Download Annette Geluk - IMMUNIDIAGNOSTIC TOOLS FOR LEPROSY

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
Early Diagnosis
Transmission
M. leprae = killed
M. leprae survives
95%
5%
2-6 yrs >10 yrs
Early
Leprosy
diagnosis
Leprosy
Diagnostic test
Transmission
Early
treatment
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
T-cells/mφ/
neutrophils
marker?
CMI
Infected
contacts
HMI
α-PGL-I IgM
Infected
contacts
Longitudinal studies are lacking
TT
BT
BB
BL
LL
Paucibacillary (PB) - Multibacillary (MB)
α-LID-1 IgG
α-Ag85 IgG
α-LAM IgG
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)
Identification of M. leprae unique sequences
Analyse T cell reactivity in endemic regions
Surplus Value of T cell assay using
M. leprae unique proteins:
85%
Average IFN-  (pg/ml)
7%
100%
71%
2500
1500
500
200
100
0
NA
PGL-I - PGL-I +
students
EC
PGL-I - PGL-I +
PB
PGL-I -PGL-I +
Rx
PGL-I - PGL-I +
(H)HC
Surplus
1st T cell study IDEAL
CSU Fiocruz
LSHTM
LUMC
Selection M. leprae
proteins & peptides
Brazil
Nepal
Fiocruz/Goiás
TLMN
Bangladesh
ICDDR,B
IDRI
Pasteur
• Immunogenic in exposed
• non-responsive in EC (Rio)
Pakistan
Aga Khan
Ethiopia
AHRI
IFN- in LST
cumulative for 5 sites
ML 2283
4000
IFN-  (pg/ml)
3000
2000
1000
200
100
0
EC Mlep+
12/43
EC Mlep-
0/7
BL/LL
BT/TT
HHC
TB
9 /50
10/50
10/46
3/50
IFN- responses in EC
• Why IFN- responses in EC & TB to M. leprae unique antigens?
• Influence of leprosy endemicity of habitat?
IFN- & M.leprae unique protein in WBA
allows detection of M. leprae exposure
14000
14000
ML2478
12000
ML0840
14000
10000
8000
8000
6000
6000
6000
4000
4000
4000
500
400
10000
8000
IFN-  (pg/ml)
2000
2000
500
400
500
400
300
200
200
200
100
100
100
0
0
0
Bangladesh
EC
EC
TB
South-Korea
p=0.0232
BT/TT HHC
Bangladesh
EC
EC
TB
South-Korea
p=0.036
2000
300
BT/TT HHC
M. leprae
12000
10000
IFN-  (pg/ml)
IFN- (pg/ml)
12000
300
BT/TT HHC
Bangladesh
EC
EC
TB
South-Korea
Ethiopia/ ML2478
Brazil/ ML2478
5000
p=0.0001
4000
20000
p=0.0021
3000
15000
2000
10000
IFN- (pg/ml)
IFN-  (pg/ ml)
IFN- to M.leprae unique Ag
in EClow and EChigh in one city
1000
1000
800
600
400
5000
500
400
300
200
200
100
0
EC low
EC high
HHC
BT/TT
0
EC low
EC high
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?
Stages of M. leprae infection
Infection stage:
Biomarkers for:
1. Elimination of M.leprae without priming
of M. leprae-specific T cells
Innate immunity
to M. leprae: IGRAneg
EChigh
2. M. leprae infection eliminated in association
with T cell priming
Adaptive immunity Innate immunity
to M. leprae
HC
3. M. leprae replication maintained at a
subclinical level by immune response
Early/ Subclinical
Innate immunity
infection
less
HC
4. Pathogenic immune response to M. leprae
Leprosy disease
TT/BT
Co-infections
Diabetes
BL/LL
Innate immunity
T cell subsets/ Cytokines involved in leprosy
Activated
T cell:
Naive
T cell
IL-12/IFN-
M.leprae
IL-4
APC
IL-1/IL-6
TGF-,
IL-23
Th2
Th17
TGF-
Treg
IFN-
TNF
IP-10
Biomarkers
Th1
(Possible)
Role in :
• Protection
• TT/BT
• RR
IL-4
IL5
IL-13
α-PGL-I
• LL/BL
• ENL
IL17A
IL-17F
IL-22
IL21
• TT/BT
• RR
• ENL
IFN-
IL-10
• LL/BL
Biomarkers for M. leprae infection
endemic vs not endemic (anymore) areas
MIP-1
MIP-1/
M. leprae
IP-10/IP-10
M. leprae
ns
16000
p=0.0007
p=0.0039
ns
10000
ns
p=0.029
14000
12000
8000
10000
8000
ns
4000
4000
pg/ ml
pg/ ml
6000
ns
6000
4000
3000
2000
2000
1000
0
0
TT/BT
HHC
Bangladesh
EC
EC
TB
South-Korea
TT/BT
HHC
Bangladesh
EC
EC
TB
South-Korea
Biomarkers for M. leprae infection
endemic vs not endemic (anymore) areas
MCP-1/
M.leprae
MCP-1
IL-1

IL-1/
M.leprae
p=0.0006
14000
p=0.0021
ns
p=0.0001
p=0.001
4000
12000
10000
3000
ns
8000
pg/ ml
pg/ ml
p=0.066
6000
p=0.044
2000
4000
1000
2000
0
0
TT/BT
HHC
Bangladesh
EC
EC
TB
South-Korea
TT/BT
HHC
Bangladesh
EC
EC
TB
South-Korea
Biomarkers for M. leprae infection
(mycobacterial disease)
ROC curve
TT/BT vs EC
MCP-1, MIP-1, IL-1
sensitivity
150
100
50
0
0
20
40
60
80
100% - specificity%
Area
Std. Error
95% confidence interval
P value
0.9900
0.01616
0.9583 to 1.022
0.0002145
100
Biomarkers for M. leprae infection
(mycobacterial disease)
ROC curve
TT/BT vs EC
MCP-1, MIP-1, IL-1
sensitivity
150
100
50
0
0
20
40
60
80
100% - specificity%
Area
Std. Error
95% confidence interval
P value
0.9900
0.01616
0.9583 to 1.022
0.0002145
100
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)
Early Diagnosis 2
Transmission
M. leprae = killed
M. leprae survives
95%
5%
2-6 yrs >10 yrs
1. Early
Leprosy
diagnosis
2. Early diagnosis
leprosy reactions
M. leprae infection
Diagnostic test
Transmission
Early
treatment
Biomarkers for Leprosy Reactions
Biomarker for T1R
2007
2008-
2010-
1.
Nepal
Anandaban Hospital
2.
Bangladesh
ICDDR,B


3.
Brazil
UFU, Uberlandia


4.
Brazil
Fiocruz, Rio

5.
Ethiopia
AHRI

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
Biomarker Study Set-up: prospective cohort
t = 0:
• at recruitment
• before MDT
• no signs of T1R
> 3months
t = x:
• onset of T1R
t = end:
• end of therapy
1. CMI
2. Serum
3. RNA
1. CMI
2. Serum
3. RNA
1. CMI
2. Serum
3. RNA
T1R
MB
T1R
T1R
MB
EC
Cellular Biomarkers: Cytokine 4
Bangladesh
Nepal
4000
20000
3000
2000
1000
pg/ ml
pg/ ml
2000
1500
1000
200
500
500
150
400
100
300
50
200
100
0
0
RR
RR
RR
t=0
t=x
t=end
RR
t=0
RR
t=x
RR
t=end
Longitudinal trial Nilphamari
Biobanking :
1. WBA samples
2. RNA samples
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
UCP-LF Diagnostic Test
Strip 1
Strip 2
flow
flow
samples
value
LUMC, NL
Jolien vd Ploeg-Schip
Elisa Tjon Kon Fat
Kees Franken
Louis Wilson
Marielle Haks
Paul Corstjens
Tom Ottenhoff
LSHTM
Hazel Dockrel
UFU, Brazil
Luiz & Isabela Goulart
Janaina Lobato
AHRI
Kidist Bobosha
ICDDR,B
Sayera Banu
Senjuti Kabir
Fiocruz, Brazil
Euzenir Sarno
Roberta Olmo Pinheiro
Cristina Pessolani
Geraldo Pereira
Marcia Brandao
Milton Moraes
KIT: Linda Oskam
CSU: John Spencer
Yonsei: Ray Cho
Anandaban Hospital, Nepal
Saraswoti Khadge
Prathiba Thapa
Chhatra B. Kunwar
Murdo McDonald
Deanna Hagge
Q.M. GastmannWichers Foundation