Proc. Nadl. Acad. Sci. USA Vol. 85, pp. 1917-1921, March 1988 Immunology Immunological significance of leprae cell walls (cell-mediated immunity/delayed-type hypersensitivty/peptidoglycan- complex//precursor frequency analysis) JOHANNE MELANCON-KAPLAN*, SHIRLEY Wu HUNTERt, MICHAEL MCNEILt, CAROL STEWARTt, ROBERT L. MODLINf, THOMAS H. REAt, JACINTO CONVIT§, PADMINI SALGAME*, VIJAY MEHRA*, BARRY R. BLOOM*, AND PATRICK J. BRENNANt¶ *Department of Microbiology and Immunology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY 10461; tDepartment of Microbiology, Colorado State University, Fort Collins, CO 80523; tSection of Dermatology, University of Southern California School of Medicine, Los Angeles, CA 90033; and 1lnstitute de Biomedicine, Caracas 101, Venezuela Contributed by Barry R. Bloom, November 2, 1987

ABSTRACT Cell walls of Mycobacterium leprae, pre- positive healthy contacts of lepromatous patients or from pared by differential solvent extraction, were shown to contain blood or lesions of patients with , only a arabinogalactan, mycolates, and peptidoglycan. In addition, small proportion of clones tested are responsive to the amino acid analysis revealed the unexpected presence of large known serologically defined recombinant (ref. 5; amounts of protein that retained potent immunological reac- J.M.-K., R.L.M., and B.R.B., unpublished data). tivity. Purified cell walls stimulated proliferation of T cells For many years, significant immune responses to purified from tuberculoid, but not from , patients cell walls of various mycobacteria have been sporadically and elicited delayed-type hypersensitivity skin reactions in reported. For example, cell walls from Mycobacterium guinea pigs and patients sensitized to M. leprae. Analysis ofthe can evoke delayed-type hypersensitivity (DTH) precursor frequency of -reactive human peripheral T responses (6), and, of course, adjuvant activity is associated cells revealed that as many cells (41/6000) proliferate to with the muramyl-dipeptide of the basic peptidoglycan (7). antigen contained in cell walls as to intact M. keprae. Sequen- The present work explores the possibility that M. leprae cell tial removal of mycolates and arabinogalactan resulted in a wall contains antigens important for cell-mediated immunity large peptidoglycan-protein complex that retained all the to this organism. immunological activity. This immunological reactivity and the inherent protein were destroyed by proteolysis. Thus, cell wall protein is a major contributor to cell-mediated immune reac- METHODS AND MATERIALS tivity to this pathogenic mycobacterium. Preparation of Cell Walls of M. eprae. Lyophilized M. leprae (1.42 g) prepared from infected livers (8) Leprosy is a chronic infectious disease that afflicts 10-13 was exhaustively extracted with CHC13/CH30H (2:1) to million people, primarily living in developing countries (1). remove all lipids, disrupted in a French press followed by Although the incidence of leprosy is declining in many parts sonication and further extraction with refluxing 70% ethanol of the world, largely due to greater control efforts, the cost in water to remove lipoarabinomannan, lipomannan, other of multidrug therapy to combat emergent drug resistance of soluble carbohydrates, and soluble (9). The residue Mycobacterium leprae is high, and the development of an was washed extensively with water to yield 671 mg of cell effective vaccine is widely agreed to offer the best hope for wall insoluble fraction (CWIF). disease eradication (2, 3). Because M. leprae is one of few Cell wall core (CWC) was prepared by stirring CWIF (10 major pathogens of man that has not been successfully mg) in 1-2 ml of 2% NaDodSO4 for 2 hr at 50'C; this cultivated in vitro, the principal available source of antigens extraction was repeated four times. The final insoluble for study and vaccines remains infected tissues of the residue was further extracted 10 times with phosphate- nine-banded armadillo novemcinctus, which is in- buffered saline (PBS), followed sequentially by water and evitably limited and costly. Consequently, immunological acetone to yield 6.5 mg of CWC. and molecular biological approaches that permit identifica- Mycolic acids were removed from CWC (10 mg) by tion and production of protective antigens are needed. Thus stirring in 5 ml of 0.5% KOH in C2H5OH at 370C for 48 hr. far, using murine monoclonal , genes for six major The reaction mixture was centrifuged, and the pellet was antigens of M. leprae have been isolated from Agtll libraries washed three times with 5 ml each of water, ethanol, and (4). ether to yield 6.3 mg of demycolated CWC. To destroy the From a variety of studies, cell-mediated immunity, which covalently bound arabinogalactan, 10 mg of demycolated is regulated by specifically sensitized T lymphocytes, is CWC was stirred into 3 ml of 0.05 M NaIO4 buffered with clearly required for protection and resistance to leprosy (1). 0.03 M sodium acetate, pH 4.0, and kept under dark condi- The disease encompasses an immunological spectrum. At tions at 250C for 42 hr. The insoluble residue after centrifu- one pole, lepromatous leprosy, patients selectively lack gation at 20,000 x g for 20 min was washed extensively with cell-mediated immunity to antigens of M. leprae and fail to water and reduced with NaBH4 (1.25 mg in 250 Al) at 250C restrict the growth of the pathogen; at the tuberculoid end of for 1 hr. The insoluble residue was again thoroughly washed the spectrum, the patients exhibit cell-mediated immunity to with water, and the residual polyols were removed by gentle antigens of M. leprae and develop one or a few sharply hydrolysis (500 1.l of HCl, pH 1.0, at 150C for 18 hr), defined local lesions that contain only few acid-fast . followed by repeated washing. Exactly 5 mg (dry weight) of However, when we and other workers derived M. leprae- specific CD4 + (helper ) clones from strongly lepromin- Abbreviations: CWIF, cell wall insoluble fraction; CWC, cell wall core; PPC, peptidoglycan-protein complex; DTH, delayed-type The publication costs of this article were defrayed in part by page charge hypersensitivity; GC, gas chromatography; PBS, phosphate- payment. This article must therefore be hereby marked "advertisement" buffered saline. in accordance with 18 U.S.C. §1734 solely to indicate this fact. ITo whom reprint requests should be addressed. 1917 Downloaded by guest on September 26, 2021 1918 Immunology: Melancon-Kaplan et al. Proc. Natl. Acad Sci. USA 85 (1988) this peptidoglycan-protein complex (PPC) free of mycolates Table 1. Chemical composition of the cell wall fractions of and arabinogalactan was recovered. M. leprae Proteolytic digestion of CWIF or CWC was accomplished Cell wall fraction,* weight % by adding protease (either one-half the substrate weight of proteinase K or Pronase from Streptomyces griseus; type Whole Demycolyl Cell wall XXI, Sigma) to the sample in 2 ml of PBS containing 0.5% Component M. leprae CWIF CWC CWC PPC Triton X-100. The mixture was sonicated and incubated at Arabinogalactan 15.9 12.9 22.5 33.1 0.6 37°C for 4 hr and centrifuged at 20,000 x g for 20 min; then Other the pellet was further digested under identical conditions. carbohydrates 20.9 2.0 0 0 0 The pellet was again recovered by centrifugation, washed Mycolates 12.8 35.0 37.0 0 0 repeatedly with water, and Iyophilized to yield 6.7 mg of Peptidoglycan 6.3 6.1 6.9 10.7 18.4 protease-digested CWIF. Protein 50.8 42.8 25.9 38.1 60.6 Analytical Procedures. The neutral sugar content and, *Arabinogalactan was estimated by hydrolysis of cell wall fractions from this content, the arabinogalactan content of cell wall with 2 M CF3COOH and GC of the alditol acetates with inositol fractions were measured by hydrolysis of 100-ug samples added as an internal standard. Arabinose represented 77.3% of the containing 9 of inositol added as internal standard with 2 neutral sugars and galactose represented 18.6% with a trace of pg mannose (0.4%). Mycolates were estimated by weighing the sapon- M CF3COOH, conversion to the corresponding alditol ace- ifiable lipids obtained after treatment of cell wall fractions with tates, and resolution by gas chromatography (GC) on a 0.5% KOH in C2HOH. Peptidoglycan and protein amino acids Durabond-1 fused silica capillary column (30 m x 0.25 mm, were estimated as described in the legend for Fig. 2; peptidoglycan 0.25-,um film thickness; J&W Scientific, Rancho Cordova, content was then calculated using the total weights ofglucosamine, CA) (10). To analyze amino acid and aminoglycoside con- muramic acid, diaminopimelic acid, and the weight of one mol tent, 100-pug samples of cell wall were hydrolyzed with 4 M equivalent of alanine, glycine, and glutamic acid as compared with or 6 M HCl at 115°C for 16 hr and converted to the diaminopimelic acid. corresponding N (O for serine and threonine)-heptafluoro- heptafluorobutyryl isobutylglycoside isobutyl esters for glu- butyryl isobutyl esters, for amino acids, and N- or O- cosamine and muramic acid (11). The derivatives were then A chromatographed on the described Durabond-1 capillary 40-0 column. a-Aminoadipic acid was included in each assay as an internal standard to allow measurement. The amino acid E and aminoglycoside assignments were confirmed by gas 30 chromatography and mass spectrometry using electron- impact ionization. CD20 Lymphocyte Transformation and Precursor Frequency Analysis. The precursor frequency of human peripheral L 0 blood T lymphocytes reactive to various antigen prepara- tions from M. leprae was determined by a modified limiting L00 dilution procedure (12). Briefly, T lymphocytes were iso- lated from the blood of lepromin-positive tuberculoid lep- LL BL BT/11 NORMAL rosy patients or contacts, and replicate cultures were estab- I~ n 27 12 25 12 AMINO ACIDS AND AMINO SUGARS OF A OF M. B ~~~CWC leprae 0 M -1 0z I-- n I ~~~z-J a) 0 0 0 w a) co 0 I'C z 0 IL 0 CO) ______Ili - 0) co Lu a) z B AMINO COMPOUNDS 0w PRONASE-TREATED CWC OF M. Iopras

Cells / Culture (xi 0-3) 0 FIG. 1. Proliferative responses to M. leprae, cell wall, and 11 Ii soluble antigens of T cells from patients across the disease spec- LA trum. (A) [3H]Thymidine incorporation at 6 days by lymphocytes I fI'u1 from patients with polar lepromatous (LL), borderline lepromatous (BL), borderline tuberculoid (BT), and tuberculoid (IT) forms of 10 15 leprosy, stimulated with 0.1 ml of M. leprae (o), CWIF (m), soluble RETENTION TIME (MIN) M. leprae extract (batch CD64) (me) (all at 10 ,ug/0.1 ml). (B) Precursor frequency analysis. (A), Dharmenda lepromin; (e), FIG. 2. Amino acid and aminoglycoside composition ofthe cell CWCs; (o), soluble antigen batch CD64; (o), soluble antigen batch wall fractions ofM. leprae. (A) CWC hydrolyzed with 6 M HCI. (B) CD78. Numbers indicate estimated frequency of antigen-reactive Pronase-digested CWC. DAP, diaminopimelic acid; GlcNH2, glucos- proliferating T cells. amine; Mur, muramic acid. Downloaded by guest on September 26, 2021 Immunology: Melancon-Kaplan et al. Proc. Nati. Acad. Sci. USA 85 (1988) 1919 lished in round-bottom microtiter wells. Identical sets con- turer's instructions. Skin tests were made intradermally taining 500, 1,000, 5,000, 10,000 and 20,000 T lymphocytes 30-45 days later using killed M. leprae, cell wall prepara- together with 104 irradiated erythrocyte-rosette-negative an- tions, and soluble antigen preparations. The soluble M. tigen-presenting mononuclear cells per well were established leprae extracts CD64 and CD78 were supplied by R. Rees with either M. leprae or medium control. Cultures were (National Institute of Medical Research, London), from the incubated at 37TC in 7% CO2. On days 6 and 9, interleukin 2 World Health Organization Immunology of Leprosy (IMM- was added to each well, and cultures were treated on day 12 LEP) Bank. Recombinant M. leprae 65-kDa antigen-f3- with 1 gCi of [3H]thymidine (1 Ci = 37 GBq) per well and galactosidase fusion protein was provided by R. Young harvested 6 hr later. Antigen-containing cultures were (Whitehead Institute, Cambridge, MA). Diameter and de- scored as positive if the proliferative response (measured in gree of induration (15) were recorded. cpm) was greater than the mean + 3 SDs of control cultures Intradermal tests with 0.1 ug of cell wall PPC (2 ug per 0.1 without antigen. The percentage of negative cultures was ml) and 0.1 ml of lepromin A (containing 3.5 x 106 bacilli) used to determine precursor frequency according to Poisson were made in five patients with borderline tuberculoid distribution and x-squared minimization (13). leprosy and five patients with lepromatous leprosy after Sensitization of Guinea Pigs and Skin Testing. Guinea pigs obtaining informed consent. (Camm, Wayne, NJ) were sensitized to 200 1kg of M. leprae in saline in five intradermal sites as described (14). Sensiti- zation to cell wall fractions was similar, except that the RESULTS antigen was incorporated into Freund's incomplete adjuvant The unique adjuvant activity of M. tuberculosis and bacillus (Difco) or into a mixture of trehalose dimycolate and detox- Calmette-Guerin has long been known to be associated with ified lipopolysaccharide (monophosphorylliposaccharide) the cell wall and not with the vast content of soluble proteins (Ribi Vaccine, Hamilton, MO), according to the manufac- (16). The present experiments were designed to learn A

ZIZJH

""I'm

- Test antigen 4 8 12 16 20 1 2 3 4 DIAMETER OF INDURATION (mm) DEGREE OF INDURATION B

M. leprae H i

CwC a-i

Pronase CWC

MZZOM PPD U BEEMOMMM

I I I I I I 0 4 8 12 16 20 24 0 1 2 3 Test antigen DIAMETER OF INDURATION (mm) DEGREE OF INDURATION FIG. 3. (A) Elicitation of DTH responses in guinea pigs sensitized to 200 ,ug of M. leprae in PBS by skin tests with cell wall fractions, Pronase-digested CWC, and recombinant 65-kDa antigen at 20 1Lg/0.1 ml. (B) Induction of DTH in guinea pigs to skin test after immunization with 200 ,ug of CWC in incomplete Freund's adjuvant or a mixture of trehalose dimycolate and monophosphoryllipopolysaccharide (detoxified endotoxin). Skin tests were made with antigens at 10,g/0.1 ml 1-2 mo after sensitization with M. leprae (2), CWC in incomplete Freund's adjuvant (m), and CWC in trehalose dimycolate and monophosphorylliposaccharide (6). Downloaded by guest on September 26, 2021 1920 Immunology: Melancon-Kaplan et al. Proc. Natl. Acad. Sci. USA 85 (1988) whether highly purified cell walls of M. leprae retained any A immunological reactivity associated with specific cell- -:...--'., mediated immunity. Accordingly, the reactivity of lympho- cytes from leprosy patients across the disease spectrum to .2I the entire leprosy bacillus and to purified cell walls was initially examined. Fig. 1A shows that lymphocytes from .: tuberculoid leprosy patients respond as well to cell walls as they do to the intact bacillus and respond more than to soluble M. leprae extract CD64. Preparation and composition of the bacillary fractions containing the cell wall insoluble residue are described in Table 1. The cell wall insoluble fraction of M. leprae was further subjected to repeated extraction with NaDodSO4 at Is 560C followed by acetone, which removed 42% of the mass and all soluble proteins detectable by NaDodSO4/PAGE and Al silver staining (9). This fraction, termed CWC, contained 26% cell wall-associated protein as determined by its content of protein amino acids (Table 1; Fig. 2A) and a considerable B amount of arabinogalactan and associated mycolates. The mycolates of the CWC and the bulk of the arabinogalactan were sequentially removed, resulting in =60% loss of the original CWC mass. Analysis of the remaining residue indi- cated that it is predominantly protein (Table 1), designated peptidoglycan-peptide complex (PPC). Perhaps the most rigorous assessment of the importance of individual antigens and epitopes within the panoply of components of any pathogen in the T-cell repertoire is analysis of antigen-reactive T-cell precursor frequency. Al- though this approach has been useful in murine systems (15, and has been used in man, 28-30) it has had only limited application in humans because of a larger number of cells proliferating to growth factors independently of antigen recognition. We recently developed a limiting-dilution assay to estimate the frequency of antigen-reactive T-cell precur- sors that virtually eliminates the background of antigen- cw Iept independent proliferation, thus rendering the assay more sensitive and accurate (12). Results (Fig. 1B) revealed that as FIG. 4. DTH reactions elicited by cell wall fractions. (A) many T cells are reactive to antigens associated with purified Reactions at 20 hr in guinea pigs sensitized to 200 1g of killed M. M. leprae cell walls as are reactive to intact bacilli, indicat- leprae in PBS followed by intradermal immunization with CWC. ing that cell wall-associated antigens are a major contributor Skin tests made at 1 mo with the following (right to left): purified to cell-mediated immune responses. Because preparations of protein derivative of tuberculin (10 j.g); M. leprae (10 ,ug); and CWC varied in ability to stimu- (100, 10, and 1 ltg). (B) Reactions at 48 hr of skin tests with lepromin soluble M. leprae extracts greatly (0.1 ml) or cell wall PPC (2 gug/0.1 ml) in patient with borderline late T cells (Fig. 1B), the question arises whether there is tuberculoid leprosy. variable release of cell wall-associated antigen into extracts by sonication. from the CWC, leaving only the peptidoglycan components, Experiments were undertaken to ascertain whether the which were present in the expected approximate unimolar highly purified CWC retained significant immunological re- relationship (Ala, 16.1; Gly, 17.5; Glu, 16.8; diaminopimelic activity in vivo. Guinea pigs were sensitized to intact M. acid, 17.5; glucosamine, 4.0; muramic acid, 18.2 mol per- leprae and tested 1-2 mo later with intact M. leprae, cell wall cent, corrected for different responses on GC, cf. Fig. 2). Of fractions, soluble M. leprae antigen and tuberculin purified particular significance, tuberculoid leprosy patients sensi- protein derivative. Results in >40 animals (Figs. 3A and 4A) reacted showed that the CWC of M. leprae was more effective than tized to M. leprae antigens by natural several soluble antigen preparations and the only available strongly with the cell wall PPC fraction (Fig. 4B). recombinant M. leprae antigen (r65kDa) and was as effective Finally, it was important to learn whether cell walls could as intact M. leprae in eliciting DTH reactions. As in the induce cell-mediated immunity to M. leprae antigens in limiting dilution analysis in vitro, this result shows that the naive animals, which would be required of any practical CWC contains a major antigen(s) recognized in vivo by vaccine against leprosy. Although poorly immunogenic lymphocytes responsible for DTH. Species-specific epitopes when given in saline, CWC (in oil adjuvant or in suspension appear to be recognized by animals sensitized to M. leprae containing trehalose dimycolate and detoxified lipopolysac- because reactions were greater to the homologous CWC charide) injected intradermally into normal guinea pigs in- than to the comparable preparation of M. tuberculosis (data duced high levels of cell-mediated immunity (Fig. 3B). not shown). Removal of the mycolates and arabinogalactan had no effect on skin test reactivity, but protease treatment DISCUSSION markedly diminished or eliminated antigenicity (Fig. 3 A and B), indicating that cell-mediated immunity is likely to be Largely because of limited quantities of bacilli available for directed to protein epitopes. This was confirmed by the study, the M. leprae cell wall is not well characterized. strong DTH reactions elicited by the purified protein deriv- However, some specific information on the basic peptido- ative of tuberculin antigen, a response that was also sensitive glycan unit is known: As in other mycobacteria, the unit is to protease digestion. Chemical analysis confirmed (Fig. 2B) based on a repeating muramyl-N-glycolyl(pl-.4)-N-acetyl- that proteolytic digestion removed all protein amino acids glucosaminyl disaccharide (17-19). However, the N-terminal Downloaded by guest on September 26, 2021 Immunology: Melancon-Kaplan et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1921 amino acid of the tetrapeptide side chain is glycine rather Research and Training in Tropical Diseases (Immunology of Lep- than the expected L-alanine (19). Attached to the muramyl rosy, IMMLEP) and the WHO Programme for Vaccine Develop- unit, presumably to the 6-OH function, is the expected ment (Immunology of Tuberculosis, IMMTUB); and support from arabinogalactan, which is composed solely of arabinofura- the Leonard Wood Foundation, Drown Foundation, Hartford Foun- nose and galactofuranose (20). The composition of the dation, and Irvington House Institute for Medical Research. mycolates of M. leprae, presumably attached to the 5-OH of 1. Bloom, B. R. & Godal, T. (1983) Rev. Infect. Dis. 5, 763-780. arabinofuranose residues, is also known (21, 22). 2. Maugh, T. H. (1982) Science 215, 1083-1086. Present data establish that the cell walls contain one or 3. Sansarricq, H. (1981) Lepr. Rev. 52, Suppl. 1, 15-31. more antigens of importance in cell-mediated immunity in 4. Young, R. A., Mehra, V., Sweetser, D., Buchanan, T., Clark- leprosy. Reactivity to CWC correlated with the cell- Curtiss, J., Davis, R. W. & Bloom, B. R. (1985) Nature mediated reactivity of patients-i.e., positive in patients (London) 316, 450-452. with tuberculoid leprosy, but not in patients with leproma- 5. Mustafa, A. S., Gill, H. K., Nerland, A., Britton, W. J., tous leprosy. Cell wall antigens represent the major propor- Mehra, V., Bloom, B. R., Young, R. A. & Godal, T. (1986) tion of the T-cell repertoire committed to M. leprae in Nature (London) 319, 63-66. sensitized individuals; 1/6000 peripheral T cells and 1/60 6. Misaki, A., Yukawa, S., Tsuchiya, K. & Yamasaki, T. (1966) CD4 + J. Biochem. (Tokyo) 59, 388-3%. cells obtained from tuberculoid lesions can proliferate 7. Adam, A., Petit, J. F., Lefrancier, P. & Lederer, E. (1981) to intact organisms, consistent with previous studies (28, 29), Mol. Cell. Biochem. 41, 27-47. or cell walls (ref. 14 and Fig. 1). The cell wall antigens are as 8. Draper, P. (1980) Report of the Fifth Meeting of the Scientific potent as intact M. leprae and stronger than some batches of Working Group on Immunology and Leprosy, 1980, Geneva soluble extracts in eliciting DTH reactions in guinea pigs (WHO Doc. TDR/IMMLEP-SWG (5)/80.3. Annex 4) (WHO, sensitized to killed M. leprae or tuberculoid patients and Geneva). contacts sensitized by natural infection. The CWC material is 9. Hunter, S. W., Gaylord, H. & Brennan, P. J. (1986) J. Biol. also highly immunogenic in guinea pigs when given in appro- Chem. 261, 12345-12351. priate adjuvant, inducing cell-mediated immunity to the intact 10. McNeil, M., Tsang, A. Y. & Brennan, P. J. (1987) J. Biol. bacillus. Finally, the antigenically active cell wall compo- Chem. 262, 2630-2635. 11. MacKenzie, S. L. & Tenaschuk, D. (1975) J. Chromatogr. nent(s) is most likely a protein because immunological reac- 111, 413-415. tivity is not diminished by extraction of soluble proteins, 12. Modlin, R. A., Melancon-Kaplan, J., Young, S. M. M., Kino, arabinogalactans, and associated mycolates, but reactivity is H., Convit, J., Rea, T. H. & Bloom, B. R. (1988) Proc. Natl. destroyed by treatment with Pronase or proteinase K. Acad. Sci. USA 85, 1213-1217. From chemical analysis of the degraded CWC (Table 1) 13. Taswell, C. (1981) J. Immunol. 126, 1614-1619. and the immunologic results in vivo, we believe the immu- 14. Mehra, V. & Bloom, B. R. (1978) Infect. Immun. 23, 787-794. nogenic cell wall protein(s) of M. leprae is probably inti- 15. Eichmann, K., Falk, I., Melchers, F. & Simon, M. M. (1980) mately associated with the peptidoglycan. The existence of J. Exp. Med. 152, 977-987. proteins covalently or noncovalently bound to the peptido- 16. Ribi, E., Granger, D. L., Milner, K. C., Yamamoto, K., glycan backbone of cell walls of both Gram-positive and Strain, S. M. & Parker, R. (1982) Immunology 46, 297-305. Gram-negative is well documented, and these pro- 17. Draper, P. (1976) Int. J. Lepr. 44, 95-98. teins play key roles in pathogenesis, immunogenicity, and 18. Wietzerbin, J., Das, B. C., Petit, J.-F., Lederer, E., Leyh- Bouille, M. & Ghuysen, J.-M. (1974) Biochemistry 13, bacterial physiology. Examples would include the M protein 3471-3476. of streptococci (23), the A protein of staphylococci (24), and 19. Draper, P. (1984) Int. J. Lepr. 52, 527-532. Braun's lipoprotein (25), which may contribute up to 40o of 20. McNeil, M., Wallner, S. J., Hunter, S. W. & Brennan, P. J. the insoluble cell wall of some enterobacteria. In mycobac- (1987) Carbohydr. Res. 166, 299-308. teria poly(L-glutamic acid), long known to be associated with 21. Kusaka, T., Kohasaka, K., Fukunishi, Y. & Akimori, H. pathogenic strains of M. tuberculosis, is covalently attached (1981) Int. J. Lepr. 49, 406-416. to the D-glutamic acid of the tetrapeptide side chain (26). 22. Draper, P., Dobson, G., Minnikin, D. E. & Minnikin, S. M. Misaki et al. (6) reported immunological reactivity associ- (1982) Ann. Microbiol. (Paris) 133B, 39-47. ated with cell walls of M. tuberculosis that was resistant to 23. Fischetti, V. A., Jones, K. F., Manjula, B. N. & Scott, J. R. periodate oxidation but was destroyed by Pronase. Ribi et (1984) J. Exp. Med. 159, 1083-1095. al. (16, 27) reported that dehydrated cell walls given in oil 24. Guss, V., Uhlan, M., Wilsson, G., Lindberg, M., Sjoquist, J. adjuvants, but not bacterial "protoplasm," were protective & Sjodahl, J. (1984) Eur. J. Biochem. 138, 413-420. against M. tuberculosis infection in mice and that the effect 25. Braun, V. & Rehn, K. (1969) Eur. J. Biochem. 10, 426-438. 26. Petit, J. F. & Lederer, E. (1984) in The Mycobacteria: A was abolished by protease treatment. We believe the present Sourcebook, eds. Kubica, G. P. & Wayne, L. G. (Dekker, results warrant a major effort to isolate and characterize the New York), Part A, pp. 301-313. cell wall-associated protein antigen(s) involved in cell- 27. Ribi, E., Araskoy, R. L., Brehmer, W., Goode, G., Larson, mediated immunity in leprosy and tuberculosis. C. L., List, R. H., Milner, K. C. & Wicht, W. C. (1966) J. Bacteriol. 92, 869-879. We are grateful to Drs. R. Rees, R. Young, and D. Sweetser for 28. van Oers, M. H. J., Pinkster, J. & Zeijlmaker, W. P. (1978) kindly providing antigens for these studies. This report from four Eur. J. Immunol. 8, 477-484. laboratories was supported by Public Health Service Grants 29. Brett, S. J., Kingston, A. E. & Colston, M. J. (1987) J. Clin. A122187, A122553, A107118, A120111 and Contract A152582 from Exp. Immunol. 68, 510-520. the National Institute of and Infectious Diseases; grants 30. Carvalho, E. M., Johnson, W. D., Barreta, E., Marsden, P., from the United Nations Development Programme/World Costa, J. L., Reed, S. & Rocha, H. (1985) J. Immunol. 135, Bank/World Health Organization (WHO) Special Programme for 4144-4148. Downloaded by guest on September 26, 2021