BRPI0903089B1 - RECOMBINANT PEPTIDES AND MIMETIC PROTEIN MOTIVES TO LEISHMANIA ANTIGENS AND THEIR APPLICATIONS - Google Patents

Abstract

recombinant peptides and protein motifs mimetic to leishmania antigens and their applications. the present invention refers to the selection, characterization and use of recombinant peptides, their reverse sequences and protein motifs that mimic partial antigenic regions of leishmania proteins and antigens of diagnostic and vaccine interest for diseases caused by parasites of the leishmania genus. the peptides object of this invention were selected by "phage display" and are targets for use in immunodiagnostics and in vaccine compositions for the control of different types of disease caused by parasites of the aforementioned genus, as they are specifically immunoreactive with the serum of infected individuals.

Description

The present invention relates to the selection, characterization and use of recombinant peptides, their reverse sequences and protein motifs that mimic partial antigenic regions of Leishmania proteins and antigens of diagnostic and vaccine interest for diseases caused by parasites of the Leishmania genus. The object peptides of this invention are targets to be used in immunodiagnostics and vaccine compositions for the control of different types of disease caused by parasites of the aforementioned genus.

Despite progress in diagnosis and treatment, leishmaniasis remains a public health problem, particularly in developing countries. The impact of leishmaniasis on human health has been grossly underestimated for many years, and this disease is now classified as one of the major neglected diseases by the World Health Organization (Zimmermann S. et al., Protist.- 160:151-8, 2009).

The causative agent of leishmaniasis, Leishmania, is an obligate intracellular trypanosomatid parasite, which has a biphasic life cycle consisting of a uniflagellate promastigote stage, which resides within the vector's digestive tract, and an amastigote stage, which multiplies within macrophage phagolysomes of mammalian cells (Wong AKC Biochem. Cell Biol.; 73:235-40, 1984). There are 30 species of Leishmania of which 10 occur in the Old World and 20 in the New World. All Old World species belong to the subgenus Leishmania and seven are known to infect man. There are also 11 species of the same subgenus in the New World, of which five are not found in man. Parasites of the Viannia subgenus occur only in the New World and of the nine known species, eight infect man (Shaw J. J. Mem. Inst. Oswaldo Cruz; 89:471-8, 1994).

Parasites of the genus Leishmania are transmitted by bites from mosquitoes of the genus Phlebotomus in the Old World and Lutzomya in the New World (Desjeux PH, Clin. Dermatol.; 80:271-4, 1996, Grimaldi G. Tesh RB, Clin. Microbiol. Rev. ; 6:230-50, 1993).

Depending on the parasite species and the host's immunological status, manifestations can range from chronic skin lesions to the lethal form of the infection (Murray HW et al., Lancet, 366:1561-1567, 2005), presenting the following spectrum of clinical manifestations : tegumentary leishmaniasis that varies from the cutaneous to the mucocutaneous form (LC and CML), representing the responsive pole for diffuse cutaneous leishmaniasis (LCD) and visceral leishmaniasis (VL), which covers the subclinical form and the fatal disease (Oliveira CI et al ., Drug. Discov. Today: Dis. Models; 1:81-6, 2004).

Leishmaniasis is a public health problem and affects 88 countries, 72 of which are classified as developing countries, including 13 from the least developed countries. Of VL cases, 90% occur in just five countries - Bangladesh, India, Nepal and Brazil; 90% of CL cases occur in just seven countries - Afghanistan, Algeria, Brazil, Iran, Peru, Saudi Arabia and Syria. The annual incidence of leishmaniasis is 1.5 to 2 million cases, with 1-1.5 million for cutaneous leishmaniasis and 500,000 for the visceral form; with urbanization as a crucial factor for incidence (WHO.77?e Weekly Epidemiological Record, 77:365-72, 2002 and www.who.int/tdr/diseases/leish).

Brazilian leishmaniasis behaved like a rural anthropozoonosis, but after the 1980s, its expansion was also observed to the periurban regions of large cities. Human-caused environmental changes have modified the epidemiological profile of leishmaniasis in areas where it is related to wildlife, as well as in areas where transmission occurs in peri-urban rural areas or urban neighborhoods and areas around homes. In each area, transmission depends on the adaptation of mosquito species (potential vectors) to these environments and involves domestic animals (L. chagasi, causing the visceral form of leishmaniasis, and L. braziliensis, causing the cutaneous and mucosal form) (Tolezano JE et al., Rev. Inst. Adolfo Lutz; 40:49-54, 1980; Marzochi MCAJ Bras. Med.; 63:82-104, 1992; Marzochi MCA et al., Cad. Saude Publica; 10:359- 75, 1994).

Several mammal species have been reported to be naturally infected with Leishmania spp., in endemic areas dogs are considered the major reservoir of Human Visceral Leishmaniasis (Palatinik-de-Souza, CB et al., Am. J. Trop. Med. Hyg. Hyg. .; 65:510-517, 2001 and WHO, Report of the Second WHO Meeting on Emerging Infectious Diseases, 1995).

The diagnosis of VL cannot be based solely on clinical symptoms because it shares its symptoms with other diseases such as malaria, typhoid and tuberculosis, commonly occurring in the same area as leishmaniasis (Goto Y. et al, Infect. Immun.', 74 :3939-45, 2006). For zoonotic VL, a quick and safe diagnosis is a necessary tool due to the great variability of clinical symptoms and the presence of asymptomatic but infective dogs (Dye C et al., Epidemiol Infect.' 103:647-56, 1993). The number of asymptomatic infected people is higher than the number of infected people who have symptoms of the disease. For this reason, it is important to know how infected people will develop the disease and how they can be diagnosed before showing clinical manifestations (Singh S. et al., J. Parasito/.; 81:1000-3, 1995).

The choice of antigens is important, as the use of whole antigens or whole parasites often results in low specificity in detecting disease-specific antibodies (Reed SG et al., J Immunol.', 138:1596-601, 1987 ).

In recent years, several antigens have been characterized with the aim of improving the vaccine potential and diagnosis of human and canine leishmaniasis. The development of polymerase chain reaction and immunoassays based on recombinant antigens are aimed at improving the sensitivity and specificity of the diagnosis of Visceral Leishmaniasis and decreasing the cross-reactivity shown by total antigens (Gomes YM et al., Vet. J.\ 175 :45-52, 2008).

The most commonly used drug, since the 1950s, is the N-methyl Glucamine antimoniate (Glucantime®) and remains the method of choice for initial treatment, which is resistant in 10% of cases. In this circumstance, Amphotericin B is used, which was recently associated with liposome particles (artificial lipid particles), reducing the systemic effects caused by the treatment (Berman JD Clin. Infect. Dis.; 24:684-703, 1997 and Lee MB et al., Infect. Med.; 16:34-45, 1999).

In the current situation, chemotherapy has not been sufficient for the zoonotic control of visceral leishmaniasis and vaccination needs to be considered; Understanding the immunological mechanisms involved in resistance and susceptibility to Leishmania infection is critical to the development of immunoprophylactic tools (Rosa R. et al., Vaccine; 25:4525-32, 2007).

In recent years, DNA cloning and the characterization of Leishmania-derived recombinant antigens and proteins have been carried out, leading to a great advance in diagnostic methods and in the search for an effective vaccine for the prophylaxis of leishmaniasis. Some of these antigens are described below.

Molecules such as Glycosylphosphatidylinositol (GPI), Glycosylphospholipids (GIPL), Lipophosphoglycans (LPG), Phosphoglycans (FG), Proteophosphoglycans (PPG), Leishmaniolysin (gp63) and Cysteine Proteases (CPs), are discriminated as crucial for Leishmania infection but not produce no pathology in the host (Chang KP et al., Kinetoplast. Biol. Dis.; 1:1,2002).

Promastigote Surface Proteins (PSP), also known as gp63, are membrane surface glycoproteins that have proteolytic activity, this protein is highly immunogenic, showing cross-reactivity between different Leishmania species, although the antibodies produced by this molecule do not are effectors in infection control (Mood SF Acta Trop.; 53:185-204, 1993 and Wright EP et al., Biochem. Cell Biol.; 525-36, 1989). Differential expression of the gp63 genes results in different amounts of the gp63 protein in the in vitro development from promastigote forms to the infectious form (Ramamoorthy R. et al., J. Biol. Chem.; 267:1888-95, 1992).

Fucose-Mannose (FML) binding glycoconjugates constitute a complex of antigenic proteins of promastigote forms of L. (L.) donovani that are used in serological tests for diagnosis, evaluation of prognosis and control of human kala azar, in addition to being proposed in screenings from blood donors from endemic regions of kala azar (Otero ACS et al., Am. J. Trop. Med. Hyg.; 62:128-131, 2000 and Palatinik de Souza CB et al., Trans. R. Soc. Trop Med. Hyg.; 89:390-3, 1995).

The main immunoprotective component of FML is the glycoprotein fraction designated GP36 (Paraguay de Souza E. et al., Vaccine, 19:3014-15, 2001), recognized only by sera from patients with human kala azar (Palatinik de Souza CB et al. , Rev. Soc. Bras. Med. Trop.; 29:153-63, 1996). The gene encoding L. (L.) donovani nucleoside hydrolase, which is a component of the GP36 antigen, was isolated demonstrating that recombinant LdNH is useful in diagnosing kala azar in dogs as it preferentially reacts with IgG subtype 1 immunoglobulin that is increased in dogs susceptible to kala azar and in advanced disease. (Santana D.M. et al., Mol. Biochem. Parasitol.; 120:315-9, 2002).

Molecular cloning, characterization and expression of the K9 and K26 antigens (Bathia A. et al., Mol. Biochem. Parasito.; 102:249-261, 1999) demonstrated that K26 (247 amino acid hydrophilic protein, containing 11 repeats) 14 amino acids, 64% of total protein) is a highly specific diagnostic marker to assess infection in the serum of individuals with visceral leishmaniasis.

The antigen called KMP-11 (the main membrane protein determinant of kinetoplastid protozoa) appears to be antigenic and shows a relatively strong antibody response during the natural course of leishmaniasis and Chagas disease. The observation of immunological cross-reaction in infections caused by Leishmania and T.cruzi is particularly important, contradicting studies that reported the high sensitivity of detection of these antibodies in patients with leishmaniasis. The mapping of antigenic determinants on KMP-11 using synthetic peptides revealed the predominant existence of conformational epitopes in Leishmania infections (Trujillo C. et al., Immunol. Lett.; 70:203-9, 1999).

A Leishmania chagasi - LcKin antigen, with homology to the motor protein superfamily (kinesins), called K39, has been shown to predominate in amastigotes, and contains an extensive repetitive domain highly related between L. chagasi and L. donovani species. High antibody titers were found to the rK39 antigen, which contains several 39 amino acid repeats, demonstrating the conservation of the repeat region among the species mentioned (Burns JM et al., Proc. Natl. Ac. Sci.; 90:775-9 , 1993). With the discovery of this antigen, many researchers have tested its viability in different diagnostic tests for Visceral Leishmaniasis, most of them showing highly promising results, in an attempt to use diagnostic methods that are less invasive and economically viable for the population. The detection of IgG antibodies to this antigen has been extremely sensitive and specific in the diagnosis of Visceral Leishmaniasis, although it is difficult to point out any protective role for these specific antibodies (Carvalho SF et al., Am. J. Trop. Med. Hyg.; 68 :231-324, 2003 and Chang KP et al. Kinetoplasti. Biol. Dis.; 1:1, 2002).

Several authors point out the feasibility of diagnostic tests using the rk39 antigen in different methodologies. Specificity and sensitivity are in most cases very promising. A total of 228 parasitologically confirmed cases were tested using rK39 in ELISA for IgG capture and immunochromatographic test (“dipstick test”), the results showed 100% sensitivity and specificity for both cases (Singh S. et al., Clin. Diag. Lab. Immunol.; 9:568-72, 2002); Similar results were obtained showing the sensitivity of the rK39 strip test and ELISA antigen of 90% and 89%, respectively, while the specificity was 100% and 98% respectively (Carvalho SF et al., Am. J. Trop. Med. Hyg.; 68:231-324, 2003). The effectiveness of the rK39 antigen in Tests of

Direct Agglutination - DAT (100% sensitivity and specificity) and dipstick test with 85.7% sensitivity and 82% specificity (Schallig HD; et al., Mymensingh Med. J.; 12:93-7, 2002 ).

Similar analyzes were performed for the ELISA tests (Salotra S. et al., Methods Enzymol.; 217:228-57, 2003; Maalej IA et al., Am. J. Trop. Med. Hyg.; 68:312- 20, 2003; Sreenivas G. et al., Br. J. Biomed. Sci.; 59:218-22, 2002; Braz RF et al., Am. J. Trop. Med. Hyg.; 67:244-348 , 2002; Singh et al., J. Parasitol.; 81:1000-3, 1995; Qu JQ et al., Trans. R. Soc. Trop. Med. Hyg.; 88:543-5, 1994; Kumar R. et al., Clin.Diag. Lab. Immunol.; 8:1220-4, 2001; Ozensoy S. et al., Am. J. Trop. Med. Hyg.; 59:363-9, 1998; Zijlstra EE et al., Am. al.; Clin. Diagn. Lab. Immunol.; 5:717-20, 1998; Medrano FJ et al., Am. J. Trop. Med. Hyg.; 59:155-62, 1998; Badaró R et al. , J. Infect. Dis.; 173:758-61, 1996).

Many authors have evaluated the use of rK39 using immunochromatographic tests (Sarker CB et al., Mymensingh Med. J.; 12:93-7, 2003; Carvalho SF et al., Am. J. Trop. Med. Hyg.; 68: 231-324, 2003; Boelaert M. et al., Am. J. Trop. Med. Hyg.; 70:72-7, 2004; Chappuis F. et al., Trop. Med. Int. Health.; 8: 277-285, 2003; Veeken H et al. Trop. Med. Int. Health.; 8:164-7, 2003; Qu JQ et al., Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Za Zhi 18:155- 8, 2000; Delgado O. et al., Parasite; 8:355-7, 2001; Berm C. et al., Am. J. Trop. Med. Hyg.; 63:153-7, 2000 and Zijlstra EE et al. al., Trop. Med. Int. Health; 6:108-3, 2001).

Although the antigen has been reported as satisfactory, results vary considerably in different endemic areas, for reasons that are not yet clear, Sudanese patients develop lower anti-rK39 antibody titers than Indian patients, although the test format may be the factor because other brands of immunochromatographic tests work best in this region (Sudar S. et al., Trop. Med. Int. Health; 12:284-9, 2007 and Titmeijer k. et al., Am. J. Trop. Med. Hyg.; 12:74-76, 2006).

An antigen from L. major, designated LACK (Leishmania homologue of receptor for activated C kinase), was identified by searching for antigens recognized by a protective Th1 clone derived from the spleen of a BALB/c mouse infected with a soluble extract of promastigotes. L. major. The deduced amino acid sequence of this antigen has substantial homology to the intracellular receptors for activated Kinase C (RACKs) and shows high homology between L major and L. chagasi (Mougneau E. et al., Science; 268:245-52, 1995) . It has been suggested that the LACK protein contains an immunodominant epitope that represents the target of the initial immune response (Requena J.M. et al., Parasitol. Today; 16:246-50, 2000). The LACK antigen is a well-conserved 36 KDa protein present in all Leishmania species and developmental stages and is one of the most immunogenic antigens (Mougneau E. et al., Science; 268:245-52, 1995). This antigen has been used to evaluate the use of DNA vaccines encoding this and other antigens (eg, LmSTH and TSA). The combination of these antigens has provided durable and complete protection against the development of skin lesions (Méndez S. et al., Vaccine; 31:3702-4, 2002).

Some of the antigens described above are objects of study in the search for effective vaccines, chemotherapeutics or immunodiagnostics for Leishmaniasis, being the subject of the patents described below:

Patent FR2844511 demonstrated the use of 16 amino acid peptides and their variations for the treatment of leishmaniasis and induction of the Th1 immune response in mammals, also describes the use of a diagnostic kit containing one of these peptides or their derivatives linked to biotin or polylysine molecules coupled to nitrocellulose or other polymers such as membranes, latex or other materials.

WO9711180 and WO0179276 describe compositions and methods for prevention, treatment and diagnosis of leishmaniasis and stimulation of the immune response in patients using immunogenic portions of the M15, Ldp23, Lbhsp83, Lt-210 and LbelF4A, Lmspla, Lmso9A and MAPS-1 antigens of Leishmania or portions of these and their variations. For the same purpose, patent WO2007121184 describes the use of polypeptides and fusion proteins containing at least an immunogenic portion of one or more Leishmania antigens and variants through the selection of "tandem" protein regions.

Patent CA2503932 demonstrates new Leishmania major proteins, which comprise secreted or excreted polypeptides and polynucleotides and their functional fragments and the use of these peptides as vaccines and therapeutics, in addition to the use of antibodies generated from them.

The patent WO2007142695 describes the use of a rapid diagnostic kit for detection of amastigotes, promastigotes or secreted or excreted proteins of Leishmania, which can be used in the field leading to faster treatment of cutaneous leishmaniasis, using anti-TSA antigen antibodies (Thiol Specific Antigen).

Patent PI0605889-2 demonstrates the use of soluble antigen in the diagnosis of canine visceral leishmaniasis through the immunoenzymatic ELISA reaction.

Patent US5965142 describes compositions and methods for the diagnosis of Leishmania tropica using one or more epitopes of the Lt-210 protein, with potential use in various types of immunoassays for the detection of this parasite, such peptides being also able to be used in vaccine compositions.

Patent US2007292847 describes new amino acid and nucleic acid sequences of Leishmania major (LmPDE - phosphodiesterases) and antibodies binding to these sequences for complex formation in the diagnosis and treatment of disorders related to diseases caused by this parasite.

Patent WO9633414 describes a method of preventing and serologically diagnosing Leishmania infection, by means of one or more polypeptides in diagnostic methods and kits for evaluating individual blood samples and identifying asymptomatic individuals. These polypeptides comprise an epitope of LcPO (Leishmnia chagasi homolog of eukaryotic acid ribosomal P-protein family) and variants thereof that differ only in conservative substitutions or modifications.

Patent WO2005063803 describes the use of compounds and methods for the detection of antibodies in individuals suspected of infection by parasites of the Leishmania genus, mainly Indian or correlated strains, and the use of a related diagnostic kit; demonstrating the isolation, characterization and use of the immunogenic region of the kinesin-related gene, isolated from the Leishmania donovai strain MHOM/IN/DD8 and MHOM/IN/KE16/1998 and correlation of nucleic acid sequences isolated from Indian strains and L. chagasi .

Patent US7008774 proposes an immunoassay for detection of IgM and IgG type antibodies and detection of circulating antigens in samples from individuals with visceral, cutaneous or canine leishmaniasis. It also reports the use of an immunocapture immunoassay to detect antigens released by parasites.

Patent ES2133236 describes the use of a recombinant antigen for serological diagnosis of canine leishmaniasis using a chimeric gene encoding L. infantum specific antigens (LiP2a, LiP2b, LIPH2A, LiPO); for this purpose, the DNA fragments of the genes of these proteins were sequentially added in the 5' region of the vector, generating a polypeptide with a molecular mass of 38K and an isoelectric point of 7.37.

Patent WO9639524 describes methods and compounds related to LbelF4A or LmelF4A antigens of Leishmania, analyzing the immune response for both the protein and the antigen encoded by DNA vaccine.

DE10156679 demonstrates the use of p36-1_ACK antigen in the construction of an expression cassette for treatment and vaccine composition for leishmania infections. Likewise, patent US2004235772 describes DNA expression using p36 LACK antigen for treatments of Leishmania infections as well as corresponding vaccine and patent WO2005039633 demonstrates DNA expression for immune response generation using p36-LACK, TSA, antigens Kmp-11 and the Leishmania infantum gp63 antigen or its derivatives with corresponding function.

Patent WO0181388 reports the use of polypeptides, proteins and nucleic acids that contain at least a portion of the protein Lip2a (acidic ribosomal protein from Leishmania infantum) or variants thereof to generate an immune response in individuals or cell groups or be used as therapeutic agents for Leishmania and as an adjuvant for other parasites.

The patent WO2007144903 uses the KMP-11 antigen as a hybrid cell vaccine against leishmaniasis, while the patent US2008145385 describes methods to immunize mammals infected with virulent strains of Leishmania by administering a KMP-11 antigen DNA vaccine (Kinetoplastid Membrane Protein-11 ).

Patent US2008026467 presents DNA sequences capable of encoding PSAs (Promastigote Surface Antigens) of Leishmania promastigote and amastigote forms to be used in vaccine compositions against Leishmania.

Patent US2004170636 describes the production of a DNA vaccine that stimulates an immune response against Leishmania infections using the A2 gene of Leishmania donovani. The use of this antigen is also the basis of patent PI0603490-0, which emphasizes its use in vaccines, ensuring the differentiation of naturally infected animals from those vaccinated. Patent PI0601225-6 also describes the use of vaccine antigens from different species, aiming at this discrimination.

Patent WO2006122382 (BRPI0503187) describes the obtainment of vaccine compositions that block the transmission of leishmaniasis in humans and animals from Leishmania fractions (Fucose-Mannose Ligand - FML of amastigotes or promastigotes of L. donovani).

The patent WO2006110915 describes the use of a vaccine comprising one or more antigens selected from a group of homologues of Leishmania chagasi or hypothetical proteins of L. infantum and protein of group K-39 of L. infantum.

The patent WO02072792 demonstrates that the use of the Leishmania TSA or MAPS (Leishmania thiol-specific thiol-specificantioxidant) proteins, LelF (L. braziliensis gene homologous to the eukaryotic ribosomal protein elF4A, also called "LbelF4A"), M15 (L. major stress-inducible 1 or LmSTII) and 6H (L. braziliensis gene homologous to the gene for the eukaryotic 83-kDa heat shock protein, also called "Lbhsp83") when fused to heterologous polynucleotide sequences are able to increase polynucleotide expression heterologous in eukaryotic cells. These isolated or purified Leishmania fusion polypeptides can be used for treating or preventing infections by Leishmania or other organisms such as M. Tuberculosis.

Patent FR2862313 describes the construction and isolation of nucleic acids that encode an immunogenic region of Leishmania amastigotes and promastigotes, which can be used in vaccine compositions or to generate antibodies and be used in diagnosis.

Patent FR2826018 describes the isolation of the gene encoding the LmPDI protein, which had two regions with identical sequences (Cys-Gly-His-Cys) recognized as a therapeutic target for drug development and a new element for the composition of vaccines against Leishmania for humans and animals.

Phage Display technology or phage display of biomolecules has shown increasing use in several areas of science since it was described in 1985 (Smith G.P. Science 228:1315-17, 1985). This author pioneered the expression of restriction enzyme Eco RI as a fusion of viral capsid protein three (pill).

Bacteriophages, or simply phages, are viruses that infect a variety of Gram-negative bacteria using the sex pilus as a receptor. The filamentous phage particles (lines M13, f1 and fd) that infect E. coli via the F pilus consist of single-stranded DNA enclosed in a protein capsule. A viable phage expresses approximately 2,700 copies of the gene 8 protein (g8 or pVIII, a 50 amino acid residue protein) and 3 to 5 copies of the gene III (p3p or pIII) 406 amino acid protein (Russel M., Mol. Microbiol. ; 5:1607-13, 1991).

In systems where all the pill or pVIII are used, the size of the protein inserted in the vector is limited, as large proteins interfere with the functions of the capsid proteins, making the phage less infective. This Phage display system was created for the display of small peptide libraries (maximum 30 amino acids) (Phizicky E.M. Fields S., Microbiol. Rev.; 59:94-123, 1995).

Filamentous phage display is based on the cloning of DNA fragments encoding millions of variants of certain ligands (Benhar I. Biotechnol. Adv.; 19:1-33, 2001) as proteins, including antibodies, or peptides. The DNA sequences of interest are inserted at a location in the genome of filamentous bacteriophage such that the encoded protein is expressed on the surface of the filamentous phage as a fusion product to one of the phage surface proteins (Azzay HME et al. Clin Biochem.; 35:425-45, 2002).

The connection between genotype and phenotype allows for the enrichment of specific phages, for example, using selection on an immobilized target (Benhar I. Biotchno. Adv.; 19:1-33, 2001).

The “Biopanning” or selection procedure is done by incubating the library of exposed phage peptides against the target. Most of the time, the target is retained on ELISA plates, but “beads”, resins and membranes are also used. Non-target-binding phages are eliminated by successive washes, and specific phages remain attached for further elution. The pool of specific phages is amplified for subsequent rounds of biological selection or biopanning (binding, elution and amplification cycles) to enrich the phage pool with specific sequences against the target. After three or four passages, individual clones are characterized by DNA sequencing, Western Blot or ELISA (Smith G.P. Science; 228:1315-17, 1985).

As an alternative to panning against a target immobilized on a surface, the library can react with a target in solution, given by the capture affinity of the target-phage complex on a specific affinity matrix (“bead”) for the target protein. The experiment requires substantially less target per experiment than surface panning, which may result in greater accessibility of the binding site for peptides expressed on phages, as well as avoiding partial target denaturation on the plastic surface (Barbas, C. et al. ., Cold Spr. Har. Lab. Press, NY; 2001).

Synthetic oligonucleotides with a constant length but unspecified codons, randomized by site-directed mutagenesis using degenerate deoxynucleotides, are cloned as a fusion to one of the M13 phage capsid proteins, where they are expressed as capsid-bound fusion proteins. Phage-bound peptides exhibit great mimetic potential to linear, conformational, or non-protein epitopes (Smith GP Curr. Opin. Biotechnol.; 2:668-73, 1991; Smith GP et al., Gene, 128:37-42, 1993 ).

Phage-fused peptide libraries have been widely used in the study of interactions between antigens and antibodies (Cortese R. et al., Trends Biotechnol.; 12:262-7, 1994; Birch-Marchin I. et al., J. Virol. Methods; 88:89-104, 2000; Christopher G. et al. Infect. Mmunol.; 67:4679-88, 1999), these works demonstrate obtaining specific peptides by selecting phage libraries with monoclonal and polyclonal antibodies , linear epitopes, as well as mimetopes, those that mimic linear, discontinuous, conformational and even non-peptide antigen epitopes.

Peptides selected against a particular target that have similar sequence play a role in identifying the motif required for binding (Stephen C.W. et al., J. Mol. Biol.; 225:577-83, 1992). In cases where the selected peptides resemble the natural ligand peptide, they are termed mimetopes (Geysen HM et al., Mol. Immunol.; 23:709-715, 1986; Salotra Smith GP et al., Methods Enzymol.; 217: 228-57, 1993). Peptide sequences identified by "Phage Display" have been shown to be receptor agonists or antagonists (Doobar J. Winter G.J., Mol. Biol.; 244:361-9, 1994).

Peptides that neutralize immunoglobulins can be employed as diagnostic reagents or used as therapeutic agents in controlling autoimmune diseases (Blank M. et al., Proc. Natl. Acad. Sci.; 96:5164-68, 1999). Random peptide libraries can be used to map polyclonal and monoclonal antibody epitopes, identifying binding peptides, and developing phages that define sites for different enzymes (Mattheus DJ et al., Science, 260:1113-7, 1993; Ohkubo et al. , Comb. Chem. High Throughput Screen.; 4:573-83, 2001).

Phages expressing epitopes or mimetopes may emerge as a useful tool for the development of effective vaccines or may serve as vaccine delivery vehicles in their own right (Benhar I. Biotechnol. Adv.; 19:1-33, 2001).

The presentation of small peptides on the surface of viral particles can increase their immunogenicity and consequently their potential as vaccine candidates. The immunogenic response to M13 phages is T-cell dependent and does not require adjuvant (Azzay H.M.E. et al., Clin. Biochem.; 35:425-45, 2002).

So far, there is no evidence in the literature revealing experiments using the “Phage Display” technique in the study of antigen-antibody interactions with the protozoan Leishmania. However, several authors report the use of this technique in the understanding of diseases caused by viruses, bacteria, protozoa and the interaction between their antigens and the host's immune response, seeking to map epitopes or develop vaccines for: Plamodium falciparum (de la Cruz VF et al., J. Biol. Chem.; 263:4318-22, 1988; Willis AE et al., Gene, 128:79-83, 1993), Taenia solium ( Gazarian KG et al., Immunol. Lett. ;42:191-5, 2000), Infectious Bursa Disease Virus (Cui X. et al., J. Virol. Methods; 109:75-83, 2003), Eimeria tenella (Abi-Ghanem D. et al. , Vet. Immunol. Immunophatol.; 121:58-67, 2008), Echinococcus granulosus (Read AJ et al., J. Chromatogr.B; 877:1516-22) and human neurocysticercosis (Hell RCR et al., Clin. Immunol.; 13:129-38, 2009) among others.

Several patents demonstrate the use of the “Phage Display” technology in the study of parasitic diseases such as the patents: US2007281302 (Bacterial binding peptides: B. anthracis, B. subitilis and B. cereus and their use for detection of B. anthracis); US2007141076 (identification of antigenic fragments of Toxiplasma gondii proteins and their use in diagnosis and as an immunogenic agent); CN101015691 (development of a universal microsphere vaccine against Infuenza virus type B by inserting the M2 region of the virus into bacteriophage T7); WO2006071896 (severe acute respiratory syndrome (SARS) vaccine, comprising antigenic epitopes of the virus) and US2006094017 (discovery of specific mimetopes for the gp120 protein of the HIV virus and their use as immunological conjugates in vaccination against the virus and also as diagnostic tools ).

Given the feasibility of the technology described above, it is important to emphasize that this technique complements the current trend of genomic projects, in which a large volume of information is generated, but with little phenotypic description. With the phage display technique, we can now test genes of interest on a large scale, in search of a set of interacting proteins, which has been called an “interatoma”. The search for these “interatomas” promises to expand the limits created with genomic projects and allows us to infer how these genes are related, explaining the individual phenotypically (Brígido M.M. Biotecnologia Ciência e Desenvolvimento; 26:44-51, 2002).

Methodologies for the use of this technique in the expression of recombinant polypeptides and improvement of the technology were described in patents: KR20070041966 (method for exposure of antibodies fused to plll protein on the surface of recombinant phage with greater stability and practicability); US2009105090 (use of mutant plll protein with amino acid insertion to promote a change in peptide conformation); WO9958655 (use of bacteriophage expressing heterologous polypeptides bearing a cleavage site within the expressed peptide that enables propagation of the intact fusion protein); WO2009024591 (alternative phage display using pVII protein and marketing kit thereof); US7083945 (expression of proteins in soluble form for interaction with tagged ligands), US2006068421 (expression vectors and new expression methods that allow efficient selection and selection of polypeptides), US2005202512 (methods for population selection of functional polypeptides), GB2408332 (specific binding methods for cell surface antigens), US2003104604 (proposes increasing the efficiency of the technique, through expression in bacteria), EP1452599 (libraries that express phages with various peptides or mutant proteins capable of binding to materials of interest ) and US2006035223 (peptides with binding capacity to inorganic materials such as silica, cobalt, iron or oxides) in which methodologies that improve the technique are proposed.

The present invention used the "Phage Display" technology for the identification and selection of specific peptides and protein motifs related to the Leishmania genus parasites, determining functional epitopes of proteins involved in the immune response against the parasite for use in platforms for a more accurate diagnosis. precision and preventive and/or therapeutic vaccine compositions against leishmaniasis seeking greater specificity due to the use of specific sites of the antigens.

The following examples show a detailed description of the experimental results obtained enabling a better understanding of the present invention. The experimental procedures involved will be detailed at the end of this descriptive report. The methodology used for the selection, characterization and use of recombinant peptides and protein motifs that mimic antigenic regions of Leishmania can be applied to other protozoa.

The invention can be better understood through the detailed description, in line with the attached Figures, where:

FIGURE 1 shows the “dot-blot” assay performed for selected recombinant peptides in specific elutions against antibodies from patients with Visceral Leishmaniasis (V) and also from Tegumentary Leishmaniasis (T) and healthy controls (N).

FIGURE 2 shows the competitive dot-blot assay for the most relevant clones in the specific selection for Visceral Leishmaniasis, the antibodies from patients affected by the disease were pre-incubated with Leishmania chagasi total antigen, thus showing what the recombinant peptides and the natural antigen compete for the same binding sites on antibodies.

Example 1:

This example refers to the selection and characterization of mimetic recombinant peptides, their protein motifs and inverse sequences, object of this invention, which were selected from antibodies from patients affected by Visceral Leishmaniasis and Tegumentary Leishmaniasis, independently. Comprehensive and specific selections were made for each of the pathologies listed above.

TABLE 1 presents the identity of the object sequences of this invention, designated by Seq. ID No. 1 to Seq. ID N2 56 (normal sequences) and Seq. ID No. 57 to Seq. ID N2 112 (inverted sequences) which refer to the experiments performed against antibodies from patients with Visceral Leishmaniasis and the frequencies observed for each of these sequences in each of the selection processes.

Three types of elution were used for the selection of these recombinant peptides, aiming at a comprehensive selection (acid elution) and two more specific ones (recombinant antigen or total antigen), all targeting IgG-type antibody ligands from patients affected by Visceral Leishmaniasis, being such elutions: Acid Elution (Type 1 Selection), represented by Seq. ID No. 1 to Seq. ID N2 24 and its inverted counterparts Seq. ID No. 57 to Seq. ID No. 80; the selection presented a total number of 28 clones, being 24 of them distinct from each other, the clone represented by the sequences Seq. ID No. 14 - Seq. ID N2 70 presented the highest frequency in this selection (10.71%), followed by the sequence clones represented by Seq. ID No. 3 - Seq. ID No. 59 and Seq. ID No. 19 - Seq. ID N2 75 with intermediate representation (7.14%), the other sequences were unique in the population presented here.

The Specific Elution against the rK39 recombinant antigen (Selection Type 2), represented by the Seq. ID No. 25 to Seq. ID N2 42 and its inverted counterparts Seq. ID No. 81 to Seq. ID No. 98; the selection presented a total of 30 clones, 18 of them distinct from each other, the clone represented by the sequences Seq. ID No. 34 - Seq. ID N2 90 showed the highest frequency in the population (20.0%), followed by clones Seq. ID No. 25 - Seq. ID No. 81 (16.67%), Seq. ID No. 31 - Seq. ID No. 87 (10.0%) and Seq. ID No. 28 - Seq. ID No. 84 (6.67%).

The Specific Elution performed against total Leishmania chagasi antigens (Selection Type 3), having as representatives the sequences enumerated Seq. ID No. 43 to Seq. ID N2 56 with its inverted counterparts designated by Seq. ID No. 100 to Seq. ID No. 112. The population is represented by 29 clones, 15 of which have distinct sequences. The most frequent clone in this population is represented by Seq. ID No. 43 - Seq. ID No. 99 (31.03%), followed by Seq. ID No. 52 - Seq. ID No. 118 (20.69%) and Seq. ID No. 4 - Seq. ID No. 60 (6.90%). The other sequences obtained in this selection had a frequency of 3.45% of the clones.TABLE 1

Peptides Inverted corresponding peptides Frequency (%) Type 1 Selection - Acid Elution - 28 clones

The alignment of the sequences presented in the Type 1 Selection showed the existence of 3 particular motifs repeated among the clones (consensus), which may represent epitopes that are represented by the following 5 sequences and their respective inverted sequences: Seq. ID No. 165 (Seq. ID No. 166), Seq. ID No. 167 (Seq. ID No. 168) and Seq. ID No. 169 (Seq. ID No. 170).

Due to the conserved representation in certain regions along the peptides, the motifs can point to important continuous or discontinuous epitopes of the antigens, as exemplified in TABLE 2, which presents the clones' alignment and the consensus sequences obtained in the different types of selections performed against antibodies from patients with Visceral Leishmaniasis, where: A: Acid Elution, B: Specific elution with rK39 antigen and C: Specific elution with L. chagasi total antigen. Highlighted, the consensus sequences identified among the clones.

Alignment of these consensuses with Leishmania chagasi restricted proteins in the GeneBank library (BLAST - Basic Local Alignment Search Tool - http://www.ncbi.nlm.nih.gov/BLAST) showed partial homology with Leishmania relevant proteins such as K39 , GP63, GP46 and the LACK antigen, among others, which is shown in TABLE 3, which presents the consensus sequences and the proteins to which they relate to their accession numbers, as well as the similarity given by the representative amino acids of the alignment, their position in the original protein and “Score” and “EValue” numbers for the alignment in question.

The experiments with Specific Elutions (Selection Type 2 and 3) could confirm the relevance of the consensus represented by the Seq sequence. ID No. 167 (Seq. ID No. 168); even with increasing elution specificity, these consensus peptides maintained a considerable frequency in the population of sequenced clones. The presence of protein motifs may be related to selection in favor of the ligand, by indicating that these amino acids could be crucial for the recognition of peptides by the paratope.

They show similarities with the consensus sequence identified as Seq. ID N2 165 (Seq. ID N2 166) the following peptide sequences and their respective inverted sequences: Seq. ID No. 12 (Seq. ID No. 268), Seq. ID No. 13 (Seq. ID No. 69) and Seq. ID No. 14 (Seq. ID No. 70).

The main protein to which this consensus sequence is related 5 when in normal position is K39 from Leishmania chagasi related to the Kinesin superfamily of motor proteins; the rK39 recombinant protein indicates high antibody titers in patients with Visceral Leishmaniasis, which may justify the success of the selection performed. The amino acid residue mentioned here is located in a region different from that of the recombinant antigen, which may point to a new epitope and be a useful tool in the development of 5 new technologies for immunodiagnosis and prophylaxis. Another relevant protein related to the two-position consensus is GP46 - a 46 KDa glycoprotein present on the surface of the promastigote forms of most Leishmania species - which presents high levels of messenger RNA in the infectious promastigote forms, the presence of this consensus sequence is related this protein may be an indication of the protein's participation in the host's immune response against the parasite and also have great diagnostic value. When inverted, this consensus sequence relates to the following Leishmania proteins: gp63 protein, dhfr-ts (Leishmania donovani chagasi), Leishmania major kinesin-like molecule and GP46 - 15 Membrane surface antigen (Leishmania donovani chagasi).

They are similar to the consensus sequence identified by the Seq sequences. ID No. 167 and its respective inverted sequence Seq. ID N2 168, with full or partial homology to consensus the following sequences for Type 1 Selection: Seq. ID No. 4 (Seq. ID No. 60), Seq. ID No. 5 (Seq. ID No. 61), 20 Seq. ID No. 6 (Seq. ID No. 62), Seq. ID No. 7 (Seq. ID No. 63), Seq. ID No. 8 (Seq. ID No. 64), Seq. ID No. 9 (Seq. ID No. 65), Seq. ID No. 10 (Seq. ID No. 66), Seq. ID No. 12 (Seq. ID No. 68), Seq. ID No. 20 (Seq. ID No. 76) and Seq. ID No. 23 (Seq. ID No. 79) and Seq. ID No. 29 (Seq. ID No. 85); for Type 2 Selection: Seq. ID No. 31 (Seq. ID No. 87), Seq. ID No. 32 (Seq. ID No. 88), Seq. ID No. 33 (Seq. ID No. 89), 25 Seq. ID No. 34 (Seq. ID No. 90) and Seq. ID No. 35 (Seq. ID No. 90) and Seq. ID No. 37 (Seq. ID No. 93), Seq. IDNo 38 (Seq. ID No 94) and Seq. ID No. 42 (Seq. ID No. 98) and for Type 3 Selection: Seq. ID No 43 (Seq. ID No 99),Seq. ID No. 44 (Seq. ID No. 100) and Seq. ID No. 248 (Seq. ID No. 104).

This consensus sequence when in normal position is related to the LACK antigen with partial homology, this antigen is a well conserved 36 KDa protein present in all species and developmental stages of Leishmania, being one of the most immunogenic antigens. The presence of this sequence in many of the object clones of this invention leads to the designation of an epitope within this protein with value in the diagnosis and prophylaxis of diseases both in man and in animals. When in inverted position, this consensus sequence does not show homology with the proteins deposited in the database.

The consensus sequence identified by the Seq. ID No. 169 (Seq. ID No. 170) has the Seq. ID No. 3 (Seq. ID No. 59), Seq. ID No. 22 (Seq. ID No. 78), Seq. ID No. 16 (Seq. ID No. 72), Seq. ID No. 19 (Seq. ID No. 75), Seq. ID N2 18 (Seq. ID N2 74) as representatives carrying the consensus and showed no homology in the alignments performed in databases.

TABLE 4 presents the sequences identified by Seq. ID No. 113 to Seq. ID N2 138 (normal sequences) and Seq. ID No. 139 to Seq. ID N2 164 (inverted sequences) which refer to the selections made against antibodies from patients with Tegumentary Leishmaniasis, also showing the observed frequency of each peptide sequence for each of the selection processes.

For antibodies from patients with Tegumentary Leishmaniasis, two selection processes were performed, namely:- Acid Elution (Type 1 Selection) represented by the clones of sequences Seq. ID No. 113 to Seq. ID N2 124 and its inverted counterparts Seq. ID No. 139 to Seq. ID N2 150 with a total of 12 clones each representing 8.33% of the population.- Specific Elution (Type 2 Selection) performed against total Leishmania amazonensis antigens, represented by Seq. ID No. 125 to Seq. ID N2 138 and its inverted counterparts Seq. ID No. 151 to Seq. ID No. 164, with a total of 14 new clones. The most frequent clone among them is represented by the sequences identified by Seq. ID No. 119 - Seq. ID No. 145 (16.67%), followed by the Seq. ID No. 114 - Seq. ID No. 140, Seq. ID No. 118 - Seq. ID No. 144 and Seq. ID No. 129 - Seq. ID No. 155 (8.33%), Seq. ID No. 124 - Seq. ID No. 150, Seq. ID No. 125 - Seq. ID No. 151, Seq. ID No. 126 - Seq. ID No. 152 and Seq. ID No. 137 - Seq. ID No. 163 (5.56%) and the others with 2.78%.

The clones selected in the selection described above, when aligned with each other, did not show a clear definition of consensus sequences, as shown in TABLE 5, which shows the alignment between the clones of the different types of elution against antibodies from patients with Tegumentary Leishmaniasis 5, where A: Elution acid and B: Specific Elution. On the other hand, it was observed between selections the presence of clones repeated in the two different types of selection (Seq. ID No. 114 - Seq. ID No. 140, Seq. ID No. 118 - Seq. ID No. 2 Seq. ID No. 144, Seq. ID No. 144, Seq. ID No. 119 - Seq. ID No. 145, Seq. ID No. 121 - Seq. ID No. 147, Seq. ID No. 122 - Seq. ID No. 148, Seq. ID No. 124 - Seq. ID No. 150) for showing high and common reactivity to the targets used in the selections, in addition to showing alignments in several hypothetical sequences of several Leishmania species deserve particular attention as targets to be used in the diagnosis and prophylaxis of Tegumentary Leishmaniasis.

Example 2:

This example refers to the confirmation of the reactivity of the peptides selected from the Specific Elutions for Visceral Leishmaniasis object of this invention.

The clones obtained from the Type 2 and 3 Specific Selections mentioned above, identified by the sequences Seq. ID N° 25 to Seq. ID Ns 56 and their corresponding inverted Seq. ID N281 to Seq. No. 112.

All clones representing each of the sequences were tested against sera from patients with Visceral Leishmaniasis (V), Leishmaniasis Tegumentary (T) and Negative Controls (N). No cross-response was verified, showing that the clones were positively selected by the specific target as seen in FIGURE 1.

Among the clones selected against the rK39 recombinant antigen showed positivity, those identified by the following sequences and their inverted counterparts: Seq. ID No. 25 (Seq. ID No. 81), Seq. ID No. 26 (Seq. ID No. 82), Seq. ID No. 31 (Seq. ID No. 87), Seq. ID No. 32 (Seq. ID No. 88), Seq. ID No. 34 (Seq. ID No. 90), Seq. ID No. 37 (Seq. ID No. 93), Seq. ID No. 41 (Seq. ID No. 97) and Seq. ID No. 42 (Seq. ID No. 98).

For clones selected against Leishmania chagasi total antigen, the following sequences and their inverted counterparts showed positivity: Seq. ID No. 43 (Seq. ID No. 99), Seq. ID No. 44 (Seq. ID No. 60), Seq. ID No. 50 (Seq. ID No. 106), Seq. ID No. 52 (Seq. ID No. 108), Seq. ID No. 53 (Seq. ID No. 109) and Seq. ID No. 54 (Seq. ID No. 110). TABLE 6 shows the positivity of the clones in this assay related to the presence of a consensus region in the molecule (indicated by a + sign), which shows that there is a relationship between the presence of consensus and the differential reactivity of the recombinant peptides.

The positive clones in the dot blot assay listed above were subjected to a competitive dot-blot assay, in which IgG-type antibodies from patients with the disease in question were incubated with Leishmania chagasi total antigen with the following concentrations: 1- control without antigen, 2 - 0.5 μg/mL, 3-1.3 μg/mL, 4 - 4.9 μg/mL, 5 - 14.8 μg/mL, 6 - 44.4 μg /mL, 7 - 133.3 μg/mL and 8 - 400 μg/mL, as shown in FIGURE 2. The previous reaction of the antibodies with the total antigen, before reacting with the recombinant peptides expressed on the surface of phages, had the to block common binding sites between these different antigens.

Among the clones selected against the rK39 recombinant antigen, had inhibited reactivity against the Leishmania antigen, those identified by the sequences mentioned below and their respective inverted sequences: Seq. ID No. 31 (Seq. ID No. 87), Seq. ID No. 32 (Seq. ID No. 88), Seq. ID No. 34 (Seq. ID No. 90) and Seq. ID No. 42 (Seq. ID No. 98).

For clones selected against Leishmania chagasi total antigen, they had inhibited reactivity against Leishmania antigen, the following sequences and their corresponding inverted sequences: Seq. ID No. 43 (Seq. ID No. 99), Seq. ID No. 4 (Seq. ID No. 60), Seq. ID No. 50 (Seq. ID No. 106) and Seq. ID No. 54 (Seq. ID No. 110).

A detailed description of the experimental procedures adopted may lead to a better understanding of the examples mentioned above.

Purification of immunoglobulins was performed by immunochromatography from a pool of these sera, using a protein A sepharose column. The column was equilibrated with 10 volumes of phosphate buffer (0.1 M, pH 8.0). Serum samples diluted in phosphate buffer (vol/vol) were added to the resin and incubated for 30 minutes at room temperature. After incubation, the column was washed with phosphate buffer, and the absorbance at 280 nm was monitored in a spectrophotometer (Ultrospec 1100 pro - Amersham Biosciences) until it equaled zero. Each sample was eluted with glycine buffer (0.1 M pH 2.7), and 1 mL fractions were collected and monitored by spectrophotometric reading. The fractions with the highest readings were grouped and the pH neutralized with 0.1 M NaOH. Thus, they were transferred to dialysis membrane, concentrated in commercial sugar (1 h, 4°C) and then dialyzed in 3L of phosphate buffer throughout at night in a cold room at 4°C. Quantification was done by direct reading in a spectrophotometer at 280 nm.

For the selection of the recombinant peptides (recombinant peptides expressed in the pIII protein of filamentous phages) a commercial library of 12 amino acids (Ph.D. - 12™ mer - New England Biolabs) was used. The experimental procedures were based on the protocol provided by the manufacturer, with adjustments for the best selection of specific clones.

For experiments designated as the Acid Elution type, both for Visceral Leishmaniasis and Tegumentary Leishmaniasis, the procedures for selection of recombinant peptides were performed in aqueous phase.

As substrate for this process, 50 μL of protein G agarose in 50% aqueous solution (Recombinat Protein G Agarose - Invitrogen™) were used, which were washed by resuspension in a microtube with 1mL of TBS-T (Tris-HCI 50 mM - pH 7.5, NaCI 150 mM - Tween 20 0.1%), the supernatant was carefully removed after centrifugation at 4,000 rpm for 30 seconds in a refrigerated centrifuge. Then, the resin was blocked for one hour with blocking buffer (0.1M NaHCO3, pH 8.6; 5mg/ml BSA; 0.02% NaN3) at 4°C, and mixed every 15 minutes. After incubation, it was washed four times, as described above, to then receive the mixture of 300 ng of purified antibody, together with 1.5 x 1011 viral particles in a final volume of 200 μL of TBS-T 0, 1%, previously incubated at room temperature for 20 minutes.

The resin - antibody - phage mixture was gently inverted three times during the 15 minute incubation at room temperature and then washed 10 more times with one ml of 0.1% TBS-T, preparing it for phage elution ligands made with one mL of elution buffer (0.2 M Glycine-HCl, pH 2.2; 1mg/mL BSA) for 10 minutes at room temperature. This mixture was centrifuged for 1 minute at 4,000 rpm, the eluate was transferred to a new tube and immediately neutralized with 150 μL of Tris-HCI (1 M, pH 9.1).

A small sample of this eluate (10 μL) was titrated and the remainder was used for re-amplification, carried out in 20 mL of E. coli culture (ER 2738) in early growth phase (ODeoo^ 0.3) containing tetracycline and incubated for 4, 5 hours in a shaker with controlled temperature at 37°C, before the phage precipitation procedure and subsequent titration.

The re-amplified phages from the first round of selection were used in a second cycle (2.0 x 1011 cfu) and so on for a total of four cycles, from the second cycle onwards the stringency of the wash buffer was increased by 0.1% to 0.5%, then using 0.5% Tween-20 in all washes. After finishing the selections, the phages containing the recombinant peptides were isolated and sequenced (the procedures will be described later).

For the experiments described as Specific Elutions with parasite antigens, for both Visceral Leishmaniasis and Tegumentary Leishmaniasis, the procedures were performed in solid phase.

In the first selection cycle, a well of a microtiter plate (Corning Incorporated Costar®) was sensitized with 150μL of IgG-type antibodies from a pool of sera from patients with Visceral Leishmaniasis (purified by immunochromatography, concentrated, dialyzed and quantified by direct spectrophotometric reading), at a concentration of 100 μg/mL in carbonate buffer (0.1 M NaHCOa, pH 8.6). Incubation took place overnight at a temperature of 4°C, under agitation in a humidified environment.

After discarding the solution and drying the plate, it was blocked with 300 μL of blocking solution (0.1 M NaHCOa, pH 8.6, 5 mg/mL BSA) for 1 hour at 4°C. The solution was discarded and the plate washed 6 times with TBST (TBS - 50 mM Tris-HCl pH 7.5, 150 mM NaCI, water, 0.1% volume/volume of Tween 20) to then add 4 x 1010 particles viral (10 μL of the original commercial library) diluted in 100 μl_ of TBST, and the plate was kept under agitation for one hour at room temperature. Non-antibody-binding phages were removed and the plate washed 10 times with TBST for the addition of 100 μL of a 100 μg/mL solution of IgG-type antibodies from purified healthy control patients for the elution of phages binding to these types of antibodies, then called negative elution. This mixture was incubated for one hour at room temperature, under agitation. Phage contained in the supernatant were discarded and the plate washed an additional 10 times with TBST. A second elution was performed, using the recombinant antigen rK39 at a concentration of 100 pg/ml in a volume of 100 µL, with an incubation period of one hour at room temperature. The phages contained in the supernatant, binding to rK39, were collected for further amplification and the plate was washed again for 10 more times to perform the third elution with 100 pL of a solution (100 pg/mL) of total L. chagasi antigen, the incubation was one hour at room temperature, and the phages in supernatant were also collected for further amplification. The phages from the elutions with the antigens were used in the subsequent rounds of selection. A small aliquot (1 pL) of both eluates were titrated to measure the number of viral particles per titration and the remainder was amplified for use in later cycles.

From the second round of selection on, two wells were sensitized with purified antibodies from Leishmaniasis positive patients as previously described. For each of these wells, negative and specific elution was performed, one for rK39 (Type 2 Specific Selection) and the other for L. chagasi total antigen (Type 3 Specific Selection). All blocking and washing procedures were similar to the ones described above, except for the concentration of the washing buffer which changed to TBST which I changed to 0.5% (TBS (50 mM Tris-HCI pH 7.5, 150 mM NaCI, water) , 0.5% volume/volume of Tween 20) in the second and other cycles.

In this way, a total of four cycles were performed, and then the non-amplified eluate was titrated and the blue colonies were individually placed in wells of "Deep Well" plates with 1.2 mL LB culture medium (20 μg/mL tetracycline ) containing ER2738 bacteria in early growth phase. The culture was incubated under vigorous shaking (250 rpm/min) for 5 hours. After this period, aliquots were made for "back-up" and the plates with the remaining culture remained in incubation overnight for extraction of individual DNA from the clones.

For all reamplification procedures and recombinant phage precipitation the procedure was as follows: The culture was transferred to a centrifuge tube and centrifuged (10 min, 4,000 rpm). Residual cells were discarded and the supernatant transferred to a new tube and recentrifuged. 80% of the upper supernatant was pipetted into a clean tube, where 20% by volume of PEG-NaCl (20% wt/volume polyethylene glycol-8000; 2.5 M NaCl) was added and the mixture was left to stand for the entire duration. night at 4°C. The next day, the precipitation product was centrifuged for 15 min at 10,000 rpm and 4°C. The supernatant was discarded and the tube was again centrifuged, under the same conditions as before, for another 5 minutes so that the residual supernatant could be removed. The phage pellet was resuspended in 1 ml of 1X TBS, which was transferred to a microtube and centrifuged (5 min, 10,000 rpm, 5 min) to remove residual cells. The supernatant was transferred to a new microtube for the addition of PEG-NaCl (1/6 of the volume). The mixture was incubated for 1 hour on ice and then centrifuged (15 min, 14,000 rpm, 4°C). The supernatant was discarded and the tube re-centrifuged for another 5 minutes to remove the residual supernatant. The pellet was resuspended in 200 µL of 1X TBS, ready for titration.

The titration procedure was as follows for both amplified and non-amplified eluates: serial dilutions of 10-1 to 10-4 and 10-8 to 10-11, respectively, were performed. To nine μL of each dilution, 200 μL of ER2738 bacteria (ODeoo5, 0.5) were added, incubating the mixture for 5 minutes before it was plated in LB medium (Tetracillin: 20 μg/mL, IPTG: 0.5 mM and X- gal 40 μg/mL) together with 3 mL of Agarose Top (10 g Bacto-Tryptone, 5g yeast extract, 5g NaCl, 1 g MgCl2 . 6H2O / liter). The plates were incubated at 37°C overnight, and the blue colonies representing the phage-infected colonies were counted to obtain the number of infecting particles (pfus) for entry into later cycles.

For DNA extraction from recombinant phages, colonies from the selection cycles were isolated in deep well plates containing 1 mL of culture medium with ER2738 bacteria (OD6oo> 0.3) and incubated under shaking at 37°C for 24 hours. After the culture period, the plates were centrifuged for 10 minutes at 3,700 rpm at 4°C, and 800 μL of the supernatant were transferred to a new plate, where 350 μL of PEG-NaCI was added and incubated for 10 minutes at temperature environment. The mixture was centrifuged for 40 minutes at 3700 rpm, at 20°C. The supernatant was discarded and the plate was inverted onto absorbent paper to remove excess culture medium. The precipitate was resuspended in 100 pL of iodide buffer (10 mM Tris-HCl, 1 mM EDTA, 4 M Nal) and 250 pL of Absolute Ethanol was added, incubating the mixture for another 10 minutes at room temperature before resubmitting it centrifugation for 40 minutes at 20°C. The supernatant was discarded and the precipitate washed with 150 µL of 70% Ethanol, and centrifuged for a further 15 minutes at 20°C. All centrifugations took place with a rotation of 3,700 rpm. The DNA was solubilized in 20 pL of sterile ultrapure water and qualified by electrophoresis on a 0.8% Agarose gel, comparing it to the M13 phage DNA standard provided by the library manufacturer.

Isolated samples were submitted to automatic sequencing using the Big Dye Terminator Kit (GE Healthcare) and MegaBace 1000 automatic sequencer (GE Healthcare). In the sequencing reaction, approximately 3 pL of DNA were used (depending on the mean quantification given by spectrophotometric reading), 4pL of premix and 10 pmoles of primer - 96 M13 - (5'-HOCCCTCATAGTTAGCGTAACG -3'- GE Healthcare), which amplifies the region of the coding amino acids of the random peptides fused in the recombinant M13 phages, and the reaction volume is completed for 10 pL. The reaction took place in a thermocycler (Mastercicler, Eppendorf) for 35 cycles, with denaturation of 30 seconds at 95°C, annealing of the primers for 30 seconds at 50°C and extension for 30 seconds at 60°C.

Sequence processing was done by the equipment's software (Sequence Analyzer, BASE CALLER, Cimarron 3.12, Phred 15). With the DNA sequences in hand, they were translated by the DNA2PRO program (http://relic.bio.anl.qov/proqrams.aspx), generating the expected 12 peptide sequences.

Alignments between the distinct sequences were made by the ClustalW program (http://www.ebi.ac.uk/clustalw/) to delineate likely consensuses. Sequence alignments with Leishmania proteins were performed by the BLAST program - Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov).

The most frequent phages in the populations of the specific selections (Selection Type 2 and Selection Type 3) for Visceral Leishmaniasis and the controls (wild phage and recombinant antigen rK39) were submitted to competitive dot-blot assays with L. chagasi total antigen and phage wild.

The 33 clones with distinct sequences were tested in a dot-blot assay against antibodies purified from patients with Visceral Leishmaniasis, Tegumentary and Healthy Control, together with the following controls: wild phage, irrelevant phage (selected from another selection) and recombinant antigen rK39.

The nitrocellulose membranes were sensitized with phages (5.0x109 viral particles diluted in two μL of TBS), allowing them to dry for 2 minutes at room temperature. Each membrane was blocked with one ml of blocking solution (TBS-M Moliço 5%), incubated with shaking for two hours and then washed three times with washing buffer (TBST - 0.5% Tween 20). 500 μL of the respective purified antibody (Visceral, Integumentary, Negative) at a concentration of 25 pg/mL diluted in blocking were added to each membrane, and then incubated for one hour. After incubation, the membranes were again washed six times with wash buffer and incubated with 500 μL of anti-IgG secondary antibody labeled with Alkaline Phosphatase diluted in block and incubated for one hour and then washed for another 6 times, developed with NBT/BCIP solution and washed with water before being dried for digitizing the images. All incubations were carried out at room temperature and under slow agitation.

Clones with higher reactivity in the previous assay were submitted to a new competitive assay (14 clones) together with controls (Wild Phage, Irrelevant Phage, L. chagasi total antigen and rK39 recombinant antigen).

The antigens (phages and controls) were added on the nitrocellulose membrane strips, leaving them to dry for two minutes, and then one mL of blocking (TBS-M - Moliço 5%) was added, which acted for two hours. During this period, antibody solution purified from Visceral Leishmaniasis patients was incubated with serially diluted L. chagasi total antigen (from 400 μg/mL to 0.5 pg/mL including no antibody control). After the blocking period, the membranes were washed three times with washing buffer (TBS-T - 0.5% Tween 20) and the pre-incubated mixture of antibodies and antigens was then added, which remained in contact with the membrane for one hour. The membranes were washed an additional six times with the wash buffer and 500 µl of alkaline phosphatase labeled anti-IgG secondary antibody was added (1:5000 blocking) and incubated for one hour. Six washes were performed as described above and later, development was carried out with one ml of developing solution (NBT/BCIP). Membranes were washed with water and dried for image digitization. All mentioned incubations took place at room temperature, under slow agitation.

Claims (5)

1. Recombinant peptides mimetic to Leishmania antigens, their protein motifs and reverse sequences, characterized by the fact that they comprise the sequences Seq ID N° 1 to Seq ID N° 170. 2. Use of mimetic recombinant peptides, their protein motifs and reverse sequences as defined in claim 1, characterized in that it is in the diagnosis of leishmaniasis, as probes for "in vitro" detection of the presence of circulating antibodies or other binding molecules against parasites of the genus Leishmania. 3. Use according to claim 2, characterized in that the detection of circulating antibodies or circulating molecules is performed by immunological assays, such as immunoenzymatic tests (such as ELISA, "Western Blot", immunofluorescence, immunohistochemistry, EIA , and others), immunoagglutination tests, electrochemical sensors, “Quantum dots”, IFL (lateral flow immunoassay) or other forms of detection directly or indirectly related to body fluid samples such as saliva, urine and blood. 4. Use of mimetic recombinant peptides, their protein motifs and reverse sequences as defined in claim 1, characterized in that it is in the preparation of vaccine compositions to stimulate the human or animal immune system against parasites of the genus Leishmania. 5. Use according to any one of claims 2 or 3, characterized in that the sequences identified by Seq ID No. 1 to Seq ID No. 112 and Seq ID No. 165 to Seq ID No. 170 are preferably for Differential diagnosis of the Visceral form of Leishmaniasis and Seq ID Nos. 113 to 164 are preferably for differential diagnosis of the Integumentary form of Leishmaniasis.

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