WO2021258179A1 - Synthetic mimotope peptides of an antigenic protein from m. tuberculosis, recombinant antibody or fragment thereof, aptamers, use thereof in the diagnosis of tuberculosis and method of diagnosis of tuberculosis - Google Patents

Synthetic mimotope peptides of an antigenic protein from m. tuberculosis, recombinant antibody or fragment thereof, aptamers, use thereof in the diagnosis of tuberculosis and method of diagnosis of tuberculosis Download PDF

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WO2021258179A1
WO2021258179A1 PCT/BR2021/050283 BR2021050283W WO2021258179A1 WO 2021258179 A1 WO2021258179 A1 WO 2021258179A1 BR 2021050283 W BR2021050283 W BR 2021050283W WO 2021258179 A1 WO2021258179 A1 WO 2021258179A1
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seq
tuberculosis
fragment
aptamers
peptides
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French (fr)
Portuguese (pt)
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Léa Duarte Da Silva MORAIS
Fabiana De Almeida Araújo SANTOS
Thulio Marquez CUNHA
Robinson Sabino Da SILVA
Aline Gomes De SOUZA
Vivian Alonso GOULART
Fernanda Van Petten de Vasconcelos AZEVEDO
Fabiane Nunes RIELLO
Luiz Ricardo Goulart Filho
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Universidade Federal de Uberlândia
Imunoscan Engenharia Molecular Ltda
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the present invention relates to the selection, characterization and synthesis of recombinant peptides that mimic Mycobacterium tuberculosis antigenic proteins binding to IgA from body fluids, for example saliva, of individuals with tuberculosis, aptamers and recombinant antibody or fragment thereof, protein-binders of M. tuberculosis.
  • Recombinant peptides (TBC10 and TB2C10) chemically synthesized based on the twelve amino acid sequence fused to phage CIO, selected from a commercial PhD-12 library by phage display, have been shown to be applicable in diagnostic methods or diagnostic platforms for tuberculosis.
  • DNA aptamers L1, L2, L3 and L4
  • monoclonal recombinant antibody or fragment of this 4B TBC10 and TB2C10 ligands 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 body fluids from patients with tuberculosis.
  • Tuberculosis is a contagious disease, caused by the bacteria Mycobacterium tuberculosis, which typically affects the lungs, but can also affect other organs and sites.
  • M. tuberculosis infection occurs by air, through inhalation of droplets containing the bacteria, when the patient with lung TB (in the bacilliferous phase) sneezes or coughs (FLYNN and CHAN, 2001).
  • M. tuberculosis is an intracellular bacillus that has the ability to escape the immune response of its host, and remain in latency inside macrophages for prolonged periods, thus characterizing latent infection (LTBI).
  • LTBI latent infection
  • This condition can be altered by an immunological stress, capable of reactivating the infection, originating the active disease, a phase in which the patient releases the bacillus through spittle when he sneezes or coughs, 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.
  • tuberculosis depends on how it will be organized and its maintenance for containment and elimination of the bacillus and involves phagocytic cells and T lymphocyte subpopulations and is mediated by cytokines and chemokines (O'GARRA et al., 2013; PORCELLI et al., 2013; PORCELLI et al. al., 2014; PIETERS, 2008).
  • LAM filtered protein culture - 10kDa (culture filtrate protein-10 kDa - CFP-10), early secreted antigenic target-6 kDa (early secreted antigenic target-6kDa ESAT-6), immunogenic protein MPT64, Ag85A, B and C, and hsp60, are able to prevent phage-lysosome fusion, inactivate intracellular autophagic pathways and inhibit recognition pathways, limiting innate immune responses and the development of adaptive immune responses (ROSEMBERG, 2001; PAI et al., 2016 ; GR ⁇ SCHEL et al., 2016) 9-11.
  • Proteins are a class of proteins with increased expression due to thermal 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 chaperonin 60.2 adhesin functionality in relation to the interaction with macrophages.
  • the disease burden 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. It is also estimated that about 1.2 million people, HIV-negative, and about 251,000 HIV-positive people, died of tuberculosis in 2018.
  • TB is curable as long as the 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.
  • X-ray radiography requires specialized equipment, but it is useful as an auxiliary method in paucibacillary infections and exclusion of other pathologies that require concomitant treatment (FERRI et al., 2014).
  • Bacterioscopy research for alcohol-acid resistant bacilli - Ziehl Neelsen staining, is the most used method for diagnosing new cases and also monitoring the reduction of bacillary load during treatment, it is cheap, has great specificity, but is not very sensitive, dependent observer and limited by the representativeness and quality of the clinical sample.
  • the culture of the bacillus considered the gold standard, is highly specific, indicated for confirming new cases and monitoring the treatment, allows for the determination of drug susceptibility, 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 Derivative) made from the intradermal application of purified protein derived from Rt23, is useful to prove infection, but not necessarily disease, positivity only indicates previous exposure to the antigen, but does not imply active infection.
  • nucleic acids more recently GeneXpert MTB/RIF
  • GeneXpert MTB/RIF is highly specific and sensitive, however, in addition to the high cost, it cannot differentiate active disease of treated infection, is not capable of detecting atypical mycobacteriosis, and is not applicable to the diagnosis of relapses or to monitoring of treatment.
  • serology it is the most practical method, but current antigens do not allow distinguishing active tuberculosis from latent infection, not being applicable to endemic areas (CAILLEAUX-CEZAR, 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.
  • BRASIL - MINISTRY OF HEALTH 2011; GOLETTI et al., 2016; WHO, 2006; GUTLAPALLI et al., 2016.
  • the challenge is the development of a diagnostic method for active tuberculosis of low effective cost and easy to use in the field (point-of-care) and which enables more agility in diagnosis and initiation of treatment, which presents good sensitivity and specificity , accuracy, and to better serve the most vulnerable population, less favored classes and countries or regions with fewer resources.
  • 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 the 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 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.
  • IgA immunoglobulin A
  • Mycobacterium tuberculosis can be directly detected 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, lipoarabinomanan (LAM), secreted protein complexes (CFP-10/ESAT-6) and antigens Ag85, MPT64, hsp60 and others (BORGSTR ⁇ M et al., 2011; FLINT et al., 2004; CHATTERJEE et al., 2011; WIKER and HARBOE, 1992; SINGH et al., 2005).
  • LAM lipoarabinomanan
  • CFP-10/ESAT-6 secreted protein complexes
  • Phage display 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 a variety of 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 the bacteriophage (SMITH, 1985).
  • Biopanning consists of incubating the library of peptides displayed on phages against the target. Non-binding or less specific phages are removed by successive washings and specific phages remain attached for further elution. The pool of specific phages is amplified for further rounds of selection. After three or four rounds of selection, individual clones are characterized by DNA sequencing, Western Blotting or ELISA (SMITH, 1985).
  • Phage display-generated peptide libraries 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 peptide presentation on phages, 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
  • This technique also allows selection of antibody fragments expressed on the surface of bacteriophages.
  • Single-chain variable fragments of antibodies represent the smallest functional domain of light and heavy chains (VL and VH) of an antibody necessary 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 gene III of phage M13, being able to encode and express the scFv in the PIII protein of the phage (HOOGENBOOM , 2005).
  • Selected recombinant antibodies can be expressed on a large scale and used in chemical, physiological and immunological assays.
  • SELEX Systematic Evolution of Ligands by EXponential Enrichment
  • aptamers 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 for target molecules, for detection of drugs, metabolites and toxins in body fluids, cell signaling, used as probes in cell markings for imaging examinations, or therapeutic nanoparticle carriers and biomolecular laboratory procedures (ACQUAH et al., 2015; DARMOSTUK et al., ., 2014; RADOM et al., 2013).
  • the molecular arrangement of the aptamers is the result of a thermodynamically favorable process and provides stability to the secondary structure and conformation.
  • the present patent application describes two new synthetic peptides.
  • the first peptide, TBC10 (with 24 amino acids - SEQ ID No: 01), synthesized from a sequence of 12 amino acids fused to a phage, CIO, previously obtained by phage display against IgA from saliva of individuals with tuberculosis.
  • 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 peptide TB2C10 (with 32 amino acids - SEQ ID No: 02) is synthesized from the duplication of the 12 amino acid protein motif, 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.
  • Peptides TBC10 and TB2C10 were able to detect M. tuberculosis specific IgA in body fluid samples. Showing a sensitivity in immunological assays of 94.12% and specificity greater than 76%, and are applicable, for example, in the use in biosensors for detection of TB IgA in body fluid samples, more particularly in saliva samples.
  • a scFv-type recombinant antibody fragment comprising the sequence SEQ ID No: 03 or the sequences SEQ ID No: 04 and 05, 4B, phago CIO linker, selected by phage display that was able to recognize M. tuberculosis proteins in assays as well as to recognize the synthetic peptides TBC10 and TB2C10 described here.
  • FIGURES la-c depict a representative scheme and a filamentous bacteriophage.
  • FIGURE 2 shows a sequential representation and the molecular structure of the peptides TBC10 and TB2C10.
  • FIGURE 3 shows the correlation of affinity of Phage CIO (described as Linear) with peptides TBC10 and TB2C10, against saliva IgA.
  • FIGURE 4 shows the representation of the percentage of target-specific IgA in relation to the total IgA in the saliva of individuals, represented between the groups phage CIO, Peptides TBC10 and TB2C10.
  • FIGURE 5 shows an evaluation in immunoenzymatic test by ELISA between the reactivity of phage CIO and peptides TBC10 and TB2C10 against IgA in the saliva of individuals.
  • Figure 6 shows the detection by differential pulse voltammetry of IgA binding in TBC10 and TBC2C10 immobilized on a graphite electrode.
  • FIGURES 7 A, B and C show the reactivity of scFv fragments in immunobiological assays.
  • FIGURES 8 A, B and C show the molecular weight, and the three-dimensional structure of scFv fragments in contact with mycobacterium tuberculosis, for molecular characterization.
  • Figure 9 shows the secondary structures of aptamers Ll, L2, L3 and L4.
  • Figure 10 demonstrates the reactivity of Aptamers L1, L2, L3 and L4 in immunoenzymatic assay by elisa.
  • Figure 11 shows a representative scheme of the steps of electrode sensitization with rhodamine and stabilized with light pulse and peptide adsorption on the sensor with subsequent reading of the saliva sample.
  • Figure 12 demonstrates tests with the electrode and what is the best concentration of the peptide for it. Three different concentrations of the TBC10 peptide (lug/ul, 500ng/ul and 100ng/ul) were tested in order to determine the best concentration to be used in the tests.
  • Figure 13 was intended to optimize the test time. Other types of sample adsorption were tested. As previously reported, the electrode was modified and the peptide adsorbed, later the saliva samples were diluted 1:5 in an electrolytic buffer Ferro-potassium ferricyanide and applied directly to the sensor, reducing an adsorption and washing step, resulting in less time for diagnosis .
  • Figure 14 demonstrates the differentiation between tuberculosis-negative saliva samples and positive saliva samples from tuberculosis patients.
  • Figure 15 demonstrates the specificity and sensitivity of the peptide TBC10 against positive samples for Leprosy (Disease caused by the same genus of Tuberculosis) and negative saliva samples for Leprosy and also for Tuberculosis.
  • Figures 16A, 16B and 16C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TBC10, association of Saliva from healthy individuals with the referred peptide (TBC10+Sal Neg) and association of the Saliva of an individual with tuberculosis with the referred peptide (TBC10+Sal Pos).
  • Figures 17A, 17B and 17C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TB2C10, association of Saliva from healthy individuals with said peptide (TB2C10+Sal Neg) and association of Saliva of an individual with tuberculosis with the aforementioned peptide (TB2C10+Sal Pos).
  • the present patent application aimed to identify and synthesize new recombinant peptides that mimic Mycobacterium antigenic proteins tuberculosis binding to IgA from individuals with tuberculosis, aptamers and a recombinant antibody fragment binding to M. tuberculosis proteins, which have improved ability to be used in a method of diagnosing TB.
  • 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 proteins of tuberculosis, as well as a method of diagnosing TB and the use of the aforementioned peptides, antibody fragment and aptamers in the diagnosis of TB.
  • Example 1 - Synthesis of new peptides [050]
  • two new synthetic peptides are described.
  • a previous selection by phage display allowed obtaining and characterization of M13 phage with 12-amino acid peptide (CIO), mimetic of Mycobacterium tuberculosis proteins, IgA ligand from saliva of individuals diagnosed with TB.
  • CIO 12-amino acid peptide
  • IgA ligand from saliva of individuals diagnosed with TB.
  • 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 fused peptide to the phage pIII viral capsid protein; and Figure 01(c) shows a peptide fused to the phage pVIII viral capsid protein.
  • the protein motifs and the 3D molecular structure of the new synthesized peptides TBC10 and TB2C10 are represented in Figure 02.
  • the peptide TBC10 is represented with the protein motif of 12 amino acids in green, spacer and part of the PIII of the phage in yellow.
  • the TB2C10 peptide is represented with the protein motif of 12 amino acids in green, duplicate protein motif in lime green and spacer in yellow.
  • the new peptides TBC10 and TB2C10 were able to detect M. tuberculosis specific IgA in body fluid samples.
  • the CIO phage and the peptides TBC10 and TB2C10 showed a sensitivity of 94.12% to discriminate positives from negatives, and specificity greater than 76.9%.
  • the ROC curves had 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 CIO origin ( Figure 03).
  • Figure 04 shows a representation of the percentage of target-specific IgA in relation to the 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 TB non-reactive to PPD (NP-) and negative controls for TB reactive to PPD (NP+).
  • the percentage of target-specific IgA in relation to total IgA was higher for the positive group at the beginning of the treatment (PNT) compared to the others, with a slight increase for the positive control subjects for the tuberculin test (NP+).
  • peptides 0.5 pg/well
  • CIO phage 1010 viral particles/well
  • Bicarbonate buffer 0.06 M, pH 9.6
  • the plates were washed with PBS buffer plus 0.05% Tween (0.05% PBS), blocked with PBS-BSA 5%, PBS buffer plus 5% BSA, for 1 hour at 37°C.
  • Figure 05 shows an ELISA assessment of the reactivity of CIO, TBC10 and TB2C10 against saliva IgA from Tb-positive individuals at the beginning of treatment (PNT), from Tb-positive individuals with treatment in progress (PT), healthy individuals PPD+ (NP+) and healthy individuals PPD- (NP-). Representation of the ROC curves generated from the
  • FIG. 06 shows differential pulse voltometric detection of IgA binding in TBC10 and TB2C10 immobilized on graphite electrodes. In green are the current values in an empty electrode, in blue the values after target immobilization and treatment with negative saliva pool (NP-), in red the values after target immobilization and treatment with pooled saliva positive for tuberculosis (PNT).
  • Figure 12 demonstrates different tests to determine the best concentration for the peptide.
  • Three different concentrations of the TBC10 peptide (lug/ul, 500ng/ul and 100ng/ul) were tested.
  • Figure 13 reports a more effective way to adserver the sample on the electrode.
  • Saliva samples were diluted 5x in an electrolytic buffer of potassium ferro-ferricyanide and applied directly to the sensor, reducing an adsorption and washing step, resulting in less time for diagnosis. It can be seen that in this way there was also peptide recognition and differentiation between positive and negative samples.
  • Figure 14 clearly demonstrates the differentiation between saliva samples negative for tuberculosis and saliva samples from patients with tuberculosis.
  • the electrode was previously modified as described, rhodamine 500mg and stabilized with a light pulse.
  • the peptide TBC10 lug/ul was adsorbed on the modified electrode and then saliva samples at a dilution of 1:5 were also adsorbed in order to be recognized.
  • the recognition of this peptide with a specific antibody unlike what can be observed in other types of recognition, provided free current passage in the system and, therefore, the oxidation and reduction currents remained higher.
  • the non-recognition of the peptide caused proteins from negative samples to bind more strongly to the sensor, resulting in a lower current flow, mainly in oxidation.
  • Figure 15 highlights the sensitivity and specificity of the TBC10 peptide against positive samples for Leprosy (Disease caused by the same genus as Tuberculosis) and negative saliva samples for Leprosy and also for Tuberculosis.
  • the peptide TBC10 lug/ul was adsorbed and then saliva samples diluted 1:5 from leprosy patients and saliva from patients negative for both leprosy and tuberculosis were applied.
  • the purpose of the test was to prove that, regardless of the type of disease, even for a pathology caused by a bacterium of the same genus (M. mycobacterium), if the sample is not specifically positive for the
  • Tuberculosis there is no differentiation in the electrochemical sensor and the disease cannot be confirmed. In this case the samples positive for leprosy and negative for both leprosy and tuberculosis were not able to differentiate by cyclic voltammetry.
  • FIGS. 16A, 16B and 16C demonstrate a mean infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TBC10, association of Saliva of a healthy individual with said peptide (TBC10+Sal Neg) and association of Saliva of an individual with tuberculosis with that peptide (TBC10+Sal Pos).
  • Infrared spectra of ATR-FTIR were obtained in a spectrophotometer
  • figures 17A, 17B and 17C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos) , Peptide TB2C10, association of Saliva from a healthy individual with the aforementioned peptide (TB2C10+Sal Neg) and association of Saliva from an individual with tuberculosis with the aforementioned peptide (TB2C10+Sal Pos).
  • the infrared spectra were obtained in a spectrophotometer ATR-FTIR Cary 630 and dried at room temperature for 5 minutes to obtain the spectrum which were normalized to Figure 17A or evaluated in second derivative system by Savitzky-Golay from the spectrum fingerprint between 1800-800 cm-1 for Figure 17B or more restricted between 900-1200 cm-1 for best visualization in figure 15C.
  • 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, binder phago CIO selected by phage display that was able to recognize M. tuberculosis proteins in assays as well as to recognize the synthetic peptides TBC10 and TB2C10 described here.
  • the selection cycle were used for extraction of plasmid DNA and subsequent transformation into TOP10 E. coli bacteria, strain supressora.A a non - plasmid DNA extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, DS, Germany) of according to the manufacturer's instructions. The extracted plasmid DNA was quantified (Nanodrop Spectrophotometer, (Thermo Fisher Scientific Inc., Waltham, MA, USA) and later 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 in 10 pL TOP10 bacteria.
  • the sequences were analyzed by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) to identify the light and heavy chains of 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 conserved and variable regions of the scFv.
  • Example 7 - Reactivity of scFv fragments [076] Analysis of the expression capacity of 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 expression of scFv clones and Figure (B) shows an ELISA assay for evaluating the expression pattern of selected clones and their target recognition capacity.
  • 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 to CIO phage, TBC10 and TB2C10 peptides and M.
  • tuberculosis proteins (PMt) than to Strongyloides sp (PSs), Brucella sp (PBs), Candida sp (PCs) proteins ) and Escherichia coli (PEc) (Figure 7C).
  • PSs Strongyloides sp
  • PBs Brucella sp
  • PCs Candida sp
  • PEc Escherichia coli
  • the scFv-type recombinant antibody fragment (comprising SEQ ID No: 03 or SEQ ID No: 04 and SEQ ID No: 05), 4B, also demonstrated affinity for phage CIO and peptides TBC10 and TB2C10, confirming the ability of the peptides TBC10 and TB2C10 to mimic the antigenic proteins of Mycobacterium tuberculosis binding to IgA from biological samples obtained from individuals with TB.
  • anti-His beads mAb Mag Beads, GenScript Transforming Biology Research, Piscataway, NJ, USA
  • His histidine tag
  • the procedure was performed according to the manufacturer's instructions. After successive washes, 100 ⁇ l of total M. tuberculosis proteins (1 mg/ml) in binding/wash buffer were added and incubated for 1 hour at RT. After three washes, acid elution was performed with 200pL of glycine (0.2M, pH 2.2) for 15 minutes.
  • Figure 8 (A) shows an SDS-PAGE gel with bands of scFv 4B molecules, ⁇ 28kDa, protein fraction immunocaptured by scFv (MSPMT4B), ⁇ 65kDa, and M. tuberculosis protein extract (PMTA), ⁇ 30kDa.
  • the bands visualized on 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 a ⁇ 65kDa band in the column referring to the protein extract (15pL of the total extract) is due to the low concentration of this protein in the total extract, which for having been concentrated in the immunoprecipitation (15pL of a 1.0 mL concentrate) appears discretely in the second column to the left of the marker (MSPMT4B) ( Figure 8A).
  • Figures 8 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, (Bl) front view of the scFv 4B, (B2) apical view of scFv 4B.
  • Figure 8 (C) shows the interaction between scFv 4B and the chaperoninôO.2/hsp65 protein of M. tuberculosis with emphasis on the binding amino acids.
  • Example 9 Selection and expression of aptamers
  • L1, L2, L3 and L4 comprising the sequences SEQ ID No: 06 (ACGCTCGGATGCCACTACAGTTGGTGATTAAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGCTCATTGAGTTGGGGG CGCTGACTGCTTGACCTGGGGGGGTGATTAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGCTCATTGAGTTGAGG CGTACGCTGATTGACCTGGCTGGG SEQ ID CGTACGTCTAGG GG CGTACGTACGTCTAGCTGGG SEQ ID CGTACGTCGA CGTA CGTACCTGGCTGGG ), SEQ ID NO: 08 (ACGCTCGGATGCCACTACAGGAGCCTGAGCCC CCAACTGCTTAATATAATATGAGAAATGAACTAC TCATGGACGTGCTGGTGAC) and SEQ ID NO:
  • the selection was performed using a plate of microtiter (Nunc MaxiSorpTM), where 4 wells (1 well per cycle) were sensitized with TBC10 peptide. After denaturation, 10 mM library was added with binding buffer and bovine serum albumin (BSA) in the first well and incubated for 60 minutes at room temperature. Non-binders were washed twice with wash buffer. Ligands were eluted by denaturing the peptides with proteinase K (10mg/ml) diluted in binding buffer, incubated at 37°C 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.
  • BSA bovine serum albumin
  • the selected aptamer pool was cloned into Escherichia coli DH5 competent. After growth in a plate, 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.
  • Example 10 Immunoreactivity of aptamers
  • the reactivity assay of the four chosen aptamers (Ll, L2, L3 and L4), with divergent secondary structures was carried out to verify their affinities in relation to the peptide TBC10, to the inactive bacillus of M. tuberculosis (Mtb), to the protein of M. tuberculosis (PMt) and to BSA.
  • the reactivity measured in absorbance (450nm) is shown in Figure 10.
  • the aptamers showed a greater affinity to the protein extract of Mycobacter ⁇ um tuberculosis (PMt) and to the peptide TBC10, than to BSA (negative control).
  • PMt Mycobacter ⁇ um tuberculosis
  • BSA negative control
  • the low affinity to the inactive bacillus (Mtb) suggests that the aptamers specifically bind to intracellular sites of membrane proteins or proteins secreted by the bacillus and that would not be exposed in the intact bacillus wall.
  • the aptamers (Ll, L2, L3 and L4) showed a greater affinity in the identification of M. tuberculosis proteins, confirming not only its potential for use in immunoassays for identification of TB in biological samples, as well as its use in TB diagnostic platforms.
  • the aptamers confirmed the ability of peptides TBC10 and TB2C10 to mimic Mycobacterium tuberculosis antigenic proteins binding to
  • the present patent application describes synthetic peptides antigenic protein mimetopes from Mycobacterium tuberculosis 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 N-01 and SEQ ID N-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.
  • 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 the SEQ ID No: 05, or a sequence having at least minus 85% identity with the sequence SEQ ID No:03 or the sequences SEQ ID No:04 or SEQ ID No:05.
  • the present application also describes aptamers for use in a method of diagnosing tuberculosis which comprise at least one of the amino acid sequences from 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 from the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09.
  • the present application further describes a method of diagnosing tuberculosis comprising the steps of: preparing the biological sample, in which the biological sample is preferably a bodily fluid from an individual, more preferably a saliva sample;
  • Said diagnostic method comprises an immunoassay by immunoenzymatic test or by a diagnostic platform, whose platform is characterized by being of electrochemical detection that comprises:
  • the electrode is an electrode selected from the group consisting of: graphite, gold, platinum, glassy carbon, carbon paste electrode, electrode with polymeric material and modified electrode, but particularly a graphite electrode.
  • the electrochemical detection diagnostic platform comprises a bioelectrode sensitized and stabilized with Rhodamine and light pulse, having adsorbed, incorporated or immobilized on its surface at least one of the peptides, antibody or fragment or of at least one of the aptamers described in the present invention, as well as comprising means for reading the result.

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Abstract

The present invention relates to the selection, characterization and synthesis of recombinant peptides that mimic antigenic proteins from Mycobacterium tuberculosis and bind to IgA of individuals with tuberculosis, and also to the selection, characterization and synthesis of aptamers and a recombinant antibody fragment that bind to proteins from M. tuberculosis. The present invention also describes the use both of the peptides and aptamers and the recombinant antibody fragment in methods of diagnosis or platforms for the diagnosis of tuberculosis using biological samples from an individual, such as, for example, body fluids from patients with tuberculosis.

Description

Relatório Descritivo da Patente de Invenção: "PEPTÍDEOS SINTÉTICOS MIMETOPOS DE PROTEÍNA ANTIGÊNICA DE M. TUBERCULOSIS , ANTICORPO RECOMBINANTE OU FRAGMENTO DESTE, APTÂMEROS , USO DESTES NO DIAGNÓSTICO DE TUBERCULOSE E MÉTODO DE DIAGNÓSTICO DE TUBERCULOSE". Descriptive Report of the Patent of Invention: "SYNTHETIC PEPTIDES MIMETHOPES OF M. TUBERCULOSIS ANTIGEN PROTEIN, RECOMBINANT ANTIBODY OR FRAGMENT THEREOF, APTAMERS, USE OF THESE IN THE DIAGNOSIS OF TUBERCULOSIS AND DIAGNOSTIC METHOD".
Campo da Invenção Field of Invention
[001] A presente invenção refere-se à seleção, caracterização e síntese de peptídeos recombinantes que mimetizam proteínas antigênicas de Mycobacteríum tuberculosís ligantes a IgA de fluidos corporais, por exemplo da saliva, de indivíduos com tuberculose, aptâmeros e anticorpo recombinante ou fragmento deste, ligantes a proteínas de M. tuberculosís. Os peptídeos recombinantes (TBC10 e TB2C10) sintetizados quimicamente com base na sequencia de doze aminoácidos fusionados ao fago CIO, selecionado de uma biblioteca comercial PhD-12 por phage display, demonstraram ser aplicáveis em métodos de diagnostico ou plataformas diagnósticas para tuberculose. Aptâmeros de DNA (Ll, L2, L3 e L4) e o anticorpo recombinante monoclonal ou fragmento deste 4B ligantes de TBC10 e TB2C10 selecionados, respectivamente, por SELEX e phage display também demonstraram ser aplicáveis em métodos diagnósticos ou plataformas para diagnosticar a tuberculose através de amostras biológicas de um indivíduo, como por exemplo de flúidos corporais de pacientes com tuberculose. Fundamentos da Invenção [001] The present invention relates to the selection, characterization and synthesis of recombinant peptides that mimic Mycobacterium tuberculosis antigenic proteins binding to IgA from body fluids, for example saliva, of individuals with tuberculosis, aptamers and recombinant antibody or fragment thereof, protein-binders of M. tuberculosis. Recombinant peptides (TBC10 and TB2C10) chemically synthesized based on the twelve amino acid sequence fused to phage CIO, selected from a commercial PhD-12 library by phage display, have been shown to be applicable in diagnostic methods or diagnostic platforms for tuberculosis. DNA aptamers (L1, L2, L3 and L4) and the monoclonal recombinant antibody or fragment of this 4B TBC10 and TB2C10 ligands 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 body fluids from patients with tuberculosis. Fundamentals of the Invention
[002] A tuberculose (TB) é uma doença contagiosa, causada pela bactéria Mycobacterium tuberculosis, que afeta tipicamente os pulmões, mas pode também afetar outros órgãos e sítios. A forma mais comum de infecção do M. tuberculosis se dá por via aerógena, pela inalação de gotículas contendo a bactéria, quando o doente com TB de pulmão (em fase bacilífera) espirra ou tosse (FLYNN e CHAN, 2001). [002] Tuberculosis (TB) is a contagious disease, caused by the bacteria Mycobacterium tuberculosis, which typically affects the lungs, but can also affect other organs and sites. The most common form of M. tuberculosis infection occurs by air, through inhalation of droplets containing the bacteria, when the patient with lung TB (in the bacilliferous phase) sneezes or coughs (FLYNN and CHAN, 2001).
[003] O M. tuberculosis é um bacilo intracelular que tem capacidade de escapar da resposta imunológica de seu hospedeiro, e permanecer em latência no interior de macrófagos por períodos prolongados, caracterizando assim a infecção latente (LTBI). Esta condição pode ser alterada por um estresse imunológico, capaz de reativar a infecção originando a doença ativa, fase em que o doente libera o bacilo através de perdigotos quando espirra ou tosse transmitindo a outros indivíduos (FLYNN e CHAN, 2001). Indivíduos com LTBI não transmitem a doença, contudo controlar esta infecção e impedir o desenvolvimento da doença ativa são medidas válidas e promissoras para redução da incidência . [003] M. tuberculosis is an intracellular bacillus that has the ability to escape the immune response of its host, and remain in latency inside macrophages for prolonged periods, thus characterizing latent infection (LTBI). This condition can be altered by an immunological stress, capable of reactivating the infection, originating the active disease, a phase in which the patient releases the bacillus through spittle when he sneezes or coughs, 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] A variedade de manifestações clínicas é decorrente dos diversos mecanismos de escape e fatores de virulência da bactéria, além da heterogeneidade da resposta imunológica dos indivíduos. A forma como a infecção se estabelece e a reativação da doença pode ser definida pela ação de proteínas secretadas pelo M. tuberculosis que interferem na regulação da resposta imune do hospedeiro, assim como um conjunto de fatores como ambiente, hospedeiro, patógeno, coinfecção por HIV, má nutrição e outras imunodeficiências (O'GARRA et al., 2013; GOLDBERG et al., 2014; BEHAR et al. 2010. [004] The variety of clinical manifestations is due to the various escape mechanisms and virulence factors of the bacteria, in addition to the heterogeneity of the immune response of individuals. The way the infection establishes itself and the disease reactivation 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 co-infection, malnutrition and other immunodeficiencies (O' GARRA et al., 2013; GOLDBERG et al., 2014; BEHAR et al. 2010.
[005] O grau de comprometimento do órgão afetado e magnitude das manifestações clinicas, e principalmente das formas subclinicas, associadas às limitações diagnósticas são fatores cruciais para a assertividade do tratamento e contenção da doença (PAI et al., 2016; CADENA et al., 2017). [006] A principal caracteristica da TB em humanos é a formação do granuloma, importante para contenção da bactéria e onde a bactéria consegue sobreviver por longos períodos e proliferar. A resposta imunológica ao M. tuberculosis depende de como será organizada e de sua manutenção para contenção e eliminação do bacilo e envolve células fagocíticas e subpopulações de linfócitos T e é mediada por citocinas e quimiocinas(O'GARRA et al., 2013; PORCELLI et al., 2014; PIETERS, 2008). [005] The degree of involvement of the affected organ and magnitude of clinical manifestations, and especially the subclinical forms, associated with diagnostic limitations are crucial factors for the assertiveness of the treatment and containment of the disease (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 for containment and elimination of the bacillus and involves phagocytic cells and T lymphocyte subpopulations and is mediated by cytokines and chemokines (O'GARRA et al., 2013; PORCELLI et al., 2013; PORCELLI et al. al., 2014; PIETERS, 2008).
[007] A capacidade do bacilo de sobreviver no interior de macrófagos do hospedeiro se dá pela interação de proteínas secretadas ou presentes na parede celular da micobactéria com a célula fagocítica. A secreção de proteínas pelo M. tuberculosis garante sua evasão da ação deletéria das células de defesa. Proteínas como, lipoproteínas, lipoarabinomanana[007] The ability of the bacillus to survive inside the host's macrophages is due to the interaction of secreted proteins or proteins present in the mycobacterial cell wall with the phagocytic cell. The secretion of proteins by M. tuberculosis ensures its avoidance of the harmful action of cells of defense. Proteins such as lipoproteins, lipoarabinomannan
(LAM), cultura filtrada de protína - lOkDa (culture filtrate protein-10 kDa - CFP-10), alvo antigênico precocemente secretado-6 kDa (early secreted antigenic target-6kDa ESAT-6), proteína imunogênica MPT64, Ag85A, B e C, e as hsp60, são capazes de impedir a fusão do fago-lisossomo, inativar caminhos autofágicos intracelulares e inibir vias de reconhecimento, limitando as respostas imunes inatas e o desenvolvimento da resposta imune adaptativa (ROSEMBERG, 2001; PAI et al., 2016; GRÕSCHEL et al., 2016) 9-11. (LAM), filtered protein culture - 10kDa (culture filtrate protein-10 kDa - CFP-10), early secreted antigenic target-6 kDa (early secreted antigenic target-6kDa ESAT-6), immunogenic protein MPT64, Ag85A, B and C, and hsp60, are able to prevent phage-lysosome fusion, inactivate intracellular autophagic pathways and inhibit recognition pathways, limiting innate immune responses and the development of adaptive immune responses (ROSEMBERG, 2001; PAI et al., 2016 ; GRÕSCHEL et al., 2016) 9-11.
[008] As proteínas de choque térmico (hsp - Heat Shock[008] Heat shock proteins (hsp - Heat Shock
Proteins) são uma classe de proteínas com expressão aumentada em decorrência de estresse térmico ou químico, e são agrupadas em famílias de acordo com suas sequências de aminoácidos (a.a) e pesos moleculares (CASTRO et al., 2013). Hickey e colaboradores (2009) observaram que uma proteína recombinante de hsp65, tinha habilidade para inibir a associação do bacilo com macrófago, demonstrando a funcionalidade de adesina da chaperonina 60.2 em relação à interação com macrófagos. Essas e outras proteínas, como ESAT-6, CFP-10, MPT64, Ag85, com papel importante na virulência bacteriana, são potenciais biomarcadores para o diagnóstico de TB (GOLDBERG et al., 2014; XU, 2007; RENSHAW et al., 2005; GAO et al., 2005). Proteins) are a class of proteins with increased expression due to thermal 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 chaperonin 60.2 adhesin functionality in relation to the interaction with macrophages. These and other proteins, such as ESAT-6, CFP-10, MPT64, Ag85, with 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] Segundo dados publicados pela Organização Mundial de Saúde OMS (WHO em inglês) no Relatório Global de[009] According to data published by the World Organization WHO (WHO) in the Global Health Report
Tuberculose - 2019, estima-se que 10 milhões de pessoas adoeceram em 2018, um número que tem sido relativamente estável nos últimos anos mas que continua impactando as pessoas. O ónus da doença varia enormemente entre os países, variando de menos de 5 casos a mais de 500 novos casos por 100 mil habitantes por ano, com média global em torno de 130 novos casos por 100 mil habitantes por ano. Estima-se ainda que cerca 1.2 milhões de pessoas, HIV negativas, e cerca de 251 mil pessoas HIV positivas, morreram de tuberculose em 2018. Tuberculosis - 2019, an estimated 10 million people became ill in 2018, a number that has been relatively stable in recent years but continues to impact people. The disease burden 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. It is also estimated that about 1.2 million people, HIV-negative, and about 251,000 HIV-positive people, died of tuberculosis in 2018.
[010] Apesar dos aumentos nas notificações de TB observadas pela OMS, ainda existe uma grande lacuna entre o número de novos casos relatados, cerca de 7 milhões, e o que é estimado, cerca de 10 milhões de casos em 2018 (WHO, 2019). Essa diferença se deve a combinação de subnotificação de casos detectados e subdiagnóstico. As pessoas com TB não acessam assistência médica ou não são diagnosticadas quando o fazem. [010] Despite the increases in TB notifications observed by WHO, there is still a large gap between the number of new cases reported, around 7 million, and the estimated around 10 million cases in 2018 (WHO, 2019 ). This difference is due to a combination of underreporting of detected cases and underdiagnosis. People with TB do not access medical care or are undiagnosed when they do.
[011] À medida que se intensificam os esforços para melhorar o diagnóstico e o tratamento da TB e fechar as brechas entre incidência e notificações, a proporção de casos notificados confirmados bacteriologicamente precisa ser monitorada, para garantir que as pessoas sejam corretamente diagnosticadas e iniciem, o mais cedo possível, o regime de tratamento mais eficaz. O objetivo deve ser aumentar a porcentagem de casos confirmados bacteriologicamente, aumentando o uso dos diagnósticos recomendados (por exemplo, testes moleculares rápidos) que são mais sensíveis que a microscopia de esfregaço (WHO, 2019). [011] As efforts are intensified to improve the diagnosis and treatment of TB 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 start, as soon as possible, the regime of more effective treatment. The goal should be to increase the percentage of bacteriologically confirmed cases by increasing the use of recommended diagnoses (eg, rapid molecular tests) that are more sensitive than smear microscopy (WHO, 2019).
[012] Além de um problema de saúde pública, a facilidade de propagação e dificuldade de contenção das vias de transmissão faz com que a TB seja um grave problema social, relacionada às más condições económicas, de moradias e nutrição deficiente, e fatores relacionados ao sistema imunológico, o que torna as populações vulneráveis mais susceptíveis a desenvolver a doença (BRASIL - MINISTÉRIO DA SAÚDE, 2011). [012] In addition to a public health problem, the ease of spread and difficulty in containing the transmission routes makes TB a serious social problem, related to poor economic conditions, poor housing and nutrition, and factors related to immune system, which makes vulnerable populations more susceptible to developing the disease (BRASIL - MINISTRY OF HEALTH, 2011).
[013] A TB é curável desde que o tratamento seja realizado, com associação apropriada de drogas antituberculose, dose e tempo de tratamento adequado (WHO, 2019). O diagnóstico precoce é indispensável para contenção e controle da doença. [013] TB is curable as long as the 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] Os achados bacteriológicos e radiológicos são os principais métodos de diagnósticos, e em algumas regiões também são usados testes imunoenzimáticos. Contudo, os métodos diagnósticos utilizados são limitados, por não agregarem, em um único teste, especificidade, sensibilidade e agilidade, que são essenciais para o início efetivo do tratamento e contenção da propagação do bacilo (BENTO et al., 2011). [014] Bacteriological and radiological findings are the main diagnostic methods, and enzyme immunoassays are also used in some regions. However, the diagnostic methods used are limited, as they do not aggregate, in a single test, specificity, sensitivity and agility, which are essential for the effective initiation of treatment and containment of the spread of the bacillus (BENTO et al., 2011).
[015] A radiografia por raios-X requer equipamento especializado, porém é útil como método auxiliar em infecções paucibacilares e exclusão de outras patologias que exigem tratamento concomitante (FERRI et al., 2014). A bacterioscopia, pesquisa de bacilos álcool-ácido resistente - coloração de Ziehl Neelsen, é o método mais utilizado para diagnóstico de casos novos e também monitoramento da redução da carga bacilar durante o tratamento, é barato, tem ótima especificidade, porém é pouco sensível, observador dependente e limitado pela representatividade e qualidade da amostra clínica. A cultura do bacilo, considerada padrão- ouro, é altamente específica, indicada para a confirmação dos casos novos e monitoramento do tratamento, permite determinar suscetibilidade à drogas, contudo demanda tempo, é semi-quantitativa, e também requer infra-estrutura adequada e especializada (CHEE et al., 2013). O teste da tuberculina, também conhecido como PPD (Proteína Purificada Derivada) feito a partir da aplicação intradérmica de proteína purificada derivada Rt23, é útil para provar a infecção, mas não necessariamente a doença, a positividade indica somente a exposição prévia ao antígeno, mas não implica em infecção ativa. A detecção de ácidos nucléicos, mais recentemente GeneXpert MTB/RIF é altamente específico e sensível, porém, além do alto custo, não pode diferenciar doença ativa de infecção tratada, não é capaz de detectar micobacterioses atípicas e não é aplicável ao diagnóstico de recidivas nem ao monitoramento do tratamento. Quanto à sorologia, é o método mais prático, mas os antigenos atuais não permitem distinguir a tuberculose ativa da infecção latente, não sendo aplicáveis a áreas endémicas (CAILLEAUX- CEZAR, 2012). O exame bioquímico ADA - Adenosina Deaminase - é aplicável para o diagnóstico de tuberculose pleural, mas de baixa acurácia a infecções micobacterianas de outros sítios. Os testes diagnósticos atualmente utilizados deixam a desejar, o que dificulta o rastreamento do doente e o monitoramento da evolução clínica (BRASIL - MINISTÉRIO DA SAÚDE, 2011; GOLETTI et al., 2016; WHO, 2006; GUTLAPALLI et al., 2016). [015] X-ray radiography requires specialized equipment, but it is useful as an auxiliary method in paucibacillary infections and exclusion of other pathologies that require concomitant treatment (FERRI et al., 2014). Bacterioscopy, research for alcohol-acid resistant bacilli - Ziehl Neelsen staining, is the most used method for diagnosing new cases and also monitoring the reduction of bacillary load during treatment, it is cheap, has great specificity, but is not very sensitive, dependent observer and limited by the representativeness and quality of the clinical sample. The culture of the bacillus, considered the gold standard, is highly specific, indicated for confirming new cases and monitoring the treatment, allows for the determination of drug susceptibility, 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 Derivative) made from the intradermal application of purified protein derived from Rt23, is useful to prove infection, but not necessarily disease, positivity only indicates 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 of treated infection, is not capable of detecting atypical mycobacteriosis, and is not applicable to the diagnosis of relapses or to monitoring of treatment. As for serology, it is the most practical method, but current antigens do not allow distinguishing active tuberculosis from latent infection, not being applicable to endemic areas (CAILLEAUX-CEZAR, 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 are lacking, 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] O desafio é o desenvolvimento de um método diagnóstico para a tuberculose ativa de baixo custo efetivo e fácil utilização em campo (point-of-care) e que viabilize mais agilidade no diagnóstico e início de tratamento, que apresente boa sensibilidade e especificidade, acurácia, e que permita melhor atender a população mais vulnerável, classes menos favorecidas e países ou regiões com menos recursos. [016] The challenge is the development of a diagnostic method for active tuberculosis of low effective cost and easy to use in the field (point-of-care) and which enables more agility in diagnosis and initiation of treatment, which presents good sensitivity and specificity , accuracy, and to better serve the most vulnerable population, less favored classes and countries or regions with fewer resources.
[017] Apesar de predominantemente utilizado em testes diagnósticos, a representatividade do escarro como espécime clinico é diretamente influenciada por fatores diversos como, horário e qualidade da coleta, expectoração e outros fatores relacionados à condição física do doente, e coopera para a baixa sensibilidade do exame bacterioscópico. Embora o soro seja uma amostra clínica com boa representatividade do estado do organismo, e utilizada amplamente em diversos diagnósticos, não oferece possibilidades satisfatórias para o diagnóstico de TB em virtude da heterogeneidade da infecção (POTTUMARTHY et ai., 2000). O fluido salivar apresenta excelente potencial diagnóstico por conter numerosas proteínas, fragmentos proteicos e anticorpos, que conferem a este fluido importante valor analítico (TABAK, 2001; LAWRENCE 2002; STRECKFUS e BIGLER, 2002; RIBEIRO et ai., 2010; LARREA et al., 2006). Cerca de 85% dos anticorpos presentes na saliva são imunoglobulinas A (IgA) que geralmente desempenham papel protetor, se ligando às bactérias bloqueando estruturas de ligação inibindo a aderência das mesmas (VAN NIEUW AMERONGEN, 2004). A saliva, portanto, é uma alternativa amostrai promissora tanto para o diagnóstico de doenças infecciosas quanto para o monitoramento da evolução do tratamento por sua produção contínua, evidenciando mais precisamente o estado atual do organismo e agrega a vantagem de coleta indolor e não invasiva. [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 patient's physical condition, and contributes to the low sensitivity of the bacterioscopic exam. Although the serum is a clinical sample with good representation of the state of the organism, and 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 the 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 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] Os produtos celulares e metabólitos de[018] The cellular products and metabolites of
Mycobacterium tuberculosis podem ser detectados diretamente no sangue, saliva, urina e outros espécimes clínicos. Alguns destes componentes celulares têm sido propostos como biomarcadores para diagnóstico de TB, tais como fragmentos de DNA, proteínas da parede celular, lipoarabinomanan (LAM), complexos proteicos secretados (CFP-10/ESAT-6) e antígenos Ag85, MPT64, hsp60 e outros (BORGSTRÕM et al., 2011; FLINT et al., 2004; CHATTERJEE et al., 2011; WIKER e HARBOE, 1992; SINGH et al., 2005). Mycobacterium tuberculosis can be directly detected 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, lipoarabinomanan (LAM), secreted protein complexes (CFP-10/ESAT-6) and antigens Ag85, MPT64, hsp60 and others (BORGSTRÕM et al., 2011; FLINT et al., 2004; CHATTERJEE et al., 2011; WIKER and HARBOE, 1992; SINGH et al., 2005).
[019] Uma técnica que pode ser utilizada para desenvolver novas moléculas com potencial é o Phage display. Essa é uma técnica eficiente para identificar peptídeos ou proteínas que se ligam a outras moléculas com diversas finalidades, como mapeamento de epítopos reconhecidos por anticorpos. A tecnologia é baseada no princípio de que polipeptídeos podem ser expressos na superfície de bacteriófagos filamentosos pela inserção de um segmento de DNA codificante no genoma dos mesmos, de modo que o peptídeo ou proteína expressado fique exposto na superfície da partícula virai fusionado a uma proteína virai naturalmente expressa no bacteriófago (SMITH, 1985). [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 a variety of 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 the bacteriophage (SMITH, 1985).
[020] A possibilidade de expressão de uma proteína de fusão no capsídeo de bacteriófagos, de maneira acessível ao reconhecimento por um ligante, demonstrada em 1985 por Smith, abriu caminho para a construção de bibliotecas conformacionais apresentadas na superfície destas partículas virais (SMITH, 1985). [020] The possibility of expression of a fusion protein in the capsid of bacteriophages, in a way accessible to the recognition by a ligand, demonstrated in 1985 by Smith, opened the way for the construction of conformational libraries presented on the surface of these particles viral (SMITH, 1985).
[021] A seleção de sequências baseada na afinidade de ligação do fago a uma molécula alvo é feita por um processo de seleção in vitro denominado bíopanníng. O biopanning consiste na incubação da biblioteca de peptideos expostos em fagos contra o alvo. Os fagos não ligantes ou menos específicos são eliminados por lavagens sucessivas e os fagos específicos permanecem ligados para posterior eluição. O pool de fagos específicos é amplificado para os ciclos posteriores de seleção. Após três ou quatro ciclos de seleção os clones individuais são caracterizados por sequenciamento de DNA, Western Blotting ou ELISA (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 library of peptides displayed on phages against the target. Non-binding or less specific phages are removed by successive washings and specific phages remain attached for further elution. The pool of specific phages is amplified for further rounds of selection. After three or four rounds of selection, individual clones are characterized by DNA sequencing, Western Blotting or ELISA (SMITH, 1985).
[022] Bibliotecas de peptideos geradas por phage display são extensivamente aplicadas na descoberta de uma grande variedade de polipeptídeos incluindo anticorpos, receptores e enzimas (ARAP, 2005) . Epítopos ou determinantes antigênicos, regiões de reconhecimento do antígeno pelos anticorpos, podem também ser identificados através desta metodologia de apresentação de peptideos em fagos, a qual tem sido extremamente importante para a identificação e caracterização de novos ligantes de alta afinidade e seus receptores de uma infinidade de doenças, incluindo câncer, doenças infecciosas, cardiovasculares e autoimunes[022] Phage display-generated peptide libraries 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 peptide presentation on phages, 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 e HOESS, 1995; MULLEN et al., 2006). (STERNBERG and HOESS, 1995; MULLEN et al., 2006).
[023] Esta técnica também permite a seleção de fragmentos de anticorpos expressos na superfície de bacteriófagos . Fragmentos de cadeia única das regiões variáveis de anticorpos (single-chain variable fragment, scFv) representa o menor domínio funcional de cadeias leve e pesada (VL e VH) de um anticorpo necessário à ligação específica ao seu antígeno. A expressão desses fragmentos é realizada na forma de fragmentos recombinantes de anticorpos, através da fusão das regiões codificadoras da porção variável do anticorpo (Fv) ao gene III do fago M13, sendo capaz de codificar e expressar o scFv na proteína PIII do fago (HOOGENBOOM, 2005). Os anticorpos recombinantes selecionados podem ser expressos em larga escala e serem utilizados em ensaios químicos, fisiológicos e imunológicos. [024] Outra técnica que pode ser utilizada é a SELEX (Systematic Evolution of Ligands by EXponential Enrichment). Essa é uma técnica in vitro desenvolvida e descrita a partir da década de 1990 (ELINGTON e SZOSTAK, 1998; TUERK e GOLD, 1990; AQUINO-JARQUIN e TOSCANO-GARIBAY, 2011) para seleção de pequenas moléculas sintéticas de ácidos nucleicos (RNA e DNA de cadeia simples - ssDNA) por PCR. [023] This technique also allows selection of antibody fragments expressed on the surface of bacteriophages. Single-chain variable fragments of antibodies (scFv) represent the smallest functional domain of light and heavy chains (VL and VH) of an antibody necessary 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 gene III of phage M13, being able to encode and express the scFv in the PIII protein of the phage (HOOGENBOOM , 2005). Selected recombinant antibodies can be expressed on a large scale and used in chemical, physiological and immunological assays. [024] Another technique that can be used is the 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 selection of small synthetic nucleic acid molecules (RNA and single-stranded DNA - ssDNA) by PCR.
[025] As moléculas selecionadas por esta técnica são denominadas aptâmeros. Esta técnica é usada como ferramenta biotecnológica com objetivos específicos nas pesquisas com aplicações diagnósticas e terapêuticas. Os aptâmeros são capazes de se ligar a pequenas moléculas, como receptores de moléculas alvo, para detecção de fármacos, metabólitos e toxinas em fluidos corporais, sinalização celular, usados como sondas em marcações celulares para exames de imagens, ou carreadores de nanoparticulas terapêuticas e procedimentos biomoleculares de laboratório (ACQUAH et al., 2015; DARMOSTUK et al., 2014; RADOM et al., 2013). O arranjo molecular dos aptâmeros é resultado de um processo termodinamicamente favorável e confere estabilidade à estrutura secundária e à conformação. São ligantes altamente sensíveis e específicos, com vantagens em relação aos outros ligantes como produção química mais rápida e fácil, menos propensa a contaminação virai ou bacteriana, estocáveis e não imunogênicos (ESPOSITO et al., 2015; SHANNON PENDERGRAST et al., 2005; GOPINATH et al.,2006; AMAYA-GONZÁLEZ et al., 2013; ZHU et al., 2014). [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 for target molecules, for detection of drugs, metabolites and toxins in body fluids, cell signaling, used as probes in cell markings for imaging examinations, or therapeutic nanoparticle carriers and biomolecular laboratory procedures (ACQUAH et al., 2015; DARMOSTUK et al., ., 2014; RADOM et al., 2013). The molecular arrangement of the 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] Desta forma, existe uma necessidade de desenvolver novos métodos de diagnóstico molecular de TB ativa que sejam mais eficientes, rápidos e de melhor custo-benefício. Uma das formas é o de desenvolver novos biomarcadores mais baratos, estáveis e de alta sensibilidade para detecção de proteínas alvo em fluídos corporais como a saliva, salgue, amostras de fluído cervical, urina, dentre outros. Em uma das modalidades aqui descritas, propomos novas moléculas biomarcadores que foram sintetizadas para uso em um método de diagnóstico molecular do TB baseado em ensaios imunológicos ou utilizando plataformas de diagnóstico para na detecção de anticorpos IgA a partir de amostras de fluido corporal, particularmente, em amostras de saliva. [026] Thus, there is a need to develop new molecular diagnostic methods for active TB that are more efficient, faster and more cost-effective. One of the ways is to develop new, cheaper, more stable and highly sensitive biomarkers for detecting target proteins in bodily fluids such as saliva, salt, cervical fluid samples, urine, among others. In one of the modalities described here, we propose new biomarker molecules that have been synthesized for use in an assay-based molecular diagnostic method for TB immunologicals or using diagnostic platforms for the detection of IgA antibodies from body fluid samples, particularly saliva samples.
Breve descrição da invenção Brief description of the invention
[027] O presente pedido de patente descreve dois novos peptideos sintéticos. O primeiro peptideo o TBC10 (com 24 aminoácidos - SEQ ID No: 01), sintetizado a partir de uma sequencia de 12 aminoácidos fusionado a um fago, CIO, obtida previamente por phage display contra IgA de saliva de indivíduos com tuberculose. A síntese peptídica foi projetada com base no motivo proteico, acrescentando um espaçador proteico e parte da proteína PIII do fago, com amidação na região C-terminal e acoplado a BSA na região N- terminal. O segundo peptideo TB2C10 (com 32 aminoácidos - SEQ ID No: 02) sintetizado a partir da duplicação do motivo proteico dos 12 aminoácidos, separados por dois espaçadores, e a inclusão de um espaçador após a segunda sequencia com amidação na região C-terminal e acoplado a BSA na região N- terminal. Os peptideos TBC10 e TB2C10 foram capazes de detectar IgA específica de M. tuberculosis em amostras de fluído corporal. Mostrando uma sensibilidade em ensaios imunológicos de 94,12% e especificidade superior a 76%, e são aplicáveis, por exemplo, na utilização em biossensores para detecção de IgA de TB em amostras de fluído corporal, mais particularmente em amostras de saliva. [028] Em outra modalidade do presente pedido de patente se descreve um fragmento de anticorpo recombinante do tipo scFv compreendendo a sequência SEQ ID No: 03 ou as sequências SEQ IDs No: 04 e 05, 4B, ligante do phago CIO, selecionado por phage display que foi capaz de reconhecer as proteínas de M. tuberculosís em ensaios bem como foi capaz de reconhecer os peptídeos sintéticos TBC10 e TB2C10 aqui descritos. [027] The present patent application describes two new synthetic peptides. The first peptide, TBC10 (with 24 amino acids - SEQ ID No: 01), synthesized from a sequence of 12 amino acids fused to a phage, CIO, previously obtained by phage display against IgA from saliva of individuals with tuberculosis. 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 peptide TB2C10 (with 32 amino acids - SEQ ID No: 02) is synthesized from the duplication of the 12 amino acid protein motif, 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. Peptides TBC10 and TB2C10 were able to detect M. tuberculosis specific IgA in body fluid samples. Showing a sensitivity in immunological assays of 94.12% and specificity greater than 76%, and are applicable, for example, in the use in biosensors for detection of TB IgA in body fluid samples, more particularly in saliva samples. [028] In another embodiment of the present patent application, a scFv-type recombinant antibody fragment is described comprising the sequence SEQ ID No: 03 or the sequences SEQ ID No: 04 and 05, 4B, phago CIO linker, selected by phage display that was able to recognize M. tuberculosis proteins in assays as well as to recognize the synthetic peptides TBC10 and TB2C10 described here.
[029] Em ainda outra modalidade do presente pedido de patente descreve-se o desenvolvimento de quatro aptâmeros (Ll, L2, L3 e L4) os quais compreendem as sequências SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 SEQ ID No: 9, respectivamente, e que também foram capazes de ligar e reconhecer as proteínas de M. tuberculosís bem como ao pepetídeo sintético TBC10 POR SELEX. [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 and recognize M. tuberculosis proteins as well as the synthetic peptide TBC10 BY SELEX.
[030] Em outra modalidade do presente pedido de patente descreve-se o uso de ao menos um dos peptídeos sintéticos TBC10 e TB2C10, da fração de anticorpo scFv (4B) e dos aptâmeros Ll, L2, L3 e L4, em um método de diagnóstico de Tuberculose, bem como o referido método de diagnóstico em si e um sistema de detação utilizando um bioeletrodo. [030] In another modality of the present patent application, the use of at least one of the synthetic peptides TBC10 and TB2C10, of the scFv antibody fraction (4B) and of the aptamers Ll, L2, L3 and L4, in a method of Tuberculosis diagnosis, as well as the said diagnostic method itself and a detection system using a bioelectrode.
Breve descrição das figuras Brief description of the figures
[031] A presente invenção será melhor compreendida com base na descrição, a seguir, tomada em conjunto com as figuras anexas, nas quais: [032] As FIGURAS la-c descrevem um esquema representativo e um bacteriófago filamentoso. (a) fago filamentoso tipo selvagem ilustrando as proteínas do capsideo virai: pIX, pVII, pVIII, pVI e pIII; (b) peptideo fusionado à pIII do fago; e (c) peptideo fusionado à pVIII do fago. [031] The present invention will be better understood based on the description below, taken in conjunction with the attached figures, in which: [032] FIGURES la-c depict 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] A FIGURA 2 mostra uma representação sequencial e a estrutura molecular dos peptideos TBC10 e TB2C10. [033] FIGURE 2 shows a sequential representation and the molecular structure of the peptides TBC10 and TB2C10.
[034] A FIGURA 3 mostra a correlação da afinidade do Fago CIO (descrito como Linear) com os peptideos TBC10 e TB2C10, frente a IgA de saliva. [034] FIGURE 3 shows the correlation of affinity of Phage CIO (described as Linear) with peptides TBC10 and TB2C10, against saliva IgA.
[035] A FIGURA 4 mostra a representação do percentual de IgA alvo-específdica em relação à IgA total na saliva de indivíduos, representado entre os grupos fago CIO, Peptideos TBC10 e TB2C10. [035] FIGURE 4 shows the representation of the percentage of target-specific IgA in relation to the total IgA in the saliva of individuals, represented between the groups phage CIO, Peptides TBC10 and TB2C10.
[036] A FIGURA 5 mostra uma avaliação em teste imunoenzimático por ELISA entre a reatividade do fago CIO e dos peptideos TBC10 e TB2C10 frente a IgA na saliva de indivíduos. [036] FIGURE 5 shows an evaluation in immunoenzymatic test by ELISA between the reactivity of phage CIO and peptides TBC10 and TB2C10 against IgA in the saliva of individuals.
[037] A Figura 6 mostra a detecção por voltametria de pulso diferencial da ligação IgA em TBC10 e TBC2C10 imobilizados em eletrodo de grafite. [037] Figure 6 shows the detection by differential pulse voltammetry of IgA binding in TBC10 and TBC2C10 immobilized on a graphite electrode.
[038] As FIGURAS 7 A, B e C mostram a reatividade dos fragmentos scFv em ensaios imunobiológicos. [038] FIGURES 7 A, B and C show the reactivity of scFv fragments in immunobiological assays.
[039] As FIGURAS 8 A, B e C mostram o peso molecular, e a estrutura tridimensional dos fragmentos de scFv em contato com mycobacterium tuberculosis, para caracterização molecular. [039] FIGURES 8 A, B and C show the molecular weight, and the three-dimensional structure of scFv fragments in contact with mycobacterium tuberculosis, for molecular characterization.
[040] A Figura 9 mostra as estruturas secundárias dos aptâmenros Ll, L2, L3 e L4. [040] Figure 9 shows the secondary structures of aptamers Ll, L2, L3 and L4.
[041] A Figura 10 demonstra a reatividade dos Aptâmeros Ll, L2, L3 e L4 em ensaio imunoenzimático por elisa . [041] Figure 10 demonstrates the reactivity of Aptamers L1, L2, L3 and L4 in immunoenzymatic assay by elisa.
[042] A Figura 11 mostra um esquema representativo das etapas de sensibilização do eletrodo com rodamina e estabilizados com pulso de luz e adsorção do peptideo no sensor com posterior leitura da amostra de saliva. [042] Figure 11 shows a representative scheme of the steps of electrode sensitization with rhodamine and stabilized with light pulse and peptide adsorption on the sensor with subsequent reading of the saliva sample.
[043] A Figura 12 demonstra testes com o eletrodo e qual a melhor concentração do peptideo para o mesmo. Foram testadas 3 concentrações diferentes do peptideo TBC10 (lug/ul, 500ng/ul e 100ng/ul) a fim de determinar qual a melhor concentração a ser utilizada nos testes. [043] Figure 12 demonstrates tests with the electrode and what is the best concentration of the peptide for it. Three different concentrations of the TBC10 peptide (lug/ul, 500ng/ul and 100ng/ul) were tested in order to determine the best concentration to be used in the tests.
[044] A Figura 13 teve o objetivo de otimizar o tempo do teste. Foram testados outros tipos de adsorção da amostra. Como relatado anteriormente, o eletrodo era modificado e o peptideo adsorvido, posteriormente as amostras de saliva foram diluídas 1:5 em tampão eletrolítico Ferro-ferricianeto de potássio e aplicadas diretamente no sensor, diminuindo uma etapa de adsorção e lavagem, acarretando menor tempo no diagnóstico . [044] Figure 13 was intended to optimize the test time. Other types of sample adsorption were tested. As previously reported, the electrode was modified and the peptide adsorbed, later the saliva samples were diluted 1:5 in an electrolytic buffer Ferro-potassium ferricyanide and applied directly to the sensor, reducing an adsorption and washing step, resulting in less time for diagnosis .
[045] A Figura 14 demonstra a diferenciação entre amostras de saliva negativas para tuberculose e amostras de saliva positivas oriundas de pacientes com tuberculose. [045] Figure 14 demonstrates the differentiation between tuberculosis-negative saliva samples and positive saliva samples from tuberculosis patients.
[046] A Figura 15 demonstra a especificidade e sensibilidade do peptídeo TBC10 frente a amostras positivas para Hanseníase (Doença causada pelo mesmo gênero da Tuberculose) e amostras de saliva negativas para Hanseníase e também para a Tuberculose. [046] Figure 15 demonstrates the specificity and sensitivity of the peptide TBC10 against positive samples for Leprosy (Disease caused by the same genus of Tuberculosis) and negative saliva samples for Leprosy and also for Tuberculosis.
[047] As Figuras 16A, 16B e 16C demonstram um espectro médio infravermelho de um pool de amostras de saliva de indivíduos saudáveis (Sal Neg), saliva de indivíduos com tuberculose (Sal Pos), Peptídeo TBC10, associação da Saliva de indivíduo saudável com o referido peptídeo (TBC10+Sal Neg)e associação da Saliva de indivíduo com tuberculose com o referido peptídeo (TBC10+Sal Pos). [047] Figures 16A, 16B and 16C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TBC10, association of Saliva from healthy individuals with the referred peptide (TBC10+Sal Neg) and association of the Saliva of an individual with tuberculosis with the referred peptide (TBC10+Sal Pos).
As figuras 17A, 17B e 17C demonstram um espectro infravermelho médio de um pool de amostras de saliva de indivíduos saudáveis (Sal Neg), saliva de indivíduos com tuberculose (Sal Pos), Peptídeo TB2C10, associação da Saliva de indivíduo saudável com o referido peptídeo (TB2C10+Sal Neg)e associação da Saliva de indivíduo com tuberculose com o referido peptídeo (TB2C10+Sal Pos). Figures 17A, 17B and 17C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TB2C10, association of Saliva from healthy individuals with said peptide (TB2C10+Sal Neg) and association of Saliva of an individual with tuberculosis with the aforementioned peptide (TB2C10+Sal Pos).
Objetivo da invenção Purpose of the invention
[048] O presente pedido de patente teve como objetivo identificar e sintetizar novos peptídeos recombinantes que mimetizam proteínas antigênicas de Mycobacterium tuberculosis ligantes a IgA de indivíduos com tuberculose, aptâmeros e um fragmento de anticorpo recombinante ligantes a proteínas de M. tuberculosis, que tivessem capacidade melhorada de serem usados em um método de diagnóstico do TB. [048] The present patent application aimed to identify and synthesize new recombinant peptides that mimic Mycobacterium antigenic proteins tuberculosis binding to IgA from individuals with tuberculosis, aptamers and a recombinant antibody fragment binding to M. tuberculosis proteins, which have improved ability to be used in a method of diagnosing TB.
Descrição detalhada da invenção [049] O presente pedido de patente descreve à seleção, caracterização e síntese de peptídeos recombinantes que mimetizam proteínas antigênicas de Mycobacteríum tuberculosis ligantes a IgA de indivíduos com tuberculose, além de aptâmeros e um fragmento de anticorpo recombinante ligantes a proteínas de M. tuberculosis, bem como um método de diagnóstico de TB e o uso dos referidos peptídeos, do fragmento de anticorpo e aptâmeros no diagnóstico de TB. Detailed description of the invention [049] 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 proteins of tuberculosis, as well as a method of diagnosing TB and the use of the aforementioned peptides, antibody fragment and aptamers in the diagnosis of TB.
Exemplo 1 - Síntese de novos peptídeos [050] Em uma modalidade do presente pedido de patente são descritos dois novos peptídeos sintéticos. Uma seleção prévia por phage display permitiu obtenção e caracterização do fago M13 com peptideo de 12 aminoácidos (CIO), mimético de proteínas de Mycobacteríum tuberculosis, ligante de IgA de saliva de indivíduos com diagnóstico de TB. Example 1 - Synthesis of new peptides [050] In one embodiment of the present patent application, two new synthetic peptides are described. A previous selection by phage display allowed obtaining and characterization of M13 phage with 12-amino acid peptide (CIO), mimetic of Mycobacterium tuberculosis proteins, IgA ligand from saliva of individuals diagnosed with TB.
[051] A Figura 01 mostra um esquema representativo de um bacteriófago filamentoso em que: a Figura 01 (a) mostra um esquema de um fago filamentoso tipo selvagem ilustrando as proteínas do capsídeo virai: pIX, pVII, pVIII, pVI e pIII; a Figura 01 (b) mostra um esquema de um peptideo fusionado à proteína do capsídeo virai pIII do fago; e a Figura 01 (c) mostra um peptídeo fusionado à proteína do capsídeo virai pVIII do fago. [051] 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 fused peptide to the phage pIII viral capsid protein; and Figure 01(c) shows a peptide fused to the phage pVIII viral capsid protein.
[052] O primeiro peptídeo, o TBC10 (com 24 aminoácidos[052] The first peptide, TBC10 (with 24 amino acids
- SEQ ID No: 01 - NH3-EYNTASIHSYRKGGGSAETVESCL-CONH2), foi sintetizado a partir da referida sequencia de 12 aminoácidos fusionada ao fago, CIO, obtido previamente por phage display contra IgA de saliva de indivíduos com tuberculose.A síntese peptídica foi projetada com base no motivo proteico, acrescentando um espaçador proteico e parte da proteína PIII do fago, com amidação na região C-terminal e acoplado a BSA na região N-terminal. - SEQ ID No: 01 - NH 3- EYNTASIHSYRKGGGSAETVESCL-CONH 2 ), was synthesized from the aforementioned 12 amino acid sequence fused to the phage, CIO, previously obtained by phage display against IgA from saliva of individuals with tuberculosis. 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.
[053] O segundo peptídeo, o TB2C10 (com 36 aminoácidos[053] The second peptide, the TB2C10 (with 36 amino acids
- SEQ ID No: 02 - NH3-EYNTASIHSYRKGGGSGGGSEYNTASIHSYRKGGGS- CONH2) , foi sintetizado a partir da duplicação do motivo proteico dos 12 aminoácidos, separados por dois espaçadores, e a inclusão de um espaçador após a segunda sequencia com amidação na região C-terminal e acoplado a BSA na região N- terminal. - SEQ ID No: 02 - NH 3- EYNTASIHSYRKGGGSGGGSEYNTASIHSYRKGGGS-CONH 2) , was synthesized from the duplication of the 12 amino acid protein motif, separated by two spacers, and the inclusion of a spacer after the second sequence with amidation in the C-region terminal and coupled to BSA in the N-terminal region.
[054] Os motivos proteicos e a estrutura molecular 3D dos novos peptídeos sintetizados TBC10 e TB2C10 estão representados na Figura 02. O peptídeo TBC10 está representado com o motivo proteico de 12 aminoácidos em verde, espaçador e parte da PIII do fago em amarelo. O peptideo TB2C10 está representado com o motivo proteico de 12 aminoácidos em verde, motivo proteico duplicado em verde limão e espaçador em amarelo. [054] The protein motifs and the 3D molecular structure of the new synthesized peptides TBC10 and TB2C10 are represented in Figure 02. The peptide TBC10 is represented with the protein motif of 12 amino acids in green, spacer and part of the PIII of the phage in yellow. The TB2C10 peptide is represented with the protein motif of 12 amino acids in green, duplicate protein motif in lime green and spacer in yellow.
Exemplo 2 - Imunorreatividade Example 2 - Immunoreactivity
[055] Os novos peptideos TBC10 e TB2C10 foram capazes de detectar IgA especifica de M. tuberculosis em amostras de fluido corporal. O fago CIO e os peptideos TBC10 e TB2C10 apresentaram sensibilidade de 94,12% para discriminar positivos de negativos, e especifidade maior que 76,9%. As curvas ROC apresentaram área maior que 0,9367, com valores de p< 0,001 (Figura 05). A análise de correlação entre os alvos apresentou valor igual a 0,94, indicando que a resposta dos peptideos sintéticos foi similar à do peptideo de origem CIO (Figura 03). [055] The new peptides TBC10 and TB2C10 were able to detect M. tuberculosis specific IgA in body fluid samples. The CIO phage and the peptides TBC10 and TB2C10 showed a sensitivity of 94.12% to discriminate positives from negatives, and specificity greater than 76.9%. The ROC curves had 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 CIO origin (Figure 03).
[056] A Figura 04 mostra uma representação do percentual de IgA alvo-especifica em relação à IgA total na saliva de cada indivíduo nos grupos positivos para TB sem tratamento (PNT), positivos para TB em curso de tratamento (PT), controles negativos para TB não reativos ao PPD (NP-) e controles negativos para TB reativos ao PPD (NP+). O percentual de IgA alvo-específica em relação à IgA total foi maior para o grupo positivo no início do tratamento (PNT) em relação aos demais, com discreto aumento pra os indivíduos controles positivos para o teste tuberculínico (NP+). [056] Figure 04 shows a representation of the percentage of target-specific IgA in relation to the 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 TB non-reactive to PPD (NP-) and negative controls for TB reactive to PPD (NP+). The percentage of target-specific IgA in relation to total IgA was higher for the positive group at the beginning of the treatment (PNT) compared to the others, with a slight increase for the positive control subjects for the tuberculin test (NP+).
[057] Para detecção de IgA específica, os peptideos (0,5 pg/poço) e o fago CIO (1010 partículas virais/poço) foram imobilizados com tampão Bicarbonato (0.06 M, pH 9.6) em placas de microtitulação (Nunc Immuno MaxiSorp, Roche Diagnostics) e incubadas a 4°C overnight. As placas foram lavadas com tampão PBS acrescido de 0,05% de Tween (PBST 0,05%), bloqueados com PBS-BSA 5%, tampão PBS acrescido de 5% de BSA, por 1 hora a 37°C. Lavadas com PBST 0,1% e acrescentou-se 50 pL de saliva diluída 1:10 em PBS-BSA 5%, incubou-se a 37°C por 01 hora, (para detecção de IgA total, 50 pL de saliva diluída 1:10 em tampão bicarbonato foram usados para sensibilizar, seguindo o mesmo protocolo de ELISA) lavou-se por 6 vezes com PBST 0,05%. Com a adição de anti-IgA/humana HRP(peroxidase humana), 01 hora a 37°C. Novamente lavadas 6 vezes com PBST 0,05%. A reação foi revelada pela adição de OPD (orthophenylenediamine) diluído em tampão citrato-fosfato (0,1 M, pH 5.0) e 3% de H202. A reação foi interrompida pela adição de 25 pL ácido sulfúrico 2,0 N. A leitura da absorbância foi realizada a 492 nm em leitor de microplacas (Multiskan Go/Thermo Scientific,Finland) . [057] For detection of specific IgA, peptides (0.5 pg/well) and CIO 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% PBS), blocked with PBS-BSA 5%, PBS buffer plus 5% BSA, for 1 hour at 37°C. Washed with 0.1% PBST and added 50 µL of saliva diluted 1:10 in PBS-BSA 5%, incubated at 37°C for 01 hour, (for detection of 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 PBST 0.05%. With the addition of anti-IgA/human HRP(human peroxidase), 01 hour at 37°C. Again washed 6 times with PBST 0.05%. The reaction was revealed by the addition of OPD (orthophenylenediamine) diluted in citrate-phosphate buffer (0.1 M, pH 5.0) and 3% H202. The reaction was stopped by the addition of 25 pL 2.0 N sulfuric acid. The absorbance reading was carried out at 492 nm in a microplate reader (Multiskan Go/Thermo Scientific,Finland).
[058] Os indicativos diagnósticos estão apresentados na Figura 05 que mostra uma avaliação em ELISA da reatividade de CIO, TBC10 e TB2C10 frente a IgA de saliva de indivíduos positivos para Tb no início do tratamento (PNT), de indivíduos positivos para Tb com tratamento em curso (PT), indivíduos sadios PPD+ (NP+) e indivíduos sadios PPD- (NP-). Representação das curvas ROC geradas a partir do[058] The diagnostic indicators are shown in Figure 05, which shows an ELISA assessment of the reactivity of CIO, TBC10 and TB2C10 against saliva IgA from Tb-positive individuals at the beginning of treatment (PNT), from Tb-positive individuals with treatment in progress (PT), healthy individuals PPD+ (NP+) and healthy individuals PPD- (NP-). Representation of the ROC curves generated from the
ELISA, com seus respectivos valores de sensibilidade (Se), especificidade (Sp), Odds Ratio (OR), Intervalo de confiança (IC), área da curva ROC (AUC) e valor de p. Como já descrito anteriormente, o fago CIO e os peptideos TBC10 e TB2C10 apresentaram sensibilidade de 94,12% para discriminar amostras positivas para tuberculose de amostras negativas para tuberculose, e especifidade maior que 76,9%. As curvas ROC apresentaram área maior que 0,9367, com valores de p< 0,001. Os resultados mostram que os peptideos TBC10 e TB2C10 podem ser usados em imunoensaios para o diagnóstico de Tuberculose . ELISA, with their respective sensitivity (Se), specificity (Sp), Odds Ratio (OR), Confidence Interval (CI), ROC curve area (AUC) and p value. As previously described, the CIO phage and the peptides TBC10 and TB2C10 showed a sensitivity of 94.12% to discriminate positive samples for tuberculosis from negative samples for tuberculosis, and a specificity greater than 76.9%. The ROC curves had an area greater than 0.9367, with values of p<0.001. The results show that the peptides TBC10 and TB2C10 can be used in immunoassays for the diagnosis of Tuberculosis.
Exemplo 3 - Plataforma de diagnóstico - BiossensorExample 3 - Diagnosis Platform - Biosensor
[059] Os peptideos TBC10 e TB2C10, uma vez que demonstraram capacidade de identificar IgA de M. tuberculosís em amostras biológicas, foram então imobilizados em eletrodos de grafite (Ds dropsens Drp-110), a fim de testar uma plataforma de diagnóstico eficiente, fácil e de rápida resposta, mais particularmente uma plataforma de detecção eletroquimica. A Figura 06 mostra a detecção por voltometria de pulso diferencial da ligação de IgA em TBC10 e TB2C10 imobilizados em eletrodos de grafite. Em verde estão os valores da corrente em eletrodo vazio, azul os valores após imobilização do alvo e tratamento com pool de saliva negativa (NP-), vermelho os valores após imobilização do alvo e tratamento com pool de saliva positiva para tuberculose (PNT). A ligação de IgA nos alvos imobilizados aumentou a resistência com consequente queda da corrente, com mínimo deslocamento do potencial (TBC10: 0,07V; TB2C10: 0,03V).A interação do bioeletrodo na presença de IgA negativa e positiva apresentou valores de Aipa de 12,216mA para TBC10 e 9,299mA para TB2C10, o que indica que o sistema discrimina o alvo positivo do alvo negativo. Desta forma, os referidos peptideos também se mostraram eficientes para uso em uma plataforma de diagnóstico usando biosenssores, permitindo então discriminar amostras biológicas positivas para TB (PNT) de amostras biológicas negativas para TB (NP-). [059] The peptides TBC10 and TB2C10, 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 differential pulse voltometric detection of IgA binding in TBC10 and TB2C10 immobilized on graphite electrodes. In green are the current values in an empty electrode, in blue the values after target immobilization and treatment with negative saliva pool (NP-), in red the values after target immobilization and treatment with pooled saliva positive 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 interaction of the bioelectrode in the presence of negative and positive IgA showed Aipa values of 12.216mA for TBC10 and 9.299mA for TB2C10, which indicates that the system discriminates the positive target from the negative target. Thus, said peptides also proved to be efficient for use in a diagnostic platform using biosensors, allowing then to discriminate biological samples positive for TB (PNT) from biological samples negative for TB (NP-).
[060] Uma vez que foi demonstrado a capacidade de identificar IgA de M. tuberculosís e com objetivo de confirmar os dados anteriores, nesse ensaio foi utilizado amostras de saliva de pacientes individuais, na diluição 1:5. O eletrodo de grafite foi preparado e sensibilizado com a Rodamina, cuja função é de ampliar o sinal da corrente no sistema e promover a ligação da amostra biológica no grafite do sensor, logo em seguida, foi estabilizado com pulso de luz e posteriormente o peptideo TBC10 foi imobilizado no eletrodo. Com isso demonstramos mais uma vez a capacidade de identificar IgA de M. tuberculosís em amostras salivares, representados pela figura 11 por meio desenho esquemático e pelas figuras 12, 13, 14 e 15, cujos resultados são descritos a seguir. [060] Since the ability to identify IgA from M. tuberculosis was demonstrated and with the aim of confirming the previous data, this assay used saliva samples from individual patients at a 1:5 dilution. The graphite electrode was prepared and sensitized with Rhodamine, whose function is to amplify the current signal in the system and promote the binding of the biological sample to the sensor graphite. Soon after, it was stabilized with a light pulse and subsequently the TBC10 peptide was immobilized on the electrode. Thus, we demonstrate once again the ability to identify M. tuberculosis IgA in salivary samples, represented by figure 11 by means of a schematic drawing and by figures 12, 13, 14 and 15, the results of which are described below.
[061] A figura 12 Demonstra diferentes testes para determinar melhor concentração para o peptideo. Foram testadas 3 concentrações diferentes do peptideo TBC10 (lug/ul, 500ng/ul e 100ng/ul). A maior concentração, sendo de lug/ul foi a que demonstrou melhor reconhecimento pelo sensor, esse fato pode ser observado pela diminuição da corrente de oxidação e redução em voltametria cíclica. [061] Figure 12 demonstrates different tests to determine the best concentration for the peptide. Three different concentrations of the TBC10 peptide (lug/ul, 500ng/ul and 100ng/ul) were tested. The highest concentration, being lug/ul, was the one that showed better recognition by the sensor, this fact can be observed by the decrease in the oxidation current and reduction in cyclic voltammetry.
[062] A Figura 13 Relata uma forma mais eficaz de adserver a amostra no eletrodo. As amostras de saliva foram diluídas 5x em tampão eletrolítico Ferro-ferricianeto de potássio e aplicadas diretamente no sensor, diminuindo uma etapa de adsorção e lavagem, acarretando menor tempo no diagnóstico. Pode-se observar que dessa maneira também houve reconhecimento do peptideo e diferenciação entre as amostras positivas e negativas. [062] Figure 13 reports a more effective way to adserver the sample on the electrode. Saliva samples were diluted 5x in an electrolytic buffer of potassium ferro-ferricyanide and applied directly to the sensor, reducing an adsorption and washing step, resulting in less time for diagnosis. It can be seen that in this way there was also peptide recognition and differentiation between positive and negative samples.
[063] E com objetivo de confirmar a eficácia do sensor e do peptideo, a Figura 14 demonstra claramente a diferenciação entre amostras de saliva negativas para tuberculose e amostras de saliva de pacientes com tuberculose. O eletrodo foi modificado anteriormente como descrito, rodamina de 500mg e estabilizado com pulso de luz.[063] And in order to confirm the effectiveness of the sensor and the peptide, Figure 14 clearly demonstrates the differentiation between saliva samples negative for tuberculosis and saliva samples from patients with tuberculosis. The electrode was previously modified as described, rhodamine 500mg and stabilized with a light pulse.
O peptideo TBC10 lug/ul foi adsorvido no eletrodo modificado e em seguida, amostras de saliva na diluição de 1:5 foram também adsorvidas a fim de serem reconhecidas. O reconhecimento desse peptideo com anticorpo especifico, diferente do que pode ser observado em outros tipos de reconhecimento, proporcionou livre passagem de corrente no sistema e com isso as corrente de oxidação e redução permaneceram mais altas. O não reconhecimento do peptideo fez com que proteínas das amostras negativas se ligassem mais fortemente ao sensor, resultando em uma passagem de corrente, principalmente na oxidação, menor. The peptide TBC10 lug/ul was adsorbed on the modified electrode and then saliva samples at a dilution of 1:5 were also adsorbed in order to be recognized. The recognition of this peptide with a specific antibody, unlike what can be observed in other types of recognition, provided free current passage in the system and, therefore, the oxidation and reduction currents remained higher. The non-recognition of the peptide caused proteins from negative samples to bind more strongly to the sensor, resulting in a lower current flow, mainly in oxidation.
[064] E mais uma vez, com objetivo de corroborar os achados encotrados nessa técnica, a Figura 15 destaca a sensibilidade e especificidade do peptideo TBC10 frente a amostras positivas para Hanseníase (Doença causada pelo mesmo gênero da Tuberculose) e amostras de saliva negativas para Hanseníase e também para a Tuberculose. No eletrodo modificado como anteriormente descrito, foi adsorvido o peptideo TBC10 lug/ul e em seguida aplicadas amostras de saliva diluídas 1:5, de pacientes com hanseníase e saliva de pacientes negativos tanto para hanseníase quanto para tuberculose. O objetivo do teste foi o de comprovar que, independente do tipo de doença, mesmo para uma patologia causada por uma bactéria do mesmo gênero (M. mycobacteríum), se a amostra não for positiva especificamente para a[064] And once again, in order to corroborate the findings found in this technique, Figure 15 highlights the sensitivity and specificity of the TBC10 peptide against positive samples for Leprosy (Disease caused by the same genus as Tuberculosis) and negative saliva samples for Leprosy and also for Tuberculosis. On the electrode modified as described above, the peptide TBC10 lug/ul was adsorbed and then saliva samples diluted 1:5 from leprosy patients and saliva from patients negative for both leprosy and tuberculosis were applied. The purpose of the test was to prove that, regardless of the type of disease, even for a pathology caused by a bacterium of the same genus (M. mycobacterium), if the sample is not specifically positive for the
Tuberculose, hão há diferenciação no sensor eletroquímico e a doença não pode ser confirmada. Nesse caso as amostras positivas para hanseniase e negativas tanto por hanseniase quanto tuberculose não foram capazes de se diferenciarem pela voltametria cíclica. Tuberculosis, there is no differentiation in the electrochemical sensor and the disease cannot be confirmed. In this case the samples positive for leprosy and negative for both leprosy and tuberculosis were not able to differentiate by cyclic voltammetry.
[065] As medidas foram realizadas utilizando um potenciostato portátil PalmSens conectado a um computador. Todas as medições foram realizadas à temperatura ambiente (25°C). Elétrodos de carbono Ds dropsens Drp-110 (DropSens, S.L. Oviedo,Espanha) foram utilizados para imobilizar 5 ng do peptídeo em 4 pL de tampão fosfato 0,1M, pH 7,4, bloqueados com tampão fosfato acrescido de 0,5% de BSA. Após secagem completa, foi adicionado 4 pL de pool saliva diluída entre 1:10 a 1:5 em tampão fosfato, após um minuto de reação a superfície foi lavada por três vezes com água ultrapura. Após a secagem. A leitura foi realizada utilizando solução de ferro/ferricianeto de potássio 5,OmM com 0,1M de KC1. [065] Measurements were performed using a portable PalmSens potentiostat connected to a computer. All measurements were carried out at room temperature (25°C). Drp-110 Ds dropsens carbon electrodes (DropSens, SL 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% of BSA. After complete drying, 4 pL of saliva pool diluted between 1:10 and 1:5 in phosphate buffer was added, after one minute of reaction the surface was washed three times with ultrapure water. After drying. The reading was carried out using a 5.0mM iron/potassium ferricyanide solution with 0.1M KCl.
Exemplo 4 - Espectofotometria infravermelho [066] As figuras 16A, 16B e 16C demonstram um espectro médio infravermelho de um pool de amostras de saliva de indivíduos saudáveis (Sal Neg), saliva de indivíduos com tuberculose (Sal Pos), Peptídeo TBC10, associação da Saliva de indivíduo saudável com o referido peptídeo (TBC10+Sal Neg)e associação da Saliva de indivíduo com tuberculose com o referido peptídeo (TBC10+Sal Pos). Os espectros no infravermelho de ATR-FTIR foram obtidos em espectofotômetroExample 4 - Infrared spectrophotometry [066] Figures 16A, 16B and 16C demonstrate a mean infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos), Peptide TBC10, association of Saliva of a healthy individual with said peptide (TBC10+Sal Neg) and association of Saliva of an individual with tuberculosis with that peptide (TBC10+Sal Pos). Infrared spectra of ATR-FTIR were obtained in a spectrophotometer
Cary 630 e secos em temperatura ambiente por 5 minutos para obtenção do espectro que foram normalizados para Figura 16A ou avaliado em sistema de segunda derivada por Savitzky- Golay do fingerprint do espectro entre 1800-800 cm-1 para Figura 16B ou mais restrito entre 900-1200 cm-1 para melhor visualização na figura 16C. Considerando que o aumento do vale na saliva de indivíduos com tuberculose (2nd dev Sal Pos) em comparação com a mesma saliva associada com o referido peptídeo (2nd dev TBC10+Sal Pos) foi totalmente bloqueado ou teve redução evidente nos modos vibracionais 1044 cm-1, 1076 cm-1, 1118 cm-1 e 1155 cm-1 (indicado pelas setas) demonstra-se a capacidade de ligação especifica ou de maior afinidade com a biofluidos relacionados a detecção da tuberculose. Em conjunto, esses dados demonstram claramente o papel e possibilidade do uso destes peptídeos por interação molecular detectada por espectrometria infravermelho para o diagnóstico da tuberculose. Cary 630 and dried at room temperature for 5 minutes to obtaining the spectrum that were normalized to Figure 16A or evaluated in second derivative system by Savitzky-Golay from the spectrum fingerprint between 1800-800 cm-1 for Figure 16B or more restricted between 900-1200 cm-1 for better visualization in figure 16C . Whereas the trough increase in the saliva of individuals with tuberculosis (2nd dev Sal Pos) compared to the same saliva associated with the aforementioned peptide (2nd dev TBC10+Sal Pos) was either totally blocked or had a clear reduction in vibrational modes 1044 cm- 1, 1076 cm-1, 1118 cm-1 and 1155 cm-1 (indicated by the arrows) demonstrate the specific binding capacity or higher affinity with biofluids related to the detection of tuberculosis. Together, these data clearly demonstrate the role and possibility of using these peptides by molecular interaction detected by infrared spectrometry for the diagnosis of tuberculosis.
[067] De forma similar a figura anterior (Figura 16), as figuras 17A, 17B e 17C demonstram um espectro infravermelho médio de um pool de amostras de saliva de indivíduos saudáveis (Sal Neg), saliva de indivíduos com tuberculose (Sal Pos), Peptídeo TB2C10, associação da Saliva de indivíduo saudável com o referido peptídeo (TB2C10+Sal Neg)e associação da Saliva de indivíduo com tuberculose com o referido peptídeo (TB2C10+Sal Pos). Da mesma forma, os espectros no infravermelho foram obtidos em espectofotômetro ATR-FTIR Cary 630 e secos em temperatura ambiente por 5 minutos para obtenção do espectro que foram normalizados para Figura 17A ou avaliado em sistema de segunda derivada por Savitzky-Golay do fingerprint do espectro entre 1800-800 cm-1 para Figura 17B ou mais restrito entre 900-1200 cm-1 para melhor visualização na figura 15C. Tendo em vista que um aumento da profundidade do vale espectral na saliva de indivíduos com tuberculose (2nd dev Sal Pos) em comparação com a mesma saliva associada com o referido peptídeo (2nd dev TB2C10+Sal Pos) teve maior redução no modo vibracional 1076 cm-1 (indicado pelas setas)em amostras com tuberculose do que de pacientes saudáveis. Isto caracteriza que ocorre uma ligação de maior afinidade com a biofluidos relacionados a detecção da tuberculose. [067] Similar to the previous figure (Figure 16), figures 17A, 17B and 17C demonstrate a mid-infrared spectrum of a pool of saliva samples from healthy individuals (Sal Neg), saliva from individuals with tuberculosis (Sal Pos) , Peptide TB2C10, association of Saliva from a healthy individual with the aforementioned peptide (TB2C10+Sal Neg) and association of Saliva from an individual with tuberculosis with the aforementioned peptide (TB2C10+Sal Pos). Likewise, the infrared spectra were obtained in a spectrophotometer ATR-FTIR Cary 630 and dried at room temperature for 5 minutes to obtain the spectrum which were normalized to Figure 17A or evaluated in second derivative system by Savitzky-Golay from the spectrum fingerprint between 1800-800 cm-1 for Figure 17B or more restricted between 900-1200 cm-1 for best visualization in figure 15C. Considering that an increase in the depth of the spectral valley in the saliva of individuals with tuberculosis (2nd dev Sal Pos) compared to the same saliva associated with the aforementioned peptide (2nd dev TB2C10+Sal Pos) had a greater reduction in the vibrational mode 1076 cm -1 (indicated by arrows) in samples with tuberculosis than in healthy patients. This characterizes that there is a higher affinity binding with biofluids related to the detection of tuberculosis.
[068] Em conjunto, os dados mostrados nos exemplos acima reforçam a capacidade dos peptídeos em serem utilizados no diagnóstico de Tuberculose, baseado na maior interação com componentes moleculares da saliva. [068] Taken together, the data shown in the examples above reinforce the ability of the peptides to be used in the diagnosis of Tuberculosis, based on greater interaction with molecular components of saliva.
Exemplo 5 - Seleção e expressão de fragmento de anticorpo recombinante do tipo scFv Example 5 - Selection and expression of scFv-type recombinant antibody fragment
[069] Em outra modalidade do presente pedido de patente se descreve um novo fragmento de anticorpo recombinante monoclonal do tipo scFv, que compreende a SEQ ID No: 03[069] In another embodiment of the present patent application, a new scFv-type recombinant monoclonal antibody fragment is described, comprising SEQ ID No: 03
(ELTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSATGIPD(ELTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSATGIPD
RFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSHLSTFGQGSKVEIKGGSSRSSEVQLV ESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGS IGYADSVRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSHLSTFGQGSKVEIKGGSSRSSEVQLV ESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGS IGYADSV
KGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK) ou compreende a cadeia leve (VL) da SEQ ID No: 04 (VL-ELTQSPGTLSLSPGERATLSCRASQSVSS SYLAWYQQKPGQAPRLLIYGASSATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQ YGSHLSTFGQGSKVEIK) e a cadeia pesada (VH) da SEQ ID No: 05 (VH-EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNS GSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAK) , 4B, ligante do phago CIO, selecionado por phage display que foi capaz de reconhecer as proteínas de M. tuberculosis em ensaios bem como foi capaz de reconhecer os peptídeos sintéticos TBC10 e TB2C10 aqui descritos. 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, binder phago CIO selected by phage display that was able to recognize M. tuberculosis proteins in assays as well as to recognize the synthetic peptides TBC10 and TB2C10 described here.
[070] Foi possível selecionar e expressar fragmentos de anticorpos do tipo scFv ligantes de proteínas de Mycobacteríum spp a partir do fago CIO. A seleção de clones de scFv frente ao fago CIO foi realizada utilizando uma biblioteca de fagos contendo aproximadamente 2xl06 sequências combinatórias de scFv, desenvolvida no Laboratório de Nanobiotecnologia da UFU. [070] It was possible to select and express scFv-like antibody fragments binding to Mycobacterium spp proteins from the phage CIO. The selection of scFv clones against the CIO phage was performed using a phage library containing approximately 2x10 6 combinatorial scFv sequences, developed at the Nanobiotechnology Laboratory at UFU.
[071] Foram realizados dois ciclos de seleção, sendo precedidos pela reamplificação da biblioteca de scFv em Escherichia coli XLl-Blue competentes e infecção do fago auxiliar VCSM13 para a montagem e replicação das proteínas virais. Um poço de uma placa de microtitulação (Nunc MaxiSorpTM, eBioscience, San Diego, CA, EUA) foi sensibilizado com 1010 partículas virais em 50pL/poço em tampão carbonato-bicarbonato 0,1M (pH 8,6) e incubado por 18 horas a 4°C. O poço foi bloqueado com 250 pL de PBST 0,05%/BSA 5% durante 1 hora a 37°C, e lavado três vezes com PBS. Em seguida, 100 pL da biblioteca de fagos-scFv foram adicionados ao poço e a placa foi incubada por 1 hora a 37°C. Posteriormente, o poço foi lavado 10 vezes com PBST 0,1%. Os fagos ligados às proteínas totais foram eluidos com 100 pL de glicina-HCl lOOmM (pH 2,2) durante 30 minutos à temperatura ambiente (TA). A suspensão foi neutralizada com 16,5 pL de Tris 2M (pH 9,1). Os fagos resultantes desta primeira seleção foram amplificados em E. coli XLl-Blue. [072] Bactérias E. coli XLl-Blue infectadas a partir do[071] Two rounds of selection were performed, being preceded by the reamplification of the scFv library in competent Escherichia coli XLl-Blue and infection of the helper phage VCSM13 for the assembly and replication of viral proteins. One well of a microtiter plate (Nunc MaxiSorpTM, eBioscience, San Diego, CA, USA) was sensitized with 10 10 viral particles at 50pL/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 phage-scFv library was added to the well and the plate was incubated for 1 hour at 37°C. Thereafter, the well was washed 10 times with 0.1% PBST. Total protein bound phages were eluted with 100 µl of 100 mM 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). Phage resulting from this first selection were amplified in E. coli XL1-Blue. [072] E. coli XLl-Blue bacteria infected from the
2o ciclo de seleção foram utilizadas para extração do DNA plasmidial e posterior transformação em bactérias E. coli TOP10, uma linhagem não supressora.A extração DNA plasmidial foi realizada utilizando o QIAprep Spin Miniprep Kit (Qiagen, Hilden, DS, Germany), de acordo com as instruções do fabricante. O DNA plasmidial extraído foi quantificado (Nanodrop Spectrophotometer, (Thermo Fisher Scientific Inc., Waltham, MA, EUA) e posteriormente analisado em gel de agarose a 0,8%. Bactérias E. coli TOP10 foram preparadas com CaCl2 para se tornarem quimiocompetentes. Aproximadamente 50 ng de DNA plasmidial foram adicionados cuidadosamente em 10 pL bactérias TOP10. A mistura foi mantida sob refrigeração em gelo durante 30 minutos e, em seguida, submetida a um choque térmico durante 90 segundos a 42°C e 60 segundos em banho de gelo. Posteriormente, 800 pL de meio SOC foram adicionados e a suspensão foi incubada a 37°C por 45 minutos. Aliquotas das células transformadas foram semeadas em placas de ágar Luria-Bertani (LB) contendo carbenicilina (100 pg/mL). As placas foram incubadas, overnight a 37°C, para o crescimento das colónias recombinantes que contêm o inserto. [073] Cada colónia foi transferida e cultivada em uma placa deep well contendo 1 mL de meio Superbroth (SB), carbenicilina (100 pg/mL) e 2% de glicose 2M (v/v). A placa foi coberta com filme plástico e incubada overnight sob agitação a 250 rpm e 37°C. Foram transferidos 50 pL de cada clone bacteriano para uma nova placa contendo 1 mL de meio SB/carbenicilina (100 pg/mL)/2% de glicose 2M (v/v) e incubados a 250 rpm a 37°C durante 4,5 horas. Após a incubação a placa foi centrifugada a 3.700 rpm durante 15 minutos a 4°C e o pellet foi ressuspenso em 1,5 mL de meio SB/carbenicilina (100 pg/mL). A expressão de scFv solúvel foi induzida com isopropil b-D-tiogalactopiranosídeo (IPTG)2 , the selection cycle were used for extraction of plasmid DNA and subsequent transformation into TOP10 E. coli bacteria, strain supressora.A a non - plasmid DNA extraction was performed using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, DS, Germany) of according to the manufacturer's instructions. The extracted plasmid DNA was quantified (Nanodrop Spectrophotometer, (Thermo Fisher Scientific Inc., Waltham, MA, USA) and later 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 in 10 pL TOP10 bacteria. The mixture was kept under refrigeration on ice for 30 minutes and then subjected to a thermal shock for 90 seconds at 42°C and 60 seconds in an ice bath. Afterwards, 800 µL of SOC medium were added and the suspension was incubated at 37°C for 45 minutes. Aliquots of the transformed cells were plated onto Luria-Bertani (LB) agar plates containing carbenicillin (100 pg/ml). The plates were incubated overnight at 37°C for the growth of recombinant colonies containing the insert. [073] Each colony was transferred and cultured in a deep well plate containing 1 ml of Superbroth medium (SB), carbenicillin (100 pg/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 medium/carbenicillin (100 pg/ml)/2% 2M glucose (v/v) and incubated at 250 rpm at 37°C for 4.5 hours. After incubation the plate was centrifuged at 3700 rpm for 15 minutes at 4°C and the pellet was resuspended in 1.5 ml of SB medium/carbenicillin (100 pg/ml). Soluble scFv expression was induced with isopropyl bD-thiogalactopyranoside (IPTG)
(Sigma-Aldrich, St. Louis, MI, EUA) a uma concentração final de 2,5 mM, overnight sob agitação a 250 rpm e 30°C. A placa deep well foi centrifugada a 3.700 rpm durante 15 minutos a 4°C. O sobrenadante contendo o scFv solúvel foi transferido para outra placa de 96 poços e armazenado a 4°C. O volume remanescente da primeira placa foi centrifugado, e o pellet de cada clone de bactérias foi utilizado para a extração do DNA e posterior sequenciamento. (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 from each bacterial clone was used for DNA extraction and subsequent sequencing.
Exemplo 6 - Sequenciamento de DNA e análise de bioinformática Example 6 - DNA sequencing and bioinformatics analysis
[074] Para a reação de sequenciamento foram utilizados os pellets de cada clone de bactéria previamente separado. As reações foram realizadas de acordo com o kit DyEnamic ET Dye Terminator Cycle Sequencing (GE Healthcare Life Sciences, Waukesha, WI, EUA). Foram utilizados os seguintes primers: MMB4 (5'- GCT TCC GGC TCG TAT GTT GTG T-3' - SEQ ID No: 10) para a cadeia leve (VL), SEQ ID No: 04, e MMB5 (5'- CGT TTG CCA TCT TTT CAT AAT C-3' - SEQ ID No: 11) para a cadeia pesada (VH), SEQ ID No: 05, em um volume final de 10 pL. As amostras foram submetidas ao sequenciador MegaBaceTM 1000 Genetic Analyzer (Amersham Biosciences, Pittsburgh, PA, EUA). [074] For the sequencing reaction, pellets from 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. The samples were submitted to the MegaBaceTM 1000 Genetic Analyzer sequencer (Amersham Biosciences, Pittsburgh, PA, USA).
[075] As sequências foram analisadas pelo IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) para identificação das cadeias leve e pesada das moléculas de scFv, assim como de suas regiões conservadas (framework regions, FRs) e variáveis (complementarity determining regions, CDRs). As sequências de aminoácidos deduzidas foram submetidas aos programas de bioinformática raptor-x (http://raptorx. uchicago.edu/) para obtenção da estrutura 3D da molécula de scFv, em formato pdb, e PyMOL (http://www.pymol.org/) para selecionar as regiões conservadas e variáveis do scFv. Para a identificação das possíveis regiões de ligação entre a proteína e o scFv realizou-se um docking (http://bioínfo3d. cs.tau.ac.il/PatchDock/). Após seleção da estrutura proteína-scFv mais estável, suas regiões foram selecionadas e delimitadas com o auxílio do programa PyMOL (http://www.pymol.org/). [075] The sequences were analyzed by IgBLAST (http://www.ncbi.nlm.nih.gov/igblast) to identify the light and heavy chains of 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 conserved and variable regions of the scFv. To identify possible binding regions between the protein and scFv, docking was performed (http://bioínfo3d.cs.tau.ac.il/PatchDock/). After selecting the most stable protein-scFv structure, its regions were selected and delimited with the help of the PyMOL program (http://www.pymol.org/).
Exemplo 7 - Reatividade dos fragmentos de scFv [076] A análise da capacidade de expressão dos fragmentos de anticorpos scFv e sua capacidade de reconhecimento do alvo (Figura 7 A e B) foi obtida para continuidade das análises. A Figura 7 (A) mostra um ensaio dot blot da expressão de clones scFv e a Figura (B) mostra um ensaio de ELISA para avaliação do padrão de expressão dos clones selecionados e sua capacidade de reconhecimento do alvo. Os clones scFv obtidos que foram capazes de reconhecer o alvo também foram triados quanto à reatividade cruzada frente a outros antígenos. 0 clone 4B foi o que apresentou maior afinidade e especificidade ao fago CIO, aos peptídeos TBC10 e TB2C10 e as proteínas de M. tuberculosis (PMt) que a proteínas de Strongyloides sp (PSs), Brucella sp (PBs), Candida sp (PCs) e Escherichia coli (PEc) (Figura 7 C). Desta forma, o clone 4B foi o que se mostrou mais seletivo e com maior afinidade na identificação de proteínas de M. tuberculosis, confirmando não somente o seu potencial de uso em imunoensaios para identificação de TB em amostras biológicas, bem como seu uso em plataformas de diagnóstico de TB. Além disso, o fragmento de anticorpo recombinante do tipo scFv (compreendendo a SEQ ID No: 03 ou a SEQ ID No: 04 e SEQ ID No: 05), 4B, também demonstrou afinidade pelo fago CIO e aos peptideos TBC10 e TB2C10, confirmando a capacidade dos peptideos TBC10 e TB2C10 de mimetizarem as proteínas antigênicas de Mycobacteríum tuberculosís ligantes a IgA de amostras biológicas obtidas de indivíduos com TB. Example 7 - Reactivity of scFv fragments [076] Analysis of the expression capacity of 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 expression of scFv clones and Figure (B) shows an ELISA assay for evaluating the expression pattern of selected clones and their target recognition capacity. 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 to CIO phage, TBC10 and TB2C10 peptides and M. tuberculosis proteins (PMt) than to Strongyloides sp (PSs), Brucella sp (PBs), Candida sp (PCs) proteins ) and Escherichia coli (PEc) (Figure 7C). Thus, clone 4B was the most selective and showed the greatest affinity in identifying M. tuberculosis proteins, confirming not only its potential for use. in immunoassays for identification of TB in biological samples, as well as its use in TB diagnostic platforms. Furthermore, the scFv-type recombinant antibody fragment (comprising SEQ ID No: 03 or SEQ ID No: 04 and SEQ ID No: 05), 4B, also demonstrated affinity for phage CIO and peptides TBC10 and TB2C10, confirming the ability of the peptides TBC10 and TB2C10 to mimic the antigenic proteins of Mycobacterium tuberculosis binding to IgA from biological samples obtained from individuals with TB.
Exemplo 8- Imunocaptura e caracterização molecular de ligantes Example 8- Immunocapture and molecular characterization of ligands
[077] Para a Imunocaptura utilizou-se beads anti-His (mAb Mag Beads, GenScript Transforming Biology Research, Piscataway, NJ, EUA), que possuem anticorpos anti-histidina acoplados na sua superfície capazes de se ligarem a cauda de histidina (His) do scFv. O procedimento foi realizado de acordo com as instruções do fabricante. Após lavagens sucessivas, foram adicionados 100 pL de proteínas totais de M. tuberculosís (1 mg/mL) em tampão de ligação/lavagem e incubados por 1 hora a TA. Após três lavagens, foi realizada eluição ácida com 200pL de glicina (0,2M, pH 2,2) 15 minutos. O sobrenadante coletado foi neutralizado com 50pL de Tris- HC1 (1,0M, pH 9,1). Os ligantes foram eluídos das beads com a utilização de aparato magnético DynaMag-2 e o sobrenadante foi submetido em gel de SDS-PAGE 15%, em condições desnaturantes e não redutoras. As bandas proteicas foram visualizadas por coloração com Comassie Blue. [077] For Immunocapture, anti-His beads (mAb Mag Beads, GenScript Transforming Biology Research, Piscataway, NJ, USA) were used, which have anti-histidine antibodies coupled on 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, acid elution was performed with 200pL of glycine (0.2M, pH 2.2) for 15 minutes. The collected supernatant was neutralized with 50µl of Tris-HC1 (1.0M, pH 9.1). Ligands were eluted from the beads using DynaMag-2 magnetic apparatus and the supernatant was submitted to 15% SDS-PAGE gel, under conditions denaturing and non-reducing. Protein bands were visualized by staining with Comassie Blue.
[078] A Figura 8 (A) mostra um gel de SDS-PAGE com bandas da moléculas scFv 4B, ~28kDa, fração proteica imunocaptura pelo scFv (MSPMT4B), ~65kDa, e extrato proteico de M. tuberculosis (PMTA), ~30kDa. [078] Figure 8 (A) shows an SDS-PAGE gel with bands of scFv 4B molecules, ~28kDa, protein fraction immunocaptured by scFv (MSPMT4B), ~65kDa, and M. tuberculosis protein extract (PMTA), ~ 30kDa.
[079] A bandas visualizadas no gel de acrilamida referem-se a extratos resultantes da imunocaptura pelo scFv, próprio scFv4B, fração antigênica de ~65kDa de ligante de scFv (MSPMT4B), e PMTA, fração do extrato proteico de M. tuberculosis utilizado para captura. O não aparecimento de banda de ~65kDa na coluna referente ao extrato proteico (15pL do extrato total) deve-se à baixa concentração desta proteína no extrato total, que por ter sido concentrada na imunoprecipitação (15pL de um concentrado de 1,0 mL) aparece de forma discreta na segunda coluna à esquerda do marcador (MSPMT4B) (Figura 8 A). [079] The bands visualized on 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 a ~65kDa band in the column referring to the protein extract (15pL of the total extract) is due to the low concentration of this protein in the total extract, which for having been concentrated in the immunoprecipitation (15pL of a 1.0 mL concentrate) appears discretely in the second column to the left of the marker (MSPMT4B) (Figure 8A).
[080] As Figuras 8 (BI - B2) mostram a predição da estrutura tridimensional da molécula do scFv 4B, com a identificação das suas cadeias leve (amarelo) e pesada (verde) e as regiões de CDR, (Bl) visão frontal da scFv 4B, (B2) visão apical da scFv 4B. [080] Figures 8 (BI - 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, (Bl) front view of the scFv 4B, (B2) apical view of scFv 4B.
[081] A Figura 8 (C) mostra a interação entre o scFv 4B e a proteína chaperoninôO.2/hsp65 de M. tuberculosis com destaque pra os aminoácidos de ligação. Exemplo 9 - Seleção e expressão de aptâmeros[081] Figure 8 (C) shows the interaction between scFv 4B and the chaperoninôO.2/hsp65 protein of M. tuberculosis with emphasis on the binding amino acids. Example 9 - Selection and expression of aptamers
[082] Em ainda outra modalidade do presente pedido de patente se descreve o desenvolvimento de quatro aptâmeros: Ll, L2, L3 e L4, compreendendo as sequências SEQ ID No: 06 (ACGCTCGGATGCCACTACAGTTGGTGATTAAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGTGAC ), SEQ ID No: 07 (CGCTCGGATGCCACT ACAGACGTTTACCGTGATTCTTATGAAACTTGTCAGAGGGGCTTTTCTGCTCATGGACG TGCTGGTGAC), SEQ ID No: 08 (ACGCTCGGATGCCACTACAGGAGCCTGAGCCC CCAACTGCTTAATATAATATGAGAAATGAACTAC TCATGGACGTGCTGGTGAC) e SEQ ID No: 9 (ACGCTCGGATGCCACTACAGTACGAGTTTTTCCTGGGAAGGGTCTG CCAGTAGGATAGCGGGTTCCTCATGGACGTGCTGGTGAC ), respectivamente, e que também foram capazes de se ligar as proteínas de M. tuberculosis bem como ao pepetideo sintético TBC10 POR SELEX. [083] O peptideo sintético TBC10, de 24 aminoácidos, mimético à proteína de M. tuberculosis, selecionado anteriormente pela técnica de phage display, foi utilizado como alvo para a seleção de aptâmeros de ssDNA, a partir de uma biblioteca comercial adquirida por Trilink Biotechnologies flanqueada por primers de 20 nucleotídeos (5'-ACGCTCGGATGCCACTACAG-45nt-CTCATGGACGTGCTGGTGAC3'). Os primers 5'-GTCACCAGCACGTCCATGAG-3 ' (SEQ ID NO: 12) e o primer 5'-ACGCTCGGATGCCACTACAG-3 ' (SEQ ID NO: 13) foram usados para amplificação em PCR assimétrica das fitas de ssDNA em cada ciclo de seleção do SELEX. [082] In yet another modality of the present patent application, the development of four aptamers is described: L1, L2, L3 and L4, comprising the sequences SEQ ID No: 06 (ACGCTCGGATGCCACTACAGTTGGTGATTAAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGCTCATTGAGTTGGGGG CGCTGACTGCTTGACCTGGGGGGGTGATTAAGGTGCCGTCGGTGGTACGATTTGATC ATTCTAGCTCATGGACGTGCTGGCTCATTGAGTTGAGGGG CGTACGCTGATTGACCTGGCTGGG SEQ ID CGTACGTCTAGG GG CGTACGTACGTCTAGCTGGG SEQ ID CGTACGTCGA CGTA CGTACCTGGCTGGG ), SEQ ID NO: 08 (ACGCTCGGATGCCACTACAGGAGCCTGAGCCC CCAACTGCTTAATATAATATGAGAAATGAACTAC TCATGGACGTGCTGGTGAC) and SEQ ID NO: 9 (ACGCTCGGATGCCACTACAGTACGAGTTTTTCCTGGGAAGGGTCTG CCAGTAGGATAGCGTTGGTGAC, respectively, were able to bind, respectively, the synthetic pegylated proteins CGTGCTGG, as well as the MCT10, the synthetic peat CGTGCTGG, respectively. [083] The synthetic peptide TBC10, 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'-ACCGCTCGGATGCCACTACAG-45nt-CTCATGGACGTGCTGGTGAC3'). Primers 5'-GTCACCAGCACGTCCATGAG-3' (SEQ ID NO: 12) and 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 SELEX.
[084] A seleção foi realizada utilizando uma placa de microtitulação (Nunc MaxiSorpTM), onde 4 poços (1 poço por ciclo) foram sensibilizados com peptideo TBC10. Após desnaturação, 10 mM da biblioteca foi adicionado com tampão de ligação e soro albumina bovina (BSA) no primeiro poço e incubado por 60 minutos a temperatura ambiente. Os não ligantes foram lavados por duas vezes com tampão de lavagem. A eluição dos ligantes foi realizada a partir da desnaturação dos peptideos com proteinase K (lOmg/mL) diluída em tampão de ligação incubadas a 37°C por 20 minutos e precipitado com acetato de sódio 3M. O eluato resultante do primeiro ciclo foi amplificado por PCR assimétrica, para obtenção do ssDNA em maior quantidade para um novo ciclo, e assim sucessivamente . [084] The selection was performed using a plate of microtiter (Nunc MaxiSorpTM), where 4 wells (1 well per cycle) were sensitized with TBC10 peptide. After denaturation, 10 mM library was added with binding buffer and bovine serum albumin (BSA) in the first well and incubated for 60 minutes at room temperature. Non-binders were washed twice with wash buffer. Ligands were eluted by denaturing the peptides with proteinase K (10mg/ml) diluted in binding buffer, incubated at 37°C 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.
[085] Após o 4o ciclo de seleção, o pool de aptâmeros selecionados foi clonado em Escherichia coli DH5 competente. Após crescimento em placa, vinte clones foram coletados para extração do DNA plasmidial, os quais foram sequenciados usando Genomelab DTCS Quick Start kit (Beckamn Coulter, USA) de acordo com instruções do fabricante. [085] After selection of the fourth cycle, the selected aptamer pool was cloned into Escherichia coli DH5 competent. After growth in a plate, 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.
[086] Após o sequenciamento dos clones do último ciclo de seleção, as sequências válidas foram analisadas para predição de suas estruturas secundárias e seus valores mínimos de energia livre que foram obtidos pelo software[086] After sequencing the clones from the last selection cycle, the valid sequences were analyzed to predict their secondary structures and their minimum free energy values that were obtained by the software
Sfold e estão apresentados na Figura 09. Sfold and are shown in Figure 09.
Exemplo 10 - Imunorreatividade dos aptâmeros [087] O ensaio de reatividade dos quatro aptâmeros escolhidos (Ll, L2, L3 e L4), com estruturas secundárias divergentes foi realizado para verificar suas afinidades em relação ao peptídeo TBC10, ao bacilo inativo da M. tuberculosís (Mtb), à proteína de M. tuberculosís (PMt) e ao BSA. A reatividade medida em absorbância (450nm) está representada na Figura 10. Example 10 - Immunoreactivity of aptamers [087] The reactivity assay of the four chosen aptamers (Ll, L2, L3 and L4), with divergent secondary structures was carried out to verify their affinities in relation to the peptide TBC10, to the inactive bacillus of M. tuberculosis (Mtb), to the protein of M. tuberculosis (PMt) and to BSA. The reactivity measured in absorbance (450nm) is shown in Figure 10.
[088] Nos testes realizados, os aptâmeros apresentaram uma maior afinidade ao extrato proteico de Mycobacteríum tuberculosís (PMt) e ao peptídeo TBC10, que ao BSA (controle negativo). A baixa afinidade ao bacilo inativo (Mtb) sugerem que os aptâmeros se ligam especificamente a sítios intracelulares de proteínas de membrana ou proteínas secretadas pelo bacilo e que não estariam expostas na parede do bacilo íntegro. [088] In the tests performed, the aptamers showed a greater affinity to the protein extract of Mycobacteríum tuberculosis (PMt) and to the peptide TBC10, than to BSA (negative control). The low affinity to the inactive bacillus (Mtb) suggests that the aptamers specifically bind to intracellular sites of membrane proteins or proteins secreted by the bacillus and that would not be exposed in the intact bacillus wall.
[089] Desta forma, os aptâmeros (Ll, L2, L3 e L4) mostraram uma maior afinidade na identificação de proteínas de M. tuberculosís, confirmando não somente o seu potencial de uso em imunoensaios para identificação de TB em amostras biológicas, bem como seu uso em plataformas de diagnóstico de TB. Além disso, os aptâmeros confirmaram a capacidade dos peptídeos TBC10 e TB2C10 em mimetizarem as proteínas antigênicas de Mycobacteríum tuberculosís ligantes a[089] In this way, the aptamers (Ll, L2, L3 and L4) showed a greater affinity in the identification of M. tuberculosis proteins, confirming not only its potential for use in immunoassays for identification of TB in biological samples, as well as its use in TB diagnostic platforms. In addition, the aptamers confirmed the ability of peptides TBC10 and TB2C10 to mimic Mycobacterium tuberculosis antigenic proteins binding to
IgA.Portanto, o presente pedido de patente descreve peptideos sintéticos mimetopos de proteína antigênica de Mycobacterium tuberculosis para uso em um método de diagnóstico de tuberculose. Os referidos peptideos compreendem uma das sequências de aminoácidos selecionada do grupo que consite de: SEQ ID N- 01 e SEQ ID N- 02, ou uma sequência tendo pelo menos 85% de identidade com umas das sequências de aminoácidos selecionada do grupo que consite de: SEQ ID N° 01 e SEQ ID N° 02. IgA. Therefore, the present patent application describes synthetic peptides antigenic protein mimetopes from Mycobacterium tuberculosis 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 N-01 and SEQ ID N-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.
[090] O presente pedido também descreve anticorpo recombinante ou fragmento deste para uso em um método de diagnóstico de tuberculose. O referido anticorpo ou fragmento compreende a sequência de aminoácidos SEQ ID No: 03 ou uma cadeia leve (VL) tendo a sequência SEQ ID No: 04 e uma cadeia pesada (VH) tendo a SEQ ID No: 05, ou uma sequência tendo pelo menos 85% de identidade com a sequência SEQ ID No: 03 ou com as sequências SEQ ID No: 04 ou SEQ ID No: 05. [090] 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 the SEQ ID No: 05, or a sequence having at least minus 85% identity with the sequence SEQ ID No:03 or the sequences SEQ ID No:04 or SEQ ID No:05.
[091] O presente pedido também descreve aptâmeros para uso em um método de diagnóstico de tuberculose que compreendem ao menos umas das sequências de aminoácidos do grupo que consite de SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 e SEQ ID No: 09 ou uma sequência tendo pelo menos 85% de identidade com umas das sequências de aminoácidos do grupo que consite de SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 e SEQ ID No: 09. [091] The present application also describes aptamers for use in a method of diagnosing tuberculosis which comprise at least one of the amino acid sequences from 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 from the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09.
[092] O presente pedido também descreve o uso dos peptideos (TBC10 e TB2C10), do anticorpo ou fragmento deste[092] This application also describes the use of peptides (TBC10 and TB2C10), of the antibody or fragment thereof
(fragmento de anticorpo recombinante do tipo scFv) ou dos aptâmeros (Ll, L2, L3 e L4) em um método de diagnóstico de tuberculose . (scFv-type recombinant antibody fragment) or the aptamers (L1, L2, L3 and L4) in a method of diagnosing tuberculosis.
[093] O presente pedido ainda descreve um método de diagnóstico de tuberculose que compreende as etapas de: preparar a amostra biológica, em que a amostra biológica é preferencialmente um fluido corporal de um indivíduo, mais preferencialmente uma amostra de saliva; [093] The present application further describes a method of diagnosing tuberculosis comprising the steps of: preparing the biological sample, in which the biological sample is preferably a bodily fluid from an individual, more preferably a saliva sample;
- colocar a amostra em contato com ao menos um dos peptideos (TBC10 e TB2C10), do anticorpo ou fragmento deste (fragmento de anticorpo recombinante do tipo scFv) ou ao menos um dos aptâmeros (Ll, L2, L3 e L4); ler o resultado por ensaio imunoenzimático, espectofotometria ou sensor eletroquímico. - 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 by immunoenzymatic assay, spectrophotometry or electrochemical sensor.
[094] O referido método de diagnóstico compreende um imunoensaio por teste imunoenzimático ou por uma plataforma de diagnóstico, cuja plataforma se caracteriza por ser de detecção eletroquímica que compreende: [094] Said diagnostic method comprises an immunoassay by immunoenzymatic test or by a diagnostic platform, whose platform is characterized by being of electrochemical detection that comprises:
- preparação de um bioeletrodo em que compreende a adsorção, incorporação e/ou imobilização de ao menos um dos peptideos (TBC10 e TB2C10), do anticorpo ou fragmento deste (fragmento de anticorpo recombinante do tipo scFv) ou ao menos um dos aptâmeros (Ll, L2, L3 e L4); por em contato com o bioeletrodo preparado e sensibilizado com a Rodamina, cuja função é de ampliar o sinal da corrente no sistema e promover a ligação da amostra biológica no grafite do sensor. preparação para leitura do conjunto bioeletrodo preparado e amostra biológica; - preparation of a bioelectrode comprising the adsorption, incorporation and/or immobilization of at least one of the peptides (TBC10 and TB2C10), the antibody or fragment thereof (recombinant antibody fragment of the scFv type) or at least one of the aptamers (Ll , L2, L3 and L4); put in contact with the prepared bioelectrode and sensitized with Rhodamine, whose function is to amplify the current signal in the system and promote the connection of the biological sample to the sensor's graphite. preparation for reading the prepared bioelectrode set and biological sample;
- leitura do resultado. - reading the result.
[095] O eletrodo é um eletrodo selecionado do grupo que consiste de: eletrodo de grafite, de ouro, de platina, de carbono vítreo, de pasta de carbono, eletrodo com material polimérico e eletrodo modificado, mas particularmente um eletrodo de grafite. [095] The electrode is an electrode selected from the group consisting of: graphite, gold, platinum, glassy carbon, carbon paste electrode, electrode with polymeric material and modified electrode, but particularly a graphite electrode.
[096] Portanto, a plataforma de diagnóstico de detecção eletroquimica compreende um bioeletrodo sensibilizado e estabilizado com Rodamina e pulso de luz, tendo adsorvido, incorporado ou imobilizado em sua superfície ao menos um dos peptídeos, anticorpo ou fragmento ou de ao menos um dos aptâmeros descritos na presente invenção, bem como compreende meios para leitura do resultado. [096] Therefore, the electrochemical detection diagnostic platform comprises a bioelectrode sensitized and stabilized with Rhodamine and light pulse, having adsorbed, incorporated or immobilized on its surface at least one of the peptides, antibody or fragment or of at least one of the aptamers described in the present invention, as well as comprising means for reading the result.
[097] Embora o presente pedido de patente tenha descrito a matéria objeto da presente invenção com um certo grau de detalhamento a título de ilustração e exemplificação para fins de clareza e compreensão, será evidente que certas alterações e modificações podem ser praticadas no escopo das reivindicações em anexo. [097] Although this patent application has described the subject matter of the present invention with a certain degree of detail by way of illustration and example for the purposes of clarity and understanding, it will be evident that certain changes and modifications may be made within the scope of the claims attached.
[098] Os exemplos descritos neste relatório não são limitativos, permitindo que um técnico no assunto altere alguns aspectos ou componentes da presente ivenção, equivalentes aos peptideos sintéticos, ao anticorpo recombinante ou fragmento deste, aos aptâmenros, aos usos destes e métodos de diagnósticos aqui descritos sem se distanciar do escopo da presente invenção. [098] 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 synthetic peptides, to recombinant antibody or fragment thereof, to aptamers, to their uses and diagnostic methods described herein without departing from the scope of the present invention.

Claims

REIVINDICAÇÕES
1 - Peptideo sintético mimetico a proteína antigênica de Mycobacterium tuberculosis caracterizado pelo fato de que compreende uma das sequências de aminoácidos selecionada do grupo que consite de: SEQ ID N- 01 e SEQ ID N- 02, ou uma sequência tendo pelo menos 85% de identidade com umas das sequências de aminoácidos selecionada do grupo que consite de: SEQ ID N° 01 e SEQ ID N° 02. 1 - Synthetic peptide mimicking the antigenic protein of Mycobacterium tuberculosis characterized by the fact that it comprises one of the amino acid sequences selected from the group consisting of: SEQ ID N-01 and SEQ ID N-02, or a sequence having at least 85% of identity with one of the amino acid sequences selected from the group consisting of: SEQ ID No. 01 and SEQ ID No. 02.
2 - Peptideo sintético de acordo com a reivindicação 1, caracterizado pelo fato de que é para uso em um método de diagnóstico de tuberculose. 2 - Synthetic peptide according to claim 1, characterized in that it is for use in a method of diagnosing tuberculosis.
3- Anticorpo recombinante ou fragmento deste caracterizado pelo fato de que compreende a sequência de aminoácidos SEQ ID No: 03 ou uma cadeia leve (VL) SEQ ID No: 04 e uma cadeia pesada (VH) SEQ ID No: 05 ou uma sequência tendo pelo menos 85% de identidade com a sequência SEQ ID No: 03 ou com as sequências SEQ ID No: 04 ou SEQ ID No: 05. 3- 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 with the sequence SEQ ID No:03 or with the sequences SEQ ID No:04 or SEQ ID No:05.
4- Anticorpo recombinante ou fragmento deste de acordo com a reivindicação 3, caracterizado pelo de que é para uso em um método de diagnóstico de tuberculose. 4- Recombinant antibody or fragment thereof according to claim 3, characterized in that it is for use in a method of diagnosing tuberculosis.
5- Aptâmero caracterizada pelo fato de que compreende ao menos umas das sequências de nucleotídeos do grupo que consite de SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 e SEQ ID No: 09 ou uma sequência tendo pelo menos 85% de identidade com umas das sequências de nucleotídeos do grupo que consite de SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 e SEQ ID No: 09. 6- Aptâmero de acordo com a reivindicação 5 caracterizada pelo fato de que é para uso em um método de diagnóstico de tuberculose. 5- Aptamer characterized in that it comprises at least one of the nucleotide sequences from 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 nucleotide sequences of the group consisting of SEQ ID No: 06, SEQ ID No: 07, SEQ ID No: 08 and SEQ ID No: 09. 6- Aptamer according to claim 5 characterized in that it is for use in a method of diagnosing tuberculosis.
7- Uso do peptideo descrito na reivindicação 1, do anticorpo ou fragmento deste descrito na reivindicação 3 ou do aptâmero descrito na reivindicação 5, caracterizada pelo fato de que é em um método de diagnóstico de tuberculose. 7- Use of the peptide described in claim 1, of the antibody or fragment thereof described in claim 3 or of the aptamer described in claim 5, characterized in that it is in a method of diagnosing tuberculosis.
8- Método de diagnóstico de tuberculose caracterizado pelo fato de que compreende as etapas de: 8- Method for diagnosing tuberculosis characterized by the fact that it comprises the steps of:
- preparação da amostra biológica, em que a amostra biológica é preferencialmente saliva; - preparation of the biological sample, in which the biological sample is preferably saliva;
- colocar a amostra em contato com ao menos um dos peptideos descritos na reivindicação 1; do anticorpo ou fragmento deste descrito na reivindicação 3 ou ao menos um dos aptâmeros descritos na reivindicação 5; ler o resultado por ensaio imunoenzimático, espectofotometria ou sensor eletroquimico. - placing 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 by immunoenzymatic assay, spectrophotometry or electrochemical sensor.
9- Método de diagnóstico de acordo com a reivindicação9- Diagnostic method according to claim
8, caracterizado pelo fato de que o referido método pode compreender um imunoensaio ou uma plataforma de diagnóstico. 8, characterized in that said method may comprise an immunoassay or a diagnostic platform.
10- Método de diagnóstico de acordo com a reivindicação10- Diagnostic method according to claim
9, caracterizado pelo fato de que o método usando a plataforma de diagnóstico de detecção eletroquimica compreende: preparação de um bioeletrodo em que compreende a sensibilização e estabilização do eletrodo com Rodamina e pulso de luz, incorporação ou imobilização de ao menos um dos peptideos descritos na reivindicação 1, do anticorpo ou fragmento deste descrito na reivindicação 3 ou de ao menos um dos aptâmeros descritos na reivindicação 5 em um eletrodo; 9, characterized by the fact that the method using the electrochemical detection diagnostic platform comprises: preparation of a bioelectrode in which it comprises the sensitization and stabilization of the electrode with Rhodamine and light pulse, incorporation or immobilization of at least one of the peptides described in claim 1, of the antibody or fragment thereof described in claim 3 or of at least one of the aptamers described in claim 5 on an electrode;
- por em contato o bioeletrodo preparado com a amostra biológica; preparação para leitura do conjunto bioeletrodo preparado e amostra biológica; - bringing the prepared bioelectrode into contact with the biological sample; preparation for reading the prepared bioelectrode set and biological sample;
- leitura do resultado. - reading the result.
11- Método de diagnóstico de acordo com a reivindicação 10, caracterizado pelo fato de que o eletrodo é um eletrodo selecionado do grupo que consiste de: eletrodo de grafite, de ouro, de platina, de carbono vítreo, de pasta de carbono, eletrodo com material polimérico e eletrodo modificado. 11- Diagnostic method according to claim 10, characterized in that the electrode is an electrode selected from the group consisting of: graphite, gold, platinum, vitreous carbon, carbon paste, electrode with polymeric material and modified electrode.
12- Plataforma de diagnóstico de detecção eletroquímica caracterizada pelo fato de que compreende um bioeletrodo sensibilizado e estabilizado com Rodamina e pulso de luz, tendo adsorvido, incorporado ou imobilizado em sua superfície ao menos um dos peptideos descritos na reivindicação 1, do anticorpo ou fragmento deste descrito na reivindicação 3 ou de ao menos um dos aptâmeros descritos na reivindicação 5. 12- Electrochemical detection diagnostic platform characterized by the fact that it comprises a bioelectrode sensitized and stabilized with Rhodamine and light pulse, having adsorbed, incorporated or immobilized on its surface at least one of the peptides described in claim 1, of the antibody or fragment thereof described in claim 3 or of at least one of the aptamers described in claim 5.
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