Use of mycobacterium to prevent, treat, or reduce adiposity and diabetes

Abstract

Use of Mycobacterium vaccae, for example strain ATCC 15483, for the treatment or prevention of weight gain, visceral adipose tissue (VAT), obesity, obesity-related metabolic diseases and stress-related disorders.

Description

[0001] USE OF MYCOBACTERIUM TO PREVENT, TREAT, OR REDUCE ADIPOSITY AND DIABETES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 556,357, filed February 21, 2024, and entitled “Use of Mycobacterium Immunization to Treat Adiposity and Diabetes,” the entire disclosure of which is hereby incorporated by reference herein in its entirety.

[0004] SEQUENCE LISTING

[0005] The instant application contains contents of the electronic sequence listing (90245.01141- Sequence-Listing.xml; Size: 31,002 bytes; and Date of Creation: February 19, 2025) is herein incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] Embodiments described herein relate to the field of microbiology, and more particularly to use of mycobacteria to prevent, treat, or reduce obesity and obesity-related disease conditions. Embodiments described herein disclosure also relate to pharmaceutical compositions, such as byproducts and metabolites, and / or consumer compositions including mycobacteria, for example, whole-cell, heat-killed preparations, and uses thereof in treating or preventing obesity and obesity- related diseases, optionally by reducing visceral adipose tissue (VAT) in a subject in need thereof. BACKGROUND

[0008] Obesity is a chronic disease implicating a number of genetic, cultural, and individual factors. Obesity is also an inducing factor to a series of diseases, (generally referred to herein as metabolic disease or conditions) such as hypertension, diabetes, coronary heart disease, gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer, such as prostate cancer, breast cancer and colon cancer. The National Institute of Health (NIH) reports that there are over 100 million Americans who are overweight or over-fat, in which about 25+ million people suffer from obesity-related type 2 diabetes, and about 300,000 people die from obesity-related diseases each year.

[0009] Obesity is generally caused by excess body fat due to functional changes in physiology or biochemistry. Fat usually includes neutral lipids, phospholipids and cholesterol. Fat increase is due to more energy intake over energy consumption. In terms of pathogenesis, obesity includes two types: (a) simple obesity and (b) secondary obesity. Simple obesity can be divided into idiopathic obesity and acquired obesity, and the number of patients with the simple obesity can account for more than 95% of the total number of patients with obesity. Idiopathic obesity is caused by adipocytes, and is common in childhood obesity. Acquired obesity is caused by adipocytes characterized by a larger size and is common in adult obesity. Secondary obesity, also known as symptomatic obesity, is usually caused by endocrine or metabolic diseases.

[0010] At present, there are five main strategies for the treatment of obesity: dieting, exercising, behavioral therapy, medical therapy, and therapeutic operation; which strategy or combination of strategies is selected primarily depends on health risk factors and the rate and effect of weight loss of a patient. The rate and effect of weight loss of the patient are affected by many factors, such as age, height, family history and risk factors. Diet-exercise therapy, i.e. diet with food with low calorie and low fat in combination with aerobic exercise, needs to be implemented for a long term, is generally considered unsuccessful for the general public, however. Surgery for removal of body fat can achieve immediate results, but with many restrictions, such as operation risk, non-persistent effect and high expensive cost.

[0011] Presently, medical therapy is the main clinical method for treating obesity and obesity- related diseases (such as diabetes). Mechanisms of medical therapy include appetite suppression, increasement of energy consumption, stimulation of fat movement, reduction of triglyceride synthesis and inhibition of fat absorption. Currently, drugs mainly including semaglutides such as OZEMPIC® and WEGOVY® have shown promise in reducing obesity and inducing weight-loss. However, these drugs have known and unknown side-effects that can limit their long-term effectiveness. Hyperglycemia in some diabetics still cannot be adequately controlled by the diet and / or exercise therapies or use of the above-described therapeutic compounds. For these patients, exogenous insulin should be used, which is not only very expensive and painful, but also will bring about a variety of complications to patients. For example, error calculation to insulin dose caused by lack of food and abnormal exercise will lead to insulin response (hypoglycemia). In addition, use of drugs may also give rise to local or systemic allergies or immune resistance to drugs. There thus exists a need for additional compositions and methods to treat obesity and metabolic diseases and disorders, such as diabetes. The composition and methods of the disclosure can also be effective for the treatment stress-related disorders, such as post-traumatic stress-disorder (PTSD), depression, and anxiety disorders. SUMMARY

[0012] One aspect of the present disclosure includes the use of A / . vaccae in the prevention, treatment, or reduction of the risk of developing obesity and obesity-related diseases. Specifically the disclosure includes use of Mycobacterium vaccae, and optionally M. vaccae strain ATCC 15483 deposited with the American Type Culture Collection (ATCC). In some embodiments, , the administration of a therapeutically or prophylactically effective amount of M. vaccae, optionally strain ATCC 15483, or constituent components, can counteract a high-calorie, high-fat diet, referred to herein as a “Western-style Diet (WD)” that is known to cause obesity. In some embodiments, for example those embodiments where M. vaccae or constituent components are administered prior to the onset of obesity or an obesity-related disease, administration of a prophylactically effective amount of M. vaccae or constituent components can prevent the development of or reduce the risk of developing obesity or an obesity-related disease.

[0013] In another aspect, the M. vaccae includes heat-killed or otherwise inactivated bacteria, or the gross of constituent components of M. vaccae strain ATCC 15483. In some embodiments, the M. vaccae includes whole cells o M. vaccae strain ATCC 15483. In some embodiments, the M. vaccae includes heat-killed or otherwise inactivated bacteria, or the gross of constituent components of M. vaccae strain NCTC 11659. In some embodiments, the AY. vaccae includes whole cells of M. vaccae strain NCTC 11659.

[0014] In another aspect, the disclosure includes use of Mycobacterium vaccae, and optionally a therapeutically effective amount of AT. vaccae strain ATCC 15483, or constituent components, for the reduction of visceral adipose tissue (VAT) in a subject in need thereof. In another aspect, the disclosure includes a therapeutically effective amount of M. vaccae strain NCTC 11659, or constituent components, for the reduction of visceral adipose tissue (VAT) in a subject in need thereof.

[0015] Some embodiments described herein include use of Mycobacterium vaccae, or constituent components, to modulate expression of leptin (lep). In some embodiments, expression of leptin is decreased upon administration of the therapeutically effective amount of M. vaccae, or constituent components. In some embodiments, expression of leptin is decreased by at least 20%, at least 30%, at least 40%, at least 50% or at least 60%. In some embodiments, the decrease in leptin expression comprises a decrease in leptin mRNA levels. In some embodiments, the decrease in leptin expression comprises a decrease in leptin protein in the plasma of the subject. In some embodiments, the M. vaccae comprises AY. vaccae strain ATCC 15483. In some embodiments, the M. vaccae comprises AY. vaccae strain NCTC 11659.

[0016] In another aspect, the disclosure includes use of Mycobacterium vaccae, and optionally a therapeutically effective amount of AY. vaccae strain ATCC 15483, or constituent components, to decrease expression of the leptin gene in a subject in need thereof, thereby reducing appetite and decreasing body weight. In another aspect, the disclosure includes use of a therapeutically effective amount of AY. vaccae strain NCTC 11659, or constituent components, to decrease expression of the leptin gene in a subject in need thereof, thereby reducing appetite and decreasing body weight.

[0017] In another aspect, the disclosure includes use of Mycobacterium vaccae, and optionally a therapeutically or prophylactically effective amount of AY vaccae strain ATCC 15483, or constituent components, to prevent, inhibit, reduce the risk of developing or treat a metabolic disorder or metabolic disease, which can include, but is not limited to: hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes-associated coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and / or cancer. In some embodiments, the metabolic disorder or metabolic disease comprises diabetes (e g., type 2. In another aspect, the disclosure includes use of AY. vaccae strain NCTC 11659, or constituent components, to prevent, inhibit, reduce the risk of or treat a metabolic disorder or metabolic disease.

[0018] In another aspect, the disclosure includes use of Mycobacterium vaccae, or constituent components, to prevent, treat or reduce the risk of developing one or more symptoms of a metabolic disorder or metabolic disease, which can include, but is not limited to: hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes associated coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and / or cancer. In some embodiments, the metabolic disorder or metabolic disease comprises diabetes (e.g., type 2). In some embodiments, the AY. vaccae comprises AY. vaccae strain ATCC 15483. In some embodiments, the AY. vaccae comprises AY. vaccae strain NCTC 11659.

[0019] In another aspect, the disclosure includes use of Mycobacterium vaccae, or constituent components, to reduce one or more symptoms of a metabolic disorder or metabolic disease, or reduce the risk of developing one or more symptoms of a metabolic disorder or metabolic disease, which can include, but not be limited to: hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes associated coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and / or cancer. In some embodiments, the metabolic disorder or metabolic disease comprises diabetes (e.g., type 2). In some embodiments, the M. vaccae comprises M. vaccae strain ATCC 15483. In some embodiments, the M. vaccae comprises M. vaccae strain NCTC 11659.

[0020] In another aspect, the disclosure includes use of Mycobacterium vaccae, or constituent components, to modulate expression of one or more VAT genes, such as allograft inflammatory factor 1 IBA1 adiponectin, C1Q and collagen domain containing ADIPOQ), tumor necrosis factor (TNF), forkhead boxP3 (F0XP3) and / or arginase I (ARG1). In some embodiments, the A vaccae comprises A vaccae strain ATCC 15483. In some embodiments, the AZ vaccae comprises AZ. vaccae strain NCTC 11659.

[0021] In another aspect, the disclosure includes use of Mycobacterium vaccae strain ATCC 15483, or constituent components, in a method for treating, preventing or reducing a risk of developing a stress-related disorder in a subject in need thereof. In some embodiments, the methods comprise administering a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M vaccae) to the subject. In some embodiments, the depression comprises major depressive disorder. In some embodiments, the anxiety disorder comprises general anxiety disorder, panic disorder, social anxiety disorder or a phobia-related disorder.

[0022] In a further aspect, the disclosure includes isolated AZ vaccae, or constituent components, optionally combined with a pharmaceutically acceptable carrier forming a pharmaceutical composition for the treatment of, or prevention of, or reducing the risk of developing any of the diseases or disorders described herein. In some embodiments, the AZ. vaccae comprises AZ vaccae strain ATCC 15483. In some embodiments, the AZ. vaccae comprises AZ vaccae strain NCTC 11659.

[0023] In a further aspect, the disclosure includes isolated AZ vaccae, or constituent components, optionally combined with a pharmaceutically acceptable carrier forming a pharmaceutical composition for the treatment of, or prevention of, or reducing the risk of developing any of the diseases or disorder described herein. In a preferred aspect, the pharmaceutical compositions of the disclosure can be configured as a food, probiotic, parabiotic, postbiotic, or nutraceutical composition, as well as configured for parenteral or oral administration to a subject in need thereof.

[0024] In a further aspect, the disclosure includes isolated AZ. vaccae, or constituent components, optionally combined with a pharmaceutically acceptable carrier forming a pharmaceutical composition for the manufacture of a medicament for the treatment, prevention, or reducing the risk of developing any of the diseases or disorders described herein. In a preferred aspect, the compositions of the disclosure can be configured as a food, probiotic, parabiotic, postbiotic, or nutraceutical composition, as well as configured for parenteral or oral administration to a subject in need thereof.

[0025] Additional embodiments of the disclosure may become evident in light of the figures and disclosure provided below.

[0026] BRIEF DESCRIPTION OF FIGURES

[0027] Figs. 1A-1D. Effects of M. vaccae ATCC 15483 and Western-style diet on body weight, weight of visceral adipose tissue, kcal consumed, and water consumed across time. A) Average body weight in grams over time. *p < 0.05, **p < 0.01, ***p < 0.001, BBS / WD compared to BBS / CD, Fisher’s LSD test. #p < 0.05, ##p < 0.01, ###p < 0.001, MV / WD compared to BBS / WD, Fisher’s LSD test. B) The collective weight of inguinal, epidydimal, and retroperitoneal fat pads in grams (± SEM). **p < 0.01, ***p < 0.001, Fisher’s LSD test. C) Average kcal consumed over time (± SEM). *p < 0.05, BBS / WD compared to BBS / CD, Fisher’s LSD test. #p < 0.05, MV / WD compared to BBS / WD, Fisher’s LSD test.ap< 0.05, MV / CD compared to BBS / CD, Fisher’s LSD test. D) Average water consumed in grams overtime (± SEM). **p < 0.01, ***p < 0.001, BBS / WD compared to BBS / CD, Fisher’s LSD test. \p < 0.05 MV / CD compared to BBS / CD, Fisher’s LSD test. \p < 0.05 and fttp < 0.001, MV / WD compared to MV / CD, Fisher’s LSD test.

[0028] Figs. 1E-1H. Effects of M. vaccae ATCC 15483 and Western-style diet on total kcal consumed, kcal / body weight, total water consumed, and water consumed / body weight. E) Average total kcal consumed (± SEM). F) Average weekly kcal consumed per gram of weight body (± SEM). *p < 0.05, **p < 0.01 BBS / WD compared to BBS / CD Fisher’s LSD test. #p < 0.05 MV / WD Fisher’s LSD test, ip < 0.05 Mv / WD compared to Mv / CD, Fisher’s LSD test.

[0029] G) Average total water consumed (± SEM). ***p < 0.001, LSD Fisher’s LSD. H) Average water consumed per gram of body weight (± SEM). *p < 0.05, ***p < 0.001, BBS / WD compared to BBS / CD, Fisher’s LSD test, ftp < 0.01 and itp < 0.001, Mv / WD compared to Mv / CD, Fisher’s LSD test.

[0030] Figs. 2A-2E. Anxiety-like behavior was evaluated in the elevated plus-maze (EPM). Bar graphs illustrate mean (± SEM) values for: A) percentage of time spent in the open arm of the EPM, B) frequency of entries into the open arm of the EPM, C) distance traveled in the EPM, D) percentage of time in the closed arm of the EPM, E) frequency of entries into the closed arm of the EPM. *p < 0.05, Fishers LSD; +p < 0.05, main effect of M. vaccae ATCC 15483 based on two-way ANOVA. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV, Mycobacterium vaccae ATCC 15483; SEM, standard error of mean; WD, Western-style diet.

[0031] Figs. 3A-3F. (A-F) M. vaccae ATCC 15483 decreased specific markers of hippocampal neuroinflammation, based on relative gene expression measured using real time RT-PCR (*p < 0.05, ***p < 0.001). All graphs depict mean relative gene expression (± SEM). Abbreviations: ATCC, Mycobacterium vaccae ATCC 15483; BBS, borate-buffered saline; g, grams; NIH-31, control diet; WD, Western-style diet.

[0032] Figs. 4A-4E. (A-E) Effects of M. vaccae ATCC 15483 and Western diet on specific markers of adipokines in visceral adipose tissue, assessed using real-time RT-PCR. Specific immune related markers based on relative gene expression assessed through real time RT-PCR in the visceral adipose tissue were elevated with treatment of Western diet (lp value < 0.05). All graphs except for 4E depict relative gene expression (± SEM). E) Bar graph illustrates the Logio transformation of relative gene expression of Argl (± SEM). Abbreviations: ATCC, Mycobacterium vaccae ATCC 15483; BBS, borate-buffered saline; NIH-31, control diet; SEM, standard error of the mean; WD, Western-style diet; Wk, week.

[0033] Figs. 5A-5F. Effects o£M. vaccae ATCC 15483 and Western diet on specific markers of neuroinflammation and appetite regulation in the hypothalamus assessed using real-time RT-PCR. In the hypothalamus Insr expression was attenuated with treatment of AL vaccae ATCC 15483 in the Western diet-treated groups (*p < 0.05). Abbreviations: BBS, borate-buffered saline; CD, control diet; MV, Mycobacterium vaccae ATCC 15483; SEM, standard error of mean; WD, Western-style diet.

[0034] Figs. 6 A-6H. Neither AL vaccae ATCC 15483 nor Western diet influenced the weight of testes (A). A two-way ANOVA revealed a main effect of diet ( (i,40) = 4.389, *p < 0.05, (B) on the ratio of testes’ weight to body weight. Post hoc pairwise comparisons indicated a trend toward significance (p = 0.063) between the BBS treated groups. Neither AL vaccae ATCC 15483 nor Westen diet influenced sperm concentration (C), seminiferous tubule area (D), seminiferous tubule perimeter (E), seminiferous tubule diameter (F), percentage of open luminal seminiferous tubules (G), or percentage of seminiferous tubule with mature sperm (H). Figs. 7A-7D. Alpha diversity of the gut microbiome was evaluated using multiple metrics. Line graphs illustrate mean (± SEM) values for: (A) Observed OTUs, (B) Shannon’s diversity index, (C) Pielou’s evenness, and (D) Faith’s phylogenetic diversity index. (B and C) I p < 0.05 Indicates significance within MV / CD subjects effects of time, relative to timepoint 1, Wilcoxon signed-rank tests with Bonferroni correction. H p < 0.01 Indicates significance within MV / WD subjects effects of time, relative to timepoint 1, Wilcoxon signed-rank tests with Bonferroni correction, ffi p < 0.01 Indicates significance within BBS / WD subjects effects of time, relative to timepoint 1, Wilcoxon signed-rank tests with Bonferroni correction. / ? < 0.05 Indicates significance between MV / WD to MV / CD, Fisher’s LSD test. *p < 0.05, **p < 0.01, *** p < 0.001 Indicates significance between BBS / WD to BBS / CD, Fisher’s LSD test. Abbreviations: BBS, borate- buffered saline; CD, control diet; MV , Mycobacterium vaccae ATCC 15483; SEM, standard error of mean; WD, Western diet; OTU: operational taxonomic unit.

[0035] Fig. 8. Taxonomy stacked bar chart shows the composition of the bacterial community at the phylum level for each subject over time. Each bar represents a single mouse at a single timepoint. The x-axis shows the sample id for each subject, with the four timepoints of collection indicated by each tick mark in chronological order. The y-axis shows the relative abundance at the phylum level. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV Mycobacterium vaccae ATCC 15483; WD, Western diet.

[0036] Fig. 9. Line graphs illustrate mean value obtained for the Firmicutes / Bacteroidetes ratio (± SEM). *** / ? < 0.001 Indicates significant difference between BBS / WD compared to BBS / CD, Fisher’s LSD test. Hl < 0.001 Indicates significant difference between MV / WD compared to MV / CD, LSD Fisher’s LSD test. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV , Mycobacterium vaccae ATCC 15483; SEM, standard error of mean; WD, Western diet. The color coding is the same as in Figs. 7A-7D.

[0037] Fig. 10. Principal coordinates analysis (PCoA) plot provides a visual representation of the pattern of proximity among all samples in the study. PCI explains 41.2% of the variability and PC2 explains 15.2% of the variability. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV , Mycobacterium vaccae ATCC 15483; SEM, standard error of mean; WD, Western diet.

[0038] Fig. 11. The figure illustrates a positive correlation between the weight of VAT (grams) and the relative gene expression of Lep within VAT. Color coding is the same as in Fig. 12. Figs. 12A-12F. Anxiety-like behavior was evaluated in the open-field test. Bar graphs illustrate mean (± SEM) values for: (A) percentage of time in the center zone of the open-field, (B) frequency of entries into the center zone of the open-field, (C) distance traveled in the open- field, (D) latency to enter the center zone of the open-field, (E) frequency of stretch attend posture, and (F) percentage of time performing stretch attend posture. *p < 0.05 and **p < 0.01, Fisher’s LSD test. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV, Mycobacterium vaccae ATCC 15483; SEM, standard error of the mean; WD, Western diet.

[0039] Fig. 13. Schematic illustration of experimental timeline.

[0040] Fig. 14. Plot that shows Lep plasma levels in pg / mL. Lep protein levels in plasma were assessed using ELISA. A two-way ANOVA revealed an AL vaccae ATCC 15483 x Western-style diet interaction for plasma leptin concentrations (F(l,40) = 5.32, *p < 0.05). Post hoc pairwise comparisons revealed that, among mice treated with BBS vehicle, Western-style diet-fed mice showed elevated plasma leptin concentrations as compared to control diet mice. Post hoc pairwise comparisons also revealed that, among mice fed a Western-style diet, M. vaccae ATCC 15483 decreased plasma leptin concentrations. Thus, M. vaccae ATCC 15483 prevented Western-style diet-induced increases in plasma leptin concentrations. A linear regression was performed and revealed a positive correlation between weight of VAT and plasma leptin concentrations.

[0041] Fig. 15. This figure shows weighted UniFrac principal coordinates analysis (PCoA) plots that provide a visual representation of the dissimilarity in phylogenetic microbiome composition across all samples in the study for each day. Each point represents one sample from one mouse. PCoA was performed on the distance matrix containing all samples, so each individual PCoA plot shows the data in the same PCoA space, such that the location of points can be compared across plots. Day 0 coincided with the inception of either a control diet or a Western-style diet. Abbreviations: BBS, borate-buffered saline; CD, control diet; MV , Mycobacterium vaccae ATCC 15483; WD, Western-style diet.

[0042] DETAILED DESCRIPTION

[0043] This disclosure provides methods of utilizing mycobacteria, and in particular AL vaccae to induce weight reduction in a subject in need thereof. Specifically, in a preferred embodiment, the disclosure provides methods of utilizing mycobacteria for the treatment, prevention, or reducing the risk of developing obesity, and obesity-related diseases such as metabolic disorders or syndromes as described herein. In some embodiments, the mycobacteria comprise AL vaccae. In some embodiments, the M. vaccae comprise M. vaccae strain ATCC 15483. Tn some embodiments, the M. vaccae comprise AT. vaccae strain NCTC 11659.

[0044] The skilled artisan will appreciate that multiple strains of M. vaccae are suitable for the compositions and methods of treating obesity and obesity-related disorders described herein. These include, without limitation, M. vaccae strain ATCC 15483 (available from the American Type Culture Collection), and M. vaccae strain NCTC 11659 (available from the National Collection of Type Cultures).

[0045] In one aspect, the disclosure is directed to a method of utilizing M. vaccae, or constituent components, for the reduction of visceral adipose tissue (VAT) in a subject in need thereof. In another embodiment, the disclosure provides methods of utilizing M. vaccae for decreasing the expression of leptin, in a subject in need thereof, as well as modulating the expression of other VAT-associated genes. In another embodiment, this disclosure technology provides methods of utilizing AT. vaccae for decreasing food intake in a subject in need thereof.

[0046] In another aspect, the disclosure is directed to a method for treating, preventing, or reducing the risk of developing obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AT vaccae, or constituent components. In some embodiments, the subject is obese or morbidly obese and is in need of reducing weight or reducing / preventing further weight gain. In some embodiments, the subject is overweight and is in need of reducing body weight to the normal range and / or reducing / preventing further increase in body weight or becoming obese. In some embodiments, the subject is normal weight and wants to reduce or prevent an increase in body weight.

[0047] In another aspect, the disclosure is directed to a method for reducing body weight or body weight gain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AT. vaccae, or constituent components.

[0048] In another aspect, the disclosure is directed to a method for treating an obesity-related disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AT vaccae strain, or constituent components.

[0049] In another aspect, the disclosure is directed to a method for preventing, treating or reducing the risk of developing a metabolic disorder or metabolic disease in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of AT. vaccae, or constituent components. The metabolic disorder or metabolic disease can be selected from hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes-associated coronary heart disease (CHD). In some embodiments, the metabolic disorder or metabolic disease comprises type 2 diabetes.

[0050] In another aspect, the disclosure is directed to a method for preventing, treating or reducing the risk of developing a stress-related disorder, comprising administering a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae) to a subject in need thereof. The stress-related disorder can be selected from the group consisting of post- traumatic stress-disorder (PTSD), depression (including major depressive disorder), and anxiety disorders.

[0051] In one embodiment, this technology provides methods of utilizing AT. vaccae strain ATCC 15483, or constituent components, for the reduction of visceral adipose tissue (VAT) in a subject in need thereof. In another embodiment, this technology provides methods of utilizing M. vaccae strain ATCC 15483 for decreasing expression of LEP, encoding the adipokine, leptin, in a subject in need thereof, as well as modulating the expression of other VAT-associated genes. In another embodiment, this technology provides methods of utilizing M. vaccae strain ATCC 15483 for decreasing food intake in a subject in need thereof.

[0052] In another aspect, the present disclosure is directed to a method for treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components. In some embodiments, the subject is obese or morbidly obese and is in need of reducing weight or reducing / preventing further weight gain. In some embodiments, the subject is overweight and is in need of reducing body weight to the normal range and / or reducing / preventing further increase in body weight or becoming obese. In some embodiments, the subject is normal weight and wants to reduce / prevent an increase in body weight.

[0053] In another aspect, the disclosure is directed to a method for preventing or reducing the risk of developing obesity in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of AT. vaccae strain ATCC 15483, or constituent components.

[0054] In another aspect the present disclosure is directed to a method for reducing body weight or body weight gain in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components. In another aspect the present disclosure is directed to a method for treating an obesity- related disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AT vaccae strain ATCC 15483, or constituent components.

[0055] In another aspect the present disclosure is directed to a method for preventing or reducing the risk of developing obesity-related disease or condition, or one or more symptoms thereof, in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of M. vaccae strain ATCC 15483, or constituent components.

[0056] In another aspect the present disclosure is directed to a method for treating a metabolic disorder or metabolic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components. In another aspect the present disclosure is directed to a method for preventing or reducing the risk of developing a metabolic disorder or metabolic disease, or one or more symptoms thereof, in a subject in need thereof, comprising administering to the subject a prophylactically effective amount of M. vaccae strain ATCC 15483, or constituent components. The metabolic disorder or metabolic disease can be selected from hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes-associated coronary heart disease (CHD). In some embodiments, the metabolic disorder or metabolic disease comprises type 2 diabetes.

[0057] In another aspect, the present disclosure is directed to the use of AT vaccae, or constituent components, for the manufacture of a medicament for the treatment, prevention or reducing the risk of a metabolic disorder or metabolic disease in a subject in need thereof, comprising administering a therapeutically or prophylactically effective amount of AT vaccae, or constituent components. In one embodiment, the AT. vaccae is strain ATCC 15483.

[0058] In another aspect, the present disclosure is directed to AT vaccae, or constituent components, for use in the treatment, prevention or reducing the risk of developing a metabolic disease or disorder in a subject in need thereof, wherein the AT. vaccae, or constituent components, is for administration to the subject in a therapeutically effective amount. In one embodiment, the AT. vaccae is strain ATCC 15483.

[0059] In another aspect, the present disclosure is directed to a method for preventing, ameliorating or reducing the risk of developing one or more symptoms associated with a metabolic disorder or metabolic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae, or constituent components. In one embodiment, the M. vaccae is strain ATCC 15483.

[0060] In another aspect, the present disclosure is directed to the use of M. vaccae, or constituent components, for the manufacture of a medicament for the prevention, amelioration or reducing the risk of developing at least one symptom associated with a metabolic disorder or metabolic disease in a subject in need thereof, wherein the M. vaccae, or constituent components, is administered to the subject in a therapeutically effective amount. In a preferred embodiment, the M. vaccae is strain ATCC 15483.

[0061] In another aspect, the present disclosure is directed to M. vaccae, or constituent components, for use in the prevention, amelioration or reducing the risk of developing at least one symptom associated with a metabolic disease or disorder in a subject in need thereof, wherein the M. vaccae, or constituent components, is for administration to the subject in a therapeutically effective amount. In one embodiment, the AT. vaccae is strain ATCC 15483.

[0062] In another aspect, the present disclosure is directed to a method for treating, preventing or reducing the risk of developing diabetes in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of AT. vaccae, or constituent components. In another aspect the present disclosure is directed to a method for treating, preventing or reducing the risk of developing diabetes in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of AT. vaccae strain ATCC 15483, or constituent components. In some embodiments, the diabetes is type 1 diabetes. In other embodiments, the diabetes is type 2 diabetes.

[0063] In another aspect, the present disclosure is directed to M. vaccae, or constituent components, for use in the treatment, prevention, or reducing the risk of developing diabetes in a subject in need thereof, wherein the AT. vaccae, or constituent components, is for administration to the subject in a therapeutically or prophylactically effective amount. In an embodiment, the AT. vaccae is strain ATCC 15483. In some embodiments, the diabetes is type 1 diabetes. In other embodiments, the diabetes is type 2 diabetes.

[0064] In another aspect, the present disclosure is directed to a method for ameliorating one or more symptoms associated with diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of AT vaccae, or constituent components. In a preferred embodiment, the M. vaccae is strain ATCC 15483. In some embodiments, the diabetes is type 1 diabetes. In other embodiments, the diabetes is type 2 diabetes.

[0065] In another aspect, the present disclosure is directed to a method for ameliorating one or more symptoms associated with diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components.

[0066] In another aspect, the present disclosure is directed to M. vaccae, or constituent components, for use in reducing body weight or body weight gain in a subject in need thereof, wherein the M. vaccae, or constituent components, is for administration to the subject in a therapeutically effective amount. In a preferred embodiment, the AT. vaccae is strain ATCC 15483.

[0067] In another aspect, the present disclosure is directed to M. vaccae, or constituent components, for use in the manufacture of a medicament for in reducing body weight or body weight gain in a subject in need thereof, wherein the M. vaccae, or constituent components, is for administration to the subject in a therapeutically effective amount. In a preferred embodiment, the M. vaccae is strain ATCC 15483.

[0068] In another aspect, the present disclosure is directed to a method for reducing food intake in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a M. vaccae strain ATCC 15483, or constituent components.

[0069] In another aspect, the present disclosure is directed to a method for reducing leptin expression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components (sometimes referred to herein as “constituent parts”). In some embodiments, expression of leptin is decreased by at least 20%, at least 30%, at least 40%, at least 50% or at least 60%. In some embodiments, the decrease in leptin expression comprises a decrease in leptin mRNA levels. In some embodiments, the decrease in leptin expression comprises a decrease in leptin protein levels. Leptin protein levels can be assayed from any suitable sample, such as blood serum or plasma, using any suitable method known in the art or described herein (for example enzyme-linked immunosorbent assays, or ELISA). Leptin RNA levels can be assayed from any suitable tissue using any suitable methods known in the art or described herein, for example real time PCR, or RT-PCR).

[0070] In another aspect, the present disclosure is directed to a method for modulating expression of one or more VAT genes, such as IBA1, ADIPOQ, TNF, F0XP3 and / or ARG1 in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of M. vaccae strain ATCC 15483, or constituent components.

[0071] In another aspect, the present disclosure relates to methods comprising identifying a subject at risk of developing obesity or an obesity-related metabolic disease or disorder, and administering a prophylactically effective amount of the isolated M. vaccae, constituent components or pharmaceutical composition comprising same described herein. Subjects at risk of developing obesity or an obesity-related metabolic disease can be identified, inter alia, through personal and family medical history, one or more risk factors such as diet and / or genetic markers, or other indicia known in the art and described herein.

[0072] In another aspect, the present disclosure relates to methods comprising identifying a subject at risk of developing a stress-related disorder, and administering a prophylactically effective amount of the isolated M. vaccae, constituent components or pharmaceutical composition comprising same described herein. Subjects at risk of developing a stress-related disorder can be identified, inter alia, through personal and family medical history, one or more risk factors such genetic background, or other indicia known in the art and described herein.

[0073] The present disclosure further includes a pharmaceutical composition containing isolated M. vaccae strain ATCC 15483, or constituent parts wherein the pharmaceutical composition promotes weight loss effects and / or reduction of visceral adipose tissue (VAT) in a subject in need thereof.

[0074] The present disclosure further includes a pharmaceutical composition containing isolated M. vaccae, or constituent parts, wherein the pharmaceutical composition treats, prevents or reduces the risk of developing a metabolic disorder or metabolic disease. In an embodiment, the metabolic disorder or metabolic disease is selected from hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes-associated coronary heart disease (CHD).

[0075] The present disclosure further includes a pharmaceutical composition containing isolated M. vaccae, or constituent parts, wherein the pharmaceutical composition promotes weight loss effects and / or reduction of visceral adipose tissue (VAT) in a subject in need thereof. The present disclosure further includes a pharmaceutical composition containing isolated M. vaccae, or constituent parts wherein the pharmaceutical composition treats and / or prevents obesity, and / or one or more symptoms associated with obesity in a subject in need thereof. In some embodiments, the isolated M. vaccae may be a whole cell. Alternatively, or additionally, the isolated M. vaccae may comprise a non-pathogenic heat-killed bacterium. In these methods, the isolated M. vaccae may comprise the constituent parts of the isolated M. vaccae, which may include organelles or biomolecules isolated from (e.g., extracted from) the M. vaccae.

[0076] In some embodiments, the pharmaceutical composition comprises isolated M. vaccae strain ATCC 15483, or constituent components. Alternatively, or additionally, the pharmaceutical composition may comprise isolated M. vaccae strain NCTC 11659, or constituent components.

[0077] In some embodiments, the pharmaceutical composition comprises substantially pure M. vaccae, or constituent components, i.e., the pharmaceutical composition does not include a carrier, diluent or excipient. For example, the pharmaceutical composition may comprise at least 90%, at least 95%, at least 96%, at least 97%, at least 98% at least 99% or at least 99.5% M. vaccae, or constituent components, as determined by weight. In some embodiments, the M. vaccae or constituent components is lyophilized. Such substantially pure compositions are suitable for inclusion, for example, in dietary supplements or medical foods.

[0078] In some embodiments, the pharmaceutical composition comprises isolated M. vaccae, or constituent components, and a pharmaceutically acceptable carrier or diluent, as described herein.

[0079] In some embodiments, the isolated M. vaccae or constituent components may be in the form of a vaccine composition optionally comprising an adjuvant. In these methods, the isolated M. vaccae may be administered in two or more repeated doses. Alternatively, or additionally, in these methods, the isolated A . vaccae may be administered in a unit dose comprising an effective amount of non-pathogenic heat-killed isolated AY vaccae from 107to 109cells. In these methods, the isolated AY. vaccae may be administered as a vaccination, which may be administered weekly, for example three separate vaccinations administered weekly to the subject. In some embodiments, the vaccine composition comprises isolated AY. vaccae strain ATCC 15483 or constituent components. In some embodiments, the vaccine composition comprises isolated AY vaccae strain NCTC 11659 or constituent components.

[0080] In some embodiments, the isolated AY. vaccae (e.g., ATCC 15483) or constituent components may be formulated for administration via the parenteral, oral, sublingual, nasal or pulmonary route. In some embodiments, the parenteral route is selected from subcutaneous, intradermal, subdermal, intraperitoneal, intravenous, or intravesicular injection. In particular, the isolated AY vaccae (e.g., ATCC 15483) or constituent components may be formulated for administration via the oral route. In some embodiments, the isolated M. vaccae (e.g., ATCC 15483) may be formulated for administration via the oral route and is heat-killed. In some embodiments, the isolated M. vaccae (e.g., ATCC 15483)may be formulated for administration via the oral route and is in its constituent components (or parts). In some embodiments, the isolated M. vaccae may be formulated for administration via the oral route and is whole cell. In some embodiments, the isolated AT. vaccae (e.g., ATCC 15483) may be formulated for administration via the oral route and is in its constituent components.

[0081] In certain embodiments, a particular dosage of isolated M. vaccae or constituent parts (e.g., ATCC 15483) be administered to a subject. Thus, in certain embodiments of the disclosure, there is provided a containment means comprising the effective amount of heat-killed AT. vaccae (e.g., ATCC 15483) foruse in the present disclosure, which typically may be from 103to 1011organisms, preferably from 104to IO10organisms, more preferably from 106to IO10organisms, and even more preferably from 106to 109organisms per unit dose. The effective amount of heat-killed AT vaccae (e.g., ATCC 15483) for use in the present disclosure may be from 103to 1011organisms, preferably from 104to IO10organisms, more preferably from 106to IO10organisms, and even more preferably from 106to 109organisms. Most preferably the amount of heat-killed AT. vaccae (e.g., ATCC 15483) for use in the present disclosure is from 107to 109cells or organisms. Typically, the composition according to the present disclosure may be administered at a dose of from 108to 109cells for human and animal use. Alternatively, the dose is from 0.01 mg to 1 mg or 0.1 mg to 1 mg organisms or constituent presented as either a suspension or dry preparation.

[0082] AT. vaccae “pharmaceutical compositions” according to the disclosure can comprise a therapeutically effective amount of AT vaccae (e.g., ATCC 15483 or NCTC 11659) or constituent parts, typically dispersed in a pharmaceutically acceptable carrier. The phrases “pharmaceutical or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains Mycobacterium ATCC 15483 will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, Moreover, for animal (e.g., human) administration, it will be understood that parenteral preparations should meet sterility, pyrogenicity, general safety and purity standards. A specific example of a pharmacologically acceptable carrier as described herein is sterile borate- buffered saline or sterile saline solution (0.9% NaCl).

[0083] As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329).

[0084] In certain embodiments of the disclosure, it is preferable that a particular dosage of isolated M. vaccae or constituent parts be administered to a subject. Thus, in certain embodiments of the disclosure, there is provided a composition comprising the effective amount of heat-killed M. vaccae for use in the methods of this disclosure, which typically may be from 103to 1011organisms, preferably from 104to IO10organisms, more preferably from 106to IO10organisms, and even more preferably from 106to 109organisms per unit dose. The effective amount of heat- killed M. vaccae for use in the methods of this disclosure is from 107to 109cells or organisms. Typically, the composition according to the present disclosure may be administered at a dose of from 108to 109cells for human and animal use.

[0085] The isolated M. vaccae may be administered to the patient via the parenteral, oral, sublingual, nasal or pulmonary route. In a preferred embodiment, the isolated M. vaccae or constituent parts is administered via a parenteral route selected from subcutaneous, intradermal, subdermal, intraperitoneal, intravenous and intravesicular injection. More preferably, administration by the parenteral route does not comprise injection of mycobacterial cell wall extract.

[0086] In another preferred embodiment, the isolated M. vaccae or constituent parts is administered orally, including by suspension, tablets and the like. Additional pharmaceutical vehicles could be used to control the duration of action of the preparation. They could be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization (hydroxymethylcellulose or gelatin microcapsules) in colloidal drug delivery systems (for example, liposomes, albumin microspheres, micro-emulsions, nanoparticles and nanocapsules) or in macro-emulsions. Excipients, for example, salts, various bulking agents, additional buffering agents, chelating agents, antioxidants, cosolvents and the like can be included in the final formulation.

[0087] A suitable dosage schedule according to this embodiment includes administration of the isolated M. vaccae or constituent parts followed by further doses of the isolated M. vaccae or constituent parts 2 weeks later and continuing every 2 weeks for the next 3 doses followed by 4 weeks without treatment. Thereafter, patients may receive the isolated M. vaccae or constituent parts every 4 weeks for up to 12 months or longer following the first dose given. Alternatively, dosing may involve weekly administration following the priming or initial dose.

[0088] In some embodiments, the isolated AT. vaccae or constituent parts may be administered as one or more aliquots each containing an effective amount of the isolated M. vaccae or constituent components. The isolated AT. vaccae or constituent parts may be administered at one or more time intervals between 4 weeks and 1 week prior to any anticipated stressful or surgical event. In some embodiments, the isolated M. vaccae or constituent parts may be administered as one or more aliquots each containing an effective amount of the isolated M. vaccae or constituent parts which may be administered at one or more time intervals between 4 weeks and 1 week before any anticipated stressful or surgical event and / or the isolated M. vaccae or constituent may be administered, and repeated on at least about 2, 4, 6, 8, 10, 12, 15, 20 or more occasions before and / or after any anticipated stressful or surgical event. In some embodiments, isolated M. vaccae or constituent parts may be administered at least once a day, at least twice a day, at least 3 times a day, at least 4 times a day, or at least 5 times a day before and / or after any anticipated stressful or surgical event. In some embodiments, isolated AT. vaccae or constituent parts may be administered at least once a day, at least twice a day, at least 3 times a day, at least 4 times a day, or at least 5 times a day after a stressful event, and administration can continue for at least a week, at least a month, at least 4 months, at least 6 months, at least a year, or until symptoms caused by the stressful or surgical event are reduced or no longer apparent.

[0089] In one embodiment of the present disclosure, the isolated M. vaccae or constituent components may be in the form of a medicament administered to the patient in a dosage form and / or in a schedule.

[0090] The effective or prophylactic amount of the isolated M. vaccae or its constituent parts may be administered as a single dose. Alternatively, the effective or prophylactic amount of the isolated M. vaccaeor constituent parts may be administered in multiple (repeat) doses, for example two or more, three or more, four or more, five or more, ten or more, or twenty or more repeat doses. Preferably, the isolated M. vaccae or constituent parts is administered between about 4 weeks and 1 day prior to an anticipated stressful event, more preferably between about 4 weeks and 1 week, or about between 3 weeks and 1 week, or about between 3 weeks and 2 weeks. Administration may be presented in single or multiple doses. Administration can continue after the stressful event, e.g. until symptoms caused by the stressful event are reduced or no longer apparent.

[0091] The isolated M. vaccae or constituent parts may be lyophilized and formulated for resuspension prior to administration. However, in other cases, the M. vaccae or constituent parts are suspended in a volume of a pharmaceutically acceptable liquid. In some of the most preferred embodiments there is provided a container comprising a single unit dose of M. vaccae or constituent parts suspended in pharmaceutically acceptable carrier wherein the unit dose comprises about 1X106to about 1 X1O10M. vaccae or constituent parts. In some very specific embodiments, the liquid comprising suspended M. vaccae or constituent parts is provided in a volume of between about 0. 1 ml and 10 ml, or about 0.5 ml and 2 ml. The composition comprising the mycobacteria or constituent parts may be frozen. The foregoing compositions provide ideal units for applications described herein.

[0092] Embodiments discussed in the context of a methods and / or composition of the disclosure may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the disclosure as well.

[0093] In some cases, attenuated AT. vaccae is administered to specific sites on or in a subject. For example, the AT vaccae compositions may be administered into the central nervous system, such as administration to the brain or spinal cord. Alternatively, sites of administration of a mycobacterial composition may be near the posterior cervical, tonsillar, axillary, inguinal, anterior cervical, sub-mandibular, sub mental or superclavicular lymph nodes. Such sites of administration may be on the right side, on the left side, or on both sides of the body.

[0094] A dosage of AT. vaccae or constituent parts may be administered to a subject by intradermal injection.

[0095] In some further embodiments of the disclosure, methods of the disclosure involve the administration of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses of AT vaccae separated by a period of one day or more. In certain embodiments, such separate doses will be separated by several days, one week, two weeks, one month or more. For example, methods according to the disclosure may comprise administering 1 to 5 doses of M. vaccae over a period of three weeks or more. In yet further embodiments, methods of the disclosure comprise administering 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 2 doses of M. vaccae over a period of about three weeks. Each dose administered may be the same or different dosage relative to a previous or subsequent dose administration. For example, in certain cases, it is preferred that a dosage of M. vaccae is lower than any dosage that was previously administered. Thus, in some specific cases, a dose of AT. vaccae will be administered at about half of the dosage that was administered in any previous treatment. Such methods may be preferred in certain instances where the subject's immune response to the M. vaccae is greater during subsequent therapies. Thus, in certain cases, the isolated M. vaccae may be administered a minimal number of times for example, in less than 10, 9, 8, 7, 6, 5, 4, 3 or fewer separate dosage administrations. In some cases, the mycobacterial composition is administered twice.

[0096] In some aspects, the M. vaccae, constituent components, or pharmaceutical composition comprising same can be administered about 1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, or 6 or more times per day. In some aspects, the M. vaccae, constituent components, or pharmaceutical composition comprising same can be administered about once every day (QD), about once every two days (Q2D), about once every three days (Q3D), about once every four days (Q4D), about once every 5 days (Q5D), about once every 6 days (Q6D), about once every 7 days (Q7D), about once every 8 days (Q8D), about once every 9 days (Q9D), about once every 10 days (Q10D), about once every 11 days (QI ID), about once every 12 days (Q12D), about once every 13 days (Q13D), about once every 14 days (Q14D), or about once every 15 days (Q15D). In some aspects, the M. vaccae, constituent components, or pharmaceutical composition comprising same can be administered about once every 7 days (Q7D). In some aspects, the M. vaccae can be administered about once every 14 days (Q14D).

[0097] In some embodiments, administration of the M. vaccae, constituent components, or pharmaceutical composition comprising same can continue for at least a week, at least a month, at least 4 months, at least 6 months, at least 8 months, at least a year, at least 1.5 years, or at least 2 years. In some embodiments, administration of the M. vaccae, constituent components, or pharmaceutical composition comprising same continues until one or more signs or symptoms of obesity or the related metabolic disease or disorder are reduced or no longer apparent. In some embodiments, administration of the M. vaccae, constituent components, or pharmaceutical composition comprising same continues after one or more signs or symptoms of obesity or the related metabolic disease or disorder are reduced or no longer apparent. In some embodiments, administration the M. vaccae, constituent components, or pharmaceutical composition comprising same continues until the subject’s weight is reduced. In some embodiments, administration the vaccae, constituent components, or pharmaceutical composition comprising same continues after the subject’s weight is reduced. In some embodiments, administration of the M. vaccae, constituent components, or pharmaceutical composition comprising same continues until one or more signs or symptoms of the stress-related disease or disorder are reduced or no longer apparent. In some embodiments, administration of the M. vaccae, constituent components, or pharmaceutical composition comprising same continues after one or more signs or symptoms of stress-related disease or disorder are reduced or no longer apparent.

[0098] Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical formulations to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc. suitable for ingestion by the patient. In other embodiments, pharmaceutical formulations for oral administration can be formulated without using any pharmaceutically acceptable carriers. Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the pharmaceutical formulation suspended in a diluent, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant, such as magnesium stearate, paraben, preserving agent, such as sorbic acid, ascorbic acid, .alpha. -tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.

[0099] Pharmaceutical preparations for oral use can be obtained through combination of the M. vaccae of the present disclosure, a preferred embodiment being M. vaccae ATCC 15483 with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fdlers and include, but are not limited to sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxymethyl cellulose, hydroxypropylmethyl - cellulose or sodium carboxymethylcellulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.

[0100] The present disclosure provides a kit comprising a therapeutically effective amount of the M. vaccae or constituent parts of the present disclosure, a preferred embodiment being M. vaccae ATCC 15483. In some embodiments, the AT. vaccae is heat-killed. In some embodiments, the M. vaccae or constituent components is formulated in a pharmaceutical composition described herein. In some embodiments, the M. vaccae or constituent components is substantially pure. In some embodiments, the kit comprises a container, and optionally, instructions for use.

[0101] The present disclosure provides a method of treating a metabolic disease or disorder in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of the M. vaccae or constituent components as provided in the kit described herein. The present disclosure provides a method of ameliorating one or more symptoms associated with a metabolic disease or disorder in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of M. vaccae or constituent components provided in the kit.

[0102] The present disclosure provides a method of treating diabetes in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount M. vaccae or constituent components provided in the kit. The present disclosure provides a method of ameliorating one or more symptoms associated with diabetes in a subject in need thereof, the method comprising administering to the subject in need thereof a therapeutically effective amount of the M. vaccae or constituent components provided in the kit. As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

[0103] As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles. As used herein, “individual” or “subject” or “animal” means a human or non-human animal selected for treatment or therapy.

[0104] As used herein, “identifying” or “selecting a subject having a metabolic or cardiovascular disease” means identifying or selecting a subject having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identifying or selecting a subject having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, above normal body weight, and / or above normal body fat content or any combination thereof. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring bloodpressure, measuring body fat content, measuring body weight, and the like.

[0105] As used herein, “identifying” or “selecting a diabetic subject” means identifying or selecting a subject having been identified as diabetic or identifying or selecting a subject having any symptom of diabetes (type 1 or type 2) such as, but not limited to, having a fasting glucose of at least 110 mg / dL, glycosuria, polyuria, polydipsia, increased insulin resistance, and / or decreased insulin sensitivity.

[0106] As used herein, “identifying” or “selecting an obese subject” means identifying or selecting a subject having been diagnosed as obese or identifying or selecting a subject with a BMI over 30 and / or a waist circumference of greater than 102 cm in men or greater than 88 cm in women.

[0107] As used herein, “identifying” or “selecting a subject having dyslipidemia” means identifying or selecting a subject diagnosed with a disorder of lipid and / or lipoprotein metabolism, including lipid and / or lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0108] As used herein, “identifying” or “selecting” a subject having increased adiposity” means identifying or selecting a subject having an increased amount of body fat (or adiposity) that includes concern for one or both the distribution of fat throughout the body and the size and mass of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computer tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese.

[0109] As used herein, “metabolic disorder” or “metabolic disease” refers to a condition characterized by an alteration or disturbance in metabolic function. “Metabolic” and “metabolism” are terms well known in the art and generally include the whole range of biochemical processes that occur within a living organism. Metabolic disorders include, but are not limited to, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes. Metabolic disorders also include coronary heart disease (CHD). As used herein, “coronary heart disease (CHD)” means a narrowing of the small blood vessels that supply blood and oxygen to the heart, which is often a result of atherosclerosis. As used herein, “metabolic syndrome” means a condition characterized by a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. In certain embodiments, metabolic syndrome is identified by the presence of any 3 of the following factors: waist circumference of greater than 102 cm in men or greater than 88 cm in women; serum triglyceride of at least 150 mg / dL; HDL-C less than 40 mg / dL in men or less than 50 mg / dL in women; blood pressure of at least 130 / 85 mmHg; and fasting glucose of at least 110 mg / dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285: 2486-2497).

[0110] As used herein, “diabetes mellitus” or “diabetes” is a syndrome characterized by disordered metabolism and abnormally high blood sugar (hyperglycemia) resulting from insufficient levels of insulin or reduced insulin sensitivity. The characteristic symptoms are excessive urine production (polyuria) due to high blood glucose levels, excessive thirst and increased fluid intake (polydipsia) attempting to compensate for increased urination, blurred vision due to high blood glucose effects on the eye's optics, unexplained weight loss, and lethargy.

[0111] As used herein, “diabetic dyslipidemia” or “type 2 diabetes with dyslipidemia” means a condition characterized by Type 2 diabetes, reduced HDL-C, elevated triglycerides, and elevated small, dense LDL particles.

[0112] As used herein, “individual” or “subject” or “animal” means a human or non-human animal selected for treatment or therapy.

[0113] As used herein, “identifying” or “selecting a subject having a metabolic or cardiovascular disease” means identifying or selecting a subject having been diagnosed with a metabolic disease, a cardiovascular disease, or a metabolic syndrome; or, identifying or selecting a subject having any symptom of a metabolic disease, cardiovascular disease, or metabolic syndrome including, but not limited to, hypercholesterolemia, hyperglycemia, hyperlipidemia, hypertriglyceridemia, hypertension, increased insulin resistance, decreased insulin sensitivity, above normal body weight, and / or above normal body fat content or any combination thereof. Such identification may be accomplished by any method, including but not limited to, standard clinical tests or assessments, such as measuring serum or circulating (plasma) cholesterol, measuring serum or circulating (plasma) blood-glucose, measuring serum or circulating (plasma) triglycerides, measuring bloodpressure, measuring body fat content, measuring body weight, and the like.

[0114] As used herein, “identifying” or “selecting a diabetic subject” means identifying or selecting a subject having been identified as diabetic or identifying or selecting a subject having any symptom of diabetes (type 1 or type 2) such as, but not limited to, having a fasting glucose of at least 110 mg / dL, glycosuria, polyuria, polydipsia, increased insulin resistance, and / or decreased insulin sensitivity.

[0115] As used herein, “identifying” or “selecting an obese subject” means identifying or selecting a subject having been diagnosed as obese or identifying or selecting a subject with a BMI over 30 and / or a waist circumference of greater than 102 cm in men or greater than 88 cm in women.

[0116] As used herein, “identifying” or “selecting a subject having dyslipidemia” means identifying or selecting a subject diagnosed with a disorder of lipid and / or lipoprotein metabolism, including lipid and / or lipoprotein overproduction or deficiency. Dyslipidemias may be manifested by elevation of lipids such as cholesterol and triglycerides as well as lipoproteins such as low-density lipoprotein (LDL) cholesterol.

[0117] As used herein, “identifying” or “selecting” a subject having increased adiposity” means identifying or selecting a subject having an increased amount of body fat (or adiposity) that includes concern for one or both the distribution of fat throughout the body and the size and mass of the adipose tissue deposits. Body fat distribution can be estimated by skin-fold measures, waist-to-hip circumference ratios, or techniques such as ultrasound, computer tomography, or magnetic resonance imaging. According to the Center for Disease Control and Prevention, individuals with a body mass index (BMI) of 30 or more are considered obese. As used herein, “identifying” or “selecting a subject having increased risk of developing obesity or an obesity-related metabolic disease or disorder” means identifying a subject having one or more risk factors that increase the likelihood that the subject will develop obesity or a metabolic disease or disorder as described herein. Risk factors include, but are not limited to, high blood pressure, elevated blood sugar, elevated triglycerides, elevated BMI, family history, age, genetic background, family history, sedentary lifestyle, a diet high in saturated fats, processed foods and / or sugar, and / or medications (e.g. corticosteroids and antipsychotics).

[0118] As used herein, a “stress-related disorder” refers to mental health conditions that develop in response to either acute or chronic stress. Exemplary stress disorders include post-traumatic stress disorder (PTSD), which develops as a response to trauma, acute stress disorder, a short term reaction to a particular stressor, adjustment disorder, which occurs when a subject has difficulty adjusting to changes in life circumstances, anxiety disorders, which are characterized by excessive fear and nervousness, mood disorders such as depression and bipolar disorder, and obsessive compulsive disorder. Anxiety disorders include both general anxiety disorder, panic disorder, social anxiety disorder and phobia-related disorders (extreme specific and persistent fears of an object, situation or activity). Symptoms of stress-related disorders include, but are not limited to, difficulty concentrating, irritability, insomnia, anorexia, nightmares and hypervigilance, as well as physical symptoms such as headache, stomachache and chest pain.

[0119] As used herein, “identifying” or “selecting a subject having increased risk of developing a stress-related disorder” means identifying a subject having one or more risk factors that increase the likelihood that the subject will develop a stress-related disorder. Risk factors include, but are not limited to history of trauma, personal mental health history, familial mental health history, physical health, genetic predisposition, and substance abuse.

[0120] As used herein, “isolated”, as used herein, refer to material, such as M. vaccae that is substantially or essentially free from components that normally accompany the material in its native state or when the material is produced. In one embodiment, “isolated”, as used herein, refer to material containing principally M. vaccae.

[0121] As used herein, “a,” “an,” or “the” can mean one or more than one. For example, “a” cell can mean a single cell or a multiplicity of cells. Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0122] The term “about,” as used herein when referring to a measurable value such as an amount of dose (e.g., an amount of a compound) and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified amount.

[0123] The terms “comprise,” “comprises,” and “comprising” as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0124] As used herein, the terms “reduce,” “reduces,” “reduced,” “reduction,” and similar terms mean a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more. In particular embodiments, the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0125] By the term “treat,” “treating,” or “treatment of’ (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder. With respect to obesity, the term refers to, e.g., a decrease in body mass index, a decrease in body weight, and / or a decrease in body fat. In some embodiments, treatment provides a reduction in body weight by at least about 5%, e.g., about 10%, 15%, or 20%.

[0126] A “therapeutically effective” amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0127] The terms “prevent,” “preventing,” and “prevention” (and grammatical variations thereof) refer to prevention and / or delay of the onset of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of the methods of the disclosure. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset is less than what would occur in the absence of the present disclosure. With respect to obesity, the term refers to, e.g., preventing obesity from occurring if the treatment is administered prior to the onset of the obese condition. In some embodiments, prevention refers to a decrease in the amount of body weight gained compared to the amount of body weight gained in the absence of administration of the compounds of the disclosure.

[0128] A “prevention effective” or “prophylactically effective” amount as used herein is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder and / or clinical symptoms in a subject and / or to reduce and / or delay the severity of the onset of a disease, disorder and / or clinical symptoms in a subject relative to what would occur in the absence of the methods of the disclosure. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0129] A “subject” of the disclosure includes any animal that has or is susceptible to obesity or is in need of reducing body weight, body weight gain, and / or food intake. Such a subject is generally a mammalian subject (e.g., a laboratory animal such as a rat, mouse, guinea pig, rabbit, primate, etc.), a farm or commercial animal (e.g., a cow, horse, goat, donkey, sheep, etc.), or a domestic animal (e.g., cat, dog, ferret, etc.). In particular embodiments, the subject is a primate subject, a non-human primate subject (e.g., a chimpanzee, baboon, monkey, gorilla, etc.) or a human. Subjects include males and / or females of any age, including neonates, juveniles, adolescents, adults and geriatric subjects.

[0130] A “subject in need” of the methods of the disclosure can be a subject known to have, suspected of having, or having an increased risk of developing overweight or obesity.

[0131] As used herein the term “body mass index” or “BMI” means the ratio of weight in Kg divided by the height in meters, squared.

[0132] As used herein the term “overweight” refers to a BMI between 25 and 30 in adult humans. For people under 20 “overweight” is defined as a BMI between the 85th and 95th percentile compared to people of the same age.

[0133] As used herein the term “obesity” refers to a BMI between 30 and 40 in adult humans. For people under 20 “obesity” is defined as a BMI above the 95th percentile compared to people of the same age. As used herein, the term can include both obesity and morbid obesity.

[0134] As used herein the term “morbid obesity” refers to a BMI greater than 40 in adult humans.

[0135] As used herein the term “body weight” refers to the weight of a subject's body. As used herein the term “body weight gain” refers to the increase in weight of a subject's body over time.

[0136] As used herein the term “food intake” refers to the intake of calories in any form, including without limitation food, drink, intravenous, or enteral.

[0137] A “vaccine” is typically understood to be a prophylactic or therapeutic material providing at least one epitope of an antigen, preferably an immunogen. “Providing at least one epitope” means, for example, that the vaccine comprises the epitope (or antigen comprising or providing said epitope) or that the vaccine comprises a molecule that, e.g., encodes the epitope or an antigen comprising or providing the epitope. The antigen preferably stimulates the adaptive immune system to provide an adaptive immune response. The (pharmaceutical) composition or vaccine provided herein may further comprise at least one pharmaceutically acceptable excipient, adjuvant or further component (e g., additives, auxiliary substances, and the like).

[0138] The term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigenspecific immune response by e.g., promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response.

[0139] In addition, in some embodiments, the mycobacteria of the disclosure can be used, either alone or in combination with each other or in combination with one or more other therapeutic medications or interventions. In some embodiments of the present disclosure, M. vaccae or constituent components suitable for use in the practice of this disclosure will be administered together with behavioral modifications, such as controlled diet and / or exercise.

[0140] EXAMPLES

[0141] Example 1 : Materials and Methods.

[0142] Animals

[0143] Male C57BL / 6N mice (N= 48, Charles River Laboratories, Raleigh, NC, USA) arrived at approximately postnatal day 21 on experimental day -80 (Fig. 15). All mice experienced a phase delay of ~2 h (Coordinated Universal Time (UTC)-05:00, Eastern Standard Time to UTC-07:00, Mountain Standard Time [MST]) and significant elevation gains during transport (1,559 m from Raleigh, NC, USA). Mice were housed individually in polysulfone mouse cages (Cat. No. PC575JHT, 75Jag Cage, Allentown; Allentown, NJ, USA) with Static Allentown micro isolator filter topped caging (186 mm L x 298 mm Wx128 mm H) and aspen chip bedding (Cat. No. 7090, Teklad sani-chips Bedding, Envigo (now Inotiv), Madison, WI, USA). For the first ten days (day -80 to day -70) all mice received the Office of Animal Resources (OAR) standard mouse chow (Cat. No. 2918, Teklad Global 18% Protein Rodent Diet; Envigo). Following the first ten days the mice were randomly assigned to : 1) standard rodent chow (Cat. No. 7017, NIH-31 Open Formula Mouse / Rat Sterilizable Diet; Envigo); or 2) a Western-Style Diet (Cat. No. TD.96132, Adjusted Fat Diet; Envigo). Water was supplied using a reverse osmosis / Hydropac system (Lab Products, Inc. Seaford, DE). Boulder city water was processed through reverse osmosis, then chlorinated to a final concentration of 0.5-1 ppm chlorine via addition of household bleach. The water was chlorinated to prevent microbial contamination and is standard practice in laboratory animal institutions. Hydropacs and water bottles are filled approximately 1 week prior to delivery to each animal facility, so the vast majority of chlorine has dissipated by the time the water reaches the animals. Beginning on experimental day -80 mice were housed ~11 weeks in a standard 12-h light:dark (LD) cycle with lights on at 0600 h MST or 0700 h Mountain Daylight Time (MDT). The mice were provided with environmental enrichment, which included nestlets (Cat. No. NES3600, Nestlets; Ancare, Corp, Bellmore, NY) and a red hut (Cat. No. K3327, Bio-Serv, Flemington, NJ, USA), for each cage.

[0144] The mice in the study arrived in the prepubescent stage postnatal day (pnd) 21-34. The start of all interventions, diet change and subcutaneous treatments, occurred in the prepubescent stage (pnd 21-34). The behavior testing, tissue collection and the end of the weekly subcutaneous injection all occurred when the mice were in the adulthood stage (pnd > 78).

[0145] Experimental timeline

[0146] This is a 2 (M. vaccae ATCC 15483 versus vehicle) x 2 (Western-Style Diet versus control diet) design; N= 48 (n = 12 per treatment group). See Fig. 13 for an experimental timeline. Fortyeightjuvenile (approximately postnatal day 21) male C57BL / 6N mice arrived on experimental day -80. For the first ten days (day -80 to day -70) all mice received the standard mouse chow (Cat. No. 2918, Teklad Global 18% Protein Rodent Diet; Envigo) provided by the University of Colorado Boulder Office of Animal Resources (OAR). Following the first ten days the mice were randomly assigned to a Western-Style Diet (Cat. No. TD.96132, Adjusted Fat Diet; Envigo) or a standard rodent chow (Cat. No. 7017, NIH-31 Open Formula Mouse / Rat Sterilizable Diet; Envigo). Tap water was provided ad libitum to all the mice. Mice were given three days to acclimate to their environment in the vivarium. After this period, they received weekly lx subcutaneous (s.c.) injection for 11 weeks with 100 pl of either M. vaccae ATCC 15483 in sterile borate-buffered saline (BBS) or sterile BBS vehicle at ZT2-4 on experimental days -77, -70, -63, -56, -49, -42, -35, -28, -21, -14, and -7 for both diet groups, i.e., Western-Style Diet or control diet. Throughout this eleven-week period we collected fecal pellets four times (days -78, -64, - 29, and -5) at ZT10-12 for analysis of gut microbiome diversity and community composition. On day 0, mice were tested in the open-field test. On day 3, the mice were tested in the elevated plusmaze. On day 4 mice were euthanized with a terminal dose of sodium pentobarbital (150 mg / kg, i.p.) followed by opening of the thoracic cavity and exsanguination using cardiac puncture for blood collection followed by transcardial perfusion with ice-cold saline in order to remove white blood cells from cerebrovascular vessels.

[0147] M. vaccae ATCC 15483

[0148] This study used a whole cell heat-killed preparation of AT. vaccae ATCC 15483 suspension. M. vaccae ATCC 15483 was purchased from American Type Culture Collection (ATCC) Bonicke and Juhasz (ATCC® 15483), Manassas, VA, USA). M vaccae ATCC 15483 was cultured in ATCC® Medium 1395: Middlebrook 7H9 broth with ADC enrichment at 37 °C, then centrifuged at 3000 * g at 4 °C for ten minutes to pellet the cells, growth media was removed, and cells were weighed and resuspended in sterile BBS to a concentration of 10 mg / ml. Cells were transferred to a sealed sterile glass container and autoclaved at 121 °C for 15 min. Heat-killed bacterial stock was stored at 4 °C. M. vaccae ATCC 15483 was further diluted to 1 mg / ml and s.c. injections consisted of 0.1 mg in 100 pl sterile BBS, estimated to be 1 x 108 bacterial cells.

[0149] Diet intervention

[0150] Prior to any intervention, the first ten days (day -80 to day -70) all mice received the University of Colorado Boulder OAR standard mouse chow (Cat. No. 2918, Teklad Global 18% Protein Rodent Diet; Envigo). On day -70, half of the mice received Western style diet (Cat. No. TD.96132, Adjusted Fat Diet; Envigo) — composed of 40.6% kcal from fat (beef tallow and vegetable shortening), 40.7% carbohydrate (including 18.2% of total caloric content as sucrose), and 18.7% protein whereas the other half received a standard rodent chow (Cat. No. 7017, NIH- 31 Open Formula Mouse / Rat Sterilizable Diet; Envigo) Normal chow Envigo 7917 (14% kcal from fat, 62% carbohydrate, 24% protein). The Western-style diet was specifically chosen to match the 75thpercentiles of US diets for total fat and sugar intake based on NHANES data (Brunt et al., 2022). Mice were permitted ad libitum access to food and water throughout the entirety of the study. The high-fat / high-sugar Western-style diet used in this study has consistently been shown in mice to induce excessive weight gain, increase percentage of body fat, and elevate fasting blood insulin concentrations compared with numerous types of control diets. Food consumption was monitored weekly by weighing the food in grams starting on Day 0 and ending on Day 70. To determine weekly calorie consumption, the weight of the food was multiplied by the provided manufacture kcal / gram value. Mice were permitted ad libitum access to food and water throughout the entirety of the study. The weekly water consumption was quantified starting on Day 0 and ending on Day 63 by measuring the weight of the Hydropacs. Weight of the Hydropacs were not measured on Day 70.

[0151] Fecal sample collection

[0152] Fecal sample collections occurred during the last two hours before lights off (1600 h-1800 h), wearing full vivarium PPE (gloves, gown, mask, hair cover). The experimental mice were moved from their home cage to a clean cage. Mice were allowed a maximum of 1 h to defecate in the sterile cage. On average researchers collected 2-4 pellets using a sterile 26 G needle, to avoid contamination of the pellet. Within treatment group gloves were cleaned using 70% ethanol. When changing to a new treatment group all researchers were required to change gloves. Pellets that were immersed in urine, water droplets and bedding were avoided. The pellets were deposited into a sterile, prelabeled 1.65 mb Eppendorf tube. The samples were stored at -80 °C and were later processed for analysis of the diversity and community composition of the gut microbiome.

[0153] Open-field test

[0154] The open-field test was conducted as described previously. The mice were housed in a separate room from the behavior testing room. The mice were transferred from their housing room to the behavior testing room an hour prior to the start of the test, to allow the mice to acclimate to the new environment. The open-field test was conducted on day 0 of the experiment between 0600- 1000 h. Test arena dimensions: 45 cm length x 27 cm width x 27 cm height; 350 lx. The test arena was subdivided into an inner zone (20.32 cm x 20.32 cm) and an outer zone. To begin, each mouse is placed in the outer zone (midway between two corners) facing the inner zone and were allowed to explore the arena for 10 minutes. In the open-field test (OFT), the percentage of time spent in the inner zone was analyzed as well as the distance traveled by each mouse. The test area was cleaned with 70% ethanol before each test was conducted and all test parameters were analyzed with Etho Vision XT (vl3.0.1220; Noldus Information Technology). A subset of mice (N = 14) was not placed in the designated starting position, but instead was placed directly in the inner zone. The study's block design ensured an equitable representation of animals placed within the inner zone in each treatment group. The animals starting in the inner zone were included in all analysis in the open-field test except for latency to enter the center zone. Using a Student’s / -test, no differences in behavioral outcomes were observed between those that started in the inner zone (N = 14) as compared to those that started in the outer zone (7V= 31). Each mouse was video recorded in the open-field test to allow for the collection and analysis of percent time spent in the inner zone, frequency of entries into the center zone, distance traveled, latency to enter the inner zone, stretch attend posture (SAP) frequency, and percentage of time performing SAP. To determine entry into a specific zone, EthoVision XT software generated a tracking spot, which was located in the center of the body. This tracking spot allowed the software to recognize when the mouse entered or spent time in a new zone.

[0155] Elevated plus-maze

[0156] The elevated plus-maze was conducted as described previously (Reber and Neumann, 2008). The mice were housed in a separate room from the behavior testing room. The mice were transferred from their housing room to the behavior testing room an hour prior to the start of test, to allow the mice to acclimate to the new environment. The elevated plus-maze (EPM) was conducted on day 3 of the experiment between 0600 and 1000 h for 5 min. The EPM that was used was roughly 50 cm tall. Each arm was 30.5 cm long and 5.1 cm wide with Plexiglas® walls on the closed arms measuring 15.2 cm high. The maze had two open arms and two closed arms. The maze was sterilized with 70% ethanol before each test was conducted and all test parameters were analyzed with EthoVision XT (vl3.0.1022; Noldus Information Technology). During behavioral testing, the intersection of the open arms and closed arms of the EPM was set at 130 lx. Each mouse was video recorded in the EPM to allow for the collection and analysis of percent time spent in and number of entries into the open arms, percent time spent in and number of entries into the closed arms, and distance traveled. To determine entry into a specific zone, EthoVision XT software generated a tracking spot, which was in the center of the body. This tracking spot allowed the software to recognize when the mouse entered or spent time in a new zone.

[0157] Euthanasia

[0158] On experimental day 4, mice were euthanized with a terminal dose of sodium pentobarbital (150 mg / kg, i.p.) followed by opening of the thoracic cavity and exsanguination using cardiac puncture for blood collection followed by transcardial perfusion with ice-cold saline in order to remove white blood cells from cerebrovascular vessels. In the event of spontaneous disease or illness, mice were euthanized by carbon dioxide asphyxiation using a flow rate of 30-70% volume displacement per minute followed by rapid decapitation as a secondary method of euthanasia.

[0159] Motile sperm concentration measurement by epididyntal squash

[0160] At PN60, male mice were anaesthetized using isoflurane and rapidly decapitated. A V- shaped abdominal incision was made, and epididymal fat pads were gently pulled to expose the testes and epididymis. The epididymis was isolated from each testis and placed in a petri dish with 2 mL of prewarmed (37 °C) phosphate-buffered saline (PBS). Both epididymides were used for this procedure. The epididymal cauda was isolated from the rest of the epididymis and vas deferens, minced with two scalpel blades and incubated in 2 ml of PBS at 35 °C for 15 minutes with gentle agitation. Following the incubation, 500 pl of sperm-containing PBS was mixed with 500 pl of 3% NaCl to immobilize sperm for consistent counting. The solution containing immobilized sperm was then vortexed and a 10-pl aliquot was used for counting by a hemacytometer. Of the 25 grids, four comer grids and one center grid were counted for number of sperm. Sperm that were not fully enclosed in the grid were only counted if they passed over the top or left line of the grid. Average number of sperm contained in the 5 grids was calculated. The average number of sperm was multiplied by the dilution factor, the number of total grids, and the volume of the hemocytometer to result in a final concentration of sperm per mL.

[0161] H&E staining for measurement of Leydig cell cross-sectional area

[0162] The testes were fixed in Bouin’s Solution (Cat. No. HT10132, Sigma- Aldrich, St. Louis, MO, USA) for 12 h, and then stored in 70% ethanol for 2 hours. Tissues were embedded in paraffin, and sliced into 12 pm-thick sections, and mounted on poly-L-lysine slides followed by dewaxing and rehydration. The slices were then stained with hematoxylin and eosin (H&E).

[0163] 16S rRNA gene amplicon sequencing for analysis of fecal microbiome

[0164] Genomic bacterial DNA collected from fecal samples was extracted using a Qiagen DNeasy PowerSoil HTP extraction kit (Qiagen, Redwood City, CA, USA) following the manufacturer’ s instructions. The marker genes in the extracted DNA were amplified via PCR using GoTaq Master Mix (Promega, Fitchburg, WI, USA). High-throughput sequencing was conducted using the Golay barcode primers 515 F (50- GTGCCAGCMGCCGCGGTAA-30)(SEQ ID NO. 1) and 806 R (50-GGACTACHVGGGTWTCTAAT-30) (SEQ ID NO. 2), respectively. The Golay barcode primers target the V4 hypervariable region of the 16S rRNA gene, which has been found to be highly conserved and useful for the taxonomic profding of the gut microbiome (Caporaso et al., 2012). PCR amplification methods include heating at 94 °C for 3 min followed by 35 x 94 °C for 45 s, 55 °C for 1 min, and 72 °C for 1.5 min, with the final extension being carried out at 72 °C for 10 min. To purify and normalize the PCR products, the SequelPrep Normalization Kit (Cat. No. Al 051001, ThermoFisher, Waltham, MA, USA) was used. Library preparation and gene sequencing for the 16S rRNA gene were carried out using the V2 300-cycle Illumina MiSeq System.

[0165] RNA extraction and real time RT-PCR for assessment of hippocampal and hypothalamic mRNA expression

[0166] The hippocampus, hypothalamus, and visceral adipose tissue were extracted for mRNA analysis and frozen at -80 °C until use. Total RNA was isolated by methods described in (Frank et al., 2007). Briefly, total RNA was homogenized using Trizol™ (Cat. No. 15596018, Thermo Fisher Scientific, Waltham, Massachusetts, USA) and incubated at room temperature for five minutes. Following incubation, chloroform (Cat. no. 288306-1L, Sigma-Aldrich, St. Louis, MO, USA) was added to the supernatant, vortexed and centrifuged at 12,000 g for 15 min at 4 °C. The resulting precipitate was washed in 75% ethanol and centrifuged at 7,500 g for 5 minutes at 4 °C. RNA concentration was determined using NanoDrop™ 2000 (Thermo Fisher Scientific).

[0167] Total RNA was reverse transcribed into cDNA using the SuperScript™ II RT protocol, as described in Frank et al. (2007). Briefly, 1 pg of RNA was incubated in a reaction mixture containing 5 ng / pl hexamer primers (Cat. No. 48190011, Thermo Fisher Scientific) and 1 mM of dNTPs (Cat. No. 18427088, Thermo Fisher Scientific) at 65 °C for 5 minutes. Samples were then chilled on ice, and a cDNA synthesis buffer as described in the protocol was added to the reaction and incubated at room temperature for two minutes. SuperScript II reverse transcriptase (Cat. No. 18064014, Thermo Fisher Scientific) was added to the reaction and incubated at 25 °C for ten minutes, followed by 42 °C for fifty minutes; the reaction was terminated by heating to 70 °C for 15 minutes.

[0168] Real time reverse transcription PCR amplification of cDNA was performed using the QuantiTect® SYBR Green PCR kit (Cat. No. 204145, Qiagen, Hilden, Germany), as described in Frank et al. (2007). Briefly, for each sample, 1 pl of cDNA was added to a master mix containing SYBR Green, nuclease free water and both forward and reverse primers. PCR product was detected by MyiQ Real-Time PCR Detection System (BioRad), capturing SYBR Green I fluorescence at 72 °C.

[0169] Microbiome data processing and statistical analysis

[0170] The microbiome data processing was performed as outlined in(Paripati et al., 2023. The 16S rRNA gene sequencing data underwent analysis using Quantitative Insights Into Microbial Ecology (QIIME) 2-2023.7. DADA2 (Callahan et al., 2016) was used to denoise the data into amplicon sequence variants (ASVs), where sequences were truncated at 150 bp for both the forward and reverse reads based on the quality score plots. Taxonomy assignment was conducted using a naive Bayes classifier trained on the latest SILVA version 138.1 16S rRNA gene database (March 2024) sourced from QIIME 2. A phylogenetic tree was created using the Sate-enabled phylogenetic placement (SEPP) fragment insertion method via QIIME 2 (Janssen et al., 2018). Diversity analyses were executed within QIIME 2, with data rarefied to an even sampling depth of 8595 reads per sample (Weiss et al., 2017). To assess alpha diversity of the gut microbiome, we measured observed OTUs as a metric of the number of distinct features, Shannon’ s diversity index to quantify species richness and evenness, Pielou’s evenness to measure species evenness, and Faith’s phylogenetic diversity, which measures the total length of branches in a reference phylogenetic tree for all species in each sample. The taxa bar plot was created using the R package Microshades 1.10 (Dahl et al., 2022). Weighted UniFrac was considered the primary metric for beta diversity analysis.

[0171] Differential abundance testing

[0172] To identify differentially abundant taxa based on treatment with M. vaccae ATCC 15483 or Western-style diet, we used Analysis of Compositions of Microbiomes (ANCOM, via Scikit- bio) (Mandal et al., 2015). Further, we also created a co-occurrence network of taxa, from which we grouped features into highly correlated modules, which were summed for analysis to identify differential groups of features. Specifically, we filtered rare taxa with total read counts <500 and average read counts <3 (Friedman and Alm, 2012). Taxa were center log-ratio transformed to account for compositionality. Pearson’s correlation coefficients were calculated pairwise between all features. Edges were drawn only between features with a Pearson’s rho > 0.5 and p < 0.05. The Louvain modularity maximization algorithm was applied to identify modules with many strong connections within modules and few connections between modules (Blondel et al., 2008). Relative abundances of all features in each module were summed, and M. vaccae ATCC 15483 or Western- Style Diet-based differences in module abundances were assessed via a Kruskal-Wallis test. Next, we utilized a phylogenetic isometric log-ratio transformation (PhILR) to identify taxa associated with Western-Style Diet consumption to test our assumption that Western-Style Diet consumption causes a phylogenetic shift in the gut microbiome. The PhILR transformation considers ratios between subtrees of the phylogenetic tree to use phylogeny to transform compositional microbiome data outside the simplex, such that each feature can change independently of the others (Silverman et al., 2017). At each branching point in the tree, the isometric log-ratio of the summed read counts in one subtree of the branch to the other subtree was calculated to create our “balances”. Least absolute shrinkage and selection operator (LASSO) regression, with lambda (penalty parameter) = 3000, was used to identify the top 5 balances associated with HFCS consumption. In R, PhILR was performed using the philr package vl .24.0, LASSO was performed using the glmnet package v4.7, and balances were visualized using ggtree v3.6.2 (Yu, 2020; Friedman et al., 2010).

[0173] Co-occurrence network analysis

[0174] Sparse Cooccurrence Network Investigation for compositional data (SCNIC) was used to conduct a co-occurrence network analysis, aiming to shed light on the impact of Western-style diet consumption on microbial co-occurrence patterns within the gut microbiome. We refrained from exploring the impact of AL vaccae ATCC 15483 on co-occurrence network analysis, as all other microbiome analyses conducted indicated no main effects of AL vaccae ATCC 15483 or AL 12 vaccae ATCC 15483 x Western-style diet interactions. In summary, SparCC was used to calculate correlations between ASVs, and the shared minimum distance algorithm in SCNIC was used to cluster and sum the relative abundances of highly co-occurring ASVs into “modules”, using a minimum R value of 0.35. Differentially abundant modules were assessed using ANCOM-BC2. Volatility analysis

[0175] Volatility was analyzed to identify a possible association with AL vaccae ATCC 15483 treatment or Western-style diet consumption to test our assumption that Western-style diet consumption causes greater volatility in the gut microbiome. Volatility was assessed by quantifying the differences between samples from the same animal at different timepoints, computed as the Unweighted or Weighted UniFrac distance between the two time points of interest and analyzed using linear mixed model analysis. Statistical analysis

[0176] Two-way analysis of variance (ANOVA) was used to determine main effects of AL vaccae ATCC 15483 or Western-style diet, or interactions between these factors, when evaluating outcomes at a single time point. Planned pairwise comparisons following two-way ANOVA were conducted using Fisher’s LSD test, if appropriate, at a two-tailed alpha level of 0.05 using a single pooled error term for Fisher’s LSD test. Briefly, for analysis of all behavior, relative gene expression, reproductive endpoints, plasma leptin concentration, and weight of VAT, a two-way ANOVA test was performed, which was followed by Fisher’s LSD test, at a two-tailed alpha level of 0.05 using a single pooled error term for Fisher’s LSD test. In cases where a significant main effect of AL. vaccae ATCC 15483 was observed, planned pairwise comparisons were carried out between the BBS control condition and the M. vaccae ATCC 15483 condition within both the control diet and Western-style diet conditions. Similarly, in instances of a significant main effect of the Western-style diet, planned pairwise comparisons were performed between the control diet and the Western-style diet condition within both the BBS and AL vaccae ATCC 15483 conditions. Moreover, when a significant interaction between AL vaccae ATCC 15483 and the Western-style diet was identified, both sets of planned pairwise comparisons were conducted.

[0177] Effects of AL. vaccae ATCC 15483 and Western-style diet on abundance of taxonomic rank over time were analyzed using analysis of composition of microbiomes with bias correction (ANCOM-BC2).

[0178] A PERMANOVA was used to assess differences in phylogenetic community composition (beta diversity). Due to the longitudinal nature of the data, PERMANOVA tests for effects of diet and treatment were stratified by timepoint. PERMANOVA tests were performed using the adonis2 function from the R package vegan, version 2.6.

[0179] Example 2: Effects ofM. vaccae ATCC 15483 on body weight gain

[0180] The high fat / high sugar Western-style diet used in this study has consistently been shown to induce obesity in male mice (Clayton et al., 2022). A linear mixed-effect model revealed a three- way interaction between Western-style diet x AL vaccae ATCC 15483 x time ( (i,X) = 6.4252, p < 0.0001; Fig. 1A). Post hoc pairwise comparisons revealed that among mice treated with BBS, Western-style diet-treated mice, relative to mice exposed to a control diet, had elevated body weight, starting at week 6 and continuing throughout the remainder of the experiment. Post hoc pairwise comparisons revealed that among mice fed a Western-style diet, AL vaccae ATCC 15483- treated mice had lower body weight, starting at week 8 and continuing throughout for the remainder of the experiment, as compared to BBS control mice. It is worth noting that there was no difference in body weight throughout the entirety of the experiment between the MV / WD group and the MV / control diet groups.

[0181] A two-way ANOVA revealed a main effect o M. vaccae ATCC 15483 (7 I,40)= 11.856, p < 0.01; Fig. IB) and a main effect of Western- style diet (F(i.40)= 24.614, p < 0.0001; Fig. IB) on weight of visceral adipose tissue. Post hoc pairwise comparisons revealed that among mice treated with BBS, Western diet-treated mice had elevated VAT weight as compared control diet mice. Post hoc pairwise comparisons revealed that among mice fed a Western diet, M. vaccae ATCC 15483 -treated mice had lower VAT weight as compared to BBS control mice. Post hoc pairwise comparisons revealed that among mice treated with AT. vaccae ATCC 15483, Western diet-treated mice had elevated VAT weight as compared to control diet mice.

[0182] A linear mixed-effect model revealed an interaction between Western-style diet x time (F(i.X) = 2.847, p < 0.01; Fig. 1C) for the average weekly kcal consumed. Post hoc pairwise comparisons revealed that among mice fed a Western diet, M. vaccae ATCC 15483-treated mice consumed less kcal compared to BBS control mice on the second week. Post hoc pairwise comparisons revealed that among mice fed a control diet, M. vaccae ATCC 15483-treated mice consumed less kcal compared to BBS control mice on the ninth week.

[0183] A linear mixed-effect model revealed a main effect for Western-style diet (F(i.X) = 35.910, p < 0.0001; Fig. ID) and a main effect of time (F(i,X) = 5.2054, / ? < 0.05; Fig. ID) for the average weekly water consumed. Post hoc pairwise comparisons revealed that among mice treated with BBS, the Western diet group consumed less water relative to control diet mice on weeks one, two, four, five, seven, eight, and nine. Post hoc pairwise comparisons revealed that among mice treated with M. vaccae ATCC 15483, the Western-style diet group consumed less water relative to control diet mice on weeks one, two, four, five, eight, and nine.

[0184] Neither M. vaccae 15483 nor Western diet influenced total kcal consumed during the entirety of the experiment. A linear mixed-effect model revealed an interaction between Westernstyle diet x time (F(i.X) = 7.1323, p < 0.001; Fig. IF) for the average weekly kcal consumed per gram of body weight. Post hoc pairwise comparisons revealed that among mice treated with BBS, Western style diet mice consumed less kcal per gram of body weight as compared to control mice on weeks two, six, seven, eight, and nine. Post hoc pairwise comparisons revealed that among mice treated with M. vaccae ATCC 15483, Western style diet mice consumed less kcal per gram of body weight as compared to control mice on weeks seven, eight, and nine. Post hoc pairwise comparisons revealed that among mice treated with M. vaccae ATCC 15483, Western style diet mice consumed less kcal per gram of body weight as compared to control mice on weeks seven, eight, and nine. Post hoc pairwise comparisons revealed that among mice on Western style diet, mice treated with M. vaccae ATCC 15483 consumed less kcal per gram of body weight as compared to BBS treated mice on week 2.

[0185] A two-way ANOVA revealed a main effect of Western-style diet (F(i,42) =75.063, p < 0.0001; Fig. 1G) on total water consumed throughout the entirety of the experiment. Post hoc pairwise comparisons revealed that among mice treated with BBS, Western style diet mice consumed less total water as compared to control diet mice. Post hoc pairwise comparisons also revealed that among mice treated with M. vaccae ATCC 15483, Western style diet mice consumed less total water as compared to control diet mice.

[0186] A linear mixed-effect model revealed a main effect of Western-style diet (F(i,X) = 76.1656, p < 0.0001; Fig. 1H) and a main effect of time ( (i,X) = 30.6210, p < 0.0001; Fig. 1H) for water consumed per gram of body weight. Post hoc pairwise comparisons revealed that among mice treated with BBS, Western style diet mice consumed less water per gram of body weight as compared to control mice on weeks one, two, three, four, five, seven, eight, and nine. Post hoc pairwise comparisons revealed that among mice treated with M. vaccae ATCC 15483, Western style diet mice consumed less water per gram of body weight as compared to control mice on weeks one, two, four, five, eight, and nine.

[0187] Example 3: Effects of M, vaccae ATCC 15483 and Western-style diet on gene expression within visceral adipose tissue.

[0188] Numerous studies have shown elevated plasma levels of leptin and blunted levels of adiponectin in persons with obesity (Fruhbeck et al., 2018). A two-way ANOVA revealed a main effect of AT. vaccae ATCC 15483 showing a decrease in relative gene expression of Lep, which encodes for leptin (F<I.38) = 6.067, *p < 0.05; Fig. 4A). Post hoc pairwise comparisons revealed that BBS / WD group had elevated gene expression of Lep as compared to the BBS / CD group. Post hoc pairwise comparison also showed that M. vaccae ATCC 15483 prevented the excessive gene expression levels of Leptin seen in the Western style-diet group (Fig. 4A). Numerous studies have shown a positive correlation between leptin expression and quantity of visceral adipose tissue. A linear regression was performed and revealed a positive correlation with weight of visceral adipose tissue (VAT) and relative gene expression of Lep within the VAT (Fig. 4A). Neither M. vaccae 15483 nor Western diet influenced relative gene expression levels of Adipoq, Tnf, or Ibal (Figs. 4B-D). A two-way ANOVA revealed a main effect of Western diet showing an increase in the loglO transformation relative gene expression of Argl (F(i,30)= 5.883, *p < 0.05; Fig. 4E). Post hoc pairwise comparisons failed to find a difference between groups of interest.

[0189] Example 4: Relationship of visceral adipose tissue and Lep gene expression.

[0190] Numerous studies have shown a positive correlation between Lep gene expression within visceral adipose tissue and weight of visceral adipose tissue. A linear regression was performed and revealed a positive correlation with weight of visceral adipose tissue (VAT) and relative gene expression of Lep within the VAT (Fig. 4A).

[0191] The significant effect of Western-style diet to increase Lep mRNA expression in VAT prompted further investigation using ELISA to assess plasma concentrations of leptin protein. A two-way ANOVA revealed an M vaccae ATCC 15483 x Western-style diet interaction for plasma leptin concentrations (F(l,40) = 5.32, *p < 0.05; Fig. 14). Post hoc pairwise comparisons revealed that, among mice treated with BBS vehicle, Western-style diet-fed mice showed elevated plasma leptin concentrations as compared to control diet mice. Post hoc pairwise comparisons also revealed that, among mice fed a Western-style diet, M. vaccae ATCC 15483 decreased plasma leptin concentrations. Thus, M. vaccae ATCC 15483 prevented Western-style diet-induced increases in plasma leptin concentrations. A linear regression was performed and revealed a positive correlation between weight of VAT and plasma leptin concentrations (F(l,41) = 16.3, p < 0.001).

[0192] These effects were prevented by treatment with vaccae ATCC 15483. Although a main effect of Western-style diet on Lep mRNA expression in VAT only approached statistical significance, planned post hoc pairwise comparisons revealed a significant increase in Lep mRNA expression among mice fed a Western-style diet, relative to a control diet. In contrast, there was a main effect of vaccae ATCC 15483 to decrease relative expression of Lep mRNA within VAT. Importantly, among mice fed a Western-style diet, treatment with M. vaccae ATCC 15483 decreased Lep mRNA expression within VAT.

[0193] This finding was further investigated by evaluating plasma leptin concentrations. Plasma leptin concentrations were elevated in the Western-style diet group, relative to the control diet-fed mice, and this effect of Western-style diet was prevented by immunization with M. vaccae ATCC 15483. Indeed, among mice fed a Western-style diet, treatment with A / , vaccae ATCC 15483 led to a highly significant decrease in plasma leptin concentrations. Increases in plasma concentrations of adipokines, especially leptin, positively correlate with VAT adipokine concentrations. Despite its role in promotion of satiety under conditions of physiological homeostasis, elevated leptin levels can contribute to the chronic low-grade inflammation that is associated with obesity by enhancing the release of bone marrow-derived monocytes and skewing monocytes towards an Ml phenotype, which is noted by the upregulation of the canonical proinflammatory cytokines IL-1 / >, IL-6, and tumor necrosis factor (TNF). In addition to its impact on the innate immune system, leptin is recognized for its role in inhibiting the proliferation of regulatory T cells (De Rosa et al., 2007), the predominant CD4+ T cell population within healthy VAT. Both the elevated leptin levels and inappropriate inflammation are regarded as significant contributors to the emergence of insulin resistance, affecting both insulin’s signaling in the periphery and in the brain. Overall, these data are consistent with previous studies showing that mice on a Western-style diet develop elevated leptin gene expression levels in VAT, as well as elevated levels of leptin in plasma or serum, which reflects the associated phenotype seen in obese humans. The ability of M. vaccae ATCC 15483 to reduce Lep mRNA expression in VAT and reduce plasma concentrations of leptin in the presence of a Western-style diet supports the hypothesis that Af. vaccae ATCC 15483 is a promising intervention for prevention and / or treatment of negative outcomes associated with consumption of a Western-style diet, including immunometabolic-related diseases.

[0194] Example 5: M. vaccae ATCC 15483 decreases biomarkers of hippocampal neuroinflammation.

[0195] The ventral hippocampus plays an important role in controlling anxiety-like defensive behavioral responses. M. vaccae NCTC 11659 has been shown to reduce basal levels of biomarkers of neuroinflammation in the hippocampus in rats through evaluation of genes like Nlrp3 and Nfkbia, which are both involved in microglial priming (Frank et al., 2018). A two-way ANOVA revealed a AL vaccae ATCC 15483 x Western-style diet interaction for relative gene expression of Nfkbia ( (i,40) = 9.559, p < 0.01; Fig. 3A). Post hoc pairwise comparisons revealed that BBS / WD group had decreased relative gene expression of Nfkbia as compared to BBS / CD. Post hoc pairwise comparisons revealed that the MV / CD group had decreased relative gene expression of Nfkbia as compared to BBS / CD. Post hoc pairwise comparisons revealed that MV / WD group had elevated relative gene expression of Nfkbia as compared to MV / CD (Fig. 3 A). A two-way ANOVA revealed a M. vaccae ATCC 15483 x Western-style diet interaction for Nlrp3 ( <I,40) = 4.830, p < 0.05; Fig. 3B). Post hoc pairwise comparisons revealed that MV / CD group had decreased relative gene expression of Nlrp3 as compared to BBS / CD (Fig. 3B). Neither M. vaccae ATCC 15483 nor Westen diet influenced relative gene expression of Illb (Fig. 3D), relative gene expression of 116 (Fig. 3D), relative gene expression of 1110 (Fig. 3E), and relative gene expression of Hmgbl (Fig. 3F).

[0196] Among the control diet-fed mice, treatment with M. vaccae ATCC 15483 reduced relative gene expression of basal biomarkers of neuroinflammation and microglial priming, Nfkbia and Nlrp3, within the hippocampus. The outcomes observed with M. vaccae ATCC 15483 in this study are consistent with previous findings showing that AT. vaccae NCTC 11659 reduces basal levels of hippocampal A / 7w and A7 / ' 3 mRNA expression in rats. Additionally, M. vaccae NCTC 11659 has been shown to prevent both age and surgery-induced increases in the expression of Nfkbia in the hippocampus in rats. The findings from the current study further underscore the similarity between the two strains of AT. vaccae. The importance of highlighting the AT. vaccae ATCC 15483 induced reduction of hippocampal Nfkbia and Nlrp3 mRNA expression lies in previous research demonstrating that Nfkbia and Nlrp3 play important roles in stress-induced microglial priming, and stress-induced exaggeration of hippocampal neuroinflammation is associated with stress induced exaggeration of anxiety-like defensive behavioral responses. Treatment with M. vaccae NCTC 11659 has been shown to prevent stress-induced priming of hippocampal microglia, as well as attenuate aging-associated priming of hippocampal microglia. The ability of M. vaccae to prevent hippocampal microglial priming may be partially mediated by its influence on the basal levels of both Nfkbia and Nlrp3. Thus, M. vaccae ATCC 15483 has potential to decrease Western diet induced microglial priming or reactivity and associated negative outcomes, including memory impairment.

[0197] Example 6: Evaluation of biomarkers of hypothalamic neuroinflammation.

[0198] A / , vaccae ATCC 15483 has been shown to reduce regional basal levels of biomarkers of neuroinflammation. In contrast to the hippocampus, neither M. vaccae ATCC 15483 nor Western diet influenced relative gene expression of Nlrp3 (Fig. 5A), relative gene expression of Nfkbia (Fig. 5B), relative gene expression of 7b / (Fig. 5C), or relative gene expression of LepR (Fig. 5D). A two-way ANOVA revealed a M. vaccae ATCC 15483 x Western-style diet interaction for relative gene expression of Insr (F(i,39) = 5.704, * p < 0.05 Fig. 5E). Post hoc pairwise comparisons revealed that among mice fed a Western diet, M. vaccae ATCC 15483-treated mice had lower relative gene expression of Insr as compared to BBS control mice. Post hoc pairwise comparisons revealed that among mice treated with BBS, there was a trend towards significantly elevated Insr gene expression in the mice on the Western diet as compared to the control diet.

[0199] M. vaccae ATCC 15483 has been shown to prevent stress-induced biomarkers of neuroinflammation in the hippocampus. However, less is known about effects oiM. vaccae ATCC 15483 on biomarkers of neuroinflammation in other brain regions, such as the hypothalamus. Neither M. vaccae ATCC 15483 nor Western-style diet influenced hypothalamic relative gene expression of Nfkbia, Nlrp3, or Tnf. Currently, there is no evidence that A . vaccae ATCC 15483 influences biomarkers of appetite regulation in the CNS. Due to the alteration observed with body weight and VAT, M. vaccae ATCC 15483 effects on biomarkers of appetite regulation were evaluated within the hypothalamus. Neither M. vaccae ATCC 15483 nor Western-style diet influenced relative gene expression of LepR (. However, analysis using two-way ANOVA revealed a AY vaccae ATCC 15483 x Western-style diet interaction for relative gene expression of Insr (F(l,39) = 5.70, *p < 0.05). Post hoc pairwise comparisons revealed that, among mice treated with BBS, an increase in relative gene expression of Insr in Western-style diet-fed mice relative to control diet-fed mice approached statistical significance (p = 0.067). Furthermore, among mice fed a Western-style diet, AY vaccae ATCC 15483-treated mice, relative to BBS control mice. Among mice fed a Western-style diet, treatment with AY vaccae ATCC 15483 reduced the relative expression of Insr within the hypothalamus, suggesting effects of AY. vaccae ATCC 15483 on expression of genes regulating central appetite and energy balance.

[0200] Example 7: AY vaccae ATCC 15483 effects on anxiety-like behavior in the open-field test.

[0201] AY. vaccae ATCC 15483 has been shown to attenuate stress-induced anxiety-like defensive behavior (Loupy et al., 2021). Neither AY. vaccae ATCC 15483 nor Western- style diet influenced percentage of time in center zone (Fig. 12A), frequency of entries into center zone (Fig. 12B), distance traveled (Fig. 12C), latency to enter the center zone (Fig. 12D), or frequency of stretch attend posture in the open-field test (Fig. 12E). It is worth noting that there was a trend towards a significant main effect of Western-style diet for frequency of entries into center zone. A two-way ANOVA revealed a AY. vaccae ATCC 15483 x Western-style diet interaction (F(i,43) = 4.932, p < 0.05; Fig. 12F) for percentage of time performing stretch attend posture (SAP). Post hoc pairwise comparisons revealed that, among mice fed a control diet, treatment with AY. vaccae ATCC 15483 decreased the percentage of time performing the stretch attend posture (Fig. 12F), consistent with an anxiolytic effect of M. vaccae ATCC 15483 under control diet conditions (Blanchard et al, 2001; Bilkei-Gorzo et al., 2002). Post hoc pairwise comparisons also revealed that, among mice treated with M. vaccae ATCC 15483, mice consuming the Western-style diet had a greater percentage of time performing SAP as compared to the control diet (Fig. 12).

[0202] Example 8: M. vaccae ATCC 15483 decreases anxiety-like behavior in the elevated plus-maze.

[0203] M. vaccae ATCC 15483 has been shown to attenuate stress-induced anxiety-like defensive behavior (Loupy et al., 2021). A two-way ANOVA revealed a main effect of M. vaccae ATCC 15483 to increase percentage of time ( (i,X) = 5.019; p < 0.05; Fig. 2A) and frequency of entries (F(i,x)= 4.882; p < 0.05; Fig. 2B) into the open arms of the EPM. Post hoc pairwise comparisons revealed that among mice fed a control diet, M. vaccae ATCC 15483-treated mice had elevated percentage of time and frequency of entries into the open arm of the EPM compared to BBS control mice. Neither M. vaccae ATCC 15483 nor Westen diet influenced distance traveled on the EPM (Fig. 2C), percentage of time in the closed arm (Fig. 2D), or frequency of entries into the closed arm of the EPM (Fig. 2E). Post hoc planned pairwise comparisons failed to reveal a difference. M. vaccae ATCC 15483 decreased anxiety-like defensive behavioral responses across both diet groups. Together, these findings demonstrate protective effects of AY. vaccae ATCC 15483 against Western-style diet-induced body weight gain, visceral adiposity, and disturbance of peripheral and central leptin and insulin signaling, while decreasing anxiety-like defensive behavioral responses regardless of diet.

[0204] Treatment with M. vaccae ATCC 15483 decreased anxiety-like defensive behavioral responses as assessed in both the open-field test and elevated plus-maze. Mice treated with M. vaccae ATCC 15483 had a greater number of entries into the closed arm as compared to the BBS vehicle control groups; however, this trend was not observed for percent of time spent in the closed arm and also not for the overall distance travelled during OF exposure, suggesting that locomotion was not or only mildly affected by M. vaccae ATCC 15483 during behavioral testing. The consistency of the effect of M. vaccae ATCC 15483 to decrease anxiety-like defensive behavioral responses, particularly among the control diet group, along with our observation that M. vaccae ATCC 15483 decreases the relative expression of Nfkbia and Nlrp3 mRNA within the hippocampus, is notable and corroborates the established concept that neuroinflammatory processes can play a partial role in mediating anxiogenic behavior. Example 9: M. vaccae ATCC 15483 effects on excessive weight gain while on Western-Style Diet.

[0205] Prior to the start of any intervention there was no difference in body weight between the treatment groups (Fig. 1 A). In line with previous studies that have used Western-Style Diet (Harlan Teklad Diet 96132) in adolescent / adult male mice, the Western-Style Diet significantly increased body weight as compared to control diet groups starting on week 6. As seen in Fig. 1A, the difference in body weight between the Western-Style Diet and control diet continued throughout the experiment. In contrast, adolescent male mice given M. vaccae ATCC 15483 and exposed to a Western-Style Diet did not experience the excessive weight gain, relative to M. vaccae ATCC 15483 treated mice given a control diet. An important note is that there was no difference in body weight throughout the entirety of the experiment between the Western-Style Diet group treated with AT. vaccae ATCC 15483 and the control diet groups.

[0206] Prior to the start of any intervention, there was no difference in body weight among the treatment groups (Fig. 1A). Figs. 1C and IF describe M. vaccae ATCC 15483 effects on kilocalories consumed.

[0207] Western-style diet-fed mice gained excessive body weight and accumulated VAT, relative to control diet-fed mice. Despite the fact that repeated s.c. administration of M. vaccae ATCC 15483 did not appear to impact the Western-style diet-induced disruption of the gut microbiome, it prevented the excessive body weight gain and ameliorated the VAT accumulation induced by the Western-style diet. The disparities in both body weight and VAT accumulation observed among the Western-style diet-fed mice is inadequately explained by the total calories or total water consumed during the experiment, as there were no differences observed in these metrics between these groups. Among the mice fed a control diet, the mice treated with AT. vaccae ATCC 15483 did not differ in body weight nor in VAT accumulation relative to the BBS-treated mice. This is worth highlighting as this indicates that M. vaccae ATCC 15483 is not impairing the mouse’s ability to gain body weight or VAT.

[0208] Finding ways to mitigate excessive body weight gain, especially accumulation of VAT, is a highly desired outcome in metabolic clinical research, as excessive VAT has been linked to increased risk of the development of impaired glucose and lipid metabolism, insulin resistance, cardiovascular disease, non-alcoholic fatty liver disease, metabolic syndrome, as well as predisposition to cancers of the colon, breast (Schapira et al., 1994), and prostate. Furthermore, excessive VAT has been identified as one of the best predictors of emergency room (ER) or intensive care unit (ICU) admission for patients with COVTD-19 (P. Many of these disturbances are believed to arise from the elevated baseline inflammation that can originate from the Westernstyle diet-associated “leaky gut”, cellular stress induced by hypertrophy of the adipocytes within VAT, and imbalance of adipokines observed in obese individual. The findings of the current study offer compelling evidence supporting its use for prevention and / or treatment of negative outcomes associated with consumption of a Western-style diet, including immunometabolic-related diseases. Example 10: Effects of AZ vaccae ATCC 15483 and Western diet on male reproductive health.

[0209] It has been speculated that M. vaccae may have similar beneficiary reproductive health effects as what has been documented with Lactobacillus reuteri ATCC PTA 6475. Neither AZ vaccae ATCC 15483 nor Western diet influenced the weight of testes (Fig. 6A). A two-way ANOVA revealed a main effect of diet (F(i.4O) = 4.389, *p < 0.05, Fig. 6B) on the ratio of testes’ weight to body weight. Post hoc pairwise comparisons indicated a trend toward significance (p = 0.063) between the BBS treated groups. Neither M. vaccae ATCC 15483 nor Westen diet influenced sperm concentration (Fig. 6C), seminiferous tubule area (Fig. 6D), seminiferous tubule perimeter (Fig. 6E), seminiferous tubule diameter (Fig. 8F), percentage of open luminal seminiferous tubules (Fig. 6G), or percentage of seminiferous tubule with mature sperm (Fig. 6H). Example 11 : Diet effects on alpha diversity of the gut microbiome.

[0210] Numerous studies have shown that diet alters metrics of alpha diversity in male mice. To assess diversity of the gut microbiome, we measured Observed OTUs as a metric of the number of distinct features, Shannon’s diversity index to quantify species richness and evenness, and Faith’s phylogenetic diversity, which measures the total length of branches in a reference phylogenetic tree for all species in each sample. As shown in Fig. 7A, neither AZ vaccae 15483 nor Western diet influenced Observed OTUs. A linear mixed-effect model revealed a main effect of time (Z(i,i72) = 3.995, p < 0.001; Fig. 7B) for Shannon’s diversity index. Post hoc pairwise comparisons revealed that BBS / WD group at timepoints 2, 3, and 4 had higher levels of Shannon’s diversity index as compared to baseline measurement, timepoint 1. Post hoc pairwise comparisons revealed that MV / WD group at timepoints 2, 3, and 4 had higher levels of Shannon’s diversity index as compared to baseline measurement, timepoint 1. Post hoc pairwise comparisons revealed that MV / CD group at timepoints 4 had higher levels of Shannon’s diversity index as compared to baseline measurement, timepoint 1. A linear mixed-effect model revealed a main effect of time ( / (i, 172) = 2.569,j> < 0.05; Fig. 7C) for Pielou’s evenness. Post hoc pairwise comparisons revealed that BBS / WD group at timepoints 2, 3, and 4 had significantly higher levels of Pielou’s evenness as compared to baseline measurement, timepoint 1. Post hoc pairwise comparisons revealed that MV / WD group at timepoints 2, 3, and 4 had significantly higher levels of Pielou’s evenness as compared to baseline measurement, timepoint 1. A linear mixed-effect model revealed an interaction between time and Western diet (t(i,i79) = -5.191, p < 0.0001; Fig. 7D) for Faith’s diversity index. Post hoc pairwise comparison revealed that at timepoint 1, prior to any intervention, there were no differences between groups with Faith’s diversity index. Post hoc pairwise comparison also revealed, after the intervention, at timepoints 2, 3, and 4 that both Western diet groups, regardless of treatment of AY. vaccae ATCC 15483 or BBS, had lower levels of Faith’s diversity index as compared to the control diet matched control groups.

[0211] Mice subjected to the Western-style diet exhibited a reduction in markers of richness of the gut microbiome. Specifically, a Western-style diet induced a reduction in observed OTUs and Faith’s diversity index, relative to the control diet. Subcutaneous injection of AY. vaccae ATCC 15483 did not prevent the reduction in richness induced by the Western-style diet. The effects of a Western-style diet on alpha diversity of the gut microbiome are consistent with previous studies, which have shown that high fat diets and Western-style diets often induce a reduction in richness within the gut microbiome. These data are also consistent with previous studies in which the related Mycobacterium, Mycobacterium vaccae NCTC 11659, prevents negative outcomes of stress exposure despite having no effect on stress-induced decreases in alpha diversity of the gut microbiota. The Western-style diet-induced decrease in alpha diversity of the gut microbiome is thought to play a role in development of “leaky gut”, leading to elevated endotoxemia levels, which is often observed to precede the development of excessive weight gain and visceral adipose tissue accumulation. Thus, decreases in the alpha diversity of the gut microbiome by consumption of a Western-style diet could exacerbate the development of obesity and immune-related inflammatory diseases. The lack of effect of AY vaccae ATCC 15483 on the alpha diversity of the gut microbiome suggests that it is acting downstream of the gut microbiome to prevent negative immunometabolic effects of a Western-style diet.

[0212] Example 12: AY. vaccae ATCC 15483 effects on excessive visceral adipose tissue while on Western-Style Diet.

[0213] Similar to the change in body weight between the Western-Style Diet and control diet, the Western-Style Diet group had greater levels of visceral adipose tissue (inguinal, epidydimal, retroperitoneal) than the control diet. It was determined that male adolescent mice given M. vaccae ATCC 15483 did not experience the excessive levels of visceral adipose tissue while on a Western- Style Diet.

[0214] Example 13: Effects of AT. vaccae ATCC 15483 and Western-Style Diet on the composition of bacterial communities at the phylum-level.

[0215] The phylum-level composition of bacterial communities in fecal samples of mice included Firmicutes, Bacteroidota, Verrucomicrobiota, Deferribacterota, and Actinobacteriota (Fig. 8). Based on relative abundance, the Firmicutes and Bacteroidota were most dominant. Numerous studies have shown that high fat diets as well as Western-Style Diets increase the Firmicutes to Bacteroidota ratio. A linear mixed-effects model revealed an interaction between time and diet (F(i.x) = 35.0227, p < 0.0001; Fig. 9) on the Firmicutes to Bacteroidota ratio. Post hoc pairwise comparisons revealed that, among mice treated with either BBS or M. vaccae ATCC 15483, the Western-Style Diet significantly increased the ratio of Firmicutes / Bacteroidota at timepoints two, three and four. Post hoc pairwise comparisons also revealed that, among mice treated with M. vaccae ATCC 15483, the Western-Style Diet also significantly increased the ratio of Firmicutes / Bacteroidota at timepoints two, three and four (Fig. 9).

[0216] ANCOMBC2 was used to analyze gut microbial composition at the phylum level, uncovering an interaction between Western-style diet and time. Mice fed a Western-style diet regardless of treatment with M. vaccae ATCC 15483 or BBS vehicle had a decreased relative abundance of Bacteroidota and an increased relative abundance of Verrucomicrobiota compared to control diet-fed groups (ANCOM-BC2, FDR-adjusted p < 0.05). ANCOM-BC2 was also employed to analyze gut microbial composition at the genus level, uncovering an interaction between Western-style diet and time. Mice fed a Western-style diet regardless of treatment with M. vaccae ATCC 15483 or BBS vehicle had increased relative abundances of Acetatifactor, Lachnospiraceae GCA-9000066575, Lactococcus, and Romboutsia, while simultaneously having decreased percent relative abundances of [Eubacterium xylanophilum_group], Lachnospiraceae UGC-001, Clostridia adin BB60 group, and Robinsoniella (ANCOM-BC2, FDR-adjusted p < 0.05) relative to the control diet fed groups. Additionally, time had a main effect for the relative abundance of Dysgonomonas, with all treatment groups experiencing a decrease from day -8 in the percent relative abundance (ANCOM-BC2, FDR-adjusted p < 0.05). Mice subjected to the Western-style diet experienced a dramatic shift in phylogenetic community composition, consistent with previous studies in mice. Notably, the Western-style diet induced a dramatic increase in the Bacillota to Bacteroidota ratio relative to the control diet. Treatment with M. vaccae ATCC 15483 did not prevent these community level shifts in the gut microbiome induced by the Western-style diet. Numerous studies have shown that high-fat diets as well as Western-style diets increase the Bacillota to Bacteroidota ratio. However, there are some inconsistent findings regarding the relationship between the Bacillota to Bacteroidota ratio and obesity. The initial study to establish this ratio observed that genetically obese mice, specifically ob / ob mice, exhibited a higher Bacillota to Bacteroidota ratio, formerly known as the Firmicutes / Bacteroidetes ratio. Subsequently, a clinical study observed that obese individuals undergoing weight loss on either fat- or carbohydrate-restricted diets showed a relative increase in Bacteroidetes and a decrease in Bacillota, and thus decreasing the Bacillota to Bacteroidota ratio. However, a subsequent meta-analysis has provided conflicting evidence for an association of the Bacillota to Bacteroidota ratio with obesity. The data from the current study support the notion that diets with high-fat content can influence the composition of murine gut microbiome composition regardless of obesity status. For example, in vehicle-treated mice fed a We stern- style diet, there was a dramatic increase in the Bacillota to Bacteroidota ratio on experimental Day 6, less than a week after the onset of the Western-style diet, but the increase in body weight gain relative to the control diet condition was not evident until experimental Day 42, which was approximately 6 weeks after the onset of the Western-style diet (a time course that is identical to previous studies in male C57BL / 6J mice. As another example of the dissociation between the Bacillota to Bacteroidota ratio and obesity status, M. vaccae ATCC 15483 had no effect on the Western-style diet-induced increases in the Bacillota to Bacteroidota ratio but completely prevented Western-style diet-induced weight gain. Overall, the present study indicates that repeated s.c. administration of vaccae ATCC 15483 did not counteract the Western-style diet- induced disruption of the gut microbiome’s community composition, in turn suggesting that M. vaccae ATCC 15483 influences the host through an alternative mechanism, such as downstream alterations of immunometabolism. Example 14: Effects of M. vacccie ATCC 15483 and Western-Style Diet on beta diversity of the gut microbiome.

[0217] Numerous studies have shown that diet alters metrics of beta diversity. Analysis of all samples using a principal coordinates analysis (PCoA) highlighted close clustering of samples based on Western diet. The PCI axis accounted for 41.2% of variation. The PC2 axis accounted for 15.2% of variation. Collectively the total amount of variation that PCI and PC2 accounted for is 56.4% of the total variation. A PERMANOVA revealed an interaction between diet and treatment (F(i,i40) = 5.4323, / ? < 0.01; Fig. 10) on principle coordinates of beta diversity. It was also revealed that time had a main effect ( (3,i40) = 9.6374, / ? < 0.01; Fig. 10) on principle coordinates of beta diversity.

[0218] Fig. 15 shows UniFrac PCoA plots across all samples in the study each day. Previous studies have shown that stressor exposure increases volatility of the gut microbiome across time. Although consumption of a high-fat / high-sugar Western-style diet had higher weighted UniFrac distances when comparing baseline to the first time point following initiation of the diet intervention, weighted Unifrac distances from one time point to the next were relatively stable, suggesting that the high-fat / high-sugar Western-style diet induced a rapid change in the community composition, which then remained relatively stable over time.

[0219] Using ANCOM-BC2 on SCNIC modules, while no differences were detected at baseline (experimental Day -8) it was determined that mice fed a Western-style diet, regardless of treatment with M. vaccae ATCC 15483, had lower relative abundances of amplicon sequence variants (ASVs) for modules 0, 2, 29, 50, and 52 on experimental Days 6, 41, and 65, while simultaneously showing higher relative abundances of ASVs for module 3 on days 6, 41, and 65. Among the most common ASVs represented in modules with decreased relative abundance following a Western style diet were those belonging to the Lachnospiraceae family, many of which play a role in production of butyrate and other short-chain fatty acids from dietary fiber.

[0220] Using LASSO regression, it was determined that Western-style diet reduced the balance value of nlO and n22 at day 6; conversely, Western-style diet increased the balance values of nl and n3 at day 6. Using LASSO regression, it was determined that Western-style diet reduced the balance value of nl9 and n8 at day 41; conversely, Western-style diet increased the balance values of nl6 and n5 at day 41. It was determined that Western-style diet reduced the balance value of n20 at day 65; conversely, Western-style diet increased the balance values of n3 at day 65. No differences were observed on Day -8, prior to the diet intervention (experimental Day -8). Consistent with analysis using ANC0M-BC2 on SCNIC modules, ASVs belonging to the Lachnospiraceae family were frequently identified in the phylogenetic balances with decreases in the Western-style diet groups.

[0221] TABLES

[0222] Table 1. Primers used for real time RT-PCR listed from 5’ to 3’

[0223] REFERENCES

[0224] 1. Bilkei-Gorzo, A., Racz, I., Michel, K., & Zimmer, A. (2002). Diminished Anxiety - and Depression- Related Behaviors in Mice with Selective Deletion of the Tael Gene. Journal of Neuroscience, 22(22), 10046-10052.

[0225] 2. Blanchard, D. C., Griebel, G., & Blanchard, R. J. (2001). Mouse Defensive Behaviors: Pharmacological and Behavioral Assays for Anxiety and Panic. Neuroscience & Biobehavioral Reviews, 25(3), 205-218.

[0226] 3. Blondel VD, Guillaume J-L, Lambiotte R, Lefebvre E (2008) Fast unfolding of communities in large networks. Journal of Statistical Mechanics: Theory and Experiment P10008.

[0227] 4. Brunt VE, Greenberg NT, Sapinsley ZJ, Casso AG, Richey JJ, VanDongen NS, Gioscia-Ryan RA, Ziemba BP, Neilson AP, Davy KP, Seals DR (2022) Suppression of trimethylamine N-oxide with DMB mitigates vascular dysfunction, exercise intolerance, and frailty associated with a Western-style diet in mice. J Appl Physiol (1985 ) 133:798-813.

[0228] 5. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJ, Holmes SP (2016) DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods 13:581- 583. 6. Clayton, Z. S., Gioscia-Ryan, R. A., Justice, J. N., Lubieniecki, K. L., Hutton, D. A., Rossman, M. J., Zigler, M. C., & Seals, D. R. (2022). Lifelong Physical Activity Attenuates Age-and Western-Style Diet-Related Declines in Physical Function and Adverse Changes in Skeletal Muscle Mass and Inflammation. Experimental Gerontology, 157, 111632.

[0229] 7. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Huntley J, Fierer N, Owens SM, Betley J, Fraser L, Bauer M, Gormley N, Gilbert JA, Smith G, Knight R (2012) Ultra-high- throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: 1621-1624.

[0230] 8. Dahl EM, Neer E, Bowie KR, Leung ET, Karstens L (2022) microshades: An R Package for Improving Color Accessibility and Organization of Microbiome Data. Microbiol Resour Announc 11 :e0079522.

[0231] 9. Frank, M. G., Baratta, M. V., Sprunger, D. B., Watkins, L. R., & Maier, S. F. (2007). Microglia Serve as a Neuroimmune Substrate for Stress-Induced Potentiation of CNS Pro- Inflammatory Cytokine Responses. Brain, Behavior, and Immunity, 21(1), 47-59.

[0232] 10. Frank, M. G., Fonken, L. K., Dolzani, S. D., Annis, J. L., Siebler, P. H., Schmidt, D., Watkins, L. R., Maier, S. F., & Lowry, C. A. (2018). Immunization with Mycobacterium Vaccae Induces an Anti-Inflammatory Milieu in the Cns: Attenuation of Stress-Induced Microglial Priming, Alarmins and Anxiety-Like Behavior. Brain, Behavior, and Immunity, 73, 352-363.

[0233] 11. Friedman J, Alm EJ (2012) Inferring correlation networks from genomic survey data. PLoS Comput Biol 8 :el 002687.

[0234] 12. Friedman J, Hastie T, Tibshirani R (2010) Regularization Paths for Generalized Linear Models via Coordinate Descent. J Stat Softw 33: 1-22.

[0235] 13. Friihbeck, G., Catalan, V., Rodriguez, A., & Gomez- Ambrosi, J. (2018). Adiponectin-Leptin Ratio: A Promising Index to Estimate Adipose Tissue Dysfunction. Relation with Obesity-Associated Cardiometabolic Risk. Adipocyte, 7(1), 57-62.

[0236] 14. Janssen S, McDonald D, Gonzalez A, Navas-Molina JA, Jiang L, Xu ZZ, Winker K, Kado DM, Orwoll E, Manary M, Mirarab S, Knight R (2018) Phylogenetic placement of exact amplicon sequences improves associations with clinical information. mSystems 3.

[0237] 15. Loupy KM, Cler KE, Marquart BM, Yifru TW, D'Angelo HM, Arnold MR, Elsayed Al, Gebert MJ, Fierer N, Fonken LK, Frank MG, Zambrano CA, Maier SF, Lowry CA (2021) Comparing the effects of two different strains of mycobacteria, Mycobacterium vaccae NCTC 1 1659 and M. vaccae ATCC 15483, on stress-resilient behaviors and lipid-immune signaling in rats. Brain Behav Immun 91 :212-229.

[0238] 16. Mandal S, Van TW, White RA, Eggesbo M, Knight R, Peddada SD (2015) Analysis of composition of microbiomes: a novel method for studying microbial composition. Microb Ecol Health Dis 26:27663.

[0239] 17. Paripati, N., Nesi, L., Sterrett, J. D., Dawud, L. a. M., Kessler, L. R., Lowry, C. A., Perez, L. J., DeSipio, J., & Phadtare, S. (2023). Gut Microbiome and Lipidome Signatures in Irritable Bowel Syndrome Patients from a Low-Income, Food-Desert Area: A Pilot Study. Microorganisms, 11(10), 2503.

[0240] 18. Reber SO, Neumann ID (2008) Defensive behavioral strategies and enhanced state anxiety during chronic subordinate colony housing are accompanied by reduced hypothalamic vasopressin, but not oxytocin, expression. Ann N Y Acad Sci 1148: 184-195.

[0241] 19. Silverman JD, Washbume AD, Mukherjee S, David LA (2017) A phylogenetic transform enhances analysis of compositional microbiota data. Elife 6.

[0242] 20. Weiss S, Xu ZZ, Peddada S, Amir A, Bittinger K, Gonzalez A, Lozupone C, Zaneveld JR, Vazquez-Baeza Y, Birmingham A, Hyde ER, Knight R (2017) Normalization and microbial differential abundance strategies depend upon data characteristics. Microbiome 5:27.

[0243] 21. Yu G (2020) Using ggtree to Visualize Data on Tree-Like Structures. Curr Protoc Bioinformatics 69:e96.

Claims

CLAIMSWhat is claimed is1. A method for treating, preventing or reducing a risk of developing obesity comprising administering a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae) to a subject in need thereof.

2. The method of claim 1, wherein the M. vaccae comprises AT. vaccae ATCC 15483.

3. The method of claim 1, wherein the M. vaccae comprises whole cell AT. vaccae ATCC 15483.

4. The method of claim 1, wherein the M. vaccae comprises heat-killed AT. vaccae ATCC 15483.

5. The method of claim 1, wherein the AT. vaccae comprises heat comprises a non-pathogenic heat- killed M. vaccae.

6. The method of claim 1, wherein the AT. vaccae comprises isolated constituent parts of AT vaccae, wherein said constituent parts of the AT vaccae comprise organelles or biomolecules isolated from AT. vaccae.

7. The method of claim 1, wherein the AT vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

8. The method of claim 1, wherein the AT vaccae is in the form of a vaccine composition optionally comprising an adjuvant.

9. The method of claim 1, wherein said method ameliorates at least one sign or symptom of an obesity -related metabolic disease or condition.

10. The method of claim 9, wherein said obesity -related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulinresistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

11. The method of claim 1, wherein the subject is a mammal.

12. The method of claim 1, wherein the subject is a human.

13. A method for reducing visceral adipose tissue (VAT) comprising administering a therapeutically effective amount of isolated Mycobacterium vaccae (M. vaccae) to a subject in need thereof.

14. The method of claim 13, wherein the AT. vaccae comprises M. vaccae ATCC 15483.

15. The method of claim 13, wherein the AT. vaccae comprises whole cell AT vaccae ATCC 15483.

16. The method of claim 13, wherein the AT vaccae comprises heat-killed AT. vaccae ATCC 15483.

17. The method of claim 13, wherein the AT. vaccae comprises heat comprises a non-pathogenic heat-killed AT. vaccae.

18. The method of claim 13, wherein the AT vaccae comprises isolated constituent parts of AT. vaccae, wherein said constituent parts of the AT vaccae comprise organelles or biomolecules isolated from AT vaccae.

19. The method of claim 13, wherein the AT vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

20. The method of claim 13, wherein said method ameliorates at least one sign or symptom of an obesity -related metabolic disease or condition.21 . The method of claim 20, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

22. The method of claim 13, wherein the subject is a mammal.

23. The method of claim 13, wherein the subject is a human.

24. A method for reducing food intake comprising administering a therapeutically effective amount of isolated Mycobacterium vaccae ( vaccae) to a subject in need thereof.

25. The method of claim 24, wherein the M. vaccae comprises M. vaccae ATCC 15483.

26. The method of claim 24, wherein the AT. vaccae comprises whole cell M. vaccae ATCC 15483.

27. The method of claim 24, wherein the A4. vaccae comprises heat-killed AY. vaccae ATCC 15483.

28. The method of claim 24, wherein the AT. vaccae comprises heat comprises a non-pathogenic heat-killed M. vaccae.

29. The method of claim 24, wherein the M. vaccae comprises isolated constituent parts of M. vaccae, wherein said constituent parts of the M. vaccae comprise organelles or biomolecules isolated from M. vaccae.

30. The method of claim 24, wherein the M. vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

31. The method of claim 24, wherein said method ameliorates at least one sign or symptom of an obesity -related metabolic disease or condition.

32. The method of claim 31 , wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

33. The method of claim 24, wherein the subject is a mammal.

34. The method of claim 24, wherein the subject is a human.

35. A method for treating, preventing, or reducing a risk of developing an obesity-related metabolic disease or condition comprising administering a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae) to a subject in need thereof.

36. The method of claim 35, wherein the M. vaccae comprises M. vaccae ATCC 15483.

37. The method of claim 35, wherein the AT. vaccae comprises whole cell AT vaccae ATCC 15483.

38. The method of claim 35, wherein the AT. vaccae comprises heat-killed AT. vaccae ATCC 15483.

39. The method of claim 35, wherein the AT. vaccae comprises heat comprises a non -pathogenic heat-killed AT. vaccae.

40. The method of claim 35, wherein the AT. vaccae comprises isolated constituent parts of AT. vaccae, wherein said constituent parts of the AT vaccae comprise organelles or biomolecules isolated from AT. vaccae.

41. The method of claim 35, wherein the AT vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

42. The method of claim 35, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulinresistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

43. The method of claim 35, wherein the subject is a mammal.

44. The method of claim 35, wherein the subject is a human.

45. A pharmaceutical composition for treating, preventing or reducing a risk of developing obesity in a subject in need thereof, comprising a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae), and a pharmaceutically acceptable carrier.

46. The pharmaceutical composition of claim 45, wherein the M. vaccae comprises M. vaccae ATCC 15483.

47. The pharmaceutical composition of claim 45, wherein the M. vaccae comprises whole cell M. vaccae ATCC 15483.

48. The pharmaceutical composition of claim 45, wherein the M. vaccae comprises heat-killed M. vaccae ATCC 15483.

49. The pharmaceutical composition of claim 45, wherein the M. vaccae comprises heat comprises a non-pathogenic heat-killed M. vaccae.

50. The pharmaceutical composition of claim 45, wherein the M. vaccae comprises isolated constituent parts of AT. vaccae, wherein said constituent parts of the M. vaccae comprise organelles or biomolecules isolated from M. vaccae.

51. The pharmaceutical composition of claim 45, wherein the composition is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

52. The pharmaceutical composition of claim 45, wherein the composition is in the form of a vaccine composition optionally comprising an adjuvant.

53. The pharmaceutical composition of claim 45, wherein said pharmaceutical composition ameliorates at least one sign or symptom of an obesity -related metabolic disease or condition.

54. The pharmaceutical composition of claim 53, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

55. The pharmaceutical composition of claim 45, wherein the subject is a mammal.

56. The pharmaceutical composition of claim 45, wherein the subject is a human.

57. A pharmaceutical composition for reducing visceral adipose tissue (VAT) in a subject in need thereof, comprising a therapeutically effective amount of isolated Mycobacterium vaccae (M. vaccae), and a pharmaceutically acceptable carrier..

58. The pharmaceutical composition of claim 57, wherein the M. vaccae comprises M. vaccae ATCC 15483.

59. The pharmaceutical composition of claim 57, wherein the M. vaccae comprises whole cell M. vaccae ATCC 15483.

60. The pharmaceutical composition of claim 57, wherein the M. vaccae comprises heat-killed M. vaccae ATCC 15483.

61. The pharmaceutical composition of claim 57, wherein the M. vaccae comprises heat comprises a non -pathogenic heat-killed AZ vaccae.

62. The pharmaceutical composition of claim 57, wherein the M. vaccae comprises isolated constituent parts of M. vaccae, wherein said constituent parts of the M. vaccae comprise organelles or biomolecules isolated from AT. vaccae.

63. The pharmaceutical composition of claim 57, wherein the composition is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

64. The pharmaceutical composition of claim 57, wherein the composition is in the form of a vaccine composition optionally comprising an adjuvant.

65. The pharmaceutical composition of claim 57, wherein said pharmaceutical composition ameliorates at least one sign or symptom of an obesity-related metabolic disease or condition.

66. The pharmaceutical composition of claim 65, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

67. The pharmaceutical composition of claim 57, wherein the subject is a mammal.

68. The pharmaceutical composition of claim 57, wherein the subject is a human.

69. A pharmaceutical composition for reducing food intake in a subject in need thereof comprising a therapeutically effective amount of isolated Mycobacterium vaccae (M. vaccae), and a pharmaceutically acceptable carrier..

70. The pharmaceutical composition of claim 69, wherein the M. vaccae comprises AT. vaccae ATCC 15483.71 . The pharmaceutical composition of claim 69, wherein the M. vaccae comprises whole cell M. vaccae ATCC 15483.

72. The pharmaceutical composition of claim 69, wherein the M. vaccae comprises heat-killed M. vaccae ATCC 15483.

73. The pharmaceutical composition of claim 69, wherein the M. vaccae comprises heat comprises a non-pathogenic heat-killed M. vaccae.

74. The pharmaceutical composition of claim 69, wherein the M. vaccae comprises isolated constituent parts of M. vaccae, wherein said constituent parts of the M. vaccae comprise organelles or biomolecules isolated from M. vaccae.

75. The pharmaceutical composition of claim 69, wherein the composition is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

76. The pharmaceutical composition of claim 69, wherein the composition is in the form of a vaccine composition optionally comprising an adjuvant.

77. The pharmaceutical composition of claim 69, wherein said pharmaceutical composition ameliorates at least one sign or symptom of an obesity-related metabolic disease or condition.

78. The pharmaceutical composition of claim 77, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type I and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes, coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

79. The pharmaceutical composition of claim 69, wherein the subject is a mammal.

80. The pharmaceutical composition of claim 69, wherein the subject is a human.

81. A pharmaceutical composition for treating, preventing or reducing a risk of developing an obesity-related metabolic disease or condition in a subject in need thereof comprising a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae), and a pharmaceutically acceptable carrier.

82. The pharmaceutical composition of claim 81, wherein the AT. vaccae comprises M. vaccae ATCC 15483.

83. The pharmaceutical composition of claim 81, wherein the AT. vaccae comprises whole cell A . vaccae ATCC 15483.

84. The pharmaceutical composition of claim 81, wherein the AY. vaccae comprises heat-killed AY. vaccae ATCC 15483.

85. The pharmaceutical composition of claim 81, wherein theAY vaccae comprises heat comprises a non-pathogenic heat-killed AY. vaccae.

86. The pharmaceutical composition of claim 81, wherein the AY vaccae comprises isolated constituent parts of AY. vaccae, wherein said constituent parts of theAY. vaccae comprise organelles or biomolecules isolated from AY. vaccae.

87. The pharmaceutical composition of claim 81, wherein the composition is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

88. The pharmaceutical composition of claim 81, wherein the composition is in the form of a vaccine composition optionally comprising an adjuvant.

89. The pharmaceutical composition of claim 81, wherein said obesity-related metabolic disease or condition is selected from: hypertension, hyperglycemia, prediabetes, diabetes (type 1 and type 2), obesity, insulin resistance, metabolic syndrome and dyslipidemia due to type 2 diabetes,coronary heart disease (CHD), gallbladder disease, osteoarthritis, sleep apnea, respiratory disorder, and cancer.

90. The pharmaceutical composition of claim 81, wherein the subject is a mammal.

91. The pharmaceutical composition of claim 81, wherein the subject is a human.

92. A method for treating, preventing or reducing a risk of developing a stress-related disorder comprising administering a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae) to a subject in need thereof.

93. The method of claim 92, wherein the AT. vaccae comprises ATI vaccae ATCC 15483.

94. The method of claim 92, wherein the AT. vaccae comprises whole cell AT vaccae ATCC 15483.

95. The method of claim 92, wherein the AT vaccae comprises heat-killed AT. vaccae ATCC 15483.

96. The method of claim 92, wherein the AT. vaccae comprises heat comprises a non-pathogenic heat-killed AT. vaccae.

97. The method of claim 92, wherein the AT vaccae comprises isolated constituent parts of AT. vaccae, wherein said constituent parts of the AT vaccae comprise organelles or biomolecules isolated from AT vaccae.

98. The method of claim 92, wherein the AT vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

99. The method of claim 92, wherein the AT. vaccae is in the form of a vaccine composition optionally comprising an adjuvant.

100. The method of claim 92, wherein said stress-related disorder comprises post-traumatic stressdisorder (PTSD), depression, or an anxiety disorder.

101. The method of claim 100, wherein the depression comprises major depressive disorder.

102. The method of claim 100, wherein the anxiety disorder comprises general anxiety disorder, panic disorder, social anxiety disorder or a phobia-related disorder.

103. The method of claim 92, wherein the subject is a mammal.

104. The method of claim 92, wherein the subject is a human.

105. A pharmaceutical composition for use in treating, preventing or reducing a risk of developing a stress-related disorder in a subject in need thereof, comprising a therapeutically or prophylactically effective amount of isolated Mycobacterium vaccae (M. vaccae).

106. The pharmaceutical composition of claim 105, wherein the M. vaccae comprises AY. vaccae ATCC 15483.

107. The pharmaceutical composition of claim 105, wherein the AY. vaccae comprises whole cell AY. vaccae ATCC 15483.

108. The pharmaceutical composition of claim 105, wherein the AY. vaccae comprises heat-killed AY. vaccae ATCC 15483.

109. The pharmaceutical composition of claim 105, wherein the AY vaccae comprises heat comprises a non-pathogenic heat-killed AY. vaccae.

110. The pharmaceutical composition of claim 105, wherein the AY. vaccae comprises isolated constituent parts of AY. vaccae, wherein said constituent parts of the AY vaccae comprise organelles or biomolecules isolated from AY. vaccae.

111. The pharmaceutical composition of claim 105, wherein the M. vaccae is formulated for administration via a parenteral, oral, sublingual, nasal, or pulmonary route.

112. The pharmaceutical composition of claim 105, wherein the M. vaccae is in the form of a vaccine composition optionally comprising an adjuvant.

113. The pharmaceutical composition of claim 105, wherein said stress-related disorder comprises post-traumatic stress-disorder (PTSD), depression, or an anxiety disorder.

114. The pharmaceutical composition of claim 113, wherein the depression comprises major depressive disorder.

115. The pharmaceutical composition of claim 113, wherein the anxiety disorder comprises general anxiety disorder, panic disorder, social anxiety disorder or a phobia-related disorder.

116. The pharmaceutical composition of claim 105, wherein the subject is a mammal.

117. The pharmaceutical composition of claim 105, wherein the subject is a human.

118. A pharmaceutical composition, comprising heat-killed M. vaccae ATCC 15483 or constituent components.

119. The pharmaceutical composition of claim 118, comprising a pharmaceutically acceptable carrier.

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