Steffen Stenger

Medical Microbiology and Hygiene

University Hospital Ulm

MyTB Lab

Principles and Approaches of

Discovering New Vaccine Antigens

Mycobacterial Lipids as an Example

14d

3d

Mechanisms of local antimicrobial activity

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

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

Advantages of the CD1-system for vaccination

CD1 is non-polymorphic

CD4-T-cells not requiredT-cell

dendritic cells as APC

(efficient priming)

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

Diacylated sulfoglycolipid:

a new candidate for a tuberculosis vaccine

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

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

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

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

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

**

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

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

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

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)

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

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

Walch et al., Cell, 157: 1309, 2014

Nami-Mancinelli & Vivier, Cell, 157: 1251, 2014

Delivering three punches to knock out intracellular bacteria

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)

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 +

Protected donor Susceptible donor

granulysin

granzyme B

perforin

+

+

+

+

-

-

+

+

-

+

-

+

The frequency of LAM-specific polycytotoxic T cells

correlates with protection against tuberculosis

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

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

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

J Exp Med, 206: 2497, 2009

J Immunol, 169: 330, 2002

Int Immunol, 15: 915, 2003

Infect Immun, 81, 311, 2013

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

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

Shuttling of LAM via liposomes improves T-cell responses

Hypothesis

LIPLAM contains lipoarabinomannan

40

35

25

55

70

LAM LIPLAM LIP

LAM Westernblot

Kallert et al., submitted for publication

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

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

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

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

Tanja Weil Yvonne Perrie Goutam PramanikSeah Ling Kuan