Progress Report
7/8/2009
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:
11. a) Isotyping immune serum
b) Purification of IgG for passive transfer
c) Mechanism of passive immunity
21. Use of human PBMC in correlate of protection assay
a) Activation of PBMC killing activity by FF-LVS
b) Correlation to IFNg production
29. Plans for confirming ASU Ft peptide library screen
a) Titration of frozen lymph node cells and splenocytes
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
4
-0.5
1:
1
1: 0
10
1: 0
20
1: 0
40
1: 0
8
1: 00
16
1: 00
32
1: 00
6
1: 400
12
1: 800
25
1: 600
51
20
0
NRS
IRS
3
0
1.5
IgG2a
NRS
IRS
1.0
0.0
Dilutions
1
OD450 nm
1
OD450 nm
2
1:
1
1: 0
10
1: 0
20
1: 0
40
1: 0
8
1: 00
16
1: 00
32
1: 00
6
1: 400
12
1: 800
25
1: 600
51
20
0
OD450 nm
5
0.4
3
2
NRS
IRS
0
-1
Dilutions
-0.5
1:
1
1: 0
10
1: 0
20
1: 0
40
1: 0
8
1: 00
16
1: 00
32
1: 00
6
1: 400
12
1: 800
25
1: 600
51
20
0
0.5
OD450 nm
1:
1
1: 0
10
1: 0
20
1: 0
40
1: 0
8
1: 00
16
1: 00
32
1: 00
6
1: 400
12
1: 800
25
1: 600
51
20
0
4
1:
40
0
1:
80
0
1:
16
00
1:
32
00
1:
64
00
1:
12
80
0
1:
25
60
0
1:
51
20
0
OD450 nm
Isotyping Immune Serum
IgM
IgG1
0.5
NRS
0.3
IRS
0.2
0.1
0.0
-0.1
Dilutions
IgG2b
1.5
IgG2c
1.0
NRS
IRS
0.5
0.0
Dilutions
5
Commercial
2X
1.5X
1X
1.25X
Melon gel resin
0.5X
post NH4 sulfate
serum
IgG Enrichment with Melon Gel
50
25
Resin
Recovery
0.5 X
5.1 mg
1.0 X
3.3 mg
1.25 X
2.9 mg
2X
2.4 mg
6
2nd Peak of Bacterial Growth in
Passively Immunized 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
CFU (log10)
CFU (log10)
Lungs
10
0
3
6
9
12
15
Days Post-challenge
18
21
0
3
6
9
12
15
18
21
Days Post-challenge
7
2nd Peak of Bacterial Growth in
Passively Immunized Rats
Lungs
10
Liver
Spleen
8
8
8
6
6
CFU (log 10)
CFU (log 10)
CFU (log10)
6
4
Ptran12B (day -1)
Ptran12C (day -1)
4
4
2
2
2
0
3
6
9
12
15
18
Days Post-challenge
21
0
3
6
9
12
Days Post-challenge
15
18
21
0
3
6
9
12
15
18
21
Days Post-challenge
8
2nd Peak of Bacterial Growth in
Passively Immunized Rats
Are these peaks due to antibody depletion?
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
CFU (log10)
CFU (log10)
Lungs
10
0
3
6
9
12
15
Days Post-challenge
18
21
0
3
6
9
12
15
18
21
Days Post-challenge
9
Effect of Multiple Antibody
Treatment on 2nd Peak
Lungs
10
10
8
6
CFU (log 10)
8
CFU (log 10)
CFU (log 10)
8
Liver
Spleen
10
6
6
4
4
4
2
2
2
0
3
6
9
12
15
Days Post-challenge
18
21
0
3
6
9
12
Days Post-challenge
15
18
21
Ptran 12D (day -1)
Ptran 12D (day -1 3 6 9 12)
IRS (0.25ml) Ptran 12B
0
3
6
9
12
15
18
21
Days Post-challenge
10
Is the 2nd Peak an Artifact of
Averaging Values?
Lungs
10
Liver
Spleen
8
8
8
6
6
CFU (log 10)
CFU (log 10)
CFU (log 10)
6
4
Ptran12B (day -1)
Ptran12C (day -1)
Ptran 12D (day -1)
Ptran 12D (day -1 3 6 9 12)
4
4
2
2
2
0
3
6
9
12
15
Days Post-challenge
18
21
0
3
6
9
12
Days Post-challenge
15
18
21
0
3
6
9
12
15
18
21
Days Post-challenge
11
Milestone 11 Humoral Immunity: Plans
• Determine whether IgG is sufficient for passive
immunization
• Analyze cytokine profile associated with passive
immunization
• Cells mediating protection
12
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
13
MS21: Background
• SCHU S4 increases 3-4 logs in human PBMC over 72 h
period
• Pre-stimulation with IFNg and TNF 48h before
infection sufficient to reduced bacterial burden 2-3
logs
• We have some evidence that this result can be
reproduced by pre-stimulation with FF-LVS
14
Experimental Design
IFN + TNF
HK / FF LVS
-48 h
Bacterial
burden
Wash
Infect w/ SCHU S4
-24 h
0h
24 h
48 h
72 h
Cytokine collection
15
FF-LVS Prestimulation of Vaccinated PBMC
Reduced SCHU S4 Burden @ 72 h
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
FF-LVS Prestimulation of Vaccinated PBMC
Reduced SCHU S4 Burden @ 72 h
8
no pretreatment
formalin-fixed LVS
total CFU / well (log 10)
7
IFNg + TNFa
6
5
4
3
2
0
72
hours post-infection
17
FF-LVS Prestimulation of Vaccinated PBMC
Induced Abundant IFNg
Uninfected
untreated
FF-LVS
IFNg + TNFa
30000
20000
20000
10000
10000
0
0
-24
0
24
48
hours post-infection
untreated
FF-LVS
IFNg + TNFa
30000
pg/mL
pg/mL
SCHU S4 infected
72
-24
0
24
48
72
hours post-infection
18
FF-LVS Prestimulation of Unvaccinated
PBMC Failed to induce IFNg
#70: Uninfected
#70: SCHU S4 infected
UNTREATED
FF-LVS
IFN + TNF
30000
UNTREATED
FF-LVS
IFN + TNF
30000
pg/mL
20000
pg/mL
20000
10000
10000
0
0
-24
0
24
48
72
-24
HOURS POST-INFECTION
0
24
48
72
HOURS POST-INFECTION
# 79: Uninfected
Activation of macrophage
killing activity by the
released IFNg leading to
reduction of bacterial
burden may be a second
measure of vaccination
status.
#79: SCHU S4 Infected
20000
30000
UNTREATED
FF-LVS
IFN + TNF
UNTREATED
FF-LVS
IFN + TNF
15000
pg/mL
pg/mL
20000
10000
These results suggest that
vaccinated PBMC can be
distinguished from
unvaccinated PBMC based
on IFNg release during prestimulation
10000
5000
0
0
-24
0
24
48
72
HOURS POST-INFECTION
-24
0
24
48
72
HOURS POST-INFECTION
19
Milestone 21: Plans
• Repeat experiment to examine cytokine production
over time and correlate to reduction in bacteria
burden
• Need more data from both vaccinated and
unvaccinated individuals
20
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
21
MS29: Background
• Screened ASU 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
• Frozen splenocytes and lymph node cells appeared to have
lost reactivity based on stimulation with HK-LVS
Lymph nodes
Spleen
Fresh
18 +/- 8
20 +/- 14
Frozen
3, 12
3, 1
22
MS29: Reactivity of Frozen Cells
Cell source
Cell number/well
Spots/well
Lymph node
1.5 x 105
5, 4
3.0 x 105
26, 36
6.0 x 105
94, 77
1.5 x 105
3, 1
3.0 x 105
6, 13
6.0 x 105
16, 24
Spleen
23
Milestone 29: Plans
• Test individual polypeptides by IFNg ELISpot assay
with 6.0 x 105 lymph node cells/well
24
Action Items
• Terry will ask statistician for help analyzing the
passive immunization data and the bacterial
organ burdens
• Terry will ask statistician how many vaccinated
vs. unvaccinated people are needed to
distinguish the two populations being
stimulated by cytokines for killing SCHU S4.
25