Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

• the invention relates to three bacterial strains belonging to the genus Enterococcus, a composition comprising them and its use as a medicament, in particular in the treatment and/or prevention of respiratory diseases.
• the human body has several microbiota such as the intestinal, oral, cutaneous, vaginal microbiota and more recently a pulmonary microbiota has been described. These microbiota gradually settle in from birth and guide immune maturation. Colonization by the digestive microbiota has a very important impact on immunity and health. Since then, many studies have described the bacteria present in human microbiota and used them for their beneficial effects on health, such as probiotics.
• Enterococcus faecalis (E. faecalis) is one of the enterococci found in the human gastrointestinal tract. In humans E. faecalis is subdominant in the intestinal microbiota in healthy adults and dominant in children just after birth. It is a ubiquitous bacterium of humans and animals, which is found in the intestine as in the oral cavity, but also in the pulmonary microbiota. However, certain strains of the species are the cause of infections, in particular nosocomial.
• E. faecalis has in particular been described for its important immunomodulatory properties for humans, in particular in the maturation of epithelial cells of the colon, in protection against relapses of chronic bronchitis, in dermatitis atopic, or in the susceptibility to developing asthma.
• a specific strain has already been described in patent FR 1650656 for its beneficial effects in the treatment and/or prevention of respiratory diseases.
• Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, rheumatoid arthritis, but also chronic inflammatory bowel disease (IBD) affect millions of people worldwide. world. These chronic diseases have a strong impact on the quality of life of patients for whom no curative treatment is available and whose prevalence is increasing sharply in Western countries. Therefore, these pathologies constitute a problem medical, social and economic major in the world.
• the objective of current treatments is essentially symptomatic and aims to treat or possibly prevent attacks occurring periodically in order to improve the quality of life of patients.
• An objective of the present invention is therefore to provide an alternative solution which is simple, more effective and economical for preventing and/or treating chronic inflammatory diseases, including respiratory diseases, and also making it possible to limit the undesirable effects associated with current treatments and thus reduce the prevalence of these diseases.
• the inventors have identified, among the multitude of bacteria of the species E. faecalis, three new bacterial strains of E. faecalis deposited on June 16, 2021 with the National Collection of Microorganism Cultures (CNCM), 25 rue du Dondel Roux, 75724 Paris Cedex 15, France under the number CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701, particularly useful to reduce inflammation, in particular in the prevention and/or treatment of respiratory pathologies.
• CNCM National Collection of Microorganism Cultures
• the three strains exhibit an anti-inflammatory effect in vitro on human cells of pulmonary origin (BEAS-2B) but also ex vivo on explants from the lungs of axenic mice. In particular, they reduce the release of IL-18, IL-2, IL- 5, TNF-a or IL-6.
• the other strains of E. faecalis tested do not exhibit such properties.
• the three strains according to the invention also exhibit an anti-inflammatory effect in vivo, in two preclinical models of allergic asthma. In particular, they make it possible to reduce the recruitment of eosinophils and the release of TH2 cytokines in the lungs.
• the strain deposited under the number CNCM 1-5699 also exhibits a protective effect on respiratory function in the asthma model in adult mice.
• the three strains according to the invention thus exhibit properties that are entirely advantageous and useful in the treatment and/or prevention of respiratory diseases.
• the invention relates to three strains of the species of E. faecalis deposited, respectively, under the numbers CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701.
• the strains deposited under the numbers CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701 have a chromosome, respectively, of 2843733 bp (base pair), 2937166 bp and 2982831 bp.
• the size of the chromosome of the strains according to the invention is therefore smaller than that of the strain described in patent FR 1650656 filed under the number CNCM 1-4969, the size of which is 3056663 bp.
• the invention also relates to the culture supernatant obtained from at least one of the bacterial strains chosen from the strains CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701.
• the supernatant can be obtained from a mixture of strains CNCM 1-5699 and/or CNCM 1-5700 and/or CNCM 1-5701.
• the invention also relates to a composition
• a composition comprising (i) at least one bacterial strain according to the invention and/or (ii) the culture supernatant according to the invention and (iii) at least one excipient acceptable.
• Said acceptable excipient preferably being a pharmaceutically acceptable excipient in the case of a product intended to be used as a medicament for human use or veterinary use.
• the composition is then a pharmaceutical composition comprising a strain chosen from the strains CNCM 1-5699, CNCM 1-5700, CNCM 1-5701 and their mixture and at least one pharmaceutically acceptable excipient.
• the invention is particularly suitable for using said bacterial strain, said supernatant or said composition according to the invention as a medicament.
• the present invention is particularly suitable in the prevention and / or treatment of inflammatory diseases, in particular respiratory diseases, more particularly chronic conditions such as allergy, asthma, chronic obstructive pulmonary disease (COPD), or allergic rhinitis.
• COPD chronic obstructive pulmonary disease
• Other chronic inflammatory pathologies are also of interest such as rheumatoid arthritis or chronic inflammatory bowel disease (IBD), excessive secondary inflammation caused by a primary infection, for example bacterial but also viral such as flu, infant bronchiolitis, or SARS.
• the strains according to the invention are also useful for preventing and/or treating chronic inflammatory bowel diseases, more preferably Crohn's disease, or ulcerative colitis.
• Figure 1 shows the anti-inflammatory properties of the strains according to the invention in particular the reduction of IL-8 induced by TNF-a in BEAS-2B cells.
• Figure 2 shows the ex-vivo anti-inflammatory properties of the strains according to the invention, in particular the reduction of several cytokines responsible for inflammation in explants from the lungs of axenic mice.
• Figure 3 represents the Heat map (or Heat Map in French) of the strains according to the invention on the production of cytokines by the lung explants.
• FIG. 4 represents the protocol of the preclinical model of allergic asthma induced by HDM according to Example 3 in young mice (Panel A) and in adult mice (Panel B).
• Figure 5 shows the protective effect of the strain deposited under number CNCM I-5699 against a drop in blood oxygenation in an asthma model induced by HDM in adult mice.
• Figure 6 shows the protective effect of the strain deposited under the number CNCM I-5699 against the increase in pulmonary resistance in a model of asthma induced by HDM in adult mice.
• bacteria within the meaning of the invention, is meant a unicellular microorganism capable of reproducing by cell division. Bacteria are classified by family, genus, species. Each bacterial species includes a diversity of bacterial strains. THE bacterial strains according to the invention belong to the family Enterococcaceae, to the genus Enterococcus and to the species faecalis.
• bacterial strain or “strain” within the meaning of the invention, we therefore mean a specific bacterial strain but also all the bacteria derived from the strain or obtained from the strain or corresponding to the bacterial strain and having the same metabolic functions, for example, at least one bacterium taken from a colony derived from the strain.
• bacterium(s) according to the invention within the meaning of the invention, is therefore also meant a bacterial strain according to the invention, in particular the strains deposited under the numbers CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701 as well as their derived strains.
• derived strain within the meaning of the invention, is meant a bacterial strain having a strong similarity with at least one of the bacterial strains deposited under the number CNCM 1-5699, CNCM 1-5700 or CNCM 1- 5701.
• the chromosomal nucleotide sequence of the derived strain comprises a nucleotide sequence having at least 99.9% ANI identity, more preferably at least 99.91%, at least 99.92%, at least 99.93% , at least 99.94%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% ANI identity with the chromosomal nucleotide sequence of at least one of the CNCM 1-5699, CNCM 1-5700 or CNCM 1-5701 strains.
• ANI is meant in the sense of the invention the average percentage of identity of the nucleotides calculated from the two-by-two comparison of all the chromosomal sequences of the two bacterial strains.
• the genomic DNA can be extracted from a pure bacterial culture derived from said strain of interest, followed by DNA sequencing using various well-known methods, for example Sanger, Roche 454, Illumina, Oxford Nanopore, then the sequenced genome is assembled by bioinformatics and the sequences obtained are analyzed. Finally, the sequences of the chromosomes of interest are compared two by two to calculate the ANI.
• the culture supernatant of the bacterial strain according to the invention optionally comprising cellular compounds of said strain and / or cellular debris of said strain, and / or metabolites and/or molecules secreted by said strain.
• patient resistant and/or refractory to treatment with corticosteroids is meant within the meaning of the invention a patient to whom a treatment based on corticosteroids is administered, who does not respond to this treatment, that is that is, the patient does not observe a reversal of the progression of the inflammation or a reversal or regression of the progression of the disease.
• prevention within the meaning of the invention, is meant the reduction to a lesser degree of the risk or the probability of occurrence of a given phenomenon, that is to say, in the context of the present invention of inflammation, preferably respiratory diseases, for example asthma.
• treatment within the meaning of the invention, is meant a reduction in the progression of the disease, a stabilization, a reversal or regression, or even an interruption or inhibition of the progression of the inflammation, preferentially of respiratory diseases , for example asthma.
• these terms also apply to one or more symptoms of said diseases of the present invention.
• respiratory disease within the meaning of the invention, here is meant diseases of the respiratory system or causing breathing disorders.
• acceptable excipient within the meaning of the invention is meant any compound making it possible to facilitate the shaping of the composition and not modifying the nature of the biological activity of the active principle.
• An acceptable excipient can be a solvent, buffer, saline solution, plasticizer, lubricant, dispersing medium, agents delaying absorption, flow agent, isotonic agent.
• they are pharmaceutically acceptable excipients which are chosen according to the pharmaceutical form and the desired mode of administration, from the usual excipients known to those skilled in the art and suitable for human and/or veterinary use. The excipient will thus be chosen according to the route of administration, for example suitable for oral, nasal, intravenous, intramuscular, topical administration, etc.
• the present invention therefore relates to a bacterial strain of E. chosen faecalis from the strains deposited under the numbers CNCM 1-5699, CNCM 1-5700, CNCM 1-5701 and their mixture.
• the inventors have discovered and characterized these bacterial strains of E. faecalis from human donors, or from cheeses and have demonstrated, in in vitro models, anti-inflammatory effects, in particular the reduction in the production of IL-8 induced by a pro-inflammatory molecule, TNF-a .
• the anti-inflammatory properties of the strains according to the invention have also been characterized on a model of lung explants which is closer to pulmonary physiology than the existing cell lines.
• the different strains were thus co-incubated with ex vivo axenic mouse lung explants in ex vivo culture and also show an anti-inflammatory effect, in particular they reduce the release of IL-18, IL-2, l IL-5, TNF-a or IL-6.
• the invention relates at least to one bacterial strain of E. faecalis chosen from the strains deposited under the numbers CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701.
• the sequence of the 16S RNA gene of the CNCM 1-5699 strain, of the CNCM I-5701 strain and of the CNCM 1-5700 strain are identical and of SEQ ID NO:1.
• 16S ribosomal RNA (16S rRNA) is the ribosomal RNA constituting the small subunit of the ribosomes of prokaryotes.
• the genes encoding this RNA are called 16S rDNA.
• the 16S rDNA sequence is widely used in phylogeny because of its highly conserved structure which makes it possible to reconstruct the evolutionary history of organisms and in particular prokaryotes and bacteria.
• the 16S rDNA sequence does not always make it possible to differentiate between two strains of the same species which nevertheless have different properties, for example anti-inflammatory properties, such as a different cytokine profile. This results in a different effectiveness for each strain. Consequently, the person skilled in the art given his general knowledge is able to characterize a bacterial strain according to other parameters such as the calculation of phylogenetic distance based on the nucleotide sequence of the chromosome (ANI) or its size.
• ANI nucleotide sequence of the chromosome
• the CNCM 1-5699, CNCM 1-5700 and CNCM 1-5701 strains have chromosomes whose respective sizes are 2843733 bp, 2937166 bp and 2982831 bp.
• the size of the chromosome of the strains according to the invention is therefore smaller than that of the strain described in FR 1650656 deposited under the number CNCM 1-4969, the size of which is 3056663 bp.
• the invention also relates to strains derived from at least one of the bacterial strains deposited under the numbers CNCM 1-5699, CNCM 1-5700 or CNCM 1-5701 which make it possible to maintain or improve the capacities described strains according to the invention.
• Said derived strains can be produced naturally or intentionally, by mutagenesis methods known in the state of the art.
• the mutagenesis methods that can be implemented in the context of the present invention are the growth of the original microorganism in the presence of mutagenic or stress-producing agents, or by genetic engineering aimed at modifying genes specific or not such as directed mutagenesis or random mutagenesis.
• said derived strain comprises a nucleotide sequence having at least 99.9% identity of ANI ("Average Nucleotide Identity", Average Nucleotide Identity in French) with the chromosomal nucleotide sequence of one of the bacterial strains deposited under the numbers CNCM 1-5699 or CNCM 1-5700 or CNCM 1-5701.
• ANI Average Nucleotide Identity
• the derived strain comprises a nucleotide sequence of at least 99.91%, at least 99.92%, at least 99.93%, at least 99.94%, at least 99.95%, at least 99 .96%, at least 99.97%, at least 99.98%, at least 99.99% identity of ANI with the chromosomal nucleotide sequence of at least one of the strains CNCM 1-5699, or CNCM 1 -5700 or CNCM 1-5701.
• the invention relates to the strain of E. faecalis filed under number CNCM 1-5699.
• the strains according to the invention can be produced by culture, for example, in a growth medium known to those skilled in the art (for example, a BHI medium: "Brain-Heart Infusion”) from 8 hours to 3 days, at a temperature of 30-37°C, with or without pH adjustment.
• the fermentation broth containing the bacterial cells is collected.
• the broth can be used as is, concentrated or freeze-dried.
• the bacteria will be collected, for example by centrifugation then resuspended in an appropriate buffer, for example PBS (“phosphate buffered saline”).
• Bacterial concentration can be established using a flow cytometer or other equivalent method.
• the strain according to the invention is particularly advantageous in that it causes a reduction in inflammation.
• the bacterium of the invention makes it possible to attenuate the effects of inflammatory diseases, in particular respiratory diseases.
• Another object of the invention relates to the culture supernatant obtained from one of the bacterial strains according to the invention, but also a strain derived from these.
• the culture supernatant comprises at least one of the constituents chosen from a bacterial cell compound, a bacterial cell debris, a metabolite and/or one (the) molecule(s) secreted by the strain and/or the derived strain , or a mixture thereof.
• the cellular compounds and/or cellular debris can be wall components, nucleic acids, membrane components, proteins, lipids, etc.
• the metabolites or molecules secreted can be any molecule produced or modified by the bacterium due to its metabolic activity during its growth, its use in technological processes (for example, but not limited to, production of food or medicine).
• the metabolites or molecules can be proteins, amino acids, enzymes, lipids, nucleic acids, etc.
• metabolite and/or one (of) molecule(s) secreted by the strain and/or the derived strain is meant a molecule produced and exported or released outside the bacterium by the bacterium.
• the invention also relates to the culture supernatant obtained from one of the bacterial strains according to the invention or their mixture.
• strain(s) according to the invention or derived strain(s) and/or the culture supernatant described above and the bacteria derived from said strains are advantageously administered in a composition.
• the invention relates to a composition
• a composition comprising: (i) at least one bacterial strain according to the invention and/or (ii) a supernatant according to the invention and (iii) at least one acceptable excipient.
• the composition may also comprise a physiologically acceptable medium, that is to say a medium which is compatible with the body of the individual to whom said composition is to be administered. It can be, for example, a non-toxic solvent such as water, buffer, saline solutions. In particular, said medium is compatible with oral administration.
• the composition comprises at least one bacterial strain chosen from one of the strains deposited under the number CNCM 1-5699, the number CNCM 1-5700, the number CNCM 1-5701 and their mixture; and/or the culture supernatant obtained from said strains and at least one acceptable excipient and/or a physiologically acceptable medium.
• the composition comprises the strain deposited under the number CNCM 1-5699.
• the composition comprises the strain deposited under the number CNCM 1-5699 and the strain deposited under the number CNCM 1-5700 and the strain deposited under the number CNCM 1-5701.
• the bacterial strain according to the invention may be present in any form producing the expected effects and the efficacy described in the present application.
• the strain can be in living form (cultivable or not), dead, semi-living, attenuated or inactivated.
• the dead, semi-living, attenuated or inactivated form induces the same effects as the live strain and therefore also has anti-inflammatory properties, allowing in particular the prevention and/or treatment of respiratory diseases.
• semi-living is meant a bacterium with low physiological activity whose ability to proliferate is reduced, temporarily or permanently.
• inactivated designates a bacterium which is no longer capable, temporarily or permanently, of proliferating.
• dead designates a bacterium which is no longer capable, definitively, of proliferating.
• Dead or inactivated bacteria may have intact or ruptured cell membranes.
• inactivated also designates the extracts and lysates of bacteria obtained. It is particularly advantageous to use such forms in the pharmaceutical compositions of the invention.
• composition according to the invention may also comprise at least one additional compound.
• the additional compound can be an ingredient, a molecule, an active principle, a microorganism, a bacterium or a mixture of bacteria.
• the additional compound is an anti-inflammatory agent, more preferably a corticosteroid, even more preferably budesonide.
• the additional compound may be a probiotic and/or a prebiotic.
• composition according to the invention is in any form acceptable for administration to a patient, preferably a human or animal (non-human) patient.
• a patient preferably a human or animal (non-human) patient.
• the composition according to the invention is also aimed at veterinary uses.
• composition according to the invention is in solid, liquid or freeze-dried form.
• composition when in liquid form, it may in particular comprise bacterial strains according to the invention and/or the derived strain and/or a physiologically acceptable culture medium for said bacteria which makes it possible to preserve them, such as example BHI medium, or an equivalent medium containing no product derived from animal origin.
• bacterial strains according to the invention and/or the derived strain and/or a physiologically acceptable culture medium for said bacteria which makes it possible to preserve them, such as example BHI medium, or an equivalent medium containing no product derived from animal origin.
• the bacterial strains according to the invention and/or the derived strain may be present in lyophilized form, and may also comprise excipients such as, for example, microcrystalline cellulose. , lactose, sucrose, fructose, levulose, starches, stachyose, raffinose, amylum, calcium lactate, magnesium sulfate, sodium citrate, calcium stearate, polyvinylpyrrolidone, maltodextrin , galactooligosaccharides, fructooligosaccharides, pectins, beta-glucans, lactoglobulins, isomaltooligosaccharides, polydextroses, mannitol, sorbitol, glycerol, silica dioxide, magnesium stearate, cysteine, mannose, galactose, anhydrous glucose, glucose monohydrate and/or mixtures thereof.
• excipients such as, for example, microcrystalline cellulose.
• the composition according to the invention is in a form suitable for administration by the oral, nasal, parenteral, rectal, sublingual, ocular, auricular, intramuscular, intravenous, inhaled or cutaneous route, more preferably by the nasal or inhaled route using a nebulizer device.
• composition can be in any suitable form.
• the composition according to the invention may be in a form chosen from a powder, powder to be nebulized, microencapsulated powder, microencapsulated powder to be nebulized, capsule, capsule, tablet, pastille, granules, solution, solution to be nebulized, emulsion, emulsion nebuliser, suspension, nebuliser suspension, ampule, suppository, inhaler and a syrup, more preferably in the form of an inhaler such as a nebuliser device.
• composition according to the invention may optionally be in a gastro-resistant form, for example a coated tablet containing microencapsulated bacteria.
• the composition comprises at least 10 3 colony-forming units (CFU) of bacteria per daily dose of composition to be administered, preferably from 10 4 to 10 9 CFU, more preferably from 10 5 to 10 7 CFU .
• CFU colony-forming units
• this corresponds to a daily dose of bacteria to be administered, regardless of the weight of the person or the animal.
• this dose is administered all at once.
• the useful composition then comprises at least 10 3 , preferably from 10 4 to 10 9 CFU, even more preferably from 10 5 to 10 7 CFU of bacteria per daily dose to be administered.
• Said bacteria are a mixture of bacteria corresponding to or derived from one of the strains according to the invention, including the derived strains.
• CFU Cold Forming Unit
• UFC Coldy Forming Unit
• the daily dose is measured per gram or milliliter of the composition according to the final invention.
• the composition according to the invention comprises a mixture of live bacteria or not, said mixture comprising at least live bacteria
• the composition preferably comprises at least 1% of live bacteria (by number), more preferably at least 10% of live bacteria (by number), even more preferably at least least 50% live bacteria (by number).
• the live bacteria are a mixture of bacteria corresponding to one of the strains according to the invention, including the derived strains.
• the composition according to the invention including it may in particular comprise a content of between 0.1 and 99% by weight. , in particular from 5 to 95% by weight, in particular from 10 to 90% by weight and more particularly from 15 to 85% by weight, relative to the total weight of the composition.
• a composition according to the invention may comprise a content of between 0.1 and 99% by weight, in particular from 5 to 95%, in particular from 10 to 90% by weight, more particularly from 15 to 85% by weight, of supernatant relative to the total weight of the composition.
• the composition When the composition is intended for therapeutic use, the composition comprises at least one pharmaceutically acceptable excipient, the composition according to the invention is then a pharmaceutical composition.
• pharmaceutically acceptable excipient here means an excipient whose administration to an individual is not accompanied by significant deleterious effects.
• Pharmaceutically acceptable excipients are well known to those skilled in the art.
• the invention also relates to a pharmaceutical composition
• a pharmaceutical composition comprising the ingredient(s) included in the composition according to the invention and according to any of the embodiments described above.
• Said pharmaceutical composition is a medicine.
• composition according to the invention may be in the form of a food product, a drink, a nutraceutical, a food additive, a food supplement or a dairy product.
• composition according to the invention when included in a food supplement for oral administration, it may be in the form of capsules, gelatin capsules, soft capsules, tablets, sugar-coated tablets, pills, pastes, lozenges, gums, drinkable solutions or emulsions, syrup or gel.
• a food supplement according to the present invention may further comprise a sweetener, a stabilizer, an antioxidant, an additive, a flavoring agent and/or a colorant.
• the formulation thereof is carried out by means of the usual methods for producing sugar-coated tablets, capsules, gels, hydrogels for controlled release, emulsions, tablets or capsules.
• a composition according to the present invention can also be in the form of a nutritional composition.
• a nutritional composition according to the present invention may be in the form of a yogurt, a fermented food, a cereal bar, a breakfast cereal, a dessert, a frozen food, a chilled food, a frozen food, a soup, pet food, liquid suspension, powder, tablet, gum or candy.
• a nutritional composition according to the present invention may also comprise at least one ingredient chosen from: antioxidants, fish oils, vegetable oils, DHA, EPA, vitamins, minerals, phytonutrients, a protein, a lipid , probiotics, prebiotics, postbiotics and combinations thereof.
• composition according to the invention is useful as a medicament for use in human or veterinary health.
• the composition according to the invention is intended to prevent and/or treat inflammation and thus be used in the treatment and/or prevention of inflammatory diseases, more preferably respiratory diseases and/or inflammatory diseases of the bowel and/or rheumatoid arthritis and/or inflammatory diseases associated with viral or bacterial infection.
• inflammatory diseases more preferably respiratory diseases and/or inflammatory diseases of the bowel and/or rheumatoid arthritis and/or inflammatory diseases associated with viral or bacterial infection.
• asthma asthma
• bronchial allergic, intrinsic, extrinsic, induced by exercise, of drug origin (including aspirin and nonsteroidal anti-inflammatory drugs - NSAIDs) and dust-induced asthma, steroid-resistant asthma, bronchitis, including infectious and eosinophilic bronchitis, chronic obstructive pulmonary disease (COPD), such as COPD (chronic obstructive pulmonary disease), cystic fibrosis, pulmonary fibrosis including cryptogenic fibrosing alveolitis, idiopathic pulmonary fibrosis, idiopathic interstitial pneumonia, fibrosis complicating antineoplastic therapy and chronic infection, including tuberculosis and aspergillosis and others fungal infections; complications of lung transplantation; vasculitis and thrombotic disorders of the pulmonary vascular system and pulmonary arterial hypertension (including pulmonary arterial hypertension
• composition according to the invention also targets respiratory pathologies having “immune dysregulations” common with those observed in asthma, such as the induction of certain cytokines (IL-6, TSLP, IL-8, IL-5, IL-13, IL-17) and mucosal dysregulation (hyper production of mucus).
• cytokines IL-6, TSLP, IL-8, IL-5, IL-13, IL-17
• mucosal dysregulation hyper production of mucus
• the uses and compositions of the present invention encompass the prevention and treatment of chronic respiratory diseases, for example asthma, COPD and rhinitis. Even more particularly, the uses and compositions of the present invention relate to the prevention and treatment of asthma.
• strains according to the invention are particularly effective in preventing asthma when they are administered before the appearance of all the symptoms of the disease.
• the use of the strain of the invention and the composition comprising it can in particular be used effectively to prevent the onset of asthma in patients known for their predisposition for this pathology (for example, patients with a predisposition family to the development of asthma).
• composition according to the invention is also useful in a disease chosen from Crohn's disease, ulcerative colitis, ulcerative-hemorrhagic recto-colitis, diverticulitis, esophagitis, gastritis , pancreatitis, peptic ulcer and irritable bowel syndrome.
• composition according to the invention is of particular interest when humans or the animal is resistant and/or refractory to corticosteroids.
• Example 1 Anti-inflammatory effect of the strains according to the invention.
• BEAS-2B cells were co-incubated with TNF-a (lpg/ml) alone or with E. faecalis bacteria according to the invention (CNCM 1-5699, CNCM 1-5700, CNCM 1-5701 ) or non-inventive (CNCM 1-4969, E. faecalis 1 or E. faecalis 2) MOI 250:1.
• concentration of IL-8 was measured in the supernatant.
• the inventors have observed that the three strains of E. faecalis according to the invention (CNCM 1-5699, CNCM 1-5700, CNCM 1-5701) exhibit a strong "anti-IL-8" effect, respectively of 93%, 75% and 70% compared to the strain CNCM 1- 4969 and two other strains of E. faecalis.
• the CNCM I-4969 strain certainly has an effect, but it is weaker than the three strains according to the invention.
• the E. faecalis strains according to the invention exhibit useful anti-inflammatory properties (FIG. 1).
• Example 2 Ex-vivo anti-inflammatory effect of the strains according to the invention.
• the inventors continued their work by comparing the 3 strains according to the invention with 5 other strains of E. faecalis (CNCM 1-4969, E. faecalis 1, E. faecalis 2, E. faecalis 3, E. faecalis 4).
• the lung explants are 200-250 ⁇ m cultured sections of lungs from axenic mice.
• the explants are co-incubated or not with 50 CFU (colony forming units) of bacteria: strains of E. faecalis.
• CFU colony forming units
• the culture supernatants are sampled and the quantity of cytokines measured by Luminex.
• the inventors have thus characterized the anti-inflammatory effects on a model of lung explants which is closer to pulmonary physiology in comparison with cell lines.
• the different strains of E. faecalis were co-incubated with explants of axenic mouse lungs in ex vivo culture. These explants have a basal production of different cytokines, which can be modified by the presence of immunomodulatory elements.
• E. faecalis have an anti-inflammatory effect on mouse lung explants. They reduce in particular the release of IL-18, IL-2, IL-5, TNF-a or even IL-6 ( Figures 2 and 3).
• the CNCM I-4969 strain exhibits a significantly weaker effect than the three strains according to the invention and the 4 other strains tested (E. faecalis 1, E. faecalis 2, E. faecalis 3, E. faecalis 4) have effects neutral or pro-inflammatory (Figure 3).
• Baby mice were sensitized by intranasal administration of HDM (House Dust Mite) (lpg) on D0 (Sensitization) then with lOpg from D7 to Dll (Challenges).
• the strains according to the invention were administered intranasally at 10 6 CFU every 2 days from D-2 to D10 (FIG. 4 Panel A).
• the inventors analyzed the growth monitoring, carried out a bronchoalveolar lavage (BAL) and analyzed the cells and the cytokines of the lung tissue.
• BAL bronchoalveolar lavage
• mice were also sensitized by intranasal administration of HDM (House Dust Mite - dust mite) (25 ⁇ g) on D0 and D7 (Sensitization) then with lOpg from D14 to D16 (Challenges).
• the strains according to the invention were administered intranasally at 10 6 CFU every 2 days from D-2 to D16.
• budesonide was administered intranasally (3mg/kg) from D14 to D16 ( Figure 4 Panel B).
• the inventors On D17, the inventors analyzed the growth monitoring, carried out a bronchoalveolar lavage of the cells and analyzed the cytokines found in the lavage and in the lung tissue.
• the immune cells found in the BAL, the TH2 cytokines present in the lungs and the BAL, as well as the histopathology are markers of the development of asthma common to mouse models of asthma and to human patients.
• the data in Table 2 come from an experiment with 8 and 10 animals per group, in mice and in adults, respectively. They indicate the protective (positive values) or aggravating (negative values) effects of the administration of strains of E. faecalis according to the invention (CNCM 1-5699, CNCM 1-5700, CNCM 1-5701) and outside the invention (CNCM 1-4969) in comparison with the groups of animals treated only with HDM. In the protocol used in adult mice, the effects were compared to those of budesonide, a reference steroid in the clinic. In the mouse protocol, this comparison was not made.
• mice were sensitized by intranasal administration of HDM (25 ⁇ g) on D0 and D7 then at 10 ⁇ g from D14 to D16 (FIG. 4 Panel B).
• the CNCM I-5699 strain was administered intranasally at 10 6 CFU every 2 days from D-2 to D16.
• Budesonide was administered intranasally (3mg/kg) from D14 to D16.
• Pulmonary function was measured every other day using a pulse oximeter on D-2 before the first treatment, then every 2 days until the end of the experiment. The measurement makes it possible to obtain the level of oxygen saturation of the blood, which can be affected in the event of impaired respiratory function, in particular during the development of asthma.
• the inventors carried out a measurement of the pulmonary dynamic resistance.
• bronchial hyperreactivity which is particularly observed in asthma, and which results in an increase in pulmonary resistance.
• the general principle of this bronchial provocation test consists of the administration by inhalation of increasing doses of methacholine, a substance indirectly inducing the contraction of bronchial smooth muscle, with the consequences of changes in the caliber of the airways, an increase in their resistance and a decrease in the gas flow through it.
• the mice were anesthetized and intubated. Increasing concentrations of aerosolized methacholine were administered. Resistance was recorded with a plethysmograph.

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