LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
2 June 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 – 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 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. Two different
methods of vaccination are being compared (scarification and
subcutaneous).
Endpoints include histopathology and bacterial CFUs of internal organs
(lung, spleen, liver, kidneys, and lymph nodes), records of clinical
symptoms post-infection, and clinical chemistry and hematology during
infection.
Slide 4
Milestone #8 – May 2009 Accomplishments






ELISPOT plates from survivors of TUL08 Part A study were retrieved
from the ABSL3 and analyzed.
The IACUC protocol and study protocol were written for the repeat
vaccination/challenge study.
15 animals were released from quarantine on May 7. Physical exams
and baseline blood draws were performed for assessment of immune
response and clinical chemistry.
Animals were vaccinated during the week of May 17 (1 control, 2
scarified and 2 s.c. vaccinated/day on 3 separate days).
The LVS was resuspended in 0.25 mL sterile water and a “drop” (60
microliters) of neat material was used for vaccination by scarification.
This was the highest dose possible from the DVC LVS Lot 16. For the
subcutaneous dose 0.12 ml of LVS was diluted 1:1 with saline and
injected. Both vaccinations were applied to the upper shaved back of
the monkeys.
Day 7 blood draws for assessment of immune response were
completed.
Slide 5
IFNγ Production by PBMCs from Naïve and LVS-vaccinated
NHPs pre- and post-SCHU S4 Challenge (three survivors from
TUL08 Part A)
Post-SCHU S4 challenge
700
600
A03152
Vaccine
naive
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
300
SCHUS4 hk Hi
SCHUS4 hk Mid
200
SCHUS4 ff Super
SCHUS4 ff Hi
500
400
SCHUS4 ff Mid
100
0
TUL61
TUL70
700
700
600
500
400
28643
LVS Scarified
(dose of 3 x 104)
600
500
400
300
300
200
200
100
100
0
0
Day 0
Day 25
Day 56
A00937
LVS by ID
(dose of 1.5
x 107)
Day 795
All PBMCs
plated at
200,000/well
Day 836
Slide 6
IFNg Spots (Mean +/- S.D.)
IFNγ Production by Spleen Cells from Naïve and LVSvaccinated NHPs post-SCHU S4 Challenge (three survivors
from TUL08 Part A)
800
700
LVS by
Scarification
56 days
earlier
LVS by ID
836 days
earlier
No LVS
Vaccination
600
500
400
300
200
100
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
SCHUS4 hk Super
SCHUS4 hk Hi
SCHUS4 hk Mid
SCHUS4 ff Super
SCHUS4 ff Hi
SCHUS4 ff Mid
0
28643
A00937
Splenocytes were cultured at 1 x 106/ml (150,000/well)
Antigen:
Lo = 0.06 x 105/ml
Mid = 0.25 x 105/ml
Hi = 1 x 105/ml
Super = 4 x 105/ml
A03152
Slide 7
TUL08B: LVS Vaccination/SCHU S4 Challenge


Animals were vaccinated with the highest dose of LVS
achievable
–
Lyophilized LVS vials were resuspended in 250 microliters
of water according to the DVC protocol
–
60 microliters were used to scarify; althernatively, a 1:1
dilution was made in saline and used to inject 120
microliters sub-cutaneously
Baseline immune responses were measured using PBMCs
prepared from blood draws before the vaccination
Slide 8
Milestone #8 – LVS Dosing Data
Preparation Date:
18-May-09
Scarification Inoculum Raw Data
Dose Data
Dilution
Vol. Plated
(mL)
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
Dilution
Mean CFU/dose
10-1
0.1
> 300
> 300
> 300
> 300
TNTC
10-1
TNTC
10-2
0.1
> 300
> 300
> 300
> 300
TNTC
10-2
TNTC
10-3
0.1
> 300
> 300
> 300
> 300
TNTC
10-3
TNTC
10-4
0.1
> 300
> 300
> 300
> 300
TNTC
10-4
TNTC
10-5
0.1
162
198
189
183
1.83E+08
10-5
1.10E+07
10-6
0.1
22
14
18
18
1.80E+08
10-6
1.08E+07
10-7
0.1
2
2
1
1.7
1.67E+08
10-7
1.02E+07
10-8
0.1
0
0
0
0
0.00E+00
10-8
0.00E+00
Slide 9
Milestone #8 – LVS Dosing Data
Preparation Date:
20-May-09
Scarification Inoculum Raw Data
Dose Data
Dilution
Vol. Plated
(mL)
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
Dilution
Mean CFU/dose
10-1
0.1
> 300
> 300
> 300
> 300
TNTC
10-1
TNTC
10-2
0.1
> 300
> 300
> 300
> 300
TNTC
10-2
TNTC
10-3
0.1
> 300
> 300
> 300
> 300
TNTC
10-3
TNTC
10-4
0.1
> 300
> 300
> 300
> 300
TNTC
10-4
TNTC
10-5
0.1
230
230
120
193
1.93E+08
10-5
1.16E+07
10-6
0.1
18
18
18
18
1.80E+08
10-6
1.08E+07
10-7
0.1
2
0
2
1.3
1.33E+08
10-7
7.80E+06
10-8
0.1
0
0
0
0
0.00E+00
10-8
7.80E+06
Slide 10
Milestone #8 – LVS Dosing Data
Preparation Date:
21-May-09
Scarification Inoculum Raw Data
Dilution
Vol. Plated
(mL)
Plate 1
Plate 2 Plate 3
Mean
Mean CFU/mL
10-1
10-2
10-3
10-4
10-5
10-6
10-7
0.1
0.1
0.1
0.1
0.1
0.1
0.1
> 300
> 300
> 300
> 300
n/a
15
1
> 300
> 300
> 300
> 300
n/a
15
1
> 300
> 300
> 300
> 300
n/a
12
3
> 300
> 300
> 300
> 300
n/a
14
1.7
TNTC
TNTC
TNTC
TNTC
n/a
1.40E+08
1.67E+08
10-8
0.1
0
0
0
0
0.00E+00
Dilution
Bad CHAB batch?
Scarification Inoculum Raw Data (after sitting at RT 2-3 hrs)
Vol. Plated
Plate 1
Plate 2 Plate 3
Mean
Mean CFU/mL
(mL)
10-1
10-2
10-3
10-4
10-5
10-6
10-7
0.1
0.1
0.1
0.1
0.1
0.1
0.1
> 300
> 300
> 300
440
26
3
1
> 300
> 300
> 300
382
25
4
0
> 300
> 300
> 300
377
46
5
0
> 300
> 300
> 300
400
32
4
0.3
TNTC
TNTC
TNTC
4.00E+07
3.23E+07
4.00E+07
3.33E+07
10-8
0.1
0
0
0
0
0.00E+00
NOTE: Vaccine was NOT administered this day
Slide 11
Milestone #8 – LVS Dosing Data
Preparation Date:
22-May-09
Scarification Inoculum Raw Data
Dose Data
Dilution
Vol. Plated
(mL)
Plate 1
Plate 2
Plate 3
Mean
Mean CFU/mL
Dilution
Mean CFU/dose
10-1
0.1
> 300
> 300
> 300
> 300
TNTC
10-1
TNTC
10-2
0.1
> 300
> 300
> 300
> 300
TNTC
10-2
TNTC
10-3
0.1
> 300
> 300
> 300
> 300
TNTC
10-3
TNTC
10-4
0.1
0
0
0
0
0.00E+00
10-4
0.00E+00
10-5
0.1
0
0
0
0.0
0.00E+00
10-5
0.00E+00
10-6
0.1
3
2
0
1.7
1.67E+07
10-6
1.02E+06
10-7
0.1
0
0
0
0.0
0.00E+00
10-7
0.00E+00
10-8
0.1
0
0
0
0
0.00E+00
10-8
0.00E+00
NOTE: Boxed dilution samples likely the result of bad CHAB batch
Slide 12
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)
Slide 13
Baseline IFNγ Responses of PBMCs from
TUL08B NHPs (Low and Mid-responders)
30
Low Responders
20
10
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
.67, A07427
40
20
0
Media
LVS hk Hi
.67, A06702
140
120
100
80
60
Mid-responders
.67, A06674
200
180
160
1.33, A07686
1.33, A07682
1.33, A07566
1.33, A07610
IFNg Spots (Mean +/- S.D.)
PBMCs were cultured at 0.67 or
1.33 x 106/ml (100,000 –
200,000/well)
Antigen:
Lo = 0.06 x 105/ml
Mid = 0.25 x 105/ml
Hi = 1 x 105/ml
Super = 4 x 105/ml
1.33, A07386
1.33, A06882
1.33, A06694
.67, A07686
.67, A07682
.67, A07610
.67, A07566
.67, A07386
NT
.67, A06882
0
1.33, A07427
40
No NHP exhibited
baseline IgG antiLVS levels higher
than normal
1.33, A06702
50
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
1.33, A06674
60
.67, A06694
IFNg Spots (Mean +/- S.D.)
70
Slide 14
Baseline IFNγ Responses of PBMCs from
TUL08B NHPs (High responders)
600
No NHP exhibited
baseline IgG antiLVS levels higher
than normal
Media
LVS hk Hi
LVS hk Mid
500
LVS ff Hi
400
LVS ff Mid
LVS ff Lo
300
LVS hk Super
200
1.33, A07418
1.33, A07395
1.33, A06873
1.33, A06693
1.33, A06675
.67, A07418
.67, A07395
.67, A06873
0
.67, A06693
100
.67, A06675
IFNg Spots (Mean +/- S.D.)
700
PBMCs were cultured at 0.67 or 1.33 x 106/ml (100,000 – 200,000/well)
Antigen:
Lo = 0.06 x 105/ml
Mid = 0.25 x 105/ml
Hi = 1 x 105/ml
Super = 4 x 105/ml
TNTC = 600 spots
Slide 15
Scarified
X indicates a
response not
different than
baseline
LVS hk Mid
300
x
LVS ff Lo
LVS hk Super
200
700
600
1.33, A07686
1.33, A07682
1.33, A07386
1.33, A06873
1.33, A06693
1.33, A06674
S.C. Inoculation
Media
LVS hk Hi
LVS hk Mid
500
LVS ff Hi
400
LVS ff Mid
LVS ff Lo
300
LVS hk Super
200
.67, A07566
.67, A07418
.67, A07395
0
.67, A06702
100
.67, A06675
IFNg Spots (Mean +/- S.D.)
PBMCs were cultured at 0.67
or 1.33 x 106/ml (100,000 –
200,000/well)
Antigen:
Lo = 0.06 x 105/ml
Mid = 0.25 x 105/ml
Hi = 1 x 105/ml
Super = 4 x 105/ml
TNTC = 600 spots
.67, A07686
.67, A07682
.67, A07386
.67, A06873
0
.67, A06693
100
1.33, A07610
LVS ff Mid
1.33, A07566
400
1.33, A07418
LVS ff Hi
1.33, A07395
500
1.33, A06702
LVS hk Hi
1.33, A06675
600
Media
.67, A07610
700
.67, A06674
IFNg Spots (Mean +/- S.D.)
IFNγ Production by PBMCs from NHPs
vaccinated with LVS one Week Earlier
Slide 16
Milestone #8 – LVS Vaccination
Plans for next month




Weekly blood draws will continue to assess immune response
post-vaccination.
Animals will be trained to pole and collars and chairs.
Animals will be moved to the ABSL3.
Pre-SCHU S4 challenge observations (three times daily) will be
initiated (temperatures and respirations).
Slide 17
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:
Works for
Con A and
LVS
ELISPOT
Determine
protein:CFU
relationship in
FF and HK LVS
antigens
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 18
Milestone #12/13 – May 2009 Accomplishments

No work was performed this month on this Milestone
Slide 19
Milestone #12/13 - Immune Responses in
Animals and Humans
Plans for next month


Our goal is 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 20
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 21
Milestone #21 May 2009 Accomplishments

Examined the IFNγ production from a single NHP that was
vaccinated with LVS in October 2008 by scarification (3 x 105
CFU) and challenged with SCHU S4 (800 CFU) by bronchoscopy
–
Day 21 post-bronchoscopy: PBMCs were processed at
LRRI; other tissues sent to UNM (PPG)
Slide 22
700
600
500
400
300
Day 21 postSCHU S4
Bronchoscopy
Media
LVS hk Hi
LVS hk Mid
LVS ff Hi
LVS ff Mid
LVS ff Lo
LVS hk Super
200
PBMCs were cultured at 0.67 or 1.33 x 106/ml (100,000 – 200,000/well)
Antigen:
Day Post-LVS Vaccination
Lo = 0.06 x 105/ml
Mid = 0.25 x 105/ml
Hi = 1 x 105/ml
Super = 4 x 105/ml
TNTC = 600 spots
1.33, Day 211
1.33, Day 175
1.33, Day 35
.67, Day 211
0
.67, Day 175
100
.67, Day 35
IFNg Spots (Mean +/- S.D.)
IFNγ Production by an LVS-vaccinated NHP
pre- and post-bronchoscopy with SCHU S4
Slide 23
Milestone #21 Upcoming work in the next
month
Repeat ICCS assay and include a positive mitogen control (Con
A); use PBMCs from newly vaccinated NHPs (for MS8)
Test 20 – 48 hours of stimulation with LVS
Slide 24
Action Items




Terry Wu: will check with Jason and Gloria regarding observations of lack of
SCHU S4 growth on CHAB.
Trevor will look at raw data and review the lot numbers of Remel commercial
CHAB plates used in the May LVS titers. Are these few bad CHAB plates from
a different lot relative to all other Remel commercial CHAB plates picked up
from UNM?
Julie is checking lot numbers of HK LVS and FF LVS antigen preps used for
ELIspots on pre-vaccination PBMC from 15 NHP for the Tul8B study. Why are
naïve PBMC responding best to HK LVS, though in past naives responded
better to FF LVS?
Julie will graph the ELIspot data differently. Will pick one or two antigens that
make sense and plot pre vs post for each NHP . So can look at comparability
between animals and between draws from same animal. Graph by recall
antigens. Do perhaps just high dose SCHU S4 on pre and post, rather than
multiple SCHU S4 doses. Will pick high dose of cells as don’t need to dilute out
anymore. Will present the LVS antigen responses. Can do HK, FF LVS on
different slides. Can do SCHU S4 FF and HK on different slides. Will do SCHU
S4 pre and post, with pre one week before challenge.
Slide 25