Progress Report
1/13/2010
TVDC team – UNM
Prepared by Terry Wu
1
Active Milestones
10. Testing vaccine candidates:
Second expt with Aduro’s Lm-based vaccines in mice
LVS outer membrane proteins from Dr. Norgard (UTSWMC)
11. Cellular and humoral immunity in LVS vaccinated Fischer rats:
Passive immunization in T cell deficient nude rats
12/13. Immunoassays:
Results from microagglutination assay
21. Correlate of protection assay using human PBMC
Non-specific activation of unvaccinated PBMC
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
Vaccination with rLM/iglC Protected Mice
Against Aerosal SCHU S4 Challenge (UCLA)
i.d. immunization – 0 and 4 weeks 107 Lm strains/mouse, 104 LVS/mouse
Aerosol challenge – 10 weeks
4
Lm-based Vaccines Protected Mice Against i.v. LVS
challenge but not i.n. SCHU S4 challenge
(Aduro/UNM)
Immunization (0 wk) – 3 x 105 Lm strains/mouse i.v. & 4 x 103 LVS/mouse i.n.
Boost (6 wk) -- 2 x 106 Lm strains/mouse i.v. & 5 x 103 LVS/mouse i.n.
Challenge (10 wk) – 1.52 x 105 LVS i.v. and ~80 SCHU i.n.
n = 10 / group
i.v. LVS challenge
i.n. Schu4 Challenge
100
Percent survival
Percent survival
100
80
60
40
20
0
80
PBS
LVS
BH2172 (Lm677 KatG-SL8)
BH2182 (Lm677 IglC-B8R)
60
40
20
0
0
5
10
Days post challenge
15
0
5
10
15
Days post challenge
5
Lm-based Vaccines Failed to Protect Mice
against i.v or i.n. SCHU S4 challenge
(Aduro/UNM)
Immunization / boost (0 / 6 wk): Lm (i.v. 2 x 106/ms) & LVS (i.n. 5 x 103/ms)
Challenge (10 wk) – SCHU (~10/ ms i.v. or i.n.)
n = 10 / group
IV Challenge
IN Challenge
100
Percent survival
Percent survival
100
80
60
40
20
0
80
PBS
LVS
BH2172 (Lm677 KatG-SL8)
BH2182 (Lm677 IglC-B8R)
60
40
20
0
0
10
20
Time
30
0
10
20
30
Time
6
Milestone 10: Fischer 344 rats
Prime
Lm: 107 / rat i.v.
LVS: 108 / rat s.c.
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: 103 / rat i.t.
(Jan 11, 2010)
7
LVS Outer Membrane Proteins (Norgard)
8
Testing LVS OMP in F344 Rats
Schedule
Vaccination: 12/22/09
Boost: 1/12/10
Boost: 1/26/10
Challenge 3/2/10
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
Immune Serum Extended Survival of
Nude Rats Challenged with SCHU S4
Percent survival
100
75
NRS
IRS
50
25
0
0
5
10
15
Days Post-challenge
n = 5 for IRS and 6 for NRS
12
Extended Survival
in Nude but not SCID Mice
i.p. LVS challenge
No T cells, higher macrophages and NK
i.n. LVS challenge
No T cells and B cells
13
Milestone 11 Cellular Immunity: Plans
• Finish manuscript describing passive immunization of
F344 rats
• Develop method for adoptive transfer of immune T
cells into naïve rats
14
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. tualrensis
Compare human vaccinees and controls
Blue: Steps in the milestone
Red: Completed
Green: In progress
Statistical analysis
15
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
16
MS21: Background
Pre-stim
FF LVS
-48 h
Measure
Burden
Wash
Infect SCHU S4
-24 h
0h
24 h
48 h
72 h
• Pre-stimulation of PBMC from LVS-vaccinated human
with FF-LVS inhibited /reduced SCHU S4 growth @ 72
h post infection
17
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)
18
Hypothesis
• Non-specific activation of unvaccinated PBMC
induced by excess antigen during prestimulation
• Antigen optimization will eliminate nonspecific activation while retaining specific
stimulation for vaccinated PBMC
19
Two Types of Responses from
Unvaccinated PBMC Samples
FT-AH-35
prevax 8.1 PBMC
FF-LVS #6
FT-AH-34
UBS PBMC
1. FF-LVS #6 titration
1.010 8
1.010 8
control (no pretreatment) (3870x increase)
FF-LVS#6 5.0% (340x increase)
FF-LVS#6 0.5% (940x increase)
FF-LVS#6 0.05% (4510x increase)
1.010 7
1.010 7
1.010 6
1.010 6
total CFU / well
total CFU / well
control (no pretreatment) (1350x increase)
FF-LVS#6 5.0% (1210x increase)
FF-LVS#6 0.5% (1960x increase)
FF-LVS#6 0.05% (1770x increase)
1.010 5
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
0
24
48
72
HOURS POST-INFECTION
20
DVC FF-LVS induced Non-specific Activation
DVC FF-LVS
UNM FF-LVS #6
(FT-AH-41)
(FT-AH-34)
1.010 8
1.010 7
1.010 7
1.010 6
1.010 6
total CFU / well
1.010
total CFU / well
8
1.010 5
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
0
24
48
HOURS POST-INFECTION
HOURS POST-INFECTION
control (3870x increase)
5.0% (340x increase)
0.5% (940x increase)
0.05% (4510x increase)
control (21,400x increase)
10% v/v (832x increase)
2.5% v/v (1770x increase)
0.6% v/v (3030x increase)
72
21
No Correlation between IFNg
Production and non-specific Activation
40000
pg/mL
30000
48h stimulation with FF-LVS
(pre-infection)
26.1 (#1.2)
20 (#3.1)
33 (#7.1) prep#5
20000
26.2 (#2.2)
10000
27.2
27.130 37 32 38
29
34
36
21.1
21.2
31 35
0
vaccinated
unvaccinated
Notes
1. All data shown here used Ly-FF-LVS-6 (5%v/v), with the exception of experiment #33 (vaccinee #7.1),
which used Ly-FF-LVS-5 (5%v/v).
22
Planned Experiments
• Determine minimum antigen dose required to
activate vaccinated but not unvaccinated PBMC
• Develop functional assay in NHP and Fischer 344 rats
23
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
24
Microagglutination - Example
Microagglutination Test for Early and Specific Serodiagnosis of Tularemia
Sato et al. J Clin Microbiol (1990) 28:2372
25
Microagglutination: Evaluation Criteria
from Sztein Lab
26
Microagglutination - UNM
• LVS Lot 16 cultured in Chamberlain’s for 48 h
– Reconstituted Lot 9 clumpy and data unclear
– Stained w/ Hematoxylin– faint blue
• Rb positive control serum from Dr. Sztein (1:2,560)
• Normal rabbit serum from Lyons lab
• Results:
– Buttons faint greyish blue, not easily visualized
– No lattice observed
– Pos ctrl 1:1,280, neg < 1:20 (based on disappearance of
button)
27
Microagglutination - UNM
•
•
•
•
•
•
Repeat with higher antigen
Increase visibility with safranin instead of hematoxylin
Documentation, reader?
Prepare a new positive control serum
Develop assays with sera from human, rat, mouse
Prepare SOP
28
Additional Points
Deliverables completed for each active milestone:
MS10: Tested Aduro’s Lm vaccines in mice
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
29
)
Action Items
• Barbara: will confirm Patrick’s availability for
the 1/20 Prime tech call at 2-3pm Eastern.
(Patrick confirmed on 1/13/10)
• Terry: will bridge LVS lot 9 and LVS 16 as
antigens in the microagglutination assay.
30