LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
7 July 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
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 – June 2009 Accomplishments




Blood draws were completed on Days 14, 21, 28 and 35 post-LVS
vaccination for assessment of immune response.
Immune response on Days 7 – 28 were analyzed and will be presented.
NHPs were conditioned to pole and collaring and chairing procedures.
NHPs were moved into the ABSL3 and underwent/are undergoing 1
week of pre-exposure clinical observations (temperatures and
respiratory rates 3 times daily).
Slide 5
Summary of LVS Vaccination Groups
LVS Route
Group 1
(5/18/09
1.1 x 107)
Group 2
(5/20/09
1.2 x107)
Group 3
(5/22/09
1 x 106)
Scarification
A06873 (F)
A06674 (M)
A06693 (M)
Scarification
A07386 (F)
A07682 (F)
A07686 (F)
Sub-cutaneous
A07395 (F)
A06675 (M)
A06702 (M)
Sub-cutaneous
A07418 (F)
A07566 (F)
A07610 (F)
Exposed to
SCHU S4
on 7/6;
Control =
A06694 (M)
Will be
exposed to
SCHU S4
on 7/8;
Control =
A06882 (F)
Will be
exposed to
SCHU S4
on 7/10;
Control =
A07427 (F)
Slide 6
0
A07686, Day 28
80
A07686, Day 21
Group 2
A07686, Day 14
500
A07686, Day 7
600
A07686, Day 0
140
A07686, Day 0
A07686, Day 7
A07686, Day 14
A07686, Day 21
A07686, Day 28
200
A06693, Day 28
160
A06693, Day 21
120
A06693, Day 14
A07682, Day 0
A07682, Day 7
A07682, Day 14
A07682, Day 21
A07682, Day 28
400
A06693, Day 7
A07386, Day 0
A07386, Day 7
A07386, Day 14
A07386, Day 21
A07386, Day 28
Group 1
A06693, Day 0
0
Group 2
A06873, Day 0
A06873, Day 7
A06873, Day 14
A06873, Day 21
A06873, Day 28
300
A06693, Day 0
A06693, Day 7
A06693, Day 14
A06693, Day 21
A06693, Day 28
700
A06674, Day 0
A06674, Day 7
A06674, Day 14
A06674, Day 21
A06674, Day 28
Scarified Group: IFNγ Production in Response to
LVS Antigens
Group 1
Media
LVS hk Hi
LVS ff Hi
Plated at 200,000/well
Group 3
Group 3
100
Plated at 100,000/well
Media
Group 3
LVS hk Hi
LVS ff Hi
100
Group 3
60
40
20
Slide 7
0
Group 1
600
500
300
A07610, Day 0
A07610, Day 7
A07610, Day 14
A07610, Day 21
A07610, Day 28
Group 3
A07566, Day 0
A07566, Day 7
A07566, Day 14
A07566, Day 21
A07566, Day 28
Group 1
A07418, Day 0
A07418, Day 7
A07418, Day 14
A07418, Day 21
A07418, Day 28
Group 2
A07395, Day 0
A07395, Day 7
A07395, Day 14
A07395, Day 21
A07395, Day 28
400
A06702, Day 0
A06702, Day 7
A06702, Day 14
A06702, Day 21
A06702, Day 28
700
A06675, Day 0
A06675, Day 7
A06675, Day 14
A06675, Day 21
A06675, Day 28
SC Group: IFNγ Production in Response to LVS
Antigens
Media
LVS hk Hi
LVS ff Hi
Group 2
Group 3
200
100
Plated at 200,000/well
Slide 8
0
40
A07686, Day 28
Group 3
A07686, Day 14
A07686, Day 21
Group 1
A07686, Day 7
A07682, Day 28
A07682, Day 21
120
A07682, Day 7
A07682, Day 14
A07386, Day 28
A07386, Day 21
60
A07386, Day 14
A07386, Day 7
A06873, Day 28
A06873, Day 21
Group 2
A06873, Day 14
A06873, Day 7
A06693, Day 21
A06693, Day 28
A06693, Day 14
80
A06693, Day 7
A06674, Day 28
A06674, Day 14
A06674, Day 21
100
A06674, Day 7
Scarified Group: IFNγ Production in Response to
SCHU S4 Antigens
Group 2
Media
SCHUS4 hk Hi
SCHUS4 ff Hi
Group 1
Group 3
20
Not tested in any group on Day 7; not tested in Group 1 on day 14; not
tested in A07686 on day 21 or 28; 200,000 cells/well plated
Slide 9
0
Not tested in any group on Day 7; not tested in Group 1 on day 14; not
tested in A06702 on day 21 or 28; not tested in A06675 on day 28;
200,000 cells/well plated
A07610, Day 28
A07610, Day 14
A07610, Day 21
Group 3
A07610, Day 7
A07566, Day 28
A07566, Day 21
Media
SCHUS4 hk Hi
SCHUS4 ff Hi
A07566, Day 7
A07566, Day 14
A07418, Day 28
Group 1
A07418, Day 21
A07418, Day 14
A07418, Day 7
A07395, Day 28
A07395, Day 21
180
A07395, Day 14
A07395, Day 7
40
A06702, Day 21
A06702, Day 28
Group 2
A06702, Day 14
60
A06702, Day 7
120
A06675, Day 28
140
A06675, Day 14
A06675, Day 21
160
A06675, Day 7
SC Group: IFNγ Production in Response to SCHU S4
Antigens
Group 1
Group 2
100
80
Group 3
20
Slide 10
0
160000
Group 1
Group 3
A07686, Day 0
A07686, Day 7
A07686, Day 14
A07686, Day 21
A07686, Day 28
A07682, Day 0
A07682, Day 7
A07682, Day 14
A07682, Day 21
A07682, Day 28
60000
Group 1
A07386, Day 0
A07386, Day 7
A07386, Day 14
A07386, Day 21
A07386, Day 28
Group 2
A06873, Day 0
A06873, Day 7
A06873, Day 14
A06873, Day 21
A06873, Day 28
80000
A06693, Day 0
A06693, Day 7
A06693, Day 14
A06693, Day 21
A06693, Day 28
140000
A06674, Day 0
A06674, Day 7
A06674, Day 14
A06674, Day 21
A06674, Day 28
Scarified group: Proliferation in Response to LVS
Antigens
180000
Group 2
Media
LVS hk Hi
LVS ff Hi
120000
100000
Group 3
40000
20000
Not tested on Day 0 in A06693, A06873, A07386 and A07686; not tested
on day 21 in A07386 or A07686; not tested in A07686 on day 28; 200,000
cells/well plated
Slide 11
0
250000
Group 1
Group 2
Group 3
A07610, Day 0
A07610, Day 7
A07610, Day 14
A07610, Day 21
A07610, Day 28
Group 1
A07566, Day 0
A07566, Day 7
A07566, Day 14
A07566, Day 21
A07566, Day 28
150000
A07418, Day 0
A07418, Day 7
A07418, Day 14
A07418, Day 21
A07418, Day 28
200000
A07395, Day 0
A07395, Day 7
A07395, Day 14
A07395, Day 21
A07395, Day 28
100000
A06702, Day 0
A06702, Day 7
A06702, Day 14
A06702, Day 21
A06702, Day 28
A06675, Day 0
A06675, Day 7
A06675, Day 14
A06675, Day 21
A06675, Day 28
SC group: Proliferation in Response to LVS
Antigens
Media
LVS hk Hi
LVS ff Hi
Group 2
Group 3
50000
Not tested on Day 0 in A06702 and A07566; not tested on day 7 in
A07418; not tested on day 14 in A07566 or A07610; not tested on day 21
in A06702, A07395 or A07610; not tested in A06675 or A06702 on day 28;
200,000 cells/well plated
Slide 12
0
200000
Group 2
Group 2
50000
A07686, Day 0
A07686, Day 7
A07686, Day 14
A07686, Day 21
A07686, Day 28
A07682, Day 0
A07682, Day 7
A07682, Day 14
A07682, Day 21
A07682, Day 28
A07386, Day 0
A07386, Day 7
A07386, Day 14
A07386, Day 21
A07386, Day 28
150000
A06873, Day 0
A06873, Day 7
A06873, Day 14
A06873, Day 21
A06873, Day 28
100000
A06693, Day 0
A06693, Day 7
A06693, Day 14
A06693, Day 21
A06693, Day 28
A06674, Day 0
A06674, Day 7
A06674, Day 14
A06674, Day 21
A06674, Day 28
Scarified Group: Proliferation in Response to SCHU
S4 Antigens
250000
Media
SCHUS4 hk Hi
SCHUS4 ff Hi
Group 1
Group 1
Group 3
Group 3
Not tested on Day 0 in A06693, A06873, A07386 and A07686; SCHU S4
antigens on tested on Day 0 in A07682; not tested on Day 14 or 21 in
A07386; not tested on Day 21 or 28 in A07686; 200,000 cells/well plated
Slide 13
SC group: Proliferation in Response to SCHU S4
Antigens
200000
175000
150000
Group 1
Group 2
Group 2
125000
Media
SCHUS4 hk Hi
SCHUS4 ff Hi
Group 1
100000
Group 3
75000
Group 3
50000
A07610, Day 0
A07610, Day 7
A07610, Day 14
A07610, Day 21
A07610, Day 28
A07566, Day 0
A07566, Day 7
A07566, Day 14
A07566, Day 21
A07566, Day 28
A07418, Day 0
A07418, Day 7
A07418, Day 14
A07418, Day 21
A07418, Day 28
A07395, Day 0
A07395, Day 7
A07395, Day 14
A07395, Day 21
A07395, Day 28
A06702, Day 0
A06702, Day 7
A06702, Day 14
A06702, Day 21
A06702, Day 28
0
A06675, Day 0
A06675, Day 7
A06675, Day 14
A06675, Day 21
A06675, Day 28
25000
Not tested on Day 0 in A06702 and A07566; not tested in response; SCHU S4
antigens not tested on Day 0 in A07395, A07418 or A07610, nor Day 28 in A07610;
not tested on Day 7 in A07418; not tested on day 14 in A07566 or A07610; not
tested on day 21 in A06702, A07395 or A07610; not tested in A06675 or A06702 on
day 28; 200,000 cells/well plated
Slide 14
IgG anti-LVS Units (Mean +/- S.D.
Scarified Group: IgG anti-LVS Units
2000
200
A06674
20
A06693
2
A06873
.2
A07386
A07682
.02
A07686
2E-3
Day 0
Day 7
Day 14
Day 21
Day 28
Day post-LVS Vaccination
Circles = Group 1; Triangles = Group 2; Boxes = Group 3
Slide 15
IgG anti-LVS Units (Mean +/- S.D.
SC Group: IgG anti-LVS Units
20000.01
2000
200
A06675
20
A06702
2
A07395
.2
A07418
.02
A07566
A07610
2E-3
Day 0
Day 7
Day 14 Day 21
Day 28
Day post-LVS Vaccination
Circles = Group 1; Triangles = Group 2; Boxes = Group 3
Slide 16
Milestone #8 – Plans for next month





12 LVS vaccinated NHPs and 3 naïve controls will be challenged with
1000 CFU SCHU S4
Post-SCHU S4 challenge observations (three times daily) will be
initiated (temperatures and respirations).
Blood will be collected for bacteriology and clinical chemistry on d3,
d10 and d17 post-aerosol exposure.
Moribund animals will undergo a complete necropsy with tissues
being collected for histology and bacteriological burden.
NHPs surviving to day 21 post-SCHU S4 challenge will be euthanized
and undergo a complete necropsy; spleen and PBMCs will be collected
for immunological assessment.
Slide 17
Milestone #8 – LVS Vaccination/SCHU S4 Challenge
Note

Signed Study Protocol suggests that SCHU S4 aerosol challenge
will be completed on Day 42 post-LVS vaccination; in fact, the
SCHU S4 challenges are being performed on Day 49 post-LVS
vaccination
–
An amendment will be submitted to correct the days listed
in the Study Protocol
Slide 18
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 19
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 20
Milestone #11- June 2009 Accomplishments



NHPs received and quarantined.
NHPs received 2 out of 3 TB tests.
Pole and collar conditioning is complete.
Slide 21
Milestone #11- Plans for next month




NHPs are scheduled to be released from quarantine Thursday
July 9, 2009.
Initiate chair conditioning.
Perform physical examinations.
Perform telemetry surgeries.
Slide 22
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 23


Milestone #12/13 – June 2009
Accomplishments
Reagents were ordered to set up the microagglutination assay
Positive control rabbit sera and test antigens were requested
from Dr. Sztein and USAMRIID respectively
Slide 24
Milestone #12/13 – Plans for next month



Initiate the Microagglutination Assay
Continue to attempt to establish the correlation between LVS CFU and protein
content
–
When fixed or heat-killed LVS is prepared, the actual CFU/ml can only be
estimated from the starting (live) material; we cannot be sure that no loss
occurred during preparation
–
By measuring the protein content of such preparations, we can relate it to
CFU/ml
Based on the data obtained in April we will repeat the LVS CFU:Protein content
assay
- Make 1:1 dilutions rather than 1:9 dilutions
- Sonicate the LVS in lysis buffer to possibly elaborate more protein from the
HK and FF preparations
Slide 25
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 26
Milestone #21 – June 2009 Accomplishments

Two more attempts at detecting intracellular cytokine expression in NHP PBMCs were
–
Day 27 post-LVS vaccination, PBMC from A06693 (group 3) re-stimulated in
tubes at 2x105/ml and Hi FF LVS, or, as positive control, 2.5 ug/ml PHA for 18h
(4h with Golgi plug).

–
Cells were stained for either CD4 or CD8 together with ICS stain mix
for IFNγ, TNFα and IL2. Surface staining for CD4+ and CD8+ was
successful. No cytokines could be detected above media control
background. Note that NHP A06693 had a very low response in
proliferation assay and ELISpot for IFNγ.
Day 35 post-LVS vaccination, PBMC from A07395 (group 1) re-stimulated for 19h
(4h Golgi plug) in 96-well plate at 4x105 cells/well with HK Hi LVS or FF Hi LVS at
2x105/well, 4x104/well or 800/well, or, as positive controls, PHA or Con A at
1ug/well.

Cells were stained for CD3 and ICS for IFNγ, TNFα and IL2. Surface
staining was successful and a CD3 positive population could be
identified. In the PHA control approx 2.8% of the CD3 cells were
making IFNγ compared to 1.9% in the media control. No cytokines
above media control could be detected after LVS stimulation.
Slide 27
Milestone #21- Plans for next month
Repeat ICS assay and optimize the conditions that will lead to detection of
intracellular cytokines
- Try frozen cells from early timepoints post-LVS vaccination (day 7 or 14)
in order to correlate with peak timepoints of IFNγ secretion by ELISPOT
- Try early timepoints of restimulation (6 h) which have been successful in
detecting cytokines from CD4 cells from lung homogenate in NHPs with active
disease (Amanda DuBois, UNM) and later timepoints (48 h) of restimulation
which were successful for detection of IFNγ by ICS in human T cells from LVS
vaccinated donors.
- Try PMA + Ionomycin as the positive control as well as a cytokine
positive cell line ordered from BD (HiCK cells, Human Intracellular CytoKine).
- New antibody conjugates have been ordered to move the assay from 4 color FACS
Calibur to the 6 color BD Canto, where T cells can be identified as CD3+CD4+ or
CD3+CD8+ and 3 cytokines (IFNγ, TNFα and IL2) can be detected in the same
staining tubes. Compensation Beads have been ordered from BD for automatic
compensation calculation by BD FACSDiva on the Canto.
Slide 28
Action Items





Julie Wilder: when immunoassay data is missing, put an asterisk over the
space for the missing data in the graph or figure
Julie: take cells from a positive responder NHP and freeze the cells, then use
as positive control with each IFN gamma ELIspot assay. NIAID is looking for a
consistent response from run to run of the assay. Julie may be able to get LVS
vaccinated NHP spleen cells from Terry Wu or Amanda Dubois at UNM. Using
positive control spleen cells is an assay acceptance criteria .
Freyja: trying to get a new contact at USAMRIID for UNM to replace Dr.
Mangiafico, whose email address at USAMRIID appears no longer valid. UNM
wishes to request Ft antigen, from same prep as used by Dr. Marcelo Sztein at
U of Maryland in his lab’s microagglutination assay.
Julie: will standardize CFU vs protein mass titration for antigen preps in July
2009
Cecilia: Will repeat the ICS assay, to optimize multiple aspects of the
conditions.
Slide 29