OM PARKASH Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, TajGanj, Agra-1, India

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1 Lepr Rev (2011) 82, Serological detection of leprosy employing Mycobacterium leprae derived serine-rich 45 kda, ESAT-6, CFP-10 and PGL-I: a compilation of data from studies in Indian populations OM PARKASH Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, TajGanj, Agra-1, India Accepted for publication 02 September 2011 Summary This article is acompilation of our findings recorded in the recent past where we have investigated the serological performance of Mycobacterium leprae antigens like-serine-rich 45 kda protein (45 kd), early secretary antigenic target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) and phenolic glycolipid-i (PGL-I) for detection (employing antibody detecting ELISA) of leprosy patients, particularly those belonging to the paucibacillary (PB) group. All of these antigens were capable of detecting, by themselves the majority (82 100%) of multibacillary (MB) patients. However, with respect to PB patients, only 18 47% (i.e. less than half) of the cases could be detected. Based on the results of serological assays for each of the four antigens separately acombinatorial approach was performed for these antigens, which increased the sensitivity for detection of PB patients to 73%, giving 36% improvement over conventional PGL-I based ELISA. Thus, the multi-antigenic serological approach is worthwhile for its establishment for detection of leprosy patients. Since ESAT-6 and CFP-10 are secreted proteins by nature, antibodies against them are worth exploring for detection of early infections and for monitoring of treatment efficiency. Nevertheless, efforts towards identification of more new antigens with serological potential are still desirable in order to further improve the detection rate of leprosy. Introduction Routinely, leprosy is diagnosed based on clinical criteria recommended by World Health Organization. 1 According to this, people with one or more ofthe following characteristic symptoms are considered leprosy patients: hypopigmented or reddened skin lesion(s) with definite loss of sensation and/or involvement of the peripheral nerves and skin smear positivity for acid-fast bacilli. Unfortunately, in a significant number of cases even Correspondence to: Om Parkash, Department of Immunology, National JALMA Institute for Leprosy and Other Mycobacterial Diseases, TajGanj, Agra-1, India ( om1234@gmail.com; op_v3@yahoo.com) /11/ $1.00 q Lepra 383

2 384 O. Parkash an experienced physician fails to diagnose leprosy due to inherent difficulties in clinical diagnosis. Moreover, integration of leprosy with general health services has resulted in non-availability of sufficiently experienced health workers to diagnose leprosy. 2 Therefore, development of diagnostic test to complement the clinical diagnosis still remains a research priority. Bacteriologically, leprosy has been grouped as highly bacillated [smear positive for acid fast bacilli; Mycobacterium leprae (M. leprae)] infectious form called multibacillary (MB) and less infectious form having a low load of M. leprae [smear negative for acid fast bacilli; M. leprae ]calledpaucibacillary (PB). 3 Further, antibody response is knowntocorrelate with the bacterial load in leprosy patients. 4 Generally, assays based on the detection of specific antibodies in serum are considered attractive, as they are simple, cost effective and easily implementable in conditions faced in developing countries. In this context, phenolic glycolipid-i (PGL-I) based antibody detecting assay has remained the most widely studied serological tool for detection of M. leprae infection (as evidenced by several other papers in this issue). Paucibacillary (PB) patients are a group in which, generally, the sensitivity for detection of anti- M. leprae specific antibodies remains low i.e %. 5 At our laboratory we have investigated, in the recent past, the serodiagnostic potential of serine-rich 45 kda protein (45 kda; ML0411), early secreted antigenic target-6 (ESAT-6; ML0049), culture filtrate protein 10 (CFP-10; ML0050) and phenolic glycolipid I (PGL-I) antigens. 6 8 Below, a brief review is provided describing serological data obtained with these four M. leprae antigens. 45 KDA ANTIGEN In 1993, Vegas-Lopez et al. 9 isolated and sequenced agene encoding for aserine rich 45 kda protein. The gene was expressed to produce a beta-galactosidase fusion protein. Using this fusion protein, 78% of serum samplesfrom MB patients and 68% of serum samplesfrom PB leprosy patients could be detected, indicating that 45 kda protein is a major M. leprae antigen. In contrast, all serum samples from endemic controls were negative, while 26% of the serum samples from tuberculosis (TB) patients were weakly positive. In southern hybridization experiments the DNA from Mycobacterium tuberculosis and atypical mycobacteria failed to hybridize with the 45 kda antigen encoding DNA probe which highlighted the specificity of the gene and thereby of its corresponding protein. Further, western blotting analysis in the same investigation showed 10 that M. leprae 45 kda protein was frequently recognised by antibodies from leprosy patients. This seroreactivity was specific since antibodies could not be detected in sera of TB patients. Summarised, these findings indicate that 45 kda based assays are likely M. leprae-specific and warrant further exploration. ESAT-6 ANDCFP-10 Recent publications on the genome sequences of M. tuberculosis 11 and M. leprae 12 have provided unprecedented opportunities to identify M. leprae unique proteins. In this process, homologues of M. tuberculosis derived ESAT-6 (Rv3874) and CFP-10 (Rv3875) have also been identified in M. leprae. M. leprae ESAT-6 and CFP-10 encoding genes (ML0049 and ML0050 respectively) are located one after the other in the same operon, homologous to their organisation in the M. tuberculosis genome. The sequence identity of M. leprae ESAT-6 and CFP-10 with their M. tuberculosis homologues is 36% and 40%, respectively. 13,14

3 Serological detection of leprosy patients 385 At the T cell level both M. leprae proteins (ESAT6; ML0049 and CFP-10; ML0050) have been documented to be potent antigens to stimulate T cell dependent IFN- g production in a large proportion of individuals exposed to M.leprae. 13,14 However, there is asignificant cross-reactivity (at the level of IFN-g production) between M. leprae ESAT-6 and CFP-10 as well as those of M. tuberculosis ESAT-6 and CFP ,14 In contrast, Spencer et al. 15,16 reported on the B cell stimulating potential of both of these M. leprae antigens. We considered these as encouraging findings togive usa lead towards pursuing the investigations on the potential of recombinant M. leprae ESAT-6 and CFP-10 for serodiagnosis ofleprosy. PHENOLIC GLYCOLIPID-1 In 1981, the identificationand characterisation of M. leprae specific glycolipid, phenolic glycolipid-i was reported, for the first time, by Hunter et al. 17 The oligosaccharide segment of this molecule is a species specific terminal trisaccharide: 3,6-di-O-methyl- b -D-glucopyranosyl-(1! 4)-2,3-di-O-methyl-a-L-rhamnopyranosyl-(1! 2)-3-O-methyl- a -L-rhamnopyranose. Payne et al. reported its antigenic nature in Both di- and trisaccharide parts of the molecule are principal antigenic determinants of PGL-I. 19,20 As M. leprae cannot be cultured in-vitro, production of sufficient quantities of PGL-Ifrom M. leprae was problematic. To overcome this problem, semi-synthetic derivatives were produced by linking the terminal di or trisaccharide part of PGL-Itoaprotein carrier such as bovine serum albumin (BSA) or human serum albumin (HSA), resulting in natural disaccharide linked to BSA/ HSA via an octyl linker (ND-O-BSA/HSA) and natural trisaccharide linked to BSA/ HSA via aphenyl linker (NT-P-BSA/HSA). 21 Over the years, using these semi-synthetic derivatives various typesofserological assays for detection of M. leprae infection have been designed. 5 In our studies, we have included ND-O-HSA (an analogue of PGL-I) to compare the performance of 45 kda or ESAT-6orCFP-10 protein with that of PGL-Ibased assays. In addition, we explored the use of PGL-Iincombination with theseotherthree proteins, with the aim to improve the detection rate of leprosy. PERFORMANCEOFANTIGENSFOR DETECTIONOFLEPROSYPATIENTS In our studies, recombinant proteins derived from serine-rich 45 kda antigen of M. leprae, 6 ESAT-6 7 and CFP-10 8 demonstrated to be serologically useful antigens. Regarding these three antigens, our reports are in line with several findingsindicating that the 45-kDa, 9,10,22,23 ESAT-6 15,22 24 and CFP-10 16,22 25 are potent B-cell stimulating antigens and that their serological behaviours are highly specific for leprosy. Among these antigens, the performance of the serine-rich 45 kda antigen was found to be the most impressive (sensitivity: 100% in MB and 47 4% in PB; specificity: 100%), 6 whereas that of phenolic glycolipid-i (PGL-I, the most widely used antigen for serological studies in leprosy) was slightly lower (sensitivity: 94 4% in MB and 36 8% in PB; specificity: 96%). 6 The performances of ESAT-6 (sensitivity: 82 4% in MB and 19 4% in PB; specificity: 100%), 7 and CFP-10 (sensitivity: 83 3% in MB and 18 4% in PB; specificity: 98%) 8 were less sensitive, but nonetheless very specific. Thus, all of these antigens (45 kda, ESAT-6 and CFP-10) were highly efficient for detection of MB patients, although they failed to detect the majority (53 82%) of PB patients (sensitivity varied from 18% 47%). Nevertheless, combination of results obtainedwith PGL-Iand those with 45 kda or ESAT-6 or CFP-10 further improved the detection rate: all three antigens showed improved detection capacity when combined with PGL-I (45 kda þ PGL-I,

4 386 O. Parkash ESAT-6 þ PGL-I and CFP-10 þ PGL-I). 6 8 With respect to PB patients the performance was mostefficient and statistically significant (sensitivity: 60 5%; specificity: 96%) when the combined results of 45 kda þ PGL-I were used resulting in an improvement of 23 7% over PGL-I alone. 6 Further analysis of the results indicated a serological heterogeneity against various antigens(pgl-i, 45 kda, ESAT-6and CFP-10). 26 The heterogeneity among MB patients was lower (29 4%) in comparison to that (69 7%) in PB patients. Prompted by this differential serological response, we analysed the results in a combined fashion considering the results of all four antigens together. Interestingly, the sensitivity for detection of PB patients rose to 73% which represented a significant (21 54% improvement in sensitivity with 94% specificity) when compared to either PGL-I or ESAT-6 or CFP-10 based assays. However, there was 21% improvement in detection rate over that of 45 kda antigen. From all these observations, it appears that multi-antigenic testing for detection of antibodies could help in detecting larger number of leprosy patients. In our studies, there were some cases when antibodies were not detectable despite the obvious presence of M. leprae inside the host. 6 7 This could bedue to situations where antibodies may not be freely available due to antigen antibody complexformation and due to genetic make-up of some individuals where antibody response against a particular antigen may be absent or it may be too weak for detection. PERSPECTIVES Since 1985 the globalleprosy programme has remained appreciably successfulrecording treatment of more than 14 million leprosy patients resulting in more than 90% fall in global case-load. However, approximately250,000 new casesofleprosy are still recorded annually indicating that the chain of transmission has notbeen broken and that interruption of leprosy transmission is one of the main challenges for leprosy control programmes. Interms of case load: India, Brazil, Indonesia, Bangladesh, Democratic Republic of the Congo, Ethiopia, Nepal and Nigeria top the list respectively. 27 With respect to India, though this country has achieved the goal of elimination (i.e. prevalence rate of less than one case per populations) of leprosy as apublic health problem at national level indecember 2005, higher prevalence {about 1 2/ as against the national prevalence rate (0 71/10 000) as documented on 1 st April 2009} ofleprosy is reported in some areas. 28 Moreover, considering newly detected cases (133,717) and existence of registered cases (87 190) of leprosy, India holds more than 50% of the global leprosy burden (new case detection ¼ 244,796; registered prevalence ¼ 211,903). 27 These numbers reflect that leprosy actually will remain apublic health problem in India for several more years to come. This highlights the need for continued commitment towards control of the disease and to leprosy researchinendemiccountries including India. Thecurrent strategy for combating leprosy is based on the early detection and treatment of patientstohalt transmission. Hence, there is need of asimple, sensitive, specific and cost effective test for diagnosis ofleprosy. Since serological assays are easy and affordable, they may prove to be potential alternatives for the conditions prevalent in mostdeveloping countries. By adopting the various approaches outlined above, we could detect, specifically, 21 54% more of the PB patients. Hence, the strategies reportedbyusare worthwhile evaluating before their incorporation into the diagnostic algorithm for patients who have clinical signs suggestive of active leprosy.

5 Serological detection of leprosy patients 387 ESAT-6 and CFP-10, being secreted proteins, may be produced in the early active phase of infection that in turn may give rise to early antibody responses. Consequently, serological assay(s) developed using M. leprae ESAT-6 and CFP-10 may be of use towards early detection of M. leprae infection and thereby in management and control of leprosy. Further, ESAT-6and CFP-10 would be produced only by metabolically active viable Mycobacterium leprae growing inside the host. Therefore, detecting antibodies against ESAT-6 and CFP-10 may provide a better alternative marker for monitoring the anti-leprosy treatment. Considering this hypothesis, it would be interesting to evaluate ESAT-6 and CFP-10 based serology for monitoring chemotherapy in seropositive (mostly multibacillary) leprosy patients. However, aneed for identification of otherpotential antigensisstill felt in search of development of more sensitive serological assays. Acknowledgements The author wishes to thank the Indian Council of Medical Research, New Delhi, India, and National JALMA Institutefor Leprosyand Other Mycobacterial Diseases (ICMR), India, for financial support for the studies described in this review. References 1 World Health Organization. WHO Expert Committee on Leprosy. World Health Organ Tech Rep Ser,1988; 874: Siddiqui MR, Velidi NR, Pati S et al. Integration of leprosy eliminationinto primary health care in Orissa, India. PLoS One, 2009; 4 : e World Health Organization. Expert Committee on Leprosy: fifth report. Technical report series 678. WHO, Geneva, Ridley DS, Jopling WH. Classification of leprosy according toimmunity. Afive-group system. Int JLep and Other Mycobact Dis, 1966; 34: Oskam L, Slim E, Bührer-Sékula S. Serology: recent developments, strengths, limitations and prospects: astate of the art overview. Lep Rev, 2003; 74: Parkash O, Kumar A, Nigam A et al. Evaluation of recombinant serine-rich 45-kDa antigen (ML0411) for detection of antibodies in leprosy patients. Scand J Immunol, 2006; 64: Parkash O, Pandey R, Kumar A, Kumar A. Performanceofrecombinant ESAT-6 antigen (ML0049) for detection of leprosy patients. Lett Appl Microbiol, 2007; 44: Parkash O, Kumar A, Nigam A, Girdhar BK. Detection of antibodies against Mycobacterium leprae culture filtrate protein-10 in leprosy patients. J Med Microbiol, 2006; 55: Vega-Lopez F, Brooks LA, Dockrell HM et al. Sequence and immunological characterization ofaserine-rich antigen from Mycobacterium leprae. Infect Immun, 1993; 61: Rinke de Wit TF, Clark-Curtis JE, Abebe F et al. A Mycobacterium leprae specific gene encoding an immunologically recognized 45 kda protein. Mol Microbiol, 1993; 10: Cole ST, Brosch R, Parkhill J et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature, 1998; 393: Cole ST, EiglmeierK,Parkhill J et al. Massive gene decay in the leprosy bacillus. Nature,2001; 409: Geluk A, van Meijgaarden KE, Franken KLMC et al. Identification and characterization of ESAT-6 homologue of Mycobacterium leprae and T-cell cross reactivity with Mycobacterium tuberculosis. Infect Immun, 2002; 70: Geluk A, van Meijgaarden KE, Franken KLMC et al. Immunological cross reactivity of the Mycobacterium leprae CFP-10 with its homologue in Mycobacterium tuberculosis. Scand J Immunol, 2004; 59: Spencer JS, Marques MM, Lima MCBS et al. Antigenic specificity of the Mycobacterium leprae homologue of ESAT-6. Infect Immun, 2002; 70: Spencer JS, Kim HJ, Marques AM et al. Comparative analysis of Band T-cell epitopes of Mycobacterium leprae and Mycobacterium tuberculosis culture filterate protein-10. Infect Immun, 2004; 72: Hunter SW, Brennan PJ. A novel phenolic glycolipid from Mycobacterium leprae possibly involved in immunogenicity and pathogenicity. JBacteriol, 1981; 147:

6 388 O. Parkash 18 Payne SN, Draper P, Rees RJ. Serological activity of purified glycolipid from Mycobacterium leprae. Int JLep and Other Mycobact Dis, 1982; 50: Brett SJ, Payne SN, Draper P, Gigg R. Analysis of the major antigenic determinantsofthe characteristic phenolic glycolipid from Mycobacterium leprae. Clin Exp Immunol, 1984; 56: Fujiwara T, Hunter SW, Cho SN et al. Chemical synthesis and serology of disaccharides and trisaccharides of phenolic glycolipid antigens from the leprosy bacillus and preparation of a disaccharide protein conjugate for serodiagnosis of leprosy. Infect Immun, 1984; 43: Chatterjee D, Cho SN, Brennan PJ, Aspinall GO. Chemical synthesis and seroreactivity of O-(3, 6-di-O-methylbeta-D-glucopyranosyl)-( )-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)-( )-oxynonanoyl-bovine serum albumin the leprosy-specific, natural disaccharide-octyl-neoglycoprotein. Carbo Res, 1986; 156: Duthie MS, Ireton GC, Kanaujia GV et al. Selection of antigens and development of prototype tests for point-ofcare leprosy diagnosis. Clin Vaccine Immunol, 2008; 15: Duthie MS, Hay MN, Morales CZ et al. Rational design and evaluation of amultiepitope chimeric fusion protein with the potential for leprosy diagnosis. Clin Vaccine Immunol, 2010; 17: 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: Spencer JS, Kim HJ, Wheat WH et al. Analysis of antibody responses to Mycobacterium leprae phenolic glycolipid I, lipoarabinomannan, and recombinant proteins to define disease subtype-specific antigenic profiles in leprosy. Clin Vaccine Immunol, 2011; 18: Parkash O, Kumar A, Pandey R et al. Serological heterogeneity against various Mycobacterium leprae antigens and its use in serodiagnosis of leprosy patients. J Med Microbiol, 2007; 56: World Health Organization. Global leprosy situation, Wkly Epidemiol Rec, 2009; 85: