Progress Report
07/30/2008
TVDC team – UNM
Prepared by Terry Wu & Amanda DuBois
1
Active Milestones
Active Milestones:
5, 11, 12/13, 14, 17, 19, 21, 27, 35
Today’s presentation will include:
5.
Small animal models of tularemia
11.
Effector mechanisms in LVS vaccinated rats
21.
Multifunctional T cells; Improving the reproducibility of
macrophage killing assays in mice and rats
27.
Testing of ivt proteins for ASU (ASU MS 26)
35.
RNA isolations for ASU
2
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.
LVS vaccination
LVS vaccination
LVS vaccination
Clearance of
vaccination strain
Clearance of
vaccination strain
Clearance of
vaccination strain
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
Statistical analyses
Response to LVS
vaccination dose
Blue: Steps in the milestone
Red: Completed
Green: In progress
3
MS5 Objective 1
• Perform statistical analyses of the Fischer
344 rat model so that we can make
recommendations on:
– Vaccination route and dose for vaccine studies
– Dose for respiratory challenge
– Group size if effect of subunit vaccine is as large
as LVS or if effect is less than LVS
4
Proportional Hazard Regression
Analyses of Fischer 344 rats
Naive
Intradermal
104
105
106
107
103
104
105
106
Intratracheal
Subcutaneous
104
105
104
106
105
107
106
107
5
Results of Statistical Analyses
• i.d. and s.c. are equally protective and both are
slightly better than i.t. LVS vaccination
• ~ 90% of vaccinated rats should survive i.t. challenge
with 104 to 105 SCHU S4
• If the vaccine candidate is as effective as LVS, then 6
rats per group is sufficient to detect a significant
improvement over unvaccinated rats
• If vaccine is not as effective as LVS (using i.t. LVS
vaccination as reference), then 7 per group is the
maximum number required to detect a significant
protective effect
6
Regression Modeling of Unvaccinated
Rats Does Not Fit Data
Percent survival
Naive
75
50
25
100
Percent survival
3 x 101
3 x 102
3 x 103
3 x 104
3 x 105
100
0
2.84 x 103
3.28 x 104
2.41 x 105
50
25
0
0
5
10
15
20
0
5
Days
10
15
Days
75
5.7 x 100
5.7 x 101
6.0 x 102
50
25
Percent survival
100
100
Percent survival
103
104
105
106
75
5.6 x 101
1.0 x 102
3.4 x 102
1.9 x 103
75
50
25
0
0
0
5
10
15
Days
20
25
0
5
10
15
20
Days
7
MS 5 Objective 2
Determine whether protection is dependent on LVS
vaccination dose -- In progress
Group
Vendor
s.c. LVS
vaccin. dose
(cfu/rat)
i.t. SCHU S4
challenge dose
(cfu/rat)
Current status
1
NCI
-
105
9/9 died
2
103
105
10/10 sick
3
105
105
10/10 sick
4
107
105
10/10 sick
8
MS 11: Characterization of Fischer 344 Rat
Fischer
344 rats
Humoral
immunity
LVS vaccination
Passive transfer
of serum
Protection
against i.t SCHU
challenge
Blue: Steps in the milestone
Red: Completed
Green: In progress
Cell mediated
immunity.
Production of
ascites fluid for
CD4 and CD8
depletion
LVS vaccination
In vivo depletion
Length of
protection
LVS vaccination
SCHU S4
challenge @
various times
post vaccination
Pretection against
i.t. SCHU SCHU
challenge
9
Milestone 11: Objectives
1. Demonstrate with all controls and sufficiently
large groups that passive immunization protects
NCI rats against i.t. SCHU S4 challenge
2. Compare the sensitivity of Fischer 344 rats from
Harlan and NCI to i.t. SCHU S4 challenge
3. Produce ascites fluids for CD4 T cell depletion
10
MS 11 Objective 1: Experimental Design
Demonstrate passive immunization protects Fischer 344 rats
against i.t. SCHU S4 challenge – Waiting for vaccinated rats
to clear LVS vaccine
Treatment
Serum
volume
(ml)
Group
Vendor
Vaccination
status
1
NCI
Vaccinated
None
-
2
Naïve
None
-
3
Naïve
Normal serum
0.5
4
Naïve
1.0
5
Naïve
1.5
6
Naïve
7
Naïve
1.0
8
Naïve
1.5
Immune serum
0.5
11
MS 11 Objective 2: Experimental Design
Compare the sensitivity of Harlan and NCI rats to i.t. SCHU
S4 challenge. Waiting for rats to be 9 wk old
Group
Vendor
SCHU S4
challenge dose
(cfu/rat)
1
Harlan
101
No
2
102
No
3
103
Yes
4
104
Yes
5
105
Yes
101
No
7
102
No
8
103
Yes
9
104
Yes
10
105
Yes
6
NCI
Lung
deposition
12
MS 11 Objective 3
Determine whether CD4 and CD8 T cells are
required for vaccine induced protection against i.t.
SCHU S4 challenge
13
Requirement for CD4 and CD8 T cells
s.c. LVS
vaccination
i.t. challenge with
105 SCHU S4
Expected
results
alive
LVS vaccinated
untreated
alive
LVS vaccinated
+ 1 mg isotype control
dead
LVS vaccinated
+ 1 mg anti-CD4
dead
LVS vaccinated
+ 1 mg anti-CD8
dead
Naive
14
List of Antibodies
• TS2/18.11
– Mouse anti-human CD2
– IgG1
• W3/25
– Mouse anti-rat CD4
– IgG1
– Non-depleting, causes
functional inactivatioin
• OX-8
– Mouse anti-rat CD8
– IgG1
– Depleting
• 55-6
– Mouse anti-HIV-1 gp120
– IgG2a
• OX-38
–
–
–
–
Mouse anti-rat CD4
IgG2a
Depleting
Currently in culture and
will send to Taconic for
ascites fluid production
15
Milestone 21: Multifunctional T cell assay
T cell responses from vaccinated
animals
Non-human primates
(LBERI/UNM)
Develop assay to detect CD4+ and CD8 +
multifunctional T cells
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
Mice (UNM)
Develop assay to detect
CD4+ multifunctional T cells
Develop assay to detect
CD8+ multifunctional T cells
Rats (UNM)
Develop assay to detect CD4+ and CD8 +
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
16
Protocol for multifunctional T cell assay
• Harvest organs and process to single cell
suspension
• Treat cells
–
–
–
–
media
-human CD28
-human CD28+1x106 cfu HK LVS
-human CD28+2x106 cfu HK LVS
• Block secretion with Brefeldin A
• Stain with antibodies to surface expressed
proteins (Staining both CD4+ and CD8+ cells)
• Fix, permeabilize, and stain with antibodies to
intracellular proteins
• Read on FACSCalibur (250,000 events)
17
Multiparameter
flow approach
IL-2-PE
IFNγ-PE*Cy7
TNFα-A488
IL-2-PE
TNFα-A488
TNFα-A488
18
IFNγ-PE*Cy7
IFNγ-PE*Cy7
IL-2-PE
Animal A00896
• Cynomolgus macaque vaccinated by intradermal
inoculation with 1.5x107 cfu LVS on 11/20/06
• Inoculated by bronchoscope instillation with 1x105
cfu LVS on 6/25/08
• Day 12 after infection: lungs, tracheobronchial
lymph nodes (TBLN), and spleen collected
– Lungs and spleen cells analyzed for intracellular
cytokine production
– TBLN cells used for IFNγ ELISPOT
– Extra cells frozen using Cerus freezing protocol
(obtained from J. Wilder at LBERI)
19
Both doses of HK-LVS stimulated a high frequency
of CD4+ lung cells producing TNF, IL-2, and IFN
20
Functionality profiles of CD4+ lung cells similar
following stimulation with two doses of HK-LVS
21
Observations
• Low frequency of CD4+ multifunctional cells
in the media and α-CD28 treated cells
• Both doses of HK-LVs stimulated a high
frequency of CD4+ multifunctional cells
• Very little cytokine production by CD8+ cells
that was not increased upon stimulation with
HK-LVS
• Insufficient events collected from spleen
assay
22
Non-infected NHP
• Tissues (lungs, TBLN, and spleen) collected
from two uninfected Cynomolgus macaques:
– Lungs and spleen cells analyzed for intracellular
cytokine production (extra cells frozen using Cerus
freezing protocol)
– TBLN cells frozen for IFNγ ELISPOT
• In addition, cells from LVS vaccinated/LVS
challenged animal were thawed and assessed
for intracellular cytokine production
23
%CD4+ lung cells secreting TNF, IL-2, and IFN
(Uninfected vs LVS treated NHP)
Uninfected NHP
LVS treated NHP (Fresh)
LVS treated NHP
(Frozen)
24
Frozen lung cells show a decrease in IFNγ+ cells
and an increase in multifunctional cells as compared
to fresh cells
Fresh
Frozen
25
Observations
• Neither CD4+ or CD8+ lung cells from 2
uninfected NHPs were stimulated to produce
TNF, IL-2, or IFN by treatment with αCD28/HKLVS
• Frozen CD4+cells from LVS vaccinated/LVS
challenged animal retained their ability to
respond to HK-LVS
• Although the response profiles were similar
between fresh and frozen CD4+ cells, differences
in IFN-only and multifunctional cells were noted
• Frozen spleen CD4+ and CD8+ cells showed no
increased cytokine production in response to
treatment with αCD28/HK-LVS
26
NHP Multifunctional assay: Ongoing
experiments and future directions
• Tissues will be collected from 2 NHPs
inoculated via bronchoscope instillation with
1x105 cfu LVS (day 28 and day 42)
• Frozen TBLN cells not used for IFNγ
ELISPOT will be analyzed for intracellular
cytokine production
27
memory
T cell
Milestone 21 – flow diagram
mf
F
T cell-induced macrophage killing
of intracellular Francisella
F
mf
F
X
X
Mouse
Rat
Generate bone marrow
derived macrophages
Generate bone marrow
derived macrophages
F
Load BMDM with LVS
Load BMDM with
SCHU S4
Load BMDM with LVS
Load BMDM with
SCHU S4
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Blue: Steps in the milestone
Red: Completed
28
Green: In progress
Improved Protocol for Infecting and
Processing SCHU S4-Infected Mouse
Macrophages
Representative of 4 independent experiments
29
MS21: Objective 1
• Determine whether addition of immune
splenocytes from LVS vaccinated rats
can induce rat macrophages to kill
intracellular SCHU S4
30
8
P = 0.011
7
6
P = 0.0005
5
4
cc
6
+1
0
va
iv
e
6
+1
0
na
cc
5
+1
0
va
5
+1
0
na
iv
e
S4
3
SC
H
U
cfu/ml recovered (log10)
Induction of Mouse Macrophage
Killing of Intracellular SCHU S4
31
memory
T cell
Milestone 21 – flow diagram
mf
F
T cell-induced macrophage killing
of intracellular Francisella
F
mf
F
X
X
Mouse
Rat
Generate bone marrow
derived macrophages
Generate bone marrow
derived macrophages
F
Load BMDM with LVS
Load BMDM with
SCHU S4
Load BMDM with LVS
Load BMDM with
SCHU S4
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Blue: Steps in the milestone
Red: Completed
32
Green: In progress
Immune Splenocytes Inducd Macrophage
Killing of Intracellular LVS
33
MS21: Objective 2
• Determine whether addition of immune
splenocytes from LVS vaccinated rats
can induce rat macrophages to kill
intracellular SCHU S4
34
P = 0.17
6
P = 0.01
5
4
3
/m
lI
FN
ng
g
/m
lI
FN
g
va
cc
+5
0
6
+1
0
+5
6
+1
0
ng
e
iv
5
na
iv
+1
0
na
5
+1
0
U
SC
H
va
cc
e
2
S4
cfu/ml recovered (log 10)
Splenocytes from LVS Vaccinated Rat
Induced Small but Significant Killing of
Intracellular SCHU S4 by Rat Macrophage
35
MS21: Planned Experiments
• Mouse: Repeat macrophage killing
assay with emphasis on reducing the
non-specific killing by unvaccinated
splenocytes
• Rat: Repeat macrophage killing assay
with emphasis on increasing the
difference between unvaccinated and
vaccinated splenocytes
36
Milestone 27 – flow diagram
SOP for detecting T cell
stimulation with ivt
proteins and peptide
Production of ivt
proteins &
peptide library
(ASU)
Production
Assay
development
(UNM)
T cell proliferation
IFN ELISpot assay
Screening
(UNM)
Identification of
stimulatory proteins &
peptides
Assay optimization
using ivt proteins
Blue: Steps in the milestone
Red: Completed
Green: In progress
37
Milestone 27 Objective 1
• Determine whether TBLN cells from LVSvaccinated NHP also crossreact with E. coli
proteins in the ivt reaction mix.
– NHP A00896
• Vaccinated i.d. on 11/20/06 with 107 LVS
• Boosted by bronchoscopy on 6/25/08 with 105 LVS
• Euthanized 7/7/08
– NHP A05279
• Vaccinated by bronchoscopy on 6/25/08 with 105 LVS
• Euthanized 7/23/08
– Tested LN cells in IFNg ELISpot – ASU share data
– Froze extra cells for future use
38
Milestone 35 – flow diagram
Optimization of RNA isolation
and microarray conditions
MS-33 (ASU)
Printing and testing
GDP confirmed
Printing arrays
Isolate RNA from
LVS and SCHU S4
Isolate RNA from
infected lungs
Infect BALB/c mice
i.n. with SCHU S4
GDP Confirmation
Isolate total RNA from
LVS and SCHU S4
Comparions of substrate
Poly-L Lysine vs Corning
Ultragaps
Compare TIGR PFGR
Arrays to in house arrays
Testing of linear
amplification of procaryotic
Transcripts (LAPT) process
and dilution testing of
Schu S4 RNA with and
without mouse lung RNA
RNA shipped 1/29/2007
Gray: (sub )milestone title
Red: completed
Green: in progress
Isolate eukaryotic and
prokaryotic RNA
Ship to ASU
Blue: Steps in the milestone
Red: Completed
39
Green: In progress
Isolate RNA from SCHU S4 Cultured
in Chamberlains broth and from
SCHU S4 Infected Mouse Lungs
SCHU
S4 Infected
mouse
lungs
SCHU S4 in
Chamberlain’s
broth
Groups
Time of harvest
No. for RNA
No. for Lung
deposition
1
Uninfected
3
-
2
1h
3
2
3
3h
3
2
4
5h
3
2
5
7h
3
2
6
24 h
3
2
Groups
Time of harvest
Culture volume (ml)
1
preinoculation
30
2
1h
30
3
3h
30
4
5h
30
5
7h
30
6
24 h
30
40
Action Items
•
•
•
•
•
•
•
•
Action Items from 7/30/08 UNM tech call
Barbara: request example Technology Transfer Plan from QA at LBERI
(done 7/30; Stephany Taulbee and Chuck Hobbs at LBERI do not have
one)
Terry: write Technology Transfer Plan for rat model
Marlene: review draft of Terry’s Technology Transfer Plan
Action Items from 7/2/08 UNM tech call
Terry: email Marlene a concise summary of the mouse and rat data and
will attach prior INI paper on mouse model, as soon as possible.
Terry will compare the current 7/2/08 cytokine response data in Hu AM
current data with the prior data
Marlene will research the name/source of the sponge-like matrices for
cell culture and Terry will search on internet
41