67, 3^ Correspo ndence^ 305

lowing treatment and these declines parai-leled declines in the BI. 6) In cases curcdwith dapsone therapy, thcre was an increaseor a persistcnce of . high OD valucs in ND-ELISA prior to the onset of a leprosy re-lapse.

In conclusion, we have compared andevaluated 12 immuno-assays and haveshown that the ND-ELISA is the most prac-tical one for use in investigating sero-im-munological epidemiology, subclinical in-fection with Al. leprae, early detection ofdisease, monitoring of antimicrobial ther-apy, and even for the prediction of leprosyrelapse.

—Qinxue WuXinyu Li

Yueping YinHuiwen ShuWanhui Wei

Qi LiuGanyun Ye

Institute ofDermatologyCAMS anui PUMCNanjing 210042Jiangsu Prot'inceChina

REFERENCES1. Wu., Q.X., Li, X. Y., Wia, W. H., SHI'.N, J. P.,

SuENc, D. M. and Yo, G. Y. [Potcntial of gelatinagglutination test in the study of leprosy.] ChinaLepr. J. 9 (1993) 138-141.

2. Wu, Q. X., Li, X. Y., Y1N, Y. P. and Hou, W. [Anew SACT mcthod and its comparison with PGL-I-ELISA.] China Lepr. J. 12 (1996) 108-112.

3. Wu, Q. X., MA, Z. X., Suo, 11. W., Zuou, L. L., Liu,Q., Yi:, G. Y., WANG, T. J. and MA, B. K. [Conipar-ison of ELA-ABS test employing sera from venousblood and blood from earlobes of 79 cases of lep-rosy.] Acta Acad. Med. Sin. 7 (1985) 69-71.

4. Wu, Q. X., WI:I, W. 1-1., Liu, Q., Li, X. Y., XiI:, Z.Y. and Yu, G. Y. Ms-ELISA for detection of serumantihody leve) in patients—establishment of Ms-ELISA.] Acta Acad. Med. Sin. 10 (1988)451-455.

5. Wo, Q. X., Xue, Z. Y., Li, X. Y., Woi , W. H. andYi:, G. Y. [Detection of specitic circulating im-mune complexes in leprosy patients by mousemonoclonal antihody against phenolic glycolipid-1.) Acta Acad. Med. Sin. 11 (1989) 293-297.

6. Wu, Q. X., Yi:, G. Y. and Li, X. Y. Serological ac-tivity of natural disaccharide octyl bovine serum al-humin (ND-O-BSA) in sera from patients with lep-rosy, tuberculosis and normal controls. Int. J. Lepr.56 (1988) 50-55.

7. Wu, Q. X., Yo_, G. Y., Li, X. Y., Liu, Q. and Zuou,L. L. A prcliminary study on serological activity ofa phenolic glycolipid from Al. leprae in sera frompatients with leprosy, tuberculosis and normal con-trols. Lepr. Rev. 57 (1986) 129-136.

8. Wu, Q. X., Yi., G. Y., YIN, Y. P., Li, X. Y., Liu, Q.and Wia, W.1-1. Rapid serodiagnosis for leprosy-a prcliminary study on the latex agglutination test.Int. J. Lepr. 58 (1990) 328-333.

9. Wu, Q. X., Yi:, G. Y., Znou, L. L., Suo, 11. W., Liu,X. Y., MA, Z. X. and Li, Z. W. Determination ofantibodies in dried blood from earlobes of leprosypatients by cnzyme-linkcd immunosorhent as-says—a prcliminary report. Int. J. Lepr. 53 (1985)565-570.

Vaccination of Mice Against the Leprosy Bacillus withSkin-Test Antigens

To 'mi: EDrroi:

The specter of approximately 700,000new leprosy cases a year, despite a preva-lence of only about 805,000 (" ' 2), hauntsus. Many, perhaps the majority, of thesecases are detected in the course of intensivecase finding ("), and are single-lesion caseswhich are difffcult to diagnose definitively.Measurement of the true incidence of lep-rosy requires new diagnostic tools, more

sensitive than physical examination sup-ported at times by slit-skin smear (''). Nei-ther serology, even the modern, user-friendly, sensitive phenolic glycolipid-I(PGL-I)-based kits (S), nor gene-amplifica-tion based on a variety of primers providesthe necessary sensitivity ( 10). A promisingapproach to the diagnosis of asymptomaticleprosy is provided by the availability oftwo new skin-test antigens—MLSA-LAM(soluble antigens of Mycobacteritun leprae

306 International Journal n/ Leprosy

TI II: TA M.E. Residis of /iarrests o/ M. leprae.

1999

'I'rcatnutnt .

No. AFI3 per foot pad (x10') Proha-bilityIndividual teci Mediar

Control 9.14, 5.50, 4.44, 2.40, 1.24, 0.888 3.42 0.0037''1FA 10.7, 10.0, 9.50', 8.70, 4.44 9.50 0.0014'MLSA-LAM in IFA 6.04, 3.73, 2.31, 1.78, 1.42, 1.18, 0.888, 0.710, 0.710, 0.710 1.78 0.00046°MLCwA in IFA 4.79', 2.40, 1.86, 0.444 4.68 0.0047 . 'MI.CwA-LAM in IFA 6.30, 5.68, 5.15, 3.20, 2.38', 0.355, 0.178 2.56 0.00089''APOI1 39.8, 13.7', 8.08, 6.83, 6.66, 5.59, 3.99, 2.04, 1.42, 1.15, 0.799 6.74 0.45"MLSA-LAM in AI'OH 26.4, 13.6, 6.57', 6.30, 5.77, 4.79, 3.37, 2.13, 2.01 6.13 0.72''MLCwA in AI'OII 17.2', 6.30, 2.84, 2.57, 0.533, 0.444 6.30 0.85''MLCwA-LAM in AI'OH 4.88, 3.46',0.799 3.46 0.091"

IFA = incomplete Freund's adjuvant; APOR = aluminum hydrogel; MLSA-LAM = M. /eprur-soluble anti-gen from which lipoarabinomannan has beco removed; MLCwA = ht. /optar° ccll-wall antigen; MLCwA-LAM= M. leprae ccll-wall antigen from which Iipoarahinomannan has been removed.

''The prohahility, determined hy the Kruskal-Wallis one-way analysis of variance, that the results from ali ofthe relevant groups of mice were drawn from a single population.

This harvest was performcd from the pooled tissues of four foot pads."The probability, determined by the Mann-Whitney U test, that the results from the group of mies indicated

werc drawn from the same population as were those from the relevant control grou!).Additional prohabilities: Control vs. IFA vs. APOH: 0.072; IFA vs. APOIL: 0.41; MLCwA in IFA vs. MLCwA-

LAM in IFA: 0.92; MLCwA in APOH vs. MLCwA-LAM in AP011: 0.55.

from which the immunosuppressivc lipoara-binomannan has been removed) and ML-CwA (M. leprae ccll-wall antigens) ( 2 ).These materiais have successfully under-gone Phase I safety studies in the U.S.A.,and Phase II studies are currently beingplanned for severa' arcas of high leprosy en-demicity. Awaiting the outcome of thesestudies, we examined the protective potencyof these products in the BALB/c mouse.

M. lepras harvested from the livers andspleens of M. lepras- infected armadilloswere disrupted by sonication and centrifugedto yield two major fractions—the solublesupernatant (MLSA) and the insoluble pei-let (MLCwA) ( 2). Much of the soluble car-bohydrates and lipids in the preparations,especially lipoarabinomannan (LAM) andthe phosphatidylinositol mannosides, wereremoved hy extraction with Triton X-114 toyicld the two fractions, MLSA-LAM andMLCwA-LAM ('). These materiais wereproduced at Colorado State University un-der the conditions rcquircd for approval oftheir use as skin-test antigens in humans (').BALB/c mice were bred under specificpathogen-free (SPF) conditions in the ani-mal facility of the Sasakawa ResearchBuilding, Ministry of Public Health, Non-thaburi, Thailand, from a nucleus purchasedfrom CLEA Japan, Inc., Kawasaki, Japan.The inoculum of M. leprae was prepared

from the foot pads of BALB/c mice thathad been inoculated approximatcly 4months earlier with a suspension of organ-isms of the "Thai 53" strain that had beenprovidcd by Dr. J. L. Krahenbuhl, BatonRouge, Louisiana, U.S.A. When the organ-isms were found to have multiplied to anumber approaching 10' per foot pad, thebacterial suspension was diluted to a con-centration of 5 x 10' organisms per 0.03 mi.

For the purpose of this experiment, themice of one group were held as untreatedcontrols, and those of other groups were ad-ministered incomplete Freund's adjuvant(IFA) or aluminum hydrogel (APOH), anadjuvant suitable for use in humans, or oneof the two antigen preparations suspendedin one of the adjuvants intracutaneously inthe flank. After 1 month, the coice werechallenged in the right hind foot pad with 5x 10' M. leprae. At intervals thercafter, theorganisms were harvested frota the pooledhind foot pads of four control mice, stainedby a standard, room temperature acid-faststain, and counted directly

The results of this experiment are pre-sented in The Table as the actual number ofacid-fast bacilli (AFB) per individual footpad as well as the median number of AFBper foot pad for each group. These resultsdemonstrate that ali three antigens wereprotective when administercd to mice after

67, 3^ Correspondelice^ 307

emulsification in 1FA; whcrcas nono of thethrcc antigens protectcd micc whcn admin-istcrcd in APOH.

Previously, we reported that immuniza-tion of mice with various ceil-wall fractionsof Al. leprae, progressiveiy depletcd oflipids, carbohydratcs, and soluble protcins,confcrred significant protection against sub-sequent infection with livc M. leprae (`),and subscqucnt studics (`) demonstratedthat a mixturc of protcins derived from theM. leprae ccll wall confcrred long-termprotection, compared to the shorter-termprotection provided by individual proteins.The conclusion from this experiment wasthat multiple Al. leprae protein epitopes arecriticai for solid vaccine protection againstthe infection. The activity of the fractionsstudied in this experiment, MLSA andMLCwA, from which LAM and other ap-parcntly imnutnosuppressivc lipids and li-poglycans have becn removed, provides ad-ditional evidence of the utility of multicom-poncnt fractions as distinct from individualproteins.

—Macya Ngamying, M.S.Louis Levy, M.D., Ph.D.

Sasakawa Research BuildingNational Institute of HealthDepartment of Medical ScienceMinistry of Public HealthTiwanond RoadNonthaburi 11000, Tlutiland

—Patrick J. Brennan, Ph.D.

Department of MicrobiologyColorado State UniversityFort Collins, Colorado 80523, U.S.A.

Reprint requests to Dr. Patrick J. Brennan at theabove address or FAX: 970-491-1815; e-mail:pbrennan@cvmbs.colostate.edu

Acknowledgment. We thank the Sasakawa Memo-rial Health Foundation for support of this research andDr. Yo Yuasa for his interest and encouragement. Spe-cial appreciation is extended to scientists Ms. PhanidaPhaknilrat, Ms. Sopa Srisungnam, and Ms. SukanyaWattanapokayakit at the Sasakawa Research Building.The antigens were produced under the terras of NIH,

NIAID Contract NOI AI-55262 with Colorado StateUniversity.

REFERENCES1. B13-IND 7938. FDA designated title Mvcobac-

teriam leprae (soluble antigen MLSA-LAM; ccllwall antigen MLCwA) skin test antigens (ar-madillo-derived). 1998.

2. BRENNAN, P. J., DoNnuul:, S. A., Suor'', D. K.,Rlvouzl:, 13. L. and WALSH, G. P. A plan for prepa-rado!) and application to humans of new lcprosyskin-test antigens. Int. J. Lcpr. 63 (1995) 645-646.

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4. GI:LuLIZ, R. H., Mi:uizA, V., BLOOM, B., MURRAY,L. P., Siu, P., TSANG, M. and BRGNNAN, P. J. Vac-cination with pure Mvcobacterium leprae proteininhibits M. leprae mulliplication in mouse footpads. Infect. Immun. 62 (1994) 4250-4255.

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6. Sio:P, iw, C. C. The experimental diseasc that fol-lows the injection of human lcprosy bacilli loto footpads of mice. J. Exp. Med. 112 (1960) 445-454.

7. Snuuaw, C. C., V,N LANDINGIIAM, R. M. andWALKI:R, L. L. Immunity to Mvcobacterium lep-rae infections in mice stimulated by M. leprae,BCG, and graft-versus-host reactions. Infect. Im-mun. 14 (1976) 919-928.

8. SIn:PAKD, C. C., V,N LANDINGIAM, R. M.,WAI.KI:IZ, L. L. and Yu, S. Z. Comparison of theimmunogenicity of vaccines prepared from viableMvcobacterium bovis BCG, heat killed Mvcobac-teriam leprae, and a mi xture of the two for normaland Mvcobacterium /eprae-tolerant mice. Infect.Immun. 40 (1983) 1096-1103.

9. Sstrrll, W. C. S. We need to know what is hap-pening to the incidence of leprosy. Lepr. Rev. 68(1997) 195-200.

10. WILLIAMS, D. L., GII.LIS, T. P., Boo -rtl, R. J.,LOOKER, D. and WATSON, J. D. The use of a spe-citic DNA probe and polymerase chain rcactionfor the detection of Mycobacterium leprae. J. In-fect. Dis. 162 (1990) 193-200.

II. Woi i.D HuxI:rii ORGAN zATION. Action Pro-gramme for the Elimination of Leprosy. Status Re-port 1998. WHO/LEP/98.2 (1998) 1-57.

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