immunodiagnostic tools for leprosy: the netherlands ... · ag discovery (2001 – 2010) ... 12/43...
TRANSCRIPT
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