LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
5 January 2010
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
Slide 1
Active Milestones
#2
Active
Vaccinations of study personnel- no work in
December
#8
Active
LVS vaccination protection of aerosol Schu4
confirmed in primates
#9
Active
Aerosol SOP developed for GLP transitionno work in December
#10
Active
Efficacy testing of vaccine candidates
(LBERI)- no work in December
#11
Active
In Vivo GLP NHP model efficacy SOPs and
efficacy testing of vaccine candidates- no
work in December
#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 December
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.)
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
Red: completed
Green: in progress
Blue: steps in the milestone
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)
– Compare 4 different LVS lots as vaccines (all delivered by
subcutaneous route)

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 – December 2009 Accomplishments






Euthanized survivors on Day 21
Necropsies were performed
Blood was collected for bacteremia and IFNγ ELISPOT analysis
Tissues were collected and cultured for SCHU S4 burden
Re-graphed IFNγ ELISPOT data using different strategies
Ordered 18 primates for the next study- comparison of SCHU S4
grown in Mueller Hinton broth versus Chamberlain’s broth.
Slide 5
Organ Burden by Animal and Group
LVS lot and Animal
Number
Presented dose
(CFU)
Study Day of
Death
Lung
TBLN
Spleen
Liver
Mesenteric LN
A07624
30015
A06872
1268
1823
636
Found day 5
4
4
3.01E+08
5.24E+08
1.75E+09
3.94E+07
4.72E+08
1.38E+09
3.11E+07
9.07E+07
2.68E+07
9.12E+05
6.29E+05
5.36E+05
1.35E+05
1.25E+04
3.88E+05
30003
29975
29992
2872
1667
1540
9
NA
NA
2.19E+08
4.86E+04
BLD
1.72E+06
BLD
6.61E+02
2.95E+06
BLD
BLD
2.35E+05
BLD
BLD
1.45E+07
BLD
BLD
A07872
A07754
A07699
1860
1897
787
7
NA
NA
1.97E+08
BLD
9.21E+04
5.31E+04
BLD
1.50E+03
9.64E+03
BLD
BLD
4.57E+03
BLD
BLD
BLD
BLD
BLD
30048
29976
A06488
A07683
29996
29991
30014
28461
1479
2510
2250
828
2148
304
728
575
8
NA
NA
NA
12
6
10
7
1.43E+08
BLD
1.33E+02
4.74E+04
2.76E+07
1.78E+07
3.66E+08
5.62E+08
2.75E+04
4.19E+02
2.98E+03
5.02E+03
5.95E+05
BLD
1.58E+03
4.74E+06
9.21E+03
BLD
BLD
BLD
BLD
1.31E+02
2.60E+05
2.04E+05
6.30E+03
BLD
BLD
BLD
BLD
5.49E+01
8.54E+04
1.26E+04
BLD
BLD
BLD
BLD
BLD
BLD
BLD
4.56E+02
1.01E+02
1.47E+03
3.45E+08
1.23E+02
1.55E+04
1.34E+03
1.63E+06
2.89E+07
6.36E+02
1.55E+04
1.90E+05
7.92E+03
2.03E+02
1.33E+04
BLD
BLD
BLD
BLD
2.32E+03
BLD
BLD
BLD
5.26E+04
4.96E+02
BLD
BLD
6.61E+02
BLD
BLD
BLD
1.12E+05
BLD
BLD
BLD
4.63E+02
BLD
BLD
BLD
BLD
BLD
Control NHP
Lot 16
Lot 17
Lot 20
Lot 4
30017
1257
NA
A07756
1669
NA
30019
1785
7
29987
1511
NA
30039
3766
NA
A07720
119
NA
A07842
1090
21
A07738
416
5
Note: 5e1 is BLD for all tissues but LN for which 2e1 is BLD
Slide 6
Log10 CFU Dose for Dead and Surviving NHPs
3,50
3,40
3,30
Log10 CFU
3,20
3,10
3,00
2,90
2,80
2,70
2,60
Slide 7
Log10 CFU Lung Titers for Dead and Surviving NHPs
10,00
9,00
8,00
7,00
Log10CFU
6,00
5,00
4,00
3,00
2,00
1,00
0,00
Slide 8
Log10 CFU TBLN Titers for Dead and Surviving NHPs
9,00
8,00
7,00
Log10 CFU
6,00
5,00
4,00
3,00
2,00
1,00
0,00
Slide 9
Log10 CFU Spleen Titers for Dead and Surviving NHPs
8,00
7,00
6,00
Log10 CFU
5,00
4,00
3,00
2,00
1,00
0,00
Slide 10
Log10 CFU Liver Titers for Dead and Surviving NHPs
7,00
6,00
Log10 CFU
5,00
4,00
3,00
2,00
1,00
0,00
Slide 11
Log10 CFU Mesenteric LN Titers for Dead and Surviving NHPs
8,00
7,00
6,00
Log10 CFU
5,00
4,00
3,00
2,00
1,00
0,00
Slide 12
Recovery of Viable SCHU S4 from
Tissues of Vaccinated NHPs
LVS lot and Animal #
Control NHP
Mean (Dead)
StDev (Dead)
N (Dead)
Mean (Survivors)
StDev (Survivors)
N (Survivors)
Lot 16
Mean (Dead)
StDev (Dead)
N (Dead)
Mean (Survivors)
StDev (Survivors)
N (Survivors)
Lot 17
Mean (Dead)
StDev (Dead)
N (Dead)
Mean (Survivors)
StDev (Survivors)
N (Survivors)
Lot 20
Mean (Dead)
StDev (Dead)
N (Dead)
Mean (Survivors)
StDev (Survivors)
N (Survivors)
Lot 4
Mean (Dead)
StDev (Dead)
N (Dead)
Mean (Survivors)
StDev (Survivors)
N (Survivors)
Log Dose
Lung
TBLN
Spleen
Liver
Mesenteric LN
3.06
0.232
3
8.81
0.391
3
8.14
1.355
3
7.29
0.744
3
5.83
0.116
3
5.06
0.838
3
0
0
0
0
0
0
3.46
8.34
6.24
6.45
5.37
7.16
1
3.20
1
4.69
1
2.82
1
1
1
2
1
1
0
0
0
3.27
8.29
4.73
3.98
3.66
1
3.09
1
4.96
1
3.18
1
1
0
2
1
1
0
0
0
2.92
0.336
5
3.22
0.265
3
8.03
0.666
5
3.40
1.804
2
5.02
1.524
4
3.27
0.569
3
4.20
1.538
4
3.65
1.364
4
2.66
0
0
0
2.97
0.3
3
3.03
0.564
5
7.40
1.2
3
2.92
0.901
5
5.28
3.59
1.0
3
3.93
2.67
2
1
0
0
0
1
3.46
0.855
5
1
Slide 13
Milestone #8 – December 2009 Accomplishments
Peak IFN-g response to LVS hk
350
300
No obvious correlations between day of
peak IFN-g response to hk LVS stimulation
and vaccine lot or time to death post challenge
250
lot 4
lot 16
200
lot 17
150
lot 20
100
Day of peak IFN-g reponse versus survival time;
hk LVS restim.
600
50
500
0
0
5
10
15
Day of peak IFN-g response
20
25
Hours to death post challenge
IFNg spots per 2x10^5 PBMC (media bg
subtracted)
400
400
lot 4
300
lot 16
lot 17
200
lot 20
100
0
0
5
10
15
Day of peak IFN-g response
20
25
Slide 14
Milestone #8 – December 2009 Accomplishments
500
Peak Stimulation index (IFN-g) to hkLVS
450
Stimulation index = # of spots in antigen
well/ # of spots in media control
350
300
lot 4
250
lot 16
200
lot 17
150
lot 20
100
50
Day of peak stimulation index versus survival
time post challenge; hk LVS
600
0
0
5
10
15
20
Day of peak stimulation index
No obvious correlation between
day of peak IFN-g stimulation
index to hk LVS stimulation and
vaccine lot or time to death post
challenge
25
Hours post challenge until death
Stimulation index
400
500
400
lot 4
300
lot 16
lot 17
200
lot 20
100
0
0
5
10
15
Day of peak stimulation index
20
25
Slide 15
Tables: IFN-g peak response day, number of
animals – hk LVS
Peak in number of IFN-g spots with media background subtracted; hk LVS
Peak day 0
Peak day 7
Day 7 %
Peak day 14
Day 14 %
Peak day 21
Day 21 %
Total NHP % Survival
Lot 4
0
1
13%
3
38%
4
50%
8
63%
Lot 16
0
1
33%
0
0%
2
67%
3
67%
Lot 17
0
2
67%
0
0%
1
33%
3
67%
Lot 20
0
5
63%
3
38%
0
0%
8
38%
Total
0
9
41%
6
27%
7
32%
22
Peak in stimulation index (antigen stimulated spots/media control spots); hkLVS
Peak day 0
Peak day 7
Day 7 %
Peak day 14
Day 14 %
Peak day 21
Day 21 %
Total NHP % Survival
Lot 4
0
2
25%
5
63%
1
13%
8
63%
Lot 16
0
1
33%
0
0%
2
67%
3
67%
Lot 17
0
1
33%
1
33%
1
33%
3
67%
Lot 20
1
5
63%
1
13%
1
13%
8
38%
Total
1
9
41%
7
32%
5
23%
22
Slide 16
Tables: IFN-g peak response day, number of
animals – ff LVS
Peak in number of IFN-g spots with media background subtracted; ffLVS
Peak day 0 Peak day 7 Day 7 % Peak day 14 Day 14 % Peak day 21 Day 21 %
Total
NHP
%
Survival
Lot 4
0
0
0%
5
63%
3
38%
8
63%
Lot 16
0
2
67%
0
0%
1
33%
3
67%
Lot 17
0
2
67%
0
0%
1
33%
3
67%
Lot 20
0
4
50%
2
25%
2
25%
8
38%
Total
0
8
36%
7
32%
7
32%
22
Peak in stimulation index (antigen stimulated spots/media control spots); ffLVS
Peak day 0 Peak day 7 Day 7 % Peak day 14 Day 14 % Peak day 21 Day 21 %
Total
NHP
%
Survival
Lot 4
0
1
13%
6
75%
1
13%
8
63%
Lot 16
0
1
33%
0
0%
2
67%
3
67%
Lot 17
0
1
33%
1
33%
1
33%
3
67%
Lot 20
1
4
50%
1
13%
2
25%
8
38%
Total
1
7
32%
8
36%
6
27%
22
Slide 17
No obvious correlations between day of
peak IFN-g response to ff LVS stimulation
and vaccine lot or time to death post
challenge
Peak IFN-g response to ff LVS
700
600
500
400
lot 4
300
lot 16
lot 17
200
lot 20
100
0
0
5
10
15
20
600
25
Day of peak IFN-g response versus survival time;
ffLVS restim.
Day of peak IFN-g reponse
500
Hours to death post challenge
IFN-g spots per 2x10^5 PBMC (media bg
subtracted)
Milestone #8 – December 2009 Accomplishments
400
lot 4
300
lot 16
lot 17
200
lot 20
100
0
0
5
10
15
Day of peak IFN-g response
20
25
Slide 18
Milestone #8 – December 2009 Accomplishments
500
Peak stimulation index (IFN-g) to ffLVS
Stimulation index = # of spots in antigen
well/ # of spots in media control
450
Peak Stimulation Index
400
350
300
lot 4
250
lot 16
200
lot 17
150
lot 20
100
50
600
0
5
10
15
20
25
Day of peak stimulation index
No obvious correlation between
day of peak IFN-g stimulation index
to ff LVS stimulation and vaccine
lot or time to death post challenge
Hours post challenge until death
0
Day of peak stimulation index versus survival
time post challenge; ffLVS
500
400
lot 4
300
lot 16
lot 17
200
lot 20
100
0
0
5
10
15
Day of peak stimulation index
20
25
Slide 19
Milestone #8 – Plans for next month

Fully analyze IFNγ ELISPOT data from survivors
Slide 20
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 21
Milestone #12/13 – December 2009
Accomplishments

Microagglutination assay was run at UNM and is being optimized
there
–


No assays run on NHP plasma as of yet
Received information from Dr. Anders Sjostedt regarding kit
used for protein determination of LVS
Received 4 FF F. tularensis preparations from DVC (2 SCHU S4
and 2 LVS)
Slide 22
Milestone #12/13 – Plans for next month

Continue to communicate with UNM personnel on the progress
on the development of the microagglutination assay
–


Run NHP plasma when it is optimized with control
positive rabbit sera
Order Bradford kit/reagent recommended by Dr. Anders Sjostedt
and try it using live LVS vs. various HK and FF preparations
Test the new 4 FF F. tularensis preparations recently received
from DVC (2 SCHU S4 and 2 LVS) in the NHP plasma IgG antiLVS ELISA
Slide 23
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- LVSvaccinated
NHPs
Establish conditions to
detect growth of LVS in
NHP PBMCs
Confirm
reduced growth
in LVSvaccinated
NHPs
Confirm growth
in non- LVSvaccinated
NHPs
Slide 24
Milestone #21 – December 2009 Accomplishments


PBMC from NHP A07610 frozen on day 7 post LVS (showing a
day 7 IFN-g ELISpot response to hkLVS and ff LVS) were
stimulated for 20 h with 3 different concentrations of hkLVS or
ffLVS or media only,
–
FcR’s were blocked with anti-CD32
–
Cells were surface stained for CD3, CD4, CD8, CD16 and
CD56 and i.c.-stained for IFN-g
In human PBMC, CD56 is expressed on NK cells but Carter, D et
al (Cytometry 37:41-50, 1999) suggest that CD56 identifies
monocytes and not NK cells in (rhesus) macaques
–
This needed to be confirmed in cynomologus macaques
Slide 25
Milestone #21 – December 2009 Accomplishments
2x10^5 hkLVS
per well
(4x10^5 PBMC)
Media only
controls
Slide 26
Milestone #21 – December 2009 Accomplishments
hk LVS stimulation 20 h; % IFN-g+ and median
fluorescence intensity
% IFN-g+ of parent population
70
60
50
40
%IFNg+ of CD3+CD4+
%IFNg+ of CD3+CD8+
30
%IFNg+ of CD3-CD16+
20
% IFNg+ of CD56+
10
MFI IFN-g hkLVS 20 h
Median Fluorescence Intensity IFN-g-FITC
% IFN-g+ PBMC, hkLVS stimulated 20
h
700
600
500
400
CD3+CD4+
300
CD3+CD8+
200
CD3-CD16+
CD56+
100
0
0,00E+00 1,25E+04 5,00E+04 2,00E+05
hkLVS per well with 4x10^5 PBMC
0
0,00E+00 1,25E+04 5,00E+04 2,00E+05
hkLVS per well with 4x10^5 PBMC
CD56+ cells are the only identified cells in the PBMC population that show
an increasing IFN-g expression with increasing hk LVS antigen
concentration.
Slide 27
Milestone #21 – December 2009 Accomplishments
ff LVS stimulation 20 h; % IFN-g+ and median
fluorescence intensity
% IFN-g+ PBMC, ffLVS stimulated 20
h
70
60
50
Median Fluorescence Intensity IFN-g-FITC
% IFN-g+ of parent population
80
MFI IFN-g ffLVS 20 h
%IFNg+ of CD3+CD4+
40
%IFNg+ of CD3+CD8+
30
%IFNg+ of CD3-CD16+
%IFNg+ of CD56+
20
10
0
0,00E+00 1,25E+04 5,00E+04 2,00E+05
ffLVS per well with 4x10^5 PBMC
800
700
600
500
CD3+CD4+
400
CD3+CD8+
300
CD3-CD16+
200
CD56+
100
0
0,00E+00
1,25E+04
5,00E+04
2,00E+05
ffLVS per well with 4x10^5 PBMC
CD56+ cells is the only identified PBMC population that shows an
increasing IFN-g expression with increasing ff LVS antigen concentration.
Slide 28
Milestone #21- Plans for next month


Repeat 20 h of restimulation for intracellular staining of IFN-g in
CD56+ cells and add a CD14 marker to investigate if CD56+ cells
are monocyte/macrophage like in macaques.
–
Also stain NK cells (CD3-,CD16+, CD8+/-).
–
The 20 h time point is similar to the ELIspot restimulation
and PBMC from an NHP with a good ELISpot IFN-g
response will be chosen for this experiment.
The F.t infection and growth assay will be tested using frozen
NHP PBMC (vaccinated versus naïve) with live LVS.
Slide 29
Additional Points
Deliverables completed for each active milestone:
No deliverables were completed during the month of
December.
List of relevant publications from the past month
2 LBERI abstracts accepted for ASM Biodefense meeting in
2010
MSCR status
MS 3: LBERI reviewing revised MSCR (BG sent edits on 12/18/09 )
MS 4: UNM reviewing MSCR (LBERI sent edits to BG on 12/7/09)
MS 7: LBERI reviewing revised MSCR (BG sent edits on 11/18/09)
Slide 30
Action Items (1 of 2)




Michelle: will correct in the 1/5/2010 meeting minutes for data in
the table on slide 6 for the NHP A07842 (completed in minutes
1/8/10)
Cecilia will add a table showing the number of NHP from each
LVS vaccine lot that are peaking on each day (7, 14, and 21) to
allow comparison of peak day from each LVS lot in a table.
Cecilia will add the table to the minutes of this 1/5/10 meeting.
(completed in minutes 1/8/10)
Julie could present on a future technical call, that LBERI often
detects a day 0 mitogen type response to FF antigen and LBERI
does not often get day 0 response to HK antigen.
Michelle: will check on status of histopathology slide review for
MS 11, MS 8B , and MS8C studies. Michelle will gather the
histopathology status for each of the “in life” completed studies
(MS 8B, MS8C, MS 11) and present it to an LBERI internal
meeting in January as well as to NIAID at the next LBERI monthly
technical call.
Slide 31
Action Items (2 of 2)


Michelle: will check on status of histopathology slide review for
MS 11, MS 8B , and MS8C studies. Michelle will gather the
histopathology status for each of the “in life” completed studies
(MS 8B, MS8C, MS 11) and present it to an LBERI internal
meeting in January as well as to NIAID at the next LBERI monthly
technical call. (Completed; see text below)
Histology status:
–
Tul 08B (in-life May/June 09)- Histology report was
provided and Julie Wilder and Michelle Valderas have
access to it.
–
Tul 08C (in-life Nov/Dec 09)- Histology is complete and
slides are to be given to Julie Hutt on 1/8/10. Michelle has
asked for a report by the end of Feb 2010.
–
Tul 11A and 11B (in-life April 8th for 11A and Sept 12th for
11B. )- Julie Hutt has the slides and said that she will have
the report complete by mid-Feb 2010.
Slide 32