Transcript LBERI NIAID Tech 090208 Minutes

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
2 September 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Milestones
#2
Active
Vaccinations of study personnel- no
work this month
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
efficacy studies in primates
#7
Active -
SCHU S4 LD50 in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
#21
Active-
Correlates of protection- in vitro assay
or other readout of effector function of
Ft developed for multiple species
Slide 2
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
Aeromist
Frozen
SCHU S4
Fresh
SCHU S4
Red: completed
Green: in progress
Blue: steps in the milestone
Frozen
LVS
Fresh
LVS
Frozen
LVS
Fresh
LVS
Down Select for
SCHU S4 Generator
Hospitak
Prepare bioaerosol SOP
Fresh
SCHU S4
Prepare MS Completion Report
[First drafts of both have been completed]
Slide 3
Milestone #3 – Bioaerosol Development
Accomplishments to date


Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Completed Collison, Aeromist, and Hospitak SCHU S4 testing
–

Based primarily on consistency, the Collison was downselected as the aerosol generator for all future bioaerosol
studies under this contract
Performed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice
Slide 4
Milestone #3 July 2008 Accomplishments


Decided upon Collison 3-jet nebulizer as F. tularensis bioaerosol
generator
–
Slightly less efficient than Aeromist and Hospitak, but
overall more consistent
–
Ease of tech transfer
–
Abundance of historical support data
Completed first draft of MS Completion Report
Slide 5
Milestone #3 – Bioaerosol Development
Plans for next month


Address comments on draft MS Completion report
Continue working on critical SOPs (all currently in draft form):
–
Bioaerosol preparation
–
Nebulizer operation
–
SCHU S4 stock preparation
–
Critical reagent preparation
Slide 6
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 and LD50
challenges
Mouse
Challenges
Cohort 1
Cohort 2
Cohort 3
Cohort 2
(n=2)
Cohort 3
(n=2)
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 7
Milestone #4 – NHP Aerosol Confirmation
Accomplishments to date


Completed 3 cohorts of mouse bioaerosols
–
Verified LVS and SCHU S4 virulence
–
Demonstrated no difference in SCHU S4 virulence based on 2
growth methods in this model
–
Demonstrated lung deposition is approximately 1-5% and was
greater when the Aeromist was used as the generator
Completed 3 cohorts of naïve NHP bioaerosols
–
N=2 each
Slide 8
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month


Awaiting pathology report
Milestone completion report has been initiated
–
Work will continue on this
Slide 9
MS#7 – Flow Diagram
MS 7: NHP SCHU S4 ED50
Round 1 (n=4 NHP each dose)
1,000 CFU
10,000 CFU Presented
(Target) Dose
100,000 CFU
Round 2 (n=4 NHP each dose)
x CFU (TBD)
y CFU (TBD)
Round 3 (n=4 NHP each dose)
x CFU (TBD)
Red: completed
Green: in progress
Blue: steps in the milestone
y CFU (TBD)
LD50/ED50
Determination
Slide 10
MS 7 Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature and respiration monitoring, body weight records, gross
necropsy, and viable bacterial blood/tissue cultures
Slide 11
Milestone #7 August 2008 Accomplishments


Received IACUC approval.
Prepared the draft study protocol.
Slide 12


Milestone #7 – NHP SCHU S4 ED50
Plans for next month
Protocol approval and sign off
Continue planning efforts for study initiation
–
Scheduled study dates:
– Round 1
23-24SEP08: Aerosol exposure
20-21OCT08: Term sacs
– Round 2
14-15OCT08: Aerosol Exposure
10-11NOV08: Term sacs
– Round 3
4-5NOV08: Aerosol exposure
1-2DEC08: Term sacs
Slide 13
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 or CTL?
Red: completed
Green: in progress
Yellow: on hold; restart if
necessary
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFNg
Proliferation
assay:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 14
Update on screening of non-LVS
vaccinated NHPs



We have determined that due to the ability of some nonLVS vaccinated NHPs to respond to LVS antigens, we need
to screen all such NHPs and choose non- or lowresponders for use in future SCHU S4 challenge studies
In the past several months, 58 NHPs have been screened
(although not all the data has been analyzed fully yet) and
they show a continuum of responsiveness in the
proliferation and IFNg ELISPOT assays
The only criteria we have defined as an exclusionary one is
the presence of an IgG anti-LVS titer > 20,000 (found in only
6/63 non-LVS vaccinated NHPs thus far
Slide 15
Some non-LVS vaccinated NHPs secrete IFNγ in
response to high doses of formalin fixed LVS or
SCHU S4 antigens
IFNg Spots (Mean +/- SEM)
160
140
120
100
80
60
40
20
0
28438
28447
28617
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
All cells plated at 1.33 x 106/ml; 10/24 newly screened (of 40)
non-LVS vaccinated NHPs show this representative pattern
Slide 16
Some non-LVS vaccinated NHPs secrete IFNγ in
response to antigens other than high doses of
formalin fixed LVS or SCHU S4 antigens
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
IFNg Spots (Mean +/- SEM)
300
250
200
150
SCHUS4 hk Super
100
SCHUS4 hk Hi
SCHUS4 hk Mid
50
SCHUS4 ff Super
0
SCHUS4 ff Hi
28547
28569
28570
SCHUS4 ff Mid
All cells plated at 1.33 x 106/ml; 7/24 non-LVS vaccinated NHPs
show this representative pattern
Slide 17
Some non-LVS vaccinated NHPs have a high
background in the IFNγ ELISPOT assay making it
difficult to determine their response to LVS or SCHU S4
antigens
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
IFNg Spots (Mean +/- SEM)
300
250
200
150
100
SCHUS4 hk Mid
50
SCHUS4 ff Super
SCHUS4 ff Hi
0
28395
28496
28559
SCHUS4 ff Mid
All cells plated at 1.33 x 106/ml; 6/24 non-LVS vaccinated NHPs
show this representative pattern; none had greater than 1.6%
RBC contamination
Slide 18
Some non-LVS vaccinated NHPs proliferate in
response to few or none of the LVS/SCHU S4
antigens
Relative Luciferase Units
(Mean +/- SEM)
45000
40000
35000
30000
25000
20000
15000
10000
5000
0
28438
28549
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
All cells plated at 1 x 106/ml; 12/34 NHPs show this
representative pattern
Slide 19
Relative Luciferase Units
(Mean +/- SEM)
Most non-LVS vaccinated NHPs proliferate in
response to at least one of the LVS/SCHU S4
antigens
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
80000
70000
60000
LVS ff Mid
50000
LVS ff Lo
40000
LVS hk Super
SCHUS4 hk Super
30000
SCHUS4 hk Hi
20000
SCHUS4 hk Mid
10000
SCHUS4 ff Super
0
SCHUS4 ff Hi
28447
28547
28565
SCHUS4 ff Mid
All cells plated at 1 x 106/ml; 22/34 NHPs show this
representative pattern
Slide 20
Most (57/63) non-LVS vaccinated NHPs
show an IgG anti-LVS titer less than
100,000
100000
10000
1000
28395
28438
28447
28464
28496
28503
28511
28525
28547
28549
28559
28565
28569
28570
28617
28651
IgG anti-LVS Titer
1000000
Slide 21
Cell Mean for OD405
IgG anti-LVS Titer does not tell the
whole story
2
1.8
1.6
28464
28503
28547
28651
896 Post-boost
896 Pre-boost
1.4
1.2
1
.8
.6
.4
.2
0
800
4000
20000 100000 500000
Plasma Dilution Factor
NHP
Titer is defined as the
highest dilution
producing: 1) an OD405
value > background
and 2) an OD405 value
that is equal to or
higher than the next
dilution (i.e. in a linear
portion of the curve)
800
4000
20000
100000
500000
Blank
28464
0.194
0.099
0.075
0.071
0.071
0.066
28503
0.257
0.112
0.077
0.072
0.071
0.066
28547
0.242
0.105
0.074
0.068
0.07
0.066
28651
0.481
0.161
0.087
0.074
0.072
0.066
896 Pre-boost
0.433
0.139
0.078
0.073
0.07
0.074
896 Post-boost
1.769
0.531
0.179
0.088
0.072
0.074
Slide 22
Update on testing Cerus Freeze/Thaw
Protocol on IFNg ELISPOT Assay Results
 We have previously determined that the Cerus
Freeze thaw protocol spares the reactivity of PBMCs
in the proliferation assay

In the past months, we have thawed frozen aliquots
of cells from three separate experiments for testing
in the IFNg ELISPOT assay
– All of these used non-LVS vaccinated NHPs
– Proliferation results were presented in the July
tech call as well as the tech report
– The following slide shows the IFNg ELISPOT
Assay results
Slide 23
Effect of Cerus Freeze/Thaw Process on
IFNg ELISPOT Assay Results
TUL 33
150
LVS hk Hi, Fresh
LVS hk Hi, Frozen
LVS hk Mid, Fresh
LVS hk Mid, Frozen
LVS ff Hi, Fresh
LVS ff Hi, Frozen
LVS ff Mid, Fresh
LVS ff Mid, Frozen
LVS ff Lo, Fresh
LVS ff Lo, Frozen
LVS hk Super, Fresh
LVS hk Super, Frozen
Fresh and
frozen/thawed
PBMCs were
plated at 1.33 x
106/ml
100
50
0
A04645
A05254
250
IFNg Spots (Mean +/- SEM)
IFNg Spots (Mean +/- SEM)
200
LVS hk Hi, Fresh
LVS hk Hi, Frozen
LVS hk Mid, Fresh
TUL 34
200
LVS hk Mid, Frozen
LVS ff Hi, Fresh
150
LVS ff Hi, Frozen
LVS ff Mid, Fresh
100
LVS ff Mid, Frozen
LVS ff Lo, Fresh
50
LVS ff Lo, Frozen
LVS hk Super, Fresh
0
A02314
A04308
A04713
LVS hk Super, Frozen
Slide 24
TUL 38 (not yet presented in
Tech report)
Fresh and
frozen/thawed
PBMCs were
plated at 1.33 x
106/ml
350
300
250
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
200
SCHUS4 hk Super
150
SCHUS4 hk Hi
100
SCHUS4 hk Mid
50
SCHUS4 ff Super
A06589, Frozen
A06589, Fresh
SCHUS4 ff Hi
A06587, Frozen
0
A06587, Fresh
IFNg Spots (Mean +/- SEM)
400
SCHUS4 ff Mid
Slide 25





Summary of the effect of Cerus
freeze/thaw procedure on assay
performance
Results are inconsistent
In general, the freeze/thaw process did not spare the
responsiveness of the cells in the IFNg ELISPOT assay
Frozen and thawed PBMCs from A04713 (TUL 34)
responded in a different pattern than the fresh cells
As all of these responses are from non-LVS vaccinated
NHPs, it is difficult to predict what proportion of a high
response to LVS antigens would be spared
We have once again begun comparing the Cerus and CTL
freeze/thaw protocols to see if the CTL protocol spares
more of the IFNγ ELISPOT response
Slide 26
MS 12/13: Work upcoming in the next month


Re-screen any naïve NHPs (6) which originally had high
background in the IFNg ELISPOT assay for their reactivity to LVS
and SCHU S4 antigens
Continue to test freeze/thaw protocol
–
Most cells will be from non-LVS NHPs
–
Spleen cells from A00896 (LVS vaccinated/boosted by
bronchoscopy) were frozen using both the Cerus and CTL
protocols and will be thawed on 9/2
Slide 27
MS #21 – Correlates of protection
Establish assays of effector function that detect correlates of
protection
Establish conditions to detect intracellular cytokines in NHP PBMCs
Confirm response in
LVS-vaccinated NHPs
Confirm low response
in non- LVS-vaccinated
NHPs
Slide 28

Intracellular cytokine staining
No experimental update on
PBMCs, but Amanda Dubois has
had success in NHP lung cells in
the past month (presented at the
UNM Tech Call)
Slide 29
MS 21: Upcoming work in the next month

Repeat ICCS assay and include a positive mitogen control (Con
A)
Slide 30