Progress Report
6/2/2010
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
10. Testing vaccine candidates:
Lm-iglC vaccine from Aduro
KBMA-iglC vaccines from Aduro
11. Cellular and humoral immunity in LVS vaccinated Fischer rats:
Effect of T cells depletion on LVS-induced protection
Mechanism of antibody-mediated protection
21. Correlate of protection assay using human PBMC
Correlation between IFNg production and suppression of bacterial
growth
2
MS 10: Testing Vaccines Candidates
Cerus/Aduro
ASU
UTSA
Others
UNM
BALB/c
mice
Blue: Steps in the milestone
Red: Completed
Green: In progress
Fischer 344
rats
3
Lm-iglC Protected F344 Rats
Against i.t. SCHU S4 challenge
6 wk
Boost #1
Lm: 5 x 107 / rat i.m.
6 wk
Boost #2
Lm: 5 x 107 / rat i.m.
Percent survival
Prime
Lm: 107 / rat i.v.
LVS: 108 / rat s.c.
100
80
PBS
LVS
BH2172 (Lm677 KatG-SL8)
BH2182 (Lm677 IglC-B8R)
60
40
20
0
0
5
10
15
20
Time
4 wk
Challenge
SCHU S4: 103 / rat i.t.
n = 12 / group
i.t. challenge dose = 200 cfu (deposition)
4
Repeat Expt and Vaccine Optimization
Feb23
Mar23
Apr6
May4
Timeline to completion
5
Lm-iglC Protected F344 Rats
Against i.t. SCHU S4 challenge
Percent survival
100
75
n = 7 (6 in ctrl groups)
200 SCHU S4 i.t.
4 wk after boost
50
25
0
0
5
10
15
20
25
Time
Naive
LVS
BH2182 108 IM 4 wk prime/boost
BH2182 108 ID 4 wk prime/boost
BH2182 106 IM 4 wk prime/boost
BH2182 108 IM 2 wk prime/boost
BH1222 108 IM 4 wk prime/boost
6
Summary of Results
• Vaccination with 108 Lm-IglC provided
significant protection (prolonged survival)
regardless of the regimen (p < 0.03)
• Vaccination with 106 Lm-IglC did not provide
significant protection (p = 0.11)
• Lower overall survival in this experiment (max
2/7 per group) than in 1st experiment (9/12)
7
Possible Reasons for Difference in Efficacy
1st Experiment
2nd Experiment
Differences
Prime
Lm: 107 / rat i.v.
Boosts: 2 vs. 1 boost
6 wk
Boost #1
Lm: 5 x 107 / rat i.m.
6 wk
Boost #2
Lm: 5 x 107 / rat i.m.
4 wk
Challenge
SCHU S4: 200 / rat i.t.
Prime
Lm: 107 / rat i.m
Priming: i.v. vs. i.m
6 wk
Boost #1
Lm: 5 x 107 / rat i.m.
4 wk
Challenge
SCHU S4: 200 / rat i.t.
8
Lm-IglC: Regimen Optimization
Mar11
Apr22
Jun3
Jul1
Timeline to completion
9
Experimental Design: KBMA-IglC
Regimens
(Groups1-2) Prime→ 8 wk → SchuS4 IT Challenge
(Groups 3-6) Prime → 2 wk → Boost → 2 wk → Boost → 4 wk → SchuS4 IT
Challenge
Groups
1) PBS - SC
2) 5x107 LVS (No boost) - SC
3) 1x108 Live BH2182 (Lm677:IglC) - IM or ID (includes 2nd boost)
4) 1x108 KBMA BH2182 (Lm677:IglC) - IM or ID, (includes 2nd boost)
5) 2x109 KBMA BH2182 (Lm677:IglC) – IV, (includes 2nd boost)
6) 2x109 KBMA BH2182 (Lm677:IglC) - IM or ID, (includes 2nd boost)
10
MS10: Plans
• Continue ongoing Lm-iglC experiment with
2nd boost (will challenge on July 1)
• Evaluate KBMA-iglC
• OMP vaccination – waiting on OMP prep
11
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
Characterizing elements
of protection
Blue: Steps in the milestone
Red: Completed
Green: In progress
Protection against i.t. SCHU
challenge
12
Background – Humoral Immunity
• T cell immunity dominate resistance of vaccinated
animals to SCHU S4 challenge
• Antibodies contribute to resistance
• Present goals
– Define how antibodies mediate protection
– Enhance T cell response
– Suppress pathological consequences of infection
13
CD4 and CD8 T cells Required for Abmediated protection
100
Percent survival
Percent survival
100
75
50
25
75
50
25
0
0
0
10
20
30
Days Post-infection
T cell subset depl. / treatment
Undpleted / normal serum
Undepleted / immune serum
CD4 depleted / immune serum
0
10
20
30
Days Post-challenge
T cells subset depl / treatment
Undepleted / Normal serum
Undepletedl / immune serum
CD8 depleted / immune serum
14
Immune Serum Reduced TUNEL Staining
(reduced apoptosis)
15
Immune Serum Reduced IL-6 Production
(reduced acute phase response)
Spleen
pg/mL (log 10)
Lungs
Liver
5
5
5
4
4
4
3
3
3
2
2
2
NRS
IRS
NRS
IRS
NRS
IRS
Day 5 post i.t. SCHU S4 challenge
2 independent experiments
16
Background – Cell-Mediated Immunity
• T cell immunity dominate resistance of vaccinated
animals to SCHU S4 challenge
• Antibodies contribute to resistance
• Present goals
– Demonstrate importance of T cells by depletion and
adoptive transfer strategies
17
Role of CD4 and CD8 T cells in LVSmediated Protection
• LVS vaccinated rats were treated with anti-CD8 and
anti-CD4 antibodies
• Depletion efficiency:
– CD4 reduced 32% to 4%
– CD8 reduced from 19% to 2%
• Challenged i.t. with 200 SCHU S4
• Day 20 survival
–
–
–
–
Naïve rats: 0/6 alive
Vaccinated + isotype control: 6/6 alive
Vaccinated + anti-CD4: 5/5 alive (clinically ill)
Vaccinated + anti-CD8: 4/6 alive (clinically ill)
18
Milestone 11: Plans
• T cell depletion in passively immunized rats
• T cell depletion in LVS-vaccinated rats
• Develop method for adoptive transfer of LVS-immune
T cells into naïve rats
19
MS 21: Assays in Vaccinated Humans
Assay to measure activation of PMBC killing
mechanisms in humans
Determine the approximate
yield of PBMC from whole blood
(1-200 ml max)
Determine and optimize cell
number and MOI
Evaluate assay with
IFNg and TNF
Develop assay with F. tularensis
Compare human vaccinees and controls
Blue: Steps in the milestone
Red: Completed
Green: In progress
Statistical analysis
20
Expected Responses from
Vaccinated Human PBMC
HK-LVS
Timeline
T
T
Wash
SCHU S4
FF LVS
Prestimulation
(expansion of
specific T cells)
growth
controlled
No Prestim
Burden
IFNg
-48 h
-24 h
0h
24 h
48 h
72 h
?
CFU -fold increase with
48-hour FF-LVS pretreatment,
normalized to no-pretreatment control
LVS Prestim
1.25
Growth
suppression
LVS prestim
PI =
(fold increase over 72 h)
No prestim
(fold increase over 72 h)
-fold increase, normalized to control
monocyte
s
1.00
0.75
0.50
0.25
0.00
vaccinated (N = 4) unvaccinated (N = 11)
21
Expected Responses from
Unvaccinated Human PBMC
monocytes
Timeline
HK-LVS
Wash
SCHU S4
T cell
Prestimulation
(expansion of
specific T cells)
X
FF LVS
T
-48 h
-24 h
0h
24 h
Ft growth NOT
controlled
48 h
72 h
CFU -fold increase with
48-hour FF-LVS pretreatment,
normalized to no-pretreatment control
LVS Prestim
1.25
LVS prestim
PI=
(fold increase over 72 h)
No prestim
(fold increase over 72 h)
-fold increase, normalized to control
No Prestim
Burden
1.00
0.75
0.50
0.25
0.00
vaccinated (N = 4) unvaccinated (N = 11)
22
Statistical Significant Difference between Control
and Vaccinated Samples (May 2010)
Added 2 unvaccinated and 1 vaccinated
5
Proliferation Index
5/27/10
IFNg
100000
IFNg pg/mL
proliferation index
75000
4
2
50000
1
25000
0
0
nonvax
vax
nonvax
vax
N=17
N=16
N=14
N=15
p=0.0002
2-tail Mann-Whitney
p=0.0004
2-tail Mann-Whitney
23
Pre- and Post-vaccination Responses
(IFNg & Suppression of bacterial growth )
FT proliferation index
IFNg
Pre / post-vax pairs
N=5
Pre / post-vax pairs
1.00
100000
10
0.75
75000
11
IFNg pg/mL
proliferation index
8
0.50
4
0.25
0.00
11
50000
25000
10
5
4
0
prevax
These 2 donors started
with “protected”
phenotype, therefore
vaccination provided no
enhancement
8
postvax
Wilcoxon p = 0.1876
(2-tail paired nonparametric)
prevax
postvax
p = 0.0197
N = 4 (donor 5 pending)
2 more pre- / post-vaccinees to be screening 6/8/10
24
Planned Experiments
• Compare pre- and post vaccination samples
• Calling back vaccinees
– Robustness of PBMC assay
– CD4, CD8 and antibody responses
– Microarray analyses
25
Additional Points
Deliverables completed for each active milestone:
MS10: Tested Aduro’s Lm vaccines in mice and rats
MS 11: Demonstrated contribution of Ab in vaccinated F344 rats
MS 12/13: ELISA for Ab titer, ELISpot for IFNg, T cell proliferation
MS 21: none
List of relevant publications from the past month:
Final internal review and revision prior to submission
MSCR status
MS 5 mouse: UNM reviewing 1/6/10 comments from NIAID
MS 5 rat: UNM draft to Barbara 11/17/09 (BG needs to review)
MS 12/13: UNM drafts in progress (6 UNM SOPs drafted; need microagglutination assay developed)
MS 34 w ASU: UNM wrote RNA isolation SOP 7/24/09; waiting on
ASU for MSCR
26
Additional Discussion
27
Action Items
• Barbara: will send UNM’s “Passive immunization”
article to NIAID , as it is submitted to the journal
(Infection and Immunity).
• Terry/Barbara/Karl/Justin will create a table of all
bacterial strains tested as vaccine candidates in
animal models to date. Will do by approximately
7/1/10. Will include bacterial strain, animal model
used, challenge used, survival results.
28