Progress Report
07/2/2008
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:
11.
Effector mechanisms in LVS vaccinated rats
19.
Human alveolar macrophage interaction with F. tularensis
21.
Improving the reproducibility of macrophage killing assays
in mice and rats
27.
Testing of ivt proteins for ASU (ASU MS 26)
Other milestones
5.
On hold until rabbits
12/13. Optimizing assay for multifunctional T cells
14.
CMI in human vaccinees
17.
Correlates of protection in human vaccinees
2
MS 11: Characterization of Fischer 344 Rat
Fischer
344 rats
Humoral
immunity
LVS vaccination
Passive transfer
of serum
Protection
against i.t SCHU
challenge
Blue: Steps in the milestone
Red: Completed
Green: In progress
Cell mediated
immunity.
Production of
ascites fluid for
CD4 and CD8
depletion
LVS vaccination
In vivo depletion
Length of
protection
LVS vaccination
SCHU S4
challenge @
various times
post vaccination
Pretection against
i.t. SCHU SCHU
challenge
3
Passive Immunization Against SCHU S4
Immune serum
s.c. LVS vaccination
Naïve + vacc. serum
LVS vaccinated
i.t. SCHU S4
challenge
Normal serum
Naïve + normal serum
Naive
4
Normal Serum Protected NCI Rats
Percent survival
100
No treatment
sc LVS vaccination
Normal rat serum
Immune rat serum
(2.5 ml)
75
50
25
0
0
5
10
15
Days after i.t. SCHU S4 Challenge
5
Normal Serum Did Not Protect
Harlan Rats
Percent survival
100
Naive
+ 2.5 ml NRS
+ 1.0 ml NRS
+ 0.5 ml NRS
+ 0.25 ml NRS
75
50
25
0
0
5
10
Days p.i.
15
Milestone 11: Objectives
1. Determine the minimum volume of immune
serum required to protect rats against i.t.
SCHU S4 challenge
2. Compare the sensitivity of Fischer 344 rats
from Harlan and NCI to i.t. SCHU S4
challenge
Decide on vendor, serum volume, and challenge dose
for future passive immunization experiments
Dose-dependent Protection of
NCI Rats by Immune Sera
†
d 20 mean burden (log)
Lung – 4.4 ± 0.7
Liver – 2.5 ± 0.1
Spleen – 3.7 ± 0.8
†
d 20 mean burden (log)
Lung – 4.1 ± 0.4
Liver – 2.3 ± 0.1
Spleen – 3.6 ± 0.1
d 20 mean burden (log)
Lung – 4.3 ± 0.4
Liver – 1.6 ± 1.5
Spleen – 3.3 ± 0.8
MS11 Objective 1: Interpretations
• 0.4 ml immune serum enabled NCI rats to
survive and maintain weight for 20 days after
i.t. SCHU S4 challenge
• Rats gained weight with larger serum volume
• Despite of the weight differences, the
bacterial burden in the lungs, liver, and
spleen were similar
MS 11 Objective 1: Experimental Design
Repeat passive immunization experiment including all controls
Treatment
Serum
volume
(ml)
Group
Vendor
Vaccination
status
1
NCI
Vaccinated
None
-
2
Naïve
None
-
3
Naïve
Normal serum
0.5
4
Naïve
1.0
5
Naïve
1.5
6
Naïve
7
Naïve
1.0
8
Naïve
1.5
Immune serum
0.5
MS 11 Objective 2: Experimental Design
Compare the sensitivity (LD50 and LD99) of Harlan and NCI
rats to i.t. SCHU S4 challenge
Group
Vendor
SCHU S4
challenge dose
(cfu/rat)
1
Harlan
101
No
2
102
No
3
103
Yes
4
104
Yes
5
105
Yes
101
No
7
102
No
8
103
Yes
9
104
Yes
10
105
Yes
6
NCI
Lung
deposition
11
MS 11 Objective 3: Experimental Design
Determine whether protection is dependent on LVS dose
LVS
vaccin. dose
(cfu/rat)
Group Vendor
SCHU S4
challenge dose
(cfu/rat)
-
105
2
103
105
3
105
105
4
107
105
1
NCI
12
Milestone 19 – flow diagram
Interaction between human
alveolar macrophages and F.
tularensis
Measure LVS and SCHU
S4 growth in AM
Determine effect of LVS
and SCHU S4 on cytokine
production by AM
Cytokine inhibition of
LVS and SCHU S4
intracellular growth
Blue: Steps in the milestone
Red: Completed
Green: In progress
MS19 Objective 1
• Determine the cytokine response of
human alveolar macrophages to LVS
and SCHU S4 infection
Cytokine Responses by Human Alveolar
Macrophages Infected with LVS and SCHU S4
MOI = 10 may be suitable for discriminating LVS from SCHU S4
MS 19: Limitations
• Low average yield ~ 107 cells/patient
• Fungal and bacterial contamination
• Cell loss to washes between steps
–
–
–
–
–
–
Adhere cells overnight on plates
Wash away non-adherent cells
Infect with Tuli
Wash to remove extracellular bacteria
Treat with Gentamycin to kill extracelular bacteria
Wash to remove Gentamycin
MS19: Objective 2
• To compare cytokine production between
adherent and suspended alveolar
macrophages after infection with LVS and
SCHU S4
• Suspension culture may stimulate the
alveolar environment more closely than
culture on adherent surfaces because the
effect of adhesion on macrophage activation
may be reduced.
Adherent Alveolar Macrophages
Produced More Cytokines
MS19 Objective 2: Interpretations
• Intensity of the cytokine response was 3-4fold stronger with adherent macrophages
• Adherent and suspended alveolar
macrophages responded similarly to infection
with SCHU S4
• Although adherent cultures may be more
sensitive, suspension cultures may be easier
to handle and more consistent and
reproducible across experiments
memory
T cell
Milestone 21 – flow diagram
mf
F
T cell-induced macrophage killing
of intracellular Francisella
F
mf
F
X
X
Mouse
Rat
Generate bone marrow
derived macrophages
Generate bone marrow
derived macrophages
F
Load BMDM with LVS
Load BMDM with
SCHU S4
Load BMDM with LVS
Load BMDM with
SCHU S4
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Blue: Steps in the milestone
Red: Completed
Green: In progress
Murine Macrophage Killing of
Intracellular SCHU S4
Problems: Lack consistency among replicates
Difficult to reproduce over experiments
MS21: Objective 1
• Improve consistency of the mouse
SCHU S4 macrophage assay by:
– Increasing SCHU S4 infection time from
1 to 4 hours
– Lysing cells with deoxycholate instead of
water
– Increasing the number of replicates
Increasing Infection Time Improved
Consistency and Reproducibility of
Mouse Macrophage SCHU Killing Assay
Representative of 4 independent experiments
memory
T cell
Milestone 21 – flow diagram
mf
F
T cell-induced macrophage killing
of intracellular Francisella
F
mf
F
X
X
Mouse
Rat
Generate bone marrow
derived macrophages
Generate bone marrow
derived macrophages
F
Load BMDM with LVS
Load BMDM with
SCHU S4
Load BMDM with LVS
Load BMDM with
SCHU S4
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Measure LVS killing
induced by vaccinated
T cells or cytokine
Measure SCHU S4
killing induced by
vaccinated T cells or
cytokine
Blue: Steps in the milestone
Red: Completed
Green: In progress
MS21: Objective 2
• Determine whether addition of
splenocytes from LVS vaccinated rats
can induce rat macrophages to kill
intracellular LVS
Immune Splenocytes Inducd Macrophage
Killing of Intracellular LVS
MS21: Planned Experiments
• Determine whether immune splenocytes
can induce murine macrophages to kill
SCHU S4
• Continue to develop the rat macrophage
killing assay with SCHU S4
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
Assay optimization
using ivt proteins
Blue: Steps in the milestone
Red: Completed
Green: In progress
Screening
(UNM)
Identification of
stimulatory proteins &
peptides
Milestone 27 objective 1
• Determine whether TBLN cells from LVSvaccinated NHP also crossreact with E. coli
proteins in the ivt reaction mix.
• Since NHP were vaccinated/boosted by
bronchoscopy, the TBLN should be enriched
for Ft-reactive cells
–
–
–
–
3 LVS-vaccinated NHP
July 7, July 16, and Aug 6
Samples to be supplied by ASU
Tested by IFNg ELISpot
Action Items
•
•
•
•
•
•
Terry: write MS#5 mouse and rat sections by end of summer 2008. Terry is
working to complete the rat model paper very soon.
Terry: email Marlene a concise summary of the mouse and rat data and will
attach prior INI paper on mouse model, as soon as possible.
Terry will compare the current 7/2/08 cytokine response data in Hu AM current
data with the prior data
Marlene will research the name/source of the sponge-like matrices for cell
culture and Terry will search on internet
Marlene will research whether the Martha’s Vineyard individuals were treated
with antibiotics, as well as other information about the PBL and sera tubes.
Barbara will share emails from DVC and Battelle with Marlene, if they ask
questions about the LRRI protocols
30