WO2023057599A1

WO2023057599A1 - Methods involving bacteroides strain replacement

Info

Publication number: WO2023057599A1
Application number: PCT/EP2022/077867
Authority: WO
Inventors: Xavier DUPORTET; Edith Hessel
Original assignee: Eligo Bioscience
Current assignee: Eligo Bioscience
Priority date: 2021-10-07
Filing date: 2022-10-06
Publication date: 2023-04-13
Anticipated expiration: 2024-04-07

Abstract

The present invention thus concerns a method for preventing and/or treating, in a subject in need thereof, acute inflammatory myocarditis associated with at least one immunogenic ꞵ-galactosidase encoding gene(s)-containing commensal Bacteroides strain and/or sequelae thereof, said method comprising administering to the subject a therapeutically efficient amount of at least one engineered Bacteroides strain, wherein said engineered Bacteroides strain(s) comprises a heterologous or engineered gene or gene set providing a competitive advantage over the at least one immunogenic ꞵ-galactosidase encoding gene(s)-containing commensal Bacteroides strain (target Bacteroides strain), and wherein said engineered Bacteroides strain (i) does not contain any gene encoding ꞵ-galactosidase, (ii) contains gene(s) encoding a non or less immunogenic ꞵ-galactosidase or (iii) contains a non-expressed gene encoding an immunogenic ꞵ-galactosidase.

Description

METHODS INVOLVING BACTEROIDES STRAIN REPLACEMENT

Field of the invention

[01] The present invention concerns methods for preventing or treating acute inflammatory myocarditis and/or sequelae thereof.

Background

[02] Myocarditis is an inflammatory heart disease that develops into lethal inflammatory cardiomyopathy in 20 - 30% of the patients (Buggey etal. (2018) Curr Opin Cardiol 33, 341 -346; Weintraub et al. (2017) Lancet 390, 400-414). Acute inflammatory myocarditis can be triggered by a wide variety of pathogens or other triggers, with viral infections as the most frequent initiators (Tschope et al. (2019) Circulation Research 124(1 1 ), 1568-1583). Most patients diagnosed with acute myocarditis recover without clinically relevant residual damage, however, in 47% of cases, permanent cardiomyopathy and damage is observed (including impaired ejection fraction) (Tschope et al. (2019) Circulation Research 124(11 ), 1568-1583). Acute myocarditis can be prolonged, worsened or progress into chronic inflammatory cardiomyopathy through the presence of persistent autoimmune reactivity, directed towards myosin heavy chain 6 (MYH6) peptide, which is released when cardiac cells are damaged by the viral or otherwise-induced infection and/or inflammation of the heart. This immune reactivity includes MYH6-specific T lymphocytes and antibodies. However, the mechanisms that govern the worsening of this disease or the progression to more permanent cardiomyopathy has remained less clear.

[03] It is well established that acute immune activation after infectious (or otherwise triggered) myocarditis is associated with the generation of autoimmune responses against myosin heavy chain 6 (MYH6) (Rose et al. (2014) FWOOPrime Rep 6, 25; Trachtenberg et al. (2017) Circ Res 121 , 803-818; Krebs et al. (2007) J. Autoimmun 28, 224-233), while subsequent chronic stimulation of MYH6-specific immune-reactivity including Th1 and Th17 cells precipitates inflammatory cardiomyopathy (Rangachari et al. (2006) J. Exp. Med 203, 2009-2019; Lv et al. (201 1 ) J. Clin. Invest 121 , 1561 -1573; Nindl et al. (2012) Eur J Immunol 42, 2311 -2321 ; Myers et al. (2016) JCI Insight 1 ). However, therapeutic approaches that mitigate the activity of such pathogenic T cells and prevent the severe consequences of inflammatory cardiomyopathy are still limited (Maisch et al. (2018) Herz. 43:423-430; Heymans et al. (2016) J Am Coll Cardiol 68, 2348-2364).

[04] A cardinal challenge in deciphering the progressive nature of autoimmune and chronic inflammatory diseases is the deconvolution of their multifactorial nature, which is determined by different degrees of genetic susceptibility and a multitude of environmental conditions (Generali et al. (2017) J Autoimmun 83, 51 -61 ; Davidson et al. (2001 ) N Engl J Med 345, 340-350). The quest for genetic determinants underlying susceptibility to myocarditis and dilated cardiomyopathy (DCM) has revealed associations with HLA-DQB1 * polymorphisms (Liu et al. (2005) Ann Hum Genet 69, 382-388; Portig et al. (2009) Autoimmunity 42, 33-40). Moreover, the development of progressive myocarditis in transgenic mice expressing the HLA-DQ8 haplotype (encoded by the DQA1*03:01/DQB1 *03:02 alleles) (Lv et al. (2011 ) J. Clin. Invest s , 1561 - 1573; Taylor et al. (2004) J. Immunol 172, 2651 -2658) indicates that antigen presentation via certain MHC class II molecules is a major determinant of myocarditis.

[05] Gil-Cruz et al. (2019) Science 366:881 -886 used transgenic mice with an engineered T cell receptor (TCR) that recognizes cardiac MYH6 peptides presented by MHC class II molecules on the surface of antigen-presenting cells, which develop spontaneous myocarditis (Nindl et al. (2012) Eur. J. Immunol. 42:2311 ). They found that Bacteroides thetaiotaomicron (B. theta), a gut commensal microbe, is required to initiate autoimmune myocarditis. B. theta contains gene(s) encoding p-galactosidase (P-gal), a microbial protein with sequence homology to MYH6. Presence of p-gal leads to induction of MYH6-specific CD4+ T cell proliferation and polarization to interleukin-17 (IL-17)— secreting cells, called Th17 cells. IL-17 is a cytokine involved in inflammatory and autoimmune responses (Patel et al. (2015) Immunity 43:1040). p-gal-specific and MYH6-cross-reactive TH17 cells expanded locally, near the gut within the colonic mucosa, and subsequently infiltrated the myocardium, driving profound cardiac tissue damage and death in mice colonized with B. theta strains that contain p-gal. Gil-Cruz et al. (2019) Science 366:881 - 886 furthermore demonstrated that B. theta strains lacking the p-gal gene were not able to induce this pathology, strongly indicating causality.

[06] One strategy to prevent acute inflammatory myocarditis in a subject would thus be to eliminate Bacteroides bacteria which contain gene(s) encoding p-galactosidase with sequence homology to MYH6 from the microbiome of said subject.

[07] However, using antibiotics to eliminate these p-galactosidase encoding genetcontaining Bacteroides bacteria would be either very challenging or dangerous. Indeed, commensal bacteria such as B. theta are strongly engrafted in the host microbiome and belong to a robust community of microorganisms. Having evolved in sometimes a symbiotic relationship, it has been demonstrated that antimicrobial treatment effects on commensal resident bacteria are often only transitory, i.e. the bacteria are repopulating their ecological niche after treatment, or even during treatment by becoming resistant to the treatment. On the other hand, large spectrum antibiotics can sometimes lead to the opposite effect, inducing dysbiosis which is damaging for the subject.

[08] One strategy can thus be to selectively remove these p-galactosidase encoding genetcontaining Bacteroides bacteria. However, even selectively removing a specific commensal bacterial strain can be damaging for the subject since it frees a niche in the microbiome which can be occupied by opportunistic pathogenic bacteria. Moreover, commensals like Bacteroides are often providing benefits to the host, and removing them will likely be detrimental to the host. [09] There is thus a need for new solutions enabling the selective removal of deleterious genes from commensal bacteria, such as gene(s) encoding p-galactosidase contained by Bacteroides bacteria in the context of AIM, while preserving the beneficial effects of the commensal bacteria and avoiding widespread microbiome dysbiosis or occupation of the corresponding niche by opportunistic pathogenic bacteria.

[010] The present invention meets this need.

[011] The present invention arises from the unexpected finding by the inventors that it is possible to significantly reduce the level of, and even replace, in a subject, Bacteroides bacteria containing at least one gene encoding p-galactosidase (“P-galactosidase gene(s)-containing Bacteroides bacteria”) by administering to the subject another Bacteroides strain which does not contain any gene encoding p-galactosidase or contains gene(s) encoding non-immunogenic p- galactosidase or contains gene(s) encoding p-galactosidase with reduced immunogenicity, and which has further been engineered to have a competitive advantage over the commensal p- galactosidase gene(s)-containing Bacteroides bacteria. Typically, this competitive advantage can be a metabolic advantage, by introducing in said engineered Bacteroides bacteria a heterologous gene involved in the import and/or metabolism of a nutrient source, such as porphyran.

[012] Due to this competitive advantage, the administered engineered Bacteroides strain is able to significantly reduce or even replace the p-galactosidase gene(s)-containing Bacteroides strain, and thus occupies the niche left by this strain, leading to a reduction in or absence of immunogenic p-galactosidase. Furthermore, by using an engineered bacterial strain from the same species as the p-galactosidase gene(s)-containing Bacteroides strain, the global composition of the microbiome is limitedly modified, avoiding any damage linked to the induction of dysbiosis.

Summary of the invention

[013] The present invention thus concerns a method for preventing and/or treating, in a subject in need thereof, acute inflammatory myocarditis associated with at least one immunogenic p- galactosidase encoding gene(s)-containing commensal Bacteroides strain and/or sequelae thereof, said method comprising administering to the subject a therapeutically efficient amount of at least one engineered Bacteroides strain, wherein said engineered Bacteroides strain(s) comprises a heterologous or engineered gene or gene set providing a competitive advantage over the at least one immunogenic p- galactosidase encoding gene(s)-containing commensal Bacteroides strain (target Bacteroides strain), and wherein said engineered Bacteroides strain (i) does not contain any gene encoding p- galactosidase, (ii) contains gene(s) encoding a non or less immunogenic p-galactosidase or (iii) contains a non-expressed gene encoding an immunogenic p-galactosidase.

[014] In a particular embodiment, said heterologous gene or gene set providing a competitive advantage is from another species than the engineered Bacteroides strain. [015] In another embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene involved in the import and/or metabolism of a nutrient source.

[016] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding a transporter of a nutrient source.

[017] In a particular embodiment, said nutrient source is a rare carbohydrate. In a more particular embodiment, less than 50% of other bacterial cells in the subject utilize said rare carbohydrate as a nutrient source.

[018] In a particular embodiment, said rare carbohydrate is a polysaccharide.

[019] In another particular embodiment, said rare carbohydrate is a sulfated carbohydrate.

[020] In a particular embodiment, said rare carbohydrate is selected from the group consisting of alginate, fucoidan, laminarin, xylan, galactans and any combination thereof.

[021] In a more particular embodiment, said rare carbohydrate is selected from the group consisting of porphyran, agarose, carrageenan, ulvan, xylan and any combination thereof.

[022] In a particular embodiment, said rare carbohydrate is a carbohydrate cleaved by a glycoside hydrolase belonging to glycoside hydrolase family GH86.

[023] In a particular embodiment, said rare carbohydrate is a sulfated polygalactan.

[024] In a particular embodiment, said rare carbohydrate is porphyran.

[025] In a particular embodiment, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene selected from the group consisting of genes encoding porphyranase, glycoside hydrolase, sulfatase, galactosidase and any combination thereof.

[026] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding protein(s) which sequence(s) is(are) at least 80% identical to at least one of BACPLE 1683-1706 from the Bacteroides plebeius genome.

[027] In a particular embodiment, said heterologous or engineered gene set providing a competitive advantage comprises at least two, three, four, five or six genes.

[028] In a particular embodiment, the nutrient source cannot be utilized as a nutrient source by the engineered Bacteroides strain in the absence of the heterologous or engineered gene or gene set providing a competitive advantage.

[029] In an alternative embodiment, the nutrient source is utilized as a nutrient source more efficiently by the engineered bacterial strain in the presence of the heterologous or engineered gene or gene set providing a competitive advantage than in the absence thereof.

[030] In a particular embodiment, the method further comprises administering the nutrient source to the subject.

[031] In a particular embodiment, no antibacterial agent is administered to the subject.

[032] In an alternative embodiment, an antibacterial agent is administered to the subject. [033] In a particular embodiment, said engineered Bacteroides strain further produces an antibacterial agent to which it is resistant, but to which said target Bacteroides strain(s) is sensitive. In a more particular embodiment, said engineered Bacteroides strain further produces an antibacterial agent to which it is resistant, but to which said target Bacteroides strain(s) and other bacteria from the subject are sensitive.

[034] In a particular embodiment, the antibacterial agent is a bacteriocin. In a more particular embodiment, the antibacterial agent is bacteroidetocin.

[035] In a particular embodiment, the engineered Bacteroides strain produces multiple antibacterial agents, at least one, two, three, four, five or more antibacterial agents.

[036] In a particular embodiment, said immunogenic p-galactosidase is expressed, secreted, and/or displayed by said target Bacteroides strain(s).

[037] In a particular embodiment, said engineered Bacteroides strain and said target Bacteroides strain(s) are from the same species.

[038] In a particular embodiment, said at least one target Bacteroides strain is a Bacteroides thetaiotaomicron strain and/or a Bacteroides faecis strain. In a particular embodiment, said at least one engineered Bacteroides strain is an engineered Bacteroides thetaiotaomicron strain and/or an engineered Bacteroides faecis strain.

[039] In a particular embodiment, said engineered Bacteroides strain comprises a genetic modification in at least one p-galactosidase encoding gene(s) or in a sequence regulating the expression of at least one p-galactosidase encoding gene. In a more particular embodiment, the genetic modification is in the p-galactosidase fragment recognized as epitope by the immune system leading to a weaker or absence of epitope recognition by the immune system. In still a more particular embodiment, the genetic modification induces the non-expression of p- galactosidase.

[040] In a particular embodiment, the subject has been diagnosed with, or is at risk of developing acute inflammatory myocarditis.

[041] In a particular embodiment, the subject is carrying a HLA-DQ haplotype able to present human MYH6 peptides including MYH6614-629.

[042] In a particular embodiment, the subject has previously been vaccinated before.

Detailed description of the invention

Engineered Bacteroides strain with competitive advantage

[043] In the context of the invention, an engineered Bacteroides strain comprising a heterologous or engineered gene or gene set providing a competitive advantage over an immunogenic p-galactosidase encoding gene(s)-containing commensal Bacteroides strain (target Bacteroides strain) is used.

[044] As used herein, the term “engineered” means that the bacterial cell of the invention has been modified by standard molecular biology techniques, typically to introduce the indicated heterologous gene or gene set or to modify the indicated gene or gene set, for example by transformation of the cell with a plasmid, by conjugation, by transduction of the cell with a bacteriophage, or by any suitable technique enabling introducing or modifying a nucleic acid sequence into a bacterial cell. As will be understood by the skilled person, engineering of a bacterial strain implies a deliberate action to introduce or modify a nucleic acid sequence and does not cover introduction or modification of a nucleic acid sequence through natural evolution of the bacterial strain.

[045] For example, the bacteria of the invention can be genetically engineered by transformation (chemical transformation or ultrasound transformation), transduction (using for example optionally engineered bacteriophages, or packaged phagemids technologies), conjugation, or electroporation.

[046] Typically, said heterologous gene or gene set providing a competitive advantage has been incorporated into the bacterial cell’s chromosomal or extrachromosomal expression system, or as extrachromosomal expression system, by genetic engineering techniques known in the art. For example, said bacteria can be genetically engineered by transformation (chemical transformation or ultrasound transformation), transduction (using for example optionally engineered bacteriophages, or packaged phagemids technologies), conjugation, or electroporation.

[047] By “engineered gene or gene set” is meant herein a gene or gene set, autologous to said bacterial strain, but which has been modified by standard molecular biology techniques, typically to introduce a mutation in the sequence of said autologous gene or gene set, in such a way that the expression of the gene or gene set or the activity of the protein encoded by said gene or gene set is modified. As will be understood by the skilled person, engineering of a gene or gene set implies a deliberate action to introduce a modification in the nucleic acid sequence and does not cover mutation of a nucleic acid sequence through natural evolution of the bacterial strain.

[048] Said engineered gene or gene set may be any piece of a gene such as a portion of an open reading frame of a gene, or a sequence involved in the regulation of the expression of a gene such as a promoter, an operator, a terminator. In this specific context, it also encompasses a nucleic acid encoding a transcription factor, a nucleic acid encoding a repressor, a nucleic acid encoding an activator, or a nucleic acid encoding an inducer.

[049] Typically, said engineered gene or gene set providing a competitive advantage has been modified into the bacterial cell’s chromosomal or extrachromosomal expression system, by genetic engineering techniques known in the art. For example, said bacteria can be genetically engineered by transformation (chemical transformation or ultrasound transformation), transduction (using for example optionally engineered bacteriophages, or packaged phagemids technologies), conjugation, or electroporation.

[050] In some embodiments, said heterologous or engineered gene or gene set providing a competitive advantage is under the control of a high expression promoter. In particular embodiments, said heterologous or engineered gene or gene set providing a competitive advantage is under the control of an inducible promoter, constitutive promoter, native promoter (e.g., native to the bacterial cell), heterologous promoter, or a promoter associated with said heterologous gene in its native form.

[051] In some embodiments, in particular when said heterologous or engineered gene or gene set providing a competitive advantage is a gene or gene set involved in the import and/or metabolism of a nutrient source, as defined below, said heterologous or engineered gene or gene set providing a competitive advantage is under the control of a promoter which is induced in the presence of the nutrient source. More particularly, in some embodiments, in particular when said heterologous or engineered gene or gene set providing a competitive advantage is a gene or gene set involved in the import and/or metabolism of a nutrient source, as defined below, the expression of said heterologous or engineered gene or gene set providing a competitive advantage is increased in the presence of the nutrient source.

[052] In some embodiments, said heterologous or engineered gene or gene set providing a competitive advantage is under the control of a burden-sensing promoter. More particularly, when said engineered Bacteroides strain contains a gene encoding a mutant or variant of said p- galactosidase, as defined below, synthesis of said mutant or variant may confer a burden and/or fitness cost on said engineered Bacteroides strain. In such embodiment, expression of said heterologous or engineered gene or gene set providing a competitive advantage can be up- regulated when said burden-sensing promoter is induced by said burden and/or fitness cost relative to a basal level expression of said heterologous or engineered gene or gene set when said burden-sensing promoter is not induced.

[053] In other embodiments, said engineered Bacteroides strain further comprises an essential gene operably linked to a burden-sensing promoter, wherein expression of said heterologous or engineered gene or gene set providing a competitive advantage, and/or when said engineered Bacteroides strain contains a gene encoding a mutant or variant of said p-galactosidase, synthesis of said mutant or variant, confer a burden and/or fitness cost on said strain, and wherein expression of said essential gene is up-regulated when said burden-sensing promoter is induced by said burden and/or fitness cost relative to a basal level expression of said essential gene when said burden-sensing promoter is not induced.

[054] Such burden-sensing promoters are typically disclosed in international application WO2021/160854.

[055] Examples of burden-sensing promoters include a o factor regulated promoter, such as o³², o^B and o^s factor regulated promoters, a ribosomal RNA promoter, an HAC1 -upregulated promoter comprising a UPR element, and a DNA-damage sensing promoter.

[056] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is chromosomally integrated upstream of an autologous essential gene, in particular as part of a single operon including said autologous essential gene. Such a location of the heterologous or engineered gene is advantageous to prevent loss of function of the heterologous or engineered gene. Indeed, in case the heterologous or engineered gene is not expressed due to a frameshift mutation and/or a point mutation leading to a STOP codon occuring in the heterologous or engineered gene, the downstream autologous essential gene will not be expressed anymore, and the engineered bacterial strain will die.

[057] In a particular embodiment, when said engineered Bacteroides strain contains a gene encoding a mutant or variant of said p-galactosidase, said gene encoding said mutant or variant is chromosomally integrated upstream of a heterologous or engineered gene providing a competitive advantage, in particular as part of a single operon including said heterologous or engineered gene providing a competitive advantage. Such a location of the gene encoding said mutant or variant is advantageous to prevent loss of function of the gene. Indeed, in case the gene encoding said mutant or variant is not expressed due to a frameshift mutation and/or a point mutation leading to a STOP codon occurring in the gene encoding said mutant or variant, the downstream heterologous or engineered gene providing a competitive advantage will not be expressed anymore, and the engineered Bacteroides strain will lose its competitive advantage Bacterial strain

[058] The engineered Bacteroides strain used in the context of the invention can be obtained from any suitable Bacteroides strain.

[059] In a particular embodiment, the engineered Bacteroides strain is obtained from an autologous strain, i.e. a strain isolated from the subject to be treated.

[060] In an alternative embodiment, the engineered Bacteroides strain is obtained from a non- autologous strain, i.e. a strain which was not isolated from the subject to be treated.

[061] As used herein, a “bacterial strain” refers to a genetic variant or subtype within a bacterial species. Therefore, a bacterial strain more particularly refers to a bacterium which remains genetically unchanged when grown or multiplied. The multiplicity of identical bacteria are included. A bacterial strain is typically obtained from the isolation of a clone, which can give birth to a population of cells obtained from a single bacterial cell or colony.

[062] Examples of Bacteroides species include, without limitation, B. acidifaciens, B. barnesiaes, B. caccae, B. caecicola, B. caecigallinarum, B. cellulosilyticus, B. cellulosolvens, B. clarus, B. coagulans, B. coprocola, B. coprophilus, B. coprosuis, B. distasonis, B. eggerthii, B. gracilis, B. faecichinchillae, B. faecis, B. finegoldii, B. fluxus, B. fragilis, B. galacturonicus, B. gallinaceumi, B. gallinarum, B. goldsteinii, B. graminisolvens, B. helcogene, B. intestinalis, B. luti, B. massiliensis, B. melaninogenicus, B. nordii, B. oleiciplenus, B. oris, B. ovatus, B. paurosaccharolyticus, B. plebeius, B. polypragmatus, B. propionicifaciens, B. putredinis, B. pyogenes, B. reticulotermitis, B. rodentium, B. salanitronis, B. salyersiae, B. sartorii, B. sedimenti, B. stercoris, B. suis, B. tectus, B. thetaiotaomicron, B. uniformis, B. vulgatus, B. xylanisolvens, and B. xylanolyticus. [063] In a preferred embodiment, said engineered Bacteroides strain is obtained from a Bacteroides species selected from the group consisting of Bacteroides faecis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides vulgatus, and Bacteroides fragilis.

[064] In a particular embodiment, said engineered Bacteroides strain is an engineered Bacteroides thetaiotaomicron strain and/or an engineered Bacteroides faecis strain. Heterologous or engineered gene or gene set providing a competitive advantage

[065] In the context of the invention, the engineered Bacteroides strain comprises a heterologous or engineered gene or gene set providing a competitive advantage over target Bacteroides strain (s).

[066] By “competitive advantage” of the engineered Bacteroides strain over target Bacteroides strain(s) is meant herein an advantage in the interaction between the engineered Bacteroides strain and target Bacteroides strain(s) within a community, typically within a microbiome. Examples of competitive advantages include a fitness advantage wherein the engineered Bacteroides strain is better suited to conquer a specific niche in the microbiome, an advantage in interference competition wherein the engineered Bacteroides strain can typically produce antagonists (such as toxins, biosurfactants, bacteriocins, volatiles or antibiotics) against other members of the microbiome or wherein the engineered Bacteroides strain can typically be resistant to administered antagonists (such as toxins, biosurfactants, bacteriocins, volatiles or antibiotics) to which other members of the microbiome are sensitive, or an advantage in exploitation competition wherein the engineered Bacteroides strain exploits more efficiently the growth substrates available to the community, more particularly a metabolic advantage wherein the engineered Bacteroides strain metabolize more efficiently some growth substrates available to the community.

[067] In a particular embodiment, said competitive advantage is a metabolic advantage.

[068] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is a gene or gene set involved in the import and/or metabolism of a nutrient source.

[069] By “gene or gene set involved in the import of a nutrient source” is meant herein a gene or set of genes encoding a molecule enabling directly and/or indirectly the active transport of the nutrient source from the extracellular medium into the cytoplasm.

[070] By “gene or gene set involved in the metabolism of a nutrient source” is meant herein a gene or set of genes encoding a molecule enabling, intracellularly and/or extracellularly after secretion in the extracellular medium or in the periplasm, the degradation of the nutrient source. [071] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding a transporter of a nutrient source.

[072] In a particular embodiment, said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding an intracellular or extracellular enzyme involved in the degradation of the nutrient source. [073] In a particularly preferred embodiment, said heterologous or engineered gene set providing a competitive advantage comprises a gene encoding a transporter of a nutrient source and a gene encoding an intracellular enzyme involved in the degradation of the nutrient source. [074] In a particular embodiment, said heterologous gene or gene set providing a competitive advantage is from another species than the engineered Bacteroides strain.

[075] In a particular embodiment, said nutrient source is a rare carbohydrate.

[076] By “rare” carbohydrate is meant herein a carbohydrate that is utilized, as a nutrient source, by less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1 %, less than 0.5%, less than 0.3%, less than 0.2%, less than 0.1 %, less than 0.03%, less than 0.01 %, less than 0.003%, less than 0.001 %, less than 0.0001 %, or none) of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome (i.e., cells “other” than the engineered Bacteroides strain of the invention, e.g., cells of the resident population prior to administration). Thus, in some embodiments, a rare carbohydrate is one that can be utilized, as a nutrient source, by less than 50% (e.g., less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3%, less than 2%, less than 1 %, less than 0.5%, less than 0.3%, less than 0.2%, less than 0.1 %, less than 0.03%, less than 0.01 %, less than 0.003%, less than 0.001 %, less than 0.0001 %, or none) of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome. In some cases, the rare carbohydrate is one that can be utilized or is utilized, as a nutrient source, by less than 20% of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome. In some embodiments, the rare carbohydrate is one that can be utilized or is utilized, as a nutrient source, by less than 5% of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome. In some embodiments, the rare carbohydrate is one that can be utilized or is utilized, as a nutrient source, by less than 2% of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome. In some embodiments, the rare carbohydrate is one that can be utilized or is utilized, as a nutrient source, by less than 0.5% of other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome. In some embodiments, the rare carbohydrate is one that can be utilized or is utilized, as a nutrient source, by none of the other bacterial cells present in the subject or in the environment, in particular present in the microbiome of the subject, more particularly present in a particular microbiome of the subject such as the gut microbiome and/or the skin microbiome.

[077] In some embodiments, the rare carbohydrate is a polysaccharide.

[078] In some embodiments, the rare carbohydrate is a sulfated carbohydrate. In more particular embodiments, the rare carbohydrate is selected from the group consisting of porphyran, ulvan, carrageenan, fucoidan and any combination thereof.

[079] In some embodiments, the rare carbohydrate is a marine carbohydrate. Examples of marine carbohydrates include but are not limited to: porphyran, agarose, agaropectin, carrageenan, ulvan, alginate, fucoidan, laminarin, and marine microbe exopolysaccharides. In some embodiments, the rare carbohydrate of interest is selected from porphyran and agarose. In some embodiments, the rare carbohydrate is porphyran. In some embodiments, the rare carbohydrate is agarose.

[080] In a particular embodiment, said rare carbohydrate is selected from the group consisting of alginate, fucoidan, laminarin, xylan, galactans (such as carrageenan and agarose), porphyran, ulvan, xylan and any combination thereof.

[081] In some embodiments, the rare carbohydrate is a carbohydrate cleaved by a glycoside hydrolase belonging to glycoside hydrolase family GH86.

[082] In some embodiments, the rare carbohydrate is a carbohydrate that contains a glycosidic linkage selected from the group consisting of p-d-galactopyranose to a-l-galactopyranose-6- sulfate, p-d-galactopyranose to 3,6-anhydro-a-l-galactopyranose.

[083] In some embodiments, the rare carbohydrate is a sulfated polygalactan. In some such embodiments, one or more of the galactose residues of the sulfated polygalactan can be a 3,6- anhydro-galactose (e.g., in some embodiments joined by alternating a-1 ,3 and p-1 ,4-glycosidic linkage). In some embodiments, one or more of the galactopyranose residues of the sulfated polygalactan can be modified by one or more ester sulfates. In some embodiments, one or more of the galactose residues of the sulfated polygalactan is a 3,6-anhydro-galactose (e.g., in some embodiments joined by alternating a-1 ,3 and p-1 ,4-glycosidic linkage); and one or more of the galactopyranose residues of the sulfated polygalactan is modified by one or more ester sulfates. [084] In a particular embodiment, when the nutrient source is a polysaccharide as defined above, the heterologous or engineered gene or gene set providing a competitive advantage can be or comprise a gene selected from the group consisting of genes encoding porphyranase, glycoside hydrolase, sulfatase, galactosidase and any combination thereof.

[085] In a particular embodiment, when the nutrient source is a polysaccharide as defined above, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding a porphyranase (e.g., one from GH family 86 (GH86)). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding an agarase (e.g., one from GH family 86 (GH86)). [086] In a particular embodiment, when the nutrient source is as defined above, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding BACPLE 1683-1706 from the Bacteroides plebeius genome (or homologs thereof) (see, e.g., Table 1 ). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding BACPLE 1683-1687 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage comprises nucleic acid(s) encoding BACPLE 1683-1687 from the B. plebeius genome (or homologs thereof) and BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding BACPLE 1683-1699 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding BACPLE 1688-1706 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acid(s) encoding BACPLE 1683-1687 from the B. plebeius genome (or homologs thereof) as well as nucleic acids encoding both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acid(s) encoding BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof) as well as nucleic acids encoding both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acid(s) encoding BACPLE 1683-1687 and BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof) as well as nucleic acids encoding both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome (or homologs thereof).

[087] As such, in some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1706 from the B. plebeius genome (see, e.g., Table 1 ). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1687 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1700-1706 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1687 from the B. plebeius genome (or homologs thereof) and/or at least one of BACPLE 1700-1706 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1699 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1688-1706 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1687 from the B. plebeius genome (or homologs thereof) as well as both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof) as well as both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of BACPLE 1683-1687 and/or at least one of BACPLE 1700-1706 from the B. plebeius genome (or homologs thereof) as well as both porphyranases from within BACPLE 1688-1699 from the B. plebeius genome.

Table 1 : SEQ ID NOs. and annotations for proteins encoded by B. plebeius genome (BACPLE 1669-1706)

[088] In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 14-34 (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 14-17 (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 28-34 (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 14-17 (or homologs thereof) and SEQ ID NOs: 28-34 (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 14-27 (or homologs thereof). In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein of sequence selected from SEQ ID NOs: 18-34 (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding a protein of sequence selected from SEQ ID NOs: 14-17 (or homologs thereof) as well as both porphyranases from within SEQ ID NOs: 18-27 (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding a protein of sequence selected from SEQ ID NOs: 28-34 (or homologs thereof) as well as both porphyranases from within SEQ ID NOs: 18-27 (or homologs thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding a protein of sequence selected from SEQ ID NOs: 14-17 and SEQ ID NOs: 28-34 (or homologs thereof) as well as both porphyranases from within SEQ ID NOs: 18-27 (or homologs thereof).

[089] As such, in some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-34. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-17. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 28-34. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-17 and/or at least one of SEQ ID NOs: 28-34. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-27. In some embodiments, the heterologous or engineered gene or gene set providing a competitive advantage is or comprises nucleic acid(s) encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 18-34. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-17 as well as both porphyranases set forth as SEQ ID NOs: 19 and 21. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 28-34 as well as both porphyranases set forth as SEQ ID NOs: 19 and 21 . In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding protein(s) that has(ve) 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with at least one of SEQ ID NOs: 14-17 and/or at least one of SEQ ID NOs: 28-34 as well as both porphyranases set forth as SEQ ID NOs: 19 and 21 .

[090] In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding (i) at least one protein of sequence selected from SEQ ID NOs: 19, 21 and 22 (or a homolog(s) thereof); (ii) at least one protein of sequence selected from SEQ ID NOs: 26 and 33 (or a homolog(s) thereof); and (iii) at least one protein of sequence selected from SEQ ID NOs: 25 and 32 (or a homolog(s) thereof). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding proteins of sequences SEQ ID NOs.: 19, 21 -22, 25, 26, and 32-33 (or homologs thereof).

[091] In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding (i) at least one protein that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with any one of SEQ ID NOs: 19, 21 and 22; (ii) at least one protein that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with any one of SEQ ID NOs: 26 and 33; and (iii) at least one protein that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with any one of SEQ ID NOs: 25 and 32. In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises nucleic acids encoding proteins having respectively 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, or 100% sequence identity) with SEQ ID NOs: 19, 21 -22, 25, 26, and 32-33.

[092] In some embodiments, said heterologous or engineered gene set providing a competitive advantage comprises at least 3 genes (e.g., at least 4, at least 5, at least 6, at least 8 genes, at least 10 genes, at least 12 genes, at least 15 genes, or at least 20 genes). In a particular embodiment, said heterologous or engineered gene set providing a competitive advantage comprises at least six genes. In some embodiments, said heterologous gene set providing a competitive advantage comprises from 3 to 30 genes (e.g., 5-30, 3-25, 3-20, 3-15, 3-10, 3-8, 5- 25, 5-20, 5-15, 5-10, 5-8, 8-30, 8-25, 8-20, 8-15, 10-30, 10-25, 10-20, 10-15, 12-30, 12-25, 12- 20, 15-30, 15-25, 20-30, or 20-25 genes). In some embodiments, the heterologous or engineered gene set providing a competitive advantage comprises 3 to 10 genes.

[093] As used herein, the percent identity is calculated in relation to polymers (e.g., polynucleotide or polypeptide) whose sequences have been aligned. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[094] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. AppL Biosci., 4: 11 -17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a BLOSUM62 matrix, a BLQSUM30 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In a specific embodiment the BLQSUM30 matrix is used with gap open penalty of 12 and gap extension penalty of 4.

[095] In a particular embodiment, the nutrient source cannot be utilized as a nutrient source by the engineered Bacteroides strain in the absence of the heterologous gene or gene set providing a competitive advantage. [096] In an alternative embodiment, the nutrient source is utilized as a nutrient source more efficiently by the engineered Bacteroides strain in the presence of the heterologous or engineered gene or gene set providing a competitive advantage than in the absence thereof. In other words, in an alternative embodiment, the engineered Bacteroides strain is able to use the nutrient source in the absence of the heterologous or engineered gene or gene set, but the presence of the heterologous or engineered gene or gene set enables the engineered Bacteroides strain to use the nutrient source more efficiently, for example by providing a gene that can ensure the transport of the nutrient source before it is metabolized by the bacterial strain.

[097] In a particular embodiment, the method further comprises administering the nutrient source, as defined above, to the subject.

[098] In a particular embodiment, said nutrient source is not used in its natural context.

[099] In a particular embodiment, said engineered Bacteroides strain and said nutrient source are administered either together or separately. For example, the nutrient source may be provided as a solution and the engineered Bacteroides strain may be provided in dry form or as an enteric- coated tablet or capsule. Alternatively, a composition comprising both the engineered Bacteroides strain and the nutrient source, for example in the form of a non-aqueous liquid or gel composition or in dry form or as an enteric-coated tablet or capsule, can be administered. Alternatively, the nutrient source may be provided in dry form or as an enteric-coated tablet or capsule and the engineered Bacteroides strain may be provided in a separate dry form or as an enteric-coated tablet or capsule.

[0100] In a particular embodiment, the nutrient source can be administered prior to the administration of the engineered Bacteroides strain, or the nutrient source can be administered contemporaneously with the administration of the engineered Bacteroides strain, and/or the nutrient source can be administered after the administration of the engineered Bacteroides strain. [0101] In a particular embodiment, the nutrient source is administered contemporaneously with the administration of the engineered Bacteroides strain and further administered after the administration of the engineered Bacteroides strain.

[0102] In a particular embodiment, said competitive advantage is or further includes an advantage in interference competition, in particular in intraspecies direct competition, wherein the engineered Bacteroides strain can typically produce antibacterials against other members of the microbiome, in particular against the target Bacteroides strain(s), as defined below, or wherein the engineered Bacteroides strain can typically be resistant to administered antibacterials to which other members of the microbiome, in particular the target Bacteroides strain(s), are sensitive.

[0103] In a more particular embodiment, said competitive advantage is provided or further provided by the production of one or more bacteriocins, as defined below, by said engineered Bacteroides strain. [0104] In a particular embodiment, the engineered Bacteroides strain thus comprises or further comprises a heterologous or engineered gene or gene set involved in intraspecies direct competition, more particularly a heterologous or engineered gene or gene set involved in the expression or synthesis of an antibacterial agent, in particular as defined in the section “Reduction of the level of immunogenic /3-galactosidase produced by a target Bacteroides strain" below, more particularly an antibacterial agent to which the engineered Bacteroides strain is resistant and the target Bacteroides strain(s) is sensitive, more particularly a heterologous or engineered gene or gene set involved in the expression or synthesis of one or more bacteriocins.

[0105] In a particular embodiment, said engineered Bacteroides strain does not comprise any antibiotic-resistance gene or marker.

[0106] In a particular embodiment, said engineered Bacteroides strain is auxotrophic. In a more particular embodiment, said engineered Bacteroides strain comprises an auxotrophic selection marker such as air (alanine racemase), thyA (Thymidylate synthase), dapA (4-hydroxy- tetrahydrodipicolinate synthase). In a more particular embodiment, said engineered Bacteroides strain is auxotrophic to the nutrient source as defined above.

[0107] In a particular embodiment, said engineered Bacteroides strain further comprises a nucleic acid, in particular a heterologous or engineered nucleic acid, involved in the expression of a molecule of interest, in particular a molecule of interest having a beneficial effect for the subject, or for the subject’s microbiome.

[0108] In a particular embodiment, the method of the invention further includes administering to the subject a prebiotic.

[0109] Prebiotics include, but are not limited to, amino acids, biotin, fructo-oligosaccharide, galacto-oligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g., guar gum, gum arabic and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans- galactooligosaccharide, pectins (e.g., homogalacturonan, citrus pectin, apple pectin, and rhamnogalacturonan-l), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber) and xylooligosaccharides.

[0110] In a particular embodiment, said prebiotic is not the nutrient source as defined above.

Reduction of the level of immunogenic /3-galactosidase produced by a target Bacteroides strain [0111] In the context of the invention, administration of the engineered Bacteroides strain of the invention enables reducing the level, in the subject, of the immunogenic p-galactosidase produced by the target Bacteroides strain because said engineered Bacteroides strain does not contain any gene encoding p-galactosidase, contains gene(s) encoding a non or less immunogenic p-galactosidase or contains a non-expressed gene encoding an immunogenic p- galactosidase.

Target Bacteroides strain

[0112] In the context of the invention, the target Bacteroides strain is bacterial strain as defined in the section “Bacteroides strain" above. In a particular embodiment, said target Bacteroides strain is a commensal Bacteroides strain. In another particular embodiment, said target Bacteroides strain is a pathogenic Bacteroides strain.

[0113] In a preferred embodiment, said target Bacteroides strain is a strain from a species selected from the group consisting of Bacteroides faecis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides vulgatus, and Bacteroides fragilis.

[0114] In a particular embodiment, said at least one target Bacteroides strain is a Bacteroides thetaiotaomicron strain and/or a Bacteroides faecis strain.

[0115] In a particular embodiment, said engineered Bacteroides strain and said target Bacteroides strain are from the same species.

P-galactosidase

[0116] By “P-galactosidase” is meant herein a family of glycoside hydrolase enzymes that catalyzes the hydrolysis of p-galactosides into monosaccharides through the breaking of a glycosidic bond, p-galactosidase is important for organisms as it is a key provider in the production of energy and a source of carbons through the breakdown of lactose to galactose and glucose, p-galactosidases can be found in many organisms (plants, mammals, yeast, bacteria...), the length and sequence thereof slightly varying according to the organisms.

[0117] Among bacteria, p-galactosidase is typically expressed (among others) by Escherichia coli, Bifidobacterium adolescentis, Enterobacter agglomerans, Bacillus subtilis, Pianococcus sp., Bacillus licheniformis, Therm us thermophilus, Arthrobactersp., Pseudoalteromonas haloplanktis, Geobacillus kaustophilus, Clostridium cellulovorans, Alicyclobacillus acidocaldarius subsp. acidocaldarius, Bacteroides ovatus, Lactobacillus acidophilus, Thermus sp., Leptotrichia buccalis, Lactococcus lactis subsp. lactis, Klebsiella pneumoniae, Vibrio cholerae, Shigella dysenteriae, Enterobacter cloacae, Lactobacillus delbrueckii subsp. bulgaricus, Clostridium perfringens, Yersinia pestis, Bacteroides thetaiotaomicron, Bacteroides cellulosilyticus, Bacteroides intestinal is, Bacteroides cellulosilyticus, Bacteroides fragilis or Bacteroides salyersiae.

[0118] In a particular embodiment, said p-galactosidase comprises or consists of the sequence SEQ ID NO: 45.

[0119] Depending on its specific sequence, the produced p-galactosidase can include MYH6 mimic peptides which can be recognized as epitope by the subject’s immune system, leading to the induction or sustainment of an autoimmune reaction in the subject. Therefore, in the context of the invention, said target p-galactosidase encoding gene(s)-containing Bacteroides strain is thus a Bacteroides strain containing at least one gene encoding an immunogenic p- galactosidase, in particular a p-galactosidase able to induce or sustain an autoimmune reaction in a subject, more particularly a p-galactosidase that comprises a peptide that mimics a human autoantigen.

[0120] By “immunogenic” is meant herein the capacity to induce or sustain an immune response, in particular an autoimmune reaction in a subject.

[0121] Techniques to determine that a p-galactosidase is immunogenic are for example disclosed in Gil-Cruz et al. (2019) Science 366:881-886, and typically include incubating the p- galactosidase or p-galactosidase peptide to be tested with splenocytes, typically 2x10⁵ splenocytes, obtained from MYH6-specific TCR transgenic acute myocarditis mice model (typically disclosed in Gil-Cruz et al. (2019) Science 366:881-886) and labeled with carboxyfluorescein succinimidyl ester (CFSE), typically with 10 pL of 5 mM CFSE in 10 ml of PBS for example for 10 min at 37°C, the staining reaction being typically stopped with 1 ml of FCS followed by washing with PBS, and assessing CFSE dilution by flow cytometry, for example after 3 days of incubation, typically at 37°C.

[0122] In a particular embodiment, said target Bacteroides strain is a Bacteroides strain comprising at least one gene encoding a p-galactosidase including MYH6 mimic peptides (in particular mimics of MYH6ei 4-629 or MYH6ei 4-628 peptides, typically of sequence SEQ ID NO: 35, 38 or 39), more particularly a Bacteroides strain comprising at least one gene encoding a p- galactosidase comprising a p-galn-25 peptide (typically of sequence SEQ ID NO: 36, 37, 40, 41 , 42, 43 or 44).

[0123] In a particular embodiment, said target Bacteroides strain produces said immunogenic p- galactosidase.

[0124] By “produced” is meant herein that the p-galactosidase is either directly or indirectly expressed, secreted, displayed or produced by said target Bacteroides strain. In particular, said produced p-galactosidase can directly be expressed by a gene comprised by said target Bacteroides strain, and then be secreted, membrane displayed or kept intracellularly by said target Bacteroides strain. Therefore, in a particular embodiment, the immunogenic p- galactosidase is expressed, secreted and/or displayed by the at least one target Bacteroides strain.

Reduction of the level

[0125] By “level” is meant herein the amount or concentration of said specific immunogenic p- galactosidase in the subject. As will be understood by the skilled person, depending on the fact that said immunogenic p-galactosidase is secreted, displayed or remained intracellular in said target Bacteroides strain, the level of said immunogenic p-galactosidase will be determined either in the subject, in particular in the subject’s organ hosting the target Bacteroides strain, or in the subject’s microbiome hosting the target Bacteroides strain.

[0126] By “reduction of the level of the immunogenic p-galactosidase” is meant herein a decrease in the level, in the subject, of the immunogenic p-galactosidase after the engineered Bacteroides strain is administered compared to the level, in the subject, of said immunogenic p- galactosidase in the absence of any administration of said engineered Bacteroides strain.

[0127] In the context of the invention, the reduction of the level of the immunogenic p- galactosidase applies to the specific immunogenic molecule produced by the target Bacteroides strain, and does not encompass any mutant or variant of said p-galactosidase. However, in some embodiments the level of any mutant or variant of said p-galactosidase may further be reduced by the method of the invention.

[0128] By “variant” or “mutant” of the p-galactosidase is meant herein a modified version of the p-galactosidase, compared to the version produced by the target Bacteroides strain(s). Preferably, said variant or mutant of the p-galactosidase does not have the same effect on the subject and/or the same activity as the version of the p-galactosidase produced by the target Bacteroides strain(s). Preferably, said variant or mutant of the p-galactosidase is non immunogenic or less immunogenic than the version of the p-galactosidase produced by the target Bacteroides strain (s).

[0129] In a particular embodiment, the reduction of the level of the immunogenic p-galactosidase is a statistically significant decrease in the level of the immunogenic p-galactosidase or a statistically significant decrease in the ratio of the level of the immunogenic p-galactosidase to the level of a non or less immunogenic mutant or variant of said p-galactosidase.

[0130] In a particular embodiment, said reduction of the level of the immunogenic p- galactosidase is observed 30 min after the first administration of the engineered Bacteroides strain and/or the first administration of the nutrient source, in particular 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 1 month after the first administration of the engineered Bacteroides strain and/or the first administration of the nutrient source.

[0131] In a particular embodiment, said reduction of the level of the immunogenic p- galactosidase is maintained (not necessarily at the same level) for 30 min after the first administration of the engineered Bacteroides strain and/or the first administration of the nutrient source, in particular for 1 hour, 2 hours, 3 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year after the first administration of the engineered Bacteroides strain and/or the first administration of the nutrient source.

[0132] In a particular embodiment, said reduction of the level of the immunogenic p- galactosidase is maintained (not necessarily at the same level) for the whole period during which the engineered Bacteroides strain and/or the nutrient source is administered, in particular is regularly administered.

[0133] The reduction of the level of the immunogenic p-galactosidase by administration of the engineered Bacteroides strain is due to the transient or permanent, partial or total replacement of the target Bacteroides strain(s) containing said gene(s) encoding said immunogenic p- galactosidase, by the engineered Bacteroides strain (which does not contain any gene encoding a p-galactosidase, contains at least one gene encoding a non or less immunogenic p- galactosidase or contains a non-expressed gene encoding an immunogenic p-galactosidase), thanks to the competitive advantage of the engineered Bacteroides strain over the target Bacteroides strain.

P-qalactosidase encoded gene in the engineered Bacteroides strain

[0134] In the context of the invention, the engineered Bacteroides strain (i) does not contain any gene encoding a p-galactosidase, or (ii) contains at least one gene encoding a non or less immunogenic p-galactosidase or (iii) contains a non-expressed gene encoding an immunogenic p-galactosidase.

[0135] In a particular embodiment, said engineered Bacteroides strain naturally does not contain any gene encoding a p-galactosidase.

[0136] By “naturally not contain” is meant herein that the engineered Bacteroides strain has not been engineered to not contain any gene encoding said p-galactosidase. In other words, the engineered Bacteroides strain is obtained from a bacterial strain, which, naturally, without any genetic engineering, does not contain any gene encoding said p-galactosidase.

[0137] In an alternative embodiment, said engineered Bacteroides strain has been engineered to not contain any gene encoding a p-galactosidase. In other words, the engineered Bacteroides strain is both engineered to comprise the heterologous or engineered gene or gene set providing a competitive advantage, and to not contain any gene encoding a p-galactosidase.

[0138] In an alternative embodiment, said engineered Bacteroides strain naturally contains at least one gene encoding a non or less immunogenic p-galactosidase (and no gene encoding an immunogenic p-galactosidase).

[0139] In still an alternative embodiment, said engineered Bacteroides strain naturally contains a non-expressed gene encoding an immunogenic p-galactosidase.

[0140] By “naturally contains” is meant herein that the engineered Bacteroides strain has not been engineered to contain said gene encoding a non or less immunogenic p-galactosidase, or said non-expressed gene encoding an immunogenic p-galactosidase. In other words, the engineered Bacteroides strain is obtained from a bacterial strain, which, naturally, without any genetic engineering, contains a gene encoding a non or less immunogenic p-galactosidase, or contains a non-expressed gene encoding an immunogenic p-galactosidase. Said presence of a non or less immunogenic p-galactosidase-encoding gene can be due to a natural mutation or evolution in the p-galactosidase gene leading to the coding of a non or less immunogenic p- galactosidase. Said absence of expression of said gene encoding an immunogenic p- galactosidase can be due to a natural mutation in the p-galactosidase gene leading to the nonexpression of the protein, or to a mutation in the sequence regulating the expression of the p- galactosidase gene, also leading to the non-expression on the protein. [0141] In an alternative embodiment, said engineered Bacteroides strain has been engineered to contain gene(s) encoding a non or less immunogenic p-galactosidase, or to contain a nonexpressed gene encoding an immunogenic p-galactosidase. In other words, the engineered Bacteroides strain is both engineered to comprise the heterologous or engineered gene or gene set(s) providing a competitive advantage, and to contain gene(s) encoding a non or less immunogenic p-galactosidase or to to contain a non-expressed gene encoding an immunogenic p-galactosidase. In a particular embodiment, said engineered Bacteroides strain thus comprises a genetic modification in the p-galactosidase gene, in particular in the p-galactosidase fragment recognized as epitope by the immune system, rendering said p-galactosidase non or less immunogenic. In an alternative embodiment, said engineered Bacteroides strain comprises a genetic modification in the p-galactosidase gene leading to the non-expression of the protein, or to a mutation in the sequence regulating the expression of the p-galactosidase gene, also leading to the non-expression on the protein.

[0142] By “non immunogenic” is meant herein that said p-galactosidase is not able to induce or sustain an immune response, in particular an autoimmune reaction in a subject. Techniques to determine that a p-galactosidase is non-immunogenic are well-known from the skilled person, and are typically similar to the ones disclosed above to determine that a p-galactosidase is immunogenic.

[0143] By “less immunogenic” is meant herein that said p-galactosidase is able to induce or sustain a weaker immune response, in particular a weaker autoimmune reaction, than the p- galactosidase produced by the target Bacteroides strain. Techniques to determine that a p- galactosidase is less immunogenic than the p-galactosidase produced by the target Bacteroides strain are well-known from the skilled person and typically include comparing the immunogenicity levels obtained with both p-galactosidases using the techniques disclosed above.

[0144] In a particular embodiment, said less immunogenic p-galactosidase is 5% less immunogenic, 10% less, 15% less, 20% less, 25% less, 30% less, 35% less, 40% less, 45% less, 50% less, 55% less, 60% less, 65% less, 70% less, 75% less, 80% less, 85% less, 90% less, 95% less, 90% less, 95% less, 100% less, 150% less, 200% less, or 500% less immunogenic than the p-galactosidase produced by the target Bacteroides strain.

[0145] In a particular embodiment, said non- or less immunogenic p-galactosidase is a p- galactosidase carrying a mutation in the p-galactosidase fragment recognized as epitope by the immune system leading to a weaker or absence of epitope recognition by the immune system.

[0146] By “non-expressed gene encoding an immunogenic p-galactosidase” is meant herein that said gene, while encoding an actual immunogenic p-galactosidase, cannot be expressed by said engineered Bacteroides strain because its transcription and/or translation is directly or indirectly prevented. Ways to directly or indirectly prevent transcription and/or translation of a gene are well-known from the skilled person, and typically include a mutation in the sequence of the promoter of said gene, the presence of a repressor of said promoter, etc... [0147] In a particular embodiment, the reduction of the level of the immunogenic p-galactosidase is only due to the administration of said engineered Bacteroides strain. In other words, in that embodiment, the reduction of the level of immunogenic p-galactosidase is not mediated by another administered additional element, such as an element degrading or sequestering said immunogenic p-galactosidase, an element modifying the target Bacteroides strain(s) in such a way that the immunogenic p-galactosidase is no longer produced or is produced in a non- or less immunogenic version, or an element that kills or reduces the growth the target Bacteroides strain. In particular, in an embodiment, no antibacterial agent is administered to the subject.

[0148] By “antibacterial agent” is meant an agent that either kills or inhibits the growth of a bacteria.

[0149] Examples of antibacterial agents include antibiotics, bacteriocins, endolysins, bacteriolytic enzymes, and phages (in particular prophages or filamentous phages), toxins, polypeptides having a/p-type SASP activity, nucleases, molecules involved in type Vl-secretion system (T6SS)-mediated effector translocation, molecules involved in type Ill-secretion system (T3SS), molecules involved in type IV-secretion system (T4SS), molecules involved in contactdependent inhibition (CDI), molecules involved in contact-dependent inhibition mediated by glycine zipper proteins (Cdz), molecules involved in microcin proximity-dependent inhibition (MccPDI) and any combination thereof.

[0150] In a particular embodiment, said antibacterial agent is a combination of a contactdependent antibacterial agent such as molecules involved in type Vl-secretion system (T6SS)- mediated effector translocation, molecules involved in type Ill-secretion system (T3SS), molecules involved in type IV-secretion system (T4SS), molecules involved in contact-dependent inhibition (CDI), molecules involved in contact-dependent inhibition mediated by glycine zipper proteins (Cdz) or molecules involved in microcin proximity-dependent inhibition (MccPDI), and of a contact-independent antibacterial agent such as antibiotics or bacteriocins.

[0151] In some embodiments, the antibiotic is selected from the group consisting of penicillins such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; cephalosporins such as cefacetrile, cefadroxil, cephalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefonicid, cefprozil, cefuroxime, cefuzonam, cefmetazole, cefotetan, cefoxitin, loracarbef, cefbuperazone, cefminox, cefotetan, cefoxitin, cefotiam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefovecin, cefpimizole, cefpodoxime, cefteram, ceftamere, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, latamoxef, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, flomoxef, ceftobiprole, ceftaroline, ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide, cefuracetime, and nitrocefin; polymyxins such as polysporin, neosporin, polymyxin B, and polymyxin E, rifampicins such as rifampicin, rifapentine, and rifaximin; Fidaxomicin; quinolones such as cinoxacin, nalidixic acid, oxolinic acid, piromidic acid, pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, fleroxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, grepafloxacin, levofloxacin, pazufloxacin, temafloxacin, tosufloxacin, clinafloxacin, gatifloxacin, gemifloxacin, moxifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, delafloxacin, nemonoxacin, and zabofloxacin; sulfonamides such as sulfafurazole, sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole, sulfisomidine, sulfadoxine, sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine, sulfametho-xypyridazine, sulfametoxydiazine, sulfadoxine, sulfametopyrazine, and terephtyl; macrolides such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin, and roxithromycin; ketolides such as telithromycin, and cethromycin; fluoroketolides such as solithromycin; lincosamides such as lincomycin, clindamycin, and pirlimycin; tetracyclines such as demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline; aminoglycosides such as amikacin, dibekacin, gentamicin, kanamycin, neomycin, netilmicin, sisomicin, tobramycin, paromomycin, and streptomycin; ansamycins such as geldanamycin, herbimycin, and rifaximin; carbacephems such as loracarbef; carbapenems such as ertapenem, doripenem, imipenem (or cilastatin), and meropenem; glycopeptides such as teicoplanin, vancomycin, telavancin, dalbavancin, and oritavancin; lincosamides such as clindamycin and lincomycin; lipopeptides such as daptomycin; monobactams such as aztreonam; nitrofurans such as furazolidone, and nitrofurantoin; oxazolidinones such as linezolid, posizolid, radezolid, and torezolid; teixobactin, clofazimine, dapsone, capreomycin, cycloserine, ethambutol, ethionamide, isoniazid, pyrazinamide, rifabutin, arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin (or dalfopristin), thiamphenicol, tigecycline, tinidazole, trimethoprim, alatrofloxacin, fidaxomycin, nalidixic acid, rifampin, derivatives and combination thereof.

[0152] Other examples of antibacterial agents include azaserine, bestatin, D-cycloserine, 1 ,10- phenanthroline, 6-diazo-5-oxo-L-norleucine, L-alanyl-L-1 -aminoethyl-phosphonic acid; aureolic acids such as chromomycin A3, mithramycin A and mitomycin C C; coumarin-glycosides such as novobiocin; diphenyl ether derivatives such as irgasan; epipolythiodixopiperazines such as gliotoxin from Gliocladium fimbriatum; cerulenin; glucosamines such as 1 -deoxymannojirimycin, 1 -deoxynojirimycin and N-methyl-1 -deoxynojirimycin; indole derivatives such as staurosporine; diaminopyrimidines such as iclaprim (AR-100); macrolactams such as ascomycin; statins such as mevastatin.; polyphenolic acids such as (+)-usnic acid; polyethers such as lasalocid A, lonomycin A, monensin, nigericin and salinomycin; picolinic acid derivatives such as fusaric acid; peptidyl nucleosides such as blasticidin S, nikkomycin, nourseothricin and puromycin; nucleosides such as adenine 9-p-D-arabinofuranoside, 5-azacytidine, cordycepin, formycin A, tubercidin and tunicamycin; pleuromutilins such as GSK-565154, GSK-275833 and tiamulin; peptide deformylase inhibitors such as LBM415 (NVP PDF-713) and BB 83698; methenamine (hexamine), doxorubicin, piericidin A, stigmatellin, actidione, anisomycin, apramycin, coumermycin A1 , L(+)-lactic acid, cytochalasins (e.g. cytochalasin B and cytochalasin D), emetine and ionomycin; antiseptic agents such as chlorhexidine, phenol derivatives (e.g. thymol and triclosan), quaternary ammonium compounds (e.g. benzalkonium chloride, cetylpyridinium chloride, benzethonium chloride, cetrimonium bromide, cetrimonium chloride and cetrimonium stearate), octenidine dihydrochloride, and terpenes (e.g. terpinen-4-ol).

[0153] In a particular embodiment, the level of the immunogenic p-galactosidase may be further reduced by the administration of the engineered Bacteroides strain by using an engineered Bacteroides strain, as defined above, which is further able to kill or inhibit the growth of said target Bacteroides strain (s).

[0154] Therefore, in a particular embodiment, said engineered Bacteroides strain produces or further produces an antibacterial agent, as defined above, to which it is resistant, but to which said target Bacteroides strain(s) is sensitive. In a more particular embodiment, said engineered Bacteroides strain produces or further produces an antibacterial agent, as defined above, to which it is resistant, but to which said target Bacteroides strain(s) and other bacteria from the subject are sensitive.

[0155] In the particular embodiment where the engineered Bacteroides strain further produces an antibacterial agent, the antibacterial agent can be for example a bacteriocin or an endolysin. Other examples of antibacterial agents include bacteriolytic enzymes, phages (in particular prophages or filamentous phages), toxins, polypeptides having a/p-type SASP activity, nucleases, molecules involved in type Vl-secretion system (T6SS)-mediated effector translocation, molecules involved in type Ill-secretion system (T3SS), molecules involved in type IV-secretion system (T4SS), molecules involved in contact-dependent inhibition (CDI), molecules involved in contact-dependent inhibition mediated by glycine zipper proteins (Cdz), molecules involved in microcin proximity-dependent inhibition (MccPDI) and any combination thereof.

[0156] By “bacteriocin” is meant herein a proteinaceous or peptidic toxin produced by bacteria to inhibit the growth of other bacterial strain(s).

[0157] Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. Such bacteriocins have been described from gram negative bacteria (e.g. microcins, colicin-like bacteriocins and tailocins) and from gram positive bacteria (e.g. Class I, Class II, Class III or Class IV bacteriocins).

[0158] In one embodiment, said at least one bacteriocin is selected from the group consisting of microcins, colicin-like bacteriocins, tailocins, Class I, Class II, Class III and Class IV bacteriocins. [0159] By “microcins” is meant herein very small bacteriocins, composed of relatively few amino acids, and typically including microcin V (MccV) produced by Escherichia coli and subtilosin A produced by Bacillus subtilis. Examples of microcins include MccB17, MccC, MccD93, MccJ25, MccL, MccV, MccS, MccE492, MccM, MccH47, Mccl47, MccN and MccPDL

[0160] By “colicin-like bacteriocin” or “CLBs" is meant herein bacteriocins found in Gramnegative bacteria, which are modular proteins between 20 and 90 kDa in size and often consist of a receptor binding domain, a translocation domain and a cytotoxic domain. Examples of CLBs typically include colicins, in particular colicins A, B, D, K, E1 , E2, E3, E4, E5, E6, E7, E8, E9, la, lb, M, N, S4, U, Y, 5, 10; klebicins, in particular klebicins A, B, C, CCL, D, KpneA, KaerA, KoxyY, Kvarla, Kpnela, KaerM, KpneM (or Kpne CHS1 10), KpneM2 (or Kpne e1602) and KvarM (or Kvar 6A2); alveicins, in particular alveicins A and B; marcescins, in particular marcescins A, B and 28B; S-type pyocins, in particular pyocins S1 , S2, S3, S5, S4, AP41 ; cloacins, in particular cloacin DF13; and pesticin.

[0161] By “tailocin” is meant herein a multisubunit bacteriocin that resembles bacteriophage tails. There are two classes of tailocin particles, the flexible noncontractile F-tailocins and the rigid contractile R-tailocins, which resemble and are evolutionarily related to Siphoviridae and Myoviridae phage tails, respectively. Examples of tailocins typically include F-type and R-type pyocins, carotovoricin, xenorhabdicin, and maltocin.

[0162] As used herein, the term “Class I bacteriocin” refers to small peptide inhibitors which include nisin and other lantibiotics. Examples of Class I bacteriocins typically include type A lantibiotics such as nisin A, nisin Z, bisin, subtilin, epidermin, gallidermin, mutacin II, mutacin I, mutacin III, pep5, epicidin 280, epilancin K7, lacticin 481 , lacticin 3147, cytolysin, staphylococcin C55, salvaricin A, lactocin S, streptococcin A-FF2, sublancin 168, carnocin U149, variacin 8 and cypemycin; and type B lantibiotics such as mersacidin, actagardine, duramycin B, duramycin C, cinnamycin, ancovenin, and plantaricin C.

[0163] As used herein, the term “Class II bacteriocin” refers to small (<10 kDa) heat-stable bacteriocins, subdivided into five subclasses: the class Ila bacteriocins (pediocin-like bacteriocins), which correspond to the largest subgroup and contain an N-terminal consensus sequence across this group and a C-terminal region responsible for species-specific activity, causing cell-leakage by permeabilizing the target cell wall; the class lib bacteriocins (two-peptide bacteriocins) which require two different peptides for activity; the class lie bacteriocins which encompass cyclic peptides, in which the N-terminal and C-terminal regions are covalently linked; the class lid bacteriocins which cover single-peptide bacteriocins, which are not post- translationally modified and do not show the pediocin-like signature; and the class He bacteriocins, which encompass those bacteriocins composed of three or four non-pediocin like peptides. Examples of class Ila bacteriocins typically include pediocin, pediocin A, pediocin AcH, pediocin PA-1 , pediocin PP-1 , pediocin SJ-1 , prepediocin AcH, prepediocin PA-1 , mesentericin Y105, mesentericin 52A, carnobacteriocin B2, carnobacteriocin BM1 , sakacin A, sakacin G, sakacin P, sakacin X, enterocin A, enterocin BC25, enterocin P, enterocin P-like, enterocin CRL35, enterocin HF, enterocin SE-K4, leucocin A, leucocin B-Ta11 a, leucocin C, leucocin C- TA33a, curvacin A, listeriocin 743A, avicin A, bavaricin A/SppA, curvaticin L442, mundticin, mundticin CRL35, mundticin KS, mundticin L, mundticin QU2, pediocin ACCEL, piscicocin CS526, piscicolin 126, piscicolin 126, piscicocin Vi a, bifidocin B, CoaA/Coagulin/CoaA, mutacin F-59.1 , PapA, weissellin A, bacteriocin 602, bavaricin MN, divercin V41 , divergicin M35, duracin GL, bacteriocin 31 /BacA, bacteriocin 1580, bacteriocin 43, bacteriocin RC714, bacteriocin T8, hiracin JM79, penocin A/PenA, bacteriocin MC4-1 , carnocin CP52, plantaricin 423, plantaricin C19, prebacteriocin SkgA2, lactococcin MMFII, ubericin A, piscicocin V1 b, bacteriocin E50-52, bacteriocin L-1077, bacteriocin 37, acidocin A, and bacteriocin OR-7. Examples of class lib bacteriocins typically include enterocin C, enterocin 1071 , gassericin T, gassericin S, lactococcin G, lactococcin Q, plantaricin E/F, plantaricin J/K, plantaricin S, plantaricin NC8, lactacin F, brochocin-C, thermophilin 13, ABP-118, salivaricin P, mutacin IV and lactocin 705. Examples of class He bacteriocins typically include enterocin AS-48, lactocyclicin Q, garvicin ML, gassericin A, acidocin B and butyrovibriocin AR10. Examples of class lid bacteriocins typically include aureocin A53, garvicin A, laterosporulinl O, lactococcin A, lactococcin 972, lacticin Q, carnobacteriocin XY, leucocin B, thuricin S, thuricin-17 and bactofensin A. Examples of class lie bacteriocins typically include aureocin A70.

[0164] As used herein, the term “Class III bacteriocin” refers to large (>10 kDa), heat-labile protein bacteriocins. This class is subdivided in two subclasses: subclass Illa (bacteriolysins) and subclass 11 lb. Subclass Illa comprises those peptides that kill bacterial cells by cell wall degradation, thus causing cell lysis, and typically include Lysostaphin. Subclass II lb, in contrast, comprises those peptides that do not cause cell lysis, killing the target cells by disrupting plasma membrane potential. Examples of class III bacteriocins typically include Lysostaphin, enterolysin A, helveticin V-1829, helveticin J, caseicin 80, lactacin A, lactacin B, zoocin A, millericin B, linocin M18 and acidophilus A.

[0165] As used herein, the term “Class IV bacteriocin” refers to complex bacteriocins containing lipid or carbohydrate moieties. Examples of class IV bacteriocins typically include sublancin 168, glycocin F, ASM1 , enterocin 96 and enterocin F4-9.

[0166] In a particular embodiment, the bacteriocin is a bacteroidetocin, as disclosed for example in Coyne et al. (2019) Nat. Commun. 10:3460.

[0167] By “endolysin” or “lysin” is meant herein enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. They are typically either P(1 ,4)-glycosylases (lysozymes), transglycosylases, amidases or endopeptidases. Examples of endolysins typically include PhiV10p30, STM0907.Fels0, epsilon15p25, YuA20, ORF23, BcepMu22, F1 16p62, STM2715.S.Fels2, gp76, SPSV3_gp23, phi32_17, HK022p54, HK97p58, HK620p36, VIP0007, Sf6p62, R (SfVp40), gp22, Nazgul38, K (P2p09), K (Wphi09), rv5_gp085, EpJS98_gp116, gp3.5 (from 13A phage), gp3.5 (from BAM phage), gp3.5 (from ECODS1 phage), CKV1 F_gp16, T3p18, gh-1 p12, gp3.5 (from K1 1 phage), ORF12, Bcep43-27, Bcep781 -27, Bcep1 -28, BcepNY3gene26, gp45, gp28, P27p30, RB49p102, phi1 -p102, lys (T5.040), Aeh1 p339, YYZgp45, cpSH2 lysin, lysin from STB12 phage, PlyP40, endolysin from phi11 phage, endolysins from the Pseudomonas aeruginosa phages DKZ and EL, endolysins of the Pseudomonas putida phage, endolysins of the E. coli phage N4, endolysins of the phage LUZ24, gp61 muramidase, STM0016 endolysin, PSP3 endolysin, phiKZgp144, ELgp188, Salmonella endolysin, Enterobacteria phage T4 endolysin, Acinetobacter baumanii endolysin, E. coli phage KIF endolysin, OBPgpLYS, PSP3 Salmonella endolysin (PSP3gp1 ), E. coli phage P2 endolysin (P2gp9), Salmonella typhimurium phage muramidase STMOO16, E. coli phage N4 muramidase N4-gp61 and KZ144. Examples of endolysins also include endolysins disclosed in Fernandez- Ruiz et al. (2018) Front. Microbiol. 9:1033.

[0168] In a particular embodiment, said antibacterial agent is produced from a gene or set of genes derived from a bacterium which is from the same species as the engineered Bacteroides strain. In another particular embodiment, said antibacterial agent is produced from a gene or set of genes derived from a bacterium which is from a different species than the engineered Bacteroides strain.

[0169] In a particular embodiment, said engineered Bacteroides strain produces several antibacterial agents targeting different bacterial strains or different bacterial species.

[0170] In a particular embodiment, said engineered Bacteroides strain produces at least one antibacterial agent targeting a bacterial strain from the same species as the engineered Bacteroides strain, and at least one different antibacterial agent targeting a bacterial species from another species than the engineered Bacteroides strain.

[0171] In a particular embodiment, said engineered Bacteroides strain further comprises a gene or gene set conferring resistance to said antibacterial agent, in particular to said bacteriocin, type Vl-secretion system (T6SS) or antibiotic.

[0172] In a particular embodiment, in particular when said antibacterial agent is a nuclease or a toxin, said antibacterial agent is produced in the target Bacteroides strain after conjugation. In other words, in a particular embodiment, said engineered Bacteroides strain is able to transduce, in the target Bacteroides strain, a plasmid encoding an antibacterial agent, as defined above.

[0173] In a particular aspect of the invention, the Bacteroides strain used in the context of the invention is engineered in situ. In other words, in all the methods of the invention, the method can alternatively comprise: administering to the subject or providing to the environment a bacterial delivery vehicle for delivery into a Bacteroides strain of interest, wherein said bacterial delivery vehicle comprises:

(A) (a1 ) a heterologous gene or gene set providing a competitive advantage, as defined above, to the Bacteroides strain of interest, or (a2) a nucleic acid encoding a gene editing enzyme/system designed to modify the genome of said Bacteroides strain of interest so that said Bacteroides strain of interest has a competitive advantage, as defined above, and

(B) optionally a nucleic acid encoding a gene editing enzyme/system designed to modify the genome of said Bacteroides strain of interest so that said Bacteroides strain of interest (i) does not contain any gene encoding p-galactosidase, (ii) contains gene(s) encoding a non- or less immunogenic p-galactosidase, or (iii) contains a non-expressed gene encoding an immunogenic p-galactosidase, as defined above, wherein said gene editing enzyme/system does not lead to the death of the bacterial strain of interest, whereby said Bacteroides strain of interest is engineered in situ to (i) not contain any gene encoding p-galactosidase, (ii) contain gene(s) encoding a non- or less immunogenic p- galactosidase, or (iii) contain a non-expressed gene encoding an immunogenic p- galactosidase, and to have a competitive advantage.

[0174] By “bacterial delivery vehicle” is meant herein any mean that allows the transfer of a payload into a bacterium.

[0175] There are several types of delivery vehicle encompassed by the present invention including, without limitation, bacteriophage scaffold, virus scaffold, chemical based delivery vehicle (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes), proteinbased or peptide-based delivery vehicle, lipid-based delivery vehicle, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (e.g., transformation, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment, cell-penetrating peptides) or donor bacteria (conjugation).

[0176] Any combination of delivery vehicles is also encompassed by the present invention.

[0177] The delivery vehicle can refer to a bacteriophage derived scaffold and can be obtained from a natural, evolved or engineered capsid.

[0178] In some embodiments, the delivery vehicle is the payload as bacteria are naturally competent to take up a payload from the environment on their own.

[0179] In a particular embodiment, said bacterial delivery vehicle is a packaged phagemid, said heterologous genes and/or nucleic acids being located on the phagemid.

Method of therapeutic treatment

[0180] The present invention concerns a method for preventing and/or treating, in a subject in need thereof, acute inflammatory myocarditis associated with at least one p-galactosidase- containing commensal Bacteroides strain, as defined above, and/or sequelae thereof, said method comprising administering to the subject a therapeutically efficient amount of at least one engineered Bacteroides strain, as defined above, wherein said engineered Bacteroides strain comprises a heterologous or engineered gene or gene set providing a competitive advantage, as defined above, over the at least one target Bacteroides strain, and wherein said engineered Bacteroides strain (i) does not contain any gene encoding a p- galactosidase, (ii) contains gene(s) encoding a non or less immunogenic p-galactosidase, as defined above, or (iii) contains a non-expressed gene encoding an immunogenic p- galactosidase, as defined above.

[0181] The present invention further concerns an engineered Bacteroides strain for use in a method for preventing and/or treating, in a subject, acute inflammatory myocarditis associated with at least one target Bacteroides strain, as defined above, and/or sequelae thereof, wherein said engineered Bacteroides strain comprises a heterologous or engineered gene or gene set providing a competitive advantage, as defined above, over the at least one target Bacteroides strain, and wherein said engineered Bacteroides strain (i) does not contain any gene encoding a p-galactosidase, (ii) contains gene(s) encoding a non or less immunogenic p-galactosidase, as defined above, or (iii) contains a non-expressed gene encoding an immunogenic p- galactosidase, as defined above.

[0182] The present invention also relates to the use of an engineered Bacteroides strain in the manufacture of a medicament intended for the prevention and/or treatment, in a subject, of acute inflammatory myocarditis associated with at least one target Bacteroides strain, as defined above, and/or sequelae thereof, wherein said engineered Bacteroides strain comprises a heterologous or engineered gene or gene set providing a competitive advantage, as defined above, over the at least one target Bacteroides strain, and wherein said engineered Bacteroides strain (i) does not contain any gene encoding a p-galactosidase, (ii) contains gene(s) encoding a non or less immunogenic p-galactosidase, as defined above, or (iii) contains a non-expressed gene encoding an immunogenic p-galactosidase.

[0183] By “acute inflammatory myocarditis” is meant herein an inflammatory heart disease which can be triggered by a wide variety of pathogens or other triggers, with viral infections as the most frequent initiators (Tschope et al. (2019) Circulation Research 124(1 1 ), 1568-1583). Most patients diagnosed with acute myocarditis recover without clinically relevant residual damage, however, in 47% of cases, permanent cardiomyopathy and damage is observed (including impaired ejection fraction) (Tschope et al. (2019) Circulation Research 124(1 1 ), 1568-1583). Acute myocarditis can be prolonged, worsened or progress into chronic inflammatory cardiomyopathy through the presence of persistently autoimmune reactivity, directed towards myosin heavy chain 6 (MYH6) peptide, which is released when cardiac cells are damaged by the viral or otherwise-induced infection and/or inflammation of the heart. This immune reactivity includes MYH6-specific T lymphocytes and antibodies. However, the mechanisms that govern the worsening of this disease or the progression to more permanent cardiomyopathy has remained less clear.

[0184] It is well established that acute immune activation after infectious (or otherwise triggered) myocarditis is associated with the generation of autoimmune responses against myosin heavy chain 6 (MYH6) (Rose et al. (2014) FWOOPrime Rep 6, 25; Trachtenberg et al. (2017) Circ Res 121 , 803-818; Krebs et al. (2007) J. Autoimmun 28, 224-233), while subsequent chronic stimulation of MYH6-specific immune-reactivity including Th1 and Th17 cells precipitates inflammatory cardiomyopathy (Rangachari et al. (2006) J. Exp. Med 203, 2009-2019; Lv et al. (201 1 ) J. Clin. Invest 121 , 1561 -1573; Nindl et al. (2012) Eur J Immunol 42, 2311 -2321 ; Myers et al. (2016) JCI Insight 1 ).

[0185] Sequelae from acute inflammatory myocarditis are well-known from the skilled person and include heart damage, and conditions resulting therefrom such as heart failure, arrhythmia, myocardial infarction, and sudden cardiac arrest.

[0186] In the context of the invention, the term "treating" or "treatment" means reversing, alleviating, or inhibiting the progress of the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.

[0187] In the context of the invention, the term "prevention" refers to any indicia of success in protecting a subject or patient (e.g. a subject or patient at risk of developing a disease or condition) from developing, contracting, or having a disease or condition, including preventing one or more symptoms of a disease or condition or diminishing the occurrence, severity, or duration of any symptoms of a disease or condition following administration of the engineered bacterial strain as described herein.

[0188] By a "therapeutically effective amount" of an engineered Bacteroides strain of the invention is meant a sufficient amount of the engineered Bacteroides strain to treat a specific disease, to contribute to the treatment of a specific disease, or to avoid side effects of a treatment of a specific disease, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the engineered Bacteroides strain of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treating and the severity of the disorder, activity of the specific engineered Bacteroides strain employed, the specific combinations employed, the age, body weight, general health, sex and diet of the subject, the time of administration, route of administration and rate of excretion of the specific engineered Bacteroides strains employed, the duration of the treatment, drugs used in combination or coincidental with the specific engineered Bacteroides strains employed, and like factors well known in the medical arts. [0189] In particular embodiments, the engineered Bacteroides strain of the invention is in a pharmaceutical composition.

[0190] The pharmaceutical composition according to the invention may further comprise a pharmaceutically acceptable excipient.

[0191] By “pharmaceutically acceptable excipient” is meant herein a non-pharmaceutically active additive used in the manufacture of a pharmaceutical composition, which allows the pharmaceutically active ingredient to be manufactured into a pharmaceutical formulation or a galenic formulation providing the necessary bioavailability of the medicament to the patient upon the administration of the pharmaceutical composition. The excipient is preferably compatible with the other ingredients of the composition and produces no adverse effect, allergic reaction or other undesirable reaction when it is administered to a human or an animal.

[0192] A solid pharmaceutically acceptable vehicle or excipient may include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.

[0193] The pharmaceutical composition may be prepared as a sterile solid composition that may be suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium. The pharmaceutical compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The engineered Bacteroides strains according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for enteral administration include sterile solutions, emulsions, and suspensions.

[0194] The engineered Bacteroides strains according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0195] In some embodiments, the invention encompasses pharmaceutical composition formulated for delayed or gradual enteric release. In preferred embodiments, formulations or pharmaceutical preparations of the invention are formulated for delivery of the engineered bacterial strain into the distal small bowel and/or the colon. The formulation can allow the engineered Bacteroides strain to pass through stomach acid and pancreatic enzymes and bile, and reach undamaged to be viable in the distal small bowel and colon.

[0196] In some embodiments, the pharmaceutical composition is micro-encapsulated, formed into tablets and/or placed into capsules, preferably enteric-coated capsules.

[0197] In some embodiments, the pharmaceutical compositions are formulated for delayed or gradual enteric release, using cellulose acetate (CA) and polyethylene glycol (PEG). In some embodiments, the pharmaceutical compositions are formulated for delayed or gradual enteric release using a hydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC) and magnesium stearate. In some embodiments, the pharmaceutical compositions are formulated for delayed or gradual enteric release using e.g., a poly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methyl methacrylate and/or a methacrylic acid ester, or a polyvinylpyrrolidone (PVP).

[0198] In some embodiments, the pharmaceutical compositions are formulated for delayed or gradual enteric release using a release-retarding matrix material such as: an acrylic polymer, a cellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil, polyvinylpyrrolidone, a vinyl acetate copolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylic acid and methacrylic acid copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylate copolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylic acid alkylamide copolymer, a poly(methyl methacrylate), a poly(methacrylic acid anhydride), a methyl methacrylate polymer, a polymethacrylate, a poly(methyl methacrylate) copolymer, a polyacrylamide, an aminoalkyl methacrylate copolymer, a glycidyl methacrylate copolymer, a methyl cellulose, an ethylcellulose, a carboxymethylcellulose, a hydroxypropylmethylcellulose, a hydroxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropyl cellulose, a crosslinked sodium carboxymethylcellulose, a crosslinked hydroxypropylcellulose, a natural wax, a synthetic wax, a fatty alcohol, a fatty acid, a fatty acid ester, a fatty acid glyceride, a hydrogenated fat, a hydrocarbon wax, stearic acid, stearyl alcohol, beeswax, glycowax, castor wax, carnauba wax, a polylactic acid, polyglycolic acid, a co-polymer of lactic and glycolic acid, carboxymethyl starch, potassium methacrylate/divinylbenzene copolymer, crosslinked polyvinylpyrrolidone, polyvinylalcohols, polyvinylalcohol copolymers, polyethylene glycols, non-crosslinked polyvinylpyrrolidone, polyvinyl acetates, polyvinylacetate copolymers or any combination thereof. [0199] In some embodiments, the pharmaceutical compositions are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 20110218216, which describes an extended release pharmaceutical composition for oral administration, and uses a hydrophilic polymer, a hydrophobic material and a hydrophobic polymer or a mixture thereof, with a microenvironment pH modifier. The hydrophobic polymer can be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, methacrylic acid-acrylic acid copolymers or a mixture thereof. The hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymers or a mixture thereof. The hydrophobic material can be a hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candelilla wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and a mixture thereof. The microenvironment pH modifier can be an inorganic acid, an amino acid, an organic acid or a mixture thereof. Alternatively, the microenvironment pH modifier can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid, mesylic acid or malic acid or a mixture thereof. [0200] In some embodiments, the pharmaceutical compositions are a powder that can be included into a tablet or a suppository. In alternative embodiments, a formulation or pharmaceutical preparation of the invention can be a “powder for reconstitution” as a liquid to be drunk or otherwise administered.

[0201] In some embodiments, the pharmaceutical compositions can be administered in a cream, gel, lotion, liquid, feed, or aerosol spray. Engineered Bacteroides strains may be immobilized onto appropriately sized polymeric beads so that the coated beads may be added to aerosols, creams, gels or liquids. The size of the polymeric beads may be from about 0.1 pm to 500 pm, for example 50 pm to 100 pm. The coated polymeric beads may be incorporated into animal feed, including pelleted feed and feed in any other format, incorporated into any other edible device used to present phage to the animals, added to water offered to animals in a bowl, presented to animals through water feeding systems. In some embodiments, the compositions are used for treatment of surface wounds and other surface infections using creams, gels, aerosol sprays and the like.

[0202] In some embodiments, the pharmaceutical compositions can be administered by inhalation, in the form of a suppository or pessary, topically (e.g., in the form of a lotion, solution, cream, ointment or dusting powder), epi- or transdermally (e.g., by use of a skin patch), orally (e.g., as a tablet, which may contain excipients such as starch or lactose), as a capsule, ovule, elixirs, solutions, or suspensions (each optionally containing flavoring, coloring agents and/or excipients), or they can be injected parenterally (e.g., intravenously, intramuscularly or subcutaneously). For parenteral administration, the compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. For buccal or sublingual administration the compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.

[0203] In some embodiments, the pharmaceutical compositions can also be dermally or transdermally administered. For topical application to the skin, the pharmaceutical composition can be combined with one or a combination of carriers, which can include but are not limited to, an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, proteins carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof. A topical mode of delivery may include a smear, a spray, a bandage, a time-release patch, a liquid-absorbed wipe, and combinations thereof. The pharmaceutical composition can be applied to a patch, wipe, bandage, etc., either directly or in a carrier(s). The patches, wipes, bandages, etc., may be damp or dry, wherein the engineered bacterial strain is in a lyophilized form on the patch. The carriers of topical compositions may comprise semi-solid and gel-like vehicles that include a polymer thickener, water, preservatives, active surfactants, or emulsifiers, antioxidants, sun screens, and a solvent or mixed solvent system. U.S. Pat. No. 5,863,560 discloses a number of different carrier combinations that can aid in the exposure of skin to a medicament, and its contents are incorporated herein.

[0204] For intranasal or administration by inhalation, the pharmaceutical composition is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, or nebuliser with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3, 3-heptafluoropropane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray, or nebuliser may contain a solution or suspension of the active compound, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the engineered Bacteroides strain of the invention and a suitable powder base such as lactose or starch.

[0205] For administration in the form of a suppository or pessary, the pharmaceutical composition can be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting powder. Compositions of the invention may also be administered by the ocular route. For ophthalmic use, the compositions of the invention can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum. [0206] Dosages and desired drug concentrations of the pharmaceutical compositions of the present invention may vary depending on the particular use. The determination of the appropriate dosage or route of administration is within the skill of an ordinary physician. Animal experiments can provide reliable guidance for the determination of effective doses in human therapy.

[0207] For transdermal administration, the pharmaceutical composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

[0208] For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compounds can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.

Subject, regimen and administration

[0209] The subject according to the invention is an animal, preferably a mammal, even more preferably a human. However, the term "subject" can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils, hamsters, chinchillas, rats, mice, guinea pigs and non-human primates, among others, or non-mammals such as poultry, that are in need of treatment.

[0210] The human subject according to the invention may be a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult at any age.

[0211] In a particular embodiment, the subject has been diagnosed with, or is at risk of developing acute inflammatory myocarditis. Diagnostic methods of such acute inflammatory myocarditis are well known by the man skilled in the art.

[0212] In a particular embodiment, the subject is carrying a MHC HLA-DQ haplotype which is able to present human MYH6 peptide including MYH6ei 4-629 (typically of sequence SEQ ID NO: 39); more particularly (i) a HLA-DQB1 *03 variant (in particular encoded by DQB1 *03:01 , DQB1 *03:02, DQB1 *03:03, DQB1 *03:04, or DQB1 *03:05 alleles) and/or (ii) a HLA-DQA1 *01 :02 or a HLA-DQA1 *05:01 variant; still particularly a HLA-DQ7 (for example encoded by DQA1 *02:01/DQB1 *03:01 , DQA1 *03:01/DQB1 *03:01 , DQA1 *03:03/DQB1 *03:01 ,

DQA1 *03:01/DQB1 *03:04, DQA1 *03:02/DQB1 *03:04, DQA1 *04:01 /DQB1 *03:01 ,

DQA1 *05:05/DQB1 *03:01 , or DQA1 *06:01/DQB1 *03:01 alleles), HLA-DQ8 (for example encoded by DQA1 *03:01/DQB1 *03:02 or DQA1 *03:02/DQB1 *03:02 alleles) or HLA-DQ9 (for example encoded by DQA1 *02:01/DQB1 *03:03 or DQA1 *03:02/DQB1 *03:03 alleles) haplotype. [0213] In a particular embodiment, the subject is suffering from the permanent sequelae, including cardiomyopathy, from acute inflammatory myocarditis.

[0214] In another particular embodiment, the subject has previously been vaccinated. In a more particular embodiment, the subject has previously been vaccinated against SARS-Cov-2. [0215] Acute myocarditis can occur in previously healthy patients following receipt of an mRNA COVID-19 vaccine (Montgomery et al. (2021 ) JAMA Cardiology, 6:1202-1206). In these rare cases of vaccine-associated acute myocarditis, no other etiologies for myocarditis were identified and symptoms observed in these patients included reduced left ventricular ejection fractions. A potential explanation for the appearance of vaccine-associated AIM in these patients is an underlying presence of B. theta induced p-gal specific immune responses, including p-gal- specific T cells, able to cross-react with MYH6-peptides potentially released by any vaccine- induced (sub-clinical) cardiac damage.

[0216] In a particular embodiment, the subject experiences a treatment- or disease-induced loss of self-recognition, resulting in acute inflammatory myocarditis pathology.

[0217] In a particular embodiment, said disease-induced loss of self-recognition resulting in acute inflammatory myocarditis is acute lupus myocarditis.

[0218] Lupus-associated myocarditis is a rare but potentially fatal complication affecting up to 10% of SLE patients (Appenzeller et al. (2011 ) Lupus 20(9):981 -988; Meridor et al. (2021 ) Medicine 100(18) :e25591 ). It may present as an acute illness or have a chronic course with the development of cardiomyopathy, with symptomatic left ventricular dysfunction as the most common clinical presentation of the cardiomyopathy (e.g. reduced left ventricular ejection fraction). A potential explanation for Lupus-associated acute inflammatory myocarditis is the presence of B. theta induced p-gal specific immune responses, including p-gal-specific T cells in these patients. The T cells are able to cross-react with MYH6-peptides potentially released by any Lupus-induced (sub-clinical) cardiac damage. The p-gal specific T cells are potentially capable of triggering and/or worsening the myocarditis.

[0219] In a particular embodiment, said treatment-induced loss of self-recognition resulting in acute inflammatory myocarditis is checkpoint inhibitor-associated myocarditis.

[0220] Such myocarditis is disclosed in Palaskas et al. (2020) J Am Heart Assoc. 21 ;9(2):e013757. A potential explanation for the appearance of checkpoint inhibitor-associated acute inflammatory myocarditis in these patients is an underlying presence of B. theta induced P-gal specific immune responses, including p-gal-specific T cells, able to cross-react with MYH6- peptides potentially released by any checkpoint inhibitor-induced (sub-clinical) cardiac damage. [0221] In a particular embodiment, the acute inflammatory myocarditis presents a resistance to treatment.

[0222] In a particular embodiment, the subject has never received any treatment prior to the administration of the engineered Bacteroides strain according to the invention.

[0223] In a particular embodiment, the subject has already received at least one line of treatment, preferably several lines of treatment, prior to the administration of the engineered Bacteroides strain according to the invention.

[0224] Preferably, the treatment is administered regularly, preferably between every day and every month, more preferably between every day and every two weeks, more preferably between every day and every week, even more preferably the treatment is administered every day. In a particular embodiment, the treatment is administered several times a day, preferably 2 or 3 times a day, even more preferably 3 times a day.

[0225] The duration of treatment according to the invention is preferably comprised between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks. In a particular embodiment, the duration of the treatment is about 1 week. Alternatively, the treatment may last as long as the acute inflammatory myocarditis persists.

[0226] In a particular embodiment, the duration of treatment according to the invention is comprised between 2 days and 20 weeks and involves (i) several successive administrations of the engineered Bacteroides strain and of the nutrient source, or (ii) a single administration of the engineered Bacteroides strain and several successive administrations of the nutrient source, or (iii) several successive administrations of the engineered Bacteroides strain and several successive administrations of the nutrient source, the administrations of the nutrient source being continued at least once after stopping administrations of the engineered Bacteroides strain.

[0227] The form of the pharmaceutical compositions comprising the engineered Bacteroides strain of the invention and/or the nutrient source, the route of administration and the dose of administration thereof can be adjusted by the man skilled in the art according to the type and severity of the disease, and to the patient or subject, in particular its age, weight, sex, and general physical condition.

[0228] Particularly, the amount of engineered Bacteroides strains according to the invention and/or the amount of nutrient source to be administered has to be determined by standard procedure well known by those of ordinary skills in the art. Physiological data of the patient or subject (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient or subject.

[0229] For example, the total amount of engineered Bacteroides strain, for each administration may be comprised between 1 billion and 100 billion cfu of engineered bacteria, from 5 billion to 50 billion cfu, or from 10 billion to 25 billion cfu of engineered bacteria.

[0230] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0231] All publications mentioned herein are incorporated herein by reference. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0232] It must be noted that as used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells (e.g., a population of such cells). Similarly, reference to “a nucleic acid” includes one or more of such nucleic acids.

Brief description of the sequences

Examples

Example 1 : Method to stop the progression of myocarditis by replacing an immunogenic- peptide producing bacterial population

[0233] It is herein disclosed a method to stop the progression of myocarditis towards debilitating cardiomyopathy, with significant mortality, by in-situ reduction or replacement of the resident Bacteroides faecis and/or Bacteroides thetaiotaomicron strains with a cocktail of one Bacteroides faecis strain and one Bacteroides thetaiotaomicron strains, both engineered to express a beta- galactosidase with decreased homology to human MYH6 cardiac peptide and therefore reduced immunogenic potential.

[0234] In the present method, patients diagnosed with Acute Inflammatory Myocarditis (AIM) are screened for the presence of Bacteroides thetaiotaomicron and/or Bacteroides faecis in their stool and the presence of antibodies against these strains in their serum.

[0235] For patients colonized with B. thetaiotaomicron, a strain of B. thetaiotaomicron with an engineered version of beta-galactosidase genes that leads to the expression of an active betagalactosidase with reduced homology to human MYH6 cardiac peptide, wherein said strain has further been engineered to be able to internalize and metabolize porphyran is administered to the patient, together with porphyran.

[0236] For patients colonized with B. faecis, a strain of B. faecis with an engineered version of beta-galactosidase genes that leads to the expression of an active beta-galactosidase with reduced homology to human MYH6 cardiac peptide, wherein said strain has further been engineered to be able to internalize and metabolize porphyran is administered to the patient, together with porphyran.

[0237] For patients colonized with both B. thetaiotaomicron and B. faecis, (i) a strain of B. thetaiotaomicron with an engineered version of beta-galactosidase genes that leads to the expression of an active beta-galactosidase with reduced homology to human MYH6 cardiac peptide, wherein said strain has further been engineered to be able to internalize and metabolize porphyran, and (ii) a strain of B. faecis with an engineered version of beta-galactosidase genes that leads to the expression of an active beta-galactosidase with reduced homology to human MYH6 cardiac peptide, wherein said strain has further been engineered to be able to internalize and metabolize porphyran, are administered to the patient, together with porphyran.

[0238] The preparation of the administered pharmaceutical composition is achieved by growing both strains separately in fermenters at high titers, followed by formulation into a single stable drug product or two separate stable drug products.

[0239] The administration results in colonization of the patient gut by the engineered B. thetaiotaomicron and/or B. faecis strains in which the immunogenicity of a peptidic sequence within the beta-galactosidase is reduced, leading to significant reduction or even total replacement of the resident population of B. thetaiotaomicron, or B. faecis, or both in the patient, which translates into a decrease of the level of highly immunogenic beta-galactosidase proteins with strong homology with human MYH6 cardiac peptide in the patient gut. Additional administrations over time further result in a diminution, in the patient, of the percentage of Bacteroides having a peptidic sequence of Bacteroides beta-galactosidase with high homology with human MYH6 cardiac peptide, while increasing, in the patient, the percentage of Bacteroides having a peptidic sequence of Bacteroides beta-galactosidase with low homology with human MYH6 cardiac peptide. In this way, the exposure of the patient to the negative auto-immune effects caused by the immune cross-reactivity of the Bacteroides thetaiotaomicron and/or Bacteroides faecis beta-galactosidase protein and the human MYH6 cardiac peptide is minimized.

Example 2: Replacement, in mice gut, of a B. thetaiotaomicron strain expressing a betagalactosidase protein with high homology to human MYH6 cardiac peptide by an engineered B. thetaiotaomicron strain expressing a beta-galactosidase protein with low homology to human MYH6 cardiac peptide and a competitive advantage

[0240] Mice comprising, in their gut, a B. thetaiotaomicron strain expressing a beta-galactosidase protein with high homology to human MYH6 cardiac peptide are used.

[0241] An engineered B. thetaiotaomicron strain wherein the gene encoding beta-galactosidase has been inactivated is produced. Said strain is further engineered to be resistant to a specific antibiotic, which confers to this strain a competitive advantage over the B. thetaiotaomicron strain present in the mice gut.

[0242] The engineered B. thetaiotaomicron strain is orally administered to the mice, together with the antibiotic, prior to or after the antibiotic.

[0243] This administration results in a diminution, in the mice gut, of the percentage of B. thetaiotaomicron expressing a beta-galactosidase protein with high homology to human MYH6 cardiac peptide, while increasing, in the mice gut, the percentage of B. thetaiotaomicron expressing a beta-galactosidase protein with low homology to human MYH6 cardiac peptide. This leads to the replacement, in the mice gut, of the B. thetaiotaomicron strain expressing a beta-galactosidase protein with high homology to human MYH6 cardiac peptide by a B. thetaiotaomicron strain expressing a beta-galactosidase protein with low homology to human MYH6 cardiac peptide.

Claims

1. A method for preventing or treating, in a subject in need thereof, acute inflammatory myocarditis associated with at least one immunogenic p-galactosidase encoding genetcontaining commensal Bacteroides strain (target Bacteroides strain) and/or sequelae thereof, said method comprising administering to the subject a therapeutically efficient amount of an engineered Bacteroides strain, wherein said engineered Bacteroides strain comprises a heterologous or engineered gene or gene set providing a competitive advantage over said at least one target Bacteroides strain, and wherein said engineered Bacteroides strain (i) does not contain any gene encoding a p- galactosidase, (ii) contains gene(s) encoding a non or less immunogenic p-galactosidase, or (iii) contains a non-expressed gene encoding an immunogenic p-galactosidase.

2. The method according to claim 1 , wherein said heterologous gene or gene set providing a competitive advantage is from another species than the engineered Bacteroides strain.

3. The method according to claim 1 or 2, wherein said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene involved in the import and/or metabolism of a nutrient source.

4. The method according to any one of claims 1 to 3, wherein said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene encoding a transporter of a nutrient source.

5. The method according to claim 3 or 4, wherein said nutrient source is a rare carbohydrate.

6. The method according to claim 5, wherein less than 50% of other bacterial cells in the subject utilize the rare carbohydrate as a nutrient source.

7. The method according to claim 5 or 6, wherein the rare carbohydrate is a polysaccharide.

8. The method according to claim 5 or 6, wherein the rare carbohydrate is a sulfated carbohydrate.

9. The method according to claim 5 or 6, wherein the rare carbohydrate is selected from the group consisting of alginate, fucoidan, laminarin, xylan, galactans, and any combination thereof.

10. The method according to claim 9, wherein the rare carbohydrate is selected from the group consisting of porphyran, agarose, carrageenan, ulvan, xylan and any combination thereof.

11. The method according to claim 9 or 10, wherein the rare carbohydrate is porphyran.

12. The method according to claim 5 or 6, wherein the rare carbohydrate is a carbohydrate cleaved by a glycoside hydrolase belonging to glycoside hydrolase family GH86.

13. The method according to claim 5 or 6, wherein the rare carbohydrate is a sulfated polygalactan.

14. The method according to any one of claims 5 to 13, wherein said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a gene selected from the group consisting of genes encoding porphyranase, glycoside hydrolase, sulfatase, galactosidase and any combination thereof.

15. The method according to any one of claims 5 to 13, wherein said heterologous or engineered gene or gene set providing a competitive advantage is or comprises a nucleic acid encoding a protein which sequence is at least 80% identical to at least one of BACPLE 1683-1706 from the Bacteroides plebeius genome.

16. The method according to any one of claims 5 to 15, wherein said heterologous or engineered gene set providing a competitive advantage comprises at least six genes.

17. The method according to any one of claims 2 to 16, wherein the nutrient source cannot be utilized as a nutrient source by the engineered Bacteroides strain in the absence of said heterologous or engineered gene or gene set providing a competitive advantage.

18. The method according to 2 to 17, said method further comprising administering said nutrient source to the subject.

19. The method according to any one of claims 1 to 18, wherein no antibacterial agent is administered to the subject.

20. The method according to any one of claims 1 to 18, wherein said engineered Bacteroides strain further produces an antibacterial agent to which it is resistant, but to which said at least one target Bacteroides strain is sensitive.

21. The method according to claim 20, wherein said engineered Bacteroides strain further produces an antibacterial agent to which it is resistant, but to which said at least one target Bacteroides strain and other bacteria from the subject are sensitive.

22. The method according to claim 20 or 21 , wherein the antibacterial agent is a bacteriocin.

23. The method according to any one of claims 1 to 22, wherein said engineered Bacteroides strain and said target Bacteroides strain are from the same species.

24. The method according to any one of claims 1 to 23, wherein said at least one target Bacteroides strain is a Bacteroides thetaiotaomicron strain and/or a Bacteroides faecis strain.

25. The method according to any one of claims 1 to 24, wherein said engineered Bacteroides strain is an engineered Bacteroides thetaiotaomicron strain and/or an engineered Bacteroides faecis strain.

26. The method according to any one of claims 1 to 25, wherein the subject has been diagnosed with, or is at risk of developing acute inflammatory myocarditis.

27. The method according to any one of claims 1 to 26, wherein the subject is suffering from the permanent sequelae, including cardiomyopathy, from acute inflammatory myocarditis

28. The method according to any one of claims 1 to 27, wherein the subject is carrying a MHC HLA-DQ haplotype capable of presenting MYH6 peptides, in particular human MYH6614-629.

29. The method according to any one of claims 1 to 28, wherein the subject has previously been vaccinated against SARS-CoV-2.

30. The method according to any one of claims 1 to 29, wherein the subject experiences a treatment or disease-induced loss of self-recognition, resulting in acute inflammatory myocarditis pathology.