LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
4 May 2010
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
1
Active Milestones
#2
Active
#8
Active
#9
Active
#10
Active
#11
Active
#12/13
Active
#21
Active
#29
Active
Vaccinations of study personnel- no work
in April
LVS vaccination protection of aerosol
Schu4 confirmed in primates
Aerosol SOP developed for GLP
transition- no work in April
Efficacy testing of vaccine candidates
(LBERI)- no work in April
In Vivo GLP NHP model efficacy SOPs
and efficacy testing of vaccine
candidates- no work in April
Assays for detecting relevant immune
responses in animals and humans
Correlates of protection- in vitro assay or
other readout of effector function of Ft
developed for multiple species
Analysis of T cells from lymph nodes and
T cell epitopes- no work in April
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.) SCHU S4 Challenge 1000 CFU
Round 4 Vaccination/Challenge (Vaccination with Lot 16 n=3; Lot 17
n=3; Lot 20 n=8; Lot 4 n=8) SCHU S4 Challenge 1000 CFU
Round 5 Vaccination/2 Broth Challenge (SC Vaccination with Lot 17,
compare CB vs. CAMHB) SCHU S4 Challenge 1000 CFU
Vaccination/Challenge Non-Telemetered Natural History Study SCHU
S4 Challenge 1000 CFU
Vaccination/Challenge Telemetered Natural History Study SCHU S4
Challenge 1000 CFU
Red: completed
Green: in progress
Blue: steps in the milestone
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) – TUL08 A and B
– Compare 4 different LVS lots as vaccines (all delivered by subcutaneous route)
– TUL08 C
– Compare SCHU S4 growth media to see if it has an effect on virulence
(Chamberlain’s broth vs. Mueller-Hinton broth) – TUL08 D
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
4
Milestone #8 – April 2010 Accomplishments
• Vaccinated NHP for Tul08D
– Vaccinated NHP over 2 days using Lot 17. In all cases save 1 NHP, vaccinations were
given within 30 minutes of preparation.
– Average post-vaccination titers were 8.75 x 108 and 2.10 x 108 CFU/mL for Days 1 and 2,
respectively.
– Pre and post titers on Day 2 differed by 2 logs, unlike day 1 where no drop was observed
– NHP were re-randomized to prevent any issues associated with differing doses
• Performed practice bioaerosols (n=6) using SCHU S4 grown in LRRIprepared supplemented Mueller-Hinton broth (MH)
– Primary goal was to evaluate bioaerosol efficacy (spray factors) for SCHU S4 grown in
MH and compare to historical data for SCHU S4 cultured in Chamberlain’s broth
– Culture procedures mimicked those established for SCHU S4 grown in Chamberlain’s
broth
– At 24h, bacteria were harvested, normalized to OD600 of 0.200 and diluted
• An OD600 of 0.200 ± 0.01 was defined as 1.4 x 109 CFU/mL
• Three (3) generator suspension concentrations were targeted:
– 1 x 106 CFU/mL
– 5 x 106 CFU/mL
– 1 x 107 CFU/mL
• [see data next slide]
5
Milestone #8 – April 2010 Accomplishments
Nebulizer
Date
23-Apr-10
F. tularensis
Strain
SCHU S4
Growth Protocol
1. 48h BCGA
2. Normalized to
OD600 of 0.100
3. Inoculate 100 mL of
MH w/ 400 mL
4. Incubate 24 at
37°C, 200 rpm, in the
dark
5. Harvest and dilute
(no centrifugation)
CFU/mL
CFU/L
CFU/3.5L
PostSpray
Factor
Brand
Operating
Pressure
Output
Target
PreActual
PostActual
Collison
22.0 psig
7.511 L/min
1.00E+06
6.75E+05
5.10E+05
5.05E+01
1.77E+02
9.90E-08
Collison
22.0 psig
7.511 L/min
1.00E+06
8.65E+05
4.75E+05
6.46E+01
2.26E+02
1.36E-07
Collison
22.0 psig
7.511 L/min
5.00E+06
1.78E+06
1.48E+06
2.87E+02
1.00E+03
1.94E-07
Collison
22.0 psig
7.511 L/min
5.00E+06
4.95E+06
1.10E+06
2.46E+02
8.61E+02
2.24E-07
Collison
22.0 psig
7.511 L/min
1.00E+07
Suspected
5.45E+06
error
2.10E+02
7.36E+02
3.86E-08
Collison
22.0 psig
7.511 L/min
1.00E+07
5.30E+06
4.54E+02
1.59E+03
7.56E-08
6.00E+06
6
Milestone #8 – Data Interpretation
• Actual generator suspensions were lower than targets, though
still within an acceptable range
– OD600 estimate was inaccurate
• Actual concentration of normalized suspension was 7.57 x 107 CFU/mL
(well below the estimated concentration of 1.4 x 109 CFU/mL)
– Data indicate the need to further evaluate SCHU S4 growth in MH,
particularly with respect to obtained concentrations at 24h
• CFU/L concentrations were optimal for Sprays 3-6
– Calculated CFU in 3.5L (target for nonhuman primate exposure) were close to
1,000 CFU for these four bioaerosol runs
• This is similar to what is observed with SCHU S4, particularly with respect to the
starting generator suspension concentrations; essentially, we can preliminarily
conclude that SCHU S4 grown in MH behaves similarly as a bioaerosol when
compared to SCHU S4 cultured in Chamberlain’s broth
7
Milestone #8 – Plans for next month
• The next set of practice bioaerosols are scheduled for
early May 2010
– The objective is to mimic procedures for the actual
nonhuman primate aerosol challenges and obtain enough
replicates to demonstrate that 1,000 CFU presented can be
achieved with SCHU S4 grown in MH
– Prior to this, we are conducting additional MH cultures in
order to better understand our starting concentration at
24h and obtain more titer data from OD600-normalized
suspensions
• Challenges are set for May 11 and 12 for Tul08D
8
Milestone #8 – Plans MS8E and MS8F
• Tul08E – disease course in vaccinated and challenged NHP,
with and without telemeters.
– Points for discussion:
• Draft Study protocol consistency with annual meeting presentation
• Timelines – for study inception and serial sacrifices
• Telemetry issues
• Tul08F – Duration study
– Some NHP at supplier are measles titer negative even after
vaccination. Do we want these NHP in order to stick to our timeline,
or would we rather wait and have measles titer positive animals.
9
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
Proliferation
assay
Red: completed
Green: In progress
Yellow: on hold; restart if necessary
Blue: steps in the milestone
Develop Immunoassay
methodologies
IFNg
ELISPOT
Determine
protein:CFU
relationship in FF
and HK LVS antigens
Plasma
IgG ELISA
Plasma
IgA ELISA
Microagglutination
assay
10
Milestone #12/13 - Objective and Endpoints
• Develop immunoassays that reliably distinguish LVS-vaccinated from nonvaccinated NHPs
– Thus far can rely on IgG anti-LVS ELISA for this purpose
• Develop immunoassays that may distinguish NHPs which survive SCHU S4
aerosol challenge from those that do not
– Thus far have not been able to refine our assays to function as such
– IgG anti-LVS plasma levels do not correlate with protection
– IFNγ production or proliferation by bulk PBMCs in response to LVS or
SCHU S4 antigens also appear not to distinguish those NHPs which
have been protected from SCHU S4-induced mortality but more data
mining may be in order (i.e. only examining responsiveness early postLVS vaccination or late; only looking at specific types of stimuli (FF vs.
HK))
11
Milestone #12/13 – April 2010 Accomplishments
0
0
-0.01
5
10
15
20
0.3
25 mg/ml 30
Standard curve diluent: PBS.
0.25
Standard curve protein sample buffer
-0.02
0.2
OD 595
OD 595
-0.03
-0.04
-0.07
-0.08
0.15
0.1
-0.05
-0.06
y = 0.0096x
R² = 0.9804
y = -0.003x
R² = -3.112
0.05
0
0
5
10
15
20
25
30
mg/ml
Attempt to create Bradford standard curves with bovine gamma globulin
standard set, ready-to-use from Fermentas and sample/lysis buffer from
Sjostedt lab, Umea, versus PBS as dilution buffer.
12
Milestone #12/13 – April 2010 Accomplishments
Substance
Lysis buffer from Sjostedt
lab
Compatibility chart for
Bradford reagent
(Fermentas), ready-to-use
thiourea
2M
1M
urea
7M
5M
CHAPS
4%
5%
ASB-14
0.5%
Not indicated or ASBC7Bz0, 0.25%
DTT
1.0%
10mM
Beta-mercaptoethanol
1%
1M
Protease inhibitor
0.5x
13
Milestone #12/13 – Data Interpretation
• Bradford assay will not be a good assay for
determination of protein content in fixed F.t.
preparations.
• LBERI is developing an ELISA method
according to results at UNM and have ordered
secondary antibodies to detect mouse a-fopA
and a-dnaK binding to plate-bound F.t
preparations.
14
Milestone #12/13 – Plans for next month
• Run ELISA described in previous slide.
15
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 nonLVS-vaccinated
NHPs
Red: completed
Green: In progress
Blue: steps in the milestone
Establish conditions to detect
growth of LVS in NHP PBMCs
Confirm reduced
growth in LVSvaccinated NHPs
Confirm growth in
non- LVS-vaccinated
NHPs
16
Milestone #21 - Objective and Endpoints
• Develop immunoassays of effector function that can detect correlates of
protection from SCHU S4 aerosol-induced morbidity
– Developing two assays for this purpose thus far:
– 1) Intracellular cytokine staining;
• Perhaps IFNγ production by particular cell types will distinguish
NHPs which are protected vs. those that are not
• Perhaps the presence of particular cells that are producing more
than one cytokine (ex. IFNγ, TNFα and IL-2) will predict protection
– 2) inhibition of in vitro growth of SCHU S4 by some cells contained in
the PBMC preparations of protected NHPs vs. those that succumb to
SCHU S4 aerosol
17
Milestone #21 – April 2010 Accomplishments –
In vitro growth assay NHP PBMC + F.t.
5.00E+03
Growth of LVS in PBMC in vitro
4.50E+03
4.00E+03
3.50E+03
3.00E+03
A07699 pre-vacc
A07699 wk2 post vacc
2.50E+03
30017 pre-vacc
2.00E+03
30017 wk 2 post vacc
1.50E+03
1.00E+03
5.00E+02
0.00E+00
0h
72 h
For both NHPs tested in this experiment, LVS bacteria were kept at a steady-state
in pre-vaccination PBMC over 72 h in vitro, whereas post vaccination PBMC eliminated
bacteria over 72 h. The assay is now ready to move to B3 and be tested with NHP PBMC
18
and SCHUS4.
Milestone #21-Data Interpretation
• For both NHPs tested in this experiment, LVS bacteria were
kept at a steady-state in pre-vaccination PBMC over 72 h in
vitro, whereas post vaccination PBMC eliminated bacteria
over 72 h.
19
Milestone #21 – April 2010 Accomplishments:
Intracellular staining of IFN-g in NHP PBMC
3 experiments: 20 h stimulation ffLVS/hkLVS/PHA
FcR block: a-CD32
Surface stain: CD3,CD8,CD14,CD16, CD56
Intracellular stain: IFN-g All freshly prepared PBMC
CD3+CD8(CD4 T cells)
CD3+CD8+
CD14+
IFN- g RESPONSE
CD3-CD16+
CD56+
ffLVS
CD56+
hkLVS
NHP ID
Post
vacc.
1) A09068
d7
+
-
-
-
+
-
1) A09088
d7
-
-
-
-
+?
-
2) A09088
d 14
-
+
-
-
+?
+
3) A09523
d 14
+
+
+
-
+
-
Only ffLVS, no hkLVS response
in T cells
To both ff
and hk
20
MS #21 – April 2010 Accomplishments: ICS IFN-g in
NHP PBMC – A09068 and A09088 day 7 post vacc.
cfu/well
1.25x104
0
0.6%
143
0.4
127
0.5
144
90
80
5x104
0.6
0.5
1.9
0.5
0.9
166
160
25
% IFN-g+ PBMC A09068
% IFN-g+ PBMC A09088
20
70
60
CD4 T cells
50
CD8 T cells
40
CD14+
30
% IFN-g+
% IFN-g+
ff LVS
A09068
151
147
153
2x105
CD4 T cells
15
CD8 T cells
CD14+
10
CD3-CD16+
20
CD56+
10
CD3-CD16+
CD56+
5
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu ffLVS/well
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu ffLVS/well
21
MS #21 – April 2010 Accomplishments: ICS IFN-g in
NHP PBMC – A09088 day 14 post vacc.
cfu/well
1.25x104
0
5x104
0.7
103
104
25
0.7
104
0.5
0.5
111
2x105
0.8
99
0.9
109
1.2
122
% IFN-g+ PBMC A09088 D14
14
12
20
CD8 T cells
10
CD14+
CD3-CD16+
5
CD56+
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
-5
cfu ffLVS/well
% IFN-g+ PBMC A09088 D14
10
CD4 T cells
% IFN-g+
% IFN-g+
15
ff LVS
A09088
CD4 T cells
8
CD8 T cells
6
CD14+
4
CD3-CD16+
2
CD56+
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu hkLVS/well
22
MS #21 – April 2010 Accomplishments: ICS IFN-g in
NHP PBMC – A09523 day 14 post vacc.
cfu/well
CD4+
cells
1.25x104
0
1.6
0.6
108
104
CD8+
cells
0.3
71
91
1.2
135
1.3
114
1.2
1.3
ff LVS
A09523
105
1.5
133
ff LVS
A09523
136
163
76
1.6
1.2
1.7
0.5
75
142
128
2x105
1.3
1.2
0.7
106
5x104
1.4
105
23
MS #21 – April 2010 Accomplishments: ICS IFN-g in
NHP PBMC – A09523 day 14 post vacc.
% IFN-g+ of CD14+ cells A09523
3.5
Day 14
50
45
% IFN-g+ PBMC A09523 D14
40
3
35
2.5
2
ffLVS
1.5
hkLVS
1
% IFN-g+
% IFN-g+ of CD14 cells
4
30
CD4 T cells
25
CD8 T cells
20
CD14+
15
CD3-CD16+
10
0.5
CD56+
5
0
0.00E+00 1.25E+04
5.00E+04
cfu/well
2.00E+05
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
cfu ffLVS/well
24
Milestone #21-Data Interpretation
• The production of IFN-g by NHP PBMCs appears to
be quite variable and different individuals have
different PBMC subpopulations that respond to F.
tularensis antigen restimulation and respond at
different time points post vaccination and to which
antigen preparations, hk or ff.
• Generally, T cells do not appear to respond to hk LVS,
only ffLVS at the time points tested.
25
Milestone #21 - Plans for next month
• Move in vitro growth assay into LBERI B3 facility and start
using SCHUS4.
• Attempt to test more time points post vaccination of IFN-g ICS
from the same NHP individuals with freshly prepared cells. If
enough cells, test CD4 staining to confirm that CD3+CD8- cells
are indeed CD4 T cells.
26
Additional Points
Deliverables completed for each active milestone:
No deliverables were completed during the month of April
List of relevant publications from the past month
None
MSCR status
MS 3: NIAID reviewing MSCR (UNM sent to NIAID on 2/18/2010 )
MS 4: NIAID reviewing MSCR (UNM sent to NIAID on 3/23/2010 )
MS 7: NIAID reviewing MSCR (UNM sent to NIAID on 4/7/2010 )
MS 11: NIAID reviewing MSCR (UNM sent to NIAID on 3/24/2010 )
MS 8 a: LBERI writing and due to UNM 5/14/10
MS 8 b: LBERI writing and due to UNM 6/30/10
MS 8 c: LBERI writing and due to UNM 5/4/10
27
Action Items (1 of 3)
• Trevor: will report on the new SCHU S4 MH growth and aerosol data from
the week of 5/4 as soon as available. (DONE 5/7/10)
• Trevor will provide the CFU data including plate counts for the SCHU S4
growth pre and post aerosol (DONE 5/7/10)
• Trevor will report on the flow cytometer data for measuring the starting
bacterial concentrations, as they set up this instrument
• NIAID wants an LBERI LD 50 study with more NHP numbers and with lower
aerosol doses (5 CFU and 25 CFU) of SCHU S4.
• NIAID wants the MS 11: natural history data separated into two reports,
for the telemetry and the non telemetry studies. NIAID wants all the data
related to the serial sacrifices.
28
Action Items (2 of 3)
• NIAID wants more than 2 weeks to review and approve a specific study
protocol. After the study protocol is approved, then UNM can order NHP.
NIAID does not want NHP ordered before the study protocols are formally
approved, with all details of the study protocol. NIAID wants the duration
of the experiments, with the date that the study will start ( dates for
vaccination, implants, challenge and then last sacrifice). NIAID wants to
know exact length and complexity of the study.
• NIAID does not want to order the study MS8E.
• Kristy will look for the impact of lead placement in the wave forms during
reanalysis of the respiratory, heart rate and temperature telemetry data.
• Kristy will give high priority to the telemetry data reanalysis and will
present at next month’s LBERI meeting or as soon as available.
29
Action Items (3 of 3)
• UNM/LBERI propose next set of experiments in writing to NIAID and then
get NIAID’s approval. NIAID requested more low dose LD50 on MS7, a
repeat of the telemetry NHP on MS11 and more Lot 17 LVS
vaccination/challenge NHP.
• UNM/LBERI will write several NHP study rationales and send to Patrick, Ed,
and Kristin for review.
• LBERI will better integrate the interpretations of the pathology report into
the MS7 MSCR. LBERI will incorporate new animals’ data in 2010 for LD50
before rewriting the MS7 LD 50 study MSCR
• LBERI will perform next fresh bloods PBMC infections with LVS and use a
different Y scale for bacterial growth measurements.
• Cecilia will write a summary of each flow cytometry experiment design,
the experiment results and the analysis for each of the flow cytometry
slides for the June LBERI technical call and all future LBERI slide sets and
discuss with Julie before submitting.
30