Method And Kit For Diagnosis Of Active Tuberculosis

Description

TECHNICAL FIELD

The invention belongs to the fields of molecular biology, immunology and disease diagnosis. In particular, the invention relates to a method for diagnosing whether a subject is suffering from active tuberculosis, a method for determining the therapeutic effect of a therapy on active tuberculosis, and a method for screening a candidate drug capable of treating active tuberculosis. The invention further relates to a kit comprising a specific stimulating agent and a reagent for detecting the level of IL-6.

BACKGROUND ART

Tuberculosis (TB), a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) infection, is a very serious health problem around the world. There are about 9 million new TB patients every year over the world, and up to 1.7 million people died of tuberculosis. In addition, it is estimated that about one-third of the world's population is latently infected with M. tuberculosis, which is the source of potential active tuberculosis (aTB) patients.

To control tuberculosis, the foremost thing is to discover and cure patients with infectious tuberculosis. After X-ray has been applied in clinical tests, it provides a simple, convenient, quick, and highly sensitive method for discovering TB patients. However, X-ray detection has disadvantages such as high demands on equipment, low specificity, and a high rate of over-diagnosis, missed diagnosis, and misdiagnosis. In addition, X-ray detection can only detect the suspicious shadows in lungs, but cannot achieve the purpose of detecting infectious patients. Therefore, it is explicitly pointed out in the 9^threport of the WHO Expert Committee on Tuberculosis (1974) that active finding of patients by chest X-ray examination in a population is not feasible, and has to be abolished not only in developed countries but also in developing countries.

The sputum smear test can detect infectious TB patients, and has advantages such as high specificity, simple equipment, low cost, and low demands on techniques. However, there are also problems concerning difficulty in sputum collection; for example, many patients suffer from dry cough without sputum, or a trace amount of bacteria excreted in sputum is not easy to be detected. The sensitivity of widely used sputum smear tests is between 34% and 80%. Although sputum culture test has a higher sensitivity than sputum smear test, it takes a long time and has high demands on diagnostic laboratories. Many countries with high burdens of tuberculosis cannot meet the laboratory conditions required for the method. Immunoassay technologies may have a great application value in the diagnosis of tuberculosis, especially after the realization of rapid diagnosis and bedside diagnosis.

IFN-γ release assays (IGRAs) are a widely used immunodiagnostic method for TB. It has been proved to have an application value in the detection of M. tuberculosis infection, and is significantly superior to TST (tuberculin skin test). However, neither of IGRAs and TST can distinguish latent tuberculosis infection (LTBI) from active tuberculosis. In countries with high burdens of tuberculosis, the application of IGRAs is restricted due to a high proportion of latently infected people, and IGRAs can only be used as an auxiliary diagnostic tool. The tuberculosis diagnosis commonly used in clinic still mainly depends on clinical symptoms, imaging diagnosis and etiological diagnosis. If tuberculosis can be diagnosed accurately and quickly in the early stage and appropriate chemotherapy is performed, it will be of great value to reduction of the harm of TB patients and control of TB infection.

Therefore, there is need in this field to develop a method with high accuracy and specificity for diagnosing active tuberculosis simply, conveniently and quickly.

Contents of Invention

In the invention, unless otherwise specified, the scientific and technical terms used herein have the meanings as generally understood by a person skilled in the art. Moreover, the laboratory operations of cell culture, biochemistry, nucleic acid chemistry and immunology used herein are the routine operations widely used in the corresponding fields. Meanwhile, in order to better understand the invention, the definitions and explanations of the relevant terms are provided as follows.

As used herein, the term “RV0183” or “RV0183 protein” refers to a protein naturally occurring in M. tuberculosis, which belongs to monoacylglycerol lipase (MAGL) or lysophospholipase. The sequence of RV0183 protein is well known in the art, and can be found in various public databases (for example, Accession No. CP009480.1, AIR12906.1, CCP42909.1, NP_214697.2, or WP_003401112.1 in NCBI database).

In the invention, when the amino acid sequence of RV0183 is mentioned, it is described by reference to the sequence as set forth in SEQ ID NO: 1. However, a person skilled in the art understands that M. tuberculosis includes not only the standard strain H37Rv, but also various isolated strains, and there might be difference in the amino acid sequence of RV0183 protein among various isolated strains. Furthermore, a person skilled in the art understands that although there might be difference in sequence, RV0183 proteins from different isolated strains of M. tuberculosis, have a very high identity in amino acid sequence, and have substantively the same biological function. In addition, a person skilled in the art understands that in the amino acid sequence of RV0183 protein, mutation or variation (including, but not limited to, substitution, deletion and/or addition) may occur naturally or be introduced artificially, without affecting the biological function thereof. Therefore, in the invention, the term “RV0183” includes not only the protein set forth in SEQ ID NO: 1, but also the RV0183 protein from various isolated strains of M. tuberculosis (for example, RV0183 protein set forth in BAL64025.1, AOE34503.1, EGE52872.1, or CDM08372.1), and the natural or artificial variants thereof. Moreover, when a sequence fragment of RV0183 protein is described, it includes not only a sequence fragment of SEQ ID NO: 1, but also the corresponding sequence fragment of the RV0183 protein from various isolated strains of M. tuberculosis, and the corresponding sequence fragment of the natural or artificial variants of SEQ ID NO: 1. Therefore, the expression “amino acid residues from positions 1 to 20 of RV0183 protein” includes the amino acid residues from positions 1 to 20 of SEQ ID NO: 1, the corresponding fragment of the RV0183 protein from various isolated strains of M. tuberculosis, and the corresponding fragment of the natural or artificial variants of RV0183 protein. In the invention, the expression “corresponding fragment” refers to the fragment that is located in equivalent positions of sequences when the sequences are subjected to optimized alignment, i.e. the sequences are aligned to obtain a highest percentage of identity.

In the invention, the term “RV0183” or “RV0183 protein” is not restricted by any particular method for synthesis of protein, and can be produced by conventional techniques known by a person skilled in the art, for example, recombinant DNA technique or chemical synthesis technique.

As used herein, the term “PlcD” or “PlcD protein” refers to a protein naturally occurring in M. tuberculosis, which belongs to phospholipase C family. The sequence of PlcD protein is well known in the art, see, for example, Andersen ST, et al., Nature, 1998. 393(6685): 537-44 (which is incorporated herein by reference), and Accession No. CCP44521.1 in NCBI database.

In the invention, when the amino acid sequence of PlcD is mentioned, it is described by reference to the sequence as set forth in SEQ ID NO: 3. However, a person skilled in the art understands that M. tuberculosis includes not only the standard strain H37Rv, but also various isolated strains, and there might be difference in the amino acid sequence of PlcD protein among various isolated strains. Furthermore, a person skilled in the art understands that although there might be difference in sequence, PlcD proteins from different isolated strains of M. tuberculosis, have a very high identity in amino acid sequence, and have substantively the same biological function. In addition, a person skilled in the art understands that in the amino acid sequence of PlcD protein, mutation or variation (including, but not limited to, substitution, deletion and/or addition) may occur naturally or be introduced artificially, without affecting the biological function thereof. Therefore, in the invention, the term “PlcD” includes not only the protein set forth in SEQ ID NO: 3, but also the PlcD protein from various isolated strains of M. tuberculosis, and its natural or artificial variants. Moreover, when a sequence fragment of PlcD protein is described, it includes not only a sequence fragment of SEQ ID NO: 3, but also the corresponding sequence fragment of the PlcD protein from various isolated strains of M. tuberculosis, and the corresponding sequence fragment of the natural or artificial variants of SEQ ID NO: 3. In the invention, the expression “corresponding fragment” refers to the fragment that is located in equivalent positions of sequences when the sequences are subjected to optimized alignment, i.e. the sequences are aligned to obtain a highest percentage of identity.

In the invention, the term “PlcD” or “PlcD protein” is not restricted by any particular method for synthesis of protein, and can be produced by conventional techniques known by a person skilled in the art, for example, recombinant DNA technique or chemical synthesis technique.

As used herein, the expression “antigenic fragment of RV0183 or PlcD” refers to the amino acid sequence fragment (i.e. polypeptide) obtained by the truncation of RV0183 or PlcD protein, which has the same biological activity as the corresponding full-length protein, i.e. capability of stimulating peripheral blood from aTB patients to generate IL-6, so as to distinguish an aTB population from a non-aTB population (for example, individuals with latent tuberculosis infection, patients with old tuberculosis or individuals without tuberculosis infection). For example, in the invention, the amino acid sequence fragments set forth in SEQ ID NOs: 5-25 are antigenic fragments of RV0183. In the invention, the antigenic fragments are not restricted by any particular method for synthesis of polypeptide, and can be produced by conventional techniques known by a person skilled in the art, for example, recombinant DNA technique or chemical synthesis technique.

In the invention, RV0183, PlcD, or antigenic fragments thereof (i.e. polypeptides) can be obtained by recombinant DNA technique, for example, obtained from polynucleotides encoding these proteins or polypeptides by using a cell-free expression system (a cell-free expression system includes, for example, a reticulocyte lysate-based expression system, a wheat germ extract-based expression system, and an E. coli extract-based expression system); or obtained from polynucleotides encoding these proteins or polypeptides by using an in vivo expression system (for example, an E. coli prokaryotic expression system, a yeast eukaryotic expression system). As an alternative, RV0183, PlcD, or antigenic fragments thereof (i.e. polypeptides) can be produced by chemical synthesis. Methods for chemical total synthesis of proteins or polypeptides are well known in the art (see, for example, Raibaut L, et al., Top Curr Chem. 2015; 363: 103-54; Thapa P, et al. Molecules. 2014; 19(9):14461-83; Dawson P E, et al., Science, 1994; 266(5186): 776-9; and Wang P, et al., Tetrahedron Lett, 1998, 39(47): 88711-14; which is incorporated herein by reference), and including, but not limited to: Solid Phase Peptide Synthesis (SPPS) or Liquid Phase Segment Ligation Synthesis (for example, Native Chemical Ligation, NCL, Azide method, Transfer Active Ester Condensation (TAEC)).

As used herein, the expression “antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19” refers to the combination of the antigenic fragment having an amino acid sequence set forth in SEQ ID NO: 13, the antigenic fragment having an amino acid sequence set forth in SEQ ID NO: 14, and the antigenic fragment having an amino acid sequence set forth in SEQ ID NO: 19. Other similar expressions have similar meanings.

As used herein, the term “a reagent capable of detecting IL-6” refers to a substance that can specifically bind to IL-6. Such substances are well known in the art, or can be prepared by methods well known in the art, for example, an antibody, a targeting polypeptide or an aptamer, etc. In general, it is particularly preferred that such reagents can determine the level of IL-6 in a sample by immunologic assay. The use of immunologic assay is particularly advantageous as it utilizes the specific interaction/binding affinity between an antigen and an antibody. Therefore, as long as a reagent retains the reactivity of specifically binding to IL-6, the reagent can determine the level of IL-6 in a sample by immunologic assay (that is, the reagent can be used as a reagent capable of detecting IL-6). Various reagents, which retain the reactivity of specifically binding to IL-6, can be readily envisaged and can be obtained easily by a person skilled in the art, and including, but not limited to, an anti-IL-6 antibody or an antigen binding fragment thereof, for example, a polyclonal or monoclonal antibody against IL-6, such as an IgG antibody or an IgM antibody. As an alternative, the reagent capable of detecting IL-6 can also be an aptamer that binds to IL-6, including, but not limited to the aptamers described in PCT international application WO02014159669 and “Gupta S, et al., J Biol Chem. 2014; 289(12): 8706-19”, which are incorporated herein by reference.

As used herein, the term “specific binding/specifically bind” refers to the non-random binding reaction between two molecules (i.e. a binding molecule and a target molecule), for example, the reaction between an antibody and an antigen that the antibody is directed to. The binding affinity between two molecules can be described by K_Dvalue. K_Dvalue refers to a dissociation constant, which is a ratio of kd (a dissociation rate of a specific binding molecule-target molecule interaction; also called koff) to ka (an association rate of a specific binding molecule-target molecule interaction; also called kon); or refers to kd/ka which is expressed as molar concentration (M). The lower the K_Dvalue is, the tighter the binding of the two molecules is, and the higher the affinity is. In some embodiments, an antibody specifically binding to a certain antigen (or an antibody specific for a certain antigen) means that the antibody binds to the antigen with an affinity (K_D) of below about 10⁻⁵M, for example, below about 10⁻⁶M, 10⁻⁷M, 10⁻⁸M, 10⁻⁹M or 10⁻¹⁰M or less. K_Dvalue can be determined by methods well known in the art, for example, by surface plasmon resonance (SPR) in BIACORE instrument.

As used herein, the term “immunologic assay” refers to an assay utilizing the specific interaction/binding affinity between an antigen and an antibody, which is generally used to determine the presence or level of a specific antigen or antibody in a sample. Such immunologic assays are well known for a person skilled in the art, including, but not limited to ELISA assay, Elispot assay, Western blot, surface plasmon resonance, and the like. The detailed description of immunologic assay, can be found in, for example, Fundamental Immunology, Ch. 7 Paul, W., ed., the 2^ndedition, Raven Press, N.Y. (1989).

As used herein, the term “antibody” generally refers to an immunoglobulin molecule consisting of two pairs of polypeptide chains (each has a light (L) chain and a heavy (H) chain). Light chain of an antibody may be classified into κ and λ light chain. Heavy chain may be classified into μ, δ, γ, α and ε, which define isotypes of an antibody as IgM, IgD, IgG; IgA and IgE, respectively. In a light chain and a heavy chain, a variable region is linked to a constant region via a “J” region of about 12 or more amino acids, and a heavy chain further comprises a “D” region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). A heavy chain constant region consists of 3 domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). A light chain constant region consists of a domain CL. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including various cells (e.g., effector cells) of an immune system and the first component (C1q) of classical complement system. VH and VL regions can also be divided into hypervariable regions (called complementary determining regions (CDR)), which are interspaced by relatively conservative regions (called framework region (FR)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N-terminal to C-terminal. The variable region (VH and VL) of each heavy/light chain pair forms an antigen binding site, respectively. Distribution of amino acids in each region or domain follows the definition in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. The term “antibody” is not restricted by any specific method for producing antibodies. For example, antibodies include particularly, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies may be of different antibody isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

As used herein, “an antigen binding fragment” of an antibody refers to one or more parts of a full-length antibody, which retain the ability of binding to the same antigen (e.g. IL-6) that a full-length antibody binds to, and compete with a full-length antibody for specifically binding to the antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., the second edition, Raven Press, N.Y. (1989), which is incorporated herein by reference for all purposes. Antigen binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Under some conditions, antigen binding fragments include Fab, Fab′, F(ab′)₂, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g. scFv), chimeric antibody, diabody and such polypeptides that comprise at least one part of an antibody sufficient to confer the specific antigen binding ability on the polypeptides.

As used herein, the term “Aptamer” refers to a single-stranded oligonucleotide that can bind to a target protein or other biological target molecule with high affinity and high specificity, which can be folded into a thermodynamically stable 3D structure such as Stem-Loop, Hairpin, Pseudoknot or G-tetramer, and specifically bind to a target protein or other biological target molecule, for example, via formation of complementary structure, base stacking force, van der Waals force, hydrogen bond or electrostatic interaction. An aptamer may be DNA or RNA, and may also include nucleic acid analogs (for example, locked nucleic acid (LNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA) or threose nucleic acid (TNA)). Methods for obtaining an aptamer binding to a specific target protein are well known in the art, for example, SELEX (Systematic evolution of ligands by exponential enrichment) screening technology.

As used herein, the term “a targeting polypeptide” refers to a polypeptide molecule that can specifically bind to a target protein. In the invention, the targeting polypeptide may include naturally occurring amino acids, synthetic amino acids, or amino acid mimetics that work in a similar manner as naturally occurring amino acids do. Naturally occurring amino acids are amino acids that are encoded by genetic codes and those that are modified later, for example, hydroxyproline, y-hydroxyglutamate, 0-phospho-serine, phosphothreonine or phosphotyrosine. In the invention, the “specificity” between a targeting polypeptide and its target protein can be determined based on affinity, and the affinity can be described by a dissociation equilibrium constant (i.e. K_Dvalue) between a targeting polypeptide and a target protein binding thereto. The lower the K_Dvalue is, the tighter the binding between a targeting polypeptide and a target protein binding thereto is. It is well known in the art that a K_Dvalue of above about 10⁻³M is generally regarded as no binding or a non-specific binding. Based on a particular target protein, a targeting polypeptide specifically binding to the target protein can be obtained by methods known by a person skilled in the art, for example, screening by phage display technology or protein microarray technology.

As used herein, the term “a detectable label” refers to any composition that can be detected by fluorescent, spectral, photochemical, biochemical, immunological, electrical, optical or chemical means. In the invention, it is particularly preferred that such labels can be applied to immunologic assay (for example, enzyme linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art, including, but not limited to, enzymes (for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclide (for example, ³H, ¹²⁵, ³⁵S, ¹⁴C or ³²P), fluorescent dyes (for example, fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dot or cyanine dye derivatives (e.g. Cy7, Alexa 750)), acridinium ester compounds, magnetic beads (e.g. Dynabeads®), calorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g. streptavidin) with which said labels are modified. The patents, which teach the use of the labels, include, but are not limited to U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all of which are incorporated herein by reference). The labels covered in the invention can be detected by methods known in the art. For example, radiolabels can be detected using photographic film or scintillation counters, and fluorescent labels can be detected by determining the emitted light using a photodetector. Enzyme labels are generally detected by providing a substrate for the enzyme and detecting the reaction product resulted from the reaction between the enzyme and the substrate, and calorimetric labels are detected by simple visualization of the stained labels.

As used herein, the term “a specific stimulating agent” refers to a substance that can only stimulate PBMC from aTB patients to generate IL-6, but cannot stimulate PBMC from a non-aTB population (for example, individuals with latent tuberculosis infection (LTBI), patients with old tuberculosis or individuals without tuberculosis infection (tested negative in IGRA)) to generate IL-6; or refers to such a substance that stimulates PBMC from aTB patients to generate IL-6 in an amount significantly higher than PBMC from non-aTB populations (for example, individuals with latent tuberculosis infection (LTBI), patients with old tuberculosis or individuals without tuberculosis infection (tested negative in IGRA)). The specific stimulating agent according to the invention includes RV0183, PlcD, or antigenic fragments thereof. In the preferred embodiments of the invention, the specific stimulating agent contains no or almost no endotoxin. Methods for removing endotoxin from said specific stimulating agent are well known in the art, for example, ion exchange chromatography, affinity chromatography or extraction.

As used herein, the term “a non-specific stimulating agent” refers to a substance that can stimulate most or all of lymphocytes, without being restricted by TCR or BCR specificity. Activated lymphocytes can secrete a lot of cytokines (e.g. IL-6). Substances that can be used as non-specific stimulating agents, are well known in the art, including, but not limited to mitogen, phytohemagglutinin (PHA), Concanavalin A (ConA), pokeweed mitogen (PWM), lipopolysaccharide (LPS), or staphylococcal protein A (SPA). In some embodiments, the non-specific stimulating agent may be comprised in a culture medium (for example, a cell culture medium, such as RPMI-1640 medium and DMEM medium).

As used herein, the term “a statistical analysis value” refers to a value obtained by statistical analysis of the results obtained by various detection methods. Various statistical analysis methods are well known in the art (see, for example, PCT international application WO2009064901), and include, but are not limited to Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM) of the detection results. In general, it is particularly preferred that the statistical analysis value is a value obtained by statistical analysis using Logistic regression model. Logistic regression model is particularly described in, for example, “Hu Chunyan. Serum level of four tumor markers in women with ovarian cancer [D]. Guangzhou: Sun Yat-sen University, 2008: 1-39”.

As used herein, the term “a reference value” (also called the optimal diagnostic critical value) refers to a value that can reflect the status of a non-aTB population. In the invention, a reference value includes, for example, a normal value or range determined depending on a difference value in the level of IL-6 of samples from a non-aTB population (for example, individuals with latent tuberculosis infection (LTBI), patients with old tuberculosis or individuals without tuberculosis infection (tested negative in IGRA)) before and after the stimulation with a specific stimulating agent; and a value (a statistical analysis value) obtained by statistical analysis of the determined values (for example, the difference value in the level of IL-6 as described above) obtained from samples of a non-aTB population. Methods for determining an optimal diagnostic critical value are well known in the art, including, but not limited to Receiver Operating Characteristic (ROC) curve analysis, which is particularly described in, for example, “Habibzadeh F, et al., Biochem Med (Zagreb). 2016; 26(3):297-307” and “Chen Weizhong et al., Selection of optimal operating point for ROC curve [J]. Chinese Journal of Health Statistics, 2006, 23:157-158”.

As used herein, the term “Peripheral blood mononuclear cell (PBMC)” is a generic name for cells having a single nucleus in peripheral blood, including, but not limited to lymphocytes (T cells, B cells, NK cells) and monocytes or dendritic cells. Methods for obtaining PBMC from peripheral blood are well known in the art, including, but not limited to Ficoll method or Percoll method.

As used herein, the term “peripheral blood buffy coat” refers to a component formed after natural sedimentation, centrifugation or density gradient centrifugation of peripheral anticoagulant blood, and consisting mainly of white blood cells (including peripheral blood mononuclear cells) and platelets. After centrifugation of anticoagulant blood, there are an upper layer of plasma, a bottom layer of red blood cells, and a white thin layer (called buffy coat) between them, which accounts for about 1% of total blood by volume.

As used herein, the term “subject” includes, but is not limited to, various animal, particularly mammal, for example, human.

As used herein, the term “an anticoagulant” refers to an agent or a substance that can prevent blood coagulation. Such substances are well known in the art, including, but not limited to heparin, EDTA, oxalates (e.g. sodium oxalate, potassium oxalate, ammonium oxalate), and sodium citrate.

In the invention, the term “a diluent” is preferably an electrolyte solution that can retain the cell osmotic pressure, and if necessary, can also act to retain the physiological pH value. Such solutions are well known in the art, including, but not limited to Alsever's solution, Earle's Balanced Salt Solution (EBSS), Gey's Balanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS), Phosphate Buffered Saline (PBS), Dulbecco's Phosphate Buffered Saline (DPBS), Puck's Balanced Salt Solution, Ringer's Balanced Salt Solution (RBSS), Simm's Balanced Salt Solution (SBSS), TRIS-buffered saline (TBS), Tyrode's Balanced Salt Solution (TBSS), physiological saline or Ringer's Solution.

As used herein, the term “a culture solution” or “a culture medium” refers to nutrients capable of maintaining the viability of cells. Usually, the nutrients contain amino acids, vitamins, carbohydrates, inorganic salts, etc. Such nutrients are well known in the art, including, but not limited to RPMI-1640 medium or DMEM medium. In the invention, the purpose of adding a culture solution or a culture medium into a sample from a subject is to maintain the viability of cells, especially PBMC in the sample, during the stimulation with a stimulating agent. Methods for maintaining the viability of cells in a blood component are well known in the art, which can be selected by a person skilled in the art depending on practical needs. In some embodiments, when the sample is whole blood, a culture solution can be added, wherein the culture solution may be, for example, phosphate buffer or physiological saline, to which an appropriate amount of glucose, sodium chloride, potassium chloride, and the like are added. In some embodiments, the culture solution is phosphate buffer, to which an appropriate amount of glucose and potassium chloride are added. In some embodiments, when the sample is peripheral blood mononuclear cell (PBMC), peripheral blood buffy coat or other blood components containing PBMC, a culture medium, for example, a cell culture medium (e.g. a cell culture medium suitable for maintaining the viability of blood cells, especially PBMC, such as RPMI-1640 medium or DMEM medium), can be added.

As used herein, the term “active tuberculosis (aTB)” means that the focus of tuberculosis is active, for example, tested positive in sputum smear test, or accompanied by related symptoms such as low fever, cough, weight loss, fatigue, poor appetite, and so on. In the invention, tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis. The extrapulmonary tuberculosis includes, for example, lymphatic tuberculosis, tuberculous meningitis, tuberculous peritonitis, intestinal tuberculosis, renal tuberculosis, epididymal tuberculosis, tuberculosis of female reproductive system (including fallopian tube, endometrial, ovarian tuberculosis), bone and joint tuberculosis, etc.

The inventors of the present application screened a large number of antigens of RD region of M. tuberculosis, and surprisingly found that RV0183, PlcD, or antigenic fragments thereof can stimulate peripheral blood from aTB patients to generate a large amount of IL-6, and therefore can distinguish an aTB population from a non-aTB population (for example, individuals with latent tuberculosis infection (LTBI), patients with old tuberculosis or individuals without tuberculosis infection). However, before the present application, the IGRAs and TST commonly used in the art cannot distinguish latent tuberculosis infection (LTBI) from active tuberculosis. Based on this discovery, the inventors develop a new method for diagnosing active tuberculosis.

Therefore, in one aspect, the invention provides a kit comprising one or more of RV0183, PlcD, or antigenic fragments thereof, and a reagent capable of detecting IL-6.

In some preferred embodiments, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3.

In some preferred embodiments, the kit comprises RV0183 and/or PlcD.

In some preferred embodiments, the kit comprises one or more antigenic fragments of RV0183. In some further preferred embodiments, the antigenic fragments have the amino acid sequences selected from: SEQ ID NOs: 5-25.

In some preferred embodiments, the kit comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19.

Optionally, the kit further comprises a combination of the following antigenic fragments:

1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22,

2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12,

3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or

4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25.

In some preferred embodiments, the kit comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25.

In some preferred embodiments, the reagent capable of detecting IL-6 is a substance that can specifically bind to IL-6, for example, an antibody, a targeting polypeptide or an aptamer.

Optionally, the reagent further comprises a detectable label.

In some preferred embodiments, the reagent determines the level of IL-6 in the sample by immunologic assay. In some further preferred embodiments, the immunologic assay is selected from the group consisting of ELISA assay, Elispot assay, Western blot, and surface plasmon resonance.

In some preferred embodiments, the reagent comprises an anti-IL-6 antibody or an antigen binding fragment thereof. In some further preferred embodiments, the reagent determines the level of IL-6 by ELISA.

In some preferred embodiments, the anti-IL-6 antibody is a monoclonal antibody or a polyclonal antibody. In some further preferred embodiments, the anti-IL-6 antibody is an IgG antibody or an IgM antibody.

In some preferred embodiments, the kit further comprises one or more devices or reagents selected from 1) to 5):

1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube;

2) an anticoagulant, e.g. heparin;

3) a culture solution or a culture medium;

4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A;

5) a diluent, e.g. phosphate buffer or physiological saline.

In some preferred embodiments, the kit is useful for diagnosing active tuberculosis, determining the therapeutic effect of a therapy on active tuberculosis or screening a candidate drug capable of treating active tuberculosis.

In another aspect, the invention provides use of a specific stimulating agent in the manufacture of a kit for diagnosing active tuberculosis; wherein, the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof.

In some preferred embodiments, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3.

In some preferred embodiments, the specific stimulating agent is selected from the group consisting of RV0183, PlcD, and a combination thereof.

In some preferred embodiments, the specific stimulating agent is selected from one or more antigenic fragments of RV0183. In some further preferred embodiments, the antigenic fragments have the amino acid sequences selected from: SEQ ID NOs: 5-25.

In some preferred embodiments, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 13, 14 and 19.

Optionally, the specific stimulating agent further comprises a combination of the following antigenic fragments:

1) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 11 and 22,

2) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 7-8 and 11-12,

3) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-7, 11-12, 22 and 24, or

4) antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5, 8-10, 12, 15 and 22-25.

In some preferred embodiments, the specific stimulating agent comprises antigenic fragments having the amino acid sequences set forth in SEQ ID NOs: 5-25.

In some preferred embodiments, the kit comprises a reagent capable of detecting IL-6, such as an antibody, a targeting polypeptide or an aptamer that can specifically bind to IL-6.

Optionally, the reagent further comprises a detectable label.

In some preferred embodiments, the kit further comprises one or more devices or reagents selected from 1) to 5):

1) a blood collection device, e.g. a pyrogen-free vacuum blood collection tube;

2) an anticoagulant, e.g. heparin;

3) a culture solution or a culture medium;

4) a non-specific stimulating agent, e.g. phytohemagglutinin or Concanavalin A;

5) a diluent, e.g. phosphate buffer or physiological saline.

In some preferred embodiments, the kit diagnoses whether the subject is suffering from active tuberculosis, by a method comprising the following steps:

(1) stimulating at least one sample from the subject with a specific stimulating agent, and using the at least one sample as a test sample, and using a non-stimulated sample from the subject, as a negative control sample, wherein the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof;

(2) determining the level of IL-6 in each of the samples in the step (1) by using a reagent capable of detecting IL-6, and calculating a difference value in the level of IL-6 between the test sample and the negative control sample; and

(3) comparing the difference value with a reference value, or subjecting the difference value to statistical analysis so as to obtain a statistical analysis value, and comparing the statistical analysis value with a reference value, and determining whether the subject is suffering from active tuberculosis;

wherein, the sample comprises peripheral blood mononuclear cell (PBMC), for example, whole blood (e.g. anticoagulated whole blood), peripheral blood mononuclear cell (PBMC), or peripheral blood buffy coat, and optionally an additional component, for example, an anticoagulant, a diluent, etc.

In some preferred embodiments, when the difference value or the statistical analysis value obtained from the difference value is greater than the reference value, it indicates that the subject providing the sample is suffering from active tuberculosis; when the difference value or the statistical analysis value obtained from the difference value is not greater than the reference value, it indicates that the subject providing the sample is not suffering from active tuberculosis.

In some preferred embodiments, in the step (3), the difference value is subjected to statistical analysis by using a statistical model selected from the group consisting of: Linear combination, Linear regression model, Logistic regression model, Linear discrimination analysis (LDA) model, The nearest neighbor model and Prediction Analysis of Microarrays (PAM). In some further preferred embodiments, in the step (3), statistical analysis of the difference value is carried out by using Logistic regression model.

In some preferred embodiments, in the step (1), stimulating one or more samples from the subject with at least two specific stimulating agents together or separately, and using the one or more samples as the test sample, wherein the specific stimulating agents are each independently selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof. In some further preferred embodiments, in the step (1), stimulating at least two samples with RV0183 and PlcD separately, and using the at least two samples as the test sample. Or, in some preferred embodiments, in the step (1), stimulating at least one sample with one or more antigenic fragments of RV0183 together, and using the at least one sample as the test sample.

In some preferred embodiments, in the step (1), the specific stimulating agent is contained in a culture medium, for example, a cell culture medium, such as RPMI-1640 medium or DMEM medium, and then is used to stimulate the sample from the subject, so as to generate a test sample.

In some preferred embodiments, the step (1) further comprises: stimulating at least one sample with a non-specific stimulating agent, and using the at least one sample as a positive control sample. In some further preferred embodiments, the non-specific stimulating agent includes phytohemagglutinin or Concanavalin A.

In some preferred embodiments, in the step (1), a sample from the subject is incubated or diluted with a culture medium that contains neither the specific stimulating agent nor a non-specific stimulating agent (for example, a cell culture medium, such as RPMI-1640 medium or DMEM medium), so as to generate a negative control sample.

In some preferred embodiments, prior to the step (1), the method further comprises one or more of the following steps: (a) obtaining the sample from the subject by using a blood collection device; (b) treating the blood collection device or the sample from the subject with an anticoagulant; (c) treating the sample from the subject with a culture solution or a culture medium; and, (d) diluting the sample from the subject with a diluent.

In some preferred embodiments, in the step (1), the sample from the subject is stimulated with the stimulating agent at a temperature (for example, 36-38° C., e.g. about 37° C.) at which cells (such as PBMC) have a high activity.

In some preferred embodiments, in the step (1), the sample from the subject is stimulated with the stimulating agent for at least 12 h, for example, 15-24 h, e.g. 20-24 h.

In another aspect, the invention provides use of a specific stimulating agent in the manufacture of a kit for determining the therapeutic effect of a therapy on active tuberculosis; wherein, the specific stimulating agent is one or more selected from the group consisting of RV0183, PlcD, and antigenic fragments thereof.

In some preferred embodiments, the RV0183 has an amino acid sequence set forth in SEQ ID NO: 1; and/or, the PlcD has an amino acid sequence set forth in SEQ ID NO: 3.

In some preferred embodiments, the specific stimulating agent is selected from the group consisting of RV0183, PlcD, and a combination thereof.

In some preferred embodiments, the specific stimulating agent is selected from one or more antigenic fragments of RV0183. In some further preferred embodiments, the antigenic fragments have amino acid sequences selected from: SEQ ID NOs: 5-25.