BR102020013235A2 - SYNTHETIC PEPTIDES MIMETOTOPES OF M. TUBERCULOSIS ANTIGEN PROTEIN, RECOMBINANT ANTIBODY OR FRAGMENT OF IT, APTAMERS, USE OF THESE IN THE DIAGNOSIS OF TUBERCULOSIS AND METHOD OF DIAGNOSIS OF TUBERCULOSIS - Google Patents

Abstract

synthetic peptides mimetopes of antigenic protein of m. tuberculosis, recombinant antibody or a fragment thereof, aptamers, their use in tuberculosis diagnosis and tuberculosis diagnosis method. The present invention relates to the selection, characterization and synthesis of recombinant peptides that mimic antigenic proteins of mycobacterium tuberculosis binding to the iga of tuberculosis from individuals with tuberculosis, as well as the selection, characterization and synthesis of aptamers and recombinant antibody fragment binding to proteins of tuberculosis. m. tuberculosis. the present invention also describes the use of both the peptides and the aptamers and the recombinant antibody fragment in diagnostic methods or platforms for diagnosing tuberculosis through biological samples from an individual, such as body fluids from tuberculosis patients.

Description

SYNTHETIC PEPTIDES MIMETOTOPES OF M. TUBERCULOSIS ANTIGEN PROTEIN, RECOMBINANT ANTIBODY OR FRAGMENT OF IT, APTAMERS, USE OF THESE IN THE DIAGNOSIS OF TUBERCULOSIS AND METHOD OF DIAGNOSIS OF TUBERCULOSIS Field of Invention

[001] The present invention relates to the selection, characterization and synthesis of recombinant peptides that mimic Mycobacterium tuberculosis IgA-binding antigenic proteins from body fluids, for example from saliva, from individuals with tuberculosis, aptamers and recombinant antibody or fragment thereof, binding to M. tuberculosis proteins. The recombinant peptides (TBC10 and TB2C10) chemically synthesized based on the sequence of twelve amino acids fused to phage C10, selected from a commercial library PhD-12 by phage display, proved to be applicable in diagnostic methods or diagnostic platforms for tuberculosis. DNA aptamers (L1, L2, L3 and L4) and the recombinant monoclonal antibody or fragment of this 4B ligands of TBC10 and TB2C10 selected, respectively, by SELEX and phage display have also been shown to be applicable in diagnostic methods or platforms to diagnose tuberculosis through biological samples from an individual, such as bodily fluids from tuberculosis patients.

Fundamentals of the Invention

[002] Tuberculosis (TB) is a contagious disease, caused by the bacterium Mycobacterium tuberculosis, which typically affects the lungs, but can also affect other organs and sites. The most common form of M. tuberculosis infection occurs via the air route, through the inhalation of droplets containing the bacteria, when the patient with lung TB (in the bacilliferous phase) sneezes or coughs (FLYNN and CHAN, 2001).

[003] M. tuberculosis is an intracellular bacillus that has the ability to escape the immune response of its host, and remain latent inside macrophages for prolonged periods, thus characterizing latent infection (LTBI). This condition can be altered by an immunological stress, capable of reactivating the infection, giving rise to active disease, a phase in which the patient releases the bacillus through droplets when sneezing or coughing, transmitting it to other individuals (FLYNN and CHAN, 2001). Individuals with LTBI do not transmit the disease, however controlling this infection and preventing the development of active disease are valid and promising measures to reduce the incidence.

[004] The variety of clinical manifestations is due to the different escape mechanisms and virulence factors of the bacteria, in addition to the heterogeneity of the immune response of individuals. The way in which the infection is established and the reactivation of the disease can be defined by the action of proteins secreted by M. tuberculosis that interfere in the regulation of the host's immune response, as well as a set of factors such as environment, host, pathogen, HIV coinfection , malnutrition and other immunodeficiencies (O'GARRA et al., 2013; GOLDBERG et al., 2014; BEHAR et al. 2010.

[005] The degree of involvement of the affected organ and magnitude of clinical manifestations, and especially of subclinical forms, associated with diagnostic limitations are crucial factors for assertive treatment and disease containment (PAI et al., 2016; CADENA et al. , 2017).

[006] The main feature of TB in humans is the formation of granuloma, important for containing the bacteria and where the bacteria can survive for long periods and proliferate. The immune response to M. tuberculosis depends on how it will be organized and its maintenance to contain and eliminate the bacillus and involves phagocytic cells and subpopulations of T lymphocytes and is mediated by cytokines and chemokines(O'GARRA et al., 2013; PORCELLI et al., 2013; PORCELLI et al. al., 2014; PIETERS, 2008).

[007] The ability of the bacillus to survive inside the host's macrophages is due to the interaction of proteins secreted or present in the cell wall of the mycobacteria with the phagocytic cell. The secretion of proteins by M. tuberculosis guarantees its evasion of the deleterious action of the defense cells. Proteins such as lipoproteins, lipoarabinomannan (LAM), filtered protein culture - 10kDa (culture filtrate protein-10 kDa - CFP-10), early secreted antigenic target-6 kDa - ESAT-6), The immunogenic protein MPT64, Ag85A, B and C, and hsp60, are capable of preventing phage-lysosome fusion, inactivating intracellular autophagic pathways and inhibiting recognition pathways, limiting innate immune responses and the development of adaptive immune responses. 2001; PAI et al., 2016; GRÖSCHEL et al., 2016) 9–11.

[008] Heat Shock Proteins (hsp) are a class of proteins with increased expression due to heat or chemical stress, and are grouped into families according to their amino acid sequences (a.a) and molecular weights ( CASTRO et al., 2013). Hickey et al. (2009) observed that a recombinant hsp65 protein had the ability to inhibit the association of the bacillus with macrophages, demonstrating the adhesin functionality of chaperonin 60.2 in relation to the interaction with macrophages. These and other proteins, such as ESAT-6, CFP-10, MPT64, Ag85, which play an important role in bacterial virulence, are potential biomarkers for the diagnosis of TB (GOLDBERG et al., 2014; XU, 2007; RENSHAW et al., 2005; GAO et al., 2005).

[009] According to data published by the World Health Organization - WHO (WHO) in the Global Tuberculosis Report - 2019, it is estimated that 10 million people became ill in 2018, a number that has been relatively stable in recent years but that continues to impact people. The burden of disease varies enormously between countries, ranging from less than 5 cases to more than 500 new cases per 100,000 population per year, with a global average around 130 new cases per 100,000 population per year. An estimated 1.2 million HIV-negative people and about 251,000 HIV-positive people died from tuberculosis in 2018.

[010] Despite the increases in TB notifications observed by the WHO, there is still a large gap between the number of new cases reported, around 7 million, and what is estimated, around 10 million cases in 2018 (WHO, 2019). ). This difference is due to the combination of underreporting of detected cases and underdiagnosis. People with TB do not access medical care or are not diagnosed when they do.

[011] As efforts to improve TB diagnosis and treatment intensify and close the gaps between incidence and reporting, the proportion of bacteriologically confirmed reported cases needs to be monitored to ensure that people are correctly diagnosed and initiated, as soon as possible, the most effective treatment regimen. The aim should be to increase the percentage of bacteriologically confirmed cases by increasing the use of recommended diagnostics (e.g. rapid molecular tests) that are more sensitive than smear microscopy (WHO, 2019).

[012] In addition to a public health problem, the ease of propagation and difficulty in containing the transmission routes make TB a serious social problem, related to poor economic conditions, housing and poor nutrition, and factors related to the immune system, which makes vulnerable populations more susceptible to developing the disease (BRAZIL – MINISTRY OF HEALTH, 2011).

[013] TB is curable as long as treatment is carried out, with appropriate association of antituberculosis drugs, dose and adequate treatment time (WHO, 2019). Early diagnosis is essential to contain and control the disease.

[014] Bacteriological and radiological findings are the main diagnostic methods, and in some regions enzyme immunoassays are also used. However, the diagnostic methods used are limited, as they do not aggregate specificity, sensitivity and agility in a single test, which are essential for the effective initiation of treatment and containment of the spread of the bacillus (BENTO et al., 2011).

[015] X-ray radiography requires specialized equipment, but is useful as an auxiliary method in paucibacillary infections and exclusion of other pathologies that require concomitant treatment (FERRI et al., 2014). Bacteroscopy, investigation of alcohol-acid resistant bacilli – Ziehl Neelsen stain, is the most used method for diagnosing new cases and also monitoring the reduction of bacillary load during treatment, it is cheap, has excellent specificity, but is not very sensitive, dependent observer and limited by the representativeness and quality of the clinical sample. Bacillus culture, considered the gold standard, is highly specific, indicated for the confirmation of new cases and monitoring of treatment, allows determining susceptibility to drugs, however it takes time, is semi-quantitative, and also requires adequate and specialized infrastructure (CHEE et al., 2013). The tuberculin test, also known as PPD (Purified Protein Derived) made from the intradermal application of purified protein derived Rt23, is useful to prove the infection, but not necessarily the disease, positivity indicates only previous exposure to the antigen, but does not imply active infection. The detection of nucleic acids, more recently GeneXpert MTB/RIF, is highly specific and sensitive, however, in addition to the high cost, it cannot differentiate active disease from treated infection, is not capable of detecting atypical mycobacteriosis and is not applicable to the diagnosis of relapses or to treatment monitoring. As for serology, it is the most practical method, but current antigens do not allow distinguishing active tuberculosis from latent infection, and are not applicable to endemic areas (CAILLEAUXCEZAR, 2012). The ADA biochemical test - Adenosine Deaminase - is applicable for the diagnosis of pleural tuberculosis, but with low accuracy for mycobacterial infections from other sites. The diagnostic tests currently used leave something to be desired, which makes it difficult to track the patient and monitor the clinical evolution (BRASIL – MINISTRY OF HEALTH, 2011; GOLETTI et al., 2016; WHO, 2006; GUTLAPALLI et al., 2016).

[016] The challenge is the development of a diagnostic method for active tuberculosis of low cost, effective and easy to use in the field (point-of-care) and that enables faster diagnosis and initiation of treatment, which has good sensitivity and specificity. , accuracy, and that allows better serving the most vulnerable population, less favored classes and countries or regions with fewer resources.

[017] Although predominantly used in diagnostic tests, the representativeness of sputum as a clinical specimen is directly influenced by several factors such as time and quality of collection, sputum and other factors related to the physical condition of the patient, and cooperates for the low sensitivity of the sputum. bacterioscopic examination. Although serum is a clinical sample with good representation of the state of the organism, and is widely used in different diagnoses, it does not offer satisfactory possibilities for the diagnosis of TB due to the heterogeneity of the infection (POTTUMARTHY et al., 2000). Salivary fluid has excellent diagnostic potential because it contains numerous proteins, protein fragments and antibodies, which give this fluid an important analytical value (TABAK, 2001; LAWRENCE 2002; STRECKFUS and BIGLER, 2002; RIBEIRO et al., 2010; LARREA et al. , 2006). About 85% of the antibodies present in saliva are immunoglobulin A (IgA) that generally play a protective role, binding to bacteria blocking binding structures and inhibiting their adherence (VAN NIEUW AMERONGEN, 2004). Saliva, therefore, is a promising sampling alternative both for the diagnosis of infectious diseases and for monitoring the evolution of the treatment due to its continuous production, showing more precisely the current state of the organism and adding the advantage of painless and non-invasive collection.

[018] The cellular products and metabolites of Mycobacterium tuberculosis can be detected directly in blood, saliva, urine and other clinical specimens. Some of these cellular components have been proposed as biomarkers for the diagnosis of TB, such as DNA fragments, cell wall proteins, lipoarabinomann (LAM), secreted protein complexes (CFP-10/ESAT-6) and antigens Ag85, MPT64, hsp60 and others (BORGSTRÖM et al., 2011; FLINT et al., 2004; CHATTERJEE et al., 2011; WIKER and HARBOE, 1992; SINGH et al., 2005).

[019] A technique that can be used to develop new molecules with potential is the Phage display. This is an efficient technique to identify peptides or proteins that bind to other molecules for various purposes, such as mapping epitopes recognized by antibodies. The technology is based on the principle that polypeptides can be expressed on the surface of filamentous bacteriophages by inserting a segment of coding DNA into their genome, so that the expressed peptide or protein is exposed on the surface of the viral particle fused to a viral protein. naturally expressed in bacteriophage (SMITH, 1985).

[020] The possibility of expressing a fusion protein in the capsid of bacteriophages, in a manner accessible to recognition by a ligand, demonstrated in 1985 by Smith, paved the way for the construction of conformational libraries presented on the surface of these viral particles (SMITH, 1985). ).

[021] The selection of sequences based on the binding affinity of the phage to a target molecule is done by an in vitro selection process called biopanning. Biopanning consists of incubating the phage-displayed peptide library against the target. Non-binding or less specific phages are eliminated by successive washes and specific phages remain bound for further elution. The pool of specific phages is amplified for later rounds of selection. After three or four rounds of selection the individual clones are characterized by DNA sequencing, Western Blotting or ELISA (SMITH, 1985).

[022] Peptide libraries generated by phage display are extensively applied in the discovery of a wide variety of polypeptides including antibodies, receptors and enzymes (ARAP, 2005). Epitopes or antigenic determinants, regions of antigen recognition by antibodies, can also be identified through this methodology of phage peptide display, which has been extremely important for the identification and characterization of new high-affinity ligands and their infinity receptors. of diseases, including cancer, infectious, cardiovascular and autoimmune diseases (STERNBERG and HOESS, 1995; MULLEN et al., 2006).

[023] This technique also allows the selection of antibody fragments expressed on the surface of bacteriophages. Single-chain fragments of antibody variable regions (single-chain variable fragment, scFv) represents the smallest functional domain of light and heavy chains (VL and VH) of an antibody required for specific binding to its antigen. The expression of these fragments is carried out in the form of recombinant antibody fragments, through the fusion of the coding regions of the variable portion of the antibody (Fv) to the gene III of the M13 phage, being able to encode and express the scFv in the phage PIII protein (HOOGENBOOM , 2005). The selected recombinant antibodies can be expressed on a large scale and be used in chemical, physiological and immunological assays.

[024] Another technique that can be used is SELEX (Systematic Evolution of Ligands by EXponential Enrichment). This is an in vitro technique developed and described in the 1990s (ELINGTON and SZOSTAK, 1998; TUERK and GOLD, 1990; AQUINO-JARQUIN and TOSCANO-GARIBAY, 2011) for the selection of small synthetic nucleic acid molecules (RNA and single-stranded DNA (ssDNA) by PCR.

[025] The molecules selected by this technique are called aptamers. This technique is used as a biotechnological tool with specific objectives in research with diagnostic and therapeutic applications. Aptamers are capable of binding to small molecules, such as receptors of target molecules, for detection of drugs, metabolites and toxins in body fluids, cell signaling, used as probes in cell markers for imaging exams, or carriers of therapeutic nanoparticles and procedures. laboratory biomolecules (ACQUAH et al., 2015; DARMOSTUK et al., 2014; RADOM et al., 2013). The molecular arrangement of aptamers is the result of a thermodynamically favorable process and provides stability to the secondary structure and conformation. They are highly sensitive and specific ligands, with advantages over other ligands such as faster and easier chemical production, less prone to viral or bacterial contamination, storable and non-immunogenic (ESPOSITO et al., 2015; SHANNON PENDERGRAST et al., 2005; GOPINATH et al., 2006; AMAYA-GONZÁLEZ et al., 2013; ZHU et al., 2014).

[026] Thus, there is a need to develop new methods of molecular diagnosis of active TB that are more efficient, faster and more cost-effective. One way is to develop new cheaper, more stable and highly sensitive biomarkers for detection of target proteins in body fluids such as saliva, salt, samples of cervical fluid, urine, among others. In one of the modalities described here, we propose new biomarker molecules that have been synthesized for use in a method of molecular diagnosis of TB based on immunological assays or using diagnostic platforms for the detection of IgA antibodies from samples of body fluid, particularly in saliva samples.

Brief description of the invention

[027] The present patent application describes two new synthetic peptides. The first peptide is TBC10 (with 24 amino acids – SEQ ID No: 01), synthesized from a sequence of 12 amino acids fused to a phage, C10, previously obtained by phage display against IgA from the saliva of individuals with tuberculosis. The peptide synthesis was designed based on the protein motif, adding a protein spacer and part of the phage PIII protein, with amidation in the C-terminal region and coupled to BSA in the N-terminal region. The second TB2C10 peptide (with 32 amino acids - SEQ ID No: 02) synthesized from the duplication of the protein motif of the 12 amino acids, separated by two spacers, and the inclusion of a spacer after the second sequence with amidation in the C-terminal region and coupled to BSA in the Nterminal region. TBC10 and TB2C10 peptides were able to detect M. tuberculosis-specific IgA in body fluid samples. Showing a sensitivity in immunological assays of 94.12% and a specificity of over 76%, and are applicable, for example, in the use in biosensors for the detection of TB IgA in body fluid samples, more particularly in saliva samples.

[028] In another embodiment of the present patent application, a fragment of recombinant antibody of the scFv type is described comprising the sequence SEQ ID No: 03 or the sequences SEQ IDs No: 04 and 05, 4B, phage C10 binder, selected by phage display that was able to recognize the M. tuberculosis proteins in assays as well as was able to recognize the synthetic peptides TBC10 and TB2C10 described here.

[029] In yet another embodiment of the present patent application, the development of four aptamers (L1, L2, L3 and L4) is described which comprise the sequences SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 SEQ ID No: 9, respectively, and which were also able to bind M. tuberculosis proteins as well as the synthetic peptide TBC10 BY SELEX.

[030] In another embodiment of the present patent application, the use of at least one of the synthetic peptides TBC10 and TB2C10, the scFv antibody fraction (4B) and the aptamers L1, L2, L3 and L4, in a diagnostic method of TB, as well as the aforementioned diagnostic method itself and a detection system using a bioelectrode.

Brief description of figures

[031] The present invention will be better understood based on the description below, taken together with the attached figures, in which:

[032] FIGURES 1a-c show a representative scheme and a filamentous bacteriophage. (a) wild-type filamentous phage illustrating the viral capsid proteins: pIX, pVII, pVIII, pVI and pIII; (b) peptide fused to phage pIII; and (c) peptide fused to phage pVIII.

[033] FIGURE 2 shows a sequential representation and molecular structure of the TBC10 and TB2C10 peptides.

[034] FIGURE 3 shows the correlation of the affinity of Phage C10 with peptides TBC10 and TB2C10, against IgA in saliva.

[035] FIGURE 4 shows the representation of the percentage of target-specific IgA in relation to total IgA in the saliva of individuals.

[036] FIGURE 5 shows an ELISA assessment of the reactivity of C10, TBC10 and TB2C10 against IgA in the saliva of individuals.

[037] Figure 6 shows the detection by differential pulse voltammetry of IgA binding in TBC10 and TBC2C10 immobilized on graphite electrode.

[038] FIGURES 7 A-C show the reactivity of scFv fragments in immunobiological assays.

[039] FIGURES 8 A-C show the molecular characterization of the scFv fragments.

[040] Figure 9 shows the secondary structures of aptamene L1, L2, L3 and L4.

[041] Figure 10 shows the reactivity of Aptamers L1, L2, L3 and L4 in immunobiological assays.

Purpose of the invention

[042] The present patent application aimed to identify and synthesize new recombinant peptides that mimic Mycobacterium tuberculosis antigenic proteins binding to IgA from individuals with tuberculosis, aptamers and a fragment of recombinant antibody binding to M. tuberculosis proteins, which had the ability improved from being used in a TB diagnostic method.

Detailed description of the invention

[043] The present patent application describes the selection, characterization and synthesis of recombinant peptides that mimic Mycobacterium tuberculosis antigenic proteins binding to IgA from individuals with tuberculosis, in addition to aptamers and a recombinant antibody fragment binding to M. tuberculosis proteins, as well as a method of diagnosing TB and the use of said peptides, the antibody fragment and aptamers in the diagnosis of TB.

Example 1 – Synthesis of new peptides

[044] In one embodiment of the present patent application, two new synthetic peptides are described. A previous selection by phage display allowed obtaining and characterizing the M13 phage with a 12 amino acid peptide (C10), a mimetic of Mycobacterium tuberculosis proteins, IgA ligand in the saliva of individuals diagnosed with TB.

[045] Figure 01 shows a representative schematic of a filamentous bacteriophage in which: Figure 01 (a) shows a schematic of a wild-type filamentous phage illustrating the viral capsid proteins: pIX, pVII, pVIII, pVI and pIII; Figure 01 (b) shows a schematic of a peptide fused to the phage pIII viral capsid protein; and Figure 01 (c) shows a peptide fused to the phage viral capsid protein pVIII.

[046] The first peptide, TBC10 (with 24 amino acids – SEQ ID No: 01 - NH3–EYNTASIHSYRKGGGSAETVESCL–CONH2), was synthesized from the aforementioned phage-fused sequence of 12 amino acids, C10, previously obtained by phage display against IgA of saliva from individuals with tuberculosis. The peptide synthesis was designed based on the protein motif, adding a protein spacer and part of the phage PIII protein, with amidation in the C-terminal region and coupled to BSA in the N-terminal region.

[047] The second peptide, TB2C10 (with 36 amino acids - SEQ ID No: 02 - NH3–EYNTASIHSYRKGGGSGGGSEYNTASIHSYRKGGGS–CONH2), was synthesized from the duplication of the protein motif of the 12 amino acids, separated by two spacers, and the inclusion of a spacer after the second sequence with amidation in the C-terminal region and coupled to BSA in the N-terminal region.

[048] The protein motifs and 3D molecular structure of the newly synthesized peptides TBC10 and TB2C10 are shown in Figure 02. The TBC10 peptide is shown with the 12 amino acid protein motif in green, spacer and part of the phage PIII in yellow. The TB2C10 peptide is represented with the 12 amino acid protein motif in green, the duplicated protein motif in lime green and the spacer in yellow.

Example 2 - Immunoreactivity

[049] The novel peptides TBC10 and TB2C10 were able to detect M. tuberculosis-specific IgA in body fluid samples. The phage C10 and the peptides TBC10 and TB2C10 showed a sensitivity of 94.12% to discriminate positives from negatives, and a specificity greater than 76.9%. The ROC curves showed an area greater than 0.9367, with p values < 0.001 (Figure 05). The correlation analysis between the targets showed a value equal to 0.94, indicating that the response of the synthetic peptides was similar to that of the peptide of C10 origin (Figure 03).

[050] Figure 04 shows a representation of the percentage of target-specific IgA in relation to total IgA in the saliva of each individual in the groups positive for untreated TB (PNT), positive for TB in course of treatment (PT), negative controls for non-PPD-reactive TB (NP-) and negative controls for PPD-reactive TB (NP+). The percentage of target-specific IgA in relation to total IgA was higher for the positive group at the beginning of treatment (PNT) in relation to the others, with a slight increase for the positive control individuals for the tuberculin skin test (NP+).

[051] For specific IgA detection, peptides (0.5 µg/well) and C10 phage (1010 viral particles/well) were immobilized with Bicarbonate buffer (0.06 M, pH 9.6) in microtiter plates (Nunc Immuno MaxiSorp , Roche Diagnostics) and incubated at 4°C overnight. The plates were washed with PBS buffer plus 0.05% Tween (0.05% PBST), blocked with 5% PBS-BSA, PBS buffer plus 5% BSA, for 1 hour at 37°C. Washed with 0.1% PBST and 50 µL of saliva diluted 1:10 in PBS-BSA 5% were added, incubated at 37°C for 01 hour (to detect total IgA, 50 µL of diluted saliva 1 :10 in bicarbonate buffer were used to sensitize, following the same ELISA protocol) washed 6 times with 0.05% PBST. With the addition of anti-IgA/human HRP (human peroxidase), 01 hour at 37ºC. Again washed 6 times with 0.05% PBST. The reaction was revealed by the addition of OPD (orthophenylenediamine) diluted in citrate-phosphate buffer (0.1 M, pH 5.0) and 3% H2O2. The reaction was stopped by the addition of 25 µL 2.0 N sulfuric acid. The absorbance reading was performed at 492 nm in a microplate reader (Multiskan Go/Thermo Scientific, Finland).

[052] The diagnostic indicators are presented in Figure 05 which shows an ELISA assessment of the reactivity of C10, TBC10 and TB2C10 against IgA from saliva of Tb positive individuals at the beginning of treatment (PNT), of Tb positive individuals with treatment ongoing (PT), PPD+ (NP+) healthy individuals and PPD- (NP-) healthy individuals. Representation of the ROC curves generated from the ELISA, with their respective sensitivity (Se), specificity (Sp), Odds Ratio (OR), Confidence Interval (CI), area of the ROC curve (AUC) and p value. As previously described, the phage C10 and the peptides TBC10 and TB2C10 showed a sensitivity of 94.12% to discriminate samples positive for tuberculosis from samples negative for tuberculosis, and a specificity greater than 76.9%. The ROC curves showed an area greater than 0.9367, with p values < 0.001. The results show that TBC10 and TB2C10 peptides can be used in immunoassays for the diagnosis of Tuberculosis.

Example 3 - Diagnostic Platform - Biosensor

[053] The TBC10 and TB2C10 peptides, once they demonstrated the ability to identify M. tuberculosis IgA in biological samples, were then immobilized on graphite electrodes (Ds dropsens Drp-110) in order to test an efficient diagnostic platform, easy and quick response, more particularly an electrochemical detection platform. Figure 06 shows the detection by differential pulse voltmetry of IgA binding in TBC10 and TB2C10 immobilized on graphite electrodes, 0.1M phosphate buffer was used as electrolyte ( pH 7.4). In green are the values of the current in empty electrode, blue values after immobilization of the target and treatment with pool of negative saliva (NP-), red values after immobilization of the target and treatment with pool of positive saliva for tuberculosis (PNT). The binding of IgA to the immobilized targets increased the resistance with a consequent drop in current, with minimal potential displacement (TBC10: 0.07V; TB2C10: 0.03V). The bioelectrode interaction in the presence of negative and positive IgA showed Δipa values of 12.216µA for TBC10 and 9.299µA for TB2C10, which indicates that the system discriminates the positive target from the negative target. In this way, these peptides also proved to be efficient for use in a diagnostic platform using biosensors, allowing to discriminate TB-positive biological samples (PNT) from TB-negative biological samples (NP-).

[054] Measurements were performed using a PalmSens portable potentiostat connected to a computer. All measurements were performed at room temperature (25ºC). Ds dropsens Drp-110 carbon electrodes (DropSens, S.L. Oviedo, Spain) were used to immobilize 5 ng of the peptide in 4 µL of 0.1M phosphate buffer, pH 7.4, blocked with phosphate buffer plus 0.5% BSA. After complete drying, 4 µL of pool saliva diluted 1:10 in phosphate buffer was added, after one minute of reaction, the surface was washed three times with ultrapure water. After drying. The reading was performed using 5.0mM iron/potassium ferricyanide solution with 0.1M KCl.

Example 4 - Selection and expression of scFv-type recombinant antibody fragment

[055] In another embodiment of the present patent application discloses a novel monoclonal recombinant antibody fragment scFv type, comprising SEQ ID NO: 03 (ELTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSATGIPD RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSHLSTFGQGSKVEIKGGSSRSSEVQLV ESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSV KGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK) or comprises the light chain (VL) of SEQ ID NO : 04 (VL-ELTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASSATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ YGSHLSTFGQGSKVEIK) and heavy chain (VH) of SEQ ID NO: 05 (VH-EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNS GSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK), 4B, C10 phago binder, selected by phage display that is capable of recognizing the protein of M. tuberculosis in assays and was able to recognize the synthetic peptides TBC10 and TB2C10 described here.

[056] It was possible to select and express Mycobacterium spp protein-binding scFv antibody fragments from phage C10. The selection of scFv clones against the C10 phage was performed using a phage library containing approximately 2x10 6 combinatorial scFv sequences, developed at the Nanobiotechnology Laboratory of the UFU.

[057] Two rounds of selection were performed, preceded by re-amplification of the scFv library in competent Escherichia coli XL1-Blue and infection of the helper phage VCSM13 for assembly and replication of viral proteins. One well of a microtiter plate (Nunc MaxiSorpTM, eBioscience, San Diego, CA, USA) was sensitized with 1010 viral particles at 50µL/well in 0.1M carbonate-bicarbonate buffer (pH 8.6) and incubated for 18 hours at 4°C. The well was blocked with 250 µl of 0.05% PBST/5% BSA for 1 hour at 37°C, and washed three times with PBS. Then, 100 µl of the scFv-phage library was added to the well and the plate was incubated for 1 hour at 37°C. Subsequently, the well was washed 10 times with 0.1% PBST. Phage bound to total proteins were eluted with 100 µl of 100mM glycine-HCl (pH 2.2) for 30 minutes at room temperature (RT). The suspension was neutralized with 16.5 µl of 2M Tris (pH 9.1). The phages resulting from this first selection were amplified in E. coli XL1-Blue.

[058] E. coli XL1-Blue bacteria infected from the 2nd round of selection were used for plasmid DNA extraction and subsequent transformation into E. coli TOP10 bacteria, a non-suppressor strain. Plasmid DNA extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, DS, Germany), according to the manufacturer's instructions. The extracted plasmid DNA was quantified (Nanodrop Spectrophotometer, (Thermo Fisher Scientific Inc., Waltham, MA, USA) and further analyzed on a 0.8% agarose gel.E. coli TOP10 bacteria were prepared with CaCl2 to become chemocompetent. Approximately 50 ng of plasmid DNA was carefully added to 10 µL TOP10 bacteria. The mixture was kept refrigerated on ice for 30 minutes and then subjected to a heat shock for 90 seconds at 42°C and 60 seconds in an ice bath. Subsequently, 800 µL of SOC medium was added and the suspension was incubated at 37°C for 45 minutes. Aliquots of the transformed cells were seeded on Luria-Bertani (LB) agar plates containing carbenicillin (100 µg/mL). were incubated overnight at 37°C for the growth of recombinant colonies containing the insert.

[059] Each colony was transferred and cultured in a deep well plate containing 1 mL of Superbroth medium (SB), carbenicillin (100 µg/mL) and 2% 2M glucose (v/v). The plate was covered with plastic film and incubated overnight under agitation at 250 rpm and 37°C. 50 µL of each bacterial clone were transferred to a new plate containing 1 mL of SB/carbenicillin (100 µg/mL)/2% 2M glucose (v/v) medium and incubated at 250 rpm at 37°C for 4.5 hours. After incubation, the plate was centrifuged at 3,700 rpm for 15 minutes at 4°C and the pellet was resuspended in 1.5 ml of SB/carbenicillin medium (100 µg/ml). Expression of soluble scFv was induced with isopropyl β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St. Louis, MI, USA) at a final concentration of 2.5 mM, overnight under agitation at 250 rpm and 30°C. . The deep well plate was centrifuged at 3700 rpm for 15 minutes at 4°C. The supernatant containing the soluble scFv was transferred to another 96-well plate and stored at 4°C. The remaining volume of the first plate was centrifuged, and the pellet of each bacterial clone was used for DNA extraction and subsequent sequencing.

Example 5 - DNA sequencing and bioinformatics analysis

[060] For the sequencing reaction, the pellets of each previously separated bacterial clone were used. Reactions were performed according to the DyEnamic ET Dye Terminator Cycle Sequencing kit (GE Healthcare Life Sciences, Waukesha, WI, USA). The following primers were used: MMB4 (5'- GCT TCC GGC TCG TAT GTT GTG T-3' – SEQ ID No: 10) for the light chain (VL), SEQ ID No: 04, and MMB5 (5'- CGT TTG CCA TCT TTT CAT AAT C-3' – SEQ ID No: 11) for the heavy chain (VH), SEQ ID No: 05, in a final volume of 10 µl. Samples were submitted to the MegaBaceTM 1000 Genetic Analyzer sequencer (Amersham Biosciences, Pittsburgh, PA, USA).

[061] The sequences were analyzed by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) to identify the light and heavy chains of the scFv molecules, as well as their conserved regions (framework regions, FRs) and variables (complementarity determining regions, CDRs). The deduced amino acid sequences were submitted to the bioinformatics programs raptor-x (http://raptorx.uchicago.edu/) to obtain the 3D structure of the scFv molecule, in pdb format, and PyMOL (http://www.pymol .org/) to select the scFv conserved and variable regions. To identify the possible binding regions between the protein and the scFv, docking was performed (http://bioinfo3d.cs.tau.ac.il/PatchDock/). After selecting the most stable protein-scFv structure, its regions were selected and delimited using the PyMOL program (http://www.pymol.org/).

Example 6 – Reactivity of scFv fragments

[062] The analysis of the expression capacity of the scFv antibody fragments and their target recognition capacity (Figure 7 A and B) was obtained for further analysis. Figure 7 (A) shows a dot blot assay of the expression of scFv clones and Figure (B) shows an ELISA assay for evaluating the expression pattern of selected clones and their target recognition capability. The scFv clones obtained that were able to recognize the target were also screened for cross-reactivity against other antigens. Clone 4B showed greater affinity and specificity for phage C10, peptides TBC10 and TB2C10 and M. tuberculosis proteins than for Strongyloides sp (PSs), Brucella sp (PBs), Candida sp (PCs) and Escherichia proteins. coli (PEc) (Figure 7 C). Thus, clone 4B was the most selective and with the highest affinity in the identification of M. tuberculosis proteins, confirming not only its potential for use in immunoassays for the identification of TB in biological samples, as well as its use in platforms of TB diagnosis. In addition, the scFv-like recombinant antibody fragment (comprising SEQ ID No: 03 or SEQ ID No: 04 and SEQ ID No: 05), 4B, also demonstrated affinity for phage C10 and peptides TBC10 and TB2C10, confirming the ability of the TBC10 and TB2C10 peptides to mimic the IgA-binding Mycobacterium tuberculosis antigenic proteins from biological samples obtained from individuals with TB.

Example 7 – Immunocapture and molecular characterization of ligands

[063] For Immunocapture, anti-His beads (mAb Mag Beads, GenScript Transforming Biology Research, Piscataway, NJ, USA) were used, which have anti-histidine antibodies attached to their surface capable of binding to the histidine tag (His ) of the scFv. The procedure was performed according to the manufacturer's instructions. After successive washes, 100 µL of total M. tuberculosis proteins (1 mg/mL) in binding/wash buffer were added and incubated for 1 hour at RT. After three washes, acidic elution was performed with 200µL of glycine (0.2M, pH 2.2) for 15 minutes. The collected supernatant was neutralized with 50µL of TrisHCl (1.0M, pH 9.1). The binders were eluted from the beads using a DynaMag-2 magnetic apparatus and the supernatant was subjected to a 15% SDS-PAGE gel under denaturing and non-reducing conditions. Protein bands were visualized by Coomassie Blue staining.

[064] Figure 8 (A) shows an SDS-PAGE gel with bands of scFv molecules 4B, ~28kDa, scFv immunocapture protein fraction (MSPMT4B), ~65kDa, and M. tuberculosis protein extract (PMTA), ~ 30kDa.

[065] The bands visualized in the acrylamide gel refer to extracts resulting from immunocapture by scFv, scFv4B itself, antigenic fraction of ~65kDa of scFv ligand (MSPMT4B), and PMTA, fraction of the protein extract of M. tuberculosis used for catch. The non-appearance of ~65kDa band in the column referring to the protein extract (15µL of the total extract) is due to the low concentration of this protein in the total extract, which, because it was concentrated in the immunoprecipitation (15µL of a 1.0 mL concentrate) appears discretely in the second column to the left of the marker (MSPMT4B) (Figure 8 A).

[066] Figures 8 (B1 – B2) show the prediction of the three-dimensional structure of the scFv 4B molecule, with the identification of its light (yellow) and heavy (green) chains and the CDR regions, (B1) front view of the scFv 4B, (B2) apical view of scFv 4B.

[067] Figure 8 (C) shows the interaction between scFv 4B and the M. tuberculosis protein chaperonin60.2/hsp65, highlighting the binding amino acids.

Example 8 - Selection and expression of aptamers

[068] In yet another embodiment the present application describes the development of four aptamers L1, L2, L3 and L4 comprising the sequences SEQ ID NO: 06 (ACGCTCGGATGCCACTACAGTTGGTGATTAAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGTGAC), SEQ ID NO: 07 (CGCTCGGATGCCACT ACAGACGTTTACCGTGATTCTTATGAAACTTGTCAGAGGGGCTTTTCTGCTCATGGACG TGCTGGTGAC ), SEQ ID No: 08 (ACGCTCGGATGCCACTACAGGAGCCTGAGCCC CCAACTGCTTAATATAATATGAGAAATGAACTACTCATGGACGTGCTGGTGAC) and SEQ ID No: 9 (ACGCTCGGATGCCACTACAGTACGAGTTTTTCCTGGGAAGGGTCTG CCAGTAGGATAGCGGGTTCCTCATGGACGTGCTGGTGAC) respectively, and which were also able to bind the proteins of M.

[069] The synthetic peptide TBC10, of 24 amino acids, mimetic to the M. tuberculosis protein, previously selected by the phage display technique, was used as a target for the selection of ssDNA aptamers, from a commercial library acquired by Trilink Biotechnologies flanked by 20 nucleotide primers (5′-ACGCTCGGATGCCACTACAG-45nt-CTCATGGACGTGCTGGTGAC3′). The primers 5′-GTCACCAGCACGTCCATGAG-3′ (SEQ ID NO: 12) and the primer 5′-ACGCTCGGATGCCACTACAG-3′ (SEQ ID NO: 13) were used for asymmetric PCR amplification of the ssDNA strands in each round of selection of the ssDNA. SELEX

[070] Selection was performed using a microtiter plate (Nunc MaxiSorpTM), where 4 wells (1 well per cycle) were sensitized with TBC10 peptide. After denaturation, 10 µM of the library was added with binding buffer and bovine serum albumin (BSA) in the first well and incubated for 60 minutes at room temperature. Non-bindings were washed twice with wash buffer. Elution of ligands was performed by denaturing the peptides with proteinase K (10mg/mL) diluted in binding buffer, incubated at 37ºC for 20 minutes and precipitated with 3M sodium acetate. The eluate resulting from the first cycle was amplified by asymmetric PCR, to obtain the ssDNA in greater quantity for a new cycle, and so on.

[071] After the 4th round of selection, the pool of selected aptamers was cloned into competent Escherichia coli DH5α. After plate growth, twenty clones were collected for plasmid DNA extraction, which were sequenced using Genomelab DTCS Quick Start kit (Beckamn Coulter, USA) according to the manufacturer's instructions.

[072] After sequencing the clones from the last selection cycle, the valid sequences were analyzed to predict their secondary structures and their minimum values of free energy that were obtained by the Sfold software and are shown in Figure 09.

Example 9 - Immunoreactivity of aptamers

[073] The reactivity assay of the four chosen aptamers (L1, L2, L3 and L4), with divergent secondary structures was performed to verify their affinities in relation to the TBC10 peptide, to the inactive M. tuberculosis bacillus (Mtb), to the protein tuberculosis (PMt) and BSA. The reactivity measured in absorbance (450nm) is represented in Figure 10.

[074] In the tests performed, the aptamers showed a greater affinity to the protein extract of Mycobacterium tuberculosis (PMt) and the peptide TBC10, than to BSA (negative control). The low affinity for the inactive bacillus (Mtb) suggests that aptamers specifically bind to intracellular sites of membrane proteins or proteins secreted by the bacillus and that would not be exposed in the wall of the intact bacillus.

[075] In this way, the aptamers (L1, L2, L3 and L4) showed a greater affinity in the identification of M. tuberculosis proteins, confirming not only their potential for use in immunoassays for the identification of TB in biological samples, as well as its use in TB diagnostic platforms. In addition, aptamers confirmed the ability of TBC10 and TB2C10 peptides to mimic IgA-binding Mycobacterium tuberculosis antigenic proteins.

[076] Therefore, the present patent application describes synthetic peptides mimetopes of Mycobacterium tuberculosis antigenic protein for use in a method of diagnosing tuberculosis. Said peptides comprise one of the amino acid sequences selected from the group consisting of: SEQ ID NO 01 and SEQ ID NO 02, or a sequence having at least 85% identity with one of the amino acid sequences selected from the group consisting of: SEQ ID No 01 and SEQ ID No 02.

[077] The present application also describes recombinant antibody or fragment thereof for use in a method of diagnosing tuberculosis. Said antibody or fragment comprises the amino acid sequence SEQ ID No: 03 or a light chain (VL) having the sequence SEQ ID No: 04 and a heavy chain (VH) having SEQ ID No: 05, or a sequence having at least least 85% identity to sequence SEQ ID No: 03 or sequences SEQ ID No: 04 or SEQ ID No: 05.

[078] The present application also describes aptamers for use in a method of diagnosing tuberculosis that comprise at least one of the amino acid sequences of the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09 or a sequence having at least 85% identity to one of the amino acid sequences of the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09.

[079] The present application also describes the use of the peptides (TBC10 and TB2C10), the antibody or fragment thereof (scFv-like recombinant antibody fragment) or the aptamers (L1, L2, L3 and L4) in a diagnostic method of tuberculosis.

• - preparar a amostra biológica, em que a amostra biológica é preferencialmente um fluído corporal de um indivíduo, mais preferencialmente uma amostra de saliva;
• - colocar a amostra em contato com ao menos um dos peptídeos (TBC10 e TB2C10), do anticorpo ou fragmento deste (fragmento de anticorpo recombinante do tipo scFv) ou ao menos um dos aptâmeros (L1, L2, L3 e L4);
• - ler o resultado.

[080] The present application further describes a method of diagnosing tuberculosis comprising the steps of:

• - preparing the biological sample, wherein the biological sample is preferably a body fluid from an individual, more preferably a saliva sample;
• - put the sample in contact with at least one of the peptides (TBC10 and TB2C10), the antibody or its fragment (scFv-type recombinant antibody fragment) or at least one of the aptamers (L1, L2, L3 and L4);
• - read the result.

• - preparação de um bioeletrodo em que compreende a incorporação ou imobilização de ao menos um dos peptídeos (TBC10 e TB2C10), do anticorpo ou fragmento deste (fragmento de anticorpo recombinante do tipo scFv) ou ao menos um dos aptâmeros (L1, L2, L3 e L4);
• - por em contato o bioeletrodo preparado com a amostra biológica;
• - preparação para leitura do conjunto bioeletrodo preparado e amostra biológica;
• - leitura do resultado.

[081] Said diagnostic method comprises an immunoassay or a diagnostic platform, wherein the diagnostic platform is an electrochemical detection platform comprising:

• - preparation of a bioelectrode comprising the incorporation or immobilization of at least one of the peptides (TBC10 and TB2C10), the antibody or fragment thereof (scFv-type recombinant antibody fragment) or at least one of the aptamers (L1, L2, L3 and L4);
• - putting the prepared bioelectrode in contact with the biological sample;
• - preparation for reading the prepared bioelectrode set and biological sample;
• - reading the result.

[082] The electrode is an electrode selected from the group consisting of: graphite electrode, gold, platinum, glassy carbon, carbon paste, electrode with polymeric material and modified electrode, but particularly a graphite electrode.

[083] Although the present patent application has described the subject matter of the present invention with a certain degree of detail by way of illustration and exemplification for the purposes of clarity and understanding, it will be evident that certain changes and modifications may be practiced within the scope of the claims attached.

[084] The examples described in this report are not limiting, allowing a person skilled in the art to change some aspects or components of the present invention, equivalent to the synthetic peptides, the recombinant antibody or fragment thereof, the aptamenes, their uses and diagnostic methods herein described without departing from the scope of the present invention.

Claims (11)

A synthetic Mycobacterium tuberculosis antigenic protein mimetope peptide characterized in that it comprises one of the amino acid sequences selected from the group consisting of: SEQ ID No. 01 and SEQ ID No. 02, or a sequence having at least 85% identity with one of the amino acid sequences selected from the group consisting of: SEQ ID NO 01 and SEQ ID NO 02. Synthetic peptide according to claim 1, characterized in that it is for use in a method of diagnosing tuberculosis. Recombinant antibody or fragment thereof characterized in that it comprises the amino acid sequence SEQ ID No: 03 or a light chain (VL) SEQ ID No: 04 and a heavy chain (VH) SEQ ID No: 05 or a sequence having at least 85% identity to sequence SEQ ID No: 03 or sequences SEQ ID No: 04 or SEQ ID No: 05. Recombinant antibody or fragment thereof according to claim 3, characterized in that it is for use in a method of diagnosing tuberculosis. Aptamer characterized in that it comprises at least one of the amino acid sequences of the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09 or a sequence having at least 85% identity to one of the amino acid sequences of the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09. Aptamer according to claim 5 characterized in that it is for use in a method of diagnosing tuberculosis. Use of the peptide described in claim 1, the antibody or fragment thereof described in claim 3 or the aptamer described in claim 5, characterized by the fact that it is in a method of diagnosing tuberculosis. Tuberculosis diagnosis method characterized by the fact that it comprises the steps of:

• - preparing the biological sample, wherein the biological sample is preferably a body fluid of an individual;
• - putting the sample in contact with at least one of the peptides described in claim 1; the antibody or fragment thereof described in claim 3 or at least one of the aptamers described in claim 5;
• - read the result.

Diagnostic method according to claim 8, characterized in that said method may comprise an immunoassay or a diagnostic platform. Diagnostic method according to claim 9, characterized in that the diagnostic platform is an electrochemical detection platform comprising:

• - preparing a bioelectrode which comprises incorporating or immobilizing at least one of the peptides described in claim 1, the antibody or fragment thereof described in claim 3 or at least one of the aptamers described in claim 5 on an electrode;
• - putting the prepared bioelectrode in contact with the biological sample;
• - preparation for reading the prepared bioelectrode set and biological sample;
• - reading the result.

Diagnostic method according to claim 10, characterized in that the electrode is an electrode selected from the group consisting of: graphite electrode, gold electrode, platinum electrode, glassy carbon electrode, carbon paste electrode, electrode with polymeric material and modified electrode.

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