Immunomodulatory control of metabolic dysregulation

Abstract

Therapeutic approaches are provided for the immunomodulatory control of metabolic dysregulation, including the treatment of subjects suffering from a metabolic dysregulation or a complication thereof, or for improving glucose and / or insulin tolerance and / or weight control in a subject, involving the administration of repeated doses of an immunogenic composition comprising whole killed or attenuated cells of an endogenous bacterial pathogen.

Description

IMMUNOMODULATORY CONTROL OF METABOLIC DYSREGULATION FIELD

[0001] The invention is in the field of medical science, relating to medicinal preparations containing organic active ingredients, including bacteria or materials therefrom, acting as immunomodulators, useful in the treatment of metabolic disorders, for example to mediate glucose homeostasis in subjects suffering from a metabolic dysregulation.BACKGROUND

[0002] The prevalence of obesity in many human populations has been increasing, with an attendant increase in the prevalence of morbidities associated with metabolic dysregulation. Obesity is anatomically characterized by significantly expanded adipose tissue and ectopic fat deposition. These anatomical features are accompanied by physiological responses that are indicative of metabolic dysregulation, including higher blood insulin levels, insulin resistance (IR) and higher blood glucose levels, all of which reflect the dysregulation of glucose level control within a normal blood glucose range. These physiological corollaries to obesity in turn lead to recognizable pathologies that are comorbid with obesogenesis, such as type 2 diabetes (T2D), metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH), liver cirrhosis, liver cancer, hypertension, cardiovascular disease, chronic kidney disease and some cancers.

[0003] Glucose homeostasis involves the concerted activity of multiple organ systems, all of which respond to changes in obesity. Glucose absorption in the gut takes place primarily in the small intestine, particularly the duodenum and the jejunum. Absorbed glucose passes from the gut via the hepatic portal vein to the liver, which is involved in glucose homeostasis via various pathways of glucose metabolism, including glycogenesis, glycogenolysis, glycolysis and gluconeogenesis. The islets of Langerhans mediate the endocrine role of the pancreas in the regulation of the blood glucose levels, secreting insulin, which acts to lower blood glucose levels, and glucagon, which acts to raise blood glucose levels. In adipose tissue, insulin stimulates glucose uptake in both the skeletal muscle and adipocytes,and communication between adipocytes and skeletal muscle in turn play a complex role in metabolic homeostasis. The small intestine, liver, pancreas, skeletal muscle, and adipose tissue are accordingly the primary organs involved in blood glucose homeostasis. These organs are all subject to the influence of attending immune cells, including tissue-resident macrophages, NK cells, T cells.

[0004] Obesity is characterized by chronic activation of various inflammatory pathways, with obesity being thereby associated with changes in the immune system that impact endocrine control of metabolism. This metabolic inflammation can impair insulin action in tissues that participate in blood glucose control, including the gut, adipose tissue, skeletal muscle, the liver and the pancreas. There is accordingly a very complex interrelationship between metabolic dysregulation and the activity of the immune system, with immune cells, including cells of the innate immune system, being involved in modulating metabolic homeostasis in obese animals. There is a similarly complex relationship relating to blood glucose control involving the concerted activity of the immune system, gut microbiome and gut barrier function, so that subversion of the gut barrier by vaccination with a microbiota-based extract may for example engage innate immunity to promote improvements in blood glucose control (see McPhee and Schertzer, 2015; Ray et al., 2016; and, Duggan et al., 2022).

[0005] Immunomodulatory therapies have revolutionized the treatment of a wide variety of diseases, including many cancers and autoimmune diseases (see WO2022198322, WO2019134036, WO2018085937, W02017185180). Opportunities remain to expand the use of immunomodulatory therapies into the treatment of additional pathologies.SUMMARY

[0006] Sustained periodic stimulation of an innate immune response to bacterial antigens is shown in the Examples herein to elicit a progressive improvement in glucose tolerance, insulin tolerance, and weight control in a murine model of obese metabolic dysfunction. Therapeutic methods are accordingly provided for the immunomodulatory control of metabolic dysregulation.

[0007] Methods are provided for treating a subject suffering from a metabolic dysregulation or a complication thereof, or for improving glucose and / or insulin tolerance and / or weight control in a subject, comprising administering to the subject repeated doses of an effective amount of an immunogenic composition comprising whole killed or attenuated cells of a bacterial species and a pharmaceutically acceptable carrier, wherein the bacterial species is an isolate of an endogenous bacterial pathogen, wherein the immunogenic composition is administered in a plurality of at least 3 successive doses given at a dosage interval of between one day and one month, over a dosage duration of at least two weeks, and wherein the plurality of successive doses elicits a progressive improvement in fasting blood glucose, and / or fasting blood insulin, and / or glucose tolerance, and / or insulin tolerance and / or weight control in the subject.

[0008] The bacterial species may for example be an isolate, such as a clinical isolate, that shares critical features of pathogen-associated molecular patterns (PAMPs) of an endogenous bacterial pathogen of the human mucosa, including the bowel, the liver and / or the pancreas, such as an E. coli.

[0009] Administration may be by subcutaneous or subdural injection. In select embodiments, medicaments may be administered at an administration site in successive doses given at a dosage interval of between one hour and one month, over a dosage duration of at least one week.

[0010] Subjects amendable to treatment may for example be obese, may be a mammal, such as a human patient, for example suffering from a metabolic dysregulation is a disease, disorder or condition that is obesity, prediabetes, type 2 diabetes, type 1 diabetes, metabolic syndrome, dyslipidemia, kidney disease, diabetic nephropathy, diabetic neuropathy, heart disease, peripheral vascular dysfunction, metabolic dysfunction-associated fatty liver disease (MAFLD, also referred to as metabolic dysfunction-associated steatotic (fatty) liver disease or steatotic (fatty) liver disease or non-alcoholic fatty liver disease), metabolic dysfunction-associated steatohepatitis (MASH), liver cirrhosis, liver fibrosis, liver inflammation, liver cancer, polycystic ovary syndrome, Cushing’s syndrome, pancreatitis, surgical trauma, trauma, Alzheimer’s disease or a cancer.

[0011] In conjunction with treatment, subjects may be monitored for clinical indicia, for example to assess the progressive improvement in glucose and / or insulin tolerance and / or weight control. Further, monitoring the subject may be carried out so as to provide an assessment of the clinical indicia, such as the progressive improvement in glucose and / or insulin tolerance and / or weight control. The treatment regimen may accordingly be adjusted, for example by adjusting a quantity of the immunogenic composition in the successive doses, and / or adjusting the dosage interval, and / or adjusting the dosage duration, based on the monitoring or the assessment of the subject.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 includes two graphs illustrating the acute effect of an immunomodulatory treatment, QBECO, on blood glucose in an obese mouse model, in which Figure 1A is a line graph illustrating blood glucose concentration and Figure 1B is a bar graph illustrating area over the curve (AOC) of blood glucose over time, both graphs illustrating data from the same cohorts. Subsequent Figures illustrate a time course of subsequent date from the same cohorts of treated mice.

[0013] Figure 2 is a bar graph illustrating the acute effect of QBECO treatment on Day 1 following the initiation of treatment.

[0014] Figure 3 includes two graphs illustrating the effect of the QBECO treatment on glucose tolerance (assessed using the glucose tolerance test; GTT) on Day 3, in which Figure 3A is a line graph illustrating blood glucose concentration and Figure 3B is a bar graph illustrating area under the curve (AUC) of blood glucose over time.

[0015] Figure 4 includes two graphs illustrating the effect of the QBECO treatment on insulin tolerance (as assessed by the insulin tolerance test; ITT) on Day 10, in which Figure 4A is a line graph illustrating blood glucose concentration and Figure 4B is a bar graph illustrating AOC of blood glucose.

[0016] Figure 5 includes two graphs illustrating the effect of the QBECO treatment on glucose tolerance (GTT) on Day 21 , in which Figure 5A is a line graph illustrating blood glucose concentration and Figure 5B is a bar graph illustrating AUC blood glucose over time.

[0017] Figure 6 is a bar graph illustrating a comparison of Day 3 and Day 21 results for fasting blood glucose in the more frequently dosed cohort (MFD), compared to placebo, with alternative 6 and 2 hour fasting respectively.

[0018] Figure 7 includes two graphs, illustrating a comparison of body mass results by week 3 for the more frequently dosed cohort (MFD) compared to placebo, with Figure 7A illustrating body mass in a line graph, and Figure 7B illustrating in a bar graph the distinct divergence in body mass between the cohorts by day 21 .

[0019] Figures 8A, 8B, and 8C are three bar graphs illustrating at Day 28: 8A body mass composition; 8B comparing fat mass and lean mass of placebo and more frequently dosed (MFD) cohorts; and 8C charting the change, delta, in body mass comparing fat mass and lean mass of placebo and MFD cohorts.

[0020] Figure 9 includes two graphs illustrating the effect of the QBECO treatment on glucose tolerance (GTT) on Day 30, in which Figure 9A is a line graph illustrating blood glucose concentration and Figure 9B is a bar graph illustrating AUC blood glucose.

[0021] Figure 10 includes two line graphs illustrating body weight of mice in placebo and more frequently dosed (MFD) cohorts showing: 10A weight through week 5 of QBECO treatment, and 10B weight gain through week 6 of QBECO treatment.

[0022] Figure 11 includes two graphs illustrating the effect of the QBECO treatment on glucose tolerance (GTT) on Day 37, in which Figure 11A is a line graph illustrating blood glucose concentration and Figure 11B is a bar graph illustrating AUC blood glucose.

[0023] Figure 12 is a bar graph illustrating the area under the curve (AUC) of blood glucose showing the effect of QBECO treatment on glucose tolerance (GTT) on Day 37 in the more frequently dosed (MFD) cohort compared to placebo.

[0024] Figure 13 includes two graphs illustrating the effect of the QBECO treatment on insulin tolerance (ITT) on Day 45, in which Figure 13A is a line graph illustrating blood glucose concentration and Figure 13B is a bar graph illustrating AUC of blood glucose.

[0025] Figure 14 includes two graphs illustrating the effect of QBECO treatment on oral glucose-stimulated insulin secretion (oGSS) on Day 66 in an obese mousemodel, evidencing a reversal of the development of type 2 diabetes, in which Figure 14A is a line graph illustrating increased blood insulin concentration compared to placebo in the 60 minute period following treatment and Figure 14B is a bar graph charting AUC blood insulin, illustrating a statistically significant increase in insulin secretion in the more frequently dosed (MFD) cohort of QBECO treated mice compared to placebo.

[0026] Figure 15 is a bar graph illustrating a statistically significant reduction in the liver to body mass ratio on Day 66 in the once a week dosed cohort treated with QBECO SSI compared to placebo, indicative of a reversal of fatty liver disease in an obese mouse model.

[0027] Figure 16 is a grouped column scatter plot illustrating a statistically significant reduction in fatty infiltration in the liver on Day 66 in the more frequently dosed (MFD) cohort treated with QBECO SSI compared to placebo, directly indicative of a reversal of fatty liver disease in an obese mouse model.

[0028] Figure 17 is a grouped column scatter plot illustrating a statistically significant reduction in hepatic triglycerides on Day 66 in the more frequently dosed (MFD) cohort treated with QBECO SSI compared to placebo, directly indicative of a reversal of fatty liver disease in an obese mouse model.

[0029] Figure 18 includes 4 micrographs of stained paraffin embedded liver samples, illustrating histological evidence of reduced liver fibrosis in the more frequently dosed (MFD) cohort treated with QBECO SSI compared to placebo, in which: row a. shows Masson’s Trichrome staining, in which lighter grey (representative of blue staining in contrast to red in the actual sample) is indicative of collagen; and row b. shows Picrosirius dye staining, in which darker grey is indicative of collagen (representative of darker red staining in the actual sample).

[0030] Figure 19 includes two bar graphs illustrating quantified reduced liver fibrosis evidenced by histological staining for collagen fiber deposition and a hydroxyproline assay, in which: graph a, left panel, quantifies the results of Picrosirius red dye staining for collagen fiber deposition in a cross-section of the liver; and graph b, right panel, quantifies the results of a hydroxyproline (major component of collagen) assay in liver tissue homogenates.

[0031] Figure 20 includes micrographs, panel a, and a bar graph, panel b, illustrating quantified histological evidence of reduced liver inflammation in the QBECO MFD cohort by macrophage staining using the macrophage cell surface marker F4 / 80, in which: micrographs and enlargements thereof in panel a. show a reduction in macrophage prevalence as detected by macrophage F4 / 80 marker staining - darker grey in the figure (darker red stain in the actual sample); and, the bar graph in panel b. quantifies the degree of staining.

[0032] Figure 21 is a bar graph illustrating area under the curve (AUC) of blood glucose showing the effect of QBECO during intraperitoneal insulin tolerance tests (ITT) on Day 10.

[0033] Figure 22 is a bar graph illustrating liver mass in different cohorts of obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0034] Figure 23 is a grouped column scatter plot illustrating the results of Oil Red O diazo dye staining of liver sections to quantify lipids in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0035] Figure 24 is a grouped column scatter plot illustrating the results of a liver triglyceride assay on a liver sample homogenate in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0036] Figure 25 is a grouped column scatter plot illustrating the results of liver picrosirius red (PSR) staining, indicative of fibrosis in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0037] Figure 26 includes two grouped column scatter plots, illustrating the results of two measures of liver fibrosis: (a) liver collagen fibrosis, and (b) liver alphasmooth muscle actin (SMA) fibrosis in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0038] Figure 27 includes three grouped column scatter plots, illustrating the results of liver nitrite (a) and arginase (b) assays and the attendant nitrite-to-arginase ratio in the liver (c) as a measure of inflammation in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0039] Figure 28 is a grouped column scatter plot illustrating the results of liver eosinophil peroxidase (EPO) assay, as a marker of liver inflammation in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline).

[0040] Figure 29 includes a grouped column scatter plot (a) illustrating the results of liver senescence-associated (SA) beta-galactosidase assays, in which a lower SA-beta-galactosidase level is indicative of lower levels of cellular senescence in obese mice treated with different doses and dose schedules of QBECO versus placebo (saline); and two scatter plots illustrating that this marker of senescence is directly correlated with the level of fibrosis as measured by collagen (b) and the level of hepatic triglycerides (c) in the liver.

[0041] Figure 30 is a bar graph illustrating the effect, compared to placebo, of biweekly injection subcutaneously on the ventral plane of a whole killed Proteus mirabilis formulation (QBPMI) in lowering liver fat (triglycerides) in the obese mouse model.

[0042] Figure 31 is a bar graph illustrating the effect, compared to placebo, of biweekly injection subcutaneously on the ventral plane of a QBPMI in lowering liver fibrosis (collagen detected by hydroxyproline) in the obese mouse model.

[0043] Figure 32 is a bar graph illustrating the effect, compared to placebo, of biweekly injection subcutaneously on the ventral plane of QBPMI in lowering liver inflammation (eosinophil peroxidase activity) in the obese mouse model.

[0044] Figure 33 is a bar graph illustrating the effect, compared to placebo, of biweekly injection subcutaneously on the ventral plane of a whole killed Bacteroides fragilis formulation (QBBFR) in improving liver inflammation evinced by lower eosinophil peroxidase (EPO) in the obese mouse model.

[0045] Figure 34 is a bar graph illustrating the effect, compared to placebo, of biweekly injection subcutaneously on the ventral plane of QBBFR in improving liver inflammation evinced by lower nitrite in the obese mouse model.

[0046] Figure 35 is a bar graph illustrating the effect, compared to placebo, of biweekly skin injection of QBBFR in improving liver fibrosis (collagen detected by hydroxyproline) in the obese mouse model.DETAILED DESCRIPTION

[0047] Animals are colonized to some degree by microorganisms, such as bacteria, which exist in symbiotic or commensal relationships with the host animal. Thus, many species of normally harmless bacteria are found in healthy animals, and are usually localized to the surface of specific organs and tissues. Often, these microbial communities aid in the normal functioning of the body, as members of what is termed the microbiota, where the genetic material of this community is termed the microbiome. Microbes that are generally harmless, such as Escherichia coii, can cause infection in healthy subjects, with results ranging from mild infection to death. Whether or not a microorganism is pathogenic (i.e. , causes infection) depends on factors such as: the route of entry and access to specific host cells, tissues, or organs; the intrinsic virulence of the microorganism; the amount of the microorganism present at the site of potential infection; or the health of the host animal. Thus, microorganisms that are normally harmless can become pathogenic given favorable conditions for infection, and even the most virulent microorganisms generally require specific circumstances to cause infection. Accordingly, microbial species that are members of the normal flora, “endogenous” microbial species, can be pathogens when they move beyond their normal ecological role in the endogenous flora. Microorganisms having this characteristic are sometimes referred to as “pathobionts”. For example, endogenous species can cause infection outside of their ecological niche in regions of anatomical proximity, for example by contiguous spread. When this occurs, these normally harmless endogenous bacteria are pathogenic, identified herein as endogenous bacterial pathogens.

[0048] The various regions of the gastrointestinal tract playing a significant role in glucose metabolism or absorption include: the bowel or intestine, including: the small intestine (which has three parts: the duodenum, the jejunum, and the ileum); the large intestine (which has three parts: the cecum; the colon, which includes the ascending colon, transverse colon, descending colon and sigmoid flexure; and the rectum). Bacterial species that form part of the normal flora of the human gut are listed in Table 1 , these are accordingly endogenous bacteria that in some circumstances may be pathogenic, so that they are endogenous bacterial pathogens when acting as causative agents of morbidity.Table 1 : Human Bacterial Normal Intestinal Flora

[0049] Specific microbial species are known to cause infections in specific cells, tissues, or organs in otherwise healthy subjects. Examples of bacteria that commonly cause infections in specific organs and tissues of the body are listed below. These examples are not limiting in the sense that a skilled person would be able to recognize and identify bacteria that cause infections, or commonly cause infections, in various organs and tissues in otherwise healthy organisms (andrecognize the relative frequency of infection with each bacterial species) based on the knowledge in the field as represented, for example, by the following publications: Manual of Clinical Microbiology 8th Edition, Patrick Murray, Ed., 2003, ASM Press American Society for Microbiology, Washington DC, USA; Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases 5th Edition, G. L. Mandell, J.E. Bennett, R. Dolin, Eds., 2000, Churchill Livingstone, Philadelphia, PA, USA, all of which are incorporated by reference herein.

[0050] Infections of the pancreas are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., or Legionella spp.

[0051] Infections of the liver are commonly caused by the following bacterial species: Escherichia coli, Klebsiella spp., Streptococcus (anginosus group), Enterococcus, spp. other viridans streptococci, or Bacteroides spp.

[0052] Infections of the colon / rectum are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri.

[0053] Infections of the small bowel are commonly caused by the following bacterial species: Escherichia coli, Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexneri.

[0054] Accordingly, Escherichia coli is an endogenous organism that is pathogenic in all of the small bowel, colon / rectum, liver and pancreas, and is accordingly a select embodiment of an endogenous bacterial pathogen for use in treatments disclosed herein.

[0055] An antigenic composition comprising killed or attenuated bacteria for use in treatments disclosed herein may be formulated using a wide variety of processes known to conventional pharmaceutical practice. A formulation for administration by injection may for example be made as follows. The bacteria may be grown in suitable media, and washed with physiological salt solution. The bacteria may then be centrifuged, resuspended in saline solution, and killed or attenuated, for examplewith heat. The suspensions may be standardized by direct microscopic count, mixed in required amounts, and stored in appropriate containers, which may be tested for safety, shelf life, and sterility in an approved manner. In addition to the pathogenic bacterial species and / or antigens thereof, a killed bacterial vaccine suitable for administration to humans may include phenol or another preservative and / or 0.9% sodium chloride. The bacterial vaccine may also include trace amounts of brain heart infusion (beef), peptones, yeast extract, agar, sheep blood, dextrose, sodium phosphate and / or other media components. A “pharmaceutically acceptable carrier” or “excipient” may be provided, which may for example include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0056] Alternative routes of administration may be employed, for example, parenteral, intravenous, intradermal, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, intraperitoneal, intranasal, inhalational, aerosol, topical, intratumoural, sublingual or oral administration. Therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; for intranasal formulations, in the form of powders, nasal drops, or aerosols; and for sublingual formulations, in the form of drops, aerosols or tablets.

[0057] Methods well known in the art for making formulations are found in, for example, “Remington’s Pharmaceutical Sciences” (20th edition), ed. A. Gennaro, 2000, Mack Publishing Company, Easton, PA. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate anddeoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

[0058] An “effective amount” of a composition according to the invention includes a therapeutically effective amount or a prophylactically effective amount. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as reduction or elimination of the metabolic dysregulation. A therapeutically effective amount of a composition may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount may also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as amelioration of metabolic dysregulation.

[0059] For any particular subject, the timing and dose of treatments may be adjusted over time (e.g., timing may be daily, every other day, weekly, monthly) according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. For example, in the context of subcutaneous or intradermal administration, the compositions may be administered every second day. An initial dose of approximately 0.05 ml may be administered subcutaneously, followed by increases from 0.01-0.02 ml every second day until an adequate skin reaction is achieved at the injection site (for example, a 1 inch to 2 inch diameter delayed reaction of visible redness at the injection site). Once this adequate immune reaction is achieved, this dosing is continued as a maintenance dose. The maintenance dose may be adjusted from time to time to achieve the desired visible skin reaction (inflammation) at the injection site. Dosing may be for a dosage duration, for example of at least 1 week, 2 weeks, 2 months, 6 months, 1 , 2, 3, 4, or 5 years or longer.

[0060] A “site specific immunotherapy” (SSI) is an immunomodulatory treatment that is effective to therapeutically or prophylactically alter an aspect of the immune state, or immune system physiology, at an anatomical site or sites, such as an organor tissue (hence the “site specific” nature of the SSI). As disclosed herein, a SSI may be adapted to ameliorate a metabolic dysregulation, or to treat a condition characterized by a metabolic dysregulation, using formulations comprising inactivated or killed bacteria, for example wherein the bacteria are of a strain that is an isolate of an endogenous bacterial pathogen.

[0061] Alternative strains of E. coli that have been demonstrated to elicit similar innate immune response when used as SSIs, are described below, identified as E. colip and E. colic. The characteristics of these strains are emblematic of the characteristics of the strains of endogenous E.coli bacterial pathogens that may be used in alternative formulations for modulating metabolic dysregulation as described herein.Escherichia coii. (prostate isolate “E. coiip”)

[0062] In select embodiments, compositions may be prepared from E. coli strains having characteristics as described herein, identified as E. colip. Strains of a sequence type having the following alleles, or homologous sequences being at least 99% identical thereto: adk-37, fumc-38, gyrb-19, icd-37, mdh-151 , pura-11 , reca-26 (sequence type 1231). Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B, nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide. Similarly, strains may include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in Table 2). The strain may also lack stx holotoxin virulence factors.Table 2: E. colip- Virulence factors present in E. colip

[0063] The serotype of the E. coli strain may for example be O18ac:H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession GU299793), and wzy (accession GU299793).

[0064] E. coli strains for use in the treatments disclosed herein may include one or more plasmids, for example having 90%, 95%, 99% or 100% identity to plasmid IncFIB (accession AP001918) and / or plasmid IncFI l(29) (accession CP003035), and / or plasmid CoIRNAI (accession DQ298019) and / or plasmid Coll 56 (accession NC009781 ).

[0065] The E. coli may for example be, or be derived from an E. coli strain having at least 80%, 90% or 95% sequence identity to E. coli UT189 (see Chen et al., 2006, Proc Natl Acad Sci U S A 103:5977-82).Escherichia coli. (colon isolate “E. colic”)

[0066] E. coli strains for use in the treatments disclosed herein may include strains of a sequence type having the following alleles, or homologous sequences being at least 99% identical thereto: adk-76, fumc-43, gyrb-9, icd-36, mdh-404, pura- 14, reca-10 (sequence type ST-5292). Strains may totally lack resistance genes to the following classes of antibiotic: aminoglycoside, beta-lactam, fluoroquinolone, fosfomycin, fusidic acid, MLS - macrolide, lincosamide and streptogramin B,nitroimidazole, oxazolidinone, phenicol, rifampicin, sulphonamide, tetracycline, trimethoprim, and glycopeptide. Alternatively, strains may have one or more resistance genes, such as the strB or strA aminoglycoside resistance genes (accession numbers M96392 or AF321551 ), and / or su / 1 sulphonamide resistance gene (accession AY224185), and / or sul2 sulphonamide resistance gene (accession GQ421466), and / or dfrA5 trimethoprim resistance (accession X12868). Similarly, strains may or may not include one or more virulence factor genes, having for example at least 90%, 95%, 99% or 100% identity to selected database sequences (identified by accession number in Table 3). The strain may also have a gene that is at least 95% or 99% or 100% identical to the stx holotoxin virulence factor gene stx1 (accession M 19437).Table 3: E. coli - Virulence factors present in E. colic

[0067] The serotype of the E. coli strain may for example be 0117:H7, for example representing the presence of H type serotype gene fliC (accession AF228492, and O type serotype genes wzx (accession EU694096).

[0068] E. coli strains for use in the treatments disclosed herein may include one or more plasmids, as set out in Table 4.Table 4: E. coli plasmids

[0069] E. coli strains for use in the treatments disclosed herein may for example be, or be derived from an E. coli strain having at least 80%, 90% or 95% sequence identity to E. coli SE15 or any 0117:H7 serotype E. coli.EXAMPLE 1 : Immunomodulatory control of metabolic dysregulation in a murine model

[0070] This Example illustrates that sustained periodic stimulation of an innate immune response to bacterial antigens in a murine model of obese metabolic dysfunction elicits a progressive improvement in glucose and insulin tolerance and weight control. Data illustrated in the Figures was obtained in accordance with the following procedures, which make use of an immunogenic formulation identified herein as “QBECO”.

[0071] QBECO is a whole killed E. coli formulation derived from an enteropathic strain of E. coli, isolated from a patient with an E. coli Gl infection, described above as E. colic, with whole killed cells at a concentration of 1 .5x109cells / mL or in alternative embodiments (0.5 - 9.0)x109cells / mL, suspended in physiological saline. As such, QBECO comprises all major macromolecules of the killed pathogenic strain of E. colic in the form of substantially intact killed bacterial cells (see Bressler B, Bethel KP, Kleef R, et al. Site-specific immunomodulator: a novel treatment for Crohn’s disease. Gastroenterol Res Pract. 2015, 2015:231243; and, Sutcliffe, S. etal., Novel Microbial-Based Immunotherapy Approach for Crohn's Disease. Frontiers in Medicine 6 (2019)). As is for example described in WO2017185180, QBECO when used as a site specific immunomodulator (SSI) stimulates a well characterized innate response by virtue of pattern recognition receptor (PRR) agonism. Alternative strains of E. coli in similarly formulated E. co / / -based SSIs, including E. colipand E. colic, have been shown to elicit comparable innate immune responses in a variety of in vivo models and in vitro assays, and the nature of the host immune response to SSIs has been characterized in detail (see Kalyan, S., Bazett, M., Sham, H.P. et al. Distinct inactivated bacterial-based immune modulators vary in their therapeutic efficacies for treating disease based on the organ site of pathology. Sci Rep 10, 5901 (2020); and, Sutcliffe S., et al., Novel Microbial-Based Immunotherapy Approach for Crohn's Disease, Frontiers in Medicine, v6, 2019).

[0072] All procedures in mice were approved by the McMaster University Animal Ethics Board (animal utilization protocols: 16-02-96 and 23-64). Specific pathogen free (SPF) male C57BL / 6J mice were purchased from Jackson laboratories, which were fed an obesogenic high fat diet (HFD; Diet formula D12492, Research Diets where 60% of the calories are derived from fat) for 11 weeks, where the high fat diet (HFD) was started in mice that were 6 weeks of age. Hence the mice were 17 weeks old and fed a HFD for 11 weeks before the start of the QBECO treatment protocol. Mice had established obesity before treatment started, and mice had a fat mass of -46-49% relative to body mass when study treatment began. HFD-fed mice were kept on HFD and provided water ad libitum for the duration of the experiments. All mice were housed in specific SPF conditions (3-4 mice per cage) under a 12h light / 12h dark cycle with lights switched off at 7:00 PM and on at 7:00 AM.

[0073] Mice were injected subcutaneously on the ventral plane with QBECO at the indicated doses and times in the experimental protocol.

[0074] Body mass and food intake were monitored weekly by measuring the mass of each mouse and food eaten. Body composition in mice, including the percentage of body fat mass and percentage of lean body mass was measured in each mouse, at the indicated time points, using whole body MRI (Bruker Minispec LF90-II).

[0075] Biochemical analysis: Plasma insulin was assessed using high sensitivity mouse insulin ELISA kit (Immunodiagnostics IMD, Cat# 32270).

[0076] Blood glucose in the random fed and fasted state of mice was measured, at the indicated time points, from the tail vein using a MediSure® glucometer.

[0077] Glucose tolerance: Intraperitoneal glucose tolerance tests: Mice were fasted for 2h ( at the time of day indicated on each figure) or 6h (at the time of day indicated on each figure), as indicated and tail blood glucose monitoring was performed before (0 min) and 20, 30, 40, 60, 90 and 120 min after intraperitoneal glucose injection (1 g / kg, or at the other indicated dose) using a MediSure® glucometer.

[0078] Glucose tolerance: Oral glucose tolerance tests: Mice were fasted 6h (8:00 AM - 2:00 PM), as indicated, and tail blood glucose monitoring was performed before (0 min) and 20, 30, 40, 60, 90 and 120 min after oral glucose (2 g / kg) using a MediSure® glucometer.

[0079] Insulin tolerance: Intraperitoneal insulin tolerance tests: Mice were fasted for 2h (8:00 AM - 10:00 AM) or 6h (8:00 AM - 2:00 PM), as indicated, and tail blood glucose monitoring was performed before (0 min) and 20, 30, 40, 60, 90 and 120 min after intraperitoneal insulin injection (1 U / kg, or at the other indicated dose using NovoRapid® from Novo Nordisk) using a MediSure® glucometer.

[0080] Glucose-stimulated insulin secretion: Oral glucose-stimulated insulin secretion tests: Mice were fasted for 12 h (9:00 PM - 9:00 AM), and tail vein blood obtained from conscious mice after an oral gavage with glucose (4 g / kg), where tail vein blood is drawn at the indicated time points after glucose administration and serum to be prepared for an insulin ELISA. Blood samples were centrifuged (2000 g, 10 min, 4°C) immediately and plasma samples were stored at -80°C for later analyses of insulin.

[0081] Insulin resistance: HOMA-IR was used to assess fasting insulin resistance and calculated as follows: (Fasting Insulin [mU / L] x Fasting Glucose [mmol / L]) / 22.5).

[0082] Gut permeability: Mice received 500 mg / Kg of body weight of 4000 Dalton Fluorescein Isothiocyanate (FITC) Dextran diluted in sterile phosphate buffered saline (80 mg / mL). Blood samples to be collected from tail vein using heparin coated capillary tubes before and 1 , 2 and 3 h after gavage with FITC-Dextran. Plasma to be obtainedfrom blood samples by centrifugation (4°C, 8,000 g, for 10 min), diluted 10 times in phosphate buffered saline, and analyzed for FITC-dextran concentration with a fluorescence spectrophotometer (Excitation / Emission wavelengths 485 / 535 nm). Standard curves for calculating FITC-Dextran concentration to be obtained by diluting FITC-dextran in phosphate buffered saline. Baseline readings to be used to determine sample specific background fluorescence, which was subtracted from all time points.

[0083] At the end of the treatment protocol, mice were anesthetized, and the liver was surgically excised and prepared for histological and biochemical analyses.

[0084] Statistical analysis: Data distribution was tested using Shapiro-Wilk test. For normally distributed datasets, unpaired t test was used to compare two groups, and oneway analysis of variance (ANOVA) followed by Tukey’s multiple comparison test were used to compare between three or more groups. For non-parametric datasets, Mann- Whitney U test was applied to compare two groups, and Kruskal-Wallis followed by Dunn’s multiple comparisons tests were used to compare three or more groups. Two- way repeated measures ANOVA with Bonferroni’s post hoc test was applied to compare between groups throughout several time-points. Statistical significance was accepted at p < 0.05.

[0085] The protocol followed to obtain the data in this Example was as follows. Mice were segregated into the following cohorts:• Vehicle control (n = 10) - placebo (30 pl);• QBECO treated0- low dose (n = 10) - 30 pl;• QBECO treated0- more frequent dose (MFD, n = 10) - 30 pl given once weekly;• QBECO treated0- max dose (n = 10) - 100 pl; o QBECO administered via a subcutaneous injection - of 30 -100 pL volume

[0086] Assays conducted in the course of this Example were as follows:• Day 0: Random fed blood glucose, inject mice with vehicle and QBECO and maintain mice on HFD, monitor blood glucose every hour for 6 hours;• Day 1 : Random fed blood glucose (24 hours after 1 st injection);• Day 3: Test glucose tolerance (GTT°) in all mice;• Day 10: Test insulin tolerance (ITT°) in all mice;• Day 21 : Test glucose tolerance (GTT°) in all mice;• Day 30: Test glucose tolerance (GTT°) in all mice. + / - 2 days include test of adiposity with the Echo-MRI;• Day 30: Second injection with vehicle and QBECO for mice not in the once weekly injection group of 30 uL of QBECO; and,• Day 37: Test glucose tolerance (GTT°) in all mice;• Day 45: Test insulin tolerance (ITT°) in all mice; in which, o GTTs and ITTs are in mice that are 2 hours or 6 hours fasted, as indicated, and are i.p. injection unless indicated as oral lavage.• Day 66: Oral glucose-stimulated insulin secretion in placebo mice and MFD mice• Day 71 : Fast mice for 6 h, collect blood serum, kill mice, and collect tissues

[0087] Figure 1 illustrates the acute effect of QBECO on blood glucose on Day 0, with hourly measurements taken from 0 to 6 hours following administration of QBECO, data from the low dose cohort and more frequent dose cohort are pooled. As illustrated in Figure 1A, blood glucose concentrations declined in a dose dependent fashion following treatment. This is reflected in Figure 1B, illustrating blood glucose over time AOC measurements for the cohorts, indicating increased glucose excursion in a dose dependent fashion. The data in Figure 1 accordingly illustrate an acute effect on blood sugar that coincides with the immunostimulatory impact of QBECO treatment. Figure 2 illustrates the blood glucose concentrations in the exemplified cohorts, on Day 1 , illustrating a return to comparable blood glucose levels in the treatment cohorts, with the values for the treatment cohorts being measurably less than the value for the placebo group.

[0088] Figure 3 illustrates the results of glucose tolerance testing (GTT) on Day 3, with Figure 3A showing blood glucose levels at time points following oral glucose (2 g / kg) dosing, and Figure 3B showing the resulting AUC results. All treatment cohorts exhibited reduced glucose excursion, with a more pronounced effect in the high dose cohort. These results indicate that by as early as Day 3, theimmunomodulatory QBECO treatment is achieving meaningful improvements in glucose tolerance.

[0089] Figure 4 illustrates the results of insulin tolerance testing (ITT) on Day 10, with Figure 4A showing blood glucose levels at time points after intraperitoneal insulin injection, and Figure 4B showing the resulting AOC results. All treatment cohorts exhibited enhanced insulin sensitivity and glucose excursion, with a more pronounced effect in the more frequent dose cohort (which at this stage had received a second QEBECO dose). These results indicate that by as early as Day 10, the immunomodulatory QBECO treatment is achieving meaningful improvements in insulin tolerance, with a more pronounced effect evident in the cohort receiving repeated QBECO treatment.

[0090] Figure 5 illustrates the results of glucose tolerance testing (GTT) on Day 21 , with Figure 5A showing blood glucose levels at time points following oral glucose (1 g / kg) dosing, and Figure 5B showing the resulting AUC results. The low dose and more frequent dose treatment cohorts exhibited reduced glucose excursion, with no meaningful effect in the high dose cohort. These results indicate that by Day 21 , a dose dependent effect of QBECO treatment is evident in achieving meaningful improvements in glucose tolerance.

[0091] Figure 6 illustrates a comparison of Day 3 and Day 21 results for fasting blood glucose in the more frequently dosed (MFD) cohort, compared to placebo, with alternative 6 and 2 hour fasting respectively, illustrating the statistically significant improvement in glucose tolerance in the cohort receiving a plurality of successive doses of QBECO.

[0092] Figure 7 illustrates a comparison of body mass results by week 3 for the more frequently dosed cohort compared to placebo, with Figure 7A illustrating a progressive improvement in limiting obesogenesis in the MFD cohort, culminating by Day 21 with the distinct divergence between the cohorts illustrated in Figure 7B.

[0093] Figure 8A,B,C illustrates the results of a body mass composition assessment at Day 28, comparing fat mass and lean mass of placebo and more frequently dosed (MFD) cohorts, showing a distinct reduction in fat mass with improved preservation of lean mass in the MFD group by as early as Day 28.

[0094] Figure 9 illustrates the results of glucose tolerance testing on Day 30, with Figure 9A showing blood glucose levels at time points following oral glucose (0.8 g / kg) dosing, and Figure 9B showing the resulting AUC results. The more frequent dose treatment cohorts exhibited reduced glucose excursion, with no meaningful effect in the low or high dose cohorts. These results indicate that by Day 30, a repeated-dose dependent effect of QBECO treatment is evident in achieving meaningful improvements in glucose tolerance.

[0095] Figure 10 illustrates the sustained effect of QBECO treatment on reducing weight gain in the more frequently dosed (MFD) cohort, compared to placebo, through week 5 of treatment.

[0096] Figure 11 illustrates the results of glucose tolerance testing (GTT) on Day 37, with Figure 11A showing blood glucose levels at time points following oral glucose (0.8 g / kg) dosing, and Figure 11B showing the resulting AUC results. The more frequent dose treatment cohort continued to exhibit reduced glucose excursion compared to all other cohorts. These results indicate that by Day 37, a repeated- dose dependent effect of QBECO treatment remains evident in achieving sustained improvements in glucose tolerance. As shown in Figure 12, by Day 37 the improvement in glucose tolerance in the MFD cohort is statistically significant compared to placebo, illustrating that the plurality of successive doses of the QBECO SSI elicits a progressive and dramatic improvement in glucose tolerance (as assessed by GTT). At Day 45, insulin sensitivity in the more frequent dose treatment cohort MFD cohort was statistically significantly better than placebo in an Insulin Tolerance Test (ITT), demonstrating continued improved insulin sensitivity, as seen in Figure 13A and B.

[0097] Figure 14 illustrates that by Day 66 the QBECO MFD group had significantly higher insulin secretion into the blood in response to an oral glucose load (oral glucose-stimulated insulin secretion; oGSIS). This data indicates that QBECO improves insulin release from the pancreas, and mitigates a key factor in the development and progression of prediabetes toward type 2 diabetes. These data indicate that the QBECO SSI has actions on the pancreas and blood insulin homeostasis in mice feed a high fat diet. The glucose-stimulated insulin release assay is as described above.

[0098] Figure 15 illustrates that the QBECO MFD group was characterized by reduced liver mass. Given that mice were fed a high fat diet as a model of obesity and fatty liver disease, this data is consistent with QBECO SSI amelioration of aspects of metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH, also sometime characterized as metabolic dysfunction-associated liver disease and metabolic dysfunction-associated steatohepatitis), resulting in lower liver fat mass in QBECO SSI treated mice. To assay liver mass, animals were killed and the livers were excised and weighed.

[0099] Figure 16 illustrates that the QBECO MFD group was characterized by substantially reduced fatty infiltration in the liver. Given that mice were fed a high fat diet as a model of obesity and fatty liver disease, this data is consistent with QBECO SSI amelioration of aspects of metabolic dysfunction-associated fatty liver disease (MAFLD, also sometimes referred to as metabolic dysfunction-associated steatohepatitis or metabolic dysfunction-associated liver disease and metabolic dysfunction-associated steatohepatitis), resulting in substantially reduced fatty infiltration in the liver in QBECO SSI treated mice. To assay hepatic fat area, paraffin embedded liver samples collected at the 71-day end-point of the study from euthanized mice were sectioned into 5 micron slices and subjected to hematoxylin and eosin staining. Images were acquired and analyzed for percentage of hepatic fat area using a Nikon eclipse microscope and Fiji software, respectively.

[0100] Figure 17 illustrates that the QBECO MFD group was characterized by substantially reduced hepatic triglycerides. Given that mice were fed a high fat diet as a model of obesity and fatty liver disease, this data is consistent with QBECO SSI amelioration of aspects of metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH, also known as metabolic dysfunction-associated liver disease and metabolic dysfunction-associated steatohepatitis), resulting in substantially reduced triglycerides in the liver in QBECO SSI treated mice. To assay hepatic triglycerides, liver samples collected at the 71- day end-point of the study from euthanized mice were subjected to homogenization with ceramic beads; triglycerides were quantified using a colorimetric ELISA assay.

[0101] Figure 18 illustrates histologically that the QBECO MFD group was characterized by substantially reduced liver fibrosis. Given that mice were fed a highfat diet as a model of obesity and fatty liver disease, this data is consistent with QBECO SSI amelioration of aspects of metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction-associated steatohepatitis (MASH, also known as metabolic dysfunction-associated liver disease and metabolic dysfunction- associated steatohepatitis), resulting in substantially reduced fibrosis in the liver in QBECO SSI treated mice. To obtain the histological images shown in Figure 18, paraffin embedded liver samples collected at the end-point of the study from euthanized mice were subjected to staining for liver fibrosis, illustrating: a. Masson’s Trichrome staining, in which lighter grey (representative of blue staining in contrast to red in the actual sample) is indicative of collagen; and b. Picrosirius dye staining, in which darker grey is indicative of collagen (representative of darker red staining in the actual sample).

[0102] Figure 19 provides data quantifying the reduction in liver fibrosis in the QBECO MFD cohort of obese mice, in two bar graphs showing, respectively, the results of histological staining for collagen deposition and a hydroxyproline (major component of collagen) assay in a homogenate prepared from a piece of the left lateral lobe of the liver from each mouse. Graph a, left panel, quantifies the results of Picrosirius red dye staining for collagen deposition (P value represented by *** = 0.0002). Graph b, right panel, quantifies the results of a hydroxyproline assay (P value represented by ** = 0.0089).

[0103] Monocytes are recruited to the liver in the context of obesity / MAFLD / MASH where they differentiate into macrophages, which may be detected by staining for the macrophage F4 / 80 marker. Figure 20 provides quantified histological evidence of reduced liver inflammation in the QBECO MFD cohort, illustrating the results of macrophage staining using the F4 / 80 marker, in which: micrographs and enlargements in panel a. show a reduction in macrophage prevalence as detected by the macrophage F4 / 80 marker staining - darker grey in the figure (darker red stain in the actual sample); and, quantification of the degree of staining is illustrated in a bar graph in panel b (P value represented by * = 0.0404).EXAMPLE 2: Correlation analysis in immunomodulatory control of metabolic dysregulation in a murine model

[0104] This Example illustrates effects related to the dosage and timing of an SSI treatment with QBECO in the obese mouse model of metabolic dysregulation.

[0105] All procedures in mice were approved by the McMaster University Animal Ethics Board (animal utilization protocols: 16-02-96 and 23-64). Specific pathogen free (SPF) male C57BL / 6J mice were purchased from Jackson laboratories, which were fed an obesogenic high fat diet (HFD; Diet formula D12492, Research Diets where 60% of the calories are derived from fat) for 11 weeks, where the HFD was started in mice that were 6 weeks of age. Hence the mice were 17 weeks old and fed a HFD for 11 weeks before the start of the QBECO treatment protocol. Mice had established obesity before treatment started, and mice had a fat mass of -46-49% relative to body mass when study treatment began. HFD-fed mice were kept on HFD and provided water ad libitum for the duration of the experiments. All mice were housed in specific SPF conditions (3- 4 mice per cage) under a 12h light / 12h dark cycle with lights switched off at 7:00 PM and on at 7:00 AM.

[0106] In this Example, dosing and timing of treatments was as follows:• 1 x 30 pL dose per week placebo;• 1 x 30 pL dose per week QBECO;• 1 x 10 pL dose per week QBECO;• 1 x 30 pL dose every 2 weeks (i.e., biweekly, BW) QBECO.

[0107] In this Example, at the end of the treatment protocol, on Day 52 after imitating injections of QBECO, mice were fasted for 6 h, anesthetized, blood serum was collected and tissues, including the liver, were surgically excised and prepared for histological and biochemical analyses.

[0108] As is disclosed in more detail below, this Example illustrates that the SSI dosage regimen affects therapeutic outcomes. Overall, the data disclosed herein illustrates that biweekly injection of QBECO (30 pL) is measurably superior in various aspects to the alternative regimens that were tested for improving features of MALFD in the obese mouse model. An aspect of the present invention accordingly involves adjusting the dosage regimen of an SSI treatment in order to optimize therapeutic efficacy. For example, treatments may involve monitoring a subject to assess clinical indicia, for example, to assess the progressive improvement inglucose and / or insulin tolerance and / or weight control. The monitoring of the subject may be carried out so as to provide an assessment of clinical indicia, such as an assessment of the progressive improvement in glucose and / or insulin tolerance and / or weight control. Treatments may the further involve adjusting a quantity of the immunogenic composition in the successive doses, and / or adjusting the dosage interval, and / or adjusting the dosage duration, based on the monitoring or the assessment of the subject.

[0109] Figure 21 shows the results of intraperitoneal insulin tolerance tests on the treatment cohorts, ipITT, on day 10 of treatment. Illustrating the most improvement in AUC blood glucose for the 30 pl biweekly treatment cohort.

[0110] Figure 22 shows a significantly lower liver mass in obese mice injected with 30 pL QBECO BW (every two weeks) or 30 pL weekly.

[0111] Figure 23 shows the results of Oil Red O diazo dye staining of liver sections to quantify lipids, illustrating that injections of QBECO of 30 pL BW or weekly significantly lower liver fat, with less-frequent BW QBECO injections lowering liver fat similarly to weekly 30 pL injections of QBECO.

[0112] Figure 24 shows the results of a liver triglyceride assay on a liver sample homogenate, illustrating that 10 pL weekly injections and 30 pL BW injections of QBECO lower liver triglycerides.

[0113] Figure 25 shows the results of liver picrosirius red (PSR) staining, indicative of fibrosis, illustrating that all injections of QBECO (30 pL BW or 30 pL weekly or 10 pL weekly) significantly lower liver fibrosis (each dot is a mouse). The effect is dramatic.

[0114] Figure 26 shows the results of two measures of liver fibrosis: (a) liver collagen fibrosis, and (b) liver alpha-smooth muscle actin (SMA) fibrosis. In which liver collagen and lower alpha-SMA assays respectively are indicative of significantly reduced liver fibrosis, in which injections of 30 pL BW QBECO injections are superior to other regimens for lowering these indicators of liver fibrosis (each dot is a mouse).

[0115] Figure 27 shows the results of liver nitrite (a) and arginase (b) assays, in which nitrite levels are indicative of pro-inflammatory “M1” polarization in liver macrophages, and arginase levels are indicative of “M2” tissue-healing / regeneration polarization in liver macrophages. The results illustrate that 10 pL weekly or 30 pLBW QBECO lowers liver nitrite more than 30 pl weekly. Impressively, 10 pL weekly or 30 pL BW QBECO injections dramatically decreased the nitrite-to-arginase ratio in the liver (c). This illustrates that 10 pL weekly or 30 pL BW QBECO skews liver macrophages away from M1 (proinflammatory phenotype) toward M2 (healing phenotype).

[0116] Figure 28 shows the results of liver eosinophil peroxidase assays, as a marker of liver inflammation. The results indicate that 10 pL weekly or 30 pL BW QBECO lowers liver eosinophil peroxidase (EPO), further evidence that a lower dose or a less frequent QBECO injection protocol can more effectively lower local inflammation in multiple types of immune cells / responses.

[0117] Figure 29 shows the results of liver senescence-associated (SA) beta(p)-galactosidase assays, in which a lower SA- p-galactosidase level is indicative of lower levels of cellular senescence. The results (a) indicate that 30 pL BW QBECO significantly lowers liver SA-p-galactosidase levels. Data was also assessed that shows that this marker of senescence is directly correlated with the level of fibrosis as measured by collagen (b) and the level of hepatic triglycerides (c) in the liver. Further analysis reveals that that SA- p-galactosidase correlates directly with eosinophil peroxidase activity (EPO) in liver homogenates (R-squared value of 0.58, p<0.05) and similarly SA-p-galactosidase levels correlate directly with nitrite levels in liver homogenates (R-squared value of 0.49, p<0.05).

[0118] Overall, this Example illustrates that biweekly injection of QBECO (30 pL) is in this model superior to other doses / timing of injection for improving features of MALFD in obese mice. This is accordingly indicative of an aspect of the present therapies that involves monitoring a subject, for clinical indicia, for example, to assess the progressive improvement in glucose and / or insulin tolerance and / or weight control. Further, monitoring the subject may be carried out so as to provide an assessment of the clinical indicia, such as the progressive improvement in glucose and / or insulin tolerance and / or weight control. The treatment regimen may accordingly be adjusted, for example by adjusting the quantity of the immunogenic composition in the successive doses, and / or adjusting the dosage interval, and / or adjusting the dosage duration, based on the monitoring or the assessment of the subject.EXAMPLE 3: Alternative Gut-Derived Bacterial Formulations For Treating Fatty Liver Disease

[0119] This Example illustrates the efficacy of alternative bacterial formulations in treating fatty liver disease in the same obese murine model in which QBECO mediates a therapeutic response as shown herein. The effective alternative bacterial formulations are made from whole killed bacterial cells, in which the bacterial strains were selected from the gut microflora and they are: inactivated Bacteroides fragilis (QBBFR) and inactivated Proteus mirabilis (QBPMI). Both QBPMI and QBBFR show efficacy in treating fatty liver disease, in contrast to the absence of a therapeutic effect when a non-gut bacterial formulation is used: inactivated Klebsiella pneumonia (QBKPN; animals used in these assays had tested negative for Klebsiella pneumonia, an organism that is however a member of the gut microflora, and a potential pathogen, in humans). QBPMI shows statistically significant efficacy in fatty liver treatment endpoints of liver fat, inflammation, and fibrosis in obese mice. QBBFR shows marked improvement in inflammation and fibrosis in obese mice. QBKPN did not mediate such efficacy in obese mice. Data in this Example was obtained in the same obese murine model as is described herein with respect to treatments using QBECO, with 1 x 30 pL dose every 2 weeks.

[0120] Compared to placebo: biweekly injection of QBPMI lowered liver fat (triglycerides), as illustrated in Figure 30; biweekly injection of QBPMI lowered liver fibrosis (collagen detected by hydroxyproline), as illustrated in Figure 31 ; biweekly injection of QBPMI lowered liver inflammation (eosinophil peroxidase activity), as illustrated in Figure 32. Similarly, biweekly injection of QBBFR shows marked improvement in liver inflammation evinced by lower eosinophil peroxidase (EPO), as shown in Figure 33, and as evinced by lower nitrite, as shown in Figure 34. Biweekly injection of QBBFR showed marked improvement in liver fibrosis (collagen detected by hydroxyproline), as illustrated in Figure 35.

[0121] These therapeutic effects in treating fatty liver disease with gut-derived bacterial formulations were not observed to a significant degree when a non-gut bacterial formulation was used, inactivated Klebsiella pneumonia (QBKPN). In conjunction with the evidence of the efficacy of the QBECO formulation, thisExample accordingly illustrates that steatosis, inflammation or fibrosis in fatty liver disease may advantageously be effectively treated, in some embodiments, using formulations made from bacterial species that are isolates of an endogenous bacterial member of the microbiota that is capable of causing infection, i.e. a gut pathogen in the sense that it can be present in the gut and can be pathogenic, such as Escherichia coli, Bacteroides fragilis or Proteus mirabilis.DEFINITIONS AND CITATIONS

[0122] Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Terms such as “exemplary” or “exemplified” are used herein to mean “serving as an example, instance, or illustration.” Any implementation described herein as “exemplary” or “exemplified” is accordingly not to be construed as necessarily preferred or advantageous over other implementations, all such implementations being independent embodiments. Unless otherwise stated, numeric ranges are inclusive of the numbers defining the range, and numbers are necessarily approximations to the given decimal. The word "comprising" is used herein as an open-ended term, substantially equivalent to the phrase "including, but not limited to", and the word "comprises" has a corresponding meaning. 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 thing" includes more than one such thing. Citation of references herein is not an admission that such references are prior art to the present invention. Any priority document(s) and all publications, including but not limited to patents and patent applications, cited in this specification, and all documents cited in such documents and publications, are hereby incorporated herein by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein and as though fully set forth herein. The invention includes all embodiments and variations substantially as hereinbefore described and with reference to the examples and drawings. Insome embodiments, the invention excludes steps that involve medical or surgical treatment.

[0123] General Codes and Abbreviations:SSI Site Specific ImmunomodulatorI.P. IntraperitonealSC SubcutaneousANOVA Analysis of varianceSD Standard deviation g GramL or I Liter pL microliter min MinuteQBECO Escherishia coli whole killed cell SSIITT Insulin tolerance testAOC Area over the curveMFD More frequent doseEcho-MRI™ Whole body MRI (Bruker Minispec LF90-II)GTT Glucose tolerance testHFD High fat diet

[0124] The following documents are hereby incorporated by reference:

[0125] McPhee and Schertzer, 2015, Immunometabolism of obesity and diabetes: microbiota link compartmentalized immunity in the gut to metabolic tissue inflammation. Clin Sci (Lond), Dec; 129(12): 1083-96.

[0126] Ray et al., 2016, Obesity: An Immunometabolic Perspective, Frontiers in Endocrinology, v. 7, Article 157.

[0127] Duggan et al., 2022, Gut microbiota-based vaccination engages innate immunity to improve blood glucose control in obese mice, Molecular Metabolism, 55:101404-101404.

Claims

CLAIMS:1 . A method of treating a subject suffering from a metabolic dysregulation or a complication thereof, comprising administering to the subject repeated doses of an effective amount of an immunogenic composition comprising whole killed or attenuated cells of a bacterial species and a pharmaceutically acceptable carrier, wherein the bacterial species is an isolate of an endogenous bacterial pathogen, wherein the immunogenic composition is administered in a plurality of at least 3 successive doses given at a dosage interval of between one day and one month, over a dosage duration of at least two weeks, and wherein the plurality of successive doses elicits a progressive improvement in glucose and / or insulin tolerance in the subject.

2. The method of claim 1 , wherein the bacterial species is an isolate of an endogenous bacterial pathogen of the gut, bowel, the liver and / or the pancreas; optionally, wherein the bacterial species is an Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., Legionella spp., Streptococcus (anginosus group), viridans streptococci, Bacteroides spp., Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, Proteus mirabilis or Shigella flexnerr, optionally, wherein the bacterial species is identified in Table 1.

3. The method of claim 1 or 2, wherein the bacterial species is an E. coli Bacteroides fragilis or Proteus mirabilis.

4. The method of any one of claims 1-3, wherein the bacterial species is a clinical isolate of a pathogen.

5. The method of any one of claims 1-4, wherein the administration is by subcutaneous, subdermal, or intradermal injection.

6. The method of any one of claims 1-5, wherein the subject is obese.

7. The method of any one of claims 1-6, wherein the subject is a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. The method of any one of claims 1-8, wherein the metabolic dysregulation is a disease, disorder or condition that is obesity, prediabetes, type 2 diabetes, type 1 diabetes, metabolic syndrome, dyslipidemia, kidney disease, diabetic nephropathy, diabetic neuropathy, heart disease, peripheral vascular dysfunction, metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction- associated steatohepatitis (MASH), metabolic dysfunction-associated steatotic liver disease (MASLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver cirrhosis, liver fibrosis, liver inflammation, polycystic ovary syndrome, Cushing’s syndrome, pancreatitis, surgical trauma, trauma, Alzheimer’s disease or a cancer, including liver, breast, colorectal, endometrial, kidney, esophageal, pancreatic, gallbladder cancer.

10. The method of any one of claims 1-9, further comprising monitoring the subject to assess the progressive improvement in glucose and / or insulin tolerance and / or weight control.

11. The method of claim 10, wherein monitoring the subject provides an assessment of the progressive improvement in glucose and / or insulin tolerance and / or weight control; and, further comprising adjusting a quantity of the immunogenic composition in the successive doses, and / or adjusting the dosage interval, and / or adjusting the dosage duration, based on the monitoring or the assessment of the subject.

12. A method of improving glucose and / or insulin tolerance and / or weight control in a subject, comprising administering to the subject repeated doses of an effective amount of an immunogenic composition comprising whole killed or attenuated cells of a bacterial species and a pharmaceutically acceptable carrier, wherein the bacterial species is an isolate of an endogenous bacterial pathogen, wherein the immunogenic composition is administered in a plurality of at least 3successive doses given at a dosage interval of between one day and one month, over a dosage duration of at least two weeks, and wherein the plurality of successive doses elicits a progressive improvement in glucose and / or insulin tolerance in the subject.

13. Use of an immunogenic composition for improving glucose and / or insulin tolerance in a subject, or for treating the subject suffering from a metabolic dysregulation or a complication thereof, wherein the immunogenic composition is for use by administration in repeated doses of an effective amount of the immunogenic composition, and the immunogenic composition comprises whole killed or attenuated cells of a bacterial species and a pharmaceutically acceptable carrier, wherein the bacterial species is an isolate of an endogenous bacterial pathogen, wherein the immunogenic composition is administered in a plurality of at least 3 successive doses given at a dosage interval of between one day and one month, over a dosage duration of at least two weeks, and wherein the plurality of successive doses elicits a progressive improvement in glucose and / or insulin tolerance in the subject.

14. The method of claim 12 or use according to claim 13, wherein the bacterial species is an isolate of an endogenous bacterial pathogen of the bowel, the liver and / or the pancreas; optionally, wherein the bacterial species is an Escherichia coli, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Staphylococcal spp., Mycoplasma spp., Salmonella typhi, Leptospirosis spp., Legionella spp., Streptococcus (anginosus group), viridans streptococci, Bacteroides spp., Clostridium difficile, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Proteus mirabilis, Clostridium perfringens, Salmonella enteriditis, Yersinia enterocolitica, or Shigella flexnerr, optionally, wherein the bacterial species is identified in Table 1.

15. The method or use of any one of claims 12-14, wherein the bacterial species is an E. coli, Bacteroides fragilis or Proteus mirabilis.

16. The method or use of any one of claims 12-15, wherein the bacterial species is a clinical isolate of a pathogen.

17. The method or use of any one of claims 12-16, wherein the administration is by subcutaneous, subdermal, or intradermal injection.

18. The method or use of any one of claims 12-17, wherein the subject is obese.

19. The method or use of any one of claims 12-18, wherein the subject is a mammal.

20. The method or use of claim 19, wherein the mammal is a human.

21. The method or use of any one of claims 12-20, wherein the metabolic dysregulation is a disease, disorder or condition that is obesity, prediabetes, type 2 diabetes, type 1 diabetes, metabolic syndrome, dyslipidemia, kidney disease, diabetic nephropathy, diabetic neuropathy, heart disease, peripheral vascular dysfunction, metabolic dysfunction-associated fatty liver disease (MAFLD), metabolic dysfunction- associated steatohepatitis (MASH), metabolic dysfunction-associated steatotic liver disease (MASLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver cirrhosis, liver fibrosis, liver inflammation, polycystic ovary syndrome, Cushing’s syndrome, pancreatitis, surgical trauma, trauma, Alzheimer’s disease or a cancer, including liver, breast, colorectal, endometrial, kidney, esophageal, pancreatic, gallbladder cancer.

22. The method or use of any one of claims 12-21 , further comprising monitoring the subject to assess the progressive improvement in glucose and / or insulin tolerance and / or weight control.

23. The method or use of claim 22, wherein monitoring the subject provides an assessment of the progressive improvement in glucose and / or insulin tolerance and / or weight control; and, further comprising adjusting a quantity of the immunogenic composition in the successive doses, and / or adjusting the dosage interval, and / oradjusting the dosage duration, based on the monitoring or the assessment of the subject.

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