Immunology Letters 70 (1999) 203–209

The humoral immune response to the kinetoplastid membraneprotein-11 in patients with American Leishmaniasis and Chagasdisease: prevalence of IgG subclasses and mapping of epitopes

Claudia Trujillo, Robinson Ramırez, Iva D. Velez, Christof Berberich *Programa de Estudio y Control de Enfermedades Tropicales, Facultad de Medicina, Uni6ersidad de Antioquia, A.A. 1226, Medellın, Colombia

Received 15 April 1999; received in revised form 5 August 1999; accepted 20 August 1999

Abstract

The kinetoplastid membrane protein-11 (KMP-11) is a major target of the humoral immune response during Leishmania-infec-tions. The majority of sera from visceral leishmaniasis, mucocutaneous leishmaniasis and even some cutaneous leishmaniasispatients contain detectable IgG antibodies against KMP-11. We also provide evidence that this protein may act as a potentantigen in T. cruzi infections, since most Chagas sera show immunological cross-reactivity. Therefore, KMP-11 cannot be used asa specific diagnostical tool for the serodiagnosis of leishmaniasis in those regions where both, Leishmania and T. cruzi infectionsoverlap geographically. When analyzing the subclass specificity of the antibody response to KMP-11 we observed the followingorder of reactivity: IgG1 \\IgG3 \IgG2 \IgG4, which is similiar to that seen in crude parasite extract. The mapping ofantigenic determinants by using synthetic 20-mer peptides revealed the existence of predominantly conformational epitopes inleishmaniasis, while 50% of sera from Chagas patients reacted with a particular KMP-11 peptide. These results therefore suggestthe presence of disease-specific B-cell epitopes. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: KMP-11; Leishmaniasis; Chagas disease; Immunglobulins; B-cell epitope

www.elsevier.com/locate/

1. Introduction

Intracellular infection by Leishmania sp. usually in-duces a complex immune response characterized byvarying degrees of cell-mediated immune reactions andcirculating antibodies. Based on clinical, histopatholog-ical and immunological aspects of disease, several formsof leishmaniasis can be distinguished. Localized cuta-neous leishmaniasis (LCL), the most frequent form, ischaracterized by the presence of one or few ulceratedlesions, few parasites and positive cellular immune re-sponses to Leishmania antigens [1,2,4]. Diffuse cuta-neous leishmaniasis (DCL), is a progressive chronicinfection with the development of numerous nodularlesions distributed all over the body and containinglarge numbers of parasites. Cell-mediated immunityappears to be absent in DCL-patients, but Leishmania-

specific antibody titers are relatively high. [2,3]. In themucocutaneous form of leishmaniasis (MCL) patientsoften present severe destructive lesions of mucous mem-branes of the oral, nasal and pharyngeal tract. MCL-patients show strong lymphoproliferative responses toleishmanial antigens which are significantly higher thanin LCL-patients [1,2,4]. With respect to the humoralimmune response, a successively higher specific anti-body titer can be observed in LCL, MCL and DCL.The intensity of the antibody response appears toreflect both, the parasite load and the chronicity of theinfection [5]. An exceptionally high antibody titeragainst Leishmania antigens can be detected in the mostsevere from of disease, visceral leishmaniasis (VL), as aconsequence of polyclonal activation of B-cells as aresult of the presence of large numbers of parasites inthe bone marrow and spleen [6]. The clinical symptomsinclude hepatosplenomegaly, hemorrhagic complica-tions and increased susceptibility to microbial infec-tions. Acute VL is also characterized by antigen-specificT-cell anergy. The importance of cell mediated immu-

* Corresponding author. Tel.: +57-4-2631930; fax: +57-4-2631930.

E-mail address: jramirez@quimbaya.udea.edu.co (C. Berberich)

0165-2478/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.PII: S 0 1 6 5 -2478 (99 )00146 -7

C. Trujillo et al. / Immunology Letters 70 (1999) 203–209204

nity for the resolution of disease is manifest in therecovery of lymphoproliferative responses in VL pa-tients subjected to a rigorous chemotherapy [7].

It is generally accepted that the difference betweenresitance and susceptibility to Leishmania infections liesat the level of expansion of Th1 and Th2 CD4+ cells[8–11]. Most prominent in the mouse model, inductionof a Th1-specific cytokine pattern with elevated levelsof IFN-g and TNF-a leads to resistance or resolutionof disease, whereas a Th2-specific response with pre-dominant production of IL-4 and IL-10 which down-regulate the macrophage effector functions is associatedwith exacerbation of infection. This paradigm holdsalso true in human leishmaniasis, although in a lessrigorous manner. Evidence for a predominant Th1-re-sponse in LCL has been obtained from in situ studiesdone in lesions of patients [12,13]. In MCL lesions amixture of Th1 and Th2 cytokines could be observed[12–14], whereas DCL patients exhibit a cytokine pat-tern characteristic for a nearly exclusive Th2 response[12,13].

Infection with T. cruzi induces a potent cell-mediatedimmunity maintained by Th1 lymphocytes and IFN-gprotecting the host against rapid parasite multiplication[15]. The role of specific antibodies in the immunity toLeishmania and T. cruzi infections is still not clear, butit is well known that the IgG subclass response isregulated by particular cytokines [16–18]. Studies onthe prevalence of IgG isotypes specific for a definedparasite antigen may therefore be of interest for ratio-nal vaccine development as they may reflect the type ofpredominant T-cell response to this particular antigen.So far, most of the seroepidemological studies on thesubclass antibody pattern to Leishmania or T. cruziantigens have been carried out with crude parasitefractions. We have recently described the cloning andantigenic characterization of the abundant, LPG-asso-ciated Kinetoplastid membrane protein-11 (KMP-11) inL. infantum and L. panamensis and showed evidence forthe strong antigenic character of this particular Leish-mania protein [19,20]. Its marked potential to inducelymphoproliferation in LCL patients [22] or humansrecovered from VL infections after chemotherapy [21]lends further support that KMP-11 may be considereda promising vaccine candidate against leishmaniasis. Inthis study, we have analyzed the IgG subclass responsesto KMP-11 in patients suffering from different forms ofAmerican leishmaniasis. Since our recombinant KMP-11 shows strong cross-reactivity with antibodies pro-duced during the course of Chagas disease, we alsoincluded a study on the IgG subclasses prominent inchronic patients infected with T. cruzi. The humoralresponse of VL and Chagas patients to synthetic pep-tides of KMP-11 has also been assessed.

2. Material and methods

2.1. Patients sera

Blood samples were obtained from four groups ofpatients. (1) VL sera: serum samples from ten VLpatients were collected during an outbreak of VL in thePiauı state of Brazil. In addition, seven VL sera wereobtained from patients living in endemic regions for L.chagasi/infantum at the Caribbean Coast in Colombiaand Venezuela. (2) MCL sera: serum samples from fiveMCL patients infected with L. braziliensis were ob-tained from the Rio de Janeiro state of Brazil and fivesamples were from the northeastern provinces ofColombia. (3) CL sera: eight sera were collected fromLCL patients living in endemic regions for L. panamen-sis and L. braziliensis in the central provinces ofColombia. (4). Seventeen Chagas sera from patients inthe chronic phase were obtained from the northeasternprovinces of Colombia and ten sera were obtained fromthe northern provinces of Chile where T. cruzi is en-demic. For the epitope mapping studies, six Spanishcanine sera from an endemic region for L. infantumwere used. In all cases, presence of Leishmania parasitesin the patients was determined by direct microscopicexamination and culture methods. Immunofluorescencemicroscopy using mAbs capable to distinguish betweenspecies-specific LPGs was applied in order to confirmthe identity of Leishmania parasites. The patients havenot received treatment at the time point when bloodsamples were obtained. Infection with T. cruzi in thechronic Chagas patients was confirmed by serologicalexamination (ELISA and hemagglutination).

In addition to the patients’ sera, 15 blood samplesfrom individuals with a positive Montenegro skin test,but without any clinical symptoms of a Leishmaniainfection and 13 samples from individuals with a nega-tive skin test were obtained.

2.2. Expression and purification of the recombinantKMP-11

The L. panamensis KMP-11 was expressed in Es-cherichia coli cells as a His-tagged, recombinant proteinand purified by affinity chromatography under nativeconditions as previously described [20].

2.3. ELISA tests

Ninty-six-well microtiter plates (Nunc, Roskilde,Denmark) were sensitized with 200 ng of recombinantKMP-11 in 0.1 x PBS overnight at 4°C. Plates wereblocked by incubating the wells with 200 ml of PBScontaining 5% nonfat dried milk powder and 1% igepalCA-630 (Sigma, St. Louis, MO). For the determinationof the total IgG antibodies present in the patients sera

C. Trujillo et al. / Immunology Letters 70 (1999) 203–209 205

samples were used in 1:100 dilutions. For the IgGsubclass measurements sera were used in 1:30 dilutionsin blocking solution. A biotin-streptavidin based systemwas used as second antibody in order to detect antibod-ies with high sensitivity. After sera exposure for 90 min,wells were subsequently incubated for 1 h with biotin-labelled anti-human IgG (Sigma) at a dilution of 1:1000or with biotin-labelled anti-human IgG1-4 (Sigma) at adilution of 1:500, respectively, and with alkaline-phos-phatase-labelled streptavidin (Sigma) at a dilution of1:3000 or 1:1500, respectively. All components of thebiotin-streptavidin second antibody system were freshand were recently supplied. Finally, after rigorouswashing, the immune complexes were revealed with 50ml of the substrate p-nitrophenyl phosphate (PNPP) (10mg of PNPP in 10 ml of 10 mM diethanolamine, pH9.5, containing 0.5 mM MgCl2). Incubation wasstopped after 40 min by addition of 50 ml of 0.1 MEDTA to each well. Absorbance values were read at405 nm in an automatic microelisa reader (Benchmark,Biorad, USA). All experiments were performed in du-plicate or triplicate form. The relative absorbance is themean absorbance value of the test sera minus the meanabsorbance of the blanks (absorbance value withoutserum exposure).

For the epitope mapping studies, the Falcon assayscreening test-ELISA (FAST-ELISA) was used insteadof the classic ELISA. The coating of the microbeadswas performed overnight at room temperature with 10mg of each peptide diluted in 1 x PBS. Successively, themicrobeads were immersed for 1 h in blocking solution(5% nonfat dried milk powder in PBS), for 2 h in serumsamples (dilution 1:100) and for 1 h in blocking solu-tion containing horseradish-peroxidase labelled anti-hu-man or anti-dog IgG (dilution 1:2000). After rigorouswashing, the immune-complexes were revealed with o-phenylenediamine as chromogenic substrate. The ab-sorbance was read at 490 nm. All experiments wereperformed in duplicate or triplicate form. The relativeabsorbance is the mean absorbance value of the testsera minus the absorbance mean of four sera fromindividuals with a negative Montenegro skin test usedas negative control. These sera do not react with anypeptide (mean absorbance value is 0.007)

2.4. Synthesis of peptides

The library of overlapping peptides (P1-5 are 20-mers, P6 is a 17-mer) covering the whole KMP-11sequence were synthezised by the simultaneous multi-ple-peptide solid phase synthetic method using apolyamide resin and the FMOC chemistry [31]. Puritywas checked by amino acid analysis, HPLC and massspectrometry showing 95% purity and the expectedmolecular mass. The individual peptide sequences are:

P1: 1MATTYEEFSAKLDRLGEEFN20,

P2: 16GEEFNRKMQEQNAKFFADKP35,

P3: 31FADKPDESTLSPEMKEHYEK50,

P4: 46EHYEKFERMIKEHTEKFNKK65,

P5: 61KFNKKMHEHSEHFKQKFAEL80,

P6: 76KFAELLEQQKAAQYPSK92.

3. Results

3.1. Recombinant expression and immunoreacti6ity ofKMP-11

As previously described [20], the open reading frameof the L. panamensis KMP-11 gene was inserted intothe bacterial expression vector pQE30 (Qiagen, Hilden,Germany). Induction of protein expression in E. coliyielded large amounts of soluble, His-tagged recombi-nant KMP-11 which was subsequently purified byaffinity chromatography on Ni-NTA (Qiagen). Fig.1(A) shows a 15% PAA gel used in order to monitorexpression and purification of the protein. A singleprotein band at approximately 14 kDa was detected inthe elution fraction ensuring the absence of bacterialcontaminant proteins. A single batch of the recombi-nant protein was used in all immunoassays in order toexclude batch to batch antigen variability. Since KMP-11 appears to be highly conserved in all Leishmaniaspecies in which it has been cloned so far (more than96% homology) [19,20,23], the L. panamensis proteinshould allow assessment of the specific antibody re-sponse in all cases of Leishmania infections. Fig. 1(B)shows a western blot analysis of the recombinantKMP-11 probed with a pool of 11 VL sera (I), with apool of eight Chagas sera (II) and with a polyclonalanti-KMP-11 antiserum (III) raised in rabbit [24]. Apredominant immunoreactive band at 14 kDa corre-sponding to the monomeric form of KMP-11 could beobserved in all three cases, but a second minor reactiveband at 28 kDa corresponding to the dimeric proteinform was also detectable [20].

3.2. KMP-11 specific IgG serum antibodies in differentforms of American leishmaniasis and Chagas disease.

In order to analyze in more detail the B-cell anti-genicity of KMP-11 in patients suffering from CL,MCL and acute VL as well as Chagas disease in thechronic state we performed a series of ELISA-tests. Fig.2 shows the reactivity of total IgGs present in eachpatient serum against the recombinant form of KMP-11. A high variability of ELISA values was detectablein each patients’ group probably reflecting different

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parasite load and chronicity of infection. However, asuccessively stronger anti-KMP-11 response could begenerally observed in LCL, MCL and VL patients.Antibody titers in some sera of the latter group areexceptionally high. Since a KMP-11 like immunoreac-tive band has also been observed in parasite lysate of T.cruzi [25], we were interested in evaluating whether theT. cruzi counterpart of the protein acts as an antigenduring Chagas disease. Our ELISA results, in fact,point out that the T. cruzi KMP-11 like protein mayalso stimulate a moderate to strong humoral response

Fig. 2. Total IgG antibody reactivity to KMP-11 as measured byELISA. Antibody levels are expressed as absorbance mean values ofduplicate assays minus the absorbance mean value of the blanks(absorbance values without serum exposure). The vertical line repre-sents the cut-off value (aborbance mean of 14 Montenegro-negativesera plus 3 S.D.=0.287). VL, OD values of 17 sera obtained frompatients with visceral leishmaniasis; MCL, OD values of ten seraobtained from patients with mucocutaneous leishmaniasis; CL, ODvalues of eight sera obtained from patients with cutaneous leishma-niasis; Ch, OD values of 27 sera obtained from patients sufferingfrom chronic Chagas disease; M+ , OD values of 15 sera obtainedfrom asymptomatic individuals with a positive Montenegro skin test;M− , OD values of sera obtained from 13 individuals with a negativeMontenegro skin test who never lived in endemic regions for leishma-niasis.

Fig. 1. SDS-PAGE and western blot analysis of the recombinantKMP-11. (A) A 15% PAA-gel was used in order to monitor inductionof recombinant expression in E. coli and purification of the His-tagged protein by affinity chromatography on Ni-NTA. At each stepan aliquot was taken and electrophoresed. (1) IPTG-induced bacteria;(2) column-flow through; (3), (4) washing fractions; (5), (6), (7), (8)elution fractions with imidazole-gradient; (M) molecular weightmarker. (B) western blot analysis with a pool of 11 VL sera: (I), witha pool of 8 Chagas sera; (II) and with a polyclonal anti-serum; (III)produced in rabbit. The patients’ sera were used at a dilution of 1:200and the polyclonal anti-serum was used at a dilution of 1:5000. Theimmunoreactive bands corresponding to the dominant 14 kDamonomer (M); and 28 kDa dimer form (D) of KMP-11 are indicated.

in Chagas patients and most of the sera contain mea-sureable levels of anti-KMP-11 specific IgGs. The anti-KMP-11 response appears to be quite specific sincehuman sera with a negative Montenegro skin test, ingeneral, react poorly. The observed cross-reactivity ofone control serum could be the result of exposure to adifferent pathogen whose antigenic proteins share epi-topes with KMP-11. A weak immunoreactivity withoptical density with absorbance values slightly in-creased over the non-infected control group could beoccasionally observed in Montenegro positive, butasymptomatic persons consistent with the low level ofcirculating anti-Leishmania antibodies.

3.3. IgG isotype responses to KMP-11

IgG1, IgG2, IgG3 and IgG4 antibody levels againstKMP-11 were measured in 12 VL, eight MCL and nineChagas sera. Only those sera were assayed which hadshown elevated titers of total IgGs in the anteriorELISA study which were higher than the cut-off value.Because of the weak overall immunoreactivity of CLsera to KMP-11, a more detailed analysis of the specificIgG isotypes elicited in these patients had to be ex-cluded. Fig. 3(A–D) shows that significant IgG sub-class responses are only detectable for IgG1 and to afar lesser extent for IgG3 and IgG2. Predominance of

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IgG1 antibodies in the KMP-11 specific antibody poolcould be equally found in VL, MCL and Chagas sera.IgG4 isotypes are practically absent in all analyzedpatients sera. These results suggest the following orderof IgG isotype reactivity to KMP-11 in both clinicalmanifestations of leishmaniasis as well as in Chagasdisease: IgG1 \\IgG3 \IgG2 \IgG4. In all threecases KMP-11 does not appear to elicit IgG4 subclassresponses which can be typically observed in patientswith DCL and appear to be associated with a pro-nounced Th2 like cellular immune response [3].

3.4. Epitope-mapping of KMP-11 using syntheticpeptides of 20 amino acids

In order to determine the immunodominant regionswithin KMP-11, five 20-mer peptides and one 17-merpeptide each overlapping in five residues were syn-thezised. These peptides span the whole sequence of 92amino acids of the protein. The reactivity of the 12 VLand 8 MCL sera which had been used in the IgGsubclass mapping experiments as well as six sera fromSpanish dogs infected with L. infantum was subse-quently assayed by FAST-ELISA-tests. In a similiarstudy the reactivity of eight Chagas sera against eachpeptide was also analyzed. No particular antigenic de-

terminant of KMP-11 as represented in the linear pep-tides was recognized by the different leishmaniasis serawhich were tested in a pool (Fig. 4(A)) and subse-quently each one individually (data not shown). Theseresults are in marked contrast to the high immunoreac-tivity observed when using the whole protein as antigenin the ELISA (Fig. 4(A)), thus suggesting that antiKMP-11 antibodies elicited during Leishmania-infec-tions are predominantly conformational. Similiar re-sults have also been obtained when testing sera fromexperimentally infected mice (data not shown). Thenegative linear epitope-mapping with leishmaniasis serawas not a consequence of insufficient coating of thepeptides to the FAST-ELISA microplates as evidencedby the surprising high activity of some Chagas seraagainst a particular peptide (Fig. 4(B). Four out ofeight sera (50%) reacted with peptide 3 which spans thecoil region in the well defined helix-loop-helix structureof KMP-11 [23]. The prevelance of antibodies to thispeptide is high in three Chagas sera with reactivityvalues normally observed when using the whole proteinas antigen. Therefore, our results point to disease-spe-cific epitopes of KMP-11. Peptide 3 appears to berecognized exclusively in Chagas sera, thus suggestingthat this peptide in combination with other specificantigens may be useful for the differential serodiagnosis

Fig. 3. IgG isotype response to KMP-11 as measured by ELISA. A–D corresponds to IgG1, IgG2, IgG3 and IgG4, respectively. Represented arethe absorbance values of each tested patients’ serum as absorbance mean of duplicate assays minus the absorbance mean of the blanks(absorbance values without serum exposure).

C. Trujillo et al. / Immunology Letters 70 (1999) 203–209208

Fig. 4. B-cell epitope mapping of KMP-11 by using a library ofoverlapping synthetic peptides. (A) The FAST-ELISA reactivities of apool of 12 VL sera, eight MCL sera, six canine leishmaniasis sera andeight Chagas sera against six individual peptides of KMP-11 (1–6)are expressed as the absorbance mean values of triplicate assaysminus the absorbance mean values of negative controls (aborbancevalues of four human sera with a negative Montenegro skin test,OD490=0.010). The reactivity against the entire protein (KMP-11) isalso represented. (B) The FAST-ELISA reactivities of eight Chagassera were evaluated individually against each peptide and the wholeprotein. Shown are the mean OD490 values of duplicate assays minusthe absorbance mean value of negative controls (aborbance values offour sera from individuals who never lived in endemic regions for T.cruzi, OD490=0.008). Data are presented as x9SD of three indepen-dent experiments.

ity to this particular antigen with ELISA values in therange of MCL and VL sera. The observation of amarked immunological cross-reactivity in infectionscaused either by Leishmania or T. cruzi is particularlyimportant, since it should exclude KMP-11, in contra-diction to what has been recently proposed as a resultof the high sensitivity of antibody detection in leishma-niasis patients [20], as a tool for specific diagnosis ofAmerican leishmaniasis.

Several immunological studies have correlated thepattern of cytokine production by CD4+ cells of theTh1 or Th2 subpopulations with the predominant iso-type response of immunglobulins [16–18] as evidencedfor example in DCL patients. IgG4 antibody levels areexceptionally high in this clinical form of leishmaniasisand production of IL-4, a major determinant of a Th2response and known to induce synthesis of the IgG4isotype appears significantly increased [1,3]. Therefore,DCL patients represent a clear example that the persis-tent antigen-specific T-cell anergy is associated with apredominant Th2 response. If high levels of IgG4 anti-bodies can be employed as a surrogate marker for Th2activity, our results would suggest that such activity isminimal in VL, MCL and Chagas disease. Those anti-bodies to KMP-11 are completely absent. The subclassresponse is, in contrast, predominantly of the IgG1and, to a lesser extent, of the IgG3 and IgG2 type. Thestudies on the predominant IgG subclass response inDCL versus VL disease [3,6] also suggest that funda-mental differences must exist in the mechanisms whichinduce transitory suppression of cell-mediated immu-nity in VL and persistent T-cell anergy in DCL pa-tients. Interestingly, a recent report on the IgG isotypepattern in dogs naturally infected with L. infantum haspointed out that IgG1 antibodies to Leishmania anti-gens markedly disappeared in sera of those dogs whichrecovered from infection following chemotherapy [27].The authors of the study concluded that IgG2 may beassociated with resistance to the parasite while IgG1may be related to the development of disease. Althoughit may be premature to extrapolate these results tohuman infections, some parallels to the immune re-sponses of Sudanese VL patients infected by L. dono-6ani have recently been found. The IgG1 and IgG3antibody levels in these VL sera appeared to fall signifi-cantly upon treatment with antimonial drugs [28]. Inthis way, the relatively high titer of IgG1 and the lowtiter of IgG2 antibodies observed in the South Ameri-can VL patients of our study could therefore point toprogress of disease as they have not undergonechemotherapy at the timepoint when blood sampleswere obtained.

The KMP-11 specific antibody reponse is similar tothat seen to crude parasite antigen. Rodriguez et al.reported predominant reactivity of IgG1, followed byIgG3 and IgG2 isotypes to soluble extracts of L.

of Chagas disease versus Leishmaniasis in those regionswhere Leishmania and T. cruzi infections overlapgeographically.

4. Discussion

KMP-11, a major protein determinant in the Kineto-plastid protozoan membrane appears to be antigenicand elicits a relatively strong antibody response duringnatural course of leishmaniasis and Chagas disease.Consistent with the varying levels of circulating anti-bodies in different clinical forms of Leishmania infec-tions, the highest titer of anti-KMP-11 specific IgGswas observed in acute VL, followed by MCL and CL.Some Chagas sera also showed strong immunoreactiv-

C. Trujillo et al. / Immunology Letters 70 (1999) 203–209 209

braziliensis in both, CL and MCL sera [26]. In MCLpatients the levels of IgG1 antibodies are particularlyhigh. The average of IgG4 reactivity, in contrast, waslow in both forms of American cutaneous leishmaniasis,thus suggesting that the antibody response is not associ-ated with a dominant Th-2 cytokine pattern consistentwith the positive lymphoproliferative responses typicallyobserved in these patients. IgG1 and IgG3 are also veryeffective in complement fixation [30] and may thereforecontribute to the lysis of some susceptible species.

In summary, although a clear correlation between theclinical manifestations of disease in each analyzed pa-tient groups and the IgG subclass response could not befound, we think that the absence of measurable IgG4antibodies in the KMP-11 specific antibody pool is animportant observation in line with its potential to stim-ulate lymphoproliferation in peripheral blood mononu-clear cells of leishmaniasis patients [21,22,29]. Thisantigen may therefore fullfill the criteria of an interestingvaccine candidate molecule. However, any strategy ofimmunization against leishmaniasis including this anti-gen should be better based on the whole protein, sincelinear peptides may be too less antigenic-at least at theB-cell level as shown in this report. The mapping of theantigenic determinants in KMP-11 revealed the existenceof predominantly conformational epitopes in Leishma-nia infections, while 50% of the sera from Chagaspatients reacted with a particular peptide, thus suggest-ing the presence of disease-specific B-cell epitopes. Thedifferential recognition of epitopes may also argue fordifferent modes of antigen-processing and presentationof the T. cruzi and Leishmania protein consistent withthe different life-cycles and intracellular survival strate-gies of both parasites and may be useful for the designof diagnostic tools which allow differential serodiagnosisof American leishmaniasis and Chagas disease.

Acknowledgements

This work was supported by the University of Antio-quia (grant CIM-9918) and Colciencias (grant 1115-04-012-99). C. Berberich is recipient of a fellowship fromColciencias. We are greatful to Dr C. Alonso and Dr E.Patarroyo for providing us with the synthetic KMP-11peptides. We also thank Dr Noris Rodriguez for provid-ing us with the VL sera from Venezuela, Dr SergioMendonca for the gift of the Brazilian VL and MCLsera, Dr Aldo Solari for the gift of the Chagas sera fromChile and Dr V.M. Angulo for providing us with theChagas sera from Colombia.

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