6
3) Observations 3x daily post exposure
4) Daily animal weights
Study specific differences:
1) Manual temperatures and respiration rates taken for Tul11A at each
observation
2) Tul11A animals (2 males and 2 females) were sacrificed on days 2, 4, 5,
and 6. A total of 16 animals were used.
3) 6 males and 6 females were infected in Tul11B and temperatures and
respiration rates were taken with telemetry.
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Milestone 11 – Presented Doses by Study
Presented Doses Tul11A:
Presented Doses Tul11B:
Presented doses ranged from: 183.3
to 8455 cfu.
Presented doses ranged from: 43 to
627 cfu.
The overall average was 3744 cfu
with a standard deviation of 2356.
The overall average was 228 cfu with
a standard deviation of 158.
Group
Average Presented
Dose (cfu)
Standard
Deviation
1 (day 2 sac)
2081.575
2079.967
2 (day 4 sac)
3608.5
1070.275
3 (day 5 sac)
3991
2564.046
4 (day 6 sac)
5296
2951.744
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Milestone 11 – Deaths by Day, Planned and Unplanned
Tul11A:
Animal
Number
Tul11B:
Presented
Dose (cfu)
Study Day of Death
Animal
Number
Presented
Dose (cfu)
Study Day
of Death
28012
183.3
2, planned
A07752
285
7
A06977
1277
2, planned
A07755
309
7
A06752
2241
4, planned
A07776
119
survived
A07129
4197
4, planned
A07801
96.2
7
A06297
2788
5, planned
A07805
102
8
A07126
2050
5, planned
A07808
121
8
A06940
5425
5, unplanned (day 6 sac)
A07783
43
7
A03697
1335
6, planned
A07784
182
7
A06712
1840
2, planned
A07788
248
6
A06834
5026
2, planned
A07811
627
6
A06243
3321
4, planned
A07828
327
7
A03597
4675
4, planned
A07841
273
6
A07031
3376
5, planned
A07132
7550
4, unplanned (day 5 sac)
A04840
8455
6, planned
A04925
5969
5, unplanned (day 6 sac)
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Milestone 11 – Tul11A Organ Bacterial Burden Averages
Terminal bacteremias were found in:
Group 1 = 0 of 4 NHP
Group 2 = 1 of 4 NHP
Group 3 = 4 of 4 NHP
Group 4 = 4 of 4 NHP
Terminal bacterial counts in the liver were
found in:
Group 1 = 1 of 4 NHP
Group 2 = 4 of 4 NHP
Group 3 = 4 of 4 NHP
Group 4 = 4 of 4 NHP
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Milestone 11 – Tul11A Organ Bacterial Burden Averages
Terminal bacterial counts in the lung were
found in:
Group 1 = 4 of 4 NHP
Group 2 = 4 of 4 NHP
Group 3 = 4 of 4 NHP
Group 4 = 4 of 4 NHP
Terminal bacterial counts in the MLN were
found in:
Group 1 = 0 of 4 NHP
Group 2 = 1 of 4 NHP
Group 3 = 2 of 4 NHP
Group 4 = 4 of 4 NHP
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Milestone 11 – Tul11A Organ Bacterial Burden Averages
Terminal bacterial counts in the TBLN were
found in:
Group 1 = 4 of 4 NHP
Group 2 = 4 of 4 NHP
Group 3 = 4 of 4 NHP
Group 4 = 4 of 4 NHP
Terminal bacterial counts in the brain stem
were found in:
Group 1 = 0 of 4 NHP
Group 2 = 1 of 4 NHP
Group 3 = 2 of 4 NHP
Group 4 = 2 of 4 NHP
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Milestone 11 – Tul11A Organ Bacterial Burden Averages
Terminal bacterial counts in the spleen were
found in:
Group 1 = 3 of 4 NHP
Group 2 = 4 of 4 NHP
Group 3 = 4 of 4 NHP
Group 4 = 4 of 4 NHP
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Milestone 11 - Tul11A Body Temperatures
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Milestone 11 - Tul11A Respiration Rates
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Milestone 11 – Tul11A Hematology
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Milestone 11 – Tul11A CRP Levels
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Milestone 11 - Tul 11B Bacterial Enumeration in
Various Organs
Terminal bacterial counts the
following organs were found in 11 of
12 NHP with the exception of lung
counts which were found in 12 of 12
NHP
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Milestone 11 - Tul11B Hematology
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Milestone 11 – Tul11B CRP Levels
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Milestone 11 - Problems, Corrective Actions, and differences
between planned and actual progress
1) Aerosol doses vary greatly between Tul11A and Tul11B
•
Great care was taken to standardized growth and aerosol method. We
have seen this kind of variation in the LD50 study. This remains a topic
for discussion.
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Milestone 11 - Plans for the next 6 months
1) Analyze serum cytokines (IL-1beta, IL-2, IL-6, IL-18, IFNgamma, GCSF, GM-CSF, and TNFalpha)
2) Analyze telemetry data, and complete remainder of data analysis
3) Complete histopathology analysis and report
4) Write MS11 report
5) Write report for the FDA
6) Submit abstract for ASM Biodefense Meeting
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J. Wilder
LVS VACCINATION AND PROTECTION FROM
SCHU S4 AEROSOL CHALLENGE
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Model of LVS Vaccination and SCHU S4 Aerosol
Challenge
 Cynomolgus macaques
 LVS (DVC Lot 16) given by scarification or sub-cutaneous inoculation
– Range of inoculum: 3 x 104 – 1 x 107 CFU
– Humoral and cellular immunity measured weekly until challenge
 SCHU S4, grown in Chamberlain’s broth, delivered by head-only
aerosol exposure on a day ranging between 35 – 49 days postvaccination
– Target dose of 500 – 1000 CFU presented in 3.5 L
– LD50 in unvaccinated NHPs determined to be 1 – 2 CFU
presented
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Experimental Design
 Experiment A
– 3 non-vaccinated controls
– LVS inoculum dose of 3.1 x 104 CFU
 3 vaccinated by scarification
 3 vaccinated by sub-cutaneous inoculation
– SCHU S4 challenge dose ranged from 27 – 1690 CFU presented
 Experiment B
– 3 non-vaccinated controls
– LVS inoculum dose of 0.1 – 1.2 x 107 CFU
 6 vaccinated by scarification
 6 vaccinated by sub-cutaneous inoculation
– SCHU S4 challenge dose ranged from 500 – 3740 CFU presented
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LVS Vaccination Induces Humoral Immunity in NHPs (B) and
Reduces Bacterial Burden in Tissues other than the Lung (A and B)
Control
Scarification
S.C.
1E10
20000
1E9
2000
200
2
A06675
A06702
A07395
2E-1
A07418
20
A07566
2E-2
A07610
2E-3
Day 0
Day 7
Day 14
Day 21
Day 28
SCHU S4 CFU/gram
(Mean +/- S.D.)
IgG anti-LVS Units (Mean +/- S.D.)
S.C.
1E8
* *
1E7
*
*
1E6
1E5
1E4
1E3
1E2
1E1
Spleen
Liver
TBLN
MLN
Lung
Day Post-LVS Vaccination
The same pattern of
responsiveness was
observed in LVSvaccinated NHPs in
Experiment A
* Indicates significantly
different than Control
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LVS Vaccinated NHPs are not Uniformly Protected from Death due
to SCHU S4 Aerosol Challenge (A and B)
SCHU S4 CFU Challenge
5000
Control
Scarification
S.C.
4000
3000
2000
1000
0
-1000
4
6
8
10
12
14
16
18
20
22
Days Survival Post-SCHU S4 Challenge
Day 21 = Scheduled euthanasia
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700
600
Media
Day 10, 1260
LVS hk Hi
LVS ff Hi
500
400
Day 6, 1690
300
Day 8, 2080
200
A07686, Day 28
A07686, Day 21
A07686, Day 14
A07686, Day 7
A07686, Day 0
A07682, Day 28
A07682, Day 21
A07682, Day 14
A07682, Day 7
A07682, Day 0
A07386, Day 28
A07386, Day 21
A07386, Day 14
0
A07386, Day 7
100
A07386, Day 0
IFNgamma Spots (Mean +/- S.D.)
Cellular Immunity to F. tularensis Develops in NHPs Subsequent to
LVS Vaccination (B)
Day of death and SCHU S4 CFU challenge noted above each NHP;
200,000 PBMCs plated/well and stimulated with 1 x 105 CFU HK or FF LVS/ml
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Challenges Encountered in Development of the Model:
Discrepancy from Efficacy Reported in the Historical Data
Historical Model
Current Model
Cynomolgus macaques
Health status uncertain
Health status certain; TB negative;
fed enrichment
LVS
Not DVC Lot 16
DVC Lot 16
Vaccination
1 x 105; Delivered intradermally
on arm after shaving and alcohol
prep
0.031 – 1.2 x 107; Delivered by
s.c. or scarification on upper back
after shaving
SCHU S4
“…cultivated in a modified case in
partial hydrolyzate liquid
medium.”; mean inhaled dose of
1 x 103
Grown in Chamberlain’s broth;
LD50 = 1 -2 by aerosol; mean
presented target dose of 0.5 – 1 x
103
Cellular Immunity
Not characterized
LVS induces increased IFNγ
production in vaccinees; however,
the amount produced varies
widely between NHPs
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Summary
 We have established a model of SCHU S4 aerosol challenge in NHPs
– Characterized by growth of bacteria in lung and systemic tissues and
reproducible clinical signs of disease (increased respiration and decreases in
sub-cutaneous temperature prior to death)
– All SCHU S4-exposed animals show signs of disease; a few recover
 Optimization of LVS delivery followed by SCHU S4 challenge in NHPs is
underway
– LVS delivered via s.c. inoculation or scarification leads to humoral and cellular
immunity
– LVS vaccination by either route results in reduced SCHU S4 burden, and
decreased severity of histological lesions, in tissues other than the lung, after
SCHU S4 aerosol challenge
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J. Hutt
HISTOPATHOLOGY IN VACCINATED AND
UNVACCINATED SCHU S4 CHALLENGED
CYNOMOLGUS MACAQUES
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MS-8: Hepatic pyogranulomatous to necrotizing
inflammation
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MS-8: Splenic pyogranulomatous to necrotizing
inflammation
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MS-8: Hepatic & splenic lesion scores in control &
vaccinated NHPs
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MS-8: Histopathology conclusions
 Vaccination does appear to provide some protection from
dissemination.
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Plans for 6 Months
 MS#8
Complete NHP LVS vaccination studies
 MS#12/13 Develop standardized Ag preparations for immunology
studies
Develop microagglutination assay
Develop cytokine intracellular staining assays
Develop in vitro functional assays
 MS#9
Complete SOP for SCHU S4 bioaerosol challenge of NHPs
 MS#10
Schedule NHP tularemia vaccine study with
experimental vaccine
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Discrepancies Between Actual and
Planned Progress
 MS#4
Bioaerosol challenges delayed confirmation of SCHU S4
pathogenicity in primates
 MS#8
Failure of LVS to protect has required 2 additional
studies to determine causes
 MS#12/13 Difficulties in accurately determining protein content of
tularemia antigens has delayed standardization of Ags
for immune assays
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 Questions?
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10/5/09: Action Items
 Michelle: For the data analysis on Tul A, move the NHP to the actual “ day
of death” for analyses rather than keeping them in the same original
group for the planned death.
 Michelle- remove zero bacterial burden values and determine the
variability of the burden in Tul11A NHP for which burden values were
detected.
 Michelle-Hematology on Tul 11A- plot as raw values rather than as total
WBC. Suspected that lymphocytes aren’t dropping but are changing as a
percentage of the total wbc cell population.
 Michelle- Tul 11B bacterial burden-add in the limit of detection as the
lowest value (approximately 10 to 20 in plating), rather than an artificial
low .0001 value in lieu of zero.
 Trevor- need to get bioaerosol more consistent on MS9
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