BRPI1106311A2 - use of mycobacterium leprae mimetic peptides for diagnosis and vaccines - Google Patents

Abstract

use of mycobacterium leprae mimetic peptides for diagnosis and vaccines. The present invention relates to the identification of mycobacterium leprae antigen mimotopes. mimotopes comprise peptides that have utility in diagnostic method and kit. Diagnostic methods are based on the interaction of such peptides with components of biological samples and subsequent detection of the resulting interaction. The diagnostic kit comprises the use of peptides and reaction detection reagents between peptides and components of biological samples. The invention also relates to immunological compositions containing peptides as active ingredient.

Description

Field of the Invention The present invention is concerned with obtaining novel antigens for use in diagnosis, prognosis (method and diagnostic kit), and in vaccines (immunological compositions) as a control measure for the diagnosis of mycobacterium leprae. leprosy from Mycobacterium leprae mimetic peptides used alone and / or in combinations.

Background of the Invention Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae, which primarily affects the skin and peripheral nerves (Britton W.J., Lockwood D.N. J. Lancet, 363: 1209-1219, 2004). In 2009, 244,796 new cases of leprosy were detected worldwide, numbers that have remained constant over the last years (WHO. Wkly Epidemiol. Rec., 85: 337348, 2010), indicating that the disease will still remain a health problem. in many countries. Leprosy has a wide spectrum of clinical manifestations. Two systems are used for disease classification. The Ridley and Jopling system is primarily established by histopathological examination of patient skin biopsies in association with clinical and bacteriological aspects. According to the classification, at the extremes of the spectrum are the forms called tuberculoid leprosy (TT) and lepromatous leprosy (LT), and among these forms are borderline borderline (BB), tuberculoid borderline (BT) and borderline lepromatous ( BL). For treatment purposes, patients are grouped into paudibacillary (TT and BT) and multibacillary (BB, BL, and LL) (Walker S., Lockwood D.N. Clin. DermatoL, 25: 165-172, 2007). The different clinical forms of the disease are determined by the individual's immune response to M. leprae. Patients with tuberculoid leprosy have a vigorous cellular immunity to the bacillus, which limits the disease and is characterized by the presence of few bacilli in the lesions and low titers of bacilli. circulating antibodies. In the other pole, lepromatous leprosy is characterized by the absence of specific cellular response, with uncontrolled proliferation of bacilli and high antibody titers. Borderline forms are characterized by a progressive reduction of the BT to BL form in the cellular response associated with increased bacillary load and antibody levels (Britton W.J., Lockwood D.N. J. Lancet, 363: 1209-1219, 2004). .

Currently, the diagnosis of leprosy is based on clinical signs and symptoms accompanied occasionally by bacilloscopic examination. Scholars argue that, in addition to bacilloscopy, other diagnostic tests such as histopathology, PCR, and immunoassays (based on humoral and cellular response) should be used concurrently with clinical examination (Aseffa A. et al. Lepr. Rev., 76 : 147-159, 2005). The evaluation of an individual's cellular immune response to M. leprae is performed by the Mitsuda test (Godal T. Prog. Allergy, 25: 211-242, 1978). The test consists of intradermal application of mitsudin, also called lepromin, to the anterior surface of the forearm (Tomimori-Yamashita J. et al. An. Bras. Dermatol., 71: 343-349, 1996). Positive Mitsuda reaction is associated with tuberculoid form (resistance to M. leprae), and negative reaction to lepromatous form of disease (susceptibility to M. leprae) (Opromolla DVA Classification. In: Opromolla DVA Notions of hansenology. Baurè: Center Reynaldo Quagliato, 2000, pp. 47-50). Mitsudin is a suspension of inactivated bacilli obtained from patients in the clinical form of lepromatous or armadillos (Dasypus novemcinctus) experimentally infected (Beiguelman B. C / en. Health Colet., 7: 117-128, 2002). that At. leprae is not cultivable in vitro (Scollar DM et al. Cln. Microbiol. Rev., 19: 338-381, 2006). Mitsudin is not used as a diagnosis because it contains many antigenic determinants common to other mycobacterial species, resulting in non-specificity (Geluk A. et al. Infect. Immun., 70: 2544-2548, 2000) and false results. positive in healthy subjects (Brahmbhatt S. et al. Clin. Exp. Immunol., 128: 140148,2002). The only immunological diagnostic test currently available is based on the detection of anti-PGL-1 (phenolic glycolipid I) antibodies by ELISA, dipstick test or ML Flow Test (Oskam L., Slim E. , Buhrer-Sékula S. Lepr. Rev., 74: 196-205, 2003; Aseffa A. et al., Lepr. Rev., 76: 147-159, 2005). The assay can detect antibodies in most multibacillary patients, but has limited value in identifying paucibacillary patients and their contacts (Geluk A. et al. Infect. Immun., 73: 5636-5644, 2005).

Therefore, the development of inputs that allow the early diagnosis of leprosy is extremely desirable, since patients diagnosed and treated late may present physical disabilities and deformities that may lead to decreased working capacity, limitation of social life and psychological problems; In addition, early diagnosis interrupts transmission, making it possible to eliminate the disease.

State of the art Several strategies have been used in the identification of antiphenes for use in the diagnosis of leprosy. Initially, through the use of leprosy patient serum or monoclonal antibodies produced against proteins from subcellular fractions it was possible to identify antigens in M. èprae (Gillis TP, Buchanan TM Infect. Immun., 37: 172-178, 1982; Britton WJ et al. J. Immunol., 135: 4171-4177, 1985). With the emergence of molecular biology, Young et al. (1985) constructed a recombinant DNA library and with the use of monoclonal antibodies antigen coding genes were isolated in M. leprae (Young R. A. et al. Nature, 316: 450-452, 1985).

Subsequent studies, such as those cited below, have identified antigens on M. leprae for their biochemical and / or immunological properties. Proteins were studied as total molecules or as peptides comprising part of the total proteins.

Mckenzie et al. (1991) demonstrated a 70KDa protein, obtained by recombinant DNA technology, that was able to induce humoral and cellular response in individuals exposed to mycobacteria (McKenzie K.R.efa / J. Immunol., 147: 312-319, 1991).

Estrada et al. (1992) synthesized peptides corresponding to protein segments 65kDa, 28kDa, 28kDa superoxide dismutase and 18kDa from M. leprae and evaluated their ability to induce cellular response in animals. Among the tested peptides, some were able to induce specific late type hypersensitivity responses (Estrada I. C. et al. Int. J. Lepr. Other Mycobact. Dis., 60: 18-27, 1992).

Rivoire et al. (1994) by recombinant DNA technology and biochemical methods identified a protein of lOKDa in M. leprae (Rivoire B. et al. Infect. Immun., 62: 2417-2425, 1994.) Chua-intrtra et al. (1998) characterized a peptide sequence of M. leprae lOKDa antigen as a stimulatory determinant of T cells in patients with tuberculosis leprosy which made the peptide an interesting reagent for skin test or blood test for tuberculoid leprosy (Chua-lntra B. et al., Infect. Immun., 66: 4903-4909), Wilkinson et al. (1999) evaluated the cellular and humoral response of four recombinant proteins, 10kDa, 28kDa, 36kDa and 65kDa. LOKDa antigen stimulated a predominantly cellular response, 65KDa antigen was the best cellular response inducer in tuberculoid patients, 36kDa antigen stimulated a high titer humoral response in lepromatous patients, and 28kDa antigen was a moderate stimulator of cellular response. cellular and humoral (Wilkinson KA et al. J. infect. Dis., 179: 1034-1037).

Macfarlane et al. (2001) compared the cellular response induced by different recombinant proteins in patients and controls. Data show that the 45KDa protein is a potent T cell antigen in leprosy patients and contacts, suggesting its use as a diagnostic reagent (Macfarlane A. et al. Clin. Diagn. Lab. Immunol., 8: 604- 611,2001).

Spencer et al. (2002) synthesized overlapping peptides encompassing the M. leprae ESAT-6 protein and two M. tuberculosis ESAT-ó peptides. M. leprae polyclonal and anti-ESAT-α monoclonal antibodies allowed the identification of B and T cell epitopes (Spencer J. S. et al. Infect. Immun., 70: 1010-1013, 2002).

Brahmbhatt et al. (2002) studied the cellular response to a set of overlapping peptides of the M. leprae 45KDa antigen in patients and controls. Some peptides have been recognized by tuberculosis leprosy patients, but have also been recognized by tuberculosis patients (Brahmbhatt S. et al. Clin. Exp. Immunol., 128: 140-148, 2002).

Geluk et al. (2004) attempted to identify CFP-10 protein peptides that should only be recognized by M. leprae-specific T cells since the total protein provides cross-reactions. None of the peptides were recognized only by leprosy patients (Geluk A. et al., Scand J. Immunol., 59: 66-70, 2004).

Hussain et al. (2004) evaluated T and B cell response to a set of M. leprae GroES protein overlapping peptides and the results showed that humoral response inducing peptides are different from those that induce cellular response. It was also observed that responses were directed to shared epitopes between M. leprae and M. tuberculosis (Hussain R. et al., Immunology, 111: 462-471,2004).

Spencer et al. (2004) produced recombinant CFP-10 proteins from M. leprae and M. tuberculosis and their overlapping peptides to identify leprosy-specific antigens. Peptides that differentiate both infections have been characterized (Spencer J. S. efal. Infect. Immun., 72: 3161-3170, 2004).

With sequencing of the M. leprae genome as well as other mycobacterial species, recent studies, as cited below, use this information to identify new antigens for diagnostic use.

Geluk et al. (2005) selected several candidate antigens in M. leprae that lack homo.logia in M. tuberculosis and other mycobacteria by comparative genomic analysis. Potential proteins were produced by recombinant DNA technology and evaluated for their ability to stimulate T cell in patients and controls. Most multibacillary patients did not respond to the antigens tested. Reactive paucibacillary and multibacillary patients were highly responsive to various proteins (Geluk A. et al. Infecf. Immun., 73: 5636-5644, 2005).

Spencer et al. (2005) identified M. leprae-specific genes by comparative genomic analysis and tested them in the form of recombinant proteins and synthetic peptides for their ability to stimulate T cells in patients and controls. Peptides have been shown to be useful in the development of diagnostic testing (Spencer JS et al. J. Immunol., 175: 7930-7938, 2005; Oswaldo Cruz Foundation (Rio De Janeiro, RJ). Pessolani, MCV et al. BR No. PI0605850 -7 A2, 2006).

Aráoz et al. (2006) used comparative genomics to identify potential antigens restricted to M. leprae. Recombinant proteins were evaluated for their ability to recognize against patient serum and to stimulate T cells. Two proteins (ML0308 and ML2498) were able to induce cellular and humoral response, with consequent possibility of diagnostic use (Aráoz R. et al. Immun., 74: 175-182, 2006), Reece et al. (2006) employed a serological expression clonning method with a pool of sera from lepromatous patients for protein identification. Two recombinant proteins (ML 0405 and ML 2331) have been used for serological diagnosis in multibacillary leprosy patients (Reece S. T. et al. Clin. Vaccine Immunol., 13: 333-340).

Aráoz et al. (2006) identified by comparative genomics studies restricted proteins to M. leprae and tested them as recombinant proteins. Humoral and cellular response were evaluated in patients and controls from two distinct regions. None of the antigens induced the same response in the studied populations, which probably reflects the heterogeneous immune response related to the genetic characteristics of each individual. The peptide derived from one of the proteins did not induce cellular immune response in animals sensitized with environmental mycobacteria, showing the specificity of the immune response to the peptide (Araóz R. ef al. Microbes Infect., 8: 2270-2276, 2006).

Groathouse et al. (2006) evaluated serum humoral immune response patterns of protein microarray patients to define protein profiles that reflect disease forms. The arrays were constructed with isolated cell wall, membrane and recombinant-s proteins, which are unique or selective for M. leprae according to comparative bioinformatics analysis. Results indicated that screening of sera from protein microarray patients can discern antigens and patterns that are specific to disease classification (Groathouse N. A. et al. Infect. Immun., 74: 6458-6466, 2006).

Duthie et al. (2007) evaluated recombinant proteins, selected by serological screening of M. leprae genomic libraries, and a recombinant fusion protein for the ability to detect infection by serological testing in patients from different regions. Differences in responses were observed in patients from different regions. The fusion protein allowed diagnosis before clinical signs appeared (Duthie M.S. et al. Clin. Vaccine Immunol., 14: 1400-1408, 2007).

Geluk et al. (2008) identified synthetic peptides comprising sequences of five M. leprae proteins, which were previously selected by comparative genomic analysis, and evaluated them for their ability to induce cell response in patients and controls. The combination of four peptides allowed the detection of the disease in paucibacillary patients (Geluk A. et al., Clin Vaccine Immunol., 15: 522-533, 2008).

Geluk et al. (2009) evaluated antigens, such as recombinant proteins and peptides, previously identified, regarding their ability to induce cellular response in patients and controls from different regions. Specific T cell responses were observed among the peptides studied (Geluk A. et al, Clin. Vaccine Immunol., 16: 352-359, 2009).

Duthie et al. (2010) analyzed synthetic peptides derived from previously identified M. leprae proteins for the presence of antibody binding sites by ELISA. A recombinant fusion protein containing the antibody binding sites was constructed. Serum antibodies from multibacillary patients recognized the new antigen, demonstrating its usefulness in diagnosis (Duthie M. S. et al. Clin Vaccine Immunol., 17: 298-303, 2010).

As mentioned, considerable efforts have been made to identify antigens for the diagnosis of leprosy. And the greatest difficulty in obtaining these antigens lies in the search for specificity, that is, in avoiding cross reactions with antigens from other species of mycobacteria.

Control measures include vaccination with M. bovis BCG, a vaccine that protects against leprosy and tuberculosis. BCG vaccination in leprosy has been shown to be effective, but the degree of protection varies between studies. Live bacterial vaccines show some degree of protection, but their use is limited in safety and production. The use of M. leprae or its antigens, which provide protection in animals, is limited by the availability of these reagents. Some recombinant antigens have shown protection in animals, but many results are inconsistent. Many of these antigens have not been evaluated in patients and will not be approved for use in humans due to their high homology to human proteins. With the genome sequencing of Λ4. leprae and other mycobacteria, tools for molecular biology and bioinformatics have allowed the identification of antigen in M. íeprae that may be useful in vaccination (Raman V. S. et al. Infect. Immun., 77: 5623-5630, 2009).

Therefore, the discovery of new antigens for use in diagnosis, prognosis and vaccines is necessary as a leprosy control measure. .....

Brief Description of the Invention The present invention is based on the identification of mycobacterial antigen specific mimotopes by the Phoge Display technique. The term "mimotope" refers to a peptide selected from random phage-expressed libraries that mimics the structure of an epitope. Meanwhile, the term "antigen" refers to any substance that induces an immune response involving κ or B cells.

Thus, the invention provides amino acid sequences in the form of peptides which may be employed in different presentations as in diagnostic method and kit or in immunological compositions. The invention provides a diagnostic method for mycobacterial infectious diseases, which method comprises the following steps: a) Sample collection; b) Place the sample, after processing, in contact with at least one of the identified peptides; c) Detection of binding between peptides and sample components. The present invention also envisages the development of a diagnostic kit for infectious diseases caused by mycobacteria. This diagnostic kit comprises at least one of the identified peptides and reaction detection reagents between antigen and sample components obtained from individuals. The present invention also relates to immunological compositions having as active ingredient at least one of the identified peptides associated with one or more physiologically acceptable carriers or adjuvants.

Attention to Flauras The accompanying figures will serve to provide a better understanding of the process of identifying peptides derived from phage peptide libraries and their uses. It should be noted that the process is not limited to M. leprae species, but is extended to other mycobacterial species, including / vi. tuberculosis. Figure 1 illustrates the reactivity of antigen-specific immunoglobulins by ELISA. An ELISA plate was sensitized with 10 μρ / ιτιί antigen (Ai. Leprae - A and B or M. tuberculosis - C) and incubated with immunoglobulins starting from 25 μρ / mL for the total (dark bars) and 5 pg / mL for antigen-specific (light bars). Reaction detection was performed with Fc-specific human anti-IgG antibody conjugated to peroxidase and OPD as chromogen. Figure 2 illustrates the reactivity of phage clones by ELIS / Ί against serum samples from paucibacillary and multibacillary leprosy patients, tuberculosis patients, leprosy contacts and healthy individuals. The evaluated clones were previously selected and showed antibody binding capacity of leprosy patients. In A, clone IA, B, clone 2A, C, clone 3A, D, clone 4A, E, clone 5A, F, clone 6A and G, clone 1B. ELISA plates were sensitized with 50 µg of anti-phage antibody (1: 800) and incubated with 2x1010 phage / mL followed by the addition of serum (1: 100). Detection of the reaction was performed with peroxidase-conjugated humus Fc-specific anti-IgG antibody and OPD as chromogen. MB, multibacillary leprosy patients; CP, paucibacillary leprosy patients; TB, tuberculosis patients; C, contacts; S, healthy individuals. Dotted line represents the cut-off value (mean OD492 of samples from healthy individuals plus twice the standard deviation, however the acceptance of each sample in the healthy group was assessed by z-score). The points on the graph represent the individuals. Figure 3 illustrates the reactivity of peptides identified as serum in paucibacillary leprosy patients, multibacillary leprosy patients, tuberculosis patients, and healthy individuals. ELISA plates were sensitized with 100 μί peptide pool at 1.5 μρ / mL and incubated with serum (1:50). The detection of the reaction was performed with Fc-specific human biotin anti-IgG antibody and peroxidase-conjugated streptavidin and OPD as chromogen. CP, paucibacillary leprosy patients; MB, multibacillary leprosy patients; TB, tuberculosis patients; C, contacts; S, healthy individuals. Dotted line represents the cuf-off value (mean OD492 of samples from healthy individuals plus three times the standard deviation). The points on the graph represent the individuals. Figure 4 illustrates the reactivity of mycodacterial antigens (crude antigen) against ELISA-produced hyperimmune sera in rabbits. ELISA plates were sensitized with 50 μΙ. 10 pg / mL crude antigen and incubated with 1: 4000 diluted serum. The detection of the reaction was performed with anti-IgG rabbit rabbit peroxidase-conjugated total molecule and OPD as chromogen. Figure 5 illustrates the reactivity of anti-peptide antibodies with the respective peptide, respective BSA-conjugated peptide, BSA-conjugated irrelevant peptide (1-BSA), KLH-conjugated irrelevant peptide (1-KLH) and nonimmune serum. An ELISA plate was sensitized with 100 μΐ peptide at 1.5 μρ / ιτιΐ. Reaction detection was performed with peroxidase-conjugated γ-specific mouse anti-IgG antibody and OPD as chromogen. Figure 6 illustrates the reactivity of M. leprae anti-peptide antibodies by Western Bloffing. (A) 40 pg of total M. leprae extract was separated by electrophoresis and reacted with 1B-1: 12000 anti-peptide serum (lane 1), 6A-1: 9000 anti-peptide serum (lane 2), anti-peptide serum. peptide 5A-1: 4000 (lane 3) and non-immune serum 1: 4000 (lane 4). (B) Western blotting of a two-dimensional gel with 200 μg of crude M. leprae extract and reacted with anti-peptide 1 B-1: 4000 serum. The two other anti-peptide sera had the same reactivity profile presented with anti-peptide 1B serum. The detection of the reaction was performed with peroxicity conjugated γ-specific mouse anti-IgG antibody and chemiluminescence. Figure 7 illustrates the identification of key residues in peptide 5A. Sequence 5A alanine analogs were prepared by Spot Synthesis and evaluated for reactivity to a serum pool of multibacillary leprosy patients. Antibody binding of leprosy patients (1: 250) to membrane peptides was detected by peroxidase-conjugated Fc-specific human anti-IgG (1: 120000). (A) Peptide reactivity pattern. Peptide 5A is at position 1, an unrelated peptide (CN-negative control) is at position 17 and the other spots correspond to alanine analogs of peptide 5A). (B) Peptide reactivity intensity. The residue substituted by alanine is indicated for each analog. When alanine was present at the analog generating position, the alanine residue was replaced with serine. The present invention is based on the identification of mycobacterial antigen-specific mimotopes by the Phage Display technique, a method of presentation of peptides or proteins, including antibodies, on the surface of filamentous phages. This technique allows the study of protein-ligand interactions, can be used in the mapping of monoclonal and polyclonal antibody epitopes, as well as in the identification of receptor ligands and in the selection of enzyme substrates (Azzazy H. M „Highsmith WE Jr„ Clin Biochem., 35: 425-445, 2002). The Phage Display technique presents an alternative for identifying mycobacterial antigens for diagnostic, immunotherapy, vaccine, or immunization use, as previously used methods, as described in the prior art, are lacking of specificity and sensitivity.

In addition, the methods used to date have been based on prior knowledge of antigens for the characterization of new diagnostic reagents. An advantage of the Phage Display technique is that it does not require prior identification of the natural antigen against which an immune response is directed (Azzazy H.M., Tghsmith W.E Jr., Clin. Biochem., 35: 425-445, 2002).

Thus, phage-presented peptide libraries were screened using antibodies and more specifically antigen-specific antibodies.

Among the antibodies found in leprosy patients, few are specific for M. leprae antigens. In the same individual, in addition to M. leproe-specific antibodies, antibodies directed to the most varied antigens that the individual came into contact with, including other mycobacteria, are found. .

Antigen-specific antibodies may be obtained not exclusively from samples of infected individuals, but may be obtained from samples of experimentally infected animals. The term "sample" refers to a variety of sample types obtained from individuals such as blood, serum, plasma, and other samples of biological origin.

Blood samples of human or animal origin may be processed for. antibody separation by known prior art procedures such as centrifugation or further processed by additional procedures such as precipitation and / or affinity chromatography for separation of specific immunoglobulin subclasses. Further, antibodies may be separated by binding to an antigen immobilized column or antibodies may be separated from immunoblots (Harlow E., Lane D. Antibodies: a laboratory manual. New York: Cold Spring Harbor Laboratory, 1988. 726 p. ).

The antibodies used in the present invention for the selection of phage library peptides were from a pool of serum samples from different M. lepràe infected individuals. The use of the pool was intended to cover a greater diversity of antibodies. The wells may be formed from patient samples of different clinical forms as exemplified in this invention, i.e. multibacillary patient pool and paucibacillary patient pool. The multibacillary patient pool comprises patients with borderline-borderline, borderline / lepromatous, and lepromatous leprosy), while the paucibacillary patient pool comprises patients with the tuberculoid and borderline-tuberculosis leprosy clinical forms. However, the pools may also constitute form-specific pools, that is, formed by samples of patients presenting a certain clinical form. ...

Antigen-specific antibodies denote in the present invention antibodies obtained from serum from patients who were subjected to ammonium sulfate precipitation with subsequent purification by G-protein affinity chromatography and further purified against M. leprae antigens after Immunoblottlng (Fig. However, obtaining antigen-specific antibodies is not limited to this example, but may involve the use of one or a combination of two or more known prior art procedures. Also, the antibodies obtained are not limited to the IgG class, but may comprise the other classes and their subclasses. The use of such antigen-specific antibodies for peptide selection in phage libraries provides a greater opportunity to find mycobacterial antigen mimetic peptides (mimotopes).

Phage libraries were screened using M. leprae antigen-specific antibodies as well as the same libraries were screened using M. tuberculosis antigen-specific antibodies. In the latter case, non-antibody-bound phages from tuberculosis patients were subsequently used with M. leprae antigen-specific antibodies to prevent selection of nonspecific mimotopes.

Through Phage Display technology phage / peptide clones were selected for recognizing antibodies from leprosy patients.

The identified peptides comprise mimotopes or also called mimotypes, as they are not natural epitopes, but are epitopes that mimic part of the natural epitope. Mimicry is due to sequence similarity or similarity in physicochemical properties and special organization (Moreau V. et al., Bioinformatics, 22: 1088-1095, 2006).

Among the objectives of the present invention was to evaluate whether the identified peptides, which were in turn selected from phage-expressed peptide libraries, had the ability to react with serum antibodies of leprosy patients. The ability of peptides to react with antibodies to leprosy patients has been demonstrated in the present invention. The analysis comprises incubating the sample, preferably serum, from individuals with leprosy in the presence of one or more peptides. Binding of the sample antibody to the peptide (s) is then detected. It is understood that in addition to serum, other samples of biological origin containing antibodies may be employed in the analysis. .

The peptides may be employed individually or in combination. The term "combined" refers to a composition consisting of two or more sequences listed in table 1. Therefore, one composition could consist of sequence No. 1 and sequence No. 3 and another composition could consist of sequences No. 5 , sequence No. 6 and sequence No. 7. The term "combined" also refers to peptide polymer, which in turn is a longer peptide formed by repeating a peptide sequence which may or may not be separated by spacers or liners. When present spacers or linkers may be located between some sequences or between all peptide sequences. The polymer may consist of any peptide sequence and further the polymer may comprise different peptide sequence combinations. Thus, a polymer may be formed by one or more repeats of the first sequence, followed by one or more repeats of the second peptide sequence.

The peptide sequences dealing with the present invention may be modified into variants, provided that such variant forms retain the antigenic and / or immunogenic characteristics of the original sequences from which they were derived. The term amphigenic refers to the ability of the antigen to recognize the T cell antibody or receptor, while the term immunogenic refers to the ability to induce a humoral and / or cellular immune response.

Modifications include, but are not limited to, acetylation, amidation, formylation, biotinylation, phosphorylation, succinylation, sulfurylation, addition of one or more fatty acids, addition of one or more dyes such as AMC (7-amino-4-methyl-coumarin ), Cy3, Cy5, Dabcyl, Dansyl, DNP (2,4-dinitrophenyl), DNP-lysine, EDANS (5-2 ((2-aminoethyl) amino) naphthalene-1-sulfonic acid), fluorescein, NBD (7- nitrobenzene-2-oxa-1,3-diazole), p-nitroaniline, rhodamine B and Tmra, substitution of one or more naturally occurring amino acids, substitution of one or more amino acids for unnatural or special amino acids such as, citrulline, ornithine, ε-acetyl lysine, β-alanine, aminobenzoic acid, o-aminocaproic acid, aminobutyric acid, dimethyl lysine, hydroxyproline, mercaptopropionic acid, methyl lysine, 3-nitro-tyrosine, norleucine, pyroglutamic acid, and many others, addition of one or more naturally occurring amino acids, addition of one or more unnatural or special amino acids, substitution of one or more amino acids for heavy amino acids, addition of one or more heavy amino acids, replacement of one or more amino acids for D-amino acids, addition of one or more D-amino acids, cyclization , addition of spacers or linkers, addition of one or more disulfide bridges, addition of polyethylene glycol (PEG), labeling with radioactive isotopes, substitution of one or more amino acids with N-methyl amino acids, addition of one or more N-methyl amino acids and deletion of one or more amino acids.

Another modification is the conjugation of peptides to carrier proteins such as bovine serum albumin (BSA), ovaibumin, KLH (keyhole cleaned hemocyanin). diphtheria toxoid, tetanus toxoid, etc. Several methods are available for peptide-protein conjugation such as EDC conjugation, MBS conjugation and glutaraldehyde conjugation.

Another modification that can be found in peptides is the formation of MAPs (Mu / tip / and Antigen Peptides), which are characterized by using the β-amino groups of lysine to form a backbone to which multiple peptide chains can be attached. MAPs are an alternative to conjugating antigens to carrier proteins.

Note that when peptide sequences are used individually or in combination, it refers to the use of both the original and variant sequences.

Peptides (original sequences or variants) may be produced by recombinant DNA technology or by chemical synthesis and both methods are known from the state of the art.

An object of the present invention is to provide a method of diagnosing infecção infection. leprae. To this end, at least one of the peptides (isolated or combined) is immobilized on a solid support and after contacting the antibody-containing sample obtained from individuals with the peptide (s) the binding between antibody and peptide (s) is detected. The antigen, which corresponds to at least one of the peptides, is placed in contact with a solid support so that it can bind and become adsorbed on that support. Solid supports include 96-well polystyrene or polyvinyl chloride (PVC) microplates, nitrocellulose membranes, polyvinylidene fluoride (PVDF) or nylon, polystyrene or agarose beads, glass, and more. The immobilized antigen is placed in contact with the sample containing antibodies for interaction. Molecules that have not specifically interacted with the solid support bound antigen are removed by washing with suitable buffer solutions. For detection of antigen-antibody interaction a second labeled antibody (secondary antibody) is added. The solid support may be washed again for removal of unbound antibodies. The reaction between antigen and antibody is revealed by a method, which depends on the labeling employed, to produce a detectable or measurable signal. Signal strength indicates the amount of antibody in the sample. The antibody that recognizes the antigen is called the primary antidorpo. If this antibody is labeled, direct antigen detection is possible. Generally, the secondary antibody is labeled, so the antigen is detected indirectly. The secondary antibody may be a polyclonal or monoclonal antibody which is in turn produced in host animals such as mice, rabbits, goats or sheep. The secondary antibody is a species-specific antibody of the primary antibody, ie if the primary antibody is a mouse monoclonal antibody, the secondary antibody should be an anti-mouse antibody produced in a non-mouse host animal. Secondary antibodies are available with specificity for total antibody molecule or antibody fragments such as Fc or Fab regions as well as for different classes and subclasses.

Markings used on secondary antibodies may be radioactive isotopes, enzymes or fluorophores. Generally, enzyme or fluorophore labeling is used. Enzymes provide a detectable signal by reacting with a specific substrate that generates a colored or fluorescent product or light release. The most commonly used enzymes are alkaline phosphatase and peroxidase (HRP). The most commonly used fluorophores for labeling are fluorescein and rhodamine.

Secondary antibodies may also be labeled with biotin. The presence of biotin allows efficient detection with enzyme or fluorophore labeled avidin or streptavidin. Biotin systems have the characteristic of signal amplification, thus increasing sensitivity. Secondary antibodies may also be conjugated to agarose or colloidal gold.

Antibody binding proteins such as Protein A, Protein G, Protein A / G and Protein L may also be useful for antibody detection. The detection and quantification of substances such as peptides, proteins and antibodies can be performed by the Enzyme-linked Immunosorbent Assays (ELISA) technique also called EIA (Enzyme Immunoassoy). The ELISA technique is performed on 96- or 384-well polystyrene or PVC plates in which proteins, peptides or antibodies are passively bound. Small molecules such as peptides can be more efficiently immobilized to plaques through peptide biotinylation and use of streptavidin coated plaques. The format of the technique can be direct, indirect, sandwich or competitive. More commonly, detection of the antigen-antibody reaction is done using fluorescent or enzymatic markings. The signal emitted after addition of enzyme substrates may be colorimetric, chemiluminescent or fluorescent. Among the colorimetric substrates are PNPP, ABST, OPD and TMB. The ELISA technique also allows detection of cytokines such as interferon gamma (IFN-γ). IFN-γ is a cytokine produced mainly by T cells in response to antigens and its detection has shown utility in the diagnosis of leprosy (Geluk A. et al., Clin Vaccine Immunol., 15: 522-533, 2008). IFN-γ analysis is based on the principle that T cells from individuals sensitized with M. leprae antigens produce IFN-γ when they rediscover mycobacterial antigens. Thus, increased levels of IFN-γ is indicative of infection. In IFN-γ analysis, whole blood (diluted or undiluted) or peripheral blood mononuclear cells, separated by Ficoll ™ density gradient, are cultured in the presence of antigens such as peptides and when present sensitized T cells secrete IFN -γΙ Cytokine-containing cell culture supernatant can then be detected by sandwich ELISA. Quantitation of IFN-γ can also be done using Enzyme-Linked Immunospot Assay (ELISPOT).

Other cytokines, besides IFN-γ, can be evaluated against antigens for leprosy diagnosis or prognosis (Aseffa A. etal. Lepr. Rev., 76: 147-159, 2005).

Therefore, in addition to antibody-containing samples, cell culture supernatants obtained from individuals may also be used as samples.

In addition to the ELISA technique, other techniques may be used for the detection of mycobacterial antigens or antibodies specific for mycobacterial antigens. Techniques include Western Blofting / immunoblofting, immunoprecipitation, agglutination, immunofluorescence, radioimmunoassay, Spot Synthesis, microarray, dipstick (immunochromatographic analysis), ΜΑΡΙΑ {Multiantigen Print Immunoassay).

In Western blotting or also called immunoblotting, antigens such as peptides, for example in the form of conjugates, polymers or MAPs, could be transferred to membranes, usually nitrocellulose or polyvinylidene fluoride following polyacrylamide gel electrophoresis. Following membrane blockage to prevent non-specific binding, the membrane is incubated with antibodies from individual samples. Anti-M antibodies. leprae if present in the sample will bind to the membrane-bound antigen and can be detected by the addition of a labeled secondary antibody. A suitable substrate is then added to produce a detectable signal such as a chromogenic precipitate, chemiluminescence or fluorescence. Several methods can be used for the transfer of antigens to membranes such as simple diffusion, vacuum transfer or electrotransference.

Commonly, α electrotransfer also called electrophoretic elution is the most widely used method and can be performed by the wet or semi-dry system. Generally, secondary antibodies are labeled with enzymes or fluorophores. Signal obtained from chemiluminescent substrates can be detected using X-ray film or digital imaging equipment.

Other techniques such as immunodiffusion and agglutination could be employed for detection of mycobacterial antigens or anti-mycobacterial antibodies. They are low sensitivity techniques, but they are presented as alternatives for field applications and in less developed countries. Immunodiffusion is based on the precipitation that occurs when antigen and antibody diffuse into agar and the antigen-antibody complex formed is in the form of a precipitation line or arc. Meanwhile, the agglutination reaction is characterized by the formation of visible aggregates as a result of the interaction of specific antibodies and insoluble particles that contain antigenic determinants on their surface. Agglutination may occur with inert particles (latex particles, polystyrene, betonite, sepharose, etc.) or with unrelated antigenic cells such as red blood cells which adsorb or attach soluble antigens. The immunofluorescence technique is used to search for antibodies and antigens. In direct immunofluorescence, a specific fluorophore labeled antibody binds to the antigen on cells or tissues forming an immunocomplex that is observed under a fluorescence microscope. Indirect immunofluorescence, antigens on cells or tissues are initially detected by incubation with a specific antiserum directed against the antigen to be screened followed by incubation with a fluorophore-labeled primary anti-antibody. Indirect immunofluorescence, for antibody screening, antigens attached to glass slides are incubated with antibodies from samples for antigen-antibody complex formation. Thereafter, fluorophore-labeled secondary antibody is added to the preparation and if there is antibody in the sample, fluorescence formation will occur. For amplification of the reaction fluorophores can be conjugated to streptadine. Thus, the secondary antibody-labeled biotin reacts with the fluorophore-conjugated streptavidin.

In radioimmunoassay reagents, such as antigen or antibody, are labeled with radioisotopes to detect unlabeled antigen or antibody in the sample. An unlabeled antigen (or antibody) is attached to a solid support, such as a plastic surface or particulate matrices, and the fraction of the antibody (or antigen) retained on the surface is then determined. The assay may be competitive or over reagent. Several radioisotopes can be used, among them the most employed are 125l and, 3.1. In addition to the solid phase system other methods such as immunoprecipitation, chemical separation and adsorbent techniques can be used to separate the free and bound fractions of the labeled substance.

In the Spot Synthesis technique, peptides are synthesized at defined positions (spot) on a cellulose membrane using the chemical strategy Fmoc / fBu. The peptide-bound cellulose membrane can be used for the detection of mycobacterial antibodies in samples following the same steps as described for Western Blotfing.

Microarray of proteins or peptides is another technology that has utility in detecting antibodies in samples. In this technique, appropriate antigen-containing glass slides are incubated with antibody-containing samples. Specific antigen-bound antibodies are detected by addition of the fluorophore-labeled secondary antibody. The immunochromatographic method also proves to be a tool for detecting antibodies in samples. In this test, the antibody-containing sample is deposited on a nitrocellulose membrane strip. Antibodies in the sample bind to the membrane-bound latex or colloidal gold-labeled detection antibody. The complex formed - sample antibodies and detection antibody - moves until it finds the antigen immobilized on the nitrocellulose membrane and if there are antigen-specific antibodies in the sample the ribbon will change color. The analysis may be qualitative or quantitative and in this case the method is combined with a detection system capable of quantifying the binding reaction. ΜΑΡΙΑ (Multiantigen Printing Immunoassay) is another antibody detection method and is based on the direct application of antigens by printing onto nitrocellulose membranes followed by classical antibody detection methods such as enzyme conjugated antibodies and chromogenic substrates. ΜΑΡΙΑ allows detection of antibodies to multiple antigens in a single analysis.

In addition to mycobacterial antibody detection methods in samples other methods such as lymphoproliferation analysis also allow exposure to infectious agents to be assessed. The lymphoproliferation test assesses, in cell culture, lymphocyte responses in contact with specific antigens and mitogens (usually concanavalin A or phytohemagglutinin). Cell cultures include separate mononuclear cells from peripheral blood or whole blood cultures. Activation of the immune response promotes cell proliferation of the culture and this activation can be identified by microscopic cell counting, incorporation of radioactive material into DNA (eg tritiated thymidine), reduction of tetrazolium blue (MTT), incorporation red dye lysosomal and by flow cytometry. Detection of humoral or cellular response to antigens, such as proteins and peptides, can be performed by different methods as set forth above, but are not limited to these. The present invention also envisages the development of a diagnostic kit * for leprosy. The kit may include any configuration and composition for performing the various analysis methods described herein. The kit will be presented in a commercially packaged form containing the essential elements required to conduct an analysis according to the above methods. Therefore, the kit comprises one or more peptides (isolated or combined / variant forms), devices, reagents and written instructions for carrying out the assay.

For example, a kit for detecting the presence of anti-M antibodies. leprae may contain one or more peptides for immobilization or already immobilized on a solid support and antibodies for detecting binding between sample antibodies and peptides such as biotin-conjugated human anti-IgG antibody and peroxidase-conjugated streptavidin. Or, another kit could consist of a device consisting of immunochromatographic strips impregnated with peptides and detection antibody and may also contain capillary pipettes and buffer solution.

The examples below demonstrate that peptides are capable of inducing antibody production, and furthermore, these anti-peptide antibodies are recognized by natural M. leproe antigens. In addition to inciting a humoral response, the peptides have the potential to induce a cellular response as shown in the late type hypersensitivity study example. The late type hypersensitivity reaction (DTH) allows to evaluate the previous exposure of the individual to the antigen as well as to evaluate the cellular immune response of individuals against an antigen. DTH tests employing specific reagents are extremely desired due to their simplicity of execution and reading, thus allowing their application in the field. The DTH test consists of intradermal application of the antigen, usually on the anterior face of the forearm and after 48-72 h the induration size is read. The amount of antigen applied will mainly depend on the composition of the composition and should be determined after extensive experimentation.

Immunological compositions for use in DTH assays comprise one or more peptides (isolated or combined / variant forms) associated with one or more physiologically acceptable carriers. An example of physiologically acceptable carriers is saline plus preservatives such as phenol.

Since peptides are capable of inducing a humoral response and / or cellular response, the present invention includes immunological compositions for use as vaccines, provided that vaccines are designed to enhance immunity and prevent infection in individuals. .

Immunological compositions for use as vaccines comprise one or more peptides (isolated or combined / variant forms), preservatives, physiologically acceptable carriers and additional agents as adjuvants, which may stimulate the individual's immune response to the immunogenic component. Examples of adjuvants include complete or incomplete Freund's adjuvant, aluminum hydroxide, liposomes, immunostimulating complexes (ISCOMS), etc. The composition of the formulation as well as the immunization route and the number of doses to be administered to induce an effective humoral and / or cellular response should be determined experimentally.

Example 1: Bioselection of Libraries Presented in Faqos Patients and controls Blood samples were obtained from leprosy patients, carriers of different forms of the disease, treated and untreated, from the Parana Hospital of Dermatology (Piraquara-PR), Centro Regional Specialties-Baron (Curitiba-PR), the Pró-Hansen Foundation (Curitiba-PR) and the Humanitas Philanthropic Society (São Jerônimo da Serra-PR). Blood samples were collected from treated and untreated patients with pulmonary tuberculosis treated at the Barão Regional Specialties Center (Curitiba-PR) and the Lapa São Sebastião Regional Hospital (Lapa-PR). Samples were also collected. blood pressure from leprosy patients and blood samples from healthy subjects (control groups). All individuals authorized the collection of blood through the Informed Consent Form. The study, described in the present invention, was approved by the Research Ethics Committee of the Federal University of Paraná as well as by the Research Ethics Committee Human Councils of the Paraná State Secretary of Health. .

Phage Displayed Peptide Libraries Four phage display peptide libraries were used for identification of mycdbacterial antigen mimotopes. Libraries were obtained from J. Scott, Simon Fraser University, Burnaby BC, Canada (Bonnycastle L.L. et al. J. Mol. Biol., 258: 747-762, 1996). Two of the libraries showed linear peptide sequences (XI5 and X8CX8) while two others presented cysteine residue flanked peptide sequences (XCX4CX and XCX8CX) fused to the N-terminal portion of pVIII protein of phage f88.4. Bioselection Phage libraries were initially depleted of immunoglobulin-reactive phages from tuberculous patients by incubating library phages with anti-M immunoglobulins. tuberculosis immobilized on immunotubes. Unbound phages were used in the first round of selection ... with anti-AA leprae immunoglobulins. In another bioselection, library phages were used directly in the first round of selection with anti-AA leprae immunoglobulins. Bioselection was performed as described (Ferrières G. et al. Eur. J. Biochem., 267: 1819-1829, 2000). Briefly, an immunotube was coated with anti-AA leprae immunoglobulins in 100 mM NaHCCb, pH 8.6 to 5μρ / 1.5 mL in the first cycle, Ιμρ / 1.5 mL in the second cycle and 0.5pg / 1.5 mL for the third and fourth cycles overnight at 4 ° C. For the first round of selection, phages from each library, titered at 100 times the library complexity in TBS-0.05% Tween-20 were incubated with the immobilized antibodies. In the other cycles 2x1011 phages were used. Unbound phages were removed by washes with TBS-0.5% Tween-20 followed by washes with TBS-0.05% Tween-20. Bound phages were eluted by addition of 0.1 M glycine, pH 2.2, 1 mg / ml BSA and immediately neutralized. Phages were amplified in Esccheríchia coli K91, precipitated from the bacterial culture supernatant with polyethylene glycol and used in the next round of selection or stored at -20 ° C.

Selection of phage clones After the fourth round of selection, phage clones were isolated and evaluated for binding capacity to anti-M IgG immunoglobulins. Leproe. To this end, ELISA plates were coated with 50 μΐ fd anti-bacteriophage antibody (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted 1: 800 in> 100 mM NaHCC, pH 8.6, overnighf at 4 ° C. The plates were washed with PBS-0.05% Tween-20 and then blocked with PBS-0.05% Tween 20-2% skimmed milk powder for one hour at 37 ° C. The culture supernatant (50 μΙ) from each of the isolated clones was added to a different plate well and incubated for 2 h at 37 ° C. The plate was washed and incubated with 100 pg / ml total IgG immunoglobulin from leprosy patients for one hour at 37 ° C. The detection of the reaction was performed with peroxity-conjugated Fc-specific human anti-IgG antibody (Sigma-Aldrich, Saint Louis, Missouri, USA) and OPD (Sigma-Aldrich, Saint Louis, Missouri, USA) as chromogen. The most reactive clones were selected for DNA sequencing. .

DNA Sequencing The DNA from each phage clone was extracted using the QIAprep Spin M13 kit (Qiagen, Hilden, Germany). Sequencing reactions were performed with the BigDye Terminator v3.1 kit (Applied Biosystems, Carlsbad, CA, USA) and ABI PRISM 377 automated sequencer (Applied Biosystems Carlsbad, CA, USA.) 5'-TCG GCA AGC TCT TTT AGG Primer Evaluation of phage clones with sera from patients and controls Selected phage clones were evaluated for reactivity to sera from patient and control groups by ELISA ELISA plates were sensitized with 50 μί antibody. anti-phage & fd (1: 800) in 100 mM ΝαΗΟΟβ pH 8.6 overnight at 4 ° C. Plates were washed with PBS-0.05% Tween-20 and then blocked with PBS-0.05% Tween 20- 2% skimmed milk powder, Phages were added at a concentration of 2x1010 phages / mL in blocking solution.After washing, human sera from the leprosy patient groups, tuberculosis patients, leprosy patient contacts and 1: 100 diluted healthy subjects Lockout solutions were added. Reaction detection was performed with peroxidase-conjugated anti-IgG Fc-specific antibody and OPD as chromogen (Fig. 2). The most reactive phage clones were selected for further studies. Synthesis of soluble peptides Soluble peptides were synthesized by PepTron (Daejon, Korea) using the solid phase 9-fluorenylmethoxycarbonyl (Fmoc) chemical strategy. Peptides were synthesized as unmodified peptides, as were also synthesized by adding the KKG sequence to the N-terminal portion to confer hydrophilicity. The peptides were supplied lyophilized and 70% pure.

Example 2: Peptide Reactivity Serum Samples Patients and Controls Serum samples from leprosy patients, tuberculosis patients, contacts from leprosy patients and healthy individuals were included in the study (see Example 1).

Anti-Mycobacterial Serum Antisera from ten species of mycobacteria (Table 2) were produced on animals to evaluate peptide specificity. Mycobacteria were cultured in Middlebrook 7H9 and Middlebrook 7H10 medium (Belisle JT, Sonnenberg MG Isolation of genomic DNA% mycobacteria. In: Parish T., Stoker NG Methods in molecular biology: mycobacteria protocols. Totowa: Humana Press, 1998. p. 31-44).

Cells were collected by centrifugation at 4500 rpm for 25 min. The pellet was washed three times with 0.9% NaCl, and resuspended in 10 mL 0.9% NaCl. The suspension was inactive at 100 ° C for 15 min. The cell suspension containing 100 μρ / ιτιί phenylmethanesulfonyl fluoride (PMSF) and 2 mM ethylenediaminetetraacetic acid (EDTA) was sonicated (Sonopuls HD 2200, Bandelin, Berlin, Germany) for four 15 min ice bath cycles ( Pessolani MC, Brennan PJ Infect. Immun., 60: 4452-4459, 1992). The protein concentration of the soluble extract obtained by centrifugation at 100,000 x g for 20 minutes at 4 ° C was determined by fluorescence using the Quant-iT ™ Protein Assay Kit (Invitrogen ™, Carlsbad, California, USA). For immunization, female New Zealand albino 2.5 to 3.0 kg female rabbits were used, two rabbits for each mycobacterial species. The animals received 100-250 μρ protein in incomplete subcutaneous Freund's adjuvant. Three booster doses were made at 14-day intervals. As control, two animals received incomplete Freund's adjuvant in 0.9% NaCI. Preimmune serum was collected before immunization and immune serum was collected one week after the last dose.

Table 2: Mycobacterial species used in the study SPECIES CODE M. kansasii ATCC 12478 M. scrofulaceum ATCC 19981 M.avium ATCC 25291 M. fortuitum subsp. fortuitum ATCC 6841 M. szulgai ATCC 35799 M. gordonae ATCC 14470 M. terrae ATCC 15755 M. phlei ATCC 11758 M. smegmatis ATCC 607 M. bovis ______________________________ BCG-MORAEU ELISA ELISA Plates (Cornig 2592, Lowell, Massachusetts , USA) were sensitized with 100 μί of a peptide pool (5A, 6A and 1B) at 1.5 pg / mL in 0.05 M carbonate buffer pH 9.6 overnight at 4 ° C. After washing (NaCl 0.9% -Tween-20 0.05%) the plates were blocked (protein free blocking buffer in PBS pH 7.4 with 0.05% Tween-20, Thermo Fisher Scientific, Rockford, Illinois, USA). for one hour at 37 ° C. The plates were then washed and incubated for one hour at 37 ° C with 1:50 diluted sera in PBS pH 7.4 with 0.1% (w / v) BSA. Plates were washed and incubated with biotin-conjugated Fc-specific human anti-IgG antibody (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted 1: 2500 in PBS with 0.1% BSA for one hour at 37 ° C . The plates were washed and incubated with peroxidase-conjugated streptavidin (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted 1: 2500 in PBS with 0.1% BSA for one hour at 37 ° C. After washing, the reaction was detected by addition of the OPD chromogenic substrate.

Of the samples analyzed, the peptides detected 12 multibacillary leprosy patients (52.2%) and five tuberculosis patients (16.7%). As expected, no tuberculosis leprosy patients were detected as this. group is characterized by a predominantly cellular response. None of the contact samples and healthy individuals was considered positive by the assay (Fig. 3). ELISA with Mycobacterial Peptides and Serum The production of anti-mycobacterial antibodies in animals was evaluated by the ELISA technique. To this end, ELISA plates (Falcon 353912, BD Biosciences, San Jose, California, USA) were sensitized with 50 µg antigen at 10 µ9 / ιτιί in 0.05 M carbonate buffer pH 9.6 overnight at 4 ° C. After washing (0.9% NaCl -Tween-20 0.05%) the plates were blocked (2% casein in PBS pH 7.4) for one hour at 37 ° C. The plates were then washed and incubated for one hour at 37Â ° C with sera from the immunized animals diluted 1: 4000 in incubation buffer (0.25% casein in PBS pH 7.4-Tween 0.05%). The plates were washed and incubated with peroxidase-conjugated rabbit anti-IgG antibody (Sigma-Aldrich, Saint Louis, Missouri, USA) diluted 1: 4000 in incubation buffer for one hour at 37 ° C. Following washes the reaction was developed using OPD as chromogen (Fig. 4).

To assess peptide reactivity with animal-produced anti-mycobacterial sera, ELISA plates (Cornig 2592, Lowell, Massachusetts, USA) were sensitized with 100 pL of a peptide pool (5A, 6A and 1B) at 1.5 pg. / ml in 0.05 M carbonate buffer pH 9.6 overnight at 4 ° C. The following steps were the same as those described above. The peptides showed no reactivity to anti-mycobacterial sera. Three rabbit serum dilutions were tested, 1: 4000, 1: 400 and 1:40 and none of the dilutions showed peptide reactivity.

Example 3: Evaluation of Peptide Immunoenicity Peptide Conjugation to Carrier Protein Peptides, 5A, 6A, and 1B plus the KKG sequence protein were conjugated to bovine serum albumin carrier protein via glutaraldehyde as described (Harlow, E .; Lane, D. Antibodies : a laboratory manual, New York: Cold Spring Harbor Laboratory, 1988. 726 p). Glutaraldehyde is a bifunctional coupling reagent that binds two components primarily through primary amino groups, including ε-amino groups in lysine and free α-amino groups. Carrier protein (0.03 µmol) dissolved in PBS was added, peptide solution (0.6 µmol) also prepared in PBS. Equal 0.2% volume of glutaraldehyde (Sigma-Aldrich, Saint Louis, Missouri, USA) in PBS was slowly added to the carrier protein / peptide solution which remained under constant stirring for one hour at room temperature. The mixture was incubated for one hour with shaking after addition of 1 M glycine in PBS (pH 7.2) to a final concentration of 200 mM.

Anti-Peptide Antibody Production Groups of five eight-week-old female Swiss mice were immunized with four doses of 20 μg BSA-peptide in incomplete Freund's adjuvant (Sigma-Aldrich, Saint Louis, Missouri, USA). The first and second doses were administered subcutaneously and the third to fifth doses were given intraperitoneally. Injections were performed at two-week intervals. Nonimmune serum was used as a negative control and immune blood was collected one week after the last immunization.

To evaluate the production of anti-peptide antibodies in mice, an ELISA plate (Cornig 2592, Lowell, Massachusetts, USA) was sensitized with 100 μΐ of 1.5 μρ / ηιί peptide in 0.05 M carbonate buffer pH 9.6 overnight. at 4 ° C. After washing (0.9% NaCl -Tween-0.05%) the plate was blocked (2% casein in PBS pH 7.4) for one hour at 37 ° C. The plate was then washed and incubated for one hour at 37 ° C with sera from immunized animals diluted in incubation buffer (0.25% casein in PBS pH 7.4-Tween 0.05%). After washing, reaction detection was performed with peroxity conjugated γ-specific mouse anti-IgG antibody (Sigma-Aldrich, Saint Louis, Missouri, USA) (1: 2500) and OPD as chromogen (Fig. 5).

Example 4: Research on Original Antigen in M. leprae SDS-PAGE and Western Blotting Freeze-dried M. leprae total cells were provided by Dr. JS Spencer of the University of Colorado, Fort Collins, USA, through the National Institute of Allergy and Infectious Diseases / National Health Institute under contract N01-AI-25469. Cells were resuspended in 0.9% NaCl containing 100 pg / ml PMSF and 2 mM EDTA and sonicated as described in Example 2. Forty micrograms of protein was separated on 15% polyacrylamide gel under reducing conditions (Laemmli UK , Nature, 227: 680-685, 1970). After electrophoresis the proteins were transferred to a PVDF membrane (1mmobiIon-PSQ, Millipore, Billerica, Massachusetts, USA) in 25 mM Tris, 192 mM glycine, 10% methanol and 0.025% SDS buffer for 16 ha. 24 V followed by one hour at 48 V. After transfer, the membrane was blocked with 0.3% Tween-20 in PBS and then washed with 0.05% Tween-20 in PBS (PBST-0, 05%) and incubated with anti-peptide serum in PBST-0.05%. The membrane was washed and incubated with γ-specific mouse anti-IgG antibody diluted 1: 5000 in 0.05% PBS-T. ECL Plus ™ chemiluminescent substrate (GE Healthcare, Litfle Chalfont, Buckinghamshire, UK) combined with Hyperfilm ™ ECL ™ autoradiographic film (GE Healthcare) was used for reaction detection (Fig. 6A).

Two-dimensional electrophoresis.

A total of 200 pg of M. leprae protein was purified by precipitation using the 2-D Clean-Up Kit (GE Healthcare). Precipitated proteins were resuspended in 250 μί of rehydration solution (8M urea, 2% CHAPS, 20 mM DTT, 0.5% IPG buffer, 0.002% bromophenol blue), which was applied to the Immobiline ™ DryStrip gel (IPG) strip. , GE Healthcare), pH 4-7 of 13 cm. The IPG strip was rehydrated by 4 h passive rehydration followed by active rehydration at 50 V for 12 h at 20 ° C in Ettan IPGphor II (GE Healthcare). Protein isoelectric focusing was performed using the following program: 500 V to accumulate 550 Vh, 500-1000 V to accumulate 880 Vh, 1000-8000 V to accumulate 12440 Vh and 8000 V to accumulate 5940 Vh, totaling 2140è Vh. The IPG strip was equilibrated in buffer A (6M urea, 50 mM Tris-HCI, pH 8.0, glycerol 30%, 2% SDS, 0.002% bromophenol blue and 0.065 M DTT) for 15 min followed by equilibration in buffer B (urea 6M, 50 mM Tris-HCl, pH 8.0, 30% glycerol, 2% SDS, 0.002% bromophenol blue and 0.14M iodoacetamide) for 15 min. Second dimension separation was performed on 15% polyacrylamide gel using the SE 600 Ruby vertical electrophoresis system (GE Healthcare). Electrophoresis was performed at 15 mA / gel for 15 min and 25 mA / gel until bromophenol blue reached the end of the gel.

After electrophoresis the proteins were transferred to a PVDF membrane. The membrane was blocked with 0.3% Tween-20 in PBS, washed with 0.05% PBST and incubated with 0.05% PBST antipeptide serum. After washing, the membrane was incubated with γ-specific mouse anti-IgG antibody diluted 1: 5000 in 0.05% PBS-T. The ECL Plus ™ chemiluminescent substrate combined with Hyperfilm ™ ECL ™ was used for reaction detection (Fig. 6B). For membrane reuse with different antibodies, the membrane was incubated with stripping solution (0.1 M glycine, 0.15 M NaCl, pH 2.8) for one hour at room temperature.

Example 5: Identification of Antibody-Linking Residues on Peptides Cellulose Membrane Peptide Synthesis Peptide 5A and its alanine analogs were prepared by Spot Synfhesis. Peptide synthesis was performed on cellulose membranes containing free amino groups and polyethylene glycol spacers (amino-PEG membranes, Intavis, Bioanalytical Instruments AG, Nattermannallee, Kön, Germany) using a MultiPep RS automatic peptide synthesizer (Intavis Bioanalytical Instruments AG) . The synthesis was conducted according to the protocol recommended by the manufacturer and employing amino acids Fmoc (9-fluorenylmethoxycarbonyl). Briefly, Fmoc amino acids were activated for coupling with DIC (Ν, Ν'-diisopropylcarbodiimide) and HOBt (t-hydroxybenzotriazole). After coupling, free amino groups were blocked by acetylation. The Fmoc group was removed by treatment with 20% piperidine in DMF (α, β-dimethylformamide) to prepare the membrane for the next coupling cycle. A final acetylation step was performed after the last cycle of synthesis. Removal of side chain protecting groups was accomplished by treating the membrane with a solution composed of 92% trifluoroacetic acid, 4% triisopropylsilane and 4% water for three hours followed by dichloromethane (DCM), DMF and ethanol washes. After drying, the membrane was stored at -20 ° C.

Membrane peptide immunoassay The membrane, after three washes with methanol and three washes with PBS, was incubated overnight with blocking solution (PBS-Tween 20-0.05%, 3% BSA). The membrane was washed three times for 10 min with 20-0.05% PBS-Tween (PBS-T) and incubated with a pool of sera from multibacillary leprosy patients diluted 1: 250 in PBS-T for one hour at room temperature. . After washing, the membrane was incubated with 1: 120000 Fc-specific human anti-IgG antibody diluted in PBS-T for one hour. The membrane was washed and reaction detection was performed using the chemiluminescent substrate ECL Plus ™ and Hyperfilm ™ ECL ™. Spot intensity was obtained with ImageJ software (v. 1.43, National Institutes of Health, USA) (Fig. 7). For membrane reuse, it was washed three times for 10 min with regeneration reagent A (8M urea, 1% SDS, 0.1% β-mercaptoethanol), followed by three 10 min washes with regeneration reagent. B (50% ethanol, 40% ultrapure water, 10% acetic acid). After three 10 min washes with methanol the membrane was dried and stored at -20 ° C.

Example 6: Late Toxic Hypersensitivity Reaction Animals Three groups of five female guinea pigs (Cavia porcellus) of 250300 g were used in the skin test. The experiment was conducted in accordance with the Research Ethics Committee of the Federal University of Paraná and the Research Ethics Committee of the Parana State Secretary of Health.

Antigens M. leprae antigen was prepared according to Example 2. Peptides 5A, 6A and 1B were the peptides evaluated for cellular response induction. Intradermally administered antigens were diluted in saline (0.9% NaCl). As positive control was used the Mitsuda antigen (4x107 bacilli / mL) that was supplied by the Center for Production and Research of Immunobiologicals, Piraquara, Paraná and as negative control, saline solution was used. .

Late type hypersensitivity test Five animals (group I) were sensitized by inoculating 200 pg of M. leprae antigen in incomplete subcutaneous Freund's adjuvant. Saline solution (0.9% NaCl) in incomplete Freund's adjuvant was administered to five animals in group II. Group III consisted of non-sensitized animals. After 30 days, each animal received 0.1 ml intradermally of the following antigens: peptide 5A (10 and 2 pg), peptide 6A (10 and 2 pg), peptide 1B (10 and 2 pg), pool of the three peptides ( 10 and 2 μρ), mitsudin (positive control), soluble extract of / 4. leprae (10 pg, positive control) saline (negative control). The diameter of the skin reaction was mediated after 24h, 48h, 72h, 21 days and 28 days. Maximum immune response was observed at 48h. No reaction was detected in animals immunized with adjuvant (group II) as well as in non-sensitized animals (group III). In the group sensitized with M. leprae, reaction was observed with peptide 5A (2/5), peptide 1B (1/5), mitsudin (2/5) and M. leprae (4/5). For peptide 1B the skin reaction was observed at doses of 2 and 10 μρ, while for peptide 5A, only the 10 pg dose generated response.

Claims (16)

1. USE OF Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES comprises a method for identifying mycobacterial antigen, characterized by the following steps: a) collecting samples; b) obtaining antigen-specific antibodies from samples collected in (a); c) selection of mycobacterial antigen mimetic peptides from phage-expressed peptide libraries (Phage Display technology). Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES, according to claim), characterized in that it contains antibodies, which can be obtained from individuals infected with mycobacteria at any stage, including the specific phase of the disease. obtained from animals experimentally infected with mycobacteria. Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES, according to claim), characterized by antibodies purified from samples by precipitation methods, including the affinity chromatography method, immunoblotting. 4. USE OF Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claim), characterized by the use of M. leprae antigen-specific antibodies (anti-ΛΊ leprae) which may contain phage depletion reactive to antigen antibodies -specific mycobacterial diseases, including those caused by / VI. tuberculosis. Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES comprises peptides identified by the method of claim 1, characterized by the sequences SEQ ID No. 1 to SEQ ID No. 12. 6. USE OF Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES has peptides, characterized in that they consist of: a) a peptide corresponding to any sequence SEQ ID No. 1 to SEQ ID No. 12 or; b) a variant of any peptide of (a) or; c) a combination of one or more peptides of (a) or variant of (b). The use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claim 6 (b), characterized in that it preserves the antigen and / or immunogen properties of the sequence of claim 6 (a). Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claim 6 (c), characterized in that it comprises a mixture of at least two sequences as claimed in claims 6 (a) and 6 (b), which may be further comprising forming polymers comprised of repeating a peptide sequence of claims 6 (a) and 6 (b) which may be separated by any separation method such as spacers and linkers. . Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claims 6 to 8, characterized in that they are produced by recombinant DNA technology and may also be produced by chemical synthesis. 10. USE OF Mvcobacterium leproe MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES comprises a method of defecting M. leproe infection, characterized by the following steps: a) collecting the sample; b) contacting the sample after processing with at least one of the peptides referred to in claims 6 to 8; c) detection of reaction between sample components and peptide (s) (b) by appropriate techniques. Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to Claim 10 (a), characterized in that it is any sample containing antibodies, including blood. Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claim 10 (b), characterized in that antibodies and mononuclear cells, not necessarily together, are separated from the samples and allow interaction between antigen and antibody. , also enabling the induction of cell proliferation and the induction of cytokine production against antigens. Use of Mvcobacterium leorae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES according to claim 10 (cj, characterized in that they are ELISA, Western Blotting, immunoprecipitation, agglutination, radioimmunoassay, immunofluorescence and other techniques which enable the detection of immunocomplexes. cell proliferation and cytokine production. Use of Mvcobacterium leoràe MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES comprises a diagnosis kit of M. leprae infection, characterized in that it consists of peptides as set forth in claims 6 to 8, reagents, devices and written instructions required for the assay. . Use of Mvcobacteríum leprae MIMETIC PEPTIDES FOR DIAGNOSIS AND VACCINES comprises immunological compositions, characterized in that they contain peptides as set forth in claims 6 to 8 in association with a preservative and, when necessary, adjuvant thiolologically acceptable carrier (s). (s). Use of mimetic A / lvcobacferium leprae PEPTIDES FOR DIAGNOSIS AND VACCINES according to claims 6 to 8, characterized in that in the preparation of immunological compositions to induce an immunogenic response, it may be protective in vivo, to allow the evaluation of exposure of individuals to M. leprae, contribute to the assessment of immune response and disease classification in individuals infected with M. leprae.