Progress Report
11/05/2008
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.
Small animal models of tularemia
14.
Assays in vaccinated humans
27.
Testing of ivt proteins for ASU (ASU MS 26)
35.
RNA and genomic DNA 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
Robustness of
Fischer 344 rat model
Blue: Steps in the milestone
Red: Completed
Green: In progress
3
Bronchopneumonia (Rats) vs. Vasculitis (Mice)
4
Histological Comparison of Mice, Rats,
and NHP infected i.t. with SCHU S4
• Infect i.t. with 1000 SCHU S4
• Collect organs and tissues
– NHP (days 1, 4, & 7)
– Mice and rats (days 1, 2, 3, 4 & 7)
– Lungs, LALN, spleen, liver, GI tract,
mesenteric LN, thymus, nasal cavity, etc
5
Quantum Dots Reduced SCHU S4
Virulence in vivo
100
80
60
3.7 x 10 3 cfu no QD
3.1 x 10 3 cfu w/ QD
40
20
Percent survival
Percent survival
100
0
80
60
4.7 x 10 4 cfu no QD
4.1 x 10 4 cfu w/ QD
40
20
0
0
5
10
15
0
5
Days
15
105
100
95
3.7 x 10 3 no QD
3.1 x 10 3 w/ QD
90
85
80
Percent starting weight
105
Percent starting weight
10
Days
100
4.7 x 10 4 no QD
4.1 x 10 4 w/ QD
95
90
85
80
0
5
10
Days
15
0
5
10
Days
15
6
Additional Plans
• Wrap up manuscript describing the
Fischer 344 rat model
• Complete MSCR – almost done with
first rough draft
7
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
8
Functional Correlate of Protection
mf
F F
naive
T cell
mf
F FF
F F F
Mouse assay optimized for
LVS and SCHU S4
immune
T cell
Working on rat and human
assays
mf
F
X
XF
9
Cell Yield from Whole Blood
• From 100 ml of whole blood
– 1.1 x 108 PBMC
– 8.5 x 106 monocytes
– 3.3 x 106 macrophages after 6 day culture
• For comparison, we have been using ~107
murine macrophage per well in the 24 well plate
• Scale down the number of macrophages per
well while maintaining assay sensitivity
10
MS14: Goals for Human Correlate of
Protection Assay
•
•
•
•
Reasonable scale
Rapid setup and readout
High sensitivity
High specificity
• Ideal to use whole blood or PBMC
11
total CFU per well
3.010 7
Monocyte-enriched
Lymphocyte-enriched
Monos + Lymphs
Ft + media only (approximate)
2.010 7
1.010 7
0
0
24
48
72
hours post-infection
12
MS 14: Plans
• Optimize infection of human monocytes
• Determine whether monocytes would be
a good target
13
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
14
Passive Immunization Protects Against i.t.
SCHU S4 challenge (240 CFU)
Survival Ratio
(No. alive/total)
Treatment
Unvaccinated
-
0/6
LVS vaccinated
-
6/6
Normal rat serum
1.5 ml
0/6
1.0 ml
0/6
0.5 ml
0/6
0.25 ml
0/6
1.5 ml
6/6
1.0 ml
6/6
0.5 ml
5/6
0.25 ml
6/6
Immune rat serum
15
Percent survival
Passive Immunization Protects Against i.t.
SCHU S4 challenge (0.25 ml & 217 CFU)
100
80
60
NAIVE
NRS
IRS
40
20
0
0
10
20
30
Days P.I.
16
Passively Immunized Rats Develop
Enhanced Immunity after Recovering
from SCHU S4 infection (2 x 104 CFU)
Naive
LVS
IRS/SCHU S4
LVS/SCHU S4
17
Kinetics of SCHU S4 Proliferation
and Dissemination in Actively- and
Passively-immunized Rats
Lungs
Liver
Spleen
10
10
8
8
Naive
LVS vaccinated
NRS
IRS
5
3
CFU (log)
7
CFU (log)
CFU (log)
9
6
4
2
1
5
10
Days post infection
15
4
2
0
0
6
0
0
5
10
Days post infection
15
0
5
10
15
Days post infection
18
MS17: Plans
• Repeat growth kinetics experiment in
actively and passively immunized rats
• Compare the histopathology
• Define the role of T cells in passive
immunity
19
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
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
MS27: Plans
• Vaccinated NHP with LVS
• Meeting with ASU over complexity of peptide pool.
– 2065 linear expression element constructs
– 1.5 x 108 LN cells (conservative estimate)
– 3 peptides/well in single wells
– 8 peptides/well in duplicates
– Splenocytes
• Boost vaccinated NHP with LVS
• Screen10 days after boost
21
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
22
Green: In progress
MS 35 – Work Accomplished
• Prepared RNA from Fischer 344 rat
lungs 1, 3, 5, 7, and 24 h after infection
with SCHU S4
• Agilent analyzer down
• Will ship when analyzed
23
Action Items:
•
•
•
The next UNM Tech call is scheduled on 12/5/08 friday noon-1pm MT (2-3pm
Eastern). Freyja, Terry, Rick and Kristin agreed to the new date. This replaces
the UNM Tech call originally scheduled on 12/3/08.
Terry will send rat manuscript draft to Barbara and Barbara will email to NIAID
for approval to send to the journal. (completed, including Freyja’s review; Kristin
also received it)
UNM/ASU scheduled a teleconference for 11/6/08 1pm (completed) to discuss
details of polypeptide pooling for testing at UNM with vaccinated and boosted
NHP lymph node and spleen cells
24