WO2022096740A1 - An immunomodulatory probiotic - Google Patents

Abstract

The present invention relates to the field of the human gut microbiome, more particularly to its role in health and disease. Provided herein is a novel commensal Treponema strain and methods to treat, prevent and/or reduce the severity of inflammation-associated disorders or conditions in a subject in need thereof.

Description

AN IMMUNOMODULATORY PROBIOTIC

Field of the invention

The present invention relates to the field of the human gut microbiome, more particularly to its role in health and disease. Provided herein is a novel commensal Treponema species and its use in methods to treat, prevent and/or reduce the severity of inflammation-associated disorders or conditions in a subject in need thereof. The invention also relates to a composition, a pharmaceutical composition and a medicament comprising the Treponema species herein disclosed for treating immune and/or inflammatory disorders.

Background

The human gut is the natural habitat for a large and dynamic bacterial community. The bacterial cells harboured within the human gastrointestinal tract (GIT) and the genes encoded by these cells significantly outnumber the cells and genes from the host. These human digestive-tract associated microbes are referred to as the gut microbiome. The human gut microbiome and its role in both health and disease has been the subject of extensive research and there is world-wide interest in modulating the gut microbiome to prevent or treat diseases.

Summary

Here, the Applicants of current application disclose a novel Treponema species with immunomodulatory properties. It is an object of the application to provide a Treponema species comprising a 16S rRNA with a homology of at least 90% to SEQ. ID No. 1. In one embodiment, the Treponema species is further characterised by the ability to produce alkaline phosphatase, acid phosphatase, naphtol-AS-BI- phosphohydrolase, beta-galactosidase and alpha-glucosidase. In a particular embodiment, the Treponema species is catalase negative. In another embodiment, the Treponema species induces proliferation or accumulation of CD4+ regulatory T-cells, CD4+ Foxp3+ T-cell, CD4+ CD8+ T-cells, eosinophils, neutrophils, monocytes, macrophages and/or dendritic cells. In a most particular embodiment, said Treponema species is Treponema peruensis, even more particularly the Treponema peruensis strain RCC2812 with deposit number LMG P-31793.

The application also provides an enriched culture of any the Treponema species disclosed in current the application as well as an extract of said enriched culture. Another object is to provide a composition comprising any of the Treponema species or strains disclosed herein. Composition of particular interest are food products or beverages. Yet another object is to provide any of the Treponema species or strains herein disclosed for use as a medicine. In a particular embodiment, said Treponema species or strains are provided for use to prevent or treat an immune or inflammatory disorder. In one embodiment, said Treponema species or strains are then formulated in probiotic or pharmaceutical compositions respectively. In a particular embodiment, said probiotic or pharmaceutical compositions are provided to prevent or treat gastrointestinal inflammation. Said gastro-intestinal inflammation may be selected from inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome (IBS), celiac disease, pouchitis, post-infection colitis, diarrhoeal disease due to an infectious bacterial, fungal or viral agent or the gastro-intestinal inflammation can be associated with gastrointestinal cancer, spondyloarthritis, rheumatoid arthritis, primary sclerosing cholangitis or multiple sclerosis.

DEPOSIT OF BIOLOGICAL MATERIAL

Purified cultures of the microbial strain described in present application were deposited at the BCCM (Belgian Coordinated Collections of Microorganisms) consortium (BCCM represented by Laboratorium voor Microbiologie- Bacterienverzameling (LMG), Universiteit Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium), recognized as an International Depositary Authority by the World Intellectual Property organization since March 1, 1992 and in accordance with the Budapest Treaty as specified in PCT Rule 13bis.l for the purpose of patent procedure and the regulations thereunder. The Treponema strain of current application has been deposited as Treponema peruensis type strain RCC2812⁷ with deposit number LMG P-31793. Original deposit has been done on 25/06/2020. The country of origin of the Treponema strain is Peru. To the best of Applicants' knowledge all ABS requirements are complied with.

Brief description of the figures

Figure 1. Maximum likelihood phylogenetic tree of 16S rRNA genes for all Treponema species described. Figure 2. Fluorescence in situ hybridization on a colon section using EUB338 probe demonstrates the presence of the administered Treponema RCC2812.

Figure 3. Most pronounced effects in the colon of Treponema RCC2812 treated mice.

Figure 4. Most pronounced effects in the ileum of Treponema RCC2812 treated mice.

Figure 5. Most pronounced effects in the colon myeloid of Treponema RCC2812 treated mice.

Figure 6. Schematic representation of the DSS colitis experiment. ABX, antibiotics treatment; vertical arrows, mock or Treponema administration. Detailed description

Definitions

The present invention is described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. The drawings described are only schematic and are nonlimiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated. Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The terms or definitions provided herein are solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art. It is to be understood that although particular embodiments, specific configurations as well as materials and/or molecules, are discussed herein for cells and methods according to the present invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The Examples described herein are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

For the purpose of current application, the term "bacterium" or "bacteria" includes any prokaryotic organism that does not have a distinct nucleus. While being both part of the group of microorganisms, bacteria and fungi are clearly distinct. The term "fungi" or "fungus" includes a wide variety of nucleated spore-bearing organisms that are devoid of chlorophyll. Examples of fungi include yeasts, molds, mildews, rusts and mushrooms. "Probiotic" refers to a microbial cell preparation (such as, for example, living microbial cells) or components of microbial cells which, when administered in an effective amount, provide a beneficial effect on the health or well-being of a subject.

"Prophylaxis" is defined as a process of guarding a subject against the development of a specific disease by a treatment or action that affects pathogenesis. "Prophylactic" as used herein is the product used in prophylaxis, similar to a medicament that is used in a therapeutic setting.

In microbiology, "16S rRNA sequence" refers to the sequence derived by characterizing the nucleotides that comprise the 16S ribosomal RNA gene(s). The bacterial 16S rRNA is approximately 1500 nucleotides in length. In order to reconstruct the evolutionary relationships and sequence identity of one bacterial isolate to another, phylogenetic approaches are used standardly exploiting the 16S rRNA sequence or a portion of the 16S rRNA sequence of the bacteria, although any other sequence or the entire genome of the microorganisms to be analyzed can also be used.

In this application "sequence similarity", "sequence identity" and "sequence homology" are interchangeably used. The term "sequence identity" as used herein refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP (Higgins & Sharp, CABIOS 5:151 (1989), BLAST and BLAST 2.0 (Altschul et al. J. Mol. Biol. 215: 403 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). In one embodiment, the sequence homology is calculated over the full length of the sequences.

"Stool sample" and "fecal sample" are used interchangeably and refer to as a sample or aliquot of the stool or feces of a subject, more particular a mammal, even more particularly a human being, most particularly a patient. The stool sample as used herein comprises the gut microbiome from a human patient to be diagnosed. As used herein, the term "microflora" refers to the collective bacteria in an ecosystem of a host (e.g. an animal, such as a human) or in a single part of the host's body, e.g. the gut. An equivalent term is "microbiota". As used herein, the term "microbiome" refers to the totality of bacteria, their genetic elements (genomes) in a defined environment, e.g. within the gut of a host, the latter then being referred to as the "gut microbiome".

As used herein, the term "patient" or "individual" or "subject" typically denotes humans, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably mammals, such as, e.g. non-human primates, rodents, canines, felines, equines, ovines, porcines, and the like.

As used herein, the term "gut" generally comprises the stomach, the colon, the small intestine, the large intestine, cecum and the rectum. In addition, regions of the gut may be subdivided, e.g. the right versus the left side of the colon may have different microflora populations due to the time required for digesting material to move through the colon, and changes in its composition in time. Synonyms of gut include the "gastrointestinal tract", or possibly the "digestive system", although the latter is generally also understood to comprise the mouth, esophagus, etc.

An immunomodulatory probiotic

Our current knowledge of the gut microbiome is biased towards an industrialized population. To get insight into gut flora not being "polluted" with the Western way of living and eating, the inventors of current application analyzed fecal samples obtained from a remote Amazonian community in Peru.

A Gram-stain-negative, obligatory anaerobic spirochaete was isolated. Based on the phylogenetic analysis of the 16S ribosomal RNA gene, the bacterium was most closely related to the pig intestinal spirochete Treponema succinifaciens. The latter Treponema strain showed the highest 16S rRNA gene sequence similarity to that of the Treponema species herein disclosed, more particularly 89.48%. Using electron microscopy, the herein disclosed Treponema species showed all typical features of the genus, being a small, helical spirochaete with four periplasmic flagella in a 2:4:2 arrangement. Additional phylogenetic analysis based on the whole genome of the bacterium, as well as phenotypic characterization using the API-ZYM system, confirmed that this bacterium was a distinct species within the genus Treponema. It showed alkaline phosphatase, acid phosphatase, naphtol-AS-BI- phosphohydrolase, beta-galactosidase and alpha-glucosidase activities. The isolated Treponema strain can grow in culture without blood or serum, is catalase negative and colonies did not cause local hemolysis. This Treponema is the first presumed commensal Treponema isolated from the human gut of remote populations that may shed lights on their impact on human health. On the basis of the information obtained from a polyphasic taxonomic analysis, it is proposed that the isolated Treponema represents a novel species within the genus Treponema. From here on, the Treponema species is called Treponema peruensis and the type strain is RCC2812⁷. However, it should be stressed that microbial taxonomy continues to change at a very rapid rate in the era of molecular diagnostics including whole genome sequencing. Also name giving can change over time, e.g. Treponema peruensis can be refer to as Treponema peruense. Therefore, although the bacterial species disclosed herein is referred to as a Treponema species, the nomenclature can change over the years. Hence, the bacterial species herein disclosed is alternatively described by means of its 16S rRNA sequence rather than by the name Treponema. In one particular embodiment of the invention herein disclosed, the Treponema species comprising a 16S rRNA sequence with at least 91% homology to SEQ ID No. 1 refers to a bacterial species comprising a 16S rRNA sequence with at least 91% homology to SEQ. ID No. 1. Consequently, the Treponema strain of current application can be interchangeably refer to the bacterial strain of current application.

In a first aspect, a bacterial species, more particularly a Treponema species, is provided comprising a 16S rRNA with at least 90% homology to SEQ ID No. 1. In one embodiment, the 16S rRNA of said bacterial species or Treponema species has at least 91%, at least 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to SEQ ID No. 1. In a more particular embodiment, the 16S rRNA of said Treponema species is identical over the full length thereof to the sequence depicted in SEQ ID No. 1. In a particular embodiment, said Treponema species is a Treponema strain. In a more particular embodiment, said Treponema strain is the strain RCC2812. In a most particular embodiment, said Treponema strain or the bacterial strain herein disclosed is the strain deposited with deposit number LMG P-31793.

SEQ ID No. 1 is the 16S rRNA sequence of Treponema peruense strain RCC2812.

In another embodiment, the bacterial or Treponema species or strain of the application is further characterised by the presence of alkaline phosphatase, acid phosphatase, naphthol-AS-BI- phosphohydrolase, beta-galactosidase and alpha-glucosidase activities.

In another embodiment, the bacterial or Treponema species or strain provided in current application is negative for catalase activity. In another embodiment, the bacterial or Treponema species or strain provided in current application is also negative for the following enzymatic activities: C4 esterase, C8 esterase lipase, C14 lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, alpha-galactosidase, beta-glucuronidase, beta-glucosidase, N-acetyl-beta-glucosamidase, alpha-mannosidase, alpha-fucosidase.

Upon administering the Treponema species of current application to germ-free mice the expression of a plethora of immune-related genes was modulated, displaying a balanced maturation of the immune system without pathological symptoms. Therefore, in yet another embodiment, the Treponema species or strain provided in current application can be further characterized as the Treponema species or strain that induces proliferation or accumulation of CD4+ Foxp3+ T-cells (or regulatory CD4+ T-cells), CD4+ CD8+ T-cells, eosinophils, neutrophils, monocytes, macrophages and/or dendritic cells.

Foxp3 (forkhead box P3) also known as scurfin is a transcription factor of the FOX protein family and a master regulator in the development, maintenance and function of regulatory T cells. Regulatory T cells prevent excessive immune responses, which could damage host tissues. Defects in regulatory T cell induction and/or activity have been demonstrated in autoimmune diseases, multiple allergy and asthma models.

Eosinophils are white blood cells and one of the immune system components responsible for combating parasites and certain infections.

Neutrophils (also known as neutrocytes or heterophils) are the most abundant type of granulocytes and make up 40% to 70% of all white blood cells in humans. Neutrophils are a type of phagocyte and are normally found in the bloodstream. During the beginning phase of inflammation, particularly as a result of bacterial infection, environmental exposure and some cancers, neutrophils are one of the first responders of inflammatory cells to migrate toward the site of inflammation.

Monocytes are a type of white blood cell that can differentiate into macrophages and myeloid lineage dendritic cells. As a part of the human innate immune system, monocytes also influence the process of adaptive immunity.

Macrophages originate from monocytes and are specialised cells involved in the detection, phagocytosis and destruction of bacteria and other harmful organisms. They can also present antigens to T cells and initiate inflammation by releasing cytokines that activate other cells.

Dendritic cells (DCs) are professional antigen-presenting cells located in the skin, mucosa and lymphoid tissues. Their main function is to process antigens and present them to T cells to promote immunity to foreign antigens and tolerance to self-antigens. They also secrete cytokines to regulate immune responses.

The above described bacterial or Treponema species and strains are isolated from the human gut, more particularly from the human gut flora. Therefore, in another embodiment, the application provides an isolated bacterial or Treponema species comprising a 16S rRNA with a homology of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ. ID No. 1. The application also provides any of the Treponema species and strains herein disclosed in an isolated form. The term "isolated" as used herein means that the bacterial strain has been removed from its natural environment. "Isolated" thus implies a purification step. However, "isolated" does not necessarily reflect the extent to which the microorganism, more particularly the bacterium has been purified. A bacterial or Treponema strain of current application is purified at least 2x, at least 5x, at least lOx, at least 50x or at least lOOx from the raw material from which it is isolated, i.e. the human gut flora.

The above described bacterial or Treponema species and strains are from herein referred to as "the bacterial or Treponema species or strains of the application" and one of said bacterial or Treponema species and strains is referred to as "one of the bacterial or Treponema species and strains of current application".

In one embodiment of the invention, living strains of one of the bacterial or Treponema species and strains of current application are used in the present invention, preferably the living strains are derived from cells in stationary phase of growth. In one embodiment of the invention, one of the bacterial or Treponema species and strains of current application may be in the form of viable cells. In another embodiment of the invention, one of the bacterial or Treponema species and strains of current application may be in the form of non-viable cells.

In a second aspect, an enriched culture of one of the bacterial or Treponema species and strains of current application is provided. The term "culture" as used herein refers to a population of microorganisms that are propagated on or in media of various kinds. An "enriched culture" of one of the bacterial or Treponema species and strains of current application refers to a culture of microorganisms, more particular a bacterial culture, wherein the total microbial population of the culture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of one of the isolated bacterial or Treponema species and strains of current application. This is equivalent as saying that a culture of microorganisms, more particularly a bacterial culture, is provided, wherein said culture is enriched with one of the bacterial or Treponema species and strains of current application and wherein "enriched" means that the total microbial (or more particularly the total bacterial) population of said culture contains more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% of one of the isolated bacterial or Treponema strains of current application.

In one embodiment, a biologically pure culture of one of the bacterial or Treponema species and strains of current application is provided. As used herein, "biologically pure" refers to a culture which contains substantially no other microorganisms than the desired strain of microorganism and thus a culture wherein virtually all of the cells present are of the selected strain. In practice, a culture is defined biologically pure if the culture contains at least more than 96%, at least more than 97%, at least more than 98% or at least more than 99% of one of the bacterial or Treponema strains of current application. When a biologically pure culture contains 100% of the desired microorganism a monoculture is reached. A monoculture thus only contains cells of the selected strain and is the most extreme form of a biologically pure culture.

In another aspect, a composition is provided comprising the bacterial species or strain herein disclosed and growth medium appropriate for the bacterial strain. Non-limiting examples of said growth medium is unsupplemented OTEB (Oral Treponeme Enrichment Broth) or fastidious anaerobic agar (FAA) supplemented with 10% foetal bovine serum (FBS) as described in the Example section. In one embodiment, said composition further comprises a cryoprotectant. A "cryoprotectant" as used herein protects the bacteria by preventing the damaging effects of water crystals when cells are frozen, more particularly at -60°C, or -70°C or -80°C or in liquid nitrogen. Non-limiting examples of a cryoprotectant is glycerol and trehalose.

In another aspect, a composition is provided comprising the bacterial species or strain herein disclosed wherein the bacterial species or strain is lyophilized, freeze dried or in the form of a dry powder. In one embodiment, the composition can further comprise a preservative.

In another aspect, a supernatant is provided wherein said supernatant is obtained from a culture of at least one of the bacterial or Treponema species or strains of current application and wherein said culture can be an enriched culture of said at least one species or strain or a biologically pure culture of said at least one species or strain. "Supernatant" refers to the liquid broth remaining when cells grown in broth are removed by centrifugation, filtration, sedimentation or other means well known in the art.

In yet another aspect, an extract is provided wherein said extract is obtained from a culture of at least one of the bacterial or Treponema species or strains of current application wherein said culture can be an enriched culture of said at least one species or strain or a biologically pure culture of said at least one species or strain. An "extract" as used herein refers to various forms of microbial products. These products are obtained by removing the cell walls and/or cell membranes of the microorganisms, a process also known as lysis, thereby obtaining one or more endogenous products of the cultured microorganisms. Non-limiting examples of such products are amino acids, peptides, enzymes, secondary metabolites, vitamins, minerals. Removing the cell walls and/or cell membranes of the cultured microorganisms can be obtained by several procedures which are well-known by the person skilled in the art. Non-limiting examples are addition of chemicals (e.g. sodium chloride) to a microbial culture, heating the microbial culture or induce lysis in a mechanical way. An extract can also be obtained by autolysis of the microorganisms. In another aspect of the invention, one of the bacterial or Treponema species and strains of current application is delivered into the gastrointestinal tract of a subject in need thereof. Therefore, a composition is provided comprising one of the bacterial or Treponema species and strains of current application. Said composition can be a probiotic composition suitable for direct oral consumption.

In particular embodiments, strain-release compositions may be provided, which are not ingested but are contacted with a liquid whereby said one of the Treponema species and strains of current application are released into said liquid and the resulting probiotic liquid composition is then consumed. For example, it may be favourable, particularly where the one or more probiotic strains are to be delivered to children, to initially provide the one or more strains in a composition, e g. an oil suspension, within a drinking straw whereby the one or more strains are transferred to a liquid drawn through the straw, e.g. a juice or milk from a drink carton. Such straws housing probiotic strains are described, for example, in EP1224128B1 (Biogaia AB).

In other particular embodiments, the composition comprising one of the bacterial or Treponema species and strains of current application may be formulated as a pharmaceutical composition including a pharmaceutically acceptable carrier. Said pharmaceutically acceptable carrier is preferably an ingestible carrier. In one embodiment, the pharmaceutical composition is a medicament comprising an effective amount of one of the Treponema species and strains of current application. As used herein the term "pharmaceutically acceptable carrier" refers to a substance that does not produce an adverse, allergic or other problematic reaction when administered to an animal, preferably a human. It may include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the biological standards of the FDA and EMA.

The herein described compositions, pharmaceutical compositions or medicaments should be formulated to provide an appropriate unit dosage of colony-forming units (cfu) of the Treponema species and strains of current application. This will vary with the form of administration and may be varied, for example, depending on whether the individual to be treated is a child or adult. For example a dosage of at least about 10⁴ cfu, more preferably at least about 10⁵ cfu or at least about 10^s cfu, e.g. about 1O⁶-1O¹⁰ cfu may be contemplated at least once per day.

In one embodiment of the invention, the composition of the invention comprises an amount of one of the bacterial or Treponema species and strains of current application ranging from about 10² to about 10¹⁵ cfu/g of the composition, preferably from about 10⁴ to about 10¹² cfu/g of the composition, more preferably from about 10⁵ to about 10¹⁰ cfu/g of the composition and even more preferably from about 10^s to about 10⁹ cfu/g of the composition. In one embodiment of the invention, the composition of the invention comprises an amount of one of the bacterial or Treponema species and strains of current application ranging from about 10² to about 10¹⁵ cfu/ml of the composition, preferably from about 10⁴ to about 10¹² cfu/ml of the composition, more preferably from about 10⁵ to about 10¹⁰ cfu/ml of the composition and even more preferably from about 10^s to about 10⁹ cfu/ml of the composition.

"CFU" or "cfu" as used herein refers to colony-forming unit. This unit is well-known by the person skilled in the art of microbiology (as well as the methodology how to determine the number of colony-forming units) and is used to estimate the number of viable bacteria or fungal cells in a sample. "Viable" is defined as the ability to multiply via binary fission under controlled conditions. Counting with colony-forming units requires culturing the microbes and counts only viable cells, in contrast with microscopic examination which counts all cells, living or dead.

In one embodiment of the invention, the composition, the pharmaceutical composition, or the medicament further comprises additional probiotic strains, such as, for example, bacterial probiotic strains; prokaryotes probiotics other than bacteria; or fungal strains, preferably yeast strains. In one embodiment, said additional probiotic strains are selected from those naturally present in the gut of the subject, preferably in the human gut, more preferably in the gut of healthy human subjects.

Examples of bacterial probiotic strains that may be used in the present invention include, but are not limited to Lactobacillus, Lactococcus, Bifidobacterium, Veillonella, Desemzia, Coprococcus, Collinsella, Citrobacter, Turicibacter, Sutterella, Subdoligranulum, Streptococcus, Sporobacter, Sporacetigenium, Ruminococcus, Roseburia, Proteus, Propionobacterium, Leuconostoc, Weissella, Pediococcus, Streptococcus, Prevotella, Parabacteroides, Papillibacter, Oscillospira, Melissococcus, Dorea, Dialister, Clostridium, Cedecea, Catenibacterium, Butyrivibrio, Buttiauxella, Bulleidia, Bilophila, Bacteroides, Anaerovorax, Anaerostopes, Anaerofilum, Enterobacteriaceae, Fermicutes, Atopobium, Alistipes, Acinetobacter, Slackie, Shigella, Shewanella, Serratia, Mahella, Lachnospira, Klebsiella, Idiomarina, Fusobacterium, Faecalibacterium, Eubacterium, Enterococcus, Enterobacter, Eggerthella.

Examples of prokaryote strains that may be used in the present invention include, but are not limited to Archaea, Firmicutes, Bacteroidetes (such as, for example, Allistipes, Bacteroides ovatus, Bacteroides splachnicus, Bacteroides stercoris, Parabacteroides, Prevotella ruminicola, Porphyromondaceae, and related genus), Proteobacteria, Betaproteobacteria (such as, for example, Aquabacterium and Burkholderia), Gammaproteobacteria (such as, for example, Xanthomonadaceae), Actinobacteria (such as, for example, Actinomycetaceae and Atopobium), Fusobacteria, Methanobacteria, Spirochaetes, Fibrobacters, Deferribacteres, Deinococcus, Thermus, Cyanobacteria, Methanobrevibacteria, Peptostreptococcus, Ruminococcus, Coprococcus, Subdolingranulum, Dorea, Bulleidia, Anaerofustis, Gemella, Roseburia, Dialister, Anaerotruncus, Staphylococcus, Micrococcus, Propionobacteria, Enterobacteriaceae, Faecalibacteria, Bacteroides, Parabacteroides, Prevotella, Eubacterium, Bacilli (such as, for example Lactobacillus salivans and related species, Aerococcus, Granulicatella, Streptococcus bovis and related genus and Streptococcus intermedius and related genus), Clostridium (such as, for example Eubacterium hallii, Eubacterium limosum and related genus) and Butyrivibrio. Examples of fungal probiotic strains, preferably yeast probiotic strains that may be used in the present invention include, but are not limited Ascomycetes, Zygomycetes and Deuteromycetes, preferably from the groups Aspergillus, Torulopsis, Zygosaccharomyces, Hansenula, Candida, Saccharomyces, Clavispora, Bretanomyces, Pichia, Amylomyces, Zygosaccharomyces, Endomycess, Hyphopichia, Zygosaccharomyces, Kluyveromyces, Mucor, Rhizopus, Yarrowia, Endomyces, Debaryomyces, and/or Penicillium.

In one embodiment of the invention, the composition, the pharmaceutical composition or the medicament further comprises a prebiotic. Examples of prebiotics that may be used in the present invention include, but are not limited to, inulin and inulin-type fructans, oligofructose, xylose, arabinose, arabinoxylan, ribose, galactose, rhamnose, cellobiose, fructose, lactose, salicin, sucrose, glucose, esculin, tween 80, trehalose, maltose, mannose, mellibiose, mucus or mucins, raffinose, fructooligosaccharides, galacto-oligosaccharides, amino acids, alcohols, and any combinations thereof. Other non-limiting examples of prebiotics include water-soluble cellulose derivatives, water-insoluble cellulose derivatives, unprocessed oatmeal, metamucil, all-bran, and any combinations thereof. Examples of water-soluble cellulose derivatives include, but are not limited to, methylcellulose, methyl ethyl cellulose, hydroxyethyl cellulose, ethyl hydroxyethyl cellulose, cationic hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, and carboxymethyl cellulose.

One of the bacterial or Treponema species and strains of current application or the composition, pharmaceutical composition, or medicament of the invention may be administered by several routes of administration. Examples of adapted routes of administration include, but are not limited to, oral administration, rectal administration, administration via esophagogastroduodenoscopy, administration via colonoscopy, administration using a nasogastric or orogastric tube and the like.

The composition, probiotic composition, prebiotic composition, pharmaceutical composition or medicament may be in the form of a beverage, e.g. drinking yoghurt, juice or milk, or other form of nutritional composition. It may be suitable for human and/or animal consumption. For example the composition may be in the form of a food product such as a dairy products, dairy drinks, yoghurt, cheese, confectionary, nutritional snack bar, fruit or vegetable juice or concentrate thereof, powders, malt or soy or cereal based beverages, breakfast cereal such as muesli flakes, cereal and/or chocolate bars, spreads, flours, milk, smoothies, chocolate, gels, ice creams, reconstituted fruit products, muesli bars, sauces, dips, drinks including dairy and non- dairy based drinks, sports supplements including dairy and non- dairy based sports supplements. In one embodiment of the invention, the composition, pharmaceutical composition, cosmetic composition or medicament is in the form of a food additive, drink additive, dietary supplement, nutritional product, medical food or nutraceutical composition. It may also be in the form of a nutritional supplement. For example, the one or more probiotic strains may be provided in a nutritional oil suspension, e.g. within a capsule, which will release the probiotic strains in the Gl tract.

The one of the bacterial or Treponema species and strains of current application or the composition, pharmaceutical composition, or medicament herein disclosed may also be in a solid form such as a tablet, pill, powder, granules, troches, suppository, capsules, soft gelatin capsules, sugarcoated pills, orodispersin tablets, effervescent tablets or other solids. It may be a controlled-release formulation for release of the one or more probiotic strains in the Gl tract. In another embodiment, the composition, pharmaceutical composition or medicament of the application may be in a liquid form, such as a drinkable solution, liposomal composition or suspension, e.g. an oil suspension. Such a liquid composition may be provided within an encapsulating substance to provide a capsule or microcapsules, again for release of the one or more probiotic strains in the Gl tract. In one embodiment, the pharmaceutical composition additionally includes a non-microbial therapeutic agent, e.g. a chemical drug entity or a therapeutic biologic, suitable for the prophylaxis and/or treatment of the undesired inflammatory activity of concern, pain or an associated condition. Non-limiting examples of said chemical drug entities are inhibitors of cyclooxygenase activity (aspirin, celecoxib, diclofenac, dif lunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, meloxicam, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac, tolmetin, among others) or corticosteroids (prednisone, dexamethasone, hydrocortisone, methylprednisolone, among others) or analgesics (acetaminophen, duloxetine, paracetamol, among others) or in any combination thereof. Non-limiting example of therapeutic biologies are TNF-alpha blockers, anti-IL17A monoclonal antibodies, anti-CD20 antibodies.

In one embodiment, the composition, pharmaceutical composition or medicament of the invention further comprises excipients, diluent and/or carriers selected with regard to the intended route of administration. Examples of excipients, diluent and/or carriers include, but are not limited to water, phosphate buffer saline, anaerobic phosphate buffer saline, sodium bicarbonate, juice, milk, yogurt, infant formula, dairy product, coloring agents, such as, for example, titane dioxide (E171), iron dioxide (El 72) and brilliant black BN (E151); flavoring agents; thickeners, such as, for example, glycerol monostearate; sweeteners; coating agents, such as, for example, refined colza oil, soya oil, peanut oil, soya lecithin or fish gelatin; diluting agents, such as, for example, lactose, monohydrated lactose or starch; binding agents, such as, for example, povidone, pregelatinized starch, gums, saccharose, polyethylene glycol (PEG) 4000 or PEG 6000; disintegrating agents, such as, for example, microcrystalline cellulose or sodium carboxymethyl starch, such as, for example, sodium carboxymethyl starch type A; lubricant agents, such as, for example, magnesium stearate; flow agent, such as, for example, colloidal anhydrous silica, etc...

The Treponema species and strains herein disclosed are strictly anaerobic bacteria. Therefore, in the embodiments where viable or living strains are used, prolonged contact with oxygen should be avoided. Examples of means for avoiding prolonged contact with oxygen include, but are not limited to freeze of the bacterial cells or packaging in a sealed container and the like.

As disclosed herein, administration of the bacterial or Treponema species of current application to germ- free mice lead to a boosted or activated immune system. Moreover, probiotic efficacy is demonstrated in a DSS colitis disease model. Therefore, in a fifth aspect, the one of any of the bacterial or Treponema species and strains of current application, or any composition or pharmaceutical composition herein disclosed is provided for use as a medicine or as a prophylactic.

In a particular embodiment, the one of any of the bacterial or Treponema species and strains of current application, or any composition, pharmaceutical composition or medicament herein disclosed is provided for use as prophylaxis or for use in the treatment of an immune or inflammatory disorder. This is equivalent as saying that methods are provided to deliver one of any of the bacterial or Treponema species and strains of current application to a subject or methods to prevent or treat an immune or inflammatory disorder in a subject in need thereof, said methods comprise the steps of administering an effective amount of one of any of the bacterial or Treponema species and strains of current application or of the composition, the pharmaceutical composition or the medicament herein disclosed to the subject.

In current application the term "treatment of a disease or disorder" or "treating a disease or disorder", includes preventing the development of the disease or disorder and/or reducing the severity of the disease or disorder or of the symptoms associated therewith.

In one embodiment, the methods further comprise a step of evaluating whether the administered Treponema was present in the gut of the subject or of measuring the abundance of the administered Treponema in the gut of the subject. Measuring can be quantitative or qualitatively. Said measuring or evaluation step can be easily done by collected a stool sample of the treated subject.

In one embodiment of the invention, one of any of the bacterial or Treponema species and strains of current application, or the composition, pharmaceutical composition or medicament is administered at least once a week, particularly at least twice a week, more particularly at least once a day, and even more particularly at least twice a day. In one embodiment of the invention, the daily amount of one of any of the bacterial or Treponema species and strains of current application administered per day ranges from 10² to about 10¹⁵ cfu/day, particularly from about 10⁴ to about 10¹² cfu/day, more particularly from about 10⁵ to about IO¹⁰ cfu/day and even more particularly from about 10^s to about 10⁹ cfu/day.

The term "inflammation", "inflammatory disorder" or "inflammatory disease" refers to complex but to the skilled person well known biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. However, inflammation is not a synonym for infection. Infection describes the interaction between the action of microbial invasion and the reaction of the body's inflammatory response — the two components are considered together when discussing an infection, and the word is used to imply a microbial invasive cause for the observed inflammatory reaction. Inflammation on the other hand describes purely the body's immunovascular response, whatever the cause may be. Inflammation is a protective response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, clear out necrotic cells and tissues damaged from the original insult and the inflammatory process, and to initiate tissue repair. The classical signs of inflammation are heat, pain, redness, swelling, and loss of function. Inflammation is a generic response, and therefore it is considered as a mechanism of innate immunity, as compared to adaptive immunity, which is specific for each pathogen. Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A series of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation, such as mononuclear cells, and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process.

In one particular embodiment, said inflammatory disorder is a gastro-intestinal inflammatory disorder, or alternatively phrased gut inflammation.

The wording "gut inflammation" is equivalent to the wording "microscopic gut inflammation" as used herein and refers to an inflammatory response in the gut as defined above. The inflammation can affect the entire gastrointestinal tract, can be more limited to for example the small intestine or large intestine but can also be limited to specific components or structures such as the bowel walls. In a more particular embodiment, said gut gastro-intestinal inflammatory disorder is selected from the list consisting of inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome (IBS), celiac disease, pouchitis, post-infection colitis, inflammation associated with gastrointestinal cancer, diarrhoeal disease due to undesirable inflammatory activity, diarrhoeal disease due to an infectious bacterial agent such as E.coli, Clostridium difficile associated diarrhoea, Rotavirus associated diarrhoea, and post-infective diarrhoea. In a most particular embodiment of the fifth aspect, the inflammatory disorder is IBD, more particularly CD and/or UC.

As used herein, the term "inflammatory bowel disease" or abbreviated "IBD" refers to an umbrella term for inflammatory conditions of the gut under which both Crohn's disease and ulcerative colitis fall. In people with IBD, the immune system mistakes food, bacteria, or other materials in the gut for foreign substances and responds by sending white blood cells into the lining of the bowels. The result of the immune system's attack is chronic inflammation. Crohn's disease and ulcerative colitis are the most common forms of IBD. Less common IBDs include microscopic colitis, diverticulosis-associated colitis, collagenous colitis, lymphocytic colitis and Behget's disease. In the case of CD, transmural inflammation commonly affects the terminal ileum, although any part of the gastrointestinal system can be affected. Discontinuous inflammation and the presence of non-caveating granulomas are also characteristic of the inflammation in patients with CD. In contrast, UC is characterized by continuous mucosal inflammation starting in the rectum and extending proximally until the caecum (Harries et al 1982 Br Med J Clin Res Ed, 284:706). These are chronic relapsing diseases originating mostly during adolescence and young adulthood and are characterized by chronic inflammation of the gastrointestinal tract leading to invalidating symptoms of bloody diarrhea, weight loss and fatigue (Wilks 1859 Med Times Gazette 2:264- 265). The latest epidemiologic data from France reported a mean incidence of 4.4 cases per 100 000 individuals (Ghione et al 2017 Am J Gastroenterol). Worldwide, the incidence and prevalence of CD range from 0.0-29.3 per 100 000 person-years and 0.6-318.5 per 100 000 persons, respectively. The incidence and prevalence of UC varies from 0.0-19.2 per 100000 person-years and 2.42-298.5 per 100000 persons, respectively (Molodecky et al 2012 Gastroenterology 142:46-54).

Several defects in innate and adaptive immunity have been described both in UC and CD (de Souza et al 2016 Nat Rev Gastroenterol Hepatol 13:13-27). In normal conditions, intestinal macrophages exhibit inflammatory anergy which allows the interaction with commensal flora without inducing strong inflammatory responses (Smythies et al 2005 J Clin Invest 115:66-75). However, CD14+ intestinal macrophages are more abundant in patients with CD than in healthy individuals. These CD14+ intestinal macrophages produce more proinflammatory cytokines, such as interleukin(IL)-6, IL-23 and tumor necrosis factor (TNF)-alpha, than the common CD14-intestinal macrophages (Kamada et al 2008 J Clin Invest 118:2269-2280). Adaptive immunity also plays a role in the pathogenesis of IBD. T helper (TH) lymphocytes are cytokine producing lymphocytes that potentiate or regulate immune responses by interacting with other immune cells such as macrophages, CD8+ T cells, eosinophils and basophils. Following an initial trigger (e.g. impaired barrier function by injury or exposure to xenobiotics) the microbe-associated molecular patterns will induce the secretion of cytokines by dendritic cells, epithelial cells and macrophages, among others. Different cytokine milieus will induce TH1, TH2, TH17 or regulatory T-cell (Treg) subsets (de Souza et al 2016 Nat Rev Gastroenterol Hepatol 13:13-27). In susceptible individuals, an interplay between TH1 and TH17 immune responses seem to be linked with inflammation associated with CD. On the other hand, UC has been described as a TH2-like condition with possible implication of a newly discovered TH9 lymphocytes (de Souza et al 2016 Nat Rev Gastroenterol Hepatol 13:13-27; Gerlach et al 2014 Nat Immunol 15:676-686). In both diseases, an insufficient Treg response seems to be involved in the impaired regulation of inflammatory responses (Maul et al 2005 Gastroenterology 128:1868-1878). In active IBD, the immune system shows an increased response to bacterial stimulation, thereby contributing even further to the chronic inflammatory state. This inflammatory state also produces an increase in the intestinal permeability, allowing bacterial antigens to contact with the immune system, hereby perpetuating the inflammatory state.

In particular embodiments, said inflammation or inflammatory disorder as used in the methods of current application is inflammation or an inflammatory disorder characterized by a TH1, TH17, TH2 and/or TH9 response. In even more particular embodiments, said inflammation or inflammatory disorder is characterized by a TH1 and/or TH17 response.

In another particular embodiment, said inflammatory disorder is inflammation associated with at least one of the following disorders or conditions: spondyloarthritis, ankylosing spondylitis, reactive arthritis, psoriatic arthritis, enteropathic arthritis, undifferentiated spondyloarthritis, juvenile idiopathic arthritis, rheumatoid arthritis, primary sclerosing cholangitis, multiple sclerosis, inflammatory bowel disease, Crohn's disease, ulcerative colitis and any combination thereof.

In further embodiments, said inflammatory disorder is a gut inflammatory disorder or gut inflammation associated with at least one disorder selected from the list consisting of celiac disease, primary sclerosing cholangitis, multiple sclerosis, spondyloarthritis, ankylosing spondylitis, reactive arthritis, psoriatic arthritis, enteropathic arthritis, undifferentiated spondyloarthritis, juvenile idiopathic arthritis and rheumatoid arthritis.

It is envisaged that the invention may also find application for use in the prophylaxis and/or treatment of any of the following: systemic inflammatory disease such as rheumatoid arthritis; undesirable inflammatory activity associated with an autoimmune disorder; undesirable skin inflammatory activity such as associated with atopic dermatitis or psoriasis and inflammatory activity associated with an infection or cancer or immunotherapy thereof. It may be applied, for example, for use in the prophylaxis and/or treatment of undesirable exacerbations of respiratory inflammatory activity due to bacterial or viral infections.

As used herein, the term "spondyloarthritis" or abbreviated "SpA" refers to a group of closely related, but clinically heterogeneous, inflammatory arthritis diseases with common features, including inflammation of the spine, eyes, skin, joints and gastrointestinal tract. This SpA group is also sometimes referred to as spondylitis and spondyloarthropathies. As used herein, SpA includes ankylosing spondylitis (including non-radiographic axial SpA, i.e. ankylosing spondylitis diagnosed using MRI), reactive arthritis, psoriatic arthritis, enteropathic arthritis (arthritis associated with inflammatory bowel disease or IBD related arthritis), undifferentiated spondyloarthritis, juvenile idiopathic arthritis and juvenile-onset SpA. Characteristics of these SpA diseases include inflammatory arthritis of the spine, peripheral arthritis that differs from rheumatoid arthritis, extra articular manifestations of inflammatory bowel disease, arthritis and uveitis, seronegativity for rheumatoid factor and some degree of heritability, including the presence of the gene HLA-B27. It differs from other types of arthritis, because it involves the sites are where ligaments and tendons attach to bones. The etiology and symptomatology differs from that of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are involved, with the same joints typically involved on both sides of the body. The disease may also affect other parts of the body. This may result in a low red blood cell count, inflammation around the lungs, and inflammation around the heart. Fever and low energy may also be present. Often, symptoms come on gradually over weeks to months.

"Primary sclerosing cholangitis" or "PSC" as used herein refers to a severe chronic liver disease characterized by progressive biliary inflammation and fibrosis. The development of multifocal bile duct structures can lead to liver fibrosis and subsequent cirrhosis. Patients with PSC are usually asymptomatic and the diagnostic work up is triggered by incidental findings of altered liver enzymes. In symptomatic patients, fatigue, pruritus, abdominal pain and jaundice are the most reported symptoms (Lazaridis et al 2016 N Engl J Med 375:1161-1170). A systematic review of the epidemiologic studies in PSC reported an incidence varying between 0 and 1.3 cases per 100 000 individuals and a prevalence of 0-16.2 cases per 100 000 individuals (Boonstra et al 2012 J Hepatol 56:1181-1188). Most commonly, PSC affects men at the age of 40 and the concomitant diagnose of IBD is very common. Between 60 to 80 % of the patients with PSC have concomitantly IBD, most frequently UC, pointing towards the possible role of the colon in the pathogenesis of PSC (Boonstra et al 2013 Hepatology 58:2045-2055). This role is further evidenced by transplantation data showing that colectomy before liver transplantation is a protective factor for recurrence of PSC after liver transplantation (Alabraba et al 2009 LiverTranspI 15:330-340). Interestingly, the absence of intestinal microbiota is associated with increased severity of the disease in mice model (Tabibian et al 2016 Hepatology 63:185-196). Therefore, intestinal microbiota may play an important role in the pathogenesis of PSC by modulating the gut-associated immune system to a more immunogenic or tolerogenic phenotype.

In patients with IBD, the prevalence of PSC varies from 0.4 to 6.4%. However, in a recent study using magnetic resonance to diagnose PSC in patients with IBD the prevalence of PSC was 3-fold higher than previously reported, mainly due to subclinical PSC without symptoms or altered liver enzymes (Lunder et al 2016 Gastroenterology 151:660-669). Genome-wide association studies suggested a role for immune-related pathways in the pathogenesis of PSC. Patients with PSC have a higher activity of TH17 cells. These lymphocytes help in the defence against bacteria and fungi by promoting inflammation and are involved in autoimmune diseases (Katt et al 2013 Hepatology 58:1084-1093. Moreover, Treg cells (CD4+CD25+FOXP3+CD127-), which suppress inflammation, are reduced in PSC (Sebode et al 2014 J Hepatol 60:1010-1016). Therefore, in a very particular embodiment, the inflammatory disorder as mentioned in the application refers to inflammatory disorders characterized by a TH17 response.

Given that it has been demonstrated that the number or the function of regulatory T cells (Treg) is affected in human autoimmune diseases (Dominguez-Villar and Hafler 2018 Nat Immunol 19: 665-673) and given that the Treponema species and strains of the application boost the abundance of Tregs, it is envisaged that the Treponema species and strains as well as the composition, pharmaceutical composition or medicament of the application can be used to prevent or to treat an autoimmune disease. Therefore, in yet another embodiment, the Treponema species and strains as well as the composition, pharmaceutical composition or medicament of the application are provided for use to prevent or to treat an autoimmune disease. An autoimmune disease as used herein is a condition in which the immune system mistakenly attacks the body. The immune system normally guards against invaders like bacteria and viruses and leave host cells untouched. In an autoimmune disease, the immune system mistakes part of the body, like the joints or skin, as foreign. It releases proteins called autoantibodies that attack healthy cells.

Some autoimmune diseases target only one organ. For example, type 1 diabetes damages the pancreas. Other diseases, like systemic lupus erythematosus (SLE), affect the whole body. Other non-limiting examples of autoimmune diseases are rheumatoid arthritis, psoriasis/psoriatic arthritis, multiple sclerosis, inflammatory bowel disease (CD, UC), Addison's disease, Graves' disease, Sjogren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, autoimmune vasculitis, pernicious anemia, celiac disease, vitiligo.

"Multiple sclerosis" or "MS" as used herein refers to a chronic inflammatory and neurodegenerative disease characterized by substantial clinical heterogeneity. Both genetic and immunologic factors, as well as environmental elements contribute to its aetiology. Most MS patients present with recurrent periods of relapses and remissions, with relapses thought to be provoked by the infiltration of adaptive immune cells into the central nervous system (CNS), hereby resulting in focal inflammation and myelin loss (Franciotta et al 2008 Lancet neurology 7:852-588). In a minority of patients, slow progression is observed from onset. Therefore, three clinical phenotypes can be distinguished: relapsing-remitting (RR), secondary progressive (SP) or primary progressive (PP) MS. Lublin et al (2014 Neurology 83:278- 286) further described these phenotypes as active, not active, and with or without progression. While not recognized as a separate phenotype, a subset of RRMS patients appears to have a mild course, often referred to as benign MS (BMS) (Amato et al 2006 J Neurol 253:1054-1059; Calabrese et al 2013 Mult Scler 19:904-911). Patients experience a wide variety of symptoms, ranging from physical and cognitive symptoms to even bowel dysfunction, with the latter being reported in more than 70% of cases (Wiesel et al 2001 Eur J Gastroenterol Hepatol 13:441-448). Studies in experimental allergic encephalomyelitis (EAE), a widely used mouse model for MS, have provided evidence for a substantial effect of gut microbiota on central nervous system (CNS)-specific autoimmune disease (Berer et al 2014 FEBS letters 588:4207-4013). The absence of gut microbes (germ-free conditions) or the alteration of the gut microbial flora composition with antibiotics resulted in a shift in T cell responses (decreased concentration of IL-17, increased number of regulatory T and B cells) and affected disease severity (Ochoa-Reparaz et al 2009 J Immunol 183:6041-6050). Additionally, mice raised in a germ-free environment were highly resistant to developing spontaneous EAE, unless exposed to specific pathogen- free condition-derived fecal material or a fecal transplant from MS twin-derived microbiota (Berer K et al 2011 Nature 479:538-541; Berer et al 2017 Proc Nat Ac Sc USA). Immune cells from mouse recipients of MS-twin samples produced less IL-10 than immune cells from mice colonized with healthy-twin samples. IL-10 may have a regulatory role in spontaneous CNS autoimmunity, as neutralization of the cytokine in mice colonized with healthy-twin fecal samples increased disease incidence. This evidence suggests that the microbiota may be capable of altering the individual at a phenotypic level and influence the onset, severity and progression of MS EXAMPLES

Example 1. A novel Treponema species

Treponema strain RCC2812^T was isolated from a human fecal sample of a seemingly healthy resident of the Remoyacu village in the Peruvian Amazonian jungle (approximate geographic coordinates are 5° 16' 18" S 73° 12' 23" W), within the framework of the Flora Intestinal Nativa (FLIN).

In order to determine the evolutionary relationships of the isolated strain with other Treponema species, a search of the 16S rRNA genes was performed against the EzBioCloud 16s rRNA gene database. This identified Treponema succinifaciens (DSM 2489^T) as the closest species with a similarity of 89.48%. The 16S rRNA genes of all the Treponema type species were aligned using MUSCLE and a maximum likelihood phylogenetic tree using FastTree was constructed (Figure 1). This analysis confirmed that isolate RCC2812 is most closely related with Treponema succinifaciens. Interestingly, the similarity value indicates that it represents a new species. This was further confirmed by computing the Average Nucleotide Identity (ANI) between the genome of RCC2812 and others Treponema spp, which were below 95%. Genome sequencing revealed that the genome of this new species comprises one single chromosome of 2,738,066 bp, with a GC content of 41.7% and 2580 predicted genes. The most closely related T. succinifaciens has a genome size of 2,897,425 bp, a GC-content of 39.13% and 2786 predicted genes.

In summary, the newly identified Treponema species can be clearly distinguished from any other Treponema species on a genetic level.

Example 2. Analytic Profile Index (API) test distinguishes Treponema strain RCC2812^T from other Treponema species

RCC2812⁷ can grow in culture without blood or serum, is catalase negative and colonies did not cause local hemolysis after 4 weeks of culture. On fastidious anaerobic agar (FAA) plates supplemented with 10% foetal bovine serum (FBS) after 48 to 72h, the colonies are punctiform with a diameter of approximately 0.5 mm, have a smooth surface, are translucent with a slight cream color and are not pigmented. The cells reach stationary phase in unsupplemented OTEB (Oral Treponeme Enrichment Broth) within 72h. These bacteria are mesophilic, growing best at temperature from 20°C to 40°C.

Further phenotypic characterization using the API-ZYM system confirmed that strain RCC2812⁷ was a distinct species within the genus Treponema (see Table 1).

It showed alkaline phosphatase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, beta-galactosidase and alpha-glucosidase activities. This is clearly different compared to publicly available data from other Treponema species. We also compared the enzymatic activities with closest available Treponema succinifaciens. T. succinifaciens strain DSM2489 demonstrated esterase lipase (C8), acid phosphatase, naphtol-AS-BI-phosphohydrolase, beta-galactosidase and beta-glucosidase activities, which is a profile distinct from Treponema peruensis strain RCC2812.

Table 1. Comparison of enzyme activity profiles of oral and gastrointestinal Treponema type strains from human, porcine and bovine hosts using the API ZYM system. Enzymes tested are 1. alkaline phosphatase, 2. C4 esterase, 3. C8 esterase lipase, 4. C14 lipase, 5. leucine arylamidase, 6. valine arylamidase, 7. cystine arylamidase, 8. trypsin, 9. alpha-chymotrypsin, 10. acid phosphatase, 11. naphtol-AS-BI-phosphohydrolase, 12. alpha-galactosidase, 13. beta-galactosidase, 14. betaglucuronidase, 15. alpha-glucosidase, 16. beta-glucosidase, 17. N-acetyl-beta-glucosaminidase, 18. alpha-mannosidase, 19. alpha-fucosidase. +, positive; -, negative.

On the basis of the data obtained from our study using a polyphasic taxonomic approach, RCC2812^T clearly merits recognition as representing a novel species within the genus Treponema.

Example 3. Treponema peruensis has an immune modulatory role in vivo

It is known that probiotic bacteria based on their interaction with the gut epithelial cells can induce a cascade of signals promoting an immune response (Amdekar et al. 2010 FEMS Immunology and Medical Microbiology 58: 299-306; Galdeano and Perdigon 2006 Clinic Vacc Immunol 13: 219-226). To assess whether T. peruensis has probiotic potential, Treponema RCC2812 was introduced in the gut of germ- free mice. First, it was tested whether Treponema RCC2812 could colonize the murine intestine after administration by oral gavage. Germ-free mice were gavaged with 10⁹ CFU of Treponema, feces were collected every week for colonization assessment by 16S rRNA sequencing as well as anaerobic agar plating and after 21 days colonization, the colon was dissected for analysis. Treponema RCC2812 could be cultured on plate and detected in the feces by 16S rRNA sequencing throughout the experiment. As an independent confirmation, immunochemistry using fluorescence in situ hybridization (FISH) tag (bacterial EUB338 probe) could clearly show that the administered Treponema RCC2812 colonizes the gut and localizes primarily in the colonic lumen (Figure 2).

Second, the in vivo effect of Treponema RCC2812 was tested on the immune system of mice. E. coli Nissle, a well-known probiotic E. coli strain was used as positive control, and suspension broth as negative control. Lamina propria immune cells were extracted from the ileum and the colon of the treated mice to evaluate priming of the immune system by the different bacteria. Flow cytometry analysis using a myeloid, T cell, and innate lymphoid cell (ILC) antibody panel allowed us to quantify monocytes, eosinophils, neutrophils, macrophages and DC's in the myeloid panel, and CD8 versus CD4 helper subsets (Thl, Thl, Thl7 and Treg) in the T cell panel and different ILC subsets. In parallel, expression analysis by RT-qPRC of immune-associated genes was performed, including IL-10, Foxp3, TGF beta, IFNgamma, IL-12, IL17 (A/F), IL-22, RORgt, IL23, TNF and TLR4. Since germ-free mice have a characteristic immature immune system, they are hypersensitive to pathogen exposure. We observed no signs of morbidity nor weight loss after Treponema colonization, indicating that the Treponema RCC2812 strain did not behave as a pathogen, but rather as a harmless commensal.

In the colon of Treponema RCC2812 colonized mice, the most striking effect was an increase (similar to that seen with E. coli Nissle) of regulatory CD4 T-cells (CD4+ Foxp3+) (Figure 3).

In the ileum, no difference in regulatory CD4 T-cells was observed compared to the control. Treponema treated mice showed a higher abundance of CD4+CD8+ T-cells, of CD8+ T-cells and a decrease (similar to that seen with E. coli Nissle) of CD4+ Tbet+ T-cells (Figure 4).

In the colonic myeloid department, an increase in eosinophils, neutrophils, monocytes, macrophages and dendritic cells could be observed (Figure 5). In conclusion, all data suggest that Treponema RCC2812 has immunomodulatory properties, favoring maturation of specific immune subsets, without causing inflammation. The observed responses resembled those of the known probiotic E.coli Nissle.

Example 4. Effect of Treponema RCC2812 in the DSS colitis model

To analyze the therapeutic potential of the herein disclosed novel Treponema species, in vivo mice experiments are set up using the DSS colitis model. DSS or dextran sulfate sodium administration is a chemical method to induce colitis, which results in similar key clinical and pathological features of IBDs to those in humans (Okayasu et al. 1990 Gastroenterology 98: 694-702). DSS administration is believed to impair intestinal epithelial cells due to its toxicity. Consequently, the destroyed barrier function allows the immune cells to be exposed to antigens (e.g. microbes), resulting in a subsequent immune response (Wirtz et al 2017 Nat Protoc 12: 1295-1309). Based on the properties of colitis, this method is suitable to investigate the impacts of the intestinal microbiota on colitis progression (Hudcovic et al 2001 Folia Microbio 46, 565-572; Hernandez-Chirlaque et al 2016 J Crohns Colitis 10: 1324-1335; Bian et al 2019 Front Microbiol 10: 2259).

Treponema RCC2812 as well as the mock treatment (8 mice per group) are administered to female C57BI6/J mice of 8 weeks old using oral gavage. 24h before the first gavage (at day -9) the mice are treated with 20 mg streptomycin to facilitate the engraftment of the bacterial strains. At day -8, -6, -4, - 2, 0, 2, 4 and 6 the mice are treated with Treponema RCC2812 (10⁸ cfu per mouse) or with vehicle (Figure 6). Every 2 days, stool is collected for 16S rRNA and/or qPCR analysis to evaluate the colonization and the effect of the bacterial strain on the gut microbiome of the mice.

Between day 0 and day 7, mice are treated with 1.5% DSS (Figure 6). As reported previously, body weight loss during DSS model establishment is a metric of colitis severity and is consistent with the inflammatory markers (Llewellyn et al 2018 Gastroenterology 154: 1037.e2-1046.e2). Administration of DSS in mice also induces loose stool and bloody diarrhea. These inflammation-associated parameters are measured on a daily basis and used to calculate the DAI score. In mock-treated mice, the DAI score increases with the time of DSS administration.

In the colitis model, disease severity is typically associated with colon length shortening due to intestinal inflammation (Wirtz et al 2017 Nat Protoc 12: 1295-1309). Therefore, colon length of all mice is measured at the end of the experiment. DSS-induced colitis also results in extended ulcerations, destroyed crypts and transmural inflammatory infiltration, with a barely complete mucosal structure (Bian et al 2019 Front Microbiol 10: 2259). The histological damage is measured in all mice at the end of the experiment. Additionally, a serum cytokine analysis and an immune cell profiling of the lamina propria is performed. (Original in Electronic Form)

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Claims

1. An isolated Treponema species comprising a 16S rRNA with a homology of at least 90% to SEQ. ID No. 1.

2. The Treponema species according to claim 1, wherein the species produces alkaline phosphatase, acid phosphatase, naphtol-AS-BI-phosphohydrolase, beta-galactosidase and alpha-glucosidase.

3. The Treponema species according to any of the preceding claims, wherein the species is catalase negative.

4. The Treponema species according to any of the preceding claims, wherein the species induces proliferation or accumulation of regulatory CD4+ T-cells, CD4+ CD8+ T-cells, eosinophils, neutrophils, monocytes, macrophages and/or dendritic cells.

5. The Treponema species according to any of the preceding claims, wherein said species is the Treponema peruense strain RCC2812 with deposit number LMG P-31793.

6. An enriched culture of the Treponema species of any of the preceding claims.

7. An extract of the enriched culture of claim 6.

8. A composition comprising the Treponema species of any of claims 1-5, the enriched culture of claim 6 or the extract of claim 7.

9. The composition according to claim 8, wherein said composition is a food product or beverage.

10. A composition comprising the Treponema species of any of claims 1-5 and a preservative, wherein the Treponema species is lyophilized, freeze dried or in the form of a dry powder.

11. A composition comprising the Treponema species of any of claims 1-5 and growth medium appropriate for the Treponema species and/or a cryoprotectant.

12. The Treponema species according to any of claims 1-5 for use as a medicine or prophylactic.

13. The Treponema species according to any of claims 1-5 for use in the prevention or treatment of an immune or inflammatory disorder.

14. The Treponema species of claim 13 for use according to claim 13, wherein said inflammatory disorder is a gastro-intestinal inflammatory disorder.

15. The Treponema species of claim 14 for use according to claim 14, wherein said gastro-intestinal inflammatory disorder is selected from inflammatory bowel disease (IBD), ulcerative colitis (UC), Crohn's disease (CD), irritable bowel syndrome (IBS), celiac disease, pouchitis, post-infection colitis, gut inflammation associated with gastrointestinal cancer, with spondyloarthritis, with rheumatoid arthritis, with primary sclerosing cholangitis or with multiple sclerosis, diarrhoeal disease due to an infectious bacterial, fungal or viral agent and post-infective diarrhoea.

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