Compositions comprising Anti-inflammatory vesicles and methods of use

Abstract

The disclosure relates to anti-inflammatory, oxygen-scavenging membrane vesicles derived from bacteria with modified or absent lipopolysaccharide. The disclosure further relates to pharmaceutical and nutritional compositions comprising these vesicles, and methods of using the same. Methods include therapeutic treatment of diseases associated with gut oxygenation and inflammation, such as inflammatory bowel disease, as well as non-therapeutic methods for supporting gastrointestinal health, promoting a healthy inflammatory response, and maintaining a balanced gut microbiota.

Description

COMPOSITIONS COMPRISING ANTI-INFLAMMATORY VESICLES AND METHODS OF USECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 697,942, filed September 23, 2024, which is incorporated by reference herein in its entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The Sequence Listing for this application is labeled “121504-5014-WO_Sequence_Listing.xml”, which was created on September 21, 2025, and is 36,472,687 bytes in size.FIELD

[0003] The disclosure relates generally to anti-inflammatory oxygen-scavenging membrane vesicles, compositions comprising such vesicles, and methods of using the same. The compositions and methods are directed to both therapeutic applications for treating and / or preventing disease, and non-therapeutic applications for supporting and maintaining health.BACKGROUND

[0004] A healthy gut is largely anaerobic. The mucosa prevents oxygen from diffusing into the gut while intestinal epithelial cells respire, reducing the gut lumen. Accordingly, human commensal bacteria are commonly anaerobic. Many pathobionts, undesirable bacteria associated with disease, can live under both anaerobic and aerobic conditions. When the functionality of epithelial cells decreases, for example with age, or the colon gets inflamed, oxygen leaches into the gut, fueling the propagation of pathobionts which often produce inflammatory lipopolysaccharide (LPS), or endotoxin. This exacerbates conditions such as gut dysbiosis, gut inflammation, and inflammatory gut diseases (e.g., IBD). Despite a deepening understanding of the role the microbiome has in disparate health conditions, the need for a therapeutic treatment ofDBl / 161990477.12diseases / conditions associated with gut oxygenation and or inflammatory LPS remains unmet in the art.SUMMARY

[0005] The present disclosure describes an exemplary method for treating a disease or condition in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacteria comprise modified lipopolysaccharide or the bacteria lack the reactogenic form of the lipopolysaccharide.

[0006] The present disclosure describes an exemplary pharmaceutical composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacteria comprise modified lipopolysaccharide or the bacteria lack the reactogenic form of lipopolysaccharide; and a pharmaceutically acceptable excipient

[0007] The disclosure further describes an exemplary method for treating and / or preventing a disease or condition in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0008] The disclosure also describes an exemplary method of manufacturing oxygenscavenging membrane vesicles derived from bacteria, wherein the bacteria comprise modified lipopolysaccharide or the bacteria lack the reactogenic form of lipopolysaccharide. Method comprising culturing the bacteria in a growth medium comprising succinate and or lactate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

[0009] The present disclosure also provides an exemplary nutritional composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacteria comprise modified lipopolysaccharide or the bacteria lack the reactogenic form of lipopolysaccharide; and a nutritionally acceptable excipient. In some embodiments, the nutritional composition is a food product, a dietary supplement, or a medical food.DBl / 161990477.12BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing summary, as well as the following detailed description of embodiments of the disclosure, will be better understood when read in conjunction with the appended drawings and figures.

[0011] FIGURES 1A, IB, 1C, ID, and IE collectively illustrate the inhibition of pathogenic Enter obacteriacea growth by membrane vesicles (MVs) through reduction of oxygen. FIG. 1A. Respiration of membrane vesicles. The medium contained 20 mM potassium phosphate, 650 mM succinate at a pH of 7 with 325 pg / mL of either wt or AhemB MVs. FIG. IB. Electrophoresis on a 0.8% agarose gel for 1 h, hemB was amplified with P3 and P4 primers. Expected amplicon sizes for AhemB (hemB::kanR) were 1466bp and wt hemB was 1114bp. FIG. 1C. Pathway of heme b biosynthesis from 5-aminolevulinare. FIG. ID. Transmission electron microscopy images of AhemB and wt MVs. FIG. IE. Cell density of Escherichia coli AR350, Klebsiella pneumoniae ATCC 43816, and Citrobacter rodentium ATCC 51459 after 7 hours of growth in LB aerobically and shaking at 37°C n=5 for all cultures. Cultures were inoculated with 1 / 400 dilution of overnight cultures, 13 mM succinate and 1.6 mg / mL of MVs. Statistical significance was determined using one-way ANOVA with Sidak's multiple comparisons test, bars represent mean ± SEM. Adjusted p values were as follows, ** p<0.01, **** p<0.0001.

[0012] FIGURES 2A, 2B, 2C, and 2D collectively illustrate amelioration of colitis by MVs. FIG. 2A. Schematic of the mouse experiment. FIG. 2B. Body weight of mice as a percent of the initial weight recorded at day 1. Points represent the mean ± SEM. Healthy group n=10, colitis control n=10, AhemB MVs n=15, and wt MVs n=15. FIG. 2C. Compiled percent of initial weight for each mouse from days 2 to 8. Healthy n=70, colitis control n=70, AhemB MVs n=105, wt MVs n=105. Line and error bars represent the mean ± SEM. Significance determined using Kruskal -Wallis test with Dunn’s multiple comparisons test. Adjusted p values were as follows, *** p=0.0005, **** p<0.0001. FIG. 2D. Length of colon distal of the cecum. Healthy n=10, colitis control n=10, AhemB MVs n=14, wt MVs n=l 5. Bars represent the mean ± SEM. Significance determined using one-way ANOVA with Tukey’s multiple comparisons test. Adjusted p values were as follows, ** p=0.0021, *** p<0.001, **** p<0.0001.DBl / 161990477.12

[0013] FIGURES 3A, 3B, and 3C collectively illustrate the reduction of the colonic epithelium and modulation of the cytokine response by membrane vesicles. FIG. 3A. Representative images of the distal colonic epithelium. Mice were injected intraperitoneally with pimonidazole 1 hour before euthanasia. Pimonidazole adducts were detected using Hypoxyprobe-1 RED ATTO 594 dye-conjugated IgGl mouse monoclonal antipimonidazole antibody (red fluorescence). Hoechst 33342 was used as a nuclear counter stain (blue fluorescence). No adjustments were made to the images. FIG. 3B. Quantification of the RED ATTO 594 dye. 3 sections of the distal epithelium were quantified and averaged per mouse, healthy n=10, colitis control n=10, AhemB MVs n=14, and wt MVs n=15. Box displays the range from the first to the third quartile, with a line at the median value, and Tukey whiskers. Significance was determined using one-way ANOVA with Sidak's multiple comparisons test. Adjusted p values were as follows, * p=0.0147, ** p=0.0024, *** p<0.001. FIG. 3C. Volcano plot displaying serum cytokines that were increased in, colitis control vs. wt membrane vesicles in red, AhemB MVs vs. wt MVs in purple, and wt MVs vs. colitis control and AhemB MVs in green.

[0014] FIGURES 4A, 4B, 4C, 4D, 4E, and 4F collectively illustrate the modification of the microbiome by membrane vesicles. FIG. 4A. PCoA of Bray-Curtis dissimilarity using bacterial genera as features. Each point represents a single sample, points are colored by groups. Healthy n=10, colitis control n=10, AhemB MVs n=9, wt MVs n=12. FIG. 4B. Phylogenetic tree of all bacterial families identified in the samples. Heatmap depicts average z-score of each bacterial family within a group. Bars represent the average relative abundance of bacterial families within a group. FIG. 4C. Bar graph showing the relative abundance of all bacterial genera identified in each sample, as well as the average for each group. FIG. 4D. Linear regression for the relative abundance of Enterob acteriaceae to Lactobacillaceae within the colitis control, Ahemb MVs and wt MVs groups. Each point represents a single sample (n=31), best-fit line in red. FIG. 4E. Top 5 LEfSe results represented by LDA-score of wt MVs group to AhemB MVs group. FIG. 4F. Relative abundance of the top 5 LEfSe results between wt MVs group and colitis control or AhemB MVs group, only significant differences shown. All boxes display the range from the first to the third quartile, with a line at the median value, and Tukey whiskers. For Lactobacillaceae comparison significance was determined using Kruskal -Wallis test with Dunn’sDBl / 161990477.12multiple comparisons with adjusted p value, * p<0.05. For all other comparisons significance was determined using a two-tailed Mann-Whitney test, p values were as follows, * p<0.05, ** p-0.0073.

[0015] FIGURES 5A, 5B, 5C, and 5D collectively depict the modification of the microbiome by membrane vesicle treatment. Relative abundance of select bacterial families between wt MVs group and colitis control group (Bacteroidaceae: FIG. 5A; Enterococcaceae: FIG. 5B; Enterob acteriaceae: FIG. 5C; and Akkermansiaceae: FIG. 5D). All boxes display the range from the first to the third quartile, with a line at the median value, and Tukey whiskers, significance was determined using a two-tailed Mann-Whitney.

[0016] FIGURES 6A, 6B, 6C, 6D and 6E collectively illustrate the alteration of the metatranscriptome and the metabolome by membrane vesicle treatment. FIG. 6A. PCoA of Bray-Curtis dissimilarity using SEED subsystems as features annotated using MEGAN. Each point represents a single sample, points are colored by groups, dashed arrows represent biplot vectors of the top two loadings. Healthy n=10, colitis control n=10, AhemB MVs n=9, wt MVs n=12. FIG. 6B. Heatmap of all SEED energy subsystems annotated by MEGAN, clustered by subsystems (rows). FIG. 6C. Selected energy subsystems that showed significant differences in number of assigned transcripts between wt MVs and either AhemB MVs or colitis control groups, organized into subcategories (top panel: central metabolism; middle panel: fermentation; bottom panel: respiration). All boxes display the range from the first to the third quartile, with a line at the median value, and Tukey whiskers. Significance was determined using one-way ANOVA test with Tukey ’s multiple comparisons. FIG. 6D. Principal component analysis from untargeted LCMS analysis with analytes as features. Each point represents a sample, and points are colored by group. Healthy n=7, AhemB MVs n=4, and wt MVs n=4. FIG. 6E. Comparison of peak area for glyceric acid between wt MVs and AhemB MVs, identified with LCMS and compound discoverer. Significance was determined using a two-tailed Mann-Whitney test, * p=0.0286.

[0017] FIGURES 7A, 7B, 7C, 7D, and 7E collectively depict the alteration of the metatranscriptome by membrane vesicle treatment, specifically 6-phosphofructokinase (FIG. 7A), pyruvate kinase (FIG. 7B), pyruvate, water dikinase (FIG. 7C), fructose-bisphosphonate (FIG. 7D), and pyruvate, phosphate kinase (FIG. 7E). Selected enzymes within theDBl / 161990477.12glycolysis / gluconeogenesis SEED subsystem. These enzymes were assigned using MEGAN and the GO terms database. All boxes display the range from the first to the third quartile, with a line at the median value, and Tukey whiskers. Significance was determined using one-way ANOVA test with Tukey ’s multiple comparisons.

[0018] FIGURES 8A, 8B, 8C, 8D, 8E, 8F, 8G, 8H, 81, 8J, and 8K collectively depict the alteration of the metatranscriptome by membrane vesicle treatment. Heatmap of all SEED subsystems annotated by MEGAN, clustered by subsystems (rows). Cl through C2=Healthy, C3 through C4= Colitis control, C5 through C7=AhemB MVs and C8 through C10=wt MVs.

[0019] FIGURE 9 illustrates the percent reduction of oxygen per m per minute by both Oxyrase and B. subtilis MVs, normalized by protein concentration. Testing was performed in a 15mM phosphate buffer with 650mM of succinate and 650mM of lactate. Readings were taken with a Clark electrode, n=3 for Oxyrase and n=3 for B. subtilis MVs. Bars represent mean ± SEM. Significance was determined using a two-tailed Welch’s t test, p-values were as follows **** p<0.0001.

[0020] FIGURE 10 illustrates the reduction of LPS induced immune response by MVs. Quantification of TNF-alpha induced upon challenging a human PBMC model (zen bio, SER- PBMC-P-F, 6 donor pooled, 3 male, 3 female) with a reactogenic LPS, with or without antagonistic ClearColi MVs. Results are expressed as a percentage of TNF-alpha stimulated by the LPS alone. Doses of lyophilized MVs were increased from 0.03 to 0.52pg / mL, each added to 0.21ng / mL of purified LPS from Escherichia coli O55:B5. LPS n=5, MVs n=3. Bars represent mean ± SEM. Significance was determined using a one-way ANOVA with Tukey correction. Adjusted p-values were as follows, ** p<0.005, *** p<0.0005, **** p<0.0001.DETAILED DESCRIPTIONDefinitions

[0021] Unless defined otherwise, all technical and scientific terms used in the description of the present disclosure have the same meaning as is commonly understood by one of skill in the art to which this disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties.DBl / 161990477.12

[0022] “Moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.

[0023] The terms “active pharmaceutical ingredient(s)” and “active pharmaceutical agent(s)” refer to the anti-inflammatory oxygen-scavenging membrane vesicles, and pharmaceutical compositions thereof, that treat a disease or condition associated with gut oxygenation, as described herein. The term “active pharmaceutical ingredient” may also include other additional agents that treat a disease or condition is associated with gut oxygenation.

[0024] As used herein, the terms “administer,” “administration,” or “administering” refer to (1) providing, giving, dosing, and / or prescribing by either a health practitioner or his authorized agent or under his or her direction according to the disclosure; and / or (2) putting into, taking or consuming by the mammal, according to the disclosure.

[0025] The terms “combination,” “pharmaceutical combination,” “co-administration,” “coadministering,” “administered in combination with,” “administering in combination with,” “simultaneous,” and “concurrent,” as used herein, encompass administration of two or more active pharmaceutical ingredients to a subject so that both active pharmaceutical ingredients and / or their metabolites are present in the subject at the same time. Co-administration includes concurrent administration and sequential administration. The simultaneous administration of two or more separate compositions is an example of concurrent co-administration. Another example of concurrent co-administration is the administration of a single composition in which two or more active pharmaceutical ingredients are present. The administration of two or more separate compositions at different times is an example of sequential co-administration.

[0026] ‘Effective amount” refers to an amount of an active agent (such as an antiinflammatory membrane vesicle formulation) sufficient to elicit a desired response. A “therapeutically effective amount” is an amount sufficient to effect an intended application for disease treatment. An effective amount for a non-therapeutic method is an amount sufficient to produce a measurable positive change in a parameter of health or wellness. A therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), the subject (e.g., the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc., which can readily be determined by one of ordinary skill in theDBl / 161990477.12art. The term also applies to a dose that induces a particular response in target cells. The specific dose varies depending on the particular compounds chosen, the dosing regimen to be followed, whether the oxygen-scavenging membrane vesicles is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which the oxygen-scavenging membrane vesicles are carried.

[0027] A “therapeutic effect” as that term is used herein, encompasses a therapeutic benefit and / or a prophylactic benefit. A prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0028] As used herein, the terms “treat,” “treatment,” and / or “treating” may refer to the management of a disease, disorder, or pathological condition, or symptom thereof with the intent to cure, ameliorate, stabilize, and / or control the disease, disorder, pathological condition or symptom thereof. Regarding control of the disease, disorder, or pathological condition more specifically, “control” may include the absence of condition progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g., placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the condition). As used herein, the terms “prevent,” “preventing,” and / or “prevention” may refer to reducing the risk of developing a disease, disorder, or pathological condition.

[0029] The term “z z vivo" refers to an event that takes place in a subject’s body.

[0030] The term “ / / / vitro" refers to an event that takes places outside of a subject’s body. In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.

[0031] The terms “subject” and “patient” are used interchangeably herein to refer to a warm blooded animal such as a mammal, such as a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and / or conditions described herein.

[0032] The term “non-human subject” includes any animal that can be treated or used in testing the membrane vesicles, and / or pharmaceutical compositions thereof, of present disclosure, including mammals such as non-human primates, rodents, sheep, dogs, cows, pigs,DBl / 161990477.12chickens, as well as amphibians, reptiles, etc. Preferred non-human animals are selected from the primate family or rodent family.

[0033] “Pharmaceutically acceptable” refers to those active pharmaceutical agents, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human and non-human animals without excessive toxicity, irritation, allergic response, or other adverse complications commensurate with a reasonable benefit / risk ratio.

[0034] “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” or “physiologically compatible” carrier or carrier medium is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient(s), its use in the therapeutic compositions of the disclosure is contemplated.

[0035] “Gram-negative” or “Gram-” bacterial cells refer to bacterial species having a thin layer of peptidoglycan covered by an outer membrane in their cell wall structure. “Grampositive” or “Gram+” bacterial cells refer to bacterial species having a thick layer of peptidoglycan, without any outer membrane.

[0036] The term “Gram -negative bacteria” is recognized in the art and refers generally to bacteria that do not retain Gram stain (e.g., the deposition of a colored complex between crystal violet and iodine). In an exemplary Gram stain, cells are first fixed to a slide by heat and stained with a basic dye (e.g., crystal violet), which is taken up by all bacteria (i.e., both Gram-negative and Gram-positive). The slides are then treated with an iodine-KI mixture to fix the stain, washed with acetone or alcohol, and finally counterstained with a paler dye of different color (e.g., Safranin). Gram-positive organisms retain the initial violet stain, while Gram-negative organisms are decolorized by the organic solvent and hence show the counterstain. Exemplary Gram-negative bacteria and cell lines include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp.,DBl / 161990477.12Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacier spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. and Vibrio spp.

[0037] The term “mutant Gram-negative bacteria,” or “LPS mutant Gram-negative bacteria,” or “kdsD and gutQ mutant Gram negative bacteria,” or “API mutant Gram-negative bacteria” or similar terms, as used herein, includes Gram-negative bacteria that have been mutated one or more times in, for example, one or more of the gutQ, k.dsD, kdsA, k.dsB. waaA, msbA, yhD genes, of any other biosynthetic, processing, or trafficking gene thereby producing an outer membrane substantially lacking the reactogenic form of LPS.

[0038] The term, “viable non-toxic bacteria” refers to a viable bacterial strain comprising an outer membrane comprising a modified LPS (as described herein) or lacking the reactogenic form of LPS. ClearColi is a non-limiting example of a viable non-toxic bacteria. As used in the examples, “ClearColi” refers to an Escherichia coli K-12 strain genetically engineered to produce a modified lipopolysaccharide consisting of the tetra-acylated precursor Lipid IVA, thereby rendering it non-endotoxic.

[0039] The term “lipopolysaccharide” or “LPS” refers to the major antigen of Gram-negative bacteria, a complex molecule containing both lipid and polysaccharide parts. Specifically, LPS consists of four parts: the O-antigen surface polysaccharide, an outer core oligosaccharide, an inner core oligosaccharide, and lipid A. LPS is also sometimes referred to as “endotoxin” and is a component of the outermost membrane of the cell envelope of gram-negative bacterial species. LPS is a potent activator of the immune system, as well as a pyrogen (fever-causing agent).

[0040] The term “modified lipopolysaccharide” or “modified LPS” refers to an altered version of the normal LPS molecule found in the outer membrane of bacteria. Bacterial cells have the ability to change the structure of their LPS in response to environmental stress, host immune factors, and other conditions. LPS may also be modified using chemical, enzymatic, and / or genetic methods to alter its structure and function. In the context of the instant disclosure, the alteration of the normal LPS molecule produces a modified LPS that is non-immunogenic or non- reactogenic (endotoxically inactive).DBl / 161990477.12

[0041] The term “lipid A” refers to the conserved hydrophobic region of LPS structurally defined as a backbone moiety ofN,O-acyl beta-l,6-D-glucosamine 1,4-biphosphate. The optimal reactogenic lipid A structure is believed to contain 6 acyl chains. Four acyl chains attached directly to the glucosamine sugars are beta hydroxy acyl chains usually from about 10 to about 16 carbons in length. Two additional acyl chains are often attached to the beta hydroxy group. E. coli lipid A, as an example, typically has four C 14 hydroxy acyl chains attached to the sugars and one C14 and one C12 attached to the beta hydroxy groups.

[0042] The term “acyl chain” or “acyl group” refers to a moiety containing a double-bonded oxygen atom and an organyl group (R-C=O) or a hydrogen atom (H-C=O).

[0043] The term “2-keto-3 -deoxy -D-manno-octulosonate” or “KDO” refers to a sugar acid or formula CSHBOS essential to the synthesis of lipopolysaccharide in Gram-negative bacteria. KDO acts as a structural linker between the lipid A component and the core oligosaccharide of LPS.

[0044] Non-immunogenic” or “non-reactogenic” refers to the attribute of microorganism (e.g., bacteria), or part thereof (e.g., the oxy gen-scavenging membrane vesicles described herein), that does not elicit an immune response in a subject, such as inflammation, fever, swelling, allergy, or any other immune response ( / .<?., endotoxically inactive).

[0045] The term “lipid IVA” refers to a tetra-acyl ated lipid A precursor produced by Gramnegative bacteria during the LPS biosynthetic process. Lipid IVA is endotoxically inactive and acts as an agonist to the mouse immune system by binding the TLR4 / MD-2 complex, but functions as an antagonist in human cells due to species-specific structural differences of the MD-2 protein.

[0046] The term “D-arabinose 5-phosphate isomerase,” or “A5P isomerase,” or “API” refers to the bacterial enzyme that converts the pentose pathway intermediate D-ribulose 5-phosphate into A5P; A5P is subsequently condensed with phosphoenolpyruvate to form Kdo 8-phosphate (Kdo8P) by the KdsA enzyme; hydrolyzed to Kdo by the KdsC enzyme; activated as the sugar nucleotide CMP-Kdo by the KdsB enzyme; and transferred from CMP -Kdo to the acceptor lipid IVA by the WaaA enzyme. In E. coli K12, there are two API genes (kdsD and gutQ).DBl / 161990477.12

[0047] ‘Membranes” or “membrane fragments” refer to the phospholipid-, glycolipid-, and glycoprotein-containing structures surrounding bacterial cells, and their fragments. While the cell wall structures of Gram-negative and Gram-positive bacteria differ, both possess a cytoplasmic membrane containing the respiratory chain enzymes essential for oxygen scavenging. The principles of vesicle formation, while requiring different extraction and preparation methods adapted for the specific cell wall type, can be applied to both bacterial types to produce vesicles that display these respiratory components. “Membrane vesicles” or “bacterial membrane vesicles” or “MVs” refer to the isolated nanoparticles derived from the membrane components of bacterial cells, including the membrane fragments described herein. Bacterial membrane vesicles (MVs) vary in size from 20 to 500 nm and are surrounded by a membrane bilayer derived from bacterial lipids and proteins. MVs have been observed for all Gram-negative bacterial species studied to date, as well as Gram-positive bacteria, including Akkermansia muciniphila, Rothia dentocariosa, Corynebacterium spp., Staphylococcus aureus , Enterococcus faecium, Clostridium perjringens Mycobacterium ulcerans, Bacillus spp., and Lactobacillius spp. among other accumulating examples.

[0048] “Oxygen scavenging” or “oxygen scavenger” refer to naturally occurring or engineered compounds, compositions, and / or organisms able to neutralize or reduce oxygen or reactive oxygen species (ROS). “Oxygen-scavenging membrane vesicles” or “oxygenscavenging MVs” refer to engineered MVs with the ability to neutralize or reduce oxygen or ROS. Oxygen-scavenging MVs may carry an enzymatic cargo, such as antioxidant enzymes, or an electron transport system, which reduce oxygen to water in the presence of a hydrogen donor. MVs may also include non-inflammatory or anti-inflammatory components.

[0049] “Dietary supplement” is a product taken by mouth that contains a "dietary ingredient" intended to supplement the diet. The dietary ingredients in these products can include vitamins, minerals, herbs or other botanical s, amino acids, and substances such as enzymes, organ tissues, glandulars, and metabolites. As used herein, the oxygen-scavenging membrane vesicles are considered a dietary ingredient for the purposes of formulation as a dietary supplement.

[0050] Gut dysbiosis” refers to an imbalance in the microbial community of the gastrointestinal tract. In the context of the present disclosure, it is particularly characterized by a decrease in the abundance and diversity of beneficial, obligately anaerobic bacteria (such asDBl / 161990477.12short-chain fatty acid producers) and a concurrent increase in the relative abundance of facultatively anaerobic bacteria, particularly pathobionts from the Enterob acteriaceae family. This imbalance is often associated with a pro-inflammatory state and compromised gut barrier function.

[0051] A “healthy subject” refers to a subject who has not been diagnosed by a medical professional as having a particular disease or condition that is the subject of a claim. A healthy subject may nevertheless experience transient or mild symptoms of discomfort or a sub-optimal health status and may seek to maintain or improve their overall state of wellness.

[0052] A “medical food” is a food which is formulated to be consumed or administered enterally under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.

[0053] A “nutritional composition” is a broad term referring to any composition that provides nourishment and can be consumed. It includes, but is not limited to, food products, dietary supplements, and medical foods.

[0054] The term “sub-optimal,” when referring to a health status (e.g., “sub-optimal immune status” or “sub-optimal gut function”), describes a state that is a deviation from peak health or homeostasis but does not rise to the level of a clinically diagnosed disease.

[0055] As used herein, the terms “support,” “maintain,” or “promote,” when used in the context of health (e g., “supporting gastrointestinal health”), refer to non -therapeutic methods of providing a benefit to a subject who may not have a diagnosed disease or condition. Such benefits can include, but are not limited to, maintaining normal physiological function, enhancing a subject's state of well-being, reducing the risk of developing a sub-optimal health state, or returning a subject from a sub-optimal state to a normal, healthy state. These terms are distinct from “treat.”

[0056] For the avoidance of doubt, it is intended herein that particular features (for example integers, characteristics, values, uses, diseases, etc.) described in conjunction with a particular aspect, embodiment or example of the disclosure are to be understood as applicable to any otherDBl / 161990477.12aspect, embodiment or example described herein unless incompatible therewith. Thus such features may be used where appropriate in conjunction with any of the definition, claims or embodiments defined herein. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of the features and / or steps are mutually exclusive. The disclosure is not restricted to any details of any disclosed embodiments. The disclosure extends to any novel one, or novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.THE EMBODIMENTS:Introduction

[0057] The oxygen-scavenging anti-inflammatory membrane vesicles (MVs) and compositions described herein are useful in both therapeutic and non-therapeutic applications. In therapeutic embodiments, the MVs and compositions can be used in methods for treating diseases by decreasing oxygen levels in the gut and or reducing LPS mediated inflammation. In parallel, in non-therapeutic embodiments, the compositions can be used in methods for supporting, maintaining, and promoting gastrointestinal health and a balanced inflammatory response.

[0058] Some embodiments of the present disclosure may be used to treat gut diseases (e.g., IBD or traveler’s diarrhea). In some embodiments, the composition can be taken as a prophylactic for inflammatory bowel disease or traveler’s diarrhea; and / or to maintain microbiome diversity. In some embodiments, prophylactic treatment is taken for the purpose of guarding against or preventing the spread or occurrence of disease or infection. In other embodiments, the composition could be taken as a method of treatment during or after a diagnosis of a gut disease.

[0059] Other embodiments are directed to non-pharmaceutical compositions and methods for supporting general health. In these non-therapeutic embodiments, the composition may be formulated as a nutritional product, such as a dietary supplement or medical food, and taken toDBl / 161990477.12support a healthy gut environment, promote a balanced gut microbiota, and maintain a healthy inflammatory response in a subject who has not been diagnosed with a specific disease.

[0060] In some embodiments, microbiome diversity is defined as the amount of individual bacteria from each of the bacterial species present in the gut microbiome. In some embodiments, this microbiome diversity maintains a healthy gut and reducing chance of disease. When there is high richness and diversity of the microbes in the gut, the immune system is stronger and more stable.

[0061] The present disclosure relates to exemplary methods for depleting / reducing oxygen in the gut.

[0062] It is generally recognized that bacterial respiration takes place in the cytoplasmic membrane. In some embodiments, introducing bacterial oxygen-scavenging membrane vesicles, e.g., the cytoplasmic membrane of respiring bacteria, into the gut depletes / reduces oxygen in the gut. In various embodiments, this leads to improved colon health in the subject to whom the vesicles are administered.

[0063] In various embodiments, there is provided a method of benefitting a subject by administering an effective amount of a composition described herein. Where the subject has a diagnosed disease or condition associated with gut oxygenation, the method comprises administering a therapeutically effective amount for the purpose of treating or preventing said disease or condition. Where the subject seeks to maintain or support their health, the method comprises administering an effective amount for the non-therapeutic purpose of supporting a healthy gut environment.

[0064] In an exemplary embodiment, there is provided a method of treating a disease alleviated by depleting oxygen levels in the gut by administering to a subject in need thereof a therapeutically effective amount of a composition described herein.

[0065] In an exemplary embodiment, the disclosure provides a method of alleviating a disease by depleting oxygen levels in the gut without triggering a gut immune response in the subject being treated. The method comprises, administering to a subject in need thereof a therapeutically effective amount of a composition described herein.DBl / 161990477.12

[0066] In some embodiments, the disclosure provides a method of treating a disease or condition associated with gut oxygenation and or inflammatory LPS, wherein the disease or condition is selected from the group consisting of: (a) Gastrointestinal and Inflammatory Bowel Diseases, including but not limited to, Inflammatory Bowel Disease (IBD) (such as Crohn’s disease and ulcerative colitis), Irritable Bowel Syndrome (IBS) (including all subtypes: IBS-D, IBS-C, and IBS-M), microscopic colitis, diversion colitis, radiation-induced enteritis, celiac disease, and gut dysbiosis; (b) Metabolic Disorders, including but not limited to, Type 1 and Type 2 diabetes, metabolic syndrome, obesity, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, and hyperlipidemia; (c) Neurological and Neuroinflammatory Disorders, including but not limited to, neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS); conditions with a neuroinflammatory component such as mood disorders (e.g., major depressive disorder, anxiety disorders); recovery from traumatic brain injury (TBI) or stroke; and age-associated cognitive decline and inflammaging; (d) Pain Disorders, including nociceptive and inflammatory pain such as that associated with Rheumatoid arthritis, osteoarthritis, and IBD; neuropathic pain such as diabetic neuropathy, fibromyalgia, complex regional pain syndrome (CRPS), and post-herpetic neuralgia; and other chronic pain conditions such as migraine and central sensitization syndromes; (e) Systemic Autoimmune and Inflammatory Disorders, including but not limited to, Rheumatoid arthritis, Ankylosing spondylitis, systemic lupus erythematosus (SLE), psoriasis, and atherosclerosis; (f) Oncology- related Conditions, including but not limited to, the prevention or treatment of colitis-associated colorectal cancer, the amelioration of side effects from cancer therapy such as chemotherapy- induced mucositis, and the treatment of cancer-associated cachexia; and (g) Disorders of Host- Pathogen Interaction, including but not limited to, Graft-versus-host disease (GVHD), traveler's diarrhea, and recurrent Clostridioides difficile infection. The method comprises, administering to a subject in need thereof a therapeutically effective amount of a composition described herein.

[0067] In some embodiments, the disclosure provides a method of treating a disease or condition associated with gut oxygenation, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g.,DBl / 161990477.12Alzheimer’s Disease, Parkinson’s Disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging. The method comprises, administering to a subject in need thereof a therapeutically effective amount of a composition described herein.

[0068] The present disclosure further provides dosages and dosing regimens for pharmaceutical compositions comprising the oxygen-scavenging membrane described herein, appropriate dosages of the present composition are readily determined by one of skill in the art. Exemplary factors determining appropriate dosage include, without limitation, whether a human or non-human subject is being treated, the nature and severity of the disorder or condition, the rate of administering the composition to the subject, the disposition of the composition and the discretion / experience of the prescribing physician.

[0069] In other embodiments, the pharmaceutical compositions may be formulated for oral, sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration.

[0070] The present disclosure also provides exemplary methods of manufacturing oxygenscavenging membrane vesicles derived from bacteria, wherein the bacteria comprise modified lipopolysaccharide or the bacteria lack the reactogenic form of lipopolysaccharide.

[0071] In some embodiments, the oxygen-scavenging membrane vesicles of the present disclosure are derived from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia miiciniphila. Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Coryne bacterium pseudodiphtheriticum Corynebacterium accolens and Rothia dentocariosa.

[0072] Particularly suitable for the methods and compositions of the present disclosure are membrane vesicles derived from Gram-negative bacteria. The unique outer and inner membrane structure of Gram-negative bacteria allows for the efficient formation of inverted inner membrane vesicles where the respiratory chain components, such as terminal oxidases, are oriented externally, maximizing the oxygen-scavenging effect in the target environment. In these preferred embodiments, the Gram-negative bacterium is selected from the group consisting ofDBl / 161990477.12Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium legnminosarnm, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia miiciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, and Phocaeicola vidgatus.

[0073] In various embodiments, the source bacterial cells used to manufacture the oxygenscavenging membrane vesicles of the current disclosure are reduced or depleted in lipopolysaccharide (LPS), or have a modified LPS to be non- or anti-inflammatory.Oxygen-scavenging membrane vesicles

[0074] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified lipopolysaccharide that is non-immunogenic. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of less than 6 acyl chains. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 1, 2, 3, 4 or 5 acyl chains. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 1 acyl chain. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 2 acyl chains. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 3 acyl chains. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 4 acyl chains. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that has a Lipid A component consisting of 5 acyl chains.

[0075] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that does not contain 2-keto 3-deoxy-D-manno- octulosonate. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles are derived from bacteria comprising modified LPS that is lipid IVA.DBl / 161990477.12

[0076] Those skilled in the art are aware of bacteria comprising modified lipopolysaccharide (LPS) and bacteria lacking the reactogenic form of LPS, and are also aware of suitable sources for obtaining them and / or techniques for their generation. See for example, U.S. Patent No. 8,303,964, which is incorporated by reference herein in its entirety.

[0077] In some embodiments, oxygen-scavenging membrane vesicles, and fragments thereof, are derived from bacteria comprising mutations in the gutQ, kdsD (yrbH), kdSA, kdsB, waaA, msbA, and / or yhjD genes, or mutations in any other biosynthetic, processing, or trafficking gene. In some embodiments, mutations of the gutQ and kdsD genes inhibit API expression within the bacterial strain, which inhibits KDO expression, which inhibits outer membrane LPS expression. In some embodiments, the disclosure provides viable Gram-negative bacteria with mutations in the kdsA gene. In some embodiments, the disclosure provides viable Gram-negative bacteria with mutations in the kdsB gene. In some embodiments, the disclosure provides viable Gram-negative bacteria with mutations in the waaA gene.

[0078] In some embodiments, the oxygen-scavenging membrane vesicles are derived from non-toxic (e.g., endotoxin free) bacteria with suppressed LPS expression in the outer membrane of said bacteria. In some embodiments, the oxygen-scavenging membrane vesicles are derived from bacteria with suppressed API protein expression. In some embodiments, the oxygenscavenging membrane vesicles are derived from bacteria with suppressed KDO protein expression. In some embodiments, KDO protein expression is suppressed through, for example, mutation of the gutQ gene and the kdsD gene. In some embodiments, the oxygen-scavenging membrane vesicles are derived from bacteria with lipid IVA expression in the outer membrane of said bacteria.

[0079] In some embodiments, mutations within the bacteria may be either constitutively expressed or under the control of inducible promoters, such as, for example, the temperature sensitive heat shock family of promoters, or the anaerobically-induced nirB promoter (see, e.g., Harborne, et al., 1992, Mol. Micro. 6:2805; herein incorporated by reference in its entirety) or repressible promoters, such as uapA (see, e.g., Gorfinkiel, et al., 1993, J. Biol. Chem., 268:23376; herein incorporated by reference in its entirety) or gcv (see, e.g., Stauffer, et al., 1994, J. Bact, 176:6159; herein incorporated by reference in its entirety). Selection of anDBl / 161990477.12appropriate promoter will depend on the host bacterial strain and will be apparent to those skilled in the art.

[0080] In some embodiments, the oxygen-scavenging bacterial cell membrane vesicles, and fragments thereof, of the present disclosure, are derived from bacteria containing a 16S rRNA sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, at least about 99%, or 100% identical to a 16S rRNA sequence set forth in Table 1A (any one of SEQ ID NOs: 9-13792). In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from bacteria comprising a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A (any one of SEQ ID NOs: 9- 13792). In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from bacteria comprising a 16S rRNA sequence that is at least 95% identical, 96%, 97%, 98%, 99%, 99.5%, at least 99.5% identical, or 100% identical to a 16S rRNA sequence set forth in Table 1 A (any one of SEQ ID NOs: 9-13792).

[0081] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Acetobacter aceti comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 9-48.

[0082] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Akkermansia muciniphila comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 49-483.

[0083] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Bacillus subtilis comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 484-5883.

[0084] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Bacteroides thetaiotaomicron comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 5884-6208.DBl / 161990477.12

[0085] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Bacteroides umformis comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 6209-6344.

[0086] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Corynebacterium accolens comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 6345-6403.

[0087] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Escherichia coli comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 6404-13283.

[0088] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Gluconobacter oxydans comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 13284-13398.

[0089] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Komagataeibacter xylinus comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 13399-13466.

[0090] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Phocaeicola vulgatus comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 13467-13720.

[0091] In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, are derived from Rothia dentocariosa comprising a 16S rRNA sequence that is at least about 95% identical (e.g., at least 96%, 97%, 98%, 99%, 99.5%, at least 99.9% identical) to one of SEQ ID NOs: 13721-13792.DBl / 161990477.12Table 1A: 16S rRNA sequences of bacteria used to produce oxygen-scavenging bacterial cell membrane vesicles.

[0092] The 16S rRNA sequences for the bacterial strains set forth in Table 1A were obtained starting from curated reference 16S sequences from the NCBI Targeted Locus Project (TLP). The NCBI Blast non-redundant nucleotide database (nt) was then used to return the top 1000 sequence accessions that matched with at least 80% query coverage and 95% sequence identity to the TLP query, using blastn with default settings. The aligned region was returned as the match, with potentially multiple alignments per accession. This technique provides reasonably specific coverage that is not limited to potential mislabeling of non-reference-quality sequences deposited into NCBI, and also avoids ambiguity from taxonomic changes over time.

[0093] In some embodiments, the oxygen-scavenging bacterial cell membrane vesicles of the present disclosure are anti-inflammatory membrane vesicles.

[0094] In some embodiments, the oxygen-scavenging membrane vesicles are derived from gram-negative bacteria, wherein the gram-negative bacteria are modified to reduce inflammation.

[0095] In some embodiments, the oxygen-scavenging membrane vesicles of the present disclosure are anti-inflammatory membrane vesicles derived from gram-negative bacteria, wherein the gram-negative bacteria are modified to reduce inflammation. In some embodiments, the anti-inflammatory membrane vesicles of the present disclosure are derived from gramnegative bacteria comprising modifications to one or more protein sequences set forth in TableDBl / 161990477.12IB. In some embodiments, the protein sequences set forth in Table IB meet or exceed both the sequence-wide and domain-level cutoff values specified by the PGAP R17.Table IB: Accession numbers of bacterial proteins for modification to reduce inflammation.

[0096] In some embodiments, the anti-inflammatory membrane vesicles of the present disclosure are derived from non-inflammatory or anti-inflammatory gram-positive bacteria. In some embodiments, the non-inflammatory or anti-inflammatory gram-positive bacteria are identified through in vitro assays, including but not limited to the PBMC exposure assay.

[0097] In some embodiments, the oxygen-scavenging bacterial cell membrane vesicles, and fragments thereof, of the present disclosure, are derived from bacteria comprising one or more terminal oxidase proteins set forth in Table 1C. In some embodiments, the protein sequences set forth in Table 1C meet or exceed both the sequence-wide and domain-level cutoff values specified by the PGAP R17.Table 1C: Accession numbers of bacterial terminal oxidase proteins.DBl / 161990477.12Pharmaceutical Compositions

[0098] In one aspect, the disclosure provides a pharmaceutical composition comprising oxygen- scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, as described herein; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises oxygen-scavenging membrane vesicles derived from bacteria comprising a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A, as described herein; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises oxygen-scavenging membrane vesicles derived from gram-negative bacteria comprising modifications to one or more protein sequences set forth inDBl / 161990477.12Table IB, as described herein; and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises oxygen -scavenging membrane vesicles derived from bacteria comprising one or more terminal oxidase proteins set forth in Table 1C; and a pharmaceutically acceptable excipient.

[0099] In some embodiments, the pharmaceutical composition comprises the oxygenscavenging membrane vesicles and fragments thereof, as described herein. With the equivalent dry mass of oxygen-scavenging membrane vesicles in power or liquid form, to the amount of about 0.000001 to about 0.001 g / unit or from about 0.001 g / unit to about 1 g / unit, or from about 0.03 g / unit to about 10 g / unit.

[0100] Described below are non-limiting pharmaceutical compositions comprising the antiinflammatory oxygen-scavenging membrane vesicles described herein.Pharmaceutical Compositions for Oral Administration

[0101] In an embodiment, the disclosure provides a pharmaceutical composition for oral administration comprising the active pharmaceutical ingredient described herein (z.e., the oxygen-scavenging membrane vesicles that are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide), and a pharmaceutical excipient suitable for oral administration.

[0102] In some embodiments, the disclosure provides a solid pharmaceutical composition for oral administration comprising: (i) a therapeutically effective amount of the active pharmaceutical ingredient described herein, or a combination of active pharmaceutical ingredients, and (ii) a pharmaceutical excipient suitable for oral administration. In selected embodiments, the composition further contains (iii) an effective amount of a third active pharmaceutical ingredient, and optionally (iv) an effective amount of a fourth active pharmaceutical ingredient.

[0103] In some embodiments, the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption. Pharmaceutical compositions of the disclosure suitable for oral administration can be presented as discrete dosage forms, such as capsules, sachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous orDBl / 161990477.12non-aqueous liquid, an oil-in-water emulsion, a water-in-oil liquid emulsion, powders for reconstitution, powders for oral consumptions, bottles (including powders or liquids in a bottle), orally dissolving films, lozenges, pastes, tubes, gums, and packs. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and / or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered active ingredient and / or excipient moistened with an inert liquid diluent.

[0104] The disclosure further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some ingredients and / or pharmaceutical excipients. For example, water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms of the disclosure which contain lactose can be made anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected. An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.

[0105] Each of the active pharmaceutical ingredients can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compoundingDBl / 161990477.12techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

[0106] Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, com starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.

[0107] Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

[0108] Disintegrants may be used in the compositions of the disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which disintegrate in the bottle. Too little may be insufficient for disintegration to occur, thus altering the rate and extent of release of the active ingredients from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the active pharmaceutical ingredient described herein (z.e., the oxygen-scavenging membrane vesicles that are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide). The amount of disintegrant used may vary based uponDBl / 161990477.12the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art. About 0.5 to about 15 weight percent of disintegrant, or about 1 to about 5 weight percent of disintegrant, may be used in the pharmaceutical composition. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

[0109] Lubricants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, calcium stearate, magnesium stearate, sodium stearyl fumarate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, silicified microcrystalline cellulose, or mixtures thereof. A lubricant can optionally be added in an amount of less than about 0.5% or less than about 1% (by weight) of the pharmaceutical composition.

[0110] When aqueous suspensions and / or elixirs are desired for oral administration, the active pharmaceutical ingredient(s) may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and / or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

[0111] The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.DBl / 161990477.12

[0112] Surfactants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.

[0113] A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10. An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions. Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable. Similarly, lipophilic (i.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10. However, HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.

[0114] Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl-lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

[0115] Within the aforementioned group, ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkyl sulfates; fatty acid salts; sodium docusate; acylactylates; mono- andDBl / 161990477.12di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and diglycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.

[0116] Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidyl serine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG- phosphatidylethanolamine, PVP -phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono / diacetylated tartaric acid esters of mono / diglycerides, citric acid esters of mono / diglycerides, cholyl sarcosine, caproate, caprylate, caprate, laurate, myristate, palmitate, oleate, ricinoleate, linoleate, linolenate, stearate, lauryl sulfate, teracecyl sulfate, docusate, lauroyl carnitines, palmitoyl carnitines, myristoyl carnitines, and salts and mixtures thereof.

[0117] Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene sterols, derivatives, and analogs thereof; polyoxyethylated vitamins and derivatives thereof; polyoxyethylene-polyoxypropylene block copolymers; and mixtures thereof; polyethylene glycol sorbitan fatty acid esters and hydrophilic transesterification products of a polyol with at least one member of the group consisting of triglycerides, vegetable oils, and hydrogenated vegetable oils. The polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.

[0118] Other hydrophilic-non-ionic surfactants include, without limitation, PEG- 10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG- 15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25DBl / 161990477.12glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glyceryl stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl laurate, PEG-40 glyceryl laurate, PEG-40 palm kernel oil, PEG-50 hydrogenated castor oil, PEG-40 castor oil, PEG-35 castor oil, PEG-60 castor oil, PEG-40 hydrogenated castor oil, PEG-60 hydrogenated castor oil, PEG-60 corn oil, PEG-6 caprat e / capryl ate glycerides, PEG-8 caprate / caprylate glycerides, polyglyceryl- 10 laurate, PEG-30 cholesterol, PEG-25 phyto sterol, PEG-30 soya sterol, PEG-20 trioleate, PEG-40 sorbitan oleate, PEG-80 sorbitan laurate, polysorbate 20, polysorbate 80, POE-9 lauryl ether, POE-23 lauryl ether, POE- 10 oleyl ether, POE-20 oleyl ether, POE-20 stearyl ether, tocopheryl PEG- 100 succinate, PEG-24 cholesterol, polyglyceryl-10 oleate, Tween 40, Tween 60, Tween 80, sucrose monostearate, sucrose monolaurate, sucrose monopalmitate, PEG 10-100 nonyl phenol series, PEG 15-100 octyl phenol series, and poloxamers.

[0119] Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxy ethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins / vitamin derivatives; and mixtures thereof. In some embodiments, the lipophilic surfactant is selected from one or more of glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.

[0120] In an embodiment, the composition may include a solubilizer to ensure good solubilization and / or dissolution of the active pharmaceutical ingredient of the present disclosure (z.e., the oxygen-scavenging membrane vesicles that are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide) and to minimize precipitation of the active pharmaceutical ingredient of the present disclosure. This can be especially important for compositions for non-oral use - e.g., compositions for injection.DBl / 161990477.12A solubilizer may also be added to increase the solubility of the hydrophilic drug and / or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[0121] Examples of suitable solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG; amides and other nitrogen-containing compounds such as 2-pyrrolidone, 2-piperidone, E-caprolactam, N-alkylpyrrolidone, N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkylcaprolactam, dimethylacetamide and polyvinylpyrrolidone; esters such as ethyl propionate, tributylcitrate, acetyl triethylcitrate, acetyl tributyl citrate, tri ethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, s-caprolactone and isomers thereof, 5-valerolactone and isomers thereof, P-butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin, diethylene glycol monoethyl ether, and water.

[0122] Mixtures of solubilizers may also be used. Examples include, but not limited to, triacetin, tri ethyl citrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Further non-limiting examples of suitable solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.

[0123] The amount of solubilizer that can be included is not particularly limited. The amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art. In some circumstances, it may be advantageous to include amounts of solubilizers far in excess of bioacceptable amounts, for example to maximize the concentration of the drug, with excess solubilizer removed prior to providing the composition to a patient using conventional techniques, such as distillation or evaporation. Thus, if present, theDBl / 161990477.12solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients. If desired, very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less. Typically, the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.

[0124] The composition can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[0125] In addition, an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like. Also suitable are bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid, and the like. Salts of polyprotic acids, such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used. When the base is a salt, the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals and alkaline earth metals. Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.

[0126] Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuricDBl / 161990477.12acid, nitric acid, boric acid, phosphoric acid, and the like. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenyl sulfonic acid, propionic acid, p- toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid and uric acid.

[0127] In some embodiments, the pharmaceutical composition comprises the oxygenscavenging membrane vesicles described herein; and a pharmaceutically acceptable acid selected from lactic acid, succinic acid, alpha-glycerol phosphate, formic acid, malic acid, pyruvic acid and corresponding salts. In some embodiments, the pharmaceutically acceptable acid may be present in the composition in the amount of about 0.1 wt % to about 5 wt %.[00128J In some embodiments, the pharmaceutical composition may further include buffering agents, polymers, and stabilizers. In some embodiments, buffering agents are used to control the pH of the composition. In some embodiments, polymers with nonpolar moieties such as polyethylene glycol can also be used as surfactants. In some embodiments, stabilizers may include polyols, sugars, amino acids, amines, and salts. In some embodiments, suitable sugars include sucrose and trehalose. In some embodiments, suitable amino acids include histidine, arginine, glycine, methionine, proline, lysine, glutamic acid, and mixtures thereof. It should be noted that particular molecules can serve multiple purposes. For example, in some embodiments, histidine can act as a buffering agent and an antioxidant.Other Pharmaceutical Compositions

[0129] Pharmaceutical compositions described herein may also be prepared from active pharmaceutical ingredients described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; and Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, N.Y., 1990, each of which is incorporated by reference herein in its entirety.DBl / 161990477.12

[0130] Administration of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients or a pharmaceutical composition thereof can be effected by any method that enables delivery of the anti-inflammatory oxygen-scavenging MVs to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (e g., transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation. The active pharmaceutical ingredient or combination of active pharmaceutical ingredients can also be administered intradiscally or intrathecally.

[0131] Exemplary parenteral administration forms include solutions or suspensions of the anti-inflammatory oxygen-scavenging MVs in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.

[0132] In some embodiments, the oxy gen-scavenging membrane vesicles and compositions thereof, described herein, may be stored in a liquid state at sub-zero temperatures to increase usable lifetime as a pharmaceutical / treatment. In some embodiments, the cryoprotectant may be any compound that decreases the freezing point of water, thereby preventing ice formation or damage. In some embodiments, the composition may contain glycerol or polyethylene glycol as a cryoprotectant. In some embodiments, the composition may contain the cryoprotectant in an amount of about 15% by weight to about 65% by weight, or from about 50 wt % to about 65 wt %.

[0133] Cryoprotectants are substances used to protect biological tissue from freezing damage. In some embodiments, in order to preserve cells and tissues, cryoprotectants must be non-toxic to the cells. In some embodiments, typical cryoprotectants may include glycerol, dimethyl sulfoxide (DMSO), and glycols, including ethylene glycol, propylene glycol, polyethylene glycol, and glycerol.

[0134] In some embodiments, higher concentrations of cryoprotectants (“high” meaning greater than 15 wt %) can be used. In some embodiments, higher concentrations of cryoprotectants prevent the formation of ice crystals, thereby precluding the freezing of cells and the damage and / or killing of preserved cells. In some embodiments, this results in higher numbers of cells being recovered after storage at sub-zero temperatures. In some embodiments, itDBl / 161990477.12has been found that cells stored at below zero degrees Celsius are still viable after over twelve (12) months or more of storage.

[0135] In some embodiments, the composition comprises glycerol. Glycerol is a small molecule that can pass through the semipermeable membrane of cells and gain entry into their interior. In some embodiments, the cryoprotectant nature of glycerol is available both inside and outside the cell. In some embodiments, glycerol forms strong hydrogen bonds with water molecules, thereby disrupting the crystal lattice formation of ice and maintaining the liquid nature of the composition.

[0136] In some embodiments, the composition comprises polyethylene glycol (PEG). Polyethylene glycol is a cryoprotectant that is impermeable to cell membranes and can be made at different molecular weights. In some embodiments, its mode of action is limited to the space “outside” a cell. In some embodiments, porins (i.e. channels within a cell membrane) allow movement of small hydrophilic molecules between the interior of a cell and the exterior of a cell, large hydrophilic molecules, such as polyethylene glycol, are too large to pass through the porins. In particular embodiments, the polyethylene glycol has about 400 glycol moieties (designated as PEG 400).

[0137] In some embodiments, the composition may contain one or more cryoprotectants in the amount of about 15% to about 65% by weight, or from about 50 wt % to about 65 wt %. This lowers the freezing point of the composition. For example, if 53 wt % of glycerol is used, the freezing temperature of the composition is decreased to -26° C.

[0138] In some embodiments, the composition may also contain water as a solvent for the various ingredients. In some embodiments, the composition is isotonic. In embodiments, the composition may have an osmolality of about 280 milliOsmoles / liter (mOsm / L) to about 300 mOsm / L.

[0139] In this regard, it is contemplated that the effective lifetime of the pharmaceutical composition containing the oxygen-scavenging membrane vesicles and fragments thereof, as described herein, can be extended by storage at sub-zero temperatures. In some embodiments, the composition can thus be stored in a self-defrosting freezer under these conditions. In such freezers, the temperature cycles up and down within a temperature range. For example, theDBl / 161990477.12temperature is brought up from -25° C. to -17° C. for about an hour, then reduced to the lower temperature. In some embodiments, this cycle can occur at about 24-hour intervals.

[0140] In practical use, after the compositions are warmed to reach room temperature, they are administered to a patient, including a human or other mammal. Thus, it should be noted that the cryoprotectant should be non-toxic to mammals (e g., humans) at low absolute dosages.N on-pharmaceutical compositions (Foods, Supplements, and Medical Foods)

[0141] In some embodiments, the compositions are intended for administration as a nutritional product. As used herein, the term “nutritional product” includes, but is not limited to, a nutritional composition, a food product, a dietary supplement, and a medical food.

[0142] In certain embodiments, the composition is a food product. The oxygen-scavenging membrane vesicles (MVs) may be incorporated into a wide variety of food matrices. The food product may be, for example, a beverage, a bar, a powder, a yogurt, a kefir, a fermented milk product, a pudding, a cereal, a baked good, or a confectionary. The MVs can be added as a liquid concentrate, a lyophilized powder, or an encapsulated powder to protect the MVs from processing conditions such as heat or pH extremes. For example, the MVs can be incorporated into a fruit smoothie, a protein shake, a nutritional bar, or a ready-to-mix powder for reconstitution in a liquid.

[0143] In other embodiments, the composition is a dietary supplement. The MVs may be formulated for oral administration in various dosage forms, including but not limited to a tablet, a caplet, a pill, a soft or hard gelatin capsule, a lozenge, a granule, a powder, a gummy, a gel, a sachet, an orally dissolving film, or a liquid. Such formulations may include excipients such as binders, fillers, lubricants, and disintegrants as described herein. The dietary supplement may be co-formulated with other beneficial agents, such as prebiotics (e.g., inulin, fructooligosaccharides), probiotics, vitamins, minerals, or botanical extracts. To enhance stability and ensure targeted delivery to the gastrointestinal tract, the MVs in the dietary supplement may be microencapsulated or formulated within an enteric-coated tablet or capsule designed to bypass the stomach and release its contents in the intestines.

[0144] In further embodiments, the composition is a medical food. A medical food is formulated to be consumed or administered enterally under the supervision of a physician andDBl / 161990477.12which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation. In this context, the MVs may be formulated as a sole source of nutrition or as a component of a nutritional formula for subjects with diseases or conditions associated with gut oxygenation and inflammation, such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or cancer-associated cachexia. The medical food may be in the form of a ready - to-drink oral supplement, a tube-feeding formula, or a powder to be reconstituted. The formulation would be designed to provide a specific, effective dose of MVs for the dietary management of the inflammatory and dysbiotic components of such conditions.

[0145] In any of these embodiments, the composition may be provided in a package, wherein the package is associated with or comprises indicia (e.g., a label) that the composition is for the use as described herein, such as for supporting gastrointestinal health, promoting a healthy inflammatory response, or maintaining a healthy balance of gut microbiota.Formulation of Non-Pharmaceutical C ompositions

[0146] The incorporation of the oxygen-scavenging membrane vesicles (MVs) into nonpharmaceutical formats requires consideration of factors such as stability, dose uniformity, and palatability. Standard manufacturing processes in the food, dietary supplement, and medical food industries may be adapted as described herein to ensure the viability and functionality of the MVs.

[0147] In some embodiments, for dry formulations such as powders, capsules, or tablets, the MVs are first prepared as a stable, dry powder. This may be achieved through lyophilization (freeze-drying), optionally with the use of cryoprotectants such as trehalose or sucrose to maintain vesicle integrity. This lyophilized MV powder can then be used as a starting ingredient. For powders intended for reconstitution, the MV powder is blended with other dry ingredients, i.e., “nutritionally acceptable excipients”, such as nutritionally acceptable fillers (e.g., maltodextrin), flow agents (e.g., silicon dioxide), flavorings, sweeteners, and other active ingredients like prebiotics (e.g., inulin) or vitamins. The final blend is then packaged into tubs or single-serving sachets. For capsules and tablets, the MV powder is blended with suitable excipients, such as binders and fillers, and can be encapsulated into hard gelatin or vegetarian capsules, or directly compressed into tablets. In a preferred embodiment, capsules or tablets areDBl / 161990477.12further processed with an enteric coating, for example using cellulose-based polymers, to protect the MVs from the acidic environment of the stomach and ensure their release in the targeted intestinal region.

[0148] To incorporate the MVs into food products, such as nutritional bars or baked goods, methods may be employed to protect the MVs from degradation during processing steps that involve heat or high shear. In one embodiment, the lyophilized MV powder is first microencapsulated by coating it with a protective layer, such as a lipid, wax, or polymer shell. This encapsulated powder can then be incorporated into a food matrix, such as a bar dough, with greater resistance to processing stresses. In another embodiment, the MVs are incorporated into the food product after any high-temperature steps have been completed. For example, the MV powder could be mixed into a yogurt-based coating, a chocolate drizzle, or a frosting that is applied to a nutritional bar or baked good after it has cooled.[00149J For liquid and semi-solid formulations, including beverages, yogurts, and kefir, maintaining MV stability in an aqueous environment is critical. The product may be formulated to a pH that is optimal for MV stability, typically in the range of pH 6.0-7.5, through the use of food-grade buffering agents. To ensure microbiological safety without damaging the MVs with heat, non-thermal processing methods may be used. Such methods include, but are not limited to, High-Pressure Processing (HPP) or sterile filtration of the liquid base before the aseptic addition of the MVs. For fermented products like yogurt or kefir, the MVs may be added postfermentation, immediately prior to packaging.Methods of Treatment

[0150] The present disclosure describes a method for reducing inflammation in the gut. In some embodiments, the method comprises the steps of providing oxygen-scavenging membrane vesicles or fragments thereof to a subject or patient in need thereof, e.g., in need of reducing oxygen content in the gut. In some embodiments, the oxygen scavenging bacterial cell membrane vesicles, and fragments thereof, of the present disclosure, are derived from bacteria containing modified lipopolysaccharide (LPS) or bacteria lacking the reactogenic form of LPS. In some embodiments the method comprises the steps for protecting against or inhibiting LPS induced inflammation with modified LPS.DBl / 161990477.12

[0151] In one aspect, the oxygen-scavenging membrane vesicles and pharmaceutical compositions described herein may be used in methods for treating diseases and / or disorders. The methods and compositions described herein are broadly applicable to diseases and conditions where gut oxygenation, dysbiosis, and associated systemic or local inflammation are part of the underlying pathophysiology. A common mechanism links these conditions: increased gut oxygenation promotes the growth of inflammatory pathobionts, whose metabolic byproducts and structural components (like LPS) can compromise the gut barrier and lead to systemic inflammation, affecting distal organs and systems including the central nervous system (the ‘gutbrain axis’) and contributing to metabolic disorders and age-related inflammatory states. In some embodiments, the method for treating a disease or condition in a subject in need thereof comprises administering to the subject a therapeutically effective amount of oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacteria comprises modified lipopolysaccharide or the bacteria lack the reactogenic form of lipopolysaccharide, or a pharmaceutical composition thereof, as described herein. In some embodiments, the oxygenscavenging membrane vesicles suitable for use in the method of treating diseases and / or disorders are derived from bacteria comprising a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A. In some embodiments, the oxygenscavenging membrane vesicles suitable for use in the method of treating diseases and / or disorders are derived from gram-negative bacteria comprising modifications to one or more protein sequences set forth in Table IB. In some embodiments, the oxygen-scavenging membrane vesicles suitable for use in the method of treating diseases and / or disorders are derived from bacteria comprising one or more terminal oxidase proteins set forth in Table 1C. In some embodiments, the disease or condition is associated with gut oxygenation.

[0152] In some embodiments, the disease or condition is associated with gut oxygenation and or inflammatory LPS. In various embodiments, the disease or condition is selected from the group consisting of: (a) Gastrointestinal and Inflammatory Bowel Diseases, including but not limited to, Inflammatory Bowel Disease (IBD) (such as Crohn’s disease and ulcerative colitis), Irritable Bowel Syndrome (IBS) (including all subtypes: IBS-D, IBS-C, and IBS-M), microscopic colitis, diversion colitis, radiation-induced enteritis, celiac disease, and gut dysbiosis; (b) Metabolic Disorders, including but not limited to, Type 1 and Type 2 diabetes,DBl / 161990477.12metabolic syndrome, obesity, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), insulin resistance, and hyperlipidemia; (c) Neurological and Neuroinflammatory Disorders, including but not limited to, neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS); conditions with a neuroinflammatory component such as mood disorders (e.g., major depressive disorder, anxiety disorders); recovery from traumatic brain injury (TBI) or stroke; and age-associated cognitive decline and inflammaging; (d) Pain Disorders, including nociceptive and inflammatory pain such as that associated with Rheumatoid arthritis, osteoarthritis, and IBD; neuropathic pain such as diabetic neuropathy, fibromyalgia, complex regional pain syndrome (CRPS), and post-herpetic neuralgia; and other chronic pain conditions such as migraine and central sensitization syndromes; (e) Systemic Autoimmune and Inflammatory Disorders, including but not limited to, Rheumatoid arthritis, Ankylosing spondylitis, systemic lupus erythematosus (SLE), psoriasis, and atherosclerosis; (f) Oncology- related Conditions, including but not limited to, the prevention or treatment of colitis-associated colorectal cancer, the amelioration of side effects from cancer therapy such as chemotherapy- induced mucositis, and the treatment of cancer-associated cachexia; and (g) Disorders of Host- Pathogen Interaction, including but not limited to, Graft-versus-host disease (GVHD), traveler's diarrhea, and recurrent Clostridioides difficile infection.

[0153] In some embodiments, the disease or condition is associated with gut oxygenation. In some embodiments, the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0154] In an embodiment, a method of treating a disease or condition associated with gut oxygenation and or inflammatory LPS (e.g. (a) Gastrointestinal and Inflammatory Bowel Diseases, including but not limited to, Inflammatory Bowel Disease (IBD) (such as Crohn’s disease and ulcerative colitis), Irritable Bowel Syndrome (IBS) (including all subtypes: IBS-D, IBS-C, and IBS-M), microscopic colitis, diversion colitis, radiation-induced enteritis, celiac disease, and gut dysbiosis; (b) Metabolic Disorders, including but not limited to, Type 1 andDBl / 161990477.12Type 2 diabetes, metabolic syndrome, obesity, non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), insulin resistance, and hyperlipidemia; (c) Neurological and Neuroinflammatory Disorders, including but not limited to, neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS); conditions with a neuroinflammatory component such as mood disorders (e.g., major depressive disorder, anxiety disorders); recovery from traumatic brain injury (TBI) or stroke; and age-associated cognitive decline and inflammaging; (d) Pain Disorders, including nociceptive and inflammatory pain such as that associated with Rheumatoid arthritis, osteoarthritis, and IBD; neuropathic pain such as diabetic neuropathy, fibromyalgia, complex regional pain syndrome (CRPS), and post-herpetic neuralgia; and other chronic pain conditions such as migraine and central sensitization syndromes; (e) Systemic Autoimmune and Inflammatory Disorders, including but not limited to, Rheumatoid arthritis, Ankylosing spondylitis, systemic lupus erythematosus (SLE), psoriasis, and atherosclerosis; (f) Oncology- related Conditions, including but not limited to, the prevention or treatment of colitis-associated colorectal cancer, the amelioration of side effects from cancer therapy such as chemotherapy- induced mucositis, and the treatment of cancer-associated cachexia; and (g) Disorders of Host- Pathogen Interaction, including but not limited to, Graft-versus-host disease (GVHD), traveler's diarrhea, and recurrent Clostridioides difficile infection) in a subject in need thereof is provided.

[0155] In some embodiments, the method comprises orally administering to the subject an orally ingestible composition in accordance with the disclosure herein. In some embodiments, the subject is a human adult. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MV composition, wherein the MVs are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide, thereby treating a disease or condition associated with gut oxygenation and or inflammatory LPS. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, and the bacteria is selected from one or more of Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium legumiriosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila,DBl / 161990477.12Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Coryiiebacterium pseudodiphtheriticum Corynebacterium accolens, and Rothia dentocariosa thereby treating a disease or condition associated with gut oxygenation and / or inflammatory LPS. In some embodiments, the subject receives, via oral administration of a pharmaceutical composition described herein, from about 0.001 mg to about 100 g of anti-inflammatory oxygen-scavenging MVs per day.

[0156] In some embodiments of the method of treating a disease or condition associated with gut oxygenation and or inflammatory LPS, the effective amount of oxygen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce gut inflammation, gut oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating a disease or condition associated with gut oxygenation and or inflammatory LPS, the therapeutically effective amount of oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In some embodiments of the method of treating a disease or condition associated with gut oxygenation and or inflammatory LPS, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking the reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject

[0157] In an embodiment, a method of treating gut dysbiosis in a subject in need thereof is provided. In some embodiments, method comprises orally administering to the subject an orally ingestible composition in accordance with the disclosure herein. In some embodiments, the subject is a human adult. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MV composition, wherein the MVs are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide, thereby treating gut dysbiosis. In some embodiments,DBl / 161990477.12the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, and the bacteria is selected from one or more of Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphlheriticum, Corynebacterium accolens, and Rothia dentocariosa, thereby treating gut dysbiosis. In some embodiments, the subject receives, via oral administration of a pharmaceutical composition described herein, from about 0.001 mg to about 100 g of anti-inflammatory oxygen-scavenging MVs per day.

[0158] In some embodiments of the method of treating gut dysbiosis, the therapeutically effective amount of oxy gen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce inflammation, oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, aerobically respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating gut dysbiosis, the therapeutically effective amount of oxy gen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In some embodiments of the method of treating gut dysbiosis, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking the reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject.

[0159] In an embodiment, a method of treating gut inflammation in a subject in need thereof is provided. In some embodiments, method comprises orally administering to the subject an orally ingestible composition in accordance with the disclosure herein. In some embodiments, the subject is a human adult. In some embodiments, the method comprises delivering to theDBl / 161990477.12subject an effective amount or concentration of an oxy gen-scavenging MV composition, wherein the MVs are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide, thereby treating gut inflammation. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, and the bacteria is selected from one or more of Komagataeibacter xylinus, Acetobacter aceli, Gluconobacter oxydans, Rhizobium leguminosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniform is, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa, thereby treating gut inflammation. In some embodiments, the subject receives, via oral administration of a pharmaceutical composition described herein, from about 0.001 mg to about 100 g of anti-inflammatory oxygen- scavenging MVs per day.

[0160] In some embodiments of the method of treating gut inflammation, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce inflammation, oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, aerobically respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating gut inflammation, the therapeutically effective amount of oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In some embodiments of the method of treating gut inflammation, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking the reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject.DBl / 161990477.12

[0161] In an embodiment, a method of treating IBD in a subject in need thereof is provided. In some embodiments, method comprises orally administering to the subject an orally ingestible composition in accordance with the disclosure herein. In some embodiments, the subject is a human adult. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MV composition, wherein the MVs are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide, thereby treating IBD. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygenscavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, and the bacteria is selected from one or more of Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosariim, Escherichia coh. Escherichia coli Nissle 1917, Akkermansia nruciniphila, Bacieroides iiriiformis, Bacteroides thetaiotaonticron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens and Rothia deniocariosa, thereby treating IBD. In some embodiments, the subject receives, via oral administration of a pharmaceutical composition described herein, from about 0.001 mg to about 100g of anti-inflammatory oxy gen-scavenging MVs per day.

[0162] In some embodiments of the method of treating IBD, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce inflammation, oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, aerobically respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating IBD, the therapeutically effective amount of oxygen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In some embodiments of the method of treating IBD, the therapeutically effective amount of oxygenscavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking theDBl / 161990477.12reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject.

[0163] In an embodiment, a method of treating IBS in a subject in need thereof is provided. In some embodiments, method comprises orally administering to the subject an orally ingestible composition in accordance with the disclosure herein. In some embodiments, the subject is a human adult. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxygen-scavenging MV composition, wherein the MVs are derived from bacteria comprising modified lipopolysaccharide or bacteria lacking the reactogenic form of lipopolysaccharide, thereby treating IBS. In some embodiments, the method comprises delivering to the subject an effective amount or concentration of an oxy genscavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, and the bacteria is selected from one or more of Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila. Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum , Corynebacterium accolens and Rothia dentocariosa, thereby treating IBS. In some embodiments, the subject receives, via oral administration of a pharmaceutical composition described herein, from about 0.001 mg to about 100g of anti-inflammatory oxygen-scavenging MVs per day.

[0164] In some embodiments of the method of treating IBS, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce inflammation, oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, aerobically respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating IBS, the therapeutically effective amount of oxy gen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In someDBl / 161990477.12embodiments of the method of treating IBS, the therapeutically effective amount of oxy genscavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking the reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject.

[0165] In some embodiments of the method of treating LPS induced inflammation, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, or a pharmaceutical composition thereof, as described herein, is an amount sufficient to reduce inflammation, oxygen levels, pro-inflammatory cytokines or other inflammatory biomarkers, respiring microbes, PBMC LPS response, in the gut, blood or tissue of a subject. In some embodiments of the method of treating LPS induced inflammation, the therapeutically effective amount of oxy gen-scavenging MVs, or a composition thereof, wherein the MVs are derived from bacteria comprising modified LPS or bacteria lacking the reactogenic form of LPS, is an amount sufficient to increase the abundance of fermenting microbes, anaerobic microbes, short chain fatty acids (SCFAs), anti-inflammatory cytokines or other anti-inflammatory biomarkers in the gut, blood or tissue of a subject. In some embodiments of the method of treating IBS, the therapeutically effective amount of oxygen-scavenging MVs derived from bacteria comprising modified LPS or from bacteria lacking the reactogenic form of LPS, or a pharmaceutical composition thereof, is an amount sufficient to reduce abdominal bloating of a subject.

[0166] In some embodiments, the method for treating a disease or condition associated with gut oxygenation and or inflammatory LPS comprises administering to the subject a therapeutically effective amount of the anti-inflammatory oxygen- scavenging membrane vesicles described herein (i.e., MVs derived from bacteria having modified lipopolysaccharide or lacking the reactogenic form of lipopolysaccharide), or a pharmaceutical composition thereof, in combination with other active pharmaceutical agents or forms of treatment. In some embodiments these active pharmaceutical agents or forms of treatment can be 2 to 5 g of 5-ASA taken daily to induce remission of active flare-ups of gut inflammation. In some embodiments these active pharmaceutical agents or forms of treatment can be 1 to 3 g of 5-ASA taken daily to maintain remission of gut inflammation. In some embodiments these active pharmaceutical agents or forms of treatment can be a fecal microbiota transplant (FMT). In some embodimentsDBl / 161990477.12this FMT consists of a selection of healthy donors through comprehensive screening, followed by collection, screening, processing and proper storage of donor stool. In some embodiments this FMT treatment consists of one or more doses of donor stool via enema, colonoscopy or oral delivery. In some embodiments this treatment consists of dosing 5 days per week for 8 weeks followed by weekly dosing for 6 weeks. In some embodiments these active pharmaceutical agents or forms of treatment can be probiotic microorganisms taken orally. In some embodiments, the method for treating a disease or condition associated with gut oxygenation and or inflammatory LPS in a subject in need thereof comprises administering to the subject: (a) a therapeutically effective amount of the anti-inflammatory oxygen-scavenging membrane vesicles, or a pharmaceutical composition thereof; and (b) a therapeutically effective amount of 5-aminosalicylic acid. In some embodiments, membrane vesicles are used to aid therapeutic interventions (e.g., fecal microbiota transplants). In some embodiments, the method for treating a disease or condition associated with gut oxygenation and or inflammatory LPS in a subject in need thereof comprises administering to the subject: (a) a therapeutically effective amount of the anti-inflammatory oxygen-scavenging membrane vesicles, or a pharmaceutical composition thereof; and (b) a fecal microbiota transplant. In some embodiments, the method for treating a disease or condition associated with gut oxygenation and or inflammatory LPS in a subject in need thereof comprises administering to the subject: (a) a therapeutically effective amount of the anti-inflammatory oxygen-scavenging membrane vesicles, or a pharmaceutical composition thereof; and (b) a probiotic microorganism.

[0167] In some aspects, the disclosure provides non-therapeutic methods for supporting, maintaining, or promoting health in a mammalian subject. These methods are directed to subjects who do not have a diagnosed disease but may be experiencing sub-optimal health or wish to maintain a state of wellness.

[0168] In some embodiments, a method is provided for supporting gastrointestinal health. This method is suitable for a subject experiencing occasional digestive discomforts such as bloating, gas, or bowel irregularity. The method comprises administering an effective amount of a composition as described herein to the subject to support a healthy gut environment, promote the integrity of the intestinal lining, support the growth of beneficial anaerobic bacteria, and maintain healthy bowel function.DBl / 161990477.12

[0169] In another embodiment, a method is provided for promoting a healthy inflammatory response. This method is suitable for subjects with a sub-optimal inflammatory status, which may be induced by factors such as diet, stress, or aging, but who have not been diagnosed with a clinical inflammatory disease. Administration of the composition helps to maintain a balanced cytokine profile and supports the body's natural processes for resolving low-grade inflammation, particularly within the gastrointestinal tract.

[0170] In a further embodiment, a method is provided for supporting a healthy balance of gut microbiota. This is suitable for subjects whose gut microbiome may be perturbed, for example, by travel, a course of antibiotics, or dietary changes. The method comprises administering the composition to help maintain an anaerobic environment in the colon, thereby favoring the maintenance of beneficial obligate anaerobes and limiting the overgrowth of facultative anaerobes and pathobionts.

[0171] In yet another embodiment, a method is provided for supporting metabolic health. This is suitable for subjects seeking to maintain healthy blood sugar levels already within the normal range or to support healthy lipid metabolism. By promoting a healthy gut environment and reducing low-grade inflammation associated with gut dysbiosis, the composition can support overall metabolic homeostasis.

[0172] The selection of subjects for these non-therapeutic methods can be based on selfreported feelings of sub-optimal wellness or based on baseline scores from the non-invasive tests and questionnaires described herein (e.g., GSRS, BSFS, PROMIS scales), where the subject's scores indicate mild or moderate deviation from an optimal state of health.Monitoring Efficacy of Treatment and Health Support

[0173] In some aspects, the efficacy of the methods and compositions described herein can be assessed by various clinical, biochemical, and patient-reported outcome measures.Improvement in a disease, condition, or state of health can be determined by a statistically significant change from baseline in one or more of the following endpoints.

[0174] In some embodiments, for Gastrointestinal and Inflammatory Conditions such as IBD, improvement can be measured by a reduction in the Crohn's Disease Activity Index (CD Al) or the Mayo Score, and by a reduction in biomarkers such as serum C-reactive proteinDBl / 161990477.12(CRP) and fecal calprotectin. For IBS and general digestive health, improvement can be measured by a reduction in the IBS Severity Scoring System (IBS-SSS) score, an improvement in the Gastrointestinal Symptom Rating Scale (GSRS), or an increase in the Gastrointestinal Quality of Life Index (GIQLI). Gut barrier integrity may be assessed by measuring serum zonulin or via the lactulose-to-mannitol ratio test.

[0175] In other embodiments, for Neurological and Pain Disorders, efficacy can be assessed by appropriate clinical scales. For Alzheimer's disease, this may include the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog). For Parkinson's disease, the Unified Parkinson's Disease Rating Scale (UPDRS) may be used. For pain disorders, efficacy is measured by a reduction in pain intensity on a Visual Analog Scale (VAS) or Numeric Rating Scale (NRS), or by a reduction in the overall score on the McGill Pain Questionnaire (MPQ) or the Brief Pain Inventory (BPI).[00176J In further embodiments, for Metabolic and Systemic Inflammatory Disorders, improvement can be measured by a reduction in hemoglobin Ale (HbAlc), fasting glucose, and insulin resistance as calculated by the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). Efficacy may also be demonstrated by a reduction in systemic inflammatory biomarkers such as high-sensitivity C-reactive protein (hs-CRP), Tumor Necrosis Factor-alpha (TNF-a), and Interleukin-6 (IL-6).

[0177] In certain embodiments, efficacy can be demonstrated by analyzing changes in the gut microbiome and metabolome. This includes, but is not limited to, an increase in microbial alpha diversity (e.g., richness and evenness), an increased relative abundance of beneficial anaerobic bacteria (e.g., Faecalibacterium prausnitzii), a decreased relative abundance of pathobionts (e.g., Enterobacteriaceae), and an increase in the concentration of short-chain fatty acids (SCFAs), such as butyrate, in stool or blood, as determined by methods such as 16S rRNA sequencing, shotgun metagenomics, or metabolomic analysis.Methods of Manufacturing

[0178] In one aspect, the disclosure provides a method of manufacturing oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, the method comprising: culturingDBl / 161990477.12the bacteria in a growth medium comprising succinate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

[0179] Any type of bacterial strain may be useful in the construction of viable bacteria comprising modified LPS or lacking the reactogenic form of LPS. Examples of bacteria useful in the present disclosure include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Akkermansia spp., Bacteroides spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetella spp., Legionella spp., Corynebacteria spp., Citrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Rothia spp., Helicobacter spp., and Vibrio spp. In some embodiments, the bacteria is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens. and Rothia dentocariosa. In some embodiments, the bacteria is selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, bacteria is Escherichia coli. Examples of Escherichia strains which may be used include, but are not limited to, Escherichia coli (E. coli) strains DH5a, HB 101, HS-4, 4608-58, 1-18468, 53638-C-17, 13-80, and 6-81 (see, e.g., Sambrook, et al., (Eds.), 1993. In: Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.); Grant, et al., 1990, Proc. Natl. Acad. Sci., USA, 87:4645; Sansonetti, et al., 1982, Ann. Microbiol. (Inst. Pasteur), 132A:351), enterotoxigenic E. coli (Evans, et al., 1975, Infect. Immun., 12:656), enteropathogenic E. coli (Donnenberg, et al., 1994, J. Infect. Dis., 169: 831; each herein incorporated by reference in their entireties) and enterohemorrhagic E. coli (see, e.g., McKee and O’Brien, 1995, Infect. Immun., 63:2070).

[0180] There are various approaches and methods for constructing bacteria with modified LPS or lacking the reactogenic form of LPS expression known to those skilled in the art, such as e.g., through suppression of D-arabinose 5-phosphate isomerase (API) expression; through mutation of the gutQ and / or kdsD genes; through suppression of 2-keto 3-deoxy-D-manno- octulosonate (KDO) expression; through inhibiting associations between KDO and lipid IVA; through mutations of the kdSA, and / or kdsB, and / or waaA and / or msbA and / or yhjD genes, or other biosynthetic, processing, or trafficking genes; through suppression of lipid IVA expression;DBl / 161990477.12through mutations of the IpxM gene, or other biosynthetic, processing, or trafficking genes for lipid IVA).

[0181] Any technique and method suitable for introducing genetic mutations within the bacteria from which the oxygen-scavenging membrane vesicles, and fragments thereof, disclosed herein, are derived is encompassed by the present disclosure. Exemplary techniques include non-specific mutagenesis, e.g., using chemical agents, e.g., N-methyl-N'-nitro N- nitrosoguanidine, acridine orange, ethidium bromide, or non-lethal exposure to ultraviolet light (see, e.g., Miller (Ed.), 1991. In: A Short Course in Bacterial Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). Alternatively, the mutations can be introduced using TnlO mutagenesis, bacteriophage-mediated transduction, lambda phage-mediated allelic exchange, or conjugational transfer, or site directed mutagenesis using recombinant DNA techniques (see, e.g., Miller (Ed.), 1991, supra; Hone, et al., 1987, J. Infect. Dis., 156:167; Noriega, et al., 1994, Infect. Immun., 62:5168; Hone, et al., 1991, Vaccine, 9:810; Chatfield, et al., 1992, Vaccine, 10:53: Pickard, et al., 1994, Infect. Immun., 62:3984;Odegaard, et al., 1997, J. Biol. Chem., 272: 19688; Lee, et al., 1995, J. Biol. Chem., 270:27151; Garrett, et al., 1998, J. Biol. Chem, 273: 12457; each herein incorporated by reference in its entirety). The mutations can be introduced in conjunction with one or more additional mutations (e.g., Gram-negative bacteria with more than one mutation such as mutations in the gutQ, kdsD, kdsA, kdsB, waaA msbA, yhjD genes, or mutations in any other biosynthetic, processing, or trafficking gene).

[0182] Bacteria suitable for use in the method of manufacturing the oxygen-scavenging membrane vesicles include any of those bacteria which have been described herein, such as, bacteria comprising modified LPS or lacking the reactogenic form of LPS (i.e., mutant bacterial strains with mutations in the kdsD and / or gutQ, kdsA, kdsB, waaA, msbA, ynjD genes, or other biosynthetic, processing, or trafficking genes). In some embodiments, bacteria suitable for use in the method of manufacturing the oxygen-scavenging membrane vesicles include bacteria comprising a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1 A. In some embodiments, bacteria suitable for use in the method of manufacturing the oxygen- scavenging membrane vesicles include gram-negative bacteria comprising modifications to one or more protein sequences set forth in Table IB. In someDBl / 161990477.12embodiments, bacteria suitable for use in the method of manufacturing the oxygen-scavenging membrane vesicles include bacteria comprising one or more terminal oxidase proteins set forth in Table 1C.

[0183] In some embodiments, the bacteria may be genetically engineered via cloning methods known to those skilled in the art (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; incorporated herein by reference in its entirety) to express, produce and display non-native proteins and peptides such as, but not limited to, modified LPS, LPS from other bacterial organisms, unique lipid derivatives, human protein or peptide production, non-human protein or peptide production, vaccine production, and the like.

[0184] In an exemplary embodiment, E. coll strain KPM22 is used as the host for chromosomal kdsA (E. coli strain KPM31) and waaA (E. coli strain KPM40) gene disruptions using the phage . Red recombinase system according to the procedure of Datsenko and Wanner (see, e.g., K. A. Datsenko, B. L. Wanner, Proc. Natl. Acad. Sci. U.S.A. 97, 6640 (2000); herein incorporated by reference in its entirety). Kanamycin and ampicillin were used at 15 g / mL and 100 ug / mL, respectively. Primer pairs P1 / P2 with pKD13(kan) or P3 / P4 with pKD4(kan) as tempplates were used to construct insert cassettes for KPM31 and KPM40, respectively.Antibiotic resistance markers were excised using the FLP recombinase system as described (see, e.g., K. A. Datsenko, B. L. Wanner, Proc. Natl. Acad. Sci. U.S.A. 97, 6640 (2000); herein incorporated by reference in its entirety), except all plasmids were cured at 37°C.Table 2: Primers used to produce viable E. coli comprising non-immunogenic LPSDBl / 161990477.12

[0185] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS or lacks LPS, and are selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS or lacks LPS, and are selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS or lacks LPS, and are Escherichia coli.

[0186] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS that is a non- immunogenic LPS, and are selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxy genscavenging MVs comprises non-immunogenic LPS, and are selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises non-immunogenic LPS and are Escherichia coli.

[0187] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS that has a Lipid A component consisting of less than 6 acyl chains (e.g., 1, 2, 3, 4, or 5 acyl chains), and is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that has a Lipid A component consisting of less than 6 acyl chains (e.g., 1, 2, 3, 4, or 5 acyl chains), and are selected from Escherichia coli and Escherichia coli Nissle 1917. InDBl / 161990477.12some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that has a Lipid A component consisting of less than 6 acyl chains (e.g., 1, 2, 3, 4, or 5 acyl chains), and are. Escherichia coll.

[0188] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS that has a Lipid A component consisting of 4 acyl chains, and is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium legummosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron , Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that has a Lipid A component consisting of 4 acyl chains, and are selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, the bacteria used for the manufacturing of oxygenscavenging MVs comprises modified LPS that has a Lipid A component consisting of 4 acyl chains, and are Escherichia coli .

[0189] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS that does not contain 2- keto 3-deoxy-D-manno-octulosonate (KDO), and is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that does not contain 2-keto 3-deoxy-D-manno-octulosonate (KDO), and are selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that does not contain 2-keto 3-deoxy-D- manno-octulosonate (KDO), and are Escherichia coli.

[0190] In some embodiments, the bacteria used in the method of manufacturing the oxygenscavenging membrane vesicles described herein comprises modified LPS that is lipid IVA, and is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, RhizobiumDBl / 161990477.12leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides unifomns, Bacteroides thetaiotaomicron Phocaeicola vulgatus, C orynehacterium pseudodiphtheriticum, C orynehacterium accolens. and Rothia dentocariosa. In some embodiments, the bacteria used for the manufacturing of oxygen-scavenging MVs comprises modified LPS that is lipid IVA, and are selected from Escherichia coli and Escherichia coli Nissle 1917. In some embodiments, the bacteria used for the manufacturing of oxygenscavenging MVs comprises modified LPS that is lipid IVA, and are Escherichia coli.

[0191] In some embodiments, the method of manufacturing the oxygen- scavenging membrane vesicles described herein comprises culturing the bacteria in a growth medium comprising succinate to obtain a culture. In some embodiments, the growth medium comprising succinate is a liquid medium, such as LB medium (Difco, Detroit Mich.), Nutrient broth (Difco), Tryptic Soy broth (Difco), or M9 minimal broth (Difco). In some embodiments, the bacteria may be cultured in the presence of succinate on solid media such as L-agar (Difco), Nutrient agar (Difco), Tryptic Soy agar (Difco), or M9 minimal agar (Difco).

[0192] In some embodiments, method of manufacturing the oxygen-scavenging membrane vesicles described herein comprises culturing the bacteria in tryptic soy broth (TSB) comprising succinate. In some embodiments, the bacteria is cultured for any suitable amount of time, such as, from about 12 hours to about 60 hours. In some embodiments, the bacteria is cultured at any suitable temperature, such as, from about 20°C to about 46°C. In some embodiments, the bacteria are grown in TSB comprising succinate without glucose. In some embodiments, the bacteria are grown in TSB comprising 50 mM to 750 mM succinate without glucose. In some embodiments, the bacteria are grown in TSB comprising 150 mM to 650 mM succinate without glucose. In some embodiments, the bacteria are grown in TSB comprising succinate and lactic acid, and / or their corresponding salt. In some embodiments, the bacteria are grown in TSB comprising 50 mM to 750 mM succinate and 50 mM to 750 mM lactic acid, and / or their corresponding salt. In some embodiments, the bacteria are grown in TSB comprising 150 mM to 650 mM succinate and 150 mM to 650 mM lactic acid, and / or their corresponding salt. Lactate may also be included as it is another key substrate for respiratory enzymes present in the membrane vesicles, thereby enhancing their total oxygen-scavenging capacity.DBl / 161990477.12

[0193] In some embodiments, the method of manufacturing the oxygen- scavenging membrane vesicles described herein comprises culturing the bacteria in a growth medium comprising succinate to obtain a culture. In some embodiments, the bacterial cells are harvested and concentrated by centrifugation. In further embodiments, the bacterial cells are concentrated by centrifugation at 10,000xG for 15 min. In other embodiments, the bacterial cell pellets are resuspended in phosphate buffered saline. In further embodiments, the bacterial cell pellets are resuspended in phosphate buffered saline containing succinic acid and lactic acid, and / or their respective salts. In some embodiments, the bacterial cell pellets are resuspended in phosphate buffered saline containing 650 mM succinic acid and / or its corresponding salt, and containing 650 mM lactic acid, and / or its respective salt.

[0194] In some embodiments, the bacterial cells are lysed by passing through a microfluidizer. In other embodiments, the bacterial cells are lysed by passing through a microfluidizer at 15,000 PSI. In some embodiments, the bacterial cells are lysed by passing twice through a microfluidizer at 15,000 PSI. In some embodiments, the bacterial cells are lysed by passing three times through a microfluidizer at 15,000 PSI.

[0195] In other embodiments, the bacterial cell lysate is purified using centrifugation and filtration. In specific embodiments, the bacterial cell lysate is purified using centrifugation followed by tangential flow filtration through a PES hollow fiber. In some embodiments, the bacterial cell lysate is purified using tangential flow filtration through a 500K Da PES hollow fiber. In some embodiments the lysate is purified using a 0.45 pM filter. In some embodiments the lysate is purified using a 0.2 pM filter, thereby isolating the oxygen-scavenging membrane vesicles from the culture.Dosages and Dosing Regimens

[0196] The amount of the anti-inflammatory oxygen- scavenging membrane vesicles, or pharmaceutical compositions thereof, administered using the methods herein, such as the dosages of the present composition, is dependent on the human or non-human mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician. In some embodiments, an effective dosage is in the range of about 0.000014 mg to about 1.43 g per kg body weight per day, such as about 0.000014 mg to about 1.43 g / kg / day, in single or dividedDBl / 161990477.12doses. For a 70 kg human, this would amount to about 0.001 mg to about 100 g / day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g., by dividing such larger doses into several small doses for administration throughout the day. The dosage of the pharmaceutical compositions and active pharmaceutical ingredients may be provided in units of mg / kg of body mass or in mg / m2of body surface area.

[0197] In some embodiments, the active pharmaceutical ingredient (z.e., the antiinflammatory oxygen-scavenging membrane vesicles) or pharmaceutical composition thereof is administered in a single dose. Such administration may be by injection, e g., intravenous injection, in order to introduce the active pharmaceutical ingredient quickly. However, other routes, including the oral route, may be used as appropriate. A single dose of a pharmaceutical composition may also be used for treatment of an acute condition.[00198J In some embodiments, the active pharmaceutical ingredient or pharmaceutical composition thereof is administered in multiple doses. In an embodiment, a pharmaceutical composition is administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be once a month, once every two weeks, once a week, or once every other day. In other embodiments, a pharmaceutical composition is administered about once per day to about 6 times per day. In some embodiments, a pharmaceutical composition is administered once daily, while in other embodiments, a pharmaceutical composition is administered twice daily, and in other embodiments a pharmaceutical composition is administered three times daily.

[0199] Administration of the active pharmaceutical ingredient or pharmaceutical composition thereof may continue as long as necessary. In some embodiments, the active pharmaceutical ingredient or pharmaceutical composition thereof is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 day(s). In some embodiments, the active pharmaceutical ingredient or pharmaceutical composition thereof is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day(s). In some embodiments, the active pharmaceutical ingredient or pharmaceutical composition thereof is administered chronically on an ongoing basis - e.g., for the treatment of chronic effects. In some embodiments, the administration of the active pharmaceutical ingredient or pharmaceutical composition thereof continues for less than about 7 days. In yet anotherDBl / 161990477.12embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.

[0200] In some embodiments, an effective dosage of the active pharmaceutical ingredient (z.e., the anti-inflammatory oxygen-scavenging membrane vesicles) or pharmaceutical composition thereof, as disclosed herein, is in the range of about 0.001 mg to about 100 g. about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg. In some embodiments, an effective dosage of the anti-inflammatory oxygen-scavenging membrane vesicles disclosed herein is less than about 1 mg, less than about 10 mg, less than about 25 mg, less than about 50 mg, less than about 75 mg, less than about 100 mg, less than about 125 mg, less than about 150 mg, less than about 175 mg, less than about 200 mg, less than about 225 mg, or less than about 250 mg. In some embodiments, an effective dosage of the active pharmaceutical ingredient disclosed herein is greater than about 25 mg, greater than about 50 mg, greater than about 75 mg, greater than about 100 mg, greater than about 125 mg, greater than about 150 mg, greater than about 175 mg, greater than about 200 mg, greater than about 225 mg, or greater than about 250 mg.

[0201] In some embodiments, an effective dosage of the MVs disclosed herein, for example the present composition, is in the range of about 0.000014 mg / kg to about 1.43 g / kg, or about 0.00014 mg / kg to about 0.7 g / kg, or about 0.0014 mg / kg to about 0.35 g / kg.

[0202] In some embodiments, MVs, or pharmaceutical composition thereof, is administered at a dosage of 0.00033 mg to 33 g once daily, twice daily, or three times daily. In some embodiments, MVs, or pharmaceutical compositions thereof, is administered as doses including 1, 10, 20, 30, 40 50, 60, 70, 80, 90, 100, 150, or 200 mg once daily, twice daily, or three timesDBl / 161990477.12daily. In some embodiments, the active pharmaceutical ingredient is administered at a dosage of 10 to 500 mg once daily, twice daily, or three times daily, including 1 , 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400, or 500 mg BID.

[0203] In some instances, dosage levels below the lower limit of the aforesaid ranges may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g., by dividing such larger doses into several small doses for administration throughout the day. Of course, as those skilled in the art appreciate, the dosage actually administered depends upon the condition being treated, the age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment. Moreover, the effective dosage amount may be determined by one skilled in the art on the basis of routine empirical activity testing to measure the bioactivity of the MVs in a bioassay, and thus establish the appropriate dosage to be administered.[00204J In some embodiments, the compositions described herein further include controlled- release, sustained release, or extended-release therapeutic dosage forms for administration of the anti-inflammatory oxygen-scavenging membrane vesicles described herein, which involves incorporation of the MVs into a suitable delivery system in the formation of certain compositions. This dosage form controls release of the MVs in such a manner that an effective concentration of the MVs in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and / or minimize side effects. Additionally, a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the MVs.

[0205] An effective amount of the anti-inflammatory oxygen-scavenging membrane vesicles, or pharmaceutical compositions thereof, may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.

[0206] For non-pharmaceutical compositions, such as dietary supplements, medical foods, or food products, the amount administered is referred to as a “serving” or “serving size” rather thanDBl / 161990477.12a “dosage.” The effective amount is an amount sufficient to provide the intended health support, such as promoting a healthy gut microbiota or supporting a balanced inflammatory response.

[0207] The recommended serving amount of the anti-inflammatory oxygen-scavenging membrane vesicles can vary depending on the intended use and the subject. In some embodiments, an effective daily serving is in the range of about 0.001 mg to about 10 g of MVs, for example from about 1 mg to about 1 g. This daily amount may be consumed in a single serving or divided into multiple servings throughout the day.

[0208] For a dietary supplement, a typical serving size might be one or two capsules, tablets, or one scoop of powder, providing from about 10 mg to about 500 mg of MVs per serving. For a medical food or functional food product, the amount of MVs may be incorporated to provide an effective amount within a standard unit of consumption (e.g., per 8 oz beverage, per nutritional bar).

[0209] Administration may be on a daily basis for continuous support of gastrointestinal health. In some embodiments, the composition is taken for a defined period to support the gut microbiome during a specific challenge, such as during travel or following a course of antibiotics. The specific regimen can be adapted by the consumer to meet their individual health and wellness goals.NUMBERED EMBODIMENTS

[0210] The paragraphs below set forth exemplary embodiments of the instant disclosure. It is to be understood that the elements of each of these embodiments can be combined in any permutation to produce further embodiments within the scope and spirit of the instant disclosure.

[0211] Embodiment 1. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of oxygen- scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

[0212] Embodiment 2. The method of embodiment 1, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Ghiconobacter oxydans, Rhizobium legumiaosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila,DBl / 161990477.12Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Coryiiebacterium pseudodiphtheriticum Corynebacterium accolens, and Rothia dentocariosa.

[0213] Embodiment 3. The method of embodiment 1 or 2, wherein the bacterium is Escherichia coli.

[0214] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0215] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0216] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0217] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0218] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0219] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the modified lipopolysaccharide is lipid IVA.

[0220] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the disease or condition is associated with gut oxygenation.

[0221] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0222] Embodiment 12. A pharmaceutical composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modifiedDBl / 161990477.12lipopolysaccharide or the bacterium lacks lipopolysaccharide; and a pharmaceutically acceptable excipient.

[0223] Embodiment 13. The pharmaceutical composition of embodiment 12, wherein the composition is formulated in an oral dosage form.

[0224] Embodiment 14. The pharmaceutical composition of embodiment 12 or 13, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coh. Escherichia coli Nissle 1917, Akkermansia nruciniphila, Bacteroides uniforntis, Bacteroides thetaiotaonticron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens and Rothia dentocariosa.

[0225] Embodiment 15. The pharmaceutical composition of any one of embodiments 12 to14, wherein the bacterium is Escherichia coli.

[0226] Embodiment 16. The pharmaceutical composition of any one of embodiments 12 to15, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0227] Embodiment 17. The pharmaceutical composition of any one of embodiments 12 to16, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0228] Embodiment 18. The pharmaceutical composition of any one of embodiments 12 to17, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0229] Embodiment 19. The pharmaceutical composition of any one of embodiments 12 to18, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0230] Embodiment 20. The pharmaceutical composition of any one of embodiments 12 to19, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno- octulosonate.DBl / 161990477.12

[0231] Embodiment 21. The pharmaceutical composition of any one of embodiments 12 to 20, wherein the modified lipopolysaccharide is lipid IVA.

[0232] Embodiment 22. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 12 to 21.

[0233] Embodiment 23. The method of embodiment 22, wherein the disease or condition is associated with gut oxygenation.

[0234] Embodiment 24. The method of embodiment 22 or 23, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0235] Embodiment 25. A method of manufacturing oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, the method comprising: culturing the bacteria in a growth medium comprising succinate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

[0236] Embodiment 26. The method of embodiment 25, wherein the bacterium is selected from Komagataeibacter xylimis, Acetobacter ciceti, Gluconobacter oxydans Rhizobium leguminosarum Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Coryne bacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

[0237] Embodiment 27. The method of embodiment 25 or 26, wherein the bacterium is Escherichia coli.

[0238] Embodiment 28. The method of any one of embodiments 25 to 27, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.DBl / 161990477.12

[0239] Embodiment 29. The method of any one of embodiments 25 to 28, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0240] Embodiment 30. The method of any one of embodiments 25 to 29, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0241] Embodiment 31. The method of any one of embodiments 25 to 30, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0242] Embodiment 32. The method of any one of embodiments 25 to 31, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0243] Embodiment 33. The method of any one of embodiments 25 to 32, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0244] Embodiment 34. The method of any one of embodiments 25 to 33, wherein the modified lipopolysaccharide is lipid IVA.

[0245] Embodiment 35. The method of embodiment 1, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

[0246] Embodiment 36. The method of embodiment 35, wherein the bacterium is Escherichia coli.

[0247] Embodiment 37. The method of embodiment 35 or 36, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0248] Embodiment 38. The method of any one of embodiments 35 to 37, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0249] Embodiment 39. The method of any one of embodiments 35 to 38, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0250] Embodiment 40. The method of any one of embodiments 35 to 39, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.DBl / 161990477.12

[0251] Embodiment 41. The method of any one of embodiments 35 to 40, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0252] Embodiment 42. The method of any one of embodiments 35 to 41, wherein the modified lipopolysaccharide is lipid IVA.

[0253] Embodiment 43. The method of any one of embodiments 35 to 42, wherein the disease or condition is associated with gut oxygenation.

[0254] Embodiment 44. The method of any one of embodiments 35 to 43, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0255] Embodiment 45. The pharmaceutical composition of embodiment 12, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

[0256] Embodiment 46. The pharmaceutical composition of embodiment 45, wherein the composition is formulated in an oral dosage form.

[0257] Embodiment 47. The pharmaceutical composition of embodiment 45 or 46, wherein the bacterium is Escherichia coli.

[0258] Embodiment 48. The pharmaceutical composition of any one of embodiments 45 to47, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0259] Embodiment 49. The pharmaceutical composition of any one of embodiments 45 to48, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0260] Embodiment 50. The pharmaceutical composition of any one of embodiments 45 to49, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.DBl / 161990477.12

[0261] Embodiment 51. The pharmaceutical composition of any one of embodiments 45 to50, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0262] Embodiment 52. The pharmaceutical composition of any one of embodiments 45 to51, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno- octulosonate.

[0263] Embodiment 53. The pharmaceutical composition of any one of embodiments 45 to52, wherein the modified lipopolysaccharide is lipid IVA.

[0264] Embodiment 54. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 45 to 53.

[0265] Embodiment 55. The method of embodiment 54, wherein the disease or condition is associated with gut oxygenation.

[0266] Embodiment 56. The method of embodiment 54 or 55, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0267] Embodiment 57. The method of embodiment 25, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

[0268] Embodiment 58. The method of embodiment 57, wherein the bacterium is Escherichia coli.

[0269] Embodiment 59. The method of embodiment 57 or 58, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.DBl / 161990477.12

[0270] Embodiment 60. The method of any one of embodiments 57 to 59, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0271] Embodiment 61. The method of any one of embodiments 57 to 60, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0272] Embodiment 62. The method of any one of embodiments 57 to 61, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0273] Embodiment 63. The method of any one of embodiments 57 to 62, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0274] Embodiment 64. The method of any one of embodiments 57 to 63, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0275] Embodiment 65. The method of any one of embodiments 57 to 64, wherein the modified lipopolysaccharide is lipid IVA.

[0276] Embodiment 35A. The method of any one of embodiments 1, 2, or embodiment 35, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

[0277] Embodiment 36A. The method of embodiment 35 A, wherein the bacterium isEscherichia coli.

[0278] Embodiment 37A. The method of embodiment 35A or 36A, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0279] Embodiment 38A. The method of any one of embodiments 35A to 37A, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0280] Embodiment 39A. The method of any one of embodiments 35A to 38 A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0281] Embodiment 40A. The method of any one of embodiments 35A to 39A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.DBl / 161990477.12

[0282] Embodiment 41A. The method of any one of embodiments 35A to 40 A, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0283] Embodiment 42A. The method of any one of embodiments 35A to 41A, wherein the modified lipopolysaccharide is lipid IVA.

[0284] Embodiment 43 A. The method of any one of embodiments 35A to 42A, wherein the disease or condition is associated with gut oxygenation.

[0285] Embodiment 44A. The method of any one of embodiments 35A to 43 A, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0286] Embodiment 45A. The pharmaceutical composition of embodiment 12 or embodiment 45, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

[0287] Embodiment 46A. The pharmaceutical composition of embodiment 45A, wherein the composition is formulated in an oral dosage form.

[0288] Embodiment 47A. The pharmaceutical composition of embodiment 45A or 46A, wherein the bacterium is Escherichia coli.

[0289] Embodiment 48A. The pharmaceutical composition of any one of embodiments 45A to 47A, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0290] Embodiment 49A. The pharmaceutical composition of any one of embodiments 45A to 48A, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0291] Embodiment 50A. The pharmaceutical composition of any one of embodiments 45A to 49A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.DBl / 161990477.12

[0292] Embodiment 51A. The pharmaceutical composition of any one of embodiments 45A to 50A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0293] Embodiment 52A. The pharmaceutical composition of any one of embodiments 45A to 51 A, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno- octulosonate.

[0294] Embodiment 53A. The pharmaceutical composition of any one of embodiments 45A to 52A, wherein the modified lipopolysaccharide is lipid IVA.

[0295] Embodiment 54A. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 45A to 53A.

[0296] Embodiment 55A. The method of embodiment 54A, wherein the disease or condition is associated with gut oxygenation.

[0297] Embodiment 56A. The method of embodiment 54A or 55A, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0298] Embodiment 57A. The method of embodiment 25 or embodiment 57, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

[0299] Embodiment 58A. The method of embodiment 57A, wherein the bacterium is Escherichia coli.

[0300] Embodiment 59A. The method of embodiment 57A or 58A, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.DBl / 161990477.12

[0301] Embodiment 60A. The method of any one of embodiments 57 A to 59A, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0302] Embodiment 61A. The method of any one of embodiments 57A to 60A, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0303] Embodiment 62A. The method of any one of embodiments 57A to 61A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0304] Embodiment 63A. The method of any one of embodiments 57A to 62A, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0305] Embodiment 64A. The method of any one of embodiments 57A to 63 A, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0306] Embodiment 65A. The method of any one of embodiments 57A to 64A, wherein the modified lipopolysaccharide is lipid IVA.

[0307] Embodiment 35B. The method of any one of embodiments 1, 2, 35, or 35A, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0308] Embodiment 36B. The method of embodiment 35B, wherein the bacterium is Escherichia coli.

[0309] Embodiment 37B. The method of embodiment 35B or 36B, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0310] Embodiment 38B. The method of any one of embodiments 35B to 37B, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0311] Embodiment 39B. The method of any one of embodiments 35B to 38B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0312] Embodiment 40B. The method of any one of embodiments 35B to 39B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0313] Embodiment 41B. The method of any one of embodiments 35B to 40B, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.DBl / 161990477.12

[0314] Embodiment 42B. The method of any one of embodiments 35B to 4 IB, wherein the modified lipopolysaccharide is lipid IVA.

[0315] Embodiment 43B. The method of any one of embodiments 35B to 42B, wherein the disease or condition is associated with gut oxygenation.

[0316] Embodiment 44B. The method of any one of embodiments 35B to 43B, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0317] Embodiment 45B. The pharmaceutical composition of any one of embodiments 12, 45, or 45 A, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0318] Embodiment 46B. The pharmaceutical composition of embodiment 45B, wherein the composition is formulated in an oral dosage form.

[0319] Embodiment 47B. The pharmaceutical composition of embodiment 45B or 46B, wherein the bacterium is Escherichia coli.

[0320] Embodiment 48B. The pharmaceutical composition of any one of embodiments 45B to 47B, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0321] Embodiment 49B. The pharmaceutical composition of any one of embodiments 45B to 48B, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0322] Embodiment 50B. The pharmaceutical composition of any one of embodiments 45B to 49B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0323] Embodiment 51B. The pharmaceutical composition of any one of embodiments 45B to 50B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.DBl / 161990477.12

[0324] Embodiment 52B. The pharmaceutical composition of any one of embodiments 45B to 5 IB, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno- octulosonate.

[0325] Embodiment 53B. The pharmaceutical composition of any one of embodiments 45B to 52B, wherein the modified lipopolysaccharide is lipid IVA.

[0326] Embodiment 54B. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 45B to 53B.

[0327] Embodiment 55B. The method of embodiment 54B, wherein the disease or condition is associated with gut oxygenation.

[0328] Embodiment 56B. The method of embodiment 54B or 55B, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0329] Embodiment 57B. The method of any one of embodiments 25, 57, or 57A, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0330] Embodiment 58B. The method of embodiment 57B, wherein the bacterium is Escherichia coli.

[0331] Embodiment 59B. The method of embodiment 57B or 58B, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0332] Embodiment 60B. The method of any one of embodiments 57B to 59B, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0333] Embodiment 61B. The method of any one of embodiments 57B to 60B, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.DBl / 161990477.12

[0334] Embodiment 62B. The method of any one of embodiments 57B to 61B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 1, 2, 3, 4, or 5 acyl chains.

[0335] Embodiment 63B. The method of any one of embodiments 57B to 62B, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0336] Embodiment 64B. The method of any one of embodiments 57B to 63B, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0337] Embodiment 65B. The method of any one of embodiments 57 to 64, wherein the modified lipopolysaccharide is lipid IVA.

[0338] Embodiment 35C. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0339] Embodiment 36C. The method of embodiment 35C, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosaru , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

[0340] Embodiment 37C. The method of embodiment 35C or 36C, wherein the bacterium is Escherichia coli.

[0341] Embodiment 38C. The method of any one of embodiments 35C to 37C, wherein the disease or condition is associated with gut oxygenation.

[0342] Embodiment 39C. The method of any one of embodiments 35C to 38C, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.DBl / 161990477.12

[0343] Embodiment 40C. A pharmaceutical composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C; and a pharmaceutically acceptable excipient.

[0344] Embodiment 41C. The pharmaceutical composition of embodiment 40C, wherein the composition is formulated in an oral dosage form.

[0345] Embodiment 42C. The pharmaceutical composition of embodiment 40C or 41C, wherein the bacterium is selected from Komagataeibacter xylinus. Acetobacter aceti, Gluconobacter oxydans, Rhizobium legiiminosanim, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides umformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accoleiis, and Rothia dentocariosa.

[0346] Embodiment 43C. The pharmaceutical composition of any one of embodiments 40C to 42C, wherein the bacterium is Escherichia coli.

[0347] Embodiment 44C. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of embodiments 40C to 43C.

[0348] Embodiment 45C. The method of embodiment 44C, wherein the disease or condition is associated with gut oxygenation.

[0349] Embodiment 46C. The method of embodiment 44C or 45C, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

[0350] Embodiment 47C. A method of manufacturing oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C, the method comprising: culturing the bacteria in a growth mediumDBl / 161990477.12comprising succinate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

[0351] Embodiment 48C. A method of embodiment 47C, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydctns. Rhizobium leguminosarum, Escherichia colt. Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

[0352] Embodiment 49C. The method of embodiment 47C or 48C, wherein the bacterium is Escherichia coli.

[0353] Embodiment 35D. A nutritional composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide; and a nutritionally acceptable excipient.

[0354] Embodiment 36D. The nutritional composition of embodiment 35D, wherein the nutritional composition is selected from a food product, a dietary supplement, or a medical food.

[0355] Embodiment 37D. The nutritional composition of embodiment 35D or embodiment 36D, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

[0356] Embodiment 38D. The nutritional composition of any one of embodiments 35D to 37D, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

[0357] Embodiment 39D. The nutritional composition of any one of embodiments 35D to 38D, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0358] Embodiment 40D. A non-therapeutic method for supporting gastrointestinal health in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.DBl / 161990477.12

[0359] Embodiment 41D. The method of embodiment 40D, wherein the subject is a healthy subject or a subject experiencing occasional digestive discomforts such as bloating, gas, or bowel irregularity.

[0360] Embodiment 42D. A non-therapeutic method for promoting a healthy inflammatory response in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

[0361] Embodiment 43D. The method of embodiment 42D, wherein the subject has a sub- optimal inflammatory status.

[0362] Embodiment 44D. A non-therapeutic method for supporting a healthy balance of gut microbiota in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

[0363] Embodiment 45D. The method of embodiment 44D, wherein the subject's gut microbiota is perturbed by factors such as travel, antibiotic use, or dietary changes.

[0364] Embodiment 46D. A non-therapeutic method for supporting metabolic health in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

[0365] Embodiment 47D. The method of any one of embodiments 40D to 46D, wherein the composition is a nutritional composition, a food product, a dietary supplement, or a medical food.

[0366] Embodiment 48D. The method of any one of embodiments 40D to 47D, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coll, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, PhocaeicolaDBl / 161990477.12vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

[0367] Embodiment 49D. The method of any one of embodiments 40D to 48D, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

[0368] Embodiment 50D. The method of any one of embodiments 40D to 49D, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

[0369] Embodiment 51D. The method of any one of embodiments 40D to 50D, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

[0370] Embodiment 52D. The method of any one of embodiments 40D to 5 ID, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

[0371] Embodiment 53D. The method of any one of embodiments 40D to 52D, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

[0372] Embodiment 54D. The method of any one of embodiments 40D to 53D, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

[0373] Embodiment 55D. The method of any one of embodiments 40D to 54D, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

[0374] Embodiment 56D. The method of any one of embodiments 40D to 55D, wherein the modified lipopolysaccharide is lipid IVA.

[0375] The following examples describe embodiments of the disclosure in further detail. These examples are provided for illustrative purposes only and should in no way be considered as limiting the disclosure.EXAMPLES

[0376] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to theseDBl / 161990477.12examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent approaches that have been found to function well in the practice of the disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are described herein and still obtain a like or similar result without departing from the spirit and scope of the disclosure.Example 1. Bacterial membrane vesicles restore gut anaerobiosis

[0377] Gut inflammation is a prevalent health concern that manifests in a host of conditions including IBS, IBD, Alzheimer’s, and inflarmnaging. While the etiology of gut inflammation is multifaceted, with risk factors including stress, genetics, infections, diet, and xenobiotics, mounting evidence has implicated the gut microbiota as having an often-pivotal role in the development, progression, and chronic persistence of gut inflammation. Loss of short chain fatty acid (SCFA)-producing obligate anaerobes and an increase in inflammatory, facultatively anaerobic Enterob acteriaceae is a common hallmark of an inflamed gut, where oxidants are thought to drive this gut dysbiosis. In a healthy gut, intestinal epithelial cells (lECs) metabolize SCFAs through -oxidation and oxidative phosphorylation, a process which converts O2 to H2O and limits the oxygen supplied by blood vesicles from diffusing into the gut. Low O2 levels also limit the production of NO3 from NO caused by the inflammatory response. Nitrate serves as an alternative electron acceptor for the respiratory chain and supports growth of Enterobacteriaceae. Diminishing SCFA or damaging lECs can shift the lECs’ metabolism towards glycolysis which does not consume oxygen, allowing it to diffuse into the gut. O2 in the intestinal lumen kills obligately anaerobic bacteria and, together with NO3, causes blooms of facultative anaerobes. The facultative anaerobes that take advantage of these environments are members of the Enterobacteriaceae family, a clade comprised of many pathogens and pathobionts which can express a multitude of virulence factors and immunogens, such as inflammatory lipopolysaccharide (LPS), also known as endotoxin. The cascade of events arising from oxygenation of the gut can cause or exacerbate gut inflammation which further disrupts IEC function and helps maintain the chronic presence of facultative microbes. Oxygen-driven dysbiosis of the gut has been confirmed in mouse models for colitis, colorectal cancer, cancerDBl / 161990477.12cachexia, and graft-versus-host disease. Oxygen induced dysbiosis and gut inflammation links an otherwise disparate set of animal models, and the similarities between these signatures and those found in diseases like Alzheimer’s and inflammaging highlights the deleterious role gut oxygenation may play across a host of conditions.

[0378] It was hypothesized that oxygen can be depleted in the gut by bacterial membrane vesicles containing the respiratory chain. Membrane vesicles (MVs) from E. coli were employed to remove oxygen from the gut in a mouse model of inflammation. This treatment improved mouse health, reduced inflammation and produced a functional shift in the microbiota, favoring symbionts producing short chain fatty acids (SCFAs).Membrane vesicles inhibit pathogenic Enterobacteriaceae growth through the reduction of oxygen.

[0379] Membrane vesicles from E. coli were used to deplete O2 and diminish the growth of facultatively anaerobic bacteria such as Enterobacteriaceae in the gut. In order to eliminate contaminating LPS, ClearColi was used because ClearColi have a modified LPS containing only the non-inflammatory lipid IVA. Succinate is commonly present in the gut, is enriched during inflammation, and is the substrate of membrane-bound succinate dehydrogenase (an enzyme of the respiratory chain); in view of this, membrane vesicles from ClearColi grown aerobically in a medium with succinate were prepared. ClearColi were disrupted at high pressure, which produced inverted vesicles of the inner membrane, with succinate dehydrogenase facing outward. The vesicles were first characterized in vitro, where they rapidly depleted O2 in the presence of succinate (Fig. 1A). A hemB knockout of ClearColi (Fig. IB) was made to produce control vesicles that lack cytochromes (Fig. 1C). As expected, AhemB vesicles do not respire (Fig. 1A), but have similar morphology to wild type vesicles (Fig. ID). Next, the ability of MVs to suppress the growth of three different species of Enterobacteriaceae - Escherichia coli, Klebsiella pneumoniae, and Citrobacter rodentium - was examined under aerobic conditions (Fig. IE). After 7 hours, the wt MVs inhibited the growth of all three strains indicating they may be effective at reducing the burden of pathogenic Enterobacteriaceae in vivo.DBl / 161990477.12Membrane vesicles ameliorate colitis.

[0380] Having designed membrane vesicles that inhibited the growth of C. rodentium through reducing oxygen, these vesicles were then tested as a therapeutic to reduce inflammation. To accomplish this, membrane vesicles were added to food in a C. rodentium- induced colitis model in C3H / HeNCrl mice (C3H) (Fig. 2A). Mice were weighed every 24 hours to assess progression of the disease. Mice treated with wt MVs had a significantly higher body weight than the colitis and AhemB MVs groups, indicating that the treatment reduced the severity of the disease (Fig. 2B, 2C). Furthermore, weight loss between the colitis control and AhemB MVs groups was nearly identical, suggesting that the improvement in health by treatment with wt MVs is due to the vesicles’ ability to reduce oxygen. C3H mice are unable to clear C. rodentium which means that unless the pathogen is fully eradicated, as with antibiotic treatment, the infection will be fatal. This is why although an improvement was seen in the health of wt MV treated mice, they still lost weight. Colon length also decreased in colitis and served as an important marker of disease. A significant increase in the colon length of mice treated with the wt MVs was observed, while there was no change in the AhemB MVs group (Fig. 2D). Taken together, these results show that treating mice with respiring MVs reduced disease severity.Membrane vesicles reduce the colonic epithelium and modulate the cytokine response.

[0381] In a healthy gut, the level of oxygen is low in the epithelial layer, but increases with inflammation. Thus, oxidation of the epithelial layer of mice with colitis was then compared to that of animals treated with MVs using pimonidazole labeling. Pimonidazole is a 2- nitroimidazole which forms covalent bonds with thiols of amino acids upon reduction. The pimonidazole-protein adducts were then probed with fluorescently-labeled antibodies allowing for visualization of oxygen levels in the tissue. Mice treated with wt MVs had less oxygen in their intestinal epithelial surfaces than mice in the colitis control or those treated with AhemB MV (Fig. 3A, 3B). The pimonidazole adducts along the epithelium of the distal colon were specifically quantified, as this is where C. rodentium establishes before moving into the proximal colon and therefore where one would expect to see the most cell damage and oxygenation.

[0382] Having observed that wt MVs reduced oxygen in the gut, it was next determined whether the treatment changed the cytokine response. Inflammation is modulated throughDBl / 161990477.12cytokines, and it was suspected that because MVs improved colitis symptoms, they may be modulating the cytokine response. The differences in cytokine concentration were visualized in a volcano plot where each point and color represent a cytokine enriched in that corresponding group (Fig. 3C). The comparisons made were between the AhemB MVs to wt MVs group in purple, colitis control to wt MVs group in red and wt MVs to both combined AhemB and colitis control groups in green. Notably, treatment with MVs decreased cytokines associated with inflammation in the C. rodentium infected mice. The pro-inflammatory TNF-a was the most significantly decreased cytokine in wt MVs treated group. In contrast, the anti-inflammatory IL- 10 was the most enriched (p-value 0.08) cytokine in the wt MVs group. TNF-a and IL-10 have been strongly associated with worsening or ameliorating colitis respectively. Moreover, the expression of these cytokines in colitis has been linked to the microbiota. In IL- 10 knockout mice the development of colitis is contingent on a gut microbiome, as germ-free IL-10 knockout mice do not develop inflammation. TNF-a was first identified through studying bacterial infections and the success of anti-TNF-a in treating IBD has been linked to patients’ microbiomes, highlighting the relationship between TNF-a and the microbiome. IL-3 is the most suppressed in the wt MV group and is elevated in IBD patients. Taken together, these data suggest that MV treatment reduces the inflammatory response, likely through interactions with the microbiome.Membrane vesicle treatment modifies the microbiome.

[0383] To determine if the MV treatment altered the active colitis microbiome, the stool microbiome was analyzed through metatranscriptomics. RNA from mouse stool was extracted and sequenced with Illumina sequencing, then taxonomy was annotated using Kaiju. A principal coordinate analysis (PCoA) of Bray-Curtis dissimilarity was generated using bacterial genera as features to visualize the similarities and differences between the microbiomes of the groups (Fig. 4A). This analysis showed that the wt MV treated group was distinct from the colitis control and AhemB MV groups which clustered closely together. To better understand these changes, the taxonomy of individual samples and the phylogeny of the groups were compared (Fig. 4B, 4C). In the phylogenetic tree it is seen that the microbiome is primarily made up of a small set of highly abundant families belonging to the clades Verrucomicrobiota Bacteroidia, Gammaproteobacteria, Bacilli and Clostridia with an overall drop in diversity in all the C.DBl / 161990477.12rodentium-infected groups relative to the healthy group. This loss of diversity as well as the PCoA separation show that in this model the wt MV treatment did not protect from colitis by reestablishing the initial diversity. To better understand the microbiome differences that may have contributed to the therapeutic effects of removing oxygen from the gut, linear discriminant analysis effect size (LEfSe) was performed. LEfSe is a method that uses both significance and effect size to rank features that are most likely to explain the differences between the groups. Using LEfSe it was seen that the top five distinguishing features between the wt MV and AhemB MV / colitis control groups at a family level were an increase in Lactobacillaceae and a decrease in Bacteroidaceae, Enterobacteriaceae, Enter ococcaceae. and Akkermansiaceae (Fig. 4E). The LEfSe comparisons were made between colitis control or AhemB MV groups to wt MV and wt MV to AhemB and colitis control combined. The differences between the colitis control and the wt MV group were less significant than the AhemB MVs and as such these comparisons were not in the top five. However, though not significant, all the families shown as enriched in the AhemB MV group were also enriched in the colitis control relative to the wt MV group (Fig. 5). The differences between taxa identified through LEfSe were also visualized to quantify relative abundance and significance changes between the groups (Fig. 4F). Based on previous studies, the relative decrease in Enterobacteriaceae and increase in Lactobacillaceae likely plays a significant role in the improvement in observed mouse health. Lactobacillaceaea have been previously shown to be inhibitory to Enterobacteriaceae and it is possible that the decrease in relative abundance of Enterobacteriaceae is due to inhibition from the increase in Lactobacillaceaea. Therefore, a Spearman correlation was performed and a weak and nonsignificant negative correlation between the abundance of Enterobacteriaceae and Lactobacillaceae was observed, indicating that the decrease of Enterobacteriaceae in the wt MV treated mice is unlikely to be primarily driven by the increase of Lactobacillaceae (Fig. 4D).MV treatment alters the metatranscriptome and metabolome.

[0384] Having established that MV treatment altered the microbiome, the metatranscriptome and metabolome of the stool microbiota was investigated to understand the mechanism behind these changes. To accomplish this, cDNA reads were annotated to proteins using the program DIAMOND and then assigned to SEED subsystems using MEGAN (Fig. 8A-8D). A PCoA of the SEED annotated subsystems was used to understand how the conditions within differentDBl / 161990477.12groups changed the expression of pathways within the microbiome (Fig. 6A). This analysis showed a similar result to that seen in the PCoA using taxonomic data; mainly that the pathways in healthy mice clustered closely together, the colitis control and AhemB MV treated mice clustered together, and the wt MVs clustered largely together. This indicates that both inducing colitis and treating with wt MVs caused an observable change in the microbial metatranscriptome. To better understand what transcriptional changes are driving the separation between these groups, biplot vectors were used to display the top two subsystems with the highest loadings and depict how they influenced the separation of the samples. Through this it was found that the 50s ribosomal subunit and mixed acid fermentation best separated the samples. Mixed acid fermentation is a metabolic pathway, while changes in expression of the 50s ribosomal subunit suggest differences in translation and likely energy expenditure between the groups. Collectively these results suggest that the main influence of the MV treatment is on pathways associated with energy. In light of this, a heatmap of all the energy subsystems in the SEED database was generated, which showed three distinct clusters associated with one of either healthy, colitis control and AhemB MV, or wt MVs groups (Fig. 6B). The clustering of pathway abundance within groups reinforced the observation that MV treatment induces transcriptomic changes within energy associated subsystems. The abundance of reads across all energy subsystems was then compared, and relevant systems were reported that were significantly different between the wt MVs treatment and the colitis control or AhemB MV groups to highlight the changes caused by wt MVs in a colitis environment (Fig. 6C). These results were then grouped into central metabolism, fermentation, and respiration to observe trends in the context of these larger systems. Three subsystems within the fermentation group were identified as significantly different: acetoin and butanediol fermentation, lactate fermentation, and mixed acid fermentation all of which were higher in the wt MV treated group than in any of the other groups. Conversely, three subsystems within respiration: cytochrome d ubiquinol oxidase, Na- translocating NADH-quinone oxidoreductase (Na+ NQR), and succinate dehydrogenase (SDH) and fumarate reductase (FRD) complexes, were all lower in the wt MV group relative to the other colitis groups. These results suggest that by reducing oxygen in the gut, the wt MVs are steering the microbial metabolism away from respiration and towards fermentation.DBl / 161990477.12

[0385] Fermentation and respiration are endpoints of metabolism, so to understand the changes upstream of these systems, the significantly different pathways within central metabolism between the groups were investigated. It was observed that enzymes associated with glycolysis and gluconeogenesis were more highly transcribed in the microbiomes of mice treated with wt MVs. While these pathways share a majority of the same reversable enzymes, they accomplish opposing goals either the catabolism or anabolism of glucose. To separate the reactions, the transcription of two irreversible enzymes were observed, 6-phosphofructokinase (EC 2.7.1.11) and pyruvate kinase (EC 2.7.1.40), which are found in the glycolytic pathway. Both enzymes were transcribed at higher relative abundance in the wt MV treated samples than in samples from the other groups (Fig. 7). 6-phosphofructokinase can be bypassed during gluconeogenesis via the irreversible fructose-bisphosphatase (EC 3.1.3.11), which is unchanged in the colitis samples, or the reversable diphosphate-fructose-6-phosphate 1 -phosphotransferase (PFP) (EC 2.7.1.90) which is transcribed at a lower relative abundance in the wt MV treated samples. Pyruvate kinase converts phosphoenolpyruvate to pyruvate and can be bypassed during gluconeogenesis via the reversable enzymes pyruvate, water dikinase (EC 2.7.9.2) or pyruvate, phosphate dikinase (EC 2.7.9.1). Alternatively, phosphoenolpyruvate can be catabolized through the irreversible enzymes phosphoenolpyruvate carboxykinase using GTP (EC 4.1.1.32) or phosphoenolpyruvate carboxykinase using ATP (EC 4.1.1.49). Notably, all four of these enzymes involved in catabolizing phosphoenolpyruvate remain relatively unchanged in the colitis treatments. Although a conclusion from the increased abundance of PFP cannot be made because it is reversable, the transcriptional abundance of the other observed enzymes suggest that glycolysis is more relatively abundant in the wt MV treated microbiomes than in the colitis control or AhemB MV groups. During anaerobic glycolysis bacteria use pyruvate formate lyase (PFL) to catalyze the conversion of coenzyme A (CoA) and pyruvate into acetyl-CoA and formate. PFL is strongly linked to anaerobic glycolysis due to its oxygen sensitivity, and its expression in E. coli increases by a magnitude when grown in anaerobic versus aerobic conditions. Viewed holistically, these data indicate that treatment with wt MVs increases anaerobic glycolysis and the generation of ATP through substrate level phosphorylation. The pyruvate from this process is then metabolized into lactate or acetyl-CoA via PFL and converted into acetate and ethanol through mixed acid fermentation. This increase in acetate can be inhibitory and is likely compensated for by utilizing the NADH from butanediol metabolism toDBl / 161990477.12further catabolize acetate into ethanol. Contrary to the increased anaerobic glycolysis and fermentation observed with the wt MV treatment, the microbiomes of mice in the colitis and AhemB MV controls have higher relative levels of transcripts associated with the tricarboxylic acid cycle (TCA). Relative to the other groups, the microbiomes within the AhemB MV and colitis control mice are using the TCA to power the ETC to generate ATP through respiration.

[0386] After discovering that wt MV treatment alters the metatranscriptome relative to the colitis control groups, it was sought to determine if this change was reflected in the metabolome. To accomplish this, the stool was analyzed using untargeted Liquid chromatography-mass spectrometry (LCMS). Healthy, and colitis groups treated with AhemB MV or with wt MV were compared. A principal component analysis of these untargeted metabolomics results shows a separation between all three groups indicating changes in the metabolome between the healthy and colitis groups and between AhemB and wt MV treatments (Fig. 6D). Sparse partial least squares-discrimination analysis was then used to identify the metabolites that best described the difference between the AhemB and wt MV treatments. While most of the metabolites were unidentified, the annotated metabolite that best described the difference between these groups was glyceric acid. Glyceric acid has been found to be enriched in the stool of colitis and IBS patients and was hypothesized to be a consequence of liberated triacylglycerols (TAGs) associated with the colon mucosa. TAGs are energy stores for the host cells and can be metabolized into glyceric acid. Because of this, glyceric acid in the large intestine can be attributed to the liberation of TAGs during host cell death. The lower levels of glyceric acid in the wt MV treated stool (Fig. 6E) suggests that by reducing oxygen in the gut, the vesicles help protect against mucosal degradation which is prevalent in gut inflammation.Discussion.

[0387] Emerging research is associating gut inflammation with an increasing range of otherwise disparate health conditions. While studies have found multiple factors can cause or predispose a person for gut inflammation, mounting evidence suggests that the microbiome can have a causal role. Gut inflammation is often correlated with an increase in proinflammatory facultative bacteria, which studies have shown is driven by the formation of NO3, and from oxygen leaking into the gut. Respiring E. coli and other Enterobacteriaceae produce proinflammatory lipopolysaccharide (LPS), exacerbating conditions such as gut dysbiosis, gutDBl / 161990477.12inflammation, and inflammatory gut diseases (e.g., IBD). Inflammation damages the epithelial cell barrier, allowing oxygen to leak into the lumen of the gut.

[0388] Under most conditions facultatively anaerobic bacteria within the family Enterobacteriaceae grow better aerobically or in the presence of alternative electron acceptors such as NO3 than anaerobically. This is due to the more efficient production of ATP generated by oxidative phosphorylation in respiration versus the substrate-level phosphorylation carried out in fermentation. It has been shown that the growth of Enterobacteriaceae members such as C. rodentium during colitis is strongly supported by the ability to use oxygen as a terminal electron acceptor. The reduction of oxygen can be achieved with reducing agents; however, these compounds are limited in their therapeutic efficacy as they can be toxic and are consumed by their reaction with oxygen. By contrast, bacterial respiration is enzymatic and produces CO2 and water. We reasoned that Enterobacteriaceae can be outcompeted by their respiring membrane vesicles. Membrane vesicles from E. coli rapidly consumed O2 in the presence of succinate commonly present in the gut.

[0389] These MVs were found to inhibit the growth of gut pathogens in vitro, including murine C. rodentium as well as E. coli and K. pneumoniae isolated from humans. C. rodentium displays an attaching and effacing pathotype causing gut inflammation in mice which mirrors the pathology of colitis in humans. C. rodentium respiration leads to a bloom during gut inflammation, and a C. rodentium induced colitis model was therefore chosen to test the effect of MVs.

[0390] Adding MVs to the food of mice with induced colitis mitigated weight loss and colon shortening. Notably, oxygen levels at the epithelial layer were significantly diminished, as measured by pimonidazole. In agreement with this, proinflammatory TNF-a was decreased in MV treated animals, while anti-inflammatory IL- 10 was elevated by comparison to mice with colitis. By contrast, MVs made from an E. coli hemB mutant lacking cytochromes had no effect.

[0391] Metatranscriptomics report both the taxonomy and the expression of functional pathways of the microbiota, and were used to evaluate changes in response to treatment with MVs. One of the main findings of the taxonomic analysis was that the MV treatment caused an increase in Lactobacillaceae which have been shown to be anti-inflammatory, and a decrease inDBl / 161990477.12pro-inflammatory Enterobacteriaceae . Lactobacillaceae do not respire and are at a disadvantage in aerobic conditions. These changes are consistent with MVs decreasing O2 in the gut.

[0392] Analysis of the expression data shows an increase in pathways for lactate and mixed acid fermentation as well as acetoin and butanediol fermentation in MV treated mice. Lactate fermentation is consistent with a significant increase in the relative abundance of Lactobacillaceae in mice treated with MVs. While it is possible that fermenting bacteria such as Lactobacillaceae are contributing to the increase in mixed acid fermentation, this metabolism is predominantly associated with the Enter obact er iaceae family indicating that by removing oxygen from the gut the MV treatment shifts the Enterobacteriaceae population towards fermentation. In agreement with this observation, a decrease was observed in the relative number of reads associated with cytochrome d oxidase with MV treatment. Cytochrome d oxidase is part of the bd family of oxidases preferentially expressed by Enterobacteriaceae during colitis. This high affinity oxidase provides an energetic advantage allowing the cells to bloom during gut inflammation. A decrease in the transcription of cytochrome d suggests a reduction in Enterobacteriaceae respiration in MV treated mice. Another notable observation is the decrease in the expression of succinate dehydrogenase (SDH) in MV treated mice. SDH is the enzyme the MVs rely upon to respire in the gut, and it is telling that the expression of this enzyme is suppressed in gut Enterobacteriaceae, apparently as a result of the anaerobiosis brought about by MVs.

[0393] A metabolome analysis was also performed, and the most notable change was observed in glyceric acid, where levels were lower in the MV treated group. Increases in glyceric acid has been reported in IBD studies and are thought to be a consequence of the release of triacylglycerols (TAGs) associated with the colonic mucosa . TAGs, the main source of energy for humans, are comprised of a glycerol conjugated to three fatty acyl chains and are synthesized ubiquitously by human cells. TAGs can be metabolized into fatty acids and glycerol, which can be oxidized into glyceric acid. Glycerols in the gut originate from either diet or the degradation of epithelial cells. Since diets are consistent between groups of mice, the increase in glyceric acid is likely a consequence of cell death and degradation of the colonic epithelium. Indicating damage to the mucosal layer which provides a critical barrier between the microbiome and the host. An increase in mixed acid fermentation and butanediol fermentation, which cells use toDBl / 161990477.12compensate for acetate overflow, indicate higher levels of acetate production by the microbiome with MV treatment. Acetate and other SCFA have been shown to reduce inflammation and help restore gut integrity.

[0394] Taken together, these observations suggest that the MV treatment may help reduce both oxygen levels and mucosal degradation during gut inflammation. The anti-inflammatory MVs derived from E. coli having modified LPS, as described herein, combat gut inflammation and associated diseases, conditions, and disorders.Methods.

[0395] Membrane vesicle preparation. For wt MVs, ClearColi™ was grown in TSB without Glucose with 200mM succinate and 1% NaCl at pH 7.5. Cells were harvested and concentrated by centrifugation at 10,000xg for 15min and pellets were resuspended in 20mM phosphate buffer with 650mM succinate and 650mM lactate at pH 8. Cells were then lysed by passing through a microfluidizer at 15,000 PSI two times. Lysate was purified with tangential flow filtration through a 500K Da PES hollow fiber. AhemB MVs were made by growing AhemB ClearColi in TSB + 1% NaCl + 0.4% glucose at pH 7.5 in anoxic conditions with constant N2 sparging and with an acid and base feed maintaining a neutral pH, all other steps were identical to wt MV preparation.

[0396] Production of hemB mutant E. coli. Deletion of hemB was first accomplished using lambda red recombineering. In brief, a kanamycin resistance cassette (kanR) was amplified from plasmid pKD13 using primers Pl and P2 (see Table 3), adding 50bp of homology to each side of kanR. This construct was electroporated into E. coli MG1655 containing the lambda recombineering plasmid pKD46. Cells were recovered at 37°C for 3 hours in SOC media and then plated on LB + kanamycin (50 ug / ml) + 0.4% glucose. Small colony transformants were visible after ~48 hours of incubation at 37°C. PCR with primers P3 and P4 was used to verify the swap of hemB with the KanR cassette. Deletion of hemB was further confirmed by Sanger Sequencing of the PCR product. The hemB::kanR was moved from E. coli MG1655 to E. coli ClearColi by Pl phage transduction and selection on LB+25mM citrate and 50pg / ml kanamycin, l%NaCl,0.4%glucose. Successful transduction was confirmed with PCR and catalase test. Primers are set forth in Table 3.DBl / 161990477.12Table 3. Primers used for hemB knockout E. Coli.

[0397] Measuring O2 in vitro. O2 concentration was monitored using Oxygraph, a Clark type polarographic sensor (Hansatech Instruments). Oxygraph uses a platinum cathode and a silver anode connected through an electrolytic solution to quantitate O2 concentration of a solution in real-time. The Oxygraph was calibrated with aerated and fully reduced 20mM potassium phosphate 650mM succinate pH 7. MVs were thawed and allowed to equilibrate to room temperature, they were then diluted into 20mM potassium phosphate 650mM succinate pH 7 and oxygen concentration was measured under constant stirring.

[0398] Transmission electron microscopy. Wt and AhemB MVs were made as previously described. MVs were washed and resuspended in PBS. Vesicles were coated in uranyl acetate and imaged with a JEOL JEM 1010 TEM microscope by William Fowle at the Boston Electron Microscopy Center.

[0399] Measuring Enterobacteriacea growth with MVs in vitro. Klebsiella pneumoniae ATCC 43816, Citrobacter rodentium ATCC 51459, and Escherichia coli AR350, were grown overnight at 37°C 200 r.p.m. shaking as a starter culture. This was then diluted 1 / 400 into LB with 13mM succinate with and without wt MVs in aerated culture tubes shaking at 200 r.p.m. at 37°C. MVs were added at a concentration of 1.623 mg / mL of protein. OD600 Readings were taken after 7 hours with a Ultrospec 10 spectrometer. Klebsiella pneumoniae ATCC 43816, and Citrobacter rodentium ATCC 51459 were obtained from ATCC while Escherichia coli AR35O was obtained through the CDC.DBl / 161990477.12

[0400] Ulcerative colitis model. Animal studies were approved by the Northeastern Institutional Animal Care and Use Committee, and performed at Northeastern University in accordance with institutional animal care and use policies. Five-week-old female C3H / HeNCrl mice were purchased from Charles River and cohoused in specific pathogen free (SPF) conditions with a 12-hour day / night cycle with temperature and humidity controls at ~21 °C and -30% respectively and fed PicoLab IsoPro RMH 3000 irradiated mouse food. Mice were mixed, and weight matched into new cages. Weight matching was done to avoid experimental differences between groups due to initial weight variance. After 48 hours of acclimation mice were fasted (to avoid the delivery of the inoculum into a full stomach which could increase variability between animals and make it less likely that the inoculum is aspirated into the lungs) overnight then orally gavaged with 1.71X108 CFU / lOOpl of Citrobacter rodentium ATCC 51459 resuspended in PBS. C. rodentium was grown overnight in aerated baffled flasks in LB shaking at 200 r.p.m. at 37°C . MVs treatments were normalized to 10,151 pg / ml of protein, washed, and then resuspended in PBS and added to food at a 1: 12 volume to weight. For MV treatments old food was removed and new treated food added every 24 hours.

[0401] Cytokine analysis. After euthanasia blood was collected from heart punctures and allowed to coagulate for 30 minutes at room temperature. It was then centrifuged at 2,000x g for 10 minutes at 4°C. Serum was then collected and diluted 1 :2 into PBS, immediately frozen and stored at -80°C until analysis. Cytokines were quantitated at Eve Technologies using the Mouse Cytokine / Chemokine 32-Plex Discovery Assay® Array. ROUT outlier test, Q=0.1% was applied to the data.

[0402] Colon hypoxia analysis. To assess colonic hypoxia, mice were administered 60mg / kg of pimonidazole HC1 through intraperitoneal injection 1 hour before euthanasia. Colons were flushed with sterile PBS then sectioned longitudinally and rolled into Swiss rolls which were then stored in 10% formalin. Samples were imbedded, stained, and imaged at Applied Pathology systems as follows; tissue was imbedded in paraffin with a Leica ASP-300 Tissue Processor then sectioned to a thickness of 5 pm. Tissue was blocked with BloxALL and Animal Free Blocker / Diluent then then stained with the nuclear stain Hoechst 33342 and Hypoxyprobe™-1 RED ATTO 594 a RED ATTO 594 dye-conjugated IgGl mouse monoclonal anti-pimonidazole antibody (clone 4.3.11.3). Slides were imaged with an Akoya PhenoImager Fusion and exposureDBl / 161990477.12times were optimized for 2 fluorescence filters (DAPI, Opal 620) and remained consistent across all images. For quantification 3 representative images of the distal colon were analyzed and the fluorescence of the colonic epithelium was quantified and averaged. Quantification was done using ImageJ with background subtraction, no adjusting to brightness was done.

[0403] Metatranscriptomics. Stool was collected immediately after euthanasia and submerged in 500pl of RNAprotect® then stored at -80°C until processing. To extract and purify RNA, samples were spun down at 2,130 x g for 4 minutes at 4°C and supernatant was removed. 600pl of Qiagen RLT was added to the stool with 1% 2-Mercaptoethanol. This was transferred to MP lysing matrix E bead tubes and shaken at 6m / second for 40 seconds twice using an MP FastPrep-24TM5G. Tubes were then centrifuged for 1 minute at 400xg and the supernatant was processed with the Qiagen RNeasy® Mini Kit following manufacture’s recommended protocol. Sequencing was done at Seqcenter, samples were DNAse treated with Invitrogen DNAse (RNAse free). Library preparation was performed using Illumina’s Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and lObp unique dual indices (UDI). Sequencing was done on a NovaSeq X Plus, producing paired end 150bp reads. Demultiplexing, quality control, and adapter trimming was performed with bclconvert (v4.1.5). Taxonomy was assigned with Kaiju and normalized to 5,958,789 features per sample. Phylogetic tree created with iTOL. For functional analysis reads were normalized to 9,539,011 reads per sample. DIAMOND was used to assign reads to proteins and MEGAN was used to assign proteins to subcategories using the SEED database.

[0404] Metabolomics. After sacrifice, mouse stool was immediately weighed and frozen at - 80°C until needed. Methanol was added at a ratio of 40pL / mg of stool in MP lysing matrix E bead tubes and shaken at 6m / second for 40 seconds twice using an MP FastPrep-24TM5G. Tubes were then centrifuged for 1 minute at 400xg and 500pL of supernatant was added to 1.5mL of methanol in a glass vial. 4mL of cold chloroform was added and the sample was vortexed for 1 minute before adding 2mL of miliQ water and again vortexing for 1 minute. Samples were then centrifuged for 10 minutes at 3,000 r.c.f.. The aqueous phase was the collected and sent to Harvard Center for Mass Spectrometry. All samples were dried under N2, resuspended in 0.9pL / mg of 50% acetonitrile in water. Samples were run on a Thermo ID-X tribid mass spectrometer on a ThermoFisher ID-X mass spectrometer (Zic pHILIC columnDBl / 161990477.12150x2.1 mm 5 micron). LC method is set forth in Table. 4. Data was median centered. Peak extraction, retention time alignment, gap filling, background subtraction, normalization, and compound identity determination were all performed by compound discoverer version 3.3.Table 4. Liquid chromatography (LC) method.Example 2. Anti-inflammatory MVs reduce the expression of pro-inflammatory TNF-alpha

[0405] Membrane vesicles produced from Bacilllus subtilis have significantly less oxygen reducing activity than those produced via E. coli in the product Oxyrase (Fig. 9). This demonstrates that A", coli is a superior source for producing MVs. However because Oxyrase is produced from E. coli, Oxyrase MVs contain the inflammatory endotoxin LPS. Using a limulus amoebocyte lysate test we found that Oxyrase contained 45 pg / mL of endotoxin, which with the FDA limit for endotoxin in drugs is 0.75 ng / kg greatly limits the therapeutic window of Oxyrase.

[0406] To overcome this, anti-inflammatory MVs derived from a modified E. coli known as ClearColi were produced. In a series of experiments, it was shown ClearColi caused no detectable LPS induced inflammation. Whole cells and purified LPS from the ClearColi- modified versions of E. coli caused no detectable humanTLR4 mediated immune response, even at 1,000 times the concentrations which caused the maximum observable immune response in the unmodified strains. This is particularly important, as Oxyrase uses E. coli to produce membrane vesicles. Because of this, membrane vesicles produced from these modified versions of E. coli are less inflammatory than those commercially available as Oxyrase. Additionally these ClearColi-derived MVs are directly anti-inflammatory owing to the presence of lipid IVa. In humans, lipid IVa acts as an antagonist to LPS induced inflammation.

[0407] As shown in Fig. 10, it was demonstrated that in a humanPBMC assay stimulated with LPS, ClearColi MVs were able to reduce the expression of pro-inflammatory TNF-alpha induced by LPS. The direct protective effects of lipid IVa acts as an additional anti-inflammatoryDBl / 161990477.12mechanism to the effects of removing oxygen in the gut and reducing the growth of inflammatory bacteria.

[0408] A number of patent and non-patent publications are cited herein in order to describe the state of the art to which this disclosure pertains.

[0409] While certain embodiments of the present disclosure have been described and / or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. The present disclosure is, therefore, not limited to the particular embodiments described and / or exemplified, but is capable of considerable variation and modification without departure from the scope and spirit of the appended claims.

[0410] Moreover, as used herein, the term “about” means that amounts, sizes, formulations, parameters, shapes and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter, shape or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such.

[0411] Furthermore, the transitional terms “comprising”, “consisting essentially of’ and “consisting of’, when used in the appended claims, in original and amended form, define the claim scope with respect to what unrecited additional claim elements or steps, if any, are excluded from the scope of the claim(s). The term “comprising” is intended to be inclusive or open-ended and does not exclude any additional, unrecited element, method, step or material. The term “consisting of’ excludes any element, step or material other than those specified in the claim and, in the latter instance, impurities ordinary associated with the specified material(s). The term “consisting essentially of’ limits the scope of a claim to the specified elements, steps or material(s) and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure. All compounds, compositions, formulations, and methods described herein that embody the present disclosure can, in alternate embodiments, be more specifically defined by any of the transitional terms “comprising,” “consisting essentially of,” and “consisting of.”

[0412] It is to be understood that the embodiments of the disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that can be employedDBl / 161990477.12are within the scope of the disclosure. Thus, by way of example, but not of limitation, alternative configurations of the present disclosure can be utilized in accordance with the teachings herein. Accordingly, the present disclosure is not limited to that precisely as shown and described.

[0413] While the present disclosure has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the disclosure is not restricted to the particular combinations of materials and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims. All references, patents, and patent applications referred to in this application are herein incorporated by reference in their entirety.References1 Enck, P. et al. Irritable bowel syndrome. Nat. Rev. Dis. Primers. 2, 16014 (2016).2 Roda, G. et al. Crohn’s disease. Nat. Rev. Dis. Primers. 6 (2020).3 Kobayashi, T. et al. Ulcerative colitis. Nat. Rev. Dis. Primers. 6 (2020).4 Heston, M. B. et al. 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Claims

CLAIMS1 . A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

2. The method of claim 1, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceli, Gluconobacter oxydans, Rhizobium legumiriosarum , Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniforms, Bacteroides thetaiotaomicron, Phocaeicola vulgatus. Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

3. The method of claim 1 or 2, wherein the modified lipopolysaccharide is a non- immunogenic lipopolysaccharide.

4. The method of any one of claims 1 to 3, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

5. The method of any one of claims 1 to 4, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

6. The method of any one of claims 1 to 5, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

7. The method of any one of claims 1 to 6, wherein the modified lipopolysaccharide is lipid IVA.

8. The method of claim 1, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.DBl / 161990477.

129. The method of claim 1, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

10. The method of claim 1, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

11. The method of any one of claims 1 to 10, wherein the disease or condition is associated with gut oxygenation.

12. The method of any one of claims 1 to 11, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

13. A pharmaceutical composition comprising: oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide; and a pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

15. The pharmaceutical composition of claim 13, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

16. The pharmaceutical composition of claim 13, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1 C.

17. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claims 13 to 16.

18. The method of claim 17, wherein the disease or condition is associated with gut oxygenation.

19. The method of claim 17 or 18, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g, gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

20. A method of manufacturing oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide, the method comprising: culturing the bacteria in a growth medium comprising succinate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

21. The method of claim 20, wherein the bacterium is selected from Komagataeibacter xylimis, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coH. Escherichia coli Nissle 1917, Akkermansia muciniphila Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Coryaebacterium pseudodiphtheriticum, Corynebacterium accolens and Rothia dentocariosa.

22. The method of claim 20 or 21, wherein the modified lipopolysaccharide is a non- immunogenic lipopolysaccharide.

23. The method of any one of claims 20 to 22, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

24. The method of any one of claims 20 to 23, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

25. The method of any one of claims 20 to 24, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

26. The method of any one of claims 20 to 25, wherein the modified lipopolysaccharide is lipid IVA.

27. The method of claim 20, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

28. The method of claim 20, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

29. The method of claim 20, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

30. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

31. The method of claim 30, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans Rhizobium leguminosanim , Escherichia coli. Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides uniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

32. The method of claim 30 or claim 31, wherein the disease or condition is associated with gut oxygenation.

33. The method of any one of claims 30 to 32, wherein the disease or condition is selected from gut dysbiosis (e.g., oxygen-induced gut dysbiosis), inflammation (e.g., gut inflammation), IBS, IBD, colitis, cancer (e.g, colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

34. A pharmaceutical composition comprising: oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C; and a pharmaceutically acceptable excipient.

35. A method for treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 34.

36. The method of claim 35, wherein the disease or condition is associated with gut oxygenation.

37. The method of claim 35 or 36, wherein the disease or condition is selected from gut dysbiosis ( .g., oxygen-induced gut dysbiosis), inflammation (e.g, gut inflammation), IBS, IBD, colitis, cancer (e.g., colon cancer), cachexia, graft-versus-host disease, neurological disease (e.g., Alzheimer’s disease, Parkinson’s disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis), atherosclerosis, Ankylosing spondylitis, Rheumatoid arthritis, type 1 and type 2 diabetes, and inflammaging.

38. A method of manufacturing oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C, the method comprising: culturing the bacteria in a growth medium comprising succinate to obtain a culture; and isolating the oxygen-scavenging membrane vesicles from the culture.

39. The method of claim 38, wherein the bacterium is selected from Komagataeibader xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia mucin iphi la, Bacteroides uniform is, Bader oides thetaiotaomicron , Phocaeicola vulgatus, Coryneba erium pseudodiphtheriticu , Corynebaderium accolens, and Rothia dentocariosa.

40. A nutritional composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide; and a nutritionally acceptable excipient.

41. The nutritional composition of claim 40, wherein the nutritional composition is selected from a food product, a dietary supplement, or a medical food.

42. The nutritional composition of claim 40 or 41, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

43. The nutritional composition of any one of claims 40 to 42, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

44. The nutritional composition of any one of claims 40 to 43, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

45. A non-therapeutic method for supporting gastrointestinal health in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

46. The method of claim 45, wherein the subject is a healthy subject or a subject experiencing occasional digestive discomforts such as bloating, gas, or bowel irregularity.

47. A non-therapeutic method for promoting a healthy inflammatory response in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

48. The method of claim 47, wherein the subject has a sub-optimal inflammatory status.

49. A non-therapeutic method for supporting a healthy balance of gut microbiota in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen-scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

50. The method of claim 49, wherein the subject's gut microbiota is perturbed by factors such as travel, antibiotic use, or dietary changes.

51. A non-therapeutic method for supporting metabolic health in a mammalian subject, the method comprising administering to the subject an effective amount of a composition comprising oxygen- scavenging membrane vesicles derived from bacteria, wherein the bacterium comprises modified lipopolysaccharide or the bacterium lacks lipopolysaccharide.

52. The method of any one of claims 45 to 51, wherein the composition is a nutritional composition, a food product, a dietary supplement, or a medical food.

53. The method of any one of claims 45 to 52, wherein the bacterium is selected from Komagataeibacter xylinus, Acetobacter aceti, Gluconobacter oxydans, Rhizobium leguminosarum, Escherichia coli, Escherichia coli Nissle 1917, Akkermansia muciniphila, Bacteroides itniformis, Bacteroides thetaiotaomicron, Phocaeicola vulgatus, Corynebacterium pseudodiphtheriticum, Corynebacterium accolens, and Rothia dentocariosa.

54. The method of any one of claims 45 to 53, wherein the bacterium comprises a 16S rRNA sequence that is at least about 95% identical to a 16S rRNA sequence set forth in Table 1A.

55. The method of any one of claims 45 to 54, wherein the bacterium is gram-negative and comprises modifications to one or more protein sequences set forth in Table IB.

56. The method of any one of claims 45 to 55, wherein the bacterium comprises one or more terminal oxidase proteins of Table 1C.

57. The method of any one of claims 45 to 56, wherein the modified lipopolysaccharide is a non-immunogenic lipopolysaccharide.

58. The method of any one of claims 45 to 57, wherein the modified lipopolysaccharide has a Lipid A component consisting of less than 6 acyl chains.

59. The method of any one of claims 45 to 58, wherein the modified lipopolysaccharide has a Lipid A component consisting of 4 acyl chains.

60. The method of any one of claims 45 to 59, wherein the modified lipopolysaccharide does not contain 2-keto 3-deoxy-D-manno-octulosonate.

61. The method of any one of claims 45 to 60, wherein the modified lipopolysaccharide is lipid IVA.

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