Volume 60, Number 3Printed in the

INTERNATIONAL JOURNAL. OF LEPROSY

Analysis of Circulating Immune Complexes fromLeprosy Patients for Mycobacterium leprae Antigens'

Sudhir Sinha, Vinita Chaturvedi, Brajendra Mishra,and Utpal Sengupta 2

There is considerable evidence implicat-ing immune complexes (ICs) in an array ofpathogenic and immunomodulatory mech-anisms in a variety of diseases ( 28). In lep-rosy also, 1Cs have been demonstrated alongthe clinico-immunopathological spectrumof the disease (I• ' 6 ' ' 9 2 '). Apart from beingheld responsible for the pathogenesis of re-actions—the acute inflammatory episodesoften seen after the initiation of antileprosychemotherapy ( 19, 22 )— these ICs are sus-pected of playing a large immunomodula-tory role in leprosy.

Antigens of Mycobacterium leprae (andcorresponding antibodies) are regarded asthe obvious constituents of ICs in leprosypatients (4 ' 6 ). Thus, the analysis of thesecomplexes is expected to throw light on theidentities of immunopathologically impor-tant antigens of Al. leprae which have large-ly remained elusive ( 9). The present study,aiming at the demonstration and character-ization of Al. leprae antigens in circulatingICs (CICs), was conducted in leprosy pa-tients suffering from the disease at both endsof the leprosy spectrum, i.e., tuberculoid andlepromatous ( 21 ). In addition to two poly-clonal antibodies, pooled lepromatous (LL)serum and anti-BCG antibody, a set of pres-ently available monoclonal antibodiesagainst Al. leprae ( 10.") was also used forthis purpose. Sera were investigated indi-vidually, and a correlation of the results was

' Received for publication on 6 March 1991; ac-cepted for publication in revised form on 27 May 1992.

= S. Sinha, Ph.D.; V. Chaturvedi, Ph.D.; B. Mishra,M.D.; U. Sengupta, Ph.D., Central JALMA Institutefor Leprosy (ICMR), Taj Ganj, Agra 282001, India.

Reprint requests to Dr. Sinha, Scientist-C, Divisionof Membrane Biology, Central Drug Research Institute(CSIR), Chattar Manzil Palace, Lucknow 226001, In-dia.

Present address for Dr. Chaturvedi: Division of Mi-crobiology, Central Drug Research Institute (CSIR),Lucknow 226001, India.

sought with the chnico-bacteriological sta-tus and circulating At. leprae-specific anti-body levels ( 5 . 22 ) of the patients.

MATERIALS AND METHODSStudy subjects. Thirty-one leprosy pa-

tients attending the clinics of the CentralJALMA Institute of Leprosy (CJIL), Agra,India, and classified as lepromatous (LL/13L, N = 1 5) or tuberculoid (TT/13T, N =16) according to Ridley and Jopling criteria

( 2 ') were selected for the study. Thirteen pa-tients were untreated and 18 (13 LL/13L and5 TT/BT) were under multidrug therapy ( 22 )for periods ranging from 2 weeks to 18months. Some of the patients were experi-encing reactional episodes [7 borderline-typereactions (type 1) and 2 erythema nodosumleprosum (ENL, type 2)] at the time of thestudy. All patients were subjected to routineclinical investigations.

Twelve healthy volunteers (8 Indian and4 European) served as controls.

Bacterial index (BI). Four slit-skinsmears from each patient (from both ear-lobes and at least two active sites, generallyon the arm and back) were used for deter-mination of the average BI, according toRidley's logarithmic scale ( 20).

Antigens and antibodies. Soluble Al. lep-rae antigen, prepared by sonication and ul-tracentrifugation of armadillo-derived Al.leprae, was supplied by WHO-IMMLEP(courtesy of Dr. R. J. W. Rees, NationalInstitute for Medical Research, London) ata concentration of 1 mg protein/ml. Naturaldisaccharide (epitope of phenolic glycolip-id-I) conjugated with BSA (ND-O-BSA) wasprovided by Dr. D. Chatterjee (ColoradoState University, Fort Collins, Colorado,U.S.A.). Monoclonal antibodies (MAbs)against M. leprae—ML04, ML06, ML30,and ML34 (10, 11

) —were provided by Dr. J.Ivanyi (Medical Research Council TB andRI Unit, London) in the form of ascites

396

60, 3^ Sinha, et al.: M. leprae Antigens in CICs^397

globulins. Pooled scrum from five lepro-matous leprosy patients showing high titersfor Al. /epme-specific antibodies ( 22) andanti-BCG antibody (Dakopatts, Denmark)served as sources for polyclonal antibodiesagainst Al. leprae. Peroxidase-conjugatedantibodies to mouse, rabbit, and human im-munoglobulins (Igs) were obtained, respec-tively, from Sigma Chemical Co. (St. Louis,Missouri, U.S.A.), Sera Lab (U.K.) andDakopatts.

In one experiment, optimal absorption ofanti-BCG antibodies was obtained as fol-lows: To 10 ml of diluted antibody (1:250in dilution bufkr, described below) 500 plof BCG sonicate (equivalent to 50 mg wetweight of BCG) was added, and the mixturewas rotated gently (5-7 rpm) for 2 hr atroom temperature.

Isolation of CICs. Freshly collected serawere subjected to IC precipitation with 3.5%PEG-6000 (o' 29 ). Briefly, 3-4 ml serum wasdiluted 1:5 with phosphate buffered saline(0.15 M, pH 7.4) (PBS) containing 0.02 Mdisodium EDTA (PBS-EDTA). An equalvolume of 7% PEG (in PBS-EDTA) wasadded to it (while stirring), and the mixturewas kept at 4°C overnight. The precipitatewas collected by cold centrifugation andwashed once with chilled 3.5% PEG. Fi-nally, the pellet (enriched CICs) was recon-stituted in PBS up to a volume which was

ofthe original volume of serum and storedat —20°C in aliquots.

SDS-PAGE and immunoblotting. SDS-PAGE (under reducing conditions) on mini-slabs of 12.5% analytical gel was performedaccording to Laemmli ( 13) in a Mini Pro-tean-II apparatus (Bio-Rad Laboratories,Richmond, California, U.S.A.). After elec-trophoresis, the gel slabs were either stainedfor proteins (with PAGE-blue 83, BDH) orsubjected to electroblotting (24) in Bio-Rad'sTrans-Blot apparatus (60 v x 4 hr) in a coldroom using S&S nitrocellulose paper (NCP;0.45-pm pore size). After the transfer, theNCP was blocked with 3% skimmed milkpowder (Anikspray; Lipton India Ltd.) pre-pared in Tris-buffered saline (TBS; 0.01 MTris, pH 7.4) containing 0.1% Tween 20 for2 hr at room temperature (RT) with gentle(5-7 rpm) rocking (Rockomat II; Techno-mora, Switzerland). Later the paper (eitheras such or after cutting into 5-mm-widestrips) was incubated (2 hr at RT with gentle

rocking) serially with primary and/or per-oxidase-conjugated secondary antibodies.Antibody dilutions (1:1000 for MAbs, 1:250for anti-BCG Ab, 1:10 for pooled LL serum,and 1:500 for peroxidase conjugates) weremade in 1% milk-TBS containing 0.05%Tween 20 (dilution buffer). Five washes of5 min each with TI3S-0.05% Tween 20 weregiven after each antibody reaction and be-fore adding substrate (0.05% 4-chloro-naphthol, 0.02% I-1 20 2 in TBS); the finalwash was given with TBS alone. Color wasdeveloped by gentle rocking, and the reac-tion was stopped by washing the strips ex-tensively with distilled water. The NCPstrips containing electroblotted molecularweight markers (I3DH) were stained sepa-rately with Amido black.

Assays for M. /eprae-specific antibodies.Species-specific antibodies to the epitopeson the 35-kDa protein and phenolicglycolipid-I (PGL-1) of IL leprae were as-sayed in the sera using standardized ELISAprocedures. Antibodies to the 35-kDa pro-tein were assayed by an antibody compe-tition ELISA (serum antibody competitiontest, SACT) ( 22, 23

). Briefly, the ELISA plates

coated with a sonic extract of A/. leprae wereincubated with peroxidase-conjugated MAbML04 (P-ML04, reactive to an epitope on35-kDa protein) in the presence or absenceof serial tenfold dilutions of a serum;o-phenylenediamine was used as substrate,and the percent MAb binding for each di-lution of serum (100% = mean OD 492 forP-ML04 binding in the absence of serum)was calculated. The results are expressed asID50 values (serum dilution causing 50%inhibition of ML04 binding). Based on thedata obtained from nonleprosy control sub-jects ( 22 and this study), a serum with anID50 value of L- 10 was regarded as posi-tive.

Anti-PGL-I antibodies were assayed byindirect ELISA ( 5 . 22 ). Sera, diluted 1:300,were incubated with ND-O-BSA coated tothe wells of an ELISA plate. Binding of theIgM type of antibodies was detected by se-rial incubations with peroxidase-conjugatedanti-human IgM Ab and o-phenylenedi-amine substrate. On the basis of the resultsobtained from nonleprosy controls ( 22 andthis study), a serum showing an OD 492value of > 0.20 was regarded as positivefor antibody.

76-78k >66.2k >

45k >

25.7k >

17.2k >12.3k

_...,mss^41448111^!piglib^

14... • -*el

a bcde f gFIG. 2. Reaction of representative CIC-electroblot

with peroxidase-labeled anti-human Ig antibody. Lanea — healthy; lanes b to d = tuberculoid; lanes c to g =

lepromatous.

398^ International Journal of Leprosy^ 1992

76-78K>66.2K>

/.5K>

25.7K>

17.2K>

12.3K>

a b c d e f

FIG. I. SDS-PAGE (under reducing conditions) and

protein staining of representative CICs from healthy

subjects (lanes a and e) and tuberculoid (lanes b to d)or lepromatous (lanes land g) patients. Molecular weight

markers are indicated on left.

RESULTSProteins in PEG precipitates

SUS-PAGE and staining with PAGE-bluerevealed a range of proteins (mol. wts.2-kDa) which were precipitated from se-

rum at 3.5% PEG concentration (Fig. I).The presence of these proteins was noticedconsistently, although with variable inten-sities, in all CIC precipitates prepared fromeither healthy controls or leprosy patients(tuberculoid or lepromatous). Upon im-munoblotting, many of these proteins (mol.wts. 40-kDa) reacted directly with per-oxidase-conjugated anti-human Ig antibod-ies (Fig. 2), indicating an immunoglobulinorigin for such constituents.

Reaction with monoclonal antibodies toM. leprae

The reactivities of ML06, ML30, andML34 Mabs with the corresponding Al. lep-rae antigens (12-kDa protein, 65-kDa pro-tein and its degradation products, and li-poarabinomannan or LAM), as reportedpreviously (3 , 0),11,15), were reconfirmed inthe present study (results not shown). Noreaction could be seen with ML04 since thecorresponding epitope on the 35-kDa pro-tein is known to become denatured in theprocess of immunoblotting ( 10. "). None ofthese MAbs reacted with the constituents ofany of the CICs upon immunoblotting.

Reaction with polyclonalantimycobacterial antibodies

With pooled LL serum. As an antibodysource, pooled LL serum reacted with anarray of antigens resolved from the sonicextract of Al. /eprac (Fig. 3, lane a). How-ever, when electroblotted CICs were treatedwith pooled LL serum, the resultant picture(Fig. 4) was difficult to distinguish from thatof a direct reaction between CICs and sec-ondary antibody (peroxidase-conjugatedanti-human Ig: Fig. 2). A faint reaction atabout the 26-kDa position was seen withsome of the CICs (Fig. 4, arrow). The factthat this reaction was also seen with CICsprepared from the control sera suggests thatit could have been caused by one of theseveral autoantibodics demonstrable in lep-romatous leprosy patients ( 19 ).

With anti-BCG antibody. The commer-cially available rabbit anti-BCG antibodystained Al. leprae antigens in a manner sim-ilar to that of pooled LL serum except fora few differences, particularly in the low mo-lecular weight region (Fig. 3, lane b).

The reaction of the electroblotted CICswith anti-BCG antibody revealed the pres-ence of an antigen with an apparent molec-ular weight of 65 kDa (Fig. 5) in 12 (about40%) of the CIC-precipitates from leprosypatients, irrespective of the type of leprosy(Table 1). In some instances, another faintand diffused reaction (mol. ■,vt. < 65 kDa)

76-78 k >66.2k >

45k >

25.7k >

17.2k >12.3k >

.111e.GAL.

p

■■■■■•••

a^b^c^d^e^f^g

60, 3^Sinha, et al.: M. leprae Antigens in CICs^399

76-78 k66.2 k >

45k >

-4me.^111 .44450 (11111111110..,-

25.7k > 00.

17.2k >12.3k >

abcde f^9FIG. 4. Reaction of representative CIC-electroblots

with a pooled LL serum. Lane a = healthy; lanes b tod = tuberculoid; lanes e to g = lepromatous; arrow onright = faint 26-kDa band seen in some of the CICs.

a bFIG. 3. Reaction of M. leprae electroblots with a

pooled LL serum (lane a) or anti-BCG antibody (laneb). Arrow on right = prominent 65-kDa band stainedby both polyclonal antibodies.

was observed in association with the main65-kDa band (Fig. 5, arrow). The myco-bacterial origin of this antigen was con-firmed in two ways: a) incubation of theCIC-blots directly with secondary antibody(peroxidase-labeled anti-rabbit Ig) did notshow any reaction, and b) optimal absorp-tion of anti-BCG antibodies with BCG son-icate (as described under Materials andMethods) abolished the reaction with 65-kDa antigen of CICs (as also with all Al.leprae antigens visible in Fig. 3, lane b).

None of the CICs obtained from healthysubjects showed the presence of 65-kDa orany other mycobacterial antigen.

Association of antigen positivity withdifferent parameters

Possible associations between positivityfor the apparent 65-kDa M. leprae antigen(in CICs) and the following parameters wereinvestigated: type of leprosy (tuberculoid orIcpromatous, with or without reactions); se-rum levels of M. leprae-specific antibodies(as determined by PGL-I-ELISA and SACT-ELISA); BI; duration of disease or treat-ment; and motor nerve involvement. Nostatistically significant association was not-

ed with any of these parameters individu-ally. However, we noticed a greater tenden-cy for patients with recent (< 3 years) diseaseand motor nerve involvement to show apositivity for the apparent Al. leprae antigenthan the rest of the group. There were 5 suchpatients, and 4 of them (3 briefly treated LLpatients with high BI values and AI. leprae-specific antibodies and 1 untreated TT pa-tient with massive motor damage resultingin drop foot) were antigen positive (Table

66.2kd >

FIG. 5. Reaction of representative CIC-electroblotswith anti-BCG antibody. Lanes a to c tuberculoid;lane d = healthy; lanes e to g = lepromatous. Some-times a diffused band (arrow, lane 0 was seen in as-sociation with the main 65-kDa band.

Type SACT(ID50)

65-kDaantigenin CICs

PGL-I ELISA(OD 492)

AntibodyTreatmentAverage BI^duration

(mo)

400^ International Journal of Leprosy^ 1992

TABLE I. Positivity for 65-kDa mycobac-terial antigen in PEG precipitates.

PositivitySera type

^No. tested^

No.

LL/131_, 15' 7 46.7TT/13T 16" 5' 31.3

Total 31 12 38.7Healthy 21' 0 0

Includes 2 patients with erythema nodosum lep-rosy.

'' Includes 7 patients with borderline-type reaction.Includes 1 patient with borderline-type reaction.Includes 8 Indians and 4 Europeans.

2). Out of the remaining 26 patients, onlyeight were antigen positive.

The observed lack of association betweenantigen positivity and the reactional stateswas of particular significance; neither of the2 lepromatous patients in reaction (ENL)and only 1 out of 7 tuberculoid patients inreaction were positive.

DISCUSSIONLepromatous leprosy patients synthesize

high titers of antibodies against a variety ofM. leprae antigens ("•"). Given that theyalso harbor a high bacterial load, one wouldexpect the circulating immune complexes(CICs) from a majority of them to showpositivity for many M. leprac antigens.Nevertheless, in our study CICs from lessthat 50% of the lepromatous patients turnedout to be positive and, at that, for only oneantigen of M. leprae origin. These obser-vations partly corroborate the findings ofRamanathan, et al. ('(') who reported posi-tivity for anti-Al. leprae antibodies in CICsfrom only 40% of lepromatous leprosy pa-

tients (with or without reaction). The CIC-associated antigen demonstrated by us (65kDa, a common mycobacterial antigen) ismost probably identical to the one (65-69-kDa antigen) reported by Chakrabarty, etal. ( 4) in PEG-precipitated CICs of pooledlepromatous sera, using anti-BCG antibod-ies.

Of considerable significance is the ob-served positivity for the CIC-associated Al.leprae antigen in about 30% of the tuber-culoid leprosy patients as well, which prob-ably indicates that the true extent of infec-tion in these patients may exceed theexternally noticeable lesions ( 12). The failureto detect anti-A/. leprae antibody in the CICsof such patients by Ramanathan, et al (''')could be due to the inability of a majorityof them to mount a good antibody responseto AI. leprae antigens (").

In view of the known ubiquitous distri-bution of the 65-kDa antigen, a stress pro-tein, across the genera and species of organ-isms including humans ( 7), the mycobacterialorigin of the detected antigen was ascer-tained. The reaction between apparent 65-kDa Al. leprac antigen of CICs and anti-BCG antibody could be abolished by priorabsorption of the antibody with BCG son-icate. The efficacy of this treatment was ev-ident from the fact that it also abolished thereaction with a whole range of Al. lepraeantigens in parallel experiments. The 65-kDa antigen is also known to exist in theform of degradation products ( 3• "), whichmost probably explains the existence of afaint and diffused band in some of the CICsinvestigated by us.

An array of proteins were precipitatedfrom individual sera with 3.5% PEG due to

TABLE 2. Data on patients with recent disease (< 3 years' duration) and motor nerveinvolvement.

LL (nodular) 4.5 18 42,000 0.60LL 4.0 1 1,150 0.75LL 5.0 4 2,400 0.97TT' 0 0 <10" 0.03"BT 0 0 <10" 0.12"

Patient with drop foot.Below the seropositivity cut-off values.

60, 3^Sinha, et al.: M. leprae Antigens in CICs^ 401

the fact that this technique, like other tech-niques for this purpose, serves only to iso-late a "CIC-enriched fraction" and not theCICs alone ( 4 ' 16 ' 29 ). Low levels of CICs oc-cur naturally, even in healthy subjects ( 16 ' 28 ),which is the reason for our using some PEGprecipitates from normal sera also.

The four anti-A/. leprac monoclonal an-tibodies used in this study did not react withany CIC constituent. The MAb ML30 isknown to identify an epitope on the 65-kDaantigen of armadillo-derived Al. leprae ( 3 ).Perhaps this epitope was absent or proteo-lytically deleted in the antigen present inCICs. It appears unlikely that the othermonoclonal antibodies ( 8), not tested by us,would have provided different results sincea potent polyclonal antibody like anti-BCGantibody (which showed strong reactionswith a large number of Al. leprae antigens)failed to identify anything except the 65-kDa antigen and, at that, only in some in-stances. The apparent inability of a pooledLL serum to distinctly identify the 65-kDaantigen in CICs, despite reacting strongly toit in electroblotted Al. leprac extract, couldbe attributed to either a) the occurrence ofdirect, overlapping reactions between cer-tain host-serum-derived constituents ofCICs and secondary antibody (Fig. 2), or b)inaccessibility of the 65-kDa antigen due toa complex formation with immunoglobu-lins.

A mycobacterial cell-wall-associated ma-jor stress protein like the 65-kDa antigen isa good candidate for predominant releasein the blood of leprosy patients harboringsubstantial quantities of viable Al. leprac.Our observations also indicate that the CICswere more frequently positive for the an-tigen in patients with recent, massive, andlargely untreated infection. Further, the an-tibody level against the 65-kDa antigen hasbeen reported to be relatively low ( 14), prob-ably due to its partial recognition by theimmune system as "self" ( 7). Such condi-tions (including low antibody levels) wouldfavor the formation of immune complexesin "antigen excess" facilitating better anti-gen recognition.

Since the immune complexes have main-ly been implicated in pathogenesis ( 28), theirantigenic analysis is likely to offer clues tothe pathogenic rather than immunoprotec-

tive moieties. Indeed, the 65-kDa stressprotein has been held responsible for thepathogenesis of adjuvant arthritis ( 26). Per-haps PGL-I, an immunosuppressive moietyof the Al. leprae cell wall ( 25), also circulatesin the blood of leprosy patients ( 6) in theform of an immune complex due to its co-existence with antibodies and its ability toactivate the complement system ( 17). Theconspicuous absence of lipoarabinomannan(LAM), yet another mycobacterial cell-wallantigen ofan immunosuppressive nature ( 15 ),in CICs analyzed by us needs to be consid-ered. Although LAM is capable of gener-ating an overwhelming antibody responsein leprosy patients, it most probably re-mains deposited in the host tissue due to itsaffinity for cell membranes ( 2), and is lesslikely to circulate in the free (or antibody-bound) form in the blood.

A specific role for M. leprae antigen-pos-itive CICs in the pathogenesis of leprosy-associated reactions could not be estab-lished in this or previous studies (16).However, it is the total load of immunecomplexes, irrespective of the type(s) ofconstituent antigen(s), which could be moreimportant in this context ( 18). Further, sincethe ICs which are formed or get depositedin the skin have been more directly impli-cated in the pathogenesis of reactions ( 28 ),the analysis of such in situ complexes mayprovide better insight into the pathogenicmoieties of Al. leprae.

SUMMARYCirculating immune complexes (CICs)

from 31 leprosy patients (16 tuberculoid, 15lepromatous) and 12 healthy volunteers,precipitated by 3.5% polyethylene glycol,were individually subjected to SDS-PAGEand immunoblotting using a variety ofmonoclonal and polyclonal antibodiesagainst Mycobacterium leprae. A commonmycobacterial antigen of an apparent mo-lecular size of 65 kDa was identified in CICsfrom about 40% of the patients. No corre-lation was observed between the positivityfor this antigen and any of the followingparameters: bacterial index, Al. /eprae-spe-cific antibody titers, motor nerve involve-ment, duration of disease or treatment.Nevertheless, patients with a relatively re-

402^ International Journal of Leprosy^ 1992

cent and massive infection were more fre-quently positive for antigen than the others.

RESUMENLos complejos inmones circulantes (('IC) separados

por precipitaciim con polictilen glicol at 3.5% del suerode pacientes con lepra (16 tuberculoides y 15 lepro-matosos) y del sucro de 12 voluntarios sanos, se ana-lizaron por PAGE-SDS e in muno-clectrotransferenciausando una variedad de anticuerpos monoclonales ypoliclonales contra el M yeobacterium leprae. Antiqueen el 40% de los pacientes se identificO un antigeno

icobacteriano comOn de 65 k Da, no hub° correlaciOnentre la positividad por este antigeno y cualquiera delos siguientes partimetros: indict bacteriano, niveles deanticuerpos especificos para .1/. /grim, afecciOn de losnervios motores, duraciOn de Ia enlerinedad, o trata-meinto. Sin embargo, los pacientes con una infecciOnmasiva relativamente reciente fueron inns frecuente-mente positivos pant este antigeno que otros pacientes.

RESUME,Des complexes immuns circulants (CIC) de 31 pa-

tients lepreux (16 tuberculoldes, 15 lepromateux) et 12volontaires en bonne sante, precipites par du polyethy-lene glycol a 3.5%, ont etc soumis individuellement auSDS-PAGE et n l'immunoblotting en utilisant une va-riet& d'anticorps monoclonaux et polyclonaux vis-a-vis de .11 yeobacterium leprae. Un antigene mycobac-terien commun dune taille moleculaire apparente de65 kDa a etc identifiee dans les CIC chez environ 40%des patients. Aucune correlation n'a etc observee entreIa positivite pour cet antigene et run quelconque desparametres suivants: l'index bacterien, les titres d'anti-corps specifiques vis-a-vis de M. leprae, l'envahisse-mem des nerfs moteurs, la duree de la maladie ou letraitement. Neanmoins, les patients avec une infectionrelativement recente et massive etaient plus frequent-mem positifs pour l'antigene que les autres. •

Acknowledgment. We are grateful to Dr. R. J. W.Rees for providing M. leprac antigen; to Dr. J. Ivanyifor monoclonal antibodies to .1/. leprae; and to Dr. D.Chatterjee for ND-O-BSA. We also thank Mr. P. N.Sharma and Mr. R. Dayal for technical assistance andMr. H. 0. Agarwal for photography. The manuscriptwas kindly reviewed by Dr. H. Srinivasan. Some ma-terials used in this study were generously donated byLEPRA, U.K.

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