BRPI0903089A2 - recombinant peptides and leishmania antigens mimetic protein motifs and their applications - Google Patents

Abstract

RECOMBINANT PEPTIDES AND MIMETIC PROTEIN REASONS TO LEISHMANIA ANTIGENS AND THEIR APPLICATIONS. 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 Leishmanian proteins and antigens of diagnostic and vaccine interest for diseases caused by Leishmania parasites. The peptides object of this invention were selected by "Phage Display" and are targets for use in immunodiagnostics and in vaccine compositions to control the different types of disease caused by parasites of the aforementioned genus because they are specifically immunoreactive with the serum of infected individuals.

Description

"RECOMBINANT PEPTIDES AND MYTHETIC PROTEIN MATTERS FOR ANTIGENS OF LEISHMANIA AND THEIR APPLICATIONS"

The present invention relates to the selection, characterization and use of recombinant peptides, their reverse sequences and protein motifs which mimic partial antigenic regions of Leishmania proteins and antigens of diagnostic and vaccine interest for diseases caused by Leishmania parasites. The subject peptides of this invention are intended for use in immunodiagnostics and vaccine compositions for the control of the different types of disease caused by the above-mentioned dog parasites:

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

The causative agent of Leishmaniasis, Leishmania, is an obligate intracellular parasitotripanosome with a biphasic life cycle consisting of a single-lymphatic promastigote stage, which resides within the digestive tract of the vector and an amastigote stage, which multiplies within macrophage macrophage 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 Leishmania subgenus 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 genus Viannia occur only in the New World and of the nine known species, eight infect humans (Shaw J. J. Mem. Inst. Oswaldo Cruz; 89: 471-8, 1994). Parasites of the genus Leishmania are transmitted by bites of Phlebotomus genus mosquitoes 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 host immune status, manifestations may range from chronic skin lesions to lethal form of infection (Murray HW et al., Lancei, 366: 1561-1567, 2005), with the following spectrum of clinical manifestations: Documentary Ieishmaniasis, which varies from cutaneous to mucocutaneous (LC and CML), representing the responsive pole for diffuse cutaneous Ieishmaniasis (LC) and visceral leishmaniasis (VL) that encompasses the subclinical form and 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 less developed countries. Of the VL cases, 90% occur in only five countries - Bangladesh, India, Nepal and Brazil; 90% of LC cases occur in only 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 visceral form, with urbanization being a crucial factor for incidence (WHO.Tfte WeeklyEpidemiological Record, 77: 365-72, 2002 and www.who.int/tdr/diseases/leish).

Brazilian Ieishmaniasis behaved like an anthropozoonoserural, but after the 1980s its expansion was also seen in the periurban regions of large cities. Environmental changes caused by humans have changed the epidemiological profile of yeishmaniasis in areas where it is related to wildlife, as well as in areas where transmission occurs in periurban or urban neighborhoods and surrounding areas. 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 skin and mucosa) (Tolezano JE et Al., Rev. Inst. Adolfo Lutz; 40: 49-54, 1980; Marzochi MCAJ Bras. Med .; 63: 82-104,1992; Marzochi C. C. et al., Cad. Saude Publica; 10 : 359-75, 1994).

Several mammalian species have been reported to be naturally infected by Leishmania spp. In endemic areas dogs are considered the largest reservoir of Human Visceral Leishmaniasis (Palatinik-de-Souza, CB et al., Am. J. Trop. Med. Hyg .; 65 : 510-517, 2001 eWHO, 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 fever, and tuberculosis, commonly occurring in the same area as heishmaniasis (Goto Y. et al, lnfect. Immun.) 74: 3939-45 , 2006). For zoonotic VL, rapid and safe diagnosis is a necessary tool because of the wide variability of clinical symptoms and the presence of asymptomatic but infectious 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 with symptoms of the disease. Because of this, 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. Parasitol .; 81: 1000-3, 1995).

Choice of antigens is important because the use of whole or whole parasitic antigens often results in poor specificity in the detection of disease-specific antibodies (Reed S. G. et al., JImmunol.] 138: 1596-601, 1987).

In recent years, several antigens have been characterized in order to improve the vaccine potential and the diagnosis of human and canine Ieishmaniose. The development of polymerase chain reaction and recombinant antigen-based immunoassays are intended to improve the sensitivity and specificity of the diagnosis of Leishmaniasis Visceral and to decrease the cross reactivity shown by total antigens (GomesΥ. M. et al., Vet. J.] 175: 45-52, 2008).

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

In the present 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 Leishmania infection resistance and susceptibility is critical for the development of immunoprophylactic tools (Rosa R. et al., Vaccine \ 25: 4525-32, 2007).

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

Molecules such as Glycosylphosphatidylinositol (GPI), Glycosylphospholipids (GIPL), Lipophosphoglycans (LPG), Phosphoglycans (FG), Proteophosphoglycans (PPG), Leishmaniolisin (gp63) and Cysteine Proteases (CPs) are non-discriminated by Leishmania, but they produce crude infections, but they do not cause infection. no host pathology (Chang et al., P. 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, presenting cross-reactivity between different Leishmania species, although the antibodies produced by this molecule are not effectors in the control. dainfection (Mood SF Acta Trop .; 53: 185-204, 1993 and Wright et al., P. et al., Biochem. Cell Biol .; 525-36, 1989). Differential expression of gp63 genes results in different amounts of gp63 protein in the development in vitro of promastigote forms for the infectious form (Ramamoorthy R. et al., J. Biol. Chem .; 267: 1888-95, 1992).

Fucose-Manose Binding Glycoconjugates (MLF) are a complex of promastigged L. (L.) donovani antigenic proteins that are used in serological tests for the diagnosis, prognostic evaluation and control of human kalazis, and are proposed in the screening of donors. blood from endemic caiazar regions (Otero ACS et al., Am. J. Trop. Med. Hyg .; 62: 128-131, 2000 and Palatinik de Souza CBet al., Trans. R. Soe. Trop. Med. Hyg .; 89: 390-3, 1995).

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

Molecular cloning, characterization and expression of 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 of 14 amino acids , 64% of total protein) is a highly specific diagnostic marker for assessing serum infection in individuals with Ieishmaniosevisceral.

The antigen called KMP-11 (major protein determinant of kinetoplastid protozoan membrane) appears to be antigenic and shows a relatively strong antibody response during the natural course of leishmaniasis and Chagas disease. The observation of cross-immunological reaction in infections caused by Leishmania and T. cruzi is particularly important, contradicting studies that reported the high sensitivity of these antibodies in leishmaniasis patients. Mapping antigenic determinants in 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 - LcKin1 antigen with motor protein (kinesin) superfamily homology, called K39, has been shown to be predominant in amastigotes, and contains a highly related extensive repeating domain between L. chagasi and L.donovani species. High antibody titers were found for the rK39 antigen, which contains several 39 amino acid repeats, demonstrating conservative region counseling among the mentioned species (Burns JMet al., Proc. Natl. Ac. Sci .; 90: 775-9, 1993). With the discovery of this antigen, many researchers have been testing its viability in different diagnostic tests for Visceral Leishmaniasis, most of them showing highly promising results in an attempt to use less invasive and economically viable diagnostic methods 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 function for these specific antibodies (Carvalho SF et al., Am. J. Trop. Med. Hyg .; 68: 231- 324, 2003 and Chang KP et al., Kinetoplasti. Bioi. 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 IgG capture rK39 ELISA and dipstick test, and the results showed 100% sensitivity and specificity for both cases (Singh S. et al., Ciin. Diag. Lab. Immunol. ; 9: 568-72, 2002); Similar results were obtained by showing the sensitivity of rK39 strip test antigen and ELISA of 90% and 89%, respectively, while specificity was 100% and 98% respectively (Carvalho SF et al., Am. J. Trop. Med. Hyg .; 68: 231-324,2003). The efficacy of rK39 antigen was also analyzed in Direct Agglutination Test - DAT (100% sensitivity and specificity) and dipsticktest with 85.7% sensitivity and 82% specificity (Schallig HD; etal., Mymensingh Med. J. ; 12: 93-7, 2002).

Similar analyzes were done for ELISA tests (Salotra S. etal., 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. Biomea. Sc / ';; 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 E. E. et al., Clin Diagnostic Lab Immunol., 5: 717-20, 1998; Medrano FJ et al., Am. J. Trop.Med. Hyg .; 59: 155-62,1998; Badaro 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. etal., 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 BingZa Zhi 18: 155-8, 2000 ; Delgado O. et al., Parasite; 8: 355-7, 2001; Berm C. etal., Am. J. Trop. Med. Hyg .; 63: 153-7, 2000 and Zijlstra et 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 unclear, Sudanese patients develop lower anti-rK39 antibody titers than Indian patients, although the shape of the test 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 L. major antigen, called LACK (Leishmania homologue of receptor for activated C kinase), was identified from the search for antigens recognized by a protective Th1 clone derived from the mouse spleen infected with a soluble extract of L. major promastigotes. . The deduced amino acid sequence of this antigen has substantial homologies to intracellular activated Kinase C receptors (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 trigger of the initial immune response (Requena J. M. et al., Parasitol. Today; 16: 246-50, 2000). The LACK antigen is a well-preserved 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 LmSTM and TSA). The combination of these antigens has provided durable and complete protection against the development of cutaneous lesions (Méndez S. et al., Vaccine; 31: 3702-4, 2002).

Some of the previously described antigens are objects of the study of effective Leishmaniasis vaccines, chemotherapy or immunodiagnosis, being objects of the patents described below:

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

W09711180 and W00179276 describe compositions and methods for preventing, treating and diagnosing leishmaniasis and stimulating immune response in patients using immunogenic portions of the antigens M15, Ldp23, Lbhsp83, Lt-210 and LbelF4A, Lmspla, Lmso9A and MAPS-1 thereof. and its variations. For the same purpose, patent W02007121184 describes the use of polypeptides and fusion proteins containing at least one immunogenic portion of one or more Leishmania antigens and variants by selecting tandem protein regions.

CA2503932 discloses novel proteins of Leishmania major which comprise secreted or secreted polypeptides and polynucleotides and their functional fragments and the use of these comovacin and therapeutic peptides, in addition to the use of antibodies generated from them.

W02007142695 describes the use of a rapid diagnostic kit for detection of secreted or excreted amastigote, promastigote, or excreted protein of Leishmania, which can be used in the field leading to faster treatment of cutaneous leishmaniasis using Thiol Specific Antigen (TSA) antigen antibodies.

PI0605889-2 demonstrates the use of soluble antigen in the diagnosis of canine visceral leishmaniasis by ELISA immunoenzyme reaction.

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

US2007292847 discloses novel nucleic acid amino acid sequences of Leishmania major (LmPDE - phosphodiesterases) and antibodies that are associated with these sequences for complex formation in the diagnosis and treatment of disease-related disorders caused by steppe parasites.

W09633414 describes a method for the prevention and serological diagnosis of Leishmania infection by one or more polypeptides in diagnostic methods and kits for evaluation of individual blood samples and identification of asymptomatic individuals. These polypeptides comprise an epitope of LcPO (Leishmnia chagasi homologof eukariotic acid ribosomal P-protein family) and variants thereof that differ only in conservative substitutions or modifications. Patent W02005063803 describes the use of methods and methods for the detection of antibodies in individuals suspected of being infected with parasites. Leishmania genus, mainly Indian or correlated strains and the use of a related diagnostic kit, demonstrating the isolation, characterization and use of the kinesin-related dogene immunogenic region isolated from the Leishmania donovai strainMHOM / IN / DD8 and MHOM / IN / KE16 / 1998 and correlation of nucleic acid sequences isolated from Indian and L. chagasi strains.

US7008774 proposes an immunoassay for detection of IgM and IgG antibodies and detection of circulating antigens in samples from individuals with visceral, cutaneous or canine leishmaniasis. Also reports the use of an immuno-capture immunoassay for detection of antigens released by parasites.

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); To this end, DNA fragments of these protein genes were sequentially added to the 5 'region of the vector generating a 38K molecular weight polypeptide with an isoelectric point of 7.37.

W09639524 describes methods and compounds related to Leishmania's LbelF4A or LmelF4A antigens, analyzing the immune response both to the protein and to the DNA vaccine encoded antigen.

DE10156679 demonstrates the use of p36-LACK antigen in the construction of an expression cassette for treatment and vaccine composition for leishmaniasis infections. Similarly, US2004235772 describes DNA expression using the p36 LACK antigen for treatment of Leishmania infections as well as corresponding vaccine, and W02005039633 demonstrates DNA expression for immune response generation using the p36-LACK, TSA, Kmp-11 and Leishmania infantum gp63 antigen or derivatives thereof with corresponding function.W00181388 discloses the use of polypeptides, proteins, and nucleic acids containing at least a portion of the Lip2a protein (acidicribosomal protein from Leishmania infantum) or variants thereof to generate immune response in individuals or groups. used as therapeutic agents for Leishmania and as an adjunct to other parasites.

W02007144903 uses the KMP-11 antigen as a hybrid vaccine against leishmaniasis, while US2008145385 describes methods for immunizing mammals infected with virulent Leishmania strains by administering a KMP-11 (Kinetoplastid Membrane Protein) DNA vaccine.

US2008026467 discloses DNA sequences capable of decoding PSA (Promastigote Surface Antigens) from Lemamania promastigote andmastigote forms for use in Leishmania vaccine compositions.

US2004170636 describes the production of a DNA vaccine that stimulates immune response against Leishmania infections using the Leishmania donovaniA2 gene. 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 of different species, aiming at this discrimination.

Patent W02006122382 (BRPI0503187) describes the obtainment of blocking vaccine decompositions for the transmission of human leishmaniasis and animals from Leishmania (Fucose-Manose Binder - FML of amastigotes or L. donovani promastigotes) fractions.

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

Patent W002072792 demonstrates that the use of the proteins of Leishmania TSA or MAPS (Leishmania thiol-specific thiol-specificantioxidant), LeIF (L braziliensis gene homologous to eukaryotic ribosomal proteinelF4A, also called "LbelF4A"), M15 (L. major stress- inducible 1 or LmSTII) and 6H (L. braziliensis gene homologous to the gene for the eukaryotic83-kDa heat shock protein, also called "Lbhsp83") when fused to heterologous polynucleotide sequences are capable of enhancing the expression of heterologous polynucleotides in eukaryotic cells. Isolated or purified Leishmania fusion agents may be used for treatment or prevention of Leishmania infections or other organisms such as M. Tuberculosis. FR2862313 describes the construction and isolation of nucleic acids encoding an imonogenic region of Leishmania amastigote andpromastigote forms, which can be used in vaccine compositions or to generate antibodies and be used in diagnosis.

FR2826018 describes the isolation of the LmPDI protein coding gene, which has two regions with identical sequences (Cys-Gly-His-Cys) recognized as a therapeutic target for the development of drugs and a novel element for the composition of Leishmania vaccines for humans and animals.

Phage display technology or phage display of biomolecules has been increasingly used in various areas of science since it was described in 1985 (Smith G. P. Science 228: 1315-17, 1985). This author pioneered the expression of the Eco R1 restriction enzyme as a fusion of capsidoviral protein three (p11).

Bacteriophages, or simply phages, are viruses that infect a variety of Gram-negative bacteria using the sexual pilus as a receptor. The filamentous phage particles (M13, f1 and fd strains) that infect E.coli via pilus F consist of a DNA of simple tape included in a protein capsule. One viable phage expresses approximately 2,700 copies of protein 8 (g8 or pVIII, a 50 amino acid residue protein) and 3 to 5 copies of the Ill (p3p or plll) 406 amino acid protein (Russell M., Mol.Microbiol .; 5 : 1607-13, 1991).

In systems where all plll or pVIII are used, the size of the protein inserted into the vector is limited because large proteins interfere with capsid protein functions, making the phage poorly infectious. This Phage display system has been created for the display of small (up to 30 amino acid) libraries (Phizicky E.M. Fieids S., MicrobioLRev. 59: 94-123, 1995).

Exposure in filamentous phages 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 bacteriophages, so 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 Η. Μ. E. etal. Clin Biochem; 35: 425-45, 2002).

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

Biopanning or selection procedure is performed by incubating the library of peptides exposed in phages 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 removed by successive washes, and specific phages remain bound for further elution. The specific phage pool is amplified for subsequent biological deletion or binding cycles for binding, elution and amplification cycles. enrichment of the phage set with specific sequences against the target. After three or four passages, individual clones are characterized by DNA sequencing, Western Blot or ELISA (SmithG. 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 target affinity-phage capture affinity in a target protein-specific bead matrix. The experiment requires substantially less target per experiment than surface panning, which may result in greater accessibility of the ligand site for phage-expressed peptides, as well as avoiding partial denaturation of the target on the plastic surface (Barbas, C. et al., Cold Har. Lab. Press, NY; 2001).

Synthetic oligonucleotides of constant length, but with unspecified codons, randomized by site-directed mutagenesis, using degenerate oxynucleotides, are cloned as fusion to one of the M13 phage capsid proteins, where they are expressed as capsid-linked fusion proteins. Phage-linked 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 antigen-antibody interactions (Cortese R. et al., Trends Biotechnol .; 12: 262-7, 1994; Birch-Marchin I. et al., J. Viroi Methods. ; 88: 89-104, 2000; Christopher G. et al., Infect. Mmunol .; 67: 4679-88 (1999), demonstrate that specific peptides are obtained by selecting phage libraries with monoclonal and polyclonal antibodies, epitoposlinear, as well as mimetopes, those that mimic linear, discontinuous, conformational antigens, and even non-peptide antigen peptides.

Peptides selected against a particular target that have similar sequences play a role in identifying the reason for binding (Stephen C. W. et al., J. Moi Biol .; 225: 577-83, 1992). Where selected peptides resemble the natural ligand peptide, they are termed mimetopes (Geysen M.. M. et al., Moi Immunol .; 23: 709-715,1986; Salotra Smith GP et al., Methods Enzymoi; 217: 228. -57, 1993). Phage Display-identified peptide sequences have been shown as receptor agonists or antagonists (Doobar J. Winter GJ, MoLS / o /.; 244: 361-9, 1994).

Peptides that neutralize immunoglobulins may be employed as diagnostic reagents or used as therapeutic agents 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, identify linker peptides, and develop 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 delivery vehicles of their own (Benhar I. Biotechnol. Adv .: 19: 1-33, 2001).

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

To date, there is no evidence in the literature revealing experiments using the "Phage Display" technique in the study of antigen-antibody interactions with the Leishmania protozoan. However, several authors report the use of this technique in understanding diseases caused by viruses, bacteria, protozoa and the interaction between their antigens and the host immune response, seeking epitope mapping or vaccine development for: Plamodium falciparum (de Ia Cruz VF etal., J. Biol. Chem .; 263: 4318-22, 1988; Willis AE et al., Gene, 128: 79-833,1993), Taenia solium (Gazarian KG et al., Immunol. Lett .; 42: 191- 5, 2000), Bursa Infectious Disease Virus (Cui X. et al., J. Viroi Methods; 109: 75-83, 2003), Eimeria tenelia (Abi-Ghanem D. et al., Vet. Immunol. Immunophatol ; 121: 58-67, 2008), Echinococcus granulosus (Read AJ et al., J. Chromatogr .; 877: 1516-22) and human neurocysticercosis (Hell RCR et al., Clin.Immunol .; 13: 129 -38, 2009) among others.

Several patents demonstrate the use of PhageDisplay technology in the study of parasitic diseases such as patents: US2007281302 (Bacteria-Binding Peptides: B. anthracis, B. subitilis and B.cereus and their use for B. antrhracis spore detection ); 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 T7 nobacteriophage virus) ; W02006071896 (SARS vaccine 1 comprising antigenic virus epitopes) and US2006094017 (discovery of HIV-virus gp120 protein-specific mimetopes and use of these 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 to find a set of interacting proteins that has been called an "interatoma." The search for these "interatomas" promises to expand the boundaries created by genomic projects and allows us to infer how these genes relate, explaining the individual phenotypically (Brígido Μ. M. Biotechnology Science and Development; 26: 44-51, 2002).

Methodologies for the use of this technique for expression of recombinant polypeptides and enhancement of technology have been described in patents: KR20070041966 (method for exposing fused proteinplll antibodies on recombinant phage surface with greater stability and practicability); US2009105090 (use of amino acid insert mutant plll protein to promote a change in peptide conformation); W09958655 (use of bacteriophage expressing heterologous polypeptides carrying a cleavage site within the expressed peptide which enables the propagation of the intact fusion protein); WO2009024591 (phage display alternative using pVII protein and commercialization kit thereof); US7083945 (protein expression in soluble forms for interaction with labeled ligands), US2006068421 (expression vectors and novel expression methods that allow efficient selection of polypeptide selection), US2005202512 (methods for selection of functional polypeptide selection), GB2408332 (specific binding methods) cell surface antigens), US2003104604 (proposes to increase the efficiency of the technique through bacterial expression), EP1452599 (libraries expressing phages with various mutant peptides or proteins capable of binding to materials of interest) and US2006035223 (peptides capable of bonding to inorganic materials such as silica, cobalt, iron or oxides) in which methodologies are proposed which improve the technique.

The present invention utilized "Phage Display" technology for the identification and selection of specific peptides and parasitic protein motifs of the genus Leishmania, determining functional epitopes of proteins involved in the parasite immune response to platform use for more accurate diagnosis and predictive vaccine compositions. and / or therapeutic against yeishmaniasis seeking greater specificity due to the use of specific antigen sites.

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 employed for the selection, characterization and use of recombinant peptides and protein motifs that mimic Leishmania antigenic regions could be applied to other protozoa.

The invention may be better understood by means of the detailed description, in accordance with the attached Figures, where:

FIGURE 1 presents the dot-blot assay performed for recombinant peptides selected for specific antibody-eluting antibodies in patients with Visceral Leishmaniasis (V) and also Tegumentary Leishmaniasis (T) and healthy controls (N).

FIGURE 2 presents the competitive dot-blot assay for clones of greater relevance in the specific selection for Visceral Leishmaniasis. The antibodies from patients with the disease were preincubated with the Leishmania chagasi total co-antigen, thus showing that recombinant peptides and natural antigen compete for the same binding sites on antibodies.

Example 1:

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

TABLE 1 presents the identity of the sequences for this invention, designated by Seq. ID No. 1 to Seq. ID No. 56 (normal sequences) eSeq. ID No. 57 to Seq. ID Ns 112 (inverted sequences) which refer to the antibody experiments performed on 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 broad selection (acid elution) and two more specific ones (recombinant antigen or total antigen), all targeting IgG-like antibodies from patients with Leishmaniasis Vital, such as:

The Acid Elution (Type 1 Selection), represented by the sequences Seq.ID N2 1 to Seq. ID No. 24 and its inverted counterparts Seq. ID NO: 57 aSeq. ID No. 80; The selection had a total number of 28 clones, 24 of which were distinct from each other, the clone represented by the sequences Seq. ID No. 14 -Seq. ID No. 70 presented the highest frequency in this selection (10.71%), followed by the sequence clones represented by Seq. ID No. 2 - Seq. ID Nos. 59 eSeq. ID No. 19 - Seq. ID No. 75 with intermediate representativeness (7.14%), the other sequences were unique in the population presented here.

Specific Elution against recombinant rK39 antigen (Type 2 Selection), represented by the sequences Seq. ID No. 25 to Seq. ID No. 42 and its inverted counterparts Seq. ID Nos. 81 to Seq. ID No. 98; The selection presented a total of 30 clones, 18 of them distinct from each other, the one represented by the sequences Seq. ID No. 34 - Seq. ID No. 90 showed the highest frequency in the population (20.0%), followed by the Seq clones. ID No. 25 - Seq. IDN2 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 Leishmaniachagasi total antigens (Type 3 Selection), having as representatives the sequences numbered Seq. ID NO: 43 to Seq. ID No. 56 with their corresponding designated inverters of Seq. ID No. 100 to Seq. ID Ns 112. The population is represented by 29 clones, 15 of which have distinct sequences. The most frequent occlone in this population is represented by the sequences Seq.ID N2 43 - Seq. ID No. 99 (31.03%), followed by those of sequences 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> table see original document page 20 </column> </row> <table> Continued from Table 1

<table> table see original document page 21 </column> </row> <table> Continued from Table 1

<table> table see original document page 22 </column> </row> <table>

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

Due to the conserved representation in certain peptide long regions, the motifs may point to important continuous or discontinuous antigen epitopes, as shown in TABLE 2, which presents the clone alignment and the consensus sequences obtained in the different types of selections made against Leishmaniasis patients' antibodies. Visceral, where: A: Acid Elution, B: Antigen specific elution rK39 and C: Specific elution with L. chagasi total antigen. In particular, the consensus sequences identified between the clones. TABLE 2

<table> table see original document page 23 </column> </row> <table> Continued from Table 1_

<table> table see original document page 24 </column> </row> <table>

Alignment of these consensus with Leishmaniachagasi restricted proteins in GeneBank bank (BLAST) has shown partial homology to relevant Leishmania proteins such as K39, GP63 , GP46 and the LACKd antigen among others, which is shown in TABLE 3, which presents the consensus consequences and the proteins to which they relate to their accession numbers, as well as the similarity given by the amino acids representative of the alignment, their position in the original protein and their numbers. "Score" and "EValue" for the alignment in question.

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

They present similarities with the consensus sequence identified as Seq. ID Ns 165 (Seq. ID N-166) the following peptide sequences and their respective inverted sequences: Seq. ID No. 12 (Seq. ID No. 68), Seq. ID No. 13 (Seq. ID No. 69) and Seq. ID No. 14 (Seq. ID No. 70).

TABLE 3

<table> table see original document page 25 </column> </row> <table>

The major protein to which this consensus sequence relates in a normal position is Leishmania chagasi K39 related to the motor protein Kinesin superfamily; The recombinant protein rK39 has high antibody titers in patients with visceral leishmaniasis, which may justify the success of the selection performed. The amino acid residue indicated here is in a different region from that of the recombinant antigen and may point to a new epitope and be a useful tool in the development of new immunodiagnostic and prophylaxis technologies. Another protein of consensus-related importance in two positions is GP46 - 46 KDa glycoproteinade present on the surface of the promastigote forms of most Leishmania species - which has high levels of messenger RNA in infectious promastigote forms, the presence of this consensor-related sequence to this protein may be a evidence of host immunological naresponse protein participation against the parasite and also have a high diagnostic value. When reversed, this consensus sequence is related to the following Leishmania protein \ gp63 protein, dhfr-ts (Leishmaniadonovani chagasi), Leishmania major kinesin-like molecule and GP46 -Membrane surface antigen (Leishmania donovani chagasi).

They are similar to the consensus sequence identified by the sequences Seq. ID No. 167 and its respective inverted sequence Seq. ID N-168, with full or partial consensus homology, the following sequences for Type 1 Selection: Seq. ID No. 4 (Seq. ID No. 60), Seq. ID No. 25 (Seq. ID No. 61), Seq. ID No. 26 (Seq. ID No. 62), Seq. ID No. 27 (Seq. ID No. 63), Seq. ID No. 28 (Seq. ID No. 64), Seq. ID No. 9 (Seq. ID No. 65), Seq. ID No. 10 (Seq. ID No. 66), Seq. IDN2 12 (Seq. ID No. 268), Seq. ID No. 20 (Seq. ID No. 76) and Seq. ID No. 23 (Seq. IDN2 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), Seq. ID No. 34 (Seq. ID No. 90) and Seq. ID No. 35 (Seq. ID No. 90) and for SelectionType 3: Seq. ID No. 37 (Seq. ID No. 93), Seq. ID No. 42 (Seq. ID No. 98), Seq. IDN2 38 (Seq. ID No.294), Seq. ID No. 43 (Seq. ID No. 99), Seq. ID No. 44 (Seq. ID No. 2100) and Seq. ID No. 48 (Seq. ID No. 104).

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

The consensus sequence identified by the sequence Seq. ID No. 169 (Seq. ID No. 170) has the sequences Seq. ID No. 3 (Seq. ID No. 59), Seq. ID No. 222 (Seq. ID No. 78), Seq. ID No. 16 (Seq. ID No. 72), Seq. ID No. 19 (Seq. ID No. 275), Seq. ID N2 18 (Seq. ID N2 74) as representatives of the consensus and did not present homology in the alignments performed data banks.

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

For antibodies of Tegumentary Leishmaniasis patients, two selection procedures were performed:

- Acid Elution (Type 1 Selection) represented by the sequence clones Seq. ID NO: 113 to Seq. ID No. 124 and its inverse counterparts Seq. ID No. 139 to Seq. ID No. 150 with a total of 12 clones each representing 8.33% of the population.

- Specific Elution (Type 2 Selection) performed against total antigens

of Leishmania amazonensis, represented by the sequences Seq. ID No. 125 aSeq. ID No. 138 and its inverted counterparts Seq. ID No. 151 to Seq. ID N2164, 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 N2145 (16.67%), followed by the sequence clones Seq. ID No. 114 - Seq. ID No. 2140, 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%.

TABLE 4

<table> table see original document page 28 </column> </row> <table> Clones selected in the selection described above, when aligned with each other, did not show clear definition of consensus sequences, as shown in TABLE 5 which shows the alignment between clones of different types of antibody elution against Tegumentary Leishmaniasis patients1 where A: Acid elution and B: Specific elution. In contrast, among the selections, the presence of repeated clones was observed 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. 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. reactivity to targets used in the selection, as well as showing alignments in various hypothetical sequences of various species of Leishmania deserve particular attention as targets to be used in the diagnosis and prophylaxis of cutaneous leishmaniasis.

TABLE 5

<table> table see original document page 29 </column> </row> <table> <table> table see original document page 30 </column> </row> <table>

Example 2:

This example relates to confirming the reactivity of the selected peptide from the Visceral Leishmaniasis Specific Elution objects of this invention.

The clones obtained from the aforementioned Type 2 and 3 Specific Selections previously identified by Seq. ID No. 25 to Seq. ID Nos. 56 and its inverted counterparts Seq. ID Ne 81 to Seq. ID No. 112.

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

Among the clones selected against the recombinant antigen rK39 were positively those identified by the following sequences and their inverted corresponding: Seq. ID Nos. 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. IDN2 34 (Seq. ID No. 90), Seq. ID No. 37 (Seq. ID No. 93), Seq. ID No. 41 (Seq. ID No. 297) and Seq. ID No. 42 (Seq. ID No. 98).

For the clones selected against Leishmaniachagasi total antigen, the following sequences and their inverted counterparts were positive: 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 clones in this assay related to the presence of consensus region in the molecule (indicated by + sign), which shows that there is a relationship of consensus presence with the differential reactivity of recombinant peptides.

TABLE 6

<table> table see original document page 31 </column> </row> <table> The positive clones in the above dot blot assay were subjected to a competitive dot-blot assay in which IgG type antibodies of patients with the disease in question were incubated with Leishmania chagasi antigen at the following concentrations: 1- semantogenous control, 2 - 0.5 pg / mL, 3-1.3 pg / mL, 4-4.9 pg / mL , 5 - 14.8 pg / mL, 6 - 44.4pg / mL, 7 - 133.3 pg / mL, and 8 - 400 pg / mL, as shown in FIGURE 2. Previous antibody priming with total antigen before In order to react with recombinant peptides expressed on the surface of phages, the aim was to block common ligand sites between these different antigens.

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

For the clones selected against Leishmaniachagasi total antigen, they had reactivity inhibited against Leishmania antigen, following sequences and their corresponding inverted sequences: Seq. IDN2 43 (Seq. ID No. 99), Seq. ID No. 4 (Seq. ID No. 60), Seq. ID No. 50 (Seq. ID No. 2106) and Seq. IDN2 54 (Seq. ID No. 110).

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

Immunoglobulin purification was performed by immunochromatography from a pool of these sera using protein A sepharose column. The column was equilibrated with 10 volumes 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 monitored by spectrophotometer (Ultrospec 1100 pro - AmershamBiosciences) to zero. Each sample was eluted with glycine buffer (0.1 M pH 2.7) and 1 mL fractions were collected which were also monitored by spectrophotometric reading. The fractions with the highest readings were pooled and the pH neutralized with 0.1 M NaOH. Thus, they were transferred to dialysis membrane, concentrated in commercial sugar (1h, 4 ° C) and then dialyzed in 3L phosphate buffer overnight in the chamber. cold at 4 ° C. Quantitation was by direct reading in spectrophotometer at 280 nm.

For the selection of recombinant peptides (recombinant peptides expressed in the filamentous phage p111 protein) a commercial 12-amino acid library (Ph.D.-12 ™ mer - New England Biolabs) was used. The experimental procedures were based on the protocol available from the manufacturer, with adjustments for the best selection of specific decones.

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

As substrate for this process, 50 μl of protein G agarose in 50% aqueous solution (recombinin ProteinG Agarose - Invitrogen ™) were used, which were washed by resuspension in 1mL TBS-T microtubes (50 mM Tris-HCI - pH 7.5, 150 mM NaCl - Tween 200.1%), the supernatant was carefully removed after centrifugation at 4,000rpm for 30 seconds in a refrigerated centrifuge. Then the resin was blocked for one hour with blocking buffer (0.1 M NaHCO3, pH 8.6; BSA5mg / mL; 0.02% NaN3) at 4 ° C and mixed every 15 minutes. After incubation, it was washed four times as described above to receive a mixture of 300 ng of purified antibody along with 1.5 χ 1011 viral particles in a final volume of 200 μί of 0.1% TBS-T, 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 an additional 10 times with one mL of 0.1% TBS-T, preparing for the ligating phage elution. one mL of elution buffer (Glycine-HCl 0.2 Μ, ρΗ 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. deTris-HCl (1 M, pH 9.1).

A small sample of this eluate (10 μΙ) was titrated and the remainder was used for reamplification in 20 ml of early-growing E. coli (ER2738) culture (OD60 x 0.3) containing 4.5-incubated tetracycline. hours at a controlled temperature shaker at 37 ° C prior to the phage precipitation procedure and subsequent titration.

The reamplified phages from the first selection cycle were used in a second cycle (2.0 χ 1011 cfu) and subsequently for a total of four cycles, and from the second cycle the wash buffer stringency was increased by 0.1. % to 0.5%, then using 0.5% Tween-20 in all washes. After the selection of the phage containing the recombinant peptides, the isolates were isolated and sequenced (the procedures will be described later).

For the experiments described as parasite-specific co-antigens for both Visceral Leishmaniasis and Cutaneous Leishmaniasis procedures were performed in solid phase.

In the first round of selection, one well of a Corning Incorporated Costar® microtiter plate was sensitized with 150pL of IgG antibodies from a pool of Visceral Leishmaniasis patient sera (immunochromatograph purified, concentrated, dialyzed and read-quantified). spectrophotometric analysis) at a concentration of 100 pg / mL in carbonate buffer (0.1 M NaHCO3, pH 8.6). Incubation occurred throughout the night at 4 ° C under shaking in a humidified environment.

After discarding the solution and drying the plate, it was blocked with 300 µl blocking solution (0.1 M NaHCO3, 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-HCI pH 7.5, 150 mM NaCl, water, 0.1% volume / volume of Tween 20) and then added 4 x 1010 viral particles. (10 pL from the original commercial library) diluted by 100 μι. TBST, and the plate was kept under agitation for one hour at room temperature. Non-antibody-binding phages were removed and the plate was washed 10 times with TBST to add 100 μΐ_ of a 100 pg / ml solution of purified healthy control IgG-like antibodies to elute the binding phages to these types of antibodies, then called of negative elution. This mixture was incubated for one hour at room temperature under stirring. The phage contained in the supernatant were discarded and the plate washed an additional 10 times with TBST. A second elution was performed using rK39 antigen-recombinant at a concentration of 100 pg / mL in a volume of 100 pL, with one hour incubation at room temperature. The phage contained in the rK39-binding supernatant were collected for further amplification and the plate was washed again 10 times to perform the third elution with 100 pL of a total (100 pg / mL) L. chagasi antigen solution, incubation was performed. one hour at room temperature, and supernatant phages were also collected for further amplification. Phages from antigen elutions were used in subsequent cycles of selection. A small aliquot (1 µl) of both eluates was titrated to measure the number of viral particles per titration and the remainder was amplified for use in subsequent cycles.

From the second round of selection, two wells were sensitized with purified antibodies from leishmaniasis positive patients as previously described. For each of these wells, negative and specific elution were performed, one for rK39 (Type 2 Specific Selection) and one for L.chagasi total antigen (Type 3 Specific Selection). All blocking and washing procedures were similar to those described above except that the concentration of the wash buffer turned to beTBST which is 0.5% (TBS (50 mM Tris-HCI pH 7.5, 150 mM NaCl, 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 amplified eluatonon was titrated and the blue colonies were individually placed in 1.2 mL Deep WeN plate wells (20 pg / mL tetracycline) containing bacteria. ER2738 in early stage decrease. The culture was incubated under vigorous shaking (250 rpm / min) for 5 hours. After this period, back-up aliquots were made and the remaining culture plates incubated throughout the night for individual DNA extraction from the clones.

For all phage-recombinant precipitation and reamplification processes the procedure was as follows: The culture was transferred to a centrifuge tube and centrifuged (10 min, 4,000 rpm). The residual cells were discarded and the supernatant transferred to a new tube and re-centrifuged. 80% of the upper supernatant was pipetted into a clean tube, where 20% of the volume of PEG-NaCl (20% weight / volume of polyethylene glycol-8000; 2.5 M NaCl) was added and the mixture remained in rest overnight. 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 centrifuged again, under the same conditions as above for a further 5 minutes so that the residual supernatant could be removed. The deflated 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 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 a further 5 minutes to remove the supernatant supernatant. The pellet was resuspended in 200 µl of 1X TBS and ready for titration.

The titration procedure was as follows for both the amplified and non-amplified eluates: serial dilutions of 10 "1 to 10" 4 and 10 ~ 8 to 10 "11, respectively, were performed. To nine pL of each dilution was added 200 pL of ER2738 bacteria (OD6oo> 0.5) incubating the mixture for 5 minutes before plating in LB medium (Tetracillin: 20pg / mL, IPTG: 0.5 mM and X-gal 40 pg / mL) along with 3 mL Agarose Top (10 g Bacto-Tryptone, 5 g yeast extract, 5 g NaCl 1 1 g MgCl 2 .6H20 / liter.) Plates were incubated at 37 ° C overnight, and the blue colonies representing phage-infected colonies were counted to obtain the number of infecting particles (pfus) for entry into subsequent 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 (Οθ6οο> 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 μΙ of the supernatant was transferred to a new plate, where 350 µl PEG-NaCl was added and incubated for 10 minutes at room temperature. The mixture was centrifuged for 40 minutes at 3,700 rpm at 20 ° C. The supernatant was discarded and the plate was inverted on absorbent paper to remove excess culture medium. The precipitate was resuspended in 100 µl iodide buffer (10 mM Tris-HCl, 1 mM EDTA, 4 M NaCl) and 250 µl Absolute Ethanol was added, incubating the mixture for a further 10 minutes at room temperature before subjecting it to centrifugation again. 40 minutes at 20 ° C. The supernatant was discarded and the precipitate washed with 150 µl 70% Etol, and centrifuged for a further 15 minutes at 20 ° C. All centrifugations occurred with a rotation of 3,700 rpm. The DNA was solubilized in 20 pL sterile ultrapure water and qualified by 0.8% Agarose gel electrophoresis, comparing it with the M13 phage DNA standard provided by the library manufacturer.

The isolated samples were subjected to automatic sequencing using the Big Dye Terminator Kit (GE Healthcare) and the MegaBace 1000 Automatic Sequencer (GE Healthcare). In the sequencing reaction, approximately 3 pL of DNA (depending on the average quantification given by spectrophotometric reading), 4pL of premix and 10 pmoles of the primer - 96 M13 - (5'-hoCCCTCATAGTTAGCGTAACG -3'- GE Healthcare) was used, which amplifies the region. of the coding amino acids of recombinant Μ13 fusion random peptides, the reaction volume being completed to 10 μL. The reaction occurred in a thermal cycler (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 software (Sequence Analyzer1 BASE CALLER, Cimarron 3.12, Phred 15). With DNA sequences, these were translated by the DNA2PRO program (http://relic.bio.anl.gov/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 outline likely consensus. Sequence alignments with Leishmania proteins were performed by BLAST - Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov).

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

The 33 clones with distinct sequences were tested in dot-blot assay against purified antibodies from patients with visceral, cutaneous and healthy control leishmaniasis, along with the following controls: phage-wild, irrelevant phage (selected from another selection) and rK39 antigen-recombinant.

Nitrocellulose membranes were phage sensitized (5, Ox109 viral particles diluted in two µl TBS), allowing them to dry for 2 minutes at room temperature. Each membrane was blocked with one mL of blocking solution (5% TBS-M Molico), incubated with shaking for two hours and then washed three times with wash buffer (TBST -0.5% Tween 20). 500 µl of the purified antibody (Visceral, Tegumentary, Negative) at the concentration of 25 pg / ml diluted in blockade was 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 μΙ_ of blocking dilute alkaline phosphatase labeled anti-IgG secondary antibody and incubated for one hour and then washed an additional 6 times with NBT solution. / BCIP and washed with water before being dried for image digitization. All incubations were performed at room temperature and under slow agitation.

The clones with the highest reactivity in the previous assay were subjected to a new competitive assay (14 clones) along with the controls (PhageWild, Irrelevant Phage, L.chagasi Total Antigen, and rK39 antigen-recombinant).

Antigens (phage and controls) were added to the nitrocellulose membrane strips, allowed to stand for drying for two minutes and then added one mL of blocking (TBS-M - Molico5%) which acted for two hours. During this time, the anti-purification solution from patients with Visceral Leishmaniasis was incubated with serially diluted L. chagasi antigen (400 pg / mL to 0.5 pg / mL including control without antibody). After the blocking period, the membranes were washed three times with wash buffer (TBS-T - 0.5% Tween 20) and then the preincubated mixture of antibodies and antigens was added, which remained in contact with the membrane for a hour. The membranes were washed six more times with the wash buffer and 500 µl of alkaline phosphatase labeled anti-IgG secondary antibody (1: 5000 blocking) was added and incubated for one hour. Six washes were performed as described above and further developed with one ml of developer solution (NBT / BCIP). The membranes were washed with water and dried for digitization of the images. All incubations cited occurred at room temperature, under slow agitation, LIST OF BIOLOGICAL SEQUENCES

(1) General Patent Application Information

(i) Applicant Data:

a) Name: Federal University of Uberlândia

b) Address: Av. João Naves de Avila, 2121 - Block 5L, Santa Monica Neighborhood, Zip Code: 38400-902 - Uberlândia, MG.

(ii) Title of the invention: "RECOMBINANT PEPTIDES AND REASONS MIMETIC TO LEISHMANIA ANTIGENS AND THEIR APPLICATIONS"

(iii) Number of sequences in the order: 170 (one hundred and seventy)

(iv) Computer readable format: Microsoft Word; Windows XP, PC type computer.

(2) General sequence information

Characteristics of sequenced molecules:

a) Type: PROTEIN

b) Protein Name: Leishmania protein mimetic recombinant proteins

c) Sequence Identity: Seq. ID № 1 to Seq ID № 170.

d) Original source of molecules: GenBank (www.ncbi.nlm.nih.gov/GenBank/)3- Information for Seq. ID № 1

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 1:

Wing Phe Thr Asp Lys Wing Be Wing Arg Pro Lys Wing 1 5 10

3- Information for Seq. ID № 2

Sequence Features:

a) Size: 12 amino acidsb) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 2:

Wing Thr Glu Wing His Pro Thr Thr Read Wing Wing Arg Lys1 5 10 12

3- Information for Seq. ID # 3

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 3:

Wing Thr Read Arg Asn Trp Ile Pro Pro Ser Be Thr1 5 10 12

3- Information for Seq. ID # 4

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 4:

Wing Thr Pro Asp Arg Be Arg Ile Glu Be Arg Leu1 5 10 12

3- Information for Seq. ID # 5

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 5:

Wing Thr Pro Arg Be His Met Leu Wing Be Ser1 5 10 12

3- Information for Seq. ID N2 6String Characteristics:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 6:

Aia Thr Pro Arg Be Read Be Thr Val Ile Be Ile1 5 10 12

3- Information for Seq. ID # 7

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 7:

Wing Thr Pro Arg Be Met His Pro Leu Pro Asp Leu1 5 10 12

3- Information for Seq. ID # 8 8

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 8:

Wing Thr Pro Arg Be Met Ile Wing Pro Pro Thr Wing 1 5 10 12

3- Information for Seq. ID # 9

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 9:

Wing Thr Pro Arg Be Ser Read Le Gln Ser Gln Ile1 5 10 123- Information for Seq. ID # 10 10

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 10:

Wing Thr Pro Arg Be Val His Be Wing Wing Gln Pro1 5 10 12

3- Information for Seq. ID # 11

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-11:

Phe Wing Read Lys Glu Asp Wing Gln Ser Seru Leu Ser1 5 10 12

3- Information for Seq. ID # 12

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 12:

Gly Leu His Lys Leu Wing Gly Leu Asn Leu Arg Ser1 5 10 12

3- Information for Seq. ID # 13 13

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 13: His Lys Leu Wing Gly Leu Asn Leu Arg Asp His Leu1 5 10 12

3- Information for Seq. ID # 14

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 14:

His Lys Leu Wing Gly Leu Asn Leu Arg Asp Gln Leu1 5 10 12

3- Information for Seq. ID # 15

Sequence Features:

a) Size: 7 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 15:

Leu Pro Met His Glu Be Pro Lys Arg Asp Gln Pro1 5 10 12

3- Information for Seq. ID # 16

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 16:

Pro Asn Wing Ward Thr Read Arg Thr Thr Lys Gln Ile1 5 10 12

3- Information for Seq. ID # 17

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID # 17:

Be Val Be Val Gly Met Lys Pro Be Pro Arg Pro1 5 10 12

3-Information for Seq. ID N-18

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 18:

Thr Pro Be Be Wing Be Be Val Wing Thr Read Arg1 5 10 12

3- Information for Seq. ID # 19

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 19:

Thr Be Wing Ward Thr Met Arg Read Leu Lys Ser Asp1 5 10 12

3- Information for Seq. Sequence ID: 20

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 20:

Thr Thr Pro Arg Be Glu Ile Pro Tyr Pro Ile Ile1 5 10 12

3- Information for Seq. ID # 21

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 21:

Val Cys Phe Cys Gly Tyr Glu Thr Glu

3- Information for Seq. ID N- 22

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 22:

Val Gln Ser Wing Thr Read Arg Asn Trp Ile Wing Ser1 5 10 12

3- Information for Seq. ID # 23

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 23:

Tyr Wing Wing Thr Pro Arg Be His Leu Thr Glu Leu1 5 10 12

3- Information for Seq. ID # 24 24

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 24:

Tyr Wing Wing Thr Thr Arg Be His Leu Thr Gly Pro1 5 10 123- Information for Seq. ID # 25

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 25:

Read Thr Gln Lys Phe Ser Gly Ile Asp Ile Lys Arg1 5 10 12

3- Information for Seq. ID # 26

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linearSequence description: Seq. ID No. 26:

Glu Lys Be Ser Gly Thr Be Serve His Gly Val Pro1 5 10 12

3- Information for Seq. ID # 27 27

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 27:

Val Val Thr Arg Tyr Asn Asp Asn Ile Arg His Ser1 5 10 12

3- Information for Seq. ID # 28

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 28: Ile Gly Thr Wing Pro Met Leu Phe Wing Gly Tyr Arg1 5 10 12

3- Information for Seq. ID N- 29

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 29:

Thr Thr Thr Leu Thr Arg Be Leu Pro Leu Ile His1 5 10 12

3- Information for Seq. ID N- 30

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 30:

Asn Read Asp Thr Arg Thr Trp Read Be Read Tyr Thr1 5 10 12

3- Information for Seq. ID # 31

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 31:

Gly Thr Pro Arg Be Gln Ile Gln Tyr Wing Pro1 5 10 12

3- Information for Seq. ID # 32

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 32:

Val Thr Pro Arg Be Read Asn Val Asp Pro Be Read Arg1 5 10 12

3- Information for Seq. ID No. 33

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linearSequence description: Seq. ID Numbers 33:

Ile Thr Pro Arg Be Asn Gln Wing Thr Tyr Leu Leu

1 5 10 12

3- Information for Seq. ID # 34

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 34:

Thr Thr Wing Gln Arg Asn Ser Ile Leu Val Gln Ser1 5 10 12

3- Information for Seq. ID N- 35

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 35:

Tyr Wing Be Wing Asn Thr Be Arg Thr Ile Tyr Thr1 5 10 12

3- Information for Seq. ID N- 36

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID # 36:

His His Phe Thr His Ile Ser Leu Pro Leu Val Arg1 5 10 12

3- Information for Seq. ID N- 37

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No 37:

Wing Thr Gln Arg Asn Wing Tyr Pro Be Gly Pro His1 5 10 12

3- Information for Seq. ID No. 38

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 38:

Ile Asn His Be Thr Wing Glu Be Arg Tyr Phe Met1 5 10 12

3- Information for Seq. Sequence ID: 39

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 39:

Ser Phe His Leu Pro Glu Trp His Wing Ser Pro1 5 10 123- Information for Seq. ID No. 40

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. iD N- 40:

Met Asn Wing Gly Ser Asn Wing Phe Ser Cys Leu Thr1 5 10 12

3- Information for Seq. ID N2 41

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 41:

Ile Asn Pro Be Tyr His Pro Phe Arg Met Pro Ala1 5 10 12

3- Information for Seq. ID # 42 42

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

B. Description of sequence: Seq. ID No. 42:

Wing Be Pro Arg Be Be Phe Leu Read Glu Lys Wing 1 5 10 12

3- Information for Seq. ID N2 43

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 43: Wing Thr Pro Arg Be Ile His Asp Be Glu Thr Leu1 5 10 12

3- Information for Seq. ID No. 44

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 44:

Read Be Pro His Wing Read Glu Lys Asp Arg Phe Val1 5 10 12

3- Information for Seq. ID No. 45

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 45:

His Met Being His Gln Asp Pro Asp Val Arg Read Le Gln1 5 10 12

3- Information for Seq. ID N- 46

Sequence Features:

a) Size; 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 46:

Ser Asp Thr Thr Thr Met Arg Wing Phe Arg Ile Thr1 5 10 12

3- Information for Seq. Sequence ID: 47

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 47:

Ser Leu Leu Asn Leu Phe Val Thr Ser Pro Wing Val1 5 10 12

3- Information for Seq. ID N- 48

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 48:

Wing Thr Ile Arg Asn Ile Gln Thr Phe Pro Met Val1 5 10 12

3- Information for Seq. ID N- 49

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 49:

Ser Wing Thr Val Thr Ile Lys Arg Leu Gln Tyr Thr1 5 10 12

3- Information for Seq. ID # 50

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 50:

Thr Pro Wing Arg Arg Phe Met Ile Leu Arg Pro Leu1 5 10 12

3- Information for Seq. ID No. 51

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 51:

Thr Pro Phe Met Lys Thr Val Leu Leu Thr Ser Ala1 5 10 12

3- Information for Seq. ID № 52

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 52:

Tyr Pro Thr Lys Wing Be Gly Asn Asp Read Arg Gly1 5 10 12

3- Information for Seq. ID № 53

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 53:

Tyr Asp Leu Asn Val His Tyr Leu Pro Pro Lys Pro1 5 10 12

3- Information for Seq. ID No. 54 54

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 54:

Thr Thr Wing Arg Ser Ile His Gly Phe Trp Arg Arg 5 5 123- Information for Seq. ID No. 55

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linearSequence description: Seq. ID No. 55:

Gly Lys Wing Pro Ile Thr Wing Leu Pro Gln Wing 1 5 10 12

3- Information for Seq. ID # 56

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 56:

Gly Thr Wing Ile Arg Asn Wing Gln Val Pro Read His1 5 10 12

3- Information for Seq. ID No. 57

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 57:

Wing Lys Pro Arg Wing Be Wing Lys Asp Thr Phe Wing 5 7 10 12

3- Information for Seq. ID No. 58

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 58: Lys Arg Wing Read Thr Thr Pro His Wing Glu Thr Wing 1 10 10 12

3- Information for Seq. ID N- 59

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 59:

Thr Be Ser Pro Pro Ile Trp Asn Arg Read Thr Ala1 5 10 12

3- Information for Seq. ID No. 60

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 60:

Leu Arg Be Glu Ile Arg Be Arg Asp Pro Thr Ala1 5 10 12

3- Information for Seq. ID No. 61

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID Numbers 61:

Be Ser Be Ala Read Met His Be Arg Pro Thr Ala1 5 10 12

3- Information for Seq. ID No. 62

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 62:

Ile Be Ile Val Thr Be Read Be Arg Pro Thr Wing

1 5 10 12

3- Information for Seq. ID N- 63

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 63:

Read Asp Pro Read Pro His Met Be Arg Pro Thr Wing

1 5 10 12

3- Information for Seq. ID Numbers 64

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 64:

Wing Thr Pro Pro Wing Ile Met Be Arg Pro Thr Wing

1 5 10 12

3- Information for Seq. ID N- 65

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 65:

Ile Gln Be Gln Pro Read Be Ser Arg Pro Thr Wing

1 5 10 12

3- Information for Seq. ID No. 66

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 66:

Pro Gln Ala Thr Be His Val Ser Arg Pro Thr Ala1 5 10 12

3- Information for Seq. ID N- 67

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 67:

Be Read Be Be Gln Wing Asp Glu Lys Leu Wing Phe1 5 10 12

3- Information for Seq. ID N- 68

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 68:

Ser Arg Leu Asn Leu Gly Wing Leu Lys His Leu Gly1 5 10 12

3- Information for Seq. ID N2 69

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 69:

Leu His Asp Arg Leu Asn Leu Gly Wing Leu Lys His1 5 10 123- Information for Seq. ID Ns 70

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. iD N2 70:

Leu Gln Asp Arg Leu Asn Leu Gly Wing Leu Lys His1 5 10 12

3- Information for Seq. ID N2 71

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 71:

Pro Gln Asp Arg Lys Pro To Be Glu His Met Pro Leu1 5 10 12

3- Information for Seq. ID N2 72

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 72:

Ile Gln Lys Leu Thr Arg Leu Thr Wing Asn Wing Pro1 5 10 12

3- Information for Seq. ID No. 73

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 73: Pro Arg Pro Ser Lys Met Gly Val Ser Val Ser1 5 10 12

3- Information for Seq. ID N2 74

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 74:

Arg Leu Thr Wing Val Ser Be Wing Ser Be Pro Thr1 5 10 12

3- Information for Seq. ID # 75

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 75:

Asp Ser Lys Leu Read Arg Met Thr Wing Wing Be Thr1 5 10 12

3- Information for Seq. ID N2 76

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 76:

Ile Ile Pro Tyr Ile Glu Be Arg Pro Thr Thr1 5 10 12

3- Information for Seq. ID No. 77

Sequence Features:

a) Size: 12 amino acidsb) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 77:

Glu Gln Being Glu Thr Glu Tyr Gly Cys Phe Cys Val

1 5 10 12

3- Information for Seq. ID # 78 78

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 78:

Ser Ala Ile Trp Asn Arg Read Thr Ala Ser Gln Val

1 5 10 12

3- Information for Seq. ID # 79 79

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 79:

Read Glu Thr Read His Ser Arg Pro Thr Wing Tyr1 Wing 5 10 12

3- Information for Seq. ID # 80

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 80:

Pro Gly Thr Read His Ser Arg Pro Thr Wing Wing Tyr1 5 10 12

3- Information for Seq. ID # 81 81

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 81:

Arg Lys Ile Asp Ile Gly Be Phe Lys Gln Thr Leu

1 5 10 12

3- Information for Seq. ID N- 82

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 82:

Pro Val Gly His Leu Be Thr Gly Be Ser Lys Glu

1 5 10 12

3- Information for Seq. ID # 83 83

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 83:

Being His Arg Ile Asn Asp Asn Tyr Arg Thr Val Val

1 5 10 12

3- Information for Seq. ID No. 84

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 84:

Arg Tyr Gly Ala Phe Leu Met Pro Ala Thr Gly Ile

1 5 10 123- Information for Seq. ID N- 85

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 85:

His Ile Leu Pro Leu Be Arg Thr LeuThr Thr Thr

1 5 10 12

3- Information for Seq. ID Numbers 86

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID Numbers 86:

Thr Tyr Leu Being Read Thr Trp Arg Thr Asp Leu Asn

1 5 10 12

3- Information for Seq. ID N2 87

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID Numbers 87:

Pro Wing Wing Tyr Gln Ile Gln Ser Arg Pro Thr Gly

1 5 10 12

3- Information for Seq. ID N- 88

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 88: Arg Be Pro Asp Val Asn Read Be Arg Pro Pro Val

1 5 10 12

3- Information for Seq. ID # 89 89

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID Numbers 89:

Leu Leu Tyr Thr Wing Gln Asn Ser Arg Pro Thr Ile1 5 10 12

3- Information for Seq. ID N- 90

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 90:

Be Gln Val Leu Ile Be Asn Arg Gln Pro Thr Ala1 5 10 12

3- Information for Seq. ID No. 91 91

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 91 91:

Thr Tyr Ile Thr Arg Be Thr Asn Wing Be Wing Tyr1 5 10 12

3- Information for Seq. ID # 92

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 92:

Arg Val Leu Pro Leu Ser Ile His Thr Phe His His1 5 10 12

3- Information for Seq. ID N- 93

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 93:

His Pro Gly Ser Pro Tyr Wing Asn Arg Gln Thr Wing 1 5 10 12

3- Information for Seq. ID N2 94String Characteristics:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 94:

Met Phe Tyr Arg Be Glu Wing Thr Be His Asn Ile1 5 10 12

3- Information for Seq. ID No. 95

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 95:

Pro Ser Ala His Trp Glu Arg Pro Read His Phe Ser1 5 10 12

3- Information for Seq. ID No. 96

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 96:

Thr Read Cys Be Phe Wing Asn Be Gly Wing Asn Met1 5 10 12

3- Information for Seq. ID No. 97

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 97:

Wing Pro Met Arg Phe Pro His Tyr Ser Pro Asn Ile1 2 10 12

3- Information for Seq. ID No. 98

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

20 Description of sequence: Seq. ID No. 98:

Wing Lys Glu Leu Leu Phe Be Ser Arg Pro Be Ala1 5 10 12

3- Information for Seq. ID No. 99

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 99:

Leu Thr Glu Ser Asp His Ile Ser Arg Pro Thr Ala1 5 10 123- Information for Seq. ID № 100String Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 100:

Val Phe Arg Asp Lys Glu Leu Wing His Pro Ser Leu1 5 10 12

3- Information for Seq. ID № 101

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 101:

Gln Read Arg Val Asp Pro Asp Gln His Ser Met His1 5 10 12

3- Information for Seq. ID № 102

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 102:

Thr Ile Arg Phe Wing Arg Met Thr Thr Trp Asp Ser1 5 10 12

3- Information for Seq. ID № 103

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 103: Val Wing Pro Ser Thr Val Phe Leu Asn Leu Leu Ser1 5 10 12

3- Information for Seq. ID No. 104

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 104:

Val Met Pro Phe Thr Gln Ile Asn Arg Ile Thr Ala1 5 10 12

3- Information for Seq. ID Numbers 105

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 105:

Thr Tyr Gln Read Arg Lys Ile Thr Val Thr Wing Ser1 5 10 12

3- Information for Seq. ID No. 106

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-106:

Leu Pro Arg Leu Ile Met Phe Arg Arg Pro Thr Ala1 5 10 12

3- Information for Seq. ID N2107

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID N-107:

Ala Ser Thr Read Leu Val Thr Lys Met Phe Pro Thr1 5 10 12

3- Information for Seq. ID No. 108

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 108:

Gly Arg Read Asp Asn Gly Ser Wing Lys Thr Pro Tyr1 5 10 12

3- Information for Seq. ID No. 109

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 109:

Pro Lys Pro Pro Read Tyr His Val Asn Read Asp Tyr1 5 10 12

3- Information for Seq. ID No. 110

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 110:

Arg Arg Trp Phe Gly His Ile Be Arg Pro Thr Ala1 5 10 12

3- Information for Seq. ID N-111

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-111:

Gln Pro Wing Read Wing Thr Ile Wing Pro Lys Wing Gly1 5 10 12

3- Information for Seq. ID No. 112

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 112:

His Leu Pro Val Gln Wing Asn Arg Ile Wing Thr Gly1 5 10 12

3-Information for Seq. ID N-113

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 113:

Asp Leu Ile Gln Thr Val Met Thr Arg Thr Wing Gly1 5 10 12

3- Information for Seq. ID No. 114

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 114:

Ser Ser Pro Tyr Ser Met Arg Leu Wing Lys Leu Glu1 5 10 123- Information for Seq. ID № 115

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 115:

Leu Arg Leu Ile Asp Glu Val Pro Wing Glu Leu Val1 5 10 12

3- Information for Seq. ID № 116

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 116:

Ile Pro Being Thr Thr Glu Ile Ile Leu Gly Leu1 5 10 12

3- Information for Seq. ID № 117

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 117:

Asp Gln Thr Read Phe Be Met Thr Arg Thr Pro Pro1 5 10 12

3- Information for Seq. ID № 118

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 118: Ser Ser Ala Read His Ala Thr Ala Thr Gln Ala Pro Gln1 5 10 12

3- Information for Seq. ID No. 119

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-119:

Be Be Wing Read Be Wing Wing Thr Thr Wing His Arg1 5 10 12

3- Information for Seq. ID No. 120

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 120:

Lys Ile Glu To Be Trp Read Leu To Be Asp Lys Arg1 5 10 12

3- Information for Seq. ID No. 121

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 121:

Gln Pro Asp Met Asn Ile Read Wing Met Ser Arg Pro1 5 10 12

3- Information for Seq. ID No. 122

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 122:

Gln Thr Wing Asn Asn Val Ile Wing Be Met Thr Arg1 5 10 12

3- Information for Seq. Id Ns 123

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 123:

Gln Asn Lys Val Ile Asp Wing Met SerArg His Pro1 5 10 12

3- Information for Seq. ID No. 124

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 124:

Met Arg Wing Ile Asp Ser Tyr Pro Glu Pro Ser Val1 5 10 12

3- Information for Seq. ID No. 125

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 125:

Thr Pro Be Gly Wing Gln Leu Trp Asp Ile Leu His1 5 10 12

3- Information for Seq. ID N2126

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 126:

Met Wing Pro Asp Gly Wing Gln Trp Trp Glu Wing Arg1 5 10 12

3- Information for Seq. ID N-127

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 127:

Thr Pro Be Gly Wing Gln Leu Trp Asn Asn Leu His1 5 10 12

3- Information for Seq. ID N2 128

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2128:

Thr Met Lys Thr Glu Leu Tyr Asn Leu Gln Leu Leu1 5 10 12

3- Information for Seq. ID N2129

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 129:

Tyr Ser Met Lys Thr Gln Gly Trp Leu Ser Ala Leu1 5 10 123- Information for Seq. ID No. 130

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 130:

Tyr Lys Read Asp Be Being His Read Leu Arg Gly Lys1 5 10 12

3- Information for Seq. ID No. 131

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-131:

Met Arg Wing Ile Asp Glu His Gly Ile Arg Pro Pro1 5 10 12

3- Information for Seq. ID N-132

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 132:

Gln Thr Read Leu Ile Asn Ile Gln Gly Wing Thr Lys1 5 10 12

3- Information for Seq. ID N-133

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 133: Leu Lys Gln Asp Leu Val Ile Ser Tyr Met Wing Arg1 5 10 12

3- Information for Seq. ID N- 134

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 134:

Asp Read Ile Thr Met Be Met Be Arg Be Pro Arg1 5 10 12

3- Information for Seq. ID No. 135

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-135:

Gln Arg Met Ile Asp Ser Val Pro Val Pro Ser Ala1 5 10 12

3- Information for Seq. ID No. 136

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID Nos: 136:

Asp Pro Val Ile Leu Ser Ile ThrArg Leu Leu Lys1 5 10 12

3- Information for Seq. ID N- 137

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID No. 137:

Being Pro Phe Asp Gln Val Leu Being Being Met Thr Arg1 5 10 12

3- Information for Seq. ID No. 138

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 138:

Asp Pro Read Ile Thr Thr Met Thr Thr Thr Phe Pro1 5 10 12

3- Information for Seq. ID No. 139

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 139:

Gly Wing Thr Arg Thr Met Val Thr Gln Ile Leu Asp1 5 10 12

3- Information for Seq. ID N-140

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N-140:

Glu Leu Lys Wing Leu Arg Met Ser Tyr Pro Ser Ser 5 5 12 12

3- Information for Seq. ID N-141

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 141:

Val Leu Glu Pro Wing Val Glu Asp Ile Leu Arg Leu1 5 10 12

3- Information for Seq. ID № 142

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 142:

Gly Leu Ile Ile Ile Pro Glu Thr Thr Be Pro Ile1 5 10 12

3- Information for Seq. ID № 143

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 143:

Pro Pro Thr Thr Thr Met Be Phe Read Thr Gln Asp1 5 10 12

3- Information for Seq. ID № 144

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID № 144:

Ser Ser Ala Read His Ala Wing Ala Thr Gln Ala Pro Gln1 5 10 123- Information for Seq. ID No. 145

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 145:

Arg His Wing Thr Thr Wing Wing Be Read Wing Be Ser1 5 10 12

3- Information for Seq. ID No. 146

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 146:

Arg Lys Asp Be Leu Read Le Trp Be Glu Ile Lys1 5 10 12

3- Information for Seq. ID N2 147

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 147:

Pro Arg Be Met Wing Read Ile Asn Met Asp Pro Gln1 5 10 12

3- Information for Seq. Id Ns 148

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 148: Arg Thr Met Ser Wing Ile Val Asn Asn Wing Thr Gln1 5 10 12

3- Information for Seq. ID No. 149

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 149:

Pro His Arg Be Met Wing Asp Ile Val Lys Asn Gln1 5 10 12

3- Information for Seq. ID No. 150

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 150:

Val Ser Pro Glu Pro Tyr Ser Asp Ile Wing Arg Met1 5 10 12

3- Information for Seq. ID No. 151

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 151:

His Leu Ile Asp Trp Leu Gln Wing Gly Ser Pro Thr1 5 10 12

3- Information for Seq. ID N2152

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID N-152:

Arg Wing Glu Trp Trp Gln Wing Gly Asp Pro Wing Met1 5 10 12

3- Information for Seq. ID N-153

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 153:

His Leu Asn Asn Trp Leu Gln Wing Gly Ser Pro Thr1 5 10 12

3- Information for Seq. ID No. 154

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 154:

Leu Leu Gln Leu Asn Tyr Leu Glu Thr Lys Met Thr1 5 10 12

3- Information for Seq. ID No. 155

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 155:

Read Ala Ser Read Le Trp Gly Gln Thr Lys Met Ser Tyr1 5 10 12

3- Information for Seq. ID No. 156

Sequence Characteristics: a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 156:

Lys Gly Arg Leu Read His Be Ser Asp Leu Lys Tyr1 5 10 12

3- Information for Seq. ID No. 157

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 157:

Pro Pro Arg Ile Gly His Glu Asp Ile Wing Arg Met1 5 10 12

3- Information for Seq. ID No. 158

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 158:

Lys Thr Wing Gly Gln Ile Asn Ile Read Leu Thr Gln1 5 10 12

3- Information for Seq. ID No. 159

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 159:

Arg Wing Met Tyr Ser Ile Val Leu Asp Gln Lys Leu1 5 10 123- Information for Seq. ID N2160

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 160:

Arg Pro Be Arg Be Met Be Met Thr Ile Read Asp1 5 10 12

3- Information for Seq. ID No. 161

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 161:

Wing Be Pro Val Pro Val Be Asp Ile Met Arg Gln1 5 10 12

3- Information for Seq. ID No. 162 162

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 162:

Lys Leu Leu Arg Thr Ile Ser Leu Ile Val Pro Asp1 5 10 12

3-Information for Seq. ID No. 163

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N2 163: Arg Thr Met Ser Be Leu Val Gln Asp Pro Phe Ser1 5 10 12

3- Information for Seq. ID N- 164

Sequence Features:

a) Size: 12 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 164:

Pro Phe Thr Arg Thr Met Met Thr Ile Leu Pro Asp1 5 10 12

3- Information for Seq. ID No. 165

Sequence Features:

a) Size: 9 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 165:

His Lys Leu Wing Gly Lys Asn Leu Arg1 5 9

3- Information for Seq. ID No. 166

Sequence Features:

a) Size: 9 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 166:

Arg Leu Asn Lys Gly Wing Leu Lys His1 5 9

3- Information for Seq. ID No. 167

Sequence Features:

a) Size: 5 amino acids

b) Type: amino acidc) Topology: linear

Description of sequence: Seq. ID N-167:

Thr Pro Wing Arg Ser1 5

3- Information for Seq. ID N- 168

Sequence Features:

a) Size: 5 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 168:

Ser Arg Pro Thr Ala1 5

3- Information for Seq. ID N-169

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID No. 169:

Wing Thr Read Wing 1 4

3- Information for Seq. ID N- 170

Sequence Features:

a) Size: 4 amino acids

b) Type: amino acid

c) Topology: linear

Description of sequence: Seq. ID N- 170:

Wing Read ThrAIa1 4

Claims (6)

1. - MIMETIC RECOMBINANT PEPTIDES TO ANTIGENS DELEISHMANIA ITS PROTECT REASONS AND REVERSE SEQUENCES, characterized by comprising the sequences Seq ID N-1 to Seq ID Ns 170. 2. - PROTECTIVE REASONS FOR LEISHMANIA ANTIGENS according to claim 1, characterized in that they include the sequences Seq ID N2 165a Seq ID N2 170. 3. - PEPTIDES, THEIR REVERSE SEQUENCES AND PROTECT GROUNDS according to claim 1 or 2, characterized in that they are used in the diagnosis of leishmaniasis as probes for the in vitro detection of the presence of circulating antibodies or other Leishmania genus counterparasite ligand molecules. 4. Synthetic or recombinant PEPTIDES, THEIR REVERSE SEQUENCES AND REPLACEMENTS According to Claims 1, 2 or 3, characterized by the detection of circulating antibodies or circulating molecules to be detected by immunoassays, such as by immunoenzymatic examples (such as ELISA, Western Blot, immunofluorescence, immunohistochemistry, EIA, and others), immunoagglutination tests, electrochemical sensors, quantum dots, IFL (lateral flow immunoassay) or other detection forms related directly or indirectly to body deflected samples such as saliva, urine and blood . 5. - PEPTIDES, THEIR REVERSE SEQUENCES AND PROTECTIVE GROUNDS according to claims 1 or 2, characterized in that they are used in the preparation of vaccine compositions to stimulate the human or animal immune system against parasites of the genus Leishmania. 6. - PEPTIDES, THEIR REVERSE SEQUENCES AND PROTECTIVE GROUNDS according to claims 3 and 4, characterized by the use of sequences identified by Seq. ID Na-I to Seq. ID No. 112 and Seq. ID No. 165 to Seq. ID No. 170- is preferably for differential diagnosis of the Visceral daleishmaniasis form and Seq. ID No. 113 to 164 preferably for differential diagnosis of the cutaneous form of leishmaniasis.