Bacterial compositions, and uses thereof for treating or preventing an immune mediated disease

Abstract

5 A composition in oral dosage form comprises tryptophan or at least one tryptophan intermediate, and at least one bacterium that (a) expresses enzymes of the shikimate pathway and is capable of producing tryptophan and / or (b) expresses enzymes of the indole pathway and is capable of producing one or more immunomodulatory metabolites. The composition may include a cohort of bacteria 10 including at least one bacterium that (a) expresses enzymes of the shikimate pathway and is capable of producing tryptophan and at least one bacterium that (b) expresses enzymes of the indole pathway and is capable of producing one or more immunomodulatory metabolites. Typically, the cohort of bacteria is capable of producing the immunomodulatory indole derivatives indole-3-lactic acid (ILA), 15 indole-3-propionic acid (IPA) and indole acrylic acid (IA). The composition finds utility in inhibition or prevention of immune-mediated inflammatory damage in subjects with an immune-mediated infectious or non-infectious disease, and in particular can prevent a severe inflammatory response or incidence of Long Covid in SARS-CoV-19 subjects. 20 (Figure 16)

Description

[0001] TITLE

[0002] Bacterial compositions, and uses thereof for treating or preventing an immune mediated disease

[0003] Technical Field

[0004] The present invention relates to bacterial compositions and uses thereof for treating or preventing an immune mediated disease, in particular a viral infection.

[0005] Technical Background

[0006] Infection with viruses such as SARS-CoV-2 leads to a wide variety of potential outcomes from asymptomatic responses to acute respiratory distress and death. Although several risk factors for severe COVID-19 have been identified (e.g. age, obesity), the pathophysiological mechanisms that contribute to disease severity are not fully understood, although two leading hypotheses have emerged. Firstly, an inability to mount an effective immune response in a timely manner seems important. Secondly, an inability to control SARS-CoV-2-driven inflammatory responses results in excessive levels of inflammatory molecules that damage the vasculature, limit organ function and restrict homeostatic and repair mechanisms. In addition, we now appreciate that the long-term consequences of viral infection (long COVID) can affect multiple organ systems for years after the initial virus infection.2

[0007] Previous studies indicate that the composition and metabolic activity of the gut microbiota may be closely related to severity of acute COVID-19 and the risk of long-term consequences. These studies support the concept that successful responses to infectious agents such as SARS-CoV-2 involve the gut microbiome and might be mediated by microbial-derived metabolites on innate and adaptive immune responses. It is an object of the invention to overcome at least one of the above-referenced problems.

[0008] Summary of the Invention

[0009] The Applicant has shown that tryptophan and its derivatives correlate well with circulating immune mediators, while altered levels of microbial genes required for tryptophan biosynthesis and metabolism are evident in hospitalized COVID-19 patients. The Applicant has also shown using in-vitro models that these microbial tryptophan metabolites modulate inflammatory responses to virus infection. The data suggests that gut microbiome production of tryptophan and its downstream metabolites restrains the devastating overproduction of inflammatory cytokines and soluble mediators, which lead to multiorgan failure or long COVID. The disclosure is therefore based on a composition for replenishing the microbiome of a subject suffering from an immune mediated disease with a live bacterium capable of producing tryptophan (e.g. expresses the enzymes of the shikimate pathway) and metabolising tryptophan into one or more immunomodulatory indole metabolites (e.g. expresses the enzymes of the indole pathway). The composition may also be supplemented with tryptophan or a tryptophan intermediate (e.g. anthranilate, chorismite or shikimate) as this has been shown to increase tryptophan production by the bacterium and increase production of immunomodulatory indole metabolites. In one embodiment, the composition includes a consortium of tryptophan-producing bacteria including a first bacterium that produces tryptophan, a second bacterium that produces a first immunomodulatory indole derivative (e.g. indole-3-propionic acid (I PA)) and a third bacterium that produces a second (and optionally a third or fourth) immunomodulatory indole derivative (e.g. indole-3-lactic acid ILA)).

[0010] In one aspect, there is provided a composition comprising: at least one bacterium that (a) expresses enzymes of the shikimate pathway and is capable of producing tryptophan and / or (b) expresses enzymes of the indole pathway and is capable of producing one or more immunomodulatory metabolites; and tryptophan and / or at least one tryptophan intermediate.

[0011] The composition may be selected from a nutritional supplement, a pharmaceutical composition, or a food or beverage.

[0012] In any embodiment, the composition is nutritional supplement.

[0013] In any embodiment, the composition comprises at least one, and typically at least two, three or four tryptophan intermediates, and optionally tryptophan.

[0014] In any embodiment, the at least one tryptophan intermediate is selected from chorismite, anthranilate and shikimate. Other tryptophan intermediates include shikimate-3-phosphate, 5-enolpyruvoyl-shikimate-3-phosphate, N-(5- phosphoribosyl)-anthranilate, (1S,2R)-1-C-(indol-3-yl)glycerol-3-phosphate, and indole.

[0015] In any embodiment, the composition comprises anthranilate and shikimate, and optionally tryptophan.

[0016] In any embodiment, the bacterium is capable of producing an immunomodulatory metabolite selected from indole acrylic acid (IA). , indole-3-lactic acid (ILA) and indole-3-propionic acid (IPA). Examples of strains that produce tryptophan-derived indoles include Clostridium species, especially strains of Clostridium sporogenes, for example Clostridium sporogenes (DSM 795 - German Collection of Microorganisms and Cell Cultures), Bifidobacterial species, especially strains of Bifidobacterium longum, for example Bifidobacterium longum (DSM 20219- German Collection of Microorganisms and Cell Cultures) and Bifidobacterium longum (DSM 20088- German Collection of Microorganisms and Cell Cultures), and stains of Bifidobacterium pseudocatelanum, for example Bifidobacterium pseudocatelanum DSM 20438

[0017] IPA is produced by bacteria capable of Strickland fermentation, especially Clostridium species (Little AS, Light SH. Stickland metabolism in the gut. Nat Microbiol 7, 603-604 (2022). ILA and IA are commonly produced by bacteria capable of metabolising tryptophan using the indole pathway, especially strains of Bifidobacterium longum.

[0018] Many bacteria express enzymes of the shikimate pathway and are capable of producing tryptophan. Examples include Bifidobacterial species, especially strains of Bifidobacterium longum, for example Bifidobacterium longum (DSM 20219- German Collection of Microorganisms and Cell Cultures) and Bifidobacterium longum (DSM 20088- German Collection of Microorganisms and Cell Cultures), stains of Bifidobacterium pseudocatelanum, for example Bifidobacterium pseudocatelanum DSM 20438, and strains of Enterococcus, especially Enterococcus faecalis.

[0019] It is a relatively straightforward matter for the skilled person to obtain a sample of deposited microorganism (e.g. a strain of B. longum, a strain of B. pseudocatelanum, a strain of C. sporogenes, a strain of E. faecalis) from a depository institution such as the DSMZ or NICMB (Aberdeen) and test the strain using the experimental techniques described herein to determine whether they (a) produce tryptophan and / or (b) metabolise tryptophan into immunomodulatory indole derivatives, and / or (c) are capable of producing IPA by means of Strickland fermentation. It is also possible to identify strains having one or more of these functionalities from literature searches and / or genomic database searches.

[0020] In any embodiment, the bacterium is selected from a strain of Bifidobacterium longum, Bifidobacterium pseudocatelanum, Enterococcus faecalis, and Clostridium sporogenes. In any embodiment, the strain of Bifidobacterium longum is characterised by production of indole-3-lactic acid (ILA) following growth supplemented with a tryptophan intermediate (for example anthranilate and shikimate).

[0021] In any embodiment, the strain of Bifidobacterium longum is characterised by increased tryptophan production following growth supplemented with tryptophan or shikimate / anthranilate compared with the same strain grown without tryptophan or shikimate / anthranilate supplementation. These strains as referenced herein as “high tryptophan producer B. longum strains”.

[0022] In any embodiment, the strain of Clostridium sporogenes is characterised by production of indole-3-pyruvate and / or indole-3-propionic acid following growth supplemented with a tryptophan intermediate (for example anthranilate and shikimate).

[0023] In any embodiment, the strain of Enterococcus faecalis is characterised by increased production of tryptophan following growth supplemented with tryptophan compared with the same strain grown without tryptophan supplementation. These strains as referenced herein as “high tryptophan producer E. faecalis strains”.

[0024] In any embodiment, the strain of Bifidobacterium pseudocatelanum, is characterised by increased production of tryptophan following growth supplemented with tryptophan compared with the same strain grown without tryptophan supplementation. These strains as referenced herein as “high tryptophan producer B. pseudocatelanum strains”.

[0025] In any embodiment, the composition comprises a consortium of bacteria.

[0026] In any embodiment, the consortium of bacteria comprises: a first bacterium that expresses enzymes of the indole pathway and is capable of producing a first immunomodulatory metabolite; and a second bacterium that expresses enzymes of the indole pathway and is capable of producing a second, and optionally a third, immunomodulatory metabolite.

[0027] In any embodiment, the first and / or second bacterium expresses enzymes of the shikimate pathway and is capable of producing tryptophan.

[0028] In any embodiment, the consortium of bacteria comprises a third bacterium that expresses enzymes of the shikimate pathway and is capable of producing tryptophan.

[0029] In any embodiment, the first immunomodulatory metabolite is ILA and the second immunomodulatory metabolite is IPA.

[0030] In any embodiment, the bacterium or consortium of bacteria is capable of producing one, two or all of indole-3-lactic acid (ILA), indole-3-propionic acid (IPA), and indole acrylic acid (IA).

[0031] In any embodiment, the at least one bacterium is a high tryptophan producer strain.

[0032] In any embodiment, the composition comprises a strain of Clostridium sporogenes, a strain of Bifidobacterium longum, and tryptophan and / or a tryptophan intermediate.

[0033] In any embodiment, the composition comprises a strain of Clostridium sporogenes, a plurality of Bifidobacterium longum strains, and tryptophan and a tryptophan intermediate.

[0034] In any embodiment, the composition comprises a strain of Clostridium sporogenes, a plurality of Bifidobacterium longum strains, and tryptophan, anthranilate and shikimate. In any embodiment, the composition comprises a strain of Clostridium sporogenes, a plurality of Bifidobacterium longum strains, and tryptophan.

[0035] In any embodiment, the composition is formulated for oral administration.

[0036] In any embodiment, the composition is provided in unit dose form.

[0037] In any embodiment, the unit dose comprises at least 106CFU (colony forming units)of the bacterium. In any embodiment, the composition comprises at least 104, 105, 106, 107or 108CFU (colony forming units) of the bacterium.

[0038] In any embodiment, the unit dose comprises at least 104to 1010or 106to 108CFU of the bacterium.

[0039] In any embodiment, the unit dose comprises at least 0.1 , 0.2, 0.3, 0.4 or 0.5 mg of tryptophan or tryptophan intermediate..

[0040] In any embodiment, the unit dose comprises 0.1 to 10, 0.1 to 5, 0.1 to 2, or 0.1 to 1.0 mg of tryptophan or tryptophan intermediate..

[0041] In any embodiment, the composition is a pharmaceutical composition and comprises a suitable pharmaceutical carrier.

[0042] In any embodiment, the bacterium capable of making tryptophan is a strain selected from the microbial genera consisting of: Acidaminococcus, Actinomyces, Anaeromassilibacillus, Anaerostipes, Bacteroides, Bifidobacterium, Blautia, Butyrivibrio, Campylobacter, Catabacter, Cellulosilyticum, Citrobacter, Clostridium, Coprobacillus, Dialister, Dorea, Eisenbergiella, Enterobacter, Enterococcus, Escherichia, Eubacterium, Faecalibacterium, Flavonifractor, Fusicatenibacter, Haemophilus, Hungatella, Intestinimonas, Klebsiella, Kluyvera, Lachnoclostridium, Lachnospira, Lactobacillus, Lactococcus, Lactonifactor, Lawsonibacter, Lelliottia, Lentisphaerae, Mediterraneibacter, Megamonas, Megasphaera, Mesosutterella, Monoglobus, Morganella, Murimonas, Neisseria, Odoribacter, Phascolarctobacterium, Porphyromonas, Prevotella, Proteus, Pseudoflavonifractor, Pseudomonas, Raoultella, Robinsoniella, Romboutsia, Roseburia, Rothia, Ruminococcus, Salmonella, Serratia, Streptococcus, Sutterella, Veillonella, Victivallis. Once specific example is Escherichia coli MG1655 (ATTC 700926).

[0043] In an embodiment in which the composition comprises a bacterium capable of producing chorismate, the composition optionally also comprises one or more of the shikimate pathway intermediates, for example an intermediate selected from selected from phosphoenolpyruvate (PEP), d-erythrose-4-phosphate (E4P), 3- deoxy-D-arabino-heptulosonate-7-phosphate (DAHP), shikimic acid (SA), 3- dehydroquinate (DHQ), 3-dehydroshikimate (DHS), shikimate-3-phosphate (S3P), and 5-enolpyruvoylshikimate 3-phosphate (EPSP). The composition of the invention may include one (or more) of the aforementioned intermediates in addition to, or instead of, chorismate. In any embodiment, the unit dose comprises at least 0.1 , 0.2, 0.3, 0.4 or 0.5 mg of intermediate. In any embodiment, the unit dose comprises 0.1 to 10, 0.1 to 5, 0.1 to 2, or 0.1 to 1.0 mg of intermediate.

[0044] In any embodiment, the composition comprises a prebiotic, for example a long chain carbohydrate. The prebiotic may comprise dietary fibre, especially soluble dietary fibre. The prebiotic material may comprise an inulin or pectin carbohydrate.

[0045] The strain in the composition may be viable or non-viable and may comprise a strain extract (e.g. bacterial cell lysate) or supernatant derived from the strain. The extract or supernatant may be in any physical form, for example liquid or dried.

[0046] The composition may comprise at least 106cfu of the bacteria per gram of the composition, for example, 106- 1010cfu of bacteria per gram of the composition. The composition may be solid or liquid. The composition may comprise a carrier for oral delivery. The carrier may be in the form of tablet, capsule, powder, granules, microparticles or nanoparticles. The composition may be configured for targeted release in the intestine (i.e. configured for gastric transit and ileal or colon release). The composition may be contained within a delivery vehicle configured for ileal or colon release, for example an enteric capsule or a microencapsulate. The carrier may be configured for controlled release in the intestine (e.g. configured for gastric transit and ileal or colon release).

[0047] The composition may be dried or lyophilised.

[0048] Various options for compositions are envisaged, including: a composition comprising tryptophan plus a strain of bacteria capable of metabolising tryptophan to produce ILA and / or IPA (e.g. a strain of C. sporogenesy a composition comprising tryptophan plus a strain of bacteria capable of metabolising tryptophan to produce ILA (e.g. a strain of B. longum a composition comprising tryptophan, a strain of bacteria capable of metabolising tryptophan to produce IPA (e.g. Clostridium species), and a strain of bacteria capable of metabolising tryptophan to produce ILA (e.g. a strain of B. longum a composition comprising anthranilate and / or shikimate plus a strain of bacteria capable of making tryptophan (e.g. a strain of B. pseudocatelanum or B. longumy a composition comprising anthranilate and / or shikimate, a strain of bacteria capable of making tryptophan (e.g. a strain of B. pseudocatelanum or B. longum and a strain of bacteria capable of metabolising tryptophan to produce ILA (e.g. a strain of B. longumy and a composition comprising anthranilate and / or shikimate, tryptophan, a strain of bacteria capable of making tryptophan (e.g. a strain of B. pseudocatelanum or B. longum), a strain of bacteria capable of metabolising tryptophan to produce ILA (e.g. a strain of B. longum); a strain of bacteria capable of metabolising tryptophan to produce ILA and I PA (e.g. a strain of C. sporogenes).

[0049] Specific examples include:

[0050] B. longum DSM 20088*

[0051] C. sporogenes DSM 795

[0052] Tryptophan

[0053] * DSM = German Collection of Microorganisms and Cell Cultures

[0054] B. longum DSM 20088

[0055] B. longum DSM 20219

[0056] C. sporogenes DSM 795

[0057] Tryptophan

[0058] B. longum DSM 20088

[0059] C. sporogenes DSM 795

[0060] Shikimate, Anthranilate

[0061] B. longum DSM 20219

[0062] C. sporogenes DSM 795

[0063] Shikimate, Anthranilate, Tryptophan

[0064] B. longum DSM 20088

[0065] C. sporogenes DSM 795

[0066] Bifidobacterium pseudocatelanum DSM 20438:

[0067] C. sporogenes DSM 795

[0068] Anthranilate

[0069] Example G

[0070] B. longum DSM 20219

[0071] Tryptophan

[0072] Also described is a composition comprising tryptophan and a tryptophan intermediate, for example anthranilate, shikimate or anthranilate and shikimate.

[0073] Also described is a composition comprising one, two or all of indole-3-lactic acid (ILA), indole-3-propionic acid (IPA) or indole acrylic acid (IA) (e.g. ILA + IPA, ILA + IA, IPA + IA).

[0074] Also described is a composition comprising (a) tryptophan and / or a tryptophan intermediate and (b) indole-3-lactic acid (ILA), indole-3-propionic acid (IPA) or indole acrylic acid.

[0075] Also described is a composition comprising (a) anthranilate and shikimate and (b) at least one, two or all of indole-3-lactic acid (ILA), indole-3-propionic acid (IPA) or indole acrylic acid (IA).

[0076] Also described is a composition comprising at least four, five or all of tryptophan, anthranilate, shikimate, indole-3-lactic acid (ILA), indole-3-propionic acid (IPA) and indole acrylic acid (IA).

[0077] In any embodiment, the composition is contained within a delivery vehicle configured to protect the composition during gastric transit and release the composition in the mammalian ileum. Examples of delivery vehicles include microparticles, microcapsules, and enteric capsules.

[0078] Also described is the use of a composition of the disclosure as a medicament.

[0079] Also described is a method of treating or preventing a disease in a subject comprising administering to the subject a therapeutically effective amount of a composition of the disclosure.

[0080] In any embodiment, the disease is an immune mediated disease.

[0081] In any embodiment, the method comprises inhibiting an immune-mediated inflammatory response in the subject.

[0082] In any embodiment, the immune mediated disease is an infectious disease in which the method inhibits pathogen-induced immune-mediated inflammatory damage in the subject.

[0083] In any embodiment, the infectious disease is a bacterial, fungal or viral disease.

[0084] In any embodiment, the infectious disease is a viral infection, in which the method inhibits viral-induced immune-mediated inflammatory damage in the subject.

[0085] In any embodiment, the viral infection comprises SARS (Severe Acute Respiratory Syndrome).

[0086] In any embodiment, the viral infection is selected from SARS-CoV-2 and influenza.

[0087] In any embodiment, the viral infection is SARS, in which the method prevents the subject developing a severe inflammatory response to SARS. In any embodiment, the viral infection is SARS-CoV-2, in which the method prevents the subject developing (or reduces the severity of) a severe inflammatory response to SARS-CoV-2 or prevents (or ameliorates, or delays, or reduces the severity) of Long Covid.

[0088] In any embodiment, the immune mediated disease is a non-infectious disease, for example an allergy or an autoimmune disease, in which the method inhibits immune-mediated inflammatory damage in the subject.

[0089] In any embodiment, the subject is a subject that is at-risk of poor outcome to the immune mediated disease.

[0090] Examples of subjects that are at-risk of poor outcome to the immune mediated disease include elderly subjects and immunocompromised subjects (e.g. pregnant women, patients with lung disease such as COPD or cystic fibrosis, patients undergoing graft rejection treatment).

[0091] In any embodiment, the composition is a nutritional supplement.

[0092] In any embodiment, the composition is administered orally to the subject.

[0093] Also described is a method of prevention of aberrant inflammatory response in a subject (typically a subject with an immune-mediated disease) by supplementing a diet of the subject with tryptophan producing microbes optionally in combination with appropriate dietary supports (e.g. chorismate and / or shikimate pathway intermediate).

[0094] Also described is a method of identifying a subject with an immune mediated disease that is at elevated risk of poor outcome, comprising assaying a subject with the immune mediated disease for deficient gut microbiome tryptophan metabolism, wherein a subject with a deficient gut microbiome tryptophan metabolism is indicated as being at elevated risk of poor outcome from the immune mediated condition.

[0095] Markers of deficient gut microbiome tryptophan metabolism include one or more of the following: reduced abundance of tryptophan producing gut microbes compared with a reference abundance; reduced abundance of indole producing gut microbes compared with a reference abundance; reduced abundance of tryptophan in the serum compared with a reference abundance; and reduced abundance of indole or indole derivatives compared with a reference abundance.

[0096] In any embodiment, the method comprises assaying a sample (e.g. gut microbiome sample) for abundance of tryptophan-producing microbes, comparing the abundance detected with a reference abundance, and classifying the risk of poor outcome based on the comparison. Typically, when the abundance of tryptophan- producing microbes is lower than the reference level, this is indicative of the subject having an elevated risk of poor outcome from the immune mediated disease.

[0097] The poor outcome may be risk of severe or fatal immune mediated inflammatory damage, or (in the case of SARS-CoV-2 subjects, the risk of Long Covid).

[0098] In any embodiment, the method comprises assaying a biological sample obtained from the subject, for example a sample of the microbiome from a gastrointestinal tract of the subject, a faeces sample from the subject, or, for metabolite assays, a blood sample (or blood derivative). One specific application is identifying SARS- CoV-2 patients that are at risk of Long Covid, or severe viral-induced inflammatory damage due to viruses other than SARS-CoV-2. In any embodiment, a subject with an immune mediated disease that is identified as being at risk of poor outcome is treated according to a method of this disclosure.

[0099] Other aspects and preferred embodiments of the invention are defined and described in the other claims set out below.

[0100] Brief Description of the Figures

[0101] FIGURE 1 : Serum tryptophan metabolites and serum cytokine levels correlate in acute COVID-19 patients (A) and long COVID patients (B) using Pearson correlation calculations and a two-tailed P value. Red indicates a negative correlation and blue indicates a positive correlation. Trp - Tryptophan; I PA - Indolepropionate; C-Trp - C-glycosyltryptophan; ILA - Indolelactate; IAA - Indoleacetate; H5S 5-hydroxyindole sulfate; I3S 3-indoxyl sulfate. *p<0.05; **p<0.01 ; ***p<0.001

[0102] FIGURE 2: The relative abundance of 19 genes associated with tryptophan metabolism in healthy volunteers and COVID-19 patients (n=621) are shown (A). E.C. numbers are included on the x-axis. (B) Pearson correlation matrix showing two clusters of genes whose relative abundance correlates with each other. IAA - Indoleacetate; lAAId - lndole-3-aldehyde.

[0103] FIGURE 3: (A) The microbial tryptophan biosynthesis pathway is illustrated and E.C. numbers are included for each gene. The genes that are significantly different in COVID-19 patients, or in those with severe disease are highlighted and dot plots are included for those that are significantly different. (B) Relative abundance of all genes in the tryptophan biosynthesis cluster were significantly decreased in COVID-19 patients compared to controls, and in those with severe disease compared to those with mild / moderate disease. Results are expressed as mean and standard deviation. Differences between groups were calculated using the Kruskal-Wallis test and Dunn’s multiple comparison test. *p<0.05; ***p<0.001 ; ****p<0.0001 FIGURE 4: The microbial tryptophan metabolism pathways are illustrated and E.C. numbers are included for each gene. The genes that are significantly different in COVID-19 patients, or in those with severe disease are highlighted and dot plots are included for those that are significantly different. Strikethrough E.C. numbers represent genes detected in less than 10% of samples and were not included in the analysis. Results are expressed as mean and standard deviation. Differences between groups were calculated using the Kruskal-Wallis test and Dunn’s multiple comparison test. *p<0.05; **p<0.01 ; ***p<0.001 ; ****p<0.0001

[0104] FIGURE 5: Metabolites were co-incubated with cells at 10, 100, or 1000 pM. (A) IRF3 activation in TH P-1 cells (n=4). (B) Poly l:C stimulated cytokine secretion by PBMCs (n=4). (C) LPS stimulated cytokine secretion by PBMCs (n=4). (D) Anti- CD3 and anti-CD28 stimulated cytokine secretion by PBMCs (n=6). (E) Anti-CD3 and anti-CD28 stimulated cytokine secretion by CD4+ T cells (n=6). Results are shown as % change from stimulated positive control. I PA - Indolepropionate; ILA - Indolelactate; IAA - Indoleacetate; I3S 3-indoxyl sulfate. *p<0.05

[0105] FIGURE 6. Gene set enrichment analysis of RNAseq data.

[0106] FIGURE 7. IA and IPA, but not I3S or ILA, preserve mitochondrial membrane potential in activated human lymphocytes.

[0107] FIGURE 8. I3S and IA activate the AhR, but ILA and IPA do not.

[0108] FIGURE 9. IL-22 secretion by activated human lymphocytes.

[0109] FIGURE 10. The shikimate pathway for tryptophan biosynthesis.

[0110] FIGURE 11. Tryptophan levels in culture supernatants following supplementation of the shikimate pathway. FIGURE 12. Tryptophan levels in culture supernatants following supplementation with tryptophan.

[0111] FIGURE 13. Tryptophan catabolism pathways.

[0112] FIGURE 14. Cell supernatant indole levels following growth supplemented with shikimate and anthranilate.

[0113] FIGURE 15. Heatmap illustrating the change in culture supernatant metabolite levels following supplementation with shikimate and anthranilate.

[0114] FIGURE 16. ILA and IPA levels in culture supernatants following supplementation with tryptophan.

[0115] FIGURE 17. Graphical abstract summarises the observation that COVID-19 disease severity correlates with lower levels of microbe-derived tryptophan metabolites in serum and deficient metabolic capacity for tryptophan production and metabolism by the gut microbiome.

[0116] Detailed Description of the Invention

[0117] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0118] Definitions and general preferences

[0119] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art: Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0120] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open- ended and does not exclude additional, unrecited integers or method / process steps.

[0121] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning or nutritional deficiencies.

[0122] As used herein, the term "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s) (for example, the reduction in accumulation of pathological levels of lysosomal enzymes). In this case, the term is used synonymously with the term “therapy”.

[0123] Additionally, the terms "treatment" or "treating" refers to an intervention (e.g. the administration of an agent to a subject) which prevents or delays the onset or progression of a disease or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term “prophylaxis”.

[0124] As used herein, an effective amount or a therapeutically effective amount of an agent (i.e. bacterium or composition of the invention) defines an amount that can be administered to a subject without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio, but one that is sufficient to provide the desired effect, e.g. the treatment or prophylaxis manifested by a permanent or temporary improvement in the subject's condition. The amount will vary from subject to subject, depending on the age and general condition of the individual, mode of administration and other factors. Thus, while it is not possible to specify an exact effective amount, those skilled in the art will be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. A therapeutic result in this context includes eradication or lessening of symptoms, reduced pain or discomfort, prolonged survival, improved mobility and other markers of clinical improvement. A therapeutic result need not be a complete cure.

[0125] The bacteria used herein are generally non-pathogenic or capable of growing in the mammalian gut, or preferably both. The or each bacterium may be a probiotic bacterium and / or approved by EFSA (European Food Safety Authority) or the FDA (Federal Drug Administration) for use in humans. “Probiotic” refers to live microorganisms that, when administered in adequate amounts to the gut of a mammal (especially a human), confer a health benefit on the host. They are described in Salminen et al. (Trends Food Sci. Technol. 1999:10 pg 107-110).

[0126] The compositions of the disclosure may include one or more prebiotics. “Prebiotic” refers to a material or composition that can promote the growth of probiotic microbes or bacteria, especially bacterial growth in the mammalian gastrointestinal tract. Examples include oligosaccharides, dietary fibres, or mixtures thereof. Exemplary prebiotics are described in WO2011 / 039176, pg 12-15. “Immune mediated disease” refers to a disease, disorder, syndrome or condition that results in inflammatory damage being caused to the subject by the subject’s immune system. Examples include infectious disease such as bacterial, viral and fungal infections, and non-infectious disease such as inflammatory disorders, allergic disease, autoimmune disorders, and metabolic diseases associated with obesity.

[0127] “Inflammatory disorder” refers to a disease or condition characterised by the subjects’ immune system damaging the body’s own cells or tissue resulting in chronic pain, redness, swelling, stiffness and damage to normal tissues. Examples include rheumatoid arthritis, Gout, Lupus, Inflammatory bowel disease, Vasculitis, Myositis, Sclerodema, Ankylosing Spondylitis and Sjogren’s Syndrome. The inflammatory disorder may be a chronic inflammatory disorder of the gastrointestinal tract, including Inflammatory bowel diseases (I BD) such as Crohn’s disease and ulcerative colitis. Symptoms of IBD include frequent bloody diarrhoea, abdominal cramping, anorexia, abdominal distension, and emesis.

[0128] “Allergic disease” results from IgE-mediated immune responses to foreign protein (allergens). The majority of such reactions are IgE-mediated (type I) reactions. Individuals who develop such reactions are allergic. Those predisposed on a genetic basis to synthesize IgE to environmental allergens are atopic. Most allergic reactions are precipitated when a specific allergen aggregates several IgE molecules attached to IgE receptors on the surfaces of mast cells and basophils. Chemical mediators are released which lead to the immediate signs and symptoms associated with allergic diseases including hives, asthma, and anaphylaxis (Mekori et al. Crit Rev Food Sci Nutr. 996;36 Suppl:S1 -18). Allergic diseases include asthma, allergy in children, atopic eczema / atopic dermatitis, drug allergy, food allergy, rhinitis, and skin allergies.

[0129] “Autoimmune disease” refers to a condition characterized by an immune system malfunction characterised by an anomalous response of the adaptive immune system, wherein it mistakenly targets and attacks healthy, functioning parts of the body as if they were foreign organisms. Examples of autoimmune include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0130] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

[0131] The invention also relates to pharmaceutical compositions which comprises pharmaceutical carriers. As used herein, the term “pharmaceutical composition” refers to a therapeutically effective amount of the composition, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. In the case of the present invention, the term “therapeutically effective amount” should be taken to mean an amount of therapeutic which results in a clinically significant reduction in immune-mediated inflammation in the subject.

[0132] As used herein, the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, carrier, or vehicle with which the Therapeutic is administered. Such pharmaceutical carriers can be sterile 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. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0133] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, oil drops, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0134] Pharmaceutical compositions formulated or configured for oral administration and gastric transit are known in the art and include those described below: Sathish et al. (Int. J. Pharm. Sci.2013; 258-269);

[0135] • Kushal et al. (Int. Res. J. Pharm. 2013, 4(3));

[0136] • Philip et al. (Oman Med J. 2010, 25(2));

[0137] • Polymers for controlled drug delivery - Peter Tarcha (CRC Press, 21 November 1990); • Pharmaceutical coating technology - Michael Aulton et al. (Taylor & Francis 27 October 1995);

[0138] • http: / / www.slideshare.net / Balimusale / oral-controlled-drug-delivery-system;

[0139] • European Patent No: 2418968 (Teagasc); and

[0140] • European Patent No: 2097072 (RCSI).

[0141] • Brayden et al. (European Journal of Pharmaceutical Sciences 79 (2015), 102-111.

[0142] • Tambuwala et al. (Journal of Controlled Release 217 (2015) 221-227.

[0143] • Zhang et al. Evaluation of alginate-whey protein microcapsules for intestinal delivery of lipophilic compounds in pigs (J. Sci. Food Agric. (2015).

[0144] • Lamprecht et al. (Journal of Controlled Release 104 (2005) 337-346.

[0145] • Hua et al. (Nanomedicine: Nanotechnology, Biology and Medicine 11 (2015) 1117-1132.

[0146] • Drug Delivery: Fundamentals and Applications (Chapter 7, Oral Drug Delivery, Hillary and Brayden)

[0147] As used herein, the term “food” refers to a man-made food product including beverages, food additives and food or nutritional supplements. Examples of foods include dairy products such as milk, yoghurt, cheese, cheese food, dairy powders, probiotic formulations, infant formula powders, follow-on milk formula, food for special medicinal purposes, meat products, soups, vegetable products, fruit juices, fruit products, breads, confectionary, cakes, sports supplements, nutritional supplements and the like.

[0148] It is preferable that the composition is administered at least once per week over a treatment period of at least 4 weeks, and preferably for at least a 5, 6, 7, 8, 9, 10, 11 , 12, 14, 16, 18 or 20 week period. Preferably, the composition is administered several times a week, and ideally once a day. Compositions of the disclosure generally comprise between 103and 1012CFU of bacteria per gram of dry weight of the composition, or per unit dose. In one embodiment, the composition compriseslO3and 1012CFU, or 104and 1012CFU, or 106and 1010CFU bacteria per gram of dry weight of the composition, or per unit dose. A daily dose generally comprises between 103and 1012CFU of bacteria. In one embodiment, the daily dose comprisesl 03and 1012CFU, or 104and 1012CFU, or 106and 1010CFU of the strain.

[0149] Exemplification

[0150] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0151] The microbes employed in Examples 5, 6 and 7 were obtained from the UCC Culture Collection, APC, UCC, Cork, Ireland.

[0152] Example 1

[0153] Figures 1 to 5 present data showing the scientific rationale for the use of the composition of the disclosure to reduce or inhibit immune-mediated inflammatory damage in one embodiment of infectious disease (patients with a viral SARS infection). This data shows that defective production of tryptophan by gut microbes, and consequently downstream host and microbial tryptophan metabolism, links host and microbiome processes with severe or fatal outcomes to SARS-CoV-2 infection, and the long-term consequences of infection (i.e. long COVID). Reduced microbial tryptophan generation will help identify at risk individuals, but also highlights an opportunity to replace tryptophan producing probiotics to prevent and treat infection-induced acute and long-term inflammatory responses.

[0154] Example 2

[0155] One group of gnotobiotic animals (germ-free) will be colonised with two microbial strains. One strain is selected from the list above as the optimal producer of chorismate, while the second strain is selected from the list above as the optimal producer of tryptophan from chorismate. A second group of animals will be colonised with isogenic mutant strains, which lack the ability to generate tryptophan from chorismate. A third group of animals will remain germ-free. In all animals, faecal and serum tryptophan levels will be measured by mass spectrometry. In addition, systemic and mucosal murine immune responses will be measured using in vitro culture, flow cytometry and RNA sequencing. Compared to control animals, we expect that colonisation with the microbial combination generating tryptophan will have potent effects on the immune system.

[0156] In an independent experiment, gnotobiotic mice will be colonised as described above, and also infected with a murine adapted coronavirus. Anti-viral responses, viral titres and murine recovery from infection will be determined. We expect that colonisation with the microbial combination generating tryptophan will protect against severe virus-induced inflammatory damage.

[0157] Example 3. Tryptophan-derived indoles exert indole-specific effects on human immune cells.

[0158] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy human donor blood samples, following density gradient centrifugation. Untouched naive CD4+ T cells were then negatively selected from PBMCs with naive CD4+ T cell Isolation Kit (Miltenyi Biotec). Purity of the sorted cells was routinely above 95%, as assessed by flow cytometry. Purified naive CD4+ T cells were activated with T Cell TransAct™ (Miltenyi Biotec). To assess the effects of indoles, 100 pM of indoleacrylic acid (IA), indole-3-lactic acid (ILA) or indole-3-propionic acid (IPA) were added at the beginning of the activation process. Four biological replicates of cell samples were collected at 0, 24, and 48 hours after activation. mRNA was extracted using RNeasy Mini Kit (Qiagen), and RNA purity was confirmed by NanoDrop™. Library was prepared using Illumina's Truseq stranded mRNA library prep kit with polyA enrichment. 150 bp paired-end sequencing was performed using Illumina PE150 technology. Quality of FASTQ files was assessed with FastQC. Adapters and low-quality segments were trimmed with Trimmomatic. Reads were aligned to the human reference genome GRCh38 (Ensembl), and gene expression quantification was performed using the STAR-Salmon option. Differentially expressed genes (DEGs) were identified using the DESeq2 package, applying thresholds of |log2 fold change| > 1 and adjusted P-value < 0.05. Functional annotation of genes and pathways was based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases, and only pathways with significant enrichment (P < 0.05) were retained for interpretation. For gene set construction and pathway analysis, gene set enrichment analysis (GSEA) was carried out using the ClusterProfiler package. Each indole induced a distinct effect on activated human lymphocytes (Figure 6). This suggests that in vivo all of these indoles would be required to induce the optimal effects on the host immune system, reinforcing the need for our invention where a metabolic niche is targeted by a strain consortium that generates all the required indoles.

[0159] Further, indole-specific effects were observed when assessing mitochondrial health in activated lymphocytes. Purified naive human CD4+ T cells were activated with T Cell TransAct™ (Miltenyi Biotec) and 100 pM of IA, ILA, IPA or indoxyl-3-sulphate (I3S) were added at the beginning of the activation process. Mitochondrial effects were assessed by monitoring mitochondrial membrane potential (MMP) by flow cytometry. Activation of the cells drove a decrease in MMP, but I A and IPA prevented the drop in MMP (Figure 7). In contrast, I3S and ILA had no effect on MMP.

[0160] Lastly, indoles can induce some effects in immune cells following activation of the aryl hydrocarbon receptor (AhR). AhR is a ligand-dependent transcriptional factor widely expressed in barrier tissues. We evaluated if IA, ILA, IPA or I3S could activate AhR using a reporter cell line. HT29-Lucia™ AhR cells (Invivogen) express the secreted Lucia luciferase reporter gene under the control of a minimal promoter coupled with the human Cyp1a1 gene's entire regulatory sequence, which contains six dioxin-responsive elements (DREs). As a result, these cells allow studying the AhR genomic signaling pathway, by monitoring the activity of Lucia luciferase in the cell culture supernatant. I A and I3S activate the AhR, but ILA or IPA did not trigger AhR signalling over the dose range tested (Figure 8).

[0161] These experiments demonstrate metabolite-specific effects on the host immune system, reinforcing the invention where a system to deliver all the immune modifying metabolites, such as a microbial consortium with appropriate substrates, will be required for optimal immune health benefits.

[0162] Example 4. Multiple indoles are required to induce optimal effects on immune cells. The beneficial effects of combining tryptophan derived metabolites on human lymphocyte cytokine secretion was evaluated. Purified naive human CD4+ T cells were activated with T Cell TransAct™ (Miltenyi Biotec) and 100 pM of ILA or IPA were added individually or both together at the beginning of the activation process. Interleukin-22 (IL-22) is a cytokine produced by activated lymphocytes that has important protective effects on non-hematopoietic cells such as stromal and epithelial cells. IL-22 secretion was measured by ELISA. ILA alone did not increase IL-22 secretion, IPA did increase IL-22 secretion while the combination of ILA and IPA led to the highest level of IL-22 secretion by human lymphocytes (Figure 9). This experiment demonstrates that there’s a synergistic effect of tryptophan-derived microbial metabolites on immune health. This supports the selection of microbes that can complement each other with regard to ILA and IPA production.

[0163] Example 5. Microbial production of tryptophan following supplementation of the shi ki mate pathway.

[0164] A defined medium containing all essential nutrients (TDYM) but a very low level of tryptophan was used. TDMY was supplemented with either 10 mM tryptophan or a mixture of 10 mM shikimate and 10 mM anthranilate, depending on the experimental group. Shikimate and anthranilate are substrates within the shikimate pathway that can lead to the generation of tryptophan by microbes that express the appropriate enzymes (Figure 10). Microbial strains were inoculated into TDYM from overnight cultures at a starting optical density OD (600 nm) of -0.01 . All strains were first cultured in TDMY for ~16h, then subcultured in TMDY, TDMY + Tryptophan, or TDMY + Shikimate / Anthranilate. Cell-free supernatants were collected after 36 hours of incubation for metabolomic analysis. Chemical isotope labelling (CIL) liquid chromatography mass spectrometry (LC-MS) was used to quantify metabolites in cell supernatants. Metabolite identification was performed using IsoMS Pro software and database (Nova Medical Testing Inc., Edmonton, Canada). Tier 1 metabolite identification (including tryptophan and related indoles) was based on accurate mass and retention time (RT) matching against a labelled standard library comprising 1 ,060 unique endogenous metabolites: 711 amines / phenols, 187 carboxyls, 85 hydroxyls, and 77 carbonyls.

[0165] Following supplementation with shikimate and anthranilate, species dependent production of tryptophan was observed. Bifidobacterium pseudocatelanum (B. pseudocatelanum) and Bifidobacterium longum (B. longum) strains produced the highest levels of tryptophan using the shikimate pathway (Figure 11). Results are shown with the background media levels already subtracted. Other species such as Enterococcus faecalis (E. faecalis) or Cutibacterium acnes (C. acnes) did not produce tryptophan, while Clostridium sporogenes (C. sporogenes) produced low levels of tryptophan (Figure 11).

[0166] Microbial generation of tryptophan was also evaluated following supplementation with tryptophan. When tryptophan is added in excess to the microbes, the level of tryptophan further increases in the culture supernatants of specific microbes (Figure 12). The background level of tryptophan in control media is already subtracted from these results. B. pseudocatelanum, B. longum and E. faecalis produce the highest levels of tryptophan when excess tryptophan is present in the media.

[0167] Example 6. Microbial production of indoles following supplementation of the shikimate pathway.

[0168] Using the same experimental methods as outlined above, we measured the production of indoles following supplementation with 10 mM shikimate and 10 mM anthranilate. We primarily focussed on the indoles highlighted in Figure 13 as these were already shown to correlate with COVID-19 severity and the in vitro immune models have examined the effects of these indoles on immune cell responses.

[0169] Supplementation of the shikimate pathway significantly altered downstream production of indoles in the culture supernatants. IA levels remained similar to background control levels, but shikimate & anthranilate supplementation increased secretion of the other indoles in a species-specific manner (Figure 14). C. sporogenes had the highest levels of indole-3-pyruvate (IP) and I PA, while production of ILA was increased dramatically by B. longum strains following supplementation of the shikimate pathway. Results are shown with the background media levels already subtracted. From these results, one consortium of microbes that would have optimal effects on restoring deficient tryptophan metabolism via the shikimate pathway might include B. Iongum_01 (high tryptophan producer), B. Iongum_07 (high ILA producer) and C. sporogenes (high I PA producer). Supplementation with shikimate and anthranilate also impacted secretion of metabolites unrelated to the tryptophan metabolic pathways. In total, levels of 135 metabolites were either significantly increased or decreased when these shikimate pathway substrates were provided to the bacterial cells (Figure 15). This data supports the inclusion of shikimate pathway substrates with the probiotic consortium to ensure optimal metabolic performance.

[0170] Example 7. Microbial production of indoles following supplementation with excess tryptophan.

[0171] Using the same experimental methods as outlined above, we measured the production of indoles following supplementation with 10 mM tryptophan. We primarily focussed on the indoles highlighted in Figure 13 as these were already shown to correlate with COVID-19 severity and the in vitro immune models have examined the effects of these indoles on immune cell responses.

[0172] When excess tryptophan is available, the production of the immunoregulatory metabolites ILA and I PA is greatly increased (Figure 16). B. longum strains and C. sporogenes produce the highest levels of ILA, while C. sporogenes produces a greatly increased level of I PA following tryptophan supplementation.

[0173] This example demonstrates that a composition including B. longum strains, C. sporogenes and tryptophan would result in maximum levels of indole production. The capsule containing this composition should have an enteric coating so the contents will be released in the colon where the tryptophan will not be absorbed by the host but will be available for microbial metabolism.

[0174] Example 8. Graphical abstract. Figure 17 is a graphical abstract (published in Yao L, et al., Gut Microbes 2024) that summarises the observation that COVID-19 disease severity correlates with lower levels of microbe-derived tryptophan metabolites in serum and deficient metabolic capacity for tryptophan production and metabolism by the gut microbiome. These metabolites exert protective effects using human in vitro models of immune responses to virus infection. Replacing in vivo production of these immunomodulatory metabolites is expected to improve acute responses to infection, limit hyperinflammatory responses that cause tissue damage and reduce the risk of long term post-infectious sequelae such as long COVID.

[0175] Equivalents

[0176] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A composition comprising: at least one bacterium that (a) expresses enzymes of the shikimate pathway and is capable of producing tryptophan and / or (b) expresses enzymes of the indole pathway and is capable of producing one or more immunomodulatory indole metabolites; and tryptophan or at least one tryptophan intermediate.

2. A composition according to Claim 1 , comprising tryptophan.

3. A composition according to Claim 1 , comprising at least one tryptophan intermediate selected from shikimate and anthranilate.

4. A composition according to Claim 3, comprising shikimate and anthranilate.

5. A composition according to Claim 1 , comprising tryptophan, shikimate and anthranilate.

6. A composition according to Claim 1 , in which the at least one bacterium comprises a strain of Bifidobacterium longum capable of producing indole-3-lactic acid (ILA).

7. A composition according to Claim 1 , in which the at least one bacterium comprises a strain of Clostridium sporogenes capable of producing indole-3- propionic acid (IPA).

8. A composition according to Claim 1 , in which the composition comprises a consortium of bacteria, and in which the at least one bacterium comprises:a first bacterium that expresses enzymes of the indole pathway and is capable of producing a first immunomodulatory metabolite; and a second bacterium that expresses enzymes of the indole pathway and is capable of producing a second immunomodulatory metabolite,9. A composition according to Claim 8, in which the first and / or second bacterium expresses enzymes of the shikimate pathway and is capable of producing tryptophan.

10. A composition according to Claim 8, in which the first bacterium is a strain of Bifidobacterium longum and the second bacterium is a strain of Clostridium sporogenes.11 . A composition according to Claim 7, in which the first bacterium is a strain of Bifidobacterium longum capable of producing indole-3-lactic acid (ILA) and the second bacterium is a strain of Clostridium sporogenes capable of producing indole-3-propionic acid (IPA).

12. A composition according to any of Claims 8 to 11 , including a third bacterium.

13. A composition according to Claim 12, in which the third bacterium is capable of producing one or more immunomodulatory indole metabolites.

14. A composition according to any of Claims 1 to 13, for use in a method of treating or preventing an immune mediated disease in a subject by inhibiting the inflammatory response in the subject, in which a therapeutically effective amount of the composition is administered to the subject.

15. A composition of Claim 14 for use of Claim 14, in which the infectious disease is a viral infection, in which the method inhibits viral-induced immune-mediated inflammatory damage in the subject.

16. A composition of Claim 14 for use of Claim 15, in which the viral infection is SARS-CoV-2, and in which the method prevents or inhibits the subject developing (a) a severe inflammatory response to SARS-CoV-2 or (b) Long Covid.

17. A composition comprising indole-3-lactic acid (ILA), indole-3-propionic acid (IPA) and optionally indole acrylic acid (IA).

18. A composition according to Claim 17, comprising indole acrylic acid (IA).

19. A composition according to Claim 17 or 18, comprising a delivery vehicle configured for oral administration and release of the composition in the ileum.

20. A composition according to any of Claims 17 to 19, further comprising tryptophan.

21. A composition according to any of Claims 17 to 19, further comprising a tryptophan intermediate selected from anthranilate and shikimate.

22. A composition according to any of Claims 17 to 19, further comprising tryptophan, anthranilate and shikimate.

23. A pharmaceutical composition comprising a composition of any of Claims 1 to 13 and 17 to 22 combined with a pharmaceutically acceptable carrier.

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