Annemieke Geluk

Dept. Infect. Dis.

LUMC

The Netherlands

Immunodiagnostic Tools for Leprosy:

Exposure, Infection & Disease

Brussels, 19th September 2013

Plenary Session

(Facing the) Challenges in Leprosy

prevalence

still millions patients suffer from leprosy (> MS)

NCD: quite static number globally (2012) 232,857 21,349 children

Transmission is ongoing

5%

2-6 yrs >10 yrs

M. leprae survives

Early Diagnosis

95%

M. leprae = killed

Transmission

Transmission Early treatment

Diagnostic test

Leprosy Early diagnosis

Leprosy

Diagnosis 2013

Based on clinical signs: # lesions, peripheral neuropathy

detection of M. leprae

=> No early detection

Based on immune responses:

1. Anti-PGL-I Ab: HMI: mostly detects MB not PB

Positivity does not indicate leprosy

2. Lepromin skin test: CMI: not leprosy-specific

Tests covering leprosy spectrum:

combined HMI & CMI

TT BT BB BL LL

Paucibacillary (PB) - Multibacillary (MB)

CMI HMI α-PGL-I IgM

Infected

contacts

Infected

contacts α-LID-1 IgG

α-Ag85 IgG

α-LAM IgG

T-cells/mφ/

neutrophils

marker?

Longitudinal studies are lacking

M. leprae-specific CMI Pre-genomic era

T-ESAT & T-CFP10: TB diagnostics : Quantiferon

L-ESAT 63% similarity to T-ESAT6

L-CFP10 40% similarity to T-CFP10

• Extensive recognition of L-ESAT-6 and L-CFP-10 by TB

• No application as diagnostic tools in TB endemic countries

Ag discovery (2001 – 2010)

Analyse T cell reactivity in endemic regions

Identification of M. leprae unique sequences

Surplus Value of T cell assay using

M. leprae unique proteins:

PGL-I -PGL-I

+PGL-I

-PGL-I

+PGL-I -PGL-I + PGL-I - PGL-I +

0

100

200

500

1500

2500

EC PB Rx (H)HC

NA

71%7% 85% 100%

Aver

age

IFN

- (

pg/m

l)

Surplus

students

LUMC

Brazil Fiocruz/Goiás

Nepal TLMN

Bangladesh

ICDDR,B

Pakistan Aga Khan

Ethiopia

AHRI

CSU Fiocruz

LSHTM

Pasteur

Selection M. leprae

proteins & peptides

IDRI

1st T cell study IDEAL

• Immunogenic in exposed

• non-responsive in EC (Rio)

IFN- in LST cumulative for 5 sites

ML 2283

EC Mlep+ EC Mlep- BL/LL BT/TT HHC TB 0

100

200

1000

2000

3000

4000

12/43 0/7 9 /50 10/50 10/46 3/50

IFN

- (

pg/m

l)

IFN- responses in EC

• Why IFN- responses in EC & TB to M. leprae unique antigens?

• Influence of leprosy endemicity of habitat?

BT/TT HHC EC EC TB

0

100

200

300

400

500

2000

4000

6000

8000

10000

12000

14000ML2478

Bangladesh South-Korea

IFN

- (

pg/m

l)

IFN- & M.leprae unique protein in WBA allows detection of M. leprae exposure

ML0840

BT/TT HHC EC EC TB

0

100

200

300

400

500

2000

4000

6000

8000

10000

12000

14000

Bangladesh South-Korea

p=0.0232

IFN

- (

pg/m

l)

BT/TT HHC EC EC TB

0

100

200

300

400

500

2000

4000

6000

8000

10000

12000

14000

Bangladesh South-Korea

M. leprae

p=0.036

IFN

- (

pg/m

l)

IFN- to M.leprae unique Ag in EClow and EChigh in one city

Ethiopia/ ML2478

EC low EC high

0

100

200

300

400

500

5000

10000

15000

20000

p=0.0001

IFN

- (

pg

/ml)

Brazil/ ML2478

EC low EC high HHC BT/TT0

200

400

600

800

10001000

2000

3000

4000

5000

p=0.0021

IFN

- (

pg/

ml)

Biomarkers for M. leprae Exposure

M.leprae unique proteins can be used as tools to identify

M.leprae exposed individuals using IFN- as a read out

What about tools for M. leprae infection?

Leprosy disease

1. Elimination of M.leprae without priming

of M. leprae-specific T cells

4. Pathogenic immune response to M. leprae

3. M. leprae replication maintained at a

subclinical level by immune response

Biomarkers for:

TT/BT BL/LL

Infection stage:

Early/ Subclinical

infection Innate immunity

Innate immunity

2. M. leprae infection eliminated in association

with T cell priming Adaptive immunity

to M. leprae Innate immunity

Innate immunity

to M. leprae: IGRAneg

EChigh

HC

less

HC

Stages of M. leprae infection

Co-infections

Adapted from Young et al.

Trends Microbiol 2009

Diabetes

Activated

T cell:

Th1

Th2

Naive

T cell

IL-12/IFN-

IL-4

Treg

Th17

TGF-

IFN-

TNF

IP-10

IL-4

IL5

IL-13

α-PGL-I

IL17A

IL-17F

IL-22

IL21

IFN-

IL-10

APC

IL-1/IL-6

TGF-,

IL-23

(Possible)

Role in :

• Protection

• TT/BT

• RR

• LL/BL

• ENL

• TT/BT

• RR

• ENL

• LL/BL

M.leprae

T cell subsets/ Cytokines involved in leprosy

Bio

mark

ers

MIP-1/ M. leprae

TT/BT HHC EC EC TB

0

2000

4000

6000

8000

10000

p=0.0007

Bangladesh South-Korea

p=0.029

ns

ns

pg

/ m

l

IP-10/ M. leprae

TT/BT HHC EC EC TB

0

1000

2000

3000

4000

4000

6000

8000

10000

12000

14000

16000

Bangladesh South-Korea

p=0.0039

ns

ns

ns

pg

/ m

l

Biomarkers for M. leprae infection endemic vs not endemic (anymore) areas

MIP-1 IP-10

Biomarkers for M. leprae infection endemic vs not endemic (anymore) areas

p=0.0021

IL-1/ M.leprae

TT/BT HHC EC EC TB

0

1000

2000

3000

4000

Bangladesh South-Korea

p=0.044

ns

ns

pg

/ m

l

MCP-1/ M.leprae

TT/BT HHC EC EC TB

0

2000

4000

6000

8000

10000

12000

14000

p=0.0001

p=0.0006

p=0.001

p=0.066

Bangladesh South-Korea

pg

/ m

l

MCP-1 IL-1

Biomarkers for M. leprae infection (mycobacterial disease)

ROC curve

TT/BT vs EC

MCP-1, MIP-1, IL-1

0 20 40 60 80 1000

50

100

150

Area

Std. Error

95% confidence interval

P value

0.9900

0.01616

0.9583 to 1.022

0.0002145

100% - specificity%

sen

siti

vit

y

Biomarkers for M. leprae infection (mycobacterial disease)

ROC curve

TT/BT vs EC

MCP-1, MIP-1, IL-1

0 20 40 60 80 1000

50

100

150

Area

Std. Error

95% confidence interval

P value

0.9900

0.01616

0.9583 to 1.022

0.0002145

100% - specificity%

sen

siti

vit

y

Summary I

• M.leprae unique Ag can identify M. leprae exposed individuals

using IFN-; importance of proper reference group as EC (same

socio-economic background, same part of town)

• Combinations of additional cytokines & chemokines can

discriminate between between M. leprae infected vs. uninfected

(but exposed) healthy individuals in highly endemic areas

• Longitudinal follow-up studies in HC from leprosy-endemic areas

will be essential for evaluation (intra-individual comparison)

5%

2-6 yrs >10 yrs

M. leprae survives

Early Diagnosis 2

95%

M. leprae = killed

Transmission

Transmission Early treatment

Diagnostic test

2. Early diagnosis

leprosy reactions

Leprosy 1. Early diagnosis

M. leprae infection

Biomarkers for Leprosy Reactions

Biomarker for T1R

2007-

1. Nepal

Anandaban Hospital

2. Bangladesh

ICDDR,B

3. Brazil

UFU, Uberlandia

4. Brazil

Fiocruz, Rio

5. Ethiopia

AHRI

2008- 2010-

Biomarkers for T1R

Overall objective:

• Identify novel biomarker signatures for early diagnosis of leprosy

reactions

Working hypothesis:

• The development of leprosy reactions coincides with changes in

the: - immunological profile and/ or

- gene expression profile

t = end:

• end of therapy

Biomarker Study Set-up: prospective cohort

T1R

MB

T1R

T1R

MB EC

t = 0:

• at recruitment

• before MDT

• no signs of T1R

t = x:

• onset of T1R

> 3months

1. CMI

2. Serum

3. RNA

1. CMI

2. Serum

3. RNA

1. CMI

2. Serum

3. RNA

Cellular Biomarkers: Cytokine 4

Nepal Bangladesh

RR RR RR

0

100

200

300

400

500

500

1000

1500

2000

20000

t=0 t=x t=end

pg

/ m

l

RR RR RR

0

50

100

150

200

1000

2000

3000

4000

t=0 t=x t=end

pg

/ m

l

Biobanking :

1. WBA samples 2. RNA samples

Longitudinal trial Nilphamari

24h

Test development: UCP-LF

• A field-friendly, LF is developed for cellular (cytokines)

and humoral (PGL-I) IR to M. leprae.

• Th1 (IFN-) & Th2 cytokines (IL-10)

• More cytokines under development

• Evaluated in Africa (AE-TBC/ EDCTP)

IFN IL10 PGL-I

samples

flow

Strip 1 Strip 2

flow

UCP-LF Diagnostic Test

value

LUMC, NL Fiocruz, Brazil

Jolien vd Ploeg-Schip Euzenir Sarno

Elisa Tjon Kon Fat Roberta Olmo Pinheiro

Kees Franken Cristina Pessolani

Louis Wilson Geraldo Pereira

Marielle Haks Marcia Brandao

Paul Corstjens Milton Moraes

Tom Ottenhoff

KIT: Linda Oskam

CSU: John Spencer

LSHTM Yonsei: Ray Cho

Hazel Dockrel

UFU, Brazil

Luiz & Isabela Goulart

Janaina Lobato

AHRI

Kidist Bobosha

ICDDR,B

Sayera Banu

Senjuti Kabir

Q.M. Gastmann-

Wichers Foundation

Anandaban Hospital, Nepal

Saraswoti Khadge

Prathiba Thapa

Chhatra B. Kunwar

Murdo McDonald

Deanna Hagge