Impairment of reactivity to lepromin by mycobacterial antigens related to, or identical with, Mycobacterium leprael Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by WA STATE UNIV LIBRARIES on 12/12/14 For personal use only.
J. B. G. KWAPINSKI, L. M. BECHELLI, N. HADDAD,A N D EMILIATAVARES SIMAO Depnrrtnent of Medicnl Microbiology, University of Mntlitobn, Wirlnipeg, Mnnitoba ntld Fnclrldnde d o Medicitla, Universidnde do Siio Palrlo, Riberiio PrPro, Brnzil Accepted February 3, 1975 K W A P I N S KJI., B. G . , L. M., BECHELLI,N. HADDAD,and E. T . S I M A O .1975. Impairment of reactivity to lepromin by mycobacterial antigens related to, o r identical with, Mycobncrerirrm leprne. Can. J . Microbiol. 21: 896-901. Three hundred and twenty young children were injected with Bacillus Calmette-Guerin (BCG) saline, o r with one of the mycobacterial cytoplasmic antigens related with M~~coO~rc~erirrtn 1c.prne. At an appropriate time thereafter they were tested for dermal hypersensitivity to the antigens and for reactions to lepromin. Whereas all the antigens induced cell-mediated immunity, the incidence and intensity of late response to lepromin were significantly reduced in children preinjected with the cytoplasmic mycobacterial antigens, as contrasted with increased lepromin reactivity in the BCG group and with the findings in saline-injected children. K W A P I N S KJI., B. G.. L. M. BECHELLI,N . HADDADe t E. T . S I M A O .1975. Impairment of reactivity to leplamin by mycobacterial antigens related to, o r identical with, Mycobacterirrm leprae. Can. J . Microbiol. 21: 896-901. Trois cent-vingt jeunes enfants ont ete injectes avec du Bacillus Calmette-Guerin (BCG), du solute salin ou encore des antigenes cytoplasmiques de mycobacteries apparenties a Myc.oboctc.ri1rt1 1c.preoe. Apres un delai approprii, o n a verifie chez ces sujets I'hypersensibilite cutanee a c e s antigenes ainsi que les reactions a la Iepromine. Bien que tous les antigenes declenchent une immunite cellulaire, on a trouvi que I'incidence e t I'intensite de la reponse tardive i la lepromine est significativement riduite chez les enfants qui ont reGu les antigenes cytoplasmiques d e mycobacteries, ce qui contraste avec la reactiviti accrue la lepromine notee chez les enfants qui ont reGu du BCG ou du solute salin. [Traduit par le journal]
Introduction Studies on the possible use of Bacillus Calmette-GuCrin (BCG) for prevention of leprosy revealed that BCG caused up to 80% conversion of the late lepromin reaction (2), but the epidemiological field trials were inconclusive in respect of the protection against leprosy (1-3, 8). In fact, the data obtained from extensive studies ~erformedin Burma and Karimui were ODDOsitely different from those obtained in uga;da (WHO Expert Committee on Leprosy (1 1)). Because of these discrepancies, the use of BCG as a possible means against leprosy was deferred until further studies, by the Committee on Leprosy Control which met in Bergen in 1973. Since close immunological relationships between the cytoplasmic antigens of Mycobacterium leprae and those of M. avium, M. gallinarum, and M. simiae were revealed by Kwapinski et al. (5, 6 ) it was proposed to examine the immune 'Received J u n e 13, 1974.
effect of ~reviousex~osureto mvcobacterial antigens, closely related to, or identical with, M. leprae, on the reactivity to lepromin. The choice of lepromin as a parameter for studying possible immunogenic effect of the antigens followed the opinion expressed by the Committee on Immunology of a number of leprosy international congresses that lepromin-reactivity indicates a relative degree of resistance to leprosy.
Materials and Methods Human Subjects T h e subjects used for the research were 6 months to 4-year-old male and female children from orphanages and playgrounds in Ribeirao PrCto, S3o Paulo, Brazil. T h e authorization for testing the children was obtained from their parents. T h e prevalence of leprosy was slightly above one per thousand. Most of the children had probably not been exposed t o M. leprae, a n d none showed signs of leprosy. T h e children were distributed to five groups, A, B, C, D, E, by randomized stratified method according t o age and sex. Although according to the calculations, a minimum total of 235 children o r 47 children per group were required, the initial number was increased t o 340 t o alleviate possible absenteeism. The
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KWAPINSKI ET AL.: MYCOBACTERIUM LEPRAE
minimum number of subjects required per group was calculated taking into account the estimated proportion of lepromin reactivity in children 6-35 months (25%), 36-47 months (40%). and 48-59 months (50%). The studies were carried out a s a "double blind trial" in which only one of the investigators (N.H.) was aware of the identity of all the factors involved in the investigation. Antigens and Plucebo The following antigen preparations and placebo were used : 1. Soluble cytoplasmic antigens, obtained from ultrasonically lysed cultures of the following mycobacteria, grown on KIM (4, 5): antigen X which contained cytoplasms (0.1% solids) of strain Nos. 701, 801, 19710, and 19711 (M. avium and M. gallinarum) and mycobacterial determinants a , b, c, d , and e ; antigen Y containing cytoplasms of strain Nos. 3016, 3055, 701, and 19710 (M. simiae, M. aviurn, and M . gallinarum) and determinants a , b, e, and y ; and antigen Z containing the cytoplasms of strains LP, 3016, 3055, and 10235 (M. leprae, M. simiue, and M . borstelen~e)and determinants a , c, d , g , and y . The antigenic determinants were characterized by a n immunodiffusion analysis on cellulose-acetate membranes and in 1% agarose gel. Cultures of cultivable mycobacteria were obtained from American Type Culture Collection and from Trudeau Institute. Mycobacterium leprae was recovered from the spleen of a deceased patient autopsied in Goyania, Brazil, and purified as reported earlier (6). 2. A BCG vaccine (40 mglml) obtained from Connaught Medical Research Laboratory, Toronto, was used as antigen BCG. 3. A sterile 0.85% sodium chloride solution was used as placebo. Purified Protein Derivative ( P P D ) Test Before the application of mycobacterial antigens, the hypersensitivity to PPD was examined by intradermal injections of 0.04 ml (2.0 T U Z )of a PPD solution (Batch CT 68, Connaught Medical Research Laboratory, Toronto). The reactions were read after 48 h by measuring (in mm) the diameter of induration. Application of Antigens The cytoplasmic antigens (X, Y, Z) or a sterile 0.85% NaCl solution were injected subcutaneously in a volume of 0.1 ml into the children of the groups designated respectively as E, A, D , and C. The antigen BCG was applied intradermally in a volume of 0.1 ml to the group B. During the ensuing week, all the children were observed as to their behavior, appetite, temperature, and local and general symptoms. If a n oedema in the injection area was noted, its diameter was measured. The Skin Hypersensitivity Tests Three weeks after injection of the antigens, the skin hypersensitivity was examined. In this assay, the children preinjected with antigens X, Y, Z received intradermal injections of 0.04 ml of the corresponding antigen, diluted 1 :200 with a sterile saline solution. Those who were preinjected with BCG or saline received an intradermal injection of PPD as in the screening test.
=TU, tuberculin units.
897
Lepromin Test The lepromin test was simultaneously performed with the above hypersensitivity test. In this assay, 0.1 ml of lepromin preparation (produced by Instituto de Leprologia, Rio de Janeiro), containing 160 million of bacilli per millilitre, was injected intracutaneously. The early Fernandez's reaction was read in 48 h by measuring two diameters of the oedema, at a right angle. The late Mitsuda's reaction was read after 30 and 45 days, by measuring the oedema or nodule and recording the necrosis if present. Early and late reactions were classified in accordance with the recommendations of International Congresses of Leprology (Madrid, 1953 and Tokyo, 1958).
Results At the end of the study, the numbers of children in each group, who were carried through all the injections and assays, were as follows: 59 children in group A, 57 in B, 63 in C, 59 in D, and 65 in E. These numbers exceeded by over 20% the minimum sample test required. The number of the 6-to-1 1 months and 11-to-23 months old was small; the proportion of children 48-59 months was above 50% in each group. The distribution of children by age, sex, and race in the five groups remained similar (Table 1). Eflect of the Antigens on the Late Mitsuda Lepromin Reaction The proportion of positive late (Mitsuda) lepromin reactions in groups A, D, and E was found to be significantly lower than in children of group B, who were preinjected with BCG and of the control group who received saline (Table 2, Fig. 1). In a qualitative analysis of the results, the differences between the proportions of the various groups, according t o the degree of intensity of the reactions, were statistically significant (cr = 0.05). For comparisons between the groups, the negative doubtful and positive (1 2 +, and 3 +) reactions were put together. The differences between groups A, D, and E are not statistically significant (cr = 0.05). However, when the data for^, D, and E groups are compared with those of the control group, the incidence of positive reactions in groups A, D, and E are significantly lower (cr = 0.05) than in the control group. Group B (BCG) had a significantly higher proportion of positive lepromin reactions than A, D, and E groups. The comparison between group B (BCG) and the control group showed that the differences in the incidence of lepromin reactivity were not significant (a = 0.05). However, when the de-
+,
CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 1 Distribution of children by age and sex at the end of the study -- -- -
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Male Group
White
A
6-35 36-47 48-59 Total
B
6-35 36-47 48-59 Total
- -
Female
Non white
White
Non white
Total
6-35 36-47 48-59 Total 6-35 36-47 48-59 Total
E
6-35 36-47 48-59 Total TABLE 2 Number and incidence (in parenthesis) of late lepromin reactions
And A B C D E Total
+ -
+
++
+++
Total
26 (44.1) 2 ( 3.5) 6 ( 9.5) 26 (44.1) 23 (35.4)
3 ( 5.1) 4 ( 7.0) 12(19.1) 8 (13.6) 13 (20.0)
23 (38.9) 33 (57.9) 37(58.7) 21 (35.6) 24 (36.9)
7 (11.9) 18 (31.6) 8 (12.7) 4 ( 6.7) 5 ( 7.7)
59 (100.0) 57 (100.0) 63 (100.0) 59 (100.0) 65 (100.0)
83
40
138
42
gree of reactivity was taken into account, the difference between the two groups was significant (a = 0.05), i.e., stronger reactions (2+ and 3 +) were significantly more frequent in the BCG-vaccinated children. In the quantitative analysis, i.e. the analysis of variance which reveals differences between the mean diameter of the Mitsuda's reactions in the five groups, the differences were statistically significant. When only two groups were com-
303
pared by Scheffk's test the results were similar to those shown by the qualitative analysis, but results for groups A and C were not significantly different.
Effect of the Antigens on tlie Early Fernandez Lepromin Reaction The number of positive reactions was very small and only in the BCG group the proportion of positive Fernandez reaction was significantly
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KWAPINSKl ET
AL.:
MYCOBACTERIUM LEPRAE
differences between groups A, D, and E and the control group were not significant (a = 0.05). The differences between group B and groups A, C, D , and E were significant ( a = 0.05). In the quantitative analysis used to test the differences between the mean diameter of Fernandez's reactions, there were significant differences among the five groups. When only two groups were considered, in the Scheffi's test, a significant difference was noted between group B and groups A, C, D, E. The differences among the latter four groups were not significant. Relations between Reactivity to PPD and to Lepromin Only a small number of children showed tuberculin reactions of 2 10 mm before the application of mycobacterial antigens and BCG vaccine. About 10% of children in groups B, C, and D and about 3% in groups A and E gave this reaction. The differences in the early or late lepromin reactivity noted in the trial groups did not appear t o be correlated to the reactivity to PPD.
GROUPS
FIG.I . Late lepromin reactions detected in the groups A, B, C, D, and E of children. Shaded areas refer to the strength of the reactions.
higher than in the control (Table 3). Antigens X, Y, and Z did not appear to have influenced the early reactivity to lepromin, since the incidence of reactions was similar to that of the control group. For the qualitative analysis the negative and doubtful results were considered together and the same was applied to 1 +, 2 +, and 3 + reactions. The global analysis of differences between the proportion of positivity in the five groups were statistically significant. The
Dermal Hypersensitivity to Antigens The analysis of variance, which reveals differences between the mean diameter of reactions to the relevant antigens, showed that the differences among the five groups were significant ( a = 0.01). When two groups were compared by Scheffi's test (Table 4), the dermal hypersensitivity in the BCG-injected group was significantly higher than in any other group. Mean diameters of the skin hypersensitivity evoked by corresponding antigens in groups A, D, and E were significantly higher than in the control C group.
TABLE 3 The incidence of early lepromin reactions
Group
Total
-
5-
+
++
Total
205
74
14
3
296
CAN. J . MICROBIOL. VOL. 21, 1975
TABLE 4 Evaluation of late dermal hypersensitivity reactions to mycobacterial antigens by Scheffe's test
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Comparison
Means
A with B A with C A with D A with E B with C B with D B with E C with D C with E D with E
Discussion The data obtained from the investigations reported here show that all the antigens X, Y, and Z and BCG induced cell-mediated immune response detected by the dermal hypersensitivity to the antigens in question. However, the cytoplasmic mycobacterial antigens X, Y, and Z not only had not enhanced the late reactivity to lepromin but had significantly decreased its incidence and intensity (if any) as contrasted with the BCG and as compared to the control group. The total number of all positive late lepromin reactions in the latter two groups were quite similar, although stronger (2+ and 3 +) reactions prevailed in the BCG group. This observation differs from that reported by Souza Campos et al. (9), who in 6- to 34-month-old children found positive reactions in 13 out of 14 children who received BCG intradermally, in 13 out of 14 children who received oral BCG, and in 4 out of 14 control subjects. This difference may be explained by the fact that 4-year-old children had been included in our study and that as shown by above authors, there exists a correlation between the age and lepromin reactivity; namely the reactions significantly intensifies with age, even within months. Unvaccinated children, as they grow older, tend to have a spontaneous increase in the proportion and degree of reactivity to lepromin (Souza Campos et al. (9); Bechelli et al. (2). In some studies (Paula Souza et al. (7)) the conversion of the lepromin reactions from negative to positive was found to be similar in the BCG vaccinated and the non-vaccinated school children.
Contrast
Results (a = 0.05) Significant Significant Not significant Not significant Significant Significant Significant Significant Significant Not significant
It appears that the three cytoplasmic mycobacterial preparations, one of which contained soluble constituents of M. leprae, caused an impairment of the lepromin reactivity. No other antigen is known to cause the impairment. Thus, the vaccines against smallpox, diphtheria, tetanus, and whooping cough had not been found to impede the conversion from negative to positive lepromin reaction (10). To verify whether the antigens X, Y, and Z have in fact acted as depressors of the lepromin reactivity it would be desirable to perform again the lepromin test on those children that were non-reactors. If the assumption is confirmed, the length of the impairment ought to be studied. In view of the worldwide observation that "reactivity to lepromin increases rapidly with age, from negativity in infancy to almost universal positivity after adolescence in endemic areas..." (WHO, Expert Committee (12)), it is probable that any possible impairment of reactivity caused by antigens is only temporary. Most of the people have the potentiality to react to lepromin and this becomes manifest with age, through exposure to M. leprae, M. tuberculosis, and possibly to nonpathogenic mycobacteria, and some unknown factors. It is rather strange that the antigens injected, one of them combined with M. leprae, could have caused an impairment of the lepromin reactivity. It remains to be investigated whether they cause a decrease of the blastic transformation of lymphocytes from healthy lepromin reactors and (or) of tuberculoid patients. To further our experience in the area above,
KWAPINSKI ET AL.: MYCOBACTERIUM LEPRAE
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additional studies using the mycobacterial antigens so as to reconstruct exactly the complex determinant composition of M. leprae are being planned. In the forthcoming investigations, assays for cell-mediated immunity may be used.
Conclusions The above reported data provided an evidence that certain cytoplasmic antigens obtained from the mycobacteria found to be immunologically closely related to, or identical with M. leprae, when injected into young children, significantly reduced the incidence and intensity of late reactions to lepromin. This contrasted with increased incidence and intensity of lepromin reactions observed in the BCG-vaccinated, and with the findings in non-vaccinated children. Interpretations of the data are temporarily withheld pending further investigations on probable causes of the impairment. Acknowledgments Appreciation is expressed to the World Health Organization for providing a Research Exchange Grant to one of us (J.B.G. Kwapinski) to carry out these studies in Brazil. The authors also express thanks to Dr. Lygia, M. C. Andrade, and Dr. Tuma (Divisiio Nacional de Lepra), who provided the lepromin for the study; to the assistants (Mr. Osvaldo Campos Borelli, Miss Maria de Lourdes Olivi, and Miss Regina Clove1 Toloy), who helped in the field trials and the municipal health authorities; and to the directors and personnel of children's institutions of Ribeiriio Pr&to. The advice received from Professor J. 0 . de Almeida, Departamento do Microbiologia and
901
Imunologia, Faculdade do Medicina, Riberdo Przto, is also gratefully acknowledged. 1. BECHELLI, L. M., P. G . GARABAJOSA, M. M. G Y I ,P. U E N M U R AW. , E N G L E R ,V. M. DOMINGUEZ, T. SUNDAROSAN, R. QUAGLIATO, and M. MATEJKA. 1971. BCG vaccination of children against leprosy. Preliminary findings of the WHO-controlled trial. Int. J. Lepr. 39: 609-642. 2. BECHELLI, L. M..GARABAJOSA, P. G.. U E N M U R A R.., E N G L E RW., , DOMINGUEZ, V. M., PAREDES,L.. SUNDAROSAN, R., KOCH,C.. A N D M. MATUKA.1970. BCG vaccination of children against leprosy. Preliminary findings of the WHO-controlled trial in Burma. Bull. WHO, 42: 235-281. 3. BROWN, J . A., and M. M. STONE.1966. BCGvaccination of children against leprosy: first results of a trial in Uganda. Br. Med. J. 5478: 7-14. 4. K W A P I N S KJI., B. G., and A. ALCASID.1972. Immunological specificity and relationships of cytoplasmic antigens of non-chromogenic mycobacteria. Can. J. Microbiol. 18: 445448. 5. K W A P I N S K I ,B. J . G., A. ALCASID, E. H. K W A P I N S K I . and V. N A I R N1972. . The immunology of cytoplasmic antigens of mycobacteria. Can. J. Microbiol. 18: 1201-121 1. 6. K W A P I N S KJ.I ,B. G., J. 0 . DE A L M E I D A and , E. H. K W A P I N S K1972. I. Immunological determination of Mycohocteri~i,,~ Ieproc, by means of cytoplasmic antigens. Bull. WHO,46: 509-513. 7. PAULA SOUZA.R. 1956. Rev. Bras. Leprol. 24: 9-22. 8. RUSSELL,D. A., G. C. SCOTT,and S. C. WIGLEY. 1964. BCG vaccination in leprosy. A preliminary report of a "blind" control trial. Int. J. Lepr. 32: 235-245. 9. S o u z ~CAMPOS,N., N. LESER,L. M. BECHELLI, R. QUAGLIATO, and ROTBERG.1962. The conversion of the lepromin reaction as a functionof different stimuli. Influence of age in this reversal in the age of 6 to 43 months. Rev. Bras. Leprol. 30: 3-20. 10. VALIS,F. D., J. MORAJCOMAS,andC. D. SELA.1951. Acta dermo-sifiliogr. (Madrid). 42: 505-510. 11. World Health Organization, Expert Committee on Leprosy. 1970. WHO. Tech. Rep. Ser. No. 459. 12. World Health Organization, Expert Committee on Leprosy. 1966. WHO, Tech. Rep. Ser. No. 319.
J. B. G. KWAPINSKI, L. M. BECHELLI, N. HADDAD,A N D EMILIATAVARES SIMAO Depnrrtnent of Medicnl Microbiology, University of Mntlitobn, Wirlnipeg, Mnnitoba ntld Fnclrldnde d o Medicitla, Universidnde do Siio Palrlo, Riberiio PrPro, Brnzil Accepted February 3, 1975 K W A P I N S KJI., B. G . , L. M., BECHELLI,N. HADDAD,and E. T . S I M A O .1975. Impairment of reactivity to lepromin by mycobacterial antigens related to, o r identical with, Mycobncrerirrm leprne. Can. J . Microbiol. 21: 896-901. Three hundred and twenty young children were injected with Bacillus Calmette-Guerin (BCG) saline, o r with one of the mycobacterial cytoplasmic antigens related with M~~coO~rc~erirrtn 1c.prne. At an appropriate time thereafter they were tested for dermal hypersensitivity to the antigens and for reactions to lepromin. Whereas all the antigens induced cell-mediated immunity, the incidence and intensity of late response to lepromin were significantly reduced in children preinjected with the cytoplasmic mycobacterial antigens, as contrasted with increased lepromin reactivity in the BCG group and with the findings in saline-injected children. K W A P I N S KJI., B. G.. L. M. BECHELLI,N . HADDADe t E. T . S I M A O .1975. Impairment of reactivity to leplamin by mycobacterial antigens related to, o r identical with, Mycobacterirrm leprae. Can. J . Microbiol. 21: 896-901. Trois cent-vingt jeunes enfants ont ete injectes avec du Bacillus Calmette-Guerin (BCG), du solute salin ou encore des antigenes cytoplasmiques de mycobacteries apparenties a Myc.oboctc.ri1rt1 1c.preoe. Apres un delai approprii, o n a verifie chez ces sujets I'hypersensibilite cutanee a c e s antigenes ainsi que les reactions a la Iepromine. Bien que tous les antigenes declenchent une immunite cellulaire, on a trouvi que I'incidence e t I'intensite de la reponse tardive i la lepromine est significativement riduite chez les enfants qui ont reGu les antigenes cytoplasmiques d e mycobacteries, ce qui contraste avec la reactiviti accrue la lepromine notee chez les enfants qui ont reGu du BCG ou du solute salin. [Traduit par le journal]
Introduction Studies on the possible use of Bacillus Calmette-GuCrin (BCG) for prevention of leprosy revealed that BCG caused up to 80% conversion of the late lepromin reaction (2), but the epidemiological field trials were inconclusive in respect of the protection against leprosy (1-3, 8). In fact, the data obtained from extensive studies ~erformedin Burma and Karimui were ODDOsitely different from those obtained in uga;da (WHO Expert Committee on Leprosy (1 1)). Because of these discrepancies, the use of BCG as a possible means against leprosy was deferred until further studies, by the Committee on Leprosy Control which met in Bergen in 1973. Since close immunological relationships between the cytoplasmic antigens of Mycobacterium leprae and those of M. avium, M. gallinarum, and M. simiae were revealed by Kwapinski et al. (5, 6 ) it was proposed to examine the immune 'Received J u n e 13, 1974.
effect of ~reviousex~osureto mvcobacterial antigens, closely related to, or identical with, M. leprae, on the reactivity to lepromin. The choice of lepromin as a parameter for studying possible immunogenic effect of the antigens followed the opinion expressed by the Committee on Immunology of a number of leprosy international congresses that lepromin-reactivity indicates a relative degree of resistance to leprosy.
Materials and Methods Human Subjects T h e subjects used for the research were 6 months to 4-year-old male and female children from orphanages and playgrounds in Ribeirao PrCto, S3o Paulo, Brazil. T h e authorization for testing the children was obtained from their parents. T h e prevalence of leprosy was slightly above one per thousand. Most of the children had probably not been exposed t o M. leprae, a n d none showed signs of leprosy. T h e children were distributed to five groups, A, B, C, D, E, by randomized stratified method according t o age and sex. Although according to the calculations, a minimum total of 235 children o r 47 children per group were required, the initial number was increased t o 340 t o alleviate possible absenteeism. The
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KWAPINSKI ET AL.: MYCOBACTERIUM LEPRAE
minimum number of subjects required per group was calculated taking into account the estimated proportion of lepromin reactivity in children 6-35 months (25%), 36-47 months (40%). and 48-59 months (50%). The studies were carried out a s a "double blind trial" in which only one of the investigators (N.H.) was aware of the identity of all the factors involved in the investigation. Antigens and Plucebo The following antigen preparations and placebo were used : 1. Soluble cytoplasmic antigens, obtained from ultrasonically lysed cultures of the following mycobacteria, grown on KIM (4, 5): antigen X which contained cytoplasms (0.1% solids) of strain Nos. 701, 801, 19710, and 19711 (M. avium and M. gallinarum) and mycobacterial determinants a , b, c, d , and e ; antigen Y containing cytoplasms of strain Nos. 3016, 3055, 701, and 19710 (M. simiae, M. aviurn, and M . gallinarum) and determinants a , b, e, and y ; and antigen Z containing the cytoplasms of strains LP, 3016, 3055, and 10235 (M. leprae, M. simiue, and M . borstelen~e)and determinants a , c, d , g , and y . The antigenic determinants were characterized by a n immunodiffusion analysis on cellulose-acetate membranes and in 1% agarose gel. Cultures of cultivable mycobacteria were obtained from American Type Culture Collection and from Trudeau Institute. Mycobacterium leprae was recovered from the spleen of a deceased patient autopsied in Goyania, Brazil, and purified as reported earlier (6). 2. A BCG vaccine (40 mglml) obtained from Connaught Medical Research Laboratory, Toronto, was used as antigen BCG. 3. A sterile 0.85% sodium chloride solution was used as placebo. Purified Protein Derivative ( P P D ) Test Before the application of mycobacterial antigens, the hypersensitivity to PPD was examined by intradermal injections of 0.04 ml (2.0 T U Z )of a PPD solution (Batch CT 68, Connaught Medical Research Laboratory, Toronto). The reactions were read after 48 h by measuring (in mm) the diameter of induration. Application of Antigens The cytoplasmic antigens (X, Y, Z) or a sterile 0.85% NaCl solution were injected subcutaneously in a volume of 0.1 ml into the children of the groups designated respectively as E, A, D , and C. The antigen BCG was applied intradermally in a volume of 0.1 ml to the group B. During the ensuing week, all the children were observed as to their behavior, appetite, temperature, and local and general symptoms. If a n oedema in the injection area was noted, its diameter was measured. The Skin Hypersensitivity Tests Three weeks after injection of the antigens, the skin hypersensitivity was examined. In this assay, the children preinjected with antigens X, Y, Z received intradermal injections of 0.04 ml of the corresponding antigen, diluted 1 :200 with a sterile saline solution. Those who were preinjected with BCG or saline received an intradermal injection of PPD as in the screening test.
=TU, tuberculin units.
897
Lepromin Test The lepromin test was simultaneously performed with the above hypersensitivity test. In this assay, 0.1 ml of lepromin preparation (produced by Instituto de Leprologia, Rio de Janeiro), containing 160 million of bacilli per millilitre, was injected intracutaneously. The early Fernandez's reaction was read in 48 h by measuring two diameters of the oedema, at a right angle. The late Mitsuda's reaction was read after 30 and 45 days, by measuring the oedema or nodule and recording the necrosis if present. Early and late reactions were classified in accordance with the recommendations of International Congresses of Leprology (Madrid, 1953 and Tokyo, 1958).
Results At the end of the study, the numbers of children in each group, who were carried through all the injections and assays, were as follows: 59 children in group A, 57 in B, 63 in C, 59 in D, and 65 in E. These numbers exceeded by over 20% the minimum sample test required. The number of the 6-to-1 1 months and 11-to-23 months old was small; the proportion of children 48-59 months was above 50% in each group. The distribution of children by age, sex, and race in the five groups remained similar (Table 1). Eflect of the Antigens on the Late Mitsuda Lepromin Reaction The proportion of positive late (Mitsuda) lepromin reactions in groups A, D, and E was found to be significantly lower than in children of group B, who were preinjected with BCG and of the control group who received saline (Table 2, Fig. 1). In a qualitative analysis of the results, the differences between the proportions of the various groups, according t o the degree of intensity of the reactions, were statistically significant (cr = 0.05). For comparisons between the groups, the negative doubtful and positive (1 2 +, and 3 +) reactions were put together. The differences between groups A, D, and E are not statistically significant (cr = 0.05). However, when the data for^, D, and E groups are compared with those of the control group, the incidence of positive reactions in groups A, D, and E are significantly lower (cr = 0.05) than in the control group. Group B (BCG) had a significantly higher proportion of positive lepromin reactions than A, D, and E groups. The comparison between group B (BCG) and the control group showed that the differences in the incidence of lepromin reactivity were not significant (a = 0.05). However, when the de-
+,
CAN. J. MICROBIOL. VOL. 21, 1975
TABLE 1 Distribution of children by age and sex at the end of the study -- -- -
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Male Group
White
A
6-35 36-47 48-59 Total
B
6-35 36-47 48-59 Total
- -
Female
Non white
White
Non white
Total
6-35 36-47 48-59 Total 6-35 36-47 48-59 Total
E
6-35 36-47 48-59 Total TABLE 2 Number and incidence (in parenthesis) of late lepromin reactions
And A B C D E Total
+ -
+
++
+++
Total
26 (44.1) 2 ( 3.5) 6 ( 9.5) 26 (44.1) 23 (35.4)
3 ( 5.1) 4 ( 7.0) 12(19.1) 8 (13.6) 13 (20.0)
23 (38.9) 33 (57.9) 37(58.7) 21 (35.6) 24 (36.9)
7 (11.9) 18 (31.6) 8 (12.7) 4 ( 6.7) 5 ( 7.7)
59 (100.0) 57 (100.0) 63 (100.0) 59 (100.0) 65 (100.0)
83
40
138
42
gree of reactivity was taken into account, the difference between the two groups was significant (a = 0.05), i.e., stronger reactions (2+ and 3 +) were significantly more frequent in the BCG-vaccinated children. In the quantitative analysis, i.e. the analysis of variance which reveals differences between the mean diameter of the Mitsuda's reactions in the five groups, the differences were statistically significant. When only two groups were com-
303
pared by Scheffk's test the results were similar to those shown by the qualitative analysis, but results for groups A and C were not significantly different.
Effect of the Antigens on tlie Early Fernandez Lepromin Reaction The number of positive reactions was very small and only in the BCG group the proportion of positive Fernandez reaction was significantly
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KWAPINSKl ET
AL.:
MYCOBACTERIUM LEPRAE
differences between groups A, D, and E and the control group were not significant (a = 0.05). The differences between group B and groups A, C, D , and E were significant ( a = 0.05). In the quantitative analysis used to test the differences between the mean diameter of Fernandez's reactions, there were significant differences among the five groups. When only two groups were considered, in the Scheffi's test, a significant difference was noted between group B and groups A, C, D, E. The differences among the latter four groups were not significant. Relations between Reactivity to PPD and to Lepromin Only a small number of children showed tuberculin reactions of 2 10 mm before the application of mycobacterial antigens and BCG vaccine. About 10% of children in groups B, C, and D and about 3% in groups A and E gave this reaction. The differences in the early or late lepromin reactivity noted in the trial groups did not appear t o be correlated to the reactivity to PPD.
GROUPS
FIG.I . Late lepromin reactions detected in the groups A, B, C, D, and E of children. Shaded areas refer to the strength of the reactions.
higher than in the control (Table 3). Antigens X, Y, and Z did not appear to have influenced the early reactivity to lepromin, since the incidence of reactions was similar to that of the control group. For the qualitative analysis the negative and doubtful results were considered together and the same was applied to 1 +, 2 +, and 3 + reactions. The global analysis of differences between the proportion of positivity in the five groups were statistically significant. The
Dermal Hypersensitivity to Antigens The analysis of variance, which reveals differences between the mean diameter of reactions to the relevant antigens, showed that the differences among the five groups were significant ( a = 0.01). When two groups were compared by Scheffi's test (Table 4), the dermal hypersensitivity in the BCG-injected group was significantly higher than in any other group. Mean diameters of the skin hypersensitivity evoked by corresponding antigens in groups A, D, and E were significantly higher than in the control C group.
TABLE 3 The incidence of early lepromin reactions
Group
Total
-
5-
+
++
Total
205
74
14
3
296
CAN. J . MICROBIOL. VOL. 21, 1975
TABLE 4 Evaluation of late dermal hypersensitivity reactions to mycobacterial antigens by Scheffe's test
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Comparison
Means
A with B A with C A with D A with E B with C B with D B with E C with D C with E D with E
Discussion The data obtained from the investigations reported here show that all the antigens X, Y, and Z and BCG induced cell-mediated immune response detected by the dermal hypersensitivity to the antigens in question. However, the cytoplasmic mycobacterial antigens X, Y, and Z not only had not enhanced the late reactivity to lepromin but had significantly decreased its incidence and intensity (if any) as contrasted with the BCG and as compared to the control group. The total number of all positive late lepromin reactions in the latter two groups were quite similar, although stronger (2+ and 3 +) reactions prevailed in the BCG group. This observation differs from that reported by Souza Campos et al. (9), who in 6- to 34-month-old children found positive reactions in 13 out of 14 children who received BCG intradermally, in 13 out of 14 children who received oral BCG, and in 4 out of 14 control subjects. This difference may be explained by the fact that 4-year-old children had been included in our study and that as shown by above authors, there exists a correlation between the age and lepromin reactivity; namely the reactions significantly intensifies with age, even within months. Unvaccinated children, as they grow older, tend to have a spontaneous increase in the proportion and degree of reactivity to lepromin (Souza Campos et al. (9); Bechelli et al. (2). In some studies (Paula Souza et al. (7)) the conversion of the lepromin reactions from negative to positive was found to be similar in the BCG vaccinated and the non-vaccinated school children.
Contrast
Results (a = 0.05) Significant Significant Not significant Not significant Significant Significant Significant Significant Significant Not significant
It appears that the three cytoplasmic mycobacterial preparations, one of which contained soluble constituents of M. leprae, caused an impairment of the lepromin reactivity. No other antigen is known to cause the impairment. Thus, the vaccines against smallpox, diphtheria, tetanus, and whooping cough had not been found to impede the conversion from negative to positive lepromin reaction (10). To verify whether the antigens X, Y, and Z have in fact acted as depressors of the lepromin reactivity it would be desirable to perform again the lepromin test on those children that were non-reactors. If the assumption is confirmed, the length of the impairment ought to be studied. In view of the worldwide observation that "reactivity to lepromin increases rapidly with age, from negativity in infancy to almost universal positivity after adolescence in endemic areas..." (WHO, Expert Committee (12)), it is probable that any possible impairment of reactivity caused by antigens is only temporary. Most of the people have the potentiality to react to lepromin and this becomes manifest with age, through exposure to M. leprae, M. tuberculosis, and possibly to nonpathogenic mycobacteria, and some unknown factors. It is rather strange that the antigens injected, one of them combined with M. leprae, could have caused an impairment of the lepromin reactivity. It remains to be investigated whether they cause a decrease of the blastic transformation of lymphocytes from healthy lepromin reactors and (or) of tuberculoid patients. To further our experience in the area above,
KWAPINSKI ET AL.: MYCOBACTERIUM LEPRAE
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additional studies using the mycobacterial antigens so as to reconstruct exactly the complex determinant composition of M. leprae are being planned. In the forthcoming investigations, assays for cell-mediated immunity may be used.
Conclusions The above reported data provided an evidence that certain cytoplasmic antigens obtained from the mycobacteria found to be immunologically closely related to, or identical with M. leprae, when injected into young children, significantly reduced the incidence and intensity of late reactions to lepromin. This contrasted with increased incidence and intensity of lepromin reactions observed in the BCG-vaccinated, and with the findings in non-vaccinated children. Interpretations of the data are temporarily withheld pending further investigations on probable causes of the impairment. Acknowledgments Appreciation is expressed to the World Health Organization for providing a Research Exchange Grant to one of us (J.B.G. Kwapinski) to carry out these studies in Brazil. The authors also express thanks to Dr. Lygia, M. C. Andrade, and Dr. Tuma (Divisiio Nacional de Lepra), who provided the lepromin for the study; to the assistants (Mr. Osvaldo Campos Borelli, Miss Maria de Lourdes Olivi, and Miss Regina Clove1 Toloy), who helped in the field trials and the municipal health authorities; and to the directors and personnel of children's institutions of Ribeiriio Pr&to. The advice received from Professor J. 0 . de Almeida, Departamento do Microbiologia and
901
Imunologia, Faculdade do Medicina, Riberdo Przto, is also gratefully acknowledged. 1. BECHELLI, L. M., P. G . GARABAJOSA, M. M. G Y I ,P. U E N M U R AW. , E N G L E R ,V. M. DOMINGUEZ, T. SUNDAROSAN, R. QUAGLIATO, and M. MATEJKA. 1971. BCG vaccination of children against leprosy. Preliminary findings of the WHO-controlled trial. Int. J. Lepr. 39: 609-642. 2. BECHELLI, L. M..GARABAJOSA, P. G.. U E N M U R A R.., E N G L E RW., , DOMINGUEZ, V. M., PAREDES,L.. SUNDAROSAN, R., KOCH,C.. A N D M. MATUKA.1970. BCG vaccination of children against leprosy. Preliminary findings of the WHO-controlled trial in Burma. Bull. WHO, 42: 235-281. 3. BROWN, J . A., and M. M. STONE.1966. BCGvaccination of children against leprosy: first results of a trial in Uganda. Br. Med. J. 5478: 7-14. 4. K W A P I N S KJI., B. G., and A. ALCASID.1972. Immunological specificity and relationships of cytoplasmic antigens of non-chromogenic mycobacteria. Can. J. Microbiol. 18: 445448. 5. K W A P I N S K I ,B. J . G., A. ALCASID, E. H. K W A P I N S K I . and V. N A I R N1972. . The immunology of cytoplasmic antigens of mycobacteria. Can. J. Microbiol. 18: 1201-121 1. 6. K W A P I N S KJ.I ,B. G., J. 0 . DE A L M E I D A and , E. H. K W A P I N S K1972. I. Immunological determination of Mycohocteri~i,,~ Ieproc, by means of cytoplasmic antigens. Bull. WHO,46: 509-513. 7. PAULA SOUZA.R. 1956. Rev. Bras. Leprol. 24: 9-22. 8. RUSSELL,D. A., G. C. SCOTT,and S. C. WIGLEY. 1964. BCG vaccination in leprosy. A preliminary report of a "blind" control trial. Int. J. Lepr. 32: 235-245. 9. S o u z ~CAMPOS,N., N. LESER,L. M. BECHELLI, R. QUAGLIATO, and ROTBERG.1962. The conversion of the lepromin reaction as a functionof different stimuli. Influence of age in this reversal in the age of 6 to 43 months. Rev. Bras. Leprol. 30: 3-20. 10. VALIS,F. D., J. MORAJCOMAS,andC. D. SELA.1951. Acta dermo-sifiliogr. (Madrid). 42: 505-510. 11. World Health Organization, Expert Committee on Leprosy. 1970. WHO. Tech. Rep. Ser. No. 459. 12. World Health Organization, Expert Committee on Leprosy. 1966. WHO, Tech. Rep. Ser. No. 319.
