Transcript TVDC LBERI Tech call minutes final 080508

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
5 August 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
Frozen
LVS
Fresh
LVS
Down Select for
SCHU S4 Generator
Fresh
SCHU S4
Prepare bioaerosol SOP and
write MS completion report
Red: completed
Green: in progress
Blue: steps in the milestone
Frozen
LVS
Fresh
LVS
Hospitak
Fresh
SCHU S4
Slide 3
Milestone #3 – Bioaerosol Development
Accomplishments to date


Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Performed numerous bioaerosols with fresh and frozen SCHU S4
using the Collison and Aeromist
–


Aeromist proved slightly better; chosen as generator for
all future bioaerosols at annual site visit
Have recently performed extensive testing using the Hospitak
nebulizer
–
Aeromist production has ceased and LBERI’s stocks have
diminished
–
Technology identical to Aeromist: disposable
compressed air jet nebulizer; one time use
Performed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice
Slide 4
Milestone #3 July 2008 Accomplishments

Completed SCHU S4 bioaerosol testing comparing the Hospitak and
Collison nebulizers
–
30JUN (not presented last month)

–
1JUL

–
n=4 each (4 different generator concentrations)
24JUL


–
n=4 each (4 different generator concentrations)
9JUL

–
n=4 each (4 different generator concentrations)
n=3 each (3 different generator concentrations)
New Collison used
25JUL


n=3 each (3 different generator concentrations)
New Collison used
Slide 5
Targeted versus Actual Prespray Concentrations (CFU) for
the Hospitak and Collison Nebulizers
1.00E+10
Colony forming Units (CFU)
1.00E+09
1.00E+08
Hospitak
Collison
1.00E+07
1.00E+06
1.00E+05
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
Slide 6
Percentage of Organisms Recovered from the
Generator Suspension After Spray Completion
(Postspray) as Compared to the Prespray Generator
Suspension for the Hospitak and Collison Nebulizers
120
Postspray as Percentage
of Prespray
100
80
Hospitak
60
Collison
40
20
0
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
Slide 7
Log AGI Concentration Compared to
Prespray Target Concentration for the
Hospitak and Collison Nebulizers
Log AGI Concentration
8.00
4.00
Hospitak
Collison
2.00
1.00
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
Slide 8
AGI Concentration Recovered as a Percentage of
the Prespray Concentration for the Hospitak and
Collison Nebulizers
0.10
0.09
AGI Recovered as Percentage
of Prespray
0.08
0.07
0.06
Hospitak
0.05
Collison
0.04
0.03
0.02
0.01
0.00
1e6
1e7
1e8
1e9
Prespray Target Concentration (CFU)
Slide 9
Data Discussion

Generator suspensions
–
Concentrations were accurate
–
Demonstrates consistent growth at 24h
–
Titer decrease observed in post-spray




Slightly more with Collison though significance is questionable
Expected: SCHU S4 is fragile
CFU/L
–
Generally higher with the Hospitak, though significance is questionable
–
Consistent increase demonstrated with increased generator concentration;
demonstrates that aerosol concentrations may be targeted (rather than only
generator suspension concentrations)
Spray factors
–
Less efficient than seen with LVS
–
Consistent with the Collison
–
Inconsistent with the Hospitak

Likely inherent to the nebulizer chosen for any given bioaerosol
testing day
Slide 10
Milestone #3 – Bioaerosol Development
Plans for next month

Continue MS Completion report
–
Filling in gaps using data from past 2 months
–
Has SCHU S4 growth method been finalized? If so, this
will expedite the completion of this report (as well as the
SOPs)
Slide 11
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 12
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 13
Milestone #4 July 2008 Accomplishments

Challenged 2 NHP to high dose of aerosolized SCHU S4:
Slide 14
Microbiology Data
Slide 15
Temperature Data
Slide 16
Respiratory Rate Data
Slide 17
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 18
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 19
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 20
Milestone #7 – NHP SCHU S4 ED50
Plans for next month

IACUC currently being reviewed
–


6AUG08 IACUC meeting
Provide draft protocol for review; Collison nebulizer will be used
Continue planning efforts for study initiation
–
Tentative study dates:
– 15SEP08: ABSL-3 move-in
– 23-24SEP08: Aerosol exposure
Slide 21
Microbiology Quality Assurance

Routine Maintenance
–
Autoclave



–

Culture plates
–
Broth
–
Dilution blanks
Certificates of Analysis received and filed
for purchased media
As needed repairs
Checked weekly
Monthly PM
Shaker incubator

–
Monthly PM
(preventive
maintenance) checks
–
CO2 and regular incubators

–
Biological indicator
check for each run
Quality control performed on all prepared
media
Eye wash stations

–

As needed PM
Freezers

Annual defrost
Slide 22
Microbiology Quality Assurance (cont.)

Temperature Monitoring
–
Freezers

–
–
Continuous w/
alarm
Continuous w/
alarm
–
–
Recorded
during use
NIST traceable –
use 2 years and
replace
Temperature Probes – all
incubators

–
Annual calibration
and when used
Thermometers

–
Annual calibration
and when used
Balances

Water baths

Pipettors

Incubators

–
Equipment Calibrations
Refrigerators

–
Continuous w/
alarm

Annual calibration
BSC

Annual
recertification
Slide 23
Aerosol Quality Assurance






Yearly recertification of the Class III biosafety cabinets (gloveboxes) in
1027 and 1028
Daily eyewash checks
Yearly calibrations of:
–
Bios flowmeter
–
TSI APS
–
Alicat Mass Flow Controllers
Daily calibration of AGIs
Daily calibration of aerosol generators
LabVIEW Program validated and reviewed annually
Slide 24
IS and Data Management Quality Assurance

Data backup
–

All data on ABSL3 server backed up daily
Microbiology data recorded and maintained in workbook format
–
Reviewed regularly
–
Scanned out of ABSL3 regularly


Moving forward with electronic data recording
Aerosol data recorded and stored via LabVIEW
–
Data sheets scanned out after each bioaerosol day

Moving forward with electronic data recording
Slide 25
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 26
Update on screening of non-LVS vaccinated
NHPs




We have determined that due to the ability of some
non-LVS vaccinated NHPs to respond to LVS
antigens, we need to screen all such NHPs and
choose non- or low-responders for use in future
SCHU S4 challenge studies
In the past three months, 34 NHPs have been
screened 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 5 non-LVS vaccinated NHPs thus far
24 additional primates will be screened in the
coming weeks (approximately 4 – 8/week)
Slide 27
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; 6/16 non-LVS vaccinated NHPs
show this representative pattern
Slide 28
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; 6/16 non-LVS vaccinated NHPs
show this representative pattern
Slide 29
Three 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; 3/16 non-LVS vaccinated NHPs
show this representative pattern; none had greater than 1.6%
RBC contamination
Slide 30
Summary of proliferative response in newly screened
non-LVS vaccinated NHPs


4/15 NHPs responded to none of the LVS or SCHU S4 antigens
11/15 NHPs respond to at least one LVS or SCHU S4 antigens
Slide 31
Most 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 32
Cell Mean for OD405
IgG anti-LVS Titer does not tell
the whole story
2
1.8
1.6
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)
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
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 33
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
– IFNg ELISPOT Assay results were presented in
the July tech report
Slide 34
TUL 38 (not yet presented in Tech report or call)
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 35
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
Most recent 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 36
MS 12/13: Work upcoming in the next month


Continue to screen naïve NHPs for their reactivity to LVS and
SCHU S4 antigens in our assays
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 37
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
No update this month; Future plan: Repeat ICCS assay and
include a positive mitogen control (Con A)
Slide 38
Update on aggressive NHP (A00896)

MS 4: one LVS- vaccinated NHP (A00896) developed an
extremely aggressive behavior pattern and was becoming a
danger to the animal care technicians
–
We were told by the Veterinary staff that he would not be
a good candidate for an ABSL3 study and asked that we
use him soon on a study that would result in his
euthanasia
–
On 6/25/08, he was administered 100,000 CFU LVS by
bronchoscopy
–
On 7/7/08, he was euthanized; we tested his blood and
spleen cells for their reactivity to LVS and SCHU S4
antigens in our assays; baseline data was obtained for
the IFNg ELISPOT assay on 6/25; plasma was also stored
Slide 39
400
350
*
Day 583
Day 595
300
*
*
*
x
x
250
200
*
x
150
x
x
100
LVS hk Super
LVS ff Lo
LVS ff Mid
LVS ff Hi
LVS hk Mid
0
LVS hk Hi
50
Media
IFNg Spots (Mean +/- SEM)
LVS bronchoscopy boosted the ability of
NHP PBMCs to respond to LVS antigens
by IFNγ
Day 583 = pre-LVS bronch; Day 595 = post-LVS bronch; All cells plated at 1.33 x
106/ml; * indicates significantly different from both media and the opposite day of
bleed by ANOVA; x indicates significantly different from media
Slide 40
Further summary of bronchoscopic boost by
LVS



Plasma IgG anti-LVS titer increased from 1/20,000 (pre-boost) to
1/100,000 (day 12 post-boost)
Spleen cells (day 12 post-LVS bronchoscopy) responded to LVS
and SCHU S4 antigens by IFNg secretion; plating at 1.33 x 106/ml
is too concentrated and many wells were TNTC
There were not enough cells to test LVS-specific proliferation on
day 583 but comparing the post-boost proliferation to a day 276
bleed did not reveal any enhancement of response
Slide 41
QA in Wilder Lab
Item
QA Performed by:
Scheduled for
testing
Last Tested
Balances
Contractor to LRRI
Yearly
6/26/08
Pipettmen
Contractor to LRRI
Yearly
1/11/08
BioSafety Cabinet
Contractor to LRRI
Yearly
7/10/08
Fume Hood
Contractor to LRRI
Yearly
7/10/07 (contacted LRRI
personnel in charge of
scheduling for update)
-80 Freezer
LRRI surveillance
Daily
8/4/08
CO2 Incubator
Lab Technician
Monthly
7/9/08
Eye Wash
Lab Technician
Weekly
7/29/08
Slide 42
Action Items






Trevor- complete first draft of MS#3 MSCR by 9/2/2008 (next LBERI tech call)
Michelle will add the axis titles and units to the figures for the sprays (completed 8/12/08)
Trevor- will include the spray factor data in the monthly written tech report of 8/7/08.
Trevor- will remove the additional bioaerosols with the Hospitak as a goal for the month
of August (completed 8/12/08)
Trevor- will correct the presented doses for each NHP, as listed on slides 14 and 15. The
values on the two slides for each NHP should match and they do not match. (completed
8/12/08)
Michelle -change foot note on slide 15, to indicate that both NHP died and were not
euthanized. (completed 8/12/08)

Michelle -LBERI will better define language for “ target generator suspension dose” and “
presented dose”, using standard terminology accepted in the broader aerosol field.

Michelle will update slide 21 and others, regarding the “target” dose values and “target”
dose terminology.

Trevor- will edit slide21, as Hospitak is not relevant to this slide (completed 8/12/08)
Trevor- will include the microbiology/aerosol QA data in the minutes of this call.
(completed 8/12/08)
Slide 43