Transcript LBERI NIAID Tech call 030408 minutes final

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
4 March 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
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
Slide 2
Milestone #2 – Vaccinations of Study Personnel

Vaccinations completed
–
33 LBERI scientists and staff received the LVS vaccination
between 9/11/07 and 1/8/08
–
33/33 completed the 1 month post vaccination followup
Slide 3
Milestone #2 – Vaccinations of Personnel
Plans for this month


Health screenings in progress in March
Preparing for Vaccinations
–
0 LBERI scientists and staff may be vaccinated on 3/18/08;
health clearance is pending on 2 and 1 opted out
–
4 UNM scientists may be vaccinated on 3/18/08, ;1 opted out, 1
not yet medically cleared and 1 out of USA currently
–
1 UNM scientist may be vaccinated on 4/1/08 if becomes
medically cleared.
Slide 4
Goals for Milestone #3 – Bioaerosol Development









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
Slide 5
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
Slide 6
Milestone #3 – Bioaerosol Development
Accomplishments




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
Slide 7
SCHU S4 Bioaerosol Data to Date


No bioaerosol optimization testing was performed in January
2008
Cumulative LVS data were presented in Baltimore at the ASM
Biodefense Meeting (25FEB08)
Slide 8
Milestone #3 – Bioaerosol Development
Plans for this month


Continue MS Completion report
Titer new working stock
Slide 9
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 3
Cohort 2
(n=2); to
follow
mouse
challenges
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 10
Milestone #4 – Confirmation of Aerosol in vivo in NHP



Awaiting NHP histopathology results
Currently compiling daily observations data; Microbiology report
near completion
Completed mouse follow-on study (see next slide)
Slide 11
Francisella tularensis SCHU S4-exposed BALB/c mice Morbidity Data
11
10
Number of Mice
9
8
7
6
5
4
3
2
1
0
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Study Day (Exposure = Day 0)
Group 1: Low dose 72h BCGA
Group 2: High dose 72h BCGA
Group 3: Low dose 48h Chamberlains
Group 4: High dose 48h Chamberlains
Data suggest further investigation into
deposition (see following slide)
Slide 12
Mouse Deposition
Group
Media
Dose
(CFU)
Animal
No.
Lung
Wt.
Mean
CFU
Diln
CFU/
mL
CFU/
Lung
% Dep
1
BCGA
661
15965
0.165
0.0
10
0.0
0
0.0%
2
BCGA
22,700
15943
0.183
1.3
100
133.3
291
1.3%
3
CB
437
15959
0.223
0.0
10
0.0
0
0.0%
4
CB
24,900
15951
0.186
3.3
10
33.3
73
0.3%
Slide 13
Mouse Deposition Discussion




Surprisingly low deposition observed
Contamination may have contributed to calculations
Expected ~5%
Question as to fate of bacteria following inhalation
–
Does the Collison damage SCHU S4 to the point of low
deposition and/or culturability following inhalation?
–
Comparison testing with Aeromist planned (see next
slide)
Slide 14
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month

Further investigate SCHU S4 deposition
–
BALB/c mice
–
Aeromist vs. Collison
–
48h Chamberlains broth
 Use new working stock

–
Similar doses to previous mouse challenge
–
n=5 per group followed by immediate Nx and lung culture
on selective media (to reduce/prevent contamination)
Schedule follow-on NHP SCHU S4 bioaerosol challenge
Slide 15
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
Slide 16
Update on IFNg ELISPOT detection from LVSvaccinated NHPs
Remaining questions:


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?
Slide 17
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
NT
NT
NT
NT
NT
All cells plated at 200,000/well
Slide 18
ID vs SC-vaccinated NHPs: Plated for IFNγ Secretion
on the same day
Cell Mean for IFNg Spots
400
Media
350
ID
LVS hk Hi
300
LVS hk Mid
250
LVS ff Hi
200
LVS ff Mid
SC
150
100
50
0
A00659
A00868
A00896
A00908
200,000 cells/well plated; SC vaccinated NHPs are 442 days
post-vaccination; ID vaccinated NHPs are 451 days postvaccination
Slide 19
160
Media
LVS hk Hi
LVS ff Hi
140
120
100
80
60
Frozen, Aliquot 3
Frozen, Aliquot 2
NT
Frozen, Aliquot 1
0
NT
Fresh, Aliquot 3
20
Fresh, Aliquot 2
40
Fresh, Aliquot 1
IFNgamma Spots (Mean +/- SEM)
Effect of Cerus freeze/thaw protocol on IFNg secretion
by one ID-vaccinated NHP
All cells were plated at 200,000/well; ID vaccinated NHPs were 237 days postLVS vaccination (n = 2 fresh; 3 aliquots from 1 NHP frozen
Slide 20
IFNgamma Spots (Mean +/- SEM)
Effect of Cerus freeze/thaw protocol on IFNg secretion
by one SC-vaccinated NHP
90
Media
LVS hk Hi
LVS ff Hi
80
70
60
50
40
30
20
10
0
Fresh
Frozen
All cells were plated at 200,000/well; SC vaccinated NHPs were 278 days
post-LVS vaccination (n = 2 fresh; 1 aliquot from 1 NHP frozen)
Slide 21
MS 12/13: Plans for the next month



Begin to work out the conditions to detect intracellular IFNγ
production by flow cytometry in LVS-vaccinated NHPs
Attend NIAID/FDA meeting (3/11 – 3/13)
Attend Tularemia conference in Albany, NY (3/30 – 4/2)
Slide 22
Action Items





Barbara will check whether all 33 LBERI had titers.
Kristin: provide followup on USAMRIID’s 6 infected NHPs,
Bob/Trevor: will continue growing SCHU S4 in Chamberlains only.
Julie:Next NHP vaccinations will include 1) earlier time points of blood
draws 2) number of cells plated per well (150,000 to 200,000/well) and
lower number is preferred for lower background levels using FF LVS
(not seen the problem with the HK LVS) 3) keep non specific responses
down in vaccinated and naïve animals 4) do day 7day, 14day, 28 day,
42day, 56 day and 6 months for IFN gamma for two different plating
doses
Julie: will try both fresh and frozen cells in the intracellular cytokine
staining assays under development. Will use naïve and vaccinated
also.
Slide 23