INTERNATIONAL JOURNAL OF LEPROSY^ Volume 51, Number 3

Printed In the U.S.A.

INTERNATIONAL (ISSN 0148-916X)

JOURNAL OF LEPROSYAnd Other Mycobacterial Diseases

VOLUME 51, NUMBER 3^ SEPTEMBER 1983

Relation Between Anti-Mycobacterium leprae AntibodyActivity and Clinical Features in Borderline

Tuberculoid (BT) Leprosy'Els J. M. Touw Langendijk, Titia Warndorff van Diepen,

Morten Harboe, and Ayele Belehu 2

For several decades, the main view hasbeen that antibodies against mycobacterialantigens occur frequently and in highamounts in lepromatous leprosy and rarelyand in low concentrations in tuberculoidleprosy. This view was based on studies em-ploying various immunological methods,such as complement fixation tests ( 4 ."), pas-sive hemagglutination tests ('), and doublediffusion precipitation reactions in gel(16.18,20,24) .

The introduction of new, sensitive ra-dioimmunoassays for the demonstrationand quantification of antibodies led to a re-vision of this view. When groups of patientswere studied, it was demonstrated that themedian antibody concentration decreasedgradually from the lepromatous to the tu-

' Received for publication on 30 November 1982;accepted for publication on 15 February 1983.

= E. J. M. Touw Langendijk, M.D.; T. Warndorff vanDiepen, M.D., All-Africa Leprosy and RehabilitationTraining Centre (ALERT), P.O. Box 165, Addis Aba-ba, Ethiopia. M. Harboe, M.D., Ph.D., University ofOslo, Institute for Experimental Medical Research, UI-levaal Hospital, Oslo 1, Norway. A. Belehu, Ph.D.,Armauer Hansen Research Institute (AHRI), P.O. Box1005, Addis Ababa, Ethiopia (deceased 17 May 1983).

berculoid end of the spectrum. It becameapparent, however, that there was a strikingvariation in antibody activity in individualsera from patients with similar clinical andhistological classification, e.g., in borderlinetuberculoid (BT) leprosy. This type of vari-ation in antibody activity has been dem-onstrated in radioimmunoassay (RIA) forthe quantification of antibodies against BCGantigen 60 ( 8) and its crossreacting antigenMycobacterium leprae antigen 7 ( 15 ' 26). Sim-ilar findings were also made in solid-phaseradioimmunoassay (sRIA) for IgG, IgA andIgM anti-M. leprac antibodies ( 13 ). The rea-son for this variation in antibody activityhas not been established.

The purpose of the present investigationwas to study groups of patients with bor-derline tuberculoid (BT) leprosy to obtainadditional information on the relationshipbetween activity and clinical features in thisform of leprosy.

MATERIALS AND METHODSPatient sera. Sera were obtained from two

groups of patients with BT leprosy at theAll-Africa Leprosy and RehabilitationTraining Centre (ALERT), Addis Ababa,

305

306^ International Journal qf Leprosy^ 1983

Ethiopia. The cases were classified clinical-ly, and in most cases histologically, accord-ing to the extended Ridley-Jopling scale( 17 . 21 ' 22 ). One group comprised the first 40patients with newly diagnosed 13T leprosywho, according to the available informa-tion, had not been treated previously andwere entered into the TUBA trial. The sec-ond group comprised the first 40 patientswith I3T leprosy entered into the SUS trial.They were suspected from their case his-tories to have dapsone (DDS) resistant dis-ease, appearing with active skin lesions ornew skin lesions despite information on pre-vious DDS treatment of long duration. Theywere put on DDS treatment under super-vision with regular clinical examinations toestablish if they were clinically resistant toDDS. Studies on antibody activity duringcontinuous treatment with DDS, includingcases with clinical DDS resistance followingchange of drug therapy, are reported in theaccompanying paper ( 6 ).

The sera used in the present study wereobtained from the first venous blood sampletaken at entry into the study. The sera wereseparated from venous blood, stored at—25°C in Addis Ababa, and subsequentlytransported in a frozen state to Oslo, Nor-way, for testing by RIA. Due to damageduring storage and transportation, three serafrom the SUS group were not included inthe study.

Mycobacteria and antibody assays. Al.leprae was provided by Dr. R. J. W. Reesfrom the IMMLEP bank as freeze-dried ba-cilli purified from liver tissue of infectedarmadillos. Two radioimmunoassays wereused.

Antibodies against M. leprae antigen 7were determined by RIA using protein Acontaining staphylococci to separate anti-body-bound labeled antigen from free an-tigen ( 15). Labeling and testing of the labeledantigen and performance of the assay aredescribed in the accompanying paper ( 6 ).Antibody activity is expressed in percentof the activity in a lepromatous scrum pool(LSP) used for reference, and the calcula-tions were made as described previously(13,14) .

A solid phase RIA was used for the quan-tification of IgG antibodies against variousantigenic components in M. leprae sonicate

as described previously (''•'') and in the ac-companying paper ( 6 ).

Statistical calculations. Wilcoxon's ranksum test for unpaired samples was used totest differences between groups ( 25 ). The sta-tistical significance of association betweenhigh and low antibody activity, as definedin the Results section, and various clinicalfeatures were evaluated by the x 2-test forsmall numbers calculated from 2 X 2 con-tingency tables ('''). Probability (p) valueslarger than 0.05 were regarded as not sig-nificant (n.s.).

RESULTSThe Figure shows the findings in the two

antibody assays. Each point corresponds toone patient, filled circles representing theSUS group of previously treated suspectedDDS resistant cases, open circles the TUBAgroup of new cases. Anti-M. leprae 7 anti-body activity varied from 550% to 1% ofthe activity in the LSP used for reference,the median value for the whole group being19% of the LSP. The activity in the SUSgroup was higher than in the TUBA group,with median values of 40% and 11.5%, re-spectively. The difference between the twogroups was close to the significance level,p 0.05.

The findings in the IgG anti-Af. lepraeassay were similar with a marked variationin activity between individual sera. The me-dian value for the whole group was 22% ofthe activity in the LSP. Again, the SUS groupshowed higher activity (median value 30%of the LSP) than the TUBA group of newcases (median value 8 % of the LSP). Thedifference between these two groups was sta-tistically significant, p < 0.01.

These findings strongly indicated that thebasic differences in clinical features whichled to the division of the borderline tuber-culoid patients into these two groups wasassociated with differences in antibody ac-tivity. To obtain further information on thispoint, two groups of sera were defined, thosewith high antibody activity and those withlow antibody activity. These groups areshown in The Figure. High antibody activ-ity was defined as being more than threetimes the median activity, meaning above57% of the activity in the LSP in the anti-M. leprae 7 assay and above 66% of the LSP

51, 3^Toutt' Langendijk, et al.: Anti-M1 Ab in 137' Leprosy^307

TABLE 1. Relationship between treatment status and antibody activity in 13T leprosy.

frprai, 7 IgG anti-M. leprae

High'^Low''^High"^Low"

Previously treated, still activecases (SUS group) 16 1 18 11 5 16

New cases (TUBA group) 8 15 23 7 17 2424 17 41 18 22 40

x= = 12.18^ 2C2 = 6.08I ds.^ 1

p < 0.001^ p < 0.02

Higher than three times median value of the whole group of 77 sera.Lower than one third of the median value of the whole group.

in the IgG anti-A/. Ieprae assay. Similarly,low antibody activity was defined as activitybelow one third of the median value of thewhole group, meaning less than 6.3% of theactivity in the LSP in the anti-.11. /eprac 7assay and less than 7.3% of the LSP in theIgG anti-Al. leprae assay.

Table 1 shows the relationship betweenantibody activity and treatment status inBT leprosy. Eighteen of the previously treat-ed but still active cases in the SUS grouphad antibody activity above three times orbelow one third of the median value of thewhole group in the anti-M. leprae 7 assay,with 16 cases in the former and only two inthe latter group. In the group of new cases,

the reverse was found with eight patientshaving high and 15 low antibody activity.This difference was statistically significantwith a p value <0.001 in the x 2-test. In theIgG leprae assay the difference be-tween these two groups was also statisticallysignificant, p < 0.02.

Table 2 shows various comparisons madeto see if differences between the two groupswere significantly correlated with antibodyactivity.

In the newly diagnosed cases, the ap-pearance of "active lesions" with signs ofinflammation was not significantly corre-lated with high antibody activity.

In the SUS group further subdivision was

TABLE 2. Relationship between antibody activity and clinical fmtures in B7' leprosy.

Signi-ficance'Group

Anti-%1. leprae 7 Ratio

High" Low"

Total material 24 17 1.4

Males 11 8 1.4^1Females 13 9 1.4^fNew cases (TUBA) 8 15 0.53Previously treated

cases (SUS) 16 2 8.0^

}

TUBA, active lesions 3 9 0.33TUBA, inactive lesions 5 6 0.83

SUS^5 yrs treatment 8 0 ccSUS < 5 yrs treatment 8 2 4.0

SUS, active lesions 13 0 coSUS, quiescent lesions 3 21 1.5

SUS, neuritis 8 0 coSUS, no neuritis 8 2 4.0

IgG anti-.11. leprae^Ratio

High' Low"

18 22 0.8210 10 1.

8 120.067

7 17 0.41

11 5 2.20

3 10 0.304 7 0.57

6 05 5 1.0^}7 04 _2 2 70 } 6 0 co^15 5 1.0^f

n.s.

p < 0.001

11.S.

n.s.

p < 0.02

n.s.

Signi-ficance"

n.s.

p < 0.02

n.s.

p < 0.05

n.s.

p < 0.05

Defined as in Table I." Calculated by x 2 test as in Table 1.

308^ International Journal of Leprosy^ 1983

Antibody Activity(in % of LSP)

TABLE 3. Relationship between antibodyactivity and length of treatment in the SUSgroup of BT leprosy.

Anti-M. leprae 7^IgG anti-M. leprae

g^

lc)8^0

cP41-4•

Duration of treatment in years

High activity'^Low activity"

Median Mean Median Mean

Anti-M. leprae 7IgG^leprae

^

4.0^5.7^2.5^2.5

^

6.0^6.4^2.0^2.2

Defined as in Table I.

it^•^in cases with active skin lesions to activityin the anti-A!. leprae 7 assay and in caseswith neuritis to the activity in the IgG anti-M. leprae assay.

10 0 0

100

1 0

.0.0

0

0

951"0

88.00f0•0088

THE FIGURE. Antibody activity in borderline tu-berculoid (BT) leprosy. The activity in the assay forantibodies against .11. leprae antigen 7 and IgG anti-bodies against various antigens of M. leprae is recordedin percent of the activity in a lepromatous serum pool(LSP) used for reference. Each point represents onepatient; • = previously treated but still active cases(the SUS group); 0 = newly diagnosed cases (the TUBAgroup).

correlated to a significant extent to variationin antibody activity. The relationship tolength of treatment is shown in Table 3. Inboth assays, high antibody activity was cor-related with longer duration of disease andtreatment. The difference was not statisti-cally significant in the anti-Al. leprae 7 assaydue to the presence of only two SUS casesin the low activity group in this assay; where-as it was statistically significant in the IgGanti-Al. leprae assay.

Signs of inflammatory activity in the le-sions were correlated to antibody activity,

DISCUSSIONThe present investigation confirmed the

previous demonstration of marked varia-tion in antibody activity in individual pa-tients with BT leprosy ( 132(.) both in the as-say for antibodies against AI. leprae antigen7 and in the polyvalent solid RIA for IgGantibodies against various antigenic com-ponents present in Al. leprae sonicates.Yoder, et al. (26) studied groups of BT lep-rosy patients treated for various periods oftime and found that DDS treatment led toa marked decrease in antibody activity andthat suspected or proven relapse was asso-ciated with renewed antibody synthesis andincreased anti-Al. leprae 7 antibody activityin the serum. These observations are prob-ably reflected in the difference observed be-tween the SUS and TUBA groups in thepresent investigation. The SUS group con-sisted of cases with a continued presence ofactive lesions or cases who developed newlesions despite DDS treatment for severalyears. As shown in the accompanying paper( 6) this group was heterogeneous. Althoughthe patients were suspected to have DDSresistant disease on the basis of their clinicalhistory, some of them responded to super-vised DDS treatment. In these cases, thepresence of active lesions or the appearanceof new lesions was probably related to ir-regular or insufficient administration ofDDS, and these cases correspond to "re-lapse cases" in the study of Yoder, et al. ( 26 ).In other cases, supervised treatment withDDS did not lead to clinical improvement,and these patients were then clinically di-

51, 3^Touw Langendijk, et al.: Anti-MI Ab in BT Leprosy^309

agnosed as having DDS resistant disease. Inboth groups, active multiplication of bacilliis expected to occur, resulting in an in-creased antigenic load in the organism andprobably in an increased liberation of an-tigen from bacilli-containing cells whichimply a potent stimulus to the immune sys-tem, resulting in increased antibody synthe-sis.

This is probably the main reason for theincreased antibody activity in the SUS groupcompared with the newly diagnosed cases.

Skin lesions with signs of inflammationand the development of acute neuritis havebeen demonstrated to be associated with in-creased activity in lymphocyte transfor-mation tests ( 2 ' 3 ' 7 ), that is, with increased de-layed-type hypersensitivity against antigensof Al. leprae. The main current view is thatthe development of active inflammation inskin lesions or nerves is not only associatedwith increased cell-mediated immune re-actions, but directly mediated by the cell-mediated hypersensitivity to mycobacterialantigens liberated in the skin lesions or pe-ripheral nerves. The correlation betweensigns of inflammation of lesions and highantibody activity observed in the presentwork indicates that the underlying processesare associated with the stimulation of bothhumoral and cellular immune responses. Ithas often been claimed that there is an in-verse relationship between cell-mediated andhumoral immune responses to M. lepraethroughout the leprosy spectrum ( 5 ' 16). Pre-vious studies by various radioimmunoas-says have shown that this view cannot beupheld since there is a wide variation inantibody activity in individual patients withsimilar clinical classification (t•13.26), and nostrict inverse relationship between cell-mediated and humoral immune responseswas observed when assayed against one de-fined antigenic component of AI. leprae ()).The present study adds a new facet to thisdiscussion by showing that increased in-flammatory activity in skin lesions andnerves is associated with high antibody levelsin patients with persisting BT leprosy, andthere is an obvious need for combined stud-ies of humoral and immune reactions dur-ing reactions in leprosy.

In newly diagnosed cases of BT leprosythere was no significant correlation betweenthe presence of active skin lesions and high

antibody activity. In such cases the basicstimulation of the immune system is prob-ably less profound, and the stimulation byantigens of actively inflamed lesions is notstrong enough to "break through" to inducea marked increase in antibody activity. Thecause of the wide variation in antibody ac-tivity in individual cases with newly diag-nosed BT leprosy has not been established.Further studies should be made in this groupwhich appears to be particularly useful asan indicator of other factors influencing theimmune response during the developmentof paucibacillary leprosy. In early cases oftuberculoid leprosy, it is expected that sev-eral other factors, such as genetic predispo-sition, extent and kind of previous exposureto environmental mycobacteria, concurrentinfection and malnutrition, may strongly in-fluence the antibody activity as measuredin the current assays. In these cases it wouldbe particularly important to correlate anti-body activity with various indicators of an-tigenic load. This load represents mycobac-terial antigen not only in skin lesions butalso elsewhere in the body, since several au-thors have provided evidence that leprosyis a generalized disease, not only in the lep-romatous forms but also in the apparentpaucibacillary forms of tuberculoid leprosy(10,23) .

SUMMARYAntibody activity against Mycobacterium

leprae antigen 7 was determined by ra-dioimmunoassay and IgG antibodies againstvarious antigens present in an M. lepraesonicate by a solid phase radioimmunoas-say in 77 patients with borderline tuber-culoid (BT) leprosy.

In both assays there was a wide variationin antibody activity in individual patientsalthough all were diagnosed as having BTleprosy. The median antibody activity waslower in newly diagnosed cases than in pa-tients appearing with active skin lesions ornew skin lesions despite dapsone (DDS)treatment of long duration. Further com-parison of patients with high and low an-tibody activity revealed that high antibodyactivity was significantly correlated statis-tically with active skin lesions, new skin le-sions and neuritis despite DDS treatmentof long duration. The reason for variationin antibody activity in newly diagnosed BT

310^ International Journal of Leprosy^ 1983

leprosy remains unclear, and this patientgroup is of particular interest for furthercharacterization of the basis for variation inantibody activity in tuberculoid leprosy.

RESUMENSe determinO la actividad de anticuerpo contra el

antigen° 7 de Mycobacterium lepra' (por radioinmu-

noensayo) y Ia actividad de anticucrpo IgG contra los

antigenos presentcs en un sonic-ado de .11. /eprac (por

radioinmunoensayo en base sOlida), en 77 pacientes con

lepra tuberculoide intermedia, 13T.En ambos ensayos Sc presentO una amplia variaciOn

en la actividad de anticuerpo en los pacientes indivi-

duales, aunque todos ellos se diagnosticaron comp B . F.

La mediana de la actividad de anticuerpo file mzis baja

en los casos de reciente diagnOstico que en los pacientescon lesiones activas en la piel (nuevas o crOnicas), in-

dependientemente del trzuamiento con dapsona (DDS).

La comparaciOn adicional de los pacientes con alta y

baja actividad de anticucrpo revel() que la alta acti-vidad de anticuerpo correlacionaba estadisticamente

con la presencia de lesiones activas cn Ia piel, de le-siones nuevas en la piel y de neuritis, siendo esto in-

dependiente del tratamiento prolongado con DDS. La

razOn de la variaciOn en la actividad de anticuerpo enpacientes I3T de reciente diagnOstico permanecc obs-cura pero este grupo de pacientes es de interes parti-cular porque su estudio detallado nos puede ayudar a

entender las causas de la variaciOn en la actividad de

anticuerpo observada en lepra tuberculoide.

RESUMEOn a procede au dosage radioimmunologique de l'ac-

tivite en anticorps contre l'antigene 7 de Mycobacte-

rium leprae, ainsi qu'au dosage radiobiologique en phase

solide des anticorps IgG contre divers antig&nes pre-

sents dans un sonicat de M. Ieprae, chez 77 malades

atteints de lepre tuberculoide dimorphe (BT).

Pour l'un et l'autre de ces dosages, on a constate une

grande variation dans l'activite en anticorps chez desmalades individuels, quoique tous aient etc diagnos-tiques comme atteints do lepre BT. L'activite moycnne

en anticorps etait plus faible chez des cas recemment

diagnostiques que chez des malades presentant des le-

sions cutanees actives, ou de nouvelles lesions cuta-flees, malgre un traitcment prolonge a la dapsone (DDS).La comparaison des malades presentant respective-

ment des activites &levees ou faibles en anticorps a

revele qu'une activite elevee en anticorps presentait

une correlation statistiquement significative avec leslesions cutanees actives, les nouvelles lesions cutanees,

et la nevrite, et ceci malgre un traitement prolong& parla DDS. La raison de cette variation dans Eactivite en

anticorps chez des malades BT recernment diagnos-

tiques, n'est pas claire. Ce groupe de malades presenteun interét particulier pour une etude ulterieure des fac-

teurs qui expliquerait les variations dans l'activite enanticorps dans la lepre tuberculoide.

Ackno The work was supported by grants

from Anders Jahre's Fund for the Promotion of Sci-

ence, the Norwegian Council for Science and the Hu-manities, and by the Immunology of Leprosy(IMMLEP) component of the UNDP/World Bank/W 110 Special Programme for Research and Training

in Tropical Diseases. We thank Tadelle Ciebretsion andh lelen Bcrgsvik Mr their excellent technical assistance.Armauer Hansen Research Institute is sponsored by

the Norwegian and Swedish Save the Children Orga-

nizations.

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