WO2025104216A1

WO2025104216A1 - Bacteriophage compositions

Info

Publication number: WO2025104216A1
Application number: PCT/EP2024/082444
Authority: WO
Inventors: Li Deng; Jinling XUE; Jinlong RU
Original assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Current assignee: Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
Priority date: 2023-11-15
Filing date: 2024-11-15
Publication date: 2025-05-22
Anticipated expiration: 2026-05-15

Abstract

The present invention relates to a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl- coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity, wherein said bacteriophage is capable of being propagated in (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells. The present invention also relates to nutritional compositions and medical compositions comprising such bacteriophages. The present invention further relates to such compositions for use in methods of treating an inflammatory or immune-mediated intestinal condition of a subject. The present invention further relates to methods for detecting a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity. Furthermore, the present invention relates to a kit comprising such compositions.

Description

Bacteriophage compositions

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority of EP Patent Application No. 23210199.8 filed 15 November 2023, the content of which is hereby incorporated by reference in its entirety for all purposes.

The present invention relates to a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyrylcoenzyme A (CoA):acetate CoA-transferase (BCoAT) activity, wherein said bacteriophage is capable of being propagated in (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells. The present invention also relates to nutritional compositions and medical compositions comprising such bacteriophages. The present invention further relates to such compositions for use in methods of treating an inflammatory or immune-mediated intestinal condition of a subject. The present invention further relates to methods for detecting a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity. Furthermore, the present invention relates to a kit comprising such compositions.

The human microbiome has a direct effect on clinical outcome in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). Besides bacteria, fungi and viruses as well as intestinal microbiota-derived metabolites are involved, but it is still unclear how dynamic shifts in these three kingdoms contribute to the production of intestinal metabolites, how metabolites are impacted by GvHD or antibiotics and whether they are associated with clinical outcome.

Allo-SCT is a curative treatment option for many hematological diseases. Its success is limited by severe morbidity and mortality associated with acute graft-versus-host disease (GvHD), still the most common complication (Zeiser et al., N Engl J Med (2017), 377(22): 2167-2179). In patients undergoing allo-SCT, the intestinal bacterial microbiome is altered by loss of diversity and domination of disease-related taxa (Holler et al., Biol Blood Marrow Transpl (2014), 20(5): 640-645; Taur et al., Clin Infect Dis (2012), 55(7): 905-914). Multiple factors contribute to this GvHDrelated microbial signature, such as use of antibiotics, administration of (radio)chemotherapy and nutrition (Weber et al., Clin Infect Dis (2018), 68(8): 1303-1310; Malard et al., Bone Marrow Transplant (2018), 53(12): 1493-1497). There is extensive evidence suggesting that alterations of bacterial communities strongly influence the pathophysiology of GvHD and affect outcome of patients undergoing allo-SCT (Pelet et al., N Eng J Med (2020), 982(9): 822-834; Shono et al., Sci Transl Med (2016), 8(339): 339ra71- 339ra71 ; Weber et al., Bio Blod Marrow Transplant (2017): 23(5): 845-852). Yet, the mediators of the bacteriome that promote these outcomes are poorly understood.

Microbiota-derived metabolites such as short-chain fatty acids (SCFAs), tryptophanderivatives such as indoles and secondary bile acids (BAs) have been shown to exert immunomodulatory effects (e.g. via modulation of regulatory T cell [T_regs] function or induction of Type I IFN [IFN- I] signaling) and tissue-homeostatic functions (e.g. via direct impact on intestinal epithelial cells) (Mathewson et al., Nat Immunol (2016): 17(5): 505-513; Swimm et al., Blood (2018), 132(23): 2506-2519). In particular, propionic and butyric acid, indole-3-carboxaldehyde (ICA) and deoxycholic acid (DCA) have been associated with protection against GvHD in mice and humans (Swim et al., loc cit). Additionally, alterations of bacteria together with metabolites, such as loss of specific anaerobic commensals and a decrease in intestinal SOFA levels, have been linked to GvHD severity in allo-SCT patients (Payen et al., Blood Adv (2020), 4(9): 1824- 1832).

Microbiota-derived metabolites have been analyzed in the blood of acute (Michomneau et al., Nat Commun (2019), 10(1): 5695) and chronic GvHD patients (Markey et al., Blood (2020), 136(1): 130-136). However, in humans, the majority of SCFA are metabolized on first pass (Peters et al., Gut (1992), 33(9): 1249-1252). Therefore, it is important to characterize not only systemic concentration but also intestinal levels, since numerous reports have demonstrated that metabolites act locally. For example: while only 2% of intestinal butyric acid becomes systemically available (Boets et al., J Physiol (2017), 595(2): 541-555), it is essential locally for maintaining of intestinal homeostasis where it fuels colonocytes, promotes mucus secretion and enhances expression of tight-junction proteins (Gaudier et al., Physiol Res (2009), doi: 10.33549/physiolres.931271 ; Wang et al., PNAS (2020), 117(21): 11648-11657.

The intestinal microbiome is comprised not only of bacteria, but also fungi, viruses, protozoa and archaea. Prokaryotic viruses have been described that encode auxiliary metabolite genes (AMG) and can modulate bacterial metabolism while imparting fitness benefits to themselves (Kieft et al., Nat Commun (2021), 12(1): 3503; Lindell et al., Nature (2005), 438(7064): 86-89). These bacteriophage infected bacteria, also named virocells, undergo phagedirected metabolism and exhibit distinct physiology compared to their uninfected peers (Howard- Varona et al., ISME J (2020), 14(4): 881-895; Zanela et al., Microbiome (2021), 9(1): 28). However, analyses of the human gut virome remain challenging due to the high degree of viralsequence diversity and continual discovery of novel viruses by metagenomics (Zanella et al., loc cit), making it difficult to find a promising treatment for inflammatory or immune- mediated intestinal conditions such as GVHD.

These and further disadvantages need to be overcome. The present invention therefore addresses these needs and technical objectives and provides a solution as described herein and as defined in the claims.

The present invention relates to a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyrylcoenzyme A (CoA):acetate CoA-transferase (BCoAT) activity, wherein said bacteriophage is capable of being propagated in (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

Preferably, said bacteriophage is propagated in (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells

As has been surprisingly found in context with the present invention, inflammatory or immune- mediated intestinal conditions such as GVHD which may occur in cancer patients undergoing allogenic stem cell therapy (allo-SCT) can be mitigated and may even prevented if such patients have in their gut bacteriophages comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA- transferase (BCoAT) activity, as well as bacteria in which such bacteriophages can be propagated (i.e. (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells). This has been found in course of several investigations and experiments which have been made in context with the present invention. In several patients undergoing allogenic stem cell therapy (allo-SCT) because of hematological malignancies, the intestinal bacteriome, fungome, virome was profiled and intestinal microbiota-derived metabolites from the start of the pre-transplantation conditioning regimen until day 28 after allo-SCT was examined. A serious medical condition that may arise with allo- SCT is graft versus host disease (GVHD). Although GVHD may affect any organ, intestinal GVHD is particularly important because of its frequency, severity and impact on the general condition of the patient. It is assumed that donor T cells are critical for the induction of GVHD, because depletion of T cells from bone marrow grafts effectively prevents GVHD but also results in an increase of leukaemia relapse. It has been shown that the gastrointestinal tract plays a major role in the amplification of systemic disease because gastrointestinal damage increases the translocation of endotoxins, which promotes further inflammation and additional gastrointestinal damage. It was found that patients who did either not suffer from intestinal GVHD or had mild symptoms had a higher abundance of microbial pathways involved in the biosynthesis of short-chain fatty acids (SCFA), specifically butyric acid via butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT). Strikingly, bacteriophages were identified that encoded BCoAT as an auxiliary metabolic gene (AMG). Such bacteriophages were more abundant in low-risk patients and were strongly correlated with intestinal butyric acid levels. More strikingly, it could be shown that transferring gut bacteria and such bacteriophages from a donor to a patient suffering from GHVD, particularly intestinal GVHD could recover from severe symptoms of GVHD. It is known that maintaining optimal butyrate levels improves gastrointestinal health in animal models by supporting colonocyte function, decreasing inflammation, maintaining the gut barrier, and promoting a healthy microbiome. Butyrate has also shown protective actions in the context of intestinal diseases such as inflammatory bowel disease, graft-versus-host disease of the gastrointestinal tract, and colon cancer, whereas lower levels of butyrate and/or the microbes which are responsible for producing this metabolite are associated with disease and poorer health outcomes. In sum, bacteriophages harboring a nucleic acid comprising a nucleotide sequence encoding butyric acid via butyrylcoenzyme A (CoA):acetate CoA-transferase (BCoAT) seem to be beneficial for butyric acid levels in the intestinal tract.

Butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) represents one of at least two mechanisms for reclaiming CoA from butyryl-CoA at the end of butyrate biosynthesis (a process performed by some colonic bacteria), namely transfer of CoA to acetate. Butanoate, acetoacetate and any of their CoA thioesters are the preferred substrates of BCoAT, but the enzyme also acts, more slowly, on the derivatives of a number of C2 to C6 monocarboxylic acids. In accordance with the present invention, BCoAT is also referenced to under EC number EC2.8.3.9. That is, in context with the present invention, a polypeptide (or fragment thereof) having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity means that such polypeptide (or fragment thereof) is capable of catalyzing the transfer of CoA to acetate. In one embodiment of the present invention, a polypeptide (or fragment thereof) having butyryl- coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity is capable of catalyzing the transfer of CoA to acetate, wherein butanoate, acetoacetate, and/or one of their CoA thioesters is/are used as substrate(s). In one embodiment of the present invention, said bacteriophage may comprise a nucleic acid having a nucleotide sequence selected from the group consisting of:

(a1) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 1 having BCoAT activity;

(b1) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1 , said fragment having BCoAT activity;

(c1) a nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a1) or (b1) and which encodes a polypeptide having BCoAT activity; and (d1) a nucleotide sequence complementary to the nucleotide sequences of any one of (a1) to (c1).

In this context, and in accordance with the present invention, where said bacteriophage comprises a nucleic acid having a nucleotide sequence according to (c1) as described herein, said nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a1) or (b1) and which encodes a polypeptide having BCoAT activity deviates from the nucleotide sequence of (a1) or (b1) preferably in a way that the majority or all of the deviations are silent mutations or substitutions, conservative mutations or substitutions, or highly conservative mutations or substitutions vis-a-vis the encoded polypeptide.

In a specific embodiment of the present invention, particularly where said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of (a1), (b1), (c1) and (d1) as described herein, said bacteriophage may be capable of being propagated in (Pseudo) Flavonifractor sp. cells, preferably, said bacteriophage ispropagated in (Pseudo)Flavonifractor sp. cells.

By “fragment” in reference to a polypeptide as described herein is meant any amino acid sequence present in a polypeptide as described herein, as long as it is shorter than the full length sequence and as long as it has BCoAT activity as described herein. Preferred fragments have at least about 20, 40, or 60 amino acids. Such preferred fragments have BCoAT activity as described herein.

In accordance with the present invention, as used herein in context with amino acid sequences, the term “similar” means that a given amino acid sequence comprises identical amino acids or only conservative or highly conservative substitutions compared to the amino acid sequence of the respective SEQ ID NO. As used herein, “conservative” substitutions mean substitutions as listed as “Exemplary Substitutions” in Table I herein. “Highly conservative” substitutions as used herein mean substitutions as shown under the heading “Preferred Substitutions” in Table I herein.

TABLE I Amino Acid Substitutions

As used herein, “silent” mutations or substitutions mean base substitutions within a nucleic acid sequence which do not change the amino acid sequence encoded by the nucleic acid sequence. “Conservative” mutations or substitutions mean substitutions vis-a-vis the encoded polypeptide as listed as “Exemplary Substitutions” in Table I. “Highly conservative” substitutions as used herein mean substitutions vis-a-vis the encoded polypeptide as shown under the heading “Preferred Substitutions” in Table I.

The term "position" when used in accordance with the present invention means the position of an amino acid within an amino acid sequence depicted herein. The term "corresponding" in this context also includes that a position is not only determined by the number of the preceding nucleotides/amino acids.

The level of identity between two or more sequences (e.g., nucleic acid sequences or amino acid sequences) can be easily determined by methods known in the art, e.g., by BLAST analysis. Generally, in context with the present invention, if two sequences (e.g., polynucleotide sequences or amino acid sequences) to be compared by, e.g., sequence comparisons differ in identity, then the term "identity" may refer to the shorter sequence and that part of the longer sequence that matches said shorter sequence. Therefore, when the sequences which are compared do not have the same length, the degree of identity may preferably either refer to the percentage of nucleotide residues in the shorter sequence which are identical to nucleotide residues in the longer sequence or to the percentage of nucleotides in the longer sequence which are identical to nucleotide sequence in the shorter sequence. In this context, the skilled person is readily in the position to determine that part of a longer sequence that matches the shorter sequence. Furthermore, as used herein, identity levels of nucleic acid sequences or amino acid sequences may refer to the entire length of the respective sequence and is preferably assessed pair-wise, wherein each gap is to be counted as one mismatch. These definitions for sequence comparisons (e.g., establishment of "identity" values) are to be applied for all sequences described and disclosed herein.

Moreover, the term “identity” as used herein means that there is a functional and/or structural equivalence between the corresponding sequences. Nucleic acid/amino acid sequences having the given identity levels to the herein-described particular nucleic acid/amino acid sequences may represent derivatives/variants of these sequences which, preferably, have the same biological function. They may be either naturally occurring variations, for instance sequences from other varieties, species, etc., or mutations, and said mutations may have formed naturally or may have been produced by deliberate mutagenesis. Furthermore, the variations may be synthetically produced sequences. The variants may be naturally occurring variants or synthetically produced variants or variants produced by recombinant DNA techniques.

“Deviations” from sequences (e.g., amino acid or nucleic acid sequences) as used herein may comprise, e.g., deletions, substitutions, additions, insertion and/or recombination. The term "addition" refers to adding a nucleic acid residue/amino acid to the end or beginning of the given sequence, whereas "insertion" refers to inserting a nucleic acid residue/amino acid within a given sequence. The term "deletion" refers to deleting or removal of a nucleic acid residue or amino acid residue in a given sequence. The term "substitution" refers to the replacement of a nucleic acid residue/amino acid residue in a given sequence. Again, these definitions as used here apply, mutatis mutandis, for all sequences provided and described herein unless specified otherwise.

As used herein, unless specifically defined otherwise, the term “nucleic acid” or “nucleic acid molecule” is used synonymously with “oligonucleotide”, “nucleic acid strand”, or the like, and means a polymer comprising one, two, or more nucleotides, arranged in single- or double stranded nucleotide chains.

Generally, as used herein, the terms ..polynucleotide", ..nucleic acid" and ..nucleic acid molecule" are to be construed synonymously. Generally, nucleic acid molecules may comprise inter alia DNA molecules, RNA molecules, oligonucleotide thiophosphates, substituted ribooligonucleotides or PNA molecules. Furthermore, the term "nucleic acid molecule" may refer to DNA or RNA or hybrids thereof or any modification thereof that is known in the art (see, e.g., US 5525711 , US 471 1955, US 5792608 or EP 302175 for examples of modifications). The polynucleotide sequence may be single- or double- stranded, linear or circular, natural or synthetic, and without any size limitation. For instance, the polynucleotide sequence may be genomic DNA, cDNA, mitochondrial DNA, mRNA, antisense RNA, ribozymal RNA or a DNA encoding such RNAs or chimeroplasts (Gamper, Nucleic Acids Research, 2000, 28, 4332 - 4339). Said polynucleotide sequence may be in the form of a vector, plasmid, cosmid, or of viral DNA or RNA. Also described herein are nucleic acid molecules which are complementary to the nucleic acid molecules described above and nucleic acid molecules which are able to hybridize to nucleic acid molecules described herein. A nucleic acid molecule described herein may also be a fragment of the nucleic acid molecules in context of the present invention. Particularly, such a fragment is a functional fragment. Examples for such functional fragments are nucleic acid molecules which can serve as primers.

In another embodiment of the present invention, said bacteriophage may comprise a nucleic acid having a nucleotide sequence selected from the group consisting of:

(a2) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 having BCoAT activity;

(b2) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 2, said fragment having BCoAT activity;

(c2) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a2) or (b2) and which encodes a polypeptide having BCoAT activity; and

(d2) a nucleotide sequence complementary to the nucleotide sequences of any one of (a2) to (c2).

In this context, and in accordance with the present invention, where said bacteriophage comprises a nucleic acid having a nucleotide sequence according to (c2) as described herein, said nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a2) or (b2) and which encodes a polypeptide having BCoAT activity deviates from the nucleotide sequence of (a2) or (b2) preferably in a way that the majority or all of the deviations are silent mutations or substitutions, conservative mutations or substitutions, or highly conservative mutations or substitutions vis-a-vis the encoded polypeptide.

In a specific embodiment of the present invention, particularly where said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of (a2), (b2), (c2) and (d2) as described herein, said bacteriophage may be capable of being propagated in Lawsonibacter sp. cells, preferably, said bacteriophage is propagated in Lawsonibacter sp. cells.

(a3) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 3 having BCoAT activity;

(b3) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 3, said fragment having BCoAT activity;

(c3) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a3) or (b3) and which encodes a polypeptide having BCoAT activity; and

(d3) a nucleotide sequence complementary to the nucleotide sequences of any one of (a3) to (c3).

In this context, and in accordance with the present invention, where said bacteriophage comprises a nucleic acid having a nucleotide sequence according to (c3) as described herein, said nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a3) or (b3) and which encodes a polypeptide having BCoAT activity deviates from the nucleotide sequence of (a3) or (b3) preferably in a way that the majority or all of the deviations are silent mutations or substitutions, conservative mutations or substitutions, or highly conservative mutations or substitutions vis-a-vis the encoded polypeptide.

In a specific embodiment of the present invention, particularly where said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of (a3), (b3), (c3) and (d3) as described herein, said bacteriophage may be capable of being propagated in Mycobacterium sp. cells, preferably said bacteriophage is propagated in Mycobacterium sp. cells.

In another embodiment of the present invention, said bacteriophage may comprise a nucleic acid having a nucleotide sequence selected from the group consisting of: (a4) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 having BCoAT activity;

(b4) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 4, said fragment having BCoAT activity;

(c4) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a4) or (b4) and which encodes a polypeptide having BCoAT activity; and

(d4) a nucleotide sequence complementary to the nucleotide sequences of any one of (a4) to (c4).

In this context, and in accordance with the present invention, where said bacteriophage comprises a nucleic acid having a nucleotide sequence according to (c4) as described herein, said nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a4) or (b4) and which encodes a polypeptide having BCoAT activity deviates from the nucleotide sequence of (a4) or (b4) preferably in a way that the majority or all of the deviations are silent mutations or substitutions, conservative mutations or substitutions, or highly conservative mutations or substitutions vis-a-vis the encoded polypeptide.

In a specific embodiment of the present invention, particularly where said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of (a4), (b4), (c4) and (d4) as described herein, said bacteriophage may be capable of being propagated in Megasphaera sp. cells, preferably, said bacteriophage is propagated in Megasphaera sp. cells.

(a5) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 5 having BCoAT activity;

(b5) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 5, said fragment having BCoAT activity;

(c5) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a5) or (b5) and which encodes a polypeptide having BCoAT activity; and

(d5) a nucleotide sequence complementary to the nucleotide sequences of any one of (a5) to (c5).

In this context, and in accordance with the present invention, where said bacteriophage comprises a nucleic acid having a nucleotide sequence according to (c5) as described herein, said nucleotide sequence which is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the nucleotide sequence of (a5) or (b5) and which encodes a polypeptide having BCoAT activity deviates from the nucleotide sequence of (a5) or (b5) preferably in a way that the majority or all of the deviations are silent mutations or substitutions, conservative mutations or substitutions, or highly conservative mutations or substitutions vis-a-vis the encoded polypeptide.

In a specific embodiment of the present invention, particularly where said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of (a5), (b5), (c5) and (d5) as described herein, said bacteriophage may be capable of being propagated in Alistipes sp. cells, preferably, said bacteriophage is propagated in Alistipes sp. cells.

In one embodiment of the present invention, expression of said nucleotide sequences may be effected by an endogenous promoter of said bacteriophage. Said endogenous promoter has preferably sequences which are recognized by the prokaryotic transcription machinery, e.g. a -35 box and/or a -10 box and a sequence with provides for transcription initiation]. Preferably, such promoter is recognized in and allow expression of said nucleotide sequence in procaryotic cells such as, e.g., (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

The term “promoter” as used throughout this document, refers to a nucleic acid sequence needed for gene sequence expression. Promoter regions vary from organism to organism, but are well known to those skilled in the art for different organisms. For example, in prokaryotes, the promoter region contains both the promoter (which directs the initiation of RNA transcription) as well as the DNA sequences which, when transcribed into RNA, will signal synthesis initiation. Such regions will normally include those 5'-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and the like. Both constitutive and inducible promoters can be used in the present invention, in accordance with the needs of a particular embodiment. A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to cistron DNA encoding a polypeptide described herein by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of choice. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of a selected nucleic acid sequence. The promoter can also be a recombinant promoter. The word “recombinant” is used in this document to describe a nucleic acid molecule that, by virtue of its origin, manipulation, or both is not associated with all or a portion of the nucleic acid molecule with which it is associated in nature. Generally a recombinant nucleic acid molecule includes a sequence which does not naturally occur in the respective wildtype organism or cell. Typically a recombinant nucleic acid molecule is obtained by genetic engineering, usually constructed outside of a cell. Generally a recombinant nucleic acid molecule is at least substantially identical and/or substantial complementary to at least a portion of the corresponding nucleic acid molecule occurring in nature. A recombinant nucleic acid molecule may be of any origin, such as genomic, cDNA, mammalian, bacterial, viral, semisynthetic or synthetic origin.

In one embodiment of the present invention, the composition described and provided herein may be a nutritional supplement. In a more specific embodiment of the present invention, such nutritional supplement may be in form of a, e.g., capsule, syrup, suspension or sustained release formulation. A nutritional supplement may contain a filler, tonicity modifier, and/or a buffering agent.

In one embodiment of the present invention, the composition described and provided herein may be a medical device. In a more specific embodiment of the present invention, such medical device may be in the form of a suppository.

Unless specified otherwise herein, the term “medical device” can be used synonymously with the term “pharmaceutical composition”. The present invention thus also relates to a pharmaceutical composition comprising the bacteriophages as defined herein. Thus, such composition of the invention can also be a medical device or pharmaceutical composition. Such medical device or pharmaceutical composition may further comprise one or more of pharmaceutically acceptable ingredients (such as at least one excipient). If at least one excipient is further comprised by the medical device or pharmaceutical composition, such excipient refers to at least one pharmaceutically acceptable excipient. Suitable pharmaceutical excipients are further described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. Said pharmaceutically acceptable excipient includes any excipient that does not itself elicit an adverse reaction harmful to the subject receiving the pharmaceutical composition. If the composition of the invention additionally comprises at least one pharmaceutically acceptable excipient, said composition refers to a pharmaceutical composition. Said pharmaceutical composition is thus used herein for therapeutic purposes. Moreover, the present invention relates to the use of said composition as disclosed herein for the preparation of a pharmaceutical composition.

In accordance with the present invention, the term “medical device” or "pharmaceutical composition" relates to a composition for administration to a subject as defined herein, preferably a human. Medical devices, pharmaceutical compositions or formulations are usually in such a form as to allow the biological activity of the active ingredient to be effective and may therefore be administered to a subject for therapeutic use as described herein. The medical device or pharmaceutical composition can be administered in a therapeutically effective amount by, e.g., inhalation, injection, infusion, rectally (preferred), or orally. The administration of said medical device or pharmaceutical composition may be performed rectally (preferred), via enema, through fecal microbiome transplantation (or, as is interchangeably used herein, fecal microbiota transplantation), intraperitoneally, intravenously, intraarterially, subcutaneously, intramuscularly, parenterally, transdermally, intraluminally, intrathecally, and/or intranasally. The medical device or pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

Suitable excipients are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and lipid aggregates such as e.g. oil droplets or liposomes. The excipient used in combination with the (pharmaceutical) composition of the present invention may be waterbased and forms an aqueous solution. An oil-based excipient solution is an alternative to the aqueous excipient solution.

Also, the medical device or pharmaceutical composition as defined herein may further comprise one or more adjuvants. The term "adjuvant" is used according to its well-known meaning in connection with medical devices or pharmaceutical compositions. Specifically, an adjuvant is an immunological agent that modifies, preferably enhances, the effect of such composition while having few, if any, desired immunogenic effects on the immune system when given per se. Suitable adjuvants can be inorganic adjuvants such as, e.g., aluminium salts (e.g., aluminium phosphate, aluminium hydroxide), monophosphoryl lipid A, or organic adjuvants such as squalene or oil-based adjuvants, as well as virosomes.

In one embodiment of the present invention, the composition described and provided herein may further comprise bacterial cells which are capable of being infected by the bacteriophages as defined herein. Additionally or alternatively, such bacterial cells may already be infected by the bacteriophages as defined herein. In a more specific embodiment of the present invention, such bacterial cells which are capable of being infected by the bacteriophages as defined herein may be, e.g., colonic bacterial cells. In an even more specific embodiment of the present invention, such bacterial cells which are capable of being infected by the bacteriophages as defined herein may be, e.g., bacterial cells from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

Methods for producing phages are known in the art and described in, e.g.,WO2012/048257.

A “composition” comprising the phages which have been isolated or produced as known in the art (see also WO2012/048257), refers to any kind of composition which comprises these isolated or produced phages. Said composition can be in the form of capsule, syrup, suspension or sustained release formulation. It may be a liquid (preferably aqueous), a solid, a gel, a powder, a paste, an ointment, a capsule, a nutritional composition, etc. Further comprised herein is a dried or frozen form of the composition as defined herein. Thus, said composition may be stored directly in liquid form for later use, stored in a frozen state and thawed prior to use, or prepared in dried form, such as a lyophilized, air-dried, or spray-dried form, for later reconstitution into a liquid form or other form prior to use. Thus, it is envisaged that a composition described herein may be stored by any method known to one of skill in the art. Non-limiting examples include cooling, freezing, lyophilizing, and spray drying the formulation, wherein storage by cooling is preferred.

The composition of the invention comprising said isolated or produced phages may further comprise one or more ingredients, by the way of illustration and not limitation, such as, e.g., an excipient, a preservative, an ingestible support, a flavour, a solubilizer, a wetting agent, a sweetener, a colorant, a pharmaceutically acceptable carrier, a coating agent, or an antioxidant. The excipients of the composition may refer to diluents such as, e.g. water, saline, glycerol, ethanol, bacteriostatic water for injection (BWFI), Ringer's solution, dextrose solution, or aqueous solutions of salts and/or buffers etc. Thus, the composition of the present invention may further comprise at least one excipient as defined herein, preferably a buffer (pH buffering agent) as excipient. Furthermore, substances necessary for formulation purposes may be comprised in said composition as acceptable excipients such as emulsifying agents, stabilizing agent, and/or surfactants known to a person skilled in the art.

The term “buffer” or “pH buffering agent” as used herein, includes those agents that maintain the pH in a desired range. A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base or a weak base and its conjugated acid. It has the property that the pH of the solution changes very little when a small amount of a strong acid or base is added. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications. A buffer when applied in the composition of the invention preferably stabilizes the isolated phages. Preferably, as a buffer being further comprised by the composition of the present invention PBS or sodium bicarbonate buffer is used.

In this context, as used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In the context of the present invention, the phages which are isolated may refer to the substance and/or entity separated from samples such as sewage water samples by applying at least any one of the defined hooks which have been explicitly designed I prepared by the inventors as explained above and in the Example section in more detail. Such “targeted phage isolation” (“hook isolation”) refers to applying viral tagging as it is disclosed in WO2021/048257, followed by phage isolation methods as defined elsewhere herein and qPCR, where the already designed hook(s) are used as primers. In the context of the present invention, for the isolation of said labeled phages (after said viral tagging with a label) from a sample, suitable bacterial target cells (e.g., (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells) may be applied, whereby such labeled phages get into contact with said target cells by binding to receptors on the surface.

Utilizing metagenomic sequencing in selected Immuno-modulatory Metabolite Risk Index low- risk patients, in accordance with this invention, the microbial pathways associated with the production of intestinal metabolites was decoded, in particular butyric acid via butyryl- CoA:acetate CoA-transferase (BCoAT). In the viral metagenome, BCoAT-coding bacteriophage contigs were detected, highlighting the potential cooperation of bacteria and their bacteriophages associated with the production of protective metabolites. Intestinal Immuno-modulatory Metabolite levels were critically impacted by the onset of GVHD but could be modulated by fecal microbiota transplantation (FMT) as described herein.

Accordingly, in one embodiment of the present invention, the composition described and provided herein may be for use in a method of increasing the amount of butyrate in the intestinal tract of a subject.

In one embodiment of the present invention, the composition described and provided herein may be for use in a method of promoting gut health of a subject.

In one embodiment of the present invention, the composition described and provided herein may be for use in a method of treating an inflammatory or immune-mediated intestinal condition of a subject. In a more specific embodiment of the present invention, said inflammatory intestinal condition may be inflammatory bowel disease. In another more specific embodiment of the present invention, said immune-mediated intestinal condition may be graft-versus-host- disease (GVHD) or colon cancer.

In another embodiment of the present invention, where the composition described and provided herein may be for use in a method of

• increasing the amount of butyrate in the intestinal tract of a subject,

• promoting gut health of a subject, and/or

• treating an inflammatory or immune-mediated intestinal condition of a subject, said composition may be administered orally, rectally, via enema or through fecal microbiome transplantation to said subject. Fecal microbiome transplantation may preferably be done by fecal microbiome transplant capsules. In this context, in a more specific embodiment of the present invention, said composition may be in the form of, e.g., a capsule, syrup, suspension, sustained release formulation or suppository.

In context with the present invention, where the composition described and provided herein may be for use in a method of

• increasing the amount of butyrate in the intestinal tract of a subject,

• promoting gut health of a subject, and/or

• treating an inflammatory or immune-mediated intestinal condition of a subject, said subject is preferably a human subject.

In one embodiment of the present invention, where the composition described and provided herein may be for use in a method of

• increasing the amount of butyrate in the intestinal tract of a subject,

• promoting gut health of a subject, and/or

• treating an inflammatory or immune-mediated intestinal condition of a subject, a nucleotide sequence (encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity) as defined herein is not detected in the intestinal microbiome of said subject.

Accordingly, in one embodiment of the present invention, the present invention provides a method for increasing the amount of butyrate in the intestinal tract of a subject, comprising administering the composition described and provided herein to a subject, wherein administering increases the amount of butyrate in the intestinal tract of a subject. In one embodiment of the present invention, there is provided a method of promoting gut health of a subject, comprising administering the composition described and provided herein to a subject, wherein administering promotes gut health of a subject.

In one embodiment of the present invention, there is provided a method of treatment of an inflammatory or immune-mediated intestinal condition of a subject, comprising administering the composition described and provided herein, wherein administering treats an inflammatory or immune-mediated intestinal condition of a subject.

Accordingly, in one embodiment of the present invention, the present invention provides a use of the composition described and provided herein for the manufacture of a medicament for increasing the amount of butyrate in the intestinal tract of a subject.

In one embodiment of the present invention, the present invention provides a use of the composition described and provided herein for the manufacture of a medicament for promoting gut health of a subject.

In one embodiment of the present invention, the present invention provides a use of the composition described and provided herein for the manufacture of a medicament for the treatment of an inflammatory or immune-mediated intestinal condition of a subject.

In a more specific embodiment of the present invention, said inflammatory intestinal condition may be inflammatory bowel disease. In another more specific embodiment of the present invention, said immune-mediated intestinal condition may be graft-versus-host-disease (GVHD) or colon cancer.

Said composition may be administered orally, rectally, via enema or through fecal microbiome transplantation to said subject. Fecal microbiome transplantation may preferably be done by fecal microbiome transplant capsules. In this context, in a more specific embodiment of the present invention, said composition may be in the form of, e.g., a capsule, syrup, suspension, sustained release formulation or suppository.

Said subject is preferably a human subject. Preferably, in the intestinal microbiome of said subject a nucleotide sequence (encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity) as defined herein is not detected. The present invention also relates to a method for detecting a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyrylcoenzyme A (CoA):acetate CoA-transferase (BCoAT) activity, comprising

(A) isolating bacteriophages from a sample obtained from a subject; and

(B) determining whether said bacteriophages comprise a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity.

Methods for isolating and producing phages are known in the art and described in, e.g.,WO2012/048257

In one embodiment of the present invention, relating to the method for detecting a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity as described herein, a nucleotide sequence as defined herein is used as a probe in step (B).

The present invention also relates to a kit comprising a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity as described and provided herein, and bacterial cells which are capable of being infected by the bacteriophages as defined herein.

The present invention also relates to a kit comprising the composition comprising the bacteriophages described herein and bacterial cells which are capable of being infected by the bacteriophages as defined herein. Thus, when a kit comprises said composition, said composition may be provided in a vial or a container, preferably also comprising in said vial or container at least one excipient as defined herein. Further, said kit may be associated with a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration or diagnostics. Said kit may comprise the composition, preferably in a vial or container, in dried form, such as a lyophilized, air-dried, or spray-dried form (in form of a powder), for later reconstitution into a liquid form or other form prior to use. Further, said kit may also comprise the composition, preferably in a vial or container, in a frozen state, being thawed prior to use. Further, said kit may also comprise the composition, preferably in a vial or container, in liquid state. In one embodiment of the present invention, relating to the kit comprising a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity as described and provided herein, said bacterial cells may be colonic bacterial cells. In a specific embodiment of the present invention, said bacterial cells may be from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

The present invention further provides a method comprising a step of infecting a colonic bacterial cell with a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity to form an infected colonic bacterial cell that expresses said polypeptide.

Said method may be carried out in vivo or in vitro.

Also, the present invention provides for a use of a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity for infecting colonic bacterial cell to form an infected colonic bacterial cell that expresses said polypeptide.

Said nucleic acid molecule is preferably one as described herein.

Said use may be carried out in vivo or in vitro.

Further, the present invention provides for a use of a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity for infecting colonic bacterial cell to form an infected colonic bacterial cell that expresses said polypeptide. Said nucleic acid molecule is preferably one as described herein.

Said use may be carried out in vivo or in vitro.

The embodiments which characterize the present invention are described herein, illustrated in the Examples, and reflected in the claims.

It must be noted that as used herein, the singular forms “a”, “an”, and “the”, include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents and reference to “the method” includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein. Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The term "and/or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% or 2% of a given value or range, and also comprise the respective exact numeric value.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term “comprising” can be substituted with the term “containing” or “including” or sometimes when used herein with the term “having”.

When used herein “consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

When used herein, the term “essentially”, e.g., “essentially free of” or “essentially non-[...]” or the like, the term also includes the absolute condition, i.e. “free of”, “non-[...]” or the like, respectively.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

The present invention refers to the following polypeptide sequences:

SEQ ID NO: 1

MDDTNDVHVIAQLDNFISINNAVDVDLFGQVNAESAGLKHISGTGGQLDFVLGAYLSKGG KSFICLSSTMTGKDRQMKSRIVPTLTNGSITTDPRSAVHYLVTEYGMVNLKGASTWERAE KIISIAHPDFREELIAQAEKIGIWRKSNR

SEQ ID NO: 2

MDYQAMYQQKLTTPEEAVKWKSGDWVDYTWCTNHPVALDKALAARKDELTDVKIRGGVT

MWMPEIAKADDAGDHFTWHSWHCSGIDRKIMKKGMGYFSPMRYSELPRFYRENLAPVDV VMLQVSPMDAHGNFNFGLAASHLADMMARAKCIIVEVNQNMPWVYGLTGTEINIQDVDFVV EGDNPPVAQLGGGGEPTDVDRAVANLVVPEIPNGACLQLGIGGMPNTIGAMIAQSDLKDL SVHTEMYVDGFVDIAAAGKITGRHKNLDKGRQVYAFAAGTQKLYDYVNRNPDVMAAPVDY TNDVRVIGQIDNFISINNAVDLDLFGQVNAESAGLKHISGTGGQLDFVMGAYLSKGGKSF ICLSSTVTGKDGTMKSRIVPTLTNGSICTDPRSCVHYIVTEYGMVNLKGLSTWERAEALI SIAHPDFREQLIADAEKMGIWRRSNK

SEQ ID NO: 3

MPLELTAEQAAARLNPVDTLGIPLGPGQPPAFLRALGVRKDWTDLRVYGALLAVGTELFS RPGVHYLSGFFGPLERALRDMGADIEFAAADFRRFGPLLERQSPRVMTTVATPPDPDGWC SLSLHAGGTIGELRRAGADPARLLIVEVSEAYPRTFGVGEQHRHALHVDEIDVLVSSTDA PLALPGGDAAPSDVDRAIARHAVSFIGPGATLQTGIGAIPNQIATLLAEGEGGGYGLHSE MFTDGCMKLHRAGKVTNTGKGQYDGVSVTTFAFGSPDLYAWLDGNADVAFLPVEIVNAPE VIGANNDMISINGALSLDIQGQVVADTINGGQFSGIGGAEDFVAGAGLELSDRSLICLPS TFEKGGALQSRIVPWFGPGAVITTPRHQVDVVITEYGAAELEGRTVRERGEALAAVAHPQ FRDALRAAAARAANGRSPVS

SEQ ID NO: 4

MSQWTDMYKQKLMTPEEAVKVVKSGDWVDYGMGTTQPVLLDQALAARRDELQDVKVRM CL

SVAPRQIIEQDPDRKAFTAMNWHMGGYDRKKCALGQMNFIPMCYRNKPSMYRDLLDVDVA LITVGPMDKHGFFNFGLAVSATEAITQKAKKVIVEVNEAMPRVLGGRGECIHISDIDGIV

EYGNHPLATIPFATGDDIDATIAKMIVEQVPNGATLQLGIGSLPNTIGALLSESDLKDLG

VHTEMLVDAFYLMYKNGQLTNRRKAFCPNKVSWSFALGTQDLYDWMDDNPYLAAYPVDVI

NDPFVIAQMDNFISINNCIEVDLFGQVSSESSGTQQISGSGGQLDFTDGAYRSPGGKSII ALRSTFHNKKTGRDESRILPTLAPGTTVTDPRSQINFVVTEYGMVNLMGASTWERAERLI

SIAHPDFREDLIKEAEKMKIWRYSNKK

SEQ ID NO: 5

MINYTTAAEAAKLIKSNDSVYIQGSVSIPEVLVKAMADRGHELRGVKVYSAFAIGREEAP YCKPEYKDSFLVYSLFVSNSVRN Wl AQGYGQAI PAFLGEI PGLFRKGI I PI DVALLNCSR

PNADGYCSFGTSADLAVSAAECAKWIAQINPHVPFSYGDALIHVSKLTAAVEVDEPLVE

LPTAQPSEIDRKIGGYIAELIPDGATLQIGVGGIPNAVLAALGDHKHLGLHTEALTDGVV

PLIRSGVIDNSQKKVLPGKNLASLALGSKRLYEYMDYNEDLIMKDVAWTNDPFRIRENPK

VMAINSALEVDLTGQICADSIGTMIYSSVGGQHDFMYGGALSEGGKTFIALPSTTSKGQS KIKALLTPGAGVVTTRFQTQYVVTEYGAAYLLGKGLAERARALIDIAHPSAREELEKAAC

ERFGYSFLRLK

The present invention may also be characterized by the following items:

1. A composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity, wherein said bacteriophage is capable of being propagated in (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

2. The composition of item 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

(b1 ) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1, said fragment having BCoAT activity;

(c1) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a1) or (b1) and which encodes a polypeptide having BCoAT activity; and

(d1) a nucleotide sequence complementary to the nucleotide sequences of any one of (a1) to (c1).

3. The composition of item 1 or 2, wherein said bacteriophage is capable of being propagated in (Pseudo) Flavonifractor sp. cells.

4. The composition of item 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

5. The composition of item 1 or 4, wherein said bacteriophage is capable of being propagated in Lawsonibacter sp. cells. The composition of item 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

(d3) a nucleotide sequence complementary to the nucleotide sequences of any one of (a3) to (c3). The composition of item 1 or 6, wherein said bacteriophage is capable of being propagated in Mycobacterium sp. cells. The composition of item 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

(a4) a nucleotide sequence encoding a polypeptide having the amino acid sequence shown in SEQ ID NO: 4 having BCoAT activity;

(d4) a nucleotide sequence complementary to the nucleotide sequences of any one of (a4) to (c4). The composition of item 1 or 8, wherein said bacteriophage is capable of being propagated in Megasphaera sp. cells. The composition of item 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

(b5) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 5, said fragment having BCoAT activity; (c5) a nucleotide sequence which is at least 70% identical to the nucleotide sequence of (a5) or (b5) and which encodes a polypeptide having BCoAT activity; and

11. The composition of item 1 or 10, wherein said bacteriophage is capable of being propagated in Alistipes sp. cells.

12. The composition of any one of items 1 to 12, wherein expression of said nucleotide sequences is effected by an endogenous promoter of said bacteriophage.

13. The composition of any one of items 1 to 12 which is a nutritional supplement.

14. The composition of item 13, wherein said nutritional supplement is in the form of a capsule, syrup, suspension or sustained release formulation.

15. The composition of any one of items 1 to 12 which is a medical device.

16. The composition of item 15, wherein said medical device is in the form of a suppository.

17. The composition of any one of the preceding items, further comprising bacterial cells which are capable of being infected by the bacteriophage as defined in any one of items 1 to 12.

18. The composition of item 17, wherein said bacterial cells are colonic bacterial cells.

19. The composition of item 17 or 18, wherein said bacterial cells are from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

20. The composition of any one of items 1 to 19 for use in a method of increasing the amount of butyrate in the intestinal tract of a subject.

21. The composition of any one of items 1 to 19 for use in a method of promoting gut health of a subject. 22. The composition of any one of items 1 to 19 for use in a method of treating an inflammatory or immune-mediated intestinal condition of a subject.

23. The composition for the use of item 22, wherein said inflammatory intestinal condition is inflammatory bowel disease.

24. The composition for the use of item 22, wherein said immune-mediated intestinal condition is graft-versus-host-disease or colon cancer.

25. The composition for the use of any one of items 20 to 24, wherein said composition is administered orally, rectally, via enema or through fecal microbiome transplantation to said subject.

26. The composition for the use of item 25, wherein said composition is in the form of a capsule, syrup, suspension, sustained release formulation or suppository.

27. The composition for the use of any one of items 20 to 26, wherein said subject is a human subject.

28. The composition for the use of any one of items 20 to 27, wherein in the intestinal microbiome of said subject a nucleotide sequence as defined in any one of items 1 to 11 is not detected.

29. A method for detecting a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA): acetate CoA-transferase (BCoAT) activity, comprising

(A) isolating bacteriophages from a sample obtained from a subject; and

(B) determining whether said bacteriophages comprise a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA): acetate CoA- transferase (BCoAT) activity.

30. The method of item 29, wherein in step (B) a nucleotide sequence as defined in any one of items 1 to 12 is used as a probe.

31 . A kit comprising a composition of any one of items 1 to 19 and bacterial cells which are capable of being infected by the bacteriophage as defined in any one of items 1 to 12. The kit of item 31 , wherein said bacterial cells are colonic bacterial cells. The kit of item 31 or 32, wherein said bacterial cells are from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells. A method comprising a step of infecting a colonic bacterial cell with a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity to form an infected colonic bacterial cell that expresses said polypeptide. Use of a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA- transferase (BCoAT) activity for infecting colonic bacterial cell to form an infected colonic bacterial cell that expresses said polypeptide. 1 Examples

The butyric acid synthesis gene butyryl-CoA:acetate CoA-transferase was encoded in bacteriophage genomes as an auxiliary metabolic gene

The BCoAT gene was searched in the viral metagenome. This dataset was obtained separately from the bacterial metagenome, following isolation of virus-like particles from stool samples (see “Extraction of viral nucleid acid”). Strikingly, detected whole length (1.34 kb) and a 450 bp fragment of BCoAT was detected in two individual bacteriophage contigs with almost complete phage genome length: VC-2 (90,756 bp) and VC-1 (77,772 bp). Phylogenetic analysis showed a close evolutionary relationship of the bacteriophage BCoAT to that of Oscillospiraceae, indicating that the gene may have been acquired from this bacterial family. In addition, discovered BCoAT was discovered in Myoviridae sp. (GenBank ID: DAV55430.1) identified in another cohort of human metagenomic samples including those from the Human Microbiome Project (Tisza et al., PNAS (2021), 118(23) :e2023202118), indicating that BCoAT- coding phages identified in this study are unlikely to be an assembly artefact. Patients which harbored VC-1 and VC-2 had significantly elevated Factor 1 and 3 values VC-2 and VC-1 were significantly more abundant and more likely to be present in IMM-RI low- vs high-risk patients.

Despite their low relative abundance, VC-2 and VC-1 were detected in more than 60% and 80% of patients across both centers at Day -7, respectively, but detection declined progressively during the peri-engraftment period. Exposure to antibiotics resulted in a significant decrease of the relative abundance of VC-2 and VC-1 . Both viral contigs exhibited a significant Spearman’s correlation with intestinal butyric acid levels. By comparing the bacteriophage BCoAT sequence against bacterial reference sequences, it was observed that it aligned most closely to members of the Oscillospiracaea family. In aligning the genomes of VC-2 and Lawsonibacter, a close match in sequence identity was observed for BCoAT and the upstream-located DEAD box helicase, which in bacteria are involved in ribosome biogenesis, RNA turnover and translation initiation43. Meanwhile, VC-1 encoded only a part of the BCoAT gene with a sequence most identical to that of Pseudoflavonifractor, another species within the Oscillospiracaea family.

VC-1 and VC-2 were characterized as temperate bacteriophages, evident by the presence of genes for integration, excision and packaging. Phage AMGs are frequently located within the auxiliary gene cassettes and are separated by structural and nucleotide metabolism cassettes (hatfull et al., CurrO pin Virol (2011), 1 (4): 298-303; Kieft et al., loc. cit.). This was the case for VC-1 and VC-2 where the tail (structural) gene cassettes were clustered together, but located separately from the BCoAT AMG. Thus, the organizational structure of the VC-1 and VC-2 genomes was characteristic of pervasive mosaicism, a hallmark feature of bacteriophages, in which horizontal genetic exchange shapes their genome architectures (Hatfull et al., loc. cit.) .

In sum, despite an overall reduction of protective metabolites in the peri-engraftment period, patients with low-risk IMM-RI displayed a higher abundance of pathways involved in the bacterial synthesis of microbiota-derived metabolites. BCoAT, a key enzyme in the terminal reaction of the acetyl-CoA pathway - the predominant biosynthesis pathway of butyric acid (Vital et al., mBio (2014), 5(2): e00889; Duncan et al., Br J Nutr (2004), 91(6): 915-923) - was elevated in IMM-RI low-risk patients. The BCoAT gene was detected in two unique temperate bacteriophages, providing first evidence of BCoAT as AMG and that bacteriophages may directly modulate bacterial metabolism in humans.

Extraction of viral nucleic acids

The stool samples were mixed with 10 ml of filtered phage SM buffer and vortexed vigorously for 4 h at 4 °C, then centrifuged at 4000 g for 30 min to collect supernatant. The supernatant was passed through 0.22 pm filters (PES Membrane, Lot No. ROCB29300, Merck Millipore, Co., Cork, Ireland) to remove bacterial-associated particles, and the volume was subsequently concentrated to less than 50 pL by Amicon® Ultra Centrifugal Filters (10 kDA, Lot No. R9EA18187, Merck Millipore, Co., Cork, Ireland). Then 1/5 volume of chloroform was mixed with the samples and centrifuged at 14,000 g for 3 min, retaining the upper phase followed by a DNAse I (1 U/pL, Lot No. 1158858, Invitrogen, Carlsbad, CA, USA) treatment for 1 h at 37 °C to remove non-phage DNA. DNase I was inactivated by adding EDTA (0.1 M).

Subsequently, lysis buffer (700 pL KOH stock (0.43 g/10 mL), 430 pL DDT stock (0.8 g/10 mL), and 370 pL H2O, pH = 12) was added to the reaction and incubated at room temperature for 10 min followed by 2 h incubation at -80 °C, and 5 min at 55 °C. Lysed VLPs were then treated for 30 min at 55 °C with Proteinase K (20 mg/mL, Lot No. 1112907, Invitrogen, Carlsbad, CA, USA) to digest remaining viral capsid and extract the virome DNA. AM Pure beads (Agencourt, Beckman Coulter, Brea, CA, USA) were added to the extracted DNA and incubated for 15 min at room temperature. DNA was eluted from beads by 35 pL Tris buffer (10 mM, pH = 9.8) and stored at -80 °C until it was sent for sequencing. Sequencing was performed on an Illumina NovaSeq-PE150 platform. Fecal microbiota transplantation

Fecal microbiota transplantation (FMT; see below) was performed within compassionate use in accordance with protocols approved by Bavarian authorities. Regulations in Germany allow for the treatment of individuals within compassionate use (Jndividueller Heilversuch"). This requires approval of the local government. We have obtained this approval (General Administration of the Free State of Bavaria, reference number „2677.Ph_3-748-1“, issued to PD Dr. med. Christian Schulz) as well as patient informed consent to treatment and informed consent to publication (according to recommendations by the Committee on Publication Ethics). FMT was performed as last-line treatment, of severe grade IV GI-GvHD that was refractory to steroids, ruxolitinib and immunosuppressive therapy. The recipient patient (received two separate FMTs at Days 0 and +8 from the same healthy, unrelated FMT donor. At Day +13, the patient was discharged in good condition.

Patient samples were aligned with clinical metadata including body temperature, white blood count (WBC), C-reactive protein (CRP), administration and timing of anti-infective medication including antibiotics as well as of immunosuppressive therapy.

Prior to transplantation, the FMT donor was screened according to site-specific standard operating procedures at the Ludwig-Maximilians University Munich and the University Hospital Regensburg in compliance with regulations issued by the German Federal Institute for Drugs and Medical Devices. The FMT product consisted of a homogenized suspension of freshly- sampled feces in sterile saline solution and was applied via colonoscopy at the MUG endoscopy suite. During and following the procedure, the patient’s vital signs (ECG, blood pressure, pulse, temperature, peripheral oxygen saturation) were monitored.

The following criteria were used to assess response to FMT:

• CR (complete response) was defined as complete resolution of GI-GvHD.

• VGPR (very good partial response) was defined as improvement of at least 2 stages in the severity of GI-GvHD except improvement to stage 0.

• PR (partial response) was defined as improvement of one stage in the severity of Gl GvHD except improvement to stage 0.

Fecal microbiota transplantation restores intestinal IMMs

FMT is a microbiome-based therapy which has achieved partial and complete responses in clinical trials in allo-SCT patients suffering from steroid-refractory GI-GvHD (van Lier et al., Sci Transl Med (2020), 12(556): eaaz8926; Qi et al., Front Immunol (2018), 9:doi: 10.3389(fimmu.2018.02195; DeFilipp et al., Blood Adv (2018), 2(7): 745-753). It was hypothesized that FMT could be used to restore protective IMMs when they were depleted. As was observed in the subgroup of GI-GvHD patients, this was the case in GvHD-related microbial signatures. As proof-of-principle, FMT was performed in the patient described above with severe steroid- and ruxolitinib refractory clinical-grade IV GI-GvHD. Grade 3 GvHD (histopathological grading) (Lerner et al., Transplant Proc (1974), 6(4): 367-371 ; Sale et al., Am J Surg Pathol (1979), 3(4): 291-300) was confirmed in tissue biopsies, noticing crypt apoptosis, exploding crypt cells and loss of crypts in the large intestinal epithelium.

On Day 0 (Day +306 after allo-SCT) the patient received the first of two FMTs via colonoscopy from a healthy, unrelated donor who previously underwent donor screening. The second FMT was performed from the same donor on Day +8.

IMMs was assessed in the FMT donor and patient at baseline: SCFAs, BCFAs and 11 Ms were undetectable in the patient at Day -1. In contrast, the FMT donor expressed high levels of these metabolites, only DAT was not evident. After the second FMT at Day +8, by Day +12 a substantial increase was observed in all protective IMMs as well as the immunomodulatory SBAs DCA and LCA. In line, at Day -1 , low bacterial alpha diversity was observed in the patient. By Day +12 a considerable increase in the patient’s bacterial diversity was observed. The patient’s viral alpha diversity did not change during this time.

Claims

2. The composition of claim 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

(b1) a nucleotide sequence encoding a fragment of the polypeptide having the amino acid sequence shown in SEQ ID NO: 1, said fragment having BCoAT activity;

3. The composition of claim 1 or 2, wherein said bacteriophage is capable of being propagated in (Pseudo) Flavonifractor sp. cells.

4. The composition of claim 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

5. The composition of claim 1 or 4, wherein said bacteriophage is capable of being propagated in Lawsonibacter sp. cells.

6. The composition of claim 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

7. The composition of claim 1 or 6, wherein said bacteriophage is capable of being propagated in Mycobacterium sp. cells.

8. The composition of claim 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

9. The composition of claim 1 or 8, wherein said bacteriophage is capable of being propagated in Megasphaera sp. cells.

10. The composition of claim 1 , wherein said bacteriophage comprises a nucleic acid having a nucleotide sequence selected from the group consisting of:

11. The composition of claim 1 or 10, wherein said bacteriophage is capable of being propagated in Alistipes sp. cells.

12. The composition of any one of claims 1 to 11 , wherein expression of said nucleotide sequences is effected by an endogenous promoter of said bacteriophage.

13. The composition of any one of claims 1 to 12 which is a nutritional supplement.

14. The composition of claim 13, wherein said nutritional supplement is in the form of a capsule, a syrup, a suspension or a sustained release formulation.

15. The composition of any one of claims 1 to 12 which is a medical device.

16. The composition of claim 15, wherein said medical device is in the form of a suppository.

17. The composition of any one of the preceding claims, further comprising bacterial cells which are capable of being infected by the bacteriophage as defined in any one of claims 1 to 12.

18. The composition of claim 17, wherein said bacterial cells are colonic bacterial cells.

19. The composition of claim 17 or 18, wherein said bacterial cells are from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

20. The composition of any one of claims 1 to 19 for use in a method of increasing the amount of butyrate in the intestinal tract of a subject.

21. The composition of any one of claims 1 to 19 for use in a method of promoting gut health of a subject.

22. The composition of any one of claims 1 to 19 for use in a method of treating an inflammatory or immune-mediated intestinal condition of a subject.

23. The composition for the use of claim 22, wherein said inflammatory intestinal condition is inflammatory bowel disease.

24. The composition for the use of claim 22, wherein said immune-mediated intestinal condition is graft-versus-host-disease or colon cancer.

25. The composition for the use of any one of claims 20 to 24, wherein said composition is administered orally, rectally, via enema or through fecal microbiome transplantation to said subject.

26. The composition for the use of claim 25, wherein said composition is in the form of a capsule, a syrup, a suspension, a sustained release formulation or suppository.

27. The composition for the use of any one of claims 20 to 26, wherein said subject is a human subject.

28. The composition for the use of any one of claims 20 to 27, wherein in the intestinal microbiome of said subject a nucleotide sequence as defined in any one of claims 1 to 12 is not detected.

(A) isolating bacteriophages from a sample obtained from a subject; and

(B) determining whether said bacteriophages comprise a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA- transferase (BCoAT) activity.

30. The method of claim 29, wherein in step (B) a nucleotide sequence as defined in any one of claims 1 to 12 is used as a probe.

31. A kit comprising the composition of any one of claims 1 to 19 and bacterial cells which are capable of being infected by the bacteriophage as defined in any one of claims 1 to 12.

32. The kit of claim 31 , wherein said bacterial cells are colonic bacterial cells.

33. The kit of claim 31 or 32, wherein said bacterial cells are from (Pseudo)Flavonifractor sp., Lawsonibacter sp., Mycobacteroides sp., Mycobacterium sp., Megashaera sp. or Alistipes sp. cells.

34. An ex vivo method comprising a step of infecting a colonic bacterial cell with a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity to form an infected colonic bacterial cell that expresses said polypeptide.

35. Use of a composition comprising a bacteriophage comprising a nucleic acid molecule having a nucleotide sequence encoding a polypeptide having butyryl-coenzyme A (CoA):acetate CoA-transferase (BCoAT) activity for infecting colonic bacterial cell to form an infected colonic bacterial cell that expresses said polypeptide.