Detection and characterization of leishmania antigens from an American cutaneous leishmaniasis vaccine for diagnosis of visceral leishmaniasis

Diagnostic Microbiology and Infectious Disease 45 (2003) 35– 43

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Parasitology

Detection and characterization of leishmania antigens from an American cutaneous leishmaniasis vaccine for diagnosis of visceral leishmaniasis Wanderley Almeida Ferreiraa, Wilson Mayrinkb, Marcos Luiz dos Mares-Guiaa, Carlos Alberto Pereira Tavaresa,* a

Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Minas Gerais, Brazil b Department of Parasitology, Institute of Biological Sciences, Federal University of Minas Gerais, Minas Gerais, Brazil Received 20 February 2002; accepted 19 August 2002

Abstract Antigens were isolated from vaccines against American Cutaneous Leishmaniasis (ACL) and their reactivity tested against nine different groups of human sera and two groups of dog sera. These antigens react specifically with human and dog visceral leishmaniasis sera when compared to sera from non-infected individuals. Sera from humans from endemic areas of ACL before, or one year after, vaccination, and ACL patients treated and cured by immunotherapy with polyvalent vaccine, did not display significant differences of reactivity to these antigens. In contrast, they displayed a significantly higher reactivity to the antigens when compared to sera from healthy humans from non-endemic areas. No sera reactivity was observed with patients carrying Chagas’ disease or tuberculosis. These antigens are polysaccharides aggregates and present molecular masses ranging from 90 to over 200 KDa. These data suggest the use of these antigens for sero-diagnosis of human and canine visceral leishmaniasis. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Leishmaniasis; Vaccine; Antigen; Diagnostic

1. Introduction Human leishmaniasis is a disease caused by several species of the protozoan parasite of the genus Leishmania. In human hosts the clinical spectrum induced by different Leishmania species can range from a single cutaneous lesion, that may undergo spontaneous cure, to mucocutaneous lesions, that can cause grossly disfiguring lesions. Severe diffuse cutaneous lesions, can also occur that are extremely difficult to treat. Moreover, the disease can evolve to visceralizing forms that are lethal in the majority of the cases. An important host affected by visceral leishmaniasis is the dog, also acting as an important reservoir for this parasite in urban centers (Berman, 1997). Leishmaniasis is a serious public health problem in several countries, according to the World Health Organization (WHO, 1997). The principle measure for control of this disease include, fast diagnostic methods to detect its initial course, followed by traditional therapeutic treatment where available. An-

* Corresponding author. Fax: 55 31 34415963. E-mail address: [email protected] (C.A.P. Tavares).

other approach is to develop vaccines capable of inducing immune protection (Berman, 1997). Several antigens have been proposed for diagnosis of leishmaniasis utilizing ELISA or immunoblot assays (Badaro´ et al., 1996; Montoya et al., 1997; Martin et al., 1998; Cabrera et al., 1999; Brito et al., 2000). Among the different methods for diagnosis, ELISA is the most widely used in epidemiologic surveys, by virtue of its low cost and easy execution. Hypersensitivity skin tests are also used for diagnosis of leishmaniasis and also indicate the presence or absence of a cellular immune response (Nascimento et al., 1990). Mayrink et al., 1979 developed a vaccine against American Cutaneous Leishmaniasis (ACL) consisting of killed promastigotes from five dermotropic strains of Leishmania: L. (Leishmania) amazonensis (IFLA/BR/67/PH8), L. (Leishmania) mexicana (MHOM/BR/BH6), L. (Viannia) guyanensis (MHOM/BR/70/M1176), and Leishmania MHOM/BR/BH49 and MHOM/BR/73/BH21, major-like strains (Silva et al., 1994). Each one was isolated in different endemic areas for ACL in Brazil. The vaccine is produced by combining sonicated (50%) and non-sonicated killed stationary-phase promastigotes (50%), in phosphate buffer 0.10M, pH 7.40, containing thimerosal 0.010%

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(Mayrink et al., 1979). Studies analyzing the stability and potential of this vaccine as a source of antigens for skin hypersensitivity tests (Da-Costa et al., 1996) led to the observation that these vaccines, in spite of long-time storage (1 to 5 years), were able to induce a positive Montenegro skin test in humans living in endemic areas to ACL. However, the authors did not identify the molecules responsible by this activity and reported the absence of high molecular weight molecules in these vaccines. As a consequence, they suggested that antigens biologically active in these vaccines could be peptides, or small non-protein molecules resulting of the degradation of large antigens present in these vaccine formulations (Da-Costa et al., 1996). The aim of this work was to isolate, identify, and characterize antigens from long-time storage vaccines against ACL and determine their utility for the control of Leishmaniasis.

2. Materials and Methods 2.1. Leishmania extracts The strains and species of parasites used in the present study were the five strains incorporated into the vaccine developed by Mayrink et al. 1979 and produced by BIOBRAS S.A, a pharmaceutical company. Antigens were isolated from vaccines prepared with the five strains of parasites produced in 1991, called long-time storage pentavalent vaccine (stock nr. 9031 004-1V). Antigens were also isolated from vaccine prepared from a single parasite strain (L. (L.) amazonensis, PH8 strain), called monovalent vaccine, recently prepared or monovalent vaccine prepared in 1994 (stock nr. 3031 012-1V). 2.2. Sample preparation Vaccines were initially submitted to centrifugation at 120,000g for 75 min at 10°C. The supernatant was separated and filtered through a Millipore (0.22␮). Supernatant from long-time storage pentavalent vaccine was named SFE and supernatant from long-time storage monovalent vaccine (PH8 strain) was named SF8. Supernatant from fresh monovalent vaccine was named (FM8), and was obtained from a vaccine recently prepared with the PH8 strain according to Mayrink et al. (1979) immediately after vaccine preparation. 2.3. Antigen isolation Antigens were obtained by molecular exclusion chromatography of the vaccine supernatants. Samples were submitted to FPLC gel filtration, using a Superose 12 HR 10/30 column (Amershan Pharmacia Biotech) and phosphate buffer 0.10M, pH 7.40 as eluent system. Samples were applied in the same buffer and eluted at a flow rate of 0.250

mL/min, with fractions of 1.25 mL. The content of protein in each fraction was estimated by Lowry and Bradford methods (Lowry et al., 1951, Bradford 1976). Sugar content was determined by the phenol/sulfuric acid method (Dubbois et al., 1956). 2.4. Antigen selection ELISA was used to select antigenic fractions and was performed as described by Tavares et al., 1984, with some modifications. Polystyrene microtitration plates were sensitized overnight at 4°C with 0.30 ␮g of antigen in each well, in duplicate, diluted in carbonate/bicarbonate buffer 0.10M, pH 9.60. After three washes with 0.01M phosphate-buffered saline (PBS) with 0.050% Tween 20 (PBS-Tween), pH 7.40, plates were incubated at 37°C for one hour with 2% bovine albumin in PBS. Plates were washed three times with PBS-Tween and incubated for one hour at 37°C with each sera tested, diluted in PBS. After washing the wells three times in PBS-Tween, plates were incubated for one hour with the appropriated conjugates, diluted in PBS. After five washes with PBS-Tween and once with PBS, 200ul of 0.0020% O-phenilenediamine 13.0 mM Na2HPO4 24.0 mM citrate containing 0.0120% H2O2 was added to each well. The reaction was left to develop at room temperature for 20 min and then stopped by addition of 20.0 ␮L 8.0N H2SO4. The absorbance at 492 nm was measured in an ELISA reader. 2.5. Immunoblot assay After separation of the antigens by SDS-polyacrylamide gel electrophoresis (PAGE) (SDS-PAGE, 10% acrylamide), electrophoretic transfer of proteins to nitrocellulose membranes was done according to Towbin et al., 1979. After transfer, lanes containing molecular mass standards were stained with Ponceau at 5% in 10% acetic acid solution. The nitrocellulose sheets were then cut into 0.50 cm wide strips and washed in PBS-tween 0.050% for 20 min under agitation. The sheets were then blocked with PBS-Tween containing 0.30% of casein and were incubated with the appropriate sera diluted in PBS-Tween 0.050% for 60 min, under agitation. Sheets were washed four times with PBS-Tween 0.050% and incubated for 60 min with anti-IgG peroxidase conjugate diluted 1:5000 in PBS-Tween 0.050%. Sheets were then washed twice with PBS-Tween for 20 min and twice with PBS alone for another 20 min. Reactive bands were revealed by incubation of the strips in 100 mM HClTRIS buffer, pH 7.4, containing 0.50 mg/ml 3,3⬘ -diaminobenzidine and 0.10% H2O2. All sera tested in immunoblot (humans and dogs) were diluted 100 times. 2.6. Analysis of the chemical nature of the reactive epitopes For this analysis a modified ELISA was conducted. After the conventional blocking step with albumin solution and

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washings, wells with or without antigens were submitted to treatment with periodic-acid (0.20M, pH 4.50), 2-mercaptoethanol (0.060M, pH 4.70) or pronase (1.0 mg/ml in acetate buffer 0.050M, pH 5.6). For each treatment, wells containing the antigen were incubated in triplicates with 0.10 mL of reagent solution for 4 h to 37°C. Next, wells were washed with PBS and the ELISA was conducted as described above. Control wells were made by sensitization of wells without antigen treatment, and in wells without antigen but submitted to the same chemical treatments. 2.7. Protein, sugars and nucleic acid detection Presence of protein or sugars in the antigen structure was primarily determined by PAGE (PAGE, 10% acrylamide). For detection of proteins, gels were stained by the silver method (Oakley et al., 1980), and for sugar detection, by periodic acid Schiff method (PAS) according to Fairbanks et al., 1971. Agarose gel electrophoresis (AGE, 0.50% agarose) stained by ethidium bromide was used for nucleic acid detection. 2.8. Aminoacid analysis Aminoacid detection was performed by the Pico-Method (Cohen et al., 1989). Antigen and standard aminoacids were submitted to acid hydrolysis in gas-phase (HCl 6.0N, 16h, 100°C) or hydrolysis in liquid-phase (NaOH 4.20N, 16h, 100°C). Samples hydrolyzed were then derivatized using phenylisothiocyanate (PITC). Aminoacid contents of samples were then analyzed by reversed-phase chromatography by HPLC using Pico-Tag column (Waters, Millipore Division). Samples were eluted by an increasing gradient of acetonitrile in trifluoroacetic acid (CH3CN 70% in TFA 0.02%). The column was previously equilibrated with TFA 0.080% in H2O. 2.9. Homogeneity of the antigenic fractions Homogeneity of the fractions was checked by reversedphase chromatography, in Aquapore (C8) RP 300 column, 250/4.6 mm (Applied BioSystems). Samples were eluted in the same eluent system described for aminoacid analysis. The column was previously equilibrated with TFA 0.080% and samples were eluted in an isocratic gradient of CH3CN (0 to 70%) and flow of 1.50 mL/min. 2.10. Sera collection A total of 90 samples of human sera, divided in groups of 10, were used. The sera were: from normal humans of an endemic area of ACL (Manaus Municipality, Amazonas, Brazil) and sera of the same 10 individuals collected one year after a vaccination program with pentavalente vaccine; sera of patients from the same endemic area, cured of ACL after immunotherapy treatment with pentavalent vaccine;

Fig. 1. Chromatographic profiles obtained by molecular exclusion chromatography of the vaccines in FPLC. SFE (䡬), SF8 (䡺), FM8 (‚). Arrows indicate the exclusion volume of the column. Insert: calibration curve of the Superose 12 column. Numbers indicate molecular weight of calibration standards, in KDa. SFE and SF8 corresponds to soluble fractions from polyvalent vaccine (a pool of five Leishmania strains) and monovalent vaccine (PH8 single strain), respectively, both from a long-time storage. FM8 corresponds to the soluble fraction from a fresh monovalent vaccine (PH8 single strain).

sera of patients with active ACL living in the same endemic area; sera of normal humans from non-endemic area (Belo Horizonte Municipality, Minas Gerais, Brazil). Sera of patients carrying visceral leishmaniasis, active mucocutaneous leishmaniasis, Chagas disease and tuberculosis (also 10 patients in each group.) A total of 20 sera of dogs were also tested, 10 sera of dogs carrying active visceral leishmaniasis and 10 sera of normal dogs. All human sera used in this work were diagnosed by direct identification of the causal agent of the diseases.

3. Results The degree of protein degradation in the stored vaccines is high, judging by the low absorption at 280 nm observed in fractions eluted in the void volume, as compared to absorption at 280 nm of the same fractions obtained from fresh vaccine (Figure 1). In spite of these results, in ELISA, antigens recognized by sera from human and dogs carrying active visceral leishmaniasis were detected only in fractions eluted in the void volume, fractions 4, 5 and 6. These same fractions do not react with sera from human cutaneous leishmaniasis patients (not shown). These fractions were pooled and denominated antigen PI and antigen P8, according to the vaccine from which they were originated, SFE and SF8 respectively.

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Fig. 2. Reactivity of human and dog leishmaniasis sera to PI and P8 antigens in immunoblot assays. 1: standard molecular weight markers stained with Ponceau. 2 and 3: reactivity of human visceral leishmaniasis sera to PI and P8, respectively. 4 and 5: reactivity of human cutaneous leishmaniasis sera to PI and P8. 6 and 7: reactivity of normal human sera to PI and P8. 8 and 9: reactivity of dog visceral leishmaniasis sera to PI and P8. 10 and 11: reactivity of normal dogs sera to PI and P8. A pool of four sera, diluted 1: 100, was used in each test, and human and dog ␣-IgG peroxidase conjugate diluted 1:5000. 1.36 ␮g of total carbohydrate was applied to each gel lane before transfer to nitrocellulose sheets. PI was isolated from SFE and P8 was isolated from SF8.

Immunoblots using PI and P8 antigens confirmed the results obtained in ELISA. PI and P8 antigens discriminate sera of humans and dogs carrying active visceral leishmaniasis from the normal ones, and are also able to discriminate sera of humans with active visceral leishmaniasis from sera of patients carrying cutaneous leishmaniasis. Cross-reactivity with sera of patients with Chagas’ disease was not observed (Figure 2). Reactivity with sera of humans and dogs with visceral leishmaniasis was observed in fractions presenting molecular weights between 90 to over 200 KDa (Fig. 2). This result was apparently incompatible with the fact that PI and P8 antigens had molecular weight above 300 KDa, when estimated by gel filtration (Fig. 1). These results suggest that these high molecular weight antigens are formed by aggregation of smaller molecules. In fact, when submitted to 10% SDS-PAGE, PI and P8 antigens dissociate in several bands in the gel (Fig. 3, lanes A4 and B2). Dissociation of PI and P8 is also seen in PAGE under reducing conditions, but the pattern of dissociation is distinct from that observed in SDS-PAGE (Fig. 3, lanes A3 and B1). Using 10% PAGE under native conditions PI and P8 present as two bands of high molecular weight, as expected from their elution volumes in the Superose 12 column (Fig. 1). These antigens were stained by silver (Fig. 3, lanes A3, A4, A5 and B1, B2, B3), but the protein content could not be detected by the Lowry and Bradford methods.

Due to the high molecular weight of these antigens, and their positive staining by silver, the presence of nucleic acids as components of these antigens was checked in AGE (0.5% agarose). No reaction of PI or P8 antigens was observed with ethidium bromide (Fig. 3, lanes D2 and D3) indicating the absence of nucleic acids. The glycidic nature of PI was revealed by PAGE and SDS-PAGE stained by PAS method. In spite of the weak staining of native PI (Fig. 3, lane C4), in the presence of SDS a remarkable staining with PAS was observed (Fig. 3, lane C1). Interestingly, although in the presence of SDS, PI and P8 appear distributed as a number of bands of high and low molecular weight (Fig. 3, lanes A4, B2), bands below 90 KDa do not react with human and dogs sera with visceral leishmaniasis in immunoblot assay (Fig. 2). Fig. 4 shows the reactivity of dog sera with visceral leishmaniasis with PI and P8 antigens, after chemical and enzymatic treatments. Periodic acid completely abolishes the reactivity of antigens with dog sera (Fig. 4). This result agrees with that observed in PAGE stained by PAS method (Fig. 3, lanes C1 and C4), where these antigens reacted positively, confirming the presence of carbohydrate in its structure. A similar effect was observed using antigen treated with mercaptoethanol, which drastically reduce antigen reactivity with dog sera (Fig. 4). Mercaptoethanol and SDS have distinctive dissociation effects upon PI and P8 (Fig. 3, lanes A3, A4 and B1,

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Fig. 3. PAGE and Agarose gel electrophoresis (AGE) of PI and P8 antigens for detection of Proteins (silver stain), sugars (PAS stain) and nucleic acids (ethidium bromide stain). In A, PI silver stained in PAGE: Lane A1- standard molecular weight markers; Lane A2- SFE; Lane A3- PI in reducing conditions; Lane A4- PI in presence of SDS and Lane A5- PI in native conditions. In B, P8 silver stained in PAGE: Lane B1- P8 in reducing conditions; Lane B2- P8 in the presence of SDS; Lane B3- P8 in native conditions; Lane B4- SF8 and Lane B5- standard molecular weight markers. In C, PI stained by PAS method in PAGE: Lane C1- PI in presence of SDS; Lane C2- egg albumin; Lane C3- trypsinogen; C4- P1 in native conditions. In D, AGE stained by ethidium bromide: Lane D1-1 Kb DNA marker; Lane D2- PI antigen; Lane D3- P8 antigen and in Lane D4- ␭ hind DNA. In each lane was applied 1.80 ␮g of total carbohydrate.

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Fig. 4. Effects of chemical and enzymatic treatments upon reactivity of visceral leishmaniasis dog sera against PI and P8 antigens, measured by ELISA. (1) PI not treated, (2) PI pronase treated, (3) PI mercaptoethanol treated, (4) PI periodic-acid treated, (5) P8 not treated, (6) P8 pronase treated, (7) P8 mercaptoethanol treated, (8) P8 periodic-acid treated. In all cases a pool of five dog sera was used at 1:320 and ␣-IgG peroxidase conjugated at 1:10000. In each well was applied 0.030 ␮g of total carbohydrate.

B2), as seen by the finding that SDS does not abolish the reactivity of the high molecular weight components of PI and P8 antigens with dog sera (Fig. 2), whereas mercaptoethanol abolishes all reactivity (Fig. 4). Pronase treatment affected the recognition of antigens by sera of dogs with visceral leishmaniasis (Fig. 4), but its effect is much smaller than the observed with other chemical treatments. This result apparently contrasts with the fact that no aminoacid residues were detected by aminoacid analysis of PI and P8 antigens, after acid and basic hydrolysis. Moreover, cysteic acid is not found in these assays (not shown). These data suggest that the effects of pronase could involve enzymatic activities other than the proteolytic ones. The fact that PI antigen stained by the PAS method (Fig. 3, lanes C1 and C4) along with the effect observed by treatment of PI and P8 with periodic acid (Fig. 4), suggest that PI and P8 are polysaccharide antigens. This is further supported by the results observed performing aminoacid analysis (not shown) and by the failure to detect protein by Lowry and Bradford methods. In reversed phase chromatography, PI and P8 always eluted early as a single peak, even under different conditions of elution. However, PI and P8 dissociate, when eluted in buffers of high ionic force, or in presence of SDS (data not shown). These results indicate that PI and P8 are highly polar aggregates of polysaccharides and acidic in their nature. The following experiments were performed to analyze the possibilities of using these polysaccharide antigens for diagnosis of visceral leishmaniasis. PI does displays differ-

Fig. 5. Reactivity of human and dog sera to PI in ELISA. (1) Sera of health humans from endemic area to ACL before vaccination; (2) Sera of health humans from endemic area to ACL one year after vaccination with pentavalente vaccine; (3) Sera of ACL patients treated and cured by immunotherapy with pentavalente vaccine, (4) Sera of patients with active ACL, (5) Sera of health humans from non-endemic area to ACL, (6) Sera from patients with active visceral leishmaniasis, (7) Sera from patients with active mucocutaneous leishmaniasis, (8) Sera from patients with Chagas disease, (9) Sera from patients with tuberculosis, (10) Sera from dogs carrying active visceral leishmaniasis, (11) sera from health dogs. Wells were sensitized with 0.030 ␮g of total carbohydrate. Each closed circle corresponds to one human serum and each closed triangle to one dog serum (N ⫽ 10 in both cases). Human sera were diluted 1:100 and ␣-IgG human IgG peroxidase conjugated was diluted 1:5000. Dog sera were diluted 1:320 and dog ␣-IgG peroxidase conjugated was diluted 1:10000. Dashes indicate the median value of the reactivity in each group tested. Doted line is the cut-off value for human sera plus three standard deviation. Full line is the cut-off value plus three standard deviation for dog sera.

ential reactivity to human sera from patients with the three distinct forms of leishmaniasis. PI showed a very strong reactivity with sera from human visceral leishmaniasis as compared to the other clinical forms (Fig. 5, groups 4, 6, 7). This antigen also clearly discriminates sera of healthy dogs from sera of dogs with visceral leishmaniasis (Fig. 5, groups 11 and 10). Interestingly, sera from the same human individuals, collected before, and one year after, vaccination with the pentavalent vaccine, do not display significant differences in intensity (p ⬎ 0.01) of reactivity with PI antigen (Fig. 5, groups 1 and 2). Moreover, they also did not differ significantly from sera of human patients cured of active ACL, by immunotherapy using the polyvalent vaccine (Fig. 5, Group 3), all originating from the same endemic areas for ACL (p ⬎ 0.01). It should be emphasized that PI does not crossreact with sera of patients with Chagas disease, which usually induces antibodies that recognize Leishmania antigens (Fig. 5, Group 8). It was observed that sera from health humans gave significantly (p ⬍ 0.01) lower

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readings than sera of persons living in an endemic area to leishmaniasis (Fig. 5, groups 1 and 5). Finally, no anti-PI reaction was observed with serum from human patients with tuberculosis (Fig. 5, Group 9).

4. Discussion The isolation and immunogenic properties of several leishmania antigens have been described. These molecules have been associated to several major characteristic of human infection by leishmania parasites (Decker-Jackson & Honigberg 1978, Russel & Wilhelm 1986, Palatnik et al., 1989, Turco & Descoteaux 1992, Burns et al., 1993, Palatnik-de-Souza et al., 1995). We have isolated polysaccharide antigens from a vaccine against American Cutaneous Leishmaniasis (ACL) that discriminates between sera of patients with the visceral form of the disease from the cutaneous ones. These antigens were isolated from extracts composed by a mixture of species of Leishmania parasites and from Leishmania amazonensis, the species related to the dermotropic form of the disease. Other antigens able to discriminate sera of patients with the visceral form of the disease from the cutaneous ones have been identified. These antigens have been isolated from Leishmania donovani, Leishmania chagasi and Leishmania infantum, species related to the visceral form of the disease (Mary et al., 1992, Montoya et al., 1997, Martin et al., 1998, Cabrera et al., 1999). It is difficult to relate one unique specie of Leishmania parasite to a single clinical form of the disease, since several species can be associated with more than one clinical form of the disease. Moreover, many antigens display extensive cross-reactivity between different species of Leishmania. This was used as the rationale behind the vaccine trial performed with L. major for control of visceral leishmaniasis in an endemic area for L. donovani in Sudan (Khalil et al., 2000). Despite similar antigenic properties of PI and P8 antigens to antigens previously described, they are different in their structure and chemical properties. Results presented here indicate that molecular interactions among components of ACL vaccines occur during the early stages of vaccine storage, generating new antigenic aggregates. Changes in the composition and physicochemical properties of several antigens, or antigenic preparations, have been described. The literature demonstrates the isolation, from L. donovani promastigotes, of molecules with antigenic similarities that differ greatly in their physical and chemical properties (Decker-Jackson & Honigberg 1978, Semprevivo 1978, El-On et al., 1979). These antigens were initially defined as distinct molecular entities, but were subsequently recognized as different molecular assemblies of the same antigen as a result of different methodologies of isolation, storage or due to the culture media used by different investigators (Semprevivo & Honigberg 1980). Earlier studies have demonstrated that Leishmania dono-

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vani derived antigens chemically related to PI and P8 could form the same type molecular aggregates from small molecules as that seen by us (Ray & Ghose 1985). Although similar to PI and P8, these antigens are not recognized by sera of patients with visceral leishmaniasis. The reactivity of sera of humans and dogs with the visceral form of the disease against PI and P8 involved molecular species of high molecular weight, above 90 KDa (Fig. 2). The results presented in Fig. 2 are in the presence of SDS and no reactivity was observed with PI or P8, even though most of the bands are concentrated in the area of low molecular weight (Fig. 3 lanes A4 and B2). These results contrast with the reactivity of human visceral leishmaniasis sera to antigens from L. infantum, which react mainly with antigens between 14-40 KDa (Mary et al., 1992). Moreover, the carbohydrate component of these antigens is responsible for the cross-reactivity with sera of patients with Chagas’ disease, and the reactivity of human visceral sera against low molecular weight antigens from L. infantum have a tendency to disappear with the clinical cure of the disease (Cardenosa et al., 1995). PI and P8 antigens do not react with sera of patients with Chagas’ disease, and their reactivity against sera of patients with ACL or the ones treated and cured using immunotherapy with polyvalent vaccine, did not show significant differences (p ⬎ 0.01). Reactivity of human sera collected before, and one year after, vaccination with polyvalent vaccine also did not show significant difference (p ⬎ 0.01). However, the reactivity of this sera to PI and P8 is significantly different from the reactivity of the sera from non-endemic area, non-infected, individuals (p ⬍ 0.01). The meaning of these basal level of antibodies in healthy humans from endemic areas is unknown, but could reflect a previous contact of these individuals with Leishmania parasites. We did not observe any significant reactivity of IgG antibodies against peptides or molecules below 45 KDa, in fractions obtained by chromatography from freshly, or longtime storage, ACL vaccines (data not shown). However, these antigens are likely degradation products, without the conformation of the original molecule. The strong reactivity of PI and P8 antigens against human and dog sera with visceral leishmaniasis, probably, involve epitopes expressed in a new conformational structure, in highly aggregate forms. These new reactive structures originated from associations of small degradation products, that dissociate in the presence of SDS (Fig. 3), however, dissociation intermediates with more than 90 KDa still are recognized (Fig. 2). The same dissociation effect upon these aggregated antigens was observed with 2-mercaptoethanol, but a new pattern of bands was observed in PAGE in comparison to the SDS effect (Fig. 3A and 3B). The effects of 2-mercaptoethanol and periodic acid upon PI and P8 involve covalent modifications, resulting in the loss of all reactivity of these antigens (Fig. 4). These results clearly indicate that, PI and P8 recognition by human and dog visceral sera is dependent on the conformational organization of small epitopes, and that

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the recognition requires these epitopes in an antigenic structure with a conformation and minimum defined size. Research related to the mapping of antigenic determinants of proteins from Leishmania, and antigen conformation, correlates with our hypothesis presented above. Trujilo et al., 1999 suggests a predominance of conformational epitopes in leishmaniasis, as compared to Chagas’ disease. This was confirmed by studies providing evidence that sera from chagasic patients recognize linear peptides, while sera from patients with visceral leishmaniasis are predominantly directed against conformational epitopes (Thomas et al., 2001). In conclusion, we have isolated antigens from vaccines for ACL, which have great potential as a tool for differential serodiagnosis and for epidemiologic studies of Leishmaniasis. These antigens are able to discriminate the visceral form of the disease from the cutaneous ones and from Chagas’ disease. Lastly, they are products of changes in molecular composition of the vaccines, formed by events of molecular aggregation. The chemical structures, as well as their monosaccharidic composition, and sequences, are currently under investigation in the laboratory. Acknowledgments We thank Dr. Odair Genaro and Dr. Alexandre Barbosa Reis for the supply of sera used in this study, Rosaˆ ngela Barbosa de Deus and Ka´ tia Morais for the assistance in the ELISA, and Dr. Kenneth J. Gollob for the critical review of the manuscript. Financial support: This work was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´ gico (CNPq) and by Bioquı´mica do Brasil S/A (BIO´ S). BRA References Badaro´ , R., Benson, D., & Eula´ lio, M. C., et al. (1996). rK39: a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis. J Infect Dis, 173, 758 –761. Berman, J. D. (1997). Human Leishmaniasis: clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis, 24, 684 –703. Bradford, M. M. (1976). Rapid and sensitive method for the quantification of microgram quantities of protein using the principle of protein dyebinding. Anal Biochem, 72, 248 –254. Brito, M. E. F., Mendonc¸ a, M. G., Gomes, Y. M., Jardim, M. L., & Abath, F. G. C. (2000). Identification of potentially diagnostic Leishmania brasiliensis antigens in human cutaneous leishmaniasis by immunoblot assays. Clin Diag Lab Immun, 7 (2), 318 –321. Burns, M. J., Shreffler, W. G., Benson, D. R., Ghalib, H. W., Badaro´ , R., & Reed, S. G. (1993). Molecular characterization of a kinesin-related antigen of Leishmania chagasi with detects specific antibody in African and American visceral leishmaniasis. Proc Natl Acad Sci USA, 90, 775–779. Cabrera, G.P.B., De-Silva, V. O., & Da-Costa, R. T., et al. (1999). The fucose-manose ligand-ELISA in the diagnosis and prognosis of canine visceral leishmaniasis in Brasil. A J Trop Med Hyg, 61 (5), 196 –301.

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