Immunological determination of Mycobacterium leprae by means of cytoplasmic antigens

Bull. Org. mond. Santt 1972, 46, 509-513Bull. Wld Hlth Org. Immunological determination of Mycobacterium leprae by means of cytoplasmic antigens J. B. G. KWAPINSKI,1 J. 0. DE ALMEIDA,2 & E. H. KWAPINSKI 3 Mycobacterium leprae was isolated and purified from lepromas, the spleen, and the liver of leprosy patients. An immunodiffusion analysis of the cytoplasms obtained from four lots of M. leprae and M. lepraemurium, 295 strains of different actinomycetales, and 12 other bacteria was performed with the use ofthe cytoplasm antisera. Immunological relationships were revealed between the cytoplasms of M. leprae, M. lepraemurium, M. avium, M. gallinarum, M. tuberculosis, M. simiae, M. kansasii, M. chitae, M. cap- sulatum, Actinomyces israelii, A. naeslundii, and some strains of saprophytic myco- bacteria. These studies led to the proposed concept of the immunological evolution of M. leprae and M. lepraemurium and an Actinomyces-like progenitor through M. avium- M. gallinarum and to a proposal for the polyvalent vaccine currently being developed by this research group. Most of the past immunological research on lep- rosy dealt with the skin or serum reactions of leprosy patients with different mycobacterial antigen prepa- rations. Almost all of these data were critically re- viewed by Bechelli (1971) and by de Almeida.4 More recently, the cross-reactions given by polysaccharide- protein complexes purified from M. leprae with the sera obtained from human leprosy, tuberculosis, and nocardiosis were revealed by Estrada-Parra (1970). Investigations related to our research (Fukui et al., 1966) have shown that some antigens isolated from mice infected with M. lepraemurium shared an immunological factor with the extra-cellular antigens of M. avium and M. kansasii. The main purpose of the research reported in the present paper was to characterize immunologically the cytoplasm antigens prepared from purified M. leprae, isolated from biopsy and autopsy mate- rials, in relation to those of other actinomycetales. It was envisaged that the data thus obtained would 1 Professor of Medical Microbiology, University of Mani- toba, Winnipeg, Manitoba, Canada. 2 WHO Reference Laboratory for Serology of Leprosy Departamento de Microbiologia e Imunologia Faculdade de Medicina, Universidade de Sao Paulo, Riberao Preto, S.P., Brazil. 3 Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada. ' Almeida, J. 0. de (1967) Serology in leprosy, unpublished WHO document LEP/69/2. help to elucidate the immunogenicity of M. leprae and would be useful for the preparation of an anti- leprosy vaccine. MATERIALS AND METHODS Sources of M. leprae and M. lepraemurium The materials from which M. leprae was isolated were: (1) three different batches of lepromas, each collected from 12-20 cases of lepromatous leprosy, obtained from Ayer's Leprosarium, Sao Paulo, and from Santa Clara Leprosarium, Goyana, Brazil, and (2) the spleen and a part of the liver obtained from an autopsy of a 59-year-old female, who had suffered from lepromatous leprosy and who had not been treated for 10 years because her illness did not re- spond to sulfone drugs (Santa Clara Leprosarium). M. kepraemurium in a partly purified form was recov- ered from infected rat tissues and was supplied by L. Kat6.5 The following cultures (a total of 310) of bacteria were employed: 164 Mycobacterium, 62 Nocardia, 57 Streptomyces, 7 Actinomyces, 2 Mycococcus, 4 Dermatophilus, 10 Streptococcus, Pneumococcus, and Staphylococcus, as well as 4 Candida strains. ' Dr L. Kat6, Institute of Hygiene and Microbiology, University of Montreal, Canada. 2826 - 509- J. B. G. KWAPINSKI ET AL. Isolation and purification of M. keprae The isolation of M. leprae from the tissues was preceded by the detection of large aggregates of the acid-fast bacteria in smears and ultrathin sections stained by the method of Fite (1940). The materials were then treated with 1% formol at 20°C for 16 hours, after which the tissues were freed from formol by repeated washing in saline and dialysis against distilled water overnight. After dialysis, the tissues were placed in a steel chamber held in crushed ice and were homogenized in a Sorvall electric omni- mixer at the highest speed for 5 min. The homo- genates were centrifuged at 4 000 g for 10 min in an International Refrigerated Centrifuge, model B-20. The sediment was washed 5 times in PBS, pH 7.2, and was finally resuspended in distilled water and redialysed against distilled water overnight at 4°C. The dialysed materials were concentrated by cen- trifugation at 4 000 g for 10 min, and the sediment was resuspended in 15 parts (w/w) of Hanks' buffered saline solution (HBSS) and adjusted to pH 7.2. In order to liberate cells from the connective material, the tissues were treated for 2 hours at 37°C with hyaluronidase, added at a final concentration of 3.0 units/ml. The digested material was treated ultrasonically for 10 min at 9 kHz in a Raytheon sonic oscillator, and then centrifuged at 6 000 g for 10 min in an International Refrigerated Centrifuge. The sediment was collected, washed 5 times with HBSS, resuspended in 15 parts (w/w) of O.O1M phos- phate buffer, pH 7.8, containing trypsin (0.1 mg/ml), and placed in a shaking water bath at 37°C for 3 h. Subsequently, the digested material was centrifuged at 6 000 g for 10 min and the sediment was washed as above. In order to release M. leprae from cells that might remain undisrupted, the tissue-cell mixtures were suspended in 10 parts (w/w) of a 10-5M solution of edetic acid, pH 7.5, homogenized for 2 min on a high-speed omnimixer and subjected for 2 min to 20 kHz oscillation in the Biosonik III Ultrasonicator, in the cold. The homogenate was examined micro- scopically, after Ziehl-Neelsen staining, for the pres- ence of mycobacteria and cell debris. In order to free the mycobacteria from cell debris, the homogenates were subjected to two cycles of sucrose-gradient centrifugation, and 1-2 cycles of continuous particle electrophoresis. In this proce- dure, after the ultrasonic treatment, the coarse cell debris was removed by centrifugation at 118 g for 5 min. The supernatant material was collected and placed on a sucrose gradient made in 50-ml plastic centrifuge tubes by sequential layering of 7 ml, 5 ml, and 5 ml of 30%, 10%, and 5% solutions of sucrose; 5 ml of the homogenate containing M. lep- rae were placed on the top of the 5 % sucrose layer. The materials were centrifuged in the cold at 1 000 g for 45 min to separate two grey bands; the upper band, which contained partly purified mycobacteria, was collected. Upper bands collected separately from each lot of the material were pooled and sub- jected to another run of the same sucrose-gradient centrifugation. To obtain further purification of the mycobacteria, the material obtained after the second sucrose-gradient centrifugation was subjected to con- tinuous particle electrophoresis in Brinkmann's Elec- trophoretic Separator Model FF-3, using a O.OlM, pH 7.3, phosphate buffer as the liquid phase. The suspensions containing purified mycobacteria, as determined microscopically after Ziehl-Neelsen stain- ing, were pooled and centrifuged at 20 000 g for 1 h to deposit the purified mycobacteria, which were finally washed 3 times in distilled water. In order to establish that no extraneous human material was adsorbed to the purified bacteria, a slide agglutination test was employed. Thus, dense sus- pensions of the purified mycobacteria in PBS (pH 7.2) were mixed with a drop of an anti-human rabbit serum on a glass slide and were incubated in a moist chamber at 37°C for 1-2 min and inspected for agglutination. Controls were set up with tissue homogenates containing no bacteria. The human antiserum was produced in rabbits by immunization with a mixture of different human tissues and groupA and group B erythrocytes, suspended in pooled nor- mal human sera, by the method of Kwapinski (1969). M. lepraemurium was purified by continuous par- ticle electrophoresis as described above. Preparation of cytoplasms and cytoplasm antisera Cytoplasm preparations were obtained by the method of Kwapinski & Alcasid (1970) from the purified M. leprae, M. lepraemurium, and cultivable microorganisms. The actinomycetales were grown at 37°C in Kwapinski's KIM, KNM, or KPM media (Kwapinski & Alcasid, 1970, 1972a, 1972b), depending on the preference of the culture, until a full growth was obtained. The antisera against cyto- plasms of M. leprae, M. lepraemurium, and represen- tative strains of the cultivable microorganisms were produced in rabbits according by the immunization procedure of Kwapinski (1969). The cytoplasms were examined with the cytoplasm antisera using an immunodiffusion method (Kwapin- 510 IMMUNOLOGICAL DETERMINATION OF M. LEPRAE Table 1. Cytoplasms and cytoplasm-antisera of actinomycetales reactive with the cytoplasm-antisera or cytoplasms of M. Ieprae, determined by the immunodiffusion test Name Number Cytoplasm factor a M. lepraemurium M. avium M. avium M. avium M. avium M. gallinarum M. gallinarum Mycobacterium sp. M. intracellulare M. simiae M. simiae M. simiae M. tuberculosis M. tuberculosis M. kansasil M. smegmatis M. smegmatis M. abscessus M. borstelense M. borstelense M. fortuitum M. chitae M. chitae M. chitae - M. capsulatum Actinomyces israeli Actinomyces naeslundii Streptomyces abikoensis Streptomyces haistedii Streptomyces cellostaticus Streptomyces fradiae Streptomyces goshikiensis Streptomyces roseus Streptomyces exfoliatus LK-1 801 701 19077 15769 19711 19710 23396 13209 3055 3056 3016 201 (H37Ra) 206 (RlRa) P-24 23019 31003 23003 19235 19237 23010 19627 19628 19629 13556,13557 236, 271, 285, 337 45, 599A, X600 5025 5068 5189 5063 5190 5076 5060 a The antigenic factors have been named according to reactions of the cytoplasm with a standard cytoplasm-antiserum (consult references for details). 511 Ill III Ill Ill Ill III Ill Illl III Ill Il IV IV IV IV IV IV IV IV IV V IV IV IV IV abcdfhi abcdefhijkl abcdfik aik abfj afk ajno ghjk gf gf 9 cdh cdj cfgh abc ac acd acd ab (c) bcd bc bc bc abcde abcde abcd abcd ab J. B. G. KWAPINSKI ET AL. ski & Alcasid, 1972a, 1972b). Each test was set up in triplicate. RESULTS It was found that the cytoplasms prepared from M. leprae recovered from all four sources of human tissue cross-reacted with each other's cytoplasm antiserum as well as with the M. lepraemurium anti- serum, producing two precipitation lines. Reactions between the cytoplasms of M. leprae and the cyto- plasm antisera of certain actinomycetales, and the reactions given by combinations between the M. lep- rae cytoplasm antisera and cytoplasms of some actinomycetales, are presented in Table 1. Since the reactions of all four M. leprae cytoplasms with the actinomycetales were practically identical, the data are presented jointly. Thus, the M. leprae cytoplasms were found to share a single antigenic factor with the following actinomycetales: the factor IlIa with M. avium and M. gailinarum, the determinant Ig with M. simiae, the factor IlIc with M. tuberculosis and an irregular M. kansasii strain, and the determinant lVc (and probably IVa, IVb) with M. chitae and some strains designated as M. smegmatis, M. borstelense, M. ab- scessus, and M. fortuitum. Cross-reactions were also revealed between the M. leprae antisera and the main antigenic factor a of the streptomycetes and with the determinants of Mycococcus and Actino- myces, not identified so far. No immunological reactions were observed when M. leprae cytoplasms and antisera were investigated with the corresponding antisera or cytoplasms of the nocardiae, dermatophili, corynebacteria, candidas, pneumococci, streptococci, and staphylococci. DISCUSSION Although the cytoplasm antigens of M. leprae have been found to be immunologically related to a few different actinomycetales, the most significant immunological relationships seem to be those be- tween M. leprae and M. avium, M. gallinarum, M. simiae, M. chitae, Mycococcus, Actinomyces, and M. lepraemurium. The finding of cross-reactions between M. eprae and M. avium are in line with the data of Fukui et al. (1966) on the relationship between M. epraemurium and M. avium. The exact immunochemical nature of these immunological relationships is the subject of our follow-up studies; but it may be assumed that these antigenic connec- tions depend on the presence in the M. leprae cyto- plasm of a supramolecular component endowed with a number of determinants identical to those occur- ring in the actinomycetales bearing a close immuno- biological relationship to M. leprae. In view of these data and the scheme of Kwapinski (1972) for the phylo-immunogenic evolution ofmicro- organisms, it is postulated that M. leprae and its closest relative, M. lepraemurium, are derived from an Actinomyces-like microorganism. Through the association with birds, this progenitor microorga- nism acquired immunobiological properties of the avium-type mycobacteria. Later in the process of evolution, as a result of the association of birds and man in nature, individual microorganisms of the M. avium or M. gallinarum type might have been transformed into M. leprae and M. epraemurium in competent human and rat cells, respectively. This postulated sequence of biological events would then have been instrumental in the establishment of the strict parasitic associations between M. leprae and susceptible human cells and between M. lepraemurium and rat cells. On the basis of the present research, it is postulated that a vaccine prepared from the cyto- plasms of M. avium, M. gallinarum, M. simiae, and M. chitae, or from a combination of these micro- organisms sharing certain antigens with M. leprae might be immunologically effective against human leprosy, and that conversely, immunization with the M. leprae cytoplasm may be protective against infec- tions with M. avium, M. gallinarum, and M. simiae. These antigens, as well as a vaccine prepared from M. leprae isolated from human tissues and a vac- cine obtained from M. lepraemurium, are currently being investigated by this research group. ACKNOWLEDGEMENTS The authors are greatly indebted to Dr A. Campos, Director, Santa Clara Leprosarium, Goyafia, for providing the lepromas from his patients and for permission to per- form the autopsy in his hospital, to Dr D. V. Oppromola, Director, Ayer's Leprosarium, Sao Paulo, for supplying the lepromas from his patients, and to Dr L. Kat6, Institute of Hygiene and Microbiology, University of Montreal, for providing partly purified M. lepraemurium. Part of this research was supported by an Exchange of Researchers Grant received from the World Health Organization. 512 IMMUNOLOGICAL DETERMINATION OF M. LEPRAE 513 REiSUMt CARACrTRISATION IMMUNOLOGIQUE DE MYCOBACTERIUM LEPRAE AU MOYEN D'ANTIGtNES CYTOPLASMIQUES L'objectif principal de la presente recherche etait d'etudier les relations immunologiques entre les antigenes cytoplasmiques de Mycobacterium leprae et ceux d'autres actinomycetales. On a pr6pare les antigenes cytoplasmiques de Myco. leprae a partir de trois lots de lepromes pr6lev6s chez des malades l6promateux et de fragments de tissus h6patique et splenique recueillis a l'autopsie d'une autre malade. Myco. lepraemurium a ete obtenu a partir de tissus de rat infecte. Les autres actinomyc6tales et un certain nombre de bacteries (310 souches au total) ont 6te r6colt6s a partir de cultures. Apres une purification tres poussee du materiel d'etude, on a proc6de a des 6preuves d'immunodiffusion 'a l'aide des pr6parations cytoplasmiques des differents antigenes et des antiserums correspondants obtenus par immuni- sation du lapin. On a observe des reactions croises entre les antigenes et les antiserums de Myco. leprae de differentes origines ainsi qu'entre Myco. leprae et l'antiserum Myco. leprae- murium. Les 6preuves ont aussi fait ressortir une parent6 immunologique entre les antigenes de Myco. leprae et ceux de Myco. avium, Myco. gailinarum, Myco. simiae, Myco. tuberculosis, Myco. kansasii, Myco. chitae, Actinomyces israelii, A. naeslundi et les streptomycetes possedant le facteur antigenique a. On n'a par contre pas constat6 de reaction entre antigenes et antis6rums de Myco. leprae et antigenes ou antis6rums de Nocardia, Dermatophilus, Candida, Pneumococcus, Streptococcus et Staphylococcus. Ces resultats amenent les auteurs a formuler une hypothese selon laquelle Myco. leprae et Myco. leprae- murium auraient pour ancetre un micro-organisme du type Actinomyces. Au cours de 1'evolution, ce dernier se serait d'abord adapte aux oiseaux, acquerant les caract6ristiques immunologiques de Myco. avium ou Myco. gallinarum, puis se serait transforme en Myco. leprae et Myco. lepraemurium, adapt6s a 1'homme et au rat, et agents de la 1lpre chez ces especes. On 6tudie actuellement la possibilite de pr6parer un vaccin contre la 1lpre humaine en utilisant les antigenes cytoplasmiques de Myco. avium, Myco. gallinarum, Myco. simiae et Myco. chitae qui presentent une parent6 immunologique avec Myco. leprae. REFERENCES Bechelli, L. M. (1971) Acta leprol., 63, 125 Estrada-Parra, S. (1970) Bact. Proc., p. 107 Fite, G. L. (1940) J. Lab. clin. Med., 25, 743-744 Fukui, Y. et al. (1966) Biken's J., 9, 63-75 Kwapinski, J. B. G. (1969) Canad. J. Microbiol., 15, 1141- 1144 Kwapinski, J. B. G. (1972) Contemporary methodological aspects of immunological classification of microorga- nisms. In: Kwapinski J. B. G., ed., Research in immu- nochemistry and immunobiology, vol. 2, Baltimore, University Park Press, pp. 1-122 Kwapinski, J. B. G. & Alcasid, A. (1970) Canad. J. Microbiol., 16, 1263-1266 Kwapinski, J. B. G. & Alcasid, A. (1972a) Canad. J. Microbiol., 18, 445448 Kwapinski, J. B. G. & Alcasid, A. (1972b) Canad. J. Microbiol., 18 (in press)