LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
4 August 2009
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Active Milestones
#2
Active
Vaccinations of study personnel- no work in
July
#8
Active
LVS vaccination protection of aerosol Schu4
confirmed in primates
#9
Active
Aerosol SOP developed for GLP transition
#10
Active
Efficacy testing of vaccine candidates
(LBERI)- no work in July
#11
Active
In Vivo GLP NHP model efficacy SOPs 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- no work in July
Slide 2
MS#8 – LVS Vaccinated NHP Challenged with
SCHU S4
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 Vaccination/Challenge (Vaccination
with Highest Dose of LVS attainable by
scarification and s.c.)
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
1000 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.
– Compare two different methods of vaccination (scarification and
subcutaneous).

Endpoints
– histopathology
– bacterial CFUs of internal organs (lung, spleen, liver, kidneys, and
lymph nodes)
– records of clinical symptoms post-infection
– clinical chemistry and hematology during infection
Slide 4
Milestone #8 – July 2009 Accomplishments





12 LVS vaccinated NHPs and 3 naïve controls were challenged with
1000 CFU SCHU S4
Post-SCHU S4 challenge observations (three times daily) were
performed (temperatures and respirations).
Blood was collected for bacteriology and clinical chemistry on d3, and
weekly thereafter on the survivors.
Complete necropsies with tissues being collected for histology and
bacteriological burden were performed on animals undergoing
moribund or scheduled euthanasia or animals found dead.
NHPs that survived to day 21 post-SCHU S4 challenge ( n= 2) were
euthanized. Complete necropsies were performed and spleen and
PBMCs were collected for immunological assessment.
Slide 5
Milestone #8- Overview of Survival Data
Vaccination
Cohort
Animal ID
Vaccine
Status
Date; Hour of
Challenge
1 (1 x 107 LVS)
A06694
Control
7/8; 9:20 A.M.
1 (1 x 107 LVS)
A06873 (F)
Scarified
7/8; 9:45 A.M.
1 (1 x 107 LVS)
A07386 (F)
Scarified
7/8; 10:40 A.M.
1 (1 x 107 LVS)
1 (1 x 107 LVS)
A07395 (F)
A07418 (F)
S.C.
S.C.
7/8; 10:00 A.M.
7/8; 11:00 A.M.
2 (1 x 107 LVS)
A06882
Control
7/8; 11:30 A.M.
2 (1 x 107 LVS)
A06674 (M)
Scarified
7/8; 11:50 A.M.
SCHU S4
Presented
Dose (CFU)
865
1240
1260
1410
791
2140
Date/Hour of Death
Notes
7/22; 9 a.m. (Day 14; 336 h)
Died during morning observations
7/15; 9 a.m. (Day 7; 167 h)
Euthanized morning of 7/15
7/18; 9 a.m. (Day 10; 238.5
hours)
7/29; Day 21 – Terminal SAC
7/29; Day 21 – Terminal SAC
Found dead at morning obs
7/14; 7:30 p.m. (Day 6; 152 h)
Found dead at evening obs
7/14; 8:40 p.m. (Day 6; 153 h)
Found moribund; collapsed after evening obs;
received anesthesia and seized before
euthanasia administered
Euthanized evening of 7/14
3740
2 (1 x 107 LVS)
A07682 (F)
Scarified
7/8; 1:30 P.M.
2 (1 x 107 LVS)
A06675 (M)
S.C.
7/8; 12:50 P.M.
2 (1 x 107 LVS)
A07566 (F)
S.C.
7/8; 2:00 P.M.
3 (1 x 106 LVS)
A07427
Control
7/10; 10:10 A.M.
3 (1 x 106 LVS)
A06693 (M)
Scarified
7/10; 10:30 A.M.
3 (1 x 106 LVS)
A07686 (F)
Scarified
7/10; 10:55 A.M.
3 (1 x 106 LVS)
A06702 (M)
S.C.
7/10; 11:20 A.M.
3 (1 x 106 LVS)
A07610 (F)
S.C.
7/10; 11:50 A.M.
1690
2610
1250
1950
1330
2080
500
519
7/14; 9:05 p.m. (Day 6; 152.5 h)
7/14; 7:45 p.m. (Day 6; 151 h)
Euthanized on Day 21 - SURVIVOR
Euthanized on Day 21 - SURVIVOR
7/14; 2:30 p.m. (Day 6; 144.5 h)
Found moribund on bottom of cage; died
before anesthesia for euthanasia administered
Died during observation period
7/15; ~7:30 p.m. (Day 5; 129 h)
Euthanized evening of 7/15
7/17; 8 p.m. (Day 7; 178.5 hours) Euthanized evening of 7/17
7/18; 3 p.m. (Day 8; 196 hours)
Euthanized afternoon of 7/18
7/18; 9 a.m. (Day 8; 189.5 hours) Euthanized morning of 7/18
7/15; ~8:30 p.m. (Day 5; 129 h)
Euthanized evening of 7/15
Slide 6
Tissue Burdens
CFU/g
Mes LN
Animal ID
Vaccine
Group
Presented
Dose (CFU)
Day to
Death
A06694
Control
865
14
2.16E+06 5.94E+03 3.92E+06 8.43E+06 1.41E+08
A06882
Control
2140
6
3.71E+08 2.75E+06 1.33E+06 6.84E+08 9.38E+08
A07427
Control
1950
5
1.91E+09 2.37E+07 4.87E+08 1.40E+09 2.34E+09
A06873
Scarified
1240
7
3.03E+07 2.19E+06 3.86E+03 6.49E+06 7.84E+08
A07386
Scarified
1260
10
2.60E+08 1.38E+06
BLD
1.68E+07 7.72E+08
A06674
Scarified
3740
6
1.16E+04 2.28E+03
BLD
6.11E+03 2.28E+08
A07682
Scarified
1690
6
2.08E+03 2.50E+03 9.08E+02 3.34E+05 7.96E+07
A06693
Scarified
1330
7
3.00E+05 4.56E+04
A07686
Scarified
2080
8
1.17E+04 1.63E+04 1.17E+04 8.23E+04 1.96E+08
A07395
S.C.
1410
21*
BLD
BLD
BLD
1.38E+04 1.36E+04
A07418
S.C.
791
21*
BLD
5.95E+01
BLD
1.37E+04 1.38E+04
A06675
S.C.
2610
6
1.24E+05 1.73E+04 5.66E+02 6.65E+05 1.32E+09
A07566
S.C.
1250
6
8.94E+03 3.10E+03
A06702
S.C.
500
8
6.44E+05 6.06E+04 2.34E+07 1.39E+07 2.66E+09
A07610
S.C.
519
5
4.92E+03 2.38E+03
Spleen
Liver
BLD
BLD
BLD
TBLN
Lung
7.82E+06 1.99E+09
3.01E+06 1.12E+08
1.73E+06 1.27E+08
a
BLD, below limit of detection; * Terminal sacrifice
Slide 7
Milestone #8- Microbiology Data
Animal ID
Presented Dose
Vaccine Group
Day to Death
(CFU)
Bacteremia (CFU/ml)
Day 3
Day 10
@ Necropsy
BLD
BLD
TNTC
A06694
Control
865
14
A06882
Control
2140
6
3.33E+00
N/A
No data
A07427
Control
1950
5
BLD
N/A
TNTC*
A06873
Scarified
1240
7
BLD
N/A
1.75E+02
A07386
Scarified
1260
10
BLD
N/A
No data
A06674
Scarified
3740
6
BLD
N/A
No data
A07682
Scarified
1690
6
BLD
N/A
BLD
A06693
Scarified
1330
7
BLD
N/A
BLD*
A07686
Scarified
2080
8
BLD
N/A
BLD
A07395
S.C.
1410
21*
BLD
BLD
BLD
A07418
S.C.
791
21*
BLD
BLD
BLD
A06675
S.C.
2610
6
BLD
N/A
No data
A07566
S.C.
1250
6
BLD
N/A
No data
A06702
S.C.
500
8
BLD
N/A
TNTC
A07610
S.C.
519
5
BLD
N/A
BLD
* Qualitative growth observed in broth
Slide 8
Milestone #8- Preliminary Data Interpretation




DVC Lot 16 fails to protect most NHPs from morbidity due to
aerosolized SCHU S4
Two survivors were both s.c. inoculated possibly suggesting
that s.c. inoculation with LVS provides slightly more protection
than scarification
Controls died between days 5 – 15
Vaccinees died between days 5 – 10 (exclusive of 2 who
survived)
Slide 9
Milestone #8 and #10 – Plans for next month


MS 8: Compile thrice daily temperature and respiration data
MS 10: Prepare for next experiment utilizing 25 NHPs



Write Study Protocol
Draw baseline blood for PBMC response to LVS in
immunological assays
Vaccinate s.c. with various forms of LVS:
–
–
–
–
–

3 Controls
3 s.c. with DVC Lot 16
3 s.c. with DVC Lot 17
8 s.c. with USAMMDA IND 157
8 s.c. with DVC Lot 20
This comparative study will be run under Milestone 10 (Testing
new vaccine candidates to elicit protection in primates)
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- July 2009 Accomplishments


Qualification Plan for aerosol was submitted to the Compliance
Group for review and approval.
Performed a practice run on July 23, 2009.
Slide 13
Milestone #9- Practice Run Data

Trevor will present data in September
–
In an email dated 7/28, Trevor stated:
–
The practice SCHU S4 qualification bioaerosols
conducted on 23JUL passed as per the most recent
Qualification Plan. The generator suspension
concentrations were uncharacteristically low, but we were
still able to obtain 100-200 CFU/L (based on AGI counts);
this would result in presented doses of about 350 to 700
CFU in 3.5 liters or, more likely, 400-800 CFU presented.
Slide 14
Milestone #9- Plan for next month


Compliance Group will complete review of the Aerosol
Qualification Plan.
Once the plan is approved, the aerosol qualification will be
executed.
Slide 15
MS#11 – GLP Model Efficacy SOPs Developed
in NHPs and Efficacy Testing of Vaccine
Candidates
GLP Model Efficacy SOPs Developed in NHPs and Efficacy
Testing of Vaccine Candidates
Non-Telemetered Natural History
SCHU S4 Challenge 1000 CFU
Telemetered Natural History Study
Red: completed
Green: in progress
Blue: steps in the milestone
SCHU S4
Challenge
1000 CFU
Slide 16
Milestone #11- Objectives and Endpoints


Describe the natural history of aerosol delivered Schu S4 in the
cynomolgus macaque.
Endpoints
Histopathology
Bacterial CFUs
Clinical symptoms
Clinical Chemistry
Hematology

Deliverables will include protocol(s) and documents necessary
for an aerosol primate model of Schu S4 compatible with Good
Laboratory Practice (GLP) that will meet FDA standards for
product development.
Slide 17
Milestone #11- July 2009 Accomplishments




NHPs received their third TB test.
Performed NHP physical exams.
Performed telemeter surgeries.
Initiated chair conditioning.
Slide 18
Milestone #11- Plans for next month

Challenges are scheduled for August 18th and August 19th.
Slide 19
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
Red: completed
Green: In progress
Yellow: on hold; restart if
necessary
Blue: steps in the milestone
Develop
Immunoassay
methodologies
IFNg
Proliferation
assay
ELISPOT
Microagglutination
assay
Determine
protein:CFU
relationship in
FF and HK LVS
antigens
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 20
Milestone #12/13 – July 2009 Accomplishments

No work was performed on this milestone in July
Slide 21
Milestone #12/13 – Plans for next month
We will consult with the UNM group on proceeding with the LVS and SCHU S4
protein:CFU assay
Terry’s group has thus far been unable to show a linear relationship
between LVS protein and CFU;
Considering a different lysis buffer and/or procedure (i.e. testing
sonication)
We requested a rabbit positive control sera from Dr. Sztein at the University of
Maryland in order to initiate the microhemagglutination protocol
UNM and LBERI will stain the LVS Lot#9 Ft antigen ourselves with
hematoxylin, using the USAMRIID protocol for staining
We can test both primate(LBERI), rat and human (UNM) LVS vaccinees
and use one of those as a positive control moving forward once the
assay is established
Slide 22
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 23
Milestone #21: Objectives and Endpoints



To establish the intracellular cytokine staining assay as a potential
correlate of protection in NHPs
Previous attempts at visualizing intracellular cytokine production by
LVS-vaccinated NHPs have been unsuccessful, potentially due to:
–
Testing at a non-optimized time point post-LVS vaccination
–
Using a non-optimized concentration of HK- or FF-LVS as
stimuli
–
Uncertainty as to performance of the anti-cytokine antibodies in
NHPs
Experiments conducted this month attempted to address some of these
factors
Slide 24
Milestone #21 – July 2009 Accomplishments











Attempt to detect intracellular cytokines in PBMC from LVS vaccinated NHP:
Cryopreserved PBMC from NHP A06873, collected and frozen at day 8 post LVS (1.1x107
by scarification on 5/18/2009), were thawed and stimulated at 4x105 cells per well for a
total of 6 h (4 h with Golgi plug) with either of:
Media only
8x103 hkLVS or ffLVS per well
4x104 hkLVS or ffLVS per well
2x105 hkLVS or ffLVS per well
1 ug PHA or Con A per well
10ng PMA/100ng ionomycin per well
As an additional control for cytokine staining HiCK-1 cells were permeabilized and
stained along with the NHP samples
For automatic compensation with FACSDiva anti-mouse Ig Compbeads were single
stained with mAb-conjugates: IFN-g-FITC, TNF-a-PE, CD3-PerCPCy5.5, CD4-PECy7, IL-2APC, CD8-APCCy7. Non-stained PBMC were used for voltage gain PMT settings.
At the first attempt to run samples the Canto machine did not pass the Canto software
calibration, but had to be serviced, so the samples, including compensation setup
samples, had to be stored in PFA buffer for 3 days which affects the fluorescence
emission, in particular from tandem flurochromes such as APC-Cy7 and PerCP-Cy5.5.
Slide 25
Milestone #21 – July 2009 Accomplishments




Compensation issues:
Fluorochrome
-% Fluorochrome
Spectral overlap
FITC (IFNg)
PerCPCy5.5 (CD3)
133.58%
PE (TNFa)
PerCPCy5.5
130.6%
APC (IL-2)
PerCPCy5.5
212.78%
IL-2-APC is a rat-antibody and did not bind to the anti-mouse Ig Compbeads which is part
of the explanation for the above compensation issues. PerCP-Cy5.5, as a tandemflurochrome is not stable for prolonged storage in PFA contaning buffer.
Anti-rat Compbeads have been purchased, as well as a CD3-PerCP conjugate to
substitute for the CD3-PerCP-Cy5.5.
Despite the above compensation issues, lymphocytes were identified by FSC/SSC and
CD4 and CD8 surface stains were normal.
Slide 26
Milestone #21 – July 2009 Accomplishments

FSC/SSC gated lymphocytes analyzed for IFN-g and TNF-a:
Media only
4.4%
8000 LVS
3.8%
4x104 LVS
2x105 LVS
8.7%
59.2%
3.7%
1.3%
hkLVS
1.4%
1.5%
fkLVS
1.4%
PMA/iono
2.0%
HiCK-1
Slide 27
Milestone #21- Plans for next month


Repeat ICS experiment and run FACS Canto immediately after
PBMC staining. Set compensation with anti-rat Compeads for IL2-APC and use CD3-PerCP instead of CD3-PerCP-Cy55 which
appeared to cause large emission spectral overlaps. PerCP is a
less intense fluorochrome than PerCP-Cy5.5 and should also be
less sensitive to storage.
Titrate CD3-PerCPCy5.5 for possible use at a more optimal
staining concentration.
Slide 28
Milestone #21- Plans for next month
Emission differences between PerCP (top spectra viewer) and
PerCP-Cy5.5 (bottom spectra viewer) as adapted from BD Biosciences
Spectrum viewer for FACSCanto with applicable filters and flurochromes
Slide 29
Action Items









Matthew Hinz /DVC will send 6 vials of LVS lot#17 to UNM during week of 8/4/09
Barbara will request 6 vials of LVS Lot#20 from BEI, to arrive in less than a week and will CC Freyja
(NOTE AFTER CALL: Barbara emailed Susan Peacock on 8/4/09 and requested vials by 8/11/09)
Barbara asked Christopher Mortorff of USAMMDA to send the 3 vials of IND 157 LVS to UNM and to
share the resuspension conditions. Christopher said he could send during week of 8/3/09. (NOTE
AFTER CALL: Christopher will email Barbara how to arrange the shipment through
FisherBioservices; UNM will pay for the shipment and charge to TVDC)
Michelle will write the study protocol and the IACUC for the comparison of 4 LVS lots (#17 DVC, #20
DVC, #16 DVC, IND157 USAMMDA)
Barbara will ask Rick to address the MS 8 vs MS10 question on the next call with NIAID. Rick
previously wanted the non lot16 LVS tested on MS 10 rather than MS8.
Trevor will present MS 9 aerosol qualification data at the next LBERI monthly technical call.
Barbara will send preliminary results from the MS 11 natural history study to Kristin and Freyja prior
to final results being QC’d at LBERI
Barbara: UNM will get unstained Ft lot#9 antigen from USAMRIID and staining protocol from
USAMRIID, for the Ft antigen for the microagglutination assay. USAMRIID (Bev Fogtman/Lynn Wilson)
agreed to sign a Simple Letter of Agreement (SLA) with UNM for the transfer of Lot#9 LVS to UNM.
NOTE AFTER CALL: Judy Holian at USAMMDA is determining whether the SLA can be used or whether
USAMMDA and UNM must sign a modification to the MTA for receipt of the LVS lot#9 for the
microagglutination assay.
Action: Barbara will follow up with Dr Sztein regarding the positive rabbit sera
Slide 30