Transcript 5 TB JW LBERI TVDC 2007 Meeting Mon session 100807

Optimization of Aerosol
Generation Techniques for
Francisella tularensis – Laying
the Groundwork for Nonhuman
Primate Exposures
Trevor Brasel, Ph.D.
Lovelace Respiratory Research Institute
2425 Ridgecrest Dr. SE, Albuquerque, NM 87108
1
Milestone 3
• Optimize and select bioaerosol technique
for Francisella tularensis
• Key achievements to date:
– Live vaccine strain of F. tularensis (LVS)
growth conditions characterized and
optimized
– Aerosol collection technology chosen
– Bioaerosol generation technology downselected to two devices (Collison and
Aeromist nebulizers)
• Result of extensive testing using Bacillus globigii
spores and LVS
2
LVS Growth Optimization
Cysteine Heart Agar with Blood
(CHAB)
Blood Cysteine Glucose Agar
(BCGA)
3
LVS Growth Optimization
48h LVS growth on CHAB
48h LVS growth on BCGA
4
Bioaerosol Generation Technology
Optimization and Down-Selection
• Numerous technologies tested:
– Nebulizing, sparging and ultrasonic generators
• Four technologies down selected for further
testing:
Collison
Sparging
Micropump
Aeromist
5
• Collison nebulizer
– 3-jet model
– Industry standard
– 7.5 L/min at 20
psig
– 10 mL optimal
working volume
– 1-2 mm particle
generation
6
• Aeromist nebulizer
– Medical device
– Cost-effective
– 8.5 L/min at 20
psig
– 5 mL optimal
working volume
– 1-2 mm particle
generation
7
Collison vs. Aeromist Bioaerosols
Collison Target vs. Actual (Frozen)
9.00
Log10 Actual CFU/mL
8.00
7.00
6.00
5.00
4.00
3.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
Log10 Target CFU/mL
8/8/2006
7/14/2006
8/15/2006
1/16/2007
2/23/2007
8/25/2006
8
Collison vs. Aeromist Bioaerosols
Aeromist: Target vs. Actual CFU/mL (Frozen)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
4/13/2007
6/21/2007
6/26/2007
8/8/2007
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
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Collison vs. Aeromist Bioaerosols
Collison: Actual CFU/mL vs. Spray Factor (Frozen)
-3.000
Log10 Spray Factor
3.00
-4.000
4.00
5.00
6.00
7.00
8.00
9.00
-5.000
-6.000
-7.000
-8.000
-9.000
-10.000
Log10 Actual CFU/mL
8/8/2006
7/14/2006
8/15/2006
2/23/2007
8/25/2006
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Collison vs. Aeromist Bioaerosols
Aeromist: Actual CFU/mL vs. Spray Factor (Frozen)
-5.00
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Spray Factor (Log10)
-5.50
-6.00
-6.50
-7.00
-7.50
Actual CFU/ml (Log10)
4/13/2007
6/21/2007
6/26/2007
8/8/2007
8/16/2007 (Pre; 10psi)
8/16/2007 (Pre; 15psi)
8/16/2007 (Pre; 20psi)
8/16/2007 (Post; 10psi)
8/16/2007 (Post; 15psi)
8/16/2007 (Post; 20psi)
8/30/2007 (Collison, Pre; 20psi)
8/30/2007 (Collison, Post; 20psi)
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Fresh LVS Results (to date)
• Both technologies performed better
• Aeromist performed as well as or better
than the Collison
– Spray factors consistently in the 10-6 range
(based on post-aerosol values) for the
Aeromist
– Lower delivered pressures resulted in
increased viability retention in the Aeromist
12
Problems Encountered
• Exposure line decontamination and microbiology
QC
– Thoroughly investigated both and now have
procedures in place
• Equipment issues
– Major repairs led to down time
• Consistency of CHAB and Chamberlain’s
– Batch to batch variability; have since replaced CHAB
with BCGA
– LVS and Schu4 occasionally do not grow in the
Chamberlain’s broth; reason unknown
• Frozen stock stability
– 6 months maximum; viability drops afterwards
– Requires regular replacement of frozen stock
13
Future Plans
• Complete LVS bioaerosols using Collison and
Aeromist
– Finish Aeromist testing with fresh LVS culture
– Focus on delivered pressure to Aeromist and Collison
• Plan and perform bioaerosols with F. tularensis
Schu4 using the methods optimized for LVS
• Confirm LVS and Schu4 pathogenicity in mice
– IACUC protocol approved
• NHP exposures
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Measuring Immunity to
Francisella tularensis (LVS) in
Non-Human Primates
Julie Wilder, Ph.D.
Lovelace Respiratory Research Institute
2425 Ridgecrest Dr. SE, Albuquerque, NM 87108
15
Overall Goal for Milestone 12/13
• Develop assays for detecting relevant
(anti-Francisella tularensis) immune
responses in vaccinated and challenged
animals (and vaccinated humans)
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Key Accomplishments
• Vaccinated 3 non-human primates (NHP;
cynomolgus macaques) with LVS via the
interdermal route and 3 via the subcutaneous
route
• Established reliable protocols for:
– Purification of PBMCs from non-human
primate (NHP) blood
– Proliferation of PBMCs to mitogen and LVS
– IFNg ELISPOT detection
– Serum IgG-anti-LVS ELISA
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400000
300000
Media
LVS hk Hi
LVS ff Hi
200000
Day 28, SC
Day 28, ID
Day 21, SC
Day 21, ID
Day 14, SC
Day 14, ID
Day 7, SC
Day 7, ID
Cells cultured at
0.25 x 106/ml
0
Day 0, SC
100000
Day 0, ID
Luciferase Units (Mean +/- SEM)
PBMCs from vaccinated NHPs
proliferate in response to LVS
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PBMCs from vaccinated NHPs
secrete IFNg in response to LVS
IFNg spots (Mean +/- SEM)
60
50
40
.33
.67
1
1.33
30
20
10
0
Media
LVS hk Hi
6 NHPs combined, 3 SC- and 3 ID-vaccinated
LVS ff Hi
19
IgG anti-LVS Titer (Mean +/- SEM)
LVS-vaccination induces increase
in serum IgG-anti-LVS titer
100000
ID
SC
10000
1000
100
Day 0
Day 7
Day 14 Day 21 Day 28
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As yet unresolved…
• Still testing two different freeze/thaw protocols to
spare NHP PBMC viability and function
• Occasional failure of PBMCs from vaccinated
NHPs to proliferate in response to LVS – doesn’t
seem to be a matter of waning response
• Possibility that “naïve” NHPs are secreting IFNg
in response to formalin-fixed LVS
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Future Plans
• Continue to test two different freezing protocols
• Test more “naïve” NHPs for response to LVS
• Challenge vaccinated (and naïve control) NHPs
with aerosolized Schu4
– Track clinical signs
– Collect lung-draining lymph nodes upon necropsy and
study their proliferation and ability to secrete IFNg by
ELISPOT in response to LVS
– Study lung histology
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Acknowledgements
• LRRI Microbiology
–
–
–
–
Bob Sherwood, Ph.D.
Liz Frye
Rebecca Wisecup
Penny Armijo
• LRRI Aerosol
–
–
–
–
Ed Barr, MSEE
Steve Storch
Gil Vigil
Veronica Gonzales
• LRRI Immunology
– Ayako Monier
• LRRI Animal Care
and Veterinarian
Technicians
• UNM
– C. Rick Lyons, MD,
Ph.D.
– Barbara Griffith,
M.S.
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Questions after presentation
• Tom: Is Julie considering another enzyme or flow assays for
detecting response to LVS antigens?
• Julie: explored interferon gamma intracellular staining but the frozen
cells had lost CD3 marker. Julie is mostly doing elispot assays. For
NHP can do interferon gamma and il 4 but there aren’t too many
reagents available for non human primates.
• Rick: intracellular flow is hard to do using GLP procedures, relative
to elispot which is reliable for GLP labs.
• Bernard: Has LBERI examined the isotypes of the antibodies
produced by the NHP?
• Julie: LBER is trying to detect igA, but they are using human igA
and this human reagent hasn’t been working so are testing with
controls now.
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Questions after presentation
• Amanda: has done some igA experiments in NHP in the
past.
• Karl : should the team ask a company to make the NHP
reagents?
• Julie: It took long to get goat anti iga reagent, from 3
different vendors.
• Bernard: there are human reagents that may cross react
with NHP.
• Rick: Chuck, what do you think of naïve response to
LVS? Are these animals coming from areas where Ft is
endemic?
• Chuck: The NHP are Vietnamese controls- don’t really
know about the presence of tularemia in Vietnman?
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Questions after presentation
• Julie : the 6 animals vaccinated, didn’t respond fortuitously while
other naives have responded to LVS as if the naives weren’t really
naïve
• Stephen: do the vaccinated NHP have an antibody response?
• Julie: LBERI is testing this now
• Ed: LBERI should definitely prebleed before vaccination in the
future.
• Julie: LBERI knew they don’t have a proliferative response, but
didn’t know if had elispot response because the elispot assay wasn’t
set up yet a year a go before the NHP were vaccinated.
• Ed: if naives have pre-response, then distribute the “responsive
naives” across all animal groups and track them.
• Julie: may not see antibody responses but see elispot for ifn
gamma
• Chuck: the previously vaccinated NHP were from a breeding colony
in Vietnam, so not from the wild truly
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Questions after presentation
• Bob: they have found malaria in NHP
• Julie: can do all 3 assays from one bleed from NHP, but they can’t
mix the PBMC from multiple NHPs.
• Stephen: : any given antibody can cross-react with many different
stimujli, as seen in their human screening at ASU
• Chuck: naïve s that don’t die cause interpretation problems in
assays… they must have been exposed and developed immunity
unknowingly or have cross reactivity.
• Ed: save archival samples of all samples, as these will be valuable
for future assays and tests.
• Julie: This is why LBERI is being so careful to choose a freezing
and thawing procedure. LBER is also keeping serum .
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