INTERNATIONAL JOURNAL OF LEPROSY^

Volume 58, Number IPrinted in the U.S.A.

Antibodies to Lipoarabinomannan Antigenin Sooty Mangabey Monkeys Experimentally

Inoculated with Mycobacterium lepraeiBobby J. Gormus, Keyu Xu, Wayne M. Meyers,

Gerald P. Walsh, William R. Levis,and Harry C. Meeker2

There is considerable interest in the pos-sibility that antibody assays for antigens ofMycobacterium leprae might provide ameans of early detection of subclinical lep-

(4, 8, 9, 11, 1 6, 18‘. Trosy ) • It is also important todetermine ifleprosy-susceptible individualscan be identified among groups in leprosy-endemic areas.

The availability of purified forms of my-cobacterial carbohydrate-containing anti-gens, such as lipoarabinomannan (LAM) andthe Al. leprae-specific (12, 14. 15

) phenolic gly-colipid-1 (PGL-I) antigen, has made it pos-sible to perfect serologic assays for the de-termination of antibodies to these cell-wallconstituents (l• 6' 1 (). The serodominant ep-itope of the PGL-I antigen has been foundto reside in the terminal trisaccharide regionof the molecule; synthetic glycoconjugatesbound to bovine serum albumin (BSA) areavailable for use in ELISA for detection ofanti-PGL-I antibodies (1, 2, 5, 6, 12, 14, 15).

Several studies utilizing these serologicmethods have suggested that antibody titersto PGL-I can be used to identify the ma-jority of multibacillary (MB) leprosy pa-tients and lesser numbers of paucibacillary(PB) subjects (1, 2, 5, 6, 17, 20. 22) Some studiessuggest that antibody titers to PGL-I deter-mined by EL1SA may be useful in identi-

' Received for publication on 30 May 1989; acceptedfor publication in revised form on 16 August 1989.

= B. J. Gormus, Ph.D., Research Scientist; K. Xu,M.D., Delta Regional Primate Research Center, Tu-lane University, Three Rivers Road, Covington, Lou-isiana 70433, U.S.A. W. M. Meyers, M.D., Ph.D., Chief,Mycobacteriology, Armed Forces Institute of Pathol-ogy, Washington, D.C. 20306, U.S.A. G. P. Walsh,Ph.D., Leonard Wood Memorial Laboratory for Lep-rosy Research, P.O. Box 727, Cebu City, The Philip-pines. W. R. Levis, M.D.; H. C. Meeker, Ph.D., BayleySeton Hospital, Staten Island, New York, New York10304, U.S.A.

fying subclinical leprosy patients amongleprosy household contacts (3,18,21 ) , but thispoint is disputed (").

In an ELISA study of IgG and IgM an-tibody titers to LAM and PGL-I, Levis, etal. found that 71% of PB and 85.5% of MBpatients were positive for at least one of thefollowing three antibodies: IgG anti-LAM,IgM anti-LAM, or IgM anti-PGL-I; onesymptomatic contact with a neuropathy waspositive for anti-LAM and anti-PGL-I IgMand for IgG antibodies to PGL-I (b6). Theseresults suggest that it might be important tomonitor each of these antibody types in or-der to detect most Al. /eprae-infected in-dividuals.

We recently observed that monitoring IgGand IgM antibody levels to PGL-I in eightsooty mangabey monkeys (('ercocebus alt's)experimentally inoculated with Al. lepraeindicated that elevated anti-PGL-I IgG cor-related with resistance to and elevated anti-PGL-1 1gM with susceptibility to leprosy(13). In the present study, we monitored theIgG and IgM antibody levels to LAM inthese same eight mangabeys to test the sug-gestion of Levis, et al. that IgG and IgMantibody titers to LAM and PGL-I can beused together for diagnosing leprosy, and todetermine if LAM antibody levels are re-lated to disease susceptibility ( 16).

MATERIALS AND METHODSAnimals and M. leprae inoculations. We

have previously reported the details con-cerning the sources of animals, routes ofinoculation, criteria for disease classifica-tion, and descriptions of leprosy progres-sion in the four pairs of mangabeys studiedhere (13)• In brief, mangabeys D171 andD172 were inoculated with the highest doseof M. leprae (4.8 x 10'° bacilli, 10% mor-

65

IgG Anti-LAM

6 6^ International Journal of Leprosy^ 1 990

Ao 0177 LLo 0174 LL

• D172 LL,

A 0176 LL

0171 LL

L.D173 Ind

1 0178 TT/neuritic

• 0175 Healthy

40

1.8

16

1.4

• AVG of Puifloectilary cases

12

LAVG of Paucibacollary cooes

0.6

0.4

0.2

X u••uhy

30^ 40Time Postinoculation (Months)

FIG. 1. Serum IgG antibody levels to lipoarabinomannan (LAM) antigen in mangabey monkeys before andat intervals after H. leprac inoculation. A = The indicated mangabcys (D171, D172, D174, D176, and D177)

58, I^Gornius, et al.: Anti-LAM Ab in Mangabeys^67

phologic index); serial, tenfold dilutions weregiven in a paired fashion to D173 and D174,D175 and D176, and D177 and D178, re-spectively. Monkey sera were obtained atthe times indicated and stored at —70°Cuntil assayed.

It is noteworthy that the animals wereage- and sex-matched (males) and were bornin captivity in a group compound at theYcrkes Regional Primate Research Center(Atlanta, Georgia, U.S.A.). After shipmentto the Delta Regional Primate ResearchCenter (Covington, Louisiana, U.S.A.) in1982 at approximately 2 years of age, theanimals were housed in individual cages.

Leprosy classifications. All classifica-tions of leprosy are according to the Ridleyand Jopling system (19).

ELISA. LAM was purified from M. tu-berculosis and provided under NIAID con-tract #1-AI-52582 by Dr. Patrick J. Bren-nan, Colorado State University, FortCollins, Colorado, U.S.A. LAM was dis-solved in carbonate buffer at 0.5 ktg/ml. Flat-bottom, 96-well plates (Immulon I; Dyna-tech Laboratories, Inc., Chantilly, Virginia,U.S.A.) were used, and a Dynatech MR-700ELISA reader was used to determine thefinal optical density (OD) values. Monkeysera were assayed at the predetermined op-timal dilutions of 1:200 for IgM and 1:100for IgG.

Anti-LAM levels were determined afteradding 50 pi of LAM or carbonate bufferalone to experimental or control wells, re-spectively, and incubation of the plates at4°C overnight. After emptying, 100 Al of 1%bovine serum albumin-phosphate bufferedsaline (BSA-PBS), pH 7.4, was added to eachwell and the plates were incubated for 1 hrat 37°C. The plates were then washed withPBS and incubated with 50 kt1 of monkeyantiserum diluted in PBS-10% heat-inacti-vated (56°C, 30 min) normal goat serum forI hr at 37°C followed by washing four timesin PBS. Fifty l of optimally diluted (1:1000,IgG; 1:500, IgM) peroxidase-conjugated,goat anti-human IgG or IgM (anti-Fe piece,

1J, - or -y-specific, affinity purified; CooperBiomedical, Malvern, Pennsylvania, U.S.A.)was then added to each well. The plates wereincubated for 1 hr at 37°C followed by fourwashes in PBS. Thereafter, 50 Al of 0-phen-ylenediamine (Sigma Chemical Co., St.Louis, Missouri, U.S.A.) at 0.4 mg/ml in0.2 M citrate-phosphate buffer, pH 5.0, con-taining 0.002% H,07, was added to eachwell. The wells were acidified with 50 il of2.5 N H2SO4, and the OD at 490 nm wasdetermined. The reported OD values rep-resent the OD of wells containing LAM an-tigen minus that of control wells lackingLAM antigen but containing all other com-ponents.

Statistics. Anti-LAM OD values forgroups of animals with lepromatous (LL)leprosy, those with paucibacillary disease,and one of the Al. /eprae-inoculated man-gabeys that remained healthy were subject-ed to an analysis of variance. The resultsshowed that values for paucibacillary lep-rosy could be grouped together with the lep-rosy-resistant Al. /eprae-inoculated man-gabey, and that the data from the fiveanimals with LL leprosy could also begrouped. A comparison of mean values forthe two groups by the analysis of varianceshowed that OD values for the LL groupdiffered significantly from that of the othergroup (p < 0.0001). Further comparison ofthe two groups by the Student's t test con-firmed that level of significance.

RESULTSFigure IA shows the time course of the

appearance of serum IgG antibody to LAMin each of the eight mangabeys beginningprior to Al. leprae inoculation. Five of theeight animals (D171, D172, D174, D176,and D177) ultimately developed leproma-tous (LL) leprosy. Preinoculation levels ofIgG antibody to LAM were highest in these5, compared to the other 3 animals, andeach of the 5 developed anti-LAM IgG levelsin excess of 1.2 OD units after Al. lepraeinoculation.

developed lepromatous (LL) or subpolar lepromatous (LLs) leprosy; D173 developed indeterminate leprosy;D178 developed TT/neuritic leprosy; D175 remained healthy. B = Average values for the group of animals thatdeveloped LL (multibacillary) forms (•) or indeterminate and TT/neuritic (paucibacillary) forms (A) of leprosyand the values for the animal that failed to develop leprosy ( x).

IgM Anti-LAM

1.6

1.4

1.2

x D171 LL

• D172 LLs

n D173 Ind

o D174 LL

• D175 Healthy

A D176 LL

o D177 LL

0.6I D178 TT/neuritic

1.8

cr) 1 . 0

00.8

68^ International Journal of Leprosy^ 1990

10^ 20^

30^ 40Time Postinoculation (Months)

Fin. 2. Serum IgM antibody levels to lipoprabinomannan (LAM) antigen in mangabey monkeys before andat intervals after Al. leprac inoculation (see Fig. IA legend for details).

Two (D173 and D178) of the remainingthree mangabeys developed paucibacillaryforms of leprosy (indeterminate and tuber-culoid/neuritic) and the other animal (D175)has remained healthy to date. These latterthree mangabeys each had preinoculationlevels of anti-LAM IgG no greater than 0.4OD unit, and the levels remained below 1.0OD unit throughout the course of obser-vation after Al. leprac inoculation except fora single transient fluctuation above that val-ue (Fig. 1A). Mangabeys D171, D172, andD176 developed clinical signs of LL leprosyby 4-6 months, D174 by 10 months, andD177 by 26 months postinoculation; D173showed clinical signs of paucibacillary lep-rosy by 12 months and D178 by 35 monthspostinoculation (13). In each animal thepreinoculation level of anti-LAM IgG hadbegun to increase prior to the clinical ap-pearance of leprosy (Fig. 1A).

Figure 1B shows the average ELISA level

for anti-LAM IgG in the group of five man-gabeys with multibacillary (LL) leprosy, thetwo animals with paucibacillary leprosy (in-determinate and TT/neuritic), and thehealthy mangabey. The average valuesclearly show that mangabcys susceptible tomultibacillary (LL) forms of leprosy haveelevated levels of anti-LAM IgG prior to ALleprae inoculation, and the levels rapidlyincrease after inoculation in conjunctionwith progression to LL leprosy (13). Man-gabeys that will ultimately develop pauci-bacillary forms of leprosy have much lowerbaseline anti-LAM IgG levels that graduallybegin to increase after inoculation and priorto the appearance of clinical signs of leprosy(13) (Fig. 1B). The leprosy-resistant man-gabey (D175) had a preinoculation anti-LAM IgG level of 0.4 OD unit which failedto undergo a significant sustained change(Fig. 1B).

Figure 2 shows the IgM anti-LAM ELISA

58, 1^Gornnts, et al.: Anti-LAM Ab in Mangabeys^69

data. Preinoculation levels were below 0.1OD unit in each of the 8 mangabeys, andthe levels rose transiently above 0.2 ODunits after AL leprae inoculation in only 3out of 5 of the animals that developed LLforms of leprosy (D171, D174, and D176).IgM anti-LAM levels ultimately fell to be-low 0.1 OD unit in all eight mangabeyswithin 3 years after M. leprae inoculation.There were no significant detectable anti-LAM IgM responses in the leprosy-resis-tant, healthy animal (D175), the two man-gabeys with paucibacillary leprosy (D173and D178), and two of those with LL lep-rosy (D172 and D177) (Fig. 2).

DISCUSSIONThe data are consistent with the obser-

vations of Levis, et al. that anti-LAM IgGantibody levels are useful for detecting in-dividuals with multibacillary and pauciba-ciliary leprosy (16). Elevated levels of anti-LAM IgG were observed in each of the fivemangabeys that developed LL forms of lep-rosy months before the appearance of clin-ical signs of leprosy (13). Significantly, thelive LL leprosy-susceptible mangabeys hadthe highest levels of anti-LAM IgG of theeight mangabeys studied, greater than 0.5OD unit, prior to Al. leprae inoculation. Af-ter inoculation, the IgG anti-LAM level in-creased to and remained greater than 1.2OD unit in each of the five animals with LLleprosy.

The two mangabeys that developed pau-cibacillary forms of leprosy (D173, indeter-minate; D178, tuberculoid/neuritic) hadanti-LAM levels of 0.32 and near 0 ODunits, respectively, prior to Al. leprae in-oculation; the levels increased gradually, be-ginning several months prior to the ap-pearance of clinical symptoms and reachingmaximal levels above 0.8 OD unit after theappearance of disease (13). Mangabey D175,which has failed to develop signs of leprosy(13), had a preinoculation anti-LAM IgGlevel of 0.4 OD unit which has fluctuatedbut remained near the preinoculation level3 years after M. leprae inoculation.

These results, together with our previousobservations on IgG and IgM anti-PGL-Iantibody levels (13), confirm that of the fourantibody types studied, IgG and IgM anti-PGL-I and IgG and IgM anti-LAM, at leastone will be detectably elevated in monkeys

after infection with Al. leprae and prior tothe appearance of signs of the disease (16).This is markedly so in the case of animalsthat will develop LL forms of leprosy. Itshould be noted, however, that our previousdata showed that elevated IgG anti-PGL-Ilevels, especially in the absence of signifi-cant IgM anti-PGL-I antibody levels, cor-relate with resistance to the development ofleprosy although a subclinical infection maybe present (13).

Anti-LAM IgM levels increased signifi-cantly but transiently in only 3 of the 5 LL-susceptible mangabeys. The remaining an-imals studied showed no significant IgManti-LAM levels. These observations sug-gest that anti-LAM IgM antibodies appearto play little role in the pathogenesis of lep-rosy.

Our observations indicate that anti-LAMIgG levels are much more sensitive in thedetection of preclinical leprosy and/or sus-ceptibility to the disease than the determi-nation of anti-PGL-I IgG or IgM levels.Anti-PGL-I IgG or IgM levels are muchmore specific for leprosy detection, how-ever, inasmuch as all eight mangabeys stud-ied had significant anti-PGL-I IgG or IgMlevels at some point after H. leprae inocu-lation but no detectable anti-PGL-I IgG orIgM levels prior to M. leprae inoculation(13). This pattern of specificity is not sur-prising since LAM antigen may be commonto most if not all mycobacterial species,whereas PGL-I appears to be a specific Al.leprae antigen (12.14,15,.

) It is significant,however, that the mangabeys with anti-LAMIgG levels greater than 0.5 OD prior to in-oculation were all susceptible to LL formsof leprosy; whereas the remaining three an-imals each had an anti-LAM IgG level be-low 0.5 OD and each of these failed to de-velop multibacillary leprosy. This resultsuggests the possibility that prior exposureto LAM-containing environmental myco-bacteria may play a role in increasing thesusceptibility to leprosy in at least some in-dividuals. Each of the animals studied,however, was age- and sex-matched and hadsimilar histories. They were born and group-housed in the same compound, followed bylater housing in individual cages in the sameroom. They each had the same sources offood and water. It seems likely, therefore,that each animal might have been exposed

70^ International Journal of Leprosy^ 1990

to the same sources of environmental my-cobacteria. Further study will be necessaryto elucidate the detailed nature of the re-lationship between anti-LAM IgG levels andsusceptibility to leprosy.

Pronounced differences in leprosy sus-ceptibility were observed between pairs ofmangabeys that were inoculated by similarroutes with identical numbers of M. leprae("). Thus, whatever the mechanism(s) maybe, the results arc consistent with the pos-sible involvement of underlying genetic in-fluences in addition to possible environ-mental factors (").

One practical consideration resulting fromour observations is that in leprosy-endemicareas, asymptomatic individuals with ele-vated anti-LAM IgG titers alone or espe-cially in combination with anti-LAM IgMor anti-PGL-I IgG or IgM might be advisedto undergo prophylactic multidrug or vac-cine therapy (7). Asymptomatic individualswho possess such antibody profiles and wholive in very close contact with LL leprosypatients might be especially advised to con-sider prophylactic therapy.

It should be noted that in the present studywe had the luxury of being able to study themonkeys prior to M. leprae infection, toknow precisely when infection occurred, andto obtain serum samples and observe theanimals longitudinally as leprosy developedand progressed. The mangabeys were bornand reared in the U.S.A. and, in all prob-ability, had never been exposed to M. lepraeprior to experimental inoculation. Thiscombination of circumstances is not-readilypossible in human populations. One cannotknow whether an individual in a leprosy-endemic area has been subclinically infectedwith M. leprae or another mycobacterium.This presents a problem in defining what a"normal" or unexposed baseline level ofanti-PGL-I or anti-LAM antibody titermight be among any given human popu-lation. For this reason, it is necessary toperform longitudinal serological ELISA forIgG and IgM antibody levels to PGL-I andto LAM in order to monitor and screen hu-man groups who are at risk for leprosy in-fection.

Human populations in leprosy-endemicareas should be carefully studied to deter-mine whether our observations apply to hu-mans. Previous studies in human leprosy

patients suggest that similar relationshipsmay exist (16). A minimal ELISA screen forIgG antibody to LAM might be recom-mended; follow up anti-PGL-I IgM deter-minations would be suggested for those whotest positive for anti-LAM. Our observa-tions in mangabey monkeys suggest that in-dividuals with high and increasing levels ofIgG anti-LAM and IgM anti-PGL-I and lowor absent IgG anti-PGL-I are highly sus-ceptible and at risk to develop LL leprosy(13).

SUMMARYIgG and IgM antibody levels to myco-

bacterial lipoarabinomannan (LAM) anti-gen were determined by ELISA in eight sootymangabey monkeys (Cercocebus alt's) priorto and at intervals after experimental in-oculation with Mycobacterium leprae. Highlevels of anti-LAM IgG were present beforeinoculation and increased thereafter in thefive mangabeys that developed lepromatous(LL) forms of leprosy; lower levels of anti-LAM IgG were observed in two mangabcysthat developed indeterminate leprosy andtuberculoid/neuritic leprosy, respectively,and in a mangabey that was leprosy resis-tant. IgM anti-LAM levels were near zerobefore M. leprae inoculation in all eight an-imals, rose significantly in only three LL-leprosy-susceptible animals after inocula-tion, and returned to near zero in all animalswithin 3 years.

Anti-LAM antibody levels appear to bepotentially valuable as an indicator of lep-rosy susceptibility, and when measured lon-gitudinally together with antibody levels toM. /eprae-specific phenolic glycolipid-I an-tigen, as a means to detect preclinicalleprae infections in high-risk individuals.

RESUMENUsando un metodo inmunoenzimatico (ELISA) se

determinaron los niveles de anticuerpos IgG e IgMcontra la lipoarabinomanana (LAM) micobacterianaen 8 monos mangabey pardos (Cercocebus atys) antesy a intervalos despues de la inoculaciOn experimentalcon Mycobacterium leprae. En 5 monos que desarrolla-ron lepra lepromatosa se encontraron niveles elevadosde IgG anti-LAM antes de la inoculaciOn con M. lepraey êstos aumentaron despues de la infecciOn; en 2 monosque desarrollaron lepra indeterminada a tuberculoide/neuritica y en un mono que fue resistente a la lepra,

58, 1^Gormus, et al.: Anti-LAM Ab in Mangabeys^71

se observaron niveles más bajos de IgG anti-LAM. Losniveles de IgM anti-LAM fueron cercanos a cero en 8monos antes de la inoculaciOn con M. leprae, en 3animales susceptibles (lepromatosos) los niveles de IgMaumentaron significativamente despues de la inocu-laciOn y regresaron a valores cercanos a cero en todoslos animales dentro de los siguientes 3 afios.

Los anticuerpos anti-LAM parecen ser potencial-mente valiosos como indicadores de susceptibilidad ala lepra, y cuando se miden paralelamente con los anti-cuerpos anti-glicolipido fenOlico-I del Al. leprae, re-sultan adecuados para detectar infecciones preclinicaspor Al. leprae en individuos de alto riesgo.

RESUMEChez huit singes mangabey (C'ercocelms alt's), on a

determine au moyen d'une inethode ELISA les tauxd'anticorps IgG et IgM au lipoarabinomannan myco-bacterien (LAM), avant inoculation experimentale parMycobacterium leprae, et ensuite ft intervalles repâtesapres cette inoculation. Des taux eleves d'IgG-LAMetaient decelables avant l'inoculation; ces taux ont en-suite augmente chez les cinq singes ayant developpedes formes lepromateuses (LL) de la lepre. Des tauxplus faiblcs d'IgG anti-LAM ont ete relevecs chez lesdeux singes mangabey qui avaient developpe respec-tivement une lepre indeterminee, et une lepre tuber-culoIde de type nevritique, de mane que chez un singequi emit resistant ft la lepre. Les taux d'IgM anti-LAM&talent proches de zero avant inoculation par Al. leprae,et ceci chez les huit animaux. Ces taux ont alors aug-ment& de maniere significative apres l'inoculation cheztrois animaux susceptibles a la lepre lepromateuse; ilssont retournes a des niveaux proches de zero chez busles animaux dans les trois annees qui ont suivi.

Les taux d'anticorps anti-LAM semblent constituerun indicateur peut-etre interessant de la susceptiblitêa la lepre. Lorsqu'on les mesure de maniere longitu-dinale, en meme temps que les taux d'anticorps ft l'anti-gene phenoglycolipidique-I specifique pour M. leprae,ils pourraient permettre de (feeder des infections pre-cliniques a M. leprae chez des individus a haut risque.

Acknowledgments. We are indebted to Mrs. CyndiTrygg for technical assistance and to Mrs. Mary AnnBennett for typing this manuscript. Graphics were pro-vided by Mr. Murphey Dowouis. Financial supportwas provided by grants from the National Institutes ofHealth #RR-00164, the National Institute for Allergyand Infectious Diseases #2R22AI19302 and from theLeonard Wood Memorial Leprosy Foundation. Thesooty mangabeys were provided by Dr. Harold Mc-Clure of the Yerkes Regional Primate Research Center,Atlanta, Georgia, U.S.A.

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