Use of bacterial outer membrane vesicles in the manufacture of a product for modulating gut homeostasis

Abstract

The present application relates to the field of medicine, and provides use of bacterial outer membrane vesicles in preparation of a product for regulating intestinal homeostasis. The product for regulating intestinal homeostasis can up-regulate beneficial microflora, avoid mass propagation of intestinal opportunistic pathogens; be transferred to Peyer's patches to activate mucosal immunoglobulin A response, regulate intestinal immune homeostasis; enter intestinal epithelial cells to stimulate expression of tight junction and mucus to maintain integrity of the intestinal barrier, thereby reversing intestinal ecological disorder. The product for regulating intestinal homeostasis improves symptoms and pathological manifestations of dextran sulfate sodium-induced colitis by reducing pathogen abundance, maintaining immune homeostasis and enhancing intestinal epithelial tight junction. The product for regulating intestinal homeostasis also overcomes drug resistance to PD-1 blockade in tumor immunotherapy by maintaining beneficial intestinal microbial structure.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to the use of bacterial outer membrane vesicles in the preparation of products that regulate intestinal homeostasis. Background Technology

[0002] The gut microbiota has become a key factor in maintaining gut and systemic homeostasis. Within the gut ecosystem, due to the epithelial barrier, communication between microbes and the host is rarely mediated through direct cell-to-cell contact. Numerous studies have shown that bacterial metabolites can mediate microbe-host interactions. For example, butyrate produced by Clostridium can stimulate colonic regulatory T cells, thereby suppressing the host's inflammatory response. Microbiome-modified bile acids have been shown to regulate the accumulation of natural killer T cells in liver tumors. Undoubtedly, the intricate crosstalk between the gut microbiota and the host is multidirectional and reciprocal, meaning that optimally characterized derivatives secreted by bacteria may be involved in their interactions. Gram-negative bacteria typically release outer membrane vesicles (OMVs) during growth. OMVs are bilayered nanostructures ranging in size from 20 to 400 nm, containing a variety of parental components such as nucleic acids, proteins, enzymes, and lipopolysaccharides. Because of these properties, OMVs may play a crucial role in microbe-microbe and host-microbe interactions. Recent studies have highlighted the immune-stimulating responses triggered by OMVs produced by pathogenic bacteria and engineered symbiotic bacteria after crossing the host barrier. Although the biological functions of OMV extend to transporting bioactive substances and defending against stressors affecting the outer membrane, such as bacteriophage predation and antimicrobial agent invasion, little is known about whether it can mediate host responses beyond the unilateral defense of the immune system. It is generally accepted that long-term evolution has fostered an advanced symbiotic relationship between the gut microbiota and the host. However, there is no clear evidence that OMV can serve as an effective extracellular mechanism for regulating gut homeostasis. Therefore, it is necessary to provide information on the use of symbiotic bacterial-derived OMVs in modulating the gut microbiota, mucosal adaptive immunity, and physicochemical barriers to treat intestinal and extraintestinal diseases. Summary of the Invention

[0003] In view of the shortcomings of the prior art described above, the object of the present invention is to provide the use of bacterial outer membrane vesicles in the preparation of products for regulating intestinal homeostasis, the use of which is to repair intestinal flora balance, maintain immune homeostasis and enhance intestinal homeostasis constituted by tight junctions in the intestine, thereby solving the problems of the prior art that currently uses live bacterial transplantation to alleviate colitis by utilizing the intestinal microbiota, and the lack of products that enhance anti-PD-1 immunotherapy for colorectal cancer by maintaining intestinal homeostasis.

[0004] To achieve the above and other related objectives, the present invention provides the use of bacterial outer membrane vesicles in the preparation of products for regulating intestinal homeostasis.

[0005] Preferably, the product for regulating intestinal homeostasis is a product for treating enteritis or a tumor treatment enhancer.

[0006] Preferably, the bacterial outer membrane vesicles are prepared by Gram-negative bacteria.

[0007] The present invention also provides a pharmaceutical composition for regulating intestinal homeostasis, the product comprising bacterial outer membrane vesicles and pharmaceutically acceptable carriers or excipients.

[0008] In some specific embodiments, the bacterial outer membrane vesicles are outer membrane vesicles produced by one or more of the following: Akkermansia myxophila, Megamonas, Koala, Bacteroides, Desulfovibrio, and Prevotella foetida.

[0009] This invention also provides the use of bacterial outer membrane vesicles in the preparation of any of the following products:

[0010] 1) Products that increase the relative abundance of beneficial or symbiotic bacteria in the gut;

[0011] 2) Products that reduce the relative abundance of bacteria that cause gut microbiota dysbiosis;

[0012] 3) Products that promote the growth of probiotics;

[0013] 4) Products that trigger mucosal immune regulatory responses;

[0014] 5) Products that repair the physical and chemical barriers of the intestinal epithelium;

[0015] 6) Products that alleviate symptoms of intestinal ecological imbalance.

[0016] As described above, the use of bacterial outer membrane vesicles of the present invention in the preparation of products for regulating intestinal homeostasis has the following beneficial effects:

[0017] 1. The present invention provides the use of bacterial outer membrane vesicles to upregulate beneficial microbiota and prevent the proliferation of opportunistic pathogens in the intestine;

[0018] It migrates to Pain's nodes to activate the mucosal immunoglobulin A response and regulate intestinal immune homeostasis; it enters intestinal epithelial cells to stimulate tight junctions and mucus expression to maintain the integrity of the intestinal barrier, thereby reversing intestinal ecological dysbiosis.

[0019] 2. The present invention provides the use of bacterial outer membrane vesicles to improve the symptoms and pathological manifestations of dextran sulfate sodium (DSS)-induced colitis by restoring intestinal flora balance, maintaining immune homeostasis, and enhancing tight junctions;

[0020] 3. The present invention provides the use of bacterial outer membrane vesicles to overcome resistance to PD-1 blockade in tumor immunotherapy by maintaining a beneficial gut microbiota structure. Attached Figure Description

[0021] Figure 1 For the regulation of the gut microbiota. ae 16S ribosomal RNA gene sequencing analysis of the gut microbiota: DSS-treated mice were administered 100 μl Akk OMV suspension containing 20 μg total protein by gavage for 5 days daily, followed by euthanasia for sampling. Healthy mice treated with phosphate-buffered saline (PBS) and DSS mice served as controls, respectively; a sparse curve (top) and Shannon curve (bottom); b bacterial genus and c Bacteroides genus species distribution; d Bacteroides acid-producing (B. acidifaciens) and e Bacteroides thetaiotaomicron (B. thetaiotaomicron); f, g Interactions between Akk OMV and Bacteroides species: 50 μl of logarithmic-phase bacterial solution and 100 μl Akk OMV suspension were co-incubated in 1 ml of culture medium at 37°C. Bacteria cultured in PBS were used as controls; 3D confocal laser scanning microscopy (CLSM) images and flow cytometry histograms of *Bacteroides* after incubation at specified time points, scale bar: 25 μm; bacterial growth curves were measured by recording OD values ​​at 600 nm, and data are mean ± standard error of mean (SEM); significance was assessed using t-tests or two-way ANOVA tests, and p-values ​​were given, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0022] Figure 2Regulation of mucosal immune response. a. Mouse Pascal's node (PP) (top) and mesenteric lymph node (MLN) (bottom) images from IVIS. Akk OMV enrichment in PP and MLN was observed 2 and 4 hours after intravenous injection (with the ends of the intestinal segment located at PP tied off); b. CLSM images of bone marrow-derived dendritic cells (BMDCs); c. Flow cytometry analysis (left) of CD80+ percentage of BMDCs (right), Cy5.5-labeled OMV was used for CLSM and flow cytometry measurements, scale bar: 10 μm; di. Intestinal mucosal immune response in mice with intestinal disorders after oral feeding of mice with 100 μl Akk OMV suspension containing 20 μg total protein or PBS for 5 days; d. Percentage of CD80+ DCs in PP; e. Representative flow cytometry scatter plots of B cells (B220+CD138-) and plasma cells (B220-CD138+) in PP (left panel) and the percentage of B cells (right panel); f Quantitative analysis of CD69+ B cells and IgA+ plasma cells in PP; g IgA concentration in the gut; h Flow cytometry histograms and quantification of IgA+ fecal bacteria, with naked bacteria used as a control; i Quantitative analysis of IFN-γ+CD4+ T cells, IL-4+CD4+ T cells, IL-17+CD4+ T cells, IL-4+ / IFN-γ+CD4+ T cells, and FOXP3+CD25+ / IL-17+CD4+ T cells in MLN; Data are mean ± SEM, significance was assessed using t-tests, and p-values ​​are given: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 3To maintain the intestinal physicochemical barrier, the repair of the intestinal physicochemical barrier in DSS-induced mice was assessed after daily oral administration of 100 μl AkkOMV suspension containing 20 μg total protein or PBS for 5 days. a) Alcian blue stained image of the proximal colon, with black and yellow arrows representing goblet cells with pathological cavities and thickened mucus layers, respectively (scale bar: 600 μm); b) Percentage of goblet cells in the intestinal epithelium; c) 3D CLSM image of intestinal epithelial cells uptake of AkkOMV, with red fluorescence indicating Cy5.5-labeled AkkOMV (scale bar: 50 μm); d) Immunofluorescence image of tight junction ZO-1 and occludin expressed on proximal colonic epithelial cells (scale bar: 20 μm); e, f) Assessment of tight junction proteins after co-culturing Caco-2 cells with 0.1 mg / ml AkkOMV and 5 μg / ml LPS at 37°C for 24 hours; Cy5.5-labeled OMV was used for CLSM. CLSM and f immunofluorescence images, scale bar: 25 μm; g Interleukin (IL)-10, IL-13 and angiotensin-converting enzyme (ACE) 2 in colon tissue and interferon (IFN)-γ and C-reactive protein (CRP) cytokine levels in serum were measured by enzyme-linked immunosorbent assay (ELISA). Samples were collected from DSS mice after treatment with Akk OMV or PBS. Data are mean ± SEM. Significance was assessed using t-tests, and p-values ​​are given, *p<0.05.

[0024] Figure 4 To improve the therapeutic value of DSS-induced colitis, mice with DSS-induced colitis were orally administered 100 μl of Akk OMV suspension containing 20 μg total protein via gavage for five consecutive days, followed by euthanasia to evaluate efficacy. 8 CFU-treated Akk and PBS-treated DSS mice served as controls; a) weight fluctuations during treatment; b) mean colon length after treatment (left panel) and digital photographs of the colon tissue removed from the cecum to the rectum (right panel), scale bar: 1 cm; c) representative myeloperoxidase (MPO) staining images of the proximal colon (left panel) and quantitative analysis of MPO-positive cells (right panel), black arrows representing MPO-positive cells, scale bar: 100 μm; d) typical hematoxylin-eosin (H&E) staining images of the proximal colon, green, yellow, and blue arrows representing inflammatory cell infiltration, mucosal edema, crypt swelling, and destruction, respectively, scale bar: 625 μm; data are mean ± SEM, significance was assessed using one-way or two-way ANOVA tests, p-values ​​are given, *p<0.05, ****p<0.0001.

[0025] Figure 5To assess its therapeutic value in colorectal cancer (CRC), mice with CRC were administered 100 μl of Akk OMVs suspension containing 20 μg of total protein for 2 days before intraperitoneal injection of PD-1 antibody every 3 days (for a total of 3 times). Under the same experimental conditions, 10... 8 CFU-treated Akk and PBS-treated DSS mice were used as controls; a) Individual tumor growth curves; b) Quantitative analysis of PD-L1+ cells, PD-L1+CD45- cells, and PD-L1+CD45+ cells in tumors; c) Immunofluorescence images of PD-L1 and CD45 infiltration in the tumor bed, scale bar: 50 μm; d) Representative immunofluorescence images of CD3 and CD8 infiltration in the tumor bed, scale bar: 50 μm; e) Typical H&E staining images of tumor tissue, scale bar: 300 mm; Data are mean ± SEM. Significance was assessed using one-way or two-way ANOVA tests, and p-values ​​are given, *p<0.05, **p<0.01. Detailed Implementation

[0026] This invention provides the use of bacterial outer membrane vesicles in the preparation of products that regulate intestinal homeostasis.

[0027] The product mentioned is for the treatment of enteritis.

[0028] The bacterial outer membrane vesicles were prepared using Gram-negative bacteria.

[0029] The bacterial outer membrane vesicles have a size of 20 nm to 400 nm. Preferably, the average size of the bacterial outer membrane vesicles is 60 nm to 160 nm.

[0030] The Gram-negative bacteria are selected from one or more of Akkermansia myxophilus, Megamonas, Koala, Bacteroides, Desulfovibrio, and Prevotella foetida, preferably Akkermansia myxophilus.

[0031] The bacterial outer membrane vesicles regulate intestinal homeostasis through one or more of the following mechanisms: increasing the relative abundance of probiotics or symbiotic bacteria, selectively interacting with specific bacteria in the gut microbiota, reducing the relative abundance of conditionally pathogenic bacteria in the gut, triggering mucosal immune regulatory responses, repairing the physicochemical barrier of the intestinal epithelium, or alleviating one or more manifestations of intestinal dysbiosis.

[0032] The probiotics or symbiotic bacteria are selected from one or more of the genera *Bacteroides*, *Lactobacillus*, *Alternaria*, *Clostridium*, *Prevotella*, *Bifidobacterium*, *Lactobacillus*, or *Trichophyton*. Preferably, the genus is selected from one or more of the genera *Bacteroides*, *Lactobacillus*, *Alternaria*, or *Trichophyton*.

[0033] Furthermore, bacterial outer membrane vesicles can increase the relative abundance of probiotics or symbiotic bacteria in the gut by 4 to 45 times.

[0034] The specific bacteria are selected from one or more of Bacteroides acidifacicidae, Bacteroides thetaiotaomicron, Bacteroides dorei, Bacteroides uniformi, and Bacteroides fragilis.

[0035] The conditionally pathogenic bacteria leading to intestinal dysbiosis are selected from one or more of Bacteroides vulgatus and bacteria of the Proteobacteria phylum (Escherichia coli, Shigella, Salmonella). Preferably, the bacteria causing intestinal microbiota dysbiosis are selected from Bacteroides vulgatus or bacteria of the Proteobacteria phylum.

[0036] Furthermore, bacterial outer membrane vesicles can reduce the relative abundance of bacteria that cause gut microbiota dysbiosis by 0.5-0.6% of their original abundance.

[0037] In some specific implementations, bacterial outer membrane vesicles can increase the proliferation of beneficial bacteria by 1.3-2.6 times compared to the PBS control group.

[0038] Furthermore, the immunomodulatory response is an immunoglobulin A (IgA) response or a T cell response.

[0039] Furthermore, the initiation of the mucosal immunomodulatory response is the initiation of a mucosal immunomodulatory response at one or more sites in the Pell's plaque or mesenteric lymph nodes.

[0040] In some specific implementations, bacterial outer membrane vesicles can increase the concentration of immunoglobulin A (IgA) by 1.1 to 2.5 times.

[0041] Furthermore, the repair of the intestinal epithelial physicochemical barrier is achieved through one or more of the following mechanisms: increasing the number of goblet cells and / or upregulating the expression of mucin Muc2, tight junction component occludin, or tight junction protein ZO-1 in intestinal epithelial cells.

[0042] In some specific implementations, bacterial outer membrane vesicles can increase the number of cells by 2 to 10 times.

[0043] In some specific implementations, bacterial outer membrane vesicles can upregulate the expression of mucin Muc2, tight junction component occludin, or tight junction protein ZO-1 in intestinal epithelial cells by 1.2-2.0 times.

[0044] Furthermore, the reduction of gut dysbiosis is achieved by reducing inflammatory cell infiltration, upregulating anti-inflammatory cytokines, and / or downregulating pro-inflammatory cytokines.

[0045] In some specific embodiments, the inflammatory cells are myeloperoxidase-positive cells. Bacterial outer membrane vesicles can reduce the number of myeloperoxidase-positive cells to 0.4-0.5 of their original level.

[0046] In some specific embodiments, the anti-inflammatory cytokines are selected from one or more of IL-10 and IL-13. Bacterial outer membrane vesicles can increase the anti-inflammatory cytokines to 1.5-1.8 of their original levels.

[0047] In some specific embodiments, the pro-inflammatory cytokines are selected from one or more of ACE2, IFN-γ, CRP, and IL-1β. And / or, bacterial outer membrane vesicles can reduce the levels of pro-inflammatory cytokines to 0.2-0.9 of their original levels.

[0048] The enteritis is selected from one or more of radiation enteritis, ulcerative colitis, Crohn's disease, immune checkpoint inhibitor-associated enteritis, and antibiotic-associated enteritis.

[0049] The product that regulates intestinal homeostasis is also a tumor treatment enhancer.

[0050] The bacterial outer membrane vesicles enhance the therapeutic effect of tumor treatment drugs by regulating intestinal homeostasis. The tumor treatment drugs vary depending on the type of tumor, and those skilled in the art can select them according to the specific circumstances.

[0051] Furthermore, the bacterial outer membrane vesicles enhance the therapeutic effect of PD-1 immunotherapy by regulating intestinal homeostasis.

[0052] The PD-1 immunotherapy comprises administering a drug targeting PD-1. The drug is selected from one or more drugs containing nivolumab, pembrolizumab, durvalumab, atezolizumab, camrelizumab, sintilimab, toripalimab, or tislelizumab as an active ingredient.

[0053] The tumor is selected from one or more of the following: breast cancer, pancreatic cancer, small intestine cancer, colon cancer, rectal cancer, lung cancer, head and neck cancer, ovarian cancer, hepatocellular carcinoma, esophageal cancer, hypopharyngeal cancer, nasopharyngeal cancer, laryngeal cancer, myeloma cell carcinoma, bladder cancer, cholangiocarcinoma, clear cell renal cancer, prostate cancer, neuroendocrine tumors, carcinogenic osteomalacia, sarcoma, primary unknown cancer, thymic cancer, glioma, neuroglioma, astrocytoma, cervical cancer, prostate cancer, leukemia, bone cancer, brain cancer, bronchial cancer, ependymoma, retinoblastoma, gastric cancer, gastrointestinal cancer, melanoma, renal cancer, lymphoma, mesothelioma, oral cancer, oropharyngeal cancer, ovarian cancer, thyroid cancer, pituitary cancer, renal cancer, salivary gland cancer, sarcoma, and skin cancer.

[0054] In some specific implementations, bacterial outer membrane vesicles can upregulate programmed death-ligand 1 (PD-L1) by 1.2-1.5 times. Bacterial outer membrane vesicles can also amplify the infiltration of CD8+ cytotoxic T lymphocytes (CTLs) by 4-10 times.

[0055] The present invention also provides a pharmaceutical composition for regulating intestinal homeostasis.

[0056] The pharmaceutical composition comprises bacterial outer membrane vesicles.

[0057] In some specific embodiments, the bacterial outer membrane vesicles are outer membrane vesicles produced by one or more of the following: Akkermansia myxophila, Megamonas, Koala, Bacteroides, Desulfovibrio, and Prevotella foetida. Preferably, the bacterial outer membrane vesicles are outer membrane vesicles produced by Akkermansia myxophila.

[0058] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0059] The pharmaceutically acceptable carrier or excipient should be compatible with the active ingredient of the tumor starvation therapy drug, i.e., it should be miscible with it without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients are selected from sodium hyaluronate gel, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, or methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid or magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, or cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, or polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffer solutions, or one or more of these. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0060] The present invention also provides a method for regulating intestinal homeostasis, the method comprising administering a therapeutically safe and effective amount of the product comprising bacterial outer membrane vesicles to a subject in need.

[0061] The safe and effective dosage should be adjustable for those skilled in the art. In some specific embodiments, the dosage of the product containing bacterial outer membrane vesicles depends on the patient's weight, the type of application, the condition and severity of the disease, for example, the dosage of the bifunctional compound as the active ingredient is 1–1000 mg / kg / day, 1–3 mg / kg / day, 3–5 mg / kg / day, 5–10 mg / kg / day, 10–20 mg / kg / day, 20–30 mg / kg / day, 30–40 mg / kg / day, 40–60 mg / kg / day, 60–80 mg / kg / day, 80–100 mg / kg / day, 100–200 mg / kg / day, 200–500 mg / kg / day, or greater than 500 mg / kg / day.

[0062] This invention also provides the use of bacterial outer membrane vesicles in the preparation of any of the following products:

[0063] 1) Products that increase the relative abundance of beneficial or symbiotic bacteria in the gut;

[0064] 2) Products that reduce the relative abundance of bacteria that cause gut microbiota dysbiosis;

[0065] 3) Products that promote the growth of probiotics;

[0066] 4) Products that trigger mucosal immune regulatory responses;

[0067] 5) Products that repair the physical and chemical barriers of the intestinal epithelium;

[0068] 6) Products that alleviate symptoms of intestinal ecological imbalance.

[0069] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0070] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0071] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0072] Example 1: Regulation of the Gut Microbiota

[0073] To assess the impact of OMVs on the gut microbiota, DSS-induced mice (a model of intestinal disease) were administered Akk OMVs containing 20 μg of total protein orally via tube feeding for 5 consecutive days. Colonic contents were extracted and analyzed for microbial composition via 16S ribosomal RNA sequencing. After incubation at 37°C with simulated gastric juice (pH 1.2) for 4 hours, the size and number of OMVs did not change significantly. Figure 1 This indicates that OMV's stability across the gastric lumen after oral ingestion is satisfactory. Figure 1As shown in figure a (above), the sparse curve did not rise sharply, indicating that the sample sequencing volume was sufficient for data analysis, and the number of OTUs in the gut microbiota of OMV-treated mice was significantly greater than that in the PBS-treated group. The Shannon index of OMV-treated mice also increased significantly, comparable to that of the healthy control group. Figure 1 a, Figure below). Overall, compared with PBS-treated mice, the OMV-treated group showed significant improvements in both the richness and diversity of the gut microbiota. Compared with PBS, Akk OMV increased the relative abundance of several probiotic or symbiotic bacterial genera, including Bacteroides, Lactobacillus, and Alternaria, as well as the NK4A136 group of Trichophytonceae and Trichophytonceae bacteria (…). Figure 1 b). In particular, beneficial members of the Bacteroides genus were significantly upregulated in the OMV-treated group ( Figure 1 The relative abundances of *B. acidifaciens* and *B. thetaiotaomicron* increased from 0.47% ± 0.16% to 1.99% ± 0.44% and 0.02% ± 0.02% to 0.64% ± 0.11%, respectively. Figure 1 (d and e). *Bacteroides acidifaciens* has been reported to improve mucosal immune function by converting immunoglobulin IgM to IgA and promoting intestinal IgA production, while *Bacteroides thetaiotaomicron* can maintain intestinal barrier function and improve colitis-related damage by stimulating mucus and IgA production and reversing epithelial cell damage. OMVs also increased the relative abundance of *Bacteroides stercorirosoris* and *Bacteroides caecimuris*, which are associated with the remission of ulcerative colitis and colitis-associated tumorigenesis, from 0.14% ± 0.14% to 2.21% ± 0.54%, and 0.10% ± 0.01% to 0.45% ± 0.13%, respectively. However, unlike the aforementioned *Bacteroides* species, the relative abundance of *Bacteroides vulgatus* decreased; it possesses the ability to induce protease activity-dependent intestinal barrier dysfunction and ulcerative colitis. Figure 1 c). Furthermore, OMV reduced the relative abundance of bacteria belonging to the Proteobacteria phylum ( Figure 1 (b) Proteobacteria, containing a large number of pathogenic bacteria, are considered a microbial signature of dysbiosis in the gut microbiota. These results suggest that Akk OMV can powerfully reverse gut microbial imbalance by upregulating beneficial bacteria, particularly Bacteroides species, while reducing opportunistic pathogens.

[0074] We covalently labeled the outer membrane of OMV with Cy5.5 and stained the internal nucleic acids with Hoechst. Representative strains of Bacteroides were selected for validation, including B. acidifaciens, B. thetaiotaomicron, B. vulgatus, and B. fragilis. Log-phase Bacteroides were cultured with labeled OMV, and bacteria were collected at specified time points. CLSM images showed that Akk OMV could fuse with all of these Bacteroides bacteria, although the level of fusion varied by species. Figure 1 f). After co-incubation for 1.5 h, *B. acidifaciens* and *B. fragilis* were strongly stained with Cy5.5, while *B. thetaiotaomicron* and *B. vulgatus* showed relatively low fluorescence signals. The fluorescence intensity of Cy5.5 in these proliferating Bacteroides increased with increasing co-incubation time, indicating that Akk OMV was continuously taken up by the bacteria. 3D CLSM images showed Cy5.5 fluorescence signals on the surface of these Bacteroides and Hoechst fluorescence signals inside the cells after co-incubation, indicating good fusion of Akk OMV with the Bacteroides. To examine the specificity of OMV fusion, we selected the typical probiotic strain *Escherichia coli* Nissle 1917 (E. coil) and the common pathogen *Salmonella typhimurium* SL1344 (S. typhimurium) to study Akk OMV uptake. After 25 hours of co-incubation, no fluorescent signal was observed in *Salmonella typhimurium*, while a weak signal was observed in some *Escherichia coli*, supporting evidence that Akk OMV preferentially fuses with specific bacterial genera. Consistent with CLSM imaging results, flow cytometry analysis further quantitatively confirmed that these symbiotic bacteria fused significantly more with Akk OMV compared to *Salmonella typhimurium*. Figure 1 f (right figure). We further explored the effect of OMV fusion on bacterial proliferation by recording the OD value at 600 nm and measuring the corresponding growth curves. Figure 1As shown in g, Akk OMV significantly promoted the growth of *B. acidifacicens*, *B. thetaiotaomicron*, and *B. fragilis*, but had no benefit on *B. vulgatus*. This suggests that their selective stimulation of the proliferation of specific strains in the gut microbiota can be explained by efficient fusion with Akk OMV. Despite similar OMV fusion levels, no beneficial effect was observed on the growth of *B. vulgatus*, indicating that Akk OMV does not promote the proliferation of potentially opportunistic Bacteroidetes species under gut dysbiosis conditions. As expected, the proliferation of *Escherichia coli* and *Salmonella typhimurium* was negligible after co-incubation with Akk OMV due to the absence or inefficiency of OMV fusion. These data suggest that one of the mechanisms by which Akk OMV restores balanced gut microbiota structure is through selective fusion and direct upregulation of the proliferation of beneficial species.

[0075] Example 2: Regulation of Mucosal Immune Response

[0076] In in vivo studies, Cy5.5-labeled Akk OMV entered PPs 2 hours after direct delivery to the intestinal lumen and migrated to MLN 4 hours after administration. Figure 2 a). This observation prompted us to investigate whether OMV could induce a mucosal immunomodulatory response to regulate intestinal dysregulation. Given that the gut microbiota can influence IgA production, and that primary IgA production mainly occurs in PP, we hypothesized that the entry of Akk OMV might induce an IgA response, which is crucial for the intestinal immune barrier to prevent pathogen invasion. To analyze the mucosal immune response, PP and MLN were collected from the intestines of mice with intestinal dysregulation after 5 consecutive days of oral administration of Akk OMV. Since the interaction between B cells and subepithelial DCs in PP is essential for IgA production, we first analyzed the role of Akk OMV in dendritic cell (DC) activation. As expected, in the PP of OMV-treated mice, the expression of CD80 on the surface of DCs was significantly increased ( Figure 2 d). Co-culture with BMDC in vitro further confirmed that AkkOMV could induce higher CD80+ DC production after internalization. Figure 2 (b and c). Subsequently, we analyzed the activation of B cells (B220+CD138-) and the production of plasma cells (B220-CD138+) in the sampled PP. The OMV treatment group significantly increased the production of CD69+ B cells, IgA+ plasma cells, and total B cells, thereby increasing the intestinal IgA concentration from 18.71±1.52 mg / ml to 29.47±2.73 mg / ml. Figure 2e.g., besides the direct effect of Akk OMV on mucosal IgA response, the improved gut microbiota structure may stimulate IgA production by upregulating the expression of activation-induced cytidine deaminase (i.e., acid bacilli) and polymerase receptors, converting IgM to IgA. B. thetaiotaomicron can transport IgA across epithelial cells to the intestinal lumen. On the other hand, IgA in the intestinal lumen can be neutralized and expelled by antibody coatings. In fact, due to increased IgA production, the number of IgA+ fecal bacteria significantly increased in the OMV-treated group. Figure 2 This may be another reason why Akk OMV can reduce the relative abundance of harmful pathogens in the gut microbiota. Furthermore, gut symbiotic bacteria can utilize IgA for mucosal colonization, meaning that robustness of host-microbe symbiosis can promote the restoration of a healthy microbial composition. Therefore, Akk OMV plays an important bidirectional role in the complex interactions between mucosal IgA responses and the gut microbiota, potentially contributing to the regulation of gut homeostasis dysregulation.

[0077] Based on these findings, we infer that Akk OMV may simultaneously regulate cellular immune responses in the MLN. Previous studies have demonstrated that mucosal immune cells constitutively probe responses to the gut environment; for example, dendritic cells (DCs) capture antigens to coordinate T cell responses. The increased levels of CD80+ DCs in the prostate gland inspired our investigation of T cell responses in the MLN. Flow cytometry analysis showed an increased ratio of IL-4+ / IFN-γ+CD4+ T cells and FOXP3+CD25+ / IL-17+CD4+ T cells in the MLN collected from mice treated with Akk OMV. Figure 2 i). The anti-inflammatory phenotype polarization exhibited by T cells and the relatively low level of immune response in the MLN suggest that Akk OMV can help the host maintain mucosal immune homeostasis. Figure 2 i) Changes in the in vivo adaptive immune response can be attributed to the evolved symbiotic relationship between the gut microbiota and its host, although in vitro studies have shown that co-culture of BMDCs with splenic CD4+ T cells exhibits an immune activation effect directly induced by Akk OMVs. Mechanistically, OMVs assist in restoring the intestinal biological barrier and inducing mucosal IgA responses, leading to a significant reduction in gut pathogens capable of generating cytokine storms, and ultimately promoting the maintenance of host immune homeostasis.

[0078] Example 3: Maintaining the Intestinal Physicochemical Barrier

[0079] We collected proximal colon tissue from treated mice and found that Alcian blue-stained samples showed a significant increase in goblet cells filled with acidic mucus in the proximal colon of OMV-treated mice. Compared with single, scattered goblet cells with pathological cavities in PBS-administered mice with intestinal disorders, OMV-treated mice showed significant improvements in both the number and function of goblet cells in the proximal colon, resulting in the formation of a thicker mucus layer in the intestine. Figure 3 a). Furthermore, the percentage of goblet cells in the intestinal epithelium increased significantly from 5.11 ± 2.34 to 19.89 ± 3.57 (a). Figure 3 b). The remarkable efficacy of Akk OMV in repairing the mucus barrier can be attributed to the internalization of OMV, stimulation of goblet cells to produce mucus, and OMV-mediated increase in the abundance of beneficial species (B. thetaiotaomicron promotes mucus production).

[0080] Unlike larger bacteria, Akk OMV can penetrate the mucus layer and be internalized by intestinal epithelial cells. Figure 3 c) indicates that OMV can directly interact with physicochemical barriers. Barrier function and intestinal permeability are closely related to tight junction complexes, which act as gatekeepers of paracellular pathways. Occludin is a major component of tight junctions, decreasing with weakened epithelial barrier function, while ZO-1 is an important tight junction-associated protein that promotes epithelial proliferation, thus facilitating mucosal repair. Notably, the expression of occludin and ZO-1 in proximal colonic epithelial cells was significantly increased in the OMV treatment group, accompanied by recovery from intestinal epithelial damage, and more overlapping and intact fluorescent signals were observed in the morphologically normal intestinal epithelial contours. Figure 3 d). In vitro immunofluorescence studies after co-culturing with the LPS-treated Caco-2 epithelial cell line further showed that Akk OMV promoted an immediate enhancement of occludin and ZO-1 expression. Figure 3 f). One mechanism by which Akk OMVs upregulate intestinal tight junctions after cell internalization may be through activation of the adenosine 5'-monophosphate activated protein kinase (AMPK) pathway to regulate the reassembly and stability of tight junctions. Figure 3 e). Furthermore, OMV-mediated reversal of gut microbiota dysbiosis and maintenance of immune homeostasis jointly promote significant upregulation of anti-inflammatory cytokines, including IL-10 and IL-13, and downregulation of pro-inflammatory cytokines, including angiotensin-converting enzyme (ACE) 2, IFN-γ, and C-reactive protein (CRP), playing a crucial role in regulating tight junction proteins and enhancing intestinal barrier integrity. Figure 3 g).

[0081] Example 4: OMV-mediated intestinal homeostasis for the intervention and treatment of enteritis

[0082] To examine the therapeutic potential, we treated mice with DSS-induced acute colitis by oral administration of Akk OMV. As previous studies have shown, Akk can attenuate colitis and associated tumorigenesis; therefore, colitis mice treated with Akk and PBS were used as controls. After 5 consecutive days of oral tube feeding, the weight gain of the OMV-treated mice was comparable to that of the Akk-treated mice and significantly exceeded that of the PBS control group. Figure 4 a). Simultaneously, oral administration of Akk OMV corrected the decrease in colon length and the increase in the colon weight-to-length ratio in colitis mice ( Figure 4 (b) Neutrophil infiltration has been demonstrated to be a characteristic feature of IBD. MPO is an intracellular protein of neutrophils, and its concentration is positively correlated with IBD activity. Figure 4 As shown in Figure c, MPO staining of colonic tissue revealed that treatment with OMV or Akk significantly reduced MPO-positive cell infiltration in the corresponding colonic tissue compared to PBS. H&E staining highlighted pathological symptoms caused by leukocyte infiltration, such as DSS-induced mucosal edema and crypt swelling and destruction in the proximal colon of mice, which were significantly reduced after OMV treatment. Figure 4 d). It has been reported that in IBD mouse models, pro-inflammatory cytokines, such as IFN-γ, increase excessive infiltration of inflammatory cells and mucosal epithelial damage, while anti-inflammatory cytokines, such as IL-10, play a crucial anti-inflammatory role. Therefore, Akk OMVs, through upregulating beneficial microbiota, preventing the proliferation of opportunistic pathogens in the gut, and promoting T cell anti-inflammatory phenotype polarization, lead to the recruitment of inflammatory cells in colonic tissue and a reduction in inflammatory pathological changes. Furthermore, OMV-enhanced tight junction proteins can promote epithelial proliferation for mucosal repair. These results demonstrate that oral administration of Akk OMV can improve the symptoms and pathological manifestations of DSS-induced colitis, which may benefit from intestinal homeostasis resulting from OMV-mediated reduction in pathogen abundance, maintenance of immune homeostasis, and enhanced tight junctions.

[0083] Example 5: OMV for tumor intervention and treatment

[0084] We explored the possibility that beneficial modulation of the gut microbiome by Akk OMV could enhance the efficacy of PD-1-targeted immunotherapy in a mouse model of CRC. Mice were administered Akk OMV orally via tube feeding for two consecutive days, followed by intraperitoneal injection of a PD-1 monoclonal antibody (aPD-1, for a total of 3 doses) every 3 days. Notably, aPD-1 achieved the most potent tumor growth inhibition across all treatment groups with the aid of OMV. Figure 5a). Immunological changes analyzed by flow cytometry and immunofluorescence imaging showed that PD-L1 was significantly upregulated in tumor cells (CD45-) and immune cells (CD45+) of mice co-treated with aPD-1 and OMV, comparable to those in Akk-treated mice, and consistent with previous findings from avatar mice receiving fecal microbiota transplantation from aPD-1 responders. Figure 5 (b, 5c). These results show that Akk OMV reversed the initially weak efficacy of PD-1 blockade in CRC mice. Furthermore, the combination of OMV and aPD-1 amplified tumor infiltration of CD8+ cytotoxic T lymphocytes (CTLs), thereby enhancing the tumor response to PD-1 blockade. Figure 5 d). H&E staining images also clarified the expansion of necrotic areas in tumor tissue after treatment with OMV and aPD-1, demonstrating enhanced anti-tumor growth efficacy. Figure 5 e). That is, Akk OMV overcomes the major resistance to PD-1 blockade in tumor immunotherapy by maintaining a beneficial gut microbiota structure.

[0085] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of bacterial outer membrane vesicles of Akkermansia myxophilus in the preparation of tumor therapy enhancer products; wherein the tumor is selected from colon cancer or rectal cancer, and the tumor treatment is PD-1 antibody therapy.

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