Progress Report
2/04/2009
TVDC team – UNM
Prepared by Terry Wu & Amanda DuBois
1
Active Milestones
Active Milestones:
5, 11, 12/13, 14, 17, 18, 19, 21, 29, 35
Today’s presentation will include:
5.
Effect of LVS vaccination dose on protection of Fischer rats against i.t. SCHU
S4 challenge
12/13.
ELISA to titer vaccinated human serum
17. Repeat of SCHU S4 growth kinetics in passively immunized Fischer 344 rats
21. a) SCHU S4 infection of human PBMC
b) Multifunctional T cells in NHP
29. Plans for screening Ft peptide library from ASU
2
Milestone 21: Multifunctional T cell assay
T cell responses from
vaccinated animals
Non-human
primates
(LBERI/UNM)
Mice (UNM)
Rats (UNM)
Develop assay to detect
CD4+ multifunctional T cells
Develop assay to
detect CD4+
multifunctional T
cells
Develop assay to
detect CD4+
multifunctional T
cells
Induction of
multifunctional T cells
after LVS vaccination
Induction of
multifunctional T cells
after LVS vaccination
Recruitment of
multifunctional cells
after Schu S4 challenge
Recruitment of
multifunctional cells
after Schu S4 challenge
Induction of
multifunctional
T cells after LVS
vaccination
Induction of
multifunctional
cells after
respiratory LVS
challenge
Recruitment of
multifunctional cells after
Schu S4 challenge
Blue: Steps in the milestone
Red: Completed
Green: In progress
3
Multiparameter
flow approach
IL-2-PE
IFNγ-PE*Cy7
TNFα-A488
IL-2-PE
TNFα-A488
TNFα-A488
4
IFNγ-PE*Cy7
IFNγ-PE*Cy7
IL-2-PE
Two doses of HK-LVS stimulated a high frequency
of CD4+ lung cells producing TNF, IL-2, and IFN
5
%CD4+ lung cells secreting TNF, IL-2, and IFN
(Uninfected vs LVS treated NHP)
Uninfected
NHP (fresh)
LVS treated NHP LVS treated NHP
(Fresh)
(Frozen)
6
Animal A00868
• Cynomolgus macaque vaccinated by
subcutaneous inoculation with 2.7x106 cfu LVS
on 11/29/06
• Inoculated by bronchoscope instillation with 1x105
cfu LVS on 1/9/2009
• Day 12 after boost/infection: samples of blood,
lungs, tracheobronchial lymph nodes (TBLN),
bronchoalveolar lavage (BAL), liver, and spleen
collected
7
Measured outcomes (data not yet available)
• Cellular recruitment to the site of infection:
– phenotyping of blood and single cell suspensions
isolated from lungs, TBLN, and BAL via FACS
analysis
– differential staining of cytospins prepared from single
cell suspensions of lungs, TBLN, and BAL
• Cytokine and chemokine response to infection:
– Cytokine and chemokine levels in supernatant from
freshly isolated, homogenized lung, TBLN, spleen
and liver tissue, as well as in BAL fluid
– Cytokine and chemokine levels in supernatant from
single cell suspensions from lungs, TBLN, and BAL
cultured overnight in the presence of HK-LVS or
mitogenic stimuli
8
Frequency of multifunctional cells in lungs
following stimulation with PMA/ionomycin
and HK-LVS
2.5
2.0
1.5
1.0
0.5
d12 PMA/Ion
d12 CD28
d12 media
d12 CD28/LVS
Uninfected NHP
(frozen)
naive PMA/Ion
naive CD28/LVS
naive media
0.0
CD4/Irr Ab
%CD4+/TNF/IL-2/IFN cells
Lungs +++
LVS treated NHP (Fresh)
9
Low but detectable population of multifunctional
cells responsive to HK-LVS in spleen of LVS
vaccinated/LVS boosted NHP
7.5
5.0
2.5
0.2
0.1
d28 PMA/Ion
d28 CD28
d28 CD28/LVS
LVS vaccinated/LVS
boosted NHP (Fresh)
d28 media
d12 PMA/Ion
d12 CD28
d12 CD28/LVS
Uninfected
NHP (frozen)
d12 media
naive PMA/Ion
naive CD28/LVS
naive media
0.0
CD4/Irr Ab
%CD4+/TNF/IL-2/IFN cells
Spleen +++
Pulmonary LVS NHP
(Frozen)
10
Response to HK-LVS undetectable in
TBLN from untreated or LVS
vaccinated/LVS boosted NHP
Only organ with
detectable bacteria
(~300 cfu in total
TBLN)
2
1
0.1
Uninfected NHP
(frozen)
d12 PMA/Ion
d12 CD28/LVS
d12 CD28
d12 media
naive PMA/Ion
naive CD28/LVS
naive media
0.0
CD4/Irr Ab
%CD4+/TNF/IL-2/IFN cells
LALN +++
LVS treated NHP (Fresh)
11
Observations
• Stimulation with HK-LVS induced robust and
slight increases in multifunctional cells present in
lung and splenocyte populations, respectively,
from LVS vaccinated/LVS boosted NHP
– No increase in multifunctional cells from TBLN
• Treatment with PMA+Ionomycin was able to
induce robust expression of all 3 cytokines and
large increases in the frequency of
multifunctional T cells in frozen “naive” and fresh
LVS vaccinated/LVS boosted cells
– Exception: fresh lung cells did not respond to
PMA+Ionomycin
12
Milestone 21: Ongoing and upcoming
experiments
• Cellular recruitment and cytokine/chemokine
response data
• Presence of multifunctional cells in the lungs,
TBLN, and spleen of LVS vaccinated/SchuS4
aerosol challenge survivors (Early March)
• Frequency of multifunctional cells in lungs,
TBLN, and spleen of LVS vaccinated NHP
before and after pulmonary SchuS4 infection
13
Milestone 5 – flow diagram
Small animal models
BALB/c
mice
Guinea pigs
Fischer 344
rats
SCHU S4
LVS
SCHU S4
LVS
SCHU S4
LVS
LD50
LD50
LD50
LD50
LD50
LD50
i.n. (i.t.)
s.c., i.d.
i.n.
i.n. (i.t.)
s.c., i.d.
i.n.
i.n. (i.t.)
s.c., i.d.
i.n.
Statistical analyses
LVS vaccination
LVS vaccination
LVS vaccination
Robustness of
Fischer 344 rat model
Clearance of
vaccination strain
Clearance of
vaccination strain
Clearance of
vaccination strain
LVS dose response
SCHU S4 challenge
(in/it)
SCHU S4 challenge
(in/it)
SCHU S4 challenge
(in/it)
Clinical signs and
survival
Clinical signs and
survival
Clinical signs and
survival
Model selection
Comparison of histopathology
mice, rats, NHP
Blue: Steps in the milestone
Red: Completed
Green: In progress
14
Protection Dependent on Vaccine Dose
103 LVS
10
10
0
0
Gain
Loss
% Starting Wt
Unvaccinated

-10

-20
0
2

-10
4
6
-20
8
0
105 LVS
10
15
107 LVS
10
10
0
0
-10
-10
-20
-20
Gain
Loss
% Starting Wt
5
Days post i.t. SCHU Challenge
Days post i.t. SCHU Challenge
0
5
10
Days post i.t. SCHU Challenge
15
0
5
10
Days post i.t. SCHU Challenge
15
15
Milestone 5: Plans
• Reduce LVS vaccination dose
• Complete histological analyses of tissues from
SCHU S4-infected mouse, rat and NHP
16
Milestone 12/13 – flow diagram
Assays for Detecting Relevant Immune
Responses in Animals and Humans
Humans (UNM)
Mice (UNM)
Rats (UNM)
Generate reagents
Generate reagents
Generate reagents
ELISA for Ab titer
ELISA for Ab titer
ELISA for Ab titer
T cell proliferation
T cell proliferation
T cell proliferation
T cell IFNg production
T cell IFNg production
T cell IFNg production
Determine and optimize
assay sensitivity
Determine and optimize
assay sensitivity
Determine and optimize
assay sensitivity
Blue: Steps in the milestone
Red: Completed
Green: In progress
17
Anti-LVS Titer in Vaccinated Humans
standard 1-27-09
5
4.500
Absorbance
3.500
Blank 450
3.000
2.500
2.000
3
2
1
0
1.500
1.000
-1
10 2
0.500
10 3
10 4
10 5
10 6
Serum Dilution
0.000
-0.001
Naive
Vaccinated
4
4.000
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
Conc
18
Milestone 12/13: Plans
• Obtain sera from additional vaccinated
individuals for standard pool?
• IFN ELISpot with vaccinated human PBMC
19
MS 17: Characterization of Fischer 344 Rat
Fischer 344
rats
Blue: Steps in the milestone
Red: Completed
Green: In progress
Humoral
immunity
Cell mediated
immunity.
LVS vaccination
Purchase and culture hybridoma
cell lines
Passive transfer of
serum
Production of ascites fluid for CD4 and
CD8 depletion
Protection against i.t
SCHU challenge
In vivo depletion
Active vs passive
immunizatioin
Pretection against i.t. SCHU
SCHU challenge
20
20
Kinetics of SCHU S4 Growth & Dissemination in
Passively-immunized Rats
Spleen
10
Liver
10
8
8
6
6
4
4
2
2
0
0
Lungs
10
Naive (0.25ml PBS)
NRS (0.25ml)
Vaccinated
IRS (0.25ml)
8
6
0
5
10
15
20
25
4
2
0
5
10
15
20
25
0
5
10
15
20
25
21
Kinetics of SCHU S4 Growth & Dissemination in
Passively-immunized Rats - Repeat
Lung
CFU (log10)
CFU (log10)
8
7
6
5
4
3
2
1
2
3
Days Post-challenge
4
9
8
7
6
5
4
3
2
1
0
CFU (log10)
9
0
Spleen
Liver
0
1
2
3
Days Post-challenge
4
9
8
7
6
5
4
3
2
1
0
Naive (0.25ml PBS)
NRS (0.25ml)
Vaccinated
IRS (0.25ml)
0
1
2
3
4
Days Post-challenge
22
Milestone 17: Plans
• Complete bacterial burden experiment
23
MS 21: Assays in Vaccinated Humans
Assay to measure activation of macrophage
killing mechanisms in humans
Naive
Immu
Monocyte-derived
macrophages
Macrophage
T cells
Determine the approximate
yield of macrophages from
whole blood (1-200 ml max)
Determine the approximate yield of PBMC
and T cells from whole blood
Monocytes
PBMC
Test PBMC
Test purified
T cells
Determine and optimize cell
number and MOI
in vitro
expansion?
Positive control w/
IFN
Blue: Steps in the milestone
Red: Completed
Green: In progress
Repeat w/ human
vaccinee
24
Proliferation of SCHU S4 in Human
Monocytes
1.010 6
CFU / well
1.010 5
1.010 4
1.010 3
MOI=0.1 (-) serum
MOI=0.1 (+) serum
MOI=1.0 (-) serum
MOI=1.0 (+) serum
1.010 2
1.010 1
1.010 0
0
24
48
hours post-infection
72
25
Infection of Human PBMC & Monocytes
10 7
Total CFU / well
10 6
10 5
10 4
10 3
FT only
+ PBMC
+ Mo
+ Ly/Mo
10 2
10 1
0
24
48
72
Hours post-infection
26
Activation of PBMC and Monocytes
with recombinant IFN
10 7
10 7
10 6
10 6
10 6
10 5
10 4
10 3
total CFU/well
10 7
total CFU/well
total CFU/well
Monocytes
PBMC
PBMC
10 5
10 4
10 3
10 2
PBMC
PBMC + IFNg
0
48
24
Hours post-infection
72
10 5
10 4
10 3
10 2
10 2
PBMC
PBMC + IFNg
0
24
48
Hours post-infection
72
Mo
Mo + IFNg
0
24
48
Hours post-infection
72
27
Milestone 21: Plans
• Determine whether human PBMC/monocyte
effector activity can be increased beyond the
level observed after IFN, eg by preactivation
• Addition of vaccinated human T cells
28
MS29: Plans
• Vaccinated NHP with LVS
• ASU prepared and shipped peptide array
– 2065 linear expression element constructs
• UNM
– Ordered needed reagents and materials
• Schedule
– Feb 11 boost with LVS by bronchoscopy
– Feb 22 assay with Lymph node cells and splenocytes
– 7 peptides/well in duplicates, 16 plates total
– IFN ELIspot with cells and ELISA on culture supe
29
Action Items
•
•
•
•
•
Julie Hutt: will convey the quality of the veterinary pathology slides that return to UNM from the North
Carolina based vendor.
Terry will send the sera titration figure to Freyja for review and will include in the 2/15/09 monthly tech
report. Terry will copy the email to Barbara. (completed 2/5/09)
Terry will purchased pooled normal human sera from a commercial vendor, if available
Freyja: will get microagglutination protocol from Marcelo and send it to UNM; NIAID will make a batch of
antigens (normal and O mutants) to share with DVC and UNM.
Freyja: will advise Barbara on which Cerus lab bench protocols to move into “gold standard format”, in
association with Cerus MS#40 MSCR .
30