Serodiagnosis of Leprosy: Relationships between Antibodies to Mycobacterium leprae Phenolic Glycolipid I and Protein Antigens

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 198, p /8/12917-$2./ Copyright 198, American Society for Microbiology Vol. 24, No. Serodiagnosis of Leprosy: Relationships between Antibodies to Mycobacterium leprae Phenolic Glycolipid I and Protein Antigens WILLIAM R. LEVIS,'* HARRY C. MEEKER,2 GEORGIA B. SCHULLER-LEVIS,2 T. P. GILLIS, LOUIS J. MARINO, JR.,4 AND JOHN ZABRISKIE4 Department of Dermatology, Bayley-Seton Hospital, New York Medical College,1 and Laboratory of Immunology,2 New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 114; Gillis W. Long Hansen's Disease Center, Carville, Louisiana 7721; and Rockefeller University, New York, New York 1214 Received February 198/Accepted 19 August 198 Sera from leprosy patients and controls were assayed for immunoglobulin M (IgM) and IgG antibodies to the Mycobacterium leprae-specific phenolic glycolipid I antigen (PG) by enzyme-linked immunosorbent assay, for IgG antibodies to M. leprae protein antigens by Western immunoblot, and for antibodies to a -kilodalton (kda) protein antigen of M. leprae by a competition antibody binding assay. Elevated levels of anti-pg IgM were seen in lepromatous and borderline lepromatous patients, and elevated levels of anti-pg IgG were seen in borderline lepromatous patients. There was a significant correlation between the bacillary index (BI) and anti-pg IgM whether all leprosy patients or only multibacillary patients were analyzed. A significant correlation was seen between anti-pg IgG and BI when all leprosy patients were used for analysis, but not when only multibacillary patients were used. IgG antibodies to protein antigens of M. leprae, as detected by Western immunoblot, were more prevalent in lepromatous and borderline lepromatous patients than in borderline tuberculoid patients, while one of eight controls showed one weak band. There were significant correlations between the number of M. leprae protein antigens detected by the sera of patients and both BI and the level of anti-pg IgM. The -kda competition antibody binding assay detected active multibacillary leprosy. Patients positive for antibody to the -kda antigen had a significantly higher BI and levels of anti-pg IgM and anti-pg IgG than did patients that were negative. In addition, the level of antibody to the -kda antigen correlated with both the BI and anti-pg IgM. We conclude that testing for antibodies to protein antigens of M. leprae may provide a useful adjunct to testing for antibodies to PG. Several major advances have taken place in the serodiagnosis of leprosy. Immunoglobulin M (IgM) class antibodies to a Mycobacterium leprae-specific glycolipid have been described previously (2, 7, 12, 2). In addition to promising a test with some degree of specificity for detecting new cases, the M. leprae-specific glycolipid is also useful for monitoring disease activity (1, 19). While the glycolipid antibodies represent a significant advance, reactivity occurs in about 4 to % of a healthy U.S. population (, 1) and is even higher in endemic and hyperendemic leprosy areas (T. M. Buchanan and D. B. Young, unpublished data). Comparable serologic progress is required for M. lepraederived protein antigens and may provide necessary adjunctive serologic tests in concert with glycolipid antibodies. In this study, we demonstrate by Western immunoblot and a competition antibody binding assay (CABA) that armadillo-derived M. leprae and a -kilodalton (kda) M. leprae protein antigen show major seroreactivity in patients with active lepromatous leprosy (LL). Relationships among the bacillary index (BI), immunoblot profiles, seroreactivity of the -kda antigen, and glycolipid antibodies were evaluated. MATERIALS AND METHODS M. leprae isolated from the livers of infected armadillos (11) was generously provided by P. J. Brennan. Leprosy patients were clinically and histologically classified by the * Corresponding author. 917 Ridley-Jopling scale (18). For the purposes of some analyses, LL and borderline lepromatous (BL) patients were combined as multibacillary, while midborderline and borderline tuberculoid (BT) patients were combined as paucibacillary. The BI, an estimation of the total bacillary load in leprosy patients, was measured on a semiquantitative scale ( to +) approximating that of Ridley (17) by the following criteria: BI of = no acid-fast bacilli (AFB) per 1 oil immersion fields (OIF); 1+ = 1 to 1 AFB per 1 OIF; 2+ = 1 to 1 AFB per 1 OIF; + = ito 1 AFB per OIF; 4+ = 1 to 1 AFB per OIF; + = 1 to 1, AFB per OIF; + = 1, or more AFB per OIF. Histology and BI were determined from punch biopsies at the Gillis W. Long Hansen's Disease Center, Carville, La. An earlier study indicated a good correlation between two pathologists (C. K. Job and J. A. Freeman) in quantitation of the BI (r =.94, n = 29, P <.1) (19). Serum samples were collected from leprosy patients, contacts of patients, and unexposed healthy control subjects and stored in aliquots at -7 C. The immunoblot technique was carried out essentially as described by Blake et al. (1). Briefly, lyophilized M. leprae preparations were suspended in phosphate-buffered saline (PBS) at 2 mg/ml. From this,. mg was mixed in,ul of double-strength sample buffer and loaded onto a 12.% gel. The material was electrophoresed for h at 4 ma and then transferred across to nitrocellulose paper for 1 h at 1 A. The nitrocellulose paper was cut into strips, and each strip was incubated with the appropriate human serum (1:1 dilution) for 2 h at room temperature. After appropriate washes, the strips were placed in the enzyme-conjugated anti-human IgG for 1 h, followed by the appropriate substrate-dye mixture. Downloaded from on January, 219 by guest

2 918 LEVIS ET AL FIG. 1. Western immunoblot for antibodies to M. leprae protein antigens. Lanes: 1 to 4, LL sera; to 7, BT sera; 8 to 1, control sera; 11, PBS. Lane shows two weak bands. Lanes 1, 2, and 4 show strong bands. Antibodies to the phenolic glycolipid (PG) antigen of M. leprae were detected by enzyme-linked immunosorbent assay as previously described (1). PG (provided by P. J. Brennan) was incorporated into liposomes with sphingomyelin, cholesterol, and dicetyl phosphate. Control liposomes were made without PG. PG and control liposome suspensions were coated onto polystyrene microtiter plates (Dynatech Laboratories, Inc., Alexandria, Va.) (2. gig of PG per ml) and incubated for 18 h at 7 C. After the plates were coated, they were washed three times with PBS and then blocked for 1. h with PBS plus % bovine serum albumin (PBS-% BSA). Sera diluted 1:2 in PBS-% BSA were added in duplicate to wells containing PG liposomes and control liposomes and incubated for 1. h at 7 C. Plates were then washed three times with PBS, and goat antihuman IgM (or IgG) peroxidase conjugate (Cooper Biomedical, Inc., West Chester, Pa.) diluted 1:1, in PBS-% BSA was added for 1. h at 7 C. Plates were washed again, and a substrate solution was added (1.8 mm 2,2'-azino-di[- ethylbenzthiazoline--sulfonic acid] [Boehringer Mannheim Biochemicals, Indianapolis, Ind.]-.1 mm H22 in phosphate buffer) for 1 h at room temperature. The reaction was stopped with.2% NaF and extinction (E) was read at 4 nm. Results were expressed as AE = E (PG liposome coat) - E (control liposome coat). Sera with very high titers of anti-pg IgM were assayed at higher dilutions to prevent the enzymatic reaction from reaching a plateau before the end of the substrate incubation period. These readings were then corrected to 1:2 by multiplying by the dilution factor. The CABA was developed by using the murine monoclonal antibody IVD8 which reacts with an M. leprae-specific epitope found on a cell wall-associated protein of M. leprae (Mr =,) (9). The assay is designed to detect antibody in human serum which effectively competes for the binding of IVD8 to the M. leprae-specific epitope. IVD8 was purified from ascites with DEAE-Affigel blue (Bio-Rad Laboratories, Richmond, Calif.) as described by Bruck et al. (4) and iodinated with carrier-free Na12I (ICN Pharmaceuticals Inc., Irvine, Calif.) by the chloramine T method as described previously (1). A 1:1 dilution of IVD8 (specific activity, 2 x 1 cpm/,lg of protein) was chosen for the CABA. This dilution resulted in the binding of 2, cpm of IVD8 to the solid-phase antigen in the standard assay. Coating conditions for solid-phase antigen were described previously (9). Briefly, M. leprae cell wall material was coated on polyvinyl chloride 9-well microtiter plates (1 gil per well) in a mm carbonate buffer, ph 9., at a protein concentration of 1,ug/ml for 18 h at 4 C. The standard assay was performed on these plates after one wash with PBS, ph 7., and a -min blocking step with PBS-% BSA. Briefly,,ul each of iodinated IVD8 and either serum (1:2) from patients or diluent (PBS-1% BSA) were added to individual wells in quadruplicate and held at 4 C for 18 h. Each plate was then washed six times with PBS (2 Rl per well) to remove unbound components and then flicked dry, and 12,ul of 1% sodium dodecyl sulfate was added to each well. The plates were held at room temperature for min, and then 1 gul from each well was transferred to scintillation vials for subsequent counting in a model 4 gamma counter (Beckman Instruments, Inc., Fullerton, Calif.). Results were expressed as percent inhibition (%I) and were calculated as follows: %I = {1 - [(cpm 1% bound - cpm test sample)/cpm 1% bound]} x 1. The 1% bound control was obtained from the counts observed when IVD8 was added to antigen plates in the presence of the PBS-1% BSA diluent. All experimental values were corrected for background due to nonspecific binding of reactants with uncoated polyvinyl chloride plates. Background counts did not exceed 2 cpm. Serum samples which gave levels of inhibition greater than or equal to 2% were considered positive in the standard CABA of the -kda antigen. This cutoff level was based on the results from two control groups. The first control group consisted of healthy adults who had no history of mycobacterial disease and were never exposed to M. leprae or patients with leprosy (mean %I + standard deviation = ± ). The second control group consisted of 19 patients with active pulmonary tuberculosis from an environment not endemic for leprosy (mean %I ± standard deviation = 1 ± 4). The %I plus 2 standard deviations was below the 2% cutoff for both control groups. A second level of reactivity, low-level detectable, was arbitrarily assigned for levels of inhibition from 1 to 19%. None of the normal controls and only 4 of the 19 tuberculosis samples tested fell within this range of inhibition. Statistical analysis. Reactivity of leprosy patients to M. leprae antigens as detected by Western immunoblot and the CABA for the -kda antigen was compared with controls and contacts by the x2 test or the Fisher exact probability test. Also, multibacillary patients were compared with TABLE 1. J. CLIN. MICROBIOL. Immunoblot results by Ridley-Jopling group Group No. positivea/no. tested LL b BL.. 7/7c BT... 2/11 Control... /8 a Positive samples exhibited at least one strong band in the immunoblot. bsignificantly different than control by x2 test (X2 = 4.9, P <.). c Significantly different than the control by the Fisher exact probability test (P <.). Downloaded from on January, 219 by guest

3 VOL. 24, 198 ci ICONTROLO --BT--- F- BL----- H-ILL FIG. 2. Number of bands in Western immunoblot versus Ridley- Jopling classification in leprosy patients and controls (weak bands =.). paucibacillary patients by the x2 test. Levels of anti-pg IgM and anti-pg IgG in leprosy patients grouped by Ridley- Jopling classification were compared with control levels by the Student t test. The relationship between immunoblot banding patterns (as measured by the number of bands) and other indicators of disease (anti-pg IgM and BI) was assessed by the calculation of correlation coefficients. The Student t test was performed on the CABA data for the -kda antigen to determine if patients who were positive by this assay had levels of BI, anti-pg IgG, and anti-pg IgM different from those who were negative by the CABA. The degree of correlation between reactivity in the CABA and BI, anti-pg IgM, and anti-pg IgG was determined by the BMDP statistical package, using the Spearman rank order correlation test. For the purposes of this analysis, patients negative by CABA were assigned a rank of, low-level.4 F a * a * a * TABLE 2. SERODIAGNOSIS OF LEPROSY 919 BI and antibodies to PG in patients and controls used for immunoblot studies Group n BI Anti-PG IgM Mean titer ± SD Anti-Pg IgG LL ± ±.a. ±.11 BL 7.7 ± ±.28b.22 ±.22a BT ±.9. ±.7.2 ±. Control 8.1 ±.1.1 ±. a Significantly different than control values (Student t test, P <.). b Significantly different than control values (Student t test, P <.1). detectable inhibition was assigned a rank of 1, and all other positive samples were ranked by %I. RESULTS Western immunoblot was carried out on 47 leprosy patients and 8 healthy control subjects. The banding pattern for antibodies to protein antigens of M. leprae as detected by Western immunoblot is shown in Fig. 1. The sera of four LL patients were positive, while three BT patients and three control subjects showed no immunoreactivity. In this study, the seroreactive bands occurred primarily in the to -kda range. Table 1 summarizes immunoblot results for 47 leprosy sera and 8 control sera. Multibacillary patients (LL and BL) exhibited significantly more positive sera than paucibacillary (BT) patients (X2 = 4.1, P <.), and LL and BL sera exhibited more bands than did BT (Fig. 2). Significantly elevated levels of serum anti-pg IgM were seen in LL and BL patients, but not in BT patients used in this study (Table 2). In addition, BL patients had significantly elevated anti-pg IgG. A significant correlation between anti-pg IgM and BI was seen when all patients were used for analysis (r =.42, P <.1) and also when only multibacillary patients were analyzed (r =.128, P <.1). A significant correlation between anti-pg IgG and BI was seen when all patients were analyzed (r =.17, P <.). When only multibacillary patients were analyzed, the correlation between BI and anti-pg IgG failed to reach statistical significance (r =.2982, P >.). * Downloaded from on January, 219 by guest c 2 1 a_ * a* a de ~ ~~~ 4 ew FIG.. Number of bands in Western immunoblot versus BI in leprosy patients (weak bands =.). BI

4 92 LEVIS ET AL. J. CLIN. MICROBIOL. la c 4 m ci 2 **@ _ * * * AE PGIgM FIG. 4. Number of bands in Western immunoblot versus anti-pg IgM in leprosy patients (weak bands =.). The immunoblot banding pattern of leprosy patients, as measured by the number of bands, correlated directly with both the BI (Fig. ) and level of anti-pg IgM (Fig. 4) (r =.18, P <.1 and r =., P <.1, respectively). A total of 74 leprosy patients, 9 contacts of leprosy patients, and 8 healthy control subjects were assayed by the CABA for the -kda antigen. Of these patients, 9 were positive (mean %I ± standard deviation =.8 ± 1.7), 1 were low-level detectable, and were negative. All contacts and controls were negative. The CABA for the -kda antigen was found to detect active multibacillary leprosy as opposed to paucibacillary disease (Table ). Patients who were positive and low-level detectable for the CABA of the -kda antigen had significantly higher BI, anti-pg IgG, and anti-pg IgM than those who were negative (Table 4). When all patients tested for reactivity in the CABA were analyzed, a significant correlation was found between seroreactivity to the -kda antigen and both BI and anti-pg IgM (Table ). When only those positive and low-level detectable for seroreactivity to the -kda antigen were used for analysis, a significant correlation was found again between the seroreactivity and both BI and anti-pg IgM, while a significant negative correlation was found between antibodies to the -kda antigen and anti-pg IgG (Table ). A group of 27 patients were assayed by both the CABA of the -kda antigen and Western immunoblot. In this group, all patients positive or low-level detectable for antibody to the -kda TABLE. Detection of leprosy by a -kda CABA No. positive + no. Group low-level detectable/total no. Controls + contacts... /17 Leprosy... 19/74a Paucibacillary (BBb + BT)... /1 Multibacillary (LL + BL) c a Significantly different than controls and contacts (X2 = 4.7, P <.). b BB, Mid-borderline leprosy. C Significantly different than paucibacillary (X2 = 4.92, P <.). antigen were also positive by immunoblot (n = ). Of these patients, 1 were negative for antibody to the -kda antigen, but positive by immunoblot, while 12 were negative by both assays. While this shows a tendency towards concordance between the assays, it was not found to be statistically significant by the x2 test (X2 = 2.9, P >.). Protein A adsorption of several sera that were highly reactive in the CABA of the -kda antigen resulted in greater than % reduction in %I, indicating that IgG is the predominant isotype for seroreactivity to the -kda antigen. DISCUSSION There were several seroreactive bands by Western immunoblot against armadillo-derived M. leprae in active LL and BL patients. Reactivity occurred predominantly in bacillary active LL and BL patients. Further, the two immunoblot-positive BT patients had evidence of bacillary activity, as both had upgraded from BL. In our study, seroreactive bands occurred in the to -kda range. Previous reports with similar immunoblotting conditions with sera of leprosy patients demonstrated strong reactions with protein antigens with molecular sizes ranging from 4. to 4 kda (), 12 and kda (), and 12 to 8 kda (14). Some of these antigens may be related to antigens previously demonstrated by crossed immunoelectrophoresis (8, 1). For instance, the - and the -kda proteins of M. leprae have been shown to be related to antigen 7 of M. TABLE 4. Anti-PG antibodies and BI in leprosy patients grouped by reactivity to the -kda antigen Mean titer ± SD Reactivity n BI Anti-PG IgM Anti-PG IgG Positive ± 2.2a b a low-level detectable Negative.8 ± 1.. ±.92. ±.12 a Significantly higher than mean negative values (Student t test, P <.1). bsignificantly higher than mean negative anti-pg IgM (P <.). Downloaded from on January, 219 by guest

5 VOL. 24, 198 TABLE. Reactivity to -kda antigen in leprosy patients: correlation with BI and anti-pg antibodies Reactivity n Correlation coefficient BI Anti-PG IgM Anti-PG IgG All a1.4b.244 Positive + low-level 19.48a.47a -.428a detectable a Significant correlation (Spearman rank order correlation, P <.). b Significant correlation (Spearman rank order correlation, P <.1). tuberculosis (H. D. Engers, Letter, Infect. Immun. 48:-, 198). Staining with Coomassie blue and separation on gels indicate that the antigens under study are protein in nature. Because of sharp banding patterns it should now be possible to isolate and characterize the material under study. Monoclonal antibody production followed by immunoprecipitation is one approach under development. Cloning the genes responsible for protein antigens of M. leprae has also recently been demonstrated (21). This approach followed by seroreactive and cellular immune functional approaches should prove rewarding for seroepidemiologic and even future therapeutic application. The use of human sera with known reactivity should prove helpful for identifying material for isolation and purification. It will be important to see which of the cloned antigens are responsible for the major seroreactivity demonstrated by immunoblot. The CABA for antibodies to the -kda antigen, like the immunoblot, detected a number of active multibacillary cases. In patients with detectable antibody to the -kda antigen, a negative correlation was found between anti-pg IgG and the level of antibody to the -kda antigen. The implications of this finding are unclear; however, it was also seen in this and an earlier study (1) that BL patients have higher levels of anti-pg IgG than do LL patients. T-cell products have been implicated in isotype switching (1); thus, a positive anti-pg IgG may be indicative of increased cell-mediated immune response. Further study will be required to determine if anti-pg IgG or antibodies to the -kda antigen may be predictive of cell-mediated immunocompetence. The specificity of the seroreactivity demonstrated by Western immunoblot and the CABA for the -kda antigen is not yet resolved. In contrast, studies with the M. lepraederived PG indicate a relative specificity for leprosy patients and a lack of similar reactivity in other mycobacteria. It should be noted, however, that some studies have indicated false-positive reactions in the 2 to % range in control sera from subjects with no known history of mycobacterial infection (, 1). Thus, serologic responses to the protein antigens, whether specific or not, may provide an adjunctive test for seroepidemiologic screening. Finally, the relative roles of glycolipid and protein antigens in the pathogenesis of leprosy need to be individually explored. Quantitative differences in exposure to these antigens in concert may be important in the type of subsequent immune response. LITERATURE CITED 1. Blake, M. S., K. H. Johnston, G. J. Russell-Jone, and E. E. Gotschlich A rapid sensitive method for detection of alkaline phosphatase conjugated anti-antibody on western blots. Anal. Biochem. 1: SERODIAGNOSIS OF LEPROSY Brett, S. J., P. Draper, S. N. Payne, and R. J. W. Rees Serological activity of a characteristic phenolic glycolipid from Mycobacterium leprae in sera from patients with leprosy and tuberculosis. Clin. Exp. Immunol. 2: Britton, W. J., L. Hellquist, A. Basten, and R. L. Rason Mvcobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies. J. Immunol. 1: Bruck, C., D. Portelle, C. Glineur, and A. 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