principles and approaches of discovering new vaccine antigens ... · liplam contains...
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Steffen Stenger
Medical Microbiology and Hygiene
University Hospital Ulm
MyTB Lab
Principles and Approaches of
Discovering New Vaccine Antigens
Mycobacterial Lipids as an Example
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14d
3d
Mechanisms of local antimicrobial activity
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protected susceptible
4
8
12
16
20
(n=7)
study site A (Ulm) study site B (Borstel)
BAL cells from protected individuals limit Mtb-growthx-f
old
mu
ltip
licati
on
p<0.01
(n=37)
protected susceptible
(n=22)
x-f
old
mu
ltip
licati
on p<0.01
(n=28)
4
8
12
16
20
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Antimicrobial activity is partially CD1 (=lipid)- mediated
x-f
old
mu
ltip
licati
on
4
8
12
16
20
MHC I MHC II CD1a
**
*
Antibody
IgG1
*
CD1b
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Advantages of the CD1-system for vaccination
CD1 is non-polymorphic
CD4-T-cells not requiredT-cell
dendritic cells as APC
(efficient priming)
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CD4-, CD8- TCR ab/gd autoreactive Porcelli, Nature 1989CD1b,c
CD4-, CD8- TCR ab mycobacterial lipid Porcelli, Nature 1992CD1b
CD4-, CD8- TCR ab lipoarabinomannan Sieling, Science 1995
CD4-, CD8- TCR ab mycolic acid Beckman, Nature 1994CD1b
CD1b
CD8+ TCR ab mycobacterial lipid Stenger, Science 1997CD1b
TCR ab sphingolipids (autoreactive)n.d. Shamshiev, Immunity 2000CD1
Antigens presented by group I CD1-molecules
TCR ab isoprenoid phosphoglycolipidsCD4-CD8- Moody, Nature 2000CD1c
TCR ab mycobactin (lipopeptides)transfectants Moody, Science 2004CD1a
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Diacylated sulfoglycolipid:
a new candidate for a tuberculosis vaccine
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CD3 CD4 CD8
granulysin
2
6
10
12 CFU ( x 105)
4
8
0 72
T-Zellen : DC
0
1
3
10
Ac2SGL induces CD8+ cytolytic effector T cells
Induction of CTL
CD1-restricted
granulysin-positive
IFN-g-production
antibacterial activity
Gilleron et. al.,J. Exp. Med., 199, 2004
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WP 1.1
Subdominant epitope discovery
3. Structure and Partners
WP 1.2
Stage specific antigen discovery
WP 1.3
Lipid antigen discovery
Jes Dietrich, Joshua Woodworth,
Peter Andersen
Kopenhagen
Stefan Stevanovic, Christina
Christ, Hans-Georg RammenseeTübingen
Steffen Stenger, Martin Busch Ulm
Tom Ottenhoff, Susana Commandeur,
Annemieke Geluk, Krista van Meijgarden
Leiden
Jes Dietrich, Joshua Woodworth Kopenhagen
Yannick Van Loubbeeck Rixenart
Germain Puzo, Martine Gilleron Toulouse
Kris Huygen, Marta Romano Brussels
Leiden
Steffen Stenger, Martin Busch Ulm
Tom Ottenhoff, Susana Commandeur,
Annemieke Geluk, Krista van Meijgarden
WP1: Antigen discovery
Gennaro DeLibero, Paula Cullen-Baumann Basel
Roberto Nisini, Sabrina Mariotti Rom
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4.2.1 – Comparison of frequency and function of T lymphocytes responding
to mycobacterial lipids in BCG vaccinees, latently infected
individuals and tuberculosis patients.
4.2.2 – Head to head comparison of the frequency of lipid- and protein
responsive T-cells in the different cohorts using standardized
assays.
4.2.3 – Evaluation of purified lipid antigens for immunogenicity in Mtb
primed individuals
Deliverables M25-42
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Response of „total lipid“-reactive donors to defined lipidsre
acti
ve
do
no
rs(%
)
20
40
60
80
100
LAM GroMM PIM Ac2SGL Mycolic
Acid
Total
Lipid
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LAM Total
Lipid
0.6
1.2
1.8
2.4
Frequency and restriction of LAM-specific T-lymphocytesfr
eq
uen
cy
of
IFN
-g-p
osit
ive c
ells
(%) p<0.001
3.0
red
ucti
on
of
IFN
-g-p
osit
ive c
ells
(%)
20
40
60
80
MHC I MHC IIIgG1
0
100
CD1a CD1c
0
CD1b
**
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LAM
GM-CSF/IL4
CD1+ APC
adherent cells
Antimicrobial activity of LAM-specific PBMC: Method
PBMC
non-adherent cells
IFN-g-PE
unsorted IFN-g negative IFN-g positive
cell sorting
IFN-g-capture
d7
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LAM-specific T cells mediate antimicrobial activity
CF
U (
10
6)
2
4
6
8
10
0 hrs 48 hrs
*
Time after Infection
no T-cells
non adherent PBMC
LAM-stimulated, IFN-g-neg.
LAM-stimulated, IFN-g-pos.
+
48hrs
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total Latent TB
(protected)
Cured Patient
(susceptible)
blood (DNA, RNA)
BAL (mRNA, SN)
1558
225
458
71
306
80
- 1 center for data management (Popgen, Kiel)
- 3 research centers (Borstel, Berlin, Ulm)
- 3 health care centers (Frankfurt, Hannover, Hamburg)
- 13 hospitals specialized on lung diseases
A German Tuberculosis Cohort
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susceptible protected
10
20
30
40
50
(n=61)
Correlation of LAM-reactivity and outcome of infectionL
AM
-reacti
ve
do
no
rs(%
)
(n=28)
0.4
0.8
1.2
1.6
freq
uen
cy
of
IFN
-g-p
osit
ive c
ells
(%)
ns2.0
susceptible potected
(n=61) (n=28)
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CD3 CD4 CD8
granulysin
2
6
10
12 CFU ( x 105)
4
8
0 72
T-Zellen : DC
0
1
3
10
Ac2SGL induces CD8+ cytolytic effector T cells
Induction of CTL
CD1-restricted
granulysin-positive
IFN-g-production
antibacterial activity
Gilleron et. al.,J. Exp. Med., 199, 2004
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15kDa
9kDa
tuberculoid lepromatous
perforin
granulysin biopsies from leprosy patients
Granulysin: Introduction
Stenger et. al., Science, 1997Stenger et. al., Science, 1998
Stenger et al., Science, 2001
Stenger et al., J. Immunol, 2000Ochoa et al. , Nat. Med., 2001
Thoma et al., Science, 2003Stegelmann et al., J. Immunol, 2008
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Walch et al., Cell, 157: 1309, 2014
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Nami-Mancinelli & Vivier, Cell, 157: 1251, 2014
Delivering three punches to knock out intracellular bacteria
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Detection of LAM-specific polycytotoxic T cells
Antibody Fluorochrome source
CD3 PerCP BD
CD4 Pacific Blue Biolegend
CD8 APC-Cy7 Biolegend
IFN-g PE Miltenyi
perforin FITC BD
granzyme B APC Invitrogen
granulysin PE-Cy7 polyclonal
rabbit serum
(A. Krensky)
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0.7%
SS
C
IFN-g
A.
IFN-g +/granulysin+
pe
rfo
rin
granzyme B
67%7%
14%12%
Protected
Susceptible
SS
C
IFN-g
0.7%
granzyme B
perf
ori
n
23%4%
12%61%
granulysin
Frequency of polycytotoxic T cells in tuberculosis
IFN-g +/granulysin+
granulysin
SS
C
granulysin
SS
CS
SC
IFN-g +
87%
60%
IFN-g +
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Protected donor Susceptible donor
granulysin
granzyme B
perforin
+
+
+
+
-
-
+
+
-
+
-
+
The frequency of LAM-specific polycytotoxic T cells
correlates with protection against tuberculosis
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Miller and Ernst, J Clin Invest, 119: 1079, 2009
Anti TNF treatment reduces CD8+ T cell-mediated
antimicrobial activity against Mtb in humans
Bruns et al., J Clin Invest, 119: 1167, 2009
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SS
C
IFN-g
0.9%
SS
C
granulysin
86%
granzyme B
pe
rfo
rin
44%
23%
1%
30%
CD4
CD
8
<1%
<1%
94%
6%
LAM-specific polycytotoxic T cells express CD8
Polycytotoxic T cells
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Conclusions
- the frequency of CD8+ LAM-specific polycytotoxic T-cells
is higher in protected than in susceptible individuals
Lipoarabinomannan-specific polycytotoxic T-cells
may contribute to protection in human tuberculosis and
are a promising target population for new vaccines
- LAM-specific T cells contribute to killing of
Mycobacterium tuberculosis by human PBMC
- Lipid-specific T-cells contribute to antimicrobial
activity of bronchoalveolar lavage cells
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J Exp Med, 206: 2497, 2009
J Immunol, 169: 330, 2002
Int Immunol, 15: 915, 2003
Infect Immun, 81, 311, 2013
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Protective Efficacy of Ac2SGL / PIM
Survival time post-challenge
0
20
40
60
80
100
0 50 100 150 200 250 300 350
Time post-challenge (days)
% s
urv
ival
Group 1
Ac2SGL in
DDA/TDB with PIM
Group 2
H56 + Ac2SGL in
DDA/TDB with PIM
Group 3
3 x H56 in IC31
(high dose)
Group 4
3 x H56 in
DDA/TDB (high
dose)Group 5
BCG Danish
(1331) control
Group 6
Saline
Ann Rawkins; Martine Gilleron, Germain Puzo
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Activation
Cytotoxic T-cell
Activation of effective adaptive immune response
Tuberculosis
CD1
TLR
Liu P. et al: Science 2006
Gilleron M. et al.: J Exp Med 2004 Bastian M. et al.: J Immunol 2008Bruns H. et al.: J Clin Invest 2009
Release of Perforin ,Granulysin and Granzyme B
Hydrophobic antigens
Activation of innate immune response
Optimize the presentation of
mycobacterial lipids to effector T-cells
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Shuttling of LAM via liposomes improves T-cell responses
Hypothesis
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LIPLAM contains lipoarabinomannan
40
35
25
55
70
LAM LIPLAM LIP
LAM Westernblot
Kallert et al., submitted for publication
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Liposomes promote LAM-specific T cell responses
IFN
-g(p
g/m
l)200
400
600
800
1000
LIPLAM LAM LIP
representative result, n=5
<32
IFN-g
SS
C
0.1%
IFN-g
SS
C
0.9%
LIPLAM
LAM
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Objective II
Determine the quantity and quality of lipid-specific immune responses
in humans, mice and guinea pigs immunized with live- or lipid-based
vaccines.
Humans
François Spertini, CHUV
WP 4
CD1b transgenic mice
Gennaro DeLibero, USB
WP1
Guinea pigs
Max Bastian, PEI
WP2
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WP1 – DiscoveryLead Dr Olivier Neyrolles, CNRS, Toulouse, France
• WP1.1 – Antigen discovery
Lead. Dr Steffen Stenger, University of Ulm, Germany
• WP1.2 – Novel delivery systems & immunization strategies
Lead. Dr Else-Marie Agger, Statens Serum Institut, Copenhagen, Denmark
• WP1.3 – Novel live vaccinesLead. Dr Olivier Neyrolles, CNRS, Toulouse, France
Expected impact: Increase the number of TB vaccine candidates, which can be tested with the same resources thus increasing the chance of discovery of an effective vaccine
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WP1.1 – Antigen DiscoverySpecific Objectives
1. Unbiased discovery of novel epitopes in infected MØ
2. Discover and evaluate novel stage-specific antigens
3. Discover and improve immunogenicity of lipid antigens
4. Evaluate and exploit antibody-mediated protection
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Tanja Weil Yvonne Perrie Goutam PramanikSeah Ling Kuan