Another mycobacterium and its use in treating asthma

Abstract

The present invention provides strains of the genus Alistipes and pharmaceutical compositions comprising the strains or extracts, cultures, or processed products thereof. The strains and pharmaceutical compositions of the present invention can significantly relieve asthma, significantly inhibit the differentiation of naive T cells into Th2 cells, reduce the number of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage fluid and lung tissue, reduce lung tissue hematomas, inhibit the ratio of eosinophils to lymphocytes in bronchoalveolar lavage fluid and lung tissue, and can also inhibit the levels of IgE and HDM-specific IgE in serum and bronchoalveolar lavage fluid. Therefore, the strains of the present invention or their active extracts, cultures, or processed products can be used to prepare pharmaceutical compositions for relieving asthma, and have very broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a novel Mycobacterium strain, a pharmaceutical composition comprising the Mycobacterium strain, and use of the Mycobacterium strain or the pharmaceutical composition in treating asthma. Background Art

[0002] Alistipes is a genus of Gram-negative, obligately anaerobic bacteria in the phylum Bacteroidetes that are commensal in the intestine. They are straight or slightly curved rods with a diameter of 0.2-0.9 μm and a length of 0.5-4 μm, with rounded ends, and do not form spores. Cells usually occur alone or in pairs, occasionally appearing as longer filaments. Taxonomically, Alistipes was described in 2003 after being found in tissue samples from children with appendicitis. From an ecological perspective, Alistipes is mainly found in the intestines of healthy people. Alistipes is closely associated with many diseases. For example, in patients with compensated and decompensated cirrhosis, fresh fecal metagenomic sequences from healthy volunteers and patients with various types of cirrhosis showed that the abundance of Alistipes was increased in healthy controls compared to patients with cirrhosis. Another study showed that when comparing the fecal microbiota of patients with decompensated cirrhosis and acute hepatic encephalopathy, Alistipes had a protective effect, and its reduced abundance was associated with an increased recurrence of hepatic encephalopathy.17 Thus, a decrease in Alistipes is associated with the progression of cirrhosis to a decompensated state.

[0003] In fibrotic diseases such as nonalcoholic fatty liver disease (NAFLD), reduced abundance of Alistipes has been observed in patients with liver fibrosis. Alistipes produces acetate and propionate, and fecal acetate and propionate concentrations are reduced in patients with NAFLD and substantial fibrosis, while butyrate concentrations are not significantly different. This suggests that a reduction in Alistipes contributes to a decrease in short-chain fatty acids, potentially exacerbating advanced fibrosis in these NAFLD patients. In recent years, the role of Alistipes in disease has garnered increasing attention.

[0004] Pulmonary inflammation is an inflammatory response in the lungs caused by various stimuli, leading to pathological changes such as congestion, edema, and exudates. Allergens and other irritants are a contributing factor to lung inflammation and are also a contributing factor to asthma.

[0005] Asthma is a chronic inflammatory disease that affects hundreds of millions of people worldwide. It significantly impacts patients' daily lives and work. Acute asthma attacks can cause nighttime awakenings, limited mobility, or mood swings. Long-term asthma can lead to decreased lung function and compromise quality of life.

[0006] The therapeutic effects of probiotics on diseases have been increasingly confirmed by research. However, while existing probiotics can alleviate asthma and lung inflammation, they require synergy with conventional therapies. Currently, there are no reports of single bacteria treating asthma and lung inflammation. Therefore, there is an urgent need for effective technologies to treat asthma and lung inflammation. Summary of the Invention

[0007] The object of the present invention is to provide a probiotic that can effectively treat lung inflammation and / or asthma.

[0008] Another object of the present invention is to provide a pharmaceutical composition prepared using the probiotics of the present invention, thereby effectively treating lung inflammation and / or asthma.

[0009] Another object of the present invention is to provide a method for treating lung inflammation and / or asthma, wherein the method comprises administering a therapeutically effective amount of the probiotics or pharmaceutical composition of the present invention to a subject in need thereof.

[0010] In a first aspect, the present invention provides another strain of the genus Alistipes, wherein the strain has a 16S rRNA sequence that is at least 98.38%, 98.65%, 99%, 99.5%, 99.9% or 100% identical to the sequence shown in SEQ ID NO:1.

[0011] In a specific embodiment, the strain is Alistipes hefei.

[0012] In a specific embodiment, the strain is Alistipes hefei ibiome018, which was deposited in the China Center for Type Culture Collection (China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on March 27, 2024 with CCTCC NO: M 2024571.

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising the strain of the genus Alistipes according to the first aspect, or an extract, culture or processed product of the strain, and a pharmaceutically acceptable excipient.

[0014] In a preferred embodiment, the pharmaceutical composition is a capsule comprising the strain or an extract, culture or processed product of the strain; more preferably a microcapsule.

[0015] In a preferred embodiment, the pharmaceutical composition does not contain strains of other genera.

[0016] In a third aspect, the present invention provides the use of another Alistipes strain or an extract, culture or processed product of the strain, or a pharmaceutical composition comprising the strain or an extract, culture or processed product of the strain, in the preparation of an inhibitor of Th2 type immune response or an inhibitor of the differentiation of initial T cells into Th2 cells.

[0017] In a preferred embodiment, the strain is the strain described in the first aspect, and the pharmaceutical composition is the pharmaceutical composition described in the second aspect.

[0018] In a fourth aspect, the present invention provides use of another Alistipes strain or an extract, culture or processed product of the strain, or a pharmaceutical composition comprising the strain or an extract, culture or processed product of the strain, in the preparation of a medicament for preventing or treating lung inflammation and / or asthma.

[0019] In a specific embodiment, the lung inflammation is exogenous infection-induced lung inflammation or non-infection-induced lung inflammation.

[0020] In a specific embodiment, the infection-induced lung inflammation is caused by the following infectious agents: bacteria, viruses, fungi, mycoplasma, chlamydia.

[0021] Bacteria: Bacterial pneumonia is the most common type of pneumonia. Common bacterial pathogens include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Legionella, and Pseudomonas aeruginosa. Pneumococcus is the most common bacterial pneumonia pathogen and often causes community-acquired pneumonia. Pseudomonas aeruginosa and Staphylococcus aureus are often associated with hospital-acquired pneumonia. In addition, Mycobacterium tuberculosis can also cause lung infections, namely tuberculosis.

[0022] Virus: Viral pneumonia is also a common type of pneumonia. Common viral pathogens include influenza virus, coronavirus (such as COVID-19), parainfluenza virus, adenovirus, and respiratory syncytial virus. Influenza virus infection can cause severe pneumonia and lead to complications.

[0023] Fungi: Fungi such as Candida and Aspergillus can also cause lung infections, especially in immunocompromised patients.

[0024] Others: Mycoplasma is one of the microorganisms that cause atypical pneumonia, which usually causes mild to moderate pneumonia. Chlamydia pneumoniae is another pathogen that causes atypical pneumonia.

[0025] The non-infectious lung inflammation is caused by the following non-infectious factors: physical and chemical factors, immune factors, allergic factors, drug factors, and genetic factors.

[0026] Physical and chemical factors: Inhalation of toxic gases, foreign objects, chemicals, smoke, radiation damage, and vasculitis can all cause lung inflammation. These substances can directly damage lung tissue and trigger an inflammatory response. Inhalation of foreign objects can damage lung tissue and cause inflammation; radiation damage is caused by prolonged exposure to radiation, leading to damage and inflammation of lung tissue; and vasculitis is a disease that affects blood vessels, potentially damaging the lungs and causing inflammation.

[0027] Immune factors: including autoimmune diseases such as rheumatic diseases and systemic lupus erythematosus, which may trigger lung inflammation as part of systemic diseases.

[0028] Allergic factors: such as allergic pneumonia, which is lung inflammation caused by allergies to certain substances (dust mites, cockroaches, pollen, fungi, pet dander, catkins, oil smoke, car exhaust, cigarettes, hot and cold air, milk, eggs, seafood, peanuts, cosmetics, flavors, sesame oils, various seasonings, paint smell, etc.).

[0029] Drug factors: Certain drugs can cause lung inflammation, which is called drug-induced pneumonia. Common drugs include antibiotics, nonsteroidal anti-inflammatory drugs, chemotherapy drugs, etc. These drugs may cause inflammation by directly acting on lung tissue or causing allergic reactions.

[0030] Genetic factors: Some inherited diseases can also cause lung inflammation. For example, cystic fibrosis is an inherited disease that causes the lungs to produce mucus and become infected, leading to inflammation.

[0031] In a preferred embodiment, the strain is the strain described in the first aspect, and the pharmaceutical composition is the pharmaceutical composition described in the second aspect.

[0032] In a preferred embodiment, the pharmaceutical composition or drug can achieve one or more of the following effects:

[0033] 1) Inhibit the differentiation of initial T cells into Th2 cells;

[0034] 2) reduce the total cell count, eosinophil count, and lymphocyte count in the subjects' bronchoalveolar lavage fluid;

[0035] 3) Reduce the total cell count, eosinophil count and lymphocyte count in the subject's tissues;

[0036] 4) reducing lung tissue hematoma in subjects;

[0037] 5) Suppressing the ratio of eosinophils and lymphocytes in the subjects' bronchoalveolar lavage fluid;

[0038] 6) Suppressing the ratio of eosinophils and lymphocytes in the lung tissue of the subject;

[0039] 7) Suppressing the levels of total IgE and HDM-specific IgE in the serum and bronchoalveolar lavage fluid of the subjects.

[0040] In a fifth aspect, the present invention provides another strain of the genus Alistipes, or an extract, culture or processed product of the strain, or a pharmaceutical composition comprising the strain or an extract, culture or processed product of the strain, for preventing or treating lung inflammation and / or asthma.

[0041] In a specific embodiment, the lung inflammation is exogenous infection-induced lung inflammation or non-infection-induced lung inflammation.

[0042] In a specific embodiment, the infection-induced lung inflammation is caused by the following infectious agents: bacteria, viruses, fungi, mycoplasma, chlamydia.

[0043] Bacteria: Bacterial pneumonia is the most common type of pneumonia. Common bacterial pathogens include Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Legionella, and Pseudomonas aeruginosa. Pneumococcus is the most common bacterial pneumonia pathogen and often causes community-acquired pneumonia. Pseudomonas aeruginosa and Staphylococcus aureus are often associated with hospital-acquired pneumonia. In addition, Mycobacterium tuberculosis can also cause lung infections, namely tuberculosis.

[0044] Virus: Viral pneumonia is also a common type of pneumonia. Common viral pathogens include influenza virus, coronavirus (such as COVID-19), parainfluenza virus, adenovirus, and respiratory syncytial virus. Influenza virus infection can cause severe pneumonia and lead to complications.

[0045] Fungi: Fungi such as Candida and Aspergillus can also cause lung infections, especially in immunocompromised patients.

[0046] Others: Mycoplasma is one of the microorganisms that cause atypical pneumonia, which usually causes mild to moderate pneumonia. Chlamydia pneumoniae is another pathogen that causes atypical pneumonia.

[0047] The non-infectious lung inflammation is caused by the following non-infectious factors: physical and chemical factors, immune factors, allergic factors, drug factors, and genetic factors.

[0048] Physical and chemical factors: Inhalation of toxic gases, foreign objects, chemicals, smoke, radiation damage, and vasculitis can all cause lung inflammation. These substances can directly damage lung tissue and trigger an inflammatory response. Inhalation of foreign objects can damage lung tissue and cause inflammation; radiation damage is caused by prolonged exposure to radiation, leading to damage and inflammation of lung tissue; and vasculitis is a disease that affects blood vessels, potentially damaging the lungs and causing inflammation.

[0049] Immune factors: including autoimmune diseases such as rheumatic diseases and systemic lupus erythematosus, which may trigger lung inflammation as part of systemic diseases.

[0050] Allergic factors: such as allergic pneumonia, which is lung inflammation caused by allergies to certain substances (dust mites, cockroaches, pollen, fungi, pet dander, catkins, oil smoke, car exhaust, cigarettes, hot and cold air, milk, eggs, seafood, peanuts, cosmetics, flavors, sesame oils, various seasonings, paint smell, etc.).

[0051] Drug factors: Certain drugs can cause lung inflammation, which is called drug-induced pneumonia. Common drugs include antibiotics, nonsteroidal anti-inflammatory drugs, chemotherapy drugs, etc. These drugs may cause inflammation by directly acting on lung tissue or causing allergic reactions.

[0052] Genetic factors: Some inherited diseases can also cause lung inflammation. For example, cystic fibrosis is an inherited disease that causes the lungs to produce mucus and become infected, leading to inflammation.

[0053] In a specific embodiment, the strain is the strain described in the first aspect, and the pharmaceutical composition is the pharmaceutical composition described in the second aspect.

[0054] In a preferred embodiment, the strain or the strain's extract, culture, processed product, or pharmaceutical composition can achieve one or more of the following effects:

[0055] 1) Inhibit the differentiation of initial T cells into Th2 cells;

[0056] 2) reduce the total cell count, eosinophil count, and lymphocyte count in the subjects' bronchoalveolar lavage fluid;

[0057] 3) reducing the total cell count, eosinophil count, and lymphocyte count in the subjects' lung tissue;

[0058] 4) reducing lung tissue hematoma in subjects;

[0059] 5) Suppressing the ratio of eosinophils and lymphocytes in the subjects' bronchoalveolar lavage fluid;

[0060] 6) Suppressing the ratio of eosinophils and lymphocytes in the lung tissue of the subject;

[0061] 7) Suppressing the levels of total IgE and HDM-specific IgE in the serum and bronchoalveolar lavage fluid of the subjects.

[0062] In a sixth aspect, the present invention provides a method for preventing or treating lung inflammation and / or asthma, comprising administering a therapeutically or prophylactically effective amount of a strain of the genus Alistipes, or an extract, culture or processed product of the strain, or a pharmaceutical composition comprising the strain, or an extract, culture or processed product of the strain, to a subject in need thereof.

[0063] In a preferred embodiment, the strain is the strain described in the first aspect, and the pharmaceutical composition is the pharmaceutical composition described in the second aspect.

[0064] In a preferred embodiment, the subject is a mammal; preferably a human.

[0065] In a preferred embodiment, the method for preventing or treating lung inflammation and / or asthma can achieve one or more of the following effects:

[0066] 1) Inhibit the differentiation of initial T cells into Th2 cells;

[0067] 2) reduce the total cell count, eosinophil count, and lymphocyte count in the subjects' bronchoalveolar lavage fluid;

[0068] 3) reducing the total cell count, eosinophil count, and lymphocyte count in the subjects' lung tissue;

[0069] 4) reducing lung tissue hematoma in subjects;

[0070] 5) Suppressing the ratio of eosinophils and lymphocytes in the subjects' bronchoalveolar lavage fluid;

[0071] 6) Suppressing the ratio of eosinophils and lymphocytes in the lung tissue of the subject;

[0072] 7) Suppressing the levels of total IgE and HDM-specific IgE in the serum and bronchoalveolar lavage fluid of the subjects.

[0073] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 A smear microscopic image (40X) of another mycobacterium of the present invention is shown;

[0075] Figure 2 The figure shows the colony morphology of another mycobacterium of the present invention on solid culture medium;

[0076] Figure 3 The results of the sodium chloride tolerance test of another mycobacterium of the present invention are shown;

[0077] Figure 4 Shows the temperature tolerance test results of another mycobacterium of the present invention;

[0078] Figure 5 The results of the pH tolerance experiment of another mycobacterium of the present invention are shown;

[0079] Figure 6 The bile salt tolerance test results of another mycobacterium of the present invention are shown;

[0080] Figure 7 The figure shows the effect of the mycobacterium of the present invention on the differentiation of T helper 2 cells (Th2), * indicates P < 0.05;

[0081] Figure 8 The results show the effect of the mycobacterium of the present invention on the cell count of mouse bronchoalveolar lavage fluid, * indicates P < 0.05;

[0082] Figure 9 The figure shows the effect of the mycobacterium of the present invention on the number of cells in the mouse lungs, ** indicates P < 0.01;

[0083] Figure 10 The results show the effect of the mycobacterium of the present invention on the pulmonary hematoma of mice;

[0084] Figure 11 The figure shows the effect of the mycobacterium of the present invention on the eosinophil ratio in the alveolar lavage fluid of mice, ** indicates P < 0.01;

[0085] Figure 12 The figure shows the effect of the mycobacterium of the present invention on the lymphocyte ratio in the alveolar lavage fluid of mice, ** indicates P < 0.01;

[0086] Figure 13 The figure shows the effect of the mycobacterium of the present invention on the proportion of eosinophils in the lungs of mice, ** indicates P < 0.01;

[0087] Figure 14 The figure shows the effect of the mycobacterium of the present invention on the proportion of lymphocytes in the lungs of mice, ** indicates P < 0.01;

[0088] Figure 15The figure shows the effects of the mycobacterium of the present invention on the levels of HDM-specific IgE and total IgE in the serum and bronchoalveolar lavage fluid of mice, and ** indicates P < 0.01. DETAILED DESCRIPTION

[0089] After extensive and in-depth research, the inventors unexpectedly discovered that Mycobacterium can strongly alleviate lung inflammation and asthma, and thus can be used to prepare a pharmaceutical composition for relieving lung inflammation and asthma. Based on this, the present invention was completed.

[0090] the term

[0091] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0092] As used herein, the terms “contain,” “have,” or “include” include “comprise,” “mainly consist of,” “substantially consist of,” and “consist of”; “mainly consist of,” “substantially consist of,” and “consist of” are subordinate concepts of “contain,” “have,” or “include.”

[0093] Lung inflammation

[0094] The term "pulmonary inflammation," as used herein, refers to an inflammatory response in lung tissue. This response, caused by various stimuli, leads to pathological changes such as congestion, edema, and exudates. This inflammatory response is a defensive response by the body to external stimuli, designed to eliminate pathogens, foreign matter, or damaged tissue, while also promoting the repair and regeneration of damaged tissue.

[0095] Typically, lung inflammation can cause the subjects to differentiate from initial T cells into Th2 cells; increase the total cell count, eosinophil count and lymphocyte count in the alveolar lavage fluid or lung tissue; increase the ratio of eosinophils and lymphocytes in the alveolar lavage fluid or lung tissue; hematoma in the lung tissue; when lung inflammation is caused by HDM allergens, it can also cause increased levels of total IgE and HDM-specific IgE in serum and bronchoalveolar lavage fluid.

[0096] Those skilled in the art will appreciate that lung inflammation can be caused by various factors, such as exogenous infection or non-infection. In a specific embodiment, the infection is caused by the following infectious agents: bacteria, viruses, fungi, mycoplasmas, or chlamydia. In another specific embodiment, the non-infection-induced lung inflammation is caused by the following non-infectious factors: physical and chemical factors, immune factors, allergic factors, drug factors, or genetic factors.

[0097] Asthma and its treatment

[0098] The term "asthma" as used herein has the meaning conventionally understood by those skilled in the art. It refers to a chronic inflammatory disease. Asthma is characterized by airway hyperresponsiveness, which leads to recurrent symptoms such as wheezing, coughing and chest tightness. Asthma is induced by a variety of factors, including environmental factors, genetic factors and immune factors. Among them, genetic factors refer to the fact that asthma has obvious familial clustering. If a first-degree relative has asthma, the probability of an individual suffering from asthma will increase significantly. Environmental factors refer to allergens (such as pollen, dust mites, animal fur, etc.), occupational exposure (such as chemicals, grain dust, etc.), air pollution, smoking, etc. Immune factors refer to abnormal reactions of the immune system that may lead to airway inflammation and airway hyperresponsiveness, thereby triggering asthma. The main symptoms of the disease are wheezing, coughing, chest tightness, and difficulty breathing.

[0099] Asthma involves a variety of immune cells, especially in the lungs. These cells include eosinophils, lymphocytes, neutrophils, and dendritic cells, as well as airway structural cells such as airway smooth muscle cells and epithelial cells. Eosinophils are a type of white blood cell that can participate in the body's immune response. When bronchial asthma attacks, the number of eosinophils increases, and they are involved in processes such as airway hyperresponsiveness, immune regulation, and airway remodeling. Programmed necrosis of eosinophils stimulates goblet cells and smooth muscle cells, leading to hypersecretion of mucus and airway hyperresponsiveness. In addition, cytotoxic basic proteins and eosinophil peroxidase released by eosinophils can damage the airway epithelium. Dendritic cells and neutrophils also play an important role during asthma attacks.

[0100] Dendritic cells are antigen-presenting cells that can take up and process antigens, including allergens such as pollen and dust mites, and then present them to T cells, triggering an immune response. During an asthma attack, neutrophils accumulate in the airways and participate in both inflammatory and immune responses. Neutrophil numbers increase in the sputum and bronchoalveolar lavage fluid of asthma patients. These neutrophils can release inflammatory mediators and proteases, leading to airway inflammation and airway smooth muscle spasm, thus triggering the clinical symptoms of asthma.

[0101] Currently, more and more studies have confirmed the therapeutic effect of probiotics on asthma. The role of probiotics in treating asthma is mainly through improving the intestinal flora, improving the balance of human immune cells, promoting Th1 immune response, inhibiting Th2 immune response, and reducing the secretion of IgE by B cells, thereby alleviating patients' allergic reactions and reducing symptoms such as cough. For example, the Bifidobacterium animalis subsp. lactis Probio-M8 (Probio-M8) developed by the Ketuo Biological Research and Development Team is a strain isolated from healthy breast milk. It has good tolerance to gastrointestinal digestive juices. The survival rate after digestion for 3 hours in artificial gastric juice at pH 2.5 is 85.38%, and the survival rate after digestion for 8 hours in artificial intestinal juice at pH 8.0 is 97.25%. This shows that Bifidobacterium lactis Probio-M8 can enter the human intestine in a living state and survive in the gastrointestinal organs, thereby exerting health benefits. Serum metabolomics changed significantly after 3 months of intervention with Probio-M8 and conventional therapy. The levels of metabolites such as dodecanoic acid, enterodiol, tryptophan and sphingomyelin in the blood were significantly increased compared with the placebo group. During the intervention period, the levels of docosahexaenoic acid, oleoylethanolamide, erythronic acid, sphingomyelin and cholesterol esters in the subjects' intestines increased. The results showed that Probio-M8 and conventional therapy acted synergistically to regulate intestinal flora through the gut-lung axis, promote the synthesis and secretion of key bioactive compounds in the blood, enhance host immunity and thus improve the clinical efficacy of asthma patients. The discovery and research of Probio-M8 not only provides new ideas for the treatment of asthma, but also further confirms the close connection between intestinal flora and immune health.

[0102] In conclusion, probiotics have broad application prospects in the treatment of asthma. However, there are currently no reports on the use of a single bacterium to treat asthma.

[0103] The strain of the present invention

[0104] To address the shortcomings of the existing technologies, the present invention provides another strain of the genus Alistipes. This strain, even as a single bacterium, has a strong asthma-relieving effect and can inhibit the proportion and number of eosinophils, alveolar macrophages, T cells, B cells, and dendritic cells. It can also suppress the levels of IgE and HDM-specific IgE in serum and bronchoalveolar lavage fluid, thereby improving lung inflammation.

[0105] Therefore, the Mycobacterium strains of the present invention can be used as microbial agents instead of drugs for the treatment of asthma and lung inflammation.

[0106] In a specific embodiment, the present invention provides another strain of the genus Alistipes, wherein the strain has a 16S rRNA sequence that is at least 98.38%, 98.65%, 99%, 99.5%, 99.9% or 100% identical to the sequence shown in SEQ ID NO:1.

[0107] In a preferred embodiment, the strain is another branch of Alistipes hefei; more preferably another branch of Alistipes hefei ibiome018, which was deposited in the China Center for Type Culture Collection (China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province) with CCTCC NO: M2024571 on March 27, 2024.

[0108] In a specific embodiment, the present invention provides another strain of the genus Alistipes, wherein the 16S rRNA gene of the strain is shown as SEQ ID NO: 1.

[0109] In a preferred embodiment, the strain was deposited in the China Center for Type Culture Collection on March 27, 2024 with CCTCC NO: M 2024571 as the deposit number, and the deposit address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and its classification name is Alistipes hefei ibiome018.

[0110] Pharmaceutical composition

[0111] Based on the Alistipes strain provided by the present invention, those skilled in the art will understand that the strain or the active extract, culture or processed product of the strain can be prepared into a pharmaceutical composition to prevent or treat lung inflammation and / or asthma.

[0112] In a specific embodiment, the pharmaceutical composition comprises the Alistipes strain or an extract, culture or processed product of the strain and a pharmaceutically acceptable excipient.

[0113] Throughout this specification, the term "extract" refers to a specific component or components isolated and extracted from bacteria. These components may include proteins, enzymes, metabolites, DNA, RNA, and others, and are used in various research and applications, such as drug development and biochemical research. The extraction process may involve steps such as cell disruption, centrifugation, filtration, and purification to obtain the desired high-purity components.

[0114] In this specification, the term "culture" refers to a population of Alistipes suspended in a culture medium under conditions suitable for the survival and / or growth of Alistipes. As will be apparent to those skilled in the art, in some aspects, these terms as used herein refer to a combination of a population of Alistipes and a culture medium in which the population is suspended. On the other hand, these terms as used herein also refer to the culture supernatant and culture components obtained after the cultivation of the Alistipes of the present invention is completed. In the present invention, the culture includes, but is not limited to, a bacterial solution, culture supernatant, or bacterial culture medium obtained by inoculating or transplanting Alistipes into a culture medium of any form (liquid or solid).

[0115] In this specification, the term "processed product" is not particularly limited as long as it is derived from a culture, and can be obtained by, for example, concentration, gelatinization, spray drying, freeze drying, vacuum drying, drum drying, liquefaction, dilution, pulverization, etc. of the culture. For these processes, known methods can be used as appropriate.

[0116] In the present invention, the microbial strain of the present invention in the culture or the processed product may be a living bacteria or a dead bacteria.

[0117] In this specification, the term "strain" may be a strain directly cultured from a deposited strain, or may be a progeny strain (offspring) or a strain cultured from the original strain (subclone strain).

[0118] Based on the teachings of the present invention and common knowledge in the art, those skilled in the art will appreciate that the pharmaceutical composition of the present invention can be formulated into a formulation suitable for various routes of administration. In a preferred embodiment, the pharmaceutical composition of the present invention can be formulated into an oral formulation, such as a capsule. To enhance the therapeutic efficacy of probiotics, the probiotics or their active extracts can be encapsulated in tiny capsules using microencapsulation technology to protect the probiotics from surviving and colonizing in the intestinal tract, thereby enhancing the therapeutic efficacy of the probiotics.

[0119] The pharmaceutical composition of the present invention may include a preventive or therapeutically effective amount of the strain of the present invention or an extract, culture or processed product of the strain. The effective amount is an amount sufficient to improve or alleviate in some way the symptoms associated with lung inflammation or asthma. Such an amount can be administered as a single dose, or can be administered according to an effective treatment regimen. The dosage may cure lung inflammation and / or asthma, but administration may also generally be for improving the symptoms of lung inflammation and / or asthma, such as significantly alleviating the symptoms of wheezing, coughing, chest tightness, dyspnea, etc., during an asthma attack, and significantly alleviating the inflammatory indicators of the subject. The dosage administered can be determined by a clinician based on the patient's age, health and weight, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.

[0120] The pharmaceutical preparation of the present invention can be administered to any mammal as long as they can obtain the therapeutic effect of the compound of the present invention. Among these mammals, the most important one is human.

[0121] The pharmaceutical compositions of the present invention can be manufactured in a known manner. Pharmaceutical compositions of the present invention include pharmaceutically acceptable excipients, such as sugars such as lactose or sucrose, mannitol or sorbitol; cellulose preparations or calcium phosphates, such as tricalcium phosphate or dibasic calcium phosphate; and binders, such as starch pastes, including corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone. If necessary, disintegrants such as the starches mentioned above, as well as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added. Auxiliary agents are particularly flow regulators and lubricants, such as silica, talc, stearates, such as magnesium calcium stearate, stearic acid, or polyethylene glycol. If necessary, the tablet cores can be provided with a suitable coating that is resistant to gastric juices. For this purpose, concentrated sugar solutions can be used. This solution may contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and / or titanium dioxide, a lacquer solution and a suitable organic solvent or solvent mixture. To prepare a coating resistant to gastric juices, a suitable cellulose solution may be used, for example, cellulose acetate phthalate or hydroxypropyl methylcellulose phthalate. Dyes or pigments may be added to the coating of the tablet or lozenge core, for example, for identification or to characterize the combination of active ingredient doses.

[0122] In a preferred embodiment, the pharmaceutical composition of the present invention can be prepared into capsules; preferably microcapsules.

[0123] Methods of preventing or treating lung inflammation and / or asthma

[0124] Based on the strain or pharmaceutical composition of the present invention, the present invention provides a method for preventing or treating pulmonary inflammation and / or asthma, comprising administering a therapeutically or prophylactically effective amount of the strain of the present invention, an extract of the strain, or the pharmaceutical composition to a subject in need thereof. The subject can be any mammal, but is preferably a human.

[0125] The dosage administered during treatment can be determined by the clinician based on the subject's age, health and weight, type of concurrent treatment, frequency of treatment, and desired therapeutic benefit.

[0126] Advantages of the present invention:

[0127] 1. This invention discovered for the first time a new species of Mycobacterium spp., Alistipes fei ibiome018;

[0128] 2. The Mycobacterium strain of the present invention significantly alleviates lung inflammation and asthma in subjects, significantly inhibits the differentiation of naive T cells into Th2 cells, reduces the number of total cells, eosinophils, and lymphocytes in bronchoalveolar lavage fluid and lung tissue, reduces lung tissue hematoma, inhibits the ratio of eosinophils to lymphocytes in bronchoalveolar lavage fluid and lung tissue, and inhibits the levels of total IgE and HDM-specific IgE in serum and bronchoalveolar lavage fluid;

[0129] 3. The Mycobacterium strains of the present invention can be used as single bacteria for treatment;

[0130] 4. The Mycobacterium strain of the present invention can be used to prepare pharmaceutical compositions for relieving lung inflammation and asthma, and has very broad application prospects.

[0131] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0132] Example

[0133] Example 1. Isolation and identification of bacterial species

[0134] 1. Separation

[0135] Alistipes hefei ibiome018 was isolated from a fecal sample of a healthy Han Chinese female volunteer in Hefei, Anhui Province, who had not taken antibiotics for the previous three months. Normal saline was dispensed into sterile 15ml centrifuge tubes in a biosafety cabinet. FAB blood plates (purchased from Solarbio, Cat. No. LA4550) and sterile saline were transferred to an anaerobic workbench 24 hours in advance.

[0136] Fecal samples from healthy volunteers were preserved in 80% volume fraction sterile glycerol buffer. The fecal samples were then diluted to 10-10 in an anaerobic workstation. 0.1 ml of the diluted bacterial solution was spread on FAB blood plates and cultured at 37°C in an anaerobic workstation for 72 hours. Single colonies were picked and cultured in GAM broth liquid culture medium.

[0137] 2. Identification

[0138] 2.1 16S rRNA sequencing

[0139] Alistipes hefei ibiome018 was amplified by PCR using 16S rRNA universal primers (upstream primer 27F: AGAGTTTG ATCCTGGCTCAG (SEQ ID NO: 2), downstream primer 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 3)).

[0140] Experimental methods:

[0141] PCR system (20 μL): 2×Taq Master Mix: 10 μL; Primer 1 (27F): 1 μL; Primer 2 (1492R): 1 μL; ddH2O: 7 μL; Bacterial solution: 1 μL.

[0142] PCR reaction program: 95°C for 10 min; 95°C for 15 s, 58°C for 30 s, 72°C for 40 s, step 2-4 30-35×; 72°C for 5 min.

[0143] The PCR product of the 16S rRNA gene was sequenced, and the result was shown as SEQ ID NO: 1.

[0144]

[0145] Comparison showed that the similarity with the species with the highest sequence similarity was only 98.37%, which is lower than the 98.65% generally recognized in the field (Kim M, et al. Towards a taxonomic coherence between average nucleotide identity and 16S rRNA gene sequence similarity for species demarcation of prokaryotes. International Journal of Systematic and Evolutionary Microbiology, 2014 Feb; 64(Pt 2): 346-351. doi: 10.1099 / ijs.0.059774-0.).

[0146] 2.2 Smear microscopy

[0147] The ibiome018 was smeared and examined under a microscope at 40X. Figure 1 As shown in the microscopic examination image, ibiome018 is Gram-negative, short rod-shaped or spherical, without spores or flagella.

[0148] 2.3 Single colony photos

[0149] After 72 hours of anaerobically culturing ibiome018 on FAB medium, photos of single colonies were taken. Figure 2 As shown, the colonies are white, round, with neat edges and a moist surface.

[0150] 2.4 Catalase test and sugar alcohol fermentation biochemical reaction detection

[0151] Liquid cultured ibiome018 was streaked onto FAB solid medium for purification, and single colonies were picked for catalase assay and sugar alcohol fermentation biochemical reaction detection.

[0152] Catalase test: Add 2-3 drops of catalase reaction reagent (purchased from Qingdao Haibo Biological, product number HB8650) to the ibiome018 colony. The result shows bubbles, which is positive. However, the catalase of the model bacteria Alistipes finegoldii, Alistipes sonderdonkii, Alistipes shahii, Alistipes ihummi, and Alistipes inops reported in the literature (Parker BJ, et al. The Genus Alistipes: Gut Bacteria With Emerging Implications to Inflammation, Cancer, and Mental Health. Front Immunol. 2020 Jun 9; 11: 906. doi: 10.3389 / fimmu.2020.00906. The same below) were all negative.

[0153] Sugar alcohol fermentation biochemical reaction detection: 60 μL of bacterial solution was added to commercial bacterial biochemical detection ampoules (purchased from Qingdao Haibo Biological, product numbers GB007, GB014, GB054, GB055, GB056, GB057, GB060, GB062, GB102-1, GB112, GB177, GB178, GB188, GB195, GB196, GB199, GB200, GB202, GB203, GS001, GB033, GS004), inoculated and placed in 37 ° C anaerobically for 48 h, and the test results were judged according to the kit instructions, as shown in Table 1:

[0154] Table 1. Biochemical identification results of Alistipes hefei ibiome018

[0155]

[0156]

[0157] Note: + indicates positive; - indicates negative.

[0158] 3. Sodium chloride, temperature, pH, and bile salt tolerance tests

[0159] 3.1 Sodium chloride tolerance test

[0160] Take the liquid culture ibiome018 with a 10% inoculation volume for sodium chloride tolerance test, and take 200 μL of bacterial solution at regular intervals to measure OD using a microplate reader. 600 The results are as follows Figure 3As shown, the bacteria can grow in a 1% NaCl concentration.

[0161] 3.2 Temperature tolerance test

[0162] Take the liquid culture of ibiome018 with a 10% inoculation volume for temperature tolerance test, and take 200 μL of bacterial solution at regular intervals to measure OD using a microplate reader. 600 The results are as follows Figure 4 As shown in the figure, the bacteria can grow in the temperature range of 30℃-42℃, and the optimal growth temperature is 37℃.

[0163] 3.3 pH tolerance test

[0164] Take the liquid culture of ibiome018 with a 10% inoculation volume for pH tolerance test, and take 200 μL of bacterial solution at regular intervals to measure OD using a microplate reader. 600 The results are as follows Figure 5 As shown, the bacteria can grow in the pH range of 7-8, and the optimal growth pH is 7-8.

[0165] 3.4 Bile salt tolerance test

[0166] Take the liquid culture of ibiome018 with a 10% inoculation volume for bile salt tolerance test, and take 200 μL of bacterial solution at regular intervals to measure OD using a microplate reader. 600 The results are as follows Figure 6 As shown in the results, the bacterium can tolerate 0.05% bile salts, while the model bacteria Alistipes putredinis and Alistipes indistinctus reported in the literature do not have the ability to tolerate bile salts.

[0167] Example 2: Mycobacterium inhibits the differentiation of Th2 cells

[0168] The spleen of the IL4-FITC fluorescent reporter mouse was removed, ground under sterile conditions, and 2 mL of red blood cell lysis buffer was added. After lysis at room temperature for 2 minutes, the pellet was centrifuged at 500 g for 5 minutes. The pellet was resuspended in 1 mL of Macs Buffer and filtered through a 70 μM cell filter. TM Mouse CD4 T Cell Isolation Kit for CD4 + For T cell sorting, add 15 μL of antibody. After 15 minutes, add 15 μL of beads and place the tube in a magnet for 5 minutes. After MACS sorting, remove the supernatant and count.

[0169] Th2 cell differentiation conditions are shown in Table 2:

[0170] Table 2

[0171] cytokines concentration Anti-IFNγ 5 μg / mL IL2 5ng / mL IL4 20 ng / mL Anti-CD3ε 2 μg / mL Anti-CD28 1 μg / mL

[0172] The cells were divided into two groups, a control group and an experimental group. 5% GAM culture medium was added to the control group, and 5% supernatant of Mycobacterium spp. was added to the experimental group. After 4 days of cell differentiation, the proportion of IL4 was detected by flow cytometry. The results are shown in Figure 2. Figure 7 As shown in the figure, 5% supernatant of Mycobacterium spp. can significantly inhibit the differentiation of Th2 and reduce the level of IL4 + The proportion of Th2.

[0173] Example 3. Mycobacterium reduces the number of cells in bronchoalveolar lavage fluid of HDM-induced asthmatic mice

[0174] C57BL / 6J (10 weeks, male) mice, SPF grade, were selected and purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. After the mice adapted for one week, they were given ordinary sterilized feed. HDM-induced asthma modeling: The mice were anesthetized by intraperitoneal injection of pentobarbital with an insulin syringe. After sufficient anesthesia, the mice were grasped by the neck and turned over with the nose tip facing down. A 20-microliter pipette was used to draw up the prepared HDM nasal drops, 25 μg of HDM was dripped into the nose of each mouse, and 10 μg of HDM was dripped into the nose of each mouse after 7 days, and the nasal drops were continued for 5 days. On the 14th day, the alveolar lavage fluid and lung tissue of the mice were collected for testing. The experiment was divided into three groups:

[0175] (1) Vehicle group: On Day 0, 25 μL of PBS (25 μg / mouse) was dripped into the nose of each mouse. From Day 7 to Day 11, 10 μL of PBS (10 μg / mouse) was dripped into the nose every day. On Day 11, the mice were sacrificed for analysis.

[0176] (2) HDM group: On Day 0, each mouse was intranasally instilled with 25 μL of HDM solution (containing 25 μg HDM), and on Day 7-Day 11, 10 μL of HDM solution (containing 10 μg HDM) was intranasally instilled every day. On Day 11, the mice were sacrificed for analysis.

[0177] (3) HDM+ Mycobacterium group: Based on the HDM group, ibiome018 bacterial solution was gavaged every two days (10 9 CFU).

[0178] After centrifugation of the alveolar lavage fluid, the cell pellet was obtained. After mincing the lung tissue, it was placed in a centrifuge tube containing RPMI 1640 (1mg / mL Collagenase I, 10% FBS) and digested at 37°C and 220rpm for 1h. The digested lung tissue was vigorously shaken for 20 seconds, 25mL PBS was added to terminate the digestion, and after mixing, it was filtered through a steel mesh (without grinding) and transferred to a centrifuge tube and centrifuged at 2200rpm for 10min. The supernatant was aspirated, 1mL of red blood cell lysis solution was added to lyse the red blood cells for 2min, 10mL of PBS was added to terminate the reaction, and the tube was centrifuged at 2200rpm for 10min. After labeling with Siglecf-APC, CD11c-P7, and CD3 / CD19-FITC flow cytometry antibodies, the tube was tested on the machine. The results are as follows. Figure 8 As shown in the results, oral administration of Mycobacterium spp. could significantly reduce the number of total cells, eosinophils and lymphocytes in the alveolar lavage fluid of mice.

[0179] Example 4. Mycobacterium reduces the number of lung tissue cells in HDM-induced asthmatic mice

[0180] The method is the same as in Example 3. The results are as follows Figure 9 As shown in the results, oral administration of Mycobacterium spp. could significantly reduce the number of total cells, eosinophils and lymphocytes in the lung tissue of mice.

[0181] Example 5. Mycobacterium alleviates lung hematoma in HDM-induced asthmatic mice

[0182] The mouse lungs were taken and photographed. Figure 10 As shown in the figure, it can be seen that after HDM-induced asthma, obvious hematoma appeared in the lungs of mice, and the hematoma in the lungs of mice was alleviated after oral administration of Mycobacterium.

[0183] Example 6. Mycobacterium spp. reduces the proportion of eosinophils in bronchoalveolar lavage fluid of asthmatic mice

[0184] After obtaining cells from mouse bronchoalveolar lavage fluid, they were labeled with Siglecf and CD11c flow cytometry antibodies and then detected using a flow cytometer. Figure 11 As shown in the results, HDM modeling significantly promoted the proportion of eosinophils, while oral administration of Mycobacterium inhibited the proportion of eosinophils.

[0185] Example 7. Mycobacterium spp. reduces the lymphocyte ratio in bronchoalveolar lavage fluid of asthmatic mice

[0186] After obtaining cells from mouse bronchoalveolar lavage fluid, they were labeled with CD3 / CD19 and CD11c flow cytometry antibodies and then detected using a flow cytometer. Figure 12As shown in the results, HDM modeling significantly promoted the proportion of lymphocytes, while oral administration of Mycobacterium inhibited the proportion of lymphocytes.

[0187] Example 8. Mycobacterium spp. reduces the proportion of eosinophils in the lung tissue of asthmatic mice

[0188] After obtaining cells from mouse lung tissue, they were labeled with Siglecf and CD11c flow cytometry antibodies and then detected using a flow cytometer. Figure 13 As shown in the results, HDM modeling significantly promoted the proportion of eosinophils, while oral administration of Mycobacterium spp. inhibited the proportion of eosinophils.

[0189] Example 9. Mycobacterium spp. reduces the lymphocyte ratio in the lung tissue of asthmatic mice

[0190] After obtaining mouse lung tissue cells, they were labeled with CD3 / CD19 and CD11c flow cytometry antibodies and then detected using a flow cytometer. Figure 14 As shown in the results, HDM modeling significantly promoted the proportion of lymphocytes, while oral administration of Mycobacterium inhibited the proportion of lymphocytes.

[0191] Example 10. Mycobacterium serovar inhibits total IgE and HDM-specific IgE levels in serum and bronchoalveolar lavage fluid of asthmatic mice

[0192] Bronchoalveolar lavage fluid was obtained from mice and the supernatant was collected by centrifugation. Blood was collected from the eye sockets and centrifuged at 3500 rpm for 15 minutes at room temperature to obtain mouse serum. Total IgE and HDM-specific IgE levels were measured using ELISA kits. The results are shown in Figure 2. Figure 15 As shown in the results, HDM modeling significantly promoted the levels of total IgE and HDM-specific IgE, while oral administration of Mycobacterium spp. significantly inhibited the levels of total IgE and HDM-specific IgE in bronchoalveolar lavage fluid and serum.

[0193] In summary, the Mycobacterium strains of the present invention can effectively inhibit the differentiation of naive T cells into Th2 cells; reduce the total cell count, eosinophil count, and lymphocyte count in the alveolar lavage fluid and lung tissue of mice; alleviate lung tissue hematomas in mice; suppress the eosinophil-to-lymphocyte ratio in the alveolar lavage fluid and lung tissue of mice; and simultaneously suppress the levels of total IgE and HDM-specific IgE in the serum and alveolar lavage fluid of mice. Therefore, the Mycobacterium strains of the present invention have excellent therapeutic and / or preventive effects on both pulmonary inflammation and asthma.

[0194] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A strain of the genus Alistipes, the strain being another Alistipes strain Alistipes hefei )ibiome018, deposited in the China Center for Type Culture Collection on March 27, 2024 with CCTCC NO: M 2024571.

2. A pharmaceutical composition comprising the strain of the genus Alistipes or a culture thereof according to claim 1, and a pharmaceutically acceptable excipient.

3. Use of the strain of the genus Alistipes according to claim 1 or the culture of the strain or the pharmaceutical composition according to claim 2 in the preparation of a medicament for preventing or treating hypersensitivity pneumonitis and / or allergic asthma.

4. The use according to claim 3, characterized in that The use is in at least one of the following aspects (a) to (d): (a) inhibiting Th2 cell differentiation, (b) decrease in bronchoalveolar lavage fluid: total cell count, eosinophil ratio, lymphocyte ratio and / or total IgE level, (c) Reduction of: total cell count, eosinophil ratio, lymphocyte ratio and / or total IgE level in lung tissue, (d) Reduce lung tissue hematoma.

5. The use according to claim 3, characterized in that The purpose is to: Reduce HDM-specific IgE levels in bronchoalveolar lavage fluid; or Reduced HDM-specific IgE levels in lung tissue.

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