LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
6 January 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
#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
Milestone #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)
25 CFU
250 CFU
Round 3 (n=4 NHP each dose)
250 CFU
Red: completed
Green: in progress
Blue: steps in the milestone
500 CFU
LD50/ED50
Determination
Slide 3
Milestone #7 - Tentative Endpoints

The endpoints for each set of exposures will be clinical observations,
temperature and respiration monitoring, body weight records, gross
necropsy, hematology, and viable bacterial blood/tissue cultures
Slide 4
Milestone #7- Wave 2 Survivor Data
•
Wave 2 lone survivor animal 28624 was euthanized on
December 2, 2008, 46 days post-challenge. There was no
culturable F. tularensis in any tissues other than the TBLN
which had 350 cfu/g. The lung and TBLN were grossly
contaminated with at least 3 colony types of bacteria.
Slide 5
Milestone #7- Wave 3 Hematology Data
Slide 6
Milestone #7 – NHP SCHU S4 ED50
Plans for next month
 Identify 3 colony types from Wave 2 lone survivor’s lung and
TBLN.
 Histopathology for all 3 waves is pending
Slide 7
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=3
previously vaccinated; 1 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 8
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 9
Milestone #8 - December 2008
Accomplishments


Finished analyzing the results from assessment of immunity to
LVS on Days 28 and 35 post-vaccination of the 5 NHPs (3 via
scarification and 2 via subcutaneous) that were vaccinated with
LVS in October 2008.
Prepared the study protocol for the challenge study which will
consist of 9 LVS vaccinated animals ( 3 by scarification and 3 by
subcutaneous route in 1/8/09, 3 previously vaccinated in 10/06, 2
by intradermal route and 1 by subcutaneous route) and 3 naïve
animals.
Slide 10
LVS Vaccination by Either Route Induces PBMC
Proliferation
8.00E5
6.00E5
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
4.00E5
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
0
Day 7, Scarification
2.00E5
Day 7, SC
RLU (Mean +/- SEM)
1.00E6
Note: No stimulus induced more than 100,000 RLU prior to vaccination
Slide 11
LVS Vaccination by Either Route Induces PBMCs to
Secrete IFNγ
Media
LVS hk Hi
LVS ff Hi
SCHUS4 hk Hi
SCHUS4 ff Hi
400
300
200
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
0
Day 7, Scarification
100
Day 7, SC
IFNgamma Spots
(Mean +/- SEM)
500
Note: No stimulus induced more than 80 spots prior to vaccination; all NHPs responded;
One scarified NHP had too many spots to count in response to LVS antigens on d15, d21
and d28 (but when PBMCs were cultured at less cells/well (0.67 – 1 x 106/well) there were
no differences in the pattern of responsiveness between SC and Scarified NHPs)
Slide 12
Milestone #8 – LVS Vaccination
Plans for next month
•
Pre-screen newly arrived NHPs to assess ability to respond to
LVS antigens (4 each scheduled for 1/5 and 1/19). Nonresponders will be placed on study as non-vaccinated controls.
•
Vaccinate 6 naïve animals with LVS (3=scarification and
3=subcutaneous); scheduled for 1/8. These NHPs have been
previously screened and are known to have low IgG anti-LVS
titers. Vaccination efficacy (LVS- and SCHU S4-specific
proliferation and IFNγ secretion) will be measured on 2/2/09 (day
25 post-vaccination).
Slide 13
Milestone #8 – LVS Vaccination
Plans for next month (continued)
•
PBMCs will be prepared on 1/22 from 3 NHPs previously
vaccinated with LVS in 10/06. This will serve as their prechallenge baseline evaluation (LVS- and SCHU S4-specific
proliferation and IFNγ secretion will be measured).
•
A00868, previously vaccinated with LVS by s.c. route in 10/06,
will receive an LVS bronchoscopy on 1/8/08; he has behavioral
problems that preclude his entry into the ABSL3 for SCHU S4
challenge; we will collect PBMCs prior to the bronchoscopy (1/5)
as well as 12 days post bronchoscopy (1/20); this protocol is
similar to that of the animal who required euthanasia due to
agreesive behavior (ate Tyvek sleeve)
Slide 14
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 Standard Growth Curve Qualification
Prepare Aerosol SOP
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 15
Milestone #9 - Objective

Develop a SOP compatible with GLP transition for aerosol delivery of
Schu S4.
Slide 16

Milestone #9 - December 2008
Accomplishments
Outlined plan to prepare and finalize an aerosol SOP
Pre-qualification data is currently being compiled and
organized.
Aerosol qualification will be performed on 3 different days by 3
different operators. Approximately 6 sprays will be performed on
each day. Based on data collected to date, the qualified range
will be 250 to 1000 cfu.
Slide 17



Milestone #9 – Aerosol SOP Development
Plans for next month
Complete qualification plan for the aerosol and perform aerosol
qualification.
Based on the results of the qualifications modify the draft
aerosol SOP.
Begin work on the Milestone Completion report.
Slide 18
Milestone #11 – In vivo GLP NHP model
efficacy SOP and testing of vaccine candidates
MS #11: NHP Model SOP Development and Vaccine Efficacy
NHP Model SOP
Vaccine Efficacy
NHP challenge (n= 32)
Vaccinate (n=TBD)
500 cfu
NHP challenge
SOP Development
500 cfu
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 19


Milestone #11 - December 2008
Accomplishments
Submitted IACUC protocol.
Submitted ES&H work review.
Slide 20
Milestone #11 - In vivo GLP NHP model
efficacy SOP and testing of vaccine candidates
Plans for next month


IACUC protocol and ES&H work review will be reviewed.
Order animals.
Slide 21
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 22


Milestone #12/13 - December 2008
Accomplishments
Continued to test freeze/thaw protocol.
Continued to test O-mutant antigens and discovered that the
protein concentrations of our preparations do not correspond to
the estimated CFU/ml and are thus not equivalent.
Slide 23
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
We obtained O-mutant organisms (LVS and SCHU S4) from Anders Sjostedt
and tested them in our assays
Given what appeared to be differences in the responses to these mutant
antigens, we tested the protein content of each antigen preparation to make
sure that we were delivering equivalent doses to the wells (currently using
organisms/ml to normalize each antigen preparation but with the fixed and
heat-killed treatments it is difficult to confirm that the same number of
organisms are still there after treatment)
– The protein content analysis (BCA) showed that the most of the antigen
preparations were NOT equivalent and thus previous results must be reanalyzed
Slide 24
Results of Protein Assay
Antigen
Protein
Concentration (in
μg/ml)
Final
Concentration in
Proliferation Assay
(in μg/ml)
Final Concentration
in ELISPOT Assay
(in μg/ml)
FF LVS WT
135.98
0.19
0.18
FF LVS Mutant
29.46
0.008
0.008
HK LVS WT
1119.83
1.9
1.8
HK LVS Mutant
1223.57
0.022
0.02
FF SCHU S4 WT
149.23
0.005
0.004
FF SCHU S4
Mutant
3.41
0.0003
0.00033
HK SCHU S4
WT
1330.84
0.04
0.04
HK SCHU S4
Mutant
859.21
0.072
0.068
Fold
Difference
from WT
(average)
23
90
14
0.57
Slide 25
IFNg Production in Response to O-mutants in Non-LVS
vaccinated NHPs
14x in conc.
Media
IFNg Spots (Mean +/- SEM)
160
LVS ff Hi
140
120
SCHUS4 ff Hi
23x in conc.
LVS ff Mutant Hi
100
SCHUS4 ff Mutant Hi
80
60
40
20
0
28461
A05403
A06199
Responses to mutants may be decreased only because the antigen
concentration is decreased
Slide 26
400
350
300
*
Media
SCHUS4 hk Hi
*
SCHUS4 hk Mutant Hi
*
250
*
200
*
150
100
*
*
Day 35, Scarification
Day 35, SC
Day 28, Scarification
Day 28, SC
Day 21, Scarification
Day 21, SC
Day 15, Scarification
Day 15, SC
0
Day 7, Scarification
50
Day 7, SC
IFNgamma Spots (Mean +/- SEM)
IFNg Production in Response to SCHU S4 HK O-mutants is often Increased
in LVS vaccinated NHPs
Response to HK SCHU S4 mutant is varied but often increased and may be due to the
1.75x increase in concentration. * indicates significant difference (p < .05) from the
other two stimuli in the group by one-way ANOVA.
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
RLU (Mean +/- SEM)
Neither Protocol Spares the Responsiveness of
PBMCs to LVS as Measured by Proliferation
350000
Media
300000
LVS hk Hi
Day 7 post-LVS * *
vaccination
LVS ff Hi
250000
200000
SCHUS4 hk Hi
SCHUS4 ff Hi
150000
100000
Scarification, None
Scarification, CTL
Scarification, Cerus
SC, None
SC, CTL
0
SC, Cerus
50000
Only Day 7 post-LVS vaccination is shown; Note that peak proliferation does not
occur until Day 28 post-LVS; * indicates significantly different (p<.05) than
media by one-way ANOVA
Slide 29
Milestone #12/13 - Immune Responses in
Animals and Humans
Plans for next month


Continue to analyze the results of freeze/thaw protocols
Re-titrate the WT and mutant antigens based on protein content
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 - December 2008
Accomplishments
Maintenance of 2 s.c. LVS-vaccinated NHPs transferred to MS 29
IACUC amended to allow for LVS bronchoscopy
Slide 32
Milestone #29 - Analysis of T cells from lymph
nodes and T cell epitopes
Plans for next month

LVS bronchoscopy will likely be scheduled for late January or
early February
Slide 33
Action Items






Julie Wilder will switch to standard deviation rather than standard deviation of the mean
in ELISspot data presentation for the future.
Tevor will convert the 2nd and 3rd boxes to red , on the MS#9 flow diagram slide , as
LBERI has plenty of data to date to use for this from prior experiments
Michelle will include in her nonTVDC talk for Kristin, the data for developing a
qualification plan for standard growth curves and the performing standard growth curve
qualifications.
Julie Wilder will titer each HK, FF , wt LVS, wt SCHU S4, o mutant LVS, O mutant SCHU
S4 antigen type by protein concentration, using cells from a vaccinated animal.
Julie Wilder will do the antigen titration and then optimize the proliferation and ELISpot
assays.
Julie Wilder will develop a plan for fresh or frozen cells based on average cell yield.
Slide 34