A giant spherical bacteria strain and its application

Abstract

The present invention provides a Megasphaera strain, including Megasphaera and its application. The application includes enhancing immune system function, treating cancer, and alleviating immune-related side effects caused by cancer immunotherapy.

Description

[0001] Depository Information

[0002] The bacterium XA-511 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2021. It is classified and named as Megasphaera, and the deposit number is CGMCC No. 23400. Technical Field

[0003] The present invention belongs to the field of microorganisms, and particularly relates to a bacterium of the genus Megasphaera and its applications. Background Art

[0004] The human microbiota is rich and diverse, mainly existing in the body and on the body surface. Research shows that the human microbiota has great potential and is directly or indirectly involved in the processes of health and disease by affecting the body's metabolic functions, resisting pathogens, and the immune system.

[0005] The gut microbiota is an indispensable part of the host's digestion and nutrient production. They can produce nutrients from substances that are difficult for the host to digest. The components secreted by the microbiota can not only prevent the colonization of foreign pathogens but also support intestinal repair by promoting cell proliferation and differentiation. For example, the microbiota can produce and release short-chain fatty acids from dietary fiber that is difficult for the human body to digest and absorb, which is an important energy source for the intestinal mucosa and is crucial for regulating intestinal immune responses and tumorigenesis. The interaction between the microbiota and the host immune system is a complex, two-way dynamic process. More and more research has begun to focus on the impact of the microbiota on immune homeostasis inside and outside the gut, so as to explore the pathogenesis and treatment of diseases. Summary of the Invention

[0006] The present invention provides a bacterium of the genus Megasphaera with a deposit number of CGMCC No. 23400.

[0007] The present invention also provides a bacterium of the genus Megasphaera, the 16S rRNA gene of which comprises the sequence shown in SEQ ID NO: 1.

[0008] The present invention also provides a pharmaceutical composition, comprising: viable bacteria, inactivated bacteria, and / or culture supernatants of the aforementioned bacterium of the genus Megasphaera.

[0009] In some embodiments, it further comprises: and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the pharmaceutically acceptable carrier is an oral preparation, an injection, a tablet, a powder, or a suspension.

[0011] In some embodiments, the administration mode of the pharmaceutical composition is to enter the digestive system, more preferably oral administration or enema; or the administration mode of the pharmaceutical composition is intratumoral injection.

[0012] In some embodiments, it further comprises: a cancer immunotherapeutic agent.

[0013] In some embodiments, the cancer immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody.

[0014] In some embodiments, the cancer is breast cancer.

[0015] The present invention also provides a method for enhancing the function of the immune system, comprising administering an effective amount of the aforementioned bacteria of the genus Megasphaera or an effective amount of the aforementioned pharmaceutical composition to a subject in need thereof.

[0016] In some embodiments, the method enhances the function of the immune system by activating macrophages.

[0017] The present invention also provides a method for treating cancer, comprising administering an effective amount of the aforementioned bacteria of the genus Megasphaera or an effective amount of the aforementioned pharmaceutical composition to a subject in need thereof.

[0018] In some embodiments, the cancer is breast cancer.

[0019] The present invention also provides a method for alleviating cytokine storm caused by a cancer immunotherapeutic agent, comprising administering the aforementioned bacteria of the genus Megasphaera or the aforementioned pharmaceutical composition to a cancer patient undergoing treatment with the immunotherapeutic agent.

[0020] In some embodiments, the cancer is breast cancer.

[0021] The present invention also provides a method for producing short-chain fatty acids, comprising:

[0022] culturing the aforementioned bacteria of the genus Megasphaera; and

[0023] isolating the short-chain fatty acids from the culture supernatant.

[0024] In some embodiments, the short-chain fatty acids include acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and / or caproic acid.

[0025] In some embodiments, the bacteria are cultured using a PYG medium or a medium containing acetic acid, and preferably using an ATCC2107 medium to culture the bacteria.

[0026] In some embodiments, the medium for culturing the bacteria contains glucose, mannitol, fructose, maltose, lactose, and / or lactic acid as a carbon source.

[0027] In some embodiments, the method comprises culturing the bacteria and isolating butyric acid from the culture supernatant.

[0028] In some embodiments, the method includes any of the following:

[0029] i) Culturing the bacterium using PYG medium and isolating butyric acid, isovaleric acid, valeric acid, and / or caproic acid from the culture supernatant;

[0030] ii) Culturing the above-mentioned bacterium using a medium containing acetic acid and isolating butyric acid, isovaleric acid, valeric acid, and / or caproic acid from the culture supernatant, and the medium containing acetic acid is preferably ATCC2107 medium;

[0031] iii) Culturing the bacterium using a medium with glucose, mannitol, and / or fructose as the carbon source and isolating butyric acid from the culture supernatant.

[0032] iv) Culturing the bacterium using a medium with maltose as the carbon source and isolating acetic acid, propionic acid, and / or butyric acid from the culture supernatant; or

[0033] v) Culturing the bacterium using a medium with lactic acid as the carbon source and isolating acetic acid and / or propionic acid from the culture supernatant.

[0034] The present invention also provides the use of the aforementioned Megasphaera bacterium or the aforementioned pharmaceutical composition in the preparation of a drug for enhancing immune system function, treating cancer, or alleviating immune-related side effects caused by cancer immunotherapeutic agents.

[0035] In some embodiments, the immune-related side effects caused by the cancer immunotherapeutic agent are cytokine storms, and the cancer immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, and / or an anti-CTLA-4 antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Shows the growth status of XA-511 strain on the plate.

[0037] Figure 2 Is the morphology map of XA-511 under scanning electron microscope.

[0038] Figure 3 Is the statistical chart of gene distribution in the gene island of XA-511 strain.

[0039] Figure 4 Is the whole genome map of XA-511-Chr1.

[0040] Figure 5 A shows the statistical results of viable cell counts of XA-511 strain after being exposed to environmental oxygen for 0 h, 0.5 h, 2 h, 8 h, and 24 h and then cultured under suitable anaerobic conditions for 48 - 72 h. Figure 5B shows the colony growth photos of XA-511 strain exposed to environmental oxygen for different times. From left to right in the figure are: 0 h, 10 -5 colony growth photos of diluted solution spread on the plate; 0.5 h, 10 -5 colony growth photos of diluted solution spread on the plate; 2 h, 10 -1 colony growth photos of diluted solution spread on the plate; 8 h, colony growth photos of undiluted original solution spread on the plate; 24 h, colony growth photos of undiluted original solution spread on the plate.

[0041] Figure 6 Shows the macrophage survival rate after co-culture of XA-511 strain samples with macrophages. N = 3, the data was statistically analyzed by One-way ANOVA, and a p-value less than 0.05 was considered to have a statistically significant difference. *p < 0.05, **p < 0.01, ****p < 0.0001.

[0042] Figure 7 Shows the situation of XA-511 strain (live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN)) stimulating macrophages to produce cytokines and regulating the immune response of macrophages induced by lipopolysaccharide. (A) XA-511 live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN) stimulate macrophages to produce tumor necrosis factor TNF-α; (B) XA-511 live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN) stimulate macrophages to produce interleukin IL-6; (C) XA-511 live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN) affect LPS-induced macrophages to produce TNF-α; (D) XA-511 live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN) affect LPS-induced macrophages to produce IL-6; (E) XA-511 live bacteria (LV), heat-inactivated bacteria (HK) and sterile supernatant (SN) affect LPS-induced macrophages to produce IL-10. N = 3, the data was statistically analyzed by One-way ANOVA, and a p-value less than 0.05 was considered to have a statistically significant difference. *p < 0.05, **p < 0.01, ****p < 0.0001.

[0043] Figure 8 A shows the content of each short-chain fatty acid produced by XA-511 strain in PYG medium, where PYG control represents the content of each short-chain fatty acid in the original PYG medium. Figure 8 B shows the content of each short-chain fatty acid produced by XA-511 strain in ATCC2107 medium, where ATCC control represents the content of each short-chain fatty acid in the original ATCC2107 medium.

[0044] Figure 9Shows the amount of short-chain fatty acids produced by strain XA-511 in TPY medium with different carbon sources.

[0045] Figure 10 Shows the tumor growth curve of mouse breast cancer 4T1. N = 10, data were statistically analyzed by One-way ANOVA, and a p-value less than 0.05 was considered to have a statistically significant difference. *p < 0.05, **p < 0.01.

[0046] Figure 11 Are the tumor photos of each group for 4T1 mouse breast cancer at 21 days.

[0047] Figure 12 Are the representative pictures of multiple immunofluorescence staining of tumor tissues in each group. Among them, T cells (red CD4, purple-red CD8), dendritic cells DC (bright blue CD11c), cytokine INF-γ (yellow), cell nuclei (DAPI, blue)

[0048] Figure 13 Shows the statistical results of the infiltration density of T cells and dendritic cells (DC) in tumor tissues (N = 5), data were analyzed using T-test, *p < 0.05 significant difference, **p < 0.01 very significant difference.

[0049] Figure 14 Shows the results of the content of cytokines in the plasma of breast cancer mice (N = 5 - 8). Data were statistically analyzed by One-way ANOVA, and a p-value less than 0.05 was considered to have a statistically significant difference. *p < 0.05,

[0050] **p < 0.01, ****p < 0.0001. Detailed implementation methods

[0051] Unless otherwise specified, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art.

[0052] The term "or" refers to a single element among the listed alternative elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more, or all of the listed alternative elements.

[0053] The term "about" as used herein refers to a value within ±10% of a given value.

[0054] The terms "comprising", "containing", "having" and similar expressions mean not excluding the unlisted elements. These terms also include the case consisting only of the listed elements.

[0055] As used herein, the term "sequence identity" refers to a measure of the degree of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, prior to calculating the percentage identity between two amino acid or nucleotide sequences, sequence alignment is performed and gaps (if any) are introduced. If at a particular alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered non-identical or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. Commonly used sequence alignment algorithms or software include DANMAN, CLUSTALW, MAFFT, BLAST, MUSCLE, etc. For the purposes of the present invention, the publicly available alignment software BLAST (obtainable from https: / / www.ncbi.nlm.nih.gov / ) can be used to obtain the optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences by using the default settings.

[0056] As used herein, the term "short-chain fatty acid", unless otherwise specified, generally refers to a fatty acid having no more than ten carbon atoms, preferably no more than six carbon atoms. A "fatty acid" is a carboxylic acid having an aliphatic tail (or aliphatic chain), and can be saturated or unsaturated. Short-chain fatty acids can include fatty acids having 1, 2, 3, 4, 5, 6 carbon atoms or more carbon atoms. Examples of short-chain fatty acids include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, hexanoic acid, and may also include their salts or esters. In the present invention, unless otherwise specified, when referring to "butyric acid", it means n-butyric acid, when referring to "valeric acid", it means "n-valeric acid", and when referring to "hexanoic acid", it means "n-hexanoic acid".

[0057] As used herein, the term "live bacteria" refers to bacteria that retain their metabolic activity and reproductive ability, i.e., bacteria that can grow and reproduce under suitable conditions. Live bacteria can be processed (such as dried) or formulated into preparations, such as oral preparations, but still retain their viability.

[0058] As used herein, the term "inactivated bacteria" refers to bacteria that have substantially lost their metabolic activity or reproductive ability. Inactivated bacteria may be treated by physical or chemical methods to lose their growth and reproductive capabilities, but still substantially retain the morphological form of the bacterial cells and retain at least a portion of the bacterial proteins, DNA / RNA, and / or other intracellular substances. Common methods for preparing inactivated bacteria include heat inactivation (e.g., using a water bath or metal bath), inactivation with bactericides (e.g., ethylene oxide, ozone), radiation (e.g., ultraviolet light) inactivation, etc. Heat inactivation can be carried out by treating at a temperature in the range of 80 - 120°C for 10 - 60 minutes, for example, treating at a temperature in the range of 100 - 110°C for 10 - 15 minutes. In a specific example, bacteria cultured are treated in a 110°C metal bath for 15 min to obtain heat-inactivated bacteria.

[0059] As used herein, the terms "supernatant", "sterile supernatant", or "culture supernatant" refer to the remaining portion after removing the bacterial cells from a bacterial culture, which contains various metabolites produced by the bacteria (e.g., various short-chain fatty acids described in the examples). Methods for obtaining the supernatant from a cell culture may include centrifugation and / or sterile filtration.

[0060] As used herein, the term "prevention" refers to avoiding, reducing, or delaying the onset of a specific disease or disease-related symptoms in a subject, and such disease or disease-related symptoms have not yet appeared prior to the administration of the relevant drug. "Prevention" does not necessarily require completely preventing the onset of the disease or disease-related symptoms. For example, after the administration of the relevant drug, if it can reduce the risk of a subject developing a specific disease or disease-related symptoms, or mitigate the severity of the subsequent related symptoms, it can be considered as "preventing" the onset or development of the disease.

[0061] As used herein, the term "treatment" refers to alleviating, reducing, improving, or inhibiting (e.g., preventing the development of) a disease that a subject has already manifested or has previously experienced. For a specific disease, "treatment" may include "curing" the disease, but in most cases, it does not require completely eliminating all its symptoms. For example, if the administration of the relevant drug results in the weakening or elimination of at least one symptom in a subject, it can be considered that the subject has been treated.

[0062] As used herein, the term "immunotherapy" refers to the process of assisting in the treatment of diseases by inducing, enhancing, or suppressing an immune response in cells, tissues, or organs. The term "immunotherapeutic agent" refers to an active substance used for immunotherapy, including but not limited to immune checkpoint molecule modulators (e.g., antibodies against immune checkpoint proteins, such as antibodies against PD-1 or PD-L1), interleukins (e.g., IL-2, IL-7, IL-12, IL-15), cytokines (e.g., interferons, G-CSF, imiquimod), chemokines (e.g., CCL3, CCL26, CXCL7), vaccines (e.g., peptide vaccines, dendritic cell (DC) vaccines, EGFRvIII vaccines, mesothilin vaccines, G-VAX, Listeria vaccines), and chimeric antigen receptor T cells (CAR-T).

[0063] As used herein, the term "cytokine storm" may also be referred to as "cytokine cascade" or "hypercytokinemia", and is a potentially fatal immune response usually caused by an uncontrolled positive feedback loop between cytokines and immune cells, accompanied by a highly elevated level of multiple cytokines (e.g., INF-γ, IL-10, IL-6, CCL2, etc.).

[0064] As used herein, the term "subject" refers to an individual suffering from a disease or at risk of developing the disease. This term can generally be used interchangeably with "patient", "test subject", "treatment subject", etc. In the present invention, the subject includes animals, such as vertebrates, especially mammals, such as poultry, pigs, cats, dogs, horses, sheep, rabbits, mice, rats, etc. In some embodiments, the subject in the present invention can be a human.

[0065] As used herein, the term "drug combination" refers to a combination of two or more drugs administered to a subject within a period of time (e.g., simultaneously, or within a few minutes to a few hours, or even within a few days or weeks). The two or more drugs can be formulated into the same preparation, or can also be formulated into different preparations separately.

[0066] As used herein, the term "co-administration" refers to the mode of administration of the above drug combination, including simultaneous or sequential administration of the active ingredients of each drug to the subject within a period of time (e.g., simultaneously, or within a few minutes to a few hours, or even within a few days or weeks).

[0067] As used herein, the term "therapeutically effective amount" or "effective amount" refers to the amount of an active compound or bacterium sufficient to cause a biological or medical response (e.g., preventing or treating a specific disease) desired by a clinician in a subject. The specific "effective amount" value can be determined by those skilled in the art according to factors such as the administration route, the body weight, age, and condition of the subject.

[0068] Bacterial Strain

[0069] The present invention provides bacteria of the genus Megasphaera. Megasphaera is a Gram-negative coccus. In the present invention, the preferred Megasphaera is Megasphaera indica, and more preferably is the one herein referred to as Megasphaerasp._XA-511 or XA-511, which belongs to Megasphaera indica. Strain XA-511 was isolated by the inventor from a fresh fecal sample of a healthy donor and was identified as Megasphaera indica through morphological, cultural, physiological and biochemical characteristics, and 16S rRNA analysis. Strain XA-511 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2021, with the deposit number of CGMCC No. 23400. The gene sequence of the 16S rRNA of strain XA-511 is shown as SEQ ID NO:1. Preferably, the bacterial strain used in the present invention has the 16S rRNA gene sequence shown as SEQ ID NO:1.

[0070] Strain XA-511 has the following characteristics:

[0071] Its morphological, cultural, physiological and biochemical characteristics include: having the following characteristics:

[0072] - On the solid medium of a bacterial plate, after anaerobic culture for 24 - 72 h, the colonies are light yellow, round, with a colony diameter of 2.0 - 5.0 mm, the surface is moist, opaque, and the edge is neat;

[0073] - Under microscopic observation, the bacterial cells are round, with a diameter of 0.8 - 1.6 μm, single or multiple in a line, Gram-negative; they are prone to aggregation and reproduce by binary fission, and surface wrinkles can be observed under a scanning electron microscope.

[0074] - It is suitable for growth under anaerobic conditions. Exposure to environmental oxygen for more than 2 h significantly affects the growth of the strain; however, when exposed to environmental oxygen for less than 0.5 h, its growth ability is basically not affected.

[0075] - Using The biochemical detection (36 items) of carbon source utilization and enzyme activity using an ANC card shows that the ELLMAN reaction result of strain XA-511 is positive, and the detection results of other items are negative.

[0076] Its drug resistance characteristics include: having a certain drug resistance to ampicillin and being sensitive to most common antibiotics, including metronidazole, moxifloxacin, clindamycin, piperacillin, imipenem, and cefotaxime, etc.

[0077] Its metabolic characteristics include: it can grow using glucose, mannitol, fructose, maltose, and / or lactic acid as carbon sources, and can metabolize to produce short-chain fatty acids such as propionic acid, butyric acid, valeric acid, isovaleric acid, and caproic acid.

[0078] - When cultured in PYG medium, it can produce short-chain fatty acids such as butyric acid, isovaleric acid, valeric acid, and caproic acid (detected from the culture supernatant);

[0079] - When cultured in a medium containing acetic acid (such as ATCC2107 medium), it can produce a large amount of butyric acid, isovaleric acid, and caproic acid, and also produce valeric acid;

[0080] - When cultured in a medium with glucose, mannitol, or fructose as the carbon source, the strain XA-511 can metabolize to produce a certain amount of butyric acid;

[0081] - When cultured in a medium with maltose as the sole carbon source, in addition to producing butyric acid, the strain XA-511 can also produce a certain amount of propionic acid and a large amount of acetic acid; when maltose is replaced with lactic acid, the amount of acetic acid produced by XA-511 decreases relatively, while the amount of propionic acid increases significantly.

[0082] Its genomic characteristics include:

[0083] It has a 16S rRNA gene sequence as shown in SEQ ID NO:1; it has a circular genome with a total genome length of approximately 2,407,426 bp, a GC content of approximately 53.67%, about 4,566 genes, 7 gene islands, 23 prophages, 1 CRISPR, and does not have TNSS; it has potential resistance genes Ugd, Nocardia, and MacB (whose functions are resistance to peptides, rifamycin, and macrolide antibiotics respectively).

[0084] Bacterial strains can be identified by any suitable method. For example, bacterial strains can be identified by 16S rRNA or 23S rRNA sequencing, or can be identified by whole-genome sequencing. When needed, bacterial strains can be identified by any one or more of the above morphological characteristics, culture characteristics, physiological and biochemical characteristics, metabolic characteristics, and genomic characteristics.

[0085] The bacterial strains of the present invention also include their offspring, or cultured subcloned strains, which may have natural mutations at the genetic level but still retain the original biological activity (such as the therapeutic activity or the activity of producing short-chain fatty acids described in the present invention).

[0086] The bacterial strain of the present invention can be cultured in any medium suitable for Megasphaera, and the medium can be a liquid medium or a solid medium. The medium includes but is not limited to Reinforced Clostridial Medium (ATCC 2107), PYG (Peptone Yeast Extract) medium, and TPY medium, etc.

[0087] The Reinforced Clostridial Medium (ATCC 2107) is a liquid medium, and each liter of it contains: 10.0 g of tryptose, 10.0 g of beef extract, 5.0 g of dextrose, 3.0 g of yeast extract, 5.0 g of sodium chloride (NaCl), 0.5 g of L-Cysteine HCl, 1.0 g of Soluble Starch, 3.0 g of sodium acetate, 0.5 g of agar, and the balance is water, adjusting the pH = 7.0 ± 0.1.

[0088] The PYG medium is a liquid medium, and each liter of it contains: 20.0 g of tryptose, 5.0 g of dextrose, 10.0 g of yeast extract, 0.08 g of sodium chloride (NaCl), 0.5 g of L-Cysteine HCl, 0.008 g of calcium chloride (CaCl 2 ), 0.008 g of magnesium sulfate (MgSO 4 ), 0.04 g of dipotassium hydrogen phosphate (K 2 HPO 4 ), 0.04 g of potassium dihydrogen phosphate (KH 2 PO 4 ), 0.5 g of sodium bicarbonate (NaHCO 3 ), and the balance is water, adjusting the pH = 7.0 ± 0.1.

[0089] The TYP medium is a liquid medium, and each liter of it contains: 10.0 g of lactose, 5.0 g of beef extract, 5.0 g of yeast extract, 10.0 g of casein peptone, 5.0 g of soy peptone, 0.25 g of Tween-80, 0.1 g of magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), 2.5 g of dipotassium hydrogen phosphate (K 2 HPO 4 ), 0.0 g of potassium dihydrogen phosphate (KH 2 PO4 ) 2.5 g, 0.5 g of L-Cysteine HCl, and the balance is water, adjusting the pH = 7.0 ± 0.1.

[0090] The bacterial strain XA-511 can grow using a variety of carbon sources. These carbon sources include, but are not limited to, any one or a combination selected from glucose, mannitol, fructose, maltose, lactose, and lactic acid. The bacterial strains of the present invention can be cultured using a medium containing these carbon sources, or the composition of the medium used can also be adjusted, for example, by adding these carbon sources to the medium, or replacing the original carbon sources in the medium with these carbon sources. In some embodiments, a PYG medium supplemented with 1% lactose can be used. In some embodiments, a medium can be used in which the lactose in the TYP medium is replaced with any one or several of glucose, mannitol, fructose, maltose, or lactic acid.

[0091] The bacterial strains of the present invention can be cultured under anaerobic conditions.

[0092] In one embodiment, the culturing method may specifically include the following steps:

[0093] (1) Pick a single clone from a single colony of the bacteria XA-511 that has multiplied itself using a sterile inoculation loop and transfer it to a 5 mL Reinforced Clostridial Medium (ATCC 2107) medium, and culture it in an anaerobic workstation for 20 - 24 h;

[0094] (2) Take 1 mL of the bacterial solution, extract the strain DNA, perform PCR amplification on the strain gene fragment using the universal primers 27F / 1492R. After amplification, purify the product by gel cutting, extract and recover the fragment, ligate the target fragment to the T vector, then transform the ligation product into TOP10 competent cells, perform blue-white screening, take the positive recombinant clones and sequence them using the universal primers T7 / SP6 to obtain the 16S rRNA sequence. Use the online Blast tool Blastn of NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), select "Standard database (Nucleotide collection (nr / nt))" for the database, and keep other selections as default. After comparison with the NCBI database, the similarity with the standard strain Megasphaera indica Megasphaerasp. NMBHI-10 is 99.6%, which is greater than 98.65%, indicating that the identification result is correct. Then, pipette 4 mL of the bacterial suspension and transfer it into 100 mL of liquid Clostridium difficile medium (ATCC 2107) and culture it in an anaerobic chamber for 24 h;

[0095] (3) Pipette 1 mL of the bacterial solution and perform 16S rRNA sequencing as above. After the identification result is correct by comparison with the NCBI database using the above method, centrifuge the bacterial solution at 8000 rpm and 4 °C for 15 min, discard the supernatant, take the precipitated bacteria and resuspend them with a mixture of medium:sterile glycerol with a volume ratio of 4:1, mix well, aliquot into sterile cryotubes, and store at -80 °C.

[0096] The Megasphaera bacteria provided by the present invention can be viable bacteria or inactivated bacteria. The present invention also provides the culture supernatant of the Megasphaera bacteria, and the culture supernatant does not contain bacteria. The culture supernatant can be obtained by culturing with any one of the liquid media described above.

[0097] Co-culture the viable bacteria strain, inactivated strain of Megasphaera XA-511 of the present invention and the strain metabolite (i.e., the culture supernatant, which includes short-chain fatty acids) with macrophages and detect the production of cytokines. The experimental results show that it can activate macrophages, relieve the excessive immune response caused by LPS, and has the potential application for treating immune-related diseases.

[0098] The Megasphaera XA-511 of the present invention can produce short-chain fatty acids, activate immune cells, and can also combine with other tumor treatment means to prevent and treat tumors. Its possible mechanism of action lies in activating the immune system of the subject and enhancing immunity, thereby preventing or treating tumors.

[0099] Therefore, the megasphaera strain bacteria provided by the present invention can be used for producing short-chain fatty acids. The megasphaera strain bacteria provided by the present invention or their culture supernatants can be used for preventing or treating diseases.

[0100] Short-chain Fatty Acid Production

[0101] The strain XA-511 can grow in a medium with carbon sources such as glucose, mannitol, fructose, maltose, lactose, and / or lactic acid, etc., and can metabolize to produce short-chain fatty acids such as propionic acid, butyric acid, valeric acid, isovaleric acid, and caproic acid, etc. And with the differences in carbon sources and the changes of other components in the medium, the types and levels of the produced short-chain fatty acids are different. The strain XA-511 has different utilization abilities for different carbon sources and converts different carbon sources into different types of short-chain fatty acids through metabolism.

[0102] For example, when the strain XA-511 is cultured in a PYG medium, short-chain fatty acids such as butyric acid, isovaleric acid, valeric acid, and caproic acid, etc. can be produced (detected from the culture supernatant). When the strain XA-511 is cultured in a medium containing acetic acid (such as ATCC2107 medium), it can consume the acetic acid in the medium to produce a large amount of butyric acid, and the butyric acid yield can reach several times that when cultured in the PYG medium. The yields of its isovaleric acid and caproic acid are also higher than the corresponding yields when cultured in the PYG medium. At the same time, valeric acid is also produced, but the valeric acid yield is reduced compared with that when cultured in the PYG medium.

[0103] When the medium contains a single carbon source of glucose, mannitol, or fructose, XA-511 can metabolize to produce butyric acid; when the single carbon source in the medium is maltose, XA-511 can produce propionic acid and a large amount of acetic acid in addition to butyric acid; and when maltose is replaced with lactic acid, the amount of acetic acid produced by XA-511 relatively decreases, and at the same time, the amount of propionic acid increases significantly.

[0104] Based on the above findings, the present invention provides a method for producing short-chain fatty acids by culturing bacteria. The method may include culturing the bacteria of the present invention in a suitable medium, and separating short-chain fatty acids from the culture supernatant. The short-chain fatty acids include, but are not limited to, a combination of one or more selected from acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and caproic acid.

[0105] The bacteria of the present invention can be cultured using any medium suitable for Megasphaera, such as those described above. Conventional culture conditions well-known to those skilled in the art can be used for culturing, such as culturing under anaerobic conditions. The culture time can be in the range of 6 hours to 12 days, for example, it can be in the range of 12 hours to 6 days, in the range of 24 hours to 3 days, or in the range of 24 hours to 48 hours. In some embodiments, the method includes culturing the bacteria of the present invention using PYG medium (Peptone Yeast Glucose Broth) and separating butyric acid, isovaleric acid, valeric acid, and / or hexanoic acid from the culture supernatant.

[0106] In some embodiments, the method includes culturing the bacteria of the present invention using a medium containing acetic acid and separating butyric acid, isovaleric acid, valeric acid, and / or hexanoic acid, especially butyric acid, from the culture supernatant. In some embodiments, the method includes culturing the bacteria of the present invention using ATCC 2107 medium and separating butyric acid, isovaleric acid, valeric acid, and / or hexanoic acid, especially butyric acid, from the culture supernatant.

[0107] In some embodiments, the method includes culturing the bacteria of the present invention using a medium with glucose, mannitol, and / or fructose as the carbon source and separating butyric acid from the culture supernatant. In some embodiments, the method includes culturing the bacteria of the present invention using glucose, mannitol, and / or fructose to replace lactose in the PYG medium as the carbon source and separating butyric acid from the culture supernatant.

[0108] In some embodiments, the method includes culturing the bacteria of the present invention using a medium with maltose as the carbon source and separating acetic acid, propionic acid, and / or butyric acid from the culture supernatant. In some embodiments, the method includes culturing the bacteria of the present invention using maltose to replace lactose in the PYG medium as the carbon source and separating acetic acid, propionic acid, and / or butyric acid from the culture supernatant.

[0109] In some embodiments, the method includes culturing the bacteria of the present invention using a medium with lactic acid as the carbon source and separating acetic acid and / or propionic acid from the culture supernatant. In some embodiments, the method includes culturing the bacteria of the present invention using lactic acid to replace lactose in the PYG medium as the carbon source and separating acetic acid and / or propionic acid from the culture supernatant.

[0110] Preventive and Therapeutic Uses

[0111] Immune Activation

[0112] According to the present invention, viable bacteria, inactivated bacteria or culture supernatants of XA-511 stimulate macrophages to produce various cytokines, especially TNF-α, IL-6 and IL-10. In particular, viable bacteria, inactivated bacteria or culture supernatants of XA-511 significantly stimulate macrophages to produce the tumor necrosis factor TNF-α, with viable bacteria being particularly significant; viable bacteria, inactivated bacteria or culture supernatants of XA-511 significantly stimulate macrophages to produce the cytokine IL-6, with viable bacteria and inactivated bacteria being particularly significant. Thus, viable bacteria, inactivated bacteria or culture supernatants of the strain XA-511 have a significant activating effect on macrophages, important members of the immune system, can activate the immune system, and can be used to enhance the function of the immune system.

[0113] According to the present invention, under the condition of inducing macrophages to produce an excessive immune response with LPS, viable bacteria or culture supernatants of XA-511 significantly reduce the production of the immune factor TNF-α by LPS-induced macrophages; viable bacteria of XA-511 increase the amount of IL-6 produced by LPS-induced macrophages, while inactivated bacteria or culture supernatants can significantly reduce the production of the inflammatory factor IL-6 in LPS-induced macrophages, showing a certain immune regulatory function; the culture supernatant of XA-511 significantly increases the production of the anti-inflammatory factor IL-10 in LPS-induced macrophages. Thus, viable bacteria, inactivated bacteria or culture supernatants of the strain XA-511 show the potential to regulate immunity.

[0114] Based on the above findings, the bacteria of the genus Megasphaera or their culture supernatants of the present invention, or the pharmaceutical combinations or pharmaceutical compositions of the present invention can be used to activate macrophages or enhance the function of the immune system. In some embodiments, activating macrophages is achieved by stimulating macrophages to produce TNF-α and / or IL-6.

[0115] Prevention and Treatment of Cancer

[0116] According to the present invention, the combination of XA-511 and an anti-PD-1 antibody in the treatment of breast cancer can achieve a significant synergistic effect, and XA-511 can significantly enhance the tumor inhibitory effect of the anti-PD-1 antibody. XA-511 can promote CD8 in tumors +Infiltration of T cells and DC cells, in combination with anti-PD-1 antibody, treats tumors. XA-511 can also alleviate the production of cytokines IL-6, IL-27, IL-17A, IL-1β, IL-12p70, IL-23, IL-1α, TNFα and MCP-1 induced by PD-1 antibody treatment, slow down systemic immune overreaction, thereby reducing side effects such as cytokine storm caused by immunotherapy. Thus, XA-511 achieves good synergistic anti-PD-1 antibody treatment effect on breast cancer by enhancing the infiltration of immune cells with anti-tumor activity in tumor tissues, while reducing side effects such as cytokine storm caused by immunotherapy.

[0117] Based on the above findings, the bacteria of the genus Megasphaera or its culture supernatant of the present invention, or the pharmaceutical combination or composition of the present invention can be used for preventing or treating cancer, especially breast cancer.

[0118] The pharmaceutical combination of the bacteria of the genus Megasphaera or its culture supernatant of the present invention and a cancer immunotherapeutic agent (such as an anti-tumor antibody, such as anti-PD-1 antibody) can be used for preventing or treating cancer, especially breast cancer, and shows a synergistic effect in the treatment of cancer. Compared with the use of the immunotherapeutic agent alone, co-administration with the bacteria of the genus Megasphaera or its culture supernatant of the present invention can result in a reduction in the dosage administered, a shortening of the treatment cycle, a lower recurrence rate or mortality.

[0119] In some embodiments, the immunotherapeutic agent includes an immune checkpoint molecule modulator or CAR-T, especially an anti-PD-1 antibody. In a specific example, compared with the administration of anti-PD-1 antibody alone, co-administration with the bacteria of the genus Megasphaera or its culture supernatant of the present invention can significantly inhibit tumor growth (determined by measuring changes in tumor volume).

[0120] The pharmaceutical combination of the present invention can promote the infiltration of CD8 + T cells and DC cells in tumors, and especially can treat cancer through this promoting effect. The pharmaceutical combination of the present invention can reduce systemic immune overreaction caused by cancer immunotherapy (such as using immune checkpoint inhibitors or CAR-T), especially reduce cytokine storm caused by immunotherapy, especially reduce the production of cytokines caused by immunotherapy. In some embodiments, the cytokines caused by the immunotherapy are selected from any one or any combination of IL-6, IL-27, IL-17A, IL-1b, IL-12p70, IL-23, IL-1α, TNFα and MCP-1. In some embodiments, the pharmaceutical combination of the present invention can result in a reduction in tumor size or tumor growth.

[0121] Drug Combinations and Kits

[0122] The Megasphaera provided in this article (live bacteria, inactivated bacteria, and / or culture supernatant) can be used alone or in combination with other drugs (such as anti-PD-1 antibodies) after forming a drug combination. The term "drug combination" as used herein refers to a combination of two or more pharmaceutically active ingredients. For a drug combination comprising two or more pharmaceutically active ingredients, the two or more pharmaceutically active ingredients can coexist in the same pharmaceutical preparation (such as a pharmaceutical composition), and they can also exist as separate pharmaceutical preparations in the same drug kit, or even the two or more pharmaceutically active ingredients can exist as separate pharmaceutical preparations in different drug kits. The drug combination can be administered such that the two or more pharmaceutically active ingredients contained therein can be present in the subject's body simultaneously at a certain time. In most cases, combining two or more pharmaceutically active ingredients to form a drug combination and administering them jointly is because the two or more pharmaceutically active ingredients will produce a synergistic effect in the subject's body. Such a synergistic effect includes that the efficacy produced by the combined administration is better than that of any one pharmaceutically active ingredient administered alone, one pharmaceutically active ingredient can reduce the side effects of another pharmaceutically active ingredient, or the combined administration of the two pharmaceutically active ingredients produces a new efficacy, such as being used to treat an original indication different from that of any one pharmaceutically active ingredient.

[0123] The present invention provides a pharmaceutical composition comprising live bacteria, inactivated bacteria, or a culture supernatant of the bacteria of the present invention, which can be used to activate macrophages, enhance immune system function, and / or treat cancer.

[0124] The live bacteria, inactivated bacteria, or the culture supernatant of the bacteria of the present invention can be freeze-dried.

[0125] The pharmaceutically active ingredient can optionally be formulated into a pharmaceutical composition together with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" as used herein refers to substances such as solid or liquid diluents, fillers, antioxidants, stabilizers, preservatives, etc. that can be safely administered, which are suitable for administering to a subject without excessive adverse side effects and are also suitable for maintaining the viability of the drug or active agent located therein. Depending on the route of administration, various different carriers well known in the art can be used, including, but not limited to, sugars, starches, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, and / or pyrogen-free water, etc.

[0126] The pharmaceutical compositions of the present invention can be formulated into clinically acceptable dosage forms such as tablets, capsules, powders, powders, suppositories, sprays, gels, injections, etc. In some embodiments, any of the pharmaceutical compositions of the present invention can be formulated into a dosage form suitable for delivery to the intestine. In particular, in some embodiments, the pharmaceutical composition containing the bacteria of the present invention is formulated into a dosage form suitable for delivery to the intestine, such as an oral drug with enteric coating.

[0127] In some embodiments, the immunotherapeutic agent is formulated into an injection.

[0128] In some embodiments, the live bacteria, inactivated bacteria or culture supernatant of the bacteria of the present invention can be separately formulated into oral drugs.

[0129] The pharmaceutical combinations or compositions of the present invention can be stored in various packaging (or containers) to form a kit or a pharmaceutical kit. These packages include, but are not limited to, boxes, ampoules, vials, tubes, bags or other suitable forms known in the art. These packages can be made of plastic, glass, laminated paper, metal foil or other materials suitable for storing drugs. If necessary, an instruction manual is also provided together with the package. The instruction manual generally may include a description of information on how to use these compositions for treating or preventing tumors (such as breast cancer); dosage regimens for treating or preventing the formation of tumors (such as breast cancer); precautions; warnings; indications; contraindications; adverse reactions; animal pharmacology; clinical studies; and / or references. The instruction manual can be printed directly on the package (if any), or as a label attached to the package, or as a separate paper, booklet, card or folded printed matter provided in or together with the package.

[0130] In some embodiments, when the pharmaceutical combination or pharmaceutical composition of the present invention is stored in a closed container at about 4°C or about 25°C and in an environment with 50% relative humidity, at least 80% of the bacterial strains (in terms of live bacteria, measured by colony forming units) still exist after at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

[0131] Administration of Drugs

[0132] The pharmaceutical combinations or pharmaceutical compositions of the present invention can be administered to a subject using any suitable route, such as by oral, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal, rectal, intranasal, sublingual, or by inhalation, transdermal, etc.

[0133] In some embodiments, the pharmaceutical combination or pharmaceutical composition of the present invention can be administered to the gastrointestinal tract so that the bacterial strains of the present invention are delivered to the intestine and / or partially or fully colonize the intestine.

[0134] The two or more active ingredients included in the pharmaceutical combination of the present invention can be administered simultaneously or sequentially. When administered simultaneously, the two active ingredients can be administered in the form of a single preparation, or in multiple different preparations simultaneously. When administered sequentially, the time interval between the administrations of different active ingredients can be determined as needed, for example, it can be in the range of about 1 minute to about 60 minutes, in the range of about 1 hour to about 24 hours, in the range of about 1 day to about 7 days, and in the range of about 1 week to about 4 weeks.

[0135] The active pharmaceutical ingredients of the present invention are administered to a subject in a therapeutically effective amount. For example, each active ingredient can be administered to the subject at a total dose of about 100 mg to about 1000 mg, for example, the total dose can be about 200 mg to about 900 mg, about 300 mg to about 800 mg, about 400 mg to about 700 mg, about 500 mg to about 600 mg. In certain embodiments, a suitable daily dose is 0.01 mg to 100 mg per kilogram of body weight for each active ingredient.

[0136] For bacterial active ingredients, especially live bacteria, a suitable effective amount can be about 1×10 3 to about 1×10 12 colony forming units (CFU) per day; for example, about 1×10 4 to about 1×10 11 CFU, about 1×10 5 to about 1×10 10 CFU, about 1×10 6 to about 1×10 9 CFU, about 1×10 7 to about 1×10 8 CFU.

[0137] For bacterial components, the effective amount can also be at least 10 9 cells per day, for example, at least 10 10 cells, at least 10 11 cells or at least 10 12 cells per day.

[0138] The present invention will be further illustrated by the following specific examples.

[0139] Example 1: Obtaining of Strains

[0140] The strains were isolated from fresh fecal samples of healthy donors recruited by Shenzhen Weizhijun Biotechnology Co., Ltd. Selection of Subcultured Samples:

[0141] A large number of healthy donors were recruited by Shenzhen Unik Bio-Tech Co., Ltd. Fresh fecal samples were obtained, and metagenomic DNA was extracted to detect the bacterial flora and abundance in the fecal samples. According to the sequencing results, donor samples with a relatively high abundance of the genus Megasphaera were selected, numbered F14 (abundance of the genus Megasphaera: 2.10%), for the purposeful screening of Megasphaera strains.

[0142] Isolation of Strains

[0143] In an anaerobic operating chamber, 1 g of the fecal sample from the donor was evenly suspended in 10 mL of PBS and serially diluted to 10 -6 dilution. 100 μL of the 10 -6 dilution was pipetted into an agar plate of reinforced Clostridium medium (ATCC 2107) and spread evenly with a disposable sterile spreading rod, and anaerobically cultured at 37 °C for 3 days.

[0144] After 3 days, colonies with a surface colony diameter of 2.0 - 5.0 mm, slightly convex, round, shiny, and faintly light yellow were purposefully selected. A single colony was picked with a sterile loop and purified and cultured by streak plate method to obtain a pure strain. This strain was designated as bacterium XA-511.

[0145] Sequencing and Identification of Strains

[0146] A single colony was picked from the single colony of bacterium XA-511 that propagated itself with a sterile inoculation loop into 5 mL of the modified ATCC2107 medium and cultured in an anaerobic operating chamber for 20 - 24 h; 1 mL of the bacterial solution was taken, and the strain DNA was extracted from the bacterial solution using a DNA extraction kit. The gene fragment of the strain was PCR amplified using the universal primers 27F / 1492R. After amplification, the product was subjected to gel cutting and purification, and the recovered fragment was extracted using a SanPrep column DNA gel recovery kit (REF: 518131-0100); the target fragment was ligated to the T vector overnight at 16 °C using a pGM-T cloning kit (Cat#VT302-02); the ligation product was transformed into TOP10 competent cells, and blue-white screening was performed. Positive recombinant clones were taken and sequenced using the universal primers T7 / SP6.

[0147] Primers for PCR:

[0148]

[0149] List of components and contents in a 50 μL reaction system:

[0150]

[0151] The PCR reaction conditions are as follows:

[0152]

[0153] The 16S ribosomal RNA gene sequence of the isolated bacterium XA-511 is shown in Sequence Listing SEQ ID NO:1.

[0154]

[0155]

[0156] Strain preservation: The bacterium XA-511 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 13, 2021, with the deposit number CGMCC No. 23400.

[0157] Example 2: Preparation, sequencing, assembly and analysis of the strain genome

[0158] Genomic DNA of bacterium XA-511 was extracted by the SDS method. Then, agarose gel electrophoresis was used to detect the purity and integrity of the DNA, and Qubit was used for quantification.

[0159] Libraries were constructed using the Nanopore platform and the Illumina platform. After passing the library inspection, different libraries were sequenced on Nanopore PromethION and Illumina NovaSeq PE150 according to the effective concentration and the target output data volume. The data was quality-controlled using the NanoPlot software with a threshold of Q>7 to obtain valid data. Starting from the valid data after quality control of each sample, the Unicycler software was used to assemble the genome using second-generation + third-generation data, screen the chromosomal and plasmid sequences, and assemble the chromosomal sequences into a circular genome (or a linear genome sequence if it is a linear genome), that is, the final 0-gap completed map sequence.

[0160] The total gene length of the genome assembled from strain XA-511 is 2,407,426 bp, the N50 length is 11,760 bp, and the GC content is 53.67%. The GeneMarkS (Version 4.17) software was used to predict the coding genes of the newly sequenced genome, and the statistical information of the gene prediction results is shown in Table 1 below:

[0161] Table 1 Statistical table of coding gene prediction results

[0162]

[0163] Based on sequence composition, the IslandPath-DIOMB software (Version 0.2) was used to predict genomic islands, which determines genomic islands and potential horizontal gene transfer by detecting dinucleotide biases and mobility genes (such as transposases or integrases) in the sequence. The statistical results are shown in Table 2 below:

[0164] Table 2 Statistical Results of Genomic Island Prediction

[0165] SampleID Glsnumber Glstotallength(bp) Averagelength(bp) XA-511 7 74817 10688

[0166] The statistical chart of gene distribution in genomic islands only shows genomic islands with a length less than 15 kb ( Figure 3 ).

[0167] The prophages on the sample genome were predicted using the phiSpy software (Version 2.3), and the prediction results are shown in Table 3.

[0168] Table 3 Statistical Results of Prophage Prediction

[0169] SampleID Prophagenumber Totallength(bp) Averagelength(bp) XA-511 23 539422 23453.1

[0170] The CRISPR prediction of the sample genome was performed using CRISPRdigger (Version 1.0), and the statistical results are shown in Table 4 below:

[0171] Table 4 Statistical Results of CRISPR Prediction

[0172] SampleID CRISPRnumber Totallength(bp) Averagelength(bp) XA-511 1 3671 3671

[0173] Pathogenic bacteria secrete such proteins into the extracellular space or host cells through the type N secretion system (TNSS, currently 7 types have been identified, types I - VII), causing pathological reactions by controlling the immune response and cell apoptosis. Among them, the T3SS of Gram-negative bacteria is usually used to study pathogenic bacteria, infection mechanisms, virulence effects, etc. at the molecular level, and is a well-studied secretion system. For the TNSS system, related proteins of the secretion system were extracted and annotated based on the results of protein sequence function database annotation.

[0174] Table 5 TNSS Statistical Results

[0175]

[0176] According to the TNSS statistical results in the above table, XA-511 does not have TNSS and does not secrete systemic proteins that can cause host pathological reactions, indicating good biosafety.

[0177] The predicted gene sequences were aligned with the virulence factor VFDB database using the blast software. The results are shown in Table 6.

[0178] Table 6 List of potential toxicity genes of XA-511

[0179]

[0180]

[0181] The potential antibiotic resistance genes in the genome were analyzed using the prokka (version 1.14.6) software. The antibiotic resistance gene database was CARD. Three resistance genes, namely Ugd, Nocardia, and MacB, were found by alignment. Their functions are resistance to peptide, rifamycin, and macrolide antibiotics, respectively. The detailed alignment results are shown in Table 7.

[0182] Table 7 Potential toxicity genes of Megasphaera_XA-511 obtained by aligning with the antibiotic resistance gene database CARD

[0183]

[0184] For the assembled genome sequence, combined with the prediction results of coding genes, non-coding genes, and the analysis of gene function annotation, the Circos software was used to display the sample genome. The whole genome map of XA-511-Chr1 is shown in Figure 4 .

[0185] Example 3: Oxygen tolerance of the strain

[0186] The method used to test the oxygen tolerance of the strain was to observe whether the strain still had the ability to grow on the growth agar plate after being completely exposed to oxygen for different times. In this experiment, the strain was exposed to environmental oxygen for different times (0 h, 0.5 h, 2 h, 8 h, and 24 h), and then placed in an anaerobic chamber for growth. The relative sensitivity of the strain to environmental oxygen was compared by comparing the total number of colonies of the strain on the growth agar plate.

[0187] Specific steps:

[0188] 0.1 mL of the test bacterial solution was added to 0.9 mL of PBS buffer and mixed into a 10 -1 dilution. Serial ten-fold dilutions were carried out to 10 -5 dilution. 100 μL was taken and spread on the growth plate; A total of 5 groups of samples for detecting different exposure times were prepared according to the above preparation method, namely: Group A (0 h exposure); Group B (0.5 h exposure); Group C (2 h exposure); Group D (8 h exposure); Group E (24 h exposure);

[0189] After exposing all groups to sterile environmental oxygen for different times according to the above design, they were placed in an anaerobic culture system (Anoxomat modified atmosphere, MART; 0% O 2 , 10% CO 2 , 5% H 2 , 85% N 2 ) and cultured at 37 °C for 48 - 72 hours; the total number of colonies on the plate was recorded, and the viable bacteria count with growth ability was calculated accordingly to compare the tolerance of the strains to different exposure times to environmental oxygen in the laminar flow bench environment.

[0190] Figure 5 A shows the viable bacteria count results after culturing for 48 - 72 hours under appropriate anaerobic conditions after exposure to environmental oxygen for 0 h, 0.5 h, 2 h, 8 h, and 24 h. Figure 5 B shows representative photos of the colony growth of each group.

[0191] The experimental results are as follows:

[0192] Under the condition of exposure to environmental oxygen for 0.5 h, the final number of growing colonies on the plate was basically the same as that of the control group (exposed to environmental oxygen for 0 h). However, when the exposure time was extended to 2 h, the number of colonies growing on the plate decreased significantly. When the exposure time was further extended to 8 - 24 h, there were basically no colonies growing on the plate.

[0193] It can be seen from this that the anaerobic strain XA - 511 has a certain tolerance to environmental oxygen. When exposed to environmental oxygen for less than 0.5 h, its cloning growth ability is basically not affected. This provides favorable conditions for the development of the strain.

[0194] Example 4: Utilization of carbon sources by the strain and determination of related enzyme activities

[0195] The physiological and biochemical characteristic tests of the strain were carried out using ANC ID card together with the identification system, following the internal routine procedures and the manufacturer's instructions. All culture treatments were carried out in a 37 °C anaerobic workbench. The strain was cultured on Brucella blood agar for 48 - 72 hours to obtain monoclonal colonies before being identified with the ANC ID card. An inoculation suspension was prepared with 0.45% NaCl aqueous solution, and the calibrated Vitek 2 Densichek instrument (bioMérieux, Marcy l’etoile, France) was used to reach a standard turbidity of 2.70 - 3.30 McFarland. There were 36 biochemical detections of carbon source utilization and enzyme activities detected by the ANC ID card. Through The COMPACT instrument detects the positive and negative results of biochemical tests for carbon source utilization and enzyme activity. The identification results and their test reports are shown in Table 8.

[0196] Table 8 Physiological and Biochemical Test Results

[0197]

[0198]

[0199] Symbol Explanation: “+”, positive; “-”, negative.

[0200] Example 5: Antibiotic Sensitivity of Strains

[0201] The antibiotic sensitivity of the strains was detected by the microbroth dilution method in the two end-point determination decision-making methods for anaerobic bacteria in the standards of the Clinical and Laboratory Standards Institute (CLSI) of the United States to determine the antibiotic sensitivity of the strains. The types, concentrations, and preparation methods of the antibiotics required for the test were all carried out in accordance with the CLSI standards. Reinforced brucella broth medium added with hemin (5 μg / ml), vitamin K1 (1 μg / ml), and lysed horse blood (5%) was used as the broth solution. After the strains were resuscitated, they were inoculated on brucella blood agar plates, and the inoculum was prepared by the direct colony suspension method. The turbidity of the inoculum should reach 0.5 McFarland turbidity.

[0202] Specific Steps:

[0203] 1) Preparation of microbroth dilution plates: Prepare the antimicrobial agent intermediate solution (10×), and dilute the concentrated antimicrobial agent stock solution by the method in the standard or perform serial two-fold dilutions. Then add one part of the 10× antimicrobial agent solution to nine parts of the broth to obtain the expected final concentration. Add the antimicrobial agent / broth solution to a plastic microdilution plate, and add 0.1 ml to each well.

[0204] 2) Inoculation of the microbroth dilution plate: Dilute the bacterial suspension with adjusted turbidity of 0.5 McFarland turbidity 1:15 with water or physiological saline within 15 minutes so that after inoculation, the bacterial liquid concentration in each tube or well is 1×10 6 CFU / ml. Inoculate 10 μl of the prepared inoculum into each well of the microdilution plate, and at the same time take 100 μl and spread it on the surface of a suitable agar medium to verify whether the inoculum concentration reaches 1×10 6 CFU / ml.

[0205] 3) Incubation of the microbroth dilution plate: Incubate at 37 °C for 46 - 48 h under anaerobic conditions.

[0206] The results are shown in Table 9 below.

[0207] Table 9 Antibiotic Sensitivity Test Results

[0208]

[0209]

[0210] The experimental results show that the strain XA-511 has certain resistance only to ampicillin, but is sensitive to most common antibiotics, including metronidazole, moxifloxacin, clindamycin, piperacillin, and imipenem. Therefore, XA-511 is a strain suitable for development as a potential live bacterial drug.

[0211] Example 6: The strain activates the immune function and regulates the immune response

[0212] Macrophages are a heterogeneous population of myeloid cells in the innate immune system and are involved in a variety of physiological and pathological processes. The human leukemia monocytic cell line THP-1 is induced to form macrophages by phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) and is a model widely used to study the immune response ability of monocytes or monocyte-derived macrophages. This experiment uses this cell line to study the effect of the strain on immune macrophages.

[0213] Preparation of viable XA-511 bacteria, heat-inactivated bacteria, and supernatant: The strain XA-511 was cultured in ATCC2107 medium in an anaerobic chamber until the late logarithmic growth phase. After adjusting OD 600 = 1, the bacterial liquid was centrifuged at 5000 g for 5 min at 4 °C, the supernatant was collected, and a cell-free supernatant (XA-511 SN ) was obtained by filtering with a 0.2 mm filter (Millipore). The supernatant was aliquoted into sterile tubes at 1 mL each and stored at -80 °C for later use. The precipitate obtained by centrifugation was resuspended in cell culture medium to obtain a bacterial viable sample (XA-511 8 ) at 1×10 LV CFU / mL. A portion of the viable sample was heat-inactivated in a metal bath at 110 °C for 15 min to obtain a heat-inactivated bacterial sample (XA-511 8 ) at 1×10 HK .

[0214] Culture and polarization treatment of THP-1 cells: THP-1 cells at passage 5 were cultured in RPMI-1640 + 10% FBS medium, seeded in 96-well plates at a density of 3×10 5 cells / mL (200 μL / well), and treated with PMA at a final concentration of 50 ηg / mL for 24 h. The fresh medium was replaced and the cells were rested for 72 h to obtain adherent macrophages.

[0215] Co - culture of XA - 511 live bacteria, heat - inactivated bacteria and supernatant samples with macrophages: The prepared XA - 511 heat - inactivated bacteria samples and supernatant samples were added to the above - mentioned adherent macrophages at MOI = 10 and 1, and co - cultured in a 5% CO 2 incubator for 24 h; for the live bacteria samples, they were added to the above - mentioned adherent macrophages at MOI = 10 and 1, co - cultured in an anaerobic chamber for 2 h, then treated with cell culture medium containing 1% gentamicin in a 5% CO 2 incubator for 1 h to kill the live bacteria, washed twice with PBS, and the medium was replaced with fresh medium, and then continued to be cultured in a 5% CO 2 incubator for 22 h. The supernatants of the above co - cultures were collected, and the contents of various cytokines were detected by ProcartaPlex Multiplex Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA). The co - cultured cells were detected for cell viability by CCK8 assay to obtain appropriate co - culture conditions. For the LPS - induced inflammation model, in the above experiment, LPS with a final concentration of 1 μg / mL was added during the co - culture process. For the supernatant samples, a bacterial medium control group was set up.

[0216] Results of co - cultured cell viability:

[0217] The results of the CCK8 assay are as Figure 6 shown. The live bacteria samples and supernatant samples had an impact on the viability of macrophages under the co - culture condition of MOI = 10. Therefore, the appropriate co - culture conditions for XA - 511 samples and macrophages are: live bacteria samples (MOI = 1), heat - inactivated samples (MOI = 10, 1), and supernatant (MOI = 1). Based on this, the supernatants under the co - culture condition of MOI = 1 were uniformly taken for cytokine detection.

[0218] The results of the supernatant cytokine experiment showed that the live bacteria (LV), heat - inactivated bacteria (HK) of strain XA - 511 and sterile supernatant (SN) significantly stimulated macrophages to produce tumor necrosis factor TNF - α, and the live bacteria (LV) were particularly significant ( Figure 7 A). The live bacteria (LV), heat - inactivated bacteria (HK) of strain XA - 511 and sterile supernatant (SN) significantly stimulated macrophages to produce cytokine IL - 6, and the live bacteria and heat - inactivated bacteria were particularly significant ( Figure 7 B).

[0219] It can be seen that strain XA - 511 has a significant activation function on macrophages, an important member of the immune system, activating the immune system. Therefore, XA - 511 can be used to enhance the immune system function.

[0220] In addition to immune activation, we further explored the regulatory function of XA-511 on the immune response. Macrophages were induced to produce an inflammatory response with LPS, and then the XA-511 samples were added. By detecting cytokines, its immune regulatory ability was observed. The experimental results showed that live bacteria and the supernatant significantly reduced the production of TNF-α by LPS-induced macrophages ( Figure 7 C). The live XA-511 bacteria increased the production of IL-6 by LPS-induced macrophages, while heat-inactivated bacteria and the supernatant significantly reduced the production of IL-6 induced by LPS, showing certain immune regulatory ability ( Figure 7 D). IL-10 is a cytokine that inhibits inflammation. In the inflammatory environment induced by LPS, the culture supernatant of XA-511 significantly increased the production of the anti-inflammatory factor IL-10, showing certain potential for immune regulation ( Figure 7 E).

[0221] Example 7: The strain metabolizes to produce short-chain fatty acids such as butyric acid, valeric acid, isovaleric acid, and caproic acid

[0222] In this example, two media, PYG and Reinforced Clostridial Medium (ATCC 2107), were used to study the ability of the strain XA-511 to produce short-chain fatty acids and the differences in the production of short-chain fatty acids in different media.

[0223] The components of the two media are as follows:

[0224] Composition table of ATCC2107:

[0225]

[0226]

[0227] 1. Preparation and collection of the supernatant: The strain XA-511 was grown in the two media respectively until the late logarithmic growth phase. After adjusting OD600 = 1, the bacterial solution was centrifuged at 5000 g for 5 min at 4 °C, the supernatant was collected, and a cell-free supernatant was obtained by filtering with a 0.2 mm filter (Millipore). The supernatant was aliquoted into sterile tubes at 1 mL each and stored at -80 °C for later use.

[0228] 2. Short-chain fatty acid extraction and derivatization: Take 200 μL of the above bacterial supernatant samples, culture medium control samples, or short-chain fatty acid standards (0, 25, 50, 100, 200, 400, 800 μM), and mix them evenly with 200 μL of the internal standard isotope hexanoic acid solution containing 20 μg / mL in a 1:1 volume ratio. Take 100 μL of the mixed solution and acidify it with 10 μL of 5M HCl. The acidified supernatant sample is extracted by adding 100 μl of anhydrous diethyl ether (DE, 1:1, v / v), vortexed, and incubated on ice for 5 minutes, then centrifuged at 10,000 g for 5 minutes. Transfer the DE layer (containing SCFA) to a new microtube containing anhydrous Na 2 SO4 (to remove residual water). The remaining aqueous layer is further extracted twice with DE. The DE layers are combined and mixed for further derivatization. Accurately transfer 160 μl of the DE extract to the glass insert in a GC sample vial, add 8 μl of bis(trimethylsilyl)trifluoroacetamide (BSTFA), vortex for 5 seconds, and then cap tightly. Keep the mixture in the GC vial and incubate overnight at room temperature. The derivatized samples are analyzed by gas chromatography-mass spectrometry (GC-MS).

[0229] Detection of short-chain fatty acid content in samples: The short-chain fatty acid content is detected by a gas chromatography-mass spectrometer (8890-7000D, Agilent) equipped with an HP-5ms capillary column (30 m × 0.25 mm × 0.25 μm). The inlet, ion source, quadrupole, and GC / MS interface temperatures are 260, 230, 150, and 280 °C, respectively. The flow rate of the helium carrier gas is maintained at 1 mL / min. Inject 1 μl of the derivatized sample with a solvent delay time of 3 minutes and a split ratio of 10:1. The initial column temperature is 40 °C and is held for 2 min, then increased to 150 °C at a rate of 15 °C / min and held for 1 min, and finally increased to 300 °C at a rate of 30 °C / min and held at this temperature for 5 minutes. Ionization is carried out in the electron impact (EI) mode at 70 eV. MS data are collected in the full scan mode from m / z 40–400 at an acquisition frequency of 12.8 scans per second. The identification of compounds is confirmed by injecting pure standards and comparing the retention times and corresponding MS spectra. The analytes are quantified using target ion pairs in the selected ion monitoring (SIM) mode and confirmed by confirmation ions. The target ions (m / z) for acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, 4-methyl-valeric acid, hexanoic acid, and heptanoic acid are 117, 131, 145, 145, 159, 159, 173, 173, and 189, respectively. The content of each short-chain fatty acid in the sample is determined and calculated based on the retention time, target ions, and peak areas.

[0230] PYG medium is used as the culture medium control ( Figure 8A), the GC-MS detection results showed consistency with the ingredient list and no short-chain fatty acids were detected. When XA-511 grew to the late logarithmic phase in PYG medium, it could produce short-chain fatty acids such as butyric acid (3461 mM), isovaleric acid (789 mM), valeric acid (2924 mM), and caproic acid (3105 mM). Figure 8 A).

[0231] Figure 8 B The results showed that the ATCC2107 medium contained acetic acid and no other short-chain fatty acids. Figure 8 B); When XA-511 grew to the late logarithmic phase in the ATCC2107 medium Figure 8 B), it would almost completely consume the 14807 mM acetic acid in the medium, leaving only 81 mM. At the same time, it would produce a large amount of butyric acid (14807 mM), which was almost 4.3 times the butyric acid production in the PYG medium (3461 mM); while the production of valeric acid decreased (1509 mM), which was 1 / 2 of the production in the PYG medium. At the same time, the production of isovaleric acid increased by 3.7 times to reach 2910 mM, and the production of caproic acid increased to 7820 mM.

[0232] It can be seen from this that XA-511 can also produce short-chain fatty acids such as butyric acid, isovaleric acid, valeric acid, and caproic acid when growing in the ATCC2107 medium. Interestingly, the amounts of short-chain fatty acids produced by XA-511 are different when the medium components are different. Notably, when acetate is present in the medium (such as in the ATCC2107 medium), XA-511 will consume the acetate in the medium and produce a large amount of butyric acid. Figure 8 B).

[0233] Example 8: The ability of strain XA-511 to produce short-chain fatty acids is different in media containing different carbon sources.

[0234] As found in Example 7 above, the amounts of short-chain fatty acids produced by the strain are different in two different media. Further, we explored the effect of different carbon sources on the production of short-chain fatty acids by the strain.

[0235] Experimental method:

[0236] Using TPY medium as the basal medium, remove the carbon source lactose from the medium and replace it successively with glucose, maltose, mannitol, lactate, and fructose. When the strain grows to the late logarithmic growth phase, collect the bacterial solution, adjust the OD of the bacterial solution 600 = 1, centrifuge at 8000 rpm for 5 min to collect the supernatant, and determine the content of short-chain fatty acids (acetic acid, propionic acid, butyric acid) in the supernatant by the above method. The results are as follows Figure 9As shown

[0237] Composition table of TPY medium:

[0238]

[0239] From the experimental results, it can be seen that when the medium does not contain simple carbon sources (such as monosaccharides, disaccharides, oligosaccharides, etc.), XA-511 can produce a small amount of acetic acid (blue). When the carbon source in the medium is glucose, mannitol or fructose, XA-511 can metabolize to produce a certain amount of butyric acid (green). When the single carbon source in the medium is maltose, XA-511 can produce a certain amount of propionic acid (red, about 20000 mM) and a large amount of acetic acid (blue, about 50000 mM) in addition to butyric acid. When maltose is replaced by lactic acid, the amount of acetic acid produced by XA-511 is relatively reduced, with a yield of about (20000 mM), and at the same time, the amount of propionic acid is greatly increased, with a yield of about 350000 mM). Therefore, the strain XA-511 has different utilization abilities for different carbon sources, and can convert different carbon sources into different types of short-chain fatty acids through metabolism, which has potential application value in many disease fields.

[0240] Generally speaking, the strain XA-511 can utilize a variety of carbon sources (including glucose, maltose, mannitol, lactic acid, fructose) to metabolize and produce butyric acid. In addition, XA-511 can utilize maltose to produce a large amount of acetic acid and propionic acid.

[0241] Example 9: The strain treats breast cancer and reduces immune-related side effects caused by immunotherapy

[0242] To verify whether the bacterium XA-511 can be used for the prevention and treatment of tumors, we used a mouse homologous tumor model to conduct an experiment on inhibiting the growth of breast cancer 4T1. This experiment has passed the ethical review of the Experimental Animal Management and Use Committee of Shenzhen Top Biological Technology Co., Ltd.

[0243] Model establishment and administration: A total of 40 healthy SPF-grade Balb / c mice, female, mouse age: 4-5 weeks, were purchased from Zhuhai Beston Biotechnology Co., Ltd. All mice were adaptively housed in separate cages in an SPF-grade animal room for 1 week, at room temperature (24±2)°C, with a 12-hour day-night cycle and free access to water. After adaptive feeding, the mice were inoculated with 4T1 tumor cells. 4T1 cells were cultured in DMEM medium containing 10% fetal bovine serum and continuously cultured in a cell culture incubator at 37°C with 5% CO 2 When the cells grew to the logarithmic growth phase, the cells were collected, washed twice with PBS, and finally resuspended with PBS for cell counting. The cell concentration was adjusted to 1×10 6Cells / mL. In a sterile environment, 0.1 mL of cell suspension was injected into the right axilla (near the back) of the forelimb of each mouse. The day of cell inoculation was defined as Day 0. The mice were randomly divided into 4 groups, with 10 mice in each group, including an experimental control group, an XA-511 bacteria treatment group, an anti-PD-1 (InVivoPlus Anti-mouse PD-1, CLONE: RMP1-14, BioXcell) treatment group, and an XA-511 + anti-PD-1 treatment group. The grouping and drug administration were carried out as shown in Table 10.

[0244] Table 10 Grouping and Drug Administration of Animal Experiments on the Treatment of Breast Cancer with Strains

[0245]

[0246] Approximately one week after cell inoculation, the length and width of the tumors were measured using a vernier caliper on D7, D9, D11, D14, D16, D18, and D21. The tumor volume V was calculated using the formula: V = 0.5 × a × b 2 (where a and b represent the length and width of the tumor respectively), and the body weight of the mice was weighed. Grouping and drug administration were carried out according to the experimental design. The experiment ended 21 days after inoculation.

[0247] Mouse whole blood was collected in an EDTA anticoagulant tube and centrifuged at 3000 rpm for 10 min at room temperature. The upper plasma sample was taken and aliquoted into 0.2 mL eight-well tubes at 0.1 mL / tube and stored at -80 °C. The concentrations of cytokines and chemokines (including IL-6, IL-27, IL-17A, IL-1β, IL-12p70, IL-23, IL-1α, TNFα, and MCP-1) in the plasma were detected using the LEGENDplex Mouse Inflammation Panel (13-plex) with V-bottom Plate kit (BioLegend).

[0248] Tumor tissues were collected, fixed with paraformaldehyde, and then embedded in paraffin and sectioned. The sections were subjected to multiplex immunofluorescence staining. The anti-mouse antibodies used included CD3 (Invitrogen, Cat#MA5-14524), CD4 (Cell Signaling, Cat#25229), CD8 (Cell Signaling, Cat#98941), CD11c (Cell Signaling, Cat97585), and IFN-γ (Abcam, Cat#ab216642). All stained sections were scanned at 20× magnification using a Vectra multiplex imaging system. High-resolution images of the whole sections were generated and quantitatively analyzed using HALOTM software. The intensity of nuclear or cytoplasmic positive staining was calculated. The positive cell / area was evaluated as follows: positive cell / area = positive cells / tumor area.

[0249] Experimental results: In the experimental control group (control), the tumors in mice grew relatively fast. By 21 days after inoculation, the average tumor volume within the group reached 963.36 mm 3 ; in the anti-PD-1 antibody treatment group, the tumor growth rate was slightly slower than that in the control group. At day 21, the average tumor volume was 765.36 mm 3 , but there was no significant difference between the two groups; in the XA-511 + anti-PD-1 treatment group, the tumor growth was significantly slowed down. Moreover, at day 14 of the treatment, the tumor volume began to be significantly smaller than that in the control group and the anti-PD-1 treatment group. At day 21, the average tumor volume was 502.02 mm 3 , and the tumor inhibition rate was approximately 48% compared with the control group, showing a good tumor inhibition effect. Figure 10 This is the growth curve of 4T1 tumors in mice with breast cancer. Figure 11 These are the tumor photos of each group of 4T1 mouse breast cancer at 21 days.

[0250] Results of multiplex immunofluorescence staining of tumor tissues ( Figure 12 and Figure 13 ) showed that the XA-511 + anti-PD-1 treatment group could significantly increase the densities of CD8 + cytotoxic T lymphocytes and dendritic cells DC in the tumor tissue area, indicating that XA-511 synergistically treated tumors with anti-PD-1 antibody by promoting the infiltration of CD8 + T cells and DC cells into the tumor.

[0251] Clinically, immunotherapy is used to treat tumors, which is likely to cause immune diseases such as cytokine storm. By detecting plasma cytokines and chemokines, it was found that the combination therapy of XA-511 can alleviate the production of cytokines IL-27, IL-17A, and IL-23 caused by PD-1 antibody therapy, slow down the systemic immune overreaction, and thus reduce side effects such as cytokine storm caused by immunotherapy( Figure 14 ).

[0252] Breast cancer is considered a tumor with weak or moderate immunogenicity, and single immunotherapy often has limited therapeutic effects on it. In this example, XA-511 can achieve good synergistic anti-PD-1 antibody therapeutic effects on breast cancer by enhancing the infiltration of immune cells with anti-tumor activity in tumor tissues( Figure 10 and 13 ), while reducing side effects such as cytokine storm caused by immunotherapy.

Claims

1. A strain of Megasphaera martensii ( Megasphaera indica ), characterized in that, Its deposit number is CGMCC No.23400.

2. A pharmaceutical composition, characterized in that include: The live and / or inactivated bacteria of Megasphaera martensii as claimed in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that Also includes: Immune checkpoint inhibitors.

4. The pharmaceutical composition according to claim 3, characterized in that The immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

5. The pharmaceutical composition according to claim 2, characterized in that Also includes: A pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition is administered into the digestive system; or the pharmaceutical composition is administered by intratumoral injection.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition is administered orally or by enema.

8. Use of the Megasphaera martensii as claimed in claim 1 in the preparation of a medicament for enhancing the function of the immune system.

9. Use of the pharmaceutical composition according to claim 3 or 4 in the preparation of a medicament for treating cancer.

10. The use according to claim 9, characterized in that The cancer is breast cancer.

11. Use of Megasphaera martensii according to claim 1 in the preparation of a drug for alleviating immune-related side effects caused by immune checkpoint inhibitors, characterized in that: The immune-related side effect caused by the immune checkpoint inhibitor is a cytokine storm, and the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody and / or an anti-CTLA-4 antibody.

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