Tularemia Vaccine Development Contract
Progress in Tularemia Bioaerosol
Characterization and Non-Human Primate
Aerosol Challenges
April 2, 2010
Lovelace Respiratory Research Institute
2425 Ridgecrest Drive SE, Albuquerque, NM 87108
AGENDA
10:00 – 10:15
Scientific Overview
C. Rick Lyons
10:15 – 10:30
Update: Goals & Milestones Achieved
R. Sherwood
10:30 – 11:30
NHP Model Development (MS 8, 9, 10,
11)
R. Sherwood, J.
Wilder, T. Brasel
11:30 – 12:00
Lunch Break
12:00 – 1:00
NHP Model Development (MS 12-15, 1722, 29)
J. Wilder
1:00 - 1:45
J. Hutt
1:45 – 1:55
NHP Histopathology with SchuS4
Infection
Afternoon Break
1:55 – 2:15
LBERI Problems Encountered
R. Sherwood
2
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AGENDA (Cont.)
2:15-2:30
Milestone Completion Reports
R. Sherwood
2:30 – 3:00
Publications
R. Sherwood
3:00 – 3:15
2010 Annual Meeting
Rick Lyons/ Barbara
Griffith
9:15 – 9:45
Administrative Discussion
B. Griffith
3
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Scientific Overview
C. Rick Lyons, UNM
4
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Update: Goals & Milestones
Achieved
R. Sherwood, LBERI
5
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Goals & Milestones Achieved
 Milestones completed
– MS#3, MS#4, MS#7, MS#11
 Goals completed
– Determined aerosol LD50 in cynos
– Determined aerosol natural history for cynos at target
dose of 1000 CFU
– Determined that LVS vaccination can protect Cynos
against aerosol tularemia challenge
6
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Remaining Challenges
 Determine optimal LVS vaccination procedure so
that LVS can be used as a positive control in future
vaccine studies
 Determine if any currently used immune parameters
may be useful in predicting immune status and
infectious challenge outcome
7
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Future Plans
 Perform broth comparison challenge study to clarify
if LVS growth conditions might alter vaccine survival
 Complete study of tularemia infection in vaccinated
animals
 Complete study of vaccine duration
 Perform one NHP vaccine efficacy study
8
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NHP Model Development
(MS 8,9,10,11)
R. Sherwood, T. Brasel, LBERI
9
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NHP Study Plans
 Broth Comparison Study
 Effect of LVS Vaccination on SCHU S4
Pathogenesis
– Telemetered and non-Telemetered arms
 LVS Vaccine Duration
 Tularemia Vaccine Efficacy (Optional)
10
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Broth Comparison Study
 Goal – Determine if infection with SCHU S4
grown in Chamberlain’s broth affects
virulence in Cynos as compared to growth in
Mueller Hinton Broth II
11
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Broth Comparison Study
 Experimental Design
Test
Group
Number
Pathogen Challenge
-Study Day
(Challenge Dose)
Group
Designation
Animals/
Group
Vaccine Dose
(cfu/Animal)
Vaccination Study Day
1
Control
(unvaccinated)
3
NA
NA
35 (~ 1000 cfu, CB)
2
Control
(unvaccinated)
3
NA
NA
35 (~1000 cfu,
CAMHB)
3
DVC LVS
Lot 17
7
1.8x108 cfu
0
35 (~1000 cfu, CB)
4
DVC LVS
Lot 17
7
1.8x108 cfu
0
35 (~1000 cfu,
CAMHB)
12
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Broth Comparison Study
 Timeline
– In life -5/11 – 6/15
– Path Report - 8/31/10
– MS Report - 9/20/10
13
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Effect of LVS Vaccination on SCHU S4 Pathogenesis
 Goal
– Determine how LVS vaccination with Lot
17 affects SCHU S4 pathogenesis in
telemetered and non-telemetered Cynos
14
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Effect of LVS Vaccination on SCHU S4 Pathogenesis
 Experimental Design
Test
Group
Number
Group
Animals/
Group
Vaccine
Dose
Vaccination
(cfu/Animal) - Study Day
1
DVC LVS
Lot 17
12
1.8x108 cfu
0
2
DVC LVS
Lot 17
24
1.8x108 cfu
0
Pathogen
Challenge -Study
Day (Challenge
Dose)
Sac Day
35 (~1000 cfu)
Moribund
35 (~1000 cfu,
CAMHB)
1, 2, 4, 6
15
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Effect of LVS Vaccination on SCHU S4 Pathogenesis
 Timeline
– In life -July/Aug 2010
– Path Report - 11/15/2010
– MS Report - 12/5/2010
16
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LVS Vaccine Duration
 Goal
– Determine the ability of Lot 17 LVS to
protect Cynos against SCHU S4 aerosol
challenge over a time period of up to 6
months
– (already have short term duration data)
17
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LVS Vaccine Duration
 Experimental Design
Test
Group
Number
Group
Animals/
Group
Vaccine Dose
(cfu/Animal)
Vaccination Study Day
Pathogen Challenge Study Day (Challenge
Dose)
1
Control
8
--
0
90, 180 (~1000 cfu)
2
DVC LVS
Lot 17
16
1.8x108 cfu
0
90, 180 (~1000 cfu)
18
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LVS Vaccine Duration
 Timeline
– In life -Fall 2010 – Spring 2011
– Path Report - 12 weeks post in-life
– MS Report - 14 week post in-life
19
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Tularemia Vaccine Efficacy (Optional)
 Goal
– Determine the ability of a tularemia
vaccine to protect Cynos against an
aerosol challenge with SCHU S4
20
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Tularemia Vaccine Efficacy (Optional)
 Experimental Design
Test
Group
Number
Group
Animals/
Group
Vaccine Dose
(cfu/Animal)
Vaccination Study Day
Pathogen Challenge Study Day (Challenge
Dose)
1
Control
6
--
0
35 (~1000 cfu)
2
DVC LVS
Lot 17
10
1.8x108 cfu
0
35 (~1000 cfu)
3
Test Vaccine
10
High Dose
0
35 (~1000 cfu)
4
Test Vaccine
10
Low Dose
0
35 (~1000 cfu)
21
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Tularemia Vaccine Efficacy (Optional)
 Timeline
– In-Life - Spring 2011
– Path Report - 12 weeks post in-life
– MS Report - 14 week post in-life
22
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MS #8 and MS #11 Results
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Milestone 8: Goal
 Determine the nature of LVS-vaccination in
NHPs and its role in protection from SCHU S4
aerosol challenge
24
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Milestone 8: Objectives and Endpoints
 Describe the character 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
- Examine the disease process in animals vaccinated with DVC Lot 17 post aerosol
challenge with a lethal dose of SCHU S4 (1000 cfu) – Tul08E
 Endpoints
– histopathology, bacteremia, bacterial CFUs of organs (lung, spleen, liver, kidneys, and
lymph nodes), clinical signs, clinical chemistry, hematology, cytokine analysis (some
studies, but not all)
25
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Milestone 8: Progress – Summary of TUL08 A - C
 LVS (DVC Lot 16, 17, 20, and USAMMDA Lot 4) given by sub-cutaneous
inoculation (on back (TUL08 A and B; or stomach TUL08 C)
– Range of inoculum: 7 x 105 – 9 x 107 CFU LVS
– Humoral and cellular immunity measured weekly until challenge
 SCHU S4, grown in Chamberlain’s broth, delivered by head-only aerosol
exposure on a day 35 - 51 days post-vaccination
– Target dose of 1000 CFU presented in 3.5 L (range = 500 – 2872)
 Euthanize any survivors 21 days after exposure.
 Measure clinical signs daily; measure bacteremia on the day of aerosol
challenge and 2, 4, 6, 10, 14 and 21 days post- SCHU S4 exposure;
measure tissue burden of SCHU S4 at necropsy.
26
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Milestone 8: Progress – LVS Vaccination Induces
Humoral Immunity in NHPs
Units/ml determined by comparison to a positive control pooled
plasma sample used as a standard curve in the LVS ELISA
27
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Milestone 8: Progress – LVS Vaccination Induces
Cellular Immunity in NHPs as Evidenced by IFNγ
Production by PBMCs
28
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Milestone 8: Progress – LVS Vaccinated NHPs are not
Uniformly Protected from Death due to SCHU S4 Aerosol
Challenge
By ANOVA, all vaccinated groups were significantly different from the
unvaccinated group, however there was no difference in survival between lots.
29
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Milestone 8: Progress – High Levels of IgG anti-LVS do not Predict
Protection from Death due to SCHU S4 Aerosol Exposure
Lot 16
Lot 17
Lot 20
Lot 4
None
40000
35000
30000
25000
20000
15000
10000
5000
0
-5000
100
150
200
250 300
350
Hours to Death
400
450
500
30
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Milestone 8: Progress – LVS Vaccination Leads to
Reduced SCHU S4 Burden in Organs other than the Lung
One Way ANOVA indicates there is
no statistically significant difference
in lung burden, however with
respect to the TBLN, all vaccine lots
are signifcantly different than the
naïve controls
31
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Milestone 8: Progress – LVS Vaccination Leads to
Reduced SCHU S4 Burden in Organs other than the Lung
One way ANOVA indicates that in
the spleen, bacterial burden in the
vaccinees
was
significantly
decreased as compared to the
controls. This is true of the liver
also, with the exception of Lot 16.
32
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Milestone 8: Progress – LVS Vaccination Leads to
Reduced SCHU S4 Bacteremia as Compared with
Control NHP
Group/ Vaccine
Lot
Bacteremic NHP
(During study)
% Positive
Bacteremia
Control
3 of 3
100
Lot 16
0 of 3
0
Lot 17
1 of 3
33
Lot 20
2 of 8
25
Lot 4
2 of 8
25
33
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Milestone 8: Progress - Summary
 LVS vaccination of NHPs leads to:
– Increased survival after SCHU S4 aerosol challenge
– Increased humoral and cellular immunity; although neither high
levels of antibody nor IFNγ production predict protection from
SCHU S4 aerosol challenge
– Decreased SCHU S4 organ burden in tissues other than the lung
(blood, spleen, liver, lymph nodes); this is accompanied by
decreased histopathology in spleen and liver
 LVS vaccination does not prevent clinical signs of illness
34
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Milestone 8: Remaining Challenges
 Thus far, LVS vaccination of NHPs provides only partial protection from
SCHU S4 aerosol challenge
– Hypothesize that growth of SCHU S4 in Chamberlain’s broth as
compared to Mueller-Hinton broth may be allowing organism to
stimulate very little innate immunity and thus grow to large
numbers in the lung and eventually disseminate to peripheral
organs where it leads to mortality
– Growth in Mueller-Hinton broth has been shown to stimulate more
inflammatory cytokine secretion by macrophages in vitro and may
do so in the lung as well, leading to better control of growth and
perhaps better overall protection from morbidity
– This hypothesis will be directly tested in TUL 08 D
35
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Milestone 11: Goal
 Describe the natural history of aerosol delivered
SCHU S4 in the cynomolgus monkey.
36
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Milestone 11: Objectives and Endpoints
 The primary objective of this study was to determine the natural course
of disease in cynomolgus macaques of Vietnamese origin (with and
without telemetry devices) that had been aerosol challenged with a
dose of F. tularensis SCHU S4 that resulted in primary pulmonic
disease. A secondary objective was to relate study results to human
disease to the extent possible.
 Endpoints = histopathology, bacteremia, bacterial CFUs of organs
(lung, spleen, liver, kidneys, and lymph nodes), clinical signs, clinical
chemistry, hematology, cytokine analysis
37
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Milestone 11: Progress
 Study is completed.
 Report has been submitted to NIAID and includes the following as
described in the SOW:
– Histopathology and bacterial CFUs of internal organs (lung,
spleen, liver, brain, and lymph nodes)
– Record of clinical symptoms
– Clinical chemistry and hematology during infection
 Data, protocols, and SOPs and documentation exist to generate a
GLP compatible aerosol model of F. tularensis SCHU S4
38
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MS11 – overview of study design
Test
Group
Number
Group
Designation
1
Sac 1
2
Sac 2
3
Sac 3
4
Sac 4
5
Sac 5
Animals/
Group
2 males/
2 females
2 males/
2 females
2 males/
2 females
2 males/
2 females
6 males/
6 females
Pathogen
Challenge Study Day
(Challenge
Dose)
Terminal
Sacrifice –
Study Day
0 (~ 1000 CFU)
2
0 (~ 1000 CFU)
4
0 (~ 1000 CFU)
5
0 (~ 1000 CFU)
6
0 (~ 1000 CFU)
Terminal
39
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MS11 – Overview of Findings
40
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MS11 – Overview of Findings
Sustained fevers were not detected in
individual animals without telemetry; however,
in animals with telemeters, prolonged fevers
were detected. Hypothermia was a common
finding prior to death with or without
telemeters.
41
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MS11 – Overview of Findings
Increases in respiration rate (~50 respirations
per minute) were routinely observed in
animals without telemetry; however, only a
minor increase in respiratory rate was
observed with telemeters. We hypothesize
that the observed difference between manual
respiratory rates could be due to telemeter
lead placement.
42
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MS11 – Overview of Findings
High levels of bacteria were found in the lung, TBLN, and spleen. Lower and
more variable numbers of bacteria were found in the liver, MLN, and brain
stem. In general, progressively higher numbers of SCHU S4 were recovered
from organs with increasing time. Appearance in the spleen, liver, brain, and
lymph nodes corresponded with bacteremia
43
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MS11 – Overview of Findings
Hematology showed leukocytosis beginning days 2 to 3 and leukopenia days
5 and 6. Elevated CRP levels were typically observed on day 2 or 3 and
appeared to correlate with dissemination from lungs.
44
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Milestone 11: Remaining Challenges and Future
Plans
 There are no challenges for this milestone
 Future plans include addressing any comments on the final
report and finalizing the report.
45
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Lunch Break
46
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NHP Model Development
(MS 12-15, 17-22, 29)
J. Wilder, LBERI
47
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Milestone 12/13: Goal
 Establish assays that detect the immune response to F.
tularensis and vaccines designed to protect from disease
induced by F. tularensis in multiple animal models
– LBERI is tasked with developing immunoassays that
detect the immune response in (LVS) vaccinated and
SCHU S4 exposed NHPs
48
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Milestone 12/13: Goal – Progress toward
Objective
 Develop immunoassays that reliably distinguish LVS-vaccinated 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 but more data mining may be in order (i.e. only examining
responsiveness early post-LVS vaccination or late; only looking at specific types of
stimuli (FF vs. HK))
49
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Milestone 12/13: Progress
 Microagglutination assay:
– Designed to test LVS-specific agglutinating antibody in plasma or sera;
likely IgM
– As this assay was used in human clinical trials of vaccinees, it can be
used to bridge the NHP data to the human data
– Assay is working and the titer of the positive rabbit serum (courtesy of
Dr. Marcelo Sztein, U. of Maryland) was determined to be 1: 2560
– The optimal antigen dilution (fixed hematoxylin-stained LVS) for use in
the assay is between 1:4 and 1:8
– Day 7 plasma samples from NHPs are currently beeing analyzed at
both antigen dilutions, 1:4 and 1:8
50
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Milestone 12/13: Progress (continued) - Performance of
Formalin-fixed F. tularensis antigens in the IgG ELISA
51
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Milestone 12/13: Progress (continued) - Performance of
Formalin-fixed F. tularensis antigens in the IgG ELISA
 Data interpretation:
– DVC WT LVS (and SCHU S4) appear to work well in the IgG antiLVS ELISA whereas the mutant (minus LPS) mutants do not
– It is unclear as to whether our ELISA detects antibodies
directed to the LPS moeity of LVS or whether the mutant
antigens fail to bind to the ELISA plate
 Recent acquisition of monoclonal antibodies specific for
non-LPS moeities on LVS may help distinguish these two
possibilities
52
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Milestone 12/13: Progress (continued)
 Protein Assay:
– We would like to establish the F. tularensis CFU:protein ratio in
live, formalin fixed (FF) and heat-killed (HK) preparations
– Establishment of this ratio will allow us to normalize antigen
preparations used to simulate NHP PBMCs in the ELISPOT and
proliferation assays, as well as those used as coating antigens
in the IgG anti-LVS assay
– Fermentas Bradford Reagent has been purchased and sample
buffer prepared according to description from Dr Anders
Sjostedt, Umea University, Sweden
 We will soon test the FF and HK antigens from both UNM
and DVC
53
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Milestone 12/13: Remaining Challenges
 No assay thus far can distinguish LVS-vaccinated NHPs that
are protected from SCHU S4 aerosol exposure from those
that succumb
 DVC FF LVS and SCHU S4 antigens are not performing as
expected; they do not stimulate IFNγ production on a
CFU/ml basis as well as FF antigens prepared by Dr. Lyons’
laboratory personnel at UNM
54
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Milestone 12/13: Future Plans
 Establish the protein content:CFU ratio of the FF and HK antigens in
order to determine whether actual differences exist in their stimulatory
capacity or differences exist in the amount of protein being used in the
assays
 Test the ability of the microagglutination assay to detect NHP anti-LVS
antibody
 Continue to explore both new assays (such as total cytokine secretion
in addition of IFNγ ELISPOT) and different ways of expressing the
cellular immunoassay data to determine whether NHPs protected from
SCHU S4 aerosol challenge can be distinguished from those that
succumb
55
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Milestone 21: Goal
 Establish assays of effector function that predict correlates
of protection from SCHU S4 aerosol challenge
– LBERI is tasked with developing immunoassays that
distinguish NHPs that are protected from SCHU S4
aerosol challenge and those that succumb to it
56
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Milestone 21: Goal – Progress toward Objective
 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, TNFαand 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
57
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Milestone 21: Progress - Intracellular Cytokine
Staining
 Several experiments have been conducted to stain CD3, CD4, CD8 (T
cells) and IFN-g, TNF-a, IL-2 to look for multifunctional T cells in
PBMC at different time points post vaccination and with different
times for restimulation (6h, 12h, 24 h, 72 h)
– HiCK-1 cell controls showed that the cytokine antibodies and
staining protocol were working
 ELISpot assay for IFN-g secretion showed that PBMCs from
vaccinated NHPs produced IFN-g above media background after 20 h
of restimulation
 Using the 20 h of restimulation with hk or ff LVS investigated what
PBMC subset produced IFN-g; NK-cells? NK-T cells?
Monocytes/macrophages?
58
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Milestone 21 Progress: Intracellular Cytokine Staining
- identifying NK cells in NHPs
 Human natural killer (NK) cells are often identified in PBMC as CD3CD16+CD56+, with CD56 being the most lineage specific marker
 Carter at al (Cytometry 37:41-50, -99) showed that CD56 identifies
monocytes and not natural killer cells in rhesus macaques
 New staining panel : CD3/CD4/CD8 for T cells, CD16 for NK-cells
and CD56 for monocytes/macrophages; IFN-g as the only cytokine;
FcR block with anti-CD32 (FcgRII)
 Following slide shows frozen and thawed PBMC from NHP A07610,
day 6 post LVS vaccination (106 LVS S.C); showed IFN-g response in
ELIspot
59
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Milestone 21: Progress - IFN-g production PBMC
from A07610 day 6 post LVS
70
% IFN-g+ PBMC, ffLVS 20 h
%IFNg+ of
CD3+CD4+
%IFNg+ of
CD3+CD8+
%IFNg+ of
CD3-CD16+
% IFNg+ of
CD56+
60
50
40
30
20
10
0
hkLVS per well with 4x10^5 PBMC
% IFN-g+ of parent population
% IFN-g+ of parent population
% IFN-g+ PBMC, hkLVS 20 h
80
70
60
%IFNg+ of
CD3+CD4+
50
40
%IFNg+ of
CD3+CD8+
30
%IFNg+ of CD3CD16+
20
10
%IFNg+ of CD56+
0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
ffLVS per well with 4x10^5 PBMC
•Similar pattern when median fluorescence intensity is graphed instead of %
IFN-g+
•CD56+ cells are the only population showing antigen dose dependent IFN-g
production
• High spontaneous IFN-g production by CD56+ cells
60
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Milestone 21: Progress - Intracellular cytokine
staining – CD56+ cell production of IFNγ
 What more can be determined about the phenotype of the CD56+
IFN-g producers? How reproducible is the finding (i.e. can they be
found in other LVS-vaccinated NHPs)?
 Next experiment: New animal with a positive LVS-stimulated
response and low media background in IFN-g ELISpot. A07566 day
7 post s.c 107 LVS
 Stimulate 20 h with increasing concentrations of hk or ff LVS
 Stain surface: CD3, CD8 for T cells, CD14 for
monocytes/macrophages, CD16 for NK-cells, CD56 (monocytes?)
and IFN-g.
61
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Milestone 21: Progress - IFN-g production as measured
by ICS from PBMC A07566 day 7 post LVS
% IFN-g+ PBMC, ffLVS stimulated 20 h
60
50
40
30
20
10
0
-10
100.0
CD3+CD8CD3+CD8+
CD3-CD16+
CD56+
CD14+
hkLVS per well with 4x10^5 PBMC
% IFN-g+ of parent population
% IFN-g+ of parent population
% IFN-g+ PBMC, hkLVS stimulated 20 h
80.0
60.0
CD3+CD8CD3+CD8+
40.0
CD3-CD16+
CD56+
20.0
CD14+
0.0
0.00E+00 1.25E+04 5.00E+04 2.00E+05
-20.0
ffLVS per well with 4x10^5 PBMC
•Similar pattern when median fluorescence intensity is graphed instead of
% IFN-g+
•CD56+ cells are the cells that have the highest reponse to the antigen in a
dose-dependent manner when measuring IFN-g day 7 post LVS
62
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Milestone 21: Progress - Cell surface phenotype
of CD56+IFN-g+ PBMC
CD8
SSC
CD8
SSC
CD3
FSC
CD14
CD16
FSC
A06944
CD14
CD3
FSC
FSC
29976
CD16
CD8
SSC
A07566
CD3
CD14
63
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Milestone 21: Progress - CD56
 Human – other names: Leu-19, NKH-1, NCAM
 Ligands: NCAM-1, heparin sulfate
 MW 140kDa
 Cellular expression: T cells, NK-cells
 Mice – other names: NCAM
 MW 120, 140,180 kDa
 Ig superfamily
 Ligands; NCAM, heparin sulfate
 Cellular expression: NK, T (sub), neural tissue
 Function: adhesion, differentiation/development
64
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Milestone 21: Progress – Fresh (non-frozen)
PBMC from A06944 day 14 post LVS
1.25x104 cfu 5x104 cfu
0 cfu
1.5%
MFI 126
9.5
138
1.3
126
10.5
0.4
103
10.4
91
4.4
Day 14 post LVS
Good T cell response to
ff LVS but not hkLVS
CD3+CD8+
CD8 T cells
122
3.3
116
CD3+CD8CD4 T cells
145
3.8
3.8
102
156
153
96
12.0
9.6
3.8
0.4
102
8.3
131
144
2x105 cfu
3.3
111
Lower media background in
T cells compared to CD56+ cells
65
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Milestone 21: Progress – Fresh (non-frozen) PBMC from
A06944 day 14 post LVS – CD56+ cells
Gated CD56+ cells
0 cfu
1.25x104 cfu 5x104 cfu
2x105 cfu
% IFN-g+ A06944 PBMC 20h ffLVS,
fresh PBMC
59.5
1399
57.3
1204
% IFN-g+ of legend populations
1270
58.5
1332
1482
62.3
50.4
1013
68.9
66.1
1427
59.2
1335
14
% IFN-g+ A06944 PBMC 20h ffLVS, fresh
12
PBMC
10
8
6
CD3+CD8-
4
CD3+CD8+
2
CD3-CD16+ (NK)
CD14+
0
% IFN-g+ CD56+ PBMC
80
70
60
50
40
CD56+
30
20
10
0
0.00E+001.25E+04 5.00E+042.00E+05
cfu/well ffLVS
•Less total CD56+ cells than in previously
tested NHPs
•Do not appear to have a dose dependent
response of IFN-g to hk or ff LVS in this NHP/at
day 14
•Lower media background in T cells compared
to CD56+ cells
66
cfu/well ff LVS
7296-66
Milestone 21: Progress – Comparison of fresh vs.
frozen/thawed PBMCs in the ICS Assay
 Questions raised by our preliminary experiments:
 Are freshly prepared cells a requirement to detect
IFN-g in T cells ?
 Are the CD56+ cells, or their production of IFN-g, a
cryopreservation artifact?
67
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Milestone 21: Progress - Frozen/thawed PBMC
can be used to detect T cell derived IFNγ
ffLVS
cfu/well 0
1.25x104
5x104
2x105
Gated CD4 T cells
0.2
96
0.8
94
0.2
101
0.6
0.1
1.7
57
1.7
Gated CD8 T cells
56
1.1
63
Fresh cells are not an absolute
requirement to detect T cell
IFN-g
101
0.5
0.4
61
97
97
59
1.8
1.1
0.7
0.1
66
93
94
60
0.6
1.5
65
A06944 day 14 post
LVS
68
7296-68
Milestone 21: Progress – CD56+ IFNγ cells are
detectable in both fresh and frozen/thawed PBMC
CD56+ gated
0 cfu
1.25x104 cfu 5x104 cfu
59.5
1399
57.3
1204
50.4
1013
68.9
1482
62.3
1270
2x105 cfu
58.5
1332
66.1
1427
Fresh PBMC
59.2
1335
Similar small total CD56+
population. Larger
spontaneously IFN-g producing
population in fresh cells.
Frozen/thawed
13.6
135
148
16.3
200
18.8
14.3
104
150
22.4
27.8
164
32.1
124
18.2
124
69
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Milestone 21: Summary - Intracellular Staining
for IFN-g
 Vaccinated NHP PBMC show a dose dependent IFN-g response from CD56+
cells as early as day 7 post vaccination.
 Some vaccinated animals show a dose dependent IFN-g response from CD4 and
CD8 T cells to ffLVS (but not hk LVS) at day 14 post LVS.
 CD56+IFN-g+ PBMC have a complex surface phenotype; Preliminary results
suggest that most of them express CD3.
 The PBMC population of CD56+ cells is variable in each individual animal. This
is a characteristic of NKT cells in humans.
 Freshly prepared cells are not an absolute requirement to detect IFN-g from T
cells.
 CD56+ cells and CD56+IFN-g+ cells can be detected in both frozen and freshly
prepared cells.
70
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Milestone 21: Progress - In vitro growth assay
using NHP PBMC and live Francisella tularensis
1.00E+05
29987 PBMC pre LVS and 2 wk post
LVS
1.00E+04
cfu/well
1.00E+03
29987 wk2
29987 pre-v
1.00E+02
1.00E+01
1.00E+00
0
72 h
culture time
Slow growth over 72 h of LVS in NHP PBMC both from pre- and post vaccination. Will
repeat assay with NHP PBMC and SCHUS4 in B3 and pre-treat PBMC with fixed LVS or
rIFN-g. Use freshly prepared NHP PBMC if possible.
71
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Milestone 21: Remaining Challenges
 Find cytokine multifunctional T cells. T cells may not
produce selected cytokines (IL-2, TNF-a and IFN-g) all at
the same time point post vaccination/restimulation.
 Determine what cell type CD56+ cells constitute and if
they are important in priming a memory T cell response.
72
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Milestone 21: Future Plans
 Further investigate if CD56+ cells produce IFN-g early
(day 7) post LVS vaccination whereas a T cell IFN-g
response may appear later (day 14 and beyond) – how is
this important for protection?
 Find out if CD56+ cells are NK-T cells by a functional
assay or staining with a-GalCer loaded CD1d tetramers
(QC:d for NHP?).
 Move the in vitro growth assay into B3 and start using
SCHUS4; pre-stimulate cells with LVS/IFN-g.
73
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Milestone 29: Goal - Analysis of T cells from NHP lymph
nodes and screening of F. tularensis epitope library
 Generate T cells from LVS–vaccinated NHPs that can be
used to screen for reactivity against F. tularensis peptide
libraries at UNM by IFNγ ELISPOT
74
7296-74
Milestone 29: Goal – Progress toward Objective
 Peptide library from ASU is prepared and UNM is awaiting
selection of NHPs to be vaccinated with LVS via the s.c.
route and then subsequently boosted with LVS via
bronchoscopy
75
7296-75
Milestone 29: Remaining Challenges
 None
76
7296-76
Milestone 29: Future Plans
 Discuss with UNM personnel the desired LVS vaccination,
boost and euthanasia dates
 Schedule the procedures
7296-77
NHP Histopathology with
Schu S4
J. Hutt, LBERI
78
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LD 50 study
 Presented doses from 1.25 cfu to over 106 cfu
 <100 cfu presented-have the longest clinical course, develop only
minimal to mild lung disease, but have more severe disseminated
disease
 >25,000 cfu presented-have a rapidly progressive clinical course,
develop severe lung disease, and die quickly, prior to the
development of severe disseminated disease
 >100 cfu to <25,000 cfu presented-have an intermediate clinical
course and develop both substantial lung disease and disseminated
disease
79
7296-79
Death at low presented doses occurs with
minimal lung involvement
#28615, FY08-074, LD 50 study, 1.33 cfu presented,
death on day 9, focal lung lesion only
80
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Minimal lung involvement
<5 % of lung
>95 % of lung
#28615, FY08-074, LD 50 study, 1.33 cfu presented,
death on day 9, focal lung lesion only
81
7296-81
Liquifaction of TBLN
#28615, FY08-074, LD 50 study, 1.33 cfu presented,
death on day 9, focal lung lesion only
82
7296-82
Sinusoidal thrombi in liver and coagulation
necrosis of entire spleen
#28615, FY08-074, LD 50 study, 1.33 cfu presented,
death on day 9, focal lung lesion only
83
7296-83
Cavitating lung lesion in survivor
#28624, FY08-074, LD 50 study
2.85 cfu presented, survivor euthanized at~46 days
84
7296-84
Evidence of immune response in draining TBLN of
survivor
#28624, FY08-074, LD 50 study
2.85 cfu presented, survivor euthanized at~46 days
85
7296-85
Minimal, resolving inflammatory response in the
liver of survivor
#28624, FY08-074, LD 50 study
2.85 cfu presented, survivor euthanized at~46 days
86
7296-86
PMN counts peak earlier in animals presented
with higher doses, but peak counts are the same
PMN Counts, 10-100 cfu presented
30
30
30
25
25
1.25
20
1.33
1.65
15
2.85
10
25
19.3
20
30.3
60.7
15
89.6
10
PMN Count (X1000
cells/microliter)
35
5
363
2
4
6
8
10
12
14
16
18
20
417
15
444
621
10
675
5
884
0
0
0
237
20
5
0
0
2
4
6
8
10
12
14
16
18
0
20
Days After Exposure
PMN Counts, 1000-10,000 cfu presented
PMN Counts, 10,000-100,000 cfu presented
35
30
30
30
1380
20
2930
15
5430
5610
10
25
25,300
20
47,800
15
52,000
73,700
10
PMN Count (X1000
cells/microliter)
35
1150
0
0
8
10
12
Days After Exposure
14
16
18
20
0
2
4
6
8
10
12
Days After Exposure
12
14
16
18
20
14
16
18
20
225,000
255,000
336,000
1,260,000
10
0
6
10
15
5
4
8
20
5
2
6
25
5
0
4
PMN Counts, >100,000 cfu presented
35
25
2
Days After Exposure
Days After Exposure
PMN Counts (X1000
cells/microliter)
PMN Count (X1000 cells/microliter)
PMN Counts, 100-1000 cfus presented
35
PMN counts (X1000
cells/microliter)
PMN counts (X1000
cells/microliter)
PMN Counts, <10 cfu presented
0
2
4
6
8
10
12
Days After Exposure
14
16
18
20
87
7296-87
PMN counts peak earlier in animals presented
with larger doses, but peak counts are the same
Peak PMN Count (X1000 cells/microliter)
Peak PMN Numbers are Independent of Dose
30
25
20
15
10
5
0
1-10 cfu, d4
11-100 cfu, d4
101-1000 cfu,
d2
1001-10000
cfu, d2
10001-100000
100,001cfu, d2
1,000,000 cfu,
d2
Presented dose
88
7296-88
Natural History Study
 Target dose~1000 cfu, equal numbers of male and
female Vietnamese cynomolgus macaques
 Part A-untelemetered, serial sacrifices planned for days
2, 4, 5 and 6 post-exposure
 Part B-telemetered, survival up to day 21 post-exposure
89
7296-89
Exposure Summary-part A-untelemetered
90
7296-90
Exposure Summary-part B-telemetered
Animal #
Death Type
Death Day
Dose(cfu)
A07752
Ms
7
285
A07755
Ms
7
309
A07801
Ms
7
96.2
A07805
Nd
8
102
A07808
Ms
8
121
A07783
Ms
7
43
A07784
Nd
7
182
A07788
Ms
6
248
A07811
Ms
6
627
A07828
Nd
7
327
A07841
Ms
6
273
A07776
Ts
22
119
91
7296-91
Natural history study-PMN & monocyte counts
peaked at day 2
Total WBCs, Neutrophils & Lymphocytes-part A
Monocytes, Eosinophils & Large Unstained Cellspart A
Monocytes
30
all WBC
1
25
0.9
0.8
20
15
10
Eosinophils
3
0.7
cells/ mL X 10
Lymphocytes
Total Cells
cells/ m L X 10
Total Cells
3
PMN
0.6
0.5
0.4
Large
Unstained
Cells
0.3
5
0.2
0
-15 (16) 0 (16)
0.1
1 (16)
2 (8)
3 (8)
4 (12)
5 (7)
Exposure Day (animal number)
6 (2)
0
-15 (16)
0 (16)
1 (16)
2 (8)
3 (8)
4 (12)
5 (7)
6 (2)
Exposure Day (animal number)
92
7296-92
CRP levels peaked between day 4 & 5 in part A
(untelemetered)
CRP (mg/L)-part A
600
Concentration
500
400
300
200
CRP
(mg/L)
100
0
0 (16)
1 (16)
2 (16)
3 (6)
4 (6)
5 (7)
6 (2)
Exposure Day
avg on d4=438 (+/-125)mg/L
avg on d5=454 (+/- 95)mg/L
93
7296-93
CRP levels peaked on day 4 in part B
(telemetered)
Comparison of CRP -part B
Average
500
survivor
(A07776)
300
200
100
-100
21
20
19
18
17
16
15
14
13
12
11
10
9
8
7(2)
6 (12)
5 (12)
4(12)
3(12)
2(12)
1 (12)
0 (12)
0
-32 (12)
Concentration
400
Exposure Day (animal number)
peak avg on d4=329 (+/-78) mg/L
94
7296-94
Presented dose & peak CRP were both lower in
part B than in part A
Peak CRP, Part A and B
600
500
CRP (mg/L)
400
300
200
100
*
**
* P=.037
**P=.006
0
Part B, d4
Part B: 43-627 cfu
(avg=228, sd=158)
Part A, d4
Part A, d5
Part A:183-8458 cfu
(avg=3733, sd=2334)
95
7296-95
Chemistry changes-consistent, gradual decrease
in Na, Cl, & phosphorus
Potassium, Phosphorus & Calcium
Changes-part A
Sodium and Chloride Changes-part A
150
12
140
10
130
NA
(mmol/L)
120
CL
(mmol/L)
110
Concentration
Concentration
160
8
4
2
90
0
0 (16)
1 (16)
2 (16)
3 (6)
4 (6)
5 (7)
PHOS
(mg/dL)
6
100
-15
(16)
K
(mmol/L)
CA
(mg/dL)
-15
(16)
0 (16) 1 (16)
2 (16)
3 (6)
4 (6)
5 (7)
Exposure Day (animal number)
Exposure Day (animal number)
96
7296-96
Chemistry changes-sporadic increases in liver
enzymes, LDH & CK
AST, ALT & GGT-part A
LDH & CK-part A
LDH
(IU/L)
700
AST
(IU/L)
600
3000
2500
CK (IU/L)
2000
400
ALT
(IU/L)
300
200
GGT
(IU/L)
100
Concentration
Concentration
500
1500
1000
500
0
-500
-1000
-15
(16)
0 (16)
1 (16)
2 (16)
3 (6)
4 (6)
5 (7)
6 (2)
0
-100
-15
(16)
0 (16) 1 (16) 2 (16)
3 (6)
4 (6)
5 (7)
6 (2)
Exposure Day (animal number)
Exposure Day (animal number)
97
7296-97
Chemistry changes-sporadic increases in BUN
Alkaline Phosphatase & Triglyceridespart A
BUN & Creatinine-part A
70
ALP
(IU/L)
900
BUN
(mg/dL)
60
800
50
TRIG
(mg/dL
)
600
500
400
Concentration
Concentration
700
40
CREA
(mg/dL)
30
20
300
10
200
100
0
0
-15 (16) 0 (16)
1 (16)
2 (16)
3 (6)
4 (6)
Exposure Day (animal numbers)
5 (7)
-10
-15
(16)
0 (16) 1 (16) 2 (16)
3 (6)
4 (6)
5 (7)
6 (2)
Exposure Day (animal number)
98
7296-98
Chemistry changes-consistent, gradual increases
in TP & albumin, & increases in globulins
Total Protein, Albumin, Globulin &
Bilirubin-part A
TBILI
(mg/dL)
9
8
ALB
(g/dL)
Concentration
7
6
GLOB
(g/dL)
5
4
3
TP
(g/dL)
2
1
0
-15
(16)
0 (16)
1 (16)
2 (16)
3 (6)
4 (6)
5 (7)
6 (2)
Exposure Day (animal number)
99
7296-99
Pneumonia severity increases over time
100
7296-100
Neutrophilic & histiocytic alveolitis-day 2
101
7296-101
Antigen localized to PMNs, macrophages & ciliated
epithelium (surface) on d2
102
7296-102
Pleuritis & lymphangitis evident on d4
103
7296-103
Necrosis & edema evident on d5
104
7296-104
Necrotozing arteritis & bronchitis, with marked
edema-d6
105
7296-105
Increased antigen in lung on day 6
106
7296-106
Inflammation & necrosis in draining lymph nodes
increase in severity over time
107
7296-107
Hepatitis progression over time
108
7296-108
Splenitis progression over time
109
7296-109
Myelitis-d6
110
7296-110
Decreased necrosis in inflammatory foci of
vaccinated animals
111
7296-111
Afternoon Break
112
7296-112
LBERI Problems
Encountered, Overcoming
Problems and Challenges in
the Upcoming Year
R. Sherwood, LBERI
113
7296-113
Overcoming Problems & Challenges
 Turn around time for histopathology
– LRRI implemented TRP® (Toxicology Resource
Planning)
 Allows resource planning much earlier, thus identifying
potential roadblocks – goal is to complete histo in 10
weeks
 LVS vaccination failed to protect NHPs
– Performed follow on vaccine challenge studies
– Scheduled broth comparison study
114
7296-114
Milestone Completion Reports:
Schedule for next 18 months
R. Sherwood, LBERI
115
7296-115
Milestone Completion Report Schedule
 MS 08c - April 9
 MS 08a – April 23
 MS 08b – May 7
 MS 08d (broth comparison) - Sept 6
 MS 08e (LVS natural history) – Oct 2010
 Tul 9 (Aerosol SOP) – Fall 2010
 MS 12/13 - Sept 2011
 MS 20/21 - Sept 2011
116
7296-116
Publications
R. Sherwood, LBERI
117
7296-117
Future Publications (manuscripts)
 Schu S4 and LVS Bioaerosols (Trevor Brasel)
– Data sufficient
 Schu S4 LD50 in Cynos (Michelle Valderas)
– Data sufficient
 Schu S4 Natural History in Cynos (Michelle Valderas and Julie Hutt)
– Data sufficient
 Immunology of LVS vaccination in Cynos (Julie Wilder)
– Data?
118
7296-118
Recent Publications (posters, talks and abstracts)
 Vaccination with Francisella tularensis strain LVS induces humoral and cellular immunity in
Cynomolgus macaques but fails to uniformly protect from pulmonary disease induced by aerosol
infection with strain SCHU S4. J Wilder. ASM Biodefense & Emerging Dis . Conf. 2010, Baltimore,
MD, Feb. 21-24, 2010.
 Optimization of aerosol generation techniques for Francisella tularensis LVS and SCHU S4 strains.
Sherwood, R.L., T. Brasel and E. Barr. 6th International Conference on Tularemia, Berlin, Germany,
Sept. 13-16, 2009 .
 The pathology of Francisella tularensis SCHU S4 in Cynomolgus macaques. Hutt, J.A., M.M.
Valderas, J.A. Wilder, T. Brasel, R.L. Sherwood and C.R. Lyons. 6 th International Conference on
Tularemia, Berlin, Germany, Sept. 13-16, 2009 .
 Characterization of aerosol infection with F. tularensis SCHU S4 in Cynomolgus macaques and
LD50 determination. M.W. Valderas, E. Zinter, T. Brasel, E. Barr, J. Hutt, J. Wilder, R. Sherwood and
C.R. Lyons. 6th International Conference on Tularemia, Berlin, Germany, Sept. 13-16, 2009 .
 Humoral immunity to Francisella tularensis strain LVS fails to uniformly protect Cynomolgus
macaques from disease induced by aerosol infection with strain SCHU S4. J.A. Wilder, M. Valderas,
A. Monier, T. Brasel, R. Sherwood and C.R. Lyson. 6 th International Conference on Tularemia, Berlin,
Germany, Sept. 13-16, 2009 .
119
7296-119
2010 Annual Meeting
C. Rick Lyons & B. Griffith, UNM
120
7296-120
Action Items?
 Close
121
7296-121
Action Items ( 1 of 5 slides)
 Bob will review the MS 8E study design again and discuss adding
naïve NHP as controls, in parallel with the LVS vaccinated and SCHU
S4 challenged NHP (with and without telemetry).
 LBERI will design the “ NHP LVS vaccine duration “ study with keeping
the aerosol challenges on a consistent date and vaccinating at
different time points to get the different durations. Will include a 35
day challenge group as a positive control.
 LBERI could perform a new vaccine candidate NHP study at two
vaccine doses if the new vaccine candidate is developed by early 2011
and is promising in rat model.
 UNM/LBERI will discuss with NIAID, which new vaccine candidates to
test in NHP. Even as low as 50% protection in the rat model, may be
enough to pursue the new candidate in the NHP model since both the
rat model and vaccine candidates are relatively new.
7296-122
Action Items (2 of 5 slides)
 Julie Wilder will re-plot slide 30 ( IgG levels vs survival) on a log scale
for the Y axis.
 LBERI will break out survivors from non-survivors on slide 31, showing
that LVS vaccination leads to reduced SCHU S4 burden in organs
 LBERI will merge all vaccinated non-survivors vs. all vaccinated
survivors, when plotting that LVS vaccination leads to reduced SCHU
S4 burden in organs other than lung in MS8C
 On MS8C, look at whether one lot 17 LVS vaccinee that was
bacteremic, also survived?
 LBERI is getting help with telemeter lead placement to improve
respiration measurements on study MS8E (relative to MS11).
7296-123
Action Items (3 of 5 slides)
 LBERI will change the bacteremia report to cover serial sacrificed NHP
in the MS 11 MSCR report.
 LBERI will address NIAID’s current questions on the MS 11 MSCR
report, once NIAID sends comments to UNM.
 LBERI will develop a composite scoring system for the multiple data
types (e.g. hematology, clinical score sheets etc) and will give to NIAID
 LBERI can try using a BCA reagent test in the ELISA plate well to titrate
the coating with the DVC antigens too.
 Cecilia: look at CD56+ cells response in naives stimulated with LVS in
the cytokine multifunctional T cell assay
 LBERI will perform in vitro stimulation assays with fresh NHP PBMC
cells and SCHU S4 as the stimulus.
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Action Items (4 of 5 slides)
 LBERI will do subcutaneous LVS vaccinations only with NHP for
peptide library testing and do not use bronchoscopy on these NHP.
 Julie: needs to look at IL 17 in the LVS vaccinated and challenged NHP
 Julie/Bob will give UNM new MSCR completion dates for the site visit
minutes for MS 8c, 8a, 8b, 8d, 8e, 9, 12/13/14/15 and 20/21.
 UNM/LBERI will focus on organizing the data, across species , across
assays etc. If get a correlate of protection that is great. But if
UNM/LBERI does not reach the correlate, the studies are still a
success, if can transfer the data to NIAID in organized fashion for future
data mining. Organize by animal number etc.
 UNM TVDC/ LBERI publish all data, including negative data, in a review
or summary article, with the data organized in a database with a
weblink to the published review article.
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Action Items (5 of 5 slides)
 LBERI will put timetables together for writing the LBERI scientific
manuscripts from TVDC data.
 Bob/LBERI will initiate publication meetings approximately once
/month and include other critics/contributors from within UNM and
LBERI.
 The TVDC final contract report might be like a major review article
7296-126