BR102014013195A2 - method and kit for diagnosis of leishmaniasis using synthetic peptides derived from the mitogen-activated protein kinase coding gene - Google Patents

Abstract

Method and Kit for Diagnosing Leishmaniasis Using Synthetic Peptides Derived from the Mitogen Activated Protein Kinase Encoding Gene The present invention describes peptides, a method and kit for immunodiagnosis of leishmaniasis, a disease caused by protozoa of the genus Leishmania, whether infection and / or disease in humans and / or dogs. The invention concerns the synthesis and use of peptides derived from mitogen-activated protein kinase to be used in the identification of individuals and animals infected with visceral and integumentary leishmaniasis, aiming at the development of a more sensitive and specific diagnostic method and kit. allowing the improvement of the reactivity of serological tests.

Description

METHOD AND KIT FOR DIAGNOSTIC DIAGNOSIS OF LEISHMANIASIS USING SYNTHETIC PEPTIDES FROM THE MYTHOGEN ACTIVATED PROTEIN KINASE CODES [001] The present invention describes peptides, a method and Kit for immunodiagnosis of the leishmaniasis infection caused by Leishmaniasis and the genesis / or the disease in humans and / or dogs. The invention concerns the synthesis and use of peptides derived from mitogen-activated protein kinase to be used in the identification of individuals and animals infected with visceral and integumentary leishmaniasis, aiming at the development of a more sensitive and specific diagnostic method and kit. allowing for improved reactivity of serum tests. Leishmaniasis encompasses a complex group of parasitic diseases caused by parasites of the genus Leishmania with estimates that about 12 million people are infected, with an annual incidence of 1-1.5 million cases of cutaneous leishmaniasis and 500,000 cases. of visceral leishmaniasis (Desjeux P et al. Comp. Immunol Microbiol Infect Dis. 2004 Sep; 27 (5): 305-18). The family of mitogen-activated protein kinases are a highly conserved serine / protein kinases group among eukaryote species and important regulators of various cellular processes such as transcriptional control, apoptosis, stress response, development and differentiation during the cell cycle, and also as possible targets for protozoan drugs (Grupta CL et al. Interdiscip Sci. 2013; 5 (2): 136-144). Mitogen-activated protein kinases, when activated, can phosphorylate a wide range of targets, including other protein kinases and gene transcription factors regulated by these proteins, playing an important role in signaling pathways and in regulating the mRNA stability of various genes, in addition to being associated with the pathological process of various diseases (Davis RJ. J Biol Chem. 1993 Jul; 268 (20): 14553-6). [004] In the search for new diagnostic targets, analyzes of antigenic epitopes of pathogenic microorganism proteins have increased the number of target epitopes and provided evidence to improve peptide antigenicity. Among proteomic selection methods, epitope mapping is a very useful procedure that has wide applications in the field of antibody production, immunodiagnosis, epitope-based vaccine design, selective protein de-immunization and associated autoimmune diseases. Identification of these targets has been facilitated by the publication of the parasite genome in conjunction with the availability of epitope prediction algorithms, which allows for the large-scale use of novel antigen identification approaches (Bryson CJ et al. BioDrugs. 2010; 24 (1): 1-8). In the prior art patent documents have been found suggesting the use of peptides in the diagnosis of leishmaniasis, as examples, document P11020120335522 and P11320120335599 describing the use of Leishmania infantum proteome derived polypeptides for the diagnosis of infection. However, all the documents found in the state of the art, among which are the ones mentioned above, present differentiated proteins and peptides from those presented in this invention and, still, do not allow such an efficient diagnosis, by which one distinguishes them from other ones. infections caused by parasites present in endemic areas for human cutaneous and visceral leishmaniasis and canine visceral ieishmaniasis, such as the protozoan species Trypanosoma cruzi, an etiological agent of Chagas disease, as in the present technology. Furthermore, methods that diagnose the human and canine integumentary and visceral forms using the same antigen mapped on the Leishmania parasite proteome are not described in the literature. Thus, despite advances in recent years, the available diagnostic methods for leishmaniasis still have several limitations. Moreover, in the absence of effective diagnostic methods, the effectiveness of treatment and any initiative to block transmission or vaccination is compromised (Ministry of Health. Infectious and Parasitic Diseases: pocket guide. Brasília. 2006). Despite extensive efforts to develop immunobiologicals for more sensitive and specific diagnostic tests against leishmaniasis, improvements in the effectiveness of these methods in diagnosing the disease in all hosts involved in the parasite life cycle are still needed. Thus, the present invention provides an alternative to solve this problem. These are synthetic peptides designed from B lymphocyte epitopes predicted by immunoinformatics in the coding gene for L. braziliensis mitogen-activated protein kinase and their use in a method and kit to diagnose leishmaniasis in dogs and / or humans.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows immunoinformatic analysis of the gene encoding Mitogen-activated protein kinase and peptide-1 (DPAEEADAP), located on chromosome 19, which shows a prediction score for B lymphocyte epitope. 1.88. [008] Figure 2 presents comparative graphs relating to humoral reactivity against peptide-1 and against crude soluble Leishmania braziliensis antigen (AgSLb) in serum from individuals with Human Leishmaniasis Tegumentari (LTH). The X axis represents the evaluated groups: control individuals (TC, n = 50), individuals with Chagas Disease (CD, n = 20) individuals with Human Tegumentary Leishmaniasis (LTH) cutaneous clinical form (LTH-C, n = 45) and mucocutaneous (LTH-MC, n = 20). The Y axis represents the mean absorbance values (450 nm) by ELISA using diluted 1: 100 sera. * Cut off was established using ROC curve. [009] Figure 3 presents comparative graphs for humoral reactivity against peptide-1 and against and soluble crude Leishmania braziliensis antigen (AgSLb) in serum from individuals with Human Visceral Leishmaniasis (LVH). The X axis represents the evaluated groups: control individuals (TC, n = 50), individuals with Chagas Disease (CD, n = 20) individuals with Human Visceral Leishmaniasis (LVH, n = 55). The Y axis represents the mean absorbance values (450 nm) by ELISA using diluted 1: 100 sera. * Cut off was established using ROC curve. [010] Figure 4 presents comparative graphs regarding humoral reactivity against peptide-1 and against and soluble crude Leishmania braziliensis antigen (AgSLb) in serum of dogs with canine Visceral Leishmaniasis (LVC). The X axis represents the evaluated groups: control dogs (TC, n = 30), individuals with Chagas Disease (DC, n = 15) individuals with Canine Visceral Leishmaniasis (LVC, n = 30). The Y axis represents the mean absorbance values (450 nm) by ELISA using diluted 1: 100 sera. * Cutoff was established using ROC curve.

DETAILED DESCRIPTION OF THE TECHNOLOGY In the present invention was identified peptide derived from the gene coding for the mitogen-activated protein kinase conserved between the different species of the genus Leishmania, and polymorphic in relation to the human and canine hosts and the parasite that causes the disease. Chagas, T. cruzi, derived from B cell epitopes, consisting of the sequence represented by SEQ ID NO: 1, whether or not modified at their ends, to help improve the serodiagnosis of canine visceral leishmaniasis and human visceral and tegumentary leishmaniasis. The method for immunodiagnosis of leishmaniasis may be selected from the group comprising ELISA, Western blot, dot blot, immunodiffusion, immunochromatography and the following steps: a) binding of antileishmanial antibodies from one sample to one or more polypeptide consisting of amino acid sequences SEQ ID No. 1 attached to a solid support or carrier, preferably gold particle; b) contacting the antibodies of step (a) with a secondary antibody or protein (protein A or G), conjugated to an enzyme or label and binding to the antibodies of step (a); wherein the enzymes may be selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase and the marker may be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent; c) detecting antileishmanial antibodies in the above sample by detecting the secondary antibody or protein specifically bound to said anti-leishmanial antibody. The method may be performed using samples of blood, serum, plasma or other body fluid from the infected animal or human. The technology further comprises a Leishmaniasis Immunodiagnostic Test Kit comprising Peptide-1 consisting of the sequence SEQ ID NO: 1 associated or isolated and a secondary antibody or protein (protein A or G), where the secondary antibody or the protein is conjugated to an enzyme (such as alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase) or marker (such as enzymes, redioisotopes, biotin, chromophores, fluorophores and chemiluminescent), and a reagent to detect said enzyme or marker. In the Kit the peptides will be attached to a solid support or carrier, which solid support may be nitrocellulose, nylon, latex, polypropylene and polystyrene and the carrier consist of gold particles. The ELISA technique was chosen in the present invention because it is less invasive, easy to perform and automate, allowing studies and application in a large number of samples. The present invention may be better understood, but not limited to, by the following examples.

Example 1 - In silico identification of linear B cell epitopes [016] Initially, linear B cell epitopes were predicted throughout the Leishmania braziliensis M2904 strain proteome using the BepiPred program (Larsen JE, Lund O and Nielsen M. Immunome Res 2006 Apr 24; 2: 2). In this analysis we used a conservative approach with a cutoff value of 1.3 that provides 96% specificity and 13% sensitivity, minimizing the selection of many false positive epitopes. Sequences with the sum of polymorphism scores above 6 and mean Bepipred prediction score above 1.3 were considered as polymorphic epitopes. 15 amino acid sequences with mean epitope prediction score between the two sequences and above 1.3 and zero polymorphism score were considered conserved epitopes. To minimize the chance of cross-reacting with other phylogenetically related parasites, peptide sequences with high protein similarity to other parasites were identified using the BLAST algorithm (Altschul SF et al. J Mol Biol. 1990 Oct 5; 215 (3): 403 -10) and discarded. The blastp version of the algorithm was used to compare polymorphic and conserved epitopes with the predicted proteome of L. braziliensis, L. infantum, H. sapiens, C. familiaris and Γ. I crossed. Epitopes with more than 70% identity over more than 70% sequence were eliminated from subsequent analyzes. [017] Evaluating the data obtained, a peptide present in the mitogen-activated protein kinase was identified, evaluating the predicted Leishmania braziliensis proteome, with potential use for leishmaniasis immunodiagnosis. Figure 1 shows the immunoinformatic analysis of peptide-1 (DPAEEADAP), located on mitogen-activated protein kinase protein on chromosome 19, which has a prediction score for B lymphocyte epitope of 1.88. The peptide was synthesized in solid phase for later performance analysis as an antigen for leishmaniasis immunodiagnosis.

Example 2: Synthesis of Soluble Peptide The selected peptide was synthesized in solid phase by the N-9-fluorenimethoxycarbonyl method (Wellings DA and Atherton E. Methods Enzymoi. 1997; 289: 44-67) in the 20-30 scale. umol. Fmoc-amino acids were activated for 30-60 minutes with 1 equivalent of HObt and 2 equivalents of DIC. After this time, the activated amino acids were placed in contact with the 0.61 substitution grade amidated Rink resin for 45-60 minutes. The Fmoc groups were then removed with 25% 4-methylpiperidine solution. Activation, incorporation into the resin and deprotection of the Fmoc group were then repeated until complete synthesis of the peptide chain. After synthesis, the peptides were cleaved from the resin along with side chain deprotection with a mixture containing 9.4% TFA, 2.4% water and 0.1% triisopropylsilane. The peptide was precipitated from the crude material with diisopropyl ether for 16 hours at 4 ° C, followed by centrifugation at 3000 G for 5-10 minutes. The synthesized peptides Peptide-1 (SEQ ID NO: 1 - DPAEEADAP) were purified by reverse phase chromatography using the Sephasil Peptide C18 (Shimadzu) column coupled to the HPLC system (Shimadzu) using 0 to 25% acetonitrile for 75-90 minutes with 0-10 minutes to 10% acetonitrile. After purification, the peptide was subjected to mass spectrometry in Autoflex Speed MALDI / TOF (Bruker) equipment to confirm its molecular mass.

Example 3 - Validation of Peptide-1 against serum from individuals with LTH, LVH, and dogs with LVC [019] ELISA assays were performed to validate and characterize peptide-1 reactivity to Leishmania parasites. Sera from individuals with LTH, LVH and dogs with LVC were tested. Assays were performed on 96-well flexible PVC plates (Costar) for peptide-1. At this stage each well was sensitized with 2-10 µg synthetic peptide and then the plate was blocked with PBS plus 1.0-5.0% BS A for 30-60 minutes at 25-37 ° C. After blockage, all solution from the wells was removed by aspiration. Then 50-100 µl of the sera diluted 1: 100 in PBS with 1.0-2.5% BSA were added to the wells and incubated at 25-37 ° C for 30-60 minutes. The plates were washed 4 times with the wash solution (PBS-0.05% Tween20) and then 50-100 µl of the 1: 5000 diluted human or canine anti-igG antibody was added to the wells in PBS-BSA 1. .0-2.5%. After incubation at 37 ° C for 30-60 minutes, the plates were again washed four times with the wash solution, and 100 µl of the developer solution containing 0.1 M citric acid, 0.2 Na Na2P04, TMB 0, 05% and 0.1% H2 O were added. The plates were incubated at 25-37 ° C in the dark for 10 to 30 minutes with the developer solution when the reaction was stopped by the addition of 50-100 µl 2M H 2 SO 4. The resulting absorbance was read on an ELISA reader at 450 nm. ELISA values using peptide-1 for diagnosis of LTH and LVH were compared to ELISA using soluble L. braziliensis antigen (AgSLb) which is commonly used for serological diagnosis of leishmaniasis. For ELISA using Peptide-1 for diagnosis of CVL, the values were compared with the reference test currently recommended by the Brazilian Ministry of Health, EIE-LVC Kit (Biomanguinhos, FIOCRUZ). [020] Table 1 describes the identity in the linear epitope of B lymphocyte present in the Mitogen-activated protein kinase gene sequence between the sequences of L infantum, the causative agent of the LV parasite T. cruzi, and between the sequences from hosts H. sapiens and C. famiiiaris.

Table 1: Identity in the linear epitope of Lymphocyte B (peptide-1) present in the L. braziliensis Mitogen-activated protein kinase gene. [021] As presented, the sequence of L. infantum presents high similarity (88.90%) with the sequence of L. braziliensis, allowing use for multiple diagnosis of leishmaniasis. Regarding T. cruzi sequences, a high divergence (22.40%) was observed, which prevents cross-reactivity with patients with CD, increasing the specificity values. Regarding the orthologists of the hosts H. sapiens (44.40%) and C. famiiiaris (44.40%), a high divergence with these sequences was also observed, which allows the sensitivity values to be high. This fact increases the likelihood of recognizing infected individuals, as it has no similarity to the host's own antigens. [022] Evaluating the data described in Figure 2, the ELISA using Peptide-1 showed excellent performance and greater simultaneous ability to detect positive LTH (cutaneous and mucocutaneous form) and negative (Chagas disease control and carriers) results. ), evaluated for sensitivity and specificity (Peptide-1: Se = 98.46% and Ep = 95.71%; AgSLb: Se = 70.77% and Ep = 68.57%, data presented in Table 2), when compared to commercially available diagnostic tests.

Table 2: Diagnostic performance in serum samples from individuals with LTH, LVH, and dogs with LVC using Peptide-1 and AgSLb.

Parameters were calculated using all samples presented in this work for LT (CT + DC + LC + LM, n = 135), VL (CT + DC + LV, n = 125) and LVC (CT + DC + LVC, n = 75). ). Abbreviations: LTH: Human Cutaneous Leishmaniasis; LVH: Human Visceral Leishmaniasis; LVC: Canine Visceral Leishmaniasis; Se: sensitivity; Ep: specificity; CI: confidence interval; PPV: positive predictive value; NPV: negative predictive value; B.C; accuracy. * Cut-off obtained by the ROC curve. # Cut-off obtained according to the manufacturer. ; [023] As described in Table 2, the results of positive (PPV) and negative (NPV) predictive values were high (Peptide-1; PPV = 95.52% and NPV = 98.53%; AgSLb; PPV = 67.65% and NPV = 71.64%), as well as the accuracy (Peptide-1; AC = 97.04%; AgSLb: AC = 69.63%), compared to the AgSLb ELISA, thus, the probability of A positive or negative result obtained by the test being true is high, classifying it as an excellent option for diagnosing LTH. Evaluating the area under the curve (AUC) (Table 3), it was observed that the test using Peptide-1 showed high AUC compared to AgSLb (Peptide-1: AUC = 0.989; AgSLb: AUC = 0.753), indicating that there was no a significant incidence of false positive and false negative results, thus being efficient in discriminating sick and healthy individuals. [024] Evaluating the data described in Figure 3, the ELISA using Peptide-1 showed excellent performance and greater simultaneous ability to detect LVH positive and negative results (control individuals and Chagas disease carriers), assessed for sensitivity and specificity ( Peptide-1: Se = 90.91% and Ep = 92.86%; AgSLb: Se = 52.73% and Ep = 50.00%, data presented in Table 2), when compared to commercially available diagnostic tests. As described in Table 2, the results of positive (PPV) and negative (NPV) predictive values were high (Peptide-1: PPV = 90.91% and NPV = 92.86%; AgSLb: PPV = 45.31). % and NPV = 57.38%), as well as the accuracy (Peptide-1: AC = 92.00%; AgSLb: AC = 51.20%), compared to the AgSLb ELISA, thus, the probability of a positive result or negative obtained by the test being true is high, classifying as an excellent option for diagnosing LTH. Evaluating the area under the curve (AUC) (Table 3), it was observed that the test using Peptide-1 showed high AUC compared to AgSLb (Peptide-1: AUC = 0.968; AgSLb: AUC = 0.510), indicating that there was no a significant incidence of false positive and false negative results, thus being efficient in discriminating sick and healthy individuals. Table 3: Analysis of the ROC curve obtained from serum samples from individuals with LTH, LVH, and dogs with LVC using Peptide-1.

Abbreviations: LTH: Human Cutaneous Leishmaniasis; LVH: Human Visceral Leishmaniasis; LVC: Canine Visceral Leishmaniasis; AUC: area below the curve; CI: confidence interval. * Cut off obtained by the ROC curve. # Cut off obtained according to the manufacturer. [025] Evaluating the data described in Figure 4, ELISA using Peptide-1 performed excellently in the ability to detect LVC positive and negative (Chagas disease control and carriers) results evaluated for sensitivity and specificity (Peptide-1). -1: Se = 93.33% and Ep = 77.78%; EIE-LVC kit: Se = 100.00% and Ep = 53.33% (data shown in Table 2). As described in Table 2, the result of positive (PPV) and negative (NPV) predictive values was high (Peptide-1: PPV = 73.68% and NPV = 94.59%; EIE-LVC kit: PPV = 58, 82% and NPV = 100.00%), as well as the accuracy (Peptide-1: AC = 84.00%; Peptide-1: AC = 72.00%), thus the probability of a positive or negative result obtained by the test to be true is high, classifying as an excellent option for diagnosis of CVL. Evaluating the area under the curve (AUC) (Table 3), it was observed that the test using Peptide-1 showed high AUC (Peptide-1: AUC = 0.883), indicating that there was no significant incidence of false positive and false negative results. being efficient in discriminating sick individuals from healthy ones. [026] The sensitivity gain obtained with Peptide-1 is possibly due to its high immunogenicity and polymorphic epitopes, as described in Figure 1, a fact that is important for the correct diagnosis of leishmaniasis, allowing the treatment of infected individuals. by leishmaniasis (LTH and LVH). Additionally, correct diagnosis is important to prevent the maintenance of infected dogs (CVL) in endemic areas and to avoid unnecessary animal sacrifice due to limitations in test specificity due to infection by other microorganisms, resulting in false positive results commonly observed in tests. of diagnoses using crude parasite antigen. Evaluating the high ability to detect infections caused by different species of Leishmania, the data allow us to conclude that the serological reactions are valid using Peptide-1 for high accuracy immunodiagnosis of human and canine tegumentary and visceral leishmaniasis.

Claims (16)

1. A synthetic peptide comprising the sequence represented by SEQ ID NO: 1. 2- Use of synthetic peptide characterized for being in the diagnosis of canine visceral leishmaniasis and human cutaneous and visceral leishmaniasis. Method for immunodiagnostic testing of leishmaniasis comprising: a) binding antileishmanial antibodies of a sample to one or more polypeptides consisting of the amino acid sequences SEQ ID Nos 1, linked to a solid support or a carrier; b) contacting the antibodies of step (a) with a secondary antibody or protein, conjugated to an enzyme or label and binding to the antibodies of step (a); c) detecting antileishmanial antibodies in the above sample by detecting the secondary antibody or protein specifically bound to said anti-leishmanial antibody. Method for immunodiagnostic testing of leishmaniasis according to claim 3, step "a", characterized in that the polypeptide SEQ ID NO: 1 may be modified at its ends. Method for Immunodiagnostic Testing of Leishmaniasis according to Claim 3, step "a", characterized in that the samples are selected from the group consisting of blood, serum, plasma and other body fluid. Method for immunodiagnostic testing of leishmaniasis according to claim 3, step "a", characterized in that the carrier is preferably a gold particle. Method for immunodiagnostic testing of leishmaniasis according to claim 3, step "b", characterized in that the protein is selected from the group consisting of protein A and protein G. Method for the Immunodiagnostic Test of Leishmaniasis according to Claim 3, step "b", characterized in that the enzyme is selected from the group consisting of alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase. 9- Method; according to claim 3, step "b", characterized in that the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescent. A kit for the immunodiagnostic test of leishmaniasis, characterized in that it comprises: - at least one peptide consisting of the sequence SEQ ID No. 1 associated or isolated; A secondary antibody or protein, wherein the secondary antibody or protein is conjugated to an enzyme or marker, and a reagent for detecting said enzyme or marker. An immunodiagnostic test kit for leishmaniasis according to claim 10, characterized in that the peptides are attached to a solid support or carrier. An immunodiagnostic test kit for leishmaniasis according to claim 10, characterized in that the solid support is selected from the group of material consisting of nitrocellulose, nylon, latex, polypropylene and polystyrene. An immunodiagnostic test kit for leishmaniasis according to claim 10, characterized in that the carrier consists of gold particles. A kit for the immunodiagnostic test of leishmaniasis according to claim 10, characterized in that the protein is selected from the group consisting of protein A and protein G. Leishmaniasis immunodiagnostic test kit according to claim 10, characterized in that said marker is selected from the group consisting of enzymes, redioisotopes, biotin, chromophores, fluorophores and chemiluminescent. An immunodiagnostic test kit for leishmaniasis according to claim 10, characterized in that the enzyme is selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.