Transcript 2 LBERI Sherwood Mon session 2008

Tularemia Vaccine Development Contract
Progress in Tularemia Bioaerosol
Characterization and Non-Human Primate
Aerosol Challenges
ASU, Biodesign Institute, Tempe, AZ
October 6-7, 2008
Robert L. Sherwood, Ph.D.
Trevor Brasel, Ph.D.
Michelle Valderas, Ph.D.
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
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Previous Work Presented in 2007
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LVS Growth Optimization
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BCGA
–
Chamberlain’s
Aerosolization of LVS
–
–
Generators tested
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Collison 3-jet Nebulizer
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Aeromist Nebulizer
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Micropump Nebulizer
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Sparging Generator
Freshly grown suspension superior to frozen and reconstituted
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Milestone Achievements
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Milestone 3 (Bioaerosol optimization) is completed
Milestone 4 (Virulence confirmation) is nearly completed
Milestone 7 (ED50 in NHP) is in progress
–
Waves 1-3 ED50
–
Natural History
Milestone 8 (LVS Vaccine Efficacy) is in progress
Milestone 9 (Bioaerosol SOP) is in progress
Milestone 12/13 (Immunological assay development) is in
progress
–
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Screening new NHP for prior LVS titer
Milestone 21 (Correlates of Protection) is in progress
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Presentation Overview
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Schu S4 Microbiology Growth Parameters (Milestone 3)
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Schu S4 Bioaerosol Characterization (Milestone 3)
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Statistical comparison of Collison and Hospitak Nebulizers
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Virulence Verification in Mice (Milestone 4)
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Schu S4 NHP Bioaerosol Challenges (Milestones 4 and 7)
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Histopathology
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Problems Encountered and Corrective Actions
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LVS growth and bioaerosol characteristics not transferable
to Schu S4
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Schu S4 growth consistency
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Standardization of inocula by optical density
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Reoptimization of growth to standard curve
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Aerosol viability optimal after 24 hours growth in
Chamberlain’s broth (late log/early stationary phase)
Nebulizer of choice (Aeromist) discontinued
–
Extensive testing with Hospitak Nebulizer
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Statistical comparisons of Collison and Hospitak
Primate aerosol dosing
–
Pilot 3
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Schu S4 Growth Optimization
48h, 150 RPM,
37°C, in dark
1 CFU
48h BCGA culture grown
at 37°C
UNM-Chamberlains
50 mL in 500 mL baffled flask
24h,
200 RPM,
37°C, in dark
~10-15
CFU
48h BCGA culture grown
at 37°C
OD600 = 0.1 ± 0.01
UNM-Chamberlains
100 mL in new 500 mL sterile
baffled flask
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Schu S4 Bioaerosol Characterization
Targeted versus Actual Prespray Concentrations (CFU) for
the Hospitak and Collison Nebulizers
1.00E+10
Colony forming Units (CFU)
1.00E+09
1.00E+08
Hospitak
Collison
1.00E+07
1.00E+06
1.00E+05
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
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Schu S4 Bioaerosol Characterization
Percentage of Organisms Recovered from the
Generator Suspension After Spray Completion
(Postspray) as Compared to the Prespray Generator
Suspension for the Hospitak and Collison Nebulizers
120
Postspray as Percentage
of Prespray
100
80
Hospitak
60
Collison
40
20
0
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
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Schu S4 Bioaerosol Characterization
Log AGI Concentration Compared to
Prespray Target Concentration for the
Hospitak and Collison Nebulizers
Log AGI Concentration
8.00
4.00
Hospitak
Collison
2.00
1.00
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
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Schu S4 Bioaerosol Characterization
AGI Concentration Recovered as a Percentage of
the Prespray Concentration for the Hospitak and
Collison Nebulizers
0.10
0.09
AGI Recovered as Percentage
of Prespray
0.08
0.07
0.06
Hospitak
0.05
Collison
0.04
0.03
0.02
0.01
0.00
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
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Percent survival
LVS and Schu S4 Bioaerosol Mouse Challenges
100
90
80
70
60
50
40
30
20
10
0
LVS (6,850 CFU)
Schu S4 (1,050 CFU)
Schu S4 (400-600 CFU)
Schu S4 (22-25,000 CFU)
Note: Presented dose
0 1 2 3 4 5 6 7 8 9 10 11 12
Study Day
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Schu S4 Lung Deposition Following
Aerosol Challenge in Mice
Percent Deposition
7
6
5
Collison (5,500 CFU)
Collison (100,000 CFU)
4
Aeromist (4,400 CFU)
3
Aeromist (33,000 CFU)
2
Note: Presented dose
1
0
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Conclusions
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Growth conditions and bioaerosol characteristics differ
dramatically between LVS and Schu S4
Though the Collison and Hospitak nebulizers perform equally in
a statistical sense, the Collison was chosen for future studies
because of ease of tech transfer and consistency
Schu S4 and LVS bioaerosols cause mortality in mice, with Schu
S4 being the more virulent of the two
Schu S4 lung deposition in mice is an average of ~2%
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Screening non-LVS vaccinated NHPs
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In order to ensure that the NHPs that are chosen to be used in
LVS vaccination and/or SCHU S4 challenge experiments exhibit
low background responses in the immunologic assays, we have
begun screening all NHPs as they arrive at LRRI prior to being
put on study
We want to avoid using NHPs on studies that might have
existing cross-reactive IgG anti-LVS antibody levels or respond
non-specifically to LVS or SCHU S4 antigens in vitro in the IFN
ELISPOT or proliferation assays
IgG anti-LVS assay: 6/63 NHPs screened had titers > 100,000;
however, antibody is relatively “weak” compared to antibody
generated by LVS vaccination (lower OD405 at lowest dilutions;
dilutes out more quickly at higher dilutions)
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Schu S4 NHP Bioaerosol Challenges
1.
2.
Confirmation of Aerosol in vivo in primates (Milestone 4)
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Objective: to evaluate the pathogenicity of F. tularensis
Schu S4 on unvaccinated Cynomologus macaques
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Pilot 1
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Pilot 2
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Pilot 3
Schu S4 ED50 in primates (Miletone 7)
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Objective: to determine the 50% effective dose (ED50) of F.
tularensis Schu S4 on unvaccinated Cynomologus
macaques (euthanize animals that appear close to death
to prevent further suffering)
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Wave 1 (complete)
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Wave 2 (just begun)
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Wave 3 (begins October 27)
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Confirmation of Aerosol in vivo in Primates
• Design
Pilot 3
- 2 animals
- target presented dose of 1e6 (Hospitak)
- 28 day duration
Pilot 3 Naïve Cynomologus macaque F. tularensis Schu S4 Challenge Data
Animal
ID
Challenge
Date
Presented Necropsy
Dose1
Date
A04643
7/15/08
2.36E+05
A04645
7/15/08
1.09E+06
Tissue Culture2
Blood
Spleen
Liver
TBLN
MLN
Lung
7/18/08
1620
5.87e4
4.15e4
2.64e8
9.46e2
5.53e8
7/18/08
125
8.88e5
6.26e4
6.28e7
2.05e2
2.35e8
•1 – Presented dose is calculated from the number of bacteria recovered in the impinger multiplied by the amount of
air inhaled by the animal
•2 – blood data is presented as CFU/mL and tissue data as CFU/g
•Target dose achieved
•Both animals died on Study Day 3
•Pathogenesis of Schu S4 confirmed
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A.
B.
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A.
B.
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•
•
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ED50 Wave 1
Design: 12 animals, target presented dose of 1000, 10,000, and 100,000 (Collison),
35 day duration
A range of presented doses were achieved
All animals died by Study Day 8
Much data recovered regarding symptomology
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Animals Averaged by Group
Individual Animals
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Schu S4 ED50 in Primates - Wave 1
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At the time this presentation was edited, data was not available for low dose animals 28511 and 28438
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Comparison of Human and NHP Histopathology
with Schu S4 Infection
Tularemia in Humans
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Pyogranulomatous to necrotizing pneumonia
Fibrinosuppurative pleuritis
Pyogranulomas: spleen, liver, lymph nodes, kidney
Suppurative leptomeningitis
Ulcerative pharyngitis
+/- Gastroenteritis
Tularemia in NHPs
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Cynomolgus macaques, 2-4 yr old, M/F
Aerosol delivery various CFUs of SCHU S4
Evaluation of clinical signs and time to death with a lethal dose of
SCHU S4
Necropsy with blood and organ culture
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Gross Pathology
Lesions of lethal SCHU S4
tularemia are similar in most
species, in spite if differences in
susceptibility
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Pneumonia
Lymphadenitis
Splenitis
+/- Pleuritis
Pyogranulomatous
pneumonia
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Pyogranulomatous to Necrotizing
Bronchopneumonia
Colonies of Coccobacilli in Lung
Necrosuppurative & Ulcerative Rhinitis
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Future Plans
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Milestone 7 – complete ED50 and begin Natural History
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Milestone 9 – after ED50 is established and SOP will be created
and techniques validated by the LBERI Quality Assurance Unit
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Milestone 8 – challenge vaccinated NHP after ED50 is
established
Milestone 12/13 - Immunological assay development will
continue, and NHP will be screened prior to use on study
Milestone 21 - Correlates of protection will be ascertained along
with continuation of Milestone 8
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