Progress Report
8/4/2009
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:
12/13. Immunoassays (discussed at the LBERI tech call)
a) Correlation between CFU and protein content
b) Micro-agglutination assay
11. Cellular and humoral immunity in rats
a) Passive immunization with purified IgG
b) Depletion of CD4 and CD8 T cells
21. Use of human PBMC in correlate of protection assay
a) Production of IFNg
b) Inhibition of SCHU S4 growth
29. Result from screening individual polypeptides
2
MS 11: 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
Production of ascites fluid for CD4 and
CD8 depletion
Protection against i.t
SCHU challenge
In vivo depletion
Adoptive transfer
Active vs passive
immunization
Blue: Steps in the milestone
Red: Completed
Green: In progress
Protection against i.t. SCHU
SCHU challenge
3
Milestone 11: Humoral Immunity
• Passive immunization protected rats against i.t. SCHU
S4 challenge
• Protection is several logs lower than LVS vaccination
• Intermediate phenotype (bacteriology &
histopathology) between NRS and LVS vaccinated rats
8
8
6
4
Liver
Spleen
10
6
4
6
4
2
2
2
0
0
0
0
3
6
9
12
15
Days Post-challenge
18
21
Naive (0.25ml PBS)
NRS (0.25ml)
Vaccinated
IRS (0.25ml)
8
CFU (log10)
10
10
CFU (log10)
CFU (log10)
Lungs
0
3
6
9
12
15
Days Post-challenge
18
21
0
3
6
9
12
15
18
21
Days Post-challenge
4
Milestone 11: Humoral Immunity
• Repeated antibody treatments over the course of
infection did not further reduce bacterial burden
compared with single treatment -- antibody not
limiting
• Is immune IgG sufficient to passively transfer
protection?
5
Purified Immune IgG Provided Partial
Protection Against SCHU S4 Challenge
(Pilot Experiment)
Percent survival
100
Normal rat IgG
Immune rat IgG
75
50
25
0
0
10
20
30
Days Post-infection
6
Purified Immune IgG Protected Against
SCHU S4 Challenge
(more IgG)
Percent survival
100
75
Immue rat serum
Purified normal IgG
50
Purified Immune IgG
25
0
0
4
8
12
Days Post-infection
7
Is Passive Immunization Dependent
on T cells?
1)
2)
3)
4)
anti-CD4
anti-CD8
anti-CD4/8
Isotype ctrl
-2
i.t. infection
(~250 SCHU S4)
-1
0h
Survival
+
Weekly antibody treatment
30
1) Normal serum
2) Immune serum
8
Effective Depletion of CD8 T cells
Rat #1
Rat #2
Isotype
CD8
Depleted
* CD4 depletion was not confirmed because inactivating antibodies were used
9
Milestone 11 Humoral Immunity: Plans
• Continue to monitor rats passively immunized with
purified IgG
• Continue to monitor passively immunized rats
depleted of T cells and challenged with SCHU S4
• Analyze cytokine profile associated with passive
immunization – mechanism of protection
• Compare the kinetics of T cell response in LVS
vaccinated and passively immunize rats
10
Milestone 11 Cellular Immunity: Plans
• Experiment in progress to determine the
requirement for T cells in LVS vaccinated rats
• Develop method for adoptive transfer of immune T
cells into naïve rats (technique reported for Fischer
344 rats)
11
MS 21: Assays in Vaccinated Humans
Assay to measure activation of macrophage
killing mechanisms in humans
Naive
Immu
Monocyte-derived
macrophages
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
Monocytes
PBMC
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
12
MS21: Background
• Human PBMC supports SCHU S4 growth
• Pre-stimulation of vaccinated human PBMC with
formalin-fixed LVS
– induced IFNg production within 24 h
– inhibited /reduced SCHU S4 growth @ 72 h post infection)
Wash
SCHU S4
FF LVS
-48 h
-24 h
0h
Burden
24 h
48 h
72 h
Cytokine collection
• Activities correlates with vaccination status
13
Production of IFNg by Vaccinated PBMC
Prestimulated with FF-LVS
Unvaccinated
LVS vaccinated
Prestimulation / infection
30000
FF-LVS / UNINFECTED
FF-LVS / SCHU4
FF-LVS / LVS
20000
pg/mL
pg/mL
30000
Pre-stimulation / Infection
20000
10000
10000
0
0
-24
0
24
Time (h)
48
72
FF-LVS / UNINFECTED
FF-LVS / SCHU S4
Cytokine
washed away
during infection
-24
0
24
48
72
Time (h)
14
Reduction of SCHU S4 by Vaccinated PBMC
Pre-stimulated with FF-LVS
Vaccinated
Unvaccinated
7
7
Vacc PBMCs:unstim
+ HK-LVS
+ FF-LVS
+ IFNg + TNFa
10)
6
total CFU / well (Log
Total CFU / well (Log 10)
6
Unvacc PBMCs
+ HK-LVS
+ FF-LVS
+ IFNg + TNFa
5
4
5
4
3
3
2
2
0
72
Hours post-infection
0
72
Hours post-infection
MS21: Background
• Limited data set
– IFNg: 2 unvaccinated + 1 vaccinated
– CFU: 1 unvaccinated + 2 vaccinated
• Last month added
– IFNg: 1 unvaccinated + 2 vaccinated
– CFU: 2 vaccinated
– Data consistent with previously observed patterns
16
Milestone 21: Plans
• Gather more data from both vaccinated and
unvaccinated individuals -- CFU and IFNg
• Meet with statistician on sample size
• Identify cell types associated with IFNg production.
– Specific -- leads to bacteria reduction
– Non-specific -- unproductive and leads to death
Non- Ag-specific response
by unvaccinated & vaccinated PBMC
Unvaccinated
Vaccinated
Ag-specifc response
by vaccinated PBMC
Pre-stimulation / Infection
20000
10000
10000
0
0
-24
0
24
Time (h)
48
72
UNTREATED
FF-LVS
IFN + TNF
30000
pg/mL
20000
untreated
FF-LVS
IFNg + TNFa
30000
FF-LVS / UNINFECTED
FF-LVS / SCHU S4
pg/mL
pg/mL
30000
20000
10000
0
-24
0
24
48
hours post-infection
72
-24
0
24
48
72
HOURS POST-INFECTION
17
Milestone 21: Plans
• Determine how/why FF-LVS pre-stimulation of
unvaccinated PBMC shuts down IFNg production
– Would LVS LPS from BEI produce the same effect?
Vaccinated
untreated
FF-LVS
IFNg + TNFa
30000
UNTREATED
FF-LVS
IFN + TNF
30000
20000
pg/mL
pg/mL
Unvaccinated
10000
20000
10000
0
0
-24
0
24
48
hours post-infection
72
-24
0
24
48
72
HOURS POST-INFECTION
18
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
Confirmation
Assay optimization
using ivt proteins
Blue: Steps in the milestone
Red: Completed
Green: In progress
19
MS29: Background
• Screened ASU pooled peptide library with splenocytes and
lymph node cells from LVS vaccinated/boosted NHP
• Based on statistical analyses by Phil Stafford and location on
plate, individual peptides were selected to be rescreened to
confirm positives
• Tested individual polypeptides by IFNg ELISpot assay with 6.0
x 105 lymph node cells/well. Frozen splenocytes did not
respond well
20
Results from Testing Individual
Polypeptides
250
200
150
1
100
2
3
4
50
0
21
Action Items
• UNM will try soon adoptive transfer of
immune T cells into naïve rats
• UNM will try intracellular cytokine staining
and look at cell types making the IFN gamma.
• Terry will try stimulating the PBMC with O
antigen minus variants from Anders Sjostedt
• Rick, Terry, Kathy and Phil will strategize about
the repeat polypeptide screening data prior to
next experiment.
22