LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
3 February 2009
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Milestones
#2
Active
Vaccinations of study personnel- no work this
month
#4
Active
Confirmation of aerosol in vivo in NHP efficacy
studies in primates
#7
Active
SCHU S4 LD50 in primates
#8
Active
LVS vaccination protection of aerosol Schu4
validated in primates
#9
Active
Aerosol SOP developed for GLP transition
#10
Active
Efficacy Testing of vaccine candidates in NHP
(USAMMDA vaccine)
#11
Active
In Vivo GLP model efficacy SOPs developed in
one small species and primate and efficacy testing
of vaccine candidates
#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
#29
Active
Analysis of T cells from lymph nodes and T cell
epitopes
Slide 2
MS#8 – Flow Diagram
MS 8: LVS Vaccinated NHP Challenged with SCHU S4
Round 1 Vaccination Practice/Challenge (n=3 scarification; n=2
subcutaneous)
Round 2 Vaccination/Challenge (n=3 by
scarification; n=3 by subcutaneous route; n=4
previously vaccinated; 2 SC, 2 ID)
SCHU S4 Challenge 500 CFU
Round 3 Challenge (USAMMDA vaccine vs. DVC
LVS Lot 16 by scarification)
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
500 CFU
Slide 3
Milestone #8 - Objective and Endpoints


Describe the natural history of aerosol delivered SCHU S4 infection in
NHPs that have been previously vaccinated with LVS. Two different
methods of vaccination will be compared (scarification and
subcutaneous).
Endpoints include histopathology and bacterial CFUs of internal organs
(lung, spleen, liver, kidneys, and lymph nodes), twice a day records of
clinical symptoms post-infection, and clinical chemistry and
hematology during infection.
Slide 4
Milestone #8 – January 2009
Accomplishments
Vaccinated 6 NHPs with LVS on 1/8/09 ( 3 by scarification and 3
by subcutaneous route)
Screened 7 newly arrived non-LVS vaccinated NHPs and chose 3
to serve as controls for the upcoming SCHU S4 challenge
3 NHPs previously vaccinated with LVS in 10/06 (2 by intradermal
route and 1 by subcutaneous route) were tested for residual
immunity to LVS
Slide 5
400
300
Media
LVS hk Hi
LVS ff Hi
250
SCHUS4 hk Hi
350
200
150
100
SCHUS4 ff Hi
*
50
0
A06626
A06688
A07046
IgG anti-LVS titers:
A06626: 20,000
A03152: 20,000
A05895: 20,000
A07058
IFNg Spots (Mean +/- S.D.)
IFNg Spots (Mean +/- S.D.)
Screening of non-LVS Vaccinated NHPs for Antigenspecific IFNγ Production
500
450
400
Media
LVS hk Hi
350
300
250
200
150
SCHUS4 hk Hi
LVS ff Hi
*
SCHUS4 ff Hi
*
100
50
0
A03152
A04840
A05895
All cells plated at 1.33 x 106/ml; * indicates NHP chosen as control
Slide 6
500000
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
400000
300000
*
200000
100000
0
A06626
A06688
A07046
A07058
Relative Light Units (Mean +/- S.D.)
Relative Light Units (Mean +/- S.D.)
Screening of non-LVS Vaccinated NHPs for Antigenspecific Proliferation
600000
Media
LVS hk Hi
500000
LVS ff Hi
400000
SCHUS4 hk Hi
SCHUS4 ff Hi
300000
*
200000
100000
*
0
A03152
A04840
A05895
All cells plated at 1 x 106/ml; * indicates NHP chosen as control
Slide 7
IFNg Spots (Mean +/- S.D)
Antigen-stimulated IFNγ Production by Previously
Vaccinated NHPs
400
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
350
300
250
SCHUS4 ff Hi
200
150
100
50
NT
0
A00659
A00908
A00937
All cells plated at 1.33 x 106/ml; Day post-LVS vaccination = 786 (A00659, SC) – 795
(A00908 and A00937, ID); A00937 had insufficient cells to test either SCHU S4 antigen
Slide 8
400
12 days postLVS boost
Media
350
LVS hk Hi
300
LVS ff Hi
250
SCHUS4 hk Hi
200
SCHUS4 ff Hi
150
100
Day 783
Day 768
Day 523
Day 495
Day 442
Day 414
Day 288
0
Day 237
50
Day 195
IFNg Spots (Mean +/- S.D)
Update on A00868: Effect of LVS Boost on IFNγ
Production
LVS Vaccination by S.C. Route in 10/06; boosted with LVS
by bronchoscopy on 1/8/09
Slide 9
Milestone #8 – LVS Vaccination
Plans for next month
•
NHPs vaccinated with LVS on 1/8/09 were bled on 2/2/09 to
assess their ability to respond to LVS and SCHU S4 antigens;
and to test their IgG anti-LVS titer
•
9 LVS vaccinees and 3 non-LVS vaccinated NHPs will be
challenged with aerosol SCHU S4 (500 CFU target) on 2/12 and
2/13
• Clinical symptoms (observations 3x daily), clinical chemistry,
hematology and bacteriology (first 6 days post SCHU S4
exposure) will be measured on all
• Any survivors will be euthanized on day 21 post-challenge and
tissues taken for pathology, microbiology and immunologic
assessments
Slide 10
Milestone #9 – Aerosol SOP Development
MS #9: Aerosol SOP Development
Develop Qualification Plan for Standard Growth Curve
Perform Standard Growth Curve Qualification
Develop Qualification Plan for Aerosol
Perform Aerosol Qualification
Prepare Aerosol SOP
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 11
Milestone #9 - Objective

Develop a SOP compatible with GLP transition for aerosol delivery of
SCHU S4.
Slide 12
Milestone #9 - January 2009 Accomplishments



Pre-qualification data compiled and organized
Draft Qualification Plan submitted
Two days of mock qualification aerosols performed in an attempt
to achieve criteria outlined in Qualification Plan
–
Criteria were defined using data from previous SCHU S4
bioaerosols (prepared using similar growth conditions) at
LBERI
 See following slides
Slide 13
SCHU S4 Bioaerosol Qualification Performance Criteria
Table 1. Exposure System Tests and Performance Criteria for the Francisella tularensis SCHU S4 Target
Atmosphere
Test
Target Value
Acceptable Performance
Criteria
Acceptable Values
Generator suspension
concentration
2.00 x 106 CFU/mL
(log10 = 6.30)
± 0.500 log10 between
estimated and actual
concentration
log10 =
5.80 to 6.80
Generator suspension
stability
n/a
± 0.800 log10 between preand post-test generator
concentration
As stated
Chamber concentration
143 CFU/L
68 to 327 CFU/L
Repeatability
 40% Relative Standard Deviation (RSD) on the chamber concentration (log10
values) between the five daily runs per qualification day and between all fifteen
runs conducted for all three qualification days.
Collection efficiency of
impinger
 90% collection efficiency
Spray factor
log10 = -6.94
± 3 SD from the historical
mean per runa
Particle size
< 4 μm median diameter
T90
To be determined
T10
To be determined
Critical
component
log10 =
-8.09 to -5.79
These criteria were defined based on a target presented dose of 500 CFU in
3.5 liters inhaled and pre-qualification data including actual NHP exposures.
Slide 14
Francisella tularensis SCHU S4 24-hour Growth Curve
CFU/mL (log10 scale)
9.8
9.6
9.4
y = 10(0.1193x + 9.188)
r 2 = 0.8095
9.2
9.0
0.000
1.000
2.000
3.000
These data are based upon
24h Chamberlains broth
cultures using the optimized
growth method. The values
are tight, most ranging from
1.6 to 2.8 x 109 CFU/mL.
4.000
OD600
July 2, 2008
July 9, 2008
July 24, 2008
July 25, 2008
November 10, 2008
November 10, 2008
November 10, 2008
September 23, 2008
September 24, 2008
October 17, 2008
November 18, 2008
January 14, 2009
January 14, 2009
January 14, 2009
Norm OD600
3.390
3.470
3.510
3.480
1.000
0.704
0.534
1.900
3.050
0.777
0.965
0.597
0.477
0.552
CFU/mL
4.00e+009
4.55e+009
3.20e+009
3.85e+009
2.17e+009
1.20e+009
1.63e+009
2.80e+009
4.00e+009
1.30e+009
2.64e+009
1.63e+009
2.47e+009
1.63e+009
Semilog line -- X is linear, Y is log
Best-fit values
Yintercept
Slope
Std. Error
Yintercept
Slope
95% Confidence Intervals
Yintercept
Slope
Goodness of Fit
Degrees of Freedom
R²
Absolute Sum of Squares
Sy.x
Number of points
Analyzed
9.188
0.1193
0.05133
0.01783
9.076 to 9.300
0.08050 to 0.1582
12
0.8095
3.110e+018
5.091e+008
14
Slide 15
Scatter Dot Plot of Francisella tularensis SCHU S4 (Working Stocks Passaged from Seed
Stock Lot 24AUG07) Bioaerosol Spray Factors using the Head-Only Exposure Chamber
6.0010 -7
Spray Factor
5.0010 -7
4.0010 -7
3.0010 -7
2.0010 -7
1.0010 -7
0
Spray Factor Data
Individual Bioaerosol Runs
Number of values
57
Minimum
25% Percentile
Median
75% Percentile
Maximum
1.19e-008
6.74e-008
1.39e-007
2.22e-007
5.72e-007
5% Percentile
95% Percentile
1.93e-008
3.82e-007
Mean
Std. Deviation
Std. Error
1.58e-007
1.15e-007
1.52e-008
Lower 95% CI of mean
Upper 95% CI of mean
1.28e-007
1.89e-007
D'Agostino & Pearson omnibus normality test
K2
P value
Passed normality test (alpha=0.05)?
P value summary
Sum
Mean: 1.58 x 10-7
Min: 1.19 x 10-8
Max: 5.72 x 10-7
16.0
0.0003
No
***
9.02e-006
Slide 16
Scatter Dot Plot of Francisella tularensis SCHU S4 (Working Stocks Passaged from Seed
Stock Lot 24AUG07) Bioaerosol Spray Factors using the Head-Only Exposure Chamber
log10 Spray Factor
-6.00
-6.50
-7.00
-7.50
Spray Factor Data (log10)
-8.00
Individual Bioaerosol Runs
Number of values
57
Minimum
25% Percentile
Median
75% Percentile
Maximum
-7.92
-7.17
-6.86
-6.65
-6.24
5% Percentile
95% Percentile
-7.72
-6.42
Mean
Std. Deviation
Std. Error
-6.94
0.383
0.0507
Lower 95% CI of mean
Upper 95% CI of mean
D'Agostino & Pearson omnibus normality test
K2
P value
Passed normality test (alpha=0.05)?
P value summary
Mean: -6.94
Min: -7.92
Max: -6.24
-7.04
-6.83
5.08
0.0791
Yes
ns
Slide 17
Scatter Dot Plot of Francisella tularensis SCHU S4
(Working Stocks Passaged from Seed Stock Lot 24AUG07)
Cynomolgus Macaque Bioaerosol Exposure Times
using the Head-Only Exposure Chamber
Exposure Time (min)
30
25
20
15
10
5
0
Individual Bioaerosol Runs
Number of values
Though most exposure
Times fell within 7-15
min, a period of 25 min
was chosen for
Qualification runs in
order to ensure
validation with animals
demonstrating low
minute volumes (i.e.,
longer exposure times).
Sample Time
30
Minimum
Maximum
6.730
24.48
Mean
Std. Deviation
Std. Error
12.77
5.000
0.9128
Slide 18
21JAN09 Mock Qualification Run Data
PASS
PASS
FAIL
PASS
PASS
PASS
PASS
Slide 19
21JAN09 Mock Qualification Run Data

3 out of 5 mock qualification runs demonstrated aerosol
concentrations within the defined criteria:
–
CFU/L
 36
 75
 111
 142
 351
Slide 20
28JAN09 Mock Qualification Run Data
PASS
PASS
FAIL
PASS
PASS
PASS
PASS
Slide 21
28JAN09 Mock Qualification Run Data

3 out of 5 mock qualification runs demonstrated aerosol
concentrations within the defined criteria:
–
CFU/L
 362
 207
 144
 228
 568
Slide 22
Conclusions and Discussion


Based on mock qualification results, we can consistently get
close to a target dose of 500 CFU (143 CFU/L). However, it has
proven difficult to attain this concentration each and every time.
What are the acceptable limits?
Slide 23
Milestone #9 – Aerosol SOP Development
Plans for next month



Final and sign Qualification Plan
Begin Qualification runs
–
Three separate days
–
Three separate Micro technicians
–
Three separate Bioaerosol technicians
–
Criteria as defined on previous slides
Based on Qualification data, initiate aerosol SOP.
Slide 24
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 25



Milestone #12/13 - January 2009
Accomplishments
Continued to test freeze/thaw protocol
Discussed with the UNM and LRRI team how to standardize the
LVS and SCHU S4 antigens (protein content vs. CFU/ml)
Discussed with Freyja Lynn how to proceed on describing the
nature of the plasma IgG anti-LVS levels
– Decided to make a positive and negative control reference
plasma stock by combining several plasma samples that
perform similarly in the ELISA assay
– aliquots of the pooled plasma would then be frozen and
thawed each time the assay was run
– arbitrary units of activity could be assigned and used as a
reference standard curve
Slide 26
Update on testing O-antigen mutants as stimuli in the IFNg ELISPOT and
Proliferation Assays




We were interested in testing whether the non-specific responses to LVS and
SCHU S4 antigens we observe in PBMCs from non-vaccinated NHPs in the
proliferation and IFNg ELISPOT assays were due to LPS moeities on the fixed
and heat-killed organisms (O- antigen mutants obtained from Anders Sjostedt
When the protein content of each antigen preparation was tested using a BCA
Kit, we realized they were not equivalent
Our plan is to construct a standard curve correlating CFU/ml and protein
content using LVS and SCHU S4; aliquots will be plated and lysed; lysates will
be measured for protein content
Preparations of heat-killed and formalin-fixed LVS will also be lysed and
measured for protein content; the standard curve will allow correlation to
CFU/ml
Slide 27
Update on Freeze/Thaw Testing



We have been comparing two protocols (Cerus and CTL) for use
in freezing and thawing PBMCs
The goal is to find a protocol which results in PBMCs whose
response mimics the response of the original fresh PBMCs in the
proliferation and IFNγ ELISPOT assay
In the past months, we have only had PBMCs from non-LVS
vaccinated NHPs to compare; we are now beginning to thaw
PBMCs from the NHPs which were vaccinated with LVS in
October 2008
Slide 28
600
500
400
300
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
200
Day 28, None
Day 28, CTL
Day 28, Cerus
Day 21, None
Day 21, CTL
Day 21, Cerus
Day 15, None
0
Day 15, CTL
100
Day 15, Cerus
IFNg Spots (Mean +/- S.D)
Freezing Protocols Appear Equivalent in Sparing the
Responsiveness of PBMCs to LVS as Measured by IFNγ
Production
All cells plated at 1.33 x 106/ml
We would like to choose Cerus as the freezing protocol moving forward
Slide 29
Milestone #12/13 - Immune Responses in
Animals and Humans
Plans for next month
Determine the relationship between LVS protein content and
CFU/ml
Begin to re-titrate the WT and mutant LVS antigens based on
protein content
Construct reference positive and negative control plasma and
test in IgG anti-LVS ELISA
Slide 30
Milestone #29 – Analysis of T cells from lymph
nodes and T cell epitopes
Vaccinate NHPs with LVS
(2 S.C. Vaccinees 10/08)
Boost LVS Immunity by Bronchoscopy with LVS
Collect LNs
Transfer to UNM for testing in Peptide Librarystimulated ELISPOT Assays
Slide 31


Milestone #29 - January 2009
Accomplishments
LVS bronchoscopy scheduled for 2/11
Harvest of tissues will be on 2/23
Slide 32
Milestone #29 - Analysis of T cells from lymph
nodes and T cell epitopes
Plans for next month


LVS bronchoscopy will take place and tissues transferred to
UNM for use in peptide library screening
We will also screen PBMC responses before and after LVS boost
and test spleen cell responses at harvest
Slide 33
Action Items




Trevor- will submit the MS 9 prequalification QA plan to UNM
Trevor will follow the ICH bioanalytical guideline for the
qualification criteria; Trevor will review Bioanalytical published
quality criteria
Trevor- will test shorter than 25 minute exposure times during
some qualifying runs, to examine the relative dose at less than
25min maximum aerosol exposure.
Julie Wilder: will summarize the pertinent January data at the
March LBERI technical call.
Slide 34