Progress Report
12/05/2008
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
Active Milestones:
5, 11, 12/13, 14, 17, 19, 21, 27, 35
Today’s presentation will include:
5.
a) Effect of LVS vaccination dose on protection in Fischer rats
b) Histolopathology of SCHU S4-infected mice, rats, and NHP
14. SCHU S4 infection of human monocytes
21. SCHU S4 infection of rat bone marrow derived macrophages
17. a) Titration of serum volume and challenge dose in passive
immunization of Fischer 344 rat
b) Kinetics of SCHU S4 growth and dissemination in passively
immunized rats
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.
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
3
Bronchopneumonia (Rats) vs. Vasculitis (Mice)
Rat: intratracheal ~400 SCHU S4 (d4)
4
Mice: intranasal ~200 NMFTA1 (d4)
Mice: Aerosol 10-20 Strain 33 (d4)
Histological Comparison of Mice, Rats,
and NHP infected i.t. with SCHU S4
• Infected with 1000 SCHU S4
– Mice by surgical i.t.
– Rats by non-surgical i.t.
– NHP by bronchoscopy
• Collected lungs, lymph nodes, spleen, liver, thymus,
GI tract, kidney, etc
– Mice and rats (days 1, 2, 3, & 4)
– NHP (days 1, 4, & 7)
– rats (days 1, 2, 3, 4 & 7)
• Processing tissues for analyses
5
Direct Relationship between LVS
Vaccination Dose & Level of Resistance
NHP
RB Hornick and HT Eigelsbach. Bacteriol Rev (1966) 30: 532
Humans
6
Determine the effect of LVS dose on
resistance to i.t. SCHU challenge
Group
s.c. LVS
vaccination
dose (cfu/rat)
i.t. SCHU S4
challenge dose
(cfu/rat)
No. per group
1
107
104
6
2
105
104
6
3
103
104
6
4
---
104
6
Rats vaccinated 11/20/2008
To be challenged early Jan 2009
7
MS5: Additional Plans
• Submit manuscript on Fischer 344 rat model
• Complete MSCR
8
MS 14: Assays in Vaccinated Humans
Assay to measure activation of
macrophage killing mechanisms in
humans
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
Isolate & differentiate monocytes to
macrophages (buffy coat)
Test PBMC
Test purified
T cells
Determine and optimize cell
number and MOI
in vitro
expansion?
Positive control w/
IFNg
Blue: Steps in the milestone
Red: Completed
Green: In progress
Repeat w/ human
vaccinee
9
Functional Correlate of Protection
F
F
+
F
monocytes
F
MOI = 1:100 LVS
Gentamicin
naive
T cell
Imm.
T cell
F F
F FF F
X
FX F
10
total CFU per well
Proliferation of SCHU S4 in
Human Monocytes
3.010 7
2.010
Monocyte-enriched
Lymphocyte-enriched
Monos + Lymphs
Ft + media only (approximate)
7
1.010 7
0
0
24
48
72
hours post-infection
11
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
12
Proliferation of SCHU S4 in
Cell-free Medium
1.010 6
CFU / well
1.010 5
1.010 4
FT only (-) 10% serum
FT only (+) 10% serum
1.010 3
1.010 2
1.010 1
1.010 0
0
24
48
72
hours post-infection
13
MS 14: Plans
• Repeat MOI optimization
• Determine whether monocytes have the
effector mechanisms to control SCHU
S4 growth
– Addition of recombinant IFNg
• Repeat with LVS vaccinated people
14
Milestone 21 – flow diagram
T cell-induced macrophage
killing of intracellular
bacteria
Mouse
Rat
Generate bone marrow
derived macrophages
Generate bone marrow
derived macrophages
Load BMDM with Ft
Load BMDM with Ft
Measure LVS 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
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Blue: Steps in the milestone
Red: Completed
15
Green: In progress
Optimizing MOI for Infecting
Rat Macrophages
MOI
(SCHU S4: macrophage)
No. of bacteria recovered
on day 2
1:50
5.26 x 106
1:100
3.88 x 106
1:200
6.13 x 105
1:1000
2.33 x 105
16
MS 21: Plans
• Repeat MOI optimization
• Determine whether infected
macrophages can be activated with
recombinant IFNg
• Repeat with LVS vaccinated rats
• Try monocytes or whole blood
17
MS 17: Characterization of Fischer 344 Rat
Fischer
344 rats
Humoral
immunity
Cell mediated
immunity.
LVS vaccination
Purchase and culture
hybridoma cell lines
Passive transfer
of serum
Blue: Steps in the milestone
Red: Completed
Green: In progress
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
18
Titration of Ft-specific Antibodies in
Immune Sera
0.3
Naive Rat Serum
0.2
0.1
1:50000
1:25000
1:16000
1:8000
1:5000
1:2500
1:1600
0.0
1:800
OD405 nm
Immune Rat Serum
19
Serum Volume Required for Protection Against
i.t. SCHU S4 challenge (130 CFU)
20
Limit of Protection conferred by 0.25 ml
Immune Serum Against i.t. SCHU S4
21
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
22
Histopathology of Passively Immunized
Rats after i.t. SCHU S4 challenge
Group No.
Treatment
Challenge dose
(CFU/rat)
Time Points (days)
Total No. of Rats
1
Naïve (250ul PBS)
1000
1, 3, 5, 7
15
2
NRS (250ul NRS)
1000
1, 3, 5, 7
15
3
S.C. LVS Vaccinated
1000
1, 3, 5, 7, 10, 14, and 21
21
4
IRS (250ul IRS)
1000
1, 3, 5, 7, 10, 14, and 21
21
5
Deps
1000
-
3
23
MS17: Plans
• Repeat growth kinetics experiment in
actively and passively immunized rats
• Complete histopathological analyses of
tissues from passively immunized rats
• Define the role of T cells in passive
immunity
24
Action Items
• Action: Freyja requested that Terry could try with serum that isn’t
heat inactivated so complement would be active
• Terry/Gopi: Freyja requests that UNM pick same symbol for
NRS vs IRS, and then use different colors. Want graphs more
simple to analyze.
• Action: Freyja, Kristin and Ed review the LBERI tech call slides
and discuss the natural history dose before 12/10.
• Barbara: send LBERI slides to Ed Nuzum (done 12/7/08)
• Freyja: wants UNM to simplify the monthly technical report.
Freyja will send comments on the monthly report for UNM to
review. She doesn’t expect editing on the 11/15/08 report.
Barbara: requested that Freyja provide an example of the
preferable intermediate level of reports (greater detail than
25
highlights and lesser detail than full report)