Progress Report
1/07/2009
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
Active Milestones:
5, 11, 12/13, 14, 17, 18, 19, 21, 29, 35
Today’s presentation will include:
Our recent problems with Teknova’s Chamberlain’s medium
5.
a) Effect of LVS vaccination dose on protection in Fischer rats
b) Histopathology of SCHU S4-infected mice, rats, and NHP
14. LVS expressing Lux operon as a reporter system
17. a) Titration of serum volume in passive immunization of
Fischer 344 rat
29. Plans for screening Ft peptide library from ASU
2
Problems with Chamberlain’s Medium
• 3 failed attempts to culture SCHU S4 using
Teknova’s Chamberlain’s broth lot C071106F0801
• Similar problems at LBERI with this and other lots
from Teknova
• 3 successful cultures with LBERI’s homemade broth
– >109 cfu/ml
• Conclusion: Teknova not a reliable source for
Chamberlain’s broth
3
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
4
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)
• Decalcifying sternum and femur
• Trim tissues for cassettes
5
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
Challenged Jan 6, 2009
6
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
7
Functional Correlate of Protection
naive
T cell
F
F
+
monocytes
F
F F
F FF F
4-5 days
F
Imm.
T cell
X
FX F
instantaneous
8
Lux Operon & Bioluminescence
RCOOH + ATP + NADPH
NADP + AMP + PPi + RCHO
fatty acid
reductase
complex
luxR
luxI
luxC
luxD
luxA LuxB luxE
luciferase
FMNH2 + RCHO + O2
FMN + H2O + RCOOH
9
Sensitivity of Lux Operon System
1.010 6
1.010 5
1.010 4
1.010 3
1.010 2
4
5
6
7
8
9
2x
10
2x
10
2x
10
2x
10
2x
10
2x
10
1.010 1
PB
S
Arbitrary Fluorescence Unit
KKF337
LVS (CFU/well)
10
Kinetics of SCHU S4 Prolifeation
in Human Monocytes
1.010 6
CFU / well
1.010 5
1.010 4
MOI=1.0 (+) serum
MOI=0.1 (+) serum
1.010 3
1.010 2
0
24
48
72
hours post-infection
in vitro application limited because of cytotoxicity on macrophage
cultures, but may be improved by optimizing infection/culture conditions
11
Kinetics of SCHU S4 Proliferation
in Naïve and Vaccinated Rats
May be useful for in vivo tracking of bacteria in naïve tissues and
vaccinated lungs in which high bacterial load can be reached
12
MS 14: Plans
• Determine whether human monocytes
have the effector mechanisms to control
SCHU S4 growth: stimulation with
recombinant IFNg
• Repeat with LVS vaccinated people
• Evaluate the usefulness of lux operon
(possibility of asking Karl to make lux-operon
expressing SCHU S4)
13
MS 21: Plans
• Optimize culture/infection of rat macrophages
• Determine whether infected macrophages
can be activated with recombinant IFNg
• Repeat with LVS vaccinated rats
• Try monocytes or whole blood
14
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
15
Summary of Previous Results
Titration of challenge dose @ fixed serum volume:
250 ml protected against i.t. challenge with ≤103
SCHU S4
Titration of serum volume @ fixed challenge dose:
50 ml protected against 130 SCHU S4
Have not been able to titrate out the protective
effects of immune sera
16
The Protection by Immune
Serum Can Be Titrated (360 cfu)
100
250ul NRS
250ul IRS
25ul NRS
75
50
25ul IRS
25
0
Percent survival
Percent survival
100
2.5ul NRS
2.5ul IRS
0.25ul NRS
0.25ul IRS
75
50
25
0
0
7
14
21
Days Postchallenge
28
35
0
7
14
21
28
35
Days Postchallenge
17
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
Tissue processing in progress
18
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
19
Milestone 29 – 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
IFNg ELISpot assay
Screening
(UNM)
Identification of
stimulatory proteins &
peptides
Assay optimization
using ivt proteins
Blue: Steps in the milestone
Red: Completed
Green: In progress
20
MS29: Plans
• Vaccinated NHP with LVS
• ASU prepared and shipped peptide array
– 2065 linear expression element constructs
• UNM
– Ordered needed reagents and materials
– Scheduling with LBERI to boost vaccinated NHP
• Screen10 days after boost
– 7 peptides/well in duplicates, 16 plates total
– Lymph node cells and splenocytes
– IFNg ELIspot with cells and ELISA on culture supe
21
Action Items
•
•
•
Terry will grow the LVS/ lux operon bacteria in macrophages for the next
experiment and could increase time, to improve sensitivity and decrease
numbers of bacteria needed for detection.
UNM will be tracking the immune serum titer in another passive immunization
experiment.
Screening the ASU full Ft library of polypeptides with LN and spleen cells from
LVS vaccinated NHP will be a major effort for UNM in next 1-2 months.
22