INTESTINAL MICROBIOME

FIELD

The invention relates to compositions and methods for administering compositions that provide improved gut health modulators that offer associated health benefit in immune system mediated conditions and diseases.

BACKGROUND OF THE INVENTION

Modification of the gut microbiome has been an area of interest in science since Ilya Metchnikov identified an association between the longevity of rural Bulgarian farmers and their consumption of fermented milk products. For his work in immunity Metchnikov was awarded the 1908 Nobel Prize in medicine and is considered to be the ‘founding father’ of innate immunity.

Gut Health is important for overall health and function of the body. Indeed, the health of the microbiota comprised within the human gastro-intestinal (Gl) tract is believed to impact nearly all aspects of host physiology (Marches! et al. Gut. 2016 Feb; 65(2): 330-339). Recent attention has shifted to a greater appreciation of the role that gut microbiota has in the promotion of health and the initiation or maintenance of different Gl and non-GI diseases.

Current approaches have focussed primarily on the gut microbiota and how to modulate its composition towards one that promotes health. The composition of the gut microbiota can be influenced by many factors, including age, genetics, host environment, stress, medications and diet. However, gut health Is not just a healthy microbiota as it also involves the way in which the host responds to the existing microbiota and overall colonic environment (Wan et al. (2019) Critical Reviews in Food Science and Nutrition, 59:12, 1927-1936; and Bischoff, S. C. (2011) BMC Medicine 9:24-37).

Chinese Patent Application No. 113564069 A describes a range of extracellular polysaccharides (EPSs) produced by Bifidobacterium species under growth conditions and in the presence of carbon sources all of which lack fucose.

Wu et al. describes the use of antimicrobial EPS obtained from Bifidobacterium longum to stimulate the immune system of a recipient in order to fight off gastrointestinal disease (Int. J. Food Microbiol. (2010) Vol. 144 (1) 104-110). Hickey et al. describes the differences in properties of a variety of EPS obtained from strains of Bifidobacterium (Front, in Microbiol. (2021) Vol. 12 Article No. 653587).

Fanning et al. describes the adaptive immune response and how Bifidobacteria evade immune function. It also explains that Bifidobacterium breve UCC2003 produces different types of EPS with different immunomodulatory properties, which depend on the growth conditions (Gut Microbes (2012) Vol. 3, no. 5:420-425).

Inturri et al. describes immunomodulatory effects of EPS derived from Bifidobacterium iongum W11 on cytokine production by PBMCs (Curr. Pharm. Biotech. (2017) Vol 18, no. 1 1 : 883-889).

Hence, in order to better promote health and wellbeing there is a need for compositions and approaches that not only target the composition of gut microbiota and/or function, but also address how the host responds to the existing colonic environment. In this way it is envisaged that improving the regulation and balance of the complex interaction between microbiota and host immune system directly can result in a wide range of therapeutic and even cosmetic benefits.

These and other uses, features and advantages of the invention should be apparent to those skilled in the art from the teachings provided herein.

SUMMARY OF THE INVENTION

In view of the above problems, the present inventors have developed compositions and methods that not only target gut microbiota composition and/or function, but also surprisingly addresses how the host responds to the existing colonic environment. This response is mediated through the use of gut microbiota derived signalling molecules that interact directly with pattern recognition receptors (PRRs) of the innate immune system in the gut. The PRRs may help to regulate and balance the interaction of microbiota and host immune system. The inventors address the aforementioned problems by providing methods and compositions to achieve better gut health by combining a gut microbiota modulator together with a ligand for innate immune system PRRs.

In a first aspect the invexxion provides a composition comprising:

(i) an immunomodulatory polysaccharide extract obtained from a culture of a Bifidobacterium species, wherein the polysaccharide extract comprises a complex polysaccharide comprised of units of fucose, rhamnose, galactose and/or glucose; and

(ii) a prebiotic galacto-oligosaccharide mixture.

In a second aspect the invention provides an immunomodulatory composition comprising an immunomodulatory polysaccharide having immune cell pattern recognition receptor (PRR) agonist activity, wherein the immunomodulatory polysaccharide is obtainable from a culture a Bifidobacterium species that is grown under anaerobic culture conditions in the presence of a carbon source that comprises fucose and glucan containing oligo- or polysaccharides.

In a third aspect the invention provides a composition as described herein, wherein the composition is for use in the treatment of an inflammatory disease.

In a fourth aspect the invention provides a composition as described herein, wherein the composition is for use in the treatment of chronic and/or low-grade inflammation.

In a fifth aspect the invention provides a composition as described herein, wherein the composition is for use in the treatment of an anxiety related disorder or condition.

In a sixth aspect the invention provides a composition as described herein for use in the treatment of insulin resistance in an individual subject that is prediabetic.

In a seventh aspect the invention provides a cosmetic method of treating skin imperfections in an individual subject, the method comprising administering to the individual a cosmetically effective amount of a composition as defined herein.

In an eighth aspect the invention provides a cosmetic method of treating the appearance of skin ageing in an individual subject, the method comprising administering to the individual a cosmetically effective amount of a composition as defined herein.

A ninth aspect of the invention provides a method of treating an inflammatory disease, the method comprising administering to an individual subject in need thereof, a composition comprising an immunomodulatory polysaccharide extract obtained from a culture of a Bifidobacterium species, wherein the polysaccharide extract comprises a complex polysaccharide comprised of units of fucose, rhamnose, galactose and/or glucose.

A tenth aspect of the invention provides a dietary supplement comprising a polysaccharide extract obtained from a culture of a Bifidobacterium species, wherein the polysaccharide extract comprises a complex polysaccharide comprised of units of fucose, rhamnose, galactose and/or glucose.

An eleventh aspect of the invention provides a method of manufacturing a polysaccharide extract obtained from a culture of Bifidobacterium breve. Bifidobacterium bifidum or Bifidobacterium longum, wherein the polysaccharide extract comprises a complex polysaccharide comprised of units of fucose, rhamnose, galactose and/or glucose, the method comprising culturing Bifidobacterium breve, Bifidobacterium bifidum or Bifidobacterium iongum under anaerobic conditions at an acidic pH in the presence of a carbon source that comprises fucose and glucan containing oligo- or polysaccharides; and isolating a resulting polysaccharide extract from the culture.

Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, ail embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Fig. 1 is a graph showing the results of an in vivo experiment utilising compositions according to embodiments of the invention to treat an induced inflammatory response.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part upon a recognition that the use of gut microbiota derived signalling molecules that interact with Pattern Recognition receptors (PRRs) of the innate immune system in the gut can regulate and balance the systemic interaction between microbiota and the host immune system.

Prior to setting forth the invention, a number of definitions are provided that will assist in the understanding of the invention.

As used herein, the term ‘comprising’ means any of the recited elements are necessarily included and other elements may optionally be included as well. ‘Consisting essentially of’ means any recited elements are necessarily included, elements that would materially affect the basic and novel characteristics of the listed elements are excluded, and other elements may optionally be included. ‘Consisting of means that all elements other than those listed are excluded. Embodiments defined by each of these terms are within the scope of this invention.

All references cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The term “subject” or “individual subject” refers to an animal, such as a mammal, including but not limited to: primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice and the like; as well as birds such as chickens or other fowl. In certain specific embodiments, the subject is a human. In other embodiments the human may be an adult, an infant, a neonate, a geriatric, or from another grouping or sub-division such as an immunocompromised or otherwise immune-distinct population - e.g. HIV positive patients, diabetic patients and/or bariatric patients.

The term “prebiotic” is intended to relate to a non-digestible food component which serves to stimulate the growth and/or activity of one or a limited number of beneficial bacteria selectively within a Gl tract of a host subject. Suitably the beneficial bacteria comprise Lactic Acid Bacteria (LAB), more suitably Bifidobacteria species, typically selected from S. breve, B. bifidum and B. iongum (including B. infantis subspecies). In this way the prebiotic can improve or maintain the health of the subject. Ln particular instances, the term “prebiotic” may refer to a mixture or extract that comprises a non-digestible component that beneficially affects the host subject via selective metabolism in the Gl tract. Without wishing to be bound by theory, it is believed that the prebiotics provided herein may selectively stimulate the growth and/or activity of one or a limited number of bacteria species, such as Bifidobacteria species, in a part of the subject’s Gl tract (e.g. small intestine or colon) that have an immunomodulatory effect.

As used herein, the term “modulation” refers to a change in activity as a direct or indirect response to the presence of at least one active agent of the invention described herein, relative to the activity in the absence of the active agent in the individual subject to which it has been administered. The change may be an increase in activity or a decrease in activity and may be due to the direct interaction of the agent with one or more PRRs, or due to the interaction of the agent with one or more other factors that in turn interact with PRRs or affect PRR signal transduction pathway activity.

The terms "treatment" and "treat" as used herein, refer to and encompass prophylactic (i.e., preventive), modifying, and curative treatments. As such, these terms including treatment of individual subjects (e.g. humans) at risk of contracting a disease/aberrant pathology or suspected to have contracted a disease, as well as those who are ill or have been diagnosed as suffering from a disease or medical condition. Treatments may be therapeutic or non-therapeutic (e.g. cosmetic) as defined further below.

As used herein, the term “inflammatory disease” is intended to refer to a disease or pathology that is characterized by acute or chronic inflammation that may result from aberrant levels of pro- inflammatory cytokines or immune cells within a subject. Chronic inflammation may include so called low-grade or persistent inflammation, which may be defined as having an elevated serum C-reactive protein (CRP) level over an extended period of days, weeks, months of even several years. In some instances, a level of CRP above around 5 to 8 mg/L or higher over at least two time points separated by a period of weeks, months or years can be considered indicative of systemic low-grade inflammation. CRP is an immune system response to toxins or injuries in systemic inflammation and, therefore, represents a biomarker of low-grade inflammation (Tao et al. (2018) JAMA Netw Open. 1 (6)). Other biomarkers of inflammation may include systemic proinflammatory cytokine concentrations (e.g. in serum or plasma) including TNF-a or various interleukins, for example, IL-6, as will be understood by the skilled person. Alternatively, an increased level of acute, chronic or low-grade inflammation is that compared to a control such as a healthy subject not suffering from a disease or to the same subject prior to onset of the disease or condition. Some non-limiting examples of inflammatory diseases include autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1 , ankylosing spondylitis, Guillain-Barre syndrome, Hashimoto’s encephalitis, Hashimoto’s thyroiditis, psoriasis, Sjogren’s syndrome, glomerulonephritis, autoimmune thyroiditis, Behcet’s disease, Crohn’s disease, ulcerative colitis, vasculitis, pemphigus, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease (IBS), Addison’s disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, pelvic inflammatory disease, reperfusion injury, ischemia reperfusion injury, stroke, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis. It will be appreciated that the aforementioned conditions are frequently interlinked with myriad other diseases, disorders and conditions. A non-limiting list of inflammatory-related diseases, disorders and conditions which may, for example, be caused by elevated or aberrant levels of pro-inflammatory cytokines, include, arthritis, kidney failure, lupus, asthma, psoriasis, colitis, pancreatitis, allergies, fibrosis, surgical complications (e.g., where inflammatory cytokines prevent healing), anaemia, and fibromyalgia. Other diseases and disorders may also be associated with chronic or low-grade inflammation include Alzheimer’s disease, congestive heart failure, stroke, aortic valve stenosis, arteriosclerosis, osteoporosis, Parkinson’s disease, infections, inflammatory bowel disease (IBD), Type I and Type II diabetes mellitus, allergic contact dermatitis and other eczemas, systemic sclerosis, transplantation and multiple sclerosis. Some of the aforementioned diseases, disorders and conditions for which the compositions described herein may be particularly efficacious.

In specific embodiments the invention provides for treatment of insulin resistance in an individual subject that is prediabetic and, therefore, susceptible to developing type 2 diabetes melitus. Type 2 diabetes (also known as non-insulin dependent diabetes mellitus (NIDDM)) occurs when muscle, fat, and liver cells are unable to respond normally to insulin. This disorder (referred to as insulin resistance) may be due to a decrease in the number of insulin receptors on the surface of these cells, dysfunction of signalling pathways within the cells, or both. This loss of competency to respond to insulin may be the result of low-grade chronic inflammation resulting from a variety of environmental and lifestyle factors. Hence, by reducing the level of systemic inflammation, the present methods and compositions of the invention allow for the treatment of this debilitating disorder.

The compositions of the present invention may also be used in the cosmetic treatment of skin imperfections, such as minor lesions of the dermis or that may include eradication of redness (rosacea), skin blotches, rhytides, wrinkles, dry patches, pimples (e.g. black heads and white heads), boils, acne, scarring, premature aging, sunburn, as well as improving skin tone and apparent luminosity. Such minor cosmetic imperfections of the skin are sometimes a result of hormonal imbalances that result from low-grade chronic inflammation. More specifically, fine lines and rhytides caused by a loss of skin elasticity are called static rhytides. During the ageing process, the skin’s collagen breaks down, and the tissues can become lax. Further pro- inflammatory factors may include genetics, diet, chronic fatigue, stress, exposure to solar radiation, medication and smoking. Examples of static rhytides may include lines at the corners of the mouth, the eyes, along the cheeks, hands and neck rhytides. Hence, by eliminating the effects of the inflammatory component via administration of the compositions of the invention, the surprising and beneficial effects on improved skin health, ageing and appearance can be achieved. In a specific embodiment, the compositions may be used to ameliorate the effects of apparent chronological ageing of the skin. Cosmetic treatment may occur via administration of a nutraceutical composition to a subject in need of such treatment.

In one embodiment the present invention provides a composition containing:

· A prebiotic galacto-oligosaccharide (GOS) mixture that has been produced through the action of a beta galactosidase activity from a Bifidobacterium spp., suitably Bifidobacterium breve, Bifidobacterium bifidum and/or Bifidobacterium longum.

In a specific embodiment, the galacto-oligosaccharide (GOS) comprises β1-3, β1-4 and β1-6 galactosidic linkages in a plurality of di-, tri-, tetra- and pentasaccharides.

· a polysaccharide mixture obtained from extract of a Bifidobacterium Spp - including Bifidobacterium breve, Bifidobacterium bifidum and/or Bifidobacterium longum - that has been grown under defined growth conditions to produce a specific polysaccharide agent that unexpectedly interacts with pattern recognition receptors (PRRs) of immune cells thereby exhibiting an immunomodulatory function.

The polysaccharide mixture may comprise a complex polysaccharide comprised of units of fucose, rhamnose, galactose and glucose, suitably with β1-3, β1-4, β1-6 and α1-6 linkages. The polysaccharide extract may comprise a complex polysaccharide comprised of units selected from one or more of the group consisting of: α1-3 fucose; α1-6 fucose; α1-4 rhamnose; α1-6 galactose; β1-3 galactose; β1-6 galactose; β1-4 galactose; β1 -3 glucose; and β1 -6 glucose. In embodiments of the invention the polysaccharide comprises one or more repeating units.

In a particular embodiment of the invention, the polysaccharide comprises at least one moiety of formula A defined as:

→ 3)- β-D-Glcp-(1 → 3)-p-D-Galf (1 →4)-a-L-Rha (1 → 4)-a-D-Galf (1 → A

X wherein X may be selected from one of the group consisting of: p-D-GIcp (1 --→6); a-L-Fucp (1 -→3); a-L-Fucp (1 → 6); a-D-Galp (1 --→6); p-D-Galp (1 --→4); and p-D-Galp (1 -→6).

Optionally, the complex polysaccharide comprises more than one moiety of formula A, suitably more than two - e.g. a plurality of moieties of formula A. In an embodiment of the invention, the complex polysaccharide comprises a plurality of repeating moieties of formula A.

Surprisingly, it has been found that the compositions comprising the complex polysaccharide act synergistically to improve the gut microbiota composition within a recipient subject by increasing the numbers of Bifidobacterium species in the gut and at the same time reduces gut derived low- grade inflammation by activating dendritic cells that leads to specific T cells polarisation. In particular, in embodiments of the present invention the compositions act to downregulate T-helper 1 (Th1) and Th17 phenotypes, whilst upregulating the Th2 T cell phenotype.

Hence, the compositions and methods of the invention provide novel mechanisms to control the accumulation of systemic low-grade inflammation in individuals, in order to prevent or delay a wide range of immune-system mediated diseases and conditions.

Toll-like receptors (TLRs) are one of the best characterised PRR families for recognition of potential pathogens. TLRs are evolutionary conserved transmembrane proteins that function by sensing extracellular and intracellular pathogens in endosomes and lysosomes. TLRs contain an N-terminal leucine-rich repeats (LRRs) and a C-terminal Toll/IL-1 R homology (TIR) domain. Whilst most TLRs form homodimers, TLR2 forms a heterodimer with TLR1 or TLR6. TLRs signal through Myeloid differentiation primary response 88 (MyD88) adapter proteins, MyD88 adapterlike (Mai), TIR-domain-containing adapter-inducing IFN-p (TRIF) and TRIF-related adaptor molecule (TRAM). TLR signalling typically occurs via two distinct pathways, the MyD88- dependent and the TRIF-dependent pathways. MyD88 is utilised by all TLRs except for TLR3. Following ligand recognition, MyD88 is recruited to the TLR and a signalling cascade is initiated that culminates in NFKB nuclear translocation and transcription of various proinflammatory cytokines. Endosomal TLRs signal via the TRIF-dependent pathway to induce IRF-3 nuclear translocation and transcription of type I IFN genes. Hence, TLRs play an important role in recognising various pathogens/ligands and controlling a host immune response.

Cytokines are a broad category of small proteins important in cell signalling. Cytokines have been shown to be involved in autocrine, paracrine and endocrine signalling as immunomoduiating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumour necrosis factors. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B lymphocytes, T lymphocytes and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells; a given cytokine may be produced by more than one type of cell. They act through cell surface receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations. Cytokines have been classed as interleukins, lymphokines, monokines, interferons, colony stimulating factors and chemokines. Interleukins (ILs) are a group of cytokines (secreted proteins and signal molecules) that were first seen to be expressed by white blood cells (leukocytes). The function of the immune system depends in a large part on interleukins, and rare deficiencies of a number of them have been described, all featuring autoimmune diseases or immune deficiency. The majority of interleukins are synthesized by helper CD4 T lymphocytes, as well as through monocytes, macrophages, and endothelial cells. They promote the development and differentiation of T and B lymphocytes, and hematopoietic cells. Interferons (IFNs) are a group of signalling proteins made and released by host cells in response to the presence of several viruses. IFN-α , IFN-β, IFN-ε, IFN-K and IFN-ω bind to the IFN-α/β receptor complex and bind to specific receptors on target ceils, which leads to expression of proteins that will prevent the virus from producing and replicating its RNA and DNA. IFN-y is released by cytotoxic T cells and type-1 T helper cells, however, IFN-y blocks the proliferation of type-2 T helper cells.

Attempts to use PRR ligands to control the cytokine immunomodulatory axis have previously been explored as therapeutics for various immune mediated conditions and diseases. However synthetic, chemistry-based ligands have shown significant side effects to the point that those therapeutics could not be successfully developed and commercialised. It is a significant advantage of the present compositions that the systemic immune-modulatory effects observed are much broader than simple microbiota modulation, but rather are based on the immune- modulatory properties of the polysaccharide agents in combination with microbiota changes. This synergy represents a significant departure from previous immunomodulatory approaches.

In a further embodiment of the invention a method for preparing a personalised composition for immunomodulation is provided. The method includes isolating a Bifidobacterium spp from a sample or biopsy taken from an individual (e.g. a faecal sample). Suitably the Bifidobacterium spp. Is selected from a B. breve, B. bifidum or B iongum species. The sample is screened in order to determine the effects of extracted polysaccharide as an immunomodulator. Selective pressure approaches may be employed by incubating one or more isolated Bifidobacterium strains in defined growth media using a range of carbon sources and physicochemical conditions. This range of conditions applies selective pressures that lead to variations in transcription of key enzymes in the genome responsible for the production of polysaccharides. These polysaccharides will vary in composition depending on the growth conditions. The extracted polysaccharides can be screened using in vitro models or in vivo models (such as described in more detail in the Example below). Suitable in vitro screening approaches may include exposing immune cells, such as human monocyte-derived dendritic ceils (mo-DCs) to the generated extracted polysaccharides and monitoring the different pattern of cytokine production. In a specific embodiment, extracted polysaccharides that induce high levels of expression of IL-10 in dendritic cells are considered particularly advantageous in compositions of the present invention. In further embodiments, extracted polysaccharides that induce low levels or suppress expression of TNF- a or IL-12 are considered particularly advantageous in compositions of the present invention. In further embodiments, the in vitro screening approaches further include determining whether the compositions promote CD4+ expression and increased CD4+/CD8+ ratio in T cells, such as ThO cells.

As used herein, the term “dietary supplement” refers to compositions consumed to affect structural or functional changes in physiology for therapeutic and/or cosmetic purposes. The term “therapeutic composition” refers to compositions administered to treat or prevent a disease or to ameliorate a sign or symptom associated with a disease, such as an inflammatory disease or condition. In embodiments, the present compositions may be used both as dietary supplements or as therapeutic compositions. The dietary supplement may take the form of a solid food; a liquid; a suspension; and a gel. A dietary supplement, may take the form of a nutraceutical composition and may further comprises one or more ingredients selected from: vitamins B1 , B2, B3, B5, B6, B8, B9, B12, C, A, D, E, K1 , and/or K2; obeticholic acid, corosoiic acid, polyunsaturated fatty acids in the Omega 6 and/or Omega 3 family, orotic acid, pangamic acid, para-amino-benzoic acid, amygdalin, beta-giucanes, carnitine, dimethylglycine, imeglimin, isoflavones, alginates, alginic acid, L-arginine, oxytocin, pectin, pyridoxamine, resveratrol, viniferine, and/or L-citrulline; stevia, sugar alcohols, sorbitol, isomalt, maltitol, mannitol, iactitol xylitol, hydrogenated starch hydrolysates, laminarin, plant extracts, probiotics, bioactives, oligo-elements, arsenic, boron, calcium, copper, iron, fluorine, iodine, lithium, manganese, magnesium, molybdenum, nickel, phosphorus, selenium, vanadium, and/or zinc; conjugated linolenic acid, lipoic acid carotenoids, carnitine, choline, Q10 coenzyme, phytosterols, polyphenols in the tannin and lignan family, and/or taurine; fructo-oligosaccharides, collagen; lactic ferments; yeasts; fungi; products derived from insects compatible with the food and pharmaceutical sector; CBD and/or THO; coating agents; aromas; acidifiers; anti-caking agents; thickeners; stabilizers; emulsifiers; filler agents; and/or excipients. The route of administration of compositions according to the invention may be any of those commoniy known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected compositions or active agents of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including orally, rectally, intranasally, parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the responsible clinician. Administration can be local (e.g., local delivery to the gut) or systemic as indicated. Typically, the compositions are administered orally or rectally (e.g. via a suppository).

Pharmaceutical compositions containing the present polysaccharide active agent or combined compositions, or a combination thereof with other drugs or biologicals can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, or some other measurable parameter, of reduced inflammation is seen as a therapeutically-effective dose.

When administered to a subject, a therapeutic agent is suitably administered as part of an in vivo delivery composition and may further comprise a pharmaceutically acceptable vehicle in order to create a pharmaceutical immunomodulatory composition. Acceptable pharmaceutical vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical vehicles can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilising, thickening, lubricating and colouring agents may be used. When administered to a subject, the pharmaceutically acceptable vehicles are preferably sterile. Water is a suitable vehicle when the agents of the invention are administered intravenously or orally. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skimmed milk, glycerol, propylene, glycol, water, ethanol and the like. Pharmaceutical compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or buffering agents.

The medicaments, supplements and pharmaceutical compositions of the invention can take the form of liquids, solutions, suspensions, gels, modified-release formulations (such as slow or sustained-release), emulsions, capsules (for example, capsules containing liquids or gels), liposomes, microparticles, nanoparticles or any other suitable formulations known in the art. Other examples of suitable pharmaceutical vehicles are described in Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995, see for example pages 1447-1676.

The effectiveness of a dosage to achieve a particular level of efficacy takes into account the amount of composition given at each administration (i.e. dose), the frequency of administration, and optionally, the total number of doses to be given. The effectiveness of a dosage can be quantitated by measuring the cumulative amount of active ingredients administered in a defined period (i.e. the "dose rate") of the dosage and that results in a particular level of efficacy, where effectiveness and potency are inversely related to the dose rate. The term "therapeutically effective amount" as used herein means the dosage (dose or amount, and frequency) of a composition as defined herein which directly or indirectly reduces the levels of inflammation and suitably the accumulation of inflammatory cells in a human or other mammal afflicted with an inflammatory disorder. A reduction in inflammation may be determined by monitoring the levels of circulating inflammatory biomarkers, such as inflammatory cytokines - e.g. plasma levels of CRP.

Studies to evaluate the cosmetic efficacy on the appearance of skin rhytides, roughness, moisture and elasticity of the compositions of the invention may be carried out in a panel of appropriate subjects at the age over a designated time period. Typical trials may occur over a period of at least one week, typically at least one month and suitably of 3+ months. Product efficacy may be evaluated using any appropriate methodology known to those in the art. By way of non-limiting example, such methods may include the use of instrumental facial imaging and complexion analysis software (e.g. Canfield's VISIA™ system); instrumental measurement (Delfin’s ElastiMeter™, Courage Khazaka’s Frictiometer™ FR 700 and Corneometer® CM 825); a dermatologist’s professional opinion; as well as the opinion of the subject panel themselves recorded using a target questionnaire. In one embodiment, the methodology includes the use of the VISIA™ system which consists of the VISIA™ imaging booth and VISIA™ software and can capture and store facial images of a subject using standard lighting, cross-polarized flash, and UV flash. After capturing such images, the software automatically isolates or “masks” specific areas of the face and then performs an extensive analysis of these areas to evaluate skin features such as apparent age and presence of environmental related skin lines. The cosmetic effect of the compositions of the invention, particularly in reducing low-grade inflammatory ageing, can then be determined on the subject over a suitable trial time period after establishing a pretreatment baseline.

The invention is further illustrated by the following non-limiting examples. EXAMPLES

Example 1 - Isolation of microbial species

Tentative Bifidobacterium species from one 70yr oid healthy adult were isolated from samples of fresh faecal matter, following anaerobic incubation on Bifidobacterium spp selective agar plates. Well isolated colonies that appeared to be bifidobacteria were tentatively identified as Bifidobacterium strains for further 16S rRNA-based taxonomic analyses. S. breve (BB091109) and B. longum (BL191206) from the samples were isolated and stored at -80°C in CryoBeads.

Example 2 - Production of the polysaccharide (VMK223 - EPS) mixture component

1) Bifidobacterium breve strain 091109 was cultured in below the medium (adjust pH to 6.5) under anaerobic conditions for 48h at 37°C. The culture was repeated twice more in the same medium.

Table 1

2) When the bacterial culture is ready, it was inoculated with 100ml (aim for inoculum in the

10 ⁷ cfu/mi) of the below medium (pH 6.5, at 37°C): Table 2

The fermentation was maintained under anaerobic conditions for 60h.

At the end of fermentation the following harvesting protocol was performed:

1) Centrifugation to separate the cells from the supernatant. Keep the supernatant (A) and weigh the cells.

2) Dissolve the cell pellet in 1 N NaOH overnight at room temperature (to break the cells). After that centrifuge to get lysed cells (B) and supernatant (C),

3) Mix supernatants (A) and (C) and precipitate with 1 volume 96% cold ethanol overnight (in cold room).

4) Centrifuge (6,000 rpm for 20 min at 4°C), keep the precipitate and repeat ethanol treatment on supernatant.

5) Centrifuge, keep the precipitate and repeat ethanol treatment on supernatant.

6) Collect ail 3 precipitates and dissolve in distilled water, before heating it at 100°C for 5 minutes.

7) Dialysis (overnight) or filtration (MWCO 10-12KDa)

8) freeze dry or spray dry the retentate. The resulting EPS extract has the following composition:

A mixture of complex polysaccharides containing fucose, rhamnose, galactose and glucose with β1-3, β1-6 and α1-6 linkages.

The EPS (VMK 223) was determined to comprise repeated units of the following moiety:

→ 3)-p-D-Glcp-(1 → 3)-p-D-Galf (1 →4)-a-L-Rha (1 → 4)-a-D-Galf (1

X wherein X may be selected from one of the group consisting of: p-D-Gicp (1 --→6); a-L-Fucp (T-~→3); a-L-Fucp (1 → 6); a-D-Galp (1 --→6); p-D-Galp (1 --→4); and p-D-Galp (1 -→6).

Example 3 - Production of the galacto-oligosaccharide (VMK-GOS) component

1) B. breve 091109 was propagated in the below medium (adjust pH to 6.5) and grown under anaerobic conditions for 48h at 37°C. The culture was repeated twice more in the same medium.

Table 3

At the end of fermentation the following harvesting protocol was performed:

2) Centrifugation to separate the cells from the supernatant.

3) Dissolve the cell pellet in 1 N NaOH overnight at room temperature (to break the cells). After that we centrifuge and keep the cell pellet

4) Wash cell pellet with phosphate buffer and freeze dry 5) Using lactose as substrate measure beta-galactosidase activity of the freeze-dried cell pellet

6) Prepare a solution of at least 40% (w/w) Lactose in phosphate buffer (pH 6.8)

7) Suspend freeze dried cell pellet (20 units) in 100 ml of Lactose solution and incubate at 45°C for 18-24 hours

8) Heat solution at 100°C for 5 minutes to inactivate beta-galactosidase

9) Use nanofiltration (MWCO 200-300 Da) to remove monosaccharides

10) Evaporate and spray or freeze dry the GOS solution

The resulting GOS composition (based on High Performance anion exchange chromatography coupled with pulsed amperometric detector):

· Monosaccharides (Glucose): 10% (w/w)

· Lactose : 23% (w/w)

· GOS: 67% (w/w) of which o 20% disaccharides o 35% trisaccharides o 28% tetrasaccharides o 17% pentasaccharides or higher

Example 4 - Formulation of composition

A formulation for in vivo administration contains 400mg of PS Mixture from Example 2 and 2100mg of GOS from Example 3.

The route of administration compositions according to the invention may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected ligands thereof of the invention can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, typically orally, rectally or via topical application to the required part of the body. Alternative routes of administration may include parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the age, sex and condition of the recipient subject, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician. Administration can be local (e.g., local delivery to the gut, e.g., oral or rectal administration) or systemic as indicated.

Example 5 - In vivo trials of compositions

All experiments were carried out with local ethical approval and in under a UK Home Office licence granted under the Animals (Scientific Procedures) Act (1986). Male CD1 mice (25-30g, 6-8-week old, Harlan Orlac, UK), were housed 3 per cage (plexiglas cages 33x15x13 cms, LxWxH) and maintained under standard controlled laboratories conditions (12-h light-dark cycle, lights on at 7a.m., 21 +/-1 °C, humidity 50+/-5%). Mice were fed with standard mouse chow ad libitum, and after 4-5 days habituation to the animal facility, they were additionally provided, (in a weightmatch, pseudo-random fashion), with either drinking water containing:

· Extracellular polysaccharide (EPS) mixture called ‘VMK 223’, or

· GOS (called VMK-GOS), or

· PS mixture + GOS (the complete VMK treatment) (1 .3%, w/v), or

· drinking water alone

After 3 weeks, all animals received drinking water alone 24h prior to LPS injections. A single injection of 0.75mg/kg LPS (E. coll 026:136, Sigma-Aldrich) in saline (0.9%), or saline alone, was administered to mice by an intraperitoneal route.

8 groups (n=5 mice/group) were therefore tested:

(1) water-fed/saline injected (Water/SAL);

(2) water-fed/LPS injected, (Water/LPS);

(3) PS -fed/saiine injected (VMK 223/SAL);

(4) PS-fed/LPS injected (VMK 223/LPS;

(5) GOS -fed/saline injected (VMK-GOS/SAL);

(6) GOS-fed/LPS injected (VMK-GOS/LPS;

(7) Complete Treatment-fed/saline injected (VMK/SAL); and

(8) CompleteTreatment-fed/LPS injected (VMK/LPS).

Animals were sacrificed between 12-1 p.m. Trunk blood was collected in Ethylene Diamine Tetra Acetic Acid, (EDTA), tubes and spun for 15min at 5000rpm. Plasma was isolated and stored at - 80°C prior to analysis.

Gut microbiota analysis

The abundance of 16S DNA encoding specific microbial genera and total bacteria in faecal pellets were analysed using QPCR and previously published primers. Bacterial DNA was extracted using QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) after mechanical disruption. The concentrations of isolated DNA were quantified spectrophotometricaliy by measuring absorbance at 260 nm. The SYBR Green methodology (Power SYBER, Life Technologies, UK) was used to amplify 20 ng DNA on a 7900HT Fast Real-Time PCR System (Applied Biosystems, US): duplicate samples were held at 95 °C for 10 min and subjected to 40 cycles of denaturation (95 °C for 15 s) and annealing/extension (60 °C for 60 s). These QPCR data were also calculated using the 2~^ ^C{ relative quantitation method and reported as fold-changes relative to the saline/water group.

Cytokines in plasma

The concentrations of TNFa, IL-1 b, IL6 and IL-10 in plasma (100-150pl) were measured using commercial ELISA kits (R&D Systems), following pre-determined dilutions in respective assay buffer. Corticosterone concentrations in mouse plasma (20pl), was also determined with a commercial ELISA kit (Enzo Life Sciences). All ELISAs were performed according to manufacturer’s recommendations.

Light-dark box

This test was used to assess anxiety behaviour and is based on the conflict mice face between their attraction for novelty and their fear for bright open arenas. The apparatus consisted of a small black covered compartment (21x16x16 cms, LxWxH, with a small opening for access to the light part, 3x2.7cms, WxH) and a brighter open compartment (46.5x21 x21 cms, LxWxH). Testing was performed under a slightly dim light (50 lux within the bright compartment), as previously described (Couch et al, Brain Behav. Immun. 2013;29:136-146). Twenty-four hours following LPS/saline injections, each animal was gently placed in the dark part of the light-dark box and let free to explore the whole box for 5min. The latency to leave the dark area, number of transitions between the dark and lights parts and the time spent in the light section were measured. The criterion to enter any compartment was 4 paws in. Mice were placed back to their home cage with cage mates at the end of the procedure.

Results

Faecal microbiota characterization

The intake of VMK-GOS, either alone or as part of the complete VMK-Treatment had a significant effect on the Bifidobacterium spp population change (p<0.05) regardless of LPS injection.

Lactobacillus spp Clostridium cocoides cluster and Escherichia/Shigella spp, were significantly affected by VMK-GOS treatment (p<0.05) but this effect was diminished following LPS injection. VMK 223 PS treatment had no effect on any of the bacterial groups tested. Results are expressed as fold-change relative to the saline/LPS group. *p < 0.05, compared to the saline/water group

The effects of LPS-injection and VMK-Treatment intake on plasma cytokines The results are shown In Table 5 and for LPS treatment also In Figure 1 . The injection of LPS increased TNF-α levels in the plasma of Water/SAL, but not VMK 223 or VMK-GOS or VMK- Treatment administered, animals (p<0.05). Only VMK223 or VMK-Treatment had statistical difference between groups for IL-6 (p<0.05).

Table 5

Values with different superscript within column statistically different (p<0.05)

The effect of LPS-injection and VMK-Treatment intake on plasma corticosterone and cortical monoamines

There was no overall statistical difference between groups in the levels of plasma corticosterone (ng/ml), 24 hours after the LPS or saline injections, and 3 hours after the light/dark box tests. There was no significant difference between groups in the concentration of cortical 5-HT and 5- HIAA.

The effects of BGOS intake on LPS-induced anxiety behaviour in the tight-dark box

The injection of LPS increased anxiety behaviour in mice receiving normal drinking water. This effect was abolished in mice with access to drinking water containing VMK223 or VMK-Treatment, as assessed by the latency to enter the lit area ( p<0.01) and time spent in the lit area (p<0.01). Post-hoc analysis revealed that Water/LPS animals displayed a significant two-fold higher latency to move into the lit area than Water/SAL mice (p<0.01), and both VMK 223/SAL and VMK 223/LPS animals (both p<0.05) and VMK-Treatment/SAL and VMK-Treatment/LPS animals (both p<0.05). Water/LPS animals also spent significantly less time in the lit area compared to all the other groups (p<0.05, Water/LPS vs. all other groups). However, there was no statistical difference between groups in the number of transitions between the dark and light areas (p=0.17). There were no differences between VMK 223/SAL and VMK-Treatment/SAL animals, in any of the parameters measured. Table 6

Values with different superscript within column statistically different (p<0.05)

Conclusion

Supplementation with VMK-223 PS or VMK-Treatment, reduces the sickness and anxiety behaviours of mice receiving a single injection of LPS. The observed changes involved a reduction in some pro-inflammatory cytokines, VMK-GOS and VMK-Treatment improved Bifidobacterium spp and Lactobacillus spp population numbers and had a significant effect on keystone species of the microbiota.

Impressive results in terms of both microbiota composition and host function was achieved with VMK-Treatment that combines a microbiota modulator (GOS/Prebiotic) together with an innate immune system modulator (VMK 223 EPS). Nevertheless, the EPS composition alone also demonstrates surprising activity in vivo.

Although particular embodiments of the invention have been disclosed herein in detail, this has been done by way of example and for the purposes of illustration only. The aforementioned embodiments are not intended to be limiting with respect to the scope of the appended claims, which follow. It is contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.