Volume 54, Number 4

Printed in the U.S.A.INTERNATIONAL JOURNAL OF LEPROSY

Separate Antigenic Determinants on Cell WallAssociated Carbohydrate Antigens of

Mycobacterium leprae Defined withMonoclonal Antibodies'

Warwick J. Britton, Linus Hellqvist, and Antony Basten2

The advent of monoclonal antibody (Mab)technology has led to a rapid expansion inthe understanding of the antigens of My-cobacterium leprae. At least six differentprotein antigens have been identified withMabs, and DNA clones encoding the genesfor five of them have been isolated(2, 13, 18, 20, 30, 3l) During the Immunology ofLeprosy (IM M LEP) Workshop on these an-tibodies, a number of others were observedto react with a broad antigenic band labeled"carbohydrate/lipid" as well (12). We haddeveloped similar Mabs which in immu-noblots bound to crossreactive determi-nants on two broad bands with apparentmolecular weight (Mr) of 30-40 kD and 4.5—6 kD (2). Inhibition assays indicated thatlepromatous leprosy sera contained a hightiter ofantibody directed against these Mab-defined determinants. This finding was con-firmed by the strong reaction of unabsorbedleprosy sera with the same antigenic bandsin immunoblots. Sera absorbed with AI.bovis and M. vaccae still reacted with a trip-let of bands of Mr 32-35 kD which wasconsistent with the presence of species-spe-cific epitopes on those molecules in additionto the Mab-defined crossrcactive determi-nant. The existence of AI. /eprae-specilic an-tigens of similar molecular weight (3' 19) hasnow been confirmed in two other studies.

We have sought to characterize the 30—40 kD and 4.5-6 kD antigens immuno-chemically, and to examine their localiza-tion within the mycobacterium. Using acombination of inhibition ELISA and an-

' Received for publication on 21 February 1986; ac-cepted for publication in revised form on 14 July 1986.

= W. J. Britton, M.B., B.S., B.Sc.(Med.), F.R.A.C.P.,M.R.C.P., D.T.NL&I-1.; L. Hellqvist, Ph.D.; A. Hasten,M.B., B.S., Ph.D.(0xon), F.T.S., F.R.A.C.P.,F.R.C.P.A., F.R.C.P., Clinical Immunology ResearchCentre, University of Sydney, Sydney, Australia 2006.

tibody capture assays, we have demonstrat-ed the relative abundance of the antigens inpreparations of Al. bolls (BCG) and Al. tu-berculosis, as well as M. leprae. Experimentswith Mab-affinity columns indicated thatthe low molecular weight (LMW) antigen of4.5-6 kD is a fragment of the larger 30-40kD molecule.

MATERIALS AND METHODSAntigens. Al. leprae bacilli were provided

through the IMMLEP component of theUNDP/World Bank/WHO Special Pro-gramme for Research and Training in Trop-ical Diseases by Dr. R. J. W. Rees. Thebacilli were sonicated, as described previ-ously (2), for 15 min to yield Al. leprae son-icate. The insoluble cell wall fraction wassonicated again for 15 min to yield a solu-bilized preparation of the cell wall, Al. /ep-rae resonicate leaving a final insoluble pel-let, Al. leprae pellet. Al. bolls (BCG) wasobtained as freeze-dried bacilli from theCommonwealth Serum Laboratories (CSL,Melbourne, Australia). Al. sinegmatis andM. tuberculosis H37Rv were grown in Du-bos broth (Difco, Detroit, Michigan, U.S.A.)supplemented with albumin and glycerol.The bacilli were then harvested, washed inphosphate buffered saline (PBS) pH 7.2, andsonicated. After measuring the protein con-tent of the sonicate by Folin's reagent, thematerial was aliquoted and stored at —70°Cuntil used. Purified protein derivative (PPD)was obtained from Statcns Seruminstitut,Copenhagen, Denmark.

Monoclonal antibodies (Mabs). A panelof Mabs to At leprae and M. bovis (BCG)was produced by fusion of immunizedBALB/c spleen cells with NS-1 cell line (2).

Six of them were found to bind to antigenswith Mr of 30-40 kD and 4.5-6 kD. Other

545

546^ International Journal of Leprosy^ 1986

Mabs bound to antigens of Mr 16-18 kDand 70 kD. Li (IgM), L9 (IgG3) and L 1 0(IgG3) reacted with a single epitope on the30-40 kD molecule. L3 (IgM) and L4(IgG2a) reacted with the 4.5-6 kD antigen.B6 (IgG3) was derived from a fusion withmice primed to M. bovis (BCG). It recog-nized the same cpitope on the LMW antigenas L3 and L4. All six Mabs reacted with 13of the 14 mycobacterial species tested inenzyme-linked immunosorbent assays(ELISA).

Immunochemical characterization. M.leprae sonicate and resonicate were ana-lyzed by polyacrylamide gel electrophoresisin the presence of sodium dodecyl sulfate(SDS-PAGE) under reducing conditions asdescribed previously (2.2'). After transfer ofthe antigens to nitrocellulose paper (30)(NCP; Biorad, Richmond, California,U.S.A.), the NCP strips were probed withMab supernatant followed by '25I-labeledsheep anti-mouse Ig (2). The NCP strips weredried and exposed to Kodak XRP-1(Kodak, Melbourne, Australia) and devel-oped after 2 days at —70°C. The effect ofproteolysis on Mab binding was examinedby incubating M. leprae sonicate (1 mg/nil)with trypsin (Bovine Pancreas Type 1; Sig-ma Chemical Co., St. Louis, Missouri,U.S.A.) at 10 mg/ml for 1 hr at 37°C (17).The samples were then boiled for 5 min inelectrophoresis buffer (2% SDS, 10% su-crose, 2% 2-mercaptoethanol, 0.05 M Tris-HC1 pH 6.8), and analyzed as describedabove. The susceptibility of the antigens toperiodate oxidation was examined in a sim-ilar way by first incubating Al. leprae son-icate with sodium periodate (Sigma) at afinal concentration of 1, 5, and 10 mM for1 hr at 37°C before boiling in electrophoresisbuffer (28).

Antigen inhibition ELISA. The inhibi-tory effect of various mycobacterial soni-cates on the binding of L9 or L4 to M. lepraeor to BCG sonicates was examined by an-tigen inhibition ELISA. Initially, the titer ofhybridoma supernatant causing 75% of themaximum optical density (OD) reading foreach Mab was determined and used as thefinal dilution in the inhibition ELISA. Dou-bling dilutions of the antigens, Al. lepraesonicate, Al. leprae resonicate, BCG or Al.tuberculosis sonicate, were then made in PBS

in a polystyrene microtiter tray (Linbro,Flow Laboratories, Edinburgh, Scotland),the wells of which had been blocked with3% bovine serum albumin (BSA). An equalvolume of hybridoma supernatant, appro-priately diluted, was added, and the trayincubated for 1 hr at 37°C in a humidifiedincubator. Fifty pl of each dilution were thentransferred in triplicate to the wells of apolystyrene tray coated with Al. leprae orBCG sonicate (50 pg/m1). After incubatingfor 1 hr, the wells were washed three timeswith PBS-azide, and sheep anti-mouse Ig-alkaline phosphate conjugate (Sigma) dilut-ed 1:1000 in 0.1% BAS-PBS was added for1 hr. The wells were washed three timeswith PBS-azide and once with sodium car-bonate buffer before adding the enzymesubstrate nitrophenyl phosphate (Sigma) at1 mg/ml in 0.15 M sodium carbonate bufferpH 9.6. The tray was read at 60 min in aTitertek Scanner (Flow Laboratories). TheOD of wells coated with BSA alone wassubtracted from each reading. Maximumbinding occurred with the Mabs superna-tant at the same dilution but without inhib-iting antigen. The percentage inhibition ofthis binding by each antigen concentrationwas calculated and plotted. From this, theantigen concentration causing 50% inhibi-tion of binding for that antigen and the par-ticular Mab was determined.

Monoclonal antibody capture assays. Therelative concentration of Mab-defined de-terminants in Al. leprae, Al. leprae resoni-cate, and other mycobacteria was examinedusing a capture assay in which the captureand tracer antibody as well as the antigencould be changed. Briefly, "capture" Mab(50 pi, 100 pg/m1) purified by protein Achromatography was diluted to 100 pg/m1in PBS before incubation in the wells of apolyvinyl chloride (PVC) microtiter tray(Linbro). After 2 hr at 37°C, the wells werewashed with PBS-azide and blocked with5% casein in PBS. After washing with PBS,50 Al of mycobacterial sonicates diluted inPBS from 100 Ag/m1to 10 ng/ml were addedin triplicate for an overnight incubation at4°C. Subsequently, the trays were washedfive times with PBS, and the "tracer" Mab,radiolabeled with 125I by the chloramine Tmethod (15) (105 cpm in 50 Al), was addedfor 4 hr at 37°C. At the end of this period,

20_14- it

.mmIRMI•

Nib 111111111

54, 4^Britton, et al.: Carbohydrate Antigens of M. leprae^547

the wells were again washed extensively inPBS before counting in a Riagamma count-er (LKB, Stockholm, Sweden). The bindingof the tracer Mab to wells coated with BSAand incubated without antigen was sub-tracted from the triplicates for each antigenconcentration. An irrelevant Mab (K-1-21,IgGI, anti-kappa light chain) was also usedas the "capture" Mab.

The assay was also used to determine ifintact bacilli could be bound by monoclonalantibodies. Whole bacilli, diluted in PBSfrom 109 to 105 ml, were used as antigenbefore washing and incubation with thetracer Mab. The binding of bacilli to L9 orL4 as capture Mab was compared to thebinding of bacilli to BSA.

Immunofluorescence of M. leprae withmonoclonal antibodies. Whole Al. lepraebacilli (107 in 50 1.d) were transferred andfixed to template slides (Sigma). The slideswere incubated with Mab supernatant orascites fluid in a moist chamber for 1 hr at37°C and then washed three times in PBS/azide. Twenty-five/11 fluorescein conjugatedgoat anti-mouse Ig (Silenus, Melbourne,Australia) 1:20 dilution in PBS was addedto the slides for 1 hr at 37°C. Following this,they were washed three times, mounted inphosphate buffered glycerol (pH 8.8), andexamined under a Zeiss fluorescent micro-scope. The control preparations consistedof slides incubated with irrelevant IgG1 andIgM Mabs. The binding of lepromatous lep-rosy sera was compared with that of theMabs. A pool of sera from 20 untreatedlepromatous leprosy patients was used withfluorescein-conjugated goat anti-human Ig(Sigma) at a dilution of 1:50 as a secondantibody.

Monoclonal antibody affinity chromatog-raphy. Purified Mabs L4 and L9 were con-jugated to cyanogen bromide activatedSepharose 4B (Pharmacia, Uppsala, Swe-den) at a ratio of 5 mg antibody to 1 ml gelin a coupling buffer of 0.25 M sodium bi-carbonate (pH 9.0) containing 0.5 M so-dium chloride. After a 2-hr incubation atroom temperature, the remaining activegroups were blocked with 1 M ethanolam-ine (pH 8.0), and the gel washed alternatelywith coupling buffer and acetate buffer (0.1M, pH 4.0) containing 0.5 M sodium chlo-ride. The gels were equilibrated with PBS

94-

67-

43-

ABC D

FIG. 1. Immunoblot of ilf. leprae sonicate with(Tracks B, D, F, H) and without prior treatment withtrypsin (Tracks A, C, E, G). Antibodies used were: L5(Tracks A. B); L4 (Tracks C, D); L9 (Tracks E. F) andL7 (Tracks G, H). Position of protein molecular weightmarkers (in kD) are shown on left hand side.

and stored at 4°C. M. leprae sonicate or M.bolls sonicate was recirculated through theL9-CNBr Sepharose column for 2 hr at flowrate 2 ml/min and then through the L4-CNBr Scpharose column for a similar pe-riod of time. Both were washed extensivelywith PBS until a stable base line- wasachieved. The bound material was elutedwith 0.05 M diethylamine (pH 11.5) fol-lowed by 1 M propionic acid (pH 2.0). Theeluates were dialyzed against ammoniumbicarbonate, freeze dried, and dissolved in1.0 ml PBS. The cluates from both columnswere then examined by SDS-PAGE and im-munoblots with L9 and L4.

RESULTSImmunochemical nature of antigens. Our

initial experiments had demonstrated thatthe 30-40 kD diffuse band defined by LI.L9, and LIO and the low molecular weight(LMW) band of 4.5-6 kD identified by L3,L4, and B6 in Al. leprae sonicate were pres-ent in the samples examined under bothreducing and nonreducing conditions. Thesebands were resistant to treatment with sub-tilisin after transfer to NCP (2). This pro-tease resistance was confirmed by incubat-

94-67-

43-30_20-14- • 111111. 0110.

AR^C!DF^F

548^ International Journal of Leprosy^ 1986

FIG. 2. Immunoblot of Al. leprae sonicate without(Tracks A, E) and with prior treatment with sodiumperiodate at a final concentration of 1 mM (Tracks B,F), 5 mM (Tracks C, G) and 10 mM (Tracks D, H).Antibodies used were L5 (Tracks A—D) and L9 (TracksE—H). Position of protein molecular weight markers(in kD) are shown on left hand side.

ing the Al. leprae sonicate with trypsin priorto SDS-PAGE. The 30-40 kD and LMWbands were unaffected whereas the 16-18kD and 70 kD protein bands defined by twoother anti-M. leprae Mabs, L5 and L7, re-spectively, were destroyed (Fig. 1). Incu-bation with sodium periodate also had adifferential effect on the antigens. The pro-tease-resistant bands were sensitive at aconcentration of 5 mM and above, withcomplete loss of reactivity in immunoblotsindicating that the Mab-defined determi-nants were predominantly carbohydrate innature (Fig. 2). In view of this, the figuresgiven for apparent molecular weight as de-fined by comparison with protein markersshould be regarded only as approximations.

100

80

60

40

20

1^4^16^64^250^1000

PROTEIN CONCENTRATION INHIBITING ANTIGEN (rim!)

FIG. 3. Inhibition ELISA in which different my-cobacterial sonicates inhibited the binding of L9 to Al.leprae sonicate. Antigens were Al. leprae sonicate (0),.11. leprae resonicate (•), Al. bolls sonicate (A), and Al.tuberculosis sonicate (0).

By contrast, binding to the determinants de-fined by L5 and L7 was less affected afterexposure to 5 mM and 10 mM sodium per-iodate, which suggests they are proteinsrather than carbohydrates.

Antigen inhibition ELISA. Both L9 andL4 had been shown to react as strongly withM. leprae resonicate as with Al. leprae son-icate in immunoblotting experiments (2).Therefore, the relative concentrations of theantigens in the two samples were examinedin an inhibition ELISA. Both the Al. lepraesonicate and resonicate inhibited the bind-

TABLE 1. Mycobacterial antigen inhibition of Mab binding to M. leprae sonicate inELISA.

Protein concentration (4/m1) required for 50%inhibition of maximum binding with

MabAl. leprae^Al. lepraesonicate^resonicate

Al. bovis(BCG) M. tuberculosis^Al. smegmatis

L4^28^40^

4^15^54L9^4^4

^3/^115^32

CAPTURE- LOTRACER-'25I-L9ANTIGEN-VARIOUS SONICATES

M leprae reeorecate

M Smegrnatls

TRIAD

14

12

10

M TBM Boyle

• PPD

0.01^0.1^1^10^100

ANTIGEN CONCENTRATION Cog/ml)

Tracer Mab bound (cpm ±

'"I-L9 1251-L4

10 pg/ml 1 pg/ml 10 pg/m1 1 pg/m1

11,19214,990

120

± 348± 459

± 60

6,5826,888

± 117± 224

0

1,828220

± 80± 12

672113

± 47± 12

Antigenconcen-tration

Capture MabL9L4

ControlK-1-21

54, 4^Britton, et al.: Carbohydrate Antigens of M. leprae^549

Fla. 4. Antigen capture assay in which L9 immo-bilized on a PVC tray bound various mycobacterialsonicates which, in turn, reacted with '"I-L9. Antigenswere Af. leprae sonicate (•), Al. leprue resonicate (0),M. boils sonicate (*),M. tuberculosis sonicate (0),Al. smegmatis sonicate (A) and PPD (•). Standard de-viation from each point was less than 10%.

ing of L9 to M. leprae sonicate to an equiv-alent degree (Fig. 3). Thus, the protein con-centration associated with 50% inhibitionof maximum binding was 4 pg/m1 for each(Table 1). In the case of L4, similar con-centrations of M. leprae sonicate and re-sonicate were required to inhibit the bindingof L4 to Al. leprae (Table 1).

Previously, differences in the intensity ofbinding of the Mabs with various myco-bacterial sonicates in immunoblots had been

TRIAD CAPTURE-L4

ANTIGEN-VARIOUS SONICATESTRACER'-25I-L4

0

0.3

Smegmatis

• IA leer.)

M lepree reeorecate

PPD

0.1^1^10^100^1000ANTIGEN CONCENTRATIONGug/m0

FIG. 5. Antigen capture assay in which L4 was thecapture and tracer antibody and the mycobacterial son-icates varied. Antigens same as in Figure 4 legend.Standard deviation from each point was less than 10%.

observed. L9 reacted weakly with Al. bovisand Al. tuberculosis sonicates and the an-tigen identified had a slightly higher Mr of35-45 kD (2). By contrast, L4 reactedstrongly with a LMW band of similar mo-bility in the three mycobacterial species.Therefore, we examined the inhibitory ef-fect of other mycobacterial sonicates on thebinding of L9 and L4 to Al. leprae. Al. bolls,Al. tuberculosis and /IL smegmatis inhibitedthe binding of L9 to Al. leprae, but at ahigher concentration of sonicate than witheither AL leprae sonicate or resonicate (Ta-ble 1). However, less Al. bovis sonicate wasrequired for a 50% inhibition of L4 bindingto Al. leprae, indicating that the LMW an-tigen was more abundant in the M. bolls

M Boyle

TABLE 2. Antigen capture assay using L9 or L4 as capture and tracer antibodies andM. leprae sonicate as antigen.

" 50 III samples tested as described in text in Materials and Methods. Mean of test triplicate minus the cpmbound to BSA control well incubated without antigen.

43-

30_

550^ International Journal of Leprosy^ 1986

U.94-

67-

PIP'

20_

14 -

a b C d e

FIG. 6. Immunoblot analysis with L9 of fractionsfrom L9 and L4 Mabs-affinity columns which werereacted with M. leprae sonicate. Tracks a = originalM. leprae sonicate; b = L9 column, pH 7.4 fractioncontaining unbound antigen; c = L9 column, pH 11.5fraction; d = L9 column, pH 2.0 fraction; e = L4 col-umn, 7.4 fraction containing unbound antigen; f=L4 column, pH 11.5 fraction. Position of protein mo-lecular weight markers (in kD) are shown on left handside.

preparation (Table 1). A similar pattern wasseen when the binding of L4 to M. ,bovissonicate was examined in the inhibitionELISA. M. bovis sonicate was more efficientthan M. leprae in inhibiting the binding ofL4 to M. bovis.

Antigen capture assay. The structural re-lationship between the 30-40 kD and LMWcarbohydrate determinants was examinedin an antigen capture assay with the corre-sponding two Mabs L9 and L4. L9 couldbe used as both capture and tracer Mab todetect the 30-40 kD antigen in Al. lepraesonicate down to a concentration of 10 ngprotein/ml (Fig. 4). L4 was as efficient as L9in capturing the 30-40 kD molecule whichcould, in turn, bind '251-L9 (Table 2). Thissuggested that the two determinants werelocated on molecules associated in theaqueous phase. In order to confirm this, thereverse experiment was conducted whichshowed that L9 could capture the LMW an-tigen as revealed by '251-L4 tracer (Table 2).However, Al. leprae antigen captured by L4did not bind further '251-L4 except at highconcentrations, indicating that there were alimited number of L4-defined determinantson the LMW molecule of the Al. leprae son-icate (Fig. 5). Therefore, although the 30-40 kD and LMW antigens appeared to beseparate antigens in SDS-PAGE, they wereclearly associated and able to be immobi-lized by either L9 or L4.

The same assay was used to examine thebinding of L9 or L4 to different son icates(Fig. 4). Equivalent amounts of L9 and L4reactive antigen were present in Al. lepraesonicate and resonicate, confirming that theseMab-defined determinants were located inthe cell wall of the mycobacterium. Less L9reactive antigen was captured from Al. bovisor Al. tuberculosis sonicates compared to M.leprae, and there was a small amount of the30-40 kD antigen in the PPD (Fig. 4). By

TABLE 3. Antigen capture assay with intact bacilli.

Bacilli^ Tracer Mab bound (cpm ±

concen-^ '251-L9^

'"I-L4tration^109/m1

^10/ml^107/m1

^10/ml^108/m1^1 07/m 1

Capture MabL9 2290 ± 144h 508 ± 18' 83 ± 48 285 ± 60 150 ± 24 86 ± 55L4 573 ± 17 172 + 22 102 ± 43 409 ± 47 234 + 92 40 ± 57

BSA control 335 ± 25 34 ± 27 477 ± 44 151 ± 21 15 ± 47

50 samples tested as described in text in Materials and Methods. Mean of test triplicates minus cpm boundto BSA control well incubated without antigen.

Differences to BSA control incubated with same concentration of bacilli significant at p < 0.005 (Mann-Whitney rank sum test).

Differences to BSA control incubated with same concentration of bacilli significant at p < 0.01 (Mann-Whitney rank sum test).

54, 4^Britton, et al.: Carbohydrate Antigens of M. leprae^55 1

contrast, more '251 -L4 bound to M. boils orAl. tuberculosis than to Al. leprae sonicate(Fig. 5).

Whole bacilli were also substituted forsonicate in order to test whether the deter-minants recognized by either Mab were onthe surface of intact organisms. When thebacilli were incubated in wells coated withL9, significantly more '25I-L9 was subse-quently bound to the immobilized bacillithan if the bacilli had been incubated incontrol wells (Table 3). This only occurredif the higher concentrations of whole bacilli,109/m1 and 108/ml, were used. By con-trast,'251-L4 did not bind to the bacilli inwells coated with either L4 or L9 (Table 3).These results indicated that only the 30-40kD antigen was exposed on the surface ofthe bacilli. The difference in appearance ofthe L9 reactive bands shown in Tracks band d (Fig. 6) of the blots is presumably dueto the fact that the 30-40 kD band is com-posed of several different antigens (2.12).

Immunofluorescence. The location of thetwo antigens was examined directly by im-munofluorescence with the same Mabs anda fluorescinated second antibody. L 1 , L9,and L 1 0, which recognized the 30-40 kDantigen, stained the majority of organismswith a granular pattern. This occurred witheither supernatant or ascites fluid diluted to10-4, but the intensity was less than thatseen with a pool of human lepromatous lep-rosy sera. On the other hand, no fluores-cence was detected with any of the threeantibodies (L3, L4, B6) directed against theLMW antigen, confirming the conclusionfrom the antigen capture assay with wholebacilli.

Affinity chromatography. The relation-ship between the two antigens was furtheranalyzed by Mab-affinity chromatography.M. leprae sonicate (5 mg) was applied se-quentially to the L9 and L4 columns. Theeluates and unbound residual fractions wereconcentrated and examined by SDS-PAGEand immunoblotting with L9 and L4 (Fig.6). The 30-40 kD antigen was eluted fromthe L9 column by both alkaline and acidicbuffers (Tracks c, d). However, the unboundfraction still contained immunoreactive an-tigen (Track b). When this unbound fractionwas passed over the L4 column, it was de-pleted of the 30-40 kD antigen (Track e)which was subsequently eluted from the col-

umn with 0.05 M diethylamine. Immuno-blotting of the fractions with L4 indicatedthat the LMW antigen had bound to bothL9 and L4 columns. A similar result wasobtained when Al. bovis sonicate was usedwith the 30-40 kD and LMW antigen beingeluted from either L9 or L4. Thus, the de-terminants recognized by the two antibod-ies on the 30-40 kD and LMW antigenswere present in a common complex.

DISCUSSIONThese six antibodies were shown to define

two different determinants on related, cell-wall carbohydrate antigens, both of whichwere present in M. boils and Al. tuberculosisas well as M. leprae. The fact that the de-terminants recognized by the Mabs werecarbohydrate in nature was demonstratedby the resistance to protease and by the sen-sitivity to oxidation with periodate at lowconcentrations (Figs. 1 and 2). Despite beingsimilar in composition the two determi-nants could be distinguished on the basis oftheir location on antigens of differing mo-lecular weights when examined by poly-acrylamide electrophoresis in the presenceof SDS. There was no difference in the Mrof the antigens in reducing and nonreducingconditions, indicating there were no cova-lent disulfide bonds between the 30-40 kDand 4.5-6 kD antigens. Furthermore, therewas no crossinhibition between the threeMabs (LI, L9, and L10) which reacted withthe 30-40 kD antigen and the three Mabs(L3, L4, and B6) recognizing the 4.5-6 kDLMW band (2). In addition, even thoughboth antigens were apparently associatedwith the cell wall, being present in equiva-lent concentrations in sonicate and resoni-cate (Figs. 3-5), only the 30-40 kD antigencould be detected by the immunofluores-cence and capture assay on the intact bacilli.Nevertheless, the two antigens were clearlyrelated in a structural sense since they couldbe eluted from either L9 or L4 Mab-affinitycolumns (Fig. 6), which suggested they wereassociated in aqueous solution presumablyby non-covalent bonds. Consistent with thisconclusion were the findings from the cap-ture assay in which L9-immobilized antigencould bind '25I-L4 and vice versa (Table 2).Thus, the LMW and 30-40 kD antigens ap-peared to be derived from a common cell-wall molecule. Covalent bonds linking the

552^ International Journal qf Leprosy^ 1986

two antigens could have been split by theson icznion process, leaving them associatednon-covalently in a molecular complexwhich could be immobilized by either L9or L4. However, when the complex was ex-amined under the denaturing conditions ofSDS-PAGE, the two antigens had differentmobilities. Thus, they probably both orig-inate from the polysaccharide side chainswhich are attached to the peptidoglycanbackbone of mycobacterial cell walls (7' ").

Both arabinogalactan (AG) and arabin-omannan (AM) have been isolated frommycobacteria ("). AG was demonstratedto be a structurally important constituent ofthe cell wall in the seven mycobacterialspecies studied by Misaki and co-workersand its general structure and major antigen-ic determinant have now been defined (25)•The molecular weight of AG derived fromM. bolls was found to be 31 kD (26). In thecase of Al. leprae, Draper showed that thecell walls contained arabinose and galactosein the molar ratio 3:1, and considered thatAG with attached mycolic acid was a majorpart of the wall (8). AM, on the other hand,was thought to be a cytoplasmic rather thana cell-wall polysaccharide and that it mayhave contaminated previous cell-wall prep-arations (""). Recent data, however, indi-cate that AM may be a significant cell-wallpolysaccharide as well. Miller and Buchan-an isolated Mab AM8-2c from a fusion us-ing mice primed with AM purified from M.smegmatis (22). The molecular weights ofthe two bands identified by AM8-2c were80-90 kD and 30-40 kD, and the latter hada similar appearance to the L9-defined bandin immunoblots of Al. leprae sonicates. Thebinding site of AM8-2c encompassed morethan one sugar residue. AM isolated fromAl. smcgmatis reacted strongly with humanleprosy sera, indicating there was crossreac-tivity between antisera to the cell-wall poly-saccharide of M. smegmatis and M. leprae(23). This indirect evidence for the presenceof AM in the cell wall of Al. leprae has nowbeen confirmed following the isolation ofphosphorylated lipoarabinomannan-B(pLAM-B) from Al. leprae (16). Thus, thepurified pLAM-B proved to have a similarmolecular weight (35 kD) and appearancein immunoblots to the L9-defined antigen,and it reacted with human antisera.

On the basis of these findings, it was pos-sible that the L9- and L4-defined determi-nants could, in fact, be located on pLAM-B.To test this, pLAM-B purified from M. tu-berculosis was reacted with L9, L4, and athird Mab, 906.4, directed against LAMfrom Al. /eprac in ELISA, inhibition RIAand antigen capture assays. Both the LAMand 906.4 were kindly provided by Dr. P.J. Brennan (Colorado State University). Theresults indicated that L9 did indeed bind toLAM, on which it reacted with a separateepitope to that recognized by 906.4. Fur-thermore, according to the antigen inhibi-tion assay, there was an increased concen-tration of the L9-defined determinant inLAM from Al. tuberculosis compared to Al.leprae sonicate. By contrast, no binding ofL4 to pLAM-B was demonstrable (data notshown). This could have been due to thelocation of the L9- and L4-reactive epitopeson different polysaccharides. However, inview of the fact that the two determinantsco-eluted from the Mab affinity columns,the more likely explanation is for the epi-topes to be on the same molecule and forthe L4-defined determinant to have beendestroyed during preparation of pLAM-B.When taken in conjunction with the datashowing the binding of L9 to intact bacilliby immunofluorescence (see results), thesefindings provide additional support in favorof the presence of AM in the cell wall.

The results described here and those ofother investigators point to differences inthe species distribution of the epitopes de-fined by L4, L9, and AM8-2c. AM8-2c wasstrongly reactive with all species except Al.tuberculosis ("). Both the inhibition ELISAand capture assay data indicated that theL4-defined epitope on the LMW antigen wasmore abundant in Al. bovis and Al. tuber-culosis (Table 1). Possibly this fragment ismore readily solubilized by sonication inthese two species. Conversely, the L9 de-fined epitope on the 30-40 kD band wasabundant in Al. leprae; whereas both Al.bolls and M. tuberculosis sonicates dis-played a lower level of reactivity with L9.Misaki and co-workers (24) have demon-strated that species variation between AMpurified from different mycobacterial speciesresided in the secondary side chains whichconsisted mainly of mannose. The LMW

54, 4^Britton, et al.: Carbohydrate Antigens of M. leprae^553

fragment would appear to be derived fromthe part or the AM molecule common tothe mycobacterial species studied.

The antigen capture assay was most ef-fective when '25I-L9 was the tracer antibodyand either L9 or L4 was the capture anti-body (Table 2). This indicated that the L9-defined epitope was a repeating determinanton the 30-40 kD molecule. By contrast, theLMW fragment in Al. leprae sonicate wasmore efficiently bound by '251-L4 if first cap-tured by L9, suggesting there are a limitednumber of L4 binding sites on this frag-ment. A similar radiometric assay with asingle Mab directed against the circumspo-rozoite proteins of malaria parasites wasequally effective because there were multi-ple common epitopes within that moleculeas well (32). The sensitivity of the captureassay with L9 was greater than for any com-bination of Mabs directed against the pro-tein antigen of Al. leprae, including the 18kD, 60-65 kD, and 70 kD antigens (unpub-lished observations). The value of this assayin measuring mycobacterial antigens in bodyfluids is currently being assessed.

The cell-wall-associated polysaccharideantigens are strong B-cell immunogens inman (7). Thus, the titer of antibody againstAl. smegmatis AM in patients with lepro-matous leprosy was found to be propor-tional to the bacterial load of the patients(23). We have shown that the mean inhibi-tory titers of leprosy sera in Mab inhibitionassays with L4 were greater than those ob-tained with Mabs directed against proteinantigens (2). Even though the L4- and L9-defined determinants were present in Al. tu-berculosis, the inhibitory titers of the tu-berculosis sera tested were not significantlyraised above that of the controls. In otherwords, the serological response in lepro-matous leprosy to these common polysac-charide antigens appeared to be an effectivemarker of disease status.

The role of cell-wall polysaccharide instimulating T cells is less clear. Arabino-galactans and arabinomannans have elicit-ed immediate-type skin reactions in sensi-tized guinea pigs (14), but with two exceptionsthey have failed to elicit delayed-type hy-persensitivity in the same species (4 5). Inhuman studies, the mycobacterial polysac-charides have failed to stimulate prolifera-

tion of lymphocytes or to elicit skin reac-tions from tuberculin-positive subjects (6.27).On the contrary, AM purified from M.tuberculosis was shown to suppress antigen-specific proliferative responses by a mac-rophage-dependent method (' 1). The capac-ity of antigenic determinants on protein-free,cell-wall polysaccharide of Al. leprae toeither stimulate or to suppress the cell-me-diated immune response to Al. leprae in manawaits elucidation.

SUMMARYMonoclonal antibodies (Mabs) raised

against Mycobacterium leprae sonicate de-fined two different determinants on related,cell-wall-associated, carbohydrate antigenscommon to Al. leprae, M. bovis (BCG), andM. tuberculosis. Antigen inhibition ELISAand antigen capture assays demonstratedthat the two antigens were present in a cell-wall fraction, Al. leprae resonicate. Therewas species variation in the distribution ofthe antigens; the 4.5-6 kD antigen was moreabundant in Al. tuberculosis and M. bovis,while the 30-40 kD antigen was more con-centrated in Al. leprae preparations. Al-though both were present in the cell wall,only determinants on the 30-40 kD antigenwere accessible on intact bacilli. The resultsfrom capture assays and Mab affinity chro-matography with both L9 and L4 indicatedthat the 4.5-6 kD antigen was probably afragment of the larger molecule. Both an-tigens are significant immunogens in the hu-man B-cell response to M. leprae.

RESUMENSc prepararon anticuerpos monoclonales contra un

extracto soluble del Mycobacterium leprae, los cualesidentificaron a 2 determinantes antigenicos di ferentespresentes en carbohidratos asociados a la pared celulardel Al. leprae, M. bolls, BCG, y Al. tuberculosis. Losensayos de ELISA para inhibiciOn por antigeno y paracaptura de antigeno, demostraron que los 2 antigenosestuvieron presentes en la fracciOn correspondiente ala pared celular del Al. leprae. Sc encontrO variaciOnen la distribuciOn de los antigenos de acuerdo a lasespecies; el antigeno 4.5-6 kD estuvo más concentradoen las preparaciones de M. tuberculosis y de Al. bovis,mientras que el antigeno 30-40 kD fue más abundanteen el Al. leprae. Aunque ambos estuvieron presentesen la pared celular, solo los determinantes del antigeno30-40 kD fueron accesibles en los bacilos intactos. Losresultados de los ensayos de captura y la cromatografia

554^ International .lournal of Leprosy^ 1986

de afinidad tanto con L9 como con L4, indicaron queel antigcno 4.5-6 kD fue probablemente un fragment°del de mayor peso molecular. Ambos antigenos soninmunogénicos en los humanos.

RESUME-Des anticorps monoclonaux (Mabs) dirigés contre

des mycobactéries de la lepre (Mycobacterium lepme)traitées aux ultrasons ont permis de mettre en evidencedeux determinants distincts sur des antigenes hydro-carbones associes a la paroi cellulaire, communs a M.leprae, a M. bolls (BCG) et a M. tuberculosis. tin testde detection d'inhibition par ELISA et un test de cap-ture d'antigene ont démontre que les deux antigênesétaient presents dans une fraction de paroi cellulaireobtenue aprés une repetition du traitement de .11. lep-rae aux ultrasons. On a constaté une variation dans ladistribution des antigenes, scion l'espece de mycobac-terie. L'antigene 4.5-6 kD emit plus abondant chez M.tuberculosis et M. bovis; l'antigene 30-40 kD était plusconcentre dans les preparations de Al. leprae. Alorsque les deux antigenes &talent presents dans la paroicellulaire, seuls les determinants mis en evidence surl'antigene de 30-40 kD etaient accessibles chez desbacilles intacts. Les resultats des essais de capture, dcmeme que l'affinité chromatographique des anticorpsmonoclonaux pour L9 et L4, indiquaient que l'antigene4.5-6 kD etait probablement un fragment d'une mo-lecule volumineuse. Les deux antigenes se sont révélésetre des immunogenes significatifs pour induire unereponse des cellulcs 13 humaines a Al. leprae.

Acknowledgments. We thank Dr. R. J. W. Rees whosupplied M. leprae bacilli through the IMMLEP pro-gram. This work was supported by the National Healthand Medical Research Council of Australia, and theLeprosy Eradication and Education Program of LionsInternational. We are grateful for the assistance ofJudyWhitehead in typing this manuscript.

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