LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
8 January 2008
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
5797-1
Milestones
#2
Active
Vaccinations of study personnel
#3
Active -
Optimization of bioaerosol methods
#4
Active -
Confirmation of aerosol in vivo in NHP
#7
Inactive -
Schu-4 aerosol LD50 in cynomolgus
model
#8
Inactive -
LVS vaccine protection in Schu-4 infected
monkeys
#9
Inactive -
Development of GLP protocols for vaccine
efficacy studies in primates
#12/13
Active -
Assays for detecting relevant immune
responses in animals and humans
5797-2
Milestone #2 – Vaccinations of Study Personnel




Overall: 40-50% complete
Including the 4 people being vaccinated on 1/8/08, 32 LBERI
participants have been vaccinated.
The UNM group of 6-7 participants are tentatively scheduled for
vaccination on 3/18/08
UNM and USAMRIID are actively using the LVS vaccine web database to
track Risk Assessment form submission and acceptance, Informed
Consent submission and acceptance, Health screening
appointments, planned dates for receipt of LVS vaccinations, dates of
medical clearance, and travel arrangements
5797-3
Milestone #2 – Vaccinations of Study Personnel
(cont.)





UNM and LBERI will use their biobubbles as additional physical
protective equipment
The LBERI work stoppage has been lifted for SCHU S4 aerosols as of
11/7/07.
In approximately 1 month, UNM may have access to a local source of
human cells from LVS vaccinated individuals.
Dr. Lyons is requesting UNM IRB approval to allow blood draws on the
vaccinated LBERI and UNM scientists after their LVS vaccinations. The
LBERI and UNM scientists and staff will be offered the opportunity to
volunteer to donate bloods for the development of immunoassays,
approximately 2 months after receiving the LVS vaccination.
UNM and LBERI are offering the LVS vaccinations to up to 14 more
scientists to total 46; USAMRIID will continue to provide the LVS
vaccinations over the next 3-4 months.
5797-4
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
5797-5
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 4 Generator
Frozen
Schu4
Red: completed
Green: in progress
Blue: steps in the milestone
Fresh
Schu4
Prepare bioaerosol SOP and
write MS completion report
5797-6
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
Near completion of bioaerosol testing with frozen and fresh S4
Schu4 using the Aeromist and Collison
Completed pathogenicity study of LVS and S4 Schu4 bioaerosols
in mice
5797-7
S4 Schu4 Bioaerosol Data to Date

The following slides show Target vs. Actual CFU/mL and Actual
CFU/mL vs. Spray Factors for fresh Schu4 cultures tested to date
–
2 culture methods have been tested:
 48h Chamberlain’s
 72h BCGA
5797-8
Aeromist and Collison: Target vs. Actual CFU/mL (Fresh Chamberlain's and BCGA)
8.00
Actual CFU/ml (Log10)
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
Target CFU/ml (Log10)
11/16/2007 (Pre, CB, Aeromist)
11/16/2007 (Post, CB, Aeromist)
12/7/2007 (Pre, BCGA, Aeromist)
12/7/2007 (Post, BCGA, Aeromist)
12/4/2007 (Pre, CB, Collison)
12/4/2007 (Post, CB, Collison)
5797-9
Aeromist and Collison: Actual CFU/mL vs. Spray Factor (Fresh Chamberlain's and BCGA)
Spray Factor (Log10)
-5.50
4.00
5.00
6.00
7.00
8.00
-6.00
-6.50
-7.00
-7.50
-8.00
Actual CFU/mL (Log10)
11/16/2007 (Pre, CB, Aeromist)
11/16/2007 (Post, CB, Aeromist)
12/7/2007 (Pre, BCGA, Aeromist)
12/7/2007 (Post, BCGA, Aeromist)
12/4/2007 (Pre, CB, Collison)
12/4/2007 (Post, CB, Collison)
5797-10
Fresh Schu4 Results


48h Chamberlain’s culture
–
Spray factors less efficient than those observed when
using LVS (independent of generator used)
–
LVS data are not be predictable for Schu4 when grown in
Chamberlain’s
–
Schu4 as a bioaerosol may be heavily dependant upon
growth media, incubation time and other factors
72h BCGA
–
Spray factors similar to those observed when using LVS
and better than those observed when using 48h
Chamberlain’s culture
–
Growth on solid media increases F. tularensis SCHU S4
bioaerosol stability
5797-11
S4 Schu4 Bioaerosol Data to Date

No testing was performed using frozen S4 Schu4 cultures in
December 2007
5797-12
Milestone #4 – Confirmation of Aerosol in vivo in NHP

Continuing husbandry of vaccinated animals
–

Beyond the 1 year post-vaccination mark
Aerosol exposures were conducted using two naïve cynomolgus
macaques
–
Challenge material: 72h S4 Schu4 BCGA culture
–
Conducted in order to confirm virulence as observed in
initial mouse pathogenicity study and to establish
endpoints for NHP bioaerosol challenges
5797-13
NHP Exposure Data
FY07-083 Naïve Cynomolgus Macaque Francisella tularensis S4 Schu4 Bioaerosol Challenge Data
Animal ID
Challenge
Date
Inhaled
Dose
(CFU)
Tissue Culturec
Nx Dateb
a
Bloodd
Spleen
Liverd
TBLN
Lung
A04339
14-Dec-07
2670
27-Dec-07
1.70E+06
1.20E+06
3.50E+04
7.20E+06
5.70E+04
A04344
14-Dec-07
5030
28-Dec-07
BLD
2.60E+02
2.20E+02
6.70E+02
8.20E+04
a
Dose is based on viable bacteria collected into an all-glass impinger following achievement of 3.5L inhaled
b
Animal A04339 died prior to euthanasia
c
Blood data presented as CFU/mL; tissue data presented as CFU/g
d
Bacteria cultured from the blood of A04339 and the liver of A04344 were inconsistent with F. tularensis
5797-14
Milestone #4 – Confirmation of Aerosol in vivo in NHP



MS3 demonstrated increased bioaerosol stability when S4 Schu4
was grown on BCGA; however, NHP exposures demonstrated
decreased virulence
Additional mouse exposures planned in near future
–
BCGA culture (72h fresh)
–
Chamberlain’s (48h fresh)
–
Chamberlain’s (frozen from 48h fresh)
Initial mouse data indicate that frozen stock may be optimal
method for NHP challenge
5797-15
Milestone #2 – Vaccinations of Personnel
Plans for this month


Fifth group of personnel to be vaccinated 8JAN08
Continue follow-ups with vaccinated personnel through day 28
post vaccination and then at 1 year post vaccination
5797-16
Milestone #3 – Bioaerosol Development
Plans for this month




Decide upon aerosol challenge approach
–
Generator
–
Culture procedure
–
Need to weigh virulence vs. aerosol stability
Plan to perform addition mouse testing with various culture
techniques
–
Confirm previous frozen stock results
–
Test BCGA
–
Test 48h Chamberlain’s
Further optimization of bioaerosol techniques?
Initiate MS Completion report
5797-17
Milestone #4 – Confirmation of Aerosol in vivo in NHP
Plans for this month

Schedule additional mouse bioaerosol challenges using
challenge material harvested from various S4 Schu4 growth
methods (see previous slide)
–
These data will be necessary in order to plan repeat naïve
and all future NHP challenges
5797-18
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
5797-19
Update on IFNg ELISPOT detection and proliferation
from LVS-vaccinated and non-LVS vaccinated NHPs

We continue to screen PBMCs from non-LVS vaccinated NHPs
for their ability to respond to LVS
–
We are varying the numbers of PBMCs/well (150,000 or
200,000 for IFNg ELISPOT) and the amount of LVS used to
stimulate the PBMCs
5797-20
450
400
Media
LVS hk Hi
LVS ff Hi
350
300
250
200
150
100
A05477
A04367
A04344
A04339
A04274
A04168
A03033
0
A03016
50
A02386
IFNgamma Spots (Mean +/- SEM)
Non-LVS Vaccinated NHPs Respond Variably to LVS
by IFNg Secretion
5797-21
Non-LVS Vaccinated NHPs Respond Variably to LVS
by Proliferation
1.00E6
8.00E5
Media
LVS hk Hi
LVS ff Hi
6.00E5
4.00E5
A05477
A04367
A04344
A04339
A04274
A04168
A03033
A03016
A02386
A00937
A00908
0
A00896
2.00E5
5797-22
Non-LVS Vaccinated NHPs Generally have Low IgG
anti-LVS Titers
5000
4000
3000
2000
A05477
A04344
A04339
A04168
A03033
A03016
A02386
A00937
A00908
A00902
0
A00896
1000
A00868
IgG anti-LVS Titer
6000
5797-23
2.00E5
Media
LVS hk Hi
LVS ff Hi
1.50E5
1.00E5
5.00E4
0
A04339
A04344
180
IgG anti-LVS Titer:
A04339 = 200
A04344 = 1000
Cell Mean for IFNg Spots
Relative Luciferase Units
(Mean +/- SEM)
NHPs chosen for SCHU S4 aerosol exposure
Media
LVS hk Hi
LVS ff Hi
160
140
120
100
80
60
40
20
0
A04339
A04344
5797-24
Remaining questions about IFNg ELISPOT assay


How does the freeze/thaw process affect LVS-stimulated IFNg
secretion?
Are there any differences when comparing ID- vs. SC-vaccinated
NHPs?
5797-25
MS 12/13: Plans for the next month


Test IFNg secretions from ID and SC vaccinated NHPs on the
same day
Screen additional non-LVS vaccinated NHPs to see if they
respond to LVS by proliferation or IFNg secretion
5797-26
Action Items
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Trevor: MS 3 Milestone completion report will be written
Trevor/Julie: Need gross necropsy summary, and histology/microbiology
report on NHP# 4334 and #4339 (Gross was provided on 1/18/08)
Bob: Need mouse data before deciding on BCGA vs Chamberlains growth
with SCHU S4?
Bob: Need to determine SCHU S4 dose range and response in NHP model
Julie: will look at RBC contamination on NHP#2386 and other 8 NHP on slide
21
Julie: Freyja requested that LBERI use log scale for future IgG Ab titer figures
Julie: Will re-bleed naïve NHP and look at consistency of IFNgamma Elispot
assay for background levels per individual animal
Vicki : will ask investigator working on NHP LD50 study if NIAID can get a
report from that study.
5797-27