Recombinant antigen for detecting mycobacterium tuberculosis-specific immune response, and use of recombinant antigen

Abstract

Provided are a new stimulus for a Mycobacterium tuberculosis-specific immune response, a composition, a kit, and a use. The stimulus comprises a cell-penetrating peptide and a Mycobacterium tuberculosis-specific protein, enables in vitro detection of a Mycobacterium tuberculosis-specific T cell immune response in a sample, and can improve the sensitivity and / or specificity of Mycobacterium tuberculosis detection.

Description

A recombinant antigen for detecting Mycobacterium tuberculosis-specific immune responses and its uses

[0001] Cross-referencing

[0002] This application is based on and claims priority to Chinese Patent Application No. 202510209129.1, filed on February 25, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to a novel stimulant for Mycobacterium tuberculosis-specific immune responses, comprising a cell-penetrating peptide, a Mycobacterium tuberculosis-specific protein, such as the eTAT-E1C0 recombinant antigen, and also relates to compositions comprising said recombinant antigen, kits, and the use of said recombinant antigen. The invention further relates to a method for using said recombinant antigen to detect Mycobacterium tuberculosis-specific T-cell immune responses in a sample in vitro. Background Technology

[0004] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is one of the most deadly human diseases worldwide. TB is primarily transmitted through the air; when an infected person coughs, sneezes, or talks, they release droplets containing Mtbacilli, which can inhale and infect others. People with compromised immune systems, those living in areas with high TB ​​prevalence, or those living in unfavorable environmental conditions are at high risk of TB infection. TB has existed for tens of thousands of years and is one of the top ten causes of death globally, particularly severe in developing countries and resource-scarce regions.

[0005] Tuberculosis (TB) has a serious impact on public health, affecting approximately one-quarter of the global population. The development of TB after infection largely depends on the body's immune status and its interaction with TB. Most people have a healthy immune system that limits the proliferation and spread of TB, but it is difficult to completely eradicate it. In this state, TB exists in a low-metabolic, inactive state, thus not causing obvious symptoms, having no clinical manifestations, and being non-infectious; this state is called latent infection. This balance can be maintained for many years, even a lifetime. If the host's immune system remains strong, TB will remain latent. When the host's immune system is weakened for some reason (such as HIV infection, old age, diabetes, or immunosuppressive therapy), this balance is disrupted, and the latent TB may be reactivated, breaching the body's immune barrier and causing the infected person to progress to active TB. Patients with active tuberculosis typically experience symptoms such as cough, fever, fatigue, loss of appetite, weight loss, and hemoptysis. It is contagious and commonly known as "consumption" or "white plague." The lifetime risk of latent tuberculosis infection in individuals is 5%-15%, with an even higher risk in immunocompromised individuals.

[0006] Tuberculosis (TB) is the second leading cause of death from a single infectious disease after COVID-19, with over 10 million new cases and over 1 million deaths annually. TB is also a common opportunistic infection in HIV / AIDS patients, leading to weakened immune function, increased risk of death, and severely impacting their quality of life; it is a significant risk factor for death in HIV / AIDS patients. Latent TB infection is a precursor to active TB and continuously leads to new TB cases. Diagnosing and providing preventative treatment for latent TB infections is crucial. However, latent TB infections often lack typical clinical features, and diagnosis relies on medical testing. Currently, there is no gold standard for diagnosing latent TB infection; commonly used methods include the tuberculin skin test (TST) and the interferon-gamma release assay (IGRA), both of which indirectly detect Mtb infection by measuring memory T cell responses. The delayed-type hypersensitivity (TST) skin test is based on the principle of delayed-type hypersensitivity. Although TST has advantages such as being rapid, simple, and low-cost, its specificity is low. Exposure to non-tuberculous mycobacteria or BCG vaccination can lead to false positives on TST. Compared to TST, the IGRA has better diagnostic performance, is not affected by BCG vaccination, and is almost unaffected by non-tuberculous mycobacteria. The principle of IGRA is as follows: After infection with Mycobacterium tuberculosis, the body produces memory T cells against the bacteria. When stimulated again by Mycobacterium tuberculosis-specific antigens, these memory T cells are activated and secrete IFN-γ. The secretion of IFN-γ can be used to determine whether the subject is infected with tuberculosis.

[0007] IGRAs can be divided into two categories based on their detection methods: enzyme-linked immunospot assay (ELISpot) and enzyme-linked immunosorbent assay (ELISA). ELISpot requires the separation of peripheral blood mononuclear cells (PBMCs) and the counting of IFN-γ-secreting T cells, making the procedure relatively cumbersome and time-consuming. ELISA, on the other hand, directly measures the IFN-γ secretion concentration in whole blood, making it easier to perform and widely adopted. Existing research indicates that there is no significant difference in detection sensitivity and specificity between ELISpot and ELISA-based IGRAs. Currently, the stimulants used in IGRA are generally ESAT6 and CFP10, or a fusion protein of the two. These are antigens secreted by Mycobacterium tuberculosis that can induce strong cellular immunity. They are proteins encoded by the region of differences (RD) of Mycobacterium tuberculosis (Andersen, P. et al., Lancet, 2000. 356(9235): p.1099-104), which are absent in BCG and environmental mycobacteria, thus IGRA has high specificity. However, the sensitivity of IGRA in HIV-infected individuals is greatly affected by the significant reduction in the number of CD4+ T cells. To improve the diagnostic performance of IGRA in HIV-infected individuals and other populations, finding antigens that can induce a stronger T-cell immune response is a key focus of IGRA improvement.

[0008] The selection of tuberculosis-specific antigens is crucial in the entire experimental system. Currently, ESAT6 and CFP10 have achieved great success in the diagnosis of tuberculosis infection. Other supplementary antigens can be sought while retaining these, and studies can be conducted to verify whether the selected antigens can improve diagnostic sensitivity. To improve the detection performance of the IGRA reagent in immunocompromised individuals such as those infected with HIV, the inventors have provided a novel recombinant antigen, eTAT-E1C0, which enhances the stimulation of T cells, particularly CD8+ T cells, thereby optimizing the detection performance of the IGRA kit. Summary of the Invention

[0009] In a first aspect, the present invention provides a recombinant antigen comprising a cell-penetrating peptide, a Mycobacterium tuberculosis-specific protein, and optionally a linker and optionally a tag.

[0010] In some embodiments, the cell-penetrating peptide is selected from penetratin, Tat-derived peptides (e.g., eTAT, Tat(48-60) or Tat(47-57)), Rev(34-50), VP22, transport peptide, Pep-1, Pep-7, and any combination thereof.

[0011] In some preferred embodiments, the cell-penetrating peptide is selected from Tat-derived peptides, such as eTAT.

[0012] In some embodiments, the eTAT comprises an amino acid sequence as shown in SEQ ID NO: 2; or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence shown in SEQ ID NO: 2.

[0013] In some embodiments, the eTAT comprises or is an amino acid sequence as shown in SEQ ID NO: 11.

[0014] In some embodiments, the eTAT comprises or is an amino acid sequence as shown in SEQ ID NO: 2.

[0015] In some embodiments, the Mycobacterium tuberculosis-specific protein is selected from E1C0 protein, ESAT-6 (Rv3875), CFP-10 (Rv3874), or any combination thereof.

[0016] In some preferred embodiments, the Mycobacterium tuberculosis-specific protein is the E1CO protein.

[0017] In some embodiments, the E1C0 protein comprises an amino acid sequence as shown in SEQ ID NO: 4; or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence shown in SEQ ID NO: 4.

[0018] In some embodiments, the E1C0 protein comprises or has an amino acid sequence as shown in SEQ ID NO: 4.

[0019] In some embodiments, the cell-penetrating peptide and Mycobacterium tuberculosis-specific protein in the recombinant antigen are linked by direct fusion, through a linker, or by chemical synthesis.

[0020] In some embodiments, the adapter comprises an amino acid sequence as shown in any one of SEQ ID NO:6-10.

[0021] In some embodiments, the recombinant antigen comprises an amino acid sequence as shown in SEQ ID NO: 1; or an amino acid sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the amino acid sequence shown in SEQ ID NO: 1.

[0022] In some embodiments, the recombinant antigen comprises or is an amino acid sequence as shown in SEQ ID NO: 1.

[0023] Secondly, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant antigen.

[0024] In some embodiments, the nucleic acid molecule encoding the E1C0 protein comprises a nucleotide sequence as shown in SEQ ID NO:5, or a corresponding degenerate nucleotide sequence encoding the same amino acid as SEQ ID NO:5; or a nucleotide sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the nucleotide sequence shown in SEQ ID NO:5.

[0025] In some embodiments, the nucleic acid molecule encoding the eTAT polypeptide comprises a nucleotide sequence as shown in SEQ ID NO:3, or a corresponding degenerate nucleotide sequence encoding the same amino acid as SEQ ID NO:3; or a nucleotide sequence having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with the nucleotide sequence shown in SEQ ID NO:3.

[0026] Thirdly, the present invention provides a carrier comprising the isolated nucleic acid molecules described above.

[0027] Fourthly, the present invention provides a host cell comprising the isolated nucleic acid molecule or the vector.

[0028] Fifthly, the present invention provides a composition comprising the recombinant antigen described in the first aspect, optionally further comprising a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutically acceptable carrier is a buffer.

[0029] In a sixth aspect, the present invention provides the use of the recombinant antigen described in the first aspect and the composition described in the fifth aspect in the preparation of a kit, the kit being used for in vitro detection of Mycobacterium tuberculosis-specific T-cell immune responses in samples, or for diagnosing whether a subject is infected with Mycobacterium tuberculosis, or for evaluating the efficacy of anti-tuberculosis treatment.

[0030] In a seventh aspect, the present invention provides a kit comprising the recombinant antigen described in the first aspect or the composition described in the fifth aspect.

[0031] In some implementations, the kit is used for: detecting Mycobacterium tuberculosis-specific T-cell immune responses in samples in vitro, diagnosing whether a subject is infected with Mycobacterium tuberculosis, or evaluating the effectiveness of anti-tuberculosis treatment.

[0032] Eighthly, the present invention provides a method for detecting Mycobacterium tuberculosis-specific T-cell immune responses in an in vitro sample, comprising the following steps:

[0033] (1) Stimulate the sample with the recombinant antigen described in the first aspect;

[0034] (2) Detect the level of effector molecules produced in the stimulated sample;

[0035] (3) Determine the infection status of Mycobacterium tuberculosis.

[0036] Compared with the control, the increased levels of the effector molecules in the stimulated sample indicated an increased level of Mycobacterium tuberculosis-specific T-cell immune response in the sample.

[0037] The sample is anticoagulated whole blood or peripheral blood mononuclear cells, preferably, the sample is anticoagulated whole blood;

[0038] The effector molecules include interferon-gamma (IFN-γ) or cells that release IFN-γ;

[0039] Preferably, the method for detecting the level of effector molecules is the interferon-gamma release assay (IGRA).

[0040] In some embodiments, the control is the sample to be tested that has not been stimulated with the recombinant antigen, a sample that has not been infected with Mycobacterium tuberculosis stimulated with the recombinant antigen, and / or the sample to be tested that has been stimulated with the E1CO protein antigen.

[0041] In a ninth aspect, the present invention provides a method for detecting and distinguishing between active and inactive tuberculosis, comprising the following steps: (1) antigen stimulation; (2) detection of effector molecules of the immune response; (3) calculation of the IFN-γ release enhancement value; (4) calculation of the optimal cutoff value for the eTAT-E1C0 group (enhancement value); and (5) determination or diagnosis of active tuberculosis infection, wherein:

[0042] (1) Antigen stimulation: The test sample is stimulated with the recombinant antigen described in the first aspect or the E1C0 protein antigen, respectively, wherein the recombinant antigen is designated as the eTAT-E1C0 group and is used as the experimental group, and the E1C0 protein antigen is used as the control group.

[0043] (2) Detection of effector molecules in immune response: Detect the level of effector molecules such as IFN-γ produced in the test sample after stimulation; IFN-γ release ≥14 pg / mL and ≥N / 4 is considered IGRA positive; where N is the IFN-γ release amount in the background control culture tube;

[0044] (3) Calculate the increase in IFN-γ release: Calculate the difference between the IFN-γ release of the eTAT-E1C0 group and the IFN-γ release of the E1C0 group, i.e., IFN-γ release (eTAT-E1C0 group) - IFN-γ release (E1C0), denoted as eTAT-E1C0 group (increase value).

[0045] (4) Calculate the optimal cutoff value for the eTAT-E1C0 group (boost value): Take test samples with known actual active tuberculosis status, and after processing by the antigen stimulation module and the effector molecule detection module of the immune response mentioned above (1) and (2), calculate and determine the optimal cutoff value for diagnosing active tuberculosis based on the IFN-γ release boost value of the eTAT-E1C0 group determined to be IGRA positive and the actual active tuberculosis status of the test samples; and

[0046] (5) Determine or diagnose active tuberculosis infection status: Determine whether the sample to be tested is infected with active tuberculosis based on the optimal cutoff value of the eTAT-E1C0 group (elevation value);

[0047] Among them, the known real active tuberculosis situation mentioned in (4) refers to the hospital's diagnosis results of active or inactive tuberculosis of the sample to be tested.

[0048] In some implementations, the optimal cutoff value is calculated as follows:

[0049] (i) Import the IFN-γ release enhancement value of the eTAT-E1C0 group of the sample and the hospital diagnosis results (active tuberculosis / inactive tuberculosis) into SPSS software to obtain the cutoff value, sensitivity and specificity of the sample to be tested;

[0050] (ii) Calculate the Youden index, where the Youden index = sensitivity + specificity - 1;

[0051] (iii) The cutoff value with the largest Yoden index is determined as the optimal cutoff value for diagnosing active tuberculosis.

[0052] In some implementations, the active tuberculosis positive determination module determines that the sample is active tuberculosis if the increase in IFN-γ release of the test sample is greater than the optimal cutoff value of the eTAT-E1C0 group (increase value).

[0053] In a tenth aspect, the present invention provides an apparatus for detecting and distinguishing between active and inactive tuberculosis, characterized in that it comprises an antigen stimulation module, an effector molecule detection module for immune response, an IFN-γ release enhancement value calculation module, an optimal cutoff value calculation module for the eTAT-E1C0 group (enhancement value), and an active tuberculosis positive judgment module, wherein:

[0054] (1) The antigen stimulation module: The test sample is stimulated by the recombinant antigen described in the first aspect or the E1C0 protein antigen respectively, wherein the recombinant antigen is referred to as the eTAT-E1C0 group and is used as the experimental group, and the E1C0 protein antigen is used as the control group.

[0055] (2) The effector molecule detection module of the immune response: detects the level of effector molecules such as IFN-γ produced in the test sample after stimulation; IFN-γ release ≥14pg / mL and ≥N / 4 is considered IGRA positive; where N is the IFN-γ release amount of the background control culture tube;

[0056] (3) The IFN-γ release increase value calculation module: calculates the difference between the IFN-γ release amount of the eTAT-E1C0 group and the IFN-γ release amount of the E1C0 group, that is, the IFN-γ release amount (eTAT-E1C0 group) - the IFN-γ release amount (E1C0), which is recorded as the eTAT-E1C0 group (increase value).

[0057] (4) The optimal cutoff value calculation module for the eTAT-E1C0 group (boost value): Take the test sample with known real active tuberculosis status, and after processing by the above (1) antigen stimulation module and (2) effector molecule detection module of immune response, calculate and determine the optimal cutoff value for diagnosing active tuberculosis based on the boost value of IFN-γ release in the eTAT-E1C0 group that is determined to be IGRA positive and the real active tuberculosis status of the test sample.

[0058] (5) Active tuberculosis positive judgment module: Determine whether the sample to be tested is infected with active tuberculosis based on the optimal cutoff value of the eTAT-E1C0 group (increase value);

[0059] Among them, the known real active tuberculosis situation mentioned in (4) refers to the hospital's diagnosis results of active or inactive tuberculosis of the sample to be tested.

[0060] In some implementations, the optimal cutoff value is calculated as follows:

[0061] (i) Import the IFN-γ release enhancement value of the eTAT-E1C0 group of the sample and the hospital diagnosis results (active tuberculosis / inactive tuberculosis) into SPSS software to obtain the cutoff value, sensitivity and specificity of the sample to be tested;

[0062] (ii) Calculate the Youden index, where the Youden index = sensitivity + specificity - 1;

[0063] (iii) The cutoff value with the largest Yoden index is determined as the optimal cutoff value for diagnosing active tuberculosis.

[0064] In some implementations, the active tuberculosis positive determination module determines that the sample is active tuberculosis if the increase in IFN-γ release of the test sample is greater than the optimal cutoff value of the eTAT-E1C0 group (increase value).

[0065] In some implementations, the optimal cutoff value for the eTAT-E1C0 group (boost value) is 76.950 pg / ml, with a fluctuation range of approximately ±10, for example, 67 pg / ml, 68 pg / ml, 69 pg / ml, 70 pg / ml, 71 pg / ml, 72 pg / ml, 73 pg / ml, 74 pg / ml, 75 pg / ml, 76 pg / ml, 77 pg / ml, 78 pg / ml, 79 pg / ml, 80 pg / ml, 81 pg / ml, 82 pg / ml, 83 pg / ml, 84 pg / ml, 85 pg / ml, 86 pg / ml, and 87 pg / ml. In some implementations, the optimal cutoff value for the eTAT-E1C0 group (boost value) is 76.950 pg / ml.

[0066] In some embodiments, compared with the E1C0 antigen alone, the eTAT-E1C0 recombinant antigen of the present invention can increase the IFN-γ release of the test sample by approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 90%, or 100%.

[0067] In some embodiments, the eTAT-E1C0 recombinant antigen of the present invention can improve the sensitivity of Mycobacterium tuberculosis detection compared with the E1C0 antigen alone, said sensitivity can be increased to 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, thereby improving the diagnostic performance of IGRA.

[0068] In some implementations, the optimal cutoff value of the eTAT-E1C0 group (enhancement value) can improve the specificity of differentiating active tuberculosis and has a good indicative effect of active tuberculosis. This specificity can be increased to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0069] In some embodiments, the recombinant antigen of the first aspect, the composition of the fifth aspect, the kit of the seventh aspect, and the antigen stimulation module of the device of the tenth aspect further include an immune adjuvant.

[0070] In some embodiments, the immune adjuvant is selected from one or more of the following: aluminum hydroxide, aluminum phosphate, nano-aluminum, layered bimetallic hydroxide (LDH), nano / mesoporous silica, nanodiamond, MF59, ASO3, ASO4, CpG, liposomes, lipid monophosphate (MPL), Freund's adjuvant, polyinosinic-polycytidylic acid, QS21, or cytokines.

[0071] In some preferred embodiments, the immune adjuvant is CpG.

[0072] In some implementations, the cells that release IFN-γ include, but are not limited to, T cells and T cell-dependent interferon-γ-releasing cells, such as natural killer (NK) cells.

[0073] In some implementations, the sources of the T cells include, but are not limited to, blood and other biological samples containing T cells.

[0074] In some implementations, the T cells include, but are not limited to, CD4+ T cells, CD8+ T cells, and γδ T cells.

[0075] In some implementations, the detection methods include, but are not limited to: ELISPOT, ELISA (enzyme-linked immunosorbent assay), chemiluminescence, flow cytometry, etc.

[0076] In some preferred embodiments, the detection method employs ELISA.

[0077] In some preferred embodiments, the ELISA method involves adsorbing a known antibody onto the surface of a solid-phase support, allowing the enzyme-labeled antigen-antibody reaction to occur on the solid-phase surface, washing away free components in the liquid phase, and measuring the specific antigen.

[0078] In some preferred embodiments, the criteria for determining the infection status of Mycobacterium tuberculosis are as follows: the amount of interferon-gamma released by the peripheral lymphocytes of the subject after in vitro stimulation with the antigen described in this invention is measured, that is, the result of the test culture tube minus the result of the negative control culture tube (i.e., TN). A TN result ≥14 pg / mL and ≥N / 4 indicates Mycobacterium tuberculosis infection (including active tuberculosis and latent tuberculosis infection), which is used to distinguish tuberculosis patients from normal people.

[0079] The Mycobacterium tuberculosis-infected population described in this invention includes: active tuberculosis patients with clinical symptoms and asymptomatic latent infected individuals. Clinical symptoms here include, but are not limited to, fever, cough, chest pain, hemoptysis, and abnormal chest X-rays.

[0080] Terminology Definition

[0081] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular biology, and immunology laboratory procedures used herein are all conventional procedures widely used in the field. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0082] As used herein, the term "recombinant antigen" refers to a recombinant protein obtained by fusing different proteins or fragments thereof, particularly fragments containing epitopes. Such recombinant proteins can be produced through genetic engineering or chemical synthesis.

[0083] As used herein, the term “cell penetrating peptide (CPP)” is also known as “cell-penetrating peptide,” “protein translocation domain (PTD),” “Trojan horse peptides,” or “transduction peptide,” and refers to a polypeptide that promotes the cellular uptake of various molecules (e.g., various macromolecules including proteins or nucleic acids). Such polypeptides are well known in the art and described, for example, Stewart KM, et al. Org Biomol Chem. 2008 Jul 7; 6(13):2242-55 and Chinese patent applications CN101490081A and CN113121702A (all of which are incorporated herein by reference); or can be obtained by methods known in the art, such as those described in detail in U.S. patent application US2008 / 0234183, all of which are incorporated herein by reference.

[0084] As used herein, the term “eTAT (enhanced TAT)” refers to a fusion protein consisting of four components: Tat (48-60), a pH-sensitive fusion peptide INF7, a protease recognition sequence, and a leucine zipper, and is described in, for example, the Xiamen University Xia Ningshao team’s Nat Commun: A New Strategy for the In vitro Entry of Biological Macromolecules—Multifunctional Chimaeric Peptide-X-MOL Information, Yu S et al. Efficient intracellular delivery of proteins by a multifunctional chimaeric peptide in vitro and in vivo. Nat Commun. 2021 Aug 26;12(1):5131. doi:10.1038 / s41467-021-25448-z.PMID:34446736;PMCID:PMC8390694., and Chinese patent application CN113121702A, all of which are incorporated herein by reference.

[0085] As used herein, the terms “Penetratin”, “Tat(48-60)”, “Tat(47-57)”, “Rev(34-50)”, “VP22”, “transportan”, “Pep-1”, or “Pep-7” are described in Chinese patent application CN113121702A, the entire contents of which are incorporated herein by reference.

[0086] As used herein, the term “ESAT-6” refers to Early Secretory Antigen-6 (ESAT-6) derived from Mycobacterium tuberculosis, also known as “Rv3875” or “Mycobacterium tuberculosis protein Rv3875”, which is well known to those skilled in the art, see, for example, Andersen, P. et al., Lancet, 2000, 356(9235): p.1099-104 (which is incorporated herein by reference), and GENEBANK accession number YP_178023.

[0087] As used herein, the term “CFP-10” refers to culture medium filter protein-10 (CFP-10) derived from Mycobacterium tuberculosis, also known as “Rv3874” or “Mycobacterium tuberculosis protein Rv3874”, which is well known to those skilled in the art, see, for example, Andersen, P. et al., Lancet, 2000, 356(9235): p.1099-104 (which is incorporated herein by reference), and GENEBANK accession number NP_218391.

[0088] As used herein, the term "E1C0" refers to a fusion protein formed by amino acid residues 1-80 of the Mycobacterium tuberculosis-specific protein ESAT-6 and amino acid residues 1-100 of the Mycobacterium tuberculosis-specific protein CFP-10, which contains, for example, the amino acid sequence shown in SEQ ID NO:4, or the amino acid sequence shown in SEQ ID NO:4.

[0089] As used herein, the term "fusion" can refer to direct fusion (i.e., linking the C-terminus of one amino acid sequence to the N-terminus of another amino acid sequence via a simple covalent bond) or fusion using a linker (i.e., linking the C-terminus of one amino acid sequence to the N-terminus of another amino acid sequence via a simple linker). As is well known to those skilled in the art, for example, the fusion of two or more proteins or peptides can be achieved by genetic engineering techniques, or by chemical synthesis techniques known in the art to achieve the conjugation of two or more proteins or peptides.

[0090] As used herein, the term "linker" refers to a short peptide or chemical bond used to connect two molecules (e.g., proteins). Such linkers are well known to those skilled in the art; for example, a linker can be the hinge region of IgG, or a polypeptide linker composed of, for example, glycine, serine, threonine, or alanine in various lengths and combinations. For example, a linker can be a polyglycine repeat interrupted at intervals by serine or threonine. For example, a linker can be Ser-Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly, Gly-Gly-Ser-Ser, Gly-Gly-Gly-Gly-Ser, and (Gly-Gly-Gly-Gly-Ser)3. The linkers of the present invention can be obtained by expression using genetic engineering techniques or by chemical synthesis using methods known in the art.

[0091] As used herein, the term "tag" is well known to those skilled in the art, such as 6×His tag, Myc tag, Flag tag, fluorescent protein tag, etc. The use of such tags facilitates the detection or purification of recombinant antigens. For example, recombinant antigens carrying a 6×His tag can be purified using metal chelating chromatography (e.g., a nickel column). The selection of such tags is within the capabilities of those skilled in the art.

[0092] As used herein, the term "immune adjuvant" or "adjuvant" refers to a class of substances that can nonspecifically alter or enhance the body's specific immune response to an antigen, thereby playing an auxiliary role. This is well known to those skilled in the art; see, for example, Coffman RL, Sher A, Seder RA. Vaccine adjuvants: putting innate immunity to work. Immunity, 2010, 33(4): 492-503.

[0093] As used herein, the Mycobacterium tuberculosis-infected population referred to in this invention includes: active tuberculosis patients with clinical symptoms and asymptomatic latent infected individuals. Clinical symptoms here include, but are not limited to, fever, cough, chest pain, hemoptysis, and abnormal chest X-rays.

[0094] As used herein, the term "active tuberculosis" refers to a state of tuberculosis in which Mycobacterium tuberculosis actively multiplies in the body, causing clinical symptoms and pathological changes. Patients' sputum usually contains a large number of Mycobacterium tuberculosis, making it highly infectious; Mycobacterium tuberculosis can be detected by sputum smears, sputum cultures, or molecular testing. Chest X-rays or CT scans typically show pulmonary infiltration, cavitation, fibrosis, and other lesions. Conversely, as is well known to those skilled in the art, "inactive tuberculosis" refers to the absence of active tuberculosis; as used herein, "inactive tuberculosis" includes latent tuberculosis infection and individuals not infected with Mycobacterium tuberculosis.

[0095] As used herein, the term "cutoff value" is well-known to those skilled in the art and refers to a cutoff value, also known as a judgment criterion. It is the threshold value used to determine whether a test is positive or negative, and is used to determine the normal value of a certain indicator (such as the amount of IFN-γ released by a subject after stimulation with Mycobacterium tuberculosis-specific reactive protein) to distinguish between normal and abnormal. Under normal circumstances, where both sensitivity and specificity are important, the most commonly used method for determining the cutoff value is the receiver operating characteristic curve (ROC curve).

[0096] As used herein, the term "sensitivity" (Sen) is known to those skilled in the art, also referred to as sensitivity or true positive rate, and measures the ability of a diagnostic method under evaluation to detect patients; that is, whether the diagnostic method can identify patients from among those who are actually ill. The calculation method, as shown in the table below, is: Sen = a / (a+c). The higher the sensitivity, the more patients the diagnostic method under evaluation can correctly identify from those who are actually ill. Corresponding to sensitivity is the false negative rate, which is the proportion of people who are incorrectly identified as not ill; false negative rate = 1 - sensitivity.

[0097] As used herein, the term "specificity (Sep)" is known to those skilled in the art, also known as the true negative rate. It measures the ability of a diagnostic method under evaluation to correctly classify individuals as disease-free; in other words, it is the proportion of healthy individuals that the evaluated method can diagnose as disease-free. The calculation method, as shown in the table below, is: Sep = d / (b + d). The higher the specificity, the more people the evaluated diagnostic method can correctly classify as disease-free. The false diagnosis rate, a concept corresponding to specificity, is the proportion of people who are actually disease-free but are incorrectly classified as having the disease. False diagnosis rate = 1 - specificity.

[0098] As used herein, the term "Youden's index" is known to those skilled in the art. The Youden's index = sensitivity + specificity - 1, which represents the combined ability of the diagnostic method being evaluated to identify true patients and non-patients minus a base value of 1. A higher Youden's index indicates a better diagnostic method.

[0099] As used in this article, the term "positive likelihood ratio (LR+)" is the ratio of the true positive rate to the false positive rate (misdiagnosis rate). It indicates the multiple by which the probability of correctly identifying a positive result is greater than the probability of incorrectly identifying a positive result. In other words, it represents how many times more likely a patient is to test positive compared to a non-patient, indicating the ratio of the chance of having the disease to not having the disease when the diagnostic test result is positive. Positive likelihood ratio = sensitivity / (1 - specificity).

[0100] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ESGolub and DRGren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0101] Beneficial effects of the invention

[0102] This invention relates to a novel stimulant for a specific immune response to Mycobacterium tuberculosis, eTAT-E1C0. Compared to the stimulant E1C0, the eTAT-E1C0 stimulant of this invention can improve the sensitivity and / or specificity of Mycobacterium tuberculosis detection. Attached Figure Description

[0103] Figure 1: SDS-PAGE identification of eTAT-E1C0 expression purification.

[0104] Figure 2: Stimulation effect of recombinant antigen eTAT-E1C0 on 127 active tuberculosis samples.

[0105] Figure 3: IFN-γ+CD4+ T cell frequencies in 11 IGRA-positive subjects and 7 IGRA-negative subjects induced by different stimulating antigens.

[0106] Figure 4: IFN-γ+CD8+ T cell frequencies in 11 IGRA-positive subjects and 7 IGRA-negative subjects induced by different stimulating antigens.

[0107] Sequence information Detailed Implementation

[0108] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. In this invention, the tuberculosis infection T-cell detection kit from Wantai Biopharmaceutical is used.

[0109] Example 1: Expression and purification of eTAT-E1C0 protein

[0110] Using a plasmid containing the eTAT gene as a template, the eTAT gene was amplified with primers. Using a plasmid containing the E1C0 gene as a template, the E1C0 gene was amplified with primers. After recovering the gene fragments, the eTAT gene fragments were ligated to the N-terminus of the E1C0 gene. Then, the protein expression plasmid pTO-T7 was ligated into the protein expression plasmid using NdeI and HindIII restriction sites. Sequencing confirmed that the plasmid expressing eTAT-E1C0 (pTO-T7 vector) was successfully constructed.

[0111] The constructed plasmid was transformed into BL21 competent cells to express the protein in large quantities. Since tuberculosis protein is relatively heat-resistant, it was purified using thermal denaturation. The protein was then purified using Ni-6FF and Q-FF columns, with DOC elution used during column purification to remove endotoxins. SDS-PAGE analysis confirmed the successful preparation of the eTAT-E1C0 protein, which has a molecular weight of approximately 33 kDa and a purity greater than 85%. The SDS-PAGE results of the eTAT-E1C0 protein are shown in Figure 1.

[0112] Example 2: Stimulation and detection experiment of Mycobacterium tuberculosis-specific immune response

[0113] A total of 257 fresh venous blood samples were collected from clinical patients, including 82 samples from patients with active tuberculosis and 175 samples from patients with non-tuberculous lung disease. The stimulation effect of culture tubes with different antigenic peptides was detected by the interferon-gamma release assay (IGRA).

[0114] The experimental materials and their main components are as follows:

[0115] ELISA plate: coated with mouse IgG subtype monoclonal antibody against human IFN-γ; ELISA reagent: horseradish peroxidase-labeled mouse IgG subtype monoclonal antibody against human IFN-γ; calibrator: containing IFN-γ positive material, matrix solution is protein buffer; culture tubes: matrix solution is phosphate buffer, test culture tube (T) contains tuberculosis-specific recombinant antigen, positive control culture tube (P) contains tuberculosis non-specific stimulating antigen, background control culture tube (N) contains matrix solution; concentrated wash buffer: containing not less than 2.5% surfactant; chromogenic agent A: containing not less than 0.3 g / L peroxide; chromogenic agent B: containing not less than 0.2 g / L TMB; stop solution: containing sulfuric acid with a concentration not exceeding 2 mol / L.

[0116] The experimental steps are described as follows:

[0117] (1) Antigen stimulation: First, collect approximately 5 mL of venous blood using heparin-anticoagulated blood collection tubes. Within 16 hours of collection, gently invert and mix the venous blood, then aliquot the whole blood into the original test culture tube (T, containing 2 μg E1C0 protein), the recombinant protein eTAT-E1C0 test culture tube (T, containing 3.3 μg eTAT-E1C0 protein), the negative control culture tube (N, containing matrix solution-phosphate buffer), and the positive control culture tube (P, containing tuberculosis non-specific stimulating antigen), 1 mL of whole blood in each tube. Gently invert and mix the culture tubes, then incubate at 37°C for 24 hours. After incubation, centrifuge the culture tubes at 5000 rpm for 10 minutes and collect the serum.

[0118] (2) ELISA detection: Add 20 μL of sample diluent to each well of the ELISA plate, followed by 50 μL of sample or calibrator to each well, and gently vortex to mix. Seal the plate with sealing film and incubate at 37°C for 1 h. Add 50 μL of enzyme-labeled reagent to each well and gently vortex to mix. Seal the plate with sealing film and incubate at 37°C for 1 h. Wash the plate 5 times with washing buffer and spin dry. Add 50 μL each of colorimetric solution A and colorimetric solution B to each well, gently vortex to mix, and incubate at 37°C for 15 min. Add 50 μL of stop solution to each well, gently vortex to mix, and detect the absorbance value at 450 nm using an ELISA reader. Plot a standard curve based on the antigen content and absorbance value of each calibrator. Calculate the IFN-γ content of N tubes, T tubes, and P tubes, and determine the results according to Table 1 below:

[0119] Table 1

[0120] Since there is no gold standard for diagnosing tuberculosis infection, it is impossible to determine whether the subjects were truly infected with tuberculosis. To examine the effect of eTAT-E1C0 as a neoantigen on improving the diagnostic performance of IGRA, the test results of 82 patients with active tuberculosis in the comparative trial were analyzed, as shown in Figure 2. Data analysis was performed using the paired-samples rank-sum test, and the p-value for the comparative trial was less than 0.001. The median IFN-γ release in the eTAT-E1C0 group was 348.02 pg / mL. Compared with the E1C0 group, the recombinant antigen eTAT-E1C0 group could enhance the stimulation effect on T cells in IGRA, increasing IFN-γ release by approximately 71% (median: 348.02 pg / mL vs. 203.59 pg / mL).

[0121] Example 3: Detection of the improved diagnostic performance of recombinant antigen eTAT-E1C0 in patients with active tuberculosis

[0122] In individuals with normal immune status, an IFN-γ release level ≥14 pg / mL and ≥N / 4 is considered a positive result for tuberculosis infection. Table 2 shows the diagnostic results of 82 individuals with active tuberculosis in the eTAT-E1C0 and E1C0 comparative trials.

[0123] As shown in Table 2, the sensitivity of E1C0 (82 cases) was 87.80%, while the sensitivity of eTAT-E1C0 was 90.24%. eTAT-E1C0 helped reduce the number of missed IGRA cases by 2, suggesting that replacing the stimuli with eTAT-E1C0 can improve the diagnostic performance of IGRA.

[0124] Table 2: Note: +: indicates a positive IGRA test result using the stimulant; -: indicates a negative IGRA test result using the stimulant.

[0125] Example 4: Flow cytometry detection of the stimulatory effect of the stimulator on CD4+ / CD8+ T cells

[0126] Direct IGRA only detects the overall stimulatory effect of the stimulating antigen on T cells. Therefore, flow cytometry was used to analyze the independent stimulatory effect of the stimulating antigen on CD4+ / CD8+ T cells. This section collected fresh whole blood samples from 18 inpatients in the Department of Pulmonary Medicine at the First Affiliated Hospital of Xiamen University (Xinglin Branch), including 6 samples with active tuberculosis (information from hospital diagnosis). Two test culture tubes, E1C0 and eTAT-E1C0, were set up, along with a negative control culture tube (containing PBS). IGRA and flow cytometry were performed on both. The IGRA experimental procedure is as described in Example 2. The flow cytometry procedure is as follows:

[0127] (1) Add 500 μL of whole blood to different culture tubes and incubate in a 37°C incubator for 6 hours.

[0128] (2) Add protein transport inhibitors at a ratio of 1:1000 and incubate in a 37°C incubator for 6 hours.

[0129] (3) Centrifuge at 2000 rpm for 5 min, carefully aspirate the supernatant, add 1 mL of red blood cell lysis buffer, incubate on ice for 10 min, and invert once during the incubation period.

[0130] (4) Centrifuge at 2000 rpm for 5 min, discard the supernatant, and resuspend the cells in 1.6 mL of red blood cell lysis buffer.

[0131] (5) Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in 200 μL of DPBS solution and wash them. Repeat this step once.

[0132] (6) Add 10 mL of fetal bovine serum to 500 mL of DPBS as FACS buffer. Dilute the dead cell staining antibody and cell surface antibody in the FACS buffer at the pre-determined optimal ratio, taking care to avoid light during the process.

[0133] (7) Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in 40 μL of FACS buffer with added antibody, and incubate at 4°C in the dark for 30 min.

[0134] (8) Add 200 μL of FACS buffer and mix well. Centrifuge at 2000 rpm for 5 min and discard the supernatant. Resuspend the cells in 200 μL of fixation / permeabilization buffer and incubate at 4°C in the dark for 30 min.

[0135] (9) Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in 200 μL of permeabilizing wash buffer and wash them.

[0136] (10) Dilute the intracellular antibody in the permeation wash solution at the optimal ratio determined in advance, and avoid light during the process.

[0137] (11) Centrifuge at 2000 rpm for 5 min, discard the supernatant, resuspend the cells in 40 μL of permeabilization wash with added antibody, and incubate at room temperature in the dark for 30 min.

[0138] (12) Add 200 μL of permeation washing solution and mix well. Centrifuge at 2000 rpm for 5 min, discard the supernatant, and repeat this step once.

[0139] (13) Resuspend the cells in 300 μL of FACS buffer, filter through a cell sieve, and transfer to a flow cytometer.

[0140] (14) The cells in each stimulation tube were analyzed after adjustment of compensation using a compensation tube stained with a single fluorescent antibody on a flow cytometer.

[0141] The IGRA test results showed that 7 cases were IGRA positive (P) and 11 cases were IGRA negative (N).

[0142] The flow cytometry results are shown in Figures 3 and 4. Compared with the E1C0 group, the frequency of IFN-γ+CD4+ T cells (Figure 3) and IFN-γ+CD8+ T cells (Figure 4) in the eTAT-E1C0 group was increased, indicating that the eTAT-E1C0 recombinant antigen promoted the stimulation effect on CD4+ / CD8+ T cells.

[0143] Example 5: Analysis of the auxiliary diagnostic value of IGRAS in active tuberculosis

[0144] Samples from subjects collected from the Xinglin Branch of the First Affiliated Hospital of Xiamen University were analyzed. To determine the cutoff value that best distinguishes between active and latent tuberculosis infection in each group, the IFN-γ release levels and hospital diagnostic results (active / inactive tuberculosis) of subjects diagnosed as IGRA positive in each group were imported into SPSS software for analysis, and receiver operating characteristic (ROC) curves for the determination of active tuberculosis were determined for each group. Based on the cutoff value provided by the software and its corresponding sensitivity and specificity, the Youden index (Youden index = sensitivity + specificity - 1) was calculated. The cutoff value with the largest Youden index was determined as the optimal cutoff value for diagnosing active tuberculosis in each group. In addition, the difference in IFN-γ release between the eTAT-E1C0 group and the E1C0 group was calculated, i.e., IFN-γ release (eTAT-E1C0 group) - IFN-γ release (E1C0), which was taken as the IFN-γ release enhancement value and denoted as eTAT-E1C0 group (enhancement value). The optimal cutoff value was determined using the same method.

[0145] To compare the diagnostic efficacy of IGRA values ​​for active tuberculosis among different groups, the sensitivity, specificity, and positive likelihood ratio for diagnosing active tuberculosis were calculated in all subjects (positive likelihood ratio = sensitivity / (1 - specificity)).

[0146] The results are shown in Table 3. The eTAT-E1C0 group (elevation value) had the highest positive likelihood ratio of 6.01, indicating that patients with active tuberculosis were 6.01 times more likely to have a positive result than individuals without active tuberculosis. This suggests that the eTAT-E1C0 group (elevation value) has a good predictive value for active tuberculosis.

[0147] Table 3. Diagnostic performance of each group's cutoff value in differentiating active tuberculosis among all subjects.

[0148] Example 6: Stimulation and Detection Experiment of Mycobacterium tuberculosis-Specific Immune Response

[0149] A total of 286 fresh venous blood samples were collected from clinical patients, including 90 samples from patients with active tuberculosis and 196 samples from patients with non-tuberculous lung disease. Stimulation and detection experiments for Mycobacterium tuberculosis-specific immune responses were performed according to the method described in Example 2.

[0150] The test results of specimens from 90 patients with active tuberculosis in the comparative trial were analyzed. Data analysis was performed using the paired-samples rank-sum test, and the p-value for the comparative trial was less than 0.001. The median IFN-γ release in the eTAT-E1C0 group was 342.83 pg / mL. Compared with the E1C0 group, the recombinant antigen eTAT-E1C0 group enhanced the stimulatory effect on T cells in IGRAS, increasing IFN-γ release by approximately 68.39% (median 342.83 pg / mL vs. 203.59 pg / mL).

[0151] Example 7: Detection of the improved diagnostic performance of recombinant antigen eTAT-E1C0 in patients with active tuberculosis

[0152] In individuals with normal immune status, an IFN-γ release level ≥14 pg / mL and ≥N / 4 is considered a positive result for tuberculosis infection. Table 4 shows the diagnostic results of 90 individuals with active tuberculosis in Example 6 across the eTAT-E1C0 and E1C0 comparative trials.

[0153] As shown in Table 4, the sensitivity of E1C0 (90 cases) was 86.67%, while the sensitivity of eTAT-E1C0 was 90.00%. eTAT-E1C0 helped reduce the number of missed IGRA cases by 3, suggesting that replacing the stimuli with eTAT-E1C0 can improve the diagnostic performance of IGRA.

[0154] Table 4: Note: +: indicates a positive IGRA test result using the stimulant; -: indicates a negative IGRA test result using the stimulant.

[0155] Example 8: Analysis of the auxiliary diagnostic value of IGRAS in active tuberculosis

[0156] The subject samples collected from the First Affiliated Hospital of Xiamen University, Xinglin Branch, as described in Example 6, were analyzed. Following the method described in Example 5, the optimal cutoff values ​​for the eTAT-E1C0 group, the eTAT-E1C0 group (elevation value), and the E1C0 group were determined to best distinguish between active and latent tuberculosis infection. The sensitivity, specificity, and positive likelihood ratio (positive likelihood ratio = sensitivity / (1 - specificity)) for diagnosing active tuberculosis were also determined.

[0157] The results are shown in Table 5. The eTAT-E1C0 group (elevation value) had the highest positive likelihood ratio of 4.36, indicating that patients with active tuberculosis were 4.36 times more likely to have a positive result than individuals without active tuberculosis. This suggests that the eTAT-E1C0 group (elevation value) has a good predictive value for active tuberculosis.

[0158] Table 5. Diagnostic performance of each group's cutoff value in differentiating active tuberculosis among all subjects.

Claims

1. A recombinant antigen comprising a cell-penetrating peptide, a Mycobacterium tuberculosis-specific protein, and optionally a linker and optional a tag.

2. The recombinant antigen according to claim 1, wherein, The cell-penetrating peptide is selected from penetratin, Tat-derived peptides (e.g., eTAT, Tat(48-60) or Tat(47-57)), Rev(34-50), VP22, transport peptide, Pep-1, Pep-7, and any combination thereof; Preferably, the cell-penetrating peptide is selected from Tat-derived peptides, such as eTAT; Preferably, the eTAT comprises or is an amino acid sequence as shown in SEQ ID NO: 11 or 2.

3. The recombinant antigen of claim 1, wherein the Mycobacterium tuberculosis-specific protein is selected from E1CO protein, ESAT-6 (Rv3875), CFP-10 (Rv3874), or any combination thereof; Preferably, the Mycobacterium tuberculosis-specific protein is the E1CO protein; Preferably, the E1C0 protein comprises or has an amino acid sequence as shown in SEQ ID NO:

4.

4. The recombinant antigen according to any one of claims 1-3, wherein the cell-penetrating peptide and the Mycobacterium tuberculosis-specific protein are linked by direct fusion or by a linker, or by chemical synthesis; Preferably, the adapter comprises an amino acid sequence as shown in any one of SEQ ID NO:6-10.

5. The recombinant antigen according to any one of claims 1-4, wherein the recombinant antigen comprises or is an amino acid sequence as shown in SEQ ID NO:

1.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the recombinant antigen of any one of claims 1-5.

7. A vector comprising the isolated nucleic acid molecule of claim 6.

8. A host cell comprising the isolated nucleic acid molecule of claim 6 or the vector of claim 7.

9. A composition comprising the recombinant antigen according to any one of claims 1-5, optionally further comprising a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutically acceptable carrier is a buffer.

10. Use of the recombinant antigen according to any one of claims 1-5 and the composition according to claim 9 in the preparation of a kit, wherein the kit is used for in vitro detection of Mycobacterium tuberculosis-specific T-cell immune responses in samples, or for diagnosing whether a subject is infected with Mycobacterium tuberculosis, or for evaluating the efficacy of anti-tuberculosis treatment.

11. A kit comprising the recombinant antigen of any one of claims 1-5 or the composition of claim 9.

12. The kit of claim 11 is used for: detecting Mycobacterium tuberculosis-specific T-cell immune responses in samples in vitro, diagnosing whether a subject is infected with Mycobacterium tuberculosis, or evaluating the efficacy of anti-tuberculosis treatment.

13. A method for detecting Mycobacterium tuberculosis-specific T-cell immune responses in an in vitro sample, comprising the following steps: (1) Stimulating the sample with the recombinant antigen according to any one of claims 1-5; (2) Detect the level of effector molecules produced in the stimulated sample; (3) Determine the Mycobacterium tuberculosis infection status of the sample; Compared with the control, the increased levels of the effector molecules in the stimulated sample indicated an increased level of Mycobacterium tuberculosis-specific T-cell immune response in the sample. in, The sample is anticoagulated whole blood or peripheral blood mononuclear cells, preferably, the sample is anticoagulated whole blood; The effector molecules include interferon-gamma (IFN-γ) or cells that release IFN-γ; Preferably, the method for detecting the level of effector molecules is the gamma-interferon release assay.

14. The method of claim 12, wherein, The controls are the sample to be tested that has not been stimulated with the recombinant antigen, a sample that has not been infected with Mycobacterium tuberculosis stimulated with the recombinant antigen, and / or the sample to be tested that has been stimulated with the E1CO protein antigen.

15. An apparatus for detecting and distinguishing between active and inactive tuberculosis, characterized in that: This includes an antigen stimulation module, an effector molecule detection module for immune response, an IFN-γ release enhancement value calculation module, an optimal cutoff value calculation module for the eTAT-E1C0 group (enhancement value), and an active tuberculosis positive assessment module, among which: (1) The antigen stimulation module: The test sample is stimulated by the recombinant antigen according to any one of claims 1-5 or the E1C0 protein antigen respectively, wherein the recombinant antigen is referred to as the eTAT-E1C0 group and is used as the experimental group, and the E1C0 protein antigen is used as the control group. (2) The effector molecule detection module of the immune response: detects the level of effector molecules such as IFN-γ produced in the test sample after stimulation; IFN-γ release ≥14pg / mL and ≥N / 4 is considered IGRA positive; where N is the IFN-γ release amount of the background control culture tube; (3) The IFN-γ release increase value calculation module: calculates the difference between the IFN-γ release amount of the eTAT-E1C0 group and the IFN-γ release amount of the E1C0 group, that is, the IFN-γ release amount (eTAT-E1C0 group) - the IFN-γ release amount (E1C0), which is recorded as the eTAT-E1C0 group (increase value). (4) The optimal cutoff value calculation module for the eTAT-E1C0 group (boost value): Take the test sample with known real active tuberculosis status, and after processing by the above (1) antigen stimulation module and (2) effector molecule detection module of immune response, calculate and determine the optimal cutoff value for diagnosing active tuberculosis based on the boost value of IFN-γ release in the eTAT-E1C0 group that is determined to be IGRA positive and the real active tuberculosis status of the test sample. (5) Active tuberculosis positive judgment module: Determine whether the sample to be tested is infected with active tuberculosis based on the optimal cutoff value of the eTAT-E1C0 group (increase value); Among them, the known real active tuberculosis situation mentioned in (4) refers to the hospital's diagnosis results of active or inactive tuberculosis of the sample to be tested.

16. The apparatus of claim 15, wherein the method for calculating the optimal cutoff value is as follows: (i) Import the IFN-γ release enhancement value of the eTAT-E1C0 group of the sample and the hospital diagnosis results (active tuberculosis / inactive tuberculosis) into SPSS software to obtain the cutoff value, sensitivity and specificity of the sample to be tested; (ii) Calculate the Youden index, where the Youden index = sensitivity + specificity - 1; (iii) The cutoff value with the largest Yoden index is determined as the optimal cutoff value for diagnosing active tuberculosis.

17. The apparatus of claim 15 or 16, wherein the active tuberculosis positive determination module: if the IFN-γ release increase value of the test sample is greater than the optimal cutoff value of the eTAT-E1C0 group (increase value), then the test sample is determined to be active tuberculosis.

18. The antigen stimulation module of any one of the recombinant antigens of claims 1-5, the composition of claim 9, the kit of claim 11, or the device of any one of claims 15-17, further comprising an immune adjuvant; Preferably, the immune adjuvant is selected from one or more of the following: aluminum hydroxide, aluminum phosphate, nano-aluminum, layered bimetallic hydroxide (LDH), nano / mesoporous silica, nanodiamond, MF59, ASO3, ASO4, CpG, liposomes, lipid monophosphate (MPL), Freund's adjuvant, polyinosinic-polycytidylic acid, QS21, or cytokines. Preferably, the immune adjuvant is CpG.

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