Use of an hck agonist in the treatment of mycobacterium tuberculosis infection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a non-receptor protein tyrosine kinase agonist in resisting mycobacterium tuberculosis infection. The application screens a tyrosine phosphorylated protein as a research target point through a bioinformatics technology, and develops cell and animal experiments, and finds that the target point agonist activates immune cells, enhances the antibacterial function of macrophages, thereby improves the host immunity, and inhibits the growth and propagation of mycobacterium tuberculosis. Compared with traditional antibiotic treatment which depends on multi-drug combination, the agonist of the application reduces the dependence on antibiotics through precise targeting of immune regulation, thereby reducing the risk of generation of drug-resistant strains.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a non-receptor protein tyrosine kinase agonist in the treatment of Mycobacterium tuberculosis infection. Background Technology

[0002] Tuberculosis is a common infectious disease in clinical practice, characterized by a long course of disease and long treatment course. It is often treated with a combination of antibacterial drugs. If the patient has poor immune function and multiple underlying diseases, repeated drug treatment may cause dysbiosis and lead to multidrug-resistant bacteria (MDRO) infection (Qi Liying, Li Xuezheng, Guo Xue, et al. Application of cluster management strategy in tuberculosis infection prevention and control management in tuberculosis specialist hospital. Qilu Nursing Journal, 2021, 27(1): 165-167.). After infection with Mycobacterium tuberculosis, there are initially no typical clinical symptoms. As the pathogen becomes clearer, the body's resistance gradually decreases. If the bacterial-mediated allergic reaction increases abnormally, obvious clinical symptoms will appear, including fever, fatigue, loss of appetite, weight loss, and respiratory changes, which seriously affect the patient's health and safety (Zhou Jiaxian, Zhang Weifen, Zhou Guijiao. Application of T-cell enzyme-linked immunospot test in the investigation of latent tuberculosis infection (LTBI) among nursing staff in general hospitals. Nursing Practice and Research, 2020, 17(4): 145-147; Zhang Lijun, Liu Yan, Song Yan. The impact of hospital-family joint supervision and continuity of care model on the family burden and social support of patients with drug-resistant tuberculosis. Nursing Practice and Research, 2022, 19(5): 631-634.). In 2020, approximately 9.9 million people worldwide were diagnosed with tuberculosis, and approximately 1.3 million patients died from tuberculosis. The emergence of anti-tuberculosis drugs has limited the spread of tuberculosis to some extent. However, anti-tuberculosis treatment still has many shortcomings, with standard chemotherapy regimens requiring at least 4 to 6 months. The economic burden of long-term treatment and the adverse reactions of anti-tuberculosis drugs lead to the discontinuation of treatment, thus accelerating the development of drug-resistant tuberculosis. Drug-resistant tuberculosis is resistant to first-line anti-tuberculosis drugs such as isoniazid (INH) and rifampicin (RMP), and a small number are resistant to second-line anti-tuberculosis drugs and fluoroquinolones, resulting in a significant decrease in the cure rate of tuberculosis. At the same time, the toxic side effects of anti-tuberculosis drugs and the increasing multidrug resistance also affect the treatment of tuberculosis patients. Therefore, there is an urgent need to develop new drugs and intervention programs to control infection. Host-targeted therapy, which targets multiple molecules or pathways in the host's immune system, may help fight bacterial infection and improve the cure rate of tuberculosis.

[0003] Tyrosine kinases and tyrosine phosphorylation are closely related to various biological processes and have become one of the most important drug targets. Tyrosine kinases are an important class of molecules widely present in the human body. The tyrosine kinase family is divided into two main categories: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases are membrane-bound proteins that receive signals transmitted by ligands. They include various molecular targets such as epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), and c-MET (mesenchymal epithelial transformation factor). Non-receptor tyrosine kinases include Ab1, Src family kinases, and focal adhesion kinases. Activation of these kinases, in turn, activates their downstream signaling molecules to regulate the growth and differentiation of tumor cells. "Under normal circumstances, the activation of these molecules is strictly controlled." However, in tumor tissues, the activation of receptor and non-receptor tyrosine kinases is dysregulated, leading to changes in cell proliferation and cell function. Protein phosphorylation is synergistically regulated by protein kinases and protein phosphatases. It is a crucial step in intercellular communication, cell growth and proliferation, cell cycle regulation, differentiation, malignant tumor changes, occurrence and metastasis, nerve conduction, angiogenesis, embryogenesis, and many genetic and acquired human diseases. Therefore, research on protein phosphorylation has become a hot topic in life sciences.

[0004] The SRC family of non-receptor protein tyrosine kinases (SFKs) consists of nine members and can be divided into two groups based on the expression patterns of individual kinases in adults. Therefore, SRC, YES, and FYN are universally expressed, while LCK, FGR, BLK, LYN, YRK, and HCK are expressed only in certain cell types and tissues (Poh AR, O'Donoghue RJ, Ernst M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells[J]. Oncotarget. 2015, 6(18):15752-15771.). Hemopoietic cell kinase (HCK) expression is limited to cells in the bone marrow and B lymphocyte lineages, and the protein exists in two isoforms. HCK can participate in various physiological and pathological processes, such as macrophage activation, secretion of myeloid growth factor and pro-inflammatory cytokines, and mediation of cell development and intracellular signal transduction (Kim J, Sinn DH, Choi MS, et al. Hepatocellular carcinoma with extrahepatic metastasis: Are there still candidates for transarterial chemoembolization as an initial treatment?[J]. PkS One, 2019, 14(3): e0213547.). HCK can participate in the course of acute myeloid leukemia by activating signaling pathways such as phosphatidylinositol-3-kinase / protein kinase B and mitogen-activated protein kinase / extracellular signal-regulated kinase. The SH2 and SH3 domains of HCK interact with Bcr. Abl interactions influence the development of chronic myeloid leukemia (Roseweir AK, Powell AGMT, Horstman SL, et al. Sre family kinases. HCK and FGR, associate with local inflammation and turnout progression in colorectal cancer[J]Cell Sisnal, 2019(56): 15-22.).HCK can promote the development of colorectal cancer through the Star6-dependent Th2 cytokine signaling pathway, and patients with high expression have a lower survival rate (antermans HC, Minderman M, Kuil A, et al. Identification of the SRC-family tyrosine kinase HCK as a therapeutic target in mantle cell lymphoma[J]. Leukemia, 2021, 35(3): 881-886.). Moreover, HCK has been found to have sequence alterations, replication number aberrations, and high expression in gastric cancer, making it a potential oncogene (Mei J, Zhou WJ, Zhu XY, et al. Suppression of autophagy and HCK signaling promotes PTGS2 highFCGR3-NK cell differentiation triggered by ectopic endometrial stromal cells[J]. Autophagy, 2018. 14(8): 1376-1397.). The occurrence of other solid malignancies such as breast cancer and pancreatic cancer is also closely related to the overexpression of HCK (Ochieng P, Nath S, Macarulay R, et al. Phospholipid transfer protein and alpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation[J]. Sci Rep, 2018, 8(1): 15394; Poh AR, O. ´ Donoghue RJ, Ernst M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells[J]. Oncotarget, 2015, 6(18): 15752-15771.). Summary of the Invention

[0005] This invention discovers that the HCK agonist EPQpYEEIPIYL can improve the clearance efficiency of macrophages against MTB infection and enhance the immune response. Based on this, this invention was completed.

[0006] In a first aspect, the present invention provides the use of an HCK agonist in the preparation of a drug for treating Mycobacterium tuberculosis infection, wherein the agonist is capable of inhibiting Mycobacterium tuberculosis infection, and the agonist is EPQpYEEIPIYL.

[0007] Furthermore, the aforementioned Mycobacterium tuberculosis includes standard strains of Mycobacterium tuberculosis, clinical isolates of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0008] Furthermore, the drug can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, and / or suppositories.

[0009] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0010] Furthermore, the drug can be introduced into the body through injection, permeation, absorption, or physical or chemical mediated methods.

[0011] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; the cavity administration includes rectal or vaginal administration; and the respiratory administration includes nasal administration.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for treating Mycobacterium tuberculosis, the pharmaceutical composition containing the HCK agonist EPQpYEEIPIYL.

[0013] Furthermore, the aforementioned Mycobacterium tuberculosis includes standard strains of Mycobacterium tuberculosis, clinical isolates of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0014] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0015] Furthermore, the carrier material is selected from one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric carrier materials.

[0016] Furthermore, the pharmaceutical composition can be formulated into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, and / or suppositories.

[0017] Furthermore, the formulation may be one or more of a conventional formulation, a sustained-release formulation, a controlled-release formulation, and / or a variety of microparticle delivery systems.

[0018] Furthermore, the pharmaceutical composition can be introduced into the body by injection, permeation, absorption, or physical or chemical mediated methods.

[0019] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intracavitary injection; the cavity administration includes rectal or vaginal administration; and the respiratory administration includes nasal administration.

[0020] Beneficial effects 1. This invention uses bioinformatics technology to screen for the tyrosine phosphorylated protein HCK, and conducts cell and animal experiments using HCK knockout, HCK overexpression, HCK reintroduction, HCK inhibitors, and the HCK agonist EPQpYEEIPIYL. It was found that this agonist can significantly improve the clearance efficiency of macrophages against Mycobacterium tuberculosis (MTB) and enhance the immune response.

[0021] 2. Compared with existing anti-tuberculosis treatments, HCK agonists enhance host immunity and inhibit the growth and spread of Mycobacterium tuberculosis by activating immune cells and enhancing the antibacterial function of macrophages.

[0022] 3. This invention also has the advantage of reducing the risk of drug resistance. Compared with traditional antibiotic treatment that relies on multiple drug combinations, HCK agonists reduce dependence on antibiotics by precisely targeting immune regulation, thereby reducing the risk of the emergence of drug-resistant strains. Attached Figure Description

[0023] Figure 1 Effect of MTB infection on HCK tyrosine phosphorylation levels in macrophages.

[0024] Figure 2 Western blot was used to verify the HCK gene knockout effect. WT cells were wild-type Thrp1 cells, and KO cells were Thrp1 cells with the HCK gene knocked out.

[0025] Figure 3 CFU was used to identify the antibacterial effect of HCK gene knockout. WT cells were wild-type Thrp1 cells, and KO cells were HCK gene knockout Thrp1 cells.

[0026] Figure 4 CFU was used to identify the antibacterial effect of HCK gene overexpression. WT cells were wild-type Thrp1 cells, and HCK cells were Thrp1 cells overexpressing the HCK gene.

[0027] Figure 5 The effect of HCK gene overexpression on autophagy regulation in MTB-infected macrophages. NC represents the control group without HCK overexpression, while HCK represents the group with overexpressed HCK genome.

[0028] Figure 6The antibacterial effect of EPQpYEEIPIYL was identified by CFU. DMSO was the control group, iHCK was the inhibitor group, and EPQ was the agonist group.

[0029] Figure 7 Evaluation of the effects of EPQpYEEIPIYL on the HCK autophagy pathway. NC was the control group, and EPQ was the agonist group.

[0030] Figure 8 Evaluation of the effect of iHCK-37 on the HCK autophagy pathway. NC was the control group, and iHCK was the inhibitor group.

[0031] Figure 9 CFU counts in the right lung tissue of mice four weeks after tail vein injection of BCG. DMSO was the control group, iHCK was the inhibitor group, and EPQ was the agonist group.

[0032] Figure 10 HE staining was used to identify the effect of HCK on BCG clearance in mice. DMSO was the control group, iHCK was the inhibitor group, and EPQ was the agonist group. Detailed Implementation

[0033] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0034] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0035] Example 1: HCK selected as the research target 1.1 Macrophages The Thp1 cell line (human acute monocytic leukemia cell line) was obtained from the Department of Bacteriology and Immunology, Beijing Chest Hospital, Capital Medical University.

[0036] 1.2 Mycobacterium tuberculosis strains The Mycobacterium tuberculosis standard strain H37Rv (ATCC 27298) was preserved by the Department of Bacteriology and Immunology, Beijing Chest Hospital.

[0037] 1.3 MTB-infected macrophage model (1) Cell plating: Cell plating density = 5 × 10 5 PMA (1:1000) stimulation overnight.

[0038] (2) Preparation of bacterial suspension: Add PBS to the culture bottle, scrape colonies from the culture medium and transfer them to the culture bottle, shake, let stand, and sonicate to disperse the bacterial suspension to adjust the OD value. 600 Up to 1 (1×10 8 (units / mL).

[0039] (3) Infection: Calculate the bacterial load according to MOI=5 and infect macrophages. The protein was lysed with RIPA at 0 h, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 14 h after infection and the samples were collected.

[0040] 1.4 Western Blot Immunoblotting After sample extraction and protein quantification, the samples were separated by SDS-PAGE electrophoresis, transferred to a PVDF membrane, and then blocked, incubated with antibodies, and detected by chemiluminescence to analyze protein expression.

[0041] 1.5 Results Analysis Western blot analysis revealed that MTB invasion of macrophages did not significantly alter HCK protein expression, while the phosphorylation level at HCKTyr 410 site initially increased and then decreased over time. Figure 1 This indicates that macrophages undergo dynamic regulation of the immune response during MTB infection, involving the activation and inhibition of HCK, which may be part of the cell's adaptation to the immune response.

[0042] Example 2: Experiments on HCK knockout, complementation, and overexpression 2.1 Construction of HCK knockout strains (1) The HCK gene was knocked out using the CRISPR RNP method (Cas9 forms a complex with sgRNA).

[0043] (2) Cell preparation: Digest cells according to the cell passage procedure; wash with DPBS solution and count cells. Take the corresponding cells for use according to the counting results.

[0044] (3) RNP preparation: Prepare the RNP complex at a ratio of 5:1 to 10:1 (sgRNA:Cas9) and let it stand.

[0045] (4) Electroporation step: The RNPs were transfected into the cell nuclei using an electroporation instrument.

[0046] (5) Single cell sorting and expansion were completed using the DispenCell Single-Cell Dispenser of Molecular Device. After expansion, cell lines with clear KO genotypes (reaching 100% KO editing) and good viability were cryopreserved.

[0047] 2.2 HCK wild-type and overexpression lentiviral packaging Lentiviral vectors and their auxiliary packaging vectors, with enhanced purity and free of endotoxins, were constructed and co-transfected into HEK-293T cells using HGTransgene™ Reagent. Enhancing buffer was added 10–12 h after transfection, and the culture medium was replaced with fresh medium after 8 h. Cells were cultured for another 48 h, and the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate. The viral titer was then measured and calibrated in HEK-293T cells. Lentiviral particles within a certain titer range can meet the needs of most in vivo and in vitro experiments.

[0048] 2.3 Construction and screening of HCK overexpression and complementation stable strains (1) Macrophages were sourced the same as in 1.1, and were added to each well in complete culture medium at a density of 4 × 10⁶ cells / well. 5 Cell suspension of cells / mL.

[0049] (2) After removing the virus, thaw it in an ice bath. Refer to relevant literature or dilute the virus to the required concentration using complete culture medium based on the MOI value obtained from the preliminary experiment. Remove the original culture medium from the cells, add the virus solution diluted according to the MOI value to the cells, and centrifuge the system for infection.

[0050] (3) Observe the cell status promptly 16-24 hours after infection. If the status changes significantly, change the medium completely after centrifugation. If the status is normal, directly replenish the culture medium to the full volume and replace it with fresh culture medium on the third day.

[0051] (4) After 72 hours of infection, the infection was considered successful when about 80% of the cells were observed under a fluorescence microscope. Cells that were successfully infected and growing well were screened with puromycin, cultured, and cryopreserved.

[0052] 2.4 Infection of HCK knockout, overexpressing, and wild-type Thrp1 cells with MTB The cell source was as described in 1.1, and the Mycobacterium tuberculosis standard strain H37Rv was sourced from the same source as in 1.2. A macrophage infection model was constructed, and samples were collected after 24 h and processed using the same method as in 1.3.

[0053] 2.6 MTB Colony Forming Unit (CFU) Count (1) Add sterile PBS to each tube beforehand, and aspirate the cell lysis buffer to a label 10. -1 Mix well in the tube, then take 10 marked with a needle. -1 The tube to mark 10 -2 Mix thoroughly by blowing and blowing into the tube, then take 10 marked with a pipette.-2 The tube to mark 10 -3 Mix thoroughly in the tube, and then dilute 10 times sequentially to prepare diluents of different concentrations.

[0054] (2) Mark 10 on 7H10 solid medium. -1 10 -2 10 -3 Take 100 μL of each diluted solution of different concentrations and add it to a petri dish, then spread it on the culture medium.

[0055] (3) Use a CFU counter to count after about 2-3 weeks.

[0056] 2.7 Western Blot Immunoblotting The experimental procedure is the same as in 1.4.

[0057] 2.8 Statistical Analysis and Graphical Plotting All statistical analyses were performed using GraphPad Prism Version 9.0 software. Comparisons between two groups were performed using the Student t-test, with P < 0.05 defined as statistically significant.

[0058] 2.9 Results Analysis After successfully constructing the HCK knockout cell line ( Figure 2 Following HCK knockout, a CFU experiment was performed, and the results showed that the colony count of Mycobacterium tuberculosis significantly increased after HCK knockout. Figure 3 Conversely, overexpression of the HCK gene significantly reduced the colony count of Mycobacterium tuberculosis. Figure 4 This indicates that the HCK gene may play an important role in inhibiting the growth of Mycobacterium tuberculosis. Further investigation into the mechanism by which the HCK gene affects Mycobacterium tuberculosis growth revealed that overexpression of the HCK gene leads to increased LC3 protein expression. Figure 5 This result further demonstrates that HCK inhibits the growth of Mycobacterium tuberculosis by regulating autophagy.

[0059] Example 3: Experiments with HCK inhibitors and agonists 3.1 Macrophages The source of macrophages is the same as in 1.1.

[0060] 3.2 Mycobacterium tuberculosis strains The source of the Mycobacterium tuberculosis standard strain H37Rv is the same as in 1.2.

[0061] 3.3 Inhibition of macrophage HCK gene with iHCK-37 or stimulation of macrophage HCK gene with EPQpYEEIPIYL (1) Add 0.1 μM of iHCK-37 (C) solution 2 h before infection. 30 H 32 RPMI 1640 medium (containing 10% FBS) or EPQpYEEIPIYL (N4O2S2) or EPQpYEEIPIYL. (2) Prepare bacterial suspension with MOI=5 and construct macrophage infection model, as in 1.3-1.4; (3) Collect the samples 24 h later and process them in the same way as in 2.3. The samples are used for subsequent CFU, WB, PCR and ELISA experiments.

[0062] 3.4 MTB Colony Forming Unit (CFU) Count The experimental procedure is the same as in 2.6.

[0063] 3.5 Western Blot Immunoblotting The experimental procedure is the same as in 2.7.

[0064] 3.6 Statistical Analysis and Graphical Plotting The analysis method is the same as in 2.8.

[0065] 3.7 Results Analysis After confirming that the HCK gene can inhibit the growth of Mycobacterium tuberculosis, the effects of the HCK agonist (EPQpYEEIPIYL) and the HCK inhibitor (iHCK-37) were further evaluated. The results showed that 10 μM of the HCK agonist significantly inhibited the growth of Mycobacterium tuberculosis, while 0.1 μM of the HCK inhibitor significantly promoted its growth. Figure 6 Further experiments showed that the HCK agonist (EPQpYEEIPIYL) could promote LC3 expression. Figure 7 This means enhancing the autophagy function of macrophages, thereby improving their bactericidal ability; while HCK inhibitors (iHCK-37) can inhibit the expression of LC3 ( Figure 8 This reduces the autophagy function of macrophages, thereby weakening their killing effect on Mycobacterium tuberculosis.

[0066] Example 4: Mouse Model Validation 4.1 BCG strain The BCG strain (strain number: ATCC35737) was preserved by the Department of Bacteriology and Immunology, Beijing Chest Hospital.

[0067] 4.2 Preparation of BCG strain lysate The frozen BCG cells were thawed, and an appropriate amount of bacterial culture was extracted and plated onto Lowenstein-Jensen (LJ) medium for culture. BCG cells in the logarithmic growth phase were collected and lysed using Fast Prep®-245G. The supernatant obtained after centrifugation was the whole-cell lysate of BCG cells. Its concentration was measured and then stored.

[0068] 4.3 Infection of mice with BCG via tail vein injection (1) Experimental grouping ① DMSO treatment group, ② iHCK-37 treatment group, ③ EPQpYEEIPIYL treatment group, 6 animals in each group.

[0069] (2) Infecting strain BCG.

[0070] (3) Mode of infection In a negative pressure animal infection laboratory, mice were infected by tail vein injection of BCG. The specific steps involved collecting BCG cells in the logarithmic growth phase and adjusting the BCG culture to OD200. 600 = 0.5, approximately 5 × 10 7 The bacterial concentration was determined by injecting 200 μL of bacterial solution into each mouse via the tail vein. After the tail vein injection, the mice were separated and housed for subsequent experiments.

[0071] 4.4 Processing of spleen and lung tissues in mice infected with BCG (1) Count of CFU in mouse lungs Four weeks after tail vein injection in mice, six mice from each of the three groups were sacrificed. Lung tissue was isolated, placed in a cell filter, and then ground to break up the tissue. The original solution was then serially diluted to 10. -3 Then take 10 -1 10 -2 10 -3 The diluted solution was plated and CFU counted after 4 weeks of incubation.

[0072] (2) HE staining of mouse lung tissue Mouse lung tissue was fixed with 4% paraformaldehyde / general-purpose tissue fixative, embedded in paraffin, and sectioned. After dewaxing and hydration, the tissue sections were stained with hematoxylin for cell nuclei and eosin for cytoplasm. After dehydration and clearing, the sections were then mounted to observe the structure and morphology of the tissue cells.

[0073] 4.5 Results Analysis In a mouse model of BCG infection, Figure 9This study demonstrated that the HCK agonist EPQpYEEIPIYL promoted the clearance of MTB in the lungs of mice. HE staining results ( Figure 10 The results showed that inflammatory cell infiltration was reduced in the DMSO and iHCK-37 groups, while it was increased in the EPQpYEEIPIYL group. This indicates that the HCK inhibitor iHCK-37 inhibited inflammatory cell infiltration and hindered BCG clearance in mice, while the HCK agonist EPQpYEEIPIYL promoted inflammatory cell infiltration and facilitated BCG clearance in mice.

Claims

1. The use of an HCK agonist in the preparation of a drug for treating Mycobacterium tuberculosis infection, wherein the agonist is capable of inhibiting Mycobacterium tuberculosis infection, and the agonist is EPQpYEEIPIYL.

2. The application as described in claim 1, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis or a clinical isolate of Mycobacterium tuberculosis.

3. The application as described in claim 1, wherein the drug dosage form includes, but is not limited to, one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, and / or suppositories.

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