Progress Report
2/3/2010
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
10. Testing vaccine candidates:
First test of Aduro’s Lm-based vaccines in F344 rats
LVS outer membrane proteins from Dr. Norgard (UTSWMC)
11. Cellular and humoral immunity in LVS vaccinated Fischer rats:
IgM response after LVS vaccination
Functional assay to measure effectiveness of CD4 inactivation
12/13. Immunoassays:
Microagglutination assay
21. Correlate of protection assay using human PBMC
Specific activation of vaccinated PBMC at reduced Ag dose
Comparison of pre- and post vaccination samples
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
• Two previous experiments in BALB/c mice
showed that the Lm-based vaccines
expressing iglC or katG failed to induce any
protection against i.n. SCHU S4 challenge
• The failure may be related to the sensitivity of
the mouse to pneumonic tularemia
• A more robust model like the F344 rats may
reveal protective efficacy
4
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.
4 wk
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
n = 12 / group
i.t. challenge dose = 200 cfu (deposition)
Challenge
SCHU S4: 103 / rat i.t.
5
Testing Lm-based Vaccines
• The Lm-iglC vaccine induced significant
protection against i.t. SCHU S4 challenge, but
was not 100%
• 100% protection would facilitate future
characterization of the vaccinated state
• More effective vaccination may be generated
with more antigens in the vaccine, different
route of vaccination and/or different platform
6
Repeat Expt and Vaccine Optimization
• Monovalent vs. bivalent vaccine
• IM vs. ID vaccination
• Vaccine platform
8 rats per group, prime (6wk) boost (4wk) i.t. SCHU S4
–
–
–
–
–
–
PBS
LVS (SC)
BH2182 (IM) – iglC / actAinlBuvrABprfAG155S
BH2292 (IM) -- iglC + katG / actAinlBuvrABprfAG155S
BH2292 (ID) -- iglC + katG / actAinlBuvrABprfAG155S
BH2316 (IM) -- iglC + katG / actAinlB
7
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 are testing whether the new vaccine formulation
would protect F344 rats and become a vaccine
candidate
8
LVS Outer Membrane Proteins in F344 Rats
Schedule
Vaccination: 12/22/09
Boost: 1/12/10
Boost: 1/26/10
Challenge 3/2/10
Completed
9
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
10
Milestone 11: Humoral Immunity
• Antibody responses develop within 7 days of s.c. LVS
vaccination; IgG2a, IgG2b > IgG1, IgG2c, IgM, no IgA
• Passive immunization with immune serum or purified IgG
protected rats against low dose i.t. SCHU S4 challenge
• Intermediate phenotype between NRS and LVS vaccinated rats:
bacteriology & histopathology, disease severity
• No increase in protection with larger serum dose or with
repeated serum treatment
• Protection eliminated by depletion of CD8 T cells and in nude
rats
11
Measuring IgM Response
• A large amount of IgM is produced after LVS
vaccination in the mouse
• To better characterize the antibody response
in the F344 rat (for publication), the IgM
response was measured after LVS vaccination
12
IgM & IgG Responses Following
s.c. LVS Vaccination
A
15
150
10
100
5
50
0
IgG Titer (x 1000)
IgM Titer (x 1000)
200
IgG
IgM
0
0
7
14
21
28
Days Post-vaccination
Serum IgM is produced abundantly in F344 rats and the titer peaked 7
days after vaccination.
13
Role of T cells in Ab-mediated Protection
• Historical studies in mice suggested that T
cells are required for Ab to mediate long-term
survival after LVS challenge
• To determine the requirement for CD4 and/or
CD8 T cells in SCHU S4 challenge, rats were
treated with depleting/inactivating antibodies
before passive immunization and challenge
14
Percent survival
W3/25 mAb Unable to Completely
Abolish Protection
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
Challenge dose 200 CFU/rat
15
Measuring CD4 T cell Responses in F344
rats Treated with W3/25
• Depletion experiment showed that CD8 T cells
was required for IRS to protect rats
• It is possible that W3/25 treatment was not
completely effective
• The W3/25 antibody is a inactivating antibody
and does not cause in vivo depletion.
Therefore, we have not been able to confirm
the antibody treatment was effective before
challenging rats.
16
W3/25 mAb failed to Completely Inactivate
CD4 T Responses in Vaccinated Rats
BrdU incorporation
MACS purified CD4+ splenocytes from LVS vaccinated rats
Restimulation with iLVS
Rat 1
Rat 2
7
Naive / Untreated
Vacc / Isotype
Vacc / W3/25
RLU
6
10
x
5
x
10
4
4
4
5
2.
x
10
3
1.
25
x
10
3
6.
25
x
10
3
12
x
3.
56
x
1.
X
39
0.
10
3
10
3
10
10
x
5
x
5
4
4
10
4
T Cell Count
2.
5
x
10
3
1.
25
x
10
3
6.
25
x
10
3
3.
12
x
10
3
10
56
x
1.
78
0.
0.
39
X
10
3
5
78
6
0.
RLU
7
T Cell Count
Based on this result, we cannot make any conclusion regarding
the relevance of CD4 T cells to antibody mediated protection
17
Milestone 11 Cellular Immunity: Plans
• Finish experiments to confirm purity of isolated IgG
• Finish manuscript describing passive immunization of
F344 rats
• Develop method for adoptive transfer of immune T
cells into naïve rats
18
Milestone 12/13 – flow diagram
Assays for Detecting Relevant Immune
Responses in Animals and Humans
Humans (UNM)
Mice (UNM)
Rats (UNM)
Generate reagents
Generate reagents
Generate reagents
ELISA for Ab titer
ELISA for Ab titer
ELISA for Ab titer
ELISpot Assay
T cell proliferation
T cell proliferation
Microagglutination
IFNg ELISpot
IFNg ELISpot
Microagglutination
Microagglutination
Blue: Steps in the milestone
Red: Completed
Green: In progress
19
Microagglutination - Example
Microagglutination Test for Early and Specific Serodiagnosis of Tularemia
Sato et al. J Clin Microbiol (1990) 28:2372
20
Microagglutination - UNM
Ctrl
1:20480
1:10240
1:5120
1:2560
1:1280
1:640
1:320
1:160
1:80
1:40
• Control serum titer at UNM
1:5,120
• Technique works
1:20
• LVS Lot 16 cultured in
Chamberlain’s for 48 h
• Rb positive control serum from
Dr. Sztein (1:2,560)
• Normal rabbit serum from Lyons
lab
Ag dilution
Serum dilution
21
MS 12/13: Plans
• Microagglutination
•
•
•
•
Make new batch of stained antigen from LVS lot 9 grown in MCPH culture
Test new batch with control rabbit serum
Assay sera from human, rat, mouse
Prepare SOP
• Ag prep normalization by FACS
• Alexa-488 stained FF-LVS and HK-LVS detectable by FACS
• Technical issues include: small size, loss during washes and degradation
over time(?)
22
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
23
Assumptions Used to Develop Assays
for Protection Status
Unvaccinated
monocytes
T cell
LVS vaccinated
monocytes
T cell
T cell
Ft growth NOT
controlled
IFNg
Ft growth
controlled
TNF
24
Mixed Responses from Unvaccinated PBMC
CFU -fold increase with
48-hour FF-LVS pretreatment,
normalized to no-pretreatment control
1.25
2-tailed Mann-Whitney nonparametric
p=0.0618
-fold increase, normalized to control
40 (UBS)
18.1 (UBS)
1.00
35 (prevax 8.1)
31 (prevax 6.1)
0.75
42 (UBS)
0.50
0.25
32 (UBS)
18.2
36 (prevac 9.1)
27.1 (UBS)
*
39
33
0.00
26.1
38 (prevax 11.1)
27.2 (UBS)
20
41(prevax 11.1)
29 (prevac 4.1)
34 (UBS)
37 (prevax 10.1)
30 (prevax 5.1)
vaccinated (N = 4) unvaccinated (N = 11)
*
39
is a retest of vaccinee#2 (#18.2)
25
Hypothesis
• Excess antigen during pre-stimulation induced
non-specific activation of unvaccinated PBMC
• Antigen optimization will eliminate nonspecific activation while retaining specific
stimulation for vaccinated PBMC
26
Ag Titration Reduced Non-specific
Stimulation of Unvaccinated PBMC
DVC FF-LVS
Pre-Vax
(FT-AH-41)
1.010 8
1.010 7
Decreasing Ag conc.
in prestimulation
total CFU / well
1.010 6
Although the non-specific
response was not completely
eliminated, this result suggested
that further titration may lead to
an optimal Ag without any nonspecific activities
1.010 5
1.010 4
1.010 3
1.010 2
0
24
48
72
HOURS POST-INFECTION
control (21,400x increase)
10% v/v (832x increase)
2.5% v/v (1770x increase)
0.6% v/v (3030x increase)
27
0.6% Ag Sufficient to Induce Specific
Responses by Vaccinated PBMC
DVC- FF-LVS
Vax 13.1 PBMC
FT-AH-43
7
control (1720x increase)
LVS 10% (38x increase)
LVS 2.5% (30x increase)
LVS 0.6% (134x increase)
6
total CFU (log) / well
Vax 4.2 PBMC
FT-AH-44
7
Vax 14.1 PBMC
FT-AH-45
7
control (647x increase)
LVS 10% (132x increase)
LVS 2.5% (221x increase)
LVS 0.6% (233x increase)
6
Vax 3.2 PBMC
FT-AH-46
CONTROL (731x fold increase)
LVS 10%v/v (41x fold increase)
LVS 2.5%v/v (4x fold increase)
LVS 0.6%v/v (11x fold increase)
6
7
6
5
5
5
5
4
4
4
4
3
3
3
3
2
0
24
48
HOURS POST-INFECTION
72
2
2
2
0
24
48
HOURS POST-INFECTION
72
CONTROL (795x-fold increase)
LVS 10%v/v (278-fold increase)
LVS 2.5%v/v (116-CFU fold increase)
LVS 0.6%v/v (392-fold increase)
0
24
48
HOURS POST-INFECTION
72
0
24
48
72
HOURS POST-INFECTION
Ag dose can be further reduced and still retain specific stimulatory response
28
Comparing pre- and post-Vaccination
Response
• So far, only tested samples that were either
vaccinated or unvaccinated
• Do not know if assay predictive of vaccination
status or individual variations
• Compare pre- and post vaccination response
in same individual
29
Inhibition Correlates with Vaccine Status
Donor #4
Prevacination (10/13/09)
1.010 7
Post-vaccination (1/12/10)
1.010 7
CONTROL (1034x increase)
UNM -FF-LVS-6 / 5%v/v (211x increase)
UNM -FF-LVS-5 / 5%v/v (437x increase)
UNM -FF-LVS-6 / 5% (657x increase)
UNM -FF-LVS-5 / 5% (45x increase)
1.010 6
*
1.010 6
total CFU / well
total CFU / well
control (647x increase)
1.010 5
1.010 4
1.010 4
1.010 3
1.010 3
1.010 2
1.010 2
0
24
48
72
HOURS POST-INFECTION
*
1.010 5
0
24
48
72
HOURS POST-INFECTION
*UNM-FF-LVS-6 samples at T72 show 60x CFU difference
between replicate wells, by far the highest variability
seen in any experiment
Statistical Significant Difference between
Control and Vaccinated Samples
CFU -fold increase with
48-hour FF-LVS pretreatment,
normalized to no-pretreatment control
1.25
2-tailed Mann-Whitney nonparametric
p=0.011
-fold increase, normalized to control
40 (UBS)
18.1 (UBS)
1.00
35 (prevax 8.1)
31 (prevax 6.1)
0.75
42 (UBS)
0.50
38 (prevax 11.1)
27.2 (UBS)
29 (prevac 4.1)
32 (UBS)
0.25
44 (postvac 4.2)
14 13.1
26.2
0.00
33
26.120
41(prevax 11.1)
36 (prevac 9.1)
27.1 (UBS)
34 (UBS)
37 (prevax 10.1)
30 (prevax 5.1)
vaccinated (N = 7) unvaccinated (N = 15)
Planned Experiments
• Continue to reduce antigen dose used to activate
vaccinated but not unvaccinated PBMC
• Compare pre- and post vaccination samples
• Develop functional assay in NHP and Fischer 344 rats
32
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:
None
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
33
)
Action Items
• Terry: will try the T cell removal/inactivation
experiment again
• Terry will try a different presentation of the
correlates of protection data for next call.
34