EA040421B1 - COMPOSITION, CELLS, METHODS FOR OBTAINING POLYPEPTIDE, APPLICATION OF POLYPEPTIDE AND CELLS, METHOD OF TREATMENT - Google Patents

Description

The technical field to which the invention belongs

The invention relates to the intestinal mucosal immune system, intestinal mucosal barrier, pharmaceutical, food or feed compositions containing polypeptides and/or host cells that are capable of modulating and/or stimulating the function of the intestinal mucosal immune system and/or maintaining and/or restoring and/or increase the physical integrity of the intestinal mucosal barrier and/or maintain and/or restore and/or improve glucose and/or cholesterol and/or triglyceride homeostasis in a mammal (eg, human). More specifically, the present invention provides compositions containing an Amuc1100 polypeptide or variants thereof. Amuc-1100 was found to be able to interact with toll-like receptor 2 (TLR2) and/or modulate TLR2 and/or NFk-B dependent signaling and/or stimulate the release of cytokines (e.g., IL-6, IL-8 and IL-10) from immune cells located near the mammalian (e.g., human) intestinal mucosal barrier and/or is capable of maintaining, restoring or enhancing the physical integrity of the intestinal mucosal barrier and/or maintaining and/or restoring and/or improving glucose and/or cholesterol and/or triglyceride homeostasis in a mammal and/or is capable of improving the metabolic or immune status of the mammal. The Amuc-1100 polypeptide can be used to prevent or treat a wide variety of diseases or conditions of the invention.

Prior Art

The increased permeability or hyperpermeability of the intestinal mucosa is believed to play a role in various diseases and conditions such as intestinal diseases, autoimmune diseases, allergies, cancer, type 2 diabetes, obesity, depression, anxiety, and many others. For this reason, there is increased interest in understanding the role of intestinal mucosal barrier dysfunction in the pathogenesis of many conditions affecting the gastrointestinal tract (GIT) in the mammal.

Under normal conditions, the intestinal mucosal barrier acts as a selective barrier, allowing absorption of nutrients, electrolytes, and water and preventing exposure to harmful macromolecules, micro-organisms, and food and microbial antigens (eg, food allergens). The intestinal mucosal barrier consists predominantly of a layer of mucus and an underlying layer of epithelial cells (hereinafter referred to as intestinal epithelial cells). Intestinal epithelial cells are closely connected to each other by so-called tight junctions, which are basically physical joints between the membranes of two intestinal epithelial cells. Maintaining the intestinal mucosal barrier, in particular maintaining the physical integrity of the intestinal epithelial cell layer (i.e., maintaining connections between cells), is critical to protecting the host from migration from the intestine into the circulation of pathogens, antigens, and other unwanted agents.

The intestinal mucosal barrier is also heavily colonized with symbiotic microorganisms (approximately 10 ¹² -10 ¹⁴ ), mostly anaerobic or microaerophilic bacteria, most of which live in symbiosis with the host organism. These bacteria are beneficial to the host organism in many ways. They provide protection against pathogenic bacteria and play a role in the nutrition of the host organism through the synthesis of vitamin K and some components of the vitamin B complex. In addition, the intestinal mucosal barrier has evolved a complex intestinal mucosal immune system to distinguish between symbiotic (i.e. beneficial) bacteria and pathogenic bacteria and other harmful agents. The intestinal mucosal immune system is an integral part of the intestinal mucosa and contains lymphoid tissues and specialized immune cells (ie, lymphocytes and plasmacytes) that are widely dispersed throughout the intestinal mucosal barrier. One microorganism that naturally colonizes the mucosa of healthy subjects is the mucin-degrading bacteria Akkermansia muciniphila, which has been shown to enhance gut barrier function (Everard et al., PNAS 110 (2013) 9066-71; Reunanen et al., Appl Environ Microbiol March 20,2015) and thereby affect diseases associated with impaired intestinal barrier function.

Under certain circumstances, the intestinal mucosal barrier may be vulnerable to a variety of infectious organisms or agents that are usually unable to penetrate the intestinal mucosal barrier, but nevertheless manage to overcome it (for example, through gaps resulting from loose tight junctions between cells intestinal epithelium). Organisms or other agents that cross the intestinal mucosal barrier can cause diseases or other undesirable conditions (eg, allergies) in the host. Examples of such diseases include obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension , heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis , neurodegenerative diseases, depression, other disorders associated with impaired barrier function, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea syndrome, osteoarthritis, gallbladder disease, and cancer.

On the contrary, diseases such as those mentioned above, as well as other conditions such as food allergies, intestinal underdevelopment, for example, due to premature birth, exposure to radiation, chemotherapy and / or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, etc. ., may alter the physical integrity of the intestinal mucosal barrier (i.e., cause loosening of the tight junctions between intestinal epithelial cells), which in turn may allow unwanted microorganisms or other agents to cross the host's intestinal mucosal barrier.

In the past, several vaccines and/or antibodies have been developed that target such microorganisms or agents. However, the success of this approach is offset by the fact that vaccines or antibodies are not able to effectively target or kill some microorganisms or agents.

Other approaches have also been explored to primarily prevent harmful microorganisms and other agents from penetrating the host's intestinal mucosal barrier and/or preventing increased permeability of the intestinal mucosal barrier. For example, compositions containing glutamic acid have been developed for the prevention and/or treatment of conditions associated with increased permeability of the intestinal mucosa (WO 01/58283). Other substances have also been used for this purpose, including spermine and spermidine and their precursors (Dorhout et al. (1997), British J. Nutrition, pp. 639-654). In order to modulate the intestinal mucosal barrier, preparations containing arabinoxylan have also been developed to promote beneficial effects on gastrointestinal bacteria living near the intestinal mucosal barrier (US 2012/0230955).

The object of the present invention is to provide agents and/or compositions containing such agents that are useful for maintaining and/or restoring and/or enhancing the physical integrity of the intestinal mucosal barrier and/or for preventing increased permeability of the intestinal mucosal barrier in a mammal (e.g., human) and/or to maintain and/or restore and/or improve glucose and/or cholesterol and/or triglyceride homeostasis in a mammal, thereby preferentially preventing or treating diseases or conditions associated with suboptimal intestinal mucosal barrier permeability and/or glucose imbalance. and/or cholesterol and/or triglyceride homeostasis in said mammal. Alternatively or additionally, it is an object of the present invention to provide agents and/or compositions containing such agents that are useful for modulating and/or stimulating the function of the intestinal mucosal immune system in a mammal.

Brief summary of the invention

The present invention provides a composition containing a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or influencing the barrier intestinal function and/or glucose and/or cholesterol and/or triglyceride homeostasis; and a pharmaceutically or nutritionally acceptable carrier.

The composition may be a food composition or a pharmaceutical composition.

The invention also provides a genetically modified host cell, the genome of which has been introduced with a nucleic acid molecule selected from the group consisting of a) a nucleic acid molecule containing a nucleic acid sequence at least 50% identical throughout the entire length of the sequence of SEQ ID NO: 2 ; and b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or affect intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis.

In addition, the invention provides a genetically modified host cell that does not belong to the Akkermansia muciniphilla species and contains a nucleic acid molecule selected from the group including a) a nucleic acid molecule containing a nucleic acid sequence that is at least 50% identical along its entire length amino acid sequence SEQ ID NO: 2; and b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or affect intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis.

In addition, the invention provides a modified host cell belonging to the species

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Akkermansia muciniphila, into whose genome is introduced a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule containing a nucleic acid sequence at least 50% identical along the entire length of the amino acid sequence of SEQ ID NO: 2; and b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or affect intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis.

The invention further provides a method for producing a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or influencing barrier function intestines and/or glucose and/or cholesterol and/or triglyceride homeostasis, the method comprising the steps of: a) culturing the host cell according to any one of claims 3 to 5 under conditions allowing said polypeptide to be obtained; and b) optionally isolating the polypeptide obtained in step (a).

The invention also provides a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical over the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or affecting the intestinal barrier function, and/or glucose and/or cholesterol and/or triglyceride homeostasis, described host cell or described composition for use as a drug, in particular for use in stimulating the function of the immune system of the intestinal mucosa, to maintain, restore and /or improve glucose and/or cholesterol and/or triglyceride homeostasis or to maintain, restore and/or increase the physical integrity of the intestinal mucosal barrier in a mammal.

Said polypeptide, composition, or host cell may be used to prevent and/or treat a disorder in a mammal, selected from the group consisting of obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, gestational diabetes, pre-eclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, dysfunction immune system associated with obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other diseases associated with impaired barrier function, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease , high level of cold esterol, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the gut mucosal barrier, such as food allergies, underdevelopment of the gut, such as due to preterm birth, exposure to radiation, chemotherapy, and/or or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, and the like.

Alternatively, or additionally, said polypeptide, host cell, or composition can be used to promote anti-inflammatory activity in the gut of a mammal and/or to promote weight loss in a mammal.

The invention also provides a method for the treatment and/or prevention in a mammal of a disorder selected from the group consisting of obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease ( IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other barrier disorders, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease, high cholesterol, elevated triglycerides c, atherosclerosis, sleep apnea syndrome, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment, such as due to premature birth, exposure to radiation, chemotherapy, and/or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, and the like, to promote weight loss in a mammal, promote anti-inflammatory activity in the mammalian intestine, stimulate the immune system function of the intestinal mucosa in a mammal, maintain, restore and/or improve glucose and/or or cholesterol and/or triglyceride homeostasis in a mammal or maintaining, restoring and/or enhancing physical integrity

- 3 040421 intestinal mucosal barrier of a mammal, the method comprising the step of administering to a mammal in need thereof an effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the length of the amino acid sequence of SEQ ID NO : 1, and capable of performing immune signaling and/or affecting intestinal barrier function and/or influencing glucose and/or cholesterol and/or triglyceride homeostasis, host cells according to the invention or compositions according to the invention.

In addition, the invention provides a method for producing a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or influencing barrier function of the intestine and/or glucose and/or cholesterol and/or triglyceride homeostasis, the method comprising the steps of: a) cultivating Akkermansia muciniphila bacteria in a suitable culture medium; and b) optionally isolating the polypeptide obtained in step (a).

General definitions

In the context of the present invention, the term polypeptide is equivalent to the term protein. The polypeptide has a specific amino acid sequence. A variant polypeptide according to the present invention preferably has an amino acid sequence at least 50% identical to the sequence of the polypeptide according to the present invention. The polypeptide of the invention is isolated when it is no longer in its natural environment, i. when it is no longer surrounded by fimbriae and/or surrounded by cells such as Akkermansia muciniphila cells.

As used herein, sequence identity or sequence similarity refers to a situation where an amino acid or nucleic acid sequence is identical or similar to another reference amino acid or nucleic acid sequence. Sequence identity or sequence similarity can be determined by aligning two polypeptides or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as predominantly identical or predominantly similar when (when optimally aligned, eg, by the GAP or BESTFIT programs using default parameters) they have at least a certain minimum percentage of identity (as defined below). GAP uses the Needleman-Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizing the number of gaps. Typically, the default GAP parameters are used, with gap creation penalty=50 (nucleotides)/8 (proteins) and gap widening penalty=3 (nucleotides)/2 (proteins). The default scoring matrix used for nucleotides is nwsgapdna and the default scoring matrix for proteins is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percent sequence identity can be determined using computer programs such as version 10.3 of the GCG Wisconsin software package from Accelrys Inc. (9685 Scranton Road, San Diego, CA 92121-3752, USA) or version 2.10.0 of EmbossWin (using a programming needle). Alternatively, percent similarity or identity can be determined by searching databases using algorithms such as FASTA, BLAST, etc. Sequence identity preferably means sequence identity over their entire length.

The term transepithelial resistance (TER) is a measure of the permeability of an epithelial cell layer in vitro. Increased epithelial permeability is associated with a weakening of tight junctions and a decrease in FER.

As used herein, the term chimeric gene refers to any non-naturally occurring gene, ie. a gene that does not normally occur naturally in any species, in particular a gene in which one or more parts of the nucleic acid sequence are not naturally related to each other. For example, in nature, the promoter is not associated fortunately with either the entire transcribed region or with another regulatory region. By the term chimeric gene is meant expression constructs whose heterologous promoter or transcriptional regulatory sequence is operatively linked to one or more coding sequences and optionally to a 3'-untranslated region (3'-UTR). Alternatively, the chimeric gene may contain a promoter encoding a sequence and optionally 3'-UTRs that are derived from the same species but do not occur naturally in this combination.

Used in the description, the term genetically modified host cell refers to cells that are genetically modified, for example, by introducing the sequence of exogenous nucleic acids (for example, SEQ ID NO: 2 according to the invention) or by specifically changing the sequence of endogenous genes. Such cells could be genetically modified

- 4 040421 us by introducing, for example, one or more mutations, insertions and/or deletions in the endogenous gene and/or introducing a genetic construct (eg, a vector or a chimeric gene) into the genome. Genetically modified host cells may refer to isolated cells or cell cultures. The genetically modified cells may be transduced cells that have been infected with, in particular, a modified virus, for example a retrovirus may be used, as well as other useful viruses such as lentiviruses. Non-viral methods such as transfections can also be used. Thus, genetically modified host cells can also be permanently transfected cells or transiently transfected cells. Transfection refers to non-viral methods of transferring DNA (or RNA) into cells, resulting in gene expression. Transfection methods such as calcium phosphate mediated, PEG mediated, liposome or lipoplex mediated transfection of nucleic acids and the like are widely known in the art. Such transfection may be temporary, but may also be permanent, and cells into which the gene construct is integrated into the genome may be selected.

Used in the description, the term effective amount refers to the amount necessary to achieve the effect described in the invention. For example, an effective amount of a polypeptide or genetically engineered host cell of the invention is an amount that is effective to modulate and/or stimulate the function of the intestinal mucosal immune system and/or maintain and/or restore and/or enhance the physical integrity of the intestinal mucosal barrier. (eg, to stimulate the formation of a tighter junction between intestinal epithelial cells) and/or to modulate and/or stimulate the signaling pathway of toll-like receptors (eg, TLR2) in the immune cell and/or to increase the production of cytokines (eg, IL-6 , IL-8 and IL-10) in an immune cell and/or for the prevention and/or treatment of disorders or conditions such as obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance goats, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other barrier disorders, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease, high cholesterol, elevated triglycerides, atherosclerosis, sleep apnea syndrome, osteoarthritis , gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment, such as due to premature birth, exposure to radiation, chemotherapy, and/or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, etc. .P.

As used, the term physiologically acceptable carrier or food acceptable carrier or nutritionally acceptable carrier or pharmaceutically acceptable carrier refers to a physiologically acceptable or nutritionally acceptable or nutritionally acceptable or pharmaceutically acceptable carrier such as a liquid or solid excipient. , diluent, excipient, solvent or encapsulating material involved in providing a form for administration of the polypeptide or host cell according to the invention. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the composition and harmless to the subject, i.e. applicable for consumption or acceptable from a nutritional point of view. The term usable for consumption or nutritionally acceptable refers to ingredients or substances that are generally considered safe for human (as well as other mammalian) consumption. Non-limiting examples of materials that can serve as physiologically acceptable carriers or nutritionally acceptable or pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; (21) other non-toxic compatible substances used in pharmaceutical compositions, and the like. In addition, as used herein, the terms nutritionally acceptable and pharmaceutically acceptable refer to those compositions or combinations of substances, materials or compositions and/or dosage forms thereof

- 5 040421 which are consistent with sound medical judgment, are applicable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction, or other problems or complications, and meet a reasonable balance of benefit and risk.

The term homeostasis refers to the property of a system whose variables are regulated in such a way that internal conditions remain stable and relatively constant. All animals regulate their blood glucose levels. The regulation of glucose in the body is the process of maintaining glucose homeostasis in the body. Mammalian blood glucose is regulated by various hormones (eg, insulin, glucagon, glucagon-like peptide 1, catecholamine, and many others) and various neural pathways (eg, nerve impulse translator, gut-brain-peripheral axis). The content of glucose in the human body is maintained constant for most of the day, even after a 24-hour fast. The glucose content is only slightly reduced even during long periods of fasting. Insulin, secreted by the beta cells of the pancreas, efficiently transports glucose into the cells of the body, instructing the cells to store more glucose for their own use. If the intracellular glucose content is high, the cells will convert it to insoluble glycogen to prevent soluble glucose from interfering with cellular metabolism. This ultimately lowers blood glucose, and insulin helps prevent hyperglycemia. Diabetes occurs when insulin is deficient or cells are resistant to insulin. Glucagon, secreted by the alpha cells of the pancreas, induces cells to break down stored glycogen or convert carbohydrate-free carbon sources into glucose through gluconeogenesis and thereby prevent hypoglycemia. Numerous other factors and hormones are involved in the control of glucose metabolism (for example, glucagon-like peptide 1, catecholamine, and many others). This complex regulation is also facilitated by various mechanisms associated with neural pathways.

Cholesterol homeostasis is a mechanism that contributes to the process of maintaining a balanced internal state of cholesterol in a living organism. Cholesterol, which is one of the important biological molecules in the human body, performs various physiological functions, such as the function of a precursor for the synthesis of bile acids, vitamin D and steroid hormones. It is also one of the important structural elements of the membrane of every cell present in the body. Despite the beneficial and necessary functions of cholesterol, disruption of cholesterol homeostasis can cause an increased risk of heart disease, as well as disrupt other homeostatic feedback systems associated with cholesterol metabolism. The most obvious organ that regulates cholesterol homeostasis is the liver, as it is not only responsible for the biosynthesis of cholesterol that is released into the circulatory system, but also breaks down potentially harmful, unbound cholesterol from the bloodstream. High-density lipoproteins (HDL) are useful in maintaining cholesterol homeostasis, as they capture and return potentially dangerous cholesterol directly to the liver, where it is synthesized into safe bile acids used by the digestive system. LDL is less beneficial because it tends to accumulate its cholesterol in the cells of the body and on the arterial walls. Excessive LDL levels have been shown to increase the risk of cardiovascular disease. In healthy subjects, cholesterol homeostasis is tightly regulated by complex feedback loops. In this case, if a healthy subject consumes copious amounts of dietary cholesterol, liver biosynthesis is greatly reduced to maintain balance. In a healthy subject with high baseline LDL levels, due to years of unhealthy eating habits or other genetic or medical conditions, the feedback loop and systemic coping mechanism can be overwhelmed by this same high intake, causing a dangerous homeostatic imbalance.

Triglyceride homeostasis is a mechanism that contributes to the process of maintaining a balanced internal state of triglycerides in a living organism. Triglyceride metabolism plays an important clinical role. Hypertriglyceridemia means high blood or serum levels of triglycerides, the most common fatty acid molecules. Elevated triglycerides are associated with atherosclerosis even in the absence of hypercholesterolemia (high cholesterol) and predispose to cardiovascular disease. High triglycerides also increase the risk of acute pancreatitis. In addition, an increase in triglycerides over time increases the risk of developing diabetes. Insulin resistance has been proven to be associated with high levels of triglycerides (TG).

The term about is used to denote intervals of standard engineering tolerance, for example, within 2 standard deviations from the mean. The term about can be understood to encompass values that deviate by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, or 0.01 % of the specified value.

As used herein, the terms containing or contain and their derivative forms refer to the situation in which said terms are used in their non-limiting sense to indicate that everything that follows is included, and that not specifically stated is not excluded. It also covers the more restrictive terms consist predominantly of and consist of.

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Mentioning any element in the singular does not exclude the possibility of having more than one element, unless the context explicitly requires the presence of one and only one of the elements.

Thus the singular form usually means at least one.

Detailed description of the invention

The inventors have found that the Amuc-1100 polypeptide or variants thereof according to the invention are able to modulate and/or stimulate the function of the intestinal immune system and/or maintain and/or restore and/or increase the physical integrity of the intestinal mucosal barrier and/or maintain and/or restore and /or improve glucose and/or cholesterol and/or triglyceride homeostasis in a mammal (eg human). The inventors also found that this polypeptide is present outside the cells encoded by the bacteria Akkermansia muciniphila, which confirms its role in signaling.

The inventors believe that these beneficial effects are due to the ability of the polypeptides of the invention to interact with the TLR2 signaling pathway present on the surface of immune cells located near the mammalian intestinal mucosal barrier. More specifically, the present inventors have found that the polypeptides described herein are capable of interacting with TLR2 present on the surface of an immune cell and/or modulating and/or stimulating the TLR2 signaling pathway in an immune cell located near the intestinal mucosal barrier, so as to stimulate secretion of cytokines (eg IL-6, IL-8 and IL-10) from said immune cells.

In addition, the authors of the present invention found that the polypeptides, including their variants according to the invention, are able to modulate and/or increase the transepithelial resistance of the intestinal mucosal barrier of a mammal. Since increased transepithelial resistance is indicative of reduced permeability of the intestinal mucosal barrier, polypeptides, including variants of the invention, are expected to be able to regulate the physical integrity of the intestinal mucosal barrier, in particular at the level of tight junctions between epithelial cells.

All of these combined are thought to result in improved or enhanced function of the gut mucosal immune system (e.g., greater release of cytokines at the gut mucosal barrier), as well as improved or enhanced physical integrity of the gut mucosal barrier, in particular at the level of communication between intestinal epithelial cells (i.e. due to tighter junctions between cells).

In addition, it was found that the treatment of mice fed a high fat diet (HFD) with Amuc-1100 caused a marked decrease in body weight and an increase in fat mass without affecting food intake. Treatment with Amuc-1100 also corrected HFD-induced hypercholesterolemia with a significant decrease in serum HDL-cholesterol and a similar trend for LDL-cholesterol. In addition, the introduction of Amuc-1100 reduced glucose intolerance to the same extent as in the case of live bacteria Akkermansia muciniphila.

Finally, since metformin is known to stimulate the growth of Akkermansia (Lee H and Ko G, Appl Environ Microbiol. 2014 Oct, 80 (19): 5935-43), it is therefore likely that Akkermansia and its extracellular peptides such as Amuc-1100 , may have an effect similar to that of metformin on gestational diabetes and preeclampsia (Syngelaki et al., N Engl J Med. 2016 Feb 4, 374 (5): 434-43).

Polypeptides.

The present invention provides an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical throughout the amino acid sequence of SEQ ID NO: 1, and capable of immune signaling and/or affecting intestinal barrier function and/or glucose and/or cholesterol homeostasis. The disclosed polypeptide may be capable of binding toll-like receptor 2 (TLR2).

In one embodiment, the polypeptides and variants thereof according to the invention are capable of stimulating the TLR2 signaling pathway in the cell, stimulating the release of cytokines from the cell (e.g., IL-6, IL-8, IL-10, etc.), and/or increasing transepithelial resistance (TER) of a mammal, eg a human, and/or improve the metabolic or immune status of a mammal, eg a mouse or a human.

The polypeptide of the invention may also be referred to as an Armuc-1100 protein or an Amuc-1100 polypeptide. It should be understood that the term Armuc-1100 protein or Armuc-1100 polypeptide or polypeptide of the invention also includes variants of the Armuc-1100 protein having the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequences of said variants are greater than 50%, preferably greater than 55% more than 60% more than 65% more than 70% preferably more than 75% more than 80% more than 85% more than 90% more than 95%, preferably greater than 96%, preferably greater than 97%, preferably greater than 98% and preferably greater than 99% amino acid identity

- 7 040421 of the sequence of SEQ ID NO: 1. Variants of the Armuc-1100 polypeptide having the amino acid sequence of SEQ ID NO: 1 also include polypeptides that, by substitution, deletion or insertion of one or more amino acids, are derived from a polypeptide having the amino acid sequence of SEQ ID NO: : 1. Such polypeptides preferably contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more to 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 substitutions, deletions or insertions of amino acids compared to a polypeptide having the amino acid sequence of SEQ ID NO: 1.

The polypeptide according to the invention may be preceded by an N-terminal signal sequence that stimulates the secretion of the polypeptide from the cell. In one embodiment, the N-terminal signal sequence may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 3, which is the predicted natural N-terminal signal sequence of the Armuc-1100 polypeptide. However, other N-terminal signal sequences capable of causing Armuc-1100 to be secreted from the cell can also be used. For example, a truncated version or an extended version of the predicted natural N-terminal signal sequence of the Amuc-1100 N-polypeptide can be used, provided that such N-terminal signal sequence can confer secretion of Amuc-1100 from the cell. Alternatively, a non-naturally occurring N-terminal signal sequence may be used. One skilled in the art is able to identify the N-terminal signal sequences useful in the present invention. Thus, a polypeptide according to the present invention may contain the amino acid sequence of SEQ ID NO: 3 at the N-terminus of its amino acid sequence.

Any applicable means available in the art can be used to determine amino acid sequence identity. For example, amino acid sequence identity can be determined by pairwise alignment using the Needleman-Wunsch algorithm and the default GAP parameters as described above. It is also contemplated that many methods, such as Western blot, immunohistochemistry, ELISA, amino acid synthesis, and the like, can be used to identify, synthesize, or isolate polypeptide variants of the invention.

It is also intended that any variants of the Amuc-1100 polypeptides of the invention exhibit the same function and/or have the same activity as the Amuc-1100 polypeptide of the invention. The functionality or activity of any Amuc-1100 polypeptides or variants thereof can be determined by any methods known in the art that the skilled person would consider applicable for this purpose.

Polynucleotides.

The present invention also provides a nucleic acid molecule, such as an isolated, synthetic or recombinant nucleic acid molecule, comprising a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence at least 50% identical throughout the amino acid sequence of SEQ ID NO: 2; and (b) a nucleic acid sequence that encodes a polypeptide of the invention.

The term isolated nucleic acid molecule (eg, cDNA, genomic DNA, or RNA) includes naturally occurring, artificial, or synthetic nucleic acid molecules. Nucleic acid molecules can encode any of the polypeptides of the invention. The nucleic acid molecule can be used to obtain polypeptides according to the invention. Due to the degeneracy of the genetic code, different nucleic acid molecules may encode the same polypeptide (eg, a polypeptide containing the amino acid sequence of SEQ ID NO: 1).

In one embodiment, the isolated nucleic acid molecules of the invention include molecules of any nucleic acid variants, which include any nucleic acid molecules containing a nucleotide sequence greater than 50%, preferably greater than 55%, preferably greater than 60% , preferably more than 65%, preferably more than 70%, preferably more than 75%, preferably more than 80%, preferably more than 85%, preferably more than 90%, preferably more than 95%, preferably greater than 96%, preferably greater than 97%, preferably greater than 98%, and preferably greater than 99% identical to the nucleotide sequence of SEQ ID NO: 2. Variants also include nucleic acid molecules that, by substitution, deletion, or one or more nucleic acid inserts are derived from a nucleic acid molecule having the nucleic acid sequence SEQ I D NO: 2. Such nucleic acid molecules preferably contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more to 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 substitutions, deletions or insertions of nucleic acids compared to SEQ ID NO: 2.

Any applicable means available in the art can be used to determine sequence identity. For example, bioinformatics can be used to perform pairwise alignment of nucleotide sequences in order to identify areas of similarity that may be due to functional, structural or evolutionary relationships.

- 8 040421 between sequences. It is also contemplated that many methods, such as nucleic acid hybridization, PCR technology, computer simulations, nucleic acid synthesis, and the like, can be used to identify, synthesize, or isolate polynucleotide variants of the invention.

In addition, it is understood that any nucleic acid molecule according to the invention can encode a polypeptide according to the invention.

In one embodiment, the nucleic acid molecule is a nucleic acid molecule having the nucleic acid sequence shown in SEQ ID NO: 2.

Alternatively, the isolated nucleic acid molecule may be a nucleic acid molecule that hybridizes under stringent conditions with nucleic acid molecules of the invention and encodes a polypeptide of the invention. For example, such a nucleic acid sequence can advantageously be used in screening to detect the presence or absence of any homologues of nucleic acid molecules of the invention in a cell or organism, or to detect a decrease or increase in the expression of nucleic acid molecules of the invention in a cell or organism.

A nucleic acid molecule of the invention may be a nucleic acid molecule encoding an N-terminal signal sequence useful for promoting secretion of a polypeptide of the invention from its host cell. The nucleic acid molecule encoding the N-terminal signal sequence may comprise the nucleic acid sequence shown in SEQ ID NO: 4.

In one embodiment, the nucleic acid molecule of the invention may be included in a chimeric gene in which it is operably linked to a promoter. Thus, the present invention also relates to a chimeric gene containing the nucleic acid molecule according to the invention.

Any promoters known in the art and useful for linking the nucleic acid molecules of the invention may be used. Non-limiting examples of useful promoters include promoters capable of constitutive or regulative expression, weak and strong expression, and the like. Any methods known in the art can be used to incorporate the nucleic acid molecule of the invention into a chimeric gene.

It may be advantageous to operatively link the nucleic acid molecule of the invention to a so-called constitutive promoter.

Alternatively, it may be advantageous to operably link the polynucleotides and variants thereof according to the invention to a so-called inducible promoter. An inducible promoter may be a promoter that is physiologically regulated (eg, by topical application of certain compounds).

The chimeric gene of the invention may be contained in a vector or nucleic acid construct. Thus, the present invention also relates to vectors containing a chimeric gene according to the invention or a nucleic acid molecule according to the invention.

In one aspect, the present invention provides a host cell genetically modified such that its genome contains a nucleic acid molecule according to the invention, a chimeric gene according to the invention, or a vector according to the invention.

The genetically modified host cell of the invention can be used to produce in vitro, ex vivo and/or in vitro polypeptides and variants thereof of the invention in the cytoplasm of host cells or released from cells by any means. The polypeptides of the invention may in particular be expressed as a soluble or secreted molecule. Genetically modified host cells according to the invention can be any host cells applicable for transformation procedures or genetic engineering. Non-limiting examples of useful host cells include cultured cells such as any prokaryotic or eukaryotic cells. In one embodiment, the AMUC-1100 polypeptide is expressed in bacteria such as Escherichia coli.

In one embodiment, the host cell of the invention may be any cell that naturally expresses the polypeptide or variants thereof of the invention. In such a case, the host cell may overexpress the polypeptide or variants thereof according to the invention.

In yet another embodiment, the host cell of the invention may be any cell that does not naturally express the polypeptide or variants thereof of the invention.

In an embodiment, the host cell of the invention is not Akkermansia muciniphila.

In another embodiment, the host cell may be of the Akkermansia muciniphila species and is genetically modified to contain additional copies of the nucleic acid molecules of the invention or a chimeric gene or vector of the invention.

- 9 040421

Such an Akkermansia muciniphila cell may overexpress the Amuc-1100 polypeptide or a variant thereof according to the invention.

The host cell of the invention may be genetically modified by any of the methods known in the art. For example, host cells or organisms of the invention may be genetically modified in a manner comprising the steps of:

a) transforming a host cell with a nucleic acid molecule according to the invention, for example, an isolated, synthetic or recombinant nucleic acid molecule containing a nucleic acid sequence selected from the group of nucleic acid sequences at least 50% identical throughout the entire length of the sequence with SEQ ID NO : 2; and a nucleic acid sequence capable of encoding polypeptides and variants thereof according to the invention;

b) culturing said host cell under conditions suitable for expression of a nucleic acid molecule according to the invention and/or production of a polypeptide or variant thereof according to the invention;

c) optionally screening host cells capable of expressing a nucleic acid molecule according to the invention and/or producing a polypeptide or variant thereof according to the invention.

In one preferred embodiment, the host cells or organisms of the invention may be transformed with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 2, or a variant thereof according to the invention.

In one embodiment, the genetically modified host cell of the invention may be of a bacterial species that occurs naturally or lives near or within the intestinal mucosal barrier of a mammal. Bacteria of this species are often referred to as intestinal mucosal bacteria. Non-limiting examples of intestinal mucosal bacteria include Akkermansia muciniphila (ATTC BAA-835), Faecalibacterium prausnitzii (A2165), Lactobacillus rhamnosus (ATCC 53103), and Bifidobacterium breve (DSM-20213).

In some embodiments, it may be advantageous to genetically modify intestinal mucosal bacteria with any of the polynucleotides and variants thereof of the invention, e.g., to express or overexpress the polynucleotides of the invention, or to produce or overexpress the polypeptides of the invention immediately adjacent to or within the mammalian intestinal mucosal barrier (e.g., person). In one preferred embodiment, the intestinal mucosal bacteria may be any of the Akkermansia muciniphilla species. Such overproduction can be achieved by genetic modification techniques using recombinant DNA technologies, genome editing, such as by using tools based on systems such as CRISPR/cas, or by classical mutation selection systems.

In one embodiment, the genetically modified host cell may be any bacteria, in particular, not of a bacterial species that occurs naturally or lives near or within the intestinal mucosa of a mammal. Non-limiting examples of such bacteria include strains isolated from any beneficial intestinal bacteria, for example, probiotic bacteria can be used, in particular strains of bacteria selected from the genera Lactococcus, Lactobacillus or Bifidobacterium. In addition, strictly anaerobic intestinal bacteria can be used, such as bacteria belonging to genera known to occur in the human intestinal tract (Rajilic-Stojanovic & de Vos, The first 1000 cultured species of the human gastrointestinal microbiota. FEMS Microbiol Rev 38:996-1047).

Methods for obtaining a polypeptide.

According to one of the additional features of the present invention, a method is provided for obtaining polypeptides, including their variants according to the invention, including the steps:

(a) culturing a host cell according to the invention under conditions that produce a polypeptide or variant thereof according to the invention; and (b) optionally isolating the polypeptide obtained in step (a).

In step (a), the host cell according to the invention may be cultured by any known methods and in any known culture medium. One of ordinary skill in the art will be able to select the applicable host cell and applicable conditions to produce the polypeptide.

Alternatively, the polypeptide can be obtained by a method comprising the steps of:

(a) culturing bacteria of the species Akkermansia muciniphila in an applicable culture medium; and (b) optionally, isolating the polypeptide obtained in step (a).

The polypeptide obtained in steps (a) of said methods may be isolated by any of the methods known in the art. One skilled in the art is able to isolate a polypeptide obtained from such a culture medium.

Useful culture media are, for example, described by Derrien et al. (2004, Int. J. Syst. Evol. Microbiol. 54:1469-76). As described by Derrien et al., the MucT Muciniphila strain was isolated and grown in a basal anaerobic medium containing porcine gastric mucus mucin as the sole source of carbon and nitrogen. The authors also write that A. muciniphila can be grown in fatty media such as Columbia Broth (CB) and Brain Heart Infusion (BHI) or basal glucose media. and high content of casitones and yeast extracts. Similarly, Lukovac et al. described growing A. muciniphila in a basal medium containing glucose and fucose, as well as large amounts of casitones (2014, mBio 01438-14)

Methods for screening bacteria.

According to one additional feature of the present invention, there is provided a method for detecting the presence or absence of a polynucleotide according to the invention in bacteria, comprising the steps of:

(a) using a nucleic acid molecule that hybridizes under stringent conditions with a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence at least 50% identical throughout the entire length of the nucleic acid sequence of SEQ ID NO: 2;

(b) detecting the nucleic acid molecule used in step (a) in order to identify bacteria containing a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2 or a nucleic acid sequence at least 50% identical along the entire length of the sequence nucleic acids SEQ ID NO: 2.

The present invention also provides a method for detecting the presence or absence of a polypeptide or variants thereof in bacteria according to the invention, comprising the steps of:

(a) using an antibody that is capable of binding a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical to the amino acid sequence of SEQ ID NO: 1;

(b) detection of the antibody used in step (a) to identify bacteria containing a polypeptide having the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 50% identical to the amino acid sequence of SEQ ID NO: 1.

In one embodiment, to facilitate detection, the nucleic acid used in step (a) and/or the antibody used in step (a) are labeled (eg, with fluorescent, radioactive labels, etc.).

Compositions.

According to one of the additional features of the present invention proposed a composition containing any of the polypeptides according to the invention. In one preferred embodiment, the polypeptide has the amino acid sequence of SEQ ID NO: 1.

According to another of the additional features of the present invention proposed a composition containing a host cell according to the invention. The host cell may be present in an amount of from about 10 ⁴ to about 10 ¹⁵ colony forming units (CFU). For example, an effective amount of the host cell may be from about 105 CFU to about 10 ¹⁴ CFU, preferably from about 105 CFU to about 10 ¹³ CFU, preferably from about 10 ⁷ CFU to about 10 ¹² CFU, more preferably from about 10 ⁸ CFU to about 10 ¹² CFU. The host cell may be viable or dead. The efficiency of the host cell is in a certain relationship with the presence of the polypeptide according to the invention.

In one embodiment, the composition of the invention further comprises a carrier, for example a physiologically acceptable carrier or a pharmaceutically acceptable carrier or a food acceptable carrier or a nutritionally acceptable carrier. The carrier may be any inert carrier. For example, non-limiting examples of applicable physiologically or pharmaceutically acceptable carriers include any of the known physiological or pharmaceutical carriers, buffers, diluents, and excipients. It should be noted that the choice of the applicable physiological or pharmaceutical carrier or food carrier or nutritional carrier will depend on the intended route of administration of the composition according to the invention (for example, orally) and the intended form of the composition (for example, drink, yogurt, powder, capsule, etc.) . Those skilled in the art will know how to select an appropriate carrier, such as a physiologically acceptable carrier, or a nutritionally acceptable carrier, or a pharmaceutically acceptable carrier, which is useful in or compatible with the compositions of the invention.

In one embodiment, the compositions of the invention may be a nutritional or nutritional composition. For example, a composition according to the invention may be a food product, food supplement, feed or feed supplement such as a dairy product, for example a fermented dairy product such as yogurt or drinkable yogurt. In this case, the composition may contain a nutritionally or nutritionally acceptable carrier, which may be a suitable food base.

In one embodiment, the compositions of the invention may be

- 11 040421 pharmaceutical composition. The pharmaceutical composition can also be used as an additive (eg food additive). The pharmaceutical composition according to the invention may contain a pharmaceutically acceptable, nutritionally or nutritionally acceptable or physiologically acceptable carrier in addition to the polypeptide according to the invention and/or host cells according to the invention. The preferred form depends on the intended route of administration and the (therapeutic) application. The carrier may be any compatible, physiologically acceptable, non-toxic substances useful for delivering a polypeptide of the invention and/or a host cell of the invention to the gastrointestinal tract of a mammal (e.g., human), preferably to an area near or within the intestinal mucosal barrier (more preferably mucosal barrier of the colon) of a mammal. For example, the carrier may be sterile water or inert solids, usually supplemented with a pharmaceutically acceptable adjuvant, buffering agent, dispersant, and the like.

The composition according to the invention may be in liquid form, for example in the form of a stabilized suspension of a polypeptide according to the invention or a host cell according to the invention, or in solid form, for example in the form of a powder of lyophilized host cells according to the invention. If the host cells according to the invention are lyophilized, a cryoprotectant such as lactose, trehalose or glycogen can be used.

In the case of oral administration, the polypeptides of the invention or the lyophilized host cells of the invention may be administered as solid dosage forms such as capsules, tablets and powders, or as liquid dosage forms such as elixirs, syrups and suspensions. The polypeptide according to the invention or the host cell according to the invention may be encapsulated, such as gelatin capsules, together with inactive ingredients and powdered carriers, such as, for example, glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate , stearic acid, saccharin, talc, magnesium carbonate, etc.

In one embodiment, the compositions of the invention may contain one or more ingredients that are useful in promoting survival and/or viability and/or maintaining the polypeptide of the invention and/or its integrity and/or the host cell of the invention, and /or its integrity during storage, and/or during exposure to bile, and/or during passage through the gastrointestinal tract of a mammal (eg, human). Non-limiting examples of such ingredients include enteric coatings and controlled release gastric transit agents. Those skilled in the art will know how to select the applicable ingredients to ensure that the active ingredient (polypeptide or host cell) reaches its intended destination where it performs its action.

In one embodiment, the compositions of the invention may further comprise a mucosal binding agent or mucosal binding polypeptide. As used herein, the term mucosal-binding agent or mucosal-binding polypeptide refers to an agent or polypeptide that is capable of adhering to mucosal surfaces that form a mammalian (eg, human) intestinal mucosal barrier.

Alternatively, specific docking systems can be used to attach the Amuc-1100 polypeptide to Amuc-1100 producing cells or even to non-Amuc-1100 producing cells that are alive or dead. Binding can occur at the C-terminus or N-terminus, depending on where it seems to be more effective, although the use of spacer peptides has also been described. Examples include the use of peptidoglycan binding systems based on LysM (Visweswaran GR et al., 2014, Appl Microbiol Biotechnol. 98: 4331-45). In addition, a variety of mucosal-binding polypeptides are known. Non-limiting examples of mucosal-binding polypeptides include bacterial toxin membrane-binding subunits such as cholera toxin-binding subunit B, heat-labile enterotoxin-binding E. coli subunit B, Bordetella pertussis toxin-binding subunits S2, S3, S4 and/or S5, diphtheria toxin-binding fragment B and membrane-binding subunits of Shiga toxin or Shiga-like toxins. Other useful mucosal binding polypeptides include bacterial pili proteins such as E. coli pili K88, K99, 987P, F41, FAIL, CFAIII ICES1, CS2 and/or CS3, CFAIIV ICS4, CS5 and/or CS6), P fimbriae, etc. Other non-limiting examples of fimbriae include Bommetella pertussis filamentous hemagglutinin, toxin-regulated pili (TCP) of vibrio cholerae, mannose sensitive hemagglutinin (MSHA), fucose sensitive hemagglutinin (PSHA), and the like. Other mucosal-binding agents include viral attachment proteins, including influenza and Sendai virus haemagglutinins, as well as animal lectins or lectin-like molecules, including immunoglobulin molecules or fragments thereof, calcium-dependent type (C-type) lectins, selectins, collectins, or snail hemagglutinin, plant lectins with mucosal binding subunits, including concanavalin A, wheat germ agglutinin, phytohemagglutinin, abrin, ricin, and the like. The advantage of this delivery method is that no living recombinant organism is used.

- 12 040421

Although not essential, it may be beneficial to add one or more mucosal binding agents or mucosal binding polypeptides to the composition of the invention to target the polypeptide of the invention or a host cell of the invention to the intestinal mucosal barrier.

Compositions of the invention may further contain ingredients selected from the group consisting of prebiotics, probiotics, carbohydrates, polypeptides, lipids, vitamins, minerals, drugs, preservatives, antibiotics, or any combination thereof.

In one of the embodiments, the implementation of the composition according to the invention may further contain one or more ingredients that further enhance the nutritional value and/or therapeutic value of the compositions according to the invention. For example, it may be useful to add one or more ingredients (e.g., nutritional ingredients, veterinary or medicinal products, etc.) selected from proteins, amino acids, enzymes, mineral salts, vitamins (e.g., thiamine hydrochloride, riboflavin, pyridoxine hydrochloride, niacin, inositol, choline chloride, calcium pantothenate, biotin, folic acid, ascorbic acid, vitamin B12, p-aminobenzoic acid, vitamin A acetate, vitamin K, vitamin D, vitamin E, etc.), sugars and complex carbohydrates (e.g. , water-soluble and water-insoluble monosaccharides, disaccharides and polysaccharides), medicinal compounds (e.g. antibiotics), antioxidants, trace elements (e.g. compounds of cobalt, copper, manganese, iron, zinc, tin, nickel, chromium, molybdenum, iodine, chlorine, silicon, vanadium, selenium, calcium, magnesium, sodium, potassium, etc.). Those skilled in the art are aware of methods and ingredients useful for enhancing the nutritional and/or therapeutic/medical value of the compositions of the invention.

In one embodiment, the host cell may be included in a lyophilized form or microencapsulated form (reviewed, for example, by Solanki et al., BioMed Res. Int. 2013, Article ID 620719) or any other form that retains cell activity and/or viability. host (eg bacterial strain).

Methods of treatment.

According to another aspect of the present invention, methods are provided for treating and/or preventing a disorder or condition in a mammal selected from the group consisting of obesity, metabolic syndrome, insulin deficiency or insulin related disorders, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, immune system dysfunction, obesity (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other barrier disorders, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease, high cholesterol a, elevated triglycerides, atherosclerosis, sleep apnea, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the gut mucosal barrier, such as food allergies, underdevelopment of the gut, such as due to preterm birth, exposure to radiation, chemotherapy, and/or or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, etc.; methods of promoting weight loss in a mammal; methods of promoting anti-inflammatory activity in the gut of a mammal; methods of stimulating the function of the immune system of the intestinal mucosa of a mammal; methods for maintaining, restoring and/or improving glucose and/or cholesterol and/or triglyceride homeostasis; and methods of maintaining, restoring and/or enhancing the physical integrity of the mammalian intestinal mucosal barrier. The methods include the step of administering to a mammal in need thereof an effective amount of a polypeptide of the invention, a host cell of the invention, or a composition of the invention.

In one embodiment, a polypeptide of the invention, a host cell of the invention, or a composition of the invention may be administered by any known means. For example, the compositions of the invention may be administered orally, intravenously, topically, enterally, or parenterally. It is intended that modes or routes of administration depend on the particular case (e.g., age of the subject, desired location of effects, disease states, etc.) as well as the intended form of the composition (e.g., tablets, liquid, powder, etc.) .

In one of the preferred embodiments, the implementation of the polypeptide according to the invention, the host cell according to the invention or the composition according to the invention is administered orally.

Application.

According to another aspect of the present invention, the use of a nucleic acid molecule according to the invention, a chimeric gene according to the invention and/or vectors according to the invention for the production of polypeptides according to the invention and/or generation of host cells according to the invention is provided. Polypeptide according to the invention and/or host cell according to the invention

- 13 040421 may have an improved ability to interact with the TLR2 receptor in the cell and/or an improved ability to stimulate the TLR2 signaling pathway in the cell and/or an improved ability to stimulate the production of cytokines from the cell, in particular ΓΕ-1β. IL-6, IL-8, IL-10, and TNF-α, and/or improved ability to increase the FER of mammalian, e.g., human cells compared to a host cell (e.g., bacteria) not genetically modified with polynucleotides, chimeric genes, or vectors according to the invention.

According to another aspect of the present invention, there is provided a polypeptide of the invention, host cells of the invention, or a composition of the invention for use as a medicament; in particular, to stimulate the function of the immune system of the intestinal mucosa or to maintain, restore and/or increase the physical integrity of the intestinal mucosal barrier in a mammal; maintaining, restoring and/or improving glucose and/or cholesterol and/or triglyceride homeostasis in a mammal; prevention and/or treatment of a disorder or condition selected from the group consisting of obesity such as malnutrition obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease ( IBD), irritable bowel syndrome (IBS), glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, heart disease, stroke, non-alcoholic fatty liver disease, alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), allergies, asthma, autism, Parkinson's disease, multiple sclerosis, neurodegenerative diseases, depression, other barrier disorders, wound healing, behavioral disorders, alcohol dependence, cardiovascular disease, high cholesterol, elevated triglyceride levels, and myosclerosis, sleep apnea syndrome, osteoarthritis, gallbladder disease, cancer, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment, such as due to premature birth, exposure to radiation, chemotherapy, and/or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis, etc., in a mammal; to promote anti-inflammatory activity in the mammalian intestine; or to promote weight loss in a mammal.

In one embodiment, a mammal, such as a human, may be of any age group (eg, infants, adults, seniors) and any gender (male and female). In one embodiment, the mammal may be a child (eg, a newborn, infant, toddler, etc.), in particular a premature baby.

The mammal may be any mammal, such as humans, non-human primates, rodents, cats, dogs, cows, horses, and the like. In one preferred embodiment, the mammal is a human.

The present invention is further illustrated, but not limited, by the following examples. From the foregoing description and these examples, one skilled in the art can recognize the essential features of the present invention and can, without departing from the spirit and scope of the present invention, make various changes and modifications to adapt it to different applications and conditions. Therefore, from the above description, those skilled in the art will recognize various modifications of the invention beyond those shown and described therein. Such modifications are also intended to be within the scope of the appended claims.

Brief description of the drawings

The drawing shows:

A) total increase in body weight (g) (n=8-10);

B) total fat mass gain (g) measured by time-resolved NMR spectroscopy (n=8-10);

C) daily food intake;

D) plasma levels of VLDL, LDL and HDL cholesterol (n=8-10);

E) plasma glucose profile (mg dl ^-1 );

F) mean area under the curve (AUC) measured between -30 min and 120 min after glucose loading (mg dl ^-1 ; min ^-1 ; n=8-10);

G) ratio between control and stimulated insulin p-IRe under load control measured with densitometry (n=3-5);

H) and I) ratios between control and stimulated insulin p-Aktt ^hr308 and p-Akt ^ser473 under exercise control measured by densitometry (n=3-5).

Examples

Example 1 Propagation of bacteria genetically modified to produce Amuc-1100 proteins.

Way.

Cloned from a polynucleotide encoding a mature Amuc-1100 (with a nucleotide sequence

- 14 040421 with SEQ ID NO: 2), E. coli TOP10 with a C-terminal His-Tag under the control of the inducible T7 promoter of pET28 derivatives and introduced into E. coli BL23 (DE3) to overproduce. To do this, the start codon ATG was added to the nucleotide sequence of SEQ ID NO: 2 so that the resulting polypeptide began with the amino acid sequence MIVNS. All structures were confirmed by Sanger sequence analysis. Amuc-1100 overexpressing structures overexpressed soluble Amuc-1100 proteins, which were purified to apparent homogeneity by nickel column affinity chromatography and used at a concentration of 100-300 μg/mL. Purified Amuc-1100 was used to propagate antibodies in rabbits, predominantly as described previously (Reunanen J et al., 2012, Appl Environ Microbiol 78: 2337-44).

Results.

The results show that E. coli transformed with a polynucleotide according to the invention (SEQ ID NO: 2) is able to produce the Amuc-1100 protein in soluble form, which can be easily isolated by the nickel column method as described in Tailford LE et al., 2015, NatCommun. 6:7624.

Example 2 Interaction and stimulation of the TLR2 signaling pathway.

Way.

Reporter cell lines expressing TLR2 and TLR4 were prepared to test the ability of Amuc-1100 to bind TLR2 and other TLRs and then stimulate TLR2 and other TLR signaling pathways. The ability of Amuc-1100 to bind cell lines expressing TLR2 or TLR4 and then to stimulate the TLR2 and/or TLR4 signaling pathway in said cells was tested in vitro by measuring NF-kB release from reporter cells.

Briefly, the hTLR2 and hTLR4 cell lines (Invivogen, CA, USA) were used. Stimulation of the receptors with the appropriate ligands activates NF-kB and AP-1, which induces the production of secreted embryonic alkaline phosphatase (SEAP), the levels of which can be measured with a spectrophotometer (Spectramax). All cell lines were grown and subcultured to 70-80% confluence using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 4.5 g/l D-glucose, 50 U/ml penicillin, 50 μg as maintenance medium. streptomycin, 100 μg/ml normocin, 2 mM L-glutamine, and 10% v/v heat-inactivated fetal bovine serum (FBS). For each cell line, an immune response experiment was performed by adding 20 μl of Amuc-1100 suspensions. Reporter cells were incubated with Amuc-1100 for 20-24 h at 37°C in a 5% CO2 incubator. The receptor ligands Pam3CSK4 (10 ng/ml for hTLR2) and LPS-EB (50 ng/ml for hTLR4) were used as a positive control, and maintenance medium without any selective antibiotics was used as a negative control. SEAP secretion was determined by measuring OD600 at 15 min, 1, 2 and 3 h after the addition of 180 μl QUANTI-Blue (Invivogen, CA, USA) to 20 μg hTLR2 and hTLR4 induced supernatant. The experiments were carried out three times.

Results.

The results show that Amuc-1100 is able to interact with TLR2. In addition, the results show that Amuc-1100 has an immunostimulatory effect on TLR2-expressing reporter cells, ie. Amuc-1100 is able to stimulate the release of NF-kB from reporter cells.

Example 3 Stimulation of cytokine release from peripheral blood mononuclear cells. Way.

Tested in vitro the ability of Amuc-1100 to stimulate the production or release of cytokines from peripheral blood mononuclear cells (PBMC). We received peripheral blood from three healthy donors from the Sanquin Blood Bank, Nijmegen, The Netherlands. Peripheral blood mononuclear cells (PBMC) were separated from the blood of healthy donors by Ficoll-Paque Plus gradient centrifugation according to the manufacturer's protocol (Amersham biosciences, Uppsala, Sweden). After centrifugation, mononuclear cells were harvested, washed in Iscove's Modified Dulbecco's Medium (IMDM) + Glutamax (Invitrogen, Breda, The Netherlands) and adjusted to 0.5x106 cells/mL in IMDM+Glutamax supplemented with penicillin (100 U/mL) (Invitrogen), streptomycin (100 g/ml) (Invitrogen) and 10% heat-inactivated FBS (Lonza, Basel, Switzerland). PBMCs were seeded (0.5x106 cells per well) in 48-well tissue culture plates. A negative control (medium only) was used for each donor.

Stimulated PBMC live or heated for 10 min to 99°C cells A. muciniphila (in a ratio of 1:10 to PBMC) or Amuc-1100 for 1 day, and then measured the production of cytokines IL-6, IL-8, IL- 10, TNF-α, II.-1 β and IL-12p70 in culture supernatants by multiple assay (CBA kit for human inflammatory cytokines manufactured by Becton, Dickinson) according to manufacturer's protocol on Cantoll FACS (manufactured by Becton Dickinson) and analyzed using BD FCAP software (manufactured by Becton Dickinson). The following detection limits were established according to the manufacturer's instructions: 3.6 pg/ml IL-8, 7.2 pg/ml IL-1e, 2.5 µg/ml IL-6, 3.3 pg/ml IL-10, 3 .7 pg/ml TNF-α, 1.9 pg/ml IL-12p70.

- 15 040421

Results.

The results show that compared to the control (medium only), Amuc-1100 is able to stimulate cytokine production, ie. there was an increase in the levels of IL-P, IL-6, IL-8, IL-10 and

TNF-α. The level of cytokines induced by 4.5 μg/ml Amuc-1100 corresponded to its level in 5 x

106 live or heat-killed cells of A. muciniphila (see table).

Cytokine levels induced by Amuc-1100 and live or heat-killed Akkermansia muciniphila cells

Cytokine (pg/ml) living cells A. muciniphila Heat-killed A. muciniphila cells Amuc-1100 (4.5 µg/ml) IL-Ιβ 894 ± 298 392±71 504 ± 227 IL-6 18029±309 13477 ±2014 12508±2362 IL-8 60018±18229 54230 ±9030 45432 ± 12507 Sh-10 823±310 638±118 526±180 TNF-a 1920 ±349 957±568 1317±885 IL-12p70 <2 <2 <2

Example 4 Modulation of transepithelial resistance (TER).

Way.

The ability of Amuc-1100 to increase the integrity of the intestinal epithelial cell layer was evaluated by measuring the ability of Amuc-1100 to stimulate or increase the TER of Caco-2 cells in vitro. Briefly, Caco-2 cells were seeded (5 x 10 ⁴ cells per insert) on Millicell inserts in cell culture plates (pore size 3 μm, Millipore) and grown for 8 days. Bacterial cells were washed once with RPMI 1640 and applied to the inserts measured at 600 nm with an optical density of 0.25 (approximately 108 cells) in RPMI 1640. Purified Amuc-1100 was applied to the inserts at concentrations of 0.05, 0.5 and 5 μg /ml The transepithelial resistance of cell cultures was determined using a Millicell ERS-2 TER counter (Millipore) 0 and 24 hours after the addition of Amuc-1100.

Results.

The results showed that already at a concentration of 0.05 μg/ml after 24 h of co-cultivation with Caco-2 cells, Amuc-1100 was able to significantly increase TER to the same extent as about 108 A. muciniphila cells.

Example 5 Modulation of Nutritional Metabolic Dysfunction.

A group of 10-11 week old C57BL/6J mice (n=10 per subgroup) received a control diet (ND) or a high fat diet (HFD; 60% fat and 20% carbohydrates (kcal/100 g) D12492i, Research Diet, New Brunswick, NJ, USA) as previously described by Everard et al. (2013, PNAS vol. 110(22): 9066-9071). A. muciniphila MucT was grown in a synthetic medium (containing 0.4 g KH2PO4, 0.669 g Na2HPO4.2H2O, 0.3 g NH4Q, 0.3 g NaCl, 0.1 g MgCM^O, 10 g casitone, 1 mM Lthreonine, 1 ml mineral solution, 5 mM L-fucose and 5 mM D-glucose per liter of deionized water) as described by Lucovac et al. (2014, mBio 01438-14) and concentrated, mixed in PBS containing 25% glycerol and stored at -80°C as described by Everard et al. above. A subset of HFD-treated mice were additionally gavaged daily with 2 x 10 ⁸ cfu/0.15 ml of A. muciniphila suspended in sterile anaerobic PBS (HFD Akk) and, as this involved a 10-fold dilution of A. muciniphila, the resulting final concentration glycerol was 2.5%. The ND and HFD treated groups were gavaged with an equivalent volume of sterile anaerobic PBS containing 2.5% glycerol daily, as previously described by Everard et al. above. Another subset of HFD-treated mice additionally received Amuc-1100 peptide by daily gavage of 3.1 μg of Amuc-1100 protein in an equivalent volume of sterile PBS containing 2.5% glycerol. Treatment of HFD-treated mice with Amuc-1100 caused a similar or even greater reduction in body weight and increase in fat mass compared to live A. muciniphila (Fig. A and B) without affecting food intake (Fig. C) . Treatment with A. muciniphila or Amuc-1100 also corrected HFD-induced hypercholesterolemia with a significant decrease in serum HDL-cholesterol, with a similar trend for LDL-cholesterol (Figure D).

Notably, Amuc-1100 treatment resulted in a significant reduction in serum triglyceride levels compared to HFD-treated untreated mice. In addition, Amuc-1100 treatment also reduced the mean adipocyte diameter from 38 µm in HFD-treated mice to 29 µm, which is similar to their diameter in untreated mice (27 µm).

Interestingly, Amuc-1100 induced glucose intolerance to the same extent as the live bacterium (FIGS. E-F).

In order to further study glucose metabolism, the authors investigated insulin sensitivity by injecting insulin into the portal vein. The induced insulin

Claims (13)

nom phosphorylation of the insulin receptor (IR) and its downstream mediator Akt in the liver at the sites of threonine (Aktthr) and serine ( ^Aktser ) (Fig. G). HFD resulted in a decrease in phosphorylation of all proteins compared to mice that received the control diet, which reached significance in the case of Akt ^thr (Fig. H). Treatment with live A. muciniphila or Amuc-1100 counteracted these effects, with mice treated with Amuc-1100 (Fig. GH) showing significantly higher p-IR and p-Akt ^thr levels, and mice treated with live bacterium (FIG. I), significantly higher levels of p-Akt ^ser were observed compared to untreated mice treated with HFD. CLAIM 1. Composition for influencing immune signaling and/or intestinal barrier function, and/or modulating metabolic status, containing an isolated polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical over the entire length of the amino acid of sequence of SEQ ID NO: 1, and capable of performing immune signaling and/or affecting intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis, and a pharmaceutically or nutritionally acceptable carrier. 2. The composition according to claim 1, which is a food composition or a pharmaceutical composition. 3. Genetically modified host cell, into the genome of which a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule containing a nucleic acid sequence at least 90% identical throughout the amino acid sequence of SEQ ID NO: 2; and (b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, while said polypeptide is capable of performing immune signaling and/or influencing intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis. 4. A genetically modified host cell other than the Akkermansia muciniphilla species and containing a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule containing a nucleic acid sequence at least 90% identical throughout the entire length of the sequence of SEQ ID NO: 2; and (b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, while said polypeptide is capable of performing immune signaling and/or influencing intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis. 5. A genetically modified host cell belonging to the Akkermansia muciniphilla species, into the genome of which a nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule containing a nucleic acid sequence at least 90% identical throughout the entire length of the sequence of SEQ ID NO: 2; and (b) a nucleic acid molecule containing a nucleic acid sequence that encodes a polypeptide containing the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, while said polypeptide is capable of performing immune signaling and/or influencing intestinal barrier function and/or glucose and/or cholesterol and/or triglyceride homeostasis. 6. A method for producing a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and / or affecting the barrier function of the intestine, and/or glucose and/or cholesterol and/or triglyceride homeostasis, the method comprising the steps of: a) culturing the host cell according to any one of claims 3 to 5 under conditions allowing said polypeptide to be obtained; And b) isolating the polypeptide obtained in step (a). 7. The use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical over the entire length of the amino acid sequence of SEQ ID NO: 1 as a drug for immune signaling and/or influencing barrier function intestines, and/or glucose and/or cholesterol and/or triglyceride homeostasis. - 17 040421 8. The use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the amino acid sequence of SEQ ID NO: 1, and capable of immune signaling and/or affecting intestinal barrier function, and / or glucose and / or cholesterol and / or triglyceride homeostasis, to stimulate the function of the immune system of the intestinal mucosa, to maintain, restore and / or improve glucose and / or cholesterol and / or triglyceride homeostasis, or to maintain, restoring and/or enhancing the physical integrity of the intestinal mucosal barrier in a mammal. 9. The use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the amino acid sequence of SEQ ID NO: 1, capable of performing immune signaling and/or affecting intestinal barrier function, and/ or glucose and/or cholesterol and/or triglyceride homeostasis, for the prevention and/or treatment in a mammal of a disorder selected from the group consisting of obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 2 diabetes 1, gestational diabetes, preeclampsia, inflammatory bowel disease, irritable bowel syndrome, glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), other diseases associated with impaired barrier function, high cholesterol, elevated triglycerides, atherosclerosis, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment, such as due to premature birth, exposure to radiation, chemotherapy, and/or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis. 10. The use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of immune signaling and/or affecting intestinal barrier function, and /or glucose and/or cholesterol and/or triglyceride homeostasis, to stimulate anti-inflammatory activity in the mammalian intestine. 11. The use of a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of immune signaling and/or affecting intestinal barrier function, and /or glucose, and/or cholesterol, and/or triglyceride homeostasis, to stimulate weight loss in a mammal. 12. A method of treating and/or preventing a disorder in a mammal selected from the group consisting of obesity, metabolic syndrome, insulin deficiency or disorders associated with insulin resistance, type 2 diabetes, type 1 diabetes, gestational diabetes, preeclampsia, inflammatory bowel disease, irritable bowel syndrome. intestines, glucose intolerance, abnormal lipid metabolism, atherosclerosis, hypertension, non-alcoholic fatty liver disease, hyperglycemia, hepatic steatosis, dyslipidemia, obesity-related immune system dysfunction (weight gain), other diseases associated with impaired barrier function, high cholesterol, elevated triglyceride levels, and conditions that alter the physical integrity of the intestinal mucosal barrier, such as food allergies, intestinal underdevelopment, such as due to premature birth, exposure to radiation, chemotherapy and/or toxins, autoimmune disorders, malnutrition or malnutrition, sepsis; to promote weight loss in a mammal; to promote anti-inflammatory activity in the mammalian intestine; to stimulate the function of the immune system of the intestinal mucosa in a mammal; to maintain, restore or improve glucose and/or cholesterol and/or triglyceride homeostasis; or to maintain, restore and/or enhance the physical integrity of the intestinal mucosal barrier of a mammal, the method comprising the step of administering to a mammal in need thereof an effective amount of a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical in the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of immune signaling and/or affecting intestinal barrier function. 13. A method for producing a polypeptide containing the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence at least 90% identical throughout the entire length of the amino acid sequence of SEQ ID NO: 1, and capable of performing immune signaling and / or affecting the barrier function of the intestine, and/or glucose and/or cholesterol and/or triglyceride homeostasis, the method comprising the steps of: a) culturing bacteria of the species Akkermansia muciniphila in an applicable culture medium; And b) optionally isolating the polypeptide obtained in step (a). -