Transcript LBERI NIAID Tech call minutes 060308 final

LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
3 June 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
Frozen
LVS
Fresh
LVS
Frozen
LVS
Fresh
LVS
Down Select for
SCHU S4 Generator
Frozen
SCHU S4
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
SCHU S4
Prepare bioaerosol SOP and
write MS completion report
Slide 3
Milestone #3 – Bioaerosol Development
Accomplishments


Completed Collison, Sparging Generator, Micropump and
Aeromist LVS testing (fresh vs. frozen)
Tested additional technologies with BG spores
–
Hospitak Nebulizer tested 30MAY08
 Almost identical to Aeromist (see following slides)


Completed frozen and fresh SCHU S4 testing with the Aeromist
and Collison
Performed pathogenicity study of LVS and SCHU S4 bioaerosols
in mice
Slide 4
SCHU S4 Bioaerosol Data to Date

Hospitak vs. Aeromist testing
conducted on 30MAY
–

Manufacturing of Aeromist
has ceased
Hospitak nearly identical to
Aeromist
–
Working volume of up to 20
mL
 Can allow for longer
exposure times
Slide 5
Spray Factors vs CFU/L: Freshly Reconstituted Bacillus globigii Spore
Testing with the Aeromist and Hospitak Nebulizers
6.0
CFU/L (log10)
5.0
4.0
3.0
2.0
1.0
0.0
-4.5
-5.0
-5.5
-6.0
-6.5
-7.0
-7.5
-8.0
Spray Factor (log10)
Aeromist, 10 psi
Aeromist = 3 runs
Hospitak = 6 runs
Hospitak, 10 psi
Mean SF = 1.30 x 10-6 ± 1.61 x 10-7
Slide 6
Hospitak Testing


Hospitak demonstrated near identical performance to the
Aeromist using BG spores
Hospitak may be substituted for Aeromist
–
SCHU S4 testing required if it becomes necessary to use
Hospitak in the future (i.e., if LRRI’s Aeromist stocks are
depleted)
Slide 7
Milestone #3 – Bioaerosol Development
Plans for this month



Continue MS Completion report
–
95% complete
–
Awaiting finalization of SCHU S4 growth procedures in
Chamberlain’s
Complete SCHU S4 growth curve with revised Chamberlain’s
growth method (see following slide)
Complete bioaerosols with SCHU S4 to verify predictability of
growth curve
–
May initiate Hospitak testing with SCHU S4
Slide 8
Time (h)
10
1.50E+10
9
1.30E+10
8
7
9.00E+09
6
5
7.00E+09
4
CFU/mL
Normalized OD600
1.10E+10
5.00E+09
3
3.00E+09
2
1.00E+09
1
0
-1.00E+09
0
2
4
6
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
OD600, 6SEP07 WS, Flask 1
OD600, 6SEP07 WS, Flask 2
OD600, 28FEB08 WS, Flask 1
OD600, 28FEB08 WS, Flask 2
CFU/mL, 6SEP07 WS, Flask 1
CFU/mL, 6SEP07 WS, Flask 2
CFU/mL, 28FEB08 WS, Flask 1
CFU/mL, 28FEB08 WS, Flask 2
Slide 9
SCHU S4 Growth Curve Data to Date


Log phase for SCHU S4 using the BCGA to Chamberlain’s
method initiates at approximately 18h post-inoculation
Stationary phase is reached at approximately 32h postinoculation
–

Next growth curve will verify this
Bacteria harvested for bioaerosols will be in log phase
–
Need to discuss early, mid, or late as this may affect the
bioaerosol characteristics
Slide 10
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
(n=2)
Cohort 2
Cohort 3
NOTE: Chamberlains growth problems have
halted Cohort 3 mouse challenges two times
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 11
MS#4 Overview

Mouse studies
–

Naïve NHP studies
–

Purpose is to verify that aerosolized LVS or SCHU S4 are
virulent in mice (concern that aerosol method may alter
virulence)
Purpose is to verify that aerosolized SCHU S4 is virulent
in NHP (concern that aerosol method may alter virulence)
Vaccinated NHP studies
–
Purpose is to demonstrate protection in NHP previously
vaccinated with LVS
–
This study will follow determination of LD50 aerosol dose
Slide 12
A05254 Blood and Tissue Culture Data
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis SCHU S4 Bioaersol Challenge
Data
Animal ID
Challenge
Date
Challenge
Dose (CFU)
Nx Date1
Blood2
Spleen
Liver
TBLN3
Mes LN
Lung
A05254
14-May-08
Unknown
23-May-08
BLD
3.12E + 03
1.28E +02
1.88E +04
BLD
1.48 E +05
1- Animal A05254 was euthanized on Study Day 9
2- Blood data presented as CFU/mL; tissue data presented as CFU/g
3- In addition to F. tularensis, one contaminant was noted in the TBLN of A05254; currently being characterized
Slide 13
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for next month


Continue to observe remaining NHP
Schedule additional 2 naïve NHP
–
Awaiting completion of growth curve and bioaerosols
Slide 14
MS#7 – Flow Diagram
MS 7: NHP SCHU S4 LD50/ED50
Round 1 (n=4 NHP each dose)
5000 CFU Delivered
500 CFU
50,000 CFU
(Based on virulence study)
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 15
MS 7 Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature monitoring, body weight records, gross necropsy, and
viable bacterial blood/tissue cultures
Slide 16
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 17
Update on screening of non-LVS
vaccinated NHPs

We have determined that due 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
Slide 18






IFNγ ELISPOT Summary
Eighteen non-LVS vaccinated NHPs were
screened for reactivity to HK- and FF-LVS and
-SCHU S4 over 5 different experimental days
All cells cultured at 1.33 x 106/ml
(200,000/well)
One NHP made no response to any stimulus
Nine NHPs responded only to FF-LVS and the
responses diminished as less stimulus was
used
Three NHPs responded to both FF-LVS and
various other stimuli, including SCHU S4
Five NHPs had a high background (spots in
unstimulated wells) making interpretation
difficult
Slide 19
IFNγ ELISPOT: No response (1 of 1
shown) or Response only to FF LVS (3/9)
Cell Mean for IFNg Spots
225
200
175
150
125
100
75
50
25
0
A04994
A05987
A05997
A06199
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
Slide 20
IFNγ ELISPOT: Response only to
FF LVS (3/9)
Media
LVS hk Hi
Cell Mean for IFNg Spots
140
120
100
80
LVS hk Mid
Note:
A02314:
high IgG
anti-LVS
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
60
SCHUS4 hk Super
40
SCHUS4 hk Hi
SCHUS4 hk Mid
20
SCHUS4 ff Super
0
A02314
A04169
A04643
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 21
Cell Mean for IFNg Spots
IFNγ ELISPOT: Response only to
FF LVS (3/9)
Media
LVS hk Hi
LVS hk Mid
Note:
A05254 used
in latest
SCHU S4
pathogenict
y test
250
225
200
175
150
125
100
75
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
50
25
0
SCHUS4 hk Mid
SCHUS4 ff Super
A04713
A05254
A06592
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 22
IFNγ ELISPOT: Response to FF
LVS and other stimuli (3/3)
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
Cell Mean for IFNg Spots
400
350
300
LVS ff Mid
250
LVS ff Lo
200
LVS hk Super
SCHUS4 hk Super
150
SCHUS4 hk Hi
100
SCHUS4 hk Mid
50
SCHUS4 ff Super
0
A04308
A04999
A06587
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 23
IFNγ ELISPOT: High background makes
interpretation of stimuli-specific
responses difficult (5/5)
Cell Mean for IFNg Spots
250
200
150
100
50
0
A04645
A05262
A05403
A05988
A06589
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
Note: A05262 used in latest SCHU S4
pathogenicty test
Slide 24
Proliferation Summary




Eighteen non-LVS vaccinated NHPs were screened for reactivity
to HK- and FF-LVS and -SCHU S4 over 5 different experimental
days
All cells cultured at 1 x 106/ml
Eight NHPs had little to no response above background
Ten NHPs had a proliferative response to some stimuli
Slide 25
Proliferation: No response to
any stimuli (4/8)
Cell Mean for RLU small
250000
200000
150000
100000
50000
0
A02314
A04169
A04308
A05254
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
Note: A02314 has a high IgG anti-LVS titer; A05254 was
used in the latest SCHU S4 pathogenicity study
Slide 26
Proliferation: No response to
any stimuli (4/8)
Media
LVS hk Hi
250000
Cell Mean for RLU small
LVS hk Mid
LVS ff Hi
200000
LVS ff Mid
LVS ff Lo
150000
LVS hk Super
SCHUS4 hk Super
100000
SCHUS4 hk Hi
50000
SCHUS4 hk Mid
SCHUS4 ff Super
0
A05987
A06199
A06589
A06592
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 27
Proliferation: Response to at
least one stimuli (3/10)
Media
LVS hk Hi
LVS hk Mid
Cell Mean for RLU small
400000
350000
LVS ff Hi
300000
LVS ff Mid
250000
LVS ff Lo
200000
LVS hk Super
150000
SCHUS4 hk Super
SCHUS4 hk Hi
100000
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04643
A04645
A04713
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 28
Proliferation: Response to at
least one stimuli (3/10)
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
Cell Mean for RLU small
450000
400000
350000
LVS ff Mid
300000
LVS ff Lo
250000
LVS hk Super
200000
SCHUS4 hk Super
150000
SCHUS4 hk Hi
100000
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04994
A05262
A06587
SCHUS4 ff Hi
SCHUS4 ff Mid
Note: A05262 was used in the latest SCHU S4 pathogenicity study
Slide 29
Proliferation: Response to at
least one stimuli (4/10)
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
Cell Mean for RLU small
250000
200000
LVS ff Lo
150000
LVS hk Super
SCHUS4 hk Super
100000
SCHUS4 hk Hi
SCHUS4 hk Mid
50000
SCHUS4 ff Super
0
A04999
A05403
A05988
A05997
SCHUS4 ff Hi
SCHUS4 ff Mid
Slide 30
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 31
Intracellular cytokine staining

The first experiment was completed on 5/6/08
– PBMCs from 3 LVS-vaccinated NHPs were
prepared, rested overnight and stimulated with
either FF- or HK-LVS or –SCHU S4 for 6 hours
(last 2 hours with Golgiplug reagent to inhibit
secretion of cytokines)
– Cells were stained with either anti-CD3, antiCD4, anti-CD8 and a cocktail of anti-cytokine
antibodies (IFNγ, TNFα, and IL-2)
– Data will be analyzed this week
– An aliquot of PBMCs were tested for IFNγ
ELISPOT activity
Slide 32
IFNγ ELISPOT: Response of
LVS-vaccinated NHPs
Cell Mean for IFNg Spots
350
300
250
200
150
100
50
0
A00659
A00868
A00908
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
A00659 and 868 are 523 days post SC-LVS vaccination; A00908 is
532 days post ID-LVS vaccination
Slide 33
Action Items









Trevor will empirically test and prove that mid log bacteria aerosolize the same
as the 48 hr late log phase did.
LBERI must stabilize the SCHU S4 growth conditions and data, before
performing the LD50 study in NHP.
Trevor will present the SCHU S4 December 2007 and May 2008 NHP data in a
single slide at the 7/1/08 Tech call.
LBERI will take blood for PBMC and spleen cells at euthanizing, from the May
2008 SCHU S4 animal that has survived 21 days. Will freeze the spleen cells.
Julie :Diluting out the antigen decreases the background response by nonvaccinated NHP PBMC cells in proliferation and ELIspot assays, but still need
to show that the vaccinated NHPS show a specific, real response to the lower
level of antigen too.
Julie will replot subsets of the non-vaccinated ELIspot/proliferation data to
further analyze it.
Barbara : will contact DVC to request the LVS and SCHU S4 O antigen minus
strains, since Rick received the USDA importation permit for tularemia
Barbara: will contact USAMRIID to request the MTA form for UNM to review,
prior to the request for the clinical lot of LVS.
Barbara will copy Marlene on communications with USAMRIID and DVC.
Slide 34