LBERI Update on Animal Model
Development
Sub-NIAID Tech Call
3 March 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
Milestone #4 – Pathology Reports for Pilot 2
A05262, F, day 29 euthanasia (study termination), presented
dose unknown.

Histopathology:
– Chronic, multifocal to coalescing, pyogranulomatous to necrotizing
bronchopneumonia.
– Chronic pyogranulomatous mesenteric lymphadenitis.
– The tracheobronchial lymph node was not available for histologic
examination.
– GI nematode identified.

Clinical Pathology:
– High normal total WBC and neutrophil counts; low normal
lymphocyte counts; normocytic, normochromic anemia.
– Decreased serum albumin (within normal range).
Slide 3
Milestone #4 – Pathology Reports for Pilot 2
A05254, F, day 10 euthanasia, presented dose unknown.
•
Histopathology:
- Subacute, multifocal to coalescing, pyogranulomatous to necrotizing
bronchopneumonia.
- TBLNs (2): a) mild sinus histiocytosis and plasmacytosis; b) marked,
pyogranulomatous lymphadenitis with coagulation necrosis.
- Pyogranulomatous splenitis with coagulation necrosis.
- Hepatocellular atrophy and vacuolization.
•
Clinical Pathology:
- High normal total WBC and neutrophil counts; low normal
lymphocyte counts and hematocrit.
- Decreased serum phosphorus and albumin (within normal range).
Slide 4
Milestone #4 – Pathology Reports for Pilot 2
Summary






Lung, TBLN, spleen- lesions are those expected for F. tularensis.
Normocytic, normochromic anemia is compatible with response
to chronic inflammatory disease.
Reductions in serum phosphorus and albumin are compatible
with sustained anorexia.
Different lesions in TBLNs from the same animal are likely due to
their relative positions with respect to lymphatic drainage of the
lungs. Suggests caution should be used in interpretation of data
from just one lymph node.
Mesenteric lymphadentis may be consistent with GI uptake of
organism, or systemic dissemination to multiple lymph nodes.
Other lymph nodes will be examined in future studies.
GI nematode of undetermined significance in one animal.
Slide 5
Milestone #4 – Pathology Reports for Pilot 3
A04643, F, natural death on day 3, 2.36 x 10e5 CFU presented.

Histopathology:
– Acute, multifocal, pyogranulomatous to necrotizing
bronchointerstitial pneumonia.
– TBLNs (2): a) mild sinus histiocytosis; b) marked, widespread
pyogranulomatous and necrotizing lymphadenitis.
– Pyogranulomatous splenitis with coagulation necrosis.
Slide 6
Milestone #4 – Pathology Reports for Pilot 3
A04645, F, natural death on day 3,1.09 x 10e6 CFU presented.
•
Histopathology:
- Acute, multifocal, pyogranulomatous to necrotizing
bronchointerstitial pneumonia.
-TBLNs (2): a)&b) Widespread pyogranulomatous and necrotizing
lymphadenitis.
- Pyogranulomatous splenitis with coagulation necrosis.
- Acute, suppurative to necrosuppurative rhinitis with necrotizing
submucosal lymphangitis.
Slide 7
Milestone #4 – Pathology Reports for Pilot 3
Summary




Lung, TBLN, spleen- lesions are those expected for F. tularensis.
Lung lesions in these animals were smaller and more uniformly
distributed (miliary) than in previous pilot studies, likely due to
the greater inhaled dose and earlier death.
Different lesions in TBLNs from the same animal are likely due to
their relative positions with respect to lymphatic drainage of the
lungs. Suggests caution should be used in interpretation of data
from just one lymph node.
Nasal cavity lesions in one animal suggest that systemic
dissemination from the nasal cavity is possible.
Slide 8
MS#8 – Flow Diagram
MS 8: LVS Vaccinated NHP Challenged with SCHU S4
Set 1 Vaccination Practice (n=3 scarification; n=2 subcutaneous)
Set 2 Vaccination (n=3 by scarification; n=3 by
subcutaneous route; n=3 previously
vaccinated; 1 SC, 2 ID)
Set 2 and October 2006 Vaccinee SCHU S4 Challenge 500 CFU
OR
Set 3 Vaccination/Challenge
(USAMMDA vaccine vs. DVC
LVS Lot 16 by scarification)
Red: completed
Green: in progress
Blue: steps in the milestone
Set 3 Vaccination/Challenge
(Vaccination Dose Titration
by scarification or s.c.)
SCHU S4
Challenge
500 CFU
Slide 9
Milestone #8 - Objective and Endpoints


Describe the outcome of aerosol delivered SCHU S4 infection in NHPs
that have been previously vaccinated with LVS. Two different methods
of vaccination will be 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 10
Milestone #8 – January and February 2009
Accomplishments
Vaccinated 6 NHPs with LVS on 1/8/09 ( 3 by scarification and 3
by subcutaneous route)
Screened 7 newly arrived non-LVS vaccinated NHPs and chose 3
to serve as controls for the SCHU S4 challenge
3 NHPs previously vaccinated with LVS in 10/06 (2 by intradermal
route and 1 by subcutaneous route) were tested for residual
immunity to LVS
12 NHPs (3 controls; 6 vaccinated with LVS in 1/09; 3 vaccinated
with LVS in 10/06) were challenged with SCHU S4 by aerosol on
2/12 and 2/13 (target dose was 500 CFU)
Slide 11
Vaccinees and SCHU S4 Challenge
Animal
Number
Vaccine Group
Presented
Dose (CFU)
Exposure Date
Date of Death (Study
Day)
A03152
Control
50
2/12/09 – Flask 1
Alive as of 3/2 (18) †
28643
Jan. 8, 2009 scarified
43
2/12/09 – Flask 1
Alive as of 3/2 (18) †
28671
Jan. 8, 2009 scarified
34
2/12/09 – Flask 1
2/21/09 (9)
A04994
Jan. 8, 2009 scarified
27
2/12/09 – Flask 1
2/21/09 (9)
A05895
Control
89
2/12/09 – Flask 2
2/18/09 (6)
28627
Jan. 8, 2009 subcutaneous
117
2/12/09 – Flask 2
2/28/09 (16) †
28587
Jan. 8, 2009 subcutaneous
293
2/12/09 – Flask 2
2/24/09 (12) †
A06587
Jan. 8, 2009 subcutaneous
1690
2/12/09 – Flask 2
2/22/09 (10) †
A06626
Control
684
2/13/09
2/18/09 (5)
A00937
Oct 2006 Intradermal
754
2/13/09
Alive as of 3/2 (17) †
A00908
Oct 2006 Intradermal
1780
2/13/09
2/17/09 (4)
A00659
Oct 2006 Subcutaneous
1270
2/13/09
2/21/09 (8)
†
indicates increased time to death as compared to animals exposed to similar
presented doses in the ED50 study
• Flasks grew to near the same optical density, dosing error on 2/12/09 was a
microbiological miscalculation.
Slide 12
Tissue Burdens
Animal ID
Vaccine
Group
Presented
Dose (CFU)
A03152
Control
50
A05895
Control
43
28463
25671
A04994
8JAN09
Scar
8JAN09
Scar
8JAN09
Scar
Nx Date
Spleen
Liver
CFU/g
Mes LN
TBLN
Lung
18-Feb-09 1.56E+07 4.34E+05 1.54E+08 7.00E+04 6.39E+08
34
27
21-Feb-09 4.60E+05 4.60E+04 1.19E+07 1.12E+06 3.83E+08
89
21-Feb-09 1.20E+06 1.20E+05 4.83E+06 9.80E+02 8.18E+08
28627
8JAN09 Sub
117
28-Feb-09
28587
8JAN09 Sub
293
24-Feb-09 4.34E+05 9.00E+03 2.73E+06 4.76E+05 7.67E+08
A06587 8JAN09 Sub
1686
22-Feb-09 7.92E+05 9.46E+04 9.80E+05 4.90E+07 2.12E+08
A06626
Control
684
18-Feb-09 7.41E+07 1.92E+06 1.61E+09 2.17E+06 4.09E+08
A00937
OCT06 ID
754
A00908
OCT06 ID
1777
17-Feb-09 4.34E+04
1274
21-Feb-09 3.07E+04 3.07E+04 6.30E+06 4.76E+03 3.32E+08
A00659 OCT06 Sub
BLD
4.62E+08 9.80E+05 1.46E+08
a
BLD, below limit of detection
Slide 13
Blood Bacterial Burden
Animal ID
Vaccine
Group
Presented
Dose
Nx Date
(CFU)
A03152
Control
50
A05895
Control
43
28463
25671
A04994
28627
28587
A06587
8JAN09
Scar
8JAN09
Scar
8JAN09
Scar
8JAN09
Sub
8JAN09
Sub
8JAN09
Sub
18-Feb-09
34
Study Dayb
0
1
2
3
4
5
6
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
3.33E+00
BLD
BLD
BLD
BLD
BLD
BLD
BLD
1.30E+02 4.87E+02 4.88E+02
27
21-Feb-09
BLD
BLD
BLD
BLD
BLD
BLD
BLD
n/a
89
21-Feb-09
BLD
BLD
BLD
BLD
1.00E+01
BLD
3.33E+00
BLD
117
28-Feb-09
BLD
BLD
BLD
BLD
3.33E+00
BLD
BLD
293
24-Feb-09
BLD
BLD
BLD
BLD
6.67E+00
BLD
BLD
BLD
1686
22-Feb-09
BLD
BLD
BLD
BLD
BLD
BLD
BLD
BLD
18-Feb-09
BLD
BLD
3.33E+
00
BLD
6.33E+01
1.53E+03
BLD
BLD
BLD
BLD
BLD
BLD
3.33E+00
BLD
2.50E+02
A06626
Control
684
A00937
OCT06 ID
754
A00908
OCT06 ID
1777
17-Feb-09
BLD
BLD
BLD
3.33E+00 3.33E+00
1274
21-Feb-09
BLD
BLD
BLD
1.27E+02
A00659 OCT06 Sub
Term
BLD
BLD
Slide 14
Antigen-specific IFNγ Production by non-LVS
Vaccinated Controls
IFNg Spots (Mean +/- S.D.)
350
89 CFU
Day 6
300
250
50 CFU
200
Media
LVS hk Hi
LVS ff Hi
664 CFU
Day 5
150
100
50
0
A03152
A05895
A06626
All cells plated at 1.33 x 106/ml; All IgG anti-LVS titers were 1/20000
“Day x” indicates the day that the NHP succumbed to SCHU S4 infection.
A03152 remains alive as of 3/2/09.
Slide 15
Antigen-stimulated IFNγ Production by LVS
Vaccinated NHPs (10/06)
IFNg Spots (Mean +/- S.D.)
350
300
250
1270 CFU
Day 8
Media
LVS hk Hi
LVS ff Hi
754 CFU
200
1740 CFU
Day 4
150
100
50
0
A00659
A00908
A00937
All cells plated at 1.33 x 106/ml; Day post-LVS vaccination = 786 (A00659, SC, 2.7 x 106
CFU LVS) – 795 (A00908 and A00937, ID, 15 x 106 CFU LVS); “Day x” indicates the day
that the NHP succumbed to SCHU S4 infection. A00937 remains alive as of 3/2/09.
Slide 16
Antigen-stimulated IFNγ Production by NHPs
Vaccinated with LVS by Scarification (1/09)
250
43 CFU
500K
200
34 CFU
Day 9
Media
LVS hk Hi
LVS ff Hi
20K
500K
150
4K
100
27 CFU
Day 9
0.8K
A04994, Day 0
28671, Day 25
28671, Day 0
0
28643, Day 25
50
100K
A04994, Day 25
300
28643, Day 0
IFNg Spots (Mean +/- S.D.)
350
Day Post-LVS Vaccination
All cells plated at 1.33 x 106/ml; IgG anti-LVS titer shown (ex. 4K); LVS vaccination dose
= 31,200; “Day x” above the bars indicates the day that the NHP succumbed to SCHU S4
infection; 28643 remains alive as of 3/2/09.
Slide 17
Antigen-stimulated IFNγ Production by NHPs
Vaccinated with LVS by S.C Inoculation (1/09)
400
350
300
283 CFU
Day 12
250
100K
4K
Day 16
117 CFU
1690 CFU
Day 10
100K
100K
4K
20K
200
150
100
A06587, Day 25
A06587, Day 0
28627, Day 25
28627, Day 0
0
28587, Day 25
50
28587, Day 0
IFNg Spots (Mean +/- S.D.)
450
Media
LVS hk Hi
LVS ff Hi
Day Post-LVS Vaccination
All cells plated at 1.33 x 106/ml; IgG anti-LVS titer shown (ex. 4K); LVS vaccination dose =
31,200; “Day x” above the bar indicates the day that the NHP succumbed to SCHU S4 infection.
Slide 18
Preliminary Data Interpretation



Delivery of SCHU S4 by aerosol was more variable than
anticipated
Survival post-SCHU S4 aerosol challenge does not correlate with
IgG anti-LVS titers
Sensitivity of recently vaccinated NHPs to SCHU S4 may be
related to their relatively weak IFNγ production post-vaccination
–
The lack of responsiveness to HK LVS has not been
observed previously in any LVS-vaccinated NHP and may
be due in this case to the low LVS inoculum (31,200 CFU)
–
Previous vaccinees (10/08) received 2.6 x 105 CFU LVS
and all 5 produced more IFNγ upon HK LVS stimulation
after vaccination as compared to before vaccination (see
next slide for historical data)
Slide 19
0
SCAR
400
SC
A06199, Day 0
A06199, Day 7
A06199, Day 15
A06199, Day 21
A06199, Day 28
A06199, Day 35
300
Media
LVS hk Hi
LVS ff Hi
A05403, Day 0
A05403, Day 7
A05403, Day 15
A05403, Day 21
A05403, Day 28
A05403, Day 35
700
A04169, Day 0
A04169, Day 7
A04169, Day 15
A04169, Day 21
A04169, Day 28
A04169, Day 35
500
28656, Day 0
28656, Day 7
28656, Day 15
28656, Day 21
28656, Day 28
28656, Day 35
600
28461, Day 0
28461, Day 7
28461, Day 15
28461, Day 21
28461, Day 28
28461, Day 35
IFNg Spots (Mean +/- S.D.)
IFNγ Production by LVS-vaccinated NHPs
SCAR
SC
SCAR
200
100
Day Post-LVS Vaccination
All cells plated at 1.33 x 106/ml; an arbitrary value of 600 was
assigned to wells which were TNTC; LVS vaccination dose =
2.6 x 105
Slide 20
Milestone #8 – LVS Vaccination
Plans for next month
Any survivors from the current SCHU S4 challenge will be
euthanized on day 20 - 21 post-challenge (3/5) and tissues taken
for pathology, microbiology and immunologic assessment
Slide 21
New Experiment Proposed under MS 8

Test three different LVS vaccination doses delivered by s.c.
inoculation followed by challenge with 500 CFU SCHU S4
–
LVS doses: 1 x 105, 1 x 106, 1 x 107 (3 NHPs/dose + 3
controls)
–
Grow LVS in broth and quantitate CFU delivered based on
OD600 growth curve
–
Deliver LVS by s.c. route to ensure that entire dose is
delivered
–
SCHU S4 challenge will be delivered by aerosol (sometime
between day 28 – 45 post LVS vaccination)
–
Screened NHPs are immediately available
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:
Works for
Con A and
LVS
ELISPOT
Plasma
IgG
ELISA
Plasma
IgA
ELISA
Slide 23



Milestone #12/13 – January - February 2009
Accomplishments
Continued to test freeze/thaw protocol
Discussed with the UNM and LRRI team how to standardize the
LVS and SCHU S4 antigens (protein content vs. CFU/ml)
Discussed with Freyja Lynn how to proceed on describing the
nature of the plasma IgG anti-LVS levels
– Decided to make a positive and negative control reference
plasma stock by combining several plasma samples that
perform similarly in the ELISA assay
– aliquots of the pooled plasma would then be frozen and
thawed each time the assay was run
– arbitrary units of activity could be assigned and used as a
reference standard curve
Slide 24
Update on testing O-antigen mutants as stimuli in the IFNg ELISPOT and
Proliferation Assays




We were interested in testing whether the non-specific responses to LVS and
SCHU S4 antigens we observe in PBMCs from non-vaccinated NHPs in the
proliferation and IFNg ELISPOT assays were due to LPS moeities on the fixed
and heat-killed organisms (O- antigen mutants obtained from Anders Sjostedt
When the protein content of each antigen preparation was tested using a BCA
Kit, we realized they were not equivalent
Our plan is to construct a standard curve correlating CFU/ml and protein
content using LVS and SCHU S4; aliquots will be plated and lysed; lysates will
be measured for protein content
Preparations of heat-killed and formalin-fixed LVS will also be lysed and
measured for protein content; the standard curve will allow correlation to
CFU/ml
Slide 25
Update on Freeze/Thaw Testing



We have been comparing two protocols (Cerus and CTL) for use
in freezing and thawing PBMCs
The goal is to find a protocol which results in PBMCs whose
response mimics the response of the original fresh PBMCs in the
proliferation and IFNγ ELISPOT assay
In the past months, we have only had PBMCs from non-LVS
vaccinated NHPs to compare; we are now beginning to thaw
PBMCs from the NHPs which were vaccinated with LVS in
October 2008
Slide 26
Freezing Protocols Appear Equivalent in Sparing the
Responsiveness of PBMCs to LVS as Measured by IFNγ
Production
700
600
500
Media
LVS hk Hi
LVS ff Hi
400
300
200
Day 28, None
Day 28, CTL
Day 28, Cerus
Day 21, None
Day 21, CTL
Day 21, Cerus
Day 15, None
Day 15, CTL
0
Day 15, Cerus
100
All cells plated at 1.33 x 106/ml; an arbitrary value of 600 was
assigned to wells which were TNTC; 2 NHPs receiving LVS
by scarification are shown
We would like to choose Cerus as the freezing protocol moving forward
Slide 27
Milestone #12/13 - Immune Responses in
Animals and Humans
Plans for next month
Determine the relationship between LVS protein content and
CFU/ml
Begin to re-titrate the WT and mutant LVS antigens based on
protein content
Construct reference positive and negative control plasma and
test in IgG anti-LVS ELISA
Slide 28
Milestone #29 – Analysis of T cells from lymph
nodes and T cell epitopes
Vaccinate NHPs with LVS
(2 S.C. Vaccinees 10/08)
Boost LVS Immunity by Bronchoscopy with LVS
Collect LNs
Transfer to UNM for testing in Peptide Librarystimulated ELISPOT Assays
Red: completed
Green: in progress
Blue: steps in the milestone
Slide 29




Milestone #29 –February 2009
Accomplishments
LVS bronchoscopy was completed on 2/11
Harvest of tissues was on 2/23
PBMC and spleen responses to HK and FF LVS are being
analyzed and will be presented next month (LRRI)
Response of spleen and LN cells to peptide library will be
presented by UNM personnel
Slide 30
Action Items






Michelle: determine whether the extended time to death for the 1/8/09
subcutaneously vaccinated NHP is significant
Barbara- will arrange another call with NIAID/UNM/LBERI to include Rick and Bob,
possibly Friday 3/6 morning. Call will cover a discussion of the aerosol generation
and the microbiology, as well as the LVS vaccinations. NIAID wants more data on
vaccination doses, such as getting the LVS counts on raw data , how the vials are
resuspended, what they are resuspended in, what the calculated vs actual LVS doses
are etc.
UNM/LBERI will discuss further the next vaccination/challenge plan with NIAID at the
upcoming ad hoc call.
Barbara: No later than 3/17/09, UNM (Terry) /LBERI (Trevor) pull together a
comparison of the LVS lot#16 vials, quantitation of CFU data in LVS lot#16 vials,
and how the LVS Lot#16 vials are behaving in each location at UNM and LBERI;
Julie Wilder: will write MS 12/13 Milestone completion report and send to Barbara.
Barbara: will ask Rick/Bob if the 3/6/09 LBERI/UNM TVDC internal meeting can be
used for an ad hoc call with NIAID.
Slide 31