Progress Report
09/17/2008
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 validated
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
MS5 Objective 1
• Determine the robustness of the Fischer 344
rat model by comparing the SCHU S4
sensitivity of rats from NCI and Harlan
4
NCI and Harlan Rats Have Similar
Sensitivity to SCHU S4
5
MS5 Objective 2
• Wrap up manuscript describing the
Fischer 344 rat model and MSCR
• Compare histopathology between rats
and mice after SCHU S4 infection
– Bronchopneumonia (rats) vs. vasculitis
(mice)
6
Bronchopneumonia in SCHU S4
Infected Rats
7
Bronchopneumonia (Rats) vs. Vasculitis (Mice)
8
MS5: Plans
• Submit manuscript
• Complete MSCR
9
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
10
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 macrophage per well in the 24 well
plate
11
MS14: Plans
• Scale down the number of macrophages per
well while maintaining assay sensitivity
• Procedure for in vitro expansion of PBMC/T
cells
12
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
13
Milestone 17: Objective 1
1. Titrate the volume of normal and immune
rat serum required to protect naïve rats
from SCHU S4 challenge
• minimum volume
• No protection by normal serum
14
Passive Immunization Protects
Against i.t. SCHU S4 challenge
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
1/6
1.5 ml
6/6
1.0 ml
6/6
0.5 ml
5/6
0.25 ml
6/6
Immune rat serum
15
MS17: Plans
• Determine the kinetics of bacterial
proliferation and dissemination and
histopathology in passively immunized
rats after SCHU S4 challenge
16
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
17
IglC Immune Responses Were Also
Detected by ICS
iglC pool2
CD4
0.5
SJL
C3H/HeJ
FVB/N
C57BL/6
-0.5
Balb/c
SJL
C3H/HeJ
FVB/N
C57BL/6
Balb/c
0.0
2
0
-2
C3H/HeJ
1.0
FVB/N
0.00
1.5
CD8
4
C57BL/6
0.05
CD4
CD8
Balb/c
0.10
2.0
% IFN- g T cells
% IFN- g T cells
% IFN- g T cells
6
CD4
CD8
0.15
-0.05
LLO pool
2.5
0.20
•Responses to IglC pool1 peptides were weak in all 5 strains of mice
•Responses to IglC pool2 peptides were CD4 (Balb/c, FVBN), CD8
(C57BL/6) or both (C3H/HeJ), SJL had very weak responses
•Responses were strongest in FVBN Mice
IM08-059
Aug 20, 2008, Page 18
SJL
iglC pool1
IglC Peptide Pools Failed to
stimulate LVS Vaccinated NHP
19
MS27: Plan
• Wait for ivt peptides to be produced
20
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
21
Green: In progress
Materials Provided to ASU
• RNA from SCHU S4 cultured in
Chamberlain’s broth 1, 3, 5, 7, and 24h
• RNA from mouse lungs 1, 3, 5, 7, and
24 h after infection with SCHU S4
• SCHU S4 genomic DNA
22
MS35: Plans this month
• Prepare RNA from SCHU S4 infected
Fischer 344 rats
• Possibility of repeating RNA isolation
from SCHU S4 and SCHU S4-infected
mouse lungs
23
Quality Assurance
•
Routine maintenance
–
–
–
•
Routine calibration
–
•
Pipetmen: scheduled 11/08
Routine monitoring
–
–
–
–
–
–
–
–
•
Biological cabinets: performed 5/2/08
Millipore water system: 4/10/08
BioQuell: performed 6/23/08
Waterbaths – every week
Millipore water system – every week
Blowers for ventilated animal racks – every week
Plater and colony counter – every month
Incubator CO2 and temperature – every month
Refrigerators and freezers – every month
Scales and analytical balance – every 3 months
Reagent expiration and lot number – before every experiment
Data
–
–
–
–
Originals and cage cards kept in BSL3
Electronic files stored on server by experimental ID
Record sheets are scanned and entered into notebooks
Notebooks are kept in fire-proof file cabinets
24
Action Items
•
•
•
Terry/Rick: send draft of Rat model paper to NIAID for review before
submission to “Vaccine” journal
Marlene: will share the Rat model paper with NIAID Public Relations, at
the time of publication.
Terry will send the 1940 serum treatment article to Marlene for
distribution at NIAID. (completed 9/17/08)
25