Inactivated bacteroides bacteria and methods of use thereof

Abstract

The technology described herein is directed to inactivated Bacteroides bacteria, including but not limited to inactivated B. salyersiae bacteria and to compositions comprising them, as well as methods of use thereof, e.g., for modification of mood or other CNS-related parameters.

Description

Attorney Docket No: 083103-000102WOPT INACTIVATED BACTEROIDES BACTERIA AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 691,078 filed September 5, 2024, the contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML format via Patent Center and is hereby incorporated by reference in its entirety. Said XML copy, created on September 1, 2025, is named 083103-000102WOPT_SL.xml and is 16,198 bytes in size. TECHNICAL FIELD

[0003] The technology described herein relates to inactivated Bacteroides bacteria formulations and uses thereof related, for example, to administration to a human subject. BACKGROUND

[0004] Depression is a mood disorder characterized by persistent feelings of sadness, often co-occurring with altered mood states like irritability, fatigue, and anhedonia. Beyond altered mood states, depression has been associated with increased systemic inflammation, which contributes to a state of neuroinflammation within the central nervous system. Altered immune function and dysbiosis in the gut microbial community are key drivers of this pathological inflammation. Depression affects up to 9% of adults in the U.S. per year and was recently classified as a leading cause of disability worldwide by the World Health Organization.

[0005] The etiology and pathology of depression are influenced by a complex interplay of external factors such as diet, life events, and stress, and internal factors such as genetic predisposition, inflammation, and neurotransmitter dysregulation, including reduced levels of central and peripheral GABA. Neurobiologically, the prefrontal cortex is implicated in depression etiologies as it is essential in emotional learning and is involved in the modulation of the stress response via the hypothalamic-pituitary-adrenal (HPA) axis.

[0006] The multifaceted etiology of depression makes it a challenging disorder to treat. Treatment regimens include selective serotonin reuptake inhibitors (SSRIs), and more 1 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT recently, ketamine. However, SSRIs have a high degree of variability in treatment response across populations, and also have been shown to influence the gut microbiome, and ketamine comes with risk of misuse and abuse. Alternative treatments are therefore needed, particularly those which can influence multiple pathways implicated in depression. Evidence from animal studies and human studies implicate the ability of the gut microbiome to influence depression through its bidirectional relationship with the nervous system and immune system, so called the “gut-brain axis.” The gut microbiome represents a key therapeutic avenue to alleviate symptoms of depression. Although the mechanisms underlying the gut-brain axis are still being fully elucidated, evidence indicates the vagus nerve as a key mediator of gut-brain interactions in depression.

[0007] Research has increasingly focused on leveraging the gut microbiome to mitigate depressive symptoms by investigating the potential of probiotics as a treatment for depression, both in clinical settings and pre-clinical models. Several clinical trials and meta- analyses have highlighted the promising role of probiotics in managing depression. For instance, a meta-analysis of randomized controlled trials found that probiotics, either alone or in combination with prebiotics (known as symbiotic), can significantly improve depressive symptoms when compared to placebos (see e.g., Zhang et al.2023, BMC Psychiatry.23(1): 477). Probiotics can reduce depressive symptoms effectively when used alongside antidepressants, and can influence depressive symptoms via modulation of inflammatory responses and enhancement of neurotrophic factors like brain-derived neurotrophic factor (see e.g., Nikolova et al.2021, J Clin Med.10(4): 647). However, most preclinical and clinical studies of probiotics have focused on Bifidobacteria and Lactobacillus, as these genera have been shown to be reduced in depressive states (see e.g., Bharwani et al.2017, BMC Med.15(1): 7; Hashikawa-Hobara et al.2022, Front Neurosci.16: 918953; Partrick et al.2021, Sci Rep.11(1): 3763; Maehata et al.2019, Biosci Biotechnol Biochem.83(7): 1239- 1247; Kosuge et al.2021, Brain Behav Immun.96: 200-211; Toyoda et al.2020, Biomed Res.41(2): 101-111; Yoda et al.2022, Nutrients 14(5): 970). The link between the gut microbiome, systemic inflammation, and CNS health is not limited to depression. A growing body of evidence indicates that neuroinflammation, the activation of the brain's innate immune system, is a common underlying factor in a wide range of neurological and psychiatric conditions. This process can lead to synaptic dysfunction, neuronal damage, and disruption of blood-brain barrier. Consequently, the gut-brain-immune axis represents a critical therapeutic target for a spectrum of neuroimmune-related conditions, including 2 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT neurodegenerative diseases (e.g., Parkinson's and Alzheimer's disease), demyelinating disorders (e.g., multiple sclerosis), and cognitive deficits associated with systemic inflammation (i.e., "brain fog"). This state of chronic neuroinflammation is also a fundamental mechanism underlying many chronic pain disorders. Conditions such as neuropathic pain, fibromyalgia, and migraine are no longer viewed simply as peripheral issues, but as disorders of the central nervous system involving central sensitization. This process, driven by pro-inflammatory cytokines, leads to a hyperexcitable state in the spinal cord and brain, causing pain to be amplified and maintained long after an initial injury has healed, or even in the absence of any injury. SUMMARY

[0008] Given the high comorbidity between chronic pain, depression, and gastrointestinal disorders like Irritable Bowel Syndrome (IBS), targeting the gut-brain-immune axis is a strategy for treating pain by addressing its inflammatory and neurological roots. Interventions capable of modulating these inflammatory pathways from the gut therefore hold promise for treating a wide array of CNS disorders. The technology described herein is directed to deliberately inactivated Bacteroides bacteria, and to compositions comprising them. Inactivated Bacteroides bacteria as described herein have been found to modulate neuroinflammatory pathways. This immunomodulatory activity brings about beneficial changes in mood, a reduction in pain, other indicia of mental well-being, and amelioration of other symptoms of neuroimmune-related disorders when administered to subjects in need thereof.

[0009] In one aspect, described herein is a composition comprising: inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to 16s rRNA of Bacteroides salyersiae HB32 as set out in one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject. 3 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0010] In one aspect, described herein is a composition comprising an inactivated bacterial strain, the genome of which comprises at least one sequence encoding at least one enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof, wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject.

[0011] In some embodiments of any of the aspects, the mammalian subject is human.

[0012] In some embodiments of any of the aspects, the bacterial strain comprises a targeted mutation that inactivates a target gene or its gene product, wherein the genome of the mutated strain remains at least 99.5% identical to that of B. salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024,as Patent Deposit No. PTA-127757 or at least 99.5% identical to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900, and wherein the 16S rRNA sequence remains at least 95% identical to one of SEQ ID NOs: 1 or 2.

[0013] In some embodiments of any of the aspects, the inactivated bacterial strain is formulated for oral delivery.

[0014] In some embodiments of any of the aspects, the composition comprises a pharmaceutically acceptable excipient.

[0015] In some embodiments of any of the aspects, the inactivated bacterial strain is deliberately inactivated by heat treatment, gamma irradiation, ultrasonic disruption, lyophilization, tyndallization, pasteurization, freeze-thaw inactivation, pulsed electric field treatment, or a combination thereof.

[0016] In some embodiments of any of the aspects, the composition further comprises at least one additional prebiotic selected from the group consisting of an amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharide, polyamine (e.g., spermidine, putrescine), polyol (e.g. maltitol, xylitol). 4 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0017] In some embodiments of any of the aspects, the composition further comprises one or more species of different viable bacteria.

[0018] In some embodiments of any of the aspects, the composition further comprises one or more additional species of inactivated bacteria.

[0019] In some embodiments of any of the aspects, the composition further comprises a purified bacterial strain that is cytotoxic or cytostatic to a GABA-consuming bacteria.

[0020] In some embodiments of any of the aspects, the GABA-consuming bacteria is Evtepia gabavorous or Firmicutes bacterium MGS:114.

[0021] In some embodiments of any of the aspects, wherein the inactivated bacterial strain is dried.

[0022] In some embodiments of any of the aspects, the composition comprises an enteric coating.

[0023] In some embodiments of any of the aspects, the composition is a nutritional composition, a food product, a dietary complement, a medical food, or a medicament.

[0024] In some embodiments of any of the aspects, the composition is formulated in a tablet, a caplet, a pill, a capsule, a troche, a lozenge, a granule, a powder, a gummy, a gel, a sachet, a food composition, a nutraceutical, a medical food, or a combination thereof.

[0025] In some embodiments of any of the aspects, the genome of the inactivated bacterial strain comprises sequence encoding two or more of glutamate decarboxylase; glutamate:GABA antiporter and glutaminase enzymes.

[0026] In some embodiments of any of the aspects, the genome of the inactivated bacterial strain comprises sequence encoding glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes.

[0027] In some embodiments of any of the aspects, the sequence encoding the glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes is at least 98% identical to, or encodes only conservative amino acid differences relative to the enzymes encoded by SEQ ID NOs 3-5.

[0028] In one aspect, described herein is a method of treating a CNS disorder, a pain disorder or a neuroimmune disorder in a human subject in need thereof, the method comprising administering a therapeutically effective amount of a composition as described herein to the subject.

[0029] In some embodiments of any of the aspects, the CNS disorder is selected from the group consisting of treatment-resistant major depressive disorder (TR-MDD), major 5 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), bipolar disorder, schizophrenia, anxiety, anxiety disorders, obsessive compulsive disorder, eating disorders, addiction, social phobia, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD).

[0030] In some embodiments of any of the aspects, the CNS disorder is major depressive disorder, and wherein the administration of the composition reduces a symptom of anhedonia with an efficacy comparable to a standard therapeutic dose of ketamine in a social defeat animal model.

[0031] In some embodiments of any of the aspects, the disorder is a pain disorder, and wherein the pain disorder is chronic pain.

[0032] In some embodiments of any of the aspects, the chronic pain is selected from the group consisting of nociplastic pain, neuropathic pain, inflammatory pain, and visceral pain, and combinations thereof.

[0033] In some embodiments of any of the aspects, the pain is associated with a condition selected from the group consisting of fibromyalgia, irritable bowel syndrome, diabetic neuropathy, rheumatoid arthritis, complex regional pain syndrome, radiculopathy, post- herpetic neuralgia, chemotherapy-induced peripheral neuropathy.

[0034] In some embodiments of any of the aspects, the disorder is a neuroimmune-related condition selected from the group consisting of Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis.

[0035] In some embodiments of any of the aspects, the method results in a reduction in the serum level of one or more biomarkers selected from the group consisting of CXCL1, IL-1b, and TNFα in the subject.

[0036] In some embodiments of any of the aspects, the method further comprises detecting a level of one or more bacterial strains selected from: Bacteroides salyersiae bacterial strain HB32 (“HB32”); a bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; a bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898- CP166900; a bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 6 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA- 127757; in the stool of the subject.

[0037] In some embodiments of any of the aspects, the detecting is performed prior to the administering.

[0038] In some embodiments of any of the aspects, the detecting comprises nucleic acid sequencing.

[0039] In some embodiments of any of the aspects, the method further comprises detecting levels of an inflammatory signature in serum prior to administering.

[0040] In some embodiments of any of the aspects, the method further comprises detecting the level of one or more fecal inflammatory markers prior to administering.

[0041] In some embodiments of any of the aspects, the method further comprises detecting a GABA level prior to administering.

[0042] In some embodiments of any of the aspects, the GABA level is a serum and / or fecal GABA level.

[0043] In some embodiments of any of the aspects, detecting a level below a threshold amount or above a threshold amount for a given detected parameter provides an indication of whether the administering will be effective and / or beneficial to the subject.

[0044] In one aspect, a composition as described herein is for use in treating a disorder selected from the group consisting of a CNS disorder, a pain disorder, and a neuroimmune- related condition in a human subject.

[0045] In some embodiments of any of the aspects, the composition is comprised by a packaged food product, wherein the package of the packaged food product is associated with or comprises indicia that the composition is for the use as described herein.

[0046] In one aspect, described herein is a method for supporting a healthy immune response in a mammalian subject with a sub-optimal immune status, the method comprising: a. selecting a subject based on a baseline biomarker measurement, said measurement being within a range selected from the group consisting of: i. a serum high-sensitivity C-reactive protein (hs-CRP) level of 1.0 mg / L to 5.0 mg / L; ii. a secretory immunoglobulin A (sIgA) level in the lower quartile of a healthy population reference range; and iii. a natural killer (NK) cell activity level in the lower tertile of a healthy population reference range; and b. administering an effective amount of a composition as described herein to the selected subject. 7 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0047] In one aspect, described herein is a method for supporting mental and emotional well- being in a mammalian subject experiencing a mild to moderate challenge, the method comprising: a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. a Patient Health Questionnaire-9 (PHQ-9) score of 5 to 14; ii. a Generalized Anxiety Disorder-7 (GAD-7) score of 5 to 14; iii. a Perceived Stress Scale (PSS) score of 14 to 26; and iv. a Liebowitz Social Anxiety Scale (LSAS) score of 30 to 89; and b. administering an effective amount of a composition as described herein to the selected subject.

[0048] In one aspect, described herein is a method for alleviating mild to moderate physical discomfort in a mammalian subject, the method comprising: a. selecting a subject based on a baseline score on a validated pain scale, said score being within a range selected from the group consisting of: i. a Numeric Rating Scale (NRS) score of 3 to 6; ii. a Fibromyalgia Impact Questionnaire (FIQ) score of 25 to 59; iii. a PROMIS Pain Interference T-score of 55 to 65; and iv. a Central Sensitization Inventory (CSI) score of 30 to 49; and b. administering an effective amount of a composition as described herein to the selected subject.

[0049] In one aspect, described herein is a method for supporting neurocognitive function in a mammalian subject experiencing a mild to moderate decline, the method comprising: a. selecting a subject based on a baseline score on a validated assessment, said score being within a range selected from the group consisting of: i. a Mini-Mental State Examination (MMSE) score of 19 to 26; ii. an Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score of 10 to 25; iii. a Unified Parkinson's Disease Rating Scale (UPDRS) Part III score of 11 to 32; and iv. an Expanded Disability Status Scale (EDSS) score of 1.0 to 4.5; and b. administering an effective amount of a composition as described herein to the selected subject.

[0050] In one aspect, described herein is a method for improving sleep quality in a mammalian subject experiencing a mild to moderate sleep challenge, the method comprising: a. selecting a subject based on a baseline measurement indicative of sub-optimal sleep, said measurement being within a range selected from the group consisting of: i. a PROMIS Sleep Disturbance T-score of 55 to 65; ii. a Pittsburgh Sleep Quality Index (PSQI) global score of 6 to 14; and iii. a sleep efficiency of 75% to 84% as measured by a wearable device; and b. administering an effective amount of a composition as described herein to the selected subject. 8 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0051] In one aspect, described herein is a method for supporting gastrointestinal health and function in a mammalian subject experiencing mild to moderate digestive discomfort, the method comprising: a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. an Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) score of 75 to 300; ii. a Gastrointestinal Symptom Rating Scale (GSRS) mean score of 3 to 5; and iii. consistent reporting of Bristol Stool Form Scale types 1, 2, 6, or 7 for at least 25% of bowel movements; and b. administering an effective amount of a composition as described herein to the selected subject.

[0052] In one aspect, described herein is a composition comprising: inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; an inactivated bacterial strain, the genome of which comprises at least one sequence encoding at least one enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA- 127757, wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject.

[0053] In one embodiment of this or any other aspect described herein, the inactivated bacterial strain has 16S rRNA with at least 96%, at least 97%, at least 98%, at least 99% or greater identity, up to and including 100% identity to one of SEQ ID NOs: 1 or 2.

[0054] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain has at least 98.55, at least 99%, at least 99.55 or greater sequence identity, up to and including 100% identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900.

[0055] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain encodes in its genome at least two enzymes selected from glutamate decarboxylase; glutamate:GABA antiporter; and glutaminase. In another embodiment of this or any other aspect described herein, the inactivated bacterial strain encodes in its genome each of glutamate decarboxylase; glutamate:GABA antiporter; and glutaminase. 9 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0056] In another embodiment of this or any other aspect described herein, the bacterial strain comprises a targeted mutation that inactivates a target gene or its gene product, wherein the genome of the mutated strain remains at least 99.5% identical to the genomic sequence of B. salyersiae bacterial strain HB32 deposited under GenBank Accession No: CP166898- CP166900, and wherein the 16S rRNA sequence remains at least 95% identical to one of SEQ ID NOs: 1 or 2. Targeted mutations can include, for example, insertions of one or more nucleotides, deletion of one or more nucleotides, transition and / or transversion mutations, introduction of a premature stop codon, introduction of a frame shift mutation or missense mutation, removal or modification of a splice site or other change that inactivates expression of a targeted gene. In another embodiment of this or any other aspect described herein, the genome of the mutated strain remains at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, at least 99.92%, at least 99.94%, at least 99.96% or at least 99.98% identical to the genomic sequence of B. salyersiae bacterial strain HB32 deposited under GenBank Accession No: CP166898-CP166900. In one embodiment of this or any other aspect described herein, the targeted mutation inactivates an antimicrobial resistance gene. In another embodiment, the antimicrobial resistance gene is a β-lactamase gene (bla). Inactivation can be via insertion, deletion or substitution of nucleic acid sequence, including but not limited to deletion of the bla coding sequence, or, for example, introduction of one or more mutations that introduce one or more premature stop codons that result in truncated inactive bla protein.

[0057] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain is formulated for oral delivery.

[0058] In another embodiment of this or any other aspect described herein, the mammalian subject is a human.

[0059] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain or formulation comprising it reduces serum levels of CXCL1 when orally administered to an animal model. In another embodiment of this or any other aspect described herein, the inactivated bacterial strain or formulation comprising it reduces serum levels of CXCL1, IL1b, and / or TNFa, when orally administered to a subject, e.g., a human, an animal model (see e.g., Fig.4-7). In another embodiment of this or any other aspect described herein, the animal model is a rat model.

[0060] In another embodiment of this or any other aspect described herein, the composition comprises a pharmaceutically acceptable excipient. 10 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0061] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain is inactivated by heat treatment, gamma irradiation, ultrasonic disruption, lyophilization, tyndallization, pasteurization, freeze-thaw inactivation, pulsed electric field treatment, or a combination thereof. Where lyophilization is applicable, it should be understood that lyophilization, for example, in the absence of a cryopreservative, leads or can lead to inactivation of a substantial proportion of the bacteria.

[0062] In another embodiment of this or any other aspect described herein, the composition further comprises at least one additional prebiotic selected from the group consisting of an amino acid (e.g., arginine, glutarate, and / or ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose-enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans-galactooligosaccharide, pectin (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and / or oat fiber), xylooligosaccharide, polyamine (e.g., spermidine, putrescine), and polyol (e.g. maltitol, xylitol).

[0063] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain or formulation comprising it reduces serum levels of IL6 and / or IL1B when orally administered to a subject.

[0064] In another embodiment of this or any other aspect described herein, the bacterial strain produces or has the capacity to produce gamma aminobutyric acid (GABA) in the mammalian gut when the bacterium is viable.

[0065] In another embodiment of this or any other aspect described herein, the glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or combination thereof, is encoded by a DNA sequence comprising any one of SEQ ID NOs: 3-5 or a DNA sequence that is at least 50% identical in DNA sequence to any one of SEQ ID NOs: 3-5.

[0066] In another embodiment of this or any other aspect described herein, the composition further comprises one or more species of different viable bacteria.

[0067] In another embodiment of this or any other aspect described herein, the composition further comprises one or more additional species of inactivated bacteria.

[0068] In another embodiment of this or any other aspect described herein, the composition further comprises a purified bacterial strain that is cytotoxic or cytostatic to a GABA- 11 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT consuming bacteria. In one embodiment of this or any other aspect described herein, the GABA-consuming bacteria is Evtepia gabavorous or Firmicutes bacterium MGS:114.

[0069] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain is dried.

[0070] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain is formulated in a tablet, a caplet, a pill, a capsule, a troche, a lozenge, a granule, a powder, a gummy, a gel, a sachet, a food composition, a nutraceutical, a medical food, or a combination thereof.

[0071] In another embodiment of this or any other aspect described herein, the inactivated bacterial strain composition comprises an enteric coating.

[0072] In another aspect, described herein is a pharmaceutical composition comprising an inactivated bacterial strain as described herein and a pharmaceutically acceptable carrier.

[0073] In another aspect, described herein is a method of improving mood, improving sleep quality, improving focus, improving memory, improving quality of life score, reducing stress, or reducing fatigue in a mammalian subject in need thereof, the method comprising administering a mood-improving, sleep quality-improving, focus-improving, memory- improving, quality of life score-improving, stress-reducing, or fatigue-reducing amount of a composition comprising an inactivated bacterial strain as described herein to the subject.

[0074] In another aspect, described herein is a method of promoting relaxation or promoting a calming mood state in a mammalian subject in need thereof, the method comprising administering a relaxation-promoting or calm mood-promoting amount of a composition comprising an inactivated bacterial strain as described herein to the subject.

[0075] In one embodiment of this or any other aspect described herein, the administering comprises orally administering.

[0076] In another aspect, described herein is a method of treating a CNS disorder in a mammalian subject in need thereof, the method comprising administering a therapeutically effective amount of a composition comprising an inactivated bacterial strain as described herein to the subject.

[0077] In one embodiment of this or any other aspect described herein, the mammalian subject is a human.

[0078] In one embodiment of this or any other aspect described herein, the CNS disorder is selected from the group consisting of treatment-resistant major depressive disorder (TR- MDD), major depressive disorder and its subtypes (melancholic depression, atypical 12 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT depression, catatonic depression, postpartum depression, and seasonal affective disorder), bipolar disorder, schizophrenia, anxiety, anxiety disorders, obsessive compulsive disorder, eating disorders (anorexia nervosa, bulimia nervosa, binge-eating disorder, avoidant / restrictive food intake disorder, other specified feeding or eating disorder, pica, and rumination disorder), addiction, social phobia, Neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's diseases), orthostatic tremor, Lafora disease, restless leg syndrome, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD), and combinations thereof.

[0079] In one embodiment of this or any other aspect described herein, the treating or improving the CNS disease or disorder comprises decreasing at least one symptom of the CNS disease or disorder.

[0080] In one embodiment of this or any other aspect described herein, the at least one symptom of the mental disease or CNS disorder is selected from the group consisting of fatigue, insomnia, motor dysfunction, stress, persistent anxiety, persistent sadness, social withdrawal, substance withdrawal, irritability, thoughts of suicide, thoughts of self-harm, restlessness, obsessive thoughts, compulsive behaviors, fear of contamination, repetitive checking, low sex drive, lack of focus, seizures, memory loss, anger, bouts of emotional reactivity, confusion, muscle spasms, loss of appetite, hyperphagia, severe food restriction, binge eating, purging behaviors, distorted body image, preoccupation with body shape and weight, altered intestinal motility, low quality of life score, chronic widespread pain, neuropathic pain (e.g., burning, shooting, or tingling sensations), visceral pain, hyperalgesia (increased sensitivity to pain), allodynia (pain from stimuli that do not normally provoke pain), and pain-related interference with daily activities, and combinations thereof.

[0081] In one embodiment of this or any other aspect described herein, the CNS disorder is depression.

[0082] In another aspect, described herein is a method of treating an immunological and / or inflammatory disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of a composition comprising an inactivated bacterial strain as described herein to the subject.

[0083] In one embodiment of this or any other aspect described herein, the immunological and / or inflammatory disease or disorder is associated with or involves pathways relevant to IL1b and / or CXCL1 signalling. In one embodiment of this or any other aspect described 13 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT herein, the immunological and / or inflammatory disease or disorder is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, alcoholic liver disease, non-alcoholic fatty liver disease, Type I diabetes mellitus, graft-versus-host disease, and combinations thereof.

[0084] In another aspect, described herein is a method of reducing inflammatory cytokine production, release or serum level in a subject in need thereof, the method comprising administering to the subject an amount of a composition comprising an inactivated bacterial strain as described herein effective to reduce production, release or level of one of more inflammatory cytokines.

[0085] In one embodiment of this or any other aspect described herein, the inflammatory cytokine or chemokine is selected from TNFα, IL1B, and CXCL-1.

[0086] In one embodiment of this or any other aspect described herein, the method further comprises detecting a level of one or more bacterial strains selected from: Bacteroides salyersiae bacterial strain HB32 (“HB32”); a bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; a bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; a bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757 in the stool of the subject.

[0087] In one embodiment of this or any other aspect described herein, the detecting is performed prior to the administering.

[0088] In one embodiment of this or any other aspect described herein, the detecting comprises nucleic acid sequencing.

[0089] In one embodiment of this or any other aspect described herein, the method further comprises detecting levels of an inflammatory signature in serum prior to administering.

[0090] In one embodiment of this or any other aspect described herein, the method further comprises detecting the level of one or more fecal inflammatory markers prior to administering.

[0091] In one embodiment of this or any other aspect described herein, the method further comprises detecting a GABA level prior to administering. 14 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0092] In one embodiment of this or any other aspect described herein, the GABA level is a serum and / or fecal GABA level.

[0093] In one embodiment of this or any other aspect described herein, detecting a level below a threshold amount or above a threshold amount for a given detected parameter provides an indication of whether the administering will be effective and / or beneficial to the subject.

[0094] In another aspect, described herein is a method of making a composition for treatment of a CNS disease or disorder, the method comprising combining: an inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; an inactivated bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; and a pharmaceutically acceptable carrier.

[0095] In one embodiment of this or any other aspect described herein, the method further comprises, before the step of combining, the step of inactivating the bacterial strain.

[0096] In another aspect, described herein is a method of making a composition for improving mood, improving sleep quality, improving focus, improving memory, improving quality of life score, reducing stress, or reducing fatigue in a mammalian subject in need thereof, the method comprising combining: an inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; an inactivated bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., 15 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757, and a pharmaceutically acceptable carrier.

[0097] In another aspect, described herein is a method of making a composition for improving mood, improving sleep quality, improving focus, improving memory, improving quality of life score, reducing stress, or reducing fatigue in a mammalian subject in need thereof, the method comprising combining: an inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; an inactivated bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024,as Patent Deposit No. PTA-127757; and a food composition, a nutraceutical, components comprising a medical food, or a combination thereof.

[0098] In another aspect, described herein is a composition comprising an inactivated bacterial strain as described herein for use in treatment of a disease or disorder of the CNS.

[0099] In one embodiment of this or any other aspect described herein, the disease or disorder of the CNS is selected from treatment-resistant major depressive disorder (TR- MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), bipolar disorder, schizophrenia, anxiety, anxiety disorders, obsessive compulsive disorder, eating disorders (anorexia nervosa, bulimia nervosa, binge-eating disorder, avoidant / restrictive food intake disorder, other specified feeding or eating disorder, pica, and rumination disorder), addiction, social phobia, Neurodegenerative amyloid disorders (Parkinson's, Alzheimer's, and Huntington's diseases), orthostatic tremor, Lafora disease, restless leg syndrome, dementia, epilepsy, stiff-person syndrome, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD), and combinations thereof. 16 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0100] In another aspect, described herein is a composition comprising an inactivated bacterial strain as described herein for use in treatment of an immunological disease or disorder.

[0101] In one embodiment of this or any other aspect described herein, the immunological and / or inflammatory disease or disorder is selected from the group consisting of inflammatory bowel disease, rheumatoid arthritis, atherosclerosis, alcoholic liver disease, non-alcoholic fatty liver disease, Type I diabetes mellitus, graft-versus-host disease, and combinations thereof.

[0102] In one embodiment of this or any other aspect described herein, the composition is comprised by a packaged food product, wherein the package of the packaged food product is associated with or comprises indicia that the composition is for a use described herein.

[0103] In another aspect, described herein is a composition comprising: a) inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; an inactivated bacterial strain, the genome of which comprises at least one sequence encoding at least one enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; and b) viable B. salyersiae that have not been treated to inactivate them; wherein the composition is formulated for delivery to the gut of a mammalian subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Fig.1: Schematic representation of the social defeat model. Timeline for social defeat and dosing of experimental treatments and controls.

[0105] Fig.2A-2B: Bacteroides salyersiae HB32 prevented depressive-like behavior in the social defeat model of depression in male rats, a model dependent on an intact vagus nerve. (Fig.2A) 21-days of chronic social defeat stress (SD) in male rats significantly reduced sucrose preference, a measurement of anhedonia. Daily treatment during the final week of stress with intraperitoneal ketamine (SD-ketamine) or oral iHB32 (SD-iHB32) prevented anhedonia. (Fig.2B) Bilateral subdiaphragmatic vagotomy (SD-vagotomy) also prevented 17 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT stress-induced anhedonia, as compared to sham surgery (SD-Sham). [* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001]

[0106] Fig.3: iHB32, but not ketamine, normalized gene expression alterations in the PFC of male rats subjected to social defeat. Differentially expressed genes (DEGs) visualized via volcano plots generated in DESeq2 for comparisons between Naive and SD-Vehicle (left), SD-Ketamine and SD-Vehicle (center), SD-iHB32 and SD-Vehicle (right). Each individual dot represents a unique gene in the dataset. Significance thresholds are reported: adjusted p- value≤0.05 (dotted line), and Log2 fold change=+ / -0.5 (vertical black lines). Representative DEGs are notated.

[0107] Fig.4: IL1b release by lipopolysaccharide (LPS)-challenged human peripheral blood mononuclear cells with and without non-viable HB32 (nvHB32) and a media control (RPMI). Statistics were performed using a Mann-Whitney test (* = p <0.05).

[0108] Fig.5: Modulation of IL1b release by non-viable HB32 (nvHB32) on human peripheral blood mononuclear cells (PBMCs). PBMCs were challenged with human microbiota-derived communities (HD-2, HD-9, HD-13, and HD-20) with 0, 10, 30, and 50% iHB32. Statistics were performed using a 2-way ANOVA, followed by Dunnett’s multiple comparison test (* = p<0.05; ** = p <0.01; *** p<0.001; **** p<0.0001; ns = not significant).

[0109] Fig.6: Modulation of TNFa release by non-viable HB32 (nvHB32) on human peripheral blood mononuclear cells challenged with human microbiota-derived communities (HD-9 and HD-26). Statistics were performed using a 2-way ANOVA, followed by Sidak’s multiple comparison test (** = p <0.01).

[0110] Fig.7: Plasma levels of CXCL1 in rats dosed with inactivated HB32 (iHB32) compared to an un-dosed control (CTR). Plasma CXCL1 levels were measured by enzyme- linked immunosorbent assay. Statistics were performed using a Wilcoxon test (** = p <0.01). DETAILED DESCRIPTION

[0111] The gut microbiome has been linked to depression via the gut-brain axis. Levels of fecal Bacteroides are inversely associated with brain signatures of depression in the prefrontal cortex of human subjects. Bacteroides are important human commensals, playing a keystone role in regulating the immune system and producing bioactive metabolites, like the neurotransmitter gamma-aminobutyric acid (GABA) and vitamin B12. To better understand the link between Bacteroides and depression - and explore a potential new therapeutic 18 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT intervention - a high-GABA producing, anti-inflammatory human-derived Bacteroides salyersiae strain, HB32, was inactivated and dosed in rats subjected to the social defeat model of stress-induced depression. Orally dosed inactivated HB32 (iHB32) reduced depressive-like behavior comparable to the drug ketamine. Mechanistically, the social defeat model appeared to require the vagus nerve for its phenotype, indicating crosstalk between the gut and the brain. In support of this, after performing a multi-omics analysis of fecal and brain samples, it was found that dosing iHB32, but not ketamine, rescued stress-induced gene expression patterns in the prefrontal cortex, including those related to GABAergic signaling and inflammation. Without wishing to be bound by theory, it is contemplated that iHB32 drives these effects with a gut-originated signal through the vagus to the brain.

[0112] In addition, without wishing to be bound by theory, the therapeutic effects of the inactivated Bacteroides strains described herein are believed to be mediated, at least in part, by their potent ability to modulate immune and neuroimmune pathways. As demonstrated in the Examples, iHB32 directly reduces the production of pro-inflammatory cytokines, including IL-1b and TNF-α, from human immune cells (see e.g., Figs.4-6). These cytokines are master regulators of neuroinflammation and are implicated in the pathology of numerous CNS disorders beyond depression.

[0113] Furthermore, systemic administration of iHB32 in an animal model was shown to significantly reduce circulating levels of the chemokine CXCL1 (see e.g., Fig.7). CXCL1 is critically involved in the recruitment of neutrophils and other immune cells to sites of inflammation and can contribute to the breakdown of the blood-brain barrier, facilitating the entry of inflammatory mediators into the CNS. By reducing systemic levels of CXCL1 and other inflammatory cytokines, the compositions described herein can quell the peripheral inflammation that often drives or exacerbates neuroinflammation, thereby providing a therapeutic benefit across a range of neuroimmune-related conditions. For reference, in particular, there is some data on IL-1b and neuroimmune-related conditions in the literature, including for Multiple Sclerosis, Alzheimer’s, and ASD.

[0114] Chronic pain is often sustained by a feed-forward cycle of systemic inflammation and central nervous system sensitization. The administration of iHB32 is uniquely suited to disrupt this cycle. Without being bound by theory, the analgesic effects of the described compositions are believed to result from at least two synergistic mechanisms. First, by reducing the release of peripheral pro-inflammatory cytokines like IL-1b and TNF-α (e.g., as shown in Figs.4-6), the composition reduces the sensitization of peripheral nociceptors (pain- 19 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT sensing nerves), decreasing the afferent pain signals sent to the spinal cord. Second, by lowering systemic inflammatory tone, the composition reduces neuroinflammation within the CNS, thereby dampening the activity of microglia and astrocytes in the spinal dorsal horn and brain. This helps to reverse or prevent central sensitization, raising the pain threshold and restoring more normal pain processing.

[0115] Considerations relating to the preparation and uses of compositions comprising iHB32 are provided in the following. Bacteroides salyersiae HB32

[0116] Certain compositions and methods described herein use inactivated Bacteroides salyersiae strain HB32. As referred to herein, “Bacteroides salyersiae strain HB32” includes: Bacteroides salyersiae bacteria having 16S rRNA with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater sequence identity to one of SEQ ID NOs: 1 or 2; the B. salyersiae HB32 bacterial strain deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va. 20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; a bacterial strain with a genome comprising at least 98%, at least 98.5%, at least 99%, at least 99.5%, or greater sequence identity to the Bacteroides salyersiae genome sequence of HB32 with GenBank Accession No: CP166898-CP166900. A Bacteroides salyersiae strain HB32 bacterium useful in the compositions and methods described herein will, at a minimum, reduce one or more symptoms or indicia of depression in the rat social defeat model of depression described and demonstrated herein.

[0117] SEQ ID NO: 1, >16S_rRNA (1519 nucleotides (nt)) ATGAAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCTACAGGCTTAACACATG CAAGTCGAGGGGCATCAGGGTGTAGCAATACGCCGCTGGCGACCGGCGCACGGG TGAGTAACACGTATCCAACCTGCCCTTTACTCGGGGATAGCCTTTCGAAAGAAAG ATTAATACCCGATGGCATAACATAACCTCCTGGTTTTGTTATTAAAGAATTTCGG TAGAGGATGGGGATGCGTTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAA CCTTCGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACA CGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGA GCCTGAACCAGCCAAGTAGCGTGAAGGATGACCGCCCTATGGGTTGTAAACTTC TTTTATATGGGAATAAAGTTTGCCACGTGTGGCATTTTGTATGTACCATATGAAT AAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGT TATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTGGACATGTAAGTCAGTTGTG 20 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT AAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGCGTGTCTTGAGTACAG TAGAGGTGGGCGGAATTCGTGGTGTAGCGGTGAAATGCTTAGATATCACGAAGA ACTCCGATTGCGAAGGCAGCTCACTGGACTGCAACTGACACTGATGCTCGAAAG TGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACACAGTAAACGATGAA TACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCATTAAGTATTCCA CCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCA CAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGG CTTAAATTGCAAATGAATATGCCGGAAACGGCATAGCCGCAAGGCATTTGTGAA GGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATA ACGAGCGCAACCCTTATCTTCAGTTACTAACAGGTTATGCTGAGGACTCTGGAGA GACTGCCGTCGTAAGATGTGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCC CTTACGTCCGGGGCTACACACGTGTTACAATGGGGGGTACAGAAGGCCGCTACA CAGCGATGTGATGCCAATCCCTAAAGCCCCTCTCAGTTCGGATCGAAGTCTGCAA CCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTG AATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGCCGGGGGT ACCTGAAGTACGTAACCGCAAGGAGCGTCCTAGGGTAAAACTGGTGACTGGGGC TAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGAACACCTCCTT

[0118] SEQ ID NO: 2, >16S_rRNA (1519 nt) ATGAAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCTACAGGCTTAACACATG CAAGTCGAGGGGCATCAGGGTGTAGCAATACACCGCTGGCGACCGGCGCACGGG TGAGTAACACGTATCCAACCTGCCCTTTACTCGGGGATAGCCTTTCGAAAGAAAG ATTAATACCCGATGGCATAACATAACCTCCTGGTTTTGTTATTAAAGAATTTCGG TAGAGGATGGGGATGCGTTCCATTAGGCAGTTGGCGGGGTAACGGCCCACCAAA CCTTCGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGGAACTGAGACA CGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGAGA GCCTGAACCAGCCAAGTAGCGTGAAGGATGACCGCCCTATGGGTTGTAAACTTC TTTTATATGGGAATAAAGTTTGCCACGTGTGGCATTTTGTATGTACCATATGAAT AAGGATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATCCGAGCGT TATCCGGATTTATTGGGTTTAAAGGGAGCGTAGGTGGACATGTAAGTCAGTTGTG AAAGTTTGCGGCTCAACCGTAAAATTGCAGTTGATACTGCGTGTCTTGAGTACAG TAGAGGTGGGCGGAATTCGTGGTGTAGCGGTGAAATGCTTAGATATCACGAAGA ACTCCGATTGCGAAGGCAGCTCACTGGACTGCAACTGACACTGATGCTCGAAAG TGTGGGTATCAAACAGGATTAGATACCCTGGTAGTCCACACAGTAAACGATGAA 21 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT TACTCGCTGTTTGCGATATACAGTAAGCGGCCAAGCGAAAGCATTAAGTATTCCA CCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCA CAAGCGGAGGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGG CTTAAATTGCAAATGAATATGCCGGAAACGGCATAGCCGCAAGGCATTTGTGAA GGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCATA ACGAGCGCAACCCTTATCTTCAGTTACTAACAGGTTATGCTGAGGACTCTGGAGA GACTGCCGTCGTAAGATGTGAGGAAGGTGGGGATGACGTCAAATCAGCACGGCC CTTACGTCCGGGGCTACACACGTGTTACAATGGGGGGTACAGAAGGCCGCTACA CAGCGATGTGATGCCAATCCCTAAAGCCCCTCTCAGTTCGGATCGAAGTCTGCAA CCCGACTTCGTGAAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTG AATACGTTCCCGGGCCTTGTACACACCGCCCGTCAAGCCATGGGAGCCGGGGGT ACCTGAAGTACGTAACCGCAAGGAGCGTCCTAGGGTAAAACTGGTGACTGGGGC TAAGTCGTAACAAGGTAGCCGTACCGGAAGGTGCGGCTGGAACACCTCCTT

[0119] In some embodiments, a composition as described herein comprises an inactivated Bacteroides bacterium which comprises nucleic acid sequence encoding at least one, at least two, or each of the enzymes selected from glutamate decarboxylase; glutamate:GABA antiporter; and glutaminase (see e.g., Table 1). In some embodiments, the glutamate decarboxylase; glutamate:GABA antiporter; and glutaminase are encoded in a single operon adjacent to each other in the Bacteroides bacterium. In some embodiments, the Bacteroides bacterium expresses (e.g., transcribes and / or translates) at least one, at least two, or each of the enzymes selected from glutamate decarboxylase; glutamate:GABA antiporter; and glutaminase (see e.g., Table 1) prior to inactivation.

[0120] Table 1: Exemplary Combinations of enzymes encoded by the inactivated Bacteroides bacterium genome; for each row, “x” indicates inclusion in the bacterial genome glutamate decarboxylase glutamate:GABA (see e SEQ ID NOs: 3 anti orter (see e SEQ glutaminase (see e.g.,22 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0121] In some embodiments, the Bacteroides bacterium is capable of GABA production via glutamate decarboxylase using a 3-step pathway that involves: (1) glutamate:GABA antiporter transport protein importing glutamine into the cell and later exporting GABA out of the cell; (2) glutaminase converting the glutamine into glutamate; and (3) glutamate decarboxylase converting glutamate into GABA. In some embodiments, the Bacteroides bacterium is capable of GABA production via glutamate decarboxylase using external glutamate.

[0122] In regard to such embodiments, the Bacteroides bacterium can, at a minimum, reduce symptoms of depression in an animal model of depression including, but not limited to the social defeat model described and demonstrated herein.

[0123] In some embodiments, the glutamate decarboxylase is encoded by SEQ ID NO: 3 or a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 3. In some embodiments, the glutamate:GABA antiporter is encoded by SEQ ID NO: 4 or a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 4. In some embodiments, the glutaminase is encoded by SEQ ID NO: 5 or a nucleic acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 5.

[0124] In some embodiments, the glutamate decarboxylase comprises SEQ ID NO: 6, or an amino acid sequence 100% similar to SEQ ID NO: 6, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 6, which maintains its enzymatic function (i.e., the ability to catalyze the same enzymatic reaction catalyzed by the polypeptide of SEQ ID NO: 6). In some embodiments, the glutamate:GABA antiporter comprises SEQ ID NO: 7, or an amino acid sequence 100% similar to SEQ ID NO: 7, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 7, which maintains its enzymatic function (i.e., the ability to catalyze the same enzymatic reaction catalyzed by the polypeptide of SEQ ID NO: 7). In some embodiments, the glutaminase comprises SEQ ID NO: 8, or an amino acid sequence 100% similar to SEQ ID NO: 8, or an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to SEQ ID NO: 8, which maintains its enzymatic function (i.e., the ability to catalyze the same enzymatic reaction catalyzed by the polypeptide of SEQ ID NO: 8). 23 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0125] SEQ ID NO: 3, Glutamate decarboxylase-encoding nucleic acid, Bacteroides salyersiae (1443 nt) ATGGAAGATTTAAATTTTAGAAACGGTGATGCCAAGACTGATGTATTTGGTTCAA ACAGAATGTTGCAACCCTCTCCGGTAGAAAAAATACCTGACGGGCCGACCACTC CGGAAATCGCCTACCAAATGGTAAAAGATGAAACATTCGCACAGACTCAGCCCC GACTGAACCTGGCTACTTTCGTCACCACTTACATGGACGAATATGCCACCAAACT GATGAATGAAGCCATCAATATCAACTACATCGATGAAACAGAATATCCCCGTAT CGCTGTAATGAACGGTAAATGCATCAACATCATGGCCAACCTGTGGAATACTCC GGAAAAAGACGACTGGAAAACCGGTGCATTAGCTATCGGTTCTTCAGAAGCTTG TATGTTGGGTGGTGTCGCTGCATGGTTACGCTGGCGCAAAAAACGTCAGGCTCAG GGTAAACCCGTAGACAAACCGAATTTTGTTATTTCTACCGGCTTCCAGGTAGTAT GGGAAAAATTTGCCCAACTGTGGCAGATCGAAATGCGCGAAGTGCCTTTGACAC TGGAAAAGACAACCCTTGATCCGGAAGAAGCACTGAAGATGTGTGATGAAAACA CCATCTGTATTGTTCCTATCCAGGGCGTTACATGGACAGGATTGAATGACGACGT AGAAGCGCTCGACAAAGCGCTCGATGCTTACAATGCCAAAACCGGCTACGACAT TCCTATCCACGTAGATGCTGCAAGTGGCGGTTTTATCCTTCCGTTCCTATATCCGG AAATCAAATGGGACTTCCGTTTGAAATGGGTACTCTCTATCAGTACCTCCGGTCA TAAATTCGGCTTGGTTTATCCGGGTCTCGGTTGGGTTTGCTGGAAAGGCAAAGAA TATCTTCCCGAAGAAATGTCATTCAGCGTCAATTACTTAGGTGCCAACATCACAC AGGTAGGTCTGAACTTTTCTCGTCCGGCTGCCCAGATTTTGGGACAATACTATCA ATTTATCCGTTTGGGATTCCAGGGATATAAAGAAGTACAGTACAACTCATTGAAG ATCGCCAAGTATCTCCACGGTGAGATTGCCAAGATGACTCCGTTCGTCAACTACT CAGAAGACGTAGTGAACCCATTGTTTATCTGGTACATGAAACCGGAATATGCCA AAGATGCCAAATGGACTTTGTATGATTTGCAAGACAAACTGGCTCAGAGCGGAT GGATGGTTCCGGCTTATACCTTGCCGTCCAAATTAGAAGATTATGTAGTGATGCG TATCGTTGTCCGTCAAGGATTCAGCCGTGATATGGCAGATATGCTGTTAGGTGAC ATCAAGAACGCCATTACCGAATTGGAAAAACTGGAATATCCTACTACCACACGT ATCGCCCAAGATAAGAATCTGCCGGTAGCCACAAAGGTATTCAACCATACAGGT AAGCCACAATCGACTAAAAAATAA

[0126] SEQ ID NO: 4, Glutamate:GABA antiporter-encoding nucleic acid, Bacteroides salyersiae (1629 nt) ATGGCAAATATTAAACAAGTAGCAAAATTAGGGGTATTCACCCTGGCTATCATG AATGTAACGGCAGTAGTATCCCTACGCGGACTTCCTGCCGAGGCCGTGTACGGTA 24 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT TGAGTTCGGCCTTCTACTATCTTTTCGCAGCCATTGTCTTTTTGATACCGACTTCG CTCGTTGCAGCGGAACTGGCTGCCATGTTTCAAGACAAGCAAGGCGGTGTATTCC GCTGGGTAGGTGAAGCCTACGGCAAAAAGTTCGGTTTCCTGGCTATCTGGGTGCA ATGGATCGAAAGTACCATCTGGTATCCTACCGTTCTGACATTCGGTGCCGTTTCC ATCGCCTTCATCGGAATGAATGACGCTCACGACATGTCACTGGCAAGTAATAAAT ACTATACGCTGGCTGTCGTACTGATCATCTACTGGCTTGCCACTTTCATCTCTCTT AAAGGTATGGGATGGGTAGGTAAAGTTGCGAAAATCGGCGGTATGGTCGGTACC ATCATCCCGGCAGCCCTGCTGATTATTCTCGGCATCGTCTATCTGGCTACCGGTG GACACTCCAACCTGGACTTCCACAGCAGTTTCTTCCCCGACTTCACGAAATTCGA CAATGTAGTTCTTGCAGCAAGTATCTTCCTCTTTTATGCCGGTATGGAAATGGGC GGTATCCACGTAAAAGATGTGGAAAACCCTACTAAGAACTATCCGAAAGCCGTA TTTATCGGTGCTTTGATTACAGTAGTCATCTTCGTATTGGGAACTTTCGCATTAGG TATCATCATCCCCGCCAATGACATCAGTCTGACACAAAGCTTGCTGGTCGGTTTT GATAACTATTTCAAATACATCCACGCTTCCTGGTTGTCACCGATCATCGCCATCG CATTGGCATTCGGTGTATTGGCCGGTGTGTTGACATGGGTTGCCGGTCCGTCAAA AGGTATCTTTGCCGTAGGTAAGGCCGGTTATATGCCTCCGTTCTTCCAGAAGACC AACAAGGTAGGCGTACAGAAAAACATTCTTTTCGTACAAGGTATTGCCGTAACC GTATTGAGTCTTCTTTTCGTCGTCATGCCTTCCGTACAGAGCTTTTACCAGATTCT GTCACAGTTGACAGTAATCCTTTATCTGGTCATGTACCTGCTCATGTTCTCCGGCG CCATCTATCTGCGTTATAACATGAAGAAGACAGATCGTCCGTTCCGTATCGGTAA GAAAGGCAACGGCCTGATGTGGCTTATCGGCGGTCTCGGTTTCTGCGGTTCATTA CTCGCATTCATATTGAGCTTTATTCCGCCCAGCCAGATTTCCACAGGTAGCAATA CCGTATGGTTCACCGTTCTTATTGTCGGCGCAATTGTCGTAGTGGTAGCTCCGTTT ATCATCTACGCTTCCAAAAAGCCGTCATGGGTAGATCCGAACAGCAACTTCGAG CCTTTCCATTGGGAAGAACAAGCTCAAGTTCAATCGCAAGCAGCCACAGCAACG GTAACCGCCGCTACTACAACTGCTACCACGGCTAAAGCAGCTTCGGCAACCAGT GCAGCAACCGATGCACCGGCAGCATCGTCCACCTCTACAAACAACGTTGGAAAC AATGCTGCAAGCAAGGATAATACTCCCAAAACATAA

[0127] SEQ ID NO: 5, Glutaminase-encoding nucleic acid, Bacteroides salyersiae (963 nt) ATGAAAAAGATTTCTATATCTCAAATAAAAGAGGTGGCACAACAGGCTTATGAG CAGGTGAAGGGCAACACGGACGGAAAGAATGCCGACTACATCCCCTACCTTGCC AATATCGACAAGAACCTGTTCGGTATTAGTATCTGCCTGCTGAATGGCCAGAAAA TCACAATAGGTGATTATAATTACCGTTTCGGCATAGAATCGGTATCCAAAGTCCA 25 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT TACAGCCATTCTGGTTCTCCGTCAATACGGTGCTCAGAAGATATTGGATATGATC GGTGCCGATGCCACCGGATTGCCCTTCAACTCTATCATCGCTATCCTTCTGGAAA ACGATCATCCCTCTACACCGTTGGTCAATGCGGGAGCGATTTCCGCTTGCTCTAT GGTGCAACCGGTCGGTGAAGCTACCCAAAAATGGAATGCAATCGTAGAGAATAT TACGGATCTTTGCGGCGATGCTCCGCAATTGATTGACGAACTGTACAAATCCGAA TCTGCCACCAACTTCAACAACCGGTCGATAGCCTGGTTATTGAAGAACTACAATC GAATTTACGATGATCCGGATATGTCGCTCGATCTGTACACGCGTCAGTGTTCACT GGGCGTTACAGCAGAAATGTTGTCCGTAGCTGCCGGTACGATAGCAAATCACGG GGTAAATCCTGTGACAAACAAACAGGTTTTCGAAGCCGAACTGACTCCGAAGAT TACGTCGATGATTGCCTCCGTGGGCTTCTATGAGCATACGGGCGACTGGATGTAT ACCTCCGGTATTCCGGCTAAAACCGGTGTAGGCGGCGGTGTAATGGGTGTATTGC CGGGCGTAATGGGAGTTTCCGCTTTTGCACCTCCACTGGATGGTTCGGGCAACTC CGTAAAGGCTCAATTGGCAATCAAATTTATTATGAATAAGCTGGGGTTGAATATA TTCAACGGCGAGCAAGTTACTGTAGTTGAATAA

[0128] SEQ ID NO: 6, Glutamate decarboxylase polypeptide (see e.g., nucleic acid in SEQ ID NO: 3), Bacteroides salyersiae (480 amino acids (aa)) MEDLNFRNGDAKTDVFGSNRMLQPSPVEKIPDGPTTPEIAYQMVKDETFAQTQPRLN LATFVTTYMDEYATKLMNEAININYIDETEYPRIAVMNGKCINIMANLWNTPEKDD WKTGALAIGSSEACMLGGVAAWLRWRKKRQAQGKPVDKPNFVISTGFQVVWEKF AQLWQIEMREVPLTLEKTTLDPEEALKMCDENTICIVPIQGVTWTGLNDDVEALDKA LDAYNAKTGYDIPIHVDAASGGFILPFLYPEIKWDFRLKWVLSISTSGHKFGLVYPGL GWVCWKGKEYLPEEMSFSVNYLGANITQVGLNFSRPAAQILGQYYQFIRLGFQGYK EVQYNSLKIAKYLHGEIAKMTPFVNYSEDVVNPLFIWYMKPEYAKDAKWTLYDLQ DKLAQSGWMVPAYTLPSKLEDYVVMRIVVRQGFSRDMADMLLGDIKNAITELEKLE YPTTTRIAQDKNLPVATKVFNHTGKPQSTKK

[0129] SEQ ID NO: 7, Glutamate:GABA antiporter polypeptide (see e.g., nucleic acid in SEQ ID NO: 4), Bacteroides salyersiae (542 amino acids (aa)) MANIKQVAKLGVFTLAIMNVTAVVSLRGLPAEAVYGMSSAFYYLFAAIVFLIPTSLV AAELAAMFQDKQGGVFRWVGEAYGKKFGFLAIWVQWIESTIWYPTVLTFGAVSIAF IGMNDAHDMSLASNKYYTLAVVLIIYWLATFISLKGMGWVGKVAKIGGMVGTIIPA ALLIILGIVYLATGGHSNLDFHSSFFPDFTKFDNVVLAASIFLFYAGMEMGGIHVKDV ENPTKNYPKAVFIGALITVVIFVLGTFALGIIIPANDISLTQSLLVGFDNYFKYIHASWL SPIIAIALAFGVLAGVLTWVAGPSKGIFAVGKAGYMPPFFQKTNKVGVQKNILFVQGI 26 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT AVTVLSLLFVVMPSVQSFYQILSQLTVILYLVMYLLMFSGAIYLRYNMKKTDRPFRIG KKGNGLMWLIGGLGFCGSLLAFILSFIPPSQISTGSNTVWFTVLIVGAIVVVVAPFIIYA SKKPSWVDPNSNFEPFHWEEQAQVQSQAATATVTAATTTATTAKAASATSAATDAP AASSTSTNNVGNNAASKDNTPKT

[0130] SEQ ID NO: 8, Glutaminase polypeptide (see e.g., nucleic acid in SEQ ID NO: 5), Bacteroides salyersiae (320 amino acids (aa)) MKKISISQIKEVAQQAYEQVKGNTDGKNADYIPYLANIDKNLFGISICLLNGQKITIGD YNYRFGIESVSKVHTAILVLRQYGAQKILDMIGADATGLPFNSIIAILLENDHPSTPLV NAGAISACSMVQPVGEATQKWNAIVENITDLCGDAPQLIDELYKSESATNFNNRSIA WLLKNYNRIYDDPDMSLDLYTRQCSLGVTAEMLSVAAGTIANHGVNPVTNKQVFE AELTPKITSMIASVGFYEHTGDWMYTSGIPAKTGVGGGVMGVLPGVMGVSAFAPPL DGSGNSVKAQLAIKFIMNKLGLNIFNGEQVTVVE Inactivation

[0131] “Inactivated” Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 cells that have been intentionally rendered non-viable through a controlled process. Non-viable cells are not able to proliferate but still have a specific function or effect derived from their structural or molecular composition (e.g. proteins and / or metabolites). These non-viable cells can be intact or can consist of cell fragments. As described herein, inactivated Bacteroides salyersiae bacterial strain HB32 are the result of an affirmative physical, chemical, or biological treatment of the bacteria as a deliberate step in their preparation. It should be understood that while any given culture or preparation of B. salyersiae HB32 bacteria can contain a proportion of cells which are non- viable, such culture or preparation is not “inactivated” unless it has been subjected to an affirmative physical, chemical, or biological treatment designed to render cells non-viable. Specific function or effect derived from structural or molecular composition includes but is not limited to a statistically significant effect on depression upon administration in an animal model (including but not limited to the social defeat model described and demonstrated herein).

[0132] In some embodiments, a composition comprising an inactivated Bacteroides salyersiae bacterial strain HB32 as described herein comprises no viable HB32 cells. In other embodiments, a composition comprising an inactivated Bacteroides salyersiae bacterial strain HB32 as described herein can include a minor proportion of viable HB32 cells. In this context, that viable proportion is less than 5%, less than 4%, less than 3%, less than 2%, less 27 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT than 1%, less than 0.5% or fewer. Such proportion can be included, e.g., through mixing of a preparation in which all HB32 cells are inactivated by chemical, physical, or biological treatment with a preparation comprising viable, untreated HB32 cells. Alternatively, such a proportion can be included by physical or chemical treatment that renders 95%, 96%, 97%, 98%, 99%, 99.5% or more of the HB32 cells non-viable.

[0133] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 wherein Bacteroides salyersiae bacterial strain HB32 microorganisms are in the form of a parabiotic. Parabiotics are inanimate or non- viable microbial cells, or their cellular components, that have been inactivated through physical, chemical, or biological methods while retaining substantial structural integrity. These inactivated microbial particles or cells are devoid of reproductive capability but preserve structural properties of the original cells and confer a health benefit. Non-limiting examples of parabiotics include pasteurized cells, tyndallized cells, or lyophilized cell fragments.

[0134] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 wherein Bacteroides salyersiae bacterial strain HB32 microorganisms are in the form of a postbiotic. Postbiotics are preparations of inanimate or non-viable microbial cells or their cellular components, optionally with their metabolites, that confer a health benefit. Cells can be rendered non-viable through physical, chemical, or biological methods. These inactivated microbial cells or cellular structural components are devoid of reproductive capability but preserve structural properties of the original cells. Non-limiting examples of non-viable cells or cellular components in postbiotics include pasteurized cells, tyndallized cells, or lyophilized cell fragments. Non- limiting examples of metabolites or end products that can optionally be combined with the non-viable microbial cells or cellular components in a postbiotic include organic acids, bacteriocins, enzymes, peptides, or neurotransmitters like gamma-aminobutyric acid.

[0135] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 wherein the microorganisms are dead. In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 wherein the microorganisms are tyndallized. In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 wherein the microorganisms are pasteurized. 28 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0136] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a heating process to render the cells non-viable. In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a heating process to a temperature of 50°C to 100°C for at least 10 minutes at least one time. In some embodiments, Bacteroides salyersiae bacterial strain HB32 submitted to a heating process is also exposed to atmospheric oxygen levels.

[0137] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a pasteurization process by heating to a temperature of 50°C to 100°C for at least 10 minutes at least one time. In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a pasteurization process by heating to a temperature of 50°C to 100°C for at least 10 minutes at least two times with a period of chilling to -80°C to 15°C for at least 10 minutes between heating cycles. In some embodiments, pasteurized Bacteroides salyersiae bacterial strain HB32 is also exposed to atmospheric oxygen levels.

[0138] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a tyndallization treatment. Tyndallization is a method of intermittent sterilization applied to bacterial cultures, wherein the treatment involves sequential cycles of heating and cooling. The process includes heating the bacterial cells to between 50°C and 100°C for a period sufficient to kill cells, followed by a cooling period to between 15°C to 30°C. This cycle is repeated multiple times, typically over three consecutive days, to progressively eliminate all viable bacteria. Cells that have undergone tyndallization can be intact or can consist of cell fragments, and they are no longer capable of metabolic activity or replication but can still perform a function derived from their structural or molecular composition.

[0139] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to a gamma irradiation treatment. Gamma irradiation is the exposure of bacterial cultures to gamma radiation. This process involves irradiating bacteria with high-energy gamma rays at doses sufficient to ensure complete inactivation of the bacteria, rendering them non-viable while maintaining structural components that elicit activity in the host. Cells that have undergone gamma irradiation can be intact or can consist of cell fragments, and they are no longer capable of replication but 29 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0140] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 submitted to ultrasonic disruption treatment. Ultrasonic disruption is the application of high-intensity ultrasonic waves to bacterial cultures. The ultrasonic energy causes cavitation and mechanical shearing, leading to the disruption of bacterial cell walls and membranes while maintaining overall cellular debris. Cells that have undergone ultrasonic disruption can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0141] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 rendered inactivated by lyophilization. Lyophilization is the freeze-drying of bacterial cultures. This process includes freezing the bacterial culture and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. Lyophilization preserves the structural integrity and biochemical components of the bacteria, rendering them non- viable. Cells that have undergone lyophilization can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0142] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 rendered inactivated by pulsed electric field treatment. Pulsed electric field treatment is the exposure of bacterial cultures to short bursts of high voltage electric fields. This process involves subjecting bacteria to pulsed electric fields at intensities sufficient to ensure complete inactivation of the bacteria, rendering them non-viable while maintaining structural components that elicit activity in the host. Cells that have undergone pulsed electric field treatment can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0143] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 rendered inactivated by pascalization. Pascalization is the exposure of bacterial cultures to high hydrostatic pressure. This process involves subjecting bacteria to pressures ranging from 100 to 800 megapascals, which is sufficient to ensure complete inactivation of the bacteria, rendering them non-viable while 30 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT maintaining structural components that elicit activity in the host. Cells that have undergone pascalization can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0144] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 rendered inactivated by freeze-thaw cycles. Freeze-Thaw Cycles involve the exposure of bacterial cultures to alternating low and ambient temperatures. This process subjects bacteria to repeated cycles of freezing, below -20°C, followed by thawing at 15°C or higher. This thermal stress is sufficient to ensure complete inactivation of the bacteria, rendering them non-viable while maintaining structural components that elicit activity in the host. Cells that have undergone freeze-thaw cycles can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins).

[0145] In some embodiments, inactivated Bacteroides salyersiae bacterial strain HB32 refers to Bacteroides salyersiae bacterial strain HB32 rendered inactivated by biological methods, either through natural or engineered processes. Non-limiting examples of biological methods rendering inactivation include phage infection, treatment with bacteriocins, treatment with antimicrobial peptides, or the induction of bacterial death using CRISPR-Cas systems. Cells that have undergone biological treatment can be intact or can consist of cell fragments, and they are no longer capable of replication but can still perform a function derived from their structural or molecular composition (e.g. metabolites and proteins). Formulations

[0146] Compositions comprising inactivated B. salyersiae HB32 bacteria for administration to a subject are described herein. In some embodiments, the inactivated B. salyersiae HB32 bacteria are prepared in combination with a carrier, including but not limited to a food, a food ingredient, a medical food, or a pharmaceutically acceptable carrier.

[0147] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising inactivated HB32 bacteria as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise a preparation of inactivated HB32 bacteria as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of a preparation of inactivated HB32 bacteria as described 31 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of a preparation of inactivated HB32 bacteria as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non- limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum component, such as serum albumin, HDL and LDL; (24) C2-C12 alcohols; and (25) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a preparation of inactivated HB32 bacteria as described herein.

[0148] Pharmaceutical compositions comprising a preparation of inactivated HB32 bacteria can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of a preparation of inactivated HB32 bacteria as described herein, and can be prepared by methods of pharmacy well known 32 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005). Controlled Release

[0149] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, a pharmaceutical composition comprising a preparation of inactivated HB32 bacteria can be administered in a sustained release formulation.

[0150] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

[0151] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace 33 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT the amount of drug being metabolized and / or excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

[0152] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS®(Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

[0153] As a non-limiting example, enteric-coated capsules can be used as a controlled- or extended-release dosage form for the delivery of inactivated bacterial cells. The enteric- coated capsule dosage form utilizes a coating that resists the harsh acidic environment of the stomach, decreasing or preventing premature dissolution and degradation of the contents. The enteric coating increases the probability that the contents remain intact until they reach the intestines. By delivering the contents directly to the intestines, enteric-coated capsules promote optimal interaction of the contents (e.g., inactivated bacterial cells) with the gut microbiota, gut cells, and the gastrointestinal-associated immune system. The enteric-coated capsule dosage form increases bioavailability of microbial metabolites or end products from the encapsulated inactivated bacterial cells, facilitating their absorption into the bloodstream. Dosing

[0154] In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a composition comprising a preparation of inactivated HB32 bacteria, in an amount effective to improve mood, improve sleep quality, improve focus, improve memory, improve quality of life score, reduce stress, or reduce fatigue in a mammalian subject in need thereof. For such embodiments, the amount effective can be in, for example, a unit amount or dose, e.g., such that a single, e.g., daily, twice daily or three times daily dose delivers a sufficient amount of the preparation to improve mood, improve sleep quality, improve focus, improve memory, improve quality of 34 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT life score, reduce stress, or reduce fatigue in the mammalian subject. Improvement in this context can be by a statistically significant amount relative to the given parameter (mood, sleep quality, focus, memory, quality of life score, stress, fatigue etc.) prior to administration and as judged, for example, by an accepted scale or standard for the parameter measured. The amount effective to achieve beneficial results for a given desired outcome or parameter will vary depending upon the desired outcome or parameter and, for example, the age, weight and sex of the subject.

[0155] In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a composition comprising a preparation of inactivated HB32 bacteria to a subject in order to alleviate a symptom of a CNS disease or disorder, an inflammatory disease or disorder or an immunological disease or disorder. As used herein, "alleviating a symptom of a disease or disorder" is ameliorating any condition or symptom associated with the disease or disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art.

[0156] The term “effective amount" as used herein refers to the amount of a composition comprising a preparation of inactivated HB32 bacteria needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of the composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a composition comprising a preparation of inactivated HB32 bacteria that is sufficient to provide a particular effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.

[0157] Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. a CNS condition, inflammatory or immunological condition, as 35 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT the case may be, by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a desired level of efficacy. The effects of any particular dosage can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

[0158] The dosage ranges for the administration of a composition comprising a preparation of inactivated HB32 bacteria according to the methods described herein depend upon, for example, the form of the inactivated HB32 preparation, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for symptoms of a CNS condition, an inflammatory or immunological condition. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0159] In some embodiments, the composition comprises at least 1×10^9 inactivated HB32 bacterial cells per dose. In some embodiments, the composition comprises at most 1×10^9 inactivated HB32 bacterial cells per dose. In some embodiments, the composition comprises about 1×10^9 inactivated HB32 bacterial cells per dose. In some embodiments, the composition comprises at least 1×10^5, at least 1×10^6, at least 1×10^7, at least 1×10^8, at least 1×10^9, at least 1×10^10, at least 1×10^11, at least 1×10^12, or at least 1×10^13 inactivated HB32 bacterial cells per dose. In some embodiments, the composition comprises at most 1×10^5, at most 1×10^6, at most 1×10^7, at most 1×10^8, at most 1×10^9, at most 1×10^10, at most 1×10^11, at most 1×10^12, or at most 1×10^13 inactivated HB32 bacterial cells per dose. In some embodiments, the composition comprises about 1×10^5, about 1×10^6, about 1×10^7, about 1×10^8, about 1×10^9, about 1×10^10, about 1×10^11, about 1×10^12, or about 1×10^13 inactivated HB32 bacterial cells per dose. It should be understood that where an inactivation method generates fragments of the bacteria, the dosage 36 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT amount of fragmented bacteria, e.g., “at least 1 x 1010bacterial cells per dose” refers to the amount of bacterial cells prior to the inactivation that generated the cellular fragments.

[0160] The efficacy of a composition comprising a preparation of inactivated HB32 bacteria in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other appropriate measurable parameter. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g., depression); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker of the modeled disease or disorder is observed.

[0161] With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. 37 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0162] In certain embodiments, an effective dose of a composition comprising a preparation of inactivated HB32 bacteria can be administered to a patient repeatedly. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the composition. The desired dose or amount can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be one or more doses and / or treatments daily over a period of weeks or months. Examples of dosing and / or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a preparation of inactivated HB32 bacteria can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

[0163] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. As but one non-limiting example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Administration

[0164] A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. In certain embodiments, administration is oral. In certain embodiments, administration is intrarectal, e.g., via enema or suppository.

[0165] In some embodiments of any of the aspects, the composition comprising a preparation of inactivated HB32 bacteria described herein is administered as a monotherapy, e.g., another treatment for a CNS disease or disorder, inflammatory or immunological disease or disorder is not administered to the subject.

[0166] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and / or treatment to the subject, e.g. as part of a combinatorial therapy with, e.g., an antidepressant agent.

[0167] By “antidepressant” is meant a psychiatric medication used to alleviate mood disorders, such as major depression disorder and dysthymic disorder. Non-limiting examples of antidepressants include the monoamine oxidase inhibitors (e.g., isocarboxazid, moclobemide, phenelzine sulfate, selegiline, and tranylcypromine sulfate), tricyclic antidepressants (e.g., amitriptyline, amitriptyline hydrochloride, amitriptylinoxide, 38 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT butriptyline, clomipramine, demexiptiline, desipramine, dibenzepin, dimetacrine, dosulepin / dothiepin, doxepin, imipramine, imipraminoxide, lofepramine, melitracen, metapramine, nitroxazepine, nortriptyline, noxiptilinc, pipofezine, propizepine, protriptyline hyrdochloride, and quinupramine), tetracyclic antidepressants (e.g., amoxapine, loxapine, maprotiline, mazindol, mianserin, mirtazapine, and setiptiline), selective serotonin reuptake inhibitors (SSRIs), and serotonin-norepinephrine reuptake inhibitors (SNRIs; e.g., desvenlafaxine, duloxetine, milnacipram, and venlafaxine hydrochloride). Additional non- limiting examples of antidepressants include bupropion hydrochloride, imipramine hydrochloride, lamotrigine, trazodone hydrochloride, and trimipramine maleate. Treatment Methods

[0168] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a CNS disease or disorder and / or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder. Subjects having a CNS disease or disorder and / or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder can be identified by a physician using current methods of diagnosis. Symptoms and / or complications of such diseases or disorders which characterize these conditions and aid in diagnosis are well known in the art. A family history of such diseases or disorders, or exposure to risk factors for them can also aid in determining if a subject is likely to have such disease or disorder or in making a diagnosis of the disease or disorder.

[0169] The compositions described herein can be administered to a subject having or diagnosed as having a CNS disease or disorder and / or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder. In some embodiments, the methods described herein comprise administering an effective amount of a composition described herein, e.g. a composition comprising a preparation of inactivated HB32 bacteria to a subject in order to alleviate a symptom of the disease or disorder. As used herein, "alleviating a symptom of a disease or disorder" is ameliorating any condition or symptom associated with the disease or disorder. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.

[0170] In certain embodiments, a subject is evaluated for the presence or amount of Bacteroides bacteria including but not limited to Bacteroides salyersiae. In certain embodiments, a subject is found to have an absence of or reduced level of Bacteroides 39 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT bacteria or Bacteroides salyersiae relative to a reference level. The reference level can be, for example, in a healthy subject who does not have a CNS disease or disorder or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder. A subject found to have such an absence or reduced level can be administered a composition comprising a preparation of inactivated HB32 bacteria as described herein. Thus, in one aspect of any of the embodiments, described herein is a method of treating a CNS disease or disorder or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder in a subject in need thereof, the method comprising: a) measuring the level of Bacteroides bacteria or the level of B. salyersiae bacteria in a sample obtained from a subject; and b) administering a composition comprising a preparation of inactivated HB32 bacteria as described herein to the subject if the level of Bacteroides or if the level of B. salyersiae is reduced relative to a reference.

[0171] In some embodiments of any of the aspects, the method comprises administering a composition comprising a preparation of inactivated HB32 bacteria as described herein to a subject previously determined to have a level of Bacteroides bacteria or B. salyersiae that is reduced relative to a reference.

[0172] In some embodiments of any of the aspects, the step of determining if the subject has a reduced level of Bacteroides or a reduced level of B. salyersiae can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of Bacteroides bacteria or the level of B. salyersiae in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a reduced level of Bacteroides bacteria or a reduced level of B. salyersiae can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of Bacteroides bacteria or the level of B. salyersiae in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a reduced level of Bacteroides bacteria or reduced level of B. salyersiae can comprise ordering or requesting an assay on a sample obtained from the subject to determine / measure the level of Bacteroides bacteria or B. salyersiae in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a reduced level can comprise receiving the results of an assay on a sample obtained from the subject to determine / measure the level of Bacteroides bacteria or B. salyersiae in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a 40 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT reduced level can comprise receiving a report, results, or other means of identifying the subject as a subject with a reduced level of Bacteroides bacteria or B. salyersiae.

[0173] The compositions described herein can be administered to a subject in need thereof, for instance for the treatment of a CNS disease or disorder, or an inflammatory or immunological disease or disorder and / or a pain disorder or a neuroimmune disorder. In some embodiments, the method of treatment can comprise first diagnosing a subject or patient who can benefit from treatment by a composition described herein. In some embodiments, such diagnosis comprises detecting or measuring a low level of Bacteroides bacteria or a low level of B. salyersiae bacteria in a sample from the subject or patient, each of which are examples of an abnormal level of each analyte. In some embodiments, the method further comprises administering to the patient a composition as described herein.

[0174] In some embodiments, the subject has previously been determined to have an abnormal level of an analyte described herein relative to a reference. In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and / or obtained from a subject of similar age, sex and other demographic parameters as the sample / subject. In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.

[0175] The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or plasma sample or a fecal sample from a subject. In some embodiments of any of the aspects, the technology described herein encompasses several examples of a biological sample. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject.

[0176] In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise performing or having performed an assay on a sample obtained from the subject to determine / measure the level of analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise ordering or 41 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT requesting an assay on a sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving the results of an assay on a sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an abnormal level of an analyte described herein can comprise receiving a report, results, or other means of identifying the subject as a subject with a decreased level of the analyte.

[0177] In some aspects, described herein are methods for treating neuroinflammation in a subject in need thereof, the method comprising administering an effective amount of a composition as described herein to the subject. In some aspects, the compositions as described herein are for use in treating neuroinflammation.

[0178] In some aspects, described herein are methods for treating a neuroinflammatory disorder in a subject in need thereof, the method comprising administering an effective amount of a composition as described herein to the subject. In some aspects, the compositions as described herein are for use in treating a neuroinflammatory disorder. Non-Therapeutic Methods

[0179] In multiple aspects, described herein are non-therapeutic methods in subjects with mild or moderate symptoms of a condition as described herein, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the condition is associated with a sub-optimal: immune status, mental and emotional well-being, physical comfort and pain, neurocognitive and neuroimmune status, sleep quality, metabolic health, and / or gastrointestinal health. Mild or moderate symptoms can be identified using the exemplary biomarkers, tests, and / or devices described below. In some embodiments, “mild” symptoms include a score of 1-3 on a 10-point score or a score above normal in the lower-tertile. In some embodiments, “moderate” symptoms include a score of 4-6 on a 10-point score or a score above normal in the middle-tertile. In some embodiments, “severe” symptoms include a score of 7-10 on a 10-point score or a score above normal in the upper-tertile. In some embodiments, the subject does not have severe symptoms of a condition described herein. 42 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT Methods for Supporting Immune System Balance

[0180] In some aspects, described herein are methods for supporting a healthy immune response in a mammalian subject with a sub-optimal immune status, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing a sub-optimal or mildly-stressed immune status. A sub-optimal or mildly-stressed immune status is a physiological condition characterized by a deviation from optimal immune homeostasis, which can be quantified by specific biomarkers. The selection of subjects for administration of the composition can be based on a baseline measurement within the ranges specified below. In some embodiments, a sub-optimal immune status is associated with systemic low- grade inflammation, sub-optimal mucosal immunity, sub-optimal innate immune readiness, and / or mild cytokine imbalance.

[0181] For subjects with systemic low-grade inflammation, High-Sensitivity C-Reactive Protein (hs-CRP) can be used as a biomarker for a sub-optimal immune status. For example, a serum level between 1.0 mg / L and 5.0 mg / L, can be indicative of a mild to moderate systemic inflammatory state.

[0182] For subjects with sub-optimal mucosal immunity, Secretory Immunoglobulin A (sIgA) can be used as a biomarker for a sub-optimal immune status. For example, a baseline level in saliva or stool that falls within the lower quartile (bottom 25%) of the established normal reference range for a healthy population can indicate sub-optimal mucosal immunity, including a need for mucosal defense support, such as a composition as described herein.

[0183] For subjects with sub-optimal innate immune readiness, Natural Killer (NK) Cell Activity can be used as a biomarker for a sub-optimal immune status. For example, a baseline cytotoxic activity level that falls within the lower tertile (bottom 33%) of a healthy population reference range can indicate a sub-optimal state of immune surveillance.

[0184] For subjects with mild cytokine imbalance, Pro-inflammatory Cytokines (e.g., TNF-α, IL-6) can be used as a biomarker for a sub-optimal immune status. For example, a baseline serum level that is 25% to 200% above the mean of a healthy, non-inflamed population cohort, but below the levels characteristic of an acute or chronic inflammatory disease, can indicate a mild cytokine imbalance.

[0185] In some aspects, described herein are methods for supporting or maintaining a healthy immune status in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject. 43 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0186] In some aspects, the compositions as described herein are for use in supporting a healthy immune response in a mammalian subject with a sub-optimal immune status. In some aspects, the compositions as described herein are for use in supporting or maintaining a healthy immune status in a healthy mammalian subject. Methods for Supporting Mental and Emotional Well-Being

[0187] In some aspects, described herein are methods for supporting mental and emotional well-being in a mammalian subject experiencing a mild to moderate mental health challenge, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing mild to moderate challenges to their mental and emotional well-being. The selection of subjects for administration of the composition can be based on a baseline score within the ranges specified below. In some embodiments, mild to moderate challenges to their mental and emotional well-being include depressive mood, anxiety, stress, or other related conditions.

[0188] In some embodiments, the following tests can be used to identify a subject with a mild or moderate Depressive Mood: Montgomery-Åsberg Depression Rating Scale (MADRS), Hamilton Depression Rating Scale (HAM-D), Beck Depression Inventory (BDI), Patient Health Questionnaire (PHQ-9) (also known as PROMIS Depression 8A), and / or World Health Organization-Five Well-Being Index (WHO-5) . With MADRS, a total score between 7 and 34 can indicate a mild or moderate depressive mood in a subject. With HAM-D, a total score on the HDRS-17 of between 8 and 23 can indicate a mild or moderate depressive mood in a subject. With BDI, a total score between 14 and 28 can indicate a mild or moderate depressive mood in a subject. With PHQ-9, a total score between 5 and 14 can indicate a mild or moderate depressive mood in a subject. With WHO-5, a raw score of 13 or below can indicate a mild or moderate depressive mood in a subject.

[0189] In some embodiments, the following tests can be used to identify Anxiety and Stress in a subject: Hamilton Anxiety Rating Scale (HAM-A), Beck Anxiety Inventory (BAI), Perceived Stress Scale (PSS), Generalized Anxiety Disorder-7 (GAD-7) (also known as PROMIS Anxiety 8A), and / or Liebowitz Social Anxiety Scale (LSAS). With HAM-A, a total score between 8 and 24 can indicate a mild or moderate stress and / or anxiety in a subject. With BAI, a total score between 8 and 25 can indicate a mild or moderate stress and / or anxiety in a subject. With PSS, a total score between 14 and 26 can indicate a mild or moderate stress and / or anxiety in a subject. With GAD-7, a total score between 5 and 14 can 44 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT indicate a mild or moderate stress and / or anxiety in a subject. With LSAS, a total score between 30 and 89 can indicate a mild or moderate stress and / or anxiety in a subject.

[0190] In some embodiments, the following tests can be used to identify a subject with a mild or moderate symptoms of other related mental or emotional health conditions: Snaith- Hamilton Pleasure Scale (SHAPS), Yale-Brown Obsessive Compulsive Scale (Y-BOCS), Young Mania Rating Scale (YMRS), Positive and Negative Syndrome Scale (PANSS), Yale Food Addiction Scale (YFAS), Eating Disorder Examination Questionnaire (EDE-Q), Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q), and / or SF-36 Health Survey. With SHAPS, a score of 2 or higher can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With Y-BOCS, a total score between 8 and 23 can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With YMRS, a score between 8 and 19 can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With PANSS, a baseline total score between 58 and 95 can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With YFAS, a symptom count of between 2 and 4 can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With EDE-Q, a global score 1 to 2 standard deviations above a healthy community norm can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With Q-LES-Q, a score in the lower quartile of a healthy population norm can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject. With SF-36 Health Survey, a score on the mental health subscale that is 1 to 2 standard deviations below a healthy population norm can indicate mild or moderate symptoms of a mental and / or emotional health condition in a subject.

[0191] In some aspects, described herein are methods for supporting or maintaining mental and emotional well-being in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0192] In some aspects, the compositions as described herein are for use in supporting mental and emotional well-being in a mammalian subject experiencing a mild to moderate challenge. In some aspects, the compositions as described herein are for use in supporting or maintaining mental and emotional well-being in a healthy mammalian subject. Methods for Promoting Physical Comfort and Alleviating Pain

[0193] In some aspects, described herein are methods for alleviating mild to moderate physical discomfort in a mammalian subject, the method comprising administering an 45 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing mild to moderate levels of occasional or chronic pain. The selection of subjects can be based on a baseline score within the ranges specified below. In some embodiments, the following tests can be used to identify a subject with mild to moderate levels of occasional or chronic pain: Visual Analog Scale (VAS) / Numeric Rating Scale (NRS), McGill Pain Questionnaire (MPQ), Brief Pain Inventory (BPI), Neuropathic Pain Symptom Inventory (NPSI), Fibromyalgia Impact Questionnaire (FIQ), Patient-Reported Outcomes Measurement Information System (PROMIS) Pain Scales, Pain Catastrophizing Scale (PCS), and / or Central Sensitization Inventory (CSI). With VAS / NRS, a score between 3 and 6 on a 10-point scale can indicate mild to moderate levels of occasional or chronic pain in a subject. With MPQ, a Pain Rating Index score between 10 and 30 can indicate mild to moderate levels of occasional or chronic pain in a subject. With BPI, a score for "pain interference" between 3 and 6 on a 10-point scale can indicate mild to moderate levels of occasional or chronic pain in a subject. With NPSI, a total score between 15 and 40 can indicate mild to moderate levels of occasional or chronic pain in a subject. With FIQ), a total score between 25 and 59 can indicate mild to moderate levels of occasional or chronic pain in a subject. With PROMIS Pain Scales, a T-score for Pain Interference or Pain Behavior between 55 and 65 can indicate mild to moderate levels of occasional or chronic pain in a subject. With PCS, a total score between 20 and 39 can indicate mild to moderate levels of occasional or chronic pain in a subject. With CSI, a total score between 30 and 49 can indicate mild to moderate levels of occasional or chronic pain in a subject.

[0194] In some aspects, described herein are methods for alleviating pain in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0195] In some aspects, the compositions as described herein are for use in alleviating mild to moderate physical discomfort in a mammalian subject. In some aspects, the compositions as described herein are for use in alleviating pain in a healthy mammalian subject. Methods for Supporting Neurocognitive and Neuroimmune Function

[0196] In some aspects, described herein are methods for supporting neurocognitive function in a mammalian subject experiencing a mild to moderate decline, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing mild to moderate declines in neurocognitive function or symptoms associated with neuroimmune conditions. 46 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT The selection of subjects can be based on a baseline score within the ranges specified below. In some embodiments, mild to moderate decline in a neurocognitive function include Parkinson's-related symptoms, cognitive impairment, multiple sclerosis (MS)-related symptoms, and / or fatigue and sleep symptoms.

[0197] In some embodiments, the following tests can be used to identify a subject with mild to moderate Parkinson's-Related Symptoms: Unified Parkinson's Disease Rating Scale (UPDRS) and / or Non-Motor Symptoms Scale (NMSS). With UPDRS, a total motor score (Part III) between 11 and 32, or Hoehn and Yahr stages 1.5 to 2.5, can indicate mild to moderate Parkinson's-related symptoms in a subject. With NMSS, a total score between 20 and 60 can indicate mild to moderate Parkinson's-related symptoms in a subject.

[0198] In some embodiments, the following tests can be used to identify a subject with mild to moderate Cognitive Impairment: Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), Mini-Mental State Examination (MMSE), Trail Making Test (TMT) Parts A and B, Stroop Test, and / or Rey Auditory Verbal Learning Test (RAVLT). With ADAS-Cog, a total score between 10 and 25 can indicate mild to moderate cognitive impairment in a subject. With MMSE, a score between 19 and 26 can indicate mild to moderate cognitive impairment in a subject. With TMT Parts A and B, a completion time between 1 and 2 standard deviations slower than age-matched norms can indicate mild to moderate cognitive impairment in a subject. With the Stroop Test, an interference score 1 to 2 standard deviations below normative data can indicate mild to moderate cognitive impairment in a subject. With RAVLT, a total recall score 1 to 2 standard deviations below normative data can indicate mild to moderate cognitive impairment in a subject.

[0199] In some embodiments, the following tests can be used to identify a subject with mild to moderate MS-Related Symptoms: Expanded Disability Status Scale (EDSS), Timed 25- Foot Walk, and / or Multiple Sclerosis Impact Scale (MSIS-29). With EDSS, a score between 1.0 and 4.5 can indicate mild to moderate MS-related symptoms in a subject. With a Timed 25-Foot Walk, a time 20% to 50% slower than a healthy control can indicate mild to moderate MS-related symptoms in a subject. With MSIS-29, a score between 25 and 65 can indicate mild to moderate MS-related symptoms in a subject.

[0200] In some embodiments, the following tests can be used to identify a subject with mild to moderate Fatigue and Sleep symptoms: Fatigue Severity Scale (FSS) and / or Pittsburgh Sleep Quality Index (PSQI). With FSS, a mean score between 4.0 and 5.9 can indicate mild 47 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT to moderate fatigue and / or sleep symptoms in a subject. With PSQI, a global score between 6 and 14 can indicate mild to moderate fatigue and / or sleep symptoms in a subject.

[0201] In some aspects, described herein are methods for supporting or maintaining neurocognitive function in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0202] In some aspects, the compositions as described herein are for use in supporting neurocognitive function in a mammalian subject experiencing a mild to moderate decline. In some aspects, the compositions as described herein are for use in supporting or maintaining neurocognitive function in a healthy mammalian subject. Methods for Supporting Sleep Quality and Metabolic Health

[0203] In some aspects, described herein are methods for improving sleep quality in a mammalian subject experiencing a mild to moderate sleep challenge, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing mild to moderate challenges with sleep and / or metabolic balance. The selection of subjects can be based on a baseline measurement within the ranges specified below.

[0204] In some embodiments, the following tests or devices can be used to identify a subject with mild to moderate sleep quality symptoms: PROMIS Sleep-Related Impairment (also referred to as Sleep Disturbance (4A or 8A)) and / or wearable device data. With PROMIS Sleep-Related Impairment / Sleep Disturbance (4A or 8A), a T-score between 55 and 65 can indicate mild to moderate sleep quality symptoms in a subject. With wearable device data (e.g., via APPLE HEALTHKIT, GOOGLE health kit, APPLE watch, FITBIT, OURA, WHOOP, etc.): one or more metrics that can indicate mild to moderate sleep quality symptoms in a subject include: a sleep efficiency between 75% and 84%, a sleep latency of 21 to 45 minutes, or a heart rate variability (HRV) in the lower quartile of the subject's personal healthy range.

[0205] In some aspects, described herein are methods for promoting sleep quality in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0206] In some aspects, the compositions as described herein are for use in improving sleep quality in a mammalian subject experiencing a mild to moderate sleep challenge. In some aspects, the compositions as described herein are for use in promoting sleep quality in a healthy mammalian subject. 48 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0207] In some embodiments, the following tests or devices can be used to identify a subject with mild to moderate metabolic health symptoms: Diet and Satiety Patient-Reported Outcomes (PROs) and / or weight and body composition measurements. With Diet and Satiety PROs, a score on a hunger / satiety VAS indicating moderate to high pre-meal hunger can indicate mild to moderate metabolic health symptoms in a subject. With Weight and Body Composition, a BMI in the overweight range (25.0-29.9) can indicate mild to moderate metabolic health symptoms in a subject.

[0208] In some aspects, described herein are methods for promoting metabolic health in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0209] In some aspects, the compositions as described herein are for use in improving metabolic in a mammalian subject experiencing a mild to moderate metabolic health challenge. In some aspects, the compositions as described herein are for use in promoting metabolic health in a healthy mammalian subject. Methods for Supporting Gastrointestinal Health

[0210] In some aspects, described herein are methods for supporting gastrointestinal health in a mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject. In some embodiments, the methods are directed to subjects experiencing mild to moderate digestive discomfort or sub-optimal gut function. This state is distinct from a clinically diagnosed gastrointestinal disease (e.g., inflammatory bowel disease) and can be quantified using validated patient-reported outcome questionnaires. The selection of subjects may be based on a baseline score within the ranges specified below.

[0211] In some embodiments, the following tests can be used to identify a subject with mild to moderate digestive discomfort or sub-optimal gut function: Gastrointestinal Symptom Rating Scale (GSRS), Irritable Bowel Syndrome Severity Scoring System (IBS-SSS), Gastrointestinal Quality of Life Index (GIQLI), and / or Bristol Stool Form Scale (BSFS). For overall GI symptom severity, GSRS and / or IBS-SSS can be used. For quality of life related to GI health, GIQLI can be used. For bowel regularity, BSFS can be used.

[0212] With GSRS, a total score or a score on a specific subscale (e.g., abdominal pain, bloating, indigestion) that indicates "moderately" or "rather much" discomfort, typically a mean score of 3 to 5 on the 7-point Likert scale can indicate mild to moderate digestive discomfort or sub-optimal gut function in a mammalian subject. IBS-SSS, while named for 49 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT IBS, is a robust measure of common gut symptoms (e.g., pain, distension, bowel dissatisfaction). A mammalian subject with mild to moderate digestive discomfort or sub- optimal gut function can be defined by a total score between 75 and 300 using IBS-SSS. With GIQLI, a total score (0-144, higher is better) in the mild to moderate impairment range of 90 to 120, can indicate mild to moderate digestive discomfort or sub-optimal gut function in a mammalian subject, e.g., GI symptoms are negatively impacting daily life. With BSFS, a mammalian subject experiencing sub-optimal regularity can be defined by consistently reporting stool forms indicative of constipation (e.g., Types 1 or 2) or diarrhea (e.g., Types 6 or 7) for at least 25% of bowel movements.

[0213] In some aspects, described herein are methods for supporting or maintaining a healthy gastrointestinal (GI) system in a healthy mammalian subject, the method comprising administering an effective amount of a composition as described herein to the subject.

[0214] In some aspects, the compositions as described herein are for use in supporting a healthy gastrointestinal (GI) system in a mammalian subject experiencing mild to moderate digestive discomfort or sub-optimal gut function. In some aspects, the compositions as described herein are for use in supporting or maintaining a healthy gastrointestinal (GI) system in a healthy mammalian subject. Definitions

[0215] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0216] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 50 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal, e.g., for an individual without a given disorder.

[0217] The term "inactivated," as used herein with reference to a bacterial strain or composition, refers to a population of bacterial cells that has been deliberately subjected to a controlled physical, chemical, or biological process for the express purpose of rendering the substantial majority of the cells non-viable and incapable of replication. This term explicitly distinguishes such treated bacteria from bacteria that may be incidentally or spontaneously non-viable within a culture due to natural processes such as senescence, nutrient depletion, or autolysis. The inactivation process is an affirmative manufacturing step designed to produce a stable composition while preserving structural or molecular components of the bacteria that are responsible for conferring a health benefit to a subject. As used herein in this context, a “substantial majority” means that at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, at least 99%,at least 99.5%, at least 99.7%, at least 99.9%, at least 99.95%, at least 99.96%, at least 99.97%, at least 99.98%, at least 99.99% or more of a given population of cells deliberately subjected to such process are rendered non-viable and incapable of replication.

[0218] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0219] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include 51 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

[0220] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of CNS diseases or disorders, inflammatory or immunological diseases or disorders and / or a pain disorder or a neuroimmune disorder. A subject can be male or female.

[0221] A subject can be one who has one or more symptoms associated with a disease or disorder or condition (e.g. a CNS disease or disorder, an inflammatory or immunological disease or disorder, and / or a pain disorder or a neuroimmune disorder, or a condition one wishes to change or improve). Non-limiting examples of such symptoms include: below normal mood, below normal sleep quality, below normal focus, below normal memory, below normal quality of life score, above normal stress, and / or above normal fatigue. A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a CNS disease or disorder, an inflammatory or immunological disease or disorder, and / or a pain disorder or a neuroimmune disorder) or one or more complications related to such a condition, and optionally, have already undergone treatment for such disease or disorder or the one or more complications related to it. Alternatively, a subject can also be one who has not been previously diagnosed as having a CNS disease or disorder, an inflammatory or immunological disease or disorder, and / or a pain disorder or a neuroimmune disorder or one or more complications related to such disease or disorder. For example, a subject can be one who exhibits one or more risk factors for the disease or disorder or one or more complications related to it or a subject who does not exhibit risk factors.

[0222] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0223] A variant amino acid or DNA sequence can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The 52 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings). In some embodiments, percent sequence identity for an amino acid or nucleic sequence is calculated by dividing the number of identical amino acids or nucleotides, respectively, by the total length of the alignment (including gaps).

[0224] In some embodiments, the variant amino acid or DNA sequence maintains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the function of the native or reference sequence. As used herein, the phrase “maintains the same function”, when used in reference to an enzyme, refers to a polypeptide that catalyzes the same reaction as a reference enzyme with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the function of the native or reference enzyme. As used herein, the phrase “maintains the same function”, when used in reference to a nucleic acid encoding a polypeptide, refers to the translated polypeptide maintaining at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the function of the native or reference polypeptide.

[0225] A variant amino acid sequence can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, similar to a native or reference sequence. As used herein, “similarity” refers to an identical amino acid or a conservatively substituted amino acid, as described herein. Accordingly, the percentage of “sequence similarity” is the percentage of amino acids which is either identical or conservatively changed; e.g., “sequence similarity” = (% sequence identity)+(% conservative changes). It should be understood that a sequence that has a specified percent similarity to a reference sequence necessarily encompasses a sequence with the same specified percent identity to that reference sequence. The skilled person will be aware of various computer programs, using different mathematical algorithms, that are available to determine the identity or similarity between two sequences. For instance, use can be made of a computer program employing the Needleman and Wunsch algorithm (Needleman et al. (1970)); the GAP program in the Accelrys GCG software package (Accelerys Inc., San Diego U.S.A.); the algorithm of E. 53 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT Meyers and W. Miller (Meyers et al. (1989)) which has been incorporated into the ALIGN program (version 2.0); or more preferably the BLAST (Basic Local Alignment Tool using default parameters); see e.g., US Patent 10,023,890, the content of which is incorporated by reference herein in its entirety.

[0226] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.

[0227] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested confirm that a desired activity and specificity of a native or reference polypeptide is retained.

[0228] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp.73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gln or 54 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or into Val; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Val, into Ile or into Leu.

[0229] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a polypeptide which retains at least 50% of the wild-type or reference polypeptide’s activity. A functional fragment can comprise conservative substitutions of the sequences disclosed herein. In some embodiments, a “functional fragment” of a nucleic acid described herein is a fragment or segment of a nucleic acid which retains at least 50% of the wild-type or reference nucleic acid’s activity or at least 50% of the activity of a polypeptide encoded by the nucleic acid.

[0230] In some embodiments, the polypeptide described herein can be a variant of a polypeptide sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide-encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a protein or fragment thereof that retains activity of the native or reference polypeptide. A wide variety of, for example, PCR-based, site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan to generate and test artificial variants.

[0231] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having 55 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT particular codons altered according to the substitution, deletion, or insertion required. A wide variety of, site-specific mutagenesis approaches, e.g., Kunkel’s method, cassette mutagenesis, PCR site-directed mutagenesis (e.g., traditional PCR, primer extension, or inverse PCR), whole plasmid mutagenesis, in vivo site-directed mutagenesis, CRISPR / Cas-guided mutagenesis, are known in the art and can be applied by the ordinarily skilled artisan to introduce mutations into specific nucleic acid loci. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); Braman, Jeff, ed. (2002) In Vitro Mutagenesis Protocols, Methods in Molecular Biology, Vol.182 (2nd ed.); Khudyakov and Fields (2002), Artificial DNA: Methods and Applications, CRC Press; Hsu et al. (2014), Cell 157 (6): 1262–78; Cerchione et al. (2020) PLOS ONE 15 (4): e0231716; and U.S. Pat. Nos.4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.

[0232] In some embodiments, nucleic acid sequencing comprises 16S rRNA gene sequencing, which can also be referred to as “16S ribosomal RNA sequencing”, “16S rDNA sequencing” or “16s rRNA sequencing”. Sequencing of the 16S rRNA gene can be used for genetic studies as it is highly conserved between different species of bacteria, but it is not present in eukaryotic species. In addition to highly conserved regions, the 16S rRNA gene also comprises nine hypervariable regions (V1-V9) that vary by species.16S rRNA gene sequencing typically comprises using a plurality of universal primers that bind to conserved regions of the 16S rRNA gene, PCR amplifying the bacterial 16S rRNA gene regions (including hypervariable regions), and sequencing the amplified 16S rRNA genes with a next-generation sequencing technology as known in the art (see also e.g., US Patents 5,654,418; 6,344,316; and 8,889,358; and US Patent Application Numbers US 2013 / 0157265 and US 2018 / 0195111, which are incorporated by reference in their entireties).

[0233] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. a CNS disease or disorder or an inflammatory or immunological disease or disorder 56 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT and / or a pain disorder or a neuroimmune disorder. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0234] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in or within nature.

[0235] As used herein, the term "administering," refers to the placement of a composition or compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the composition, compound, or metabolite thereof at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and / or manipulation of a delivery device or machine. 57 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT Such activity can be performed, e.g., by a medical professional and / or the subject being treated.

[0236] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference or a p-value of less than 0.05.

[0237] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

[0238] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0239] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0240] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0241] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[0242] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. 58 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT

[0243] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in cell biology, immunology, and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081- 569-8); Immunology by Werner Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0244] Other terms are defined herein within the description of the various aspects of the invention.

[0245] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and 59 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0246] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0247] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0248] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A composition comprising: inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to 16s rRNA of Bacteroides salyersiae HB32 as set out in one of SEQ ID NOs: 1 or 2; 60 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898- CP166900; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject. 2. The composition of paragraph 1, wherein the mammalian subject is human. 3. The composition of paragraph 1, wherein the bacterial strain comprises a targeted mutation that inactivates a target gene or its gene product, wherein the genome of the mutated strain remains at least 99.5% identical to that of B. salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024,as Patent Deposit No. PTA-127757 or at least 99.5% identical to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900, and wherein the 16S rRNA sequence remains at least 95% identical to one of SEQ ID NOs: 1 or 2. 4. The composition of paragraph 1, wherein the inactivated bacterial strain is formulated for oral delivery. 5. The composition of any one of paragraphs 1-4, wherein the composition comprises a pharmaceutically acceptable excipient. 6. The composition of any one of paragraphs 1-5, wherein the inactivated bacterial strain is deliberately inactivated by heat treatment, gamma irradiation, ultrasonic disruption, lyophilization, tyndallization, pasteurization, freeze-thaw inactivation, pulsed electric field treatment, or a combination thereof. 7. The composition of any one of paragraphs 1-6, wherein the composition further comprises at least one additional prebiotic selected from the group consisting of an amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose- 61 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans- galactooligosaccharide, pectin (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharide, polyamine (e.g., spermidine, putrescine), polyol (e.g. maltitol, xylitol). 8. The composition of any one of paragraphs 1-7, further comprising one or more species of different viable bacteria. 9. The composition of any one of paragraphs 1-8, further comprising one or more additional species of inactivated bacteria. 10. The composition of any one of paragraphs 1-9, further comprising a purified bacterial strain that is cytotoxic or cytostatic to a GABA-consuming bacteria. 11. The composition of paragraph 10, wherein the GABA-consuming bacteria is Evtepia gabavorous or Firmicutes bacterium MGS:114. 12. The composition of any one of paragraphs 1-11, wherein the inactivated bacterial strain is dried. 13. The composition of any one of paragraphs 1-12, wherein the composition comprises an enteric coating. 14. The composition of any one of paragraphs 1-13, wherein the composition is a nutritional composition, a food product, a dietary complement, a medical food, or a medicament. 15. The composition of any one of paragraphs 1-14, which is formulated in a tablet, a caplet, a pill, a capsule, a troche, a lozenge, a granule, a powder, a gummy, a gel, a sachet, a food composition, a nutraceutical, a medical food, or a combination thereof. 16. A composition comprising an inactivated bacterial strain, the genome of which comprises at least one sequence encoding at least one enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof, wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject. 17. The composition of paragraph 16, wherein the genome of the inactivated bacterial strain comprises sequence encoding two or more of glutamate decarboxylase; glutamate:GABA antiporter and glutaminase enzymes. 62 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 18. The composition of paragraph 16, wherein the genome of the inactivated bacterial strain comprises sequence encoding glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes. 19. The composition of any one of paragraphs 16-18, wherein the sequence encoding the glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes is at least 98% identical to, or encodes only conservative amino acid differences relative to the enzymes encoded by SEQ ID NOs 3-5. 20. A method of treating a CNS disorder, a pain disorder or a neuroimmune disorder in a human subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of any one of paragraphs 1-19 to the subject. 21. The method of paragraph 20, wherein the CNS disorder is selected from the group consisting of treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), bipolar disorder, schizophrenia, anxiety, anxiety disorders, obsessive compulsive disorder, eating disorders, addiction, social phobia, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD). 22. The method of paragraph 20, wherein the CNS disorder is major depressive disorder, and wherein the administration of the composition reduces a symptom of anhedonia with an efficacy comparable to a standard therapeutic dose of ketamine in a social defeat animal model. 23. The method of paragraph 20, wherein the disorder is a pain disorder, and wherein the pain disorder is chronic pain. 24. The method of paragraph 23, wherein the chronic pain is selected from the group consisting of nociplastic pain, neuropathic pain, inflammatory pain, and visceral pain, and combinations thereof. 25. The method of paragraph 24, wherein the pain is associated with a condition selected from the group consisting of fibromyalgia, irritable bowel syndrome, diabetic neuropathy, rheumatoid arthritis, complex regional pain syndrome, radiculopathy, post-herpetic neuralgia, chemotherapy-induced peripheral neuropathy. 63 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 26. The method of paragraph 20, wherein the disorder is a neuroimmune-related condition selected from the group consisting of Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis. 27. The method of paragraph 20, wherein the method results in a reduction in the serum level of one or more biomarkers selected from the group consisting of CXCL1, IL-1b, and TNFα in the subject. 28. The method of any one of paragraphs 20-27, further comprising detecting a level of one or more bacterial strains selected from: Bacteroides salyersiae bacterial strain HB32 (“HB32”); a bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; a bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; a bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; in the stool of the subject. 29. The method of paragraph 28, wherein the detecting is performed prior to the administering. 30. The method of paragraph 28, wherein the detecting comprises nucleic acid sequencing. 31. The method of any one of paragraphs 23-30, further comprising detecting levels of an inflammatory signature in serum prior to administering. 32. The method of any one of paragraphs 23-31, further comprising detecting the level of one or more fecal inflammatory markers prior to administering. 33. The method of any one of paragraphs 23-32, further comprising detecting a GABA level prior to administering. 34. The method of paragraph 33, wherein the GABA level is a serum and / or fecal GABA level. 64 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 35. The method of any one of paragraphs 23-24, wherein detecting a level below a threshold amount or above a threshold amount for a given detected parameter provides an indication of whether the administering will be effective and / or beneficial to the subject. 36. A composition of any one of paragraphs 1-19 for use in treating a disorder selected from the group consisting of a CNS disorder, a pain disorder, and a neuroimmune- related condition in a human subject. 37. The composition of any one of paragraphs 1-19, wherein the composition is comprised by a packaged food product, wherein the package of the packaged food product is associated with or comprises indicia that the composition is for the use of any one of paragraphs 20-36. 38. A method for supporting a healthy immune response in a mammalian subject with a sub-optimal immune status, the method comprising: a. selecting a subject based on a baseline biomarker measurement, said measurement being within a range selected from the group consisting of: i. a serum high-sensitivity C-reactive protein (hs-CRP) level of 1.0 mg / L to 5.0 mg / L; ii. a secretory immunoglobulin A (sIgA) level in the lower quartile of a healthy population reference range; and iii. a natural killer (NK) cell activity level in the lower tertile of a healthy population reference range; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject. 39. A method for supporting mental and emotional well-being in a mammalian subject experiencing a mild to moderate challenge, the method comprising: a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. a Patient Health Questionnaire-9 (PHQ-9) score of 5 to 14; ii. a Generalized Anxiety Disorder-7 (GAD-7) score of 5 to 14; iii. a Perceived Stress Scale (PSS) score of 14 to 26; and iv. a Liebowitz Social Anxiety Scale (LSAS) score of 30 to 89; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject. 65 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 40. A method for alleviating mild to moderate physical discomfort in a mammalian subject, the method comprising: a. selecting a subject based on a baseline score on a validated pain scale, said score being within a range selected from the group consisting of: i. a Numeric Rating Scale (NRS) score of 3 to 6; ii. a Fibromyalgia Impact Questionnaire (FIQ) score of 25 to 59; iii. a PROMIS Pain Interference T-score of 55 to 65; and iv. a Central Sensitization Inventory (CSI) score of 30 to 49; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject. 41. A method for supporting neurocognitive function in a mammalian subject experiencing a mild to moderate decline, the method comprising: a. selecting a subject based on a baseline score on a validated assessment, said score being within a range selected from the group consisting of: i. a Mini-Mental State Examination (MMSE) score of 19 to 26; ii. an Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score of 10 to 25; iii. a Unified Parkinson's Disease Rating Scale (UPDRS) Part III score of 11 to 32; and iv. an Expanded Disability Status Scale (EDSS) score of 1.0 to 4.5; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject. 42. A method for improving sleep quality in a mammalian subject experiencing a mild to moderate sleep challenge, the method comprising: a. selecting a subject based on a baseline measurement indicative of sub-optimal sleep, said measurement being within a range selected from the group consisting of: i. a PROMIS Sleep Disturbance T-score of 55 to 65; ii. a Pittsburgh Sleep Quality Index (PSQI) global score of 6 to 14; and iii. a sleep efficiency of 75% to 84% as measured by a wearable device; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject. 43. A method for supporting gastrointestinal health and function in a mammalian subject experiencing mild to moderate digestive discomfort, the method comprising: 66 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. an Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) score of 75 to 300; ii. a Gastrointestinal Symptom Rating Scale (GSRS) mean score of 3 to 5; and iii. consistent reporting of Bristol Stool Form Scale types 1, 2, 6, or 7 for at least 25% of bowel movements; and b. administering an effective amount of the composition of any one of paragraphs 1- 19 to the selected subject.

[0249] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. EXAMPLES Example 1: A human-derived Bacteroides strain rescued depressive-like behavior in a social defeat-induced stress model

[0250] To explore the ability of a human gut bacterium to influence depression-like symptoms, it was tested whether orally dosing a high GABA producing, anti-inflammatory human Bacteroides salyersiae strain, HB32, could prevent depression in rats using the social defeat paradigm. In this model, rats are exposed to chronic social subordination, which induces anhedonia-like behaviors in rodents, such as decreased sucrose preference, impaired reward learning, and deficits in brain reward system function. Animals were treated with iHB32 or with the drug ketamine, a rapid acting antidepressant. To support mechanistic assessment, vagotomy and multi-omic analysis of brain tissue and fecal samples were leveraged. Methods Animal Work

[0251] Subjects: Adult male Wistar rats (approximately 9 weeks old at the beginning of each experiment) were used as experimental subjects (IMOTIV INC., formerly ENVIGO, Indianapolis, IN, USA) and male Long Evans retired breeders were used as residents for the social defeat procedure (CHARLES RIVER, Wilmington, MA, USA). Non-stress control animals were pair-housed throughout the experiment in standard polycarbonate cages, while 67 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT animals exposed to social defeat were housed individually with male residents. All rats were housed in vivarium rooms with a 12-hr reverse light-dark cycle (lights off between 7:00 AM and 7:00 PM), and they received food and water ad libitum. All procedures were conducted in accordance with national guidelines and approved by an Institutional Animal Care and Use Committee.

[0252] Interventions: The interventions used in these experiments included the NMDA receptor antagonist ketamine hydrochloride (SIGMA-ALDRICH, St. Louis, MO, USA) and an inactivated version of the human gut Bacteroides salyersiae HB32 strain. Ketamine was mixed in sterile saline and administered intraperitoneally (IP) at a subanesthetic dose of 10 mg / kg / day for 7 days. HB32 was previously isolated from a fecal sample of healthy human adult male. The strain was grown anaerobically (5% H2, 10% CO2 and balanced with N2) at 37°C in a COY chamber (COY LABORATORY PRODUCTS) in animal product free media to an OD600 of 3.5. Bacterial cells were harvested by centrifugation at 8,000 × g for 30 min, washed with reduced phosphate-buffered saline (PBS), resuspended in 20% glycerol in PBS, aliquoted and frozen at −80°C. To prepare inactivated cells, the biomass was thawed, heated at 56°C for 30 min and cooled down for 30 min. The procedure was repeated two times. Aliquots were frozen at −80°C until use. Bacterial suspensions of heat-treated cultures (iHB32) at the intended intake levels were prepared daily before dosing. Rats received 1×10^9 cells / rat / day for 7 days, resuspended in sterile saline and delivered via oral gavage. Sterile saline was used as oral vehicle control.

[0253] Procedures

[0254] Social Defeat and Drug Administration: Wistar rats were randomly assigned to receive either social defeat or no stress. Wistar rats receiving social defeat (i.e., intruders) were transported to a separate vivarium room housing Long-Evans rats (i.e., residents) selected for aggressive behavior. Each intruder was placed inside the residents’ cage (61×43×20 cm) behind a perforated PLEXIGLASS partition physically separating the rats, with food and water available ad libitum. At 9:00 AM the following day, the residents and partitions were removed. Social defeat was defined as the intruder displaying a defensive, supine posture for 3 consecutive seconds. After social defeat or 3 minutes (whichever occurred first), intruders were transferred to a different resident’s cage (separated by a partition) and remained there until the following day’s social defeat session, which was repeated for 21 days. Intruders were never paired with the same residents twice. Immediately after the social defeat sessions on days 15-21, stressed rats were administered either vehicle 68 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT (SD-vehicle), ketamine (SD-ketamine), or iHB32 (SD-iHB32). Unstressed rats served as a control (Naive) and were briefly handled daily in a separate vivarium room and were administered vehicle (see e.g., Fig.1).

[0255] Sucrose Preference: After the final social defeat session, stressed and non-stressed rats were single-housed in standard polycarbonate rat cages in their respective vivarium rooms and provided with a single bottle filled with a 1% sucrose solution for approximately 20 hr without access to tap water. The following morning, at 7:00 AM, the sucrose bottles were refilled, weighed, and added to each cage along with a bottle of tap water (also weighed). Twelve hr later, at 7:00 PM, the bottle positions were alternated in each cage. The following morning, at 7:00 AM, the sucrose and water bottles were removed and weighed. Sucrose preference was calculated as the change in sucrose bottle weights over the 24 hr test period divided by the total change in sucrose and tap water bottle weights.

[0256] Sample Collection: After the social defeat session on day 15, baseline fecal samples were collected, while post-treatment samples were collected on day 21. Rats were gently restrained in a towel and fecal samples were collected in sterile cryogenic tubes (#430659; CORNING, INC., USA), placed on dry ice, and transferred to a -80° C freezer for storage.

[0257] After the sucrose preference test, rats were rapidly decapitated without anesthesia. Brains were removed from the skull and placed in ice-cold PBS for approximately 30 seconds. Brains were then sliced with a sterile straight-edge blade in a brain block and the prelimbic and infralimbic cortices were manually dissected and placed into sterile EPPENDORF tubes on dry ice. All samples were then transferred to a -80° C freezer for storage until processing.

[0258] Vagotomy: In a separate cohort, male rats received either bilateral subdiaphragmatic vagotomy or sham surgery prior to social defeat and sucrose preference testing. During the surgery, rats were briefly anesthetized with isoflurane (1-2%), and the abdominal area was shaved and cleaned using aseptic procedures. A small midline incision was made to expose the esophagus. In the vagotomy group, the ventral and dorsal truncal branches of the vagus nerve were severed, while in the sham surgery group, they were briefly manipulated with sterile forceps without damage. The abdominal muscles and skin were sutured, and animals were administered Meloxicam (analgesic) and allowed one week to recover before testing.

[0259] Data Analysis: A Robust Outlier Identification (ROUT) test with a maximum desired false discovery rate (FDR) set at 1% was performed to identify potential outliers in the sucrose preference readout. Two outliers in the dataset were removed prior to analysis. For 69 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT the sucrose preference test, data were analyzed with a one-way between-subjects ANOVA followed by Turkey’s multiple comparisons test with the following groups: Naive, SD- Vehicle, SD-Ketamine, SD-iHB32. For the vagotomy experiment, data were analyzed with a two-way between-subjects ANOVA with Dunn–Šidák correction for multiple comparisons with Stress and Surgery as independent variables. The level of significance was set at 0.05. Brain Transcriptomics

[0260] Sample Processing: Frozen samples of the rat prefrontal cortices (combined left and right regions) were processed by AZENTA for RNA extraction, and QC was performed using QUBIT RNA quantification and BIOANALYZER. Library preparation was done with rRNA depletion and PolyA selection, and sequencing via ILLUMINA HISEQ 2x150bp (20-30M reads / sample).

[0261] Computational Analysis: The integrated Differential Expression & Pathway analysis (iDEP) version 0.96 was used for RNAseq processing and analysis pipeline. Outliers were identified via principal component analysis (PCA), and analysis of differentially expressed genes (DEGs) were visualized using volcano plots. Following this, the DEGs were determined using DESeq2, a DEG analysis pipeline. Pathway and Network analysis was performed utilizing a FDR cutoff of 0.05 and mapped to the Gene Ontology (GO) Biological Process and GO Molecular Function databases. Significance was set at adjusted p- value≤0.05, and a fold change of + / - 1.5 (log2 fold change of + / - 0.5). Results Inactivated Bacteroides salyersiae Reduced Depressive-like Behavior in Male Rats with Comparable Efficacy to Ketamine

[0262] In order to test the ability of inactivated HB32 (iHB32) to rescue depressive-like symptoms, rats first underwent social defeat daily for three weeks (see e.g., Fig.1). Naive controls (Naive) were handled daily for three weeks (see e.g., Fig.1). During the final week of stress induction, all groups excluding Naive animals underwent daily dosage of treatments. Anhedonia was measured as a metric of depression-like behavior, and the sucrose preference test measured alterations to the depressive-like phenotype to determine the efficacy of treatments in rescuing symptoms. Ketamine (SD-Ketamine) and vehicle (SD-Vehicle) served as positive and negative controls, respectively (see e.g., Fig.1). Social defeat significantly decreased sucrose preference in SD-Vehicle animals compared to Naive controls, indicating 70 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT the model induced anhedonia (p=0.018; Tukey post-hoc test) (see e.g., Fig.2A). Compared to SD-Vehicle animals, SD-Ketamine animals showed a reversal of the anhedonia phenotype (Tukey post-hoc; p<0.0001) (see e.g., Fig.2A). Dosage of inactivated HB32 (SD-iHB32; Tukey post-hoc, p<0.006) significantly increased sucrose preference comparable to what was observed in the ketamine group, indicating that treatment with Bacteroides salyersiae can reverse depressive-like behavior in this model. The Social Defeat-Induced Depression Paradigm Required an Intact Vagus Nerve

[0263] To determine if the behavioral effects of the social defeat paradigm were dependent on the vagus nerve, a separate cohort of male rats were subjected to vagotomy (SD- Vagotomy) or sham surgery (SD-Sham) prior to three weeks of social defeat and sucrose preference testing (see e.g., Fig.2B). Vagotomy abolished the anhedonia phenotype (see e.g., Fig.2B). A two-way ANOVA revealed a significant effect of stress [F(1,31)=5.545, p=0.025], with post-hoc tests revealing a significant decrease in sucrose preference in the SD- Sham (p<0.012), but not in the SD-vagotomy group, which indicates that this paradigm can require an intact vagus nerve. Social Defeat-Induced Stress Altered the Prefrontal Cortex Transcriptomic Profile Compared to Controls, and iHB32 administration rescued the phenotype

[0264] The social defeat paradigm induces depressive-symptoms in animals through chronic stress. Given that the prefrontal cortex (PFC) has been implicated both in depression and chronic stress like what is induced by social defeat, it was explored if the PFC can provide mechanistic insights underlying how iHB32 administration can alleviate depressive-like behaviors. To this end, following social defeat and treatment, animals were sacrificed, and the PFC was dissected out for bulk RNA sequencing.

[0265] To identify if the social defeat paradigm induced differential expression profiles in the PFC, DESeq2 was used, which is a differentially expressed gene (DEG) analytical pipeline. The SD-Vehicle group was used as the baseline for all comparisons. A total of 29 genes were significantly differentially expressed in the Naive vs SD-Vehicle comparison, and 403 significantly DEGs were detected in the SD-iHB32 vs SD-Vehicle comparison, reflecting the impact of the social defeat model and of B. salyersiae treatment, respectively (see e.g., Fig. 3). No genes were significantly differentially expressed when comparing the SD-iHB32 to the Naive groups (not shown), indicating the bacterial treatment broadly normalized changes in gene expression induced by the social defeat model to naïve. There were no significant 71 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT DEGs detected between the SD-Vehicle and SD-Ketamine groups (see e.g., Fig.3), indicating that the antidepressant effect of Ketamine in this model is not mediated by transcriptional changes in the PFC.

[0266] Taken together, the social defeat model induced transcriptional changes in the PFC, which can be largely reversed through oral treatment with iHB32, but not ketamine. This transcriptional rescue phenotype can contribute to the antidepressant effect of iHB32 administration. Discussion

[0267] As described herein, treatment with the inactivated commensal strain Bacteroides salyersiae HB32 had efficacy in the social defeat model of depression, with the primary endpoint being anhedonia measured by sucrose preference. iHB32 was able to reduce depression-like behavior comparable to ketamine. Other studies have reported similar success using primarily Lactobacillus spp. and / or Bifidobacteria probiotic strains for relief of depressive-like symptoms induced by social defeat paradigms in a variety of animal models, including mice, and hamsters, supporting probiotic administration as a therapeutic option in humans. Heat inactivated probiotic studies in animal models also demonstrated the ability of a probiotic to induce reversal of depressive symptoms regardless of living status. Vagus nerve dependent

[0268] Whether the vagus nerve was required for this phenotype was investigated. The social defeat paradigm required an intact vagus nerve for its anhedonia phenotype, indicating in this model, communication between the gut and the vagus was involved. The dependence of the model’s phenotype on the vagus explicitly prevents determination if iHB32 efficacy is mediated by the vagus, as severing it made the model dysfunctional for testing this hypothesis. These results are consistent with existing literature: mouse studies that induced chronic stress via the social defeat model used fecal microbiome transplants into antibiotic- depleted mice to identify specific microbes that were associated with anhedonia symptoms, which were subsequently blocked by vagotomy (see e.g., Wang et al.2020, “Ingestion of Lactobacillus intestinalis and Lactobacillus reuteri causes depression- and anhedonia-like phenotypes in antibiotic-treated mice via the vagus nerve,” J Neuroinflammation.17: 241; Wang et al.2021, “Ingestion of Faecalibaculum rodentium causes depression-like phenotypes in resilient Ephx2 knock-out mice: A role of brain–gut–microbiota axis via the 72 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT subdiaphragmatic vagus nerve,” Journal of Affective Disorders 292: 565-573). Taken together, these indicate that the vagus nerve plays a key role in mediating depression. Prefrontal Cortex and RNAseq

[0269] To further explore the mechanisms behind iHB32 phenotype rescue, RNAseq was performed on the prefrontal cortex - a brain region implicated in depression and chronic stress. Prolonged stress causes overexposure to cortical stress hormones which induces negative structural changes in the PFC like dendritic atrophy, which is strongly associated with impaired synaptic connectivity. Differential gene expression profiles in the PFC include altered expression of neurotrophic factors, glucocorticoid receptor signaling, inflammatory pathways, and neurotransmitter signaling.

[0270] A large portion of DEGs in the PFC of SD-iHB32 were reverted to a naïve expression profile. These included those involved in antioxidant defense and immune responses (e.g., metallothionein 2A (Mt2A), glutathione peroxidase 3 (Gpx3), glutathione peroxidase 1 (Gpx1), CD46 (membrane cofactor protein (MCP)), SH2 Domain Containing 1A (Sh2d1a)), stress responses, and neurotransmission, including serotonergic pathways (e.g., neuropeptide FF (Npff), G protein-coupled receptor 171 (Gpr171), Adenosine A2a Receptor (Adora2a), 5- hydroxytryptamine receptor (Htr2b, also known as the serotonin 2B receptor)). Dysregulation of several of these functions has been previously associated with mood disorders, anxiety, repetitive behaviors, and fear conditioning (1-11). Specifically, in clinically depressed patients, chronic stress has been linked to elevation of monoamine oxidase activity and neuroinflammation (12-13) and a reduction of antioxidant defenses like glutathione (14), which can persist even after treatment (15). Changes were also found in serotonin pathways involved in the regulation of mood, sleep, and appetite (16-17). Serotonin signaling has been previously shown to be modulated the microbiome, with germ free mice exhibiting anxiety- like behaviors and altered serotonin levels in the brain (18). Vagal stimulation by the gut microbiota has also been reported to alter brain concentrations of neurotransmitters, including serotonin, GABA and glutamate (19-20). Many of these genes address components of the PFC stress response, indicating a role for the bacterial treatment in alleviating PFC-specific responses induced by social defeat.

[0271] Ketamine treatment did not affect the PFC transcriptome as compared to naive animals. Other studies have reported effects of ketamine in the PFC in animals who have undergone social defeat using different metrics to describe the effects. One study subjected mice to social defeat for 5 minutes daily for 10 days followed by injection of ketamine, and 73 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT reported restoration of social defeat-induced alterations to neuronal and astroglial signaling (see e.g., Mishra et al.2018, “Subanesthetic ketamine reverses neuronal and astroglial metabolic activity deficits in a social defeat model of depression,” J Neurochem.146(6): 722- 734). Another study investigated the PFC as well as the entire neural circuit that includes the amygdala, nucleus accumbens, and hippocampus in a mouse model of social defeat examining transcriptional differences related to resilience and susceptibility to chronic stress reported that ketamine exerted the majority of its effort in the amygdala and hippocampus (see e.g., Bagot et al.2017, “Ketamine and Imipramine Reverse Transcriptional Signatures of Susceptibility and Induce Resilience-Specific Gene Expression Profiles,” Biol. Psychiatry 81(4):285-295), so investigations can consider transcriptional differences along the entire neural circuit to demonstrate the mechanism underlying ketamine-mediated rescue of depressive-like symptoms in chronic stress models. Conclusions

[0272] Overall, this study provides insights on the ability of an inactivated human-derived Bacteroides species to mitigate depressive symptoms in a model of chronic stress, in part through alterations of the stress response in the prefrontal cortex. References for Example 1 1) Sequeira A, Morgan L, Walsh DM, Cartagena PM, Choudary P, Li J, et al. Gene expression changes in the prefrontal cortex, anterior cingulate cortex and nucleus accumbens of mood disorders subjects that committed suicide. PLoS One. 2012;7(4):e35367. 2) Bevilacqua L, Doly S, Kaprio J, Yuan Q, Tikkanen R, Paunio T, et al. A population- specific HTR2B stop codon predisposes to severe impulsivity. Nature.2010 Dec 23;468(7327):1061–6. 3) Nguyen T, Marusich J, Li JX, Zhang Y. Neuropeptide FF and its receptors: therapeutic applications and ligand development. J Med Chem.2020 Nov 12;63(21):12387–402. 4) Zhang L, Koller J, Ip CK, Gopalasingam G, Bajaj N, Lee NJ, et al. Lack of neuropeptide FF signalling in mice leads to reduced repetitive behavior, altered drinking behavior, and fuel type selection. FASEB J.2021 Nov;35(11):e21980. 5) Choileain SN, Astier AL. CD46 processing: a means of expression. Immunobiology. 2012 Feb;217(2):169–75. 74 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 6) Hron JD, Caplan L, Gerth AJ, Schwartzberg PL, Peng SL. SH2D1A regulates T- dependent humoral autoimmunity. J Exp Med.2004 July 19;200(2):261–6. 7) Panyard DJ, Reus LM, Ali M, Liu J, Deming YK, Lu Q, et al. Post-GWAS multiomic functional investigation of the TNIP1 locus in Alzheimer’s disease highlights a potential role for GPX3. Alzheimers Dement.2024 July;20(7):5044–53. 8) Sharma G, Shin EJ, Sharma N, Nah SY, Mai HN, Nguyen BT, et al. Glutathione peroxidase-1 and neuromodulation: Novel potentials of an old enzyme. Food Chem Toxicol.2021 Feb;148:111945. 9) Bobeck EN, Gomes I, Pena D, Cummings KA, Clem RL, Mezei M, et al. The BigLEN- GPR171 Peptide Receptor System Within the Basolateral Amygdala Regulates Anxiety- Like Behavior and Contextual Fear Conditioning. Neuropsychopharmacology.2017 Dec;42(13):2527–36. 10) Raddatz MC, Newson CM, Stott M, Campbell C, Bobeck EN. GPR171 is necessary for normal physiological functions and mood-related behaviors in males, but not females. Behav Brain Res.2025 May 1;490:115618. 11) Garcia CP, Licht-Murava A, Orr AG. Effects of adenosine A2A receptors on cognitive function in health and disease. Int Rev Neurobiol.2023;170:121–54. 12) Belleau EL, Treadway MT, Pizzagalli DA. The Impact of Stress and Major Depressive Disorder on Hippocampal and Medial Prefrontal Cortex Morphology. Biol Psychiatry. 2019 Mar 15;85(6):443–53. 13) Michel TM, Frangou S, Thiemeyer D, Camara S, Jecel J, Nara K, et al. Evidence for oxidative stress in the frontal cortex in patients with recurrent depressive disorder--a postmortem study. Psychiatry Res.2007 May 30;151(1–2):145–50. 14) Gawryluk JW, Wang JF, Andreazza AC, Shao L, Young LT. Decreased levels of glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients with psychiatric disorders. International Journal of Neuropsychopharmacology.2011 Feb 1;14(1):123–30. 15) Meyer JH, Wilson AA, Sagrati S, Miler L, Rusjan P, Bloomfield PM, et al. Brain monoamine oxidase A binding in major depressive disorder: relationship to selective serotonin reuptake inhibitor treatment, recovery, and recurrence. Arch Gen Psychiatry. 2009 Dec;66(12):1304–12. 75 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 16) Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis. Cell.2015 Apr 9;161(2):264. 17) Hung LY, Alves ND, Colle AD, Talati A, Najjar SA, Bouchard V, et al. Intestinal Epithelial Serotonin as a Novel Target for Treating Disorders of Gut-Brain Interaction and Mood. Gastroenterology.2025 Apr 1;168(4):754–68. 18) Clarke G, Grenham S, Scully P, Fitzgerald P, Moloney RD, Shanahan F, et al. The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner. Mol Psychiatry.2013 June;18(6):666–73. 19) Caspani G, Kennedy S, Foster JA, Swann J. Gut microbial metabolites in depression: understanding the biochemical mechanisms. Microb Cell.6(10):454–81. 20) Ressler KJ, Mayberg HS. Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic. Nat Neurosci.2007 / 08 / 30 ed.2007 Sept;10(9):1116–24. Example 2

[0273] The ability of non-viable HB32 (nvHB32) to modulate the release of the pro- inflammatory cytokine IL1b by human immune cells was explored. Human peripheral blood mononuclear cells (PBMCs) pooled from six unique donors were challenged with lipopolysaccharide (LPS) to stimulate a pro-inflammatory response. When exposed to nvHB32, PBMC release of IL1b into supernatant was approximately 50% of the LPS-induced release of IL1b (see e.g., Fig.4).

[0274] Next, the ability of nvHB32 to reduce the release of IL1b when dosed in conjunction with human microbiome-derived communities (hMDCs) was assessed. hMDCs were prepared from five human donors from the HOLOBIOME Donor Vault, Human Donor 2 (HD-2), HD-9, HD-13, HD-20, and HD-26 under anaerobic conditions (5% CO2, 5%H2, 90% N2) in an anaerobic chamber (COY LABORATORY PRODUCTS; Michigan, USA). nvHB32 was spiked into hMDCs at increasing concentrations, resulting in 10%, 30% or 50% relative abundance within each human donor community. Compared to the community without nvHB32 (0%), nvHB32 reduced levels of the release in supernatant of IL1b in HD-2, HD-9, HD-13, and HD-20 (see e.g., Fig.5). Additionally, nvHB32’s ability to modulate release of the pro-inflammatory cytokine TNFa was explored at a single percent relative 76 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT abundance of the total community. The release of TNFa was significantly reduced in HD-9 and HD-26 (see e.g., Fig.6).

[0275] iHB32’s immunomodulatory potential was further explored in vivo. Adult male Wistar rats (approximately nine-weeks-old) (ENVIGO, Indianapolis, IN, USA) were administered iHB32 or a vehicle control. HB32 was grown under anaerobic conditions (5% CO2, 5%H2, 90% N2) in an anaerobic chamber (COY LABORATORY PRODUCTS; Michigan, USA). Bacterial cells were harvested and resuspended in 20% glycerol in PBS, aliquoted and frozen at −80°C. To prepare inactivated cells, the biomass was thawed, heated at 56°C for 30 min and cooled down for 30 min. The procedure was repeated two times. Rats received 1×10^9 inactivated cells / rat / day for 7 days via oral gavage. Post-treatment blood samples were collected and the plasma level of the chemokine CXCL1 was measured by enzyme-linked immunosorbent assay. Compared to the control, iHB32 significantly reduced plasma levels of CXCL1 (see e.g., Fig.7). 77 4930-7248-2659.5

Claims

Attorney Docket No: 083103-000102WOPT CLAIMS What is claimed herein is:

1. A composition comprising: inactivated Bacteroides salyersiae bacterial strain HB32 (“HB32”); an inactivated bacterial strain having 16S rRNA with at least 95% sequence identity to 16s rRNA of Bacteroides salyersiae HB32 as set out in one of SEQ ID NOs: 1 or 2; an inactivated bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898- CP166900; or inactivated Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject.

2. The composition of claim 1, wherein the mammalian subject is human.

3. The composition of claim 1, wherein the bacterial strain comprises a targeted mutation that inactivates a target gene or its gene product, wherein the genome of the mutated strain remains at least 99.5% identical to that of B. salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024,as Patent Deposit No. PTA-127757 or at least 99.5% identical to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900, and wherein the 16S rRNA sequence remains at least 95% identical to one of SEQ ID NOs: 1 or 2.

4. The composition of claim 1, wherein the inactivated bacterial strain is formulated for oral delivery.

5. The composition of any one of claims 1-4, wherein the composition comprises a pharmaceutically acceptable excipient.

6. The composition of any one of claims 1-5, wherein the inactivated bacterial strain is deliberately inactivated by heat treatment, gamma irradiation, ultrasonic disruption, 78 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT lyophilization, tyndallization, pasteurization, freeze-thaw inactivation, pulsed electric field treatment, or a combination thereof.

7. The composition of any one of claims 1-6, wherein the composition further comprises at least one additional prebiotic selected from the group consisting of an amino acid (e.g., arginine, glutarate, and ornithine), biotin, fructooligosaccharide, galactooligosaccharide, hemi cellulose (e.g., arabinoxylan, xylan, xyloglucan, glucomannan), inulin, chitin, lactulose, mannan oligosaccharide, oligofructose- enriched inulin, gum (e.g., guar gum, gum arabic, and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans- galactooligosaccharide, pectin (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-I), dietary fiber (e.g., soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber), xylooligosaccharide, polyamine (e.g., spermidine, putrescine), polyol (e.g. maltitol, xylitol).

8. The composition of any one of claims 1-7, further comprising one or more species of different viable bacteria.

9. The composition of any one of claims 1-8, further comprising one or more additional species of inactivated bacteria.

10. The composition of any one of claims 1-9, further comprising a purified bacterial strain that is cytotoxic or cytostatic to a GABA-consuming bacteria.

11. The composition of claim 10, wherein the GABA-consuming bacteria is Evtepia gabavorous or Firmicutes bacterium MGS:

114.

12. The composition of any one of claims 1-11, wherein the inactivated bacterial strain is dried.

13. The composition of any one of claims 1-12, wherein the composition comprises an enteric coating.

14. The composition of any one of claims 1-13, wherein the composition is a nutritional composition, a food product, a dietary complement, a medical food, or a medicament.

15. The composition of any one of claims 1-14, which is formulated in a tablet, a caplet, a pill, a capsule, a troche, a lozenge, a granule, a powder, a gummy, a gel, a sachet, a food composition, a nutraceutical, a medical food, or a combination thereof.

16. A composition comprising an inactivated bacterial strain, the genome of which comprises at least one sequence encoding at least one enzyme selected from glutamate 79 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof, wherein the inactivated bacterial strain is formulated for delivery to the gut of a mammalian subject.

17. The composition of claim 16, wherein the genome of the inactivated bacterial strain comprises sequence encoding two or more of glutamate decarboxylase; glutamate:GABA antiporter and glutaminase enzymes.

18. The composition of claim 16, wherein the genome of the inactivated bacterial strain comprises sequence encoding glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes.

19. The composition of any one of claims 16-18, wherein the sequence encoding the glutamate decarboxylase, glutamate:GABA antiporter and glutaminase enzymes is at least 98% identical to, or encodes only conservative amino acid differences relative to the enzymes encoded by SEQ ID NOs 3-5.

20. A method of treating a CNS disorder, a pain disorder or a neuroimmune disorder in a human subject in need thereof, the method comprising administering a therapeutically effective amount of the composition of any one of claims 1-19 to the subject.

21. The method of claim 20, wherein the CNS disorder is selected from the group consisting of treatment-resistant major depressive disorder (TR-MDD), major depressive disorder and its subtypes (melancholic depression, atypical depression, catatonic depression, postpartum depression, and seasonal affective disorder), bipolar disorder, schizophrenia, anxiety, anxiety disorders, obsessive compulsive disorder, eating disorders, addiction, social phobia, premenstrual dysphoric disorder, autism spectrum disorder, sleep disorders, and attention deficit hyperactivity disorder (ADHD).

22. The method of claim 20, wherein the CNS disorder is major depressive disorder, and wherein the administration of the composition reduces a symptom of anhedonia with an efficacy comparable to a standard therapeutic dose of ketamine in a social defeat animal model.

23. The method of claim 20, wherein the disorder is a pain disorder, and wherein the pain disorder is chronic pain. 80 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 24. The method of claim 23, wherein the chronic pain is selected from the group consisting of nociplastic pain, neuropathic pain, inflammatory pain, and visceral pain, and combinations thereof.

25. The method of claim 24, wherein the pain is associated with a condition selected from the group consisting of fibromyalgia, irritable bowel syndrome, diabetic neuropathy, rheumatoid arthritis, complex regional pain syndrome, radiculopathy, post-herpetic neuralgia, chemotherapy-induced peripheral neuropathy.

26. The method of claim 20, wherein the disorder is a neuroimmune-related condition selected from the group consisting of Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis, and Multiple Sclerosis.

27. The method of claim 20, wherein the method results in a reduction in the serum level of one or more biomarkers selected from the group consisting of CXCL1, IL-1b, and TNFα in the subject.

28. The method of any one of claims 20-27, further comprising detecting a level of one or more bacterial strains selected from: Bacteroides salyersiae bacterial strain HB32 (“HB32”); a bacterial strain having 16S rRNA with at least 95% sequence identity to one of SEQ ID NOs: 1 or 2; a bacterial strain with a genome comprising at least 98% sequence identity to the genome sequence of HB32 with GenBank Accession No: CP166898-CP166900; a bacterial strain, the genome of which comprises sequence encoding an enzyme selected from glutamate decarboxylase; glutamate:GABA antiporter; glutaminase; or a combination thereof; or Bacteroides salyersiae bacterial strain HB32 deposited under the provisions of the Budapest Treaty with the American Type Culture Collection (ATCC, 10801 University Blvd., Manassas, Va.20110-2209) on May 22, 2024, as Patent Deposit No. PTA-127757; in the stool of the subject.

29. The method of claim 28, wherein the detecting is performed prior to the administering.

30. The method of claim 28, wherein the detecting comprises nucleic acid sequencing.

31. The method of any one of claims 23-30, further comprising detecting levels of an inflammatory signature in serum prior to administering. 81 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 32. The method of any one of claims 23-31, further comprising detecting the level of one or more fecal inflammatory markers prior to administering.

33. The method of any one of claims 23-32, further comprising detecting a GABA level prior to administering.

34. The method of claim 33, wherein the GABA level is a serum and / or fecal GABA level.

35. The method of any one of claims 23-24, wherein detecting a level below a threshold amount or above a threshold amount for a given detected parameter provides an indication of whether the administering will be effective and / or beneficial to the subject.

36. A composition of any one of claims 1-19 for use in treating a disorder selected from the group consisting of a CNS disorder, a pain disorder, and a neuroimmune-related condition in a human subject.

37. The composition of any one of claims 1-19, wherein the composition is comprised by a packaged food product, wherein the package of the packaged food product is associated with or comprises indicia that the composition is for the use of any one of claims 20-36.

38. A method for supporting a healthy immune response in a mammalian subject with a sub-optimal immune status, the method comprising: a. selecting a subject based on a baseline biomarker measurement, said measurement being within a range selected from the group consisting of: i. a serum high-sensitivity C-reactive protein (hs-CRP) level of 1.0 mg / L to 5.0 mg / L; ii. a secretory immunoglobulin A (sIgA) level in the lower quartile of a healthy population reference range; and iii. a natural killer (NK) cell activity level in the lower tertile of a healthy population reference range; and b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject.   82 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT 39. A method for supporting mental and emotional well-being in a mammalian subject experiencing a mild to moderate challenge, the method comprising: a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. a Patient Health Questionnaire-9 (PHQ-9) score of 5 to 14; ii. a Generalized Anxiety Disorder-7 (GAD-7) score of 5 to 14; iii. a Perceived Stress Scale (PSS) score of 14 to 26; and iv. a Liebowitz Social Anxiety Scale (LSAS) score of 30 to 89; and b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject.

40. A method for alleviating mild to moderate physical discomfort in a mammalian subject, the method comprising: a. selecting a subject based on a baseline score on a validated pain scale, said score being within a range selected from the group consisting of: i. a Numeric Rating Scale (NRS) score of 3 to 6; ii. a Fibromyalgia Impact Questionnaire (FIQ) score of 25 to 59; iii. a PROMIS Pain Interference T-score of 55 to 65; and iv. a Central Sensitization Inventory (CSI) score of 30 to 49; and b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject.

41. A method for supporting neurocognitive function in a mammalian subject experiencing a mild to moderate decline, the method comprising: a. selecting a subject based on a baseline score on a validated assessment, said score being within a range selected from the group consisting of: i. a Mini-Mental State Examination (MMSE) score of 19 to 26; ii. an Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog) score of 10 to 25; iii. a Unified Parkinson's Disease Rating Scale (UPDRS) Part III score of 11 to 32; and iv. an Expanded Disability Status Scale (EDSS) score of 1.0 to 4.5; and 83 4930-7248-2659.5Attorney Docket No: 083103-000102WOPT b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject.

42. A method for improving sleep quality in a mammalian subject experiencing a mild to moderate sleep challenge, the method comprising: a. selecting a subject based on a baseline measurement indicative of sub-optimal sleep, said measurement being within a range selected from the group consisting of: i. a PROMIS Sleep Disturbance T-score of 55 to 65; ii. a Pittsburgh Sleep Quality Index (PSQI) global score of 6 to 14; and iii. a sleep efficiency of 75% to 84% as measured by a wearable device; and b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject.

43. A method for supporting gastrointestinal health and function in a mammalian subject experiencing mild to moderate digestive discomfort, the method comprising: a. selecting a subject based on a baseline score on a validated instrument, said score being within a range selected from the group consisting of: i. an Irritable Bowel Syndrome Severity Scoring System (IBS-SSS) score of 75 to 300; ii. a Gastrointestinal Symptom Rating Scale (GSRS) mean score of 3 to 5; and iii. consistent reporting of Bristol Stool Form Scale types 1, 2, 6, or 7 for at least 25% of bowel movements; and b. administering an effective amount of the composition of any one of claims 1-19 to the selected subject. 84 4930-7248-2659.5

Images