BR102014004107B1 - method and kit for the diagnosis of leishmaniasis using synthetic peptides - Google Patents

Abstract

METHOD AND KIT FOR DIAGNOSING LEISHMANIASIS USING SYNTHETIC PEPTIDES The present invention describes a method and kit for immunodiagnosing leishmaniasis and the synthetic peptides used in this method. The peptides developed here were able to identify individuals and animals infected by visceral and tegumentary leishmaniasis, allowing the development of a diagnostic method and kit with greater sensitivity and specificity, and improving the reactivity of serological tests. For this, a search was carried out in the proteome of parasites of the Leishmania genus of regions that present epitopes for B lymphocytes with polymorphism with orthologs present in the etiological agent of Chagas disease, Trypanossomacruzi, where serological cross-reactivity is often observed.

Description

[001] The present invention describes a method and Kit for immunodiagnosis of leishmaniasis and the synthetic peptides used in this method. The peptides developed here were able to identify individuals and animals infected by visceral and tegumentary leishmaniasis, allowing the development of a diagnostic method and kit with greater sensitivity and specificity, and improving the reactivity of serological tests. For this, a search was carried out in the proteome of parasites of the Leishmania genus of regions that present epitopes for B lymphocytes with polymorphism with orthologs present in the etiological agent of Chagas disease, Trypanossomacruzi, where serological cross-reactivity is often observed.

[002] Leishmaniasis is present in 88 countries and is widely distributed in Latin America and Brazil, with an estimated 12 million people infected, being considered among the ten infectious diseases with the greatest impact on public health and the main neglected diseases present in developing countries (Desjeux P et a/.ComplmmunolMicrobiollnfectDis. 2004 Sep; 27(5): 305-18). Infections caused by parasites belonging to the Leishmania genus are considered a serious public health problem in Brazil and the correct and early diagnosis is one of the most relevant challenges for the control and eradication of the disease.

[003] The use of recombinant antigens and synthetic peptides in the diagnosis of leishmaniasis have been preferentially used to replace parasite lysates. These new methodologies have greater reproducibility of results and elimination of steps that involve the maintenance and processing of live parasites (Frasch AC et a/.Parasitol Today. 1991 Jun;7(6):148-51; Da-Silveira JF et a/ .Trends Parasitol. 2001 Jun;17(6):286-91; Meira WSeía/.J Clin Microbiol. 2002 0ct;40(10):3735-40).

[004] In the prior art, patent documents were found that suggest the use of peptides in the diagnosis of leishmaniasis, as examples, the document P11020120335522 and P11320120335599 that describe the use of polypeptides derived from the proteome of Leishmaniainfantum for the diagnosis of infection. However, all documents found in the prior art, among which are those mentioned above, present peptides different from those presented in this invention and, still, do not allow such an effective diagnosis, through which distinction is made from other infections caused. by parasites present in endemic areas for human tegumentary and visceral leishmaniasis and canine visceral leishmaniasis, such as the protozoan species Trypanosoma cruzi, the etiological agent of Chagas disease, as in the present technology. Furthermore, there is no description in the literature of methods that diagnose the integumentary and visceral form, human and canine, using the same antigen mapped in the proteome of parasites of the Leishmania genus. Thus, despite the advances obtained in recent years, the available diagnostic methods for leishmaniasis still have several limitations. Furthermore, in the absence of effective diagnostic methods, the efficiency of treatment and any initiative to block transmission or vaccination is compromised (Ministry of Health. Infectious and Parasitic Diseases: pocket guide. Brasília. 2006).

[005] Despite the extensive search 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 life cycle of the parasite are still needed. Thus, the present invention presents an alternative to solve this problem. These are synthetic peptides designed from B lymphocyte epitopes predicted by immunoinformatics and their use in a method and kit to diagnose leishmaniasis in dogs and/or humans.

BRIEF DESCRIPTION OF THE FIGURES

[006] Figure 1 shows comparative graphs regarding humoral reactivity against peptide-1, 2 and 3in serum of individuals with Human Tegumentary Leishmaniasis. The X axis represents the evaluated groups: control individuals (CT, n=50), individuals individuals with Chagas Disease (CD, n=20) individuals with Human Tegumentary Leishmaniasis (LTH) clinical cutaneous form (LTH-C, n=45) and mucocutaneous (LTH-MC, n=20). The Y axis represents the mean absorbance values (450 nm) by the ELISA technique using 1:100 diluted sera. The cut off was established using the ROC curve.

[007] Figure 2 shows comparative graphs regarding the humoral reactivity against peptide-1, 2 and 3 in serum from individuals with Human Visceral Leishmaniasis. The X axis represents the evaluated groups: control individuals (CT, 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 the ELISA technique using 1:100 diluted sera. The cut off was established using the ROC curve.

[008] Figure 3 shows comparative graphs regarding humoral reactivity against peptides-1, 2 and 3 in serum from dogs with canine Visceral Leishmaniasis. The X axis represents the evaluated groups: control dogs (CT, n=30), individuals with Chagas Disease (DC, n=15) individuals with Canine Visceral Leishmaniasis (CVL, n=30). The Y axis represents the mean absorbance values (450 nm) by the ELISA technique using 1:100 diluted sera. The cut off was established using the ROC curve.

[009] Figure 4 presents a graph comparing the ROC curves obtained from the ELISA for peptide-1, 2 and 3 in the immunodiagnosis of Human Tegumentary Leishmaniasis. These curves were used to determine the cut off, sensitivity (Se), specificity (Ep) and area under the curve (AUC) of the ELISA technique.

[010] Figure 5 presents a graph comparing the ROC curves obtained from the ELISA for peptide-1, 2 and 3 in the immunodiagnosis of Human Visceral Leishmaniasis. These curves were used to determine the cut off, sensitivity (Se), specificity (Ep) and area under the curve (AUC) of the ELISA technique.

[011] Figure 6 presents a graph comparing the ROC curves obtained from the ELISA for peptide-1, 2 and 3 in the immunodiagnosis of Canine Visceral Leishmaniasis. These curves were used to determine the cut off, sensitivity (Se), specificity (Ep) and area under the curve (AUC) of the ELISA technique.

DETAILED DESCRIPTION OF THE TECHNOLOGY

[012] In the present invention conserved and polymorphic peptides were identified among the different species of the genus Leishmania, derived from B cell epitopes consisting of the sequences represented by SEQ ID No. 1, 2 and 3, modified or not at their ends, in order to contribute to improve the serodiagnosis of canine visceral leishmaniasis and human cutaneous and human visceral leishmaniasis. The method for immunodiagnosis of leishmaniasis can be selected from the group comprising ELISA, Western blot, dotblot, immunodiffusion, immunochromatography and have the following steps: a) binding of anti-leishmanial antibodies from a sample to one or more polypeptides consisting of the amino acid sequences SEQ ID Nos 1, 2 and 3, attached to a solid support or a carrier, preferably a gold particle; b) contacting the antibodies of step (a) with a secondary antibody or a protein (protein A or G), conjugated to an enzyme or a label and which bind to the antibodies of step (a); the enzymes can be selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase and the marker can be selected from the group comprising enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents; c) detecting the anti-leishmanial antibodies in the aforementioned sample by detecting the secondary antibody or protein specifically bound to said anti-leishmanial antibody.

[013] The method can be performed using blood, serum, plasma or other body fluid samples from the infected animal or human.

[014] The technology further comprises a Kit for immunodiagnostic test of leishmaniasis that comprises the peptides consisting of the sequences SEQ IDN0S 1, 2 and 3 associated or isolated and a secondary antibody or a 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, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents), and a reagent to detect said enzyme or label. In the Kit, the recombinant proteins or peptides will be linked to a solid support or carrier, and this solid support may be nitrocellulose, nylon, latex, polypropylene and polystyrene and the carrier will consist of gold particles.

[015] The ELISA technique was chosen in the present invention for being less invasive, easy to perform and automation, allowing studies and application in large numbers of samples.

[016] The present invention can be better understood, but in a non-limiting way, with the following examples.

Example 1- In silico identification of linear B-cell epitopes

[017] Initially, linear B-cell epitopes were predicted throughout the proteome of Leishmania braziliensis strain M2904 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 average Bepipred prediction score above 1.3 were considered as polymorphic epitopes. Sequences of 15 amino acids with a mean epitope prediction score equal between the two sequences and above 1.3 and a polymorphism score equal to zero were considered conserved epitopes. To minimize the chance of cross-reacting with other phylogenetically related parasites, peptide sequences with high similarity to proteins from other parasites were identified using the BLAST algorithm (AltschulSF 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, L. major and T. cruzi. Epitopes with greater than 70% identity over greater than 70% of the sequence were eliminated from subsequent analyses.

[018]Evaluating the data obtained, 3 peptides were identified in the predicted proteome of Leishmania braziliensis with potential use for immunodiagnosis of leishmaniasis. The immunoinformatics analysis of peptide-1 (EEDESKKKSCGDEGEPKVE), located on chromosome 33, showed a prediction score for B lymphocyte epitope of 1.81 in a region with a disorder score of 0.62. The immunoinformatics analysis of peptide-2 (VTEGGEDKKK), located on chromosome 33, showed a B lymphocyte epitope prediction score of 1.71 in a region with a disorder score of 0.62. The immunoinformatics analysis of peptide-3 (EEVAEAPPAEAAPA), located on chromosome 33, showed a B lymphocyte epitope prediction score of 1.80 in a region with a disorder score of 0.75. The peptides were synthesized in solid phase to be later analyzed for performance as an antigen for immunodiagnosis of leishmaniasis.

Example 2-Synthesis of Soluble Peptides

[019] The selected peptides were synthesized in solid phase by the N-9-fluorenylmethoxycarbonyl method (Wellings DAeAtherton E.MethodsEnzymol. 1997;289:44-67) in the 20-30 μmol scale. All synthesis was performed on the PSSM8 equipment. 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 Rinkamidated resin with a degree of substitution of 0.61 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 (SEQIDN°1- EEDESKKKSCGDEGEPKVE), Peptide-2 (SEQ ID N°2- VTEGGEDKKK) and Peptide-3 (SEQIDN°3 - EEVAEAPPAEAAPA), were purified by reverse phase chromatography using a Sephasil Peptide C18 column (Shimadzu) coupled to an HPLC system (Shimadzu) using a gradient from 0 to 25% of acetonitrile over 75-90 minutes with 0-10 minutes to 10% of acetonitrile. After purification, the peptides were submitted to mass spectrometry in an AutoflexSpeed MALDI/TOF (Bruker) equipment to confirm their molecular mass. Example 3 - Validation of peptides against serum from individuals with LTH, LVH and dogs with CVL

[020] ELISA assays were performed to validate and characterize the reactivity of the peptides against parasites of the genus Leishmania. Sera from individuals carrying LTH, LVH and dogs carrying CVL were tested. Assays were performed in 96-well flexible PVC plates (Costar). Each well was sensitized with 2-1 Oμg of synthetic peptide. After sensitization, the plate was blocked with PBS plus 1.0-5.0% BSA for 30-60 minutes at 25-37°C. After blocking, 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 washing solution (PBS-0.05% Tween20) and then 50-100 µL of anti-human or canine IgG antibody diluted 1:5000 in PBS-BSA 1 was added to the wells. 0.0-2.5%. After incubation at 37°C for 30-60 minutes, the plates were again washed four times with the washing solution, and 100 µl of the developing solution, containing 0.1 M citric acid, 0.2 M Na2PO4, 0 TMB, 0.5% and 0.1% H2O2 were added. The plates were incubated at 25-37°C in the dark for 10 to 30 minutes with the developing solution, when the reaction was stopped by the addition of 50-100 pL of 2M H2SO4. The resulting absorbance was read in an ELISA reader at 450 nm.

[021] Evaluating the data described in Figure 1, the ELISA using peptide-1, 2 and 3 showed excellent performance in the simultaneous ability to detect positive results for Human Tegumentary Leishmaniasis (cutaneous and mucocutaneous form) and negative (control subjects) and patients with Chagas disease), evaluated by sensitivity and specificity (Peptide-1: Se=63.08% and Ep=94.29%; Peptide-2: Se=63.08% and Ep=90.00%; Peptide-3: Se=89.23% and Ep=91.43%; data presented in Table 1). As described in Table 1, the result of positive (PPV) and negative (VPN) predictive values was high (Peptide-1: VPP=91.11% and NPV=73.33%; Peptide-2: VPP=85.42 % and NPV=72.41%; Peptide-3: VPP=90.63% and NPV=90.14%; data presented in Table 1), as well as accuracy (Peptide-1: AC=79.26%; Peptide-2: AC=77.04%; Peptide-3: AC=90.37%; data presented in Table 1), thus, the probability of a positive or negative result obtained by peptide-1, 2 and 3 is true is high, classifying it as an excellent option for diagnosing LTH. Assessing the area under the curve (AUC) (Table 2 and Figure 4), it was observed that the tests using peptides-1, 2 and 3 presented high AUC (Peptide-1: AUC=0.8810; Peptide-2: AUC=0.8187; Peptide-3: AUC=0.9608; data presented in Table 2), indicating that there was no significant incidence of false positive and false negative results, thus being efficient in discriminating sick and healthy individuals.

[022] Assessing the data described in Figure 2, the ELISA using peptide-1, 2 and 3 showed excellent performance in the simultaneous ability to detect positive results for negative Visceral Leishmaniasis Humanae (controls and individuals with Chagas Disease), evaluated by sensitivity and specificity (Peptide-1: Se=83.64% and Ep=95.71%; Peptide-2: Se=85.45% and Ep=92.86%; Peptide-3: Se=87.27% and Ep=94.29%; data presented in Table 1). As described in Table 1, the results of positive (PPV) and negative (VPN) predictive values were high (Peptide-1: VPP=93.88% and NPV=88.16%; Peptide-2: VPP=90.38% and VPN=89.60%; Peptide-3: VPP=92.31% and VPN=91.20%; data presented in Table 1), as well as accuracy (Peptide-1: AC=90.40%; Peptide -2: AC=89.60%; Peptide-3: AC=91.20%; data presented in Table 1), thus, the probability of a positive or negative result obtained by peptide-1, 2 and 3 being true is high, classifying it as an excellent option for diagnosing LVH. Assessing the area under the curve (AUC) (Table 2 and Figure 5), it was observed that the tests using peptides-1, 2 and 3 presented high AUC (Peptide-1: AUC=0.9338; Peptide-2: AUC=0.9240; Peptide-3: AUC=0.9434; data presented in Table 2), indicating that there was no significant incidence of false positive and false negative results, thus being efficient in discriminating sick and healthy individuals.

Abreviações: LTH: Leishmaniose Tegumentar Humana; LVH: Leishmaniose Visceral Humana; LVC: Leishmaniose Visceral Canina; Se: sensibilidade; Ep: especificidade; IC: intervalo de confiança; VPP: valor preditivo positivo; VPN: valor preditivo negativo; AC: acurácia. Tabela 2: Análise da curva ROC obtida em amostras de soro de indivíduos portadores de LTH, LVH e cães portadores de LVC utilizando os peptídeos 1, 2 e3.

Abreviações: LTH: Leishmaniose Tegumentar Humana; LVH: Leishmaniose Visceral Humana; LVC; Leishmaniose Visceral Canina; AUC: área abaixo da curva; IC: intervalo de confiança.[023] Assessing the data described in Figure 3, the ELISA using peptide-1, 2 and 3 showed excellent performance in the simultaneous ability to detect positive results for Canine Visceral Leishmaniasis and negative (controls and individuals with Chagas Disease), evaluated by sensitivity and specificity (Peptide-1: Se=96.67% and Ep=93.33%; Peptide-2: Se=63.33% and Ep=80.00%; Peptide-3: Se=93.33 % and Ep=97.78%; data presented in Table 1). As described in Table 1, the results of positive (PPV) and negative (VPN) predictive values were high (Peptide-1: VPP=90.63% and NPV=97.67%; Peptide-2: VPP=67.86 % and NPV=76.60%; Peptide-3: VPP=96.65% and NPV=95.65%; data presented in Table 1), as well as accuracy (Peptide-1: AC=94.67%; Peptide-2: AC=73.33%; Peptide-3: AC=96.00%; data presented in Table 1), thus, the probability of a positive or negative result obtained by peptide-1, 2 and 3 is true is high, classifying it as an excellent choice for the diagnosis of CVL. Assessing the area under the curve (AUC) (Table 2 and Figure 6), it was observed that the tests using peptides-1, 2 and 3 presented high AUC (Peptide-1: AUC=0.9496; Peptide-2: AUC=0.7548; Peptide-3: AUC=0.9867; data presented in Table 2), indicating that there was no significant incidence of false positive and false negative results, thus being efficient in discriminating sick and healthy individuals. Table 1: Diagnostic performance in serum samples from individuals carrying LTH, LVH and dogs carrying CVL using peptides 1, 2 and 3.

Abbreviations: LTH: Human Tegumentary Leishmaniasis; LVH: Human Visceral Leishmaniasis; LVC: Canine Visceral Leishmaniasis; If: sensitivity; Ep: specificity; CI: confidence interval; PPV: positive predictive value; VPN: negative predictive value; AC: accuracy. Table 2: Analysis of the ROC curve obtained in serum samples from individuals carrying LTH, LVH and dogs carrying CVL using peptides 1, 2 and3.

Abbreviations: LTH: Human Tegumentary Leishmaniasis; LVH: Human Visceral Leishmaniasis; LVC; Canine Visceral Leishmaniasis; AUC: area under the curve; CI: confidence interval.

[024] The gain in sensitivity in the diagnostic test for leishmaniasis, obtained by using peptides-1, 2 and 3, possibly due to the fact that these peptides have epitopes with high immunogenicity and polymorphic, is important to make the correct diagnosis of leishmaniasis enabling the treatment of infected individuals. Additionally, correct diagnosis is important to prevent the maintenance of infected dogs (LVC) in endemic areas and avoid unnecessary sacrifice of animals due to limitations in test specificity due to infection by other microorganisms, generating false positive results, commonly observed in tests of diagnoses using crude parasite antigen. Assessing the high capacity to detect infections caused by different Leishmania species, the data allow us to conclude that the serological reactions using peptides-1, 2 and 3 are valid in the immunodiagnostic with high accuracy of human and canine integumentary and visceral leishmaniasis.

Claims (14)

1. Use of synthetic peptides defined by SEQ ID No. 1, 2 and 3, characterized by being in the diagnosis of canine visceral leishmaniasis and human cutaneous and human visceral leishmaniasis. 2. Method for immunodiagnostic test of leishmaniasis characterized by comprising: a. Binding of anti-leishmanial antibodies from a sample to at least one of the polypeptides consisting of the amino acid sequences SEQ ID Nos 1, 2 and 3, isolated and linked to a solid support or a carrier; B. Contacting the antibodies from step (a) with a secondary antibody or protein, conjugated to an enzyme or label, and which bind to the antibodies from step (a); ç. Detect anti-leishmanial antibodies in the sample from step (a) by detecting the secondary antibody or protein specifically bound to anti-leishmanial antibodies. 3. Method for immunodiagnostic test of leishmaniasis, according to claim 2, step "a", characterized in that samples are selected from the group consisting of blood, serum, plasma and other body fluid. 4. Method for immunodiagnostic test of leishmaniasis, according to claim 2, step "a", characterized in that the carrier is a gold particle. 5. Method for immunodiagnostic test of leishmaniasis, according to claim 2, step "b", characterized in that the protein is selected from the group consisting of protein A and protein G. 6. Method for immunodiagnostic test of leishmaniasis, according to claim 2, 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. 7. Method for immunodiagnostic test of leishmaniasis, according to claim 2, step "b", characterized in that the marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 8. Kit for immunodiagnostic test of leishmaniasis, characterized in that it comprises: at least one of the isolated peptides defined by SEQ ID Nos. 1, 2 and 3, a secondary antibody or a protein, where the secondary antibody or protein is conjugated to an enzyme or label, and a reagent for detecting said enzyme or label. 9. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that the peptides are linked to a solid support or carrier. 10. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that the solid support is selected from the group of material consisting of nitrocellulose, nylon, latex, polypropylene and polystyrene. 11. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that the carrier consists of gold particles. 12. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that the protein is selected from the group consisting of protein A and protein G. 13. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that said marker is selected from the group consisting of enzymes, radioisotopes, biotin, chromophores, fluorophores and chemiluminescents. 14. Kit for immunodiagnostic test of leishmaniasis, according to claim 8, characterized in that the enzyme is selected from the group comprising alkaline phosphatase, peroxidase, β-galactosidase, urease, xanthine oxidase, glucose oxidase and penicillinase.

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