Lepr Rev (2011) 82, 402 421

Postgenomic Mycobacterium leprae antigens for cellular and serological diagnosis of M. leprae exposure, infection and leprosy disease
ANNEMIEKE GELUK*, MALCOLM S. DUTHIE** & JOHN S. SPENCER*** *Department of Infectious Diseases, Leiden University Medical Center, The Netherlands **Infectious Disease Research Institute, Seattle, WA, USA ***Department of Microbiology, Immunology & Pathology, Colorado State University, Ft. Collins, CO, USA Accepted for publication 09 September 2011
Due to changes in leprosy control programs and the special expertise required for diagnosis, the need for simple rapid diagnostic tests that could be applied in non-expert settings may now be greater than ever before. Since the sequencing of the M. leprae genome, many research groups have investigated the potential of M. leprae antigens in either serologic or cell mediated assays. Here we provide an overview of the nearly 200 recombinant single proteins that were investigated during the last decade for their potential to be applied in eld-friendly tests for the early diagnosis of leprosy.

Introduction Leprosy and its associated disabilities are unlikely to disappear soon.1,2 Despite a spectacular decrease in global prevalence since 1982, transmission of leprosy is sustained as evidenced by the hundreds of thousands of new cases of leprosy that are still detected globally every year: 244,796 new cases of leprosy were detected during 2009 amongst whom 22 485 were children, and the registered prevalence at the beginning of 2010 was 211,903 cases.3 In addition, a study conducted in India reported that inadequate monitoring of a policy of new case validation, in which treatment was not initiated until primary diagnosis had been veried by a leprosy expert, may have led to approximately 26% of suspect cases awaiting conrmation of diagnosis 1 8 months after their initial primary health care visit.4 These gures demonstrate that M. leprae infected contacts and people with preclinical, undiagnosed leprosy, likely the major sources of unidentied transmission, remain an incessant source of active transmission. These factors make the early detection of leprosy and M. leprae infection, followed by prompt multidrug treatment (MDT), of utmost importance
Correspondence to: Annemieke Geluk, Dept. of Infectious Diseases, LUMC, PO Box 9600, 2300 RC Leiden, The Netherlands (Tel: 31 71 5261974; Fax 31 71 5266758; e-mail: a.geluk@lumc.nl)

402

0305-7518/11/064053+20 $1.00

q Lepra

M. leprae antigens for leprosy diagnostics

403

for control of the disabling effects of leprosy. Notwithstanding this urgency, there are no tests available that can detect asymptomatic M. leprae infection or predict progression of infection to clinical disease.
DIAGNOSTIC ASSAYS FOR LEPROSY

Over recent years, several lateral ow-, dipstick-, and particle agglutination tests that incorporate the synthetic di- or trisaccharide epitope of phenolic glycolipid-I (PGL-I) have been in use in eld-based studies.5,6 While the existence of high titer IgM antibodies to PGL-I has allowed the development of user-friendly kit-based tests, these are applicable largely to multibacillary (MB)7 leprosy cases and have minor relevance to those with PB or asymptomatic leprosy showing vigorous cellular rather than humoral immune responses.6 Positive antibody responses to PGL-I range from 20-40% in paucibacillary (PB) patients. In part, the anti-PGL-I tests are useful as prognostic indicators in contacts of multibacillary (MB) patients, since those who have antibody to PGL-I have an estimated 8-fold or greater risk of developing clinical symptoms compared to those without an anti-PGL-I antibody response.7 10 Nevertheless, almost half of those individuals who have antibodies to PGL-I never develop leprosy, and many of those who develop leprosy are lacking anti-PGL-I antibodies. Thus, in order to complement current clinical methods, especially for PB patients, and to allow informed decision making on who needs treatment at a preclinical stage, improved diagnostic tools are required. These tools would specically detect M. leprae infection and/or measure biomarkers that predict disease development in infected individuals before clinical manifestations arise. Such tools for early diagnosis of M. leprae infection would facilitate informed decision making on who needs treatment, especially in individuals that have an increased risk to develop leprosy (e.g. contacts of leprosy patients). Consequently, such diagnostic tests could contribute to the prevention of leprosy disability and further transmission in otherwise undiagnosed and untreated index cases.
POST-GENOMIC DIAGNOSTIC APPROACH

With the completion of the M. leprae and M. tuberculosis genome sequences,11,12 attempts have been made to identify T cell-based biomarkers for detecting M. leprae infection using post-genomic approaches. Through comparative analysis of annotated mycobacterial genomes, several investigators selected putative open reading frames that were only found in the M. leprae genome and lacked homologues in any of the (myco)bacterial databases available at that time.13 23 Further bio-informatic analyses of these M. leprae-unique sequences identied several (hypothetical) antigens that were tested for their ability to induce in vitro T cell responses in M. leprae infected individuals. In addition many studies have examined serological responses to a variety of M. leprae recombinant proteins.24 28 Information on M. leprae antigens that have been tested in view of the design of new diagnostic tests, based on both cellular- or serological immune responses, is described in Table 1.
T CELL BASED ASSAYS

Previous cellular tests have relied on the use of complex and usually incompletely dened mixtures of M. leprae components.29 31 In addition, antigens were identied by screening

Table 1. Overview of M. leprae recombinant proteins tested in vitro for human immune responses

404

Gene acession # Homolog in Mtb Antiserum3 LST, WBA, array WBA array, ELISA, WBA blot LST, WBA ELISA, LST Phil Brazil Phil Brazil Nepal LST Brazil 19 Assay4 Field sites5 References6 Rv0007 NH NH Rv0020 NH NH Rv3875 Rv3874 Rv3873 NH Rv3835 Rv3810 Rv3806 Rv3804c Rv3803 CSU mouse polyclonal mouse polyclonal mouse polyclonal mouse polyclonal IDRI/LUMC IDRI/LUMC IDRI CSU/LUMC LUMC

Function1

Production2 site

ML0007

A. Geluk et al.

ML0008

putative conserved membrane hypothetical unknown CSU/IDRI/ LUMC LUMC IDRI CSU LUMC mAb and polyclonal mouse polyclonal Brazil Phil Nepal Ethiopia Bdesh SKorea Phil Nepal Brazil Phil Ethiopia

18, 23, in prep subm 18, 22 in prep* in prep 54, 53 18, 55, 60, 52, 28 18, 22 in prep 18 24, 18, 27 18 27 18, 22, in prep this issue

ML0009 ML0022 ML0023 ML0025

hypothetical unknown conserved hypothetical hypothetical unknown hypothetical unknown

ML0049

ESAT-6

ML0050

CFP-10

ML0051 ML0070 ML0081 ML0091

CSU/IDRI/ LUMC IDRI LUMC IDRI IDRI

mAb, rabbit polyclonal mAb, rabbit polyclonal ELISA, blot, array, LST array, ELISA, WBA LST, WBA array array, ELISA, WBA, blot array ELISA, LST, WBA

ML0095

Phil Nepal Brazil Ethiopia Phil Nepal Bdesh Phil Nepal Brazil Phil Nepal

ML0097

PPE-family hypothetical unknown conserved membrane exported repetitive (surface exposed) possible conserved integral membrane Ag85A

ML0098

Ag85C

ML0121

array, ELISA, LST, WBA WBA LST, array WBA

ML0126

conserved hypothetical unknown hypothetical unknown CSU/IDRI/ LUMC CSU Rv0983 Rv1000 CSU IDRI IDRI IDRI

18, 19, 23 this issue 18 18, 22 Ethiopia Phil Nepal this issue 18

ML0141

hypothetical unknown

ML0142 ML0176

probable membrane probable serine protease

Phil Brazil Ethiopia Nepal Phil Nepal Brazil Ethiopia Bdesh SKorea Ethiopia Brazil Ethiopia Phil Nepal Bdesh SKorea Ethiopia Phil Nepal Phil Nepal Brazil array array, ELISA, WBA WBA array

ML0188 ML0190

hypothetical unknown conserved hypothetical unknown

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal rabbit polyclonal rabbit polyclonal mAb CS-01 mouse polyclonal part of LID-I mouse polyclonal mouse polyclonal array array ELISA Phil Phil Nepal Phil Nepal Brazil Phil Nepal Phil Phil Nepal Brazil array, WBA array, WBA blot Phil in prep* in prep* 13, 18 17, 18 27 18 18 19 18 28 18, 22 13, 18, 23 array 18 17 26, 18, 60, 27, 22 13, 18 array, blot, LST blot blot 15, 18 Phil Phil in prep* in prep* 17, 18, 22 Assay4 Field sites5 References6 Rv0390 NH Rv0363c NH Rv0067 Rv0440 Rv3443 Rv3442 NH NH Rv3418c Rv3400 NH Rv0046 NH Rv3616 NH Rv2108 NH NH CSU CSU IDRI, LUMC IDRI/IPP IDRI IDRI IDRI CSU/IDRI/IPP LUMC IDRI CSU IDRI IDRI IDRI CSU IDRI IDRI, IPP CSU CSU IDRI Production2 site

Gene acession #

Function1

ML0276

ML0283

WBA, array, ELISA blot

Ethiopia Phil Nepal Brazil Phil

ML0286

ML0308

ML0316

Phil Nepal Mali Bdesh Phil Nepal Brazil

ML0317

ML0364

ML0365

ML0369 ML0376 ML0380 ML0393

conserved hypothetical unknown possible cation-efux transporter probable fructose bisphosphate aldolase conserved hypothetical unknown possible transcriptional regulator 65 kDa chaperonin 2 GroEL2 probable 50S ribosomal protein probable 30S ribosomal protein hypothetical unknown possible membrane 10 kD chaperonin GroES probable hydrolase LST array blot array, ELISA, WBA array, WBA

ML0394

hypothetical unknown

ML0396

Phil Nepal Mali Bdesh Phil Nepal

ML0398 ML0405

probable myo-inositol1-phosphate synthase possible D-ribose-binding conserved hypothetical

ML0410

PE-family

WBA array, ELISA, blot, MAPIA, WBA array, WBA

M. leprae antigens for leprosy diagnostics

ML0411

Brazil Brazil Phil Nepal Japan Venezuela India China Phil Nepal Mali Bdesh Phil Nepal Pakistan

ML0448 ML0458

45 kDa serine rich Ag, PPE-family hypothetical unknown possible oxidoreductase

405

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal array array, WBA Phil Nepal Brazil Phil Nepal Phil Nepal Brazil Phil Phil Nepal Phil Nepal Phil Nepal Brazil Phil Nepal Brazil 18, 22 in prep* 18 18 18, 22 17, 18 18 17, 18 Phil Nepal Brazil 18, 22 Assay4 Field sites5 References6 Rv2585 Rv2583 NH Rv2107 Rv2108 Rv1388 Rv1389 Rv1390 Rv1391 IDRI IDRI IDRI CSU IDRI IDRI IDRI IDRI IDRI Production2 site

406

Gene acession #

Function1

ML0489

A. Geluk et al.

ML0491

ML0527 ML0538 ML0539 ML0540

ML0541

array, ELISA, WBA array, ELISA, WBA blot array array array, ELISA, WBA array, WBA

ML0542 ML0543

ML0557 NH NH NH NH NH NH Rv2376 Rv3193 NH NH IDRI IDRI mouse polyclonal mouse polyclonal CSU LUMC LUMC mouse polyclonal LUMC LUMC LST LST LST blot LST array array LUMC LST

Rv1411c

CSU

blot

Phil

in prep* 19, this issue 19, this issue 19, this issue 19, this issue in prep* unpublished Phil Nepal Phil Nepal 18 18 13, 18, in prep subm

ML0573

possible conserved lipoprotein probable GTP pyrophosphokinase hypothetical unknown PE-family PPE-family, serine rich putative integration host factor mihF probable guanylate kinase Gmk conserved hypothetical probable DNA/ pantothenate metabolism avoprotein probable conserved lipoprotein hypothetical unknown

ML0574

hypothetical unknown

ML0575

hypothetical unknown

ML0576

hypothetical unknown

ML0588 ML0614

Brazil Ethiopia Nepal Brazil Ethiopia Nepal Brazil Ethiopia Nepal Brazil Ethiopia Nepal Phil Brazil

ML0620 ML0644

ML0678

hypothetical unknown possible conserved membrane secreted probable conserved transmembrane hypothetical unknown CSU/IDRI/IPP/ LUMC CSU

ML0755

hypothetical unknown

array, LST, WBA WBA

Phil Nepal Ethiopia Mali Bdesh Bdesh

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal mouse polyclonal mAb CS-38 mouse polyclonal mouse polyclonal array array LST, WBA LST, WBA LST, WBA array LST, WBA array, WBA array array, WBA array array array array array array array array Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Brazil array, WBA 13, 18 17, 18, 22 17, 18, 22 Assay4 Field sites5 References6 NH Rv3212 Rv3211 NH NH NH Rv2785 Rv2216 Rv0015 Rv2175 Rv1459 NH NH NH NH NH NH NH NH NH NH Rv2732 Rv2731 IDRI IDRI LUMC IDRI IDRI IDRI IDRI IDRI IDRI IDRI IDRI CSU/IDRI/IPP Production2 site

Gene acession #

Function1

ML0757

hypothetical unknown

ML0810

possible secreted

Phil Nepal Mali Bdesh Phil Nepal Brazil

ML0811

ML0840

probable ATP-dependent RNA helicase hypothetical unknown CSU/IDRI/ LUMC CSU/IDRI Brazil Bdesh Ethiopia SKorea Phil Nepal Phil Nepal

ML0841

array, ELISA, WBA array, ELISA, WBA array, ELISA, LST, WBA array, blot 18 18 18 18 18 18

17, 18, 22, this issue 18, 28, 37

ML0845

ML0853 ML0860 ML0897

ML0898

ML0899

ML0922 ML0927

35 kDa, major membrane protein I (MMP1) conserved hypothetical unknown probable 30S ribosomal conserved hypothetical probable transmembrane Ser/Thr kinase L conserved hypothetical regulatory possible conserved integral membrane conserved hypothetical hypothetical unknown

18 19, this issue in prep in prep 18 in prep 18 18 18 18 18 Phil Nepal 18

M. leprae antigens for leprosy diagnostics

ML0928 ML0939 ML0947 ML0950 ML0953 ML0956 ML0957

hypothetical unknown hypothetical unknown hypothetical unknown hypothetical unknown hypothetical unknown putative integral membrane hypothetical unknown

ML0958 ML0990

LUMC LUMC IDRI LUMC CSU/IDRI IDRI CSU/IDRI/ LUMC IDRI IDRI

Phil Nepal Brazil Bdesh SKorea Ethiopia Ethiopia Ethiopia Phil Nepal Ethiopia Bdesh Phil Nepal Phil Nepal Bdesh SKorea Phil Nepal Phil Nepal Phil Nepal

ML1002

putative membrane probable conserved transmembrane probable conserved transmembrane

407

Table 1. continued Homolog in Mtb Antiserum3 mAb and polyclonal mouse polyclonal array, WBA array, WBA array, WBA array Phil Nepal array Phil Nepal 18 Assay4 Field sites5 References6 Rv2719 NH Rv2709 Rv2707 Rv1386 Rv1197 Rv1198 NH Rv1303 Rv1337 Rv1342 NH Rv2108 NH Rv1565 Rv1566 Rv1183 Rv3821 Rv2484 Rv2091 Rv1694 LUMC IDRI IDRI IDRI IDRI LUMC IPP CSU IDRI IDRI IDRI IDRI IDRI CSU/IDRI/IPP IDRI/IPP IDRI/IPP IDRI/IPP IDRI IDRI IDRI/LUMC IDRI Production2 site

408

Gene acession #

Function1

ML1004

ML1011

probable conserved membrane hypothetical unknown array, ELISA, WBA, LST array 18 13, 18 13, 18 13, 18 Phil Nepal Brazil Ethiopia Phil Nepal

A. Geluk et al.

ML1015

17, 18, in prep 18

ML1017

ML1053

probable conserved transmembrane probable conserved integral membrane PE-family

ML1055

ESAT-6 like protein esxK

ML1056

ESAT-6 like protein esxL

ML1057

hypothetical unknown

13, 18, 23 18 18 18 18, 22 Mali Bdesh Phil Phil Nepal Brazil 13 in prep* 17, 18, 27

ML1138 ML1171 ML1176 ML1181

possible integral membrane possible integral membrane conserved membrane hypothetical protein

Phil Nepal Mali Bdesh Phil Nepal Mali Bdesh Phil Nepal Mali Bdesh Brazil Phil Nepal Mali Bdesh Phil Nepal Phil Nepal Phil Nepal Phil Nepal Brazil

ML1182 ML1189 ML1213

LST, array, WBA array array array array, ELISA, WBA WBA blot array, WBA LST, WBA array array array array LST, WBA

ML1214

Ethiopia Phil Nepal Phil Nepal Phil Nepal Phil Nepal Ethiopia

in prep 18 18 18 18 in prep

ML1231

ML1233

ML1244 ML1334

ML1358

PPE-family hypothetical unknown conserved hypothetical membrane hypothetical invasion protein conserved transmembrane transport conserved integral membrane conserved hypothetical possible conserved membrane cytotoxin/hemolysin homologue

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal mouse polyclonal mouse polyclonal mouse polyclonal mouse polyclonal array LST, WBA LST LST LST array array, WBA array, WBA Phil Nepal Phil Phil Nepal in prep* 18 13, 18, in prep 18 18, 22 18 13, 18 24, 18, 22 Phil Nepal Phil Nepal Phil Nepal 18 18 18 19, this issue 19, this issue 19, this issue 19, this issue 18 17, 18 18, 22 Phil Nepal Brazil Phil Nepal 24, 18, 22 18 Phil Nepal Brazil 18, 22 Assay4 Field sites5 References6 Rv1631 NH NH NH NH Rv2462 Rv1158 NH Rv0684 NH Rv2869c Rv2876 NH NH NH NH Rv2904 Rv2299 Rv2223c Rv2224c Rv2235 IDRI IDRI IDRI LUMC LUMC LUMC IDRI IDRI LUMC IDRI IDRI IDRI IDRI IDRI/IPP IDRI IDRI CSU CSU/IDRI IDRI Production2 site

Gene acession #

Function1

ML1383

ML1384 ML1419

array, ELISA, WBA blot array

ML1420

probable dephospho-CoA kinase hypothetical unknown possible regulatory protein hypothetical unknown CSU/IDRI/IPP/ LUMC IDRI Phil Nepal Brazil array, LST, WBA array Phil Nepal Ethiopia Mali Bdesh Phil Nepal

ML1430

ML1481

possible integral membrane probable trigger factor array, ELISA, WBA array

ML1505

ML1553

ML1556

conserved hypothetical Ala-, Pro-rich probable prolyl tRNA synthetase initiation factor IF-2 ELISA, array, WBA array array

Phil Nepal Mali Bdesh Brazil

ML1575 ML1582

ML1584

ML1601

hypothetical unknown possible conserved transmembrane possible conserved transmembrane hypothetical unknown

ML1602 ML1603 ML1604 ML1613 ML1623

hypothetical unknown hypothetical unknown hypothetical unknown probable 50S ribosomal Hsp90 family

M. leprae antigens for leprosy diagnostics

ML1632

Brazil Bdesh SKorea Ethiopia Brazil Ethiopia Brazil Ethiopia Brazil Ethiopia Phil Nepal Phil Nepal Brazil Ethiopia Phil Nepal Brazil

ML1633

ML1644

possible exported protease probable exported protease probable conserved transmembrane

array, ELISA, WBA array, ELISA, WBA array

409

Table 1. continued Homolog in Mtb Antiserum3 mAb mouse polyclonal mouse polyclonal mouse polyclonal LST, WBA array WBA array array LST blot, array array Phil Nepal Brazil Ethiopia Phil Nepal Pakistan array, ELISA, WBA Phil Nepal Brazil array Phil Nepal 18 18, 22 18 array Phil Nepal 18 Assay4 Field sites5 References6 Rv2968 Rv2969 Rv2988 Rv3005c NH NH Rv1478 NH Rv0720 Rv0718 Rv0716 Rv0714 Rv0708 Rv0703 Rv0702 Rv2533 Rv0685 Rv0683 Rv0637 NH Rv1111 NH Rv3460 NH NH Rv0093 LUMC IDRI LUMC IDRI IDRI IDRI IDRI IDRI IDRI IDRI IDRI IDRI IDRI CSU/IDRI IDRI IDRI CSU/IDRI IDRI IDRI IDRI IDRI IDRI Production2 site

410

Gene acession #

Function1

ML1666

ML1667

A. Geluk et al.

ML1685

ML1698

ML1788 ML1795

probable conserved integral membrane probable conserved membrane or secreted 3-isopropylmalate dehydratase large subunit conserved membrane protein very hypothetical 18 kDa hsp LUMC CSU/IDRI/ LSHTM IDRI CSU/IDRI/IPP array, ELISA array, blot, WBA array array array array array array array array blot, array array array array array

ML1812 ML1829

hypothetical invasion hypothetical unknown

19, this issue 34, 18, 31 in prep* 18 13, 18, in prep* Phil Phil Nepal Mali Bdesh Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Phil Nepal Phil Nepal Phil Nepal Phil Nepal Phil Nepal Ethiopia Phil Nepal Bdesh SKorea Phil Nepal Phil Nepal 18 18 18 18 18 18 18 18 18, 28 18 18 18 18 in prep 18 subm 18 18

ML1843 ML1845 ML1847 ML1849 ML1856 ML1861 ML1862 ML1864 ML1877 ML1879 ML1910 ML1915 ML1937

ML1949 ML1960 ML1976 ML1982 ML1988

probable 50S ribosomal probable 30S ribosomal probable 50S ribosomal probable 50S ribosomal probable 50S ribosomal probable 50S ribosomal probable 50S ribosomal 30S ribosomal tuf elongation factor-TU probable 30S ribosomal conserved hypothetical hypothetical unknown probable integral membrane hypothetical unknown probable 30S ribosomal hypothetical unknown putative integral membrane probable conserved membrane

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal mouse polyclonal mAb CS-90, polyclonal mAb CS-41 mouse polyclonal mouse polyclonal mouse polyclonal mouse polyclonal array LST, WBA, array LST, WBA 19, 21 19, 21, 18 18 17, 18, 27, 22, 28 60, 28 17, 18, 22 18 Bdesh Phil Nepal Brazil Ethiopia Phil Nepal 24, 17, 27, 22, in prep 18 13, 18 18, 22 13, 18 blot array array array LST, WBA in prep* 18 18 18 Brazil Bdesh Pakistan Nepal Ethiopia SKorea 19, 21, in prep Assay4 Field sites5 References6 NH NH Rv0970 Rv1886c Rv1876 NH Rv1861 Rv1860 Rv1845 NH Rv0811 NH NH NH Rv0543 Rv0559 LUMC CSU IDRI IDRI IDRI CSU/IDRI/IPP IDRI IDRI/IPP IDRI IDRI/LUMC IDRI CSU/IDRI IDRI IDRI/LUMC LUMC Production2 site

Gene acession #

Function1

ML1989

hypothetical unknown

ML1990

putative integral membrane

ML1997

Brazil Bdesh Pakistan Nepal Ethiopia SKorea Brazil Bdesh Pakistan Nepal Ethiopia SKorea Phil Phil Nepal

ML2028

probable conserved integral membrane Ag85B mycolyltransferase ELISA, blot, array, WBA, LST ELISA, blot

ML2038

bacterioferritin, bfrA

ML2044

hypothetical unknown

CSU/IDRI/ LUMC/NIID CSU/IDRI WBA, array, ELISA array

ML2054

Phil Nepal Brazil Ethiopia Bdesh Indonesia Phil Japan Indonesia Vietnam Bdesh Ethiopia Phil Nepal Brazil Phil Nepal

ML2055

ML2064

WBA, LST, blot, array, ELISA array array, WBA

ML2177

probable integral membrane probable cell surface protein, secreted, modD conserved hypothetical transmembrane uridine phosphorylase

ML2203

conserved hypothetical

Phil Nepal Mali Bdesh Phil Nepal Brazil array, ELISA, WBA array, WBA

ML2244

hypothetical unknown

M. leprae antigens for leprosy diagnostics

ML2249 ML2253 ML2258 ML2274

Phil Nepal Mali Bdesh Phil Phil Nepal Phil Nepal Phil Nepal

ML2283

hypothetical unknown conserved hypothetical conserved hypothetical possible conserved secreted hypothetical unknown

411

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal mouse polyclonal mouse polyclonal array ELISA array, WBA LST & WBA, array LST & WBA, array array, WBA ELISA, WBA LST & WBA Brazil Phil Nepal Brazil Phil Nepal Phil Nepal WBA, array Bdesh Phil Nepal 17, 18 subm 26, 18, 60, 27, 22 24, 17, 18, 22 22 18, 22 18 18 subm 18 18 13, 18 17, 18 18 13, 18 22 Assay4 Field sites5 References6 Rv3219 NH Rv3717 NH Rv1185c Rv0455 Rv1083 Rv1078 NH Rv0351 Rv2373 NH Rv0288 Rv0287 Rv0285 Rv0275 IDRI IDRI/IPP IDRI/LUMC IDRI/LUMC IDRI/IPP IDRI IDRI LUMC IDRI IDRI IDRI IDRI IDRI CSU IDRI CSU/IDRI Production2 site

412

Gene acession #

Function1

ML2307

A. Geluk et al.

ML2313 ML2331

probable transcritional regulatory protein hypothetical unknown possible secreted Bdesh Brazil Phil Venezuela Japan Nepal India China Brazil Bdesh Pakistan Nepal Ethiopia Phil

ML2346

hypothetical unknown

ML2358

WBA array, ELISA, blot, MAPIA, WBA array, ELISA, MAPIA, WBA, LST ELISA, WBA

ML2380

probable fatty-acid-CoA synthetase fadD26 possible secreted protein array, ELISA, WBA array array

ML2390 ML2395

ML2478

conserved membrane probable proline-rich antigen homolog hypothetical unknown

ML2495 ML2496

Brazil Bdesh SKorea Ethiopia Phil Nepal Phil

ML2498

Hsp70 cofactor probable chaperone protein Enoyl-CoA hydratase

ML2531

potential virulence; ESAT-6-like; CFP7

ML2532

ML2534

potential virulence; PE-family protein; TB9.8 PE-family

ML2541

possible transcriptional regulatory protein

Phil Nepal Mali Bdesh Brazil Bdesh SKorea Ethiopia Phil Nepal Brazil Bdesh SKorea Ethiopia Phil Nepal Phil Nepal Mali Bdesh Brazil

Table 1. continued Homolog in Mtb Antiserum3 mouse polyclonal LST & WBA 19, 20, 23, in prep* 18 18 18, 22 18, 22 19 Phil Nepal Brazil Phil Nepal Brazil Bdesh SKorea Phil Nepal Phil Nepal array array array array, WBA Phil Nepal Phil Nepal Phil Nepal Phil Nepal Bdesh SKorea Phil Nepal Brazil array, ELISA, MAPIA, WBA array Phil Nepal 18, 22 17, 18, 22 subm 18 18 18 18 18 17, 18 18 18 Assay4 Field sites5 References6 NH CSU/LUMC Production2 site

Gene acession #

Function1

ML2567

hypothetical unknown

ML2581 Rv0180 Rv0183 Rv0164 Rv1096 Rv0129c Rv0125 NH NH Rv3910 Rv3921 NH NH NH NH NH IDRI IDRI IDRI IDRI IDRI IDRI IDRI array CSU IDRI mouse polyclonal IDRI IDRI LUMC Brazil IDRI CSU/IDRI mouse polyclonal Phil Nepal Brazil Phil Nepal Brazil IDRI array Phil Nepal

Rv0227

IDRI

array

Brazil Bdesh Pakistan Nepal Ethiopia SKorea Phil Nepal

ML2600

ML2603

probable conserved membrane probable conserved transmembrane possible lysophospholipase

ML2629

conserved hypothetical

ML2649

array, ELISA, WBA array, ELISA, WBA LST

ML2655

ML2659

putative oligosaccharide deacetylase Ag85C mycolyl transferase probable serine protease array, ELISA, WBA array, ELISA, WBA WBA array

ML2666 ML26f

ML2700

ML2710

ML33f

ML41f

ML46f

M. leprae antigens for leprosy diagnostics

ML5049

hypothetical unknown fusion of ML0050, ML0568 probable conserved transmembrane probable conserved transmembrane fusion of ML2044, ML1862 fusion of ML0050, ML0405 fusion of ML0568, ML0405 fusion of ML0050, ML0049

ML57f

fusion of ML0050, ML0568, ML0405

413

Table 1. continued Homolog in Mtb Antiserum3 ELISA WBA, LST array, MAPIA array Phil Nepal array Phil Nepal 18 18 25, 26, 18, 60 18 18 in prep 25 array Phil Nepal 18 array Phil Nepal 18 Assay4 Field sites5 References6 NH NH NH NH NH NH NH NH NH IDRI LUMC IDRI IDRI IDRI IDRI IDRI IDRI IDRI Production2 site

414

Gene acession #

Function1

ML73f

ML75f

A. Geluk et al.

ML82f

ML92f

LID-1

fusion of ML2028, ML2346, ML2044 fusion of ML2028, ML2346, ML2307 fusion of ML2346, ML0840 fusion of ML2028, ML2346, ML1862 fusion of ML0405, ML2331 array, ELISA, blot, MAPIA, SLF, DPP array, MAPIA Phil Nepal Brazil Phil Nepal China Bdesh India Japan Venezuela Phil Nepal

LID-2

LID-3

PE-ML0701

PADL

fusion of ML0405, ML1556 fusion of ML0405, ML1556, ML2346 Tcell epitopes ML1989, ML1990, ML2283, ML2567 Bcell epitopes ML0405, ML2331, ML2055, ML0411, ML0091

Bdesh SKorea Brazil Ethiopia Nepal Brazil Phil

1 According to Leproma Web Server (http://genolist.pasteur.fr/Leproma) and Sanger database (http://www.sanger.ac.uk/). NH indicate no homolog known in M. tuberculosis (H37Rv). 2 CSU: Colorado State University; IDRI: Infectious Disease Research Institute; IPP: Institute Pasteur Paris; LSHTM: London School of Hygiene and Tropical Medicine; LUMC: Leiden University Medical Center; NIID: National Institute of Infectious Diseases, Japan. 3 Indicates anti-serum produced at CSU. 4 Assays conducted on these proteins are: blot Western blot analysis; array protein array; LST 6 d lymphocyte stimulation test; WBA 24 h whole blood assay; ELISA serum ELISA for detection of Ab against M. leprae Ag; SLF single lateral ow RDT; DPP dual path platform RDT; MAPIA multi-antigen print assay. 5 Bdesh: Bangladesh; Phil: Philippines; SKorea South Korea. 6 References are indicated by number or by: subm: manuscript submitted, Geluk et al.; in prep: manuscript in preparation, Bobosha et al.; in prep*: manuscript in preparation, Spencer et al.; this issue: Bobosha et al. this issue.

M. leprae antigens for leprosy diagnostics

32,33

415

expression libraries with sera and applied in immune response screening studies as recombinant antigen such as the M. leprae 10 kDa (GroES), 18 kDa,34,35 35 kDa,36 38 45 kDa,15,39 hsp65,40 42 70 kDa,43 and Ag85 (30/31 kDa).44 48 These antigens have had limited value for diagnosis of leprosy due to their inherent high cross reactivity with other mycobacteria, which is particularly problematic in countries with high incidence rates of tuberculosis, routine BCG vaccination, and high levels of exposure to environmental mycobacteria. Despite such extensive cross-reactivity within whole proteins of M. leprae, particular regions can show sequence divergence leading to specic T cell epitopes.49,16 In our attempts to develop simple assays based on cellular mediated immune (CMI) responses particularly for the identication of asymptomatic leprosy, we were encouraged by the recent development of two commercially available IFN-g release assays (IGRAs) for specic diagnosis of M. tuberculosis infection50,51 that exploit antigens (ESAT-6, CFP-10, TB77) that are selectively expressed by M. tuberculosis and deleted in all nonvirulent BCG vaccine strains and most other non-tuberculous mycobacteria (NTM). However, despite limited sequence homology (36% and 40%, respectively) and no crossreactivity on serological level,52,53 the M. leprae homologues of ESAT-6 and CFP-10 (ML0049 and ML0050) were well recognised by T cells from M. tuberculosis infected individuals, thereby limiting the diagnostic potential of ESAT-6 and CFP-10 in leprosy endemic areas with a high prevalence of tuberculosis.54,55 Similar data were reported for the M. leprae-specic 45 kDa serine rich antigen (ML0411) which was also recognised by TB patients probably due to sequence homology with Rv2108.15 Three initial studies that provided a basis for further collaborative efforts within the IDEAL (Initiative for Diagnostic and Epidemiological Assays for Leprosy) consortium were performed in a Brazilian population (Rio de Janeiro, South East Brazil) using M. leprae unique proteins and peptides arising from the above described post-genomic approach.19,20,23 Together, these studies identied several proteins [e.g. ML0840, ML1601, ML1990, ML2283, and ML2567]19 and peptides [e.g. ML1419c-derived HLA class I and class II epitopes23 and peptides derived from ML228320] with the potential to specically identify M. leprae infected subjects. These antigens were later re-tested in ve endemic sites in Ethiopia, Bangladesh, Pakistan, Nepal and Brazil.21 Despite the fact that all these M. leprae proteins had been selected based on their unique sequence in M. leprae, IFN-g production by T cells from endemic controls (EC) was observed in response to most of these M. leprae proteins.21 However, since these EC also responded to M. leprae sonicate, the observed T cell reactivity towards the M. leprae unique proteins could still indicate M. leprae-specicity. Therefore, screening of the (at that time) most promising M. leprae antigens was performed in an area highly endemic for leprosy (Dhaka, Bangladesh) and a low endemic area (Seoul, South Korea) by analysis of IFN-g production after 24 h incubation of whole blood cultures. For some M. leprae proteins (ML2478, ML0840 in particular, but also ML0957 and ML1601), signicant IFN-g levels in Bangladeshi EC were observed, whereas these were completely absent in EC and TB patients from South Korea (Geluk et al., ms submitted). Since other proteins included in the analysis did not show similar specicities, these data indicate that the responses observed in EC most likely indicate exposure to M. leprae and are not due to nonspecic responses to these proteins. In addition to worldwide screening of M. leprae antigens described above, other studies nia, Central-West Brazil) in their screening have focused only on one particular region (Goia for leprosy specic IFN-g responses.22 A recent report showed that, despite the in silico

416

A. Geluk et al.

prediction that selected proteins possessed T cell and B cell epitopes, fewer than half of the single proteins tested (16 of 33; 48%) were immunogenic [as estimated by production of IFN-g in whole blood assays (WBA)]. Among these 16 immunogenic proteins, nine were considered leprosy specic, inducing cell-mediated IFN-g secretion from TT/BT patients.22 The most promiscuous protein predicted was ML0405 protein, containing T cell epitopes predicted to be recognised by 50 out of 51 HLA-DR alleles along with 24 potential B cell epitopes. Consistent with this prediction, ML0405 protein induced in vitro IFN-g release in WBA using TT/BT leprosy patients blood as well as production of IgG directed against ML0405 in sera from LL/BL leprosy patients. Since the predictions for this study employed basic parameters, the use of more sophisticated in silico prediction software that not only include the probability of the antigen being presented in the context of a certain HLA allele, but also of being processed and recognised by TCR, could lead to different conclusions. In fact, a recent study conducted in Ethiopia describes immunogenicity screening of yet another set of M. leprae antigens: these newly identied M. leprae peptides and proteins were selected based on bio-informatic searches for the presence of HLA-binding motifs, CD4- or CD8-restricted predicted T cell epitopes and protease cleavage sites (Table 1 and Bobosha et al., in preparation). In summary, IFN-g secretion in response to M. leprae proteins has typically also been observed in blood cultures of healthy household contacts (HHC) and EC, thereby limiting the ability of IGRA to provide a true diagnosis of disease.21,22 As the vast majority of contacts do not progress to manifest disease symptoms, the utility of WBA for prognostic purposes remains in question. It is possible that M. leprae antigens that could distinguish PB from HHC remain undiscovered. An alternative possibility is that other, as yet to be determined biomarker proles, composed of alternate cytokines, chemokines and/or genetic expression markers, will be able to provide a distinction between progression to disease and containment of M. leprae infection (Geluk et al., ms submitted). Given the current knowledge and limitations surrounding the identication of T cell-activating antigens, it is plausible that such biomarker proles could be used in more dened situations, such as assessing the response of PB patients to treatment. Several investigators have employed such an approach within TB control programmes.56,57
SEROLOGICAL BASED ASSAYS

Across the clinical forms based on Ridley-Jopling classication, the magnitude of the antibody response to the M. leprae-specic antigen, PGL-I correlates very well with the bacillary index, with high titers in almost all patients with a more lepromatous response (BB, BL and LL), while the titer is low or undetectable in those responding with a more tuberculoid response (TT, BT).6 Several protein antigens have been shown to induce potent and highly specic antibody responses in MB patients, e.g. the hypothetical proteins ML0405 and ML233127 which have been engineered into a fusion protein called Leprosy IDRI Diagnostic (LID)-1.24 Antibody responses towards LID-1 have been shown to be positive in over 95% of those at the lepromatous end of the spectrum.26 In a prospective study involving household contacts of MB patients whose serum antibody responses were analysed retrospectively to 4 years prior to clinical diagnosis of MB leprosy, 7/11 (64%) showed an IgG antibody response to LID1 up to one year prior to developing clinical symptoms, and the responses were strikingly more elevated and occurred much earlier than the increases in the anti-PGL-I IgM response in these same individuals.24 Although the numbers are small,

M. leprae antigens for leprosy diagnostics

417

a heightened IgG titer towards LID-1 may provide an early indication of infection and active disease, as the antibody response in contacts who did not go on to develop disease was generally low. We recently conducted analyses of responses in experimentally infected armadillos in an attempt to dene the antibody targets that are recognised earliest in a controlled setting.58 Results indicated that, in general, the antibody responses develop in a heterogenous manner and suggest that a combination of antigens, such as LID-1 and ND-O-BSA, may be best suited to early diagnosis.26 The utility of M. leprae-derived major membrane protein-II (MMP-II) in the serodiagnosis of leprosy has also been examined. The percent positivity by an anti-MMPII antibody ELISA was 824% for MB, and 390% PB leprosy, with a specicity of 901%.59 The MMP-II antigen was further evaluated for detecting antibodies in leprosy patients and patients contacts in the mid-region of Vietnam and compared to the results to those for the PGL-I method (PGL-I ELISA). Results from that study indicated that 85% of MB patients and 48% of PB patients were positive by MMP-II ELISA. Variance was observed in contacts, with HHC demonstrating low positivity rates but medical staff members possessing comparatively high positivity rates. Furthermore, monitoring of results for leprosy patients and HHC showed that MMP-II is a better index marker than PGL-I, suggesting that serological testing with MMP-II would be benecial in detecting leprosy.60 Serological assays may not only be benecial for classical diagnosis, but could have signicant utility in the management of patients and within trials of novel treatments as it is well documented that anti-PGL-I antibody levels decline as bacterial burden is reduced.5,61 64 In a recent study, data indicated that the IgG responses to LID-1 decreased more rapidly than the IgM response to PGL-I in the months following MDT therapy.26 The reasons for this disparity are unclear, but one suggestion would be that protein is cleared more rapidly from the infection site than glycolipid, removing an antigen reservoir that could perpetuate antibody production. Individuals who successfully completed therapy and showed no clinical signs of disease were found to have no or very low antibody titer to LID-1 or its components. Of 57 former patients analysed 10 years after treatment, four exhibited responses that were still positive. Upon review, three of these individuals received irregular treatment. In the other case, an individual previously diagnosed with the indeterminate form of the disease who had received the shorter MDT course of therapy was identied and found to have a very high LID-1 response with active MB disease. Taken together, the IgG response to LID-1 could potentially be used to effectively monitor the efcacy of treatment by antibody clearance, and can be used to detect treatment failure or relapsing disease. Although the antibody response to LID-1 is very strong in virtually all MB cases, its ability to detect responses in PB cases is poor, and is comparable to the percentage of antiPGL-I positivity, which is usually around 20-40%.24 Other protein antigens have been described that are capable of detecting antibody responses in the majority of PB patients, including ML2028 (Ag85B) and ML2038 (bacterioferritin).28 In addition, developing preliminary data indicates that PB patients almost universally serologically detect ML0286, a fructose bisphosphate aldolase shown to be critical in the survival of persistent M. tuberculosis bacilli within the necrotic core of primary lung granulomas (Mary Jackson, personal communication; JS Spencer, unpublished data). It may be possible to engineer a fusion protein that combines critical epitopes from these three proteins which are recognised by the majority of PB cases to facilitate better detection within this group.

418

Table 2. Overview of M. leprae peptides tested in vitro human responses Production site1 CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU CSU IPP IPP LSHTM LSHTM LSHTM LSHTM LSHTM LSHTM LUMC LUMC LUMC LUMC LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST LST Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Brazil, Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Pakistan, Nepal, Ethiopia, UK Pakistan, Nepal, Ethiopia, UK Pakistan, Nepal, Ethiopia, UK Pakistan, Nepal, Ethiopia, UK Pakistan, Nepal, Ethiopia, UK Pakistan, Nepal, Ethiopia, UK Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Bdesh, Pakistan, Nepal, Ethiopia Assay2 Field sites3 References4 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 21, 23 13, 21 21 16 16 16 16 16 16 20, 21 20, 21 20, 21 20, 21

A. Geluk et al.

Gene acession #

Peptide

Amino acid sequence

ML2452c ML1420 ML1057 ML1829 ML0757c ML0840c ML1189c ML0394c ML0126 ML0008c ML2567 ML1915 ML1419c ML0308 ML0638 ML1057 ML0308 ML1420 ML1217 ML0987 ML0411 ML0411 ML0726 ML0375 ML1990 ML1989 ML2283 ML2283

p92 p70 p51 p73 p63 p86 p88 p48 p81 p40 p54 p74 p69 p56 p85 p49 Ep1-308 p2 P8975 Q9021 S9048 S9047 K8948 D8963 p40 p43 p19 p20

LQAYSNLFGRTSAMQ MQEYRGLTSHTPCCR AAALEQLLGQTADVA DAEWLKLTSLGLRPR GINLPKDELTAFGRK VYLYNYLLAETSHVL DDIWRTLASAVITGN QLMYLIEITSETKAL GDVWKSIVHLRSTRH RVSYGSECRSGNCLR NHAVSSDFKTRSTNT VKAVVDDVNAILLTGR RLDGTTLEV FDEYRAMFALSAMDL NYEVSPIFARWPRNR GRYYAEINSAKMYFG SRGFDEYRAMFALSAMDLNATI SLLMQEYRGLTSHTP RPEAVLQHARTLAKI MFGCMNYSTRVTLAD GLDSIISSASASLLT YDLMWVQNSTAMTTY VKVAVAEGQTVMTGD QYPAMLTALQRAVVE VGFVNAWSLLFYPPQGWESP STNYVNLSTIGIKLKI PLARELYRKIDCYSEEDLVE DCYSEEDLVEIAAKSLRATI

CSU: Colorado State University; IPP: Institute Pasteur Paris; LSHTM: London School of Hygiene and Tropical Medicine; LUMC: Leiden University Medical Center. assays conducted on these proteins are: LST 6 d lymphocyte stimulation test; WBA 24 h whole blood assay. 3 Bdesh: Bangladesh; Phil: Philippines. 4 references are indicated by number.

M. leprae antigens for leprosy diagnostics

SUMMARY

419

The widespread provision of WHO-MDT has had a dramatic impact on the global prevalence of leprosy, and leprosy control programmes are now being integrated into standard public health services. However, due to changes in leprosy control programmes and the special expertise required for diagnosis, the need for simple rapid diagnostic tests that could be applied in non-expert settings may now be greater than ever before. Since the sequencing and annotation of the M. leprae genome was rst published a decade ago, there has been much progress in the investigation of M. leprae protein antigens that might be useful in either serologic or cell mediated assays for the early diagnosis of leprosy. The principal research laboratories that together examined nearly 200 recombinant single proteins during the last decade have identied a number of antigens with the potential to be applied in a simple eld-friendly test that can be used to screen those (healthy) individuals who are either in a high risk category (contacts of MB index cases) or are showing possible early signs of the disease. Such tests would also have the ability to track the effectiveness of MDT treatment over time. Despite this progress, there are still issues with sensitivity and specicity, particularly when cell mediated responses to these antigens are examined. These problems are magnied in regions where rates of infection with TB and leprosy and/or exposure to non-pathogenic mycobacteria are much higher, as is the case in many developing countries. It is likely that additional antigens will be identied over time, but to ensure the success of developing a cheap and rapid test for the early diagnosis of leprosy, continued funding for these efforts is critical. Acknowledgements The authors of this article received funding from the Netherlands Leprosy Relief Foundation (AG; ILEP#: 702.02.65 & ILEP#: 701.02.49), the Turing Foundation (AG), the Q.M. Gastmann-Wichers Foundation (AG), the Order of Malta-Grants-for-Leprosy-Research (AG), American Leprosy Missions (MD), The New York Community Trust (Heiser Foundation) (MD), National Institutes of Health (1R43AI066613-01A1 and 2R44AI06661302) (MD) and NIH/NIAID Leprosy Contract N01-AI-25469 and Grant R01-AI-47197 (JS). LUMC, IDRI and CSU are part of the IDEAL (Initiative for Diagnostic and Epidemiological Assays for Leprosy) Consortium. AG wishes to thank Prof. Tom Ottenhoff and Prof. Hazel Dockrell for stimulating discussions. References
1 2 3 4 5 6 7

Fine PEM. Leprosy: what is being eliminated? Bull World Health Organ, 2007; 85: 2. Meima A, Smith WCS, van Oortmarssen GJ et al. The future incidence of leprosy: a scenario analysis. Bull World Health Organ, 2004; 82: 373380. Global Leprosy Situation, Wkly Epidemiol Rec 2010; 85: 337 348. Siddiqui MR, Velidi NR, Pati S et al. Integration of leprosy elimination into primary health care in orissa India. PLoS ONE, 2009; 4: e8351. Cho SN, Cellona RV, Fajardo TT et al. Detection of phenolic glycolipid-I antigen and antibody in sera from new and relapsed lepromatous patients treated with various drug regimens. Int J Lepr Other Mycobact Dis, 1991; 59: 2531. Oskam L, Slim E, Buhrer-Sekula S. Serology: recent developments, strengths, limitations and prospects: a state of the art overview. Lepr Rev, 2003; 74: 196205. Douglas JT, Cellona RV, Fajardo TT et al. Prospective study of serological conversion as a risk factor for development of leprosy among household contacts. Clin Diagn Lab Immunol, 2004; 11: 897900.

420
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

A. Geluk et al.

27 28

29 30 31 32 33 34 35 36 37

Beers VSM, Hatta M, Klatser PR. Seroprevalence rates of antibodies to phenolic glycolipid-I among school children as an indicator of leprosy endemicity. Int J Lepr Other Mycobact Dis, 1999; 67: 243249. Goulart IM, Bernardes Souza DO, Marques CR et al. Risk and protective factors for leprosy development determined by epidemiological surveillance of household contacts. Clin Vaccine Immunol, 2008; 15: 101 105. Moet FJ, Meima A, Oskam L, Richardus JH. Risk factors for the development of clinical leprosy among contacts, and their relevance for targeted interventions. Lepr Rev, 2004; 75: 310 326. Cole ST, Brosch R, Parkhill J et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998; 393: 537 544. Cole ST, Eiglmeier K, Parkhill J et al. Massive gene decay in the leprosy bacillus. Nature, 2001; 409: 1007 1011. Araoz R, Honore N, Banu S et al. Towards an immunodiagnostic test for leprosy. Microbes Infect, 2006; 8: 22702276. Araoz R, Honore N, Cho S et al. Antigen discovery: a postgenomic approach to leprosy diagnosis. Infect Immun, 2006; 74: 175182. Brahmbhatt S, Hussain R, Zafar S et al. Human T cell responses to peptides of the Mycobacterium leprae 45-kD serine-rich antigen. Clin Exp Immunol, 2002; 128: 140148. Dockrell HM, Brahmbhatt S, Robertson BD et al. A postgenomic approach to identication of Mycobacterium leprae-specic peptides as T-cell reagents. Infect Immun, 2000; 68: 58465855. Duthie MS, Goto W, Ireton GC et al. Antigen-specic T-cell responses of leprosy patients. Clin Vaccine Immunol, 2008; 15: 16591665. Duthie MS, Ireton GC, Kanaujia GV et al. Selection of antigens and prototype test development for a point-of-care leprosy diagnosis. Clin Vaccine Immunol, 2008; 15: 15901597. Geluk A, Klein MR, Franken KL et al. Postgenomic approach to identify novel Mycobacterium leprae antigens with potential to improve immunodiagnosis of infection. Infect Immun, 2005; 73: 56365644. Geluk A, Ploeg J, Teles RO et al. Rational combination of peptides derived from different Mycobacterium leprae proteins improves sensitivity for immunodiagnosis of M. leprae infection. Clin Vaccine Immunol, 2008; 15: 522533. Geluk A, Spencer JS, Bobosha K et al. From genome-based in silico predictions to ex vivo verication of leprosy diagnosis. Clin Vaccine Immunol, 2009; 16: 352 359. Sampaio LH, Stefani MM, Oliveira RM et al. Immunologically reactive M. leprae antigens with relevance to diagnosis and vaccine development. BMC Infect Dis, 2011; 11: 26. Spencer JS, Dockrell HM, Kim HJ et al. Identication of specic proteins and peptides in Mycobacterium leprae suitable for the selective diagnosis of leprosy. J Immunol, 2005; 175: 79307938. Duthie MS, Goto W, Ireton GC et al. Use of protein antigens for early serological diagnosis of leprosy. Clin Vaccine Immunol, 2007; 14: 14001408. Duthie MS, Hay MN, Morales CZ et al. Rational design and evaluation of a multiepitope chimeric fusion protein with the potential for leprosy diagnosis. Clin Vaccine Immunol, 2010; 17: 298 303. Duthie MS, Hay MN, Rada EM et al. Specic IgG antibody responses may be used to monitor leprosy treatment efcacy and as recurrence prognostic markers. Eur J Clin Microbiol Infect Dis, 2011; Published online, May, 2011. DOI: 10.1007/s10096-011-1221-2. Reece ST, Ireton G, Mohamath R et al. ML0405 and ML2331 are antigens of Mycobacterium leprae with potential for diagnosis of leprosy. Clin Vaccine Immunol, 2006; 13: 333340. Spencer JS, Kim HJ, Wheat WH et al. Analysis of antibody responses to Mycobacterium leprae phenolic glycolipid I, lipoarabinomannan, and recombinant proteins to dene disease subtype-specic antigenic proles in leprosy. Clin Vaccine Immunol, 2011; 18: 260 267. Brennan PJ. Skin test development in leprosy: progress with rst-generation skin test antigens, and an approach to the second generation. Lepr Rev, 2000; 71(Suppl): S50S54. Dockrell HM, Young SK, Britton K et al. Induction of Th1 cytokine responses by mycobacterial antigens in leprosy. Infect Immun, 1996; 64: 4385 4389. Weir RE, Brennan PJ, Butlin CR, Dockrell HM. Use of a whole blood assay to evaluate in vitro T cell responses to new leprosy skin test antigens in leprosy patients and healthy subjects. Clin Exp Immunol, 1999; 116: 263 269. Mustafa AS, Gill HK, Nerland A et al. Human T-cell clones recognize a major M. leprae protein antigen expressed in E. coli. Nature, 1986; 319: 6366. Rinke de Wit TF, Clark-Curtiss JE, Abebe F et al. A Mycobacterium leprae-specic gene encoding an immunologically recognized 45 kDa protein. Mol Microbiol, 1993; 10: 829 838. Dockrell HM, Stoker NG, Lee SP et al. T-cell recognition of the 18-kilodalton antigen of Mycobacterium leprae. Infect Immun, 1989; 57: 19791983. Hussain R, Dockrell HM, Kifayet A et al. Recognition of Mycobacterium leprae recombinant 18-kDa proteins in leprosy. Int J Lepr Other Mycobact Dis, 1992; 60: 368375. Chua-Intra B, Wattanapokayakit S, Srisungngam S et al. T-cell recognition of peptides from the Mycobacterium leprae 35 kDa protein in Thai leprosy patients, healthy contacts, and non-contacts. Immunol Lett, 2003; 88: 71 76. Triccas JA, Roche PW, Winter N et al. A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae. Infect Immun, 1996; 64: 51715177.

M. leprae antigens for leprosy diagnostics

38 39

421

40

41

42 43 44 45

46 47 48 49 50 51 52 53 54

55 56 57 58

59 60

61 62 63

64

Wilkinson RJ, Wilkinson KA, Jurcevic S et al. Specicity and function of immunogenic peptides from the 35-kilodalton protein of Mycobacterium leprae. Infect Immun, 1999; 67: 15011504. Macfarlane A, Mondragon-Gonzalez R, Vega-Lopez F et al. Presence of human T-cell responses to the Mycobacterium leprae 45-kilodalton antigen reects infection with or exposure to M. leprae. Clin Diagn Lab Immunol, 2001; 8: 604 611. Kale AbB, Kiessling R, Van Embden JD et al. Induction of antigen-specic CD4 HLA-DR-restricted cytotoxic T lymphocytes as well as nonspecic nonrestricted killer cells by the recombinant mycobacterial 65-kDa heatshock protein. Eur J Immunol, 1990; 20: 369377. Thole JE, Janson AA, Kie A et al. Analysis of T-cell and B-cell responses to recombinant M. leprae antigens in leprosy patients and in healthy contacts: signicant T-cell responses to antigens in M. leprae nonresponders. Int J Lepr Other Mycobact Dis, 1995; 63: 369380. Wilkinson KA, Katoch K, Sengupta U et al. Immune responses to recombinant proteins of Mycobacterium leprae. J Infect Dis, 1999; 179: 1034 1037. Oftung F, Geluk A, Lundin KE et al. Mapping of multiple HLA class II-restricted T-cell epitopes of the mycobacterial 70-kilodalton heat shock protein. Infect Immun, 1994; 62: 54115418. Launois P, NDiaye MN, Cartel JL et al. Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts. Infect Immun, 1995; 63: 88 93. Launois P, Niang MN, De BJ et al. The major secreted antigen complex (Ag 85) from Mycobacterium bovis bacille Calmette-Guerin is associated with protective T cells in leprosy: a follow-up study of 45 household contacts. J Infect Dis, 1993; 167: 11601167. Launois P, Niang NM, Sarthou JL et al. T cell reactivity against antigen 85 but not against the 18- and 65-kD heat shock proteins in the early stages of acquired immunity against Mycobacterium leprae. Clin Exp Immunol, 1994; 96: 8690. Launois P, Vandenbussche P, MBayame NN et al. IL-6 production in response to puried mycobacterial heatshock proteins and to antigen 85 in leprosy. Cell Immunol, 1993; 148: 283290. Weir RE, Butlin CR, Neupane KD. Use of a whole blood assay to monitor the immune response to mycobacterial antigens in leprosy patients: a predictor for type 1 reaction onset? Lepr Rev, 1998; 69: 279293. Chua-Intra B, Peerapakorn S, Davey N et al. T-cell recognition of mycobacterial GroES peptides in Thai leprosy patients and contacts. Infect Immun, 1998; 66: 49034909. Ferrara G, Losi M, DAmico R et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet, 2006; 367: 13281334. Pai M, Kalantri S, Dheda K. New tools and emerging technologies for the diagnosis of tuberculosis: part I. Latent tuberculosis. Expert Rev Mol Diagn, 2006; 6: 413422. Spencer JS, Kim HJ, Marques AM et al. Comparative analysis of B- and T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture ltrate protein 10. Infect Immun, 2004; 72: 31613170. Spencer JS, Marques MA, Lima MC et al. Antigenic specicity of the Mycobacterium leprae homologue of ESAT-6. Infect Immun, 2002; 70: 10101013. Geluk A, van Meijgaarden KE, Franken KL et al. Identication and characterization of the ESAT-6 homologue of Mycobacterium leprae and T-cell cross-reactivity with Mycobacterium tuberculosis. Infect Immun, 2002; 70: 25442548. Geluk A, van Meijgaarden KE, Franken KL et al. Immunological crossreactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis. Scand J Immunol, 2004; 59: 6670. Chegou NN, Black GF, Kidd M et al. Host markers in QuantiFERON supernatants differentiate active TB from latent TB infection: preliminary report. BMC Pulm Med, 2009; 9: 21. Ruhwald M, Bjerregaard-Andersen M, Rabna P et al. IP-10, MCP-1, MCP-2, MCP-3, and IL-1RA hold promise as biomarkers for infection with M. tuberculosis in a whole blood based T-cell assay. BMC Res Notes, 2009; 2: 19. Duthie MS, Truman RW, Goto W et al. Insight toward early diagnosis of leprosy through analysis of the developing antibody responses of Mycobacterium leprae-infected armadillos. Clin Vaccine Immunol, 2011; 18: 254 259. Maeda Y, Mukai T, Kai M. Evaluation of major membrane protein-II as a tool for serodiagnosis of leprosy. FEMS Microbiol Lett, 2007; 272: 202205. Kai M, Nguyen Phuc NH, Hoang Thi TH et al. Serological diagnosis of leprosy in patients in vietnam by enzymelinked immunosorbent assay with Mycobacterium leprae-derived major membrane protein II. Clin Vaccine Immunol, 2008; 15: 17551759. Rada E, Ulrich M, Aranzazu N et al. A follow-up study of multibacillary Hansens disease patients treated with multidrug therapy (MDT) or MDT immunotherapy (IMT). Int J Lepr Other Mycobact Dis, 1997; 65: 320 327. Roche PW, Britton WJ, Failbus SS. Serological monitoring of the response to chemotherapy in leprosy patients. Int J Lepr Other Mycobact Dis, 1993; 61: 3543. Silva EA, Iyer A, Ura S et al. Utility of measuring serum levels of anti-PGL-I antibody, neopterin and C-reactive protein in monitoring leprosy patients during multi-drug treatment and reactions. Trop Med Int Health, 2007; 12: 14501458. Silva RC, Lyon S, Araos R et al. The result patterns of ML Flow and ELISA (PGL-I) serologic tests in leprosyendemic and non-endemic areas. Rev Soc Bras Med Trop, 2008; 41(Suppl 2): 1922.