Progress Report
5/5/2010
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
10. Testing vaccine candidates:
Lm-iglC vaccine from Aduro
LVS outer membrane proteins from Dr. Norgard (UTSWMC)
Preliminary proteome array results
11. Cellular and humoral immunity in LVS vaccinated Fischer rats:
Effect of CD4 T cells depletion for Ab-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
Testing Lm-based Vaccines
Percent survival
• An earlier experiment showed that Lm-iglC
protected Fischer 344 rats against i.t. SCHU S4
challenge
100
80
PBS
LVS
BH2172 (Lm677 KatG-SL8)
BH2182 (Lm677 IglC-B8R)
60
40
20
0
0
5
10
15
20
Time
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: Challenge Dose Escalation
Adjust based on
result from last slide
Mar11
Apr22
Jun3
Jul1
Timeline to completion
6
Take 1 vaccinated rat to optimize ASU peptide library production (iglC)
Testing LVS Outer Membrane Proteins
• Dr. Norgard’s lab published that LVS outer membrane
proteins emulsified in complete Freund’s adjuvant
protected ~50% of mice against a low dose (40 cfu)
i.n. SCHU S4 challenge
• The vaccine has since been improved using CpGISCOM as adjuvant
• We tested whether the new vaccine formulation
would protect F344 rats and become a vaccine
candidate
7
LVS Outer Membrane Proteins Provided
Complete Protection in F344 Rats
Percent survival
100
Naive
Adjuvant Only
LVS
OMP
50
0
0
5
10
15
20
25
Days Post Challenge
Drs. Norgard and Huntley are preparing more LVS outer membrane
proteins for a repeat experiment in 3 weeks
Submitted pre-challenge serum from Adjuvant only and OMP vax groups
for proteome array analyses
8
9
0.E+00
0.E+00
6000000
5000000
1.E+06
2.E+06
Mean Signal Intensity, mouse
3.E+06
4.E+06
5.E+06
6.E+06
7.E+06
3000000
2000000
FTT0901
FTT0106c
FTT0119
FTT0831c
FTT1125
FTT1250
FTT0385
FTT0835
FTT1035c
FTT1303c
FTT1016c
FTT1136c noss
FTT0060
FTT0135
FTT0289c
FTT0682c
FTT0474
FTT1381
FTT0509c
FTT1364
FTT0399c
FTT0470
FTT1182c
FTT0614c
FTT0941c
FTT0770
FTT1034c
FTT0808
FTT1464c
FTT1246
FTT0280c
FTT0220c
7000000
6.E+06
6000000
4.E+06
3.E+06
2.E+06
Mean Signal Intensity
5.E+06
Reactive antigens in mouse sera
reactive: SIag>SIc+2SD
4000000
mouse
rat
Mean Signal Intensity
Mean Signal Intensity, rat
7.E+06
1.E+06
5000000
7000000
6000000
5000000
1000000
1000000
0
0
FTT1441
FTT0196c
FTT0873c
ORF1167
FTT1107
FTT0924
FTT0869
FTT0230c
FTT0806
FTT1025c
FTT1591 noss
FTT1362
FTT1012
FTT1378
FTT1588c
FTT0302
FTT1004c
FTT0731c
FTT1635
FTT0851
FTT1514c
FTT0744c
FTT0294
FTT0620
FTT1561
FTT0830c
FTT0082
FTT0116
FTT0669
FTT1298
FTT0727
FTT0218
FTT0657
ORF81
FTT0640
FTT1447c
7000000
FTT0625
FTT0623
FTT1731c
FTT0074
FTT0665c
FTT1269c
FTT1526c
FTT0374c
FTT0547
FTT0438
FTT1681c
FTT1257
FTT0473
FTT1241
FTT0172
FTT0019
FTT0646c
ORF760
FTT1313c
FTT1569c
FTT0785
FTT0894
FTT0296 noss
FTT0792
FTT1058c
FTT0687c
FTT0299
FTT1112c
FTT1801
FTT0054
FTT0256c
FTT0075
FTT1530
Mean Signal Intensity
LVS OMP Vaccination Mouse vs. Rat
Common reactive antigens
reactive: SIag>SIc+2SD
4000000
3000000
2000000
mouse
1000000
rat
0
Reactive antigens in rat sera
reactive: SIag>SIc+2SD
4000000
3000000
2000000
mouse
rat
Preliminary Analyses of Vaccinated
Human and NHP Samples
Samples collected > 28 d after LVS vaccination
1. For each vaccinated sample, identified wells which
stimulated a "significant" response (> mean + 2StdDev of no
DNA control)
2. After step 1, select only proteins where at least 2 of the
triplicate set showed a significant response
3. After step 2, select proteins that stimulated significant
response in >33% of vaccinees (which translates to 3 of the 9
human vaccinee samples,
and 5 of the 17 total d28 NHP samples)
Unlike the rat samples, there is more heterogeneity with human
and NHP samples
11
Immunodominant Ft Antigens in
Mouse, Rat, NHP & Human
12
MS10: Plans
• Optimization of vaccination – challenged
• Dose escalation – waiting on optimization
results to decide on boost and challenge
• OMP vaccination – waiting on OMP prep
• Proteome array – Phil Stafford
13
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
14
Role of T cells in Vaccine-induced and
Ab-mediated Protection
Percent survival
• Anti-CD8 antibody OX-8 worked very effectively in vivo
• Anti-CD4 antibody W3/25 had partial effect in reducing Ab-mediated
protection, but no way to confirm treatment was optimized
100
Depletion / treatment
80
None / NRS
Isotype Ctrl / IRS
CD4 / IRS
CD8 / IRS
CD4+CD8 / IRS
60
40
20
0
0
5
10
15
20
25
30
Days Post-challenge
15
OX-38 Depletes CD4 T cells in vivo
5 mg/kg
12 3 4 5
CD3+CD4+ Day 11
OX-38 55-6
4.4% 38.9%
9.4% 42.5%
7.4% 41.2%
1 mg/rat
12
1 mg/rat
19
Day 15
OX-38 55-6
3.4% 37.2%
7.4% 38.5%
3.3% 34.2%
22 24
Day 19 (pre tx)
OX-38 55-6
8.0% 35.6%
11.2%
Day 26
OX-38 55-6
5.2% 45.1%
2.6% 34.2%
6.0% 42.3%
• 5 consecutive treatments @ 5 mg/kg followed by 2 weekly
treatments @ 1 mg/rat led to sustained CD4 T cell depletion
• These rats were treated with IRS and challenged with SCHU S4 – in
progress
16
CD4 T cells Required for
Ab-mediated Protection
Percent survival
100
75
NRS
IRS/Anti-CD4 (OX-38)
IRS/Isotype Control(55-6)
50
25
0
0
10
20
30
40
Days Post-infection
n = 3 per group
CD4 T cells at time of challenge: isotype control 40 ± 5%; OX-38 4 ± 2%
425 SCHU S4/rat intratracheal
Ascites fluid given every 3 d post challenge
17
Adoptive Transfer of LVS-Immune T cells
• 1st experiment:
– Adoptively transferred of LVS immune splenocytes into
untreated, naive F344 rats
– 24 h after transfer, challenged i.t. with 1000 SCHU S4
– No increased resistance
– Need to irradiate or treat with chemotherapeutic agents
to free space
– Need method to track “take”
18
Milestone 11 Cellular Immunity: Plans
• Repeat CD4 T cell depletion in passive immunization
experiments
• Test depletion strategy in vaccinated rats
• If successful, determine importance of CD4 and CD8 T cells
for 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 (Mar 2010)
Added 3 additional vaccinated donors
Statistical Significant Difference between Control
and Vaccinated Samples (April 2010)
24
Pre- and Post-vaccination Responses
(IFNg & Suppression of bacterial growth )
25
Assay Reproducibility for Vaccinees
Bacterial Proliferation Index
1st test (date)
2nd test (date)
4
0.04 (1/20/2010)
2.1 (3/23/2010)
14
0.005 (1/19/2010)
0.03 (4/7/2010)
16
0.01 (2/9/2010)
0.28 (4/7/2010)
Donor
LVS prestim
(fold increase over 72 h)
Proliferation index =
No prestim
(fold increase over 72 h)
PI > 1 = No suppression (unvaccinated phenotype)
PI < 1 = Suppression (vaccinated phenotype)
26
Planned Experiments
• Intracellular cytokine staining to identify IFNg
producing cells
• Compare pre- and post vaccination samples
• Calling back vaccinees
– Robustness of PBMC assay
– Matrix: CD4 and CD8 responses (IFNg & perforin ELISpot,
proliferation), antibody responses (Ab titer,
microagglutination)
• Microarray analyses
27
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
28
Action Items: 1 of 2
• Terry will add to the minutes, what was examined in the lower two panels
of the figure on slide 10
• Terry will email Barbara the number of new sera samples that UNM
requests permission to send to Immport. Barbara will send the numbers of
samples to Patrick.
• Terry will add the gene names to the minutes for slide 12
• Terry: Y axis on right panel needs correction on slide 23
• Terry change the cutoff on slide 26 and will call back the 4 individuals who
are in pairs, to get the most interpretable data for reproducibility of the PI.
• Gopi included all Patrick’s suggestions in the manuscript and will assure
that it all flows well, then will send the revised manuscript to Rick.
• Rick will ask Julie how to incorporate the LVS Lot 17 vaccinations into her
needs for bloods for immunoassays and correlates of protection.
29
Action Items: 2 of 2
•
Patrick: wants the NHP prescreened for antibody, before the NHP is used
on low dose LD 50 study.
• LBERI will gets stats for numbers of NHP needed for low dose LD50, plan
on 6 NHP for the vaccination for immunoassays/correlates and later
challenge in 35 to 37 day (tell patrick the protocol being used) (challenge
schedule must fit into the LBERI BSL3 rooms), lot 17 challenge study will
need controls w/o vaccination, Patrick wants the protocol numbers to
assure that all is followed. Will get telemeterized planned under natural
history, but if reanalyzed natural history, then could do with
vaccinated/telemetered after modifying the protocol. Permit for the
studies. Need emails for all these studies and many NHP are not yet used.
Need final numbers of NHP on TVDC and won’t release NHP until know
what TVDC needs
30