Comparative studies of Leishmania soluble antigens by crossed-immunoelectrophoresis: demonstration of specific antigens to L. mexicana and L. braziliensis*

Bulletin of the World Health Organization, 64 (1): 113-119 (1986) © World Health Organization 1986 Comparative studies of Leishmania soluble antigens by crossed-immunoelectrophoresis: demonstration of specific antigens to L. mexicana and L. braziliensis* LEONOR L. LEON,' MICHEL VAN HOEGAERDEN, & BERNARDO GALVAO-CASTRO3 The antigenic relationship between two species of Leishmania (L. mexicana amazonensis and L. braziliensis braziliensis) was analysed using crossed-immunoelectro- phoresis with an intermediate gel. Rabbit hyperimmune sera were prepared by sub- cutaneous inoculations of soluble antigens obtained from each species, emulsified in Freund's adjuvant. The antisera were then tested by immunodiffusion and electrophoresis; soluble antigens obtainedfrom promastigoteforms were submitted to ultrasonication and ultracentrifugation. Immunoelectrophoresis with homologous and heterologous antisera demonstrated the existence of some antigens which were species specific and some which were common to both species. The importance of these antigens is discussed. In the Americas, the cutaneous and muco- cutaneous leishmaniases are caused by species be- longing respectively to the Leishmania mexicana and L. braziliensis complexes. The cutaneous lesion is single or limited, with usually an abundance of para- sites, and recovery is often spontaneous without any treatment. On the other hand, L. braziliensis is responsible for a disease generally referred to as American mucocutaneous leishmaniasis which is believed to be confined to the Latin American countries, e.g., in Brazil where it is called espundia. This disease usually produces a metastatic lesion in the mucous membrane of the nasopharyngeal cavity, with tissue destruction. In both types of leishmaniasis the organism is transmitted to man by an insect vector as the flagellated promastigote. In the vertebrate host, the parasites are restricted to the mononuclear phagocytic system as aflagellated amastigotes (2). The parasites from different Leishmania species are almost indistinguishable morphologically, but the results of molecular and immunological analysis- such as electrophoretic patterns of isoenzymes (9), * From the Department of Immunology and the WHO Collab- orating Centre for Research and Training in the Immunology of Parasitic Diseases, Fundaqao Oswaldo Cruz, Cx. Postal 926, CEP 21040, Rio de Janeiro, Brazil. Requests for reprints should be addressed to Dr L. L. Leon. ' Research Assistant. 2 WHO Consultant. Present address: Centre international de Recherches m6dicales de Franceville, Gabon. 3Head of the Department of Immunology and Director of the WHO Collaborating Centre, Fundacao Oswaldo Cruz, Rio de Janeiro, Brazil. buoyant density of k-DNA and nuclear-DNA (14), serology of excreted factors (13), and reactions using monoclonal antibodies (5, 11 ) -have recently helped in their identification and classification. However, a general study comparing several characteristics of Leishmania species has not yet been carried out. For instance, it is well known that monoclonal antibodies are highly specific not only for one antigen but also for one epitope, which for certain studies is the great advantage of hybridoma technology over conven- tional methods using antisera. However, even the former is not the method of choice for a more general comparison between the antigenic constitution of the two parasite species because of the very high limi- tation in its specificity. Kohanteb et al. (7) compared different species of leishmanias using crossed electro- immunodiffusion and electroimmunodiffusion and found common antigens for the species studied as well as some indication of antigenic heterogeneity. However, the identification of species-specific antigens should be facilitated by preabsorption with heterologous antisera. We therefore chose the method of crossed immunoelectrophoresis with an intermediate gel employing polyclonal antibodies raised in rabbits, because it allows analysis of a high number of antigens with greater resolution than the other available immunoelectrophoretic methods. In the present study, the antigenic constitutions of strains of L. mexicana amazonensis and L. braziliensis braziliensis were compared using this technique. -113- L. L. LEON ET AL. MATERIALS AND METHODS Parasites Two different species of tegumentary Leishmania were used, L. braziliensis braziliensis (M2903) and L. mexicana amazonensis (H21), which were pro- vided by the Wellcome Parasitology Unit, Evandro Chagas Institute, Belem, PA, Brazil. The promasti- gotes were maintained at 26 °C by weekly passages in a biphasic medium consisting of a rabbit-blood agar base and a liquid phase of brain heart infusion (BHI-Difco). Antigen extraction Cells obtained from bulk growth were harvested on the seventh day, washed six times with 0.15 mol/l phosphate-buffered saline at pH 7.2, and disrupted by sonication using the Branson Sonifier Disruptor at 95 W, five times (3 minutes each). The sonicated material was ultracentrifuged at 100 000 g for 30 min and the supernatant was filtered through a 0.2 um Millipore membrane. In order to minimize proteo- lytic degradation, all the procedures were carried out at 4 °C and the antigenic preparations were aliquoted and stored at - 70 °C until use. Protein content was measured by Lowry's method (8) using bovine albumin as a standard. Antisera Antisera were obtained from 3 New Zealand rabbits after subcutaneous inoculation according to the following schedule. The first injection with the soluble antigen consisted of 1.5 mg of protein emulsified in Freund's complete adjuvant. After six weeks, the animals received the same dose of protein emulsified in Freund's incomplete adjuvant. The third dose (5 mg of protein) was given one week later, also in Freund's incomplete adjuvant. The rabbits were bled on days 5, 7 and 9 after the last injection. Antisera were tested for polyvalency and titre by immunodiffusion and electrophoresis. The sera with the highest titres from the 3 rabbits were pooled and stored at - 20 'C. Crossed-immunoelectrophoresis For crossed-immunoelectrophoresis (3), glass plates (9 x 6 cm) were covered with a slab (6 x 1.5 cm) of agarose 1.2% in Tris barbital buffer at pH 8.6 (ionic strength 0.02) and the soluble antigen (0.16 mg of protein) was applied to the first dimension gel and submitted to electrophoresis (8 V/cm for 50 min at 4 'C) so that the antigens migrated towards the anode. Second dimension electrophoresis was carried out in a 1.2% agarose gel in the same buffer, in which the antiserum had been diluted (2 V/cm for 18 h at 4 'C). The slab containing the antigens separated in the first dimension was located on the glass plate in a way that permitted the antigens to migrate both towards the anode (upper side of the plate) and the cathode (lower side of the plate) (Fig. 1). The homologous and the heterologous antisera were compared by crossed-immunoelectrophoresis with an intermediate gel (1). In this case, a 1-cm slab con- taining no antiserum was introduced between the 3-cm slab of intermediate (heterologous antiserum) and the 3.5-cm slab of the upper (homologous anti- serum) gels, in order to improve the resolution. The slab containing the antigens separated in the first dimension was located at the cathodic pole to permit migration towards the anode. After electrophoresis the plates were washed, dried, and stained with Coomassie brilliant blue R-250. RESULTS The soluble extracts of the two species of Leishmania, belonging to different complexes, were submitted to crossed-immunoelectrophoresis in order to determine their antigenic profiles. The immuno- electrophoresis performed with the homologous and the heterologous antisera demonstrated the existence of both similarities and differences between them. In the homologous system, we found 28 and 27 precipitin arcs for L. braziliensis braziliensis and L. mexicana amazonensis, respectively, all of them of anodic migration. In addition to the anodic arcs, L. braziliensis braziliensis presented another arc, with migration to the cathodic pole (Fig. 1). A marked antigenic similarity between the species was revealed when their soluble extracts were reacted with the heterologous antisera. In this system, an intermediate gel containing the heterologous anti- serum was used between the upper gel mixed with the homologous antiserum and the gel with the electro- phoretically separated antigen. In the intermediate gel 15 and 13 common antigens were detected when the soluble antigens of L. braziliensis braziliensis and L. mexicana amazonensis, respectively, were sub- jected to electrophoresis (Fig. 2 and Table 1). The antigens, which were detected exclusively in the upper gel, were considered to be specific to each species. We observed 7 of them for the L. braziliensis braziliensis soluble antigens; 5 of these particular antigens (No. 3 to 7, Fig. 2 top) had the highest electrophoretic mobilities while 2 (No. 1 and 2, Fig 2 top) had intermediate mobility. When the soluble antigens of L. mexicana amazonensis were analysed by crossed- 114 CROSSED IMMUNOELECTROPHORESIS AND SPECIFICITY OF LEISHMANIA ANTIGENS Table 1. Antigens in extracts of Leishmania braziliensis braziliensis (L.b.b.) and Leishmania mexicana amazonensis (L.m.a.) as detected by crossed-immunoelectrophoresis and by crossed-immunoelectrophoresis with intermediate gel Number of precipitin lines observed in the upper gel, with the following antigens: Antiserum in L.b.b. pre-absorbed L.m.a. pre-absorbed the upper gel L.b.b. with anti-L.m.a.a L.m.a. with anti-L.b.b.' Anti-L.b.b. 28 7 1 3b ND' Anti-L.m.a. 15h NDC 27 2d a Heterologous antiserum dissolved in the intermediate gel. bLines corresponding to common antigens. c Not done. d Lines corresponding to specific antigens. immunoelectrophoresis using the anti-L. braziliensis braziliensis-containing intermediate gel, 2 particular antigens were found (Table 1); these antigens (No. 1 and 2, Fig. 2 bottom) were among those with the highest electrophoretic mobility. DISCUSSION The taxonomic separation of Leishmania into well defined species and the characterization of the relevant antigens leaves much to be desired. The com- plexity of the situation led various investigators to search for simple ways of classifying these parasites on an immunological basis. Antigenic differences among a number of Leishmania species have been detected by a variety of methods, but the nature and number of such species-specific antigens are still unknown. The results of our experiments using crossed- immunoelectrophoresis with an intermediate gel to analyse the etiological agents of cutaneous and muco- cutaneous leishmaniasis (L. mexicana amazonensis and L. braziliensis braziliensis) showed that several antigens are shared by both species. The presence of these common antigens does not necessarily imply that cross-protection can be readily achieved. In fact, as demonstrated before (10), human infections by L. mexicana do not protect against L. braziliensis although the latter induces protection against the former. We also detected the presence of antigens particular to each species being studied; some of these antigens seem to be quantitatively important, such as L. braziliensis braziliensis No. 3 and 5 (Fig. 2 top). However, since the crossed-immunoelectrophoresis technique is not quantitative, no definite conclusion can be drawn on their relative amounts. As there is evidence for a certain degree of cross- reaction between L. donovani and Mycobacterium tuberculosis (15), which is a component of the ad- juvant employed in this study, it is possible that some of the common antigens reported here reflect in fact mycobacterial cross-reactivity. However, this would not cause the antigens to be wrongly identified as par- ticular antigens, and there can be no doubts about this. On the other hand, although crossed-immuno- electrophoresis with intermediate gel is a highly sensitive method for the analysis of complex mixtures, this technique reflects only the potency and multispecificity of the antisera used. In the present study, this limitation was somehow minimized by using pooled antisera from three rabbits which produced the highest antibody titres and the most heterogeneous antibody responses, in terms of leading to maximum numbers of lines in double immunodiffusion. Moreover, in our work comparing promastigote forms of the two species, we found for L. mexicana a similar number of precipitin arcs as de- scribed by Hunter & Cook. in an homologous system (6). Thus, we have an indication that any variation due to the immune response of the immunized rabbits was negligible. As we used only one stock of each parasite, we cannot exclude antigenic variation due to heterogeneity of the parasite within a taxon in a given geographic area. However, previous reports have indicated stability of antigenic patterns for Leishmania (4). Studies analysing the antigenic make-up of Leishmania species are generally carried out using the promastigote form, because the amastigote form is difficult to be obtained pure in the quantities needed for analytical studies. Hunter & Cook. (6) compared the promastigote and amastigote forms of L. mexi- cana and detected considerable cross-reactivity; these findings suggest that a large number of antigens are shared by the infective promastigote form and the amastigote form, which is present in the vertebrate 115 116 L. L. LEON ET AL. host during the development of the disease. There- fore, the study of antigens extracted from the infective promastigote form should be of great value for immunoprophylaxis and immunopathology. Furthermore, immunological studies of the cross- reaction between Leishmania spp. and Trypanosoma cruzi, which are co-endemic in some areas of Brazil, have been carried out. Based on our results and those of Morgado et al. (12) with T. cruzi, we tried to compare these parasites, again using crossed- immunoelectrophoresis with an intermediate gel; in preliminary results, we found that approximately 20% of the soluble antigens of T. cruzi were shared by the parasites of the genus Leishmania. Many problems still remain to be solved in leishmaniasis research. We believe the isolation and characterization of the specific antigens described here might be useful for taxonomical, clinical and immunological investigations. ACKNOWLEDGEMENTS The authors wish to thank Dr L. C. Pontes de Carvalho, Dr V. Bongertz and Dr M. G. Morgado for comments on this manuscript and Mrs Herminia Coelho Santos for her excellent secretarial services. This work was supported by the Brazilian National Research Council (CNPq-Grant no. 40.3769/82) and by the UNDP/World Bank/WHO Special Pro- gramme for Research and Training in Tropical Diseases. RESUME ETUDES COMPARATIVES PAR IMMUNOELECTROPHORESE CROISEE D'ANTIGENES SOLUBLES DE LEISHMANIA: MISE EN EVIDENCE DES ANTIGENES SPECIFIQUES DE L. MEXICANA ET DE L. BRASILIENSIS La relation antigenique entre deux especes de Leishmania (L. mexicana amazonensis et L. brasiliensis brasiliensis) a et analysee par immunoelectrophorese croisee avec un milieu gelifie intermediaire. Des serums hyperimmuns de lapin ont ete prepares par inoculation sous-cutanee d'anti- genes solubles obtenus a partir de chaque espece et emul- sionnes dans l'adjuvant de Freund. Puis les immunserums ont et eprouves par immunodiffusion et electrophorese; les antigenes solubles obtenus a partir de formes promastigotes ont e soumis aux ultrasons et a l'ultracentrifugation. L'immunoelectrophorese avec des immunserums homo- logues et heterologues a revele l'existence de certains anti- genes specifiques d'espece et d'autres communs aux deux especes. 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