Progress towards development of immunoassays for detection of Mycobacterium leprae infection, employing 35kDa antigen: an update

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1 Lepr Rev (2002) 73, 9±19 REVIEW Progress towards development of immunoassays for detection of Mycobacterium leprae infection, employing 35kDa antigen: an update OM PARKASH Immunology Laboratory, Central JALMA Institute for Leprosy, Tajganj, Agra , India Accepted for publication 23 November 2001 Summary The 35kDa antigen of Mycobacterium leprae is a membrane component that contains both B and T-cell stimulating epitopes. Monoclonal antibodies, primarily speci c to M. leprae, have been developed against this antigen. Moreover, this antigen has been genetically engineered. Using recombinant 35kDa antigen and/ or a monoclonal antibody against an epitope on 35kDa, a variety of antibody/antigen detecting tests have been described for detection of M. leprae infection. 35kDa protein also stimulates peripheral blood mononuclear cells (PBMCs) from the majority of paucibacillary (PB) patients. Approaches using combined antibody and T cell are discussed. Introduction Leprosy, a disease caused by Mycobacterium leprae, has been classi ed into various clinical forms resulting in a spectrum. 1 At one pole of the spectrum lies tuberculoid leprosy (TT) while at the other lies lepromatous leprosy (LL). Between these two polar types, various borderline forms [i.e. borderline tuberculoid (BT), mid-borderline (BB), borderline lepromatous leprosy (BL)] have been described. In tuberculoid leprosy there is a localized skin and/or nerve lesion(s), with a low bacterial load and strong cell mediated immunity. In contrast, lepromatous leprosy has generalized lesions with a high bacterial load and strong humoral immunity. To assign the patients to treatment, WHO has classi ed 2,3 leprosy into two groups, namely, paucibacillary (PB) and multibacillary (MB). Early detection and treatment of leprosy patients is essential to reduce the pool of infection and transmission of disease in community and also to reduce disability. The gold standard for diagnosis of leprosy remains clinical examination followed by con rmation by demonstration of acid fast bacilli (AFB) in slit-skin smears. 4 The main drawback of the skin smear technique is low sensitivity, due to low bacterial load, in BT/TT patients. Therefore, more sensitive and speci c assays to detect M. leprae antigen(s) and/or speci c host immune response(s) might complement or substitute this traditional laboratory method to support the Correspondence to: O. Parkash ( JALMA@nde.vsnl.net.in) /02/ $1.00 q Lepra 9

2 10 O. Parkash clinical diagnosis. Although molecular tests are far more sensitive 5,6 than conventional methods, their use in routine diagnosis of leprosy, especially for countries with low economic status, has been hindered because this technology requires skilled personnel, high cost and well equipped specialized laboratories. At this juncture, immunoassays remain an affordable alternative. This review assesses the developments made towards 35kDa-based immunoassays for detection of M. leprae infection. 35kDa protein of M. leprae The presence of 35kDa protein in leprosy bacillus has been established by a series of studies following immuno-biochemical puri cation techniques. 7±9 It is one of the membrane components of M. leprae and comprises 0.05% of the total Mycobacterium leprae protein. In 1995, the gene encoding the 35kDa protein of M. leprae was characterized 10 and successfully cloned in Mycobacterium smegmatis to produce recombinant 35kDa protein (r35kda). Since M. leprae is not cultivable in vitro and has to be isolated from infected tissues of armadillo, the major advantage of r35kda antigen is its availability. Though, the gene encoding the 35kDa protein is not found in the Mycobacterium tuberculosis complex or Mycobacterium bovis BCG, its homologue with 82.0% DNA and 90.0% amino acid identity, respectively, has been found in Mycobacterium avium. 11 Therefore, to make r35kda protein more speci c, cross-reactive protein moieties need to be removed. Since the 35kDa protein is an immunogen stimulating both B and T cell mediated immune responses, 9,12,13 it has been assessed as a reagent for detecting M. leprae infection. Detection of anti 35kDa antibodies in leprosy patients (Tables 1 and 2) Detection of anti-m. leprae antibodies is a widely used approach for detecting, indirectly, M. leprae infection. 14±16 Several versions of immunoassays for detection of anti-35kda antibodies have been described. 35kDa based immunoassays for detection of anti-m. leprae antibodies started with the production of monoclonal antibodies against M. leprae. MLO4 is a monoclonal antibody against M. leprae that reacts with an epitope on 35kDa antigen. Though it is primarily speci c to M. leprae, marginal reactivity with Mycobacterium kansasii, Mycobacterium avium and Mycobacterium paratuberculosis has also been reported. 17 Using this antibody, a novel inhibition radio-immunoassay (RIA) for detection of anti-35kda antibodies was reported by Sinha et al. 18 With this assay, the antibody positivity was observed to be 98.5% for MB and 46.7% for PB patients. 19 Sera belonging to 128 non-leprosy subjects (i.e. from patients with pulmonary tuberculosis, autoimmune disease, cancer and healthy individuals) were negative. In a further development, the use of whole blood on lter paper has made the test suitable for eld studies. 20 The monoclonal antibody (MLO4) used in the above mentioned assay was employed after labeling with isotope 125 I. This isotope has a short half-life, which results in instability of the reagents. Additionally, the isotope emits penetrating g radiation, which may be biohazardous. In contrast, enzyme-labelled antibodies are more stable and safe. Therefore, RIA was successfully substituted, 21±24 without any signi cant change in sensitivity and speci city, with more convenient and user-friendly inhibition enzyme linked immunosorbent assay (ELISA). The reliability of the assay has recently been evaluated. 25 The assay has been

3 Immunoassays using 35kDa antigen of M. leprae to detect leprosy 11 Table 1. A summary of performance of various immunoassays for detection of anti-35kda antibodies/antigens in serum samples from multibacillary leprosy patients Reference Patients Controls Sensitivity Speci city Ef ciency Assay (%) (%) (%) Antibodies in serum I-RIA I-ELISA I-ELISA I-ELISA RI-ELISA D-ELISA D-ELISA CT-ELISA Antibodies in CSF I-RIA Antibodies in urine I-RIA Antibodies in skin I-ELISA Antigen in serum S-RIA Antigen in skin IHS Antigen in CSF S-RIA Antigen in urine S-RIA I-RIA = inhibition radioimmunoassay; I-ELISA = M. leprae sonicate based inhibition enzyme linked immunosorbent assay; RI-ELISA = recombinant antigen based inhibition ELISA; D-ELISA = direct ELISA; CT-ELISA = card test ELISA; S-RIA = sandwich RIA; IHS = immunohistochemical stain. Ef ciency (index of usefulness) = (No. of true positive + no. of true negative)/total no. of subjects studied. found to be highly reproducible (100%) for strongly positive and negative sera, whereas, for sera containing antibodies near the cut-off point, the reproducibility was lower (79.0% for intra-assay and 77.0% for inter-assay). Hence, for accurate results, tests with such sera must be repeated at least three times. r35kda protein obtained by expression in M. smegmatis has been used for detecting antibody responses in sera from leprosy patients. 13 Following this, r35kda antigen was demonstrated to be suitable for its use in monoclonal inhibition ELISA. 26 The sensitivity for detection of MB patients was 90.0% (54/60), whereas for PB patients it was 17.0% (5/30). The speci city of the assay was 97.5%. The assay compared favourably with the M. leprae sonicate (a crude preparation of antigen) based inhibition ELISA. The same investigators have used a direct ELISA using r35kda antigen, where the sensitivities for MB and PB were 83.0 and 17.0%, respectively. The speci city of the assay was recorded to be 94.3%. Later, Roche et al. 27 showed the sensitivities of this direct ELISA to be 72.0 and 50.0% for MB and PB patients; the speci city was slightly lower (84.0%). The discrepancies in sensitivities and speci cities of these two direct ELISAs 26,27 could be due to variation in clinical diagnosis and in classi cation of patients. Very recently, employing r35kda antigen, a simple and rapid test card assay for detection of anti-35kda antibodies has also been developed. 27 The sensitivity of the test was 84.0% for MB and 59.0% for PB patients. The speci city of the assay was

4 12 O. Parkash Table 2. A summary of performance of various immunoassays for detection of anti-35kda antibodies/antigens in serum samples from paucibacillary leprosy patients Reference Patients Controls Sensitivity Speci city Ef ciency Assay (%) (%) (%) Antibodies in serum I-RIA RI-ELISA I-ELISA D-ELISA D-ELISA CT-ELISA Antibodies in urine I-RIA Antibodies in skin I-ELISA Antigen in serum S-RIA Antigen in skin IHS Antigen in urine S-RIA I-RIA = inhibition radioimmunoassay; I-ELISA = M. leprae sonicate based inhibition enzyme linked immunosorbent assay; RI-ELISA = recombinant antigen based inhibition ELISA; D-ELISA = direct ELISA; CT-ELISA = card test ELISA; S-RIA = sandwich RIA; IHS = immunohistochemical stain. Ef ciency (index of usefulness) = (No. of true positive + no. of true negative)/total no. of subjects studied. 85.0%. The reason for comparatively low speci city has been attributed to the contamination by mycobacterial components (derived from recombinant M. smegmatis) in the puri ed r35kda antigen. Alternatively, it could be due to presence of anti-m. leprae antibodies (due to subclinical M. leprae infection) in the controls. Though, the overall ef ciency of the assay is promising, ways to improve the performance are needed. Nevertheless, the test is simple, easy and can be performed using whole blood instead of serum. Thus, the rapid card test might be worth applying in places where there is a lack of skill for clinical diagnosis and no well-equipped laboratory. Additionally, in regions with low prevalence of leprosy, the test can be useful for timely/early diagnosis. Attempts have also been made to demonstrate anti-35kda antibodies in CSF, urine and slit-skin scrapes from leprosy patients. 28±30 However, these approaches were not found to be promising either due to poor sensitivity and/or speci city, even for the well established clinically diagnosed cases. Much evidence indicates the correlation of anti-m. leprae antibodies to bacillary load. 1,23,31 In the light of this information, the inhibition ELISA was evaluated for its utility in monitoring treatment in leprosy patients and results were encouraging. 22 During treatment, the decrease in antibody level largely paralleled the decrease in M. leprae load. Thus, measuring antibody level could help in monitoring the ef cacy of chemotherapy. In addition, it is thought that the elevation in antibody level might predict the non-responsiveness to treatment and rise of antibody level in treated patients might re ect the occurrence of relapse. However, to establish these assumptions, detailed studies are needed.

5 Immunoassays using 35kDa antigen of M. leprae to detect leprosy 13 Immunoassays for detection of 35kDa antigen of M. leprae in various specimens from leprosy patients Using antibody-detecting criteria, it is possible to detect infection but it is dif cult to differentiate between past and current infection. To determine an incubating infection, the test has to be repeated on a series of samples taken at various time intervals. An increasing trend in the antibody titre may point out the ongoing infection in the host. On the other hand, detection of antigen would indicate the current infection prevailing inside the body. In leprosy, efforts have been made to demonstrate 35kDa antigen in a variety of clinical samples. A summary of the performances of the various assays has been shown in Tables 1 and 2. In an investigation using sandwich radioimmunoassay, presence of 35kDa antigen in free and complex form was looked for in sera of leprosy patients. 32 Free antigen could be detected in 19.0% (3/16) of tuberculoid and 6.0% (1/18) of lepromatous patients. After sodium dodecyl treatment to release antigens from immune complexes, comparatively pronounced positivity (69.0% and 72.0%) for both untreated tuberculoid and lepromatous patients have been described. The ndings were speci c as none of the 14 tuberculosis patients and 22 healthy individuals was found to be positive. Though highly ef cient, the technique relies on dialysis and so would not be practical for routine purpose. However, it would be interesting to devise some simple and time saving method to break the immune-complexes for antigen detection preferably in more patients. Skin lesions are active sites harbouring M. leprae. 4 Mycobacterial antigens including M. leprae speci c ones have been found in skin tissues of leprosy patients. 33±35 There is only one report relating to demonstration of 35kDa antigen of M. leprae in skin tissues from leprosy patients. 36 For this purpose, immunoperoxidase staining technique involving MLO4 was adopted and 35kDa antigen was recognized in 100.0% (13/13) of the patients, out of which six belonged to BT/TT and seven to BL/LL form of the disease. None of the control tissue sections (i.e. from four normal skins, three lymph nodes from TB patients, and three skin tissues from patients with psoriasis) was positive. Though the approach seems to be highly encouraging for detection of M. leprae infection, it needs more studies for its establishment. Further, presence of 35kDa antigen in CSF and urine of leprosy patients has been reported by Patil et al. 37,38 However, due to weak sensitivity and/or speci city, these specimens do not seem promising for detection of M. leprae infection. Assays to measure cellular immunity against 35kDa antigen of M. leprae Cell mediated immunity is known to be dominant in tuberculoid patients. 1 Therefore, testing of sensitized T cells by M. leprae speci c antigen(s) could provide an alternative approach to detect M. leprae infection in such individuals. In fact, M. leprae is known to induce proliferation of human peripheral blood mononuclear cells (PBMCs), and interferon-g production mainly from tuberculoid patients. 39 However, studies for measurement of CMI, employing various preparations from M. leprae, have not been very successful for detection of M. leprae infection, the reason being lack of sensitivity and speci city. 40 Therefore, research has aimed at developing potential and speci c T cell stimulating reagent(s), as a substitute for crude M. leprae antigen.

6 14 O. Parkash Mohagheghpour et al. 9 gave the rst evidence for the T cell stimulating property of 35kDa protein, showing that this molecule could stimulate cells of TT/BT leprosy patients and M. bovis BCG-vaccinated healthy individuals. Another study 13 with r35kda antigen indicated proliferative response and interferon-g production with PBMCs, especially and predominantly from PB leprosy patients and healthy contacts of leprosy. For proliferation of PBMCs the percentages of responders were: 83.0% (10/12). 78.0% (14/18), 38.0% (5/13) and 20.0% (2/10) for contacts of leprosy, PB, MB and tuberculosis, respectively, whereas for interferon-g production the responders were 83.0% (10/12), 72.0% (13/18), 46.0% (6/13) and 40.0% (4/10) again in case of leprosy contacts, PB, MB and tuberculosis patients, respectively. The control group for these investigations comprised 10 BCG vaccinated but non-leprosy exposed healthy individuals whose cells did not respond. Further experiments on laboratory animals indicated that six guinea pigs sensitized with M. leprae responded to give delayed type of hypersensitivity response against 35kDa protein. In contrast, seven out of eight animals sensitized with M. tuberculosis and four out of ve sensitized with BCG, failed to show a response against r35kda. These results imply that r35kda antigen might prove to be a potential skin test reagent for detection of M. leprae infection. Since M. leprae 35kDa protein has close structural homologues in M. avium, 35kDa protein lacks speci city. To identify the speci c moieties, 14 synthetic peptides of 35kDa antigen were evaluated for stimulation of T cells from leprosy patients and healthy controls. 41 The basis for selection of these peptides was high af nity and permissive binding to HLA-DR molecules. Unfortunately, none of the peptides (including the cross-reactive ones) was noticed to be promising for its use in measurement (employing PBMCs proliferation and interferon-g assays) of CMI against M. leprae. Since 35kDa protein has been demonstrated to be a stimulator of CMI, the plausible reason for the failure of these peptides could be either the absence or the prevalence of low frequency of circulating T cells sensitized to these individual peptides, whereas whole molecule of 35kDa antigen has multiple T cell epitopes which together can induce detectable immune response. In comparison to in vitro tests, a skin test for measuring CM1 would be less convenient as after injection of the antigen the patients have to revisit the clinic (3/21 days for early/delayed response respectively) for the response to be measured. It may cause large dropouts. Therefore, it would be wise to evaluate the 24-h whole blood culture to detect interferon-g production against 35kDa antigen. Such approaches have already been developed for bovine and human tuberculosis. 42,43 By doing so, the results can be available within 2 days. Furthermore, use of phytohaemagglutinin in such cultures could help in identifying the non-responders due to immunosuppression as in AIDS patients. Combined studies between 35kDa based and other immunoassays for detection of M. leprae infection Phenolic glycolipid-1 (PGL-1), a unique component of M. leprae, has also been studied extensively for detection of M. leprae infection. 14±16 Since biochemical natures of both of these components are different, antibody responses against these antigens show some heterogeneity between the individuals. 21,23,24 Therefore, combining the ndings of immunoassays (employing 35kDa and PGL-1) to increase the overall performance is an interesting idea. The potential of such a combinatorial (positivity by either one or both the assays) approach is illustrated by the following reports. The decrease or increase in sensitivities and

7 Immunoassays using 35kDa antigen of M. leprae to detect leprosy 15 Table 3. A summary of performance of combined approaches for detection of anti35kda antibodies in sera from multibacillary leprosy patients Reference Assay Sensitivity Speci city Ef ciency (%) (%) (%) 23 I-ELISA P-ELISA I-ELISA P-ELISA 27 D-ELISA P-ELISA D-ELISA P-ELISA 27 CT-ELISA PD-ELISA CT-ELISA PD-ELISA I-ELISA = M. leprae sonicate based inhibition ELISA; D-ELISA = direct ELISA; P-ELISA = phenolic glycolipid (PGL) based ELISA; PD-ELISA = PGL-based dipstick ELISA. Ef ciency (index of usefulness) = (No. of true positive + no. of true negative)/ Total no. of subjects studied. speci cities of these approaches has been computed with reference to the higher values of sensitivities and speci cities, respectively, in the respective combination groups (Tables 3 and 4). Two independent studies 23,24 from same laboratory have analysed antibody responses to 35kDa and PGL-1 in MB and in PB patients. With MB patients, the sensitivity of combined strategy between 35kDa based MLO4 inhibition ELISA and PGL-ELISA showed 5.0% rise over the more sensitive MLO4 based ELISA without any change in speci city. For PB patients, the enhancement in sensitivity was of the order of 10.0%. Roche and colleagues 27 have published their ndings on a combination between r35kda inhibition ELISA and PGL- ELISA. This approach resulted in an improvement (18.0 and 13.0% for MB and PB, respectively) in sensitivity, while decrease in speci city was of the order of 8.5%. In the same report, the combination between two simple and rapid tests (viz. a rapid test card assay to detect anti-35kda antibodies and a dipstick assay to detect anti-pgl-1 antibodies) caused 4.0% increase in sensitivity for detection of MB as well as PB patients while there was 3.5% decrease in speci city. In summary, on combination of 35kDa and PGL-1 based assays, the increase in sensitivity ranged from 4.0 to 18.0%, whereas decrease in speci city varied from 0 to 8.5%. Thus, gains in combinatorial approaches between these serological assays do not seem to be appreciable. Nevertheless, these strategies might help in detection of 4±18% (depending upon the combination) more cases of leprosy. In leprosy there exists a dichotomy between antibody and cellular immune responses against 35kDa antigen of M. leprae. MB patients have strong antibody response and weak cellular immune response and vice versa for PB patients. 9,13,19,26 Therefore, assays to detect antibody or cellular immunity against M. leprae cannot detect all types of leprosy patients.

8 16 O. Parkash Table 4. A summary of performance of combined approaches for detection of anti35kda antibodies in sera from paucibacillary leprosy patients Reference Assay Sensitivity Speci city Ef ciency (%) (%) (%) 24 I-ELISA 33.0 ± ± 24 P-ELISA 20.0 ± ± I-ELISA ± ± P-ELISA 27 D-ELISA P-ELISA D-ELISA P-ELISA 27 CT-ELISA PD-ELISA CT-ELISA PD-ELISA I-ELISA = M. leprae sonicate based inhibition ELISA; D-ELISA = direct ELISA; P-ELISA = phenolic glycolipid (PGL) based ELISA; PD-ELISA = PGL-based dipstick ELISA. Ef ciency (index of usefulness) = (No. of true positive + no. of true negative)/ Total no. of subjects studied. Hence, a combined approach between assays to detect antibody as well as cellular responses might enhance the sensitivity. Interestingly, when B and T cell responses (as seen by antibody reactivity and proliferation of PBMCs) to 35kDa antigen were considered together, 13 more than 90.0% (in contrast to positivity rates of 58.0% for CMI and 41.5% for antibody responses in leprosy patients i.e. MB + PB patients) of leprosy patients recognized the antigen (Table 5). On the other hand, only 20.0% of the tuberculosis patients responded which may be due to their earlier exposure to M. leprae. Thus, this combined approach seems to make dramatic improvements for detection of M. leprae infection. Since sensitivity of this approach is better than either antibody or cellular response, it may act as a valuable tool to support the clinical diagnosis and for epidemiological studies. Table 5. Results of combined cell-mediated and antibody response against 35kDa antigen of M. leprae in leprosy patients Reference Assay Sensitivity Speci city Ef ciency (%) (%) (%) 13 Lymphoproliferation D-ELISA Lymphoproliferation 13 + > D-ELISA D-ELISA = direct ELISA. Ef ciency (index of usefulness) = (No. of true positive + no. of true negative)/total no. of subjects studied.

9 Immunoassays using 35kDa antigen of M. leprae to detect leprosy 17 Table 6. Detection of leprosy contacts by various immunoassays Reference Assay Subjects Positivity (%) 19 I-RIA CT-ELISA D-ELISA D-ELISA Lymphoproliferation Interferon g production Conclusions I-RIA = inhibition radioimmunoassay; CT-ELISA = card test ELISA; D-ELISA = direct ELISA. It is evident from the above-described data, that the performances (as judged by criteria like sensitivity, speci city and ef ciency) of anti-35kda antibody detecting inhibition ELISA/ RIA for MB patients are very high. Regarding PB patients, the ef ciency of inhibition RIA was best. Combined approaches between various serological assays caused some improvement in sensitivities but at the cost of slight decrease in speci cities. However, the drawback with all the serological assays has remained poor performance for detection of PB patients. Thus, antibody-detecting assays have limited supportive application in clinical diagnosis. Besides such an application, the antibody detecting assay promises to act as a tool to investigate the progress during treatment. A completely different eld of investigation in which 35kDa based serology has not been applied, so far, is epidemiology, where the extent and transmission of the M. leprae infection could be studied which in turn might help in monitoring disease control. An ideal test for detection of M. leprae infection would be one that would be capable of diagnosing all types of leprosy patients. The lack of such sensitivity for serological assays has prompted the researchers to develop alternative strategies. The promising but indicative ndings obtained out of three investigations: (i) detection of 35kDa antigen in serum, 30 (ii) detection of antigen in skin 35 and (iii) measuring antibody as well as cell mediated immune responses simultaneously 13 give hope for some future test(s) that would cover the majority of MB and PB type of patients. Nevertheless, critical assessment is warranted on these lines. Additionally, both lymphoproliferation and interferon-g production could detect 83.0% of contact cases, which was highest rate among all the assays (Table 6). Therefore, these assays may be better tools for contact tracing especially in low endemic regions. In regions of low endemicity, positive individuals can be followed up for progress of the infection. Lastly, whatever approach is used, the emphasis must be laid on development, improvement and use of simple as well as cost effective assays. Such tests are very much required for their use in non-specialized peripheral health facilities and for epidemiological/operational studies, particularly in developing/underdeveloped countries. Acknowledgements I wish to extend my thanks to Dr U. Sengupta for his critical suggestions extended for preparation of the manuscript.

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