Peptides, method and kit for immunodiagnostic tegumentary leishmaniose and use

Abstract

This technology concerns peptides, a method, a kit, and their use in the diagnosis of cutaneous leishmaniasis. The peptides are epitopes of immunogenic proteins abundant in the species *Leishmania amazonensis*, *L. braziliensis*, and *L. infantum*, and are defined by SEQ IDs No. 1 to 46. The immunodiagnostic kit contains at least one of these peptides and / or the metallopeptidase proteins of the MA(E) clan of the M3 family (SEQ IDs No. 47 and / or 48), and / or the hypothetical conserved protein gi|134063939 (SEQ ID No. 49). The diagnostic method using these peptides and / or proteins allows the detection of a specific serological response for cutaneous leishmaniasis in the serum of infected individuals.

Description

1 / 15 Peptides, Method and Kit for Immunodiagnosis of Cutaneous Leishmaniasis and Use

[001] This technology relates to peptides, method, kit and their use in the diagnosis of cutaneous leishmaniasis. The peptides are epitopes of immunogenic proteins abundant in the species Leishmania amazonensis, L. braziliensis and L. infantum, and are defined by SEQ ID No. 1 to 46. The immunodiagnostic kit contains at least one of these peptides and / or the metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48) and / or the hypothetical conserved protein gi|134063939 (SEQ ID No. 49). The diagnostic method using such peptides and / or proteins allows the detection of a specific serological response for cutaneous leishmaniasis in the serum of infected individuals.

[002] Leishmaniasis is a parasitic disease caused by flagellated protozoa belonging to the genus Leishmania (Kinetoplastida: Trypanosomatidae). Parasites of the genus Leishmania are digenetic, with their life cycle divided between vertebrate and invertebrate hosts. Transmission of the protozoan occurs during the blood meal of infected female sandflies of the genera Lutzomyia and Phlebotomus. Metacyclic promastigotes are regurgitated into the vertebrate host, and subsequently, this form of the protozoan is phagocytosed by cells of the mononuclear phagocytic system. In these cells, the promastigote forms transform into amastigotes, proliferate, and thus establish the infection.

[003] Depending on the Leishmania species and the host's immune response, two clinical forms can be highlighted: Cutaneous and Visceral, with the cutaneous form presenting a wide spectrum of manifestations. In Brazil, both forms of the disease, cutaneous and visceral, have a wide geographical distribution, including overlapping areas. The most frequent and widespread species Petition 870160012171, dated 01 / 04 / 2016, page 12 / 32 Two out of every 15 cases are L. (V.) braziliensis and L.(L.) amazonensis, causing the cutaneous form, and L. (L.) infantum, causing the visceral form. Effective treatment and control of leishmaniasis depend on accurate and early diagnosis in order to prevent the development of chronic disease and / or disfiguring lesions.

[004] The low sensitivity of serological tests in the diagnosis of Cutaneous Leishmaniasis and the frequent cross-reactions with Visceral Leishmaniasis and other parasitoses make their use in the routine diagnosis of the cutaneous form impossible.

[005] Among the most commonly used methods for diagnosing cutaneous leishmaniasis are skin in-print microscopy and the Montenegro test. The first method is widely used due to the ease of sample collection; however, it has low sensitivity and requires considerable professional skill (Ashford, RW. Int. J. Parasitol. 2000; 30: 1269-128; Marfurt J, Nasereddin A, et al. J. Clin Microbiol. 2003; 41(7):3147-3153; Van der Meide WF, Schoone GJ et al. J. Clin. Microbiol. 2005; 43(11):5560-5566). The second test is based on the development of a more or less intense cellular response, depending on the clinical form, and is a simple method to perform and interpret. However, in endemic areas, a positive reaction may be due to the disease, inoculation previous antigen exposure, exposure to the parasite without disease, allergy to the diluent, or even cross-reaction with other diseases (Carvalho EM, Correa-Filho D, et al.American Journal of Tropical Medicine and Hygiene. 1995; 53: 273-277.). Furthermore, the reaction may be exacerbated in the case of the mucocutaneous form or even negative between four and six weeks after the appearance of the skin lesion, in the diffuse form and in immunosuppressed patients (Melo MN, Mayrink W, et al. Rev. Inst. Med. Trop. São Paulo. 1977; 9:161-164.; Souza WJ, Sabroza PC, et al. Acta Trop. 1992; 52:111-119). Another disadvantage of the method is that it requires... Petition 870160012171, dated 01 / 04 / 2016, page 13 / 32 3 / 15 culture of the parasite for antigen production, since preparation of a different origin may influence the sensitivity of the technique (Reithinger R & Dujardin JC. Journal of Clinical Microbiology. 2007; 45 (1):21-25).

[006] For the diagnosis of visceral leishmaniasis, serological tests are the most used, due to the high levels of antibodies in patients who present this form of the disease. Despite the satisfactory sensitivity of serological tests, they have low specificity, and cross-reactivity with other parasitoses is common (Ferreira AW, Belem, ZR, et al. J Clin Microbiol. 2001; 39(12): 4390-4395).

[007] Several recombinant proteins are being tested for the development of enzyme-linked immunosorbent assays (ELISA) for the diagnosis of visceral leishmaniasis, considering the high antibody levels in patients. The recombinant antigen K39, belonging to the kinesin superfamily, is used to perform the technique, presenting a lower number of cross-reactions when compared to the use of the crude antigen (Carvalho SF, Lemos EM, et al. Am. J. Trop. Med. Hyg. 2003; 68(3):321-4.). The prevalence of specific antibodies against various molecules was tested to evaluate the immunogenicity of kinesins and their potential for the diagnosis of human visceral leishmaniasis. The result highlights, among the proteins tested, the recombinant LicTXNPx, which demonstrated a sensitivity of 78%, specificity of 94%, and also a 60% ability to distinguish between infection and cure (Santarém N, Tomás A, et al. Immunology Letters. 2005; 101:18-23).

[008] For cutaneous leishmaniasis, despite the low antibody levels in patients, studies using recombinant proteins have demonstrated greater sensitivity of the ELISA method when compared to parasitological methods (Weigle KA, de Dávalos M, et al. Am J Trop Med Hyg. 1987; 36: 489-496; Goto H & Lindoso JA. Expert Rev Anti Infect Ther. 2010; 8(4):419-33). Sousa and colleagues (2013) used Petition 870160012171, dated 01 / 04 / 2016, page 14 / 32 4 / 15 A panel of human sera was used to test various recombinant proteins using ELISA, with the aim of developing a tool for the serological diagnosis of human cutaneous leishmaniasis. Among the proteins tested, rHSP 70 showed a sensitivity between 52-80% and a specificity between 81.7-92.2%. This variation was due to the use of patients with the cutaneous and mucocutaneous forms; however, cross-reactivity with patients with the visceral form of the disease was not evaluated (Souza AP, Soto M, et al. PLOS ONE. 2013 Volume 8 | Issue 6 | e66110).

[009] Celeste and colleagues (2014) evaluated the use of rHSP-83 for the diagnosis of cutaneous, mucocutaneous, and visceral leishmaniasis, as well as its potential for monitoring disease therapy. The sensitivity of rHSP-83 was higher when compared to values ​​using crude L. major-like antigen, as was the specificity, at approximately 97.47% for rHSP-83 and 70.89% for crude L. major-like antigen. However, the recombinant was not efficient in evaluating infection cure (Celeste BJ, Sanchez MCA, et al. Am. J. Trop. Med. Hyg. 2014; 90(5), pp. 860-865).

[010] Document US20050646496 describes an immunodiagnostic kit for the detection of Leishmaniasis containing some variations of the “elongation factor 1” protein isolated from Leishmania infantum. In addition to requiring recombinant synthesis of long amino acid sequences, the immunodiagnostic kits proposed in the aforementioned document are suitable for identifying visceral Leishmaniasis, more specifically caused by L. infantum from the Tunisian region and other Mediterranean countries.

[011] Documents US6607731B1 and US6365165 refer to various amino acid sequences used for the treatment and diagnosis of Leishmaniasis without, however, specifying the clinical form or species of Leishmania to which they refer. The aforementioned diagnostic kits Petition 870160012171, dated 01 / 04 / 2016, p. 15 / 32 5 / 15 include polypeptides containing at least one epitope from one or more Leishmania species. Of the sequences listed in the documents, the use of those containing one or more of the amino acid sequences that make up the peptides of the present invention in diagnostic kits has not been reported. Despite evidence of immunogenicity of the sequences from use in vaccine compositions, analogous to the technology of the aforementioned US20050646496 document, recombinant synthesis of long amino acid sequences is necessary for the use of said technology, and the clinical form diagnosed by the kit is not well defined.

[012] Although relatively simple, low-cost, and efficient tests exist for diagnosing cutaneous leishmaniasis, such as the Montenegro test, these are not commercially available and are used for individual diagnosis, making them difficult to use in epidemiological surveys. The development of a specific serological test for the cutaneous form can contribute to a better understanding of the distribution and frequency of disease cases and the evaluation of control measures. Furthermore, serological tests can be automated, evaluate a large number of samples, and obtain accurate results, considering the objective reading and the rigor of the validation and interpretation criteria. Additionally, it does not require highly qualified personnel or the maintenance of parasite cultures, reducing the risk of contamination and eliminating the need for a biosafety-level laboratory.

[013] The present invention comprises a serological test that uses antigens identified as highly immunogenic only in patients with the cutaneous form of the disease. These antigens are selected epitopes of proteins from the group comprising a probable trypanothione synthase, a metallopeptidase; the Clan protein Petition 870160012171, dated 01 / 04 / 2016, page 16 / 32 6 / 15 MA(E), an M3 family protein, enolase, elongation factor 2, two hypothetical proteins and m20 / m25 / m40 peptidases from Leishmania amazonensis; a hypothetical protein and a probable heat shock protein from L. braziliensis; probable aconitase, a probable beta chain of succinyl-CoA ligase [GDP-forming], probable mitochondrial peroxiredoxin, probable IgE-dependent histamine-releasing factor; paraflagellar rod 1 protein; a small myristoylated protein 1; zeta-crystalline-like protein / NADPH-oxidoreductase and the putative hsp70 heat shock protein from L. infatum. The immunodiagnostic kit contains one or more of these peptides and / or one or both of the metallopeptidase proteins, Clan MA(E), an M3 family protein, and the hypothetical conserved protein gi|134063939. The diagnostic method using these peptides and / or proteins allows the detection of a specific serological response for cutaneous leishmaniasis in the serum of infected individuals.

[014] This high immunogenicity is due to the fact that these proteins are abundant only in species that cause the cutaneous form compared to the visceral form. Thus, this type of antigen can then be detected in a serological test, even in patients with low antibody titers, characteristic of this form of the disease. BRIEF DESCRIPTION OF THE FIGURES

[015] Figure 1 is a graphical representation of the relative intensity (RI) values ​​found in the immunoassay of peptides 1 to 10. LV / C (RI between a pool of sera from patients with Visceral Leishmaniasis (VL) and a pool of sera from healthy individuals (C)), LTA / C (RI between a pool of sera from patients with American Cutaneous Leishmaniasis (ACL) and a pool of C sera), LTA / LV (RI between a pool of sera from ACL patients and a pool of patients with VL). Petition 870160012171, dated 01 / 04 / 2016, page 17 / 32 7 / 15

[016] Figure 2 represents the results found for immunoassays with patient serum pools, performed with peptides synthesized by spot synthesis. DETAILED DESCRIPTION OF THE TECHNOLOGY

[017] The present technology deals with peptides, method, kit and their use in the diagnosis of cutaneous leishmaniasis. The peptides are epitopes of immunogenic proteins abundant in the species Leishmania amazonensis, L. braziliensis and L. infantum, and are defined by SEQ ID No. 1 to 46. The immunodiagnostic kit contains at least one of these peptides and / or the metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48), and / or the hypothetical conserved protein gi|134063939 (SEQ ID No. 49). The diagnostic method using such peptides and / or proteins allows the detection of a specific serological response for cutaneous leishmaniasis in the serum of infected individuals.

[018] The peptides of the present invention comprise the primary sequences defined by SEQ ID Nos. 1 to 46. Such peptides are epitopes selected from proteins of the group comprising a probable trypanothione synthase, a Clan MA(E) metallopeptidase-protein, an M3 family protein, enolase, elongation factor 2, two hypothetical proteins and m20 / m25 / m40 peptidases from Leishmania amazonensis; a hypothetical protein and a probable heat shock protein from L. braziliensis; the probable aconitase, a probable beta chain of succinyl-CoA ligase [GDP-forming], the probable mitochondrial peroxiredoxin, the probable IgE-dependent histamine-releasing factor; the paraflagellar rod 1 protein; a small myristoylated protein 1; Zeta-crystalline / NADPH-oxidoreductase-like protein and the putative hsp70 heat shock protein of L. infantum.The immunodiagnostic kit contains one or more of these peptides and / or one or more of the metallopeptidase proteins, Clan MA(E), an M3 family protein, and the protein. Petition 870160012171, dated 01 / 04 / 2016, page 18 / 32 8 / 15 conserved hypothetical gi|134063939. The diagnostic method using such peptides and / or proteins allows the detection of a specific serological response for cutaneous leishmaniasis in the serum of infected individuals.

[019] The proposed method for diagnosing cutaneous leishmaniasis is characterized by comprising the following steps: a) Exposure of a biological sample to at least one of the recombinant metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48) and / or hypothetical conserved protein gi|134063939 (SEQ ID No. 49), and / or to the peptides defined by SEQ ID No. 1 to 46, together, individually or in various combinations, with each other and / or with other antigens; b) Addition of a secondary antibody, or a protein, conjugated to an enzyme or a marker and that binds to any antibodies that may be present in the sample from step (a); c) Detection of anti-Leishmania antibodies in the biological sample mentioned in step (a) using reagents capable of detecting the enzyme or marker mentioned in step (b).

[020] In step “a” the proteins and / or peptides are attached to a solid support or a carrier.

[021] In step “b” the antibody or protein is preferentially conjugated with peroxidase.

[022] In step “c”, the reagent, capable of detecting the enzyme or marker mentioned in step “b”, is preferably hydrogen peroxide and / or o-phenylenediamine dihydrochloride (OPD).

[023] The proposed kit for diagnosing cutaneous leishmaniasis is characterized by comprising: a) a solid support containing the antigens; b) the solutions to be used during the examination process. Petition 870160012171, dated 01 / 04 / 2016, page 19 / 32 9 / 15

[024] The antigens in item “a” are selected from the group comprising recombinant metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48) and / or the hypothetical conserved protein gi|134063939 (SEQ ID No. 49); and / or the peptides defined by the primary sequences SEQ ID No. 1 to 46, together, individually or in various combinations.

[025] The solid support defined in item “a” can preferably be a microtiter plate for ELISA or chromatographic strips.

[026] The solutions defined in item “b” are preferably a secondary antibody solution conjugated with an enzyme or marker, a reagent capable of detecting the enzyme or marker, and positive and negative control samples. The antibody is preferably conjugated with peroxidase. The reagent capable of detecting the enzyme or marker is preferably hydrogen peroxide or phenylenediamine dihydrochloride (OPD).

[027] The proposed diagnostic kit preferably belongs to the group comprising ELISA, Western blot, Dot blot and Immunochromatography.

[028] The present invention can be better understood through the following examples, which are not limiting.

[029] Example 1: Selection of differentially abundant proteins in Leishmania species that cause Cutaneous Leishmaniasis, L. amazonensis and L. braziliensis, and in L. infantum, the etiological agent of Visceral Leishmaniasis.

[030] The differentially abundant proteins in the most frequent and widely distributed Leishmania species in L. amazonensis and L. braziliensis, which cause American Cutaneous Leishmaniasis, and Petition 870160012171, dated 01 / 04 / 2016, page 20 / 32 10 / 15 in L. infantum, the etiological agent of visceral leishmaniasis, were evaluated using the Differential Gel Electrophoresis (DIGE) technique.

[031] The pool of promastigote protein extracts from each of the species was labeled with the fluorophore CyDye™ (GE Healthcare).

[032] After two-dimensional electrophoresis (2DE), images were acquired on the Typhoon Trio scanner (GE Healthcare England) and analyzed using the DeCYDER program (GE Healthcare). Subsequently, spots selected as differentially intense in a species with a P-value < 0.05 were removed from the gels, processed, and identified by MALDI-TOF / TOF mass spectrometry.

[033] Spots of interest were removed from the gels and processed individually. Samples were mixed with a matrix [10 mg / mL R-cyano-4-hydroxycinnamic acid (Aldrich, Milwaukee, WI) in 50% acetonitrile / 0.1% trifluoroacetic acid] and applied to an MTP AnchorChip™ 600 / 384 plate (Bruker Daltonics) for MALDI / ToF-Tof analysis (Bruker, AUTOFLEX, 3.0). Both MS and MS / MS were acquired using a 200-Hz neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. Spectra were edited using FlexAnalysis and Biotools software. The search was performed using the MASCOT program (http: / / www.matrixscience.com), using the National Center for Biotechnology (NCBI) database. The search parameters were molecular weight deviation tolerance between 100-200 ppm, carbamidomethylation, maximum of an uncleaved tryptic cleavage site, and methionine oxidation. Only proteins with P-score values ​​< 0.05 were considered.

[034] An immunoproteomic approach was carried out with the aim of selecting immunogenic proteins from among those differentially abundant among the species studied. After determining the best western blot conditions, the selection of immunogenic proteins was carried out from fractionation of the protein extracts by 2DE, Petition 870160012171, dated 01 / 04 / 2016, page 21 / 32 11 / 15 transferred to nitrocellulose membranes, Amersham Hybond - ECL, using the Semidry-transfer Unit (GE Healthcare) and then revealed for three serum conditions: cutaneous leishmaniasis (CL), visceral leishmaniasis (VL), and control, according to standardization. The membranes were digitized using ImageScanner (Amersham Biosciences) and the images analyzed using ImageMaster 2D Platinum 5.0 software (GE Healthcare). From the superimposition of the membrane images revealed in the western blot, using isoelectric point and molecular mass markers as references, with DIGE gels, the position of the immunogenic spots was determined, which also showed differences in intensity and consequently the identification of the corresponding proteins.

[035] After analyzing the identified proteins, eighteen were considered immunogenic and more abundant in one of the species, with seven exclusive to L. amazonensis (a probable trypanothione synthase, a metallopeptidase; the Clan MA(E) protein, an M3 family protein, enolase, elongation factor 2, two hypothetical proteins, and a partial m20 / m25 / m40 family-like peptidase), two in L. braziliensis (a hypothetical protein and a probable heat shock protein), and nine in L. infatum (the probable aconitase, a probable beta chain of succinyl-CoA ligase [GDP-forming], putative mitochondrial peroxiredoxin, the probable putative IgE-dependent histamine-releasing factor; paraflagellar rod 1 protein; myristoylated protein 1; zeta-crystalline / NADPH-oxidoreductase-like protein). and the putative hsp70 heat shock protein).

[036] Example 2: Choice of proteins and determination of amino acid sequences of peptides for immunodiagnosis.

[037] The most abundant proteins in L. amazonensis and L. braziliensis that showed exclusive reactivity to were selected. Petition 870160012171, dated 01 / 04 / 2016, page 22 / 32 12 / 15 serum from patients diagnosed with cutaneous leishmaniasis, for the prediction of B cell epitopes. The amino acid sequences of the proteins were acquired from the NCBI databases, using the accession number of each one. Subsequently, these sequences were submitted to the software: BCPreds (http: / / ailab.cs.iastate.edu / bcpreds, accessed on September 4, 2015) and ABCPred (http: / / www.imtech.res.in / raqhava / abcpred, accessed on September 4, 2015). These programs were selected for use because they analyze sequence similarity and alignment, using mathematical models that predict the location of linear B cell epitopes (Yang X & Yu X. Rev Med Virol. 2009; 19: 77-96).

[038] A search was conducted on GeneDB (http: / / www.genedb.org / , accessed on September 14, 2015) to verify the homology of the mapped epitopes in relation to their orthologs in Trypanosoma cruzi. The epitopes found, as well as the homology found for orthologs in T. cruzi, are listed in Table 1.

[039] Table 1: Epitopes found by bioinformatics for each protein analyzed and homology found for orthologs in T. cruzi. Protein Epitopes Homology Elongation Factor VSPVVRVAVEAKNP 100% 2 FVKTLQNVNVVV 92% (845aa) GAPLKISEPVVS 92% EDMPCGNVV 89% HRGGGQIIPTARRV 100% CVEGVCVQTE 100% EAGDKRIMDTR 91% MGDVQVSPEKGTVA 93% LADNYEWDVQEAR 69% FGVDELKMRERLW 85% Petition 870160012171, dated 01 / 04 / 2016, page 23 / 32 13 / 15 metallo-peptidase, SHACCHSVVPPV 0% Clan MA(E), a VRDEATNRVVE 0% GIAPPNRTFQ family protein 0% M3 EIQEYFPMD 0% (685aa) MAKKPEMAQ 0% PEEEFQKVVQLQ 50% GKAPREYTYW 0% LKEVCATPAYEAV 0% STQPRDSLNTTQL 0% Pretein QRKAKEAQAAAQK 63% conserved RKQELERAQRE 0% hypothetical KAKKEAPAN 56% (322aa) EVRVPLPAVNV 80% Enolase AERKMYNLTF 60% (429aa) ATSFREALRM 80% ACELRDGDKARY 83% MMCELDGTKNKS 67% GDEGGFAPPIK 90% peptidase YGHMDKQPPL 100% similar to PQFDPEWAT 78% family AIASLQRHGVPHG 62% m20 / m25 / m40, GGVVPDTFRIT 82% partial (343aa) Probable protein EVEEAPAPAAAPA 0% heat shock AATTDPRVKADV 0% (821aa) GVKEIVQSCNY 0% LDLGYGATDF 70% ASLKETESEGQVV 77% Probable VSYYESNHEF 50% trypanothione DLLLGDDWEIL 45% synthase NHPEHPAILKAE 58% (652aa) Protein YRGADVCND 0% hypothetical conserved (199 aa) Petition 870160012171, dated 01 / 04 / 2016, page 24 / 32 14 / 15 Protein HYCAALQETRE 0% conserved MRAQGDEEAAR 0% hypothetical EQRAASAAQAS 0% (1081 aa) PVPRSQWVESA 82% LAHMTTVEK 0%

[040] In addition, the peptides selected by bioinformatics analysis were subjected to membrane synthesis (“spot synthesis”) on the ResPep SL apparatus (IntavisAG, Bioanalytical Instruments). The cellulose membranes containing the peptides (three replicates) were washed for five minutes with phosphate buffered saline (PBS) pH 7.4, three times. Subsequently, they were incubated with a blocking solution (PBS / Phosphate buffered saline / Tween 20 - 0.1%, 3% BSA - Bovine Serum Albumin - and 5% sucrose) for 18 hours. After a 10-minute wash with 0.1% PBS / T, each membrane replicate was incubated for two hours under constant agitation with one of the serum pools (patients with ATL - American Cutaneous Leishmaniasis-, patients with VL - Visceral Leishmaniasis- and healthy individuals) diluted in blocking solution at concentrations of 1:160, 1:640 and 1:160, respectively.Next, the membranes were washed three times for 10 minutes with 0.1% PBS / T, and incubated with a blocking solution containing the peroxidase-conjugated antibody diluted to 1:100,000. The membranes were then washed three times, for 10 minutes each time, with 0.1% PBS / T, followed by development with ECL (Enhanced chemiluminescence).

[041] After performing the immunoassays, the relative intensity (RI) of the reaction of the sera with each peptide was calculated and the ratio of the intensity values ​​of the same peptide with the pools of sera used was determined using Image J software (Figure 1). All peptides with RI > 2.0 were selected. Petition 870160012171, dated 01 / 04 / 2016, page 25 / 32 15 / 15

[042] Table 3: Peptides selected by relative intensity greater than or equal to 2. Protein____________________________Epitopes__________________________ Elongation factor 2 1 - VSPVVRVAVEAKNP -FVKTLQNVNVVV 3 - GAPLKISEPVVS 4 - LADNYEWDVQEAR metallo-peptidase, Clan MA(E), 5 - GIAPPNRTFQ an M3 family protein Enolase 6 - AERKMYNLTF - GDEGGFAPPIK family-like peptidase 8 - PQFDPEWAT m20 / m25 / m40, partial____________9 - AIASLQRHGVPHG___________

[043] Peptides exhibiting IR > 2.0 considering the reaction ratios of the LTA / negative sera pool and the LTA / LV sera pool were selected as potential for the selective diagnosis of Cutaneous Leishmaniasis. Nine epitopes (Table 3), derived from the proteins: elongation factor 2 (epitopes 1, 2, 3 and 4), metallopeptidase; the Clan MA(E) protein, an M3 family protein (epitope 5), Enolase (epitopes 5 and 6) and a partial m20 / m25 / m40 family-like peptidase (epitopes 8 and 9), showed potential for use as antigens in the serological diagnosis of cutaneous leishmaniasis in humans. Of these, peptides 1, 3, 5, 7, and 9 would be potential specific antigens for LTA, capable of distinguishing this clinical form from visceral leishmaniasis. As shown in Figure 2. Petition 870160012171, dated 01 / 04 / 2016, page 26 / 32

Claims

1 / 2 CLAIMS 1. A method for diagnosing cutaneous leishmaniasis characterized by comprising the following steps: a) Exposure of a biological sample to at least one of the recombinant metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48) and / or the hypothetical conserved protein gi| 134063939 (SEQ ID No. 49), and / or to the peptides defined by SEQ ID No. 1 to 46, together, individually or in various combinations, with each other and / or with other antigens; b) Addition of a secondary antibody, or a protein, conjugated to an enzyme or a marker and which binds to the antibodies possibly present in the sample from step (a); c) Detection of anti-Leishmania antibodies in the biological sample mentioned in step (a) using reagents capable of detecting the enzyme or marker mentioned in step (b).

2. The method according to claim 1, characterized in that, in step “a”, proteins and / or peptides are linked to a solid support or carrier.

3. The method according to claim 1, characterized in that, in step “b”, the antibody or protein is conjugated with the peroxidase.

4. The method according to claim 1, characterized in that, in step “c”, the reagent capable of detecting the enzyme or marker mentioned in step “b” is hydrogen peroxide or phenylenediamine dihydrochloride.

5. Kit for the diagnosis of cutaneous leishmaniasis, characterized by comprising: a) a solid support containing at least one of the recombinant metallopeptidase proteins of Clan MA(E) of the M3 family (SEQ ID No. 47 and / or 48) and / or the hypothetical conserved protein gi|134063939 (SEQ ID No. 49), and / or the peptides defined by SEQ ID No. 1 to 46, together, individually or in various combinations, with each other and / or with other antigens; b) a solution of secondary antibody conjugated with an enzyme or marker, a reagent capable of detecting the enzyme or marker, and positive and negative control samples.

6. Diagnostic kit, according to claim 5, item “a”, characterized by the solid support being a microtiter plate for ELISA or chromatographic strips.

7. Diagnostic kit, according to claim 5, characterized in that the antibody is conjugated with peroxidase.

8. Diagnostic kit, according to claim 5, characterized in that the reagent capable of detecting the enzyme or marker is hydrogen peroxide or o-phenylenediamine dihydrochloride (OPD).

9. Diagnostic kit, according to any one of claims 5 to 8, characterized by belonging to the group comprising ELISA, Western blot, dot blot and immunochromatography.

10. Use of the peptides defined by SEQ ID Nos. 1 to 46, characterized by being in the diagnosis and / or in kits for the diagnosis of cutaneous leishmaniasis. Petition 870260069871, dated 07 / 14 / 2026, p. 8 / 9

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