Conformational peptide, method and kit for diagnosis of visceral leishmaniasis.

Abstract

The present technology deals with a diagnostic method and kit using a novel conformational epitope, defined by SEQ ID No. 1 and highly specific for B cells, obtained from a hypothetical protein of Leishmania infantum and used in the immunological diagnosis of canine and human visceral leishmaniasis.

Description

1 / 20 “CONFORMATIONAL PEPTIDE, METHOD AND KIT FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS”

[01] The present technology relates to a conformational peptide, the method and the diagnostic kit using this new conformational epitope defined by SEQ ID No. 1 which is highly specific for B cells, having been obtained from a hypothetical protein of Leishmania infantum and used in the immunological diagnosis of canine and human visceral leishmaniasis.

[02] Leishmaniasis is a complex of diseases distributed worldwide, being found in at least 98 tropical and subtropical countries. Depending on the form of involvement in humans, the disease can manifest in two groups: cutaneous leishmaniasis (CL) and visceral leishmaniasis (VL) (Altamirano-Enciso AJ et al. On the origin and dispersion of cutaneous and mucosal leishmaniasis based on pre- and post-Columbian historical sources. Hist. Ciências da Saúde Manguinhos [online]. 2003;10(3):853-82.).

[03] VL is considered the most serious infection among leishmaniases, as it can be fatal in its acute form if left untreated. The disease affects internal organs of the body, such as the spleen, liver, lymph nodes, and bone marrow. It is systemic and characterized by prolonged fever, weight loss, asthenia, adynamia, and anemia, among other manifestations. VL is considered by the World Health Organization to be one of the six most relevant tropical diseases worldwide, affecting people of all ages and ranking second only to malaria among protozoan infections affecting humans, thus being a cause for concern in several countries (Silva DA et al. Laboratory tests). Petition 870160075869, dated 12 / 15 / 2016, pp. 33 / 62 2 / 20 performed on Leishmania seroreactive dogs euthanized by the leishmaniasis control program. Veter. Parasite. 2011;(179):257-61).

[04] The infection is caused by the species L. infantum. In dogs, considered the main reservoirs of the transmitting parasite in the domestic cycle, VL can present clinical variations, and it has been proposed that infected animals can be classified as: asymptomatic, oligosymptomatic and symptomatic (Chávez-Fumagalli MA et al. Specific Serodiagnosis of Canine Visceral Leishmaniasis using Leishmania Species Ribosomal Protein Extracts. Clin. Vaccine Immunol. 2009;(16):74-80). It is worth noting that approximately 20 to 60% of infected animals may exhibit slow disease progression, which can take up to four years to manifest, but even so, during this period, the dogs remain infectious to humans, being able to transmit the parasites to them (Baneth G, koutinas, AF, Solano-Gallego L, Ourdeau P, Ferrer L. Canine leishmaniasis - new concepts and insights on an expanding zoonosis: part one. Trends Parasitol. 2008;(24):24-30).For this reason, zoonosis control of canine health becomes necessary, since the animal may be transmitting the disease even without showing clinical symptoms. It is also worth highlighting that the number of registered cases of the disease in dogs is significantly higher when compared to the number of cases reported in humans, in the various regions of the country, even though the incidence of canine visceral leishmaniasis (CVL) is related to cases of the human disease (Nicolato RC et al. Clinical forms of canine visceral leishmaniasis in naturally Leishmania infantum-infected dogs and related myelogram and hemogram changes. PLoS One. 2013: 8(12) e82947). Petition 870160075869, dated 12 / 15 / 2016, pp. 34 / 62 3 / 20

[05] Serological tests used in the diagnosis of CVL aim to detect specific antibodies to parasite antigens present in animal sera (Coelho EA et al. Specific serodiagnosis of canine visceral leishmaniasis using Leishmania species ribosomal protein extracts. Clin Vaccine Immunol. 2009, 16(12):1774-80). However, these techniques have variable sensitivity and / or specificity and, in the early stages of the disease, infected animals may be seronegative and others, even clinically cured, may remain seropositive for long periods of time (Ferrer L et al. Serological diagnosis and treatment of canine leishmaniasis. Vet. Rec. 1995; 136(20):514-6).It is also worth noting that the specificity of serological tests is compromised, mainly when they are applied in endemic areas of the disease; in healthy but vaccinated animals; and in dogs infected with other parasitoses, also common in our environment, such as Chagas disease, babesiosis, and ehrlichiosis (Costa LE et al. Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic Leishmania infantum antigens with potential serodiagnosis applications. Clin Vaccine Immunol. 2013;(21):96-106), which may be seroreactive against the antigens used in the available diagnostic kits. Therefore, a diagnostic test that can be considered a gold standard, allowing the distinction between symptomatic and asymptomatic cases of canine visceral leishmaniasis (CVL) from other animal classes, and that is also available at a low cost to the population, has not yet been found. Petition 870160075869, dated 12 / 15 / 2016, pp. 35 / 62 4 / 20

[06] Hypothetical proteins are defined as those previously revealed in the genome of organisms, but which do not yet have a clear biological application (Fernandes AP et al. Making an anti-amastigote vaccine for visceral leishmaniasis: rational, update and perspectives. Curr OpinMicrobiol. 2012;15(4):476-85). Recently, through an immunoproteomic study developed using a combination of two-dimensional electrophoresis, immunoblotting, and mass spectrometry techniques, using stationary-phase promastigote and amastigote-like forms of Leishmania infantum, several antigenic proteins of this species were identified by antibodies present in serum samples from dogs with symptomatic and / or asymptomatic VL (Coelho VTS et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach. PLOS Neglected Tropical Diseases. 2012;(6):1430).After bioinformatics studies, it was found that some of these proteins are also found in other Leishmania species, but not in other trypanosomatids, such as Trypanosoma cruzi, so their applicability in the serological diagnosis of visceral leishmaniasis (VL) could be evaluated. Among these proteins, some considered hypothetical have come to be considered as possible targets for potential biological use. These proteins proved to be specific to Leishmania and were not found in other parasites, and therefore could be considered as candidate targets for the diagnosis of the disease.

[07] Epitopes are defined as molecules formed by groups of amino acids derived from a protein and that interact with Petition 870160075869, dated 12 / 15 / 2016, pp. 36 / 62 5 / 20 B or T cell receptors, resulting in the activation of a cellular and humoral immune response (De Groot As. immunomics: discovering new targets for vaccines and therapeutics. drug discov. Today. 2006; 11(56): 203-209). From the point of view of pattern recognition in the immune system, the most important characteristic of B and T cells is that both possess receptor molecules on their surfaces capable of recognizing antigens. These receptors recognize these molecules with distinct characteristics: the B cell receptor can interact with free antigenic molecules in solution, while the T cell receptor recognizes processed antigens associated with cell surface molecules, called major histocompatibility complex (MHC) class I and II.Thus, for diagnostic purposes, specific epitopes for B cells are always preferred (Mendel L, Kerlero de Rosbo N, Ben-Nun A, Delineation of the minimal encephalitogenic epitope within the immunodominant region of myelin oligodendrocyte glycoprotein: diverse V beta gene usage by T cells recognizing the core encephalitogenic epitope for T cell receptor V beta b and T cell receptor V beta a H-2b mice. Eur. J. Immunol. 1996;(10):2470-9).

[08] A protein epitope can be classified as continuous or discontinuous. A continuous epitope, also called linear, is a sequential fragment of a protein. A discontinuous epitope, also called conformational, is composed of several fragments dispersed along the protein sequence that are brought together in spatial proximity when the protein is folded (Machado-de-Avila RA ET AL. Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhage Petition 870160075869, dated 12 / 15 / 2016, pp. 37 / 62 6 / 20 inhibitory antibodies upon vaccination of rabbits. Peptides. 2011;(8):1640-6).

[09] Most epitopes recognized by B cells are conformational, often composed of small continuous sequence elements that are spatially associated. Strategies employed in epitope identification are of great importance for the molecular understanding and recognition of the protein analyzed. These epitopes can be useful in disease diagnosis, drug formulation, vaccine development, among other biological applications (Machado-de-Ávila RA et al. Induction of Neutralizing Antibodies against Mutalysin-II from Lachesis muta Snake Venom Elicited by a Conformational B-cell Epitope Predicted by Blue Star Sting Data Base. Immunome Res. 2015;(10):83).

[010] Patent document PI10050337, filed on December 13, 2010, entitled “Recombinant peptides, method and kit for immunodiagnostic testing of visceral leishmaniasis”, deals with a method and kit for diagnosing VL through the association of recombinant antigens and their epitopes. The invention differs from the present application by dealing with a larger number of peptide sequences, all of which differ from that contained in the present technology.

[011] Patent document BR1020140281720, filed on November 11, 2014, entitled “Kit and method for immunodiagnosis of leishmaniasis and use of a Leishmania protein and a derived peptide” deals with a kit and method for immunodiagnosis of leishmaniasis comprising the recombinant protein CathepsinL-Like protease and a peptide, alone or in combination. A Petition 870160075869, dated 12 / 15 / 2016, pp. 38 / 62 7 / 20 The invention differs from the present application in that it deals with different peptide sequences and the method for producing them is different.

[012] Patent document WO2014111207, filed on January 18, 2013, entitled “Diagnosis of Leishmania infection,” deals with a polypeptide isolated from Leishmania donovani used for the diagnosis of visceral leishmaniasis. The technology differs from the present application in that the peptide sequences are not identical, with the sequence contained in the present technology having a smaller size, allowing for greater production practicality and lower cost.

[013] The present technology deals with a new sensitive and specific diagnostic antigen for the diagnosis of VL that can be made available for composition of a laboratory kit. The synthesis of the conformational peptide is cheaper than the production of a recombinant protein, the final product has a higher yield in terms of concentration, and peptides are more stable when compared to recombinant proteins.More specifically, the technology uses a novel approach through different bioinformatics computational programs to predict a conformational peptide of a hypothetical L. infantum protein and validate it as an antigen to be used in a sensitive and specific diagnostic kit for symptomatic and asymptomatic canine visceral leishmaniasis (CVL).

[014] In the state of the art, no similar technology was found using the antigenic and specific peptide sequence for B lymphocytes contained in the present application; which was obtained through bioinformatics analyses from a hypothetical L. protein. Petition 870160075869, dated 12 / 15 / 2016, pp. 39 / 62 8 / 20 infantum. The peptide in this patent application has a novel sequence and is smaller in size compared to other peptides in the state of the art, which facilitates its faster production, on a larger scale, and at a lower cost. BRIEF DESCRIPTION OF THE FIGURES

[015] Figure 1 represents the result of the analyses by the IEDB program using the Bepipred Linear Epitope Prediction algorithm, with the selected sequences marked in yellow.

[016] Figure 2 represents the analyses by the Kolaskar and Tongaorkar Antigenicity algorithm, with the selected amino acids in yellow.

[017] Figure 3 represents the analysis by the Emili Surface Accessibility prediction algorithm, with the selected amino acids in yellow.

[018] Figure 4 represents the analysis by the Hydrophilicity algorithm, with the amino acids considered satisfactory marked in yellow.

[019] Figure 5 represents the analysis by the ABCpred program.

[020] Figure 6 shows the primary sequence of the protein under study and the results of the analysis by ABCpred.

[021] Figure 7 presents a comparison and overlay of the results obtained considering the parameters of (A) accessibility (IEDB), (B) antigenicity (IEDB) and (C) ABCpred.

[022] Figure 8 represents the sequence resulting from the overlapping analysis of the results generated by the bioinformatics programs.

[023] Figure 9 represents the selection of the potential B cell epitope sequence generated by the Epitopia program. Petition 870160075869, dated 12 / 15 / 2016, pp. 40 / 62 9 / 20

[024] Figure 10 represents the optical density (OD) by means of box-plots and absorbance of canine serum samples in relation to the synthesized peptide. In (A), the tests with the predicted peptide against the following groups of canine serums are shown: SPA, sera from positive and asymptomatic dogs; SPS, sera from positive and symptomatic dogs; SNAE, sera from negative dogs from endemic areas; SNANE, sera from negative dogs from non-endemic areas; SVLT, sera from dogs vaccinated with Leish-Tec®; STC, T. cruzi serum; SEC, sera from dogs infected with Ehrlichia canis; SBC, sera from dogs infected with Babesia canis. In (B), the tests with the predicted peptide against the following groups of canine serums are shown: SPA, sera from positive and asymptomatic dogs; SPS, sera from positive and symptomatic dogs; SNAE, sera from dogs that are negative and from endemic areas; SNANE, sera from dogs that are negative and from non-endemic areas; SVLT, sera from dogs vaccinated with Leish-Tec®; STC, T. serum.cruzi; SEC, sera from dogs infected with Ehrlichia canis; SBC, sera from dogs infected with Babesia canis.

[025] Figure 11 represents the optical density (OD) by means of box-plots and absorbance of canine serum samples in relation to SLA. In (A), the tests with SLA against the following groups of canine serums are shown: SPA, sera from positive and asymptomatic dogs; SPS, sera from positive and symptomatic dogs; SNAE, sera from negative dogs from endemic areas; SNANE, sera from negative dogs from non-endemic areas; SVLT, sera from dogs vaccinated with Leish-Tec®; STC, T. cruzi serum; SEC, sera from dogs infected with Ehrlichia canis; SBC, sera from dogs infected with Babesia canis. In (B), the tests with SLA against the following groups are shown: Petition 870160075869, dated 12 / 15 / 2016, pp. 41 / 62 10 / 20 canine sera: SPA, sera from positive and asymptomatic dogs; SPS, sera from positive and symptomatic dogs; SNAE, sera from negative dogs from endemic areas; SNANE, sera from negative dogs from non-endemic areas; SVLT, sera from dogs vaccinated with Leish-Tec®; STC, T. cruzi serum; SEC, sera from dogs infected with Ehrlichia canis; SBC, sera from dogs infected with Babesia canis. DETAILED DESCRIPTION OF THE TECHNOLOGY

[026] The present invention relates to a conformational peptide (SEQ ID No. 1), method and kit for sensitive and specific immunological diagnosis of human and canine visceral leishmaniasis comprising the use of an antigenic conformational peptide sequence from a Leishmania infantum peptide.

[027] More specifically, the peptide used in the proposed method and kit is defined by SEQ ID No. 1.

[028] The proposed method for diagnosing visceral leishmaniasis comprises the following steps: a) exposure of a sample to the peptide defined by the sequence SEQ ID No. 1, or its corresponding epitopes, with such antigens bound to a solid support or carrier; b) addition of an antibody or a protein, which are conjugated to an enzyme or a marker and which bind to the antibodies in the sample from step (a); c) detection of specific antibodies for visceral leishmaniasis in the sample mentioned in step (a), using reagents capable of detecting the enzyme or marker mentioned in step (b). Petition 870160075869, dated 12 / 15 / 2016, pp. 42 / 62 11 / 20

[029] In step “a”, the sample may be selected from the group comprising blood, serum, plasma or other body fluid, without the need for large quantities thereof. The solid support may comprise materials consisting of nitrocellulose, nylon, latex, polypropylene, polystyrene or other used for the same purpose as those described above.

[030] In step “b”, the secondary antibody may comprise IgG, IgM, IgA, IgE and their respective subclasses. The protein related to this step may be Protein A and / or Protein G. The enzyme described in this step may be selected from the group consisting of peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Furthermore, the marker may be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

[031] In step “c”, the detection antibody can be selected from the group comprising fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection.

[032] The kit for the diagnosis of visceral leishmaniasis proposed in the present invention comprises the peptide defined in SEQ ID No. 1.

[033] This kit is characterized by comprising: a) the peptide defined by SEQ ID No. 1; b) a secondary antibody or a protein, wherein the secondary antibody (IgG, IgM, IgA, IgE and / or their respective subclasses), or the protein (protein A and / or protein G), is conjugated to an enzyme or a marker; c) a reagent to detect the enzyme or marker. Petition 870160075869, dated 12 / 15 / 2016, pp. 43 / 62 12 / 20

[034] The peptide must be attached to a solid support, selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene; or to a carrier, preferably gold particles.

[035] In step “b”, the marker can be selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents.

[036] In step “b”, the enzyme can be selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.

[037] The peptide contained in the present invention may be used for the diagnosis not only of human VL, but also of canine VL, due to the similarity of its recognition by the immune system of these mammalian hosts.

[038] The present invention can be better understood through the following examples, which are not limiting in the technology. EXAMPLE 1. IDENTIFICATION OF THE CONFORMATIONAL EPITOPE

[039] The hypothetical protein (XP_001468941.1) of L. infantum used for predicting conformational epitopes was previously identified by Coelho et al. (2012) (Coelho VTS et al. Identification of proteins in promastigote and amastigote-like Leishmania using an immunoproteomic approach. PLOS Neglected Tropical Diseases. 2012;(6):1430) in protein extracts of the amastigote forms of the parasites and in sera from dogs with asymptomatic VL. Thus, this antigen was positioned as a candidate for the diagnosis of the disease. Initially, the IEDB program (www.iedb.org) was used, with the following algorithms being analyzed: Petition 870160075869, dated 12 / 15 / 2016, pp. 44 / 62 13 / 20 • Bepipred Linear Epitope Prediction: performs an ROC curve analysis with 80% specificity through combinations of scales as cited by Larsen et al (2006) (Larsen JEP, Lund O, Nillsen M. Improved method for predicting linear B-cell epitopes immunone Res; 2006. 2:2) • Kolaskar and Tongaonkar Antigenicity: performs physicochemical analyses to identify amino acids with antigenic potential, as described by Kolaskar and Tongaonkar (1990) (Kolaskar AS and Tongaonkar PC. A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBSLett. 1990;(1-2):76-172). • Emili Surface Accessibility Prediction: identifies amino acid sequences capable of inducing a humoral response (Emili EA et al. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J virol.1985;55(3):836-9). • Parker Hydrophilicity Prediction: identifies hydrophilic and hydrophobic residues of protein sequences, as described by Parker et al. (1986) (Parker JM, Guo D, Hodges RS. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X ray-derived accessible sites. Biochem. 1986;25(19):25-32) .

[040] The second program used was ABCpred (http: / / www.imtech.res.in / raghava / abcpred / ), which aims to predict B cell epitopes in a given protein sequence using an artificial neural network, which consists of fixing length patterns to test possible B cell epitopes, ranging from 5 to 30 amino acid residues. This server has 65.93% of Petition 870160075869, dated 12 / 15 / 2016, pages 45 / 62 14 / 20 accuracy in predicting B-cell epitopes. The results found were compared and combined and then confirmed from an analysis of the results obtained by the Epitopia program (bmcbioinformatics.biomedcentral.com), which detects antigenic regions in protein sequences (Rubinstein ND et al. Epitopia: a web-server for predicting B-cell epitopes. BMC Bioinformatics. 2009 (10): 287).

[041] From the analyses performed by the IEDB program using the Bepipred Linear Epitope Prediction algorithm, the following parameters were used: window size of 7 and threshold of 0.70. Only amino acids that presented values ​​above what was stipulated in these parameters were selected. We can observe in Figure 1 the result of the analyses, with the selected sequences marked in yellow.

[042] The next algorithm evaluated was Kolaskar and Tongaorkar Antigenicity, coloring in yellow the amino acids that obtained results above the parameters: window size of 7 and threshold of 1.0, as shown in figure 2.

[043] Subsequently, the Emili Surface Accessibility prediction algorithm was analyzed, following the same methodology employed in the previous analyses; thus, the amino acids that presented satisfactory results when using the parameters: window size of 7 and threshold of 1.0 were marked. The selected amino acids can be observed in figure 3.

[044] The last algorithm evaluated using the IEDB program was Hydrophilicity, and the amino acids considered satisfactory were also marked in the sequence below, according to the parameters evaluated: window size of 7 and threshold of 1.0. Petition 870160075869, dated 12 / 15 / 2016, pages 46 / 62 15 / 20

[045] The results shown in Figure 4 were compared with those obtained by another website program, ABCpred. This program performs the analysis of the complete protein sequence; in this case, the program separated sequences that presented a score greater than 0.85, as shown in Figure 5.

[046] Figure 6 shows the primary sequence of the protein under study and the results of the ABCpred analysis.

[047] After using the programs, a comparison and overlay of the results obtained was performed using the following parameters: Accessibility x Antigenicity x ABCpred, with (A) accessibility (IEDB), (B) antigenicity (IEDB) and (C) ABCpred indicated in figure 7.

[048] In this way, the sequential composition shown in Figure 8 was obtained. The amino acids colored pink refer to the comparison between Accessibility and Antigenicity, and those colored red indicate the amino acids found in the 3 analyses: Accessibility, Antigenicity, and ABCpred. After these procedures, we can define the isolated sequences that will be used as epitopes. Therefore, to confirm which sequence would be most suitable for synthesis, the results were submitted to analysis by the Epitopia program, which allowed the selection of the potential B cell epitope sequence. Thus, the Epitopia program presented the sequence defined in Figure 9. The result shown displays the amino acids that have a color scale according to the immunogenicity score, and also the exposed residues of the amino acid identified with the letter “e”. Petition 870160075869, dated 12 / 15 / 2016, pp. 47 / 62 16 / 20 (orange color) and the unexposed residue identified with the letter “b” (green color).

[049] In this analysis, only amino acids with scores of 4 and 5 (lilac and purple coloration, respectively), which are exposed in the protein sequence, were taken into consideration. A final comparison was performed, using the results of the Accessibility, Antigenicity and ABCpred comparisons, and the results obtained by the Epitope analysis. After all analyses, the common amino acids were combined to obtain a unique conformational peptide that can be considered as a potential antigen, which is defined by the sequence: RLEQDNVVPVKTP.

[050] In order to verify if this peptide is water-soluble, its sequence was analyzed for hydrophilicity using the Peptide Property Calculate program. The results showed that the molecule is only soluble when diluted with organic solvents, thus making it unsuitable for use in serological diagnostic assays via ELISA. Therefore, in order to make this peptide water-soluble, neighboring amino acids were sought that, when added to the peptide, would transform it into a soluble form. Thus, the amino acid arginine was selected, causing the peptide to assume a basic character, conferring water solubility and generating the sequence RLEQDNVVRPVKTP. EXAMPLE 2. CHARACTERIZATION AND SYNTHESIS OF THE CONFORMATIONAL PEPTIDE Petition 870160075869, dated 12 / 15 / 2016, pages 48 / 62 17 / 20

[051] With the prediction defined, the peptide was manually synthesized using the F-moc synthesis technique. The physicochemical characteristics of the synthesized peptide were determined by the Expasy Server program, which showed molar mass values ​​of 1650.80 Da and an isoelectric point (pI) of 9.82.

[052] From the primary structure of the selected hypothetical protein, a bioinformatics analysis was performed to determine the physicochemical properties (hydrophilicity and solvent accessibility) using the Peptide Property Calculate program and the molar mass and isoelectric point values ​​of the peptide were obtained using the Expasy Server program (Gasteiger E et al. ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res. 2005;(31):3784-88).

[053] For the synthesis of the predicted peptide, the F-moc chemical technique, as described by (Merrifield, RB Automated synthesis of peptides, Science.1969; 150(3693):178-185) and adapted by (Machado-de-Avila RA et al. Mimotopes of mutalysin-II from Lachesis muta snake venom induce hemorrhagic inhibitory antibodies on rabbit vaccination, was used. The method consists of fixing the C-terminal amino acid of the peptide onto an insoluble solid support and then elongating the peptide chain by successive additions of residues from the C-terminal to the N-terminal portion. For this, the F-moc group is deprotected, washed, Kaiser test performed, and the amino acid is linked in a ring. Thus, the synthesis begins with soaking the resin with 25% piperidine solvent in a syringe containing a filter, initially for 5 minutes, with a 10-minute repetition to allow for the unprotected area to be removed. Petition 870160075869, dated 12 / 15 / 2016, pp. 49 / 62 18 / 20 F-moc group (fluorenyl methyl oxycarbonyl). After these 15 minutes, 3 washes are performed with DMF (dimethylformamide), one with DCM (dichloromethane), and the Kaiser test is performed by placing resin grains and 2 drops of 10% bromophenol blue solution in a test tube.

[054] In this first step, if the resin turns blue, it means that deprotection has occurred, and the procedure can continue. If deprotection does not occur, all steps must be repeated up to the 25% piperidine soaking. If this occurs correctly, it is time to prepare the amino acid by adding 500 pL of oxyma and DIPC in quantities determined by the molar mass of the peptide to be synthesized. The solution should be homogenized and reacted for 30 minutes for binding to occur. After 30 minutes, the solution should be drained, and washes should be performed with DMF (dimethylformamide) and MET (methanol), alternating twice each. Finally, a wash with DCM (dichloromethane) should be performed. These solvents have different polarities, removing any amino acid residues that did not bind. After the washes, the Kaiser test is performed again, but at this stage, the resin is expected to show a yellow color, confirming the binding of the amino acid to the resin.These procedures are performed cyclically until the last amino acid is reached.

[055] Upon completion of the synthesis, the F-moc group must be deprotected again, and then cleavage must be performed to separate the peptide from the resin. For this, cleavage is performed with a solution containing 25 pL (25%) distilled water, 25 pL (25%) TES (triethylsilane), 950 pL (95%) TFA (trifluoroacetic acid), Petition 870160075869, dated 12 / 15 / 2016, pp. 50-62 19 / 20 totaling 1 mL in a 100% solution. Leave this solution in the syringe for 3 hours, drain the liquid into a tube and add ethyl ether, leaving it to incubate for 18 hours. After this procedure, centrifuge at 4°C for 10 minutes and at 4,500 xg, carefully remove the liquid to avoid losing the peptide, add ethyl ether again and repeat the centrifugation. Remove the ethyl ether again, perform the lyophilization process and analyze by spectrophotometer to ensure the efficiency of the synthesis. The synthetic peptide was then subjected to mass spectrometry to confirm its molecular mass and verify the purity of the final product. EXAMPLE 3. VALIDATION OF THE CONFORMATIONAL PEPTIDE FOR THE DIAGNOSIS OF LV.

[056] After the synthesis of the peptide, in order to identify a new serological method for diagnosing VL that is more sensitive and specific, an ELISA test was performed using soluble Leishmania antigen (SLA) and the conformational peptide as antigens. For this purpose, the reactivity of antibodies from symptomatic and asymptomatic canine sera was evaluated, as well as sera from dogs from endemic and non-endemic regions, and from those diagnosed with babesiosis, ehrlichiosis or Chagas disease, in addition to those vaccinated with the commercial vaccine Leish-Tec®. In the analysis of the results, when ROC curves were constructed to evaluate the sensitivity and specificity of the diagnostic antigens, it can be observed that the conformational peptide presented a value around “0” when samples considered as “false-negatives” were evaluated, while the SLA presented a value close to “0.3”, demonstrating Petition 870160075869, dated 12 / 15 / 2016, pages 51 / 62 20 / 20 lower specificity in relation to the peptide. The synthesized peptide showed accuracy values ​​of 0.992, while the SLA showed a value of 0.850, with the test using the peptide also being considered more efficient. The OD results of the serological test for Leishmania are shown in Figures 10 and 11, with Figure 10 showing the reactivities against the synthesized peptide, and Figure 11, against the SLA of L. infantum.

[057] Figure 2 shows the average OD values ​​of the peptide for the positive sera: SPA 0.335 and SPS 0.496. The negative sera showed average OD values ​​of: SNAE 0.044; SNANE 0.033; SVLT 0.037; STC 0.043; SEC 0.036 and SBC 0.043; demonstrating distinct average values ​​between the groups of positive sera, with higher OD, and the groups of negative sera, with lower OD values.

[058] Figure 3 shows the mean OD values ​​of the synthesized peptide for the positive sera: SPA 0.745 and SPS 1.273. The negative sera presented mean OD values: SNAE 0.586; for SNANE 0.271; for SVLT 0.514; STC, 0.979; SEC 0.886 and for SBC 0.708. We observed that for SPS the mean was high when compared to the mean OD values ​​of the other groups. However, the STC, SEC, and SBC negative dog groups presented mean OD values ​​higher than or similar to the SPA positive dog group. Petition 870160075869, dated 12 / 15 / 2016, pp. 52 / 62

Claims

1 / 3 CLAIMS 1- CONFORMATIONAL PEPTIDE, characterized by being defined by SEQ ID No.

1. 2- METHOD FOR DIAGNOSING VISCERAL LEISHMANIASIS characterized by comprising the following steps: a) exposure of a sample to the peptide defined by the sequence SEQ ID No. 1, such peptide being bound to a solid support or a carrier; b) addition of an antibody or a protein, which are conjugated to an enzyme or a marker and which bind to the antibodies in the sample from step (a); c) detection of specific antibodies for visceral leishmaniasis in the sample mentioned in step (a), using reagents capable of detecting the enzyme or marker mentioned in step (b). 3- METHOD FOR DIAGNOSING VISCERAL LEISHMANIASIS, according to claim 2, characterized in that, in step “a”, the sample is selected from the group comprising blood, serum, plasma or other body fluid, without the need for large quantities thereof, and the solid support comprises materials consisting of nitrocellulose, nylon, latex, polypropylene, polystyrene. 4- METHOD FOR DIAGNOSING VISCERAL LEISHMANIASIS, according to claim 2, characterized in that, in step “b”, the secondary antibody comprises IgG, IgM, IgA, IgE and their respective subclasses; the protein is Protein A and / or Protein G; the enzyme is selected from the group consisting of peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase; the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. Petition 870260029081, dated 03 / 27 / 2026, page 7 / 10 2 / 3 5- METHOD FOR DIAGNOSING VISCERAL LEISHMANIASIS, according to claim 2, characterized in that, in step “c”, the detection antibody is selected from the group comprising fluorescence, immunofluorescence, immunoluminescence, absorbance or radioisotope detection. 6- KIT FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS, characterized by comprising: a) the peptide defined by SEQ ID No. 1; b) a secondary antibody or a protein, wherein the secondary antibody, selected from IgG, IgM, IgA, IgE and / or their respective subclasses, or the protein, selected from Protein A and / or Protein G, are conjugated to an enzyme or a marker; c) a reagent for detecting the enzyme or marker. 7- A kit for diagnosing visceral leishmaniasis, according to claim 6, characterized in that the peptide is linked to a solid support, selected from the group of materials comprising nitrocellulose, nylon, latex, polypropylene and / or polystyrene; or to a carrier, preferably gold particles. 8- A kit for diagnosing visceral leishmaniasis, according to claim 6, characterized in that, in item “b”, the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent agents. 9- KIT FOR DIAGNOSIS OF VISCERAL LEISHMANIASIS, according to claim 6, characterized in that, in item “b”, the enzyme is selected from the group comprising peroxidase, alkaline phosphatase, beta-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. Petition 870260029081, dated 03 / 27 / 2026, page 8 / 10 3 / 3 10- USE OF THE CONFORMATIONAL PEPTIDE DEFINED BY SEQ ID No. 1, characterized by its use in the diagnosis of visceral leishmaniasis in humans and / or dogs. Petition 870260029081, dated 03 / 27 / 2026, page 9 / 10

Images