Transcript LBERI NIAID Tech call 5FEB08 minutes 020808

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
5 February 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
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Milestones
#2
Active
Vaccinations of study personnel
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
efficacy studies in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
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Milestone #2 – Vaccinations of Study Personnel

Vaccinations completed
–
32 LBERI scientists and staff received the LVS vaccination
between 9/11/07 and 1/8/08
–
28/32 completed the 1 month post vaccination followup
–
4/32 vaccinated on 1/8/08; their 1 month post vaccination
followup is pending
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Milestone #2 – Vaccinations of Personnel
Plans for this month


Health screenings in progress in February
Preparing for Vaccinations
–
3 LBERI scientists and staff may be vaccinated on 3/18/08, but
health clearance is pending
–
Up to 7 UNM scientists may be vaccinated on 3/18/08, but health
clearances and non-US citizen clearances are pending
–
USAMRIID will take up to 10 people on 3/18/08
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Goals for Milestone #3 – Bioaerosol Development
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Characterize the LVS bioaerosol using the Collison nebulizer

Determine optimum medium for aerosol dispersal (protein conc. &
antifoam)

Determine optimum medium for aerosol recovery (AGI)

Determine spray factors at various challenge concentrations

Determine lowest spray concentration & how to quantitate

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
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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 S4 Generator
Frozen
Schu4
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
Schu4
Prepare bioaerosol SOP and
write MS completion report
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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
Completed frozen SCHU S4 testing with the Aeromist and
Collison; Near completion of bioaerosol testing with fresh SCHU
S4 using the Aeromist and Collison
Completed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice
–
Completed follow-on study with fresh SCHU S4 in
BALB/c; need to organize bioaerosol data; still awaiting
in-life data
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SCHU S4 Bioaerosol Data to Date

No bioaerosol optimization testing was performed in January
2008
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Milestone #3 – Bioaerosol Development
Plans for this month
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Continue MS Completion report
Complete ASM Biodefense Meeting 2008 poster by week of
10FEB for review and finalization
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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
challenges
Mouse
Challenges
Cohort 1
Cohort 2
Cohort 2
(n=2); to
follow
mouse
challenges
Red: completed
Green: in progress
Blue: steps in the milestone
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Milestone #4 – Confirmation of Aerosol in vivo in NHP
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Awaiting histopathology results
Currently compiling daily observations data and completing
Microbiology report
Complete mouse follow-on study, compile data, and decide upon
approach for next 2 NHP challenges (MS4)
Awaiting mouse follow-on bioaerosol and in-life data in order to
confirm ABSL-3 move-in and challenge dates for additional 2
NHP.
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Milestone #4 – Confirmation of Aerosol in vivo in NHP
A04339 lungs 13 days post-challenge
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Milestone #4 – Confirmation of Aerosol in vivo in NHP
A04344 lungs 13 days post-challenge
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NHP Lung Pathology Conclusions
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Conclusions available in gross pathology report
Lungs clearly indicative of damage
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Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month
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Complete mouse follow-on study
–
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Suggest teleconference upon completion of mouse study
to decide next approach with NHPs
Schedule follow-on NHP SCHU S4 bioaerosol challenge
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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
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Update on IFNg ELISPOT detection from LVSvaccinated NHPs
Remaining questions:
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Do NHPs vaccinated with LVS via the SC vs. the ID route secrete
different levels of IFNg?
What is the effect of the Cerus freeze/thaw protocol on IFNg
secretion?
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0
1
175
1.33
A00937, LVS ff Mid
200
SC, 9.7% RBCs
A00937, LVS ff Hi
A00937, LVS hk Mid
A00937, LVS hk Hi
50
A00937, Media
A00868, LVS ff Mid
A00868, LVS ff Hi
A00868, LVS hk Mid
A00868, LVS hk Hi
125
A00868, Media
A00659, LVS ff Mid
A00659, LVS ff Hi
A00659, LVS hk Mid
225
A00659, LVS hk Hi
A00659, Media
Cell Mean for IFNg Spots
Comparison of IFNg Secretion by NHPs vaccinated
with LVS via the SC or ID route
150
SC, 1.0% RBCs
100
75
ID, 2.4% RBCs
25
Assay run on the same day; complicated by high RBC content (A00659) and poor
response to LVS (A00868)
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Day 203
Day 195
Day 237
Day 278
Day 400
Day 203
Day 195
Day 237
Day 278
Day 400
Day 203
Day 195
Day 237
Day 278
Day 400
Day 203
Day 195
Day 237
Day 278
Day 400
Day 203
Day 195
Day 237
Day 278
Day 400
A00868,
A00868,
A00868,
A00868,
A00868,
A00896,
A00896,
A00896,
A00896,
A00896,
A00908,
A00908,
A00908,
A00908,
A00908,
A00937,
A00937,
A00937,
A00937,
A00937,
0
A00659,
A00659,
A00659,
A00659,
A00659,
IFNg Spots (Mean +/- SEM)
Historical IFNg Secretion by NHPs vaccinated via the
ID vs. SC route
300
SC
250
200
Media
LVS hk Hi
LVS ff Hi
150
100
ID
50
All cells plated at 200,000/well
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Cell Mean for IFNg Spots
Effect of Cerus freeze/thaw protocol on IFNg secretion
100
90
80
Media
LVS hk Hi
LVS ff Hi
70
60
50
40
30
20
10
0
Fresh, ID
Fresh, SC
Frozen, ID
Frozen, SC
All cells were plated at 1.33 x 106/ml; ID vaccinated NHPs were 237 days
post-LVS vaccination (n = 2 fresh; 4 aliquots from 1 NHP frozen); SC
vaccinated NHPs were 278 days post-LVS vaccination (n = 2 fresh; 1 aliquot
from 1 NHP frozen)
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Follow-up from January tech call with NIAID (1/8/08)

Does RBC contamination also account for high background
values with non-LVS vaccinated NHPs when measuring IFNg
secretion?
–
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Is the background reproducible and NHP-specific?
Re-plot IgG anti-LVS titers on a log scale
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0.5%
450
400
2%
350
Media
LVS hk Hi
LVS ff Hi
0%
300
0%
250
200
0.8%
150
6.3 %
0.6%
2.2%
100
A05477
A04367
A04339
A04274
A04168
A03033
A03016
0
A04344
0.3%
50
A02386
IFNgamma Spots (Mean +/- SEM)
RBC content does not account for small differences
in background on IFNg ELISPOT plates
All NHPs are non-LVS vaccinated and plated at 200,000/well
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Is a high background response NHP-specific?

Analyze by looking at different experiments using the same
animal
–
Most non-LVS vaccinated animals have only been bled
one time during the screening process – most have now
been dedicated to another investigation; new NHPs have
been ordered
–
Can compare LVS vaccinated animals
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IFNg Spots (Mean +/- SEM)
RBC content has the greatest effect on background
responses in the IFNg ELISPOT assay but does not
account for all background responses
200
180
160
140
120
100
80
60
40
20
0
9.7%
TUL12
TUL14
TUL15
TUL16
TUL17
TUL18
TUL19
TUL27
7.4 %
1.0%
1.3%
0.4 %
2.7%
3.5%
A00659
0.4 %
A00868
A00896
0.8 %
1.4 %
A00908
A00937
1.3%
All cells were plated at 200,000/well and left unstimulated
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Small RLU (Mean +/- SEM)
High background in the proliferation assay is neither
RBC content- nor NHP-specific
700000
500000
TUL11
TUL12
TUL14
400000
TUL15
1.4%
600000
300000
200000
9.7%
0.8%
2.8%
TUL16
TUL17
2.4%
100000
TUL18
TUL19
TUL27
0
A00659
A00868
A00896
A00902
A00908
A00937
All cells were plated at 1 x 106/ml and left unstimulated
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Cell Mean for IgG anti-LVS Titer
1
A05477
A04344
A04339
A04168
A03033
A03016
A02386
A00937
A00908
A00902
A00896
A00868
Non-LVS Vaccinated NHPs Generally have Low IgG
anti-LVS Titers
10000
1000
100
10
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MS 12/13: Plans for the next month

Test IFNg secretions from ID and SC vaccinated NHPs on the
same day
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Action Items
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Trevor and Julie will send their ASM posters in powerpoint by 2/11/08 or
2/12 to UNM and UNM will request NIAID’s review.
Trevor will prepare an organized presentation of the mouse SCHU S4
data in the 2/7 tech report
UNM/LBERI need a teleconference or local meeting to decide on the
SCHU S4 virulence testing approach together before next NHP
experiment.
DON’T use the term “natural death”. NIAID said that “death from
suspected tularemia” is the correct terminology
UNM/LBERI will discuss the mouse follow on study at the Internal
weekly UNM/LBERI tech meetings on fridays from 10:45am to noon.
Julie will label the missing bars as “not performed” to differentiate from
“not detected” on the IFN gamma figures , somehow using the analysis
software.
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