Progress Report
5/6/2009
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:
5. a) Sensitivity of Fischer 344 rats to s.c. SCHU S4 infection
b) LVS dose dependence
11. a) Cytokine production by passively immunized rats post SCHU S4 challenge
b) IgG purification from immune serum
c) Protocol for depleting CD4 T cells from rats
21. Use of human PBMC in correlate of protection assay
29. Plans for confirming ASU Ft peptide library screen
35. Plans to purify RNA from mice and rats 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.
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
3
Sensitivity of Fischer 344 Rats to
Subcutaneous SCHU S4 Challenge
103 CFU
% Weight loss/gain
101 CFU
105 CFU
10
10
10
5
5
5
0
0
0
-5
-5
-5
-10
-10
-10
-20
-20
*
-20
-30
-30
0
5
10
15
Days post infection
20
0
5
10
*15
Days post infection
*
* *
-30
20
0
5
10
15
20
Days Post Infection
4
Small LVS Vaccination Dose
Sufficient to Protect Rats
B
Percent wt gain/loss (%)
Percent survival (%)
A
100
80
60
40
20
0
0
4
8
Days post i.t. challenge
12
5
0
Unvaccinated
103 LVS
-5
105 LVS
107 LVS
-10
-15
0
4
8
12
Days post i.t. challenge
5
MS 11: Characterization of Fischer 344 Rat
Fischer 344
rats
Blue: Steps in the milestone
Red: Completed
Green: In progress
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
Active vs passive
immunizatioin
Pretection against i.t. SCHU
SCHU challenge
6
Milestone 11: Background
• Passive immunization protected rats against i.t. SCHU
S4 challenge
• Intermediate phenotype (bacteriology &
histopathology) between NRS and LVS vaccinated rats
• Peak bacterial burden similar to naïve rats, but rats
survived to clear infection
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
Days Post-challenge
18
21
7
Intermediate Cytokine Levels in
Passively Immunized Rats
Day 7 Lung Cytokines
8
MS 11 Objective 1
Prove that transfer of anti-tuli IgG is sufficient for
passive immunization


Rat IgG has moderate affinity for Protein G and low
affinity for Protein A
Enriched for IgG from immune rat serum by negative
selection using Melon Gel IgG purification kit (Pierce)
9
Percent survival
Purified IgG Protected Rats Against
i.t. SCHU S4 Challenge
100
80
Naive (0.25ml PBS)
IRS (0.25ml)
Immune IgG
60
40
20
0
0
5
10
15
20
Days P.I.
10
MS 11: Objective 2
Determine the role of T cells in protection by
LVS vaccination and immune serum transfer
Percent survival
100
80
Isotype control
CD4 (W3/25)
60
CD8 (OX-8)
CD4 + CD8
40
20
0
0
10
20
30
40
Days post challenge
SCHU S4 challenge dose = 5.84 x 10 4 cfu/rat
11
List of Available Antibodies
Anti CD4 T cells
Anti-CD8 T cells
W3/25
OX-8
– IgG1
– Non-depleting, causes
functional inactivatioin
– Mouse anti-rat CD8
– IgG1
– Depleting
OX-38
– IgG2a
– Depleting
– Currently in culture and will
send to Taconic for ascites
fluid production
Isotype control
55-6
– Mouse anti-HIV-1 gp120
– IgG2a
TS2/18.11
– Mouse anti-human CD2
– IgG1
12
Depletion of CD8 but not CD4 T cells
CD4 + CD8-depleted
0.03 %
13
12
13
10 3
10 4
27.37%
12
10 3
10 4
LVS Vaccinated
23.56%
14
15
10 1
10 2
CD4-FITC
10 3
10 0
10 1
10 1
10 0
CD8-PE
10 2
R12
15.72%
10 0
10 4
14
10 1
10 2
CD4-FITC
10 3
10 4
CD8-depleted
13
10 4
10 4
12
15
10 0
CD4-depleted
12
Depletion checked 1
wk after treatment
13
1.22%
10 3
10 3
34.27%
R12
CD8-PE
10 2
CD8-PE
10 2
R12
25.14%
14
10 0
15
10 1
10 2
CD4-FITC
10 3
10 4
10 0
10 1
10 1
16.29%
10 0
CD8 T cells
CD8-PE
10 2
R12
14
10 0
CD4 T cells
15
10 1
10 2
CD4-FITC
10 3
10 4
13
Milestone 11: Plans
• Optimize IgG purification
– Resin:serum ratio
– Repeat passive immunization with IgG from
normal and immune rat sera
• Optimize CD4 T cell depletion procedure
– Repeat depletion in LVS vaccinated rats
– Perform depletion in passively immunized rats
14
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
15
MS21: Background
• Human PBMC supported SCHU S4 growth of
3-4 logs over 72 h period
• Continuous stimulation with IFNg and TNF
starting 48h prior to infection reduced
bacterial burden – showing that IFNg and TNF
sufficient to induce bacterial control
16
Continuous Activation with IFNg/TNF
Controlled SCHU S4 Burden
IFN/TNF IFN/TNF
-24 h
IFN/TNF
IFN/TNF
Harvest
0h
24 h
48 h
72 h
6
total CFU / well (Log 10)
-48 h
Infect
IFN/TNF
5
4
PBMC
+ IFNg
+ TNF
+ IFNg + TNF
3
2
1
0
3
48
72
Time post-infection (hours)
17
48 h Preactivation Sufficient to
Control SCHU S4 Burden
IFN/TNF
-24 h
total CFU / well (log 10)
-48 h
Infect
0h
Harvest
24 h
48 h
7
6
5
4
3
2
1
0
72 h
PBMCs:control
+ IFNg + TNFa
0
72
Hours post-infection
18
Milestone 21: Plans
• Compare SCHU S4 growth in PBMC from unvaccinated and LVS
vaccinated individuals before and after stimulation with HK
LVS
• Determine the minimum amount of recombinant IFNg and
TNF required to activate PBMC control and compare with
amount produced by PBMC stimulated with HK or FF-LVS
• Determine conditions for maximum stimulation of PBMC
IFNg/TNF production
• Develop analogous assay in rats
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
Confirmation
Assay optimization
using ivt proteins
Blue: Steps in the milestone
Red: Completed
Green: In progress
20
MS29
• Screened ASU peptide library with splenocytes and lymph
node cells from LVS vaccinated/boosted NHP
• IFNg ELISpot assay detected several potential stimulatory
peptides (2SD above plate mean) but not reproducible in
replicate well or 2nd tissue
• Concern about false positives and analysis method
• Sent raw data to Phil Stafford @ ASU to select positives
• Repeat IFNg ELISpot on individual peptides with frozen
splenocytes or lymph node cells
• Enough LN cells for 300 wells and splenocytes for 600 wells
• Confirm with additional vaccinated NHP at LBERI
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
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
Gray: (sub )milestone title
Red: completed
Green: in progress
Isolate RNA from
infected lungs
Infect BALB/c mice
i.n. Or Fischer 344
rats i.t. with SCHU S4
Isolate eukaryotic and
prokaryotic RNA
Ship to ASU
Blue: Steps in the milestone
Red: Completed
Green: In progress
22
MS 35
• Prepare RNA from BALB/c mice (2 exp) and
Fischer 344 rats (1 exp) lungs 1, 3, 5, 7, and 24
h after infection with SCHU S4.
23
Action Items
•
•
•
•
•
•
•
Terry- will review the clinical symptoms of the naïve rats that were low dose SCHU S4
infected. Were the SCHU S4 infected, naïve rats ever sick and then survived?
Terry /Gopi will optimize the CD4/CD8 depletion procedure and then repeat experiment to
determine whether protection is dependent on these T cells.
Terry/Gopi will optimize the ratio of resin to sera to enrich for IgG in rat sera.
Notes added after the call:
Terry will test a combination of the two CD4 antibodies (one depleting and one inactivating)
to try to further deplete the CD4 T cells
Terry/Gopi will serially enrich for IgG with the Pierce resin, empirically determining the
enrichment for IgG using a Western/PAGE
Terry will review the Lm vaccine candidates transferred from Cerus/Anza to UNM. Terry will
discuss with Rick, testing the potential vaccine efficacy of the Lm platform producing Kat G,
the Lm producing IglC and the Lm platform alone.
24