Specificity of the 48-hour reaction to Mitsuda antigen

LEPROSY Specificity of the 48-hour reaction to Mitsuda antigen Use of a soluble antigen from human and armadillo lepromin * J. CONVIT,' M. E. PINARDI,2 J. L. AVILA,3 & N. ARANZAZU 4 Two antigens were tested and compared in relation to the 48-h Fernandez reaction. They were obtained from standard human and from standard armadillo lepromin. All the tests were negative in patients with lepromatous leprosy and highly positive in those with tuber- culoid leprosy and in lepromin-positive contacts. There was total agreement in all tests done with the two types of antigen. The antigenic component has the following basic properties: it precipitates with 80 % saturated ammonium sulfate; it is not destroyed by autoclaving or by treatment with 0.4 % phenol; it is non-dialysable; and it is destroyed by treatment with trypsin. The cutaneous response that appears 48 h after the injection of a suspension of Mycobacterium leprae in saline was described by Fernandez (1). This reaction had been observed previously by several workers (2-4) but they did not consider it to be especially important and did not suggest an explanation for it. Others (5-7) considered it a tuberculin-type reaction and suggested the possi- bility of using it for epidemiological procedures when enough M. leprae had been made available either by in vitro culture or by experimental production. Fernandez (1) studied this reaction in patients with leprosy and in their contacts with great detail, since he considered it important. From observations over many years, we have come to the conclusion that the Fernandez reaction should be studied further, since it has wide potential not only for the study of infection by M. leprae, but also as a tool for studying the epidemiology of leprosy. * From the PAHO Center for Training and Research in Leprosy and Related Diseases, Instituto Nacional de Der- matologia, Caracas, Venezuela. This work was supported by Grant 0278 of the Consejo Nacional de Investigaciones Cientificas y Tecnol6gicas, Caracas, Venezuela, and by the World Health Organization. 1 Director. 2 Auxiliar Docente. 3 Jefe de la Secci6n de Bioquimica. 4 Medico del Servicio Central de Dermatologia Sanitaria. Recently Storrs (8) and Kirchheimer & Storrs (9) have reported the experimental transmission of leprosy to the armadillo. The fact that the bacteria that produce lesions in the armadillo have been shown to be identical to M. leprae from human lesions (10) means that in the very near future large amounts of M. leprae will be available to prepare antigen for studies related to leprosy. The main objective of the present work has been the clarification of three important questions in relation to the Fernandez reaction, namely: (a) the characteristics of the antigenic substance that evokes the reaction; (b) whether the antigen prepared with bacteria obtained from armadillo lesions behaves exactly like that prepared with human leprosy lesions; and (c) whether the Fer- nandez reaction is specific for leprosy. The first two questions are covered in this paper; the third will be dealt with in a subsequent paper. MATERIALS AND METHODS Standard lepromin was obtained by grinding material from the lesions of patients with leproma- tous leprosy or infected armadillos in saline, performing a bacterial count of the macerate, and diluting to obtain a concentration of 160 x 106 acid-fast bacteria per ml. The dilution was prepared with saline to which 0.4%o phenol had been added. 3338 187 BULL. WORLD HEALTH ORGAN., Vol. 52, 1975 J. CONVIT ET AL. This suspension was then autoclaved for 30 min at 125°C. This standard lepromin was centrifuged at 192 000 g for 2 h. The supernatant was then filtered through a Millipore filter with a 0.45-jsm mem- brane to ensure that there were no bacteria in the suspension. During the first stage of the investigation most of the work was done with supernatant from human lepromin. In order to study some biochemical characteristics of the antigen, the supernatant was treated with ammonium sulfate and separated into three fractions. The first fraction, P25, was the precipitate obtained by adding saturated ammonium sulfate to the supernatant to give a final concentra- tion of 25 %; the second fraction, P80, was the precipitate obtained by adding saturated ammonium sulfate to give a final concentration of 80%, and the third fraction, S, was the supernatant of this second precipitation. After fractionation, the precipitates were re- suspended in a given volume of saline and dialysed against saline at 4°C until the dialysate did not show any more ammonium sulfate. The volume of each fraction was made up to the original volume of the supernatant. The protein concentrations of the supernatant and of the three fractions obtained by precipitation with ammonium sulfate were determined by the method of Lowry (11). The protein concentration of the supernatant varied between 550 and 557 jig/ml, in samples prepared at different times, and the concentrations of the fractions were: for the P25 fraction, 30-34 ,ug/ml; for the P80 fraction, 235-275 ,ug/ml; and for the S fraction, 21-24 ,ug/ml. A second type of antigen was also prepared, by treating the standard lepromin from both human and armadillo material with ultrasound, before centrifugation, in an MSH apparatus for 2 h at 2 A under ice. The lepromins were then centrifuged and filtered as described above. To further characterize the antigen, the super- natants from both the ultrasonically-treated and non-treated human and armadillo lepromin were treated with trypsin at a final concentration of 0.32% for 30 min at 37°C. The trypsin was then inactivated by dilution, at room temperature, for 2 h. The fractions obtained from human lepromin, after treatment with ammonium sulfate, were also treated with trypsin, under the same conditions. Control antigen was prepared from the super- natant of a suspension of skin from patients with lepromatous leprosy who had become negative after several years of sulfone treatment. This suspension was prepared at a skin concentration equal to that used for the preparation of the standard human lepromin. At this stage the supernatant from standard human lepromin and the control antigen were injected into 31 patients with lepromatous leprosy, into 12 patients with tuberculoid leprosy, and into 48 contacts of patients with lepromatous leprosy classified as lepromin-positive by previous testing. The antigens were injected in doses of 0.1 ml, one on the right forearm and the other on the left forearm. The reactions were read after 48 h by the same person who had given the injections. The antigens were numbered so that the persons giving the injections did not know which antigen was being used. The fractions obtained by precipitation with am- monium sulfate were tested in 12 patients with lepromatous leprosy, 12 patients with tuberculoid leprosy, and 12 lepromin-positive contacts. The tests were given on both forearms, in amounts of 0.1 ml. All these persons were also tested with the unfractionated supernatant as control. The antigen obtained by treating fraction P80 with trypsin was tested in 8 patients with tuberculoid leprosy, and untreated P80 fraction was injected simultaneously as control. As before, the antigens were injected in 0.1-ml doses on both forearms. In the second part of the investigation the supernatant from standard human lepromin was compared with that from standard armadillo lepromin. Each of these antigens was injected intradermally, in 0.1-ml doses on the volar surface of each forearm, into 46 patients with lepromatous leprosy at the Cabo Blanco Leprosy Hospital and in 59 normal employees of the hospital who had been in contact with the patients for a long time. In addition, the following antigens were injected into 10 healthy hospital employees: (a) supernatant from standard human lepromin; (b) supernatant from ultrasonically-treated standard human lepro- min; (c) supernatant from standard armadillo lepromin; and (d) supernatant from ultrasonically- treated standard armadillo lepromin. All these antigens were injected in both the trypsinized and the non-trypsinized form. RESULTS Table 1 shows the results obtained following the first test of the supernatant antigen in patients with 188 SPECIFICITY OF REACTION TO MITSUDA ANTIGEN Table 1. Number of persons showing reactions to standard lepromin supernatant and to a control antigen neaction Lepromatous Tuberculoid L cpromin-positive size (mm) standard standard control standard controllepromin control lepromin lepromin 0-4 31 31 1 12 - 48 5-9 - - - - - - 10-14 - - - - 2 - 15-19 - - 3 - 11 - 20-24 - - 3 - 11 - 25-29 - - 2 - 5 - >30 - - 3 - 19 - total 31 12 48 lepromatous and tuberculoid leprosy and in lepromin-positive contacts. None of the patients with lepromatous leprosy had a reaction larger than 5 mm in diameter and only one of those with tuberculoid leprosy had a reaction under 10 mm in diameter, which is the limit over which the Fer- nandez reaction is considered positive. All the lepromin-positive contacts had a reaction over 10 mm in diameter, most of them being between 15 and 25 mm in diameter. The antigen prepared with skin from bacterio- logically negative patients with lepromatous leprosy gave negative results in all persons tested; there Table 2. Number of persons showing reactions to the P25, P80, and S fractions of standard lepromin super- natant Reaction Lepromatous Tuberculoid Lepromin-positive size contacts (mm) P25 P80 S P25 P80 S P25 P80 S 0-4 12 10 12 - - 12 - - 12 5-9 - 2 - 2 - - - - - 10-14 - - - 10 6 - 12 - - 15-19 - - - - 4 - - 5 - 20-24 - - - - 2 - - 6 - 25-29 - - - - - - - 1 - >30 - - - - - total 12 12 12 was either no reaction, or there was a very small papule, which never exceeded 3-4 mm in diameter. Table 2 shows the results obtained with the three fractions separated with saturated ammonium sulfate. Fraction P80 gave similar results to the unfractionated supernatant, with only slightly smaller reactions, in those with tuberculoid leprosy and lepromin-positive contacts. Fraction P25 gave weakly positive results both in patients with tuberculoid leprosy and in lepromin-positive contacts, whereas fraction S gave negative results. In those with lepromatous leprosy, the three fractions and the untreated supernatant gave consistently negative results. The antigen prepared by treating fraction P80 with trypsin gave negative results in all 8 patients with tuberculoid leprosy in which it was tested. The untreated P80 fraction gave the same type of response as in previous tests (Table 3). The injections made to compare the supernatant from human and armadillo lepromin gave similar results in all instances, with small variations in size. All patients with lepromatous leprosy were negative to both supernatants, and almost all healthy employees of the hospital were strongly positive; in the few cases where they were negative, they were negative to both antigens. The reactions were always similar and comparable (Table 4). In the group of 12 hospital employees injected with the ultrasonically-treated and untreated supernatants (both trypsinized and non-trypsinized) the results were also very consistent. Both the ultrasonically-treated and untreated supernatants from human and armadillo lepromin were positive in all cases (these employees were chosen from a group of previously determined lepromin positives), Table 3. Number of patients with tuberculoid leprosy showing reactions to the trypsin-treated and untreated P80 fraction of standard lepromin supernatant Reaction size Untreated Trypsin-treated (mm) P80 fraction P80 fraction 0-5 - 8 5-9 _ 10-14 3 - 15-19 4 - 20-24 1 - 25-29 - - >30 - 189 J. CONVIT ET AL. Table 4. Reactions to the supernatant from standard human and standard armadillo lepromin in patients with lepromatous leprosy and lepromin-positive contacts Lepromatous Lepromin-positive contacts Reaction human armadillo human armadillo (mm) _ _ _ _ _ _ _ _ _ _ _ _ No. % No. % No. % No. % 0-4 23 100 23 100 6 10.4 6 10.4 5-9 - - - - 5 8.6 4 6.8 10-14 - - - - 8 13.7 7 12.0 15-19 - - - - 15 25.8 11 18.9 20-24 - - - - 9 15.8 11 18.9 25-29 - - - - 11 18.9 9 15.8 > 30 - - - - 4 6.8 10 17.2 total 23 100 23 100 58 100.0 58 100.0 and where the antigens had been treated with trypsin they were all negative. COMMENTS The antigen prepared by separating the super- natant from standard lepromin by centrifugation appeared to be quite adequate for eliciting positive tuberculin-type 48-h reactions in those with tuberculoid leprosy and in lepromin-positive contacts. This same antigen gave negative res- ponses in those with lepromatous leprosy. It would appear, therefore, that the 48-h reaction elicited by this antigen is similar in every respect to the Fernandez reaction produced with complete lepro- min. In subsequent studies, the results of which will be published later, this antigen was used simultane- ously with complete lepromin. The reactions pro- duced by both antigens were very similar; the only difference seemed to be that the supernatant gave slightly smaller reactions in some of the persons tested. The advantages of using this type of antigen for epidemiological studies are clear. Apart from the fact that the reaction may be read after only 48 h there is another important advantage in that this antigen does not leave a scar after a positive reaction, and therefore may be more acceptable. However, it must be kept in mind that this antigen, even if it is proved as specific as the 30-day lepro- min reaction for testing sensitization with M. leprae, probably reads a different parameter than the one reflected by the 30-day lepromin reaction. Precipitation with different concentrations of ammonium sulfate is used to separate simple proteins from solutions. A partially saturated solution of ammonium sulfate separates globulins, and total saturation separates albumins. Since the active fraction of the antigen was separated with a saturated solution of ammonium sulfate it may be assumed that it is an albumin and, therefore, soluble in water. The fact that this protein is not inactivated by autoclaving or by treatment with 0.4%. phenol indicates that it is of low molecular weight, possibly a polypeptide, although the fact that it is non-dialysable does not seem to support this last hypothesis. Nevertheless, the inactivation of the antigen by trypsin treatment seems to reinforce the proposal that it is a protein or poly- peptide. Since separation with ammonium sulfate is a very incomplete purification method, further work will be carried out to purify and characterize the anti- genic component that produces the 48-h reaction. Since it has been shown that the supernatants from human and armadillo lepromin produce identical results both in patients with leprosy and in normal contacts, further studies will be carried out, mainly with armadillo material. We have large amounts of this at our disposal, and it is undoubt- edly this antigen that will be used in the future for epidemiological and immunological studies. ;UME SPiCIFICITt DE LA REACTION Ak L'ANTIGENE MITSUDA APRES 48 HEURES. UTILISATION D'UN ANTIG'ENE SOLUBLE PROVENANT DE LEPROM1NE D'ORIGINE HUMAINE ET ANIMALE (TATOU) Deux antigenes ont et6 essayes et compares par le moyen de la r6action de Fernandez apres 48 heures. Ces antigenes provenaient de lepromine d'origine humaine et d'origine animale (tatou). Tous les tests se sont reveles negatifs chez les malades atteints de lepre lepromateuse et hautement positifs chez les malades atteints de lepre tuberculoide et chez les contacts dont la reaction a la lepromine dtait positive. Dans tous les essais realises 190 SPECIFICITY OF REACTION TO MITSUDA ANTIGEN 191 avec les deux types d'antigene, la concordance a ete totale. Les propriet6s fondamentales de cet antigene que nous avons pu determiner sont les suivantes: F'antigene est pr6cipite par le sulfate d'ammonium sature concentr6 A 80 %; il n'est pas detruit par l'autoclavage ou par traite- ment au phenol a 0,4%. I1 n'est pas dialysable. Le traite- ment a la trypsine detruit F'antigene. Puisque nous disposons actuellement de mat6riel prove- nant de lesions lepreuses experimentales provoqu6es chez le tatou, les auteurs entendent poursuivre l'examen de I'anti- gene qui produit cette reaction et en etudier la specificite. REFERENCES 1. FERNANDEZ, J. M. M. Internationaljournal ofleprosy, 8: 1-14 (1940). 2. HAYASHI, F. International journal ofleprosy, 1: 30-39 (1933). 3. CUMMINS, S. L. & WILLIAMS, E. M. British medical journal, 1: 702-703 (1934). 4. RODRIGUEZ, J. N. International journal of leprosy, 6: 11-32 (1938). 5. STEIN, A. A. & STERPERIN, M. I. Nederlandsch tijd- schrift voor hygiene, microbiologie en serologie, 1: 209-218 (1934). 6 STEIN, A. A. & STERPERIN, M. I. Urologic and cutaneous review, 38: 860-863 (1934). 7. RABELLO, F. E. JR. Revista brasiliera de leprologia, 5: 465-479 (1937). 8. STORRS, E. E. International journal of leprosy, 39: 703-714 (1971). 9. KIRCHHEIMER, W. F. & STORRS, E. E. International journal of leprosy, 39: 693-702 (1971). 10. CoNvrr, J. & PINARDI, M. E. Science, 184: 1191-1192 (1974). 11. LOWRY, J. Journal of biological chemistry, 193: 265 (1951).