LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
6 July 2010
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
1
Active Milestones
#2
Active
#8
Active
#9
Active
#10
Active
#12/13
Active
#21
Active
#29
Active
Vaccinations of study personnel- no work
in June
LVS vaccination protection of aerosol
Schu4 confirmed in primates
Aerosol SOP developed for GLP
transition- no work in June
Efficacy testing of vaccine candidates
(LBERI)- no work in June
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 June
2
MS#8 – LVS Vaccinated NHP Challenged
with SCHU S4
LVS Vaccinated NHP Challenged with SCHU S4
MS8: Vaccination Practice/Challenge (n=3 scarification; n=2
subcutaneous)
MS8A: Vaccination/Challenge (n=3 by scarification; n=3 by
subcutaneous route; n=4 previously vaccinated; 2 SC, 2 ID) SCHU S4
Challenge 500 CFU
MS8B: Vaccination/Challenge (Vaccination with Highest Dose of LVS
attainable by scarification and s.c.) SCHU S4 Challenge 1000 CFU
MS8C: 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
MS8D: Vaccination/2 Broth Challenge (SC Vaccination with Lot 17,
compare CB vs. CAMHB) SCHU S4 Challenge 1000 CFU
MS8E:Telemetery Study on Vaccinated NHP SCHU S4 Challenge 1000
CFU
Red: completed
Green: in progress
Blue: steps in the milestone
MS8F: Repeat Vaccination and broth challenge with additional
immunology parameters
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) – MS08 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) – MS08 D
– Examine if there are differences in telemetry patterns in vaccinated NHP as
compared to control NHP (MS08E)
– Conduct additional immunology on vaccinated NHP and expose to SCHU S4
grown in different media to build data from the broth comparison study
(MS08F)
Endpoints
– histopathology
– bacterial CFUs of internal organs (lung, spleen, liver, kidneys, and lymph nodes)
– records of clinical symptoms post-infection
4
– clinical chemistry and hematology during infection
Milestone #8 June 2010 Accomplishments
Bacteremia (MS8D)
Animal ID
Vaccine Culture Presented
Nx Date
Group Brotha Dose (CFU)
38
NG
Ft
NG
39
Ft
Ft
NG
40
Ft
Ft
Ft
41
Ft
Ft
Bacteremia
Study Daya
42
43
-
A09011 Control
A09410 Control
A09139 Control
CB
CB
CB
2,823
447
1,107
16-May
18-May
17-May
35
NG
NG
NG
A09064
A09097
A09088
A09094
A09169
A09487
A09164
CB
CB
CB
CB
CB
CB
CB
1,455
1,492
2,766
1,741
2,178
1,344
2,067
17-May
8-Jun
8-Jun
8-Jun-10
18-May
8-Jun
8-Jun
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
980
2,341
1,441
17-May
18-May
19-May
NG
NG
NG
NG
NG
Ft
NG
Ft
Ft
Ft
Ft
Ft
Ft
-
-
-
-
-
-
-
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
Lot 17
Lot 17
Lot 17
Lot 17
Lot 17
Lot 17
Lot 17
A09079 Control CAMHB
A09518 Control CAMHB
A09072 Control CAMHB
A09086 Lot 17 CAMHB
2,266
9-Jun
NG
NG
NG
A09066 Lot 17 CAMHB
1,050
9-Jun
NG
NG
NG
A09068 Lot 17 CAMHB
1,329
9-Jun
NG
NG
NG
A09523 Lot 17 CAMHB
2,010
9-Jun
NG
NG
NG
A08739 Lot 17 CAMHB
2,150
9-Jun
NG
NG
NG
A09140 Lot 17 CAMHB
1,630
9-Jun
NG
NG
NG
A09511 Lot 17 CAMHB
1,801
9-Jun
NG
NG
NG
a
CB, Chamberlain's broth; CAMHB, Cation-adjusted Mueller-Hinton broth
NG = no growth on plates or in liquid medium
Ft= growth on plates or in liquid medium
44
-
45
-
49
-
56
-
63, Term
-
Milestone #8 June 2010 Accomplishments
Tissue Burden (MS8D)
16-May-10
18-May-10
17-May-10
Spleen
6.06E+07
6.74E+06
5.36E+08
CFU/g Tissueb
Lung
Mes. LN
Liver
4.33E+08 2.98E+04 3.53E+05
3.88E+08 1.10E+07 1.19E+06
2.23E+06 5.21E+07 4.96E+06
TBLN
1.68E+08
8.15E+07
3.13E+08
1,455
1,492
2,766
1,741
2,178
1,344
2,067
17-May-10
8-Jun-10
8-Jun-10
8-Jun-10
18-May-10
8-Jun-10
8-Jun-10
7.00E+01
BLD
BLD
BLD
1.28E+04
BLD
BLD
1.22E+07
BLD
BLD
1.35E+05
6.23E+07
3.94E+03
3.02E+02
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
2.54E+02
2.54E+03
BLD
BLD
7.07E+02
BLD
7.19E+02
4.96E+05
3.50E+03
3.37E+01
BLD
980
2,341
1,441
17-May-10
18-May-10
19-May-10
2.47E+05
1.21E+07
4.33E+07
1.42E+08
1.03E+08
4.68E+08
1.20E+04
5.27E+05
4.74E+06
3.12E+04
7.65E+04
2.25E+04
1.05E+07
7.17E+07
2.57E+08
9-Jun-10
9-Jun-10
9-Jun-10
9-Jun-10
9-Jun-10
9-Jun-10
9-Jun-10
BLD
BLD
BLD
BLD
BLD
BLD
BLD
1.03E+03
BLD
3.10E+02
8.70E+02
8.00E+02
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
1.19E+02
BLD
3.59E+03
7.20E+02
BLD
BLD
5.61E+02
Animal ID
Challenge
Date
Vaccine
Group
Culture Brotha
Presented Dose
(CFU)
Nx Date
A09011
A09410
A09139
11-May-10
11-May-10
11-May-10
Control
Control
Control
CB
CB
CB
2,823
447
1,107
A09064
A09097
A09088
A09094
A09169
A09487
A09164
11-May-10
11-May-10
11-May-10
11-May-10
11-May-10
11-May-10
11-May-10
LVS Lot 17
LVS Lot 17
LVS Lot 17
LVS Lot 17
LVS Lot 17
LVS Lot 17
LVS Lot 17
CB
CB
CB
CB
CB
CB
CB
A09079
A09518
A09072
12-May-10
12-May-10
12-May-10
Control
Control
Control
CAMHB
CAMHB
CAMHB
A09086
12-May-10
LVS Lot 17
CAMHB
2,266
A09066
12-May-10
LVS Lot 17
CAMHB
1,050
A09068
12-May-10
LVS Lot 17
CAMHB
1,329
A09523
12-May-10
LVS Lot 17
CAMHB
2,010
A08739
12-May-10
LVS Lot 17
CAMHB
2,150
A09140
12-May-10
LVS Lot 17
CAMHB
1,630
A09511
12-May-10
LVS Lot 17
CAMHB
1,801
a
CB, Chamberlain's broth; CAMHB, Cation-adjusted Mueller-Hinton broth
b
BLD, below limit of detection
6
Milestone #8 June 2010 Accomplishments
CRP Levels (MS8D)
Broth
CB
MHB
Animal #
Status
A09011
Ctrl
Pres.
Dose
2,823
A09410
Ctrl
A09139
A09088
Day 0
Day 3
Day 5
Day 7
Day 10
Day 14
Day 21
Day 28
0.8
264.6
340.2
Dead
Dead
Dead
Dead
Dead
339
2.1
302.4
324.3
Dead
Dead
Dead
Dead
Dead
Ctrl
1,107
2.3
403.8
487.8
Dead
Dead
Dead
Dead
Dead
Vx
1,580
1.9
309.6
79.9
30.5
35.6
9.8
A09094
Vx
1,741
2.9
258
388.5
341.1
258.3
336.4
280.2
147.6
A09487
Vx
1,344
1.8
171.6
247.5
387.6
155.6
252.9
185.4
71
A09164
Vx
2,097
1.6
240
106.7
225.3
155.1
14.8
4.3
A09064
Vx
1,455
2.7
206.7
602
338.7
Dead
Dead
Dead
Dead
A09097
Vx
971
1.9
310.8
173.6
33.1
7.6
6
3.8
2.7
A09169
Vx
2,178
2.8
414.6
429
489.3
Dead
Dead
Dead
Dead
A09079
Ctrl
642
4.1
350.7
339.6
Dead
Dead
Dead
Dead
Dead
A09518
Ctrl
2,341
2.3
438.3
282.6
Dead
Dead
Dead
Dead
Dead
A09072
Ctrl
1,439
3.3
405.6
515.1
Dead
Dead
Dead
Dead
Dead
A09066
Vx
1,050
1.9
169.1
348.3
395.1
25.9
7.3
3.4
A09068
Vx
1,329
2.5
120.3
130.5
37.8
128.4
77.3
13.1
6.2
A08739
Vx
2,150
0.5
331.2
493.5
457.2
265.2
60.7
12.4
5
A06086
Vx
2,266
6.3
255.6
440.7
372.6
155.9
40.8
24.3
7.6
A09523
Vx
2,010
0.6
143.5
99.6
63.5
6.1
6.9
1.5
4.8
A09140
Vx
1,630
0.9
169.3
168.9
154.2
5.2
2.1
15.4
2.4
A09511
Vx
1,801
0.9
200.1
248.7
42
66.3
8.7
1.9
1.5
not enough sample to run CRP
7
Milestone #8 – June 2010 Accomplishments on
MS8D
Completed histopathology on slides received from 8 moribund animals on
MS08D
Microbiology data has been qc’d
Clinical pathology data qc is near completion
8
Milestone #8 – Plans for next month
• Measure IFN-γ production by PBMCs and
splenocytes of TUL08D survivors by analysis of
the ELISPOT plates recently removed from the
ABSL3
• Continue data analysis
9
Milestone #12/13 - Objective and Endpoints
• Develop immunoassays that reliably distinguish LVSvaccinated from non-vaccinated 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
10
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
Develop Immunoassay
methodologies
Proliferation
assay
Red: completed
Green: In progress
Yellow: on hold; restart if necessary
Blue: steps in the milestone
IFNg
ELISPOT
Plasma
IgG ELISA
Plasma
IgA ELISA
Determine
protein:CFU
relationship in FF
and HK LVS
antigens
Microagglutination
assay
11
Milestone #12/13 – June 2010 Accomplishments
• Ordered and received anti-monkey IgM-HRP
antibody in order to test whether plasma from
LVS-vaccinated NHPs have detectable IgM
anti-LVS levels by ELISA
– Will use this data to identify potential positive
plasma samples and test these in the microagglutination assay
12
Milestone #12/13-Plans for next
month
• Run IgM anti-LVS ELISA and repeat microagglutination assay if positive plasma samples
can be identified
• Test newly received (from UNM) FF LVS using
frozen PBMCs to determine whether it
stimulates IFN-γ production as measured by
ELISPOT or proliferation in LVS-vaccinees but
not in PBMCs from naïve NHPs
13
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
14
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
15
Milestone #21 – June 2010 Accomplishments
• Measured IFN-g intracellular staining in fresh
PBMCs from one naïve NHP (A07993)
• PBMC (4x105) stimulated 20 h with media only
or UNM prep ffLVS (1.25x104, 5x104 or 2x105
cfu/well)
• Stained with antibodies to detect CD3-, CD8-,
CD14-, CD16- and CD56-positive cells
16
Milestone #21-Data Interpretation
• No antigen dependent IFN-g response in CD4
(CD3+CD8-) or CD8 (CD3+CD8+) T cells, CD14+
(monocytes) or CD3-CD16+ (NK cells).
Background IFN-g staining for T cells was <
0.5% IFN-g+, CD14+ < 0.8% and NK cells 0.1%.
• CD56+ cells (monocytes in macaques?)
showed an increase from 10% IFN-g+ CD56+
cells in media control to approx 17 % IFN-g+ in
the wells with the two highest antigen
concentrations.
17
Milestone #21 - Plans for next month
• Test additional fresh PBMCs from naïve NHPs
in the ICS assay if they become available
(excess from other studies conducted in the
laboratory) OR draw blood from TVDC NHPs
specifically for this assay before they are
placed on upcoming studies
18
Action Items
• LBERI needs discussion on LD 50 exposure
days: 1 or 2
• Status of NIAID review of MS08E protocol
• Status of NIAID review of MS08F protocol
19
Additional Points
Deliverables completed for each active milestone:
No deliverables were completed during the month of June
List of relevant publications from the past month
Bob Sherwood submitted abstract “Characterization of a Nonhuman Primate Cynomolgus
Macaque Model of Pulmonary Tularemia for Vaccine Screening” For presentation at the 2010
Chemical and Biological Defense Science and Technology Conference, 15-19 November 2010,
Orlando, Florida. Sponsored by DTRA.
MSCR status
MS 3: NIAID accepted on 3/9/10 as Final.
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 reviewed 6/15/10 (LBERI will revise by end July 2010)
MS 8 a: LBERI writing and due to UNM TBD, once revised MS11 is submitted
MS 8 b: LBERI writing and due to UNM TBD, once revised MS11 is submitted
MS 8 c: LBERI writing and due to UNM 7/23-27/10
20
Action Items (1 of 2)
• UNM suggested that LBERI use the MSXX format rather than “round” or
and “Tul 08X” (Note LBERI will use the MSXX format for future tech calls
and reports)
• Julie Wilder- Will gather the data for comparing the UNM FF LVS
stimulation vs DVC FF LVS stimulation and will email to Barbara
(completed 7/7/10)
• Michelle Valderas: will check the IACUC to determine whether the NHP
blood draws for Julie Wilder could occur before the NHP are assigned to a
study protocol and before the study protocol is approved by NIAID.
• LBERI will write the LD50 amendment for all aerosol exposures on 1 day
for all 12 NHP. Patrick :Will send the reviewed amended version to LBERI
on 7/7. (NIAID canceled this MS7 study 7/8/10)
21
Action Items (2 of 2)
• LBERI (Trevor Brasel) should keep with Chamberlains grown
SCHU S4 on the MS9, since all of LBERI’s work from the
beginning of the TVD contract is with SCHU S4 grown in
Chamberlains.
• Patrick Sanz: Tul 08E: vaccinated/telemetry: will review and
get it back to LBERI by end of week on 7/9. Please email to
Barbara and Bob Sherwood, as Michelle Valderas is on annual
leave from 7/9 to 7/16.
• Patrick Sanz: Tul 08 F: immuno/ potential two broth
comparison for the challenge: Patrick will get back to UNM
on the turnaround time on this one.
22