BR102018073101A2 - SYNTHETIC PEPTIDE AND ITS APPLICATIONS RELATED TO SCHISTOSOMOSIS - Google Patents

Abstract

synthetic peptide and its applications related to schistosomiasis. The present invention comprises an antigenic synthetic peptide consisting of two potential schistosome epitopes. This structural characteristic could give this peptide greater sensitivity, since two promising epitopes are fused, increasing the potential of the peptide. the selected epitopes do not show similarity with phylogenetically related species, which can provide good specificity in the recognition of anti-schistosome antibodies, avoiding the occurrence of cross-reactions. Additionally, the synthetic peptide described in this invention may induce a broader and more desirable immune response to meet different applications regarding the detection, immunization, prevention and treatment of schistosomiasis, making it useful in the development of different products of interest to the pharmaceutical and biotechnology, such as diagnostic kits for detecting schistosomiasis from fluids, and also immunogenic compositions, vaccines, medicines or biopharmaceuticals to prevent and treat the disease. Furthermore, the synthetic peptide can be used as an input for the production of monoclonal antibodies.

Description

SYNTHETIC PEPTIDE AND ITS APPLICATIONS RELATED TO SCHISTOSOMOSIS FIELD OF INVENTION

[01] The present invention comprises the rational construction of a synthetic peptide containing an epitope proven to be recognized by antibodies present in the sera of infected patients and a hypothetical epitope, both of which were shown to be highly antigenic. These epitopes come from the Schistosoma genus parasites, more specifically from the hypothetical protein Smp_150390.1 and from the anchor surface glycoprotein GPI 200-kDa (Sm200), with antigenicity values of 1.39 and 1.68, respectively. The Smp_150390.1 protein epitope is conserved in Schistosoma mansoni (S. mansoni) and Schistosoma haematobium (S. haematobium) species and the Sm200 protein epitope in S. mansoni, S. haematobium and Schistosoma japonicum (S. japonicum) species. The synthetic peptide of the present invention has biotechnological potential as it presents antigenic activity and can be used in different applications in this field. Its obtaining process is based on the chemical synthesis of the fused epitopes through a linker. The synthetic peptide may be useful in the development of vaccines, drugs or biopharmaceuticals to diagnose, prevent and treat schistosomiasis. Additionally, this peptide can also be used as an input in schistosomiasis detection methods and in the production of monoclonal antibodies.

TECHNICAL STATUS Schistosoma and schistosomiasis

[02] Schistosomiasis is a chronic and acute disease caused by trematodes of the genus Schistosoma (COLLEY, DG; SECOR, WE Immunology of human schistosomiasis. Parasite Immunology, v. 36, n. 8, p. 347–357, 2014) . The three main species of schistosomes that infect humans are: Schistosoma haematobium (S. haematobium), causing urinary schistosomiasis in Africa and the Arabian peninsula and having snails of the genus Bulinus as an intermediate host; Schistosoma japonicum (S. japonicum) causing intestinal and hepatosplenic schistosomiasis in countries such as China, Philippines and Indonesia, whose vectors are molluscs of the genus Oncomelania and Schistosoma mansoni (S. mansoni) transmitted by snails of the genus Biomphalaria and responsible for the disease in its form hepatic and intestinal in the African continent, Arabian Peninsula and South America (GRYSEELS, B. et al. Human schistosomiasis. Lancet, v. 368, n. 9541, p. 1106–18, 2006). Diseases caused by parasites are a serious public health problem in several countries around the world, especially in emerging countries, and can be seen as a reflection of the economic and social situation of the country (World Health Organization (WHO), 2017. Available at: http ://www.who.int/mediacentre/factsheets/fs115/en/ Accessed: 25/06/2018). In this context, schistosomiasis is one of the most prevalent parasitic infections in the world, comprising 78 countries and territories in Africa, Asia and the Americas, with Africa being the most affected and covering 92% of cases that require treatment (World Health Organization ( WHO), 2017. Available at: http://www.who.int/mediacentre/factsheets/fs115/en/. Accessed: 25/06/2018). With 240 million people infected worldwide and 800 million people residing in areas at risk of infection, schistosomiasis is a serious public health problem and stands out among neglected diseases (WEERAKOON, KGAD et al. Advances in the diagnosis of human schistosomiasis. Clinical Microbiology Reviews, v. 28, n. 4, p. 939–967, 2015; World Health Organization (WHO), 2017. Available at: http://www.who.int/mediacentre/ factsheets/fs115/en/. Accessed on: 06/25/2018). It is estimated that currently six million people are infected and that approximately 25 million live in areas at risk of contracting the disease (ESPÍRITO-SANTO, MCC et al. Comparative Study of the Accuracy of Different Techniques for the Laboratory Diagnosis of Schistosomiasis Mansoni in Areas of Low Endemicity in Barra Mansa City, Rio de Janeiro State, Brazil. BioMed Research International, v. 2015, p. 1-16, 2015). In Brazil, schistosomiasis mansoni was identified as an endemic disease mainly of rural character, presenting a more chronic aspect and affecting low-income populations (Enk, MJ; Amorim, A.; Schall, VT Acute schistosomiasis in the metropolitan area of Belo outbreak Horizonte, Minas Gerais: alert about the risk of unnoticed transmission increased by growing rural tourism. Mem Inst Oswaldo Cruz., v. 98, n. 6, p. 745-750, 2003). It is estimated that in the country 4 to 6 million people are infected by S. mansoni and that 70% of cases are concentrated in the states of Minas Gerais and Bahia (Drummond, SC; Silva, LC; Amaral, RS; Sousa-Pereira, SR; Antunes, CM; Lambertucci, JR Morbidity of Schistosomiasis mansoni in the state of Minas Gerais, Brazil. Mem Inst Oswaldo Cruz., v. 101, n. 1, p. 37-44, 2006). The State of Minas Gerais is considered to be of great epidemiological importance for the disease in Brazil, as 61% of its municipalities showed active transmission of the infection and more than 10 million people live in endemic areas (DRUMMOND, SC et al. Schistosomiasis control program in the state of Minas Gerais in Brazil. Mem Inst Oswaldo Cruz., v. 105, n. 4, p. 519-523, 2010). Many cases of the disease have been reported in the state due to the growth of rural tourism (Enk, MJ; Amorim, A.; Schall, VT Acute schistosomiasis outbreak in the metropolitan area of Belo Horizonte, Minas Gerais: alert about the risk of unnoticed transmission by growing rural tourism. Mem Inst Oswaldo Cruz., v. 98, no. 6, p. 745-750, 2003; Massara, CL; Amaral, GL; Caldeira, RL; Drummond, SC; Enk, MJ; Carvalho, OS Schistosomiasis in an ecotourism area in the state of Minas Gerais, Brazil. Cad Saude Pública., v. 24, n. 7, p. 1709-1712, 2008; Enk, MJ; Amaral, GL; Costa e Silva, MF; Silveira- Lemos, D.; Teixeira-Carvalho, A.; Martins-Filho, OA; Correa-Oliveira, R.; Gazinnelli, G.; Coelho, PM; Massara, CL Rural tourism: a risk factor for schistosomiasis transmission in Brazil. Inst Oswaldo Cruz., v. 105, no. 4, p. 537-540, 2010; Murta, FLG; Massara, CL; Nogueira, JFC; Dos Santos Carvalho, O.; de Mendonça, CLF; Pinheiro , THEY GO.; Enk, M.J. Ecotourism as a source of infection with Schistosoma mansoni in Minas Gerais, Brazil. Trop Dis Travel Med Vaccines, v. 2, n. 3, 2016). In addition, 14 cases of schistosomiasis were reported in Divinópolis/Minas Gerais in 2010, followed by 9 cases in 2011, possibly due to the same reason mentioned above, or due to probable changes in habits, associated with risk factors (Santana , KTO; Pego, KVT; Pereira, VV; Rodrigues, LP; Ferreira, JV; Oliveira, KN; Trindade, MJF; Oliveira, H.; Silva, ES; Santos, LL; Lopes, DO Occurrence of schistosomiasis in Divinópolis-MG based on a study of schoolchildren and surveys of disease notification. J. Bras. Patol. Med. Lab., v. 50, n. 4, p. 265-271, 2014).

[03] The disease remains prevalent in Africa, Asia and South America, where the current scenario over the years tends to lead to continued endemicity, which may be due to poverty in the affected areas, limiting control measures, if not as a result of the strategies adopted being precarious and ineffective (Sokolow, SH; Wood, CL; Jones, IJ; Swartz, SJ; Lopez, M.; Hsieh, MH; Lafferty, KD; Kuris, AM; Rickards, C.; De Leo, GA Global Assessment of Schistosomiasis Control Over the Past Century Shows Targeting the Snail Intermediate Host Works Best. PLoS Negl Trop Dis., v. 10, n. 7, e0004794, 2016). Although effective drug treatments and large-scale drug distribution programs exist, most endemic areas remain without effective control of schistosomiasis (Sokolow, SH; Wood, CL; Jones, IJ; Swartz, SJ; Lopez, M.; Hsieh, MH; Lafferty, KD; Kuris, AM; Rickards, C.; De Leo, GA Global Assessment of Schistosomiasis Control Over the Past Century Shows Targeting the Snail Intermediate Host Works Best. PLoS Negl Trop Dis., v. 10, n. 7, e0004794, 2016). Factors such as the possibility of the emergence of drug resistance of the parasite, the constant reinfection of individuals, the lack of basic sanitation and especially the large extension of endemic areas, mean that the transmission levels are not affected only with the treatment, being necessary the application of other measures (KING, CH et al. Transmission control for schistosomiasis – why it matters now. Trends Parasitol., v. 22, n. 12, p. 575-582, 2006). Currently, the focus of disease control strategies is chemotherapy with anthelmintic drugs, mainly to prevent morbidity in school-age children, who usually have the highest levels of Schistosoma infection (World Health Organization (WHO). use of molluscicides in schistosomiasis control programmes: an operational manual for program managers. Geneva: World Health Organization; 2017. License: CC BY-NC-SA 3.0 IGO). Praziquantel is the drug of choice for the treatment of schistosomiasis, recommended by the World Health Organization (WHO), 2017. Accessed at: http://www.who.int/mediacentre/factsheets/fs115/en /. Accessed on: 06/25/2018), at population as well as individual level, due to its various benefits such as activity against all Schistosoma species, efficacy against adult forms of the parasite, absence of severe short-term side effects and long-term administration as a single oral dose and because it is affordable (Doenhoff, MJ; Kusel, JR; Coles, GC; Cioli, D. Resistance of Schistosoma mansoni to praziquantel: is there a problem? Trans R Soc Trop Med Hyg., v. 96, n. 5, p. 465-469, 2002). Over the last few decades, this isoquinoline derivative has significantly contributed to reducing both morbidity and prevalence of schistosomiasis, but the drug is ineffective in juvenile parasites (GONNERT, R.; ANDREWS, P. Praziquantel, a new broad-spectrum antischistosomal agent Z. Parasitenkd., v. 52, pp. 129-150, 1977; DOENHOFF, MJ et al. Praziquantel: mechanisms of action, resistance and new derivatives for schistosomiasis, Curr. Opin. Infect. Dis. , v. 21, no. 6, p. 659-667, 2008). However, only preventive chemotherapy campaigns (WHO). Preventive chemotherapy in human helminthiasis: coordinated use of anthelminthic drugs in control interventions: a manual for health professionals and program managers. Geneva: World Health Organization; mass drug administration has not been sufficient to limit transmission in high-risk regions (Barbosa, FS; Pinto, R.; Souza, OA Control of schistosomiasis mansoni in a small north east Brazilian community. Trans R Soc Trop Med Hyg., v. 65, no. 2, pp. 206-213, 1971; Sturrock, RF; Kinyanjui, H.; Thiongo, FW; Tosha, S.; Ouma, JH; King, CH; Koech, D.; Siongok, TK; Mahmoud, AA Chemotherapy-based control of haematobia schistosomiasis. 3. Snail studies monitoring the effect of chemotherapy on transmission in the Msambweni area, Kenya. Trans R Soc Trop Med Hyg., v. 84, n. 2, p. 257-261, 1990; Pieri, OS; Goncalves, JF; Sarquis, O. Repe ated focal mollusciciding for snail control in a sugar-cane area of northeast Brazil. Mem Inst Oswaldo Cruz., v. 90, no. 4, p. 535-536, 1995; Bustinduy, A.L.; Sousa-Figueiredo, J.C.; Adriko, M.; Betson, M.; Fenwick, A.; Kabatereine, N.; Stothard, J.R. Fecal occult blood and fecal calprotectin as point-of-care markers of intestinal morbidity in Ugandan children with Schistosoma mansoni infection. PLoS Negl Trop Dis., v. 7, n. 11, e2542, 2013; Ross, A.G.; Olveda, R.M.; Chy, D.; Olveda, D.U.; Li, Y.; Harn, D.A.; Gray, D.J.; McManus, D.P.; Tallo, V.; Chau, T.N.; Williams, MG Can mass drug administration lead to the sustainable control of schistosomiasis? J Infect Dis., v. 211, no. 2, p. 283-289, 2015; King, C.H.; Sutherland, L.J.; Bertsch, D. Systematic Review and Meta-analysis of the Impact of Chemical-Based Mollusciciding for the Control of Schistosoma mansoni and S. haematobium Transmission. PLoS Negl Trop Dis., v. 9, n. 12, e0004290, 2015). In addition, infection control programs based only on mass treatment and not linked to the individual diagnosis of the population, present provisional results, as they reduce the prevalence and incidence of severe forms of the disease, but there is a limitation with regard to the control of the disease. transmission, resulting in the maintenance of the disease and the appearance of new foci (Lambertucci, JR; Serufo, JC; GerspacherLara, R.; Rayes, AA; Teixeira, R.; Nobre, V.; Antunes, CM Schistosoma mansoni: assessment of morbidity before and after control. Acta Trop., v. 77, n. 1, p. 101-109, 2000). In addition to drugs, vaccines can also be used in treatment. Unlike other vaccines, such as viruses and bacteria, which activate immunological elimination and memory mechanisms, a vaccine against Schistosoma that is not fully sterilizing, but that results in a significant reduction in the parasite load, proves to be a promising strategy, as it this could be used in conjunction with chemotherapy (BERGQUIST, NR et al. Vaccine-linked chemotherapy: can schistosomiasis control benefit from an integrated approach? Trends in Parasitol., v. 21, n. 3, p. 112-117, 2005; FONSECA, CT; OLIVEIRA, SC; ALVES, CC Eliminating schistosomes through vaccination: What are the best immune weapons? Frontiers in Immunology, v. 6, p. 95, 2015). Additionally, a sensitive diagnosis represents a relevant tool for knowing the real prevalence, through the detection of active infections and also for the accurate assessment of the cure rate after therapeutic interventions (Berhe, N.; Medhin, G.; Erko, B.; Smith, T.; Gedamu, S.; Bereded, D.; Moore, R.; Habte, E.; Redda, A.; Gebre-Michael, T.; Gundersen, SG Variations in helminth faecal egg counts in Kato–Katz thick smears and their implications in assessing infection status with Schistosoma mansoni. Acta Trop., v. 92, n. 3, p. 205-212, 2004).

[04] Patent CN101624422 reports on the construction of multiepitope proteins, BSjGCP-BSj23 and BSjGCP-BSj23-BSj28, formed by the antigens of S. japonicum. The patent proposed here has the advantage over this technology because it contains epitopes that are conserved in other species of the genus Schistosoma, not just in S. japocum. This means that the technology described in the present invention has the possibility of diagnosing a wider spectrum of Schistosomes as well as biotechnological applications such as vaccine production and monoclonal antibody production.

[05] Patent CN106608917 reports the construction of a multi-epitope protein formed only by epitopes of a very specific species, S. japonicum katsurada, which has no incidence in Brazil. Thus, a protein formed only by this species could not be effective for diagnosis in Brazil, where the most prevalent species here is S. mansoni. In addition to the advantages of the present invention mentioned above, the present invention has the advantage of being a short antigenic peptide, not being a large multi-epitope protein, with the possibility of presenting a steric hindrance in its molecular conformation, preventing the interaction of epitopes with antibodies in the patient sera.

DISEASE DETECTION METHODS

[06] The diagnosis of schistosomiasis can be performed using parasitological, serological and molecular methods (CARVALHO, GBF New Schistosoma mansoni antigens for serological diagnosis of active infection and cure control. Doctoral Thesis. Fundação Oswaldo Cruz. 2016) . Some parasitological techniques used consist of spontaneous sedimentation (HPJ) (Lutz, A. Schistossoma mansoni and schistosomatosis according to observations made in Brazil. Mem.Inst. Oswaldo Cruz., v. 11, n. 1, p. 121-155, 1919) which was rediscovered by Hoffmann et al. (Hoffman, VA; Pons, JS; Janer, JL Sedimentation concentration method in the schistosomiasis mansoni. PRJ Public Health Trop. Med., v. 9, p. 283-298, 1934); the original Kato-Katz technique described by Kato and Miura (Kato, K.; Miura, M. Comparative examinations. Jpn J Parasitol., v. 3, n. 35, 1954) and modified by KATZ et al. (Katz, N. .; Chaves, A.; Pellegrino, J. A simple device for quantitative stool thick-smear technique in Schistosomiasis mansoni. Rev. Inst. Med. Trop. São Paulo., v. 14, n. 6, p. 397-400 , 1972) and the centrifugation technique with ethyl acetate formalin (TF-Test®) (Three Fecal Test) (Gomes. JF; Hoshino-Shimizu, S.; Dias, LC; Araújo, AJ; Castilho, VL; Neves. FA Evaluation of a novel kit (TF-Test) for the diagnosis of intestinal parasitic infections. J Clin Lab Anal., v. 18, n. 2, p. 132-138, 2004). The Kato-Katz technique is based on microscopic detection of parasite eggs in feces and is the method recommended by the World Health Organization because it is quantitative, low cost and easy to perform (World Health Organization (WHO). Control of schistosomiasis. (Technical Report, 830) Geneva: WHO 1994). However, in individuals with a low parasite load, the diagnosis is difficult due to a smaller amount of eggs excreted in the feces, daily fluctuations in oviposition by female worms and the limited amount of sample to be examined in a single slide, which is about 41.7 mg of feces (Kongs, A.; Marks, G.; Verlé, P.; Van der Stuyft, P. Limitations of Kato-Katz technique for evaluating S. mansoni infections. Trop Med Int Health, v. 6, p. 163-169, 2001). Although there are constant improvements in parasitological methods, they are not able to consistently point out Schistosoma infection in areas of low endemicity (Colley, DG; Bustinduy, AL; Secor, WE; King, CH Human schistosomiasis. Lancet, v. 383, n. 9936, p. 2253–2264, 2014; Wami, WM; Nausch, N.; Bauer, K.; Midzi, N.; Gwisai, R.; Simmonds, P.; Mduluza T.; Woolhouse, M.; Mutapi , F. Comparing parasitological vs serological determination of Schistosoma haematobium infection prevalence in preschool and primary school-aged children: implications for control programmes. Parasitology., v. 141, n. 14, p. 1962-1970, 2014). Thus, the development of other methods for diagnosis and surveillance are required. Antigen and antibody detection assays are promising techniques and can be associated with parasitological techniques (Cavalcanti, MG; Silva, LF; Peralta, RH; Barreto, MG; Peralta, JM Schistosomiasis in areas of low endemicity: a new era in diagnosis Trends Parasitol., v. 29, no. 2, p. 75-82, 2013). The diagnosis of schistosomiasis through molecular detection based on the polymerase chain reaction (PCR) is performed in urine, feces, or organ biopsy samples to detect DNA released from eggs (PONTES, LA et al. Detection by polymerase chain reaction of Schistosoma mansoni DNA in human serum and feces Am J Trop Med Hyg., v. 66, n. 2, p. 157-162, 2002; SANDOVAL, N. et al., A new PCR-based approach for the specific amplification of DNA from different Schistosoma species applicable to human urine samples. Parasitol., v. 133, p. 581-587, 2006). The target DNA consists of a repetitive sequence of 121 base pairs highly represented in S. mansoni genomes of both sexes (Hamburger, J.; Turetski, T.; Kapeller, I.; Deresiewicz, R. Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species-specific probe. Mol Biochem Parasitol., v. 44, p. 73-80, 1991). Although the diagnosis of S. mansoni by the conventional PCR technique reaches specificities and sensitivities that vary from 85.1% to 88% and 96.7% to 97.4%, respectively, there is still room for improvement (Cavalcanti, MG; Silva; , LF; Peralta, RH; Barreto, MG; Peralta, JM Schistosomiasis in areas of low endemicity: a new era in diagnosis. Trends Parasitol., v. 29, n. 2, p. 75-82, 2013). The technique requires several steps after DNA amplification, such as gel electrophoresis and hybridization, which limits the amount of samples to be correctly analyzed (Gomes, LI; Dos Santos Marques, LH; Enk, MJ; de Oliveira, MC; Coelho, PM; Rabello, A. Development and evaluation of a sensitive PCR-ELISA system for detection of schistosoma infection in feces. PLoS Negl Trop Dis., v. 4, n. 4, e664, 2010; Siqueira, LMV; Gomes, LI; Oliveira, E.; Oliveira, ER; Oliveira, AA; Enk, MJ; Carneiro, NF; Rabello, A.; Coelho, PMZ Evaluation of parasitological and molecular techniques for the diagnosis and assessment of cure of schistosomiasis mansoni in a low transmission area. Mem. Inst. Oswaldo Cruz., v. 110, n. 2, p. 209-214, 2015). Regarding immunological methods, although they only provide indirect evidence of the presence of the parasite, they are relevant techniques for the evaluation of infected people in epidemiological investigations (Chieffi, PP; Kanamura, H. Laboratory diagnosis of schistosomiasis mansoni. Rev Bras Malariol Doencas Trop., v. 30, p. 77-97, 1978). Indirect methods for detecting anti-Schistosoma antibodies and for detecting antigens are promising tools that complement the traditional parasitological method (Cavalcanti, MG; Silva, LF; Peralta, RH; Barreto, MG; Peralta, JM Schistosomiasis in areas of low endemicity : a new era in diagnosis. Trends Parasitol., v. 29, n. 2, p. 75-82, 2013). The indirect immunofluorescence reaction, periovular reaction and the Enzyme-linked immunosorbent assay (ELISA) are serological tests used for the diagnosis of the disease. The ELISA assay consists of a technique that uses enzyme-conjugated antigens and antibodies to detect antibodies and antigens, respectively (Engvall, E.; Jonsson, K.; Perlmann, P. Enzyme-linked immunosorbent assay II. Quantitative assay of protein antigen , immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes. Biochimica et Biophysica Acta, v. 251, p. 424-434, 1971; Van Weemen, BK; Schuurs, AHWM Immunoassay using antigen-enzyme conjugates. Febs Letters, v. 15, p. 232-236, 1971). It is the most used serological test for the diagnosis of schistosomiasis, allowing the detection of different classes of antibodies in addition to the use of a wide range of antigens (Gomes, LI; Enk, MJ; Rabello, A. Diagnosing schistosomiasis: where are we? Rev. Soc Bras Med Trop., v. 47, n. 1, p. 3-11, 2014). In general, in the detection of circulating antigens, the capture of the antigen is carried out by monoclonal antibodies. Two antigens derived from the epithelium of the parasites' digestive tract and detected in the serum and urine of patients and animals infected with S. mansoni, the AAC (Anode Circulating Antigen) and the ACC (Cathodic Circulating Antigen) have been more studied (Disch, J .; Garcia, MM; Krijger, GW; Amorim, MN; Katz, N.; Deelder, AM; Gryseels, B.; Rabello, A. Day to day fluctuation of Cca levels in urine of Schistosoma mansoni children infected in Brazil. R Soc Trop Med Hyg., v. 91, no. 2, p. 222-225, 1997; Polman, K.; Engels, D.; Fathers, L.; Deelder, AM; Gryseels, B. Day-to- day fluctuation of schistosome circulating antigen levels in serum and urine of humans infected with Schistosoma mansoni in Burundi. Am J Trop Med Hyg., v. 59, n. 1, p. 150-154, 1998). Despite the high specificity of the method of serological detection of circulating antigens, in regions of low endemicity and cases of low parasite load, the technique may be insufficiently sensitive (Rabello, A. Diagnosing schistosomiasis. Mem Inst Oswaldo Cruz., v. 92, n. n. 5, p. 669-676, 1997; Gryseels, B. Schistosomiasis. Infect Dis Clin North Am., v. 26, n. 2, p. 383-397, 2012). Even present in the serum, a better detection of ACC in urine and a slight decrease (one week) in the concentration of the antigen after chemotherapy treatment (Van't Wout, AB; De Jonge, N.; Tiu, WU; Garcia, EE; Mitchell, GF; Deelder, AM Schistosome circulating anodic antigen in serum of individuals infected with Schistosoma japonicum from The Philippines before and after chemotherapy with praziquantel Trans R Soc Trop Med Hyg, London, v. 86, no. 4, p. 410-413, 1992; Van Lieshout, L.; De Jonge, N.; Mansour, MM; Bassily, S.; Krijger FW; Deelder, AM Circulating cathodic antigen levels in serum and urine of schistosomiasis Trans R Soc Trop Med Hyg, London, v. 87, n. 3, p. 311-312, 1993) and can therefore be used for assessment of cure (El-Morshedy, H.; Kinosien, B.; Barakat , R.; Omer, E.; Khamis, N.; Deelder, AM; Phillips, M. Circulating anodic antigen for detection of Schistosoma tame ni infection in Egyptian patients. Am J Trop Med Hyg., v. 54, no. 2, p. 149-153, 1996). In this context, an immunological method that provided satisfactory results is the rapid urine test (POC-CCA), a qualitative immunochromatographic test for the detection of ACC in urine samples (Rapid Medical Diagnostics, Pretoria, South Africa). In laboratory epidemiological assessments and in endemic areas, this test has shown promise, exhibiting greater sensitivity when compared to the KatoKatz technique (Colley, DG; Binder, S.; Campbell, C.; King, CH; Tchuem Tchuenté, LA; N' Goran, EK; Erko, B.; Karanja, DM; Kabatereine, NB; van Lieshout, L.; Rathbun, S. A five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of Schistosoma mansoni Am J Trop Med Hyg., v. 88, no. 3, p. 426-432, 2013; Lamberton, PH; Kabatereine, NB; Oguttu, DW; Fenwick, A.; Webster, JP Sensitivity and specificity of multiple Kato -Katz thick smears and a circulating cathodic antigen test for Schistosoma mansoni diagnosis pre- and post-repeated-praziquantel treatment. PLoS Negl Trop Dis., v. 8, n. 9, p. e3139, 2014; Adriko, M.; Standley , CJ; Tinkitina, B.; Tukahebwa, EM; Fenwick, A.; Fleming, FM; Sousa-Figueiredo, JC; Stothard, JR; Kabatereine, NB Evaluation of cir culating cathodic antigen (CCA) urine-cassette assay as a survey tool for Schistosoma mansoni in different transmission settings within Bugiri District, Uganda. Acta Trop., v. 136, p. 50-57, 2014; Greter, H.; Krauth, S.J.; Ngandolo, B.N.; Alfaroukh, I.O.; Zinsstag, J.; Utzinger, J. Validation of a Point-of-Care Circulating Cathodic Antigen Urine Cassette Test for Schistosoma mansoni diagnosis in the Sahel, and potential cross-reaction in pregnancy. Am J Trop Med Hyg., v. 94, no. 2, p. 361-364, 2016; Siqueira, L.M.V; Couto, F.F.B; Taboada, D.; Oliveira, Á. THE.; Carneiro, N.F.F; Oliveira, E.; Coelho, M.M.Z; Katz, N. Performance of POCCCA® in diagnosis of schistosomiasis mansoni in individuals with low parasite burden. Rev Soc Bras Med Trop., v. 49, no. 3, p. 341-347, 2016). However, more studies are essential, especially in areas of low endemicity (Legesse, M; Erko, B. Field-based evaluation of a reagent strip test for diagnosis of schistosomiasis mansoni by detecting circulating cathodic antigen (CCA) Ethiopia. Parasite, v. 15, no. 2, p. 151-155, 2008; Lodh, N.; Mwansa, JC; Mutengo, MM; Shiff, CJ Diagnosis of Schistosoma mansoni without the stool: comparison of three diagnostic tests to detect Schistosoma [corrected] mansoni infection from filtered urine in Zambia. Am J Trop Med Hyg., v. 89, n. 1, p. 46-50, 2013). Coelho and collaborators (Coelho, PMZ; Siqueira, LMV; Grenfell, RFQ; Almeida, NBF; Katz, N.; Almeida Á, Carneiro, NFF; Oliveira, E. Improvement of POC-CCA Interpretation by Using Lyophilization of Urine from Patients with Patients Schistosoma mansoni Low Worm Burden: Towards an Elimination of Doubts on the Concept of Trace. PLoS Negl Trop Dis., v. 10, n. 6, p. e0004778, 2016) improved the interpretation of POC-CCA test results through the lyophilization of urine. Diagnosis of schistosomiasis based on antibody detection, on the other hand, is stimulated by the high sensitivity of the method, being applicable not only to individuals from an endemic area, but also to infected tourists who return to non-endemic areas (Doenhoff, MJ; Chiodini, PL; Hamilton, Hamilton, J, V. Specific and sensitive diagnosis of Schistosoma infection: can it be done with antibodies? Trends Parasitol., v. 20, p. 35-39, 2004). However, low specificity was found in early studies, and the method was not able to diagnose cure after treatment (Derouin, F.; Heyer F.; Lariviere, M.; Petithory, J. ELISA in schistosomiasis. Limits. Possibility of application. Pathol. Biol., v. 28, n. 7, p. 465-468, 1980; Da Silva, RM; Kanamura, HY; Camargo, ED; Chiodelli, SG; Nakamura, PM; Gargioni, C. ; Vellosa, SA; Antunes, JL A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area or schistosomiasis. Mem. Inst. Oswaldo Cruz., v. 93, n. 1 , pp. 279-282, 1998; Lin, DD; Xu, JM; Zhang, YY; Liu, YM; Hu, F.; Xu, XL; Li, JY; Gao, ZL; Wu, HW; Kurtis, J. ; Wu, GL Evaluation of IgG-ELISA for the diagnosis of Schistosoma japonicum in a high prevalence, low intensity endemic area of China. Acta Trop., v. 107, n. 2, p. 128-133, 2008). Initially, antigen detection was based on the use of Schistosoma egg homogenate, known as soluble egg antigen (SEA) and soluble S. mansoni adult worm antigen (SWAP) (Lunde, MN;Ottesen, EA Enzyme-linked immunosorbent assay (ELISA) for detecting IgM and IgE antibodies in human schistosomiasis Am J Trop Med Hyg., v. 29, n. 1, p. 82-85, 1980; Hillyer, GV; Gómez de Rios, I. The enzyme-linked immunosorbent assay (ELISA) for the immunodiagnosis of schistosomiasis. Am J Trop Med Hyg., v. 28, n. 2, p. 237-241, 1979; Valli, LC; Kanamura, HY; Da Silva, RM; Silva, MI; Vellosa, SA; Garcia, ET Efficacy of an enzyme-linked immunosorbent assay in the diagnosis of and serologic distinction between acute and chronic Schistosoma mansoni infection. Am J Trop Med Hyg., v. 57, No. 3, p. 358-362, 1997; Noya, O.; Fermin, Z.; Alarco´n De Noya, B.; Losada, S.; Colmenares, C.; Hermoso T. Humor al immune response of children with schistosomiasis. Isotype recognition of adult worm antigens. Parasite Immunol., v. 17, p. 319-328, 1995). The main advantage of using soluble antigen from adult worms compared to the use of soluble antigen from egg is the greater ease of obtaining and the high yield of the antigenic material (Hamilton, JV; Klinkert, M.; Doenhoff, MJ Diagnosis of schistosomiasis antibody detection, with notes on parasitological and antigen detection methods. Parasitology., v. 117, p. 41-57, 1998). However, the disadvantage of using raw antigens is the occurrence of cross-reactions with antigens shared with other parasites (McLaren, M.; Draper, CG; Roberts, JM; Minter-Goedbloed, E.; Ligthart, GS; Teesdale, GH; Teesdale, GH ; Amin, MA; Omer, AH; Bartlett, A.; Voller, A. Studies on the enzyme linked immunosorbent assay for Schistosoma mansoni. Ann Trop Med Parasitol., v. 72, n. 3, p. 243-253, 1978; Correa-Oliveira, R.; Dusse, LM; Viana, IR; Colley, DG; Santos Carvalho, O.; Gazzinelli, G. Human ntibody responses against schistosomal antigens. I. Antibodies from patients with Ancylostoma, Ascaris lumbricoides or Schistosoma mansoni infections react with schistosome antigens Am J Trop Med Hyg., v. 38, n. 2, p. 348-355, 1988; Ishida, MM; Rubinsky-Elefant, G.; Ferreira, AW; Hoshino-Shimizu, S .; Vaz, AJ Helminth antigens (Taenia solium, Taenia crassiceps, Toxocara canis, Schistosoma mansoni and Echinococcus granulosus) and crossreactivities in human infections and immunized animals. Acta Trop., v. 89, no. 1, p. 73-84, 2003; Luo, Q.L.; Qiao, Z.P.; Zhou, Y.D.; Li, X.Y.; Zhong, Z.R.; Yu, Y.J.; Zhang, S.H.; Liu, M.; Zheng, M.J.; Bian, M.H.; Shen, J.L. Application of signaling protein 14-3-3 and 26 kDa glutathione-S-transferase to serological diagnosis of schistosomiasis japonica. Acta Trop., v. 112, no. 2, p. 91-96, 2009). An additional disadvantage of soluble egg antigen is its laborious production, as egg collection must be done from infected laboratory animals (Chand, MA; Chiodinia, PL; Doenhoff, MJ Development of a new assay for the diagnosis of schistosomiasis, using cercarial antigens. Trans R Soc Trop Med Hyg., v. 104, n. 4, p. 255-258, 2010).

[07] Recombinant antigens and those originating from proteomic studies represent a promising tool in the diagnosis of schistosomiasis, however awaiting constant improvements (Gomes, LI; Enk, MJ; Rabello, A. Diagnosing schistosomiasis: where are we? Rev Soc Bras Med Trop. , v. 47, no. 1, p. 3-11, 2014). Relevant efforts were made in order to identify new antigenic targets for a more effective immunological diagnosis (Zhang, M.; Fu, Z.; Li, C.; Han, Y.; Cao, X.; Han, H.; Liu, Y.; Lu, K.; Hong, Y.; Lin, J. Screening diagnostic candidates for schistosomiasis from tegument proteins of adult Schistosoma japonicum using an immunoproteomic approach. PLoS Negl. Trop. Dis., v. 9, n. 2, p. e0003454, 2015). Several proteins have already been tested in ELISA assays and shown to be promising candidates (De Oliveira, EJ; Kanamura, HY; Takei, K.; Hirata, RDC; Valli, LCP; Nguyen, NY; de Carvalho Rodrigues, I.; de Carvalho Rodrigues, I.; de Jesus, AR; Hirata, MH Synthetic peptides as an antigenic basis in an ELISA for laboratory diagnosis of schistosomiasis mansoni. Trans R Soc Trop Med Hyg., v. 102, n. 4, p. 360-366, 2008; De La TorreEscudero , E.; Manzano-Roman, R.; Perez-Sanchez, R.; Barrera, I.; SilesLucas, M.; Oleaga, A. Molecular cloning, characterization and diagnostic performance of the Schistosoma bovis 22.6 antigen. Vet Parasitol., v. 190, n. 3-4, p. 530-540, 2012; Carvalho, GB F; Pacifico, LG G; Pimenta, DL F; Siqueira, LM V; Teixeira-Carvalho, A.; Coelho, PM Z; Pinheiro, CS; Fujiwara, RT; Oliveira, SC; Fonseca, CT Evaluation of the use of Cterminal part of the Schistosoma mansoni 200kDa tegumental protein in schistosomiasis diagnosis and vaccine formulation. p Parasitol., v. 139, p. 24-32, 2014; Zhang, Y.; Zhao, J.; Wang, X.; Xu, X.; Pan, W. Evaluation of six novel antigens as potential biomarkers for early immunodiagnosis of schistosomiasis. Parasit. Vectors., v. 8, n. 447, 2015). A new multidisciplinary tool for identifying new targets for the diagnosis of various diseases, with the deposit of information in public domain databases, immunoinformatics (Carvalho, GBF; Resende, DM; Siqueira, LMV; Lopes, MD; Lopes, DO; Coelho, PMZ; TeixeiraCarvalho, A.; Ruiz, JC; Fonseca, CT Selecting targets for the diagnosis of Schistosoma mansoni infection: An integrative approach using multi-omic and immunoinformatics data. PLoS One, v. 12, n. 8, p. e0182299, 2017), combined with advances in molecular biology and the result of the entire sequencing of the S. mansoni genome (BERRIMAN, M. et al. The genome of the blood fluke Schistosoma mansoni. Nature., v. 460, n. 7253, p. 352–358, 2009), have provided research for immunogenic and specific S. mansoni molecules that can be used as recombinant proteins or as synthetic peptides for the diagnosis of schistosomiasis mansoni (Rabello, A.; Pontes; , LA; Enk, M.J.; Montenegro, S.M.L.; Morais, C. N. L. Parasitological, Immunological and Molecular Diagnosis of Schistosomiasis Mansoni. In: Carvalho, O.S.; Coelho, P.M.Z.; Lenzi, HL Schistosoma mansoni and schistosomiasis: a multidisciplinary view. Rio de Janeiro: Editora Fiocruz, 2008. p. 895-925).

[08] Parasitological methods are not able to consistently pinpoint Schistosoma infection in areas of low endemicity (Colley, DG; Bustinduy, AL; Secor, WE; King, CH Human schistosomiasis. Lancet, v. 383, n. 9936, p. 2253–2264, 2014; Wami, WM; Nausch, N.; Bauer, K.; Midzi, N.; Gwisai, R.; Simmonds, P.; Mduluza T.; Woolhouse, M.; Mutapi, F. Comparing parasitological vs serological determination of Schistosoma haematobium infection prevalence in preschool and primary school-aged children: implications for control programmes. Parasitology., v. 141, n. 14, p. 1962-1970, 2014). Molecular detection through polymerase chain reaction (PCR), although it has high sensitivity and specificity, is costly. Serological tests currently available generally have reduced specificity due to cross-reactivity, making the distinction between schistosomiasis and infections caused by other helminths impractical (Sarhan, RM; Aminou, HA; Saad, GA; Ahmed , OA Comparative analysis of the diagnostic performance of adult, cercarial and egg antigens assessed by ELISA, in the diagnosis of chronic human Schistosoma mansoni infection. Xu, X.; Zhang, Y.; Lin, D.; Zhang, J.; Xu, J.; Liu, YM; Hu, F.; Qing, X.; Xia, C.; Pan, W. Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China. Lancet Infect. Dis., v. 14, n. 6, p. 489-497, 2014; Hinz, R.; Schwarz, NG, Hahn, A.; Frickmann, H. Serological approaches for the diagnosis of schistosomiasis - A review. Mol Cell Probes., v. 31, p. 2-21, 2017). The Enzyme-linked Immnosorbent Assay (ELISA) is currently the most used serological test, however, choosing the appropriate antigen for the development of a test with high sensitivity and specificity is one of the obstacles faced (Rabello, A.; Pontes, LA ; Enk, MJ; Montenegro, SML; Morais, CNL Parasitological, Immunological and Molecular Diagnosis of Schistosomiasis Mansoni. In: Carvalho, OS; Coelho, PMZ; Lenzi, HL Schistosoma mansoni and schistosomiasis: a multidisciplinary view. Rio de Janeiro: Editora Fiocruz , 2008. p. 895-925). Several factors can influence the screening of a candidate antigen for diagnosis, such as size, production and ease of obtainment, adequate stability in simple storage conditions, antigenic capacity, specificity and the possibility of use in accessible and simple serological methods ( Doenhoff, MJ; Chiodini, PL; Hamilton, J, V. Specific and sensitive diagnosis of Schistosoma infection: can it be done with antibodies? Trends Parasitol., v. 20, p. 35-39, 2004). With limited sensitivity and specificity, parasitological and serological methods can become inaccurate. Recombinant antigens have been used as an alternative to soluble egg antigens (SEA) and soluble adult worm antigens (SWAP), aiming at increasing the sensitivity and specificity of the test and consequently reducing the occurrence of cross-reactions that are present in assays using crude antigens . Additionally, the methodology allows for large-scale production, enabling the process of development and commercialization of the diagnostic test (Cavalcanti, MG Schistosomiasis in areas of low endemicity: a new era in diagnosis. Trends Parasitol., v. 29, n. 2 , p. 75-82, 2013; GOMES, LI et al. Diagnosing schistosomiasis: where are we? Rev. Soc. Bras. Med. Trop., v. 47, n. 1, p. 3-11, 2014; Xu , X.; Zhang, Y.; Lin, D.; Zhang, J.; Xu, J.; Liu, YM; Hu, F.; Qing, X.; Xia, C.; Pan, W. Serodiagnosis of Schistosoma japonicum infection: genome-wide identification of a protein marker, and assessment of its diagnostic validity in a field study in China. Lancet Infect. Dis., v. 14, n. 6, p. 489-497, 2014).

ADVANTAGES OF THE PRESENT INVENTION

[09] The present invention provides promising epitopes, one demonstrably recognized by antibodies present in the serum of patients with schistosomiasis, demonstrated by the ELISA assay, which showed 100% specificity and 96.5% sensitivity. This epitope has potential to be used in monitoring cure in epidemiological studies, as it was able to discriminate individuals infected with schistosomiasis from an endemic area from those from a non-endemic area after 30 and 180 days of treatment (Carvalho, GBF; Resende, DM; Siqueira , LMV; Lopes, MD; Lopes, DO; Coelho, PMZ; TeixeiraCarvalho, A.; Ruiz, JC; Fonseca, CT Selecting targets for the diagnosis of Schistosoma mansoni infection: An integrative approach using multi-omic and immunoinformatics data. PLoS One , v. 12, no. 8, p. e0182299, 2017). In addition, this epitope showed homology with the species S. haematobium, thus being able to be used in the diagnosis of the species S. mansoni and S. haematobium. The second epitope that constitutes the synthetic peptide was selected from the anchor surface glycoprotein GPI 200-kDa (Sm200), which is a glycoprotein with unknown function, linked to the membrane of adult worms, initially identified in the integument of the parasite. The sorted epitope showed homology with the species S. haematobium, S. japonicum, and can thus be used in the diagnosis of the 3 species, S. mansoni, S. haematobium and S. japonicum. Given the above, the two epitopes can be used as biotechnological inputs.

[010] The results of the in silico screening showed that the epitopes are antigenic, being recognized by cells of the immune system, such as receptors for B cell antigens and molecules of the human leukocyte antigen (HLA, English: Human leukocyte antigen). also potential immunogenic epitopes. Additionally, these epitopes did not show significant homology with human proteins or with proteins from other helminths that commonly result in cross-reactions, such as Ascaris suum and Ancylostoma ceylanicum, thus avoiding the occurrence of false-negative or false-positive results, respectively. Another factor that corroborates the peptide's potential is the fact that the Smp_150390.1 protein is expressed in the schistosomule, lung schistosomule and adult worm stage (Carvalho, GBF; Resende, DM; Siqueira, LMV; Lopes, MD; Lopes, DO; Coelho, DO; Coelho , PMZ; Teixeira-Carvalho, A.; Ruiz, JC; Fonseca, CT Selecting targets for the diagnosis of Schistosoma mansoni infection: An integrative approach using multi-omic and immunoinformatics data. PLoS One, v. 12, n. 8, p. . e0182299, 2017) and the Sm200 protein is expressed in all phases of the parasite, with a higher level of expression in the 24h schistosomule and adult worm phase. The production of a synthetic peptide consisting of the fusion of these two promising epitopes of the Smp_150390.1 and Sm200 proteins through a flexible linker, has great potential for innovation, since the Smp_150390.1 protein is hypothetical, that is, it has not yet been characterized and the potential of a peptide constituted by the fusion of an epitope originating from a hypothetical protein and an epitope originating from a non-hypothetical protein through a linker, is not recognized. In this context, the peptide originated through the union of these epitopes can be used not only in the diagnosis of schistosomiasis, but can also be used for the production of monoclonal antibodies, vaccines, among others, with the potential to help control the disease, as well as in monitoring cure in epidemiological studies.

DESCRIPTION OF THE FIGURES

[011] Figure 1 shows the prediction of linear epitopes with affinity for the BCR. All epitopes present in the figure were considered for the next step.

[012] Figure 2 shows the antigenicity prediction by the VaxiJen program of the Sm200 protein epitope and the Smp_150390.1 protein epitope selected for the construction of the synthetic peptide. The prediction shows the probability value of antigenicity, with the program threshold being 0.5 (epitopes with values below 0.5 are considered non-antigenic).

[013] Figure 3 shows the prediction of Sm200 protein expression in the different life stages of the parasite. C: cercariae; 3S: 3h schistosome; 24S: 24h schistosome; A: adult worm.

[014] Figure 4 presents an illustrative schematic of the amino acid sequence containing the selected epitopes for the construction of the synthetic peptide, joined by the flexible linker constituted by glycine and serine residues. The amino acid sequences corresponding to the epitopes are in bold.

[015] Figure 5 shows the result of the ELISA assay for the assessment of recognition of the synthetic peptide by antibodies present in the serum of infected patients and standardization of the amount of protein, in which the sensitization was performed with 17.5, 52.5, 157.5 and 472.5 ng of the synthetic peptide per well, at 4°C and for 16 to 18 hours. Blocking was performed with 5% skimmed milk powder at 37ºC for 1 hour. The dilution of positive sera (11, 7, 5 and 2) was 1:100 in blocking solution and the commercial negative control was 1:40 in diluent solution, incubation was carried out at 37°C for 2 hours. The secondary anti-IgG antibody was diluted 1:5,000 in PBST and incubated at 37°C for 1 hour. Development was done with TMB for 30 minutes. After time, the reaction was stopped with 0.2 mol/L sulfuric acid and the reading was taken at 450nm. The * symbol denotes that all concentrations of the synthetic peptide were statistically different from the negative control. Different letters denote significant difference between concentrations in the same group. Caption: CN-commercial negative control; SP11-Positive serum 11; SP7-Positive serum 7; SP5-Positive serum 5; SP2-Positive serum 2.

[016] Figure 6 shows the result of the ELISA assay against different positive sera and comparison of the type of microplate. Sensitization was performed with 52.5 ng of synthetic peptide in 100 µL of adsorption buffer per well, at 4°C/16-18h and blocking with 5% skimmed milk powder at 37°C/1h. The dilution of positive sera (1-20) was 1:100 in blocking solution and the commercial negative control was 1:40 in diluent solution, these were incubated at 37°C for 2 hours. The secondary anti-IgG antibody was diluted 1:5,000 in PBST and incubated at 37°C for 1 hour. Development was done with TMB for 30 minutes. After time, the reaction was stopped with 0.2 mol/L sulfuric acid and the reading was taken at 450nm. The symbol * denotes a significant difference between the negative control and the positive sera. Caption: B- White; CN negative commercial control; 1-20- Positive sera from 1 to 20.

DETAILED DESCRIPTION OF THE TECHNOLOGY

[017] For the construction of the synthetic peptide, two epitopes were selected (Table 1) originating from proteins of Schistosoma mansoni. These epitopes come from proteins already described as being promising in the recognition of antibodies. For the Smp_150390.1 protein, an epitope with high capacity for recognition by antibodies in ELISA has already been described, showing a promising result given that it had 100% specificity and 96.5% sensitivity (Carvalho, GBF; Resende, DM ; Siqueira, LMV; Lopes, MD; Lopes, DO; Coelho, PMZ; Teixeira-Carvalho, A.; Ruiz, JC; Fonseca, CT Selecting targets for the diagnosis of Schistosoma mansoni infection: An integrative approach using multi-omic and immunoinformatics date. PLoS One, v. 12, n. 8, p. e0182299, 2017). Therefore, this epitope was selected for the construction of the synthetic peptide due to its potential for a sensitive and specific diagnosis. Based on the scientific literature, it was found that the Sm200 protein also has great potential, as it is proven to be recognized by antibodies. However, as it is a relatively large protein, in silico analyzes were used to predict and select the best epitope. For this purpose, only part of the protein was used, comprising amino acid 1069 to amino acid 1520, justified by the fact that this region has been described as the most promising, according to Carvalho et al. (Carvalho, GB F; Pacifico, LG G; Pimenta , DL F; Siqueira, LM V; Teixeira-Carvalho, A.; Coelho, PM Z; Pinheiro, CS; Fujiwara, RT; Oliveira, SC; Fonseca, CT Evaluation of the use of C-terminal part of the Schistosoma mansoni 200kDa tegumental protein in schistosomiasis diagnosis and vaccine formulation. Exp Parasitol., v. 139, p. 24-32, 2014). Through bioinformatics screening, it was found that the selected epitope from Sm200 has a high probability of being recognized by B cell antigen receptors, being considered as antigenic by in silico analysis. Furthermore, this epitope does not have significant homology with proteins from other species that are phylogenetically related or not, thus avoiding the occurrence of possible cross-reactions and/or false-negatives in diagnostic tests. Given the above, the synthetic peptide constituted by the fusion of the two selected promising epitopes, through a linker, has great potential in constituting future diagnostic kits, vaccines and production of monoclonal antibodies.

Search for protein sequences and selection of epitopes Searching for protein sequences in public databases

[018] The protein sequence of the Sm200 protein and the epitope from the Smp_150390.1 protein were obtained from scientific articles available in the literature and from GenBank (http://www.genedb.org). Attention was primarily paid to sequences that were demonstrably promising.

In silico analysis of Sm200 protein for epitope selection

[019] The protein sequence Sm200 was analyzed with the aid of bioinformatics tools for prediction and selection of epitopes promising in the recognition of immune system cells. For this, only the protein sequence from amino acid 1069 to amino acid 1520 was submitted to in silico analysis, since this C-terminal portion of the protein was proven to be recognized by antibodies present in the serum of individuals with schistosomiasis.

[020] First, the internal region corresponding to amino acid 1069 to amino acid 1520 was analyzed by the BCpred program (http://ailab.ist.psu.edu/bcpred/) (CHEN, J.; LIU, H.; YANG , J.; CHOU, K. Prediction of linear B-cell epitopes using amino acid pair antigenicity scale. Amino Acids, v. 33, p. 423-4 , 2007) for the prediction of linear epitopes with affinity for the antigen receptor of B cell (BCR, English: B cell antigen receptor). For this screening, the search parameter was adjusted for epitopes containing 16 amino acids and all above the threshold established by the program were considered.

[021] After the prediction and selection of epitopes that were recognized by the BCR, antigenicity prediction was performed using the VaxiJen program (http://ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) (DOYTCHINOVA, IA; FLOWER, DR Bioinformatic Approach for Identifying Parasite and Fungal Candidate Subunit Vaccines. The Open Vaccine Journal, v. 1, n. 1, p. 22–26, 2008). The result indicates the probability of the epitope being antigenic or non-antigenic, based on a predefined cutoff value (0.5) and only those predicted as antigenic were considered. Even though the epitope of the Smp_150390.1 protein was proven to be promising, the prediction of its antigenicity was carried out in order to corroborate its potential for diagnosis.

[022] In order to prevent the occurrence of possible cross reactions and possible false negatives in diagnostic kits, it was verified through the NCBI Blast+ program (http://www.ebi.ac.uk/Tools/sss/ncbiblast /) (CAMACHO, C. et al. BLAST+: architecture and applications. BMC Bioinformatics, v. 10, n. 1, p. 421, 2009) if the epitopes predicted as antigens presented homology with proteins from other phylogenetically related species or not .

[023] After the final selection of the best epitope, the prediction of Sm200 protein expression in different life stages was analyzed: cercaria, schistosome and adult worm of the parasite. This information is available in the GenBank database (http://www.genedb.org).

In-house ELISA assays In-house ELISA assay to assess the recognition of the synthetic peptide by antibodies present in the sera of infected patients and standardization of the protein quantity

[024] In the Greiner brand ELISA microplate (adsorption medium) the protein amount of 17.5, 52.5, 157.5 and 472.5 ng in 100μL of carbonate bicarbonate adsorption buffer pH 9.6 was added to each well. The plate was incubated at 4°C for 16-18 hours for sensitization. After the sensitization time, the plate was washed 3 times with 200 µl of PBST wash buffer (PBS + 0.05% Tween) per well. After washing, the plate was rubbed on absorbent paper to remove excess buffer. Subsequently, 300 µL of blocking solution consisting of 5% skimmed milk powder in PBST was added to each well and the plate was incubated at 37°C for 1 hour. Subsequently, the plate was washed 3 times with 300 µL of wash buffer in each well and then poured onto absorbent paper to remove excess buffer. After washing, 100 μL of positive sera (11, 7, 5 and 2) was added at a 1:100 dilution in blocking solution and 100 μL of the commercial negative control at a 1:40 dilution in diluent solution and the plate was then incubated at 37°C for 2 hours. After incubation with serum, the plate was washed 3 times with 200 µL of wash buffer per well, and again after the wash was completed, the plate was rubbed on absorbent paper to remove excess wash buffer. Subsequently, 100 µL of secondary antibody (IgG conjugated to peroxidase) diluted 1:5,000 in PBST was added to each well and the plate was incubated at 37°C for 1 hour. After time, the plate was washed 3 times with 200 µl of wash buffer per well, poured onto absorbent paper to remove excess buffer. Finally, 100 µL per well of 3.3', 5,5'-tetramethylbenzidine (TMB) developer solution was added, the plate was incubated at room temperature and protected from light for 30 minutes. After the development time had elapsed, the reaction was stopped by adding 100 μL of 0.2 mol/L sulfuric acid to each well. Then, the reading of absorbances was performed in a microplate reader at 450 nm.

In-house ELISA assay with synthetic peptide against different positive sera and comparison of microplate type

[025] In the Greiner (medium adsorption) and Nunc (MaxiSorp) brand ELISA microplates, the protein amount of 52.5 ng in 100μL of adsorption buffer carbonate bicarbonate pH 9.6 was added to each well. Plates were incubated at 4°C for 16-18 hours for sensitization. After the sensitization time, the plates were washed 3 times with 200 µl of PBST wash buffer (PBS + 0.05% Tween) per well. After washing, the plates were rubbed on absorbent paper to remove excess buffer. Subsequently, 300 µL of blocking solution consisting of 5% skimmed milk powder in PBST was added to each well and the plates were incubated at 37°C for 1 hour. Subsequently, the plates were washed 3 times with 300 µL of wash buffer in each well and then poured onto absorbent paper to remove excess buffer. After washing, 100 μL of the positive sera (1-20) was added at a 1:100 dilution in blocking solution and 100 μL of the commercial negative control at a 1:40 dilution in a diluent solution, and the plates were then incubated at 37° C for 2 hours. After incubation with serum, the plates were washed 3 times with 200 μL of wash buffer per well and, again after the end of the wash, they were rubbed on absorbent paper to remove excess wash buffer. Subsequently, 100 µL of secondary antibody (peroxidase-conjugated IgG) diluted 1:5,000 in PBST was added to each well and the plates were incubated at 37°C for 1 hour. After time, the plates were washed 3 times with 200 µl of wash buffer per well, poured onto absorbent paper to remove excess buffer. Finally, 100 µL per well of 3,3',5,5'-tetramethylbenzidine (TMB) developer solution was added, the plates were incubated at room temperature and protected from light for 30 minutes. After the development time had elapsed, the reaction was stopped by adding 100 μL of 0.2mol/L sulfuric acid to each well. Then, the reading of absorbances was performed in a microplate reader at 450 nm.

Results Search for protein sequences and selection of epitopes Searching for protein sequences in public databases

[026] The protein sequence of the Sm200 protein was obtained from the GenBank database (http://www.genedb.org). The internal region from amino acid 1069 to amino acid 1520 was used for the prediction of promising epitopes. For the Smp_150390.1 protein, a promising epitope in the recognition of antibodies was described in a scientific article and this was selected for the construction of the synthetic peptide of the present invention, not being necessary the screening of epitopes by bioinformatics for this protein. The only bioinformatics analysis that was performed for this epitope was the prediction of antigenicity to prove its diagnostic potential.

In silico analysis of Sm200 protein for epitope selection The BCpred program was used to predict linear epitopes with affinity for BCR. Epitopes with a sequence of 16 amino acids were predicted and those that presented a threshold above the established ones were considered. Figure 1 represents the result generated by the program.

[027] After the prediction and selection of epitopes that were recognized by the BCR, the VaxiJen program was used to make the prediction of antigenicity of the selected epitopes. Only epitopes predicted to be antigenic were selected and those said to be non-antigenic were discarded. Furthermore, the VaxiJen program was used to predict the antigenicity of the selected epitope of the Smp_150390.1 protein. Figure 2 represents the result of the prediction of antigenity of the two epitopes selected for the construction of the synthetic peptide.

[028] Using the NCBI Blast+ program, it was verified that none of the selected epitopes of the Sm200 protein has homology with proteins from other species phylogenetically related or not, thus avoiding the occurrence of cross-reactions and false-negative results in diagnostic tests.

[029] At the end of the analyses, the probability of expression of the Sm200 protein was verified in the different life stages of the parasite. It was found that the protein is expressed in all phases of the parasite, with different levels of expression in different phases of life. However, the protein showed a higher level of expression in the 24h schistosome and adult worm phase. Figure 3 represents the prediction of Sm200 protein expression.

In-house ELISA assays In-house ELISA assay to assess the recognition of the synthetic peptide by antibodies present in the sera of infected patients and standardization of the amount of protein

[030] According to the graph, it appears that the protein amounts of 52.5 ng and 472.5 ng resulted in the highest absorbance against positive sera 11 and 5 and the amounts of 17.5 ng and 52.5 ng against positive sera 7 and 2. Since there was no statistical difference between them, the amount of 52.5 ng per well was standardized and used in the subsequent assay. Furthermore, it is noted that there was a statistical difference between the reactivity of the positive serum and the commercial negative control, verifying the veracity of the absorbance of the positive serum and evidencing the ability of the synthetic peptide to discriminate between positive and negative samples. The test result is shown in Figure 5.

In-house ELISA assay with synthetic peptide against different positive sera and comparison of microplate type

[031] After standardizing the protein amount of 52.5 ng per well, a new assay was performed in order to assess the recognition of the synthetic peptide by antibodies present in the serum of different individuals infected with S. mansoni and standardize the type of microplate. It appears that there was a significant difference between the absorbances of positive sera and commercial negative control, demonstrating the reactivity of the synthetic peptide against different positive samples and demonstrating its ability to discriminate between positive and negative samples. Furthermore, it is verified that using the Nunc brand microplate (MaxiSorp) obtained higher absorbances against all positive sera, except for positive serum 5. Therefore, the Nunc brand microplate was standardized. The test result is shown in Figure 6.

Claims (19)

SYNTHETIC PEPTIDE characterized by comprising 1 or more hypothetical antigenic epitopes of Schistosoma flatworm; SYNTHETIC PEPTIDE, according to claim 1, characterized in that it comprises epitopes selected from the amino acid sequences represented by SEQ ID. No.1 and SEQ ID. No.2; SYNTHETIC PEPTIDE, according to claims 1 and 2, characterized in that it comprises two fused epitopes regardless of the rearrangement order represented by SEQ ID. No.1 and SEQ ID. No.2; SYNTHETIC PEPTIDE, according to claims 1 and 2, characterized in that it comprises the amino acid sequence represented by SEQ ID No.3; SYNTHETIC PEPTIDE, according to claims 1 to 4, characterized in that it comprises variants or modifications in the primary structure of the synthetic protein, such as addition, deletion or substitution of amino acids or other chemical groups, mainly one or more substitutions, where an amino acid with a certain property physical and/or chemical exchanged by other amino acids with the same or similar physical and/or chemical and functional properties, or modifications in amino acid residues such as acetylation, glycosylation, methylation, phosphorylation, lipidization, esterification, carboxylation, ribosylation, hydroxylation, sulfation, amidation, polymerized and/or conjugated in any part; SYNTHETIC PEPTIDE, according to claims 1 to 5, characterized in that the peptide consists of the amino acid sequence SEQ ID No.5; METHOD OF DIAGNOSIS of Schistosoma or detection of positive or negative serology in individuals carrying the parasite that causes schistosomiasis, according to claims 1 to 7, characterized in that it comprises the use of a synthetic peptide; DIAGNOSTIC METHOD according to claim 7, characterized in that it preferably comprises the synthetic peptide represented by SEQ ID No.3; IMMUNOGENIC COMPOSITION, according to claims 1 to 8, characterized in that it comprises a synthetic peptide as a pharmaceutically acceptable vehicle; IMMUNOGENIC COMPOSITION, according to claim 9, characterized in that it preferably comprises a synthetic peptide represented by the sequence and amino acids SEQ ID No.3; IMMUNOGENIC COMPOSITION according to claims 9 and 10 characterized in that parts of the peptide function as an adjuvant; DIAGNOSTIC KIT, characterized in that it comprises a synthetic peptide and a secondary anti-human IgG antibody labeled with a substance or enzyme for detecting anti-schistosome antibodies; DIAGNOSTIC KIT according to claim 10, characterized in that it preferably comprises a synthetic peptide represented by the amino acid sequence SEQ ID No.3; DIAGNOSTIC KIT, according to claims 12 and 13, characterized in that it comprises the use in physiological samples of human beings, preferably blood; USE of the synthetic peptide, according to claims 1 to 14, characterized in that it is a diagnostic kit prepared for the detection of schistosomiasis; USE of the synthetic peptide according to claims 1 to 15, characterized in that it is useful in the preparation of monoclonal antibodies; USE of the immunogenic composition, according to claims 1 to 16, characterized in that it is useful in the preparation of a drug or drug to prevent or treat schistosomiasis; USE of the synthetic peptide, according to claims 1 to 17, characterized in that it is useful in the preparation of an anti-schistosomiasis vaccine; USE of the immunogenic composition, according to claims 1 to 18, characterized in that it is useful in the preparation of an anti-schistosomiasis vaccine.

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