Transcript LBERI NIAID Tech call 120407 minutes final

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
4 December 2007
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
5797-1
Milestones
#2
Active
Vaccinations of study personnel
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
#7
Inactive -
Schu-4 aerosol LD50 in cynomolgus
model
#8
Inactive -
LVS vaccine protection in Schu-4 infected
monkeys
#9
Inactive -
Development of GLP protocols for vaccine
efficacy studies in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
5797-2
Milestone #2 – Vaccinations of Study Personnel
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Overall: >70% complete
Group 1:
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Day 28 titers completed; approaching Day 56
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No re-vaccinations needed at this time
Groups 2 and 3:
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Titers being analyzed
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Completing follow-ups
–
For those who have received titer data, no re-vaccinations are
needed at this time
Group 4:
–
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Recently completed vaccinations
Positive titers have been observed for all participating individuals for
whom samples have been analyzed
5797-3
Goals for Milestone #3 – Bioaerosol Development
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Characterize the LVS bioaerosol using the Collison nebulizer
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Determine optimum medium for aerosol dispersal (protein conc. &
antifoam)
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Determine optimum medium for aerosol recovery (AGI)
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Determine spray factors at various challenge concentrations
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Determine lowest spray concentration & how to quantitate
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Determine differences in spray factor for reconstituted, vs. thawed, vs.
fresh
Compare Collison to sparging generator
Compare Collison to micropump generator
Compare Collison to Aeromist generator
Consider additional bioaerosol generators (Aeroeclipse II, ultrasonic generators,
others)
Determine optimum method for LVS bioaerosol generation
Perform bioaerosol studies with Schu4 as described above to determine if LVS data
are predictive
Compile SOP for Schu4 bioaerosol studies
Write Milestone Completion Report
Red: completed
Green: in progress
5797-4
MS#3 – Flow Diagram
MS 3: Bioaerosol Development
Collison Nebulizer
Aeromist
Micropump
Order & receive
instrument
Order & receive
instrument
Order & receive
instrument
Set up instrument
Set up instrument
Set up instrument
Frozen
LVS
Fresh
LVS
Lyophilized
LVS
Frozen
LVS
Fresh
LVS
Frozen
LVS
Fresh
LVS
Down Select for
Schu 4 Generator
Frozen
Schu4
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
Schu4
Prepare bioaerosol SOP and
write MS completion report
5797-5
Milestone #3 – Bioaerosol Development
Accomplishments
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Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Tested additional technologies (e.g., ultrasonic, Aeroeclipse II,
others) with BG spores; F. tularensis testing not pursued due to
difficult setup, practicality and/or no significant differences vs.
Collison
Initiated bioaerosol testing with frozen and fresh S4 Schu4 using
the Aeromist; plans underway to continue testing with the
Collison
Completed pathogenicity study of LVS and S4 Schu4 bioaerosols
in mice
5797-6
Aeromist S4 Schu4 Data to Date
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The following slides show Target vs. Actual CFU/mL and Actual
CFU/mL vs. Spray Factors for fresh Schu4 cultures tested to date
NOTE: All tested with a delivered pressure of 10psi to the
nebulizer
5797-7
Aeromist: Target vs. Actual CFU/mL (Fresh)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Post)
11/16/2007 (Post)
11/16/2007 (Post)
5797-8
Aeromist: Actual CFU/mL vs. Spray Factor (Fresh)
-6.00
4.00
-6.20
5.00
6.00
7.00
8.00
Spray Factor (Log10)
-6.40
-6.60
-6.80
-7.00
-7.20
-7.40
-7.60
-7.80
-8.00
Actual CFU/mL (Log10)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Pre)
11/16/2007 (Post)
11/16/2007 (Post)
11/16/2007 (Post)
5797-9
Aeromist Results: Fresh Schu4
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Spray factors less efficient than those observed when using LVS
LVS data may not be predictable for Schu4
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Schu4 as a bioaerosol may be heavily dependant upon
growth media, incubation time and other factors
Drop in titer observed in generator suspension post-aerosol
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Consistent with LVS
5797-10
Aeromist S4 Schu4 Data to Date
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The following slides show Target vs. Actual CFU/mL and Actual
CFU/mL vs. Spray Factors for frozen Schu4 cultures tested to
date
5797-11
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
4.00
4.50
5.00
5.50
6.00
6.50
Target CFU/ml (Log10)
11/7/2007 (Pre, 7.5psi)
11/7/2007 (Pre, 10psi)
11/7/2007 (Pre, Collison, 25psi)
11/7/2007 (Post, 7.5psi)
11/7/2007 (Post, 10psi)
11/7/2007 (Post, Collison, 25psi)
5797-12
Aeromist: Actual CFU/mL vs. Spray Factor (Frozen)
-5.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Spray Factor (Log10)
-5.50
-6.00
-6.50
-7.00
-7.50
-8.00
Actual CFU/ml (Log10)
11/7/2007 (Pre, 7.5 psi)
11/7/2007 (Pre, 10psi)
11/7/2007 (Pre, Collison, 25psi)
11/7/2007 (Post, 7.5psi)
11/7/2007 (Post, 10psi)
11/7/2007 (Post, Collison, 25psi)
5797-13
Aeromist Results: Frozen Schu4
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Spray factors, again, less efficient than those observed when
using LVS
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This holds true even with the Collison
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Efficacy higher than with fresh Schu4
 Further testing needed, but initial thought is that
Schu4 bioaerosol stability is highly dependant on
the growth procedures
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Drop in titer observed in generator suspension postaerosol
 Consistent with LVS though 7.5 psi is too low
 Optimal delivered pressure appears to be around
10 psi
5797-14
Mouse Pathogenicity Results
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High LVS and Schu4 doses successfully delivered to Swiss
Webster mice via nose-only challenge
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LVS: 650 LD50 (assuming 1 LD50 = 10 CFU)
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Schu4: 100 LD50 (assuming 1 LD50 = 10 CFU)
5797-15
Effect of Aerosol Tularemia on Mouse Mortality
% Survival
95
75
LVS - M
55
LVS - F
Schu-4 M
35
Schu-4 F
15
-5
D1 D2 D3 D4 D5 D6 D7 D8 D9 D10 D11
Study Day
5797-16
Mouse Pathogenicity Results
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Schu4 highly lethal in a rapid fashion
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All mice dead by day 5 post-challenge
LVS less lethal than Schu4
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…even at a higher delivered dose
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Survivors noted 10 days post-challenge
Pathogenicity of aerosolized LVS and Schu4 positively
demonstrated
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Indicates possibility of moving forward with NHPs
5797-17
Milestone #4 – Confirmation of Aerosol in vivo in NHP
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Continuing husbandry of vaccinated animals
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Have reached 1 year post-vaccination mark
Currently scheduling ABSL3 move-in dates and aerosol
exposures
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Naive NHP exposure (n=2) to be performed by the end of
the year
 Discussion needed on desired endpoints,
challenge dose, and bioaerosol specifics
5797-18
Milestone #2 – Vaccinations of Personnel
Plans for this month
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Continue regular personnel checks and follow-up on any revaccinations that may be required
5797-19
Milestone #3 – Bioaerosol Development
Plans for this month
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Continue testing with Schu4
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Confirm “instability” using Collison and fresh
Chamberlain’s broth culture
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Re-test Aeromist using Schu4 grown on solid BCGA
 Y. pestis stability increased on solid media; may be
same for F. tularensis
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Focus to be on Collison and Aeromist
 Collison operated under normal operating
conditions (~20-30 psi delivered)
 Aeromist testing at 10 psi
5797-20
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for this month
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Confirm dates for ABSL3 move-in and Schu4 exposure
Decide upon dose and aerosol technology (dependant upon
mouse pathogenicity and MS#3)
Prepare exposure protocol
5797-21
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
Phenotype Blood
and PBMCs
Test whether method
results in loss of B
cells
Red: completed
Green: in progress
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFNg
Proliferation
assay:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
IFNg
Intracellular
Staining
5797-22
Update on IFNg ELISPOT detection from LVSvaccinated NHPs
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We are attempting to optimize the ELISPOT assay that detects
IFNg secretion by LVS-stimulated PBMCs from previously
vaccinated NHPs
Thus far we have determined that 200,000 cells/well, 15 mg/ml
coating antibody and 1 mg/ml detection antibody is optimal
We have also determined that high RBC contamination increases
spot detection in all wells, including unstimulated wells
5797-23
Remaining questions about IFNg ELISPOT assay
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How does the freeze/thaw process affect LVS-stimulated IFNg
secretion?
Are there any differences when comparing ID- vs. SC-vaccinated
NHPs?
What is the response of PBMCs from non-LVS vaccinated NHPs?
5797-24
Responsiveness of PBMCs to FF LVS is dependent
upon cell concentration when measuring IFNg
secretion
Media
LVS hk Hi
LVS ff Hi
250
200
150
100
A04344, 1.33
A04344, 1
A04274, 1.33
A04274, 1
A00868, 1.33
A00868, 1
0
A00659, 1.33
50
A00659, 1
Cell Mean for IFNg Spots
300
5797-25
PBMCs from non-LVS vaccinated NHPs respond to
LVS by proliferation
Cell Mean for RLU small
300000
250000
Media
LVS hk Hi
LVS ff Hi
New LVS hk 5x10^6
New LVS ff 5x10^6
200000
150000
100000
50000
0
Note: All stimuli
significantly
different than
Media by unpaired
t-tests
A04168
Cell Mean for RLU small
A03033
A05477
Con A (added for comparison)
1.50E6
Media
Con A
1.00E6
LVS hk Hi
LVS ff Hi
New LVS hk 5x10^6
5.00E5
New LVS ff 5x10^6
0
A03033
A04168
A05477
5797-26
Problem encountered
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S.C. vaccinated NHP (A00902) was used on another protocol
accidentally and was euthanized
5797-27
MS 12/13: Plans for the next month
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Test IFNg secretions from ID and SC vaccinated NHPs on the
same day
Screen non-LVS vaccinated NHPs to see if they respond to LVS
by proliferation or IFNg secretion
Test whether sera from non-LVS vaccinated NHPs contains IgG
anti-LVS (using both FF-LVS and HK-LVS as coating antigens)
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Will include Day 0 pre-bleed and day 28 post-LVS
vaccination sera from 6 original NHPs as a control;
5797-28
Action Items- 1
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Trevor/Bob need to determine how long SCHU S4 is stable
frozen.
Trevor/Bob will use 10psi standardly with frozen SCHU S4
in the Aeromist generator
Trevor/Bob will report doses as CFU rather than relative
LD50s in all future experiments
Bob/ Trevor are ready to proceed with bioaersols to NHPs
and the experimental plans will be discussed at a
UNM/LBERI internal meeting on 12/7/07 Friday
Julie has chosen Cerus as the final freezing method to be
used in the future for NHP PBMC.
Julie wants to develop intraceullar staining assay to
explore the cell types in LVS vaccinated vs nonLVS
vaccinated NHP that are responding to LVS in the Elispot
assay. Could be a few naive cells making a lot of IFN
gamma in response to LVS
5797-29
Action Items-2
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Julie is currently screening 9 non LVS vaccinated animals for responsiveness, then
will choose specific animals to use for pathogenecity studies. Julie will totally
characterize naïve NHPanimals before placing into different experiments.
Julie can titrate cell numbers down and also titrate the LVS numbers down in the
proliferation or Elispot assay, to minimize background with naives but need to show
retention of vaccinated animals responsiveness to LVS by elispot or proliferation
Bob: LBERI has established that the correct report must be used before assigning
animals to a study so that previously assigned animals are not moved to a different
study.
Bob :LBERI is purchasing colored aluminum tags to bolt to the cages to mark the
studies
Julie is using IFN gamma elispot and proliferation assays to pre-test the 9 nonLVS
vaccinated NHPs for background responses to LVS
Julie will test the ID and SC vaccinated NHP PBMCs on the same day to determine
whether they still have difference in response to LVS, as these animals will be gone
after the challenge in the next 1-2 months.
Julie will order some new animals, to vaccinate some in the next few months- for
length of protection studies; want to vaccinate and challenge within 1 month and
within 6 months post vaccination, rather than wait 1 year.
5797-30