A vesicle system and its preparation method, a vaccine adjuvant and its application

Abstract

This invention discloses a vesicle system and its preparation method, a vaccine adjuvant and its application, relating to the field of vaccine development technology. The vesicle system includes hard mixed vesicles and / or soft mixed vesicles, which are obtained by membrane fusion of hard liposomes and outer membrane vesicles, and soft liposomes and outer membrane vesicles, respectively. The hard liposomes comprise the following components in parts by weight: DPPC 45.9–62.1 parts, DOTAP 28.1–38 parts, PE 8.5–11.5 parts, and DMG-PEG2000 2.6–3.5 parts; the soft liposomes comprise the following components in parts by weight: DOPE 45.9–62.1 parts, DOTAP 28.1–38 parts, DOPC 8.5–11.5 parts, and DMG-PEG2000 2.6–3.5 parts. This invention provides a vesicle system that can improve the delivery efficiency of outer membrane vesicles and regulate immune responses.

Description

Technical Field

[0001] This invention relates to the field of vaccine development technology, and in particular to a vesicle system and its preparation method, a vaccine adjuvant and its application. Background Technology

[0002] In the field of vaccinology, the application of outer membrane vesicles (OMVs) is becoming increasingly important. OMVs typically function as vaccine adjuvants in two forms: first, vaccines based on pathogen-specific OMVs; and second, vaccines made by binding OMVs isolated from safe, non-pathogenic bacteria with antigens. Despite the promising future of OMVs in vaccine development, all researchers and companies studying OMVs face a common challenge: low efficiency of OMV uptake by antigen-presenting cells (APCs) and a lack of tunability in the immune response.

[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a vesicle system and its preparation method, a vaccine adjuvant and its application, in order to solve the problems of low efficiency of OMVs being taken up by APC and lack of tunability of immune response.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a vesicle system comprising hard hybrid vesicles and / or soft hybrid vesicles, wherein the hard hybrid vesicles are obtained by membrane fusion of hard liposomes and OMVs, and the soft hybrid vesicles are obtained by membrane fusion of soft liposomes and OMVs.

[0007] The hard liposomes comprise the following components in parts by weight:

[0008] 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline 45.9–62.1 parts, (2,3-dioleoyl-propyl)-trimethylammonium chloride 28.1–38 parts, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine 8.5–11.5 parts, and 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000 2.6–3.5 parts;

[0009] The soft liposomes comprise the following components in parts by weight:

[0010] 45.9–62.1 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 28.1–38 parts of (2,3-dioleoyl-propyl)-trimethylammonium chloride, 8.5–11.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate choline, and 2.6–3.5 parts of 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000.

[0011] Optionally, in the hard mixed vesicles, the mass ratio of the OMVs to the hard liposomes is (25-75):(25-75);

[0012] In the soft mixed vesicles, the mass ratio of the OMVs to the soft liposomes is (25-75):(25-75).

[0013] Optionally, the OMVs are obtained by culturing and centrifuging a mutant strain of E. coli;

[0014] The Escherichia coli mutant strain was obtained by inactivating the pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain, or the Escherichia coli mutant strain was obtained by inactivating the ompA, pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain.

[0015] A second aspect of the present invention provides a method for preparing the vesicle system described above, wherein the method for preparing the vesicle system includes a method for preparing hard mixed vesicles and / or a method for preparing soft mixed vesicles;

[0016] The method for preparing the hard hybrid vesicles includes the following steps:

[0017] At room temperature, OMVs are mixed with hard liposomes, and then hard hybrid vesicles are prepared by one of the following methods: passive incubation, mechanical extrusion, ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method.

[0018] The method for preparing the soft hybrid vesicles includes the following steps:

[0019] At room temperature, OMVs are mixed with soft liposomes, and then soft mixed vesicles are prepared by one of the following methods: passive incubation, mechanical extrusion, ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method.

[0020] Optionally, the method for preparing the hard liposomes includes the following steps:

[0021] The components of the hard liposomes are mixed with a first organic solvent to obtain a first homogeneous lipid solution;

[0022] The first homogeneous lipid solution is transferred to a container, the first organic solvent is evaporated, and a lipid film is formed on the inner wall of the container.

[0023] Phosphate buffer solution was added to the container, followed by vortex hydration and sonication to obtain the hard liposomes;

[0024] The method for preparing the soft liposomes includes the following steps:

[0025] The components of the soft liposomes were mixed with a second organic solvent to obtain a second homogeneous lipid solution;

[0026] The second homogeneous lipid solution is transferred to a container, the second organic solvent is evaporated, and a lipid film is formed on the inner wall of the container.

[0027] Phosphate buffer was added to the container, followed by vortex hydration and sonication to obtain the soft liposomes.

[0028] Optionally, the preparation method of the OMVs includes the following steps:

[0029] Based on the Escherichia coli BL21(DE3) strain, the pagP and msbB genes were inactivated, or the ompA, pagP and msbB genes were inactivated on the Escherichia coli BL21(DE3) strain, to obtain Escherichia coli mutant strains;

[0030] After culturing and centrifuging the Escherichia coli mutant strain, OMVs were obtained.

[0031] Optionally, the passive incubation time is greater than or equal to 1 hour.

[0032] In a third aspect, the present invention provides a vaccine adjuvant, wherein the vaccine adjuvant comprises the vesicle system of the present invention as described above, or the vaccine adjuvant comprises a vesicle system prepared by the preparation method of the present invention as described above.

[0033] Optionally, the vaccine adjuvant further includes at least one of an antigen, a TLR agonist, a cytokine, and a stabilizing excipient.

[0034] Optionally, the vaccine adjuvant further includes a plasmid encapsulated in the hard mixed vesicles and / or soft mixed vesicles, the plasmid encoding a cytokine including at least one of IL-2, IL-17, TNF-α, and IFN-γ.

[0035] A fourth aspect of the invention provides the use of the vaccine adjuvant of the invention as described above in the preparation of preventive and therapeutic vaccines against infectious diseases, cancer, and chronic inflammatory diseases.

[0036] Beneficial Effects: In the vesicle system of this invention, two different liposomes are fused with OMVs via membrane fusion to obtain two types of mixed vesicles: hard mixed vesicles and soft mixed vesicles. This improves the delivery efficiency of OMVs and the efficiency of OMV uptake by APCs. Both mixed and soft mixed vesicles are internalized by APCs through a dual mechanism of phagolysosomal uptake and cytoplasmic delivery. Due to the mixed nature of hard and soft mixed vesicles (i.e., different liposomes are fused with and mixed with OMVs), the mechanism of APC internalization of OMVs can be selectively regulated. Hard and soft mixed vesicles can be internalized via the phagolysosomal / endosome pathway, promoting the processing of MHC class II (major histocompatibility complex II) molecules and CD4. + T cell initiation; delivery can also be achieved via a fusion-driven cytoplasmic pathway, facilitating cross-presentation via MHC class I (major histocompatibility complex I) molecules and activating cytotoxic CD8. + T-cell responses. This dual-pathway capability provides significant versatility for tailoring immune responses to a wide range of targets, including intracellular pathogens and tumors. However, hard mixed vesicles tend to shift the focus of substance delivery mechanisms towards the lysosomal pathway, while soft mixed vesicles tend to fuse with the cell membrane for direct delivery in the cytoplasm. Therefore, the balance between these two internalization mechanisms can be modulated and altered through hard and soft mixed vesicles to regulate the immune response. Furthermore, while both mixed and soft mixed vesicles can deliver OMV components to the cytosol, supporting cross-presentation of MHC class I molecules, and simultaneously process them intracellularly in the phagolysosomal region for MHC class II molecule presentation, hard mixed vesicles, due to their stronger membrane rigidity, are more efficient at promoting phagolysosomal maturation. In contrast, soft mixed vesicles exhibit slower uptake kinetics and induce less phagolysosomal maturation. These differences in intracellular transport kinetics affect the quality and intensity of the immune response, which can be modulated by adjusting the ratio of hard to soft mixed vesicles. Therefore, this vesicle system possesses immunomodulatory properties, providing a platform that can be adjusted according to desired immunological outcomes. Attached Figure Description

[0037] Figure 1 Pareto plots showing the normalized effects of fusing liposomes with OMVs using different parameters.

[0038] Figure 2 The figure shows the PCR verification results for the inactivation of the ompA, msbB, and pagP genes.

[0039] Figure 3The images show the atomic force microscopy nanomechanical characterization results of the co-deposition of soft and hard liposomes. In the images, A is the morphology diagram; B is the membrane rupture force diagram; C is the logarithmic Young's modulus diagram; and D is the distribution diagram of Young's modulus values.

[0040] Figure 4 Figure 1 shows the results of the fusion performance study of soft liposomes and OMVs. In this figure, A is the result of flow cytometry analysis; B is the result of confocal microscopy analysis; C is the result of fluorescence resonance energy transfer test; and D is the result of DLS test.

[0041] Figure 5 Confocal microscopy images of human macrophages after 3 hours of treatment with different substances. In the images, A is the negative control; B is OMVs treatment; C is hard liposome treatment; D is soft liposome treatment; E is hard mixed vesicle 1 treatment; F is hard mixed vesicle 2 treatment; G is hard mixed vesicle 3 treatment; H is soft mixed vesicle 1 treatment; I is soft mixed vesicle 2 treatment; and L is soft mixed vesicle 3 treatment.

[0042] Figure 6 Confocal microscopy images of human macrophages after 4 hours of treatment with different substances. In the images, A is the negative control; B is OMV treatment; C is hard liposome treatment; D is soft liposome treatment; E is hard mixed vesicle 1 treatment; F is hard mixed vesicle 2 treatment; G is hard mixed vesicle 3 treatment; H is soft mixed vesicle 1 treatment; I is soft mixed vesicle 2 treatment; and L is soft mixed vesicle 3 treatment.

[0043] Figure 7 Confocal microscopy quantitative results of human macrophages treated with different substances at different time points. In the figure, A represents hard liposome treatment, B represents OMVs treatment, and C represents hard mixed vesicle 2 treatment. Detailed Implementation

[0044] This invention provides a vesicle system and its preparation method, a vaccine adjuvant and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0046] If the embodiments of the present invention involve descriptions such as "first" or "second", such descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated.

[0047] This invention provides a vesicle system, wherein the vesicle system includes hard hybrid vesicles and / or soft hybrid vesicles, wherein the hard hybrid vesicles are obtained by membrane fusion of hard liposomes and OMVs, and the soft hybrid vesicles are obtained by membrane fusion of soft liposomes and OMVs.

[0048] The hard liposomes comprise the following components in parts by weight:

[0049] 1,2-Dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) 45.9–62.1 parts, 2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP, also known as 1,2-dioleoyl-3-trimethylammonium-propane (chloride)) 28.1–38 parts, 1,2-distearate-sn-glycerol-3-phosphoethanolamine (18:0, where 18 indicates that there are 18 carbon atoms in the fatty acid chain and 0 indicates that the fatty acid chain is fully saturated; denoted as PE) 8.5–11.5 parts, and 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000) 2.6–3.5 parts;

[0050] The soft liposomes comprise the following components in parts by weight:

[0051] 45.9–62.1 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 28.1–38 parts of DOTAP, 8.5–11.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC), and 2.6–3.5 parts of DMG-PEG2000.

[0052] To improve the delivery efficiency and immunomodulatory properties of OMVs, this embodiment fused two different types of liposomes with OMVs to obtain two types of hybrid vesicles. One type of hybrid vesicle used rigid liposomes (hard liposomes), while the other type used more fluid liposomes (soft liposomes). Hard and soft liposomes were classified based on the biophysical properties of the lipid bilayer, including membrane fluidity, elasticity, and phase behavior at physiological temperatures. Soft liposomes possess high lateral lipid fluidity and a liquid disordered phase that reduces membrane stiffness, promoting their deformability and dynamic interactions with the cell membrane. In contrast, hard liposomes maintain a more ordered, tightly packed bilayer structure, resulting in reduced fluidity and enhanced mechanical stability. These differences significantly affect biological behaviors such as cellular uptake, intracellular transport, and immune activation.

[0053] In this embodiment, hard liposomes and soft liposomes with specific content components can better fuse with OMVs, have higher fusion efficiency, and enable the hard mixed vesicles and soft mixed vesicles obtained after fusion to have the required properties (such as ensuring the final immunogenicity).

[0054] For hard liposomes fused with OMVs to form hard hybrid vesicles, DPPC and PE in the components of the hard liposomes have high gel-liquid crystal transition temperatures (DPPC's high gel-liquid crystal transition temperature is approximately 41°C, and PE's is approximately 74°C). Therefore, using fully saturated lipids such as DPPC and PE imparts rigidity and thermal stability to the hard hybrid vesicle membrane structure. Furthermore, DPPC can enhance the mechanical strength of hard hybrid vesicles, reduce permeability and deformation under physiological conditions, while PE, with its smaller head groups and saturated chains, can further improve membrane packing density and structural integrity. DOTAP can maintain its role as a cationic fusion agent and promote electrostatic interactions with OMVs. DMG-PEG2000, as a steric stabilizer, enhances pharmacokinetic properties and reduces aggregation, while maintaining the rigidity of the hard hybrid vesicles due to the anchoring of its saturated tail.

[0055] For the formation of soft hybrid vesicles by fusing soft liposomes with OMVs, the biophysical synergy of unsaturated polyethylene glycol-modified lipids was employed. DOPE, a component of the soft liposomes, possesses cis-unsaturated acyl chains and a conical geometry, introducing high negative curvature and promoting membrane fusion by forming fusion intermediates (such as stalks and semi-fused states). This property facilitates efficient fusion between liposomes and OMVs. DOTAP, a cationic lipid, provides a positive surface charge to promote electrostatic interactions with the typically negatively charged OMVs and enhances membrane fusion due to its fluid, disordered structure. DOPC, an unsaturated phosphatidylcholine, enhances structural stability and further promotes bilayer membrane fluidity. Furthermore, the addition of polyethylene glycol-modified lipids (such as DMG-PEG2000) significantly improves the colloidal stability of soft liposomes (or soft hybrid vesicles), preventing self-aggregation of soft hybrid vesicles during preparation and storage. DMG-PEG2000 also provides spatial barriers and surface hydrophilicity, reducing non-specific interactions with biological components and contributing to prolonged systemic circulation. The steric hindrance provided by the PEG chain in DMG-PEG2000 makes the soft mixed vesicles more stable by minimizing the interaction between them.

[0056] In this embodiment, the vesicle system includes hard mixed vesicles and soft mixed vesicles, which can improve the delivery efficiency of OMVs and the efficiency of OMV uptake by APCs. Both hard and soft mixed vesicles can be internalized by APCs through a dual mechanism of phagolysosome uptake and cytoplasmic delivery. Due to the mixed nature of hard and soft mixed vesicles (i.e., different liposomes fuse and mix with OMVs respectively), the mechanism of APC internalization of OMVs can be selectively modulated. Mixed vesicles (hard and soft mixed vesicles) can be internalized via the phagolysosome / endosome pathway (promoting MHC-II molecule processing and CD4). + T cell initiation) can also be achieved through a fusion-driven cytoplasmic pathway (which facilitates cross-presentation via MHC-I molecules and activation of cytotoxic CD8). + (T cell response). This dual-pathway capability provides significant versatility for tailoring immune responses to a wide range of targets, including intracellular pathogens and tumors. However, hard mixed vesicles tend to shift the focus of substance delivery mechanisms towards the lysosomal pathway, while soft mixed vesicles tend to fuse with the cell membrane for direct delivery in the cytoplasm. Therefore, the balance between these two internalization mechanisms can be modulated and altered through hard and soft mixed vesicles to regulate immune responses. Furthermore, while both hard and soft mixed vesicles can deliver OMV components to the cytosol, supporting cross-presentation of MHC class I molecules, and simultaneously process them within the phagolysosomal region for MHC class II molecule presentation, hard mixed vesicles, due to their stronger membrane rigidity, are more effective at promoting phagolysosomal maturation. In contrast, soft mixed vesicles exhibit slower uptake kinetics and induce less phagolysosomal maturation. These differences in intracellular transport dynamics affect the quality and intensity of the immune response. Therefore, the vesicle system provides a platform that can be adjusted according to the desired immunological outcome (which can be achieved by controlling the ratio of hard mixed vesicles to soft mixed vesicles. That is, only hard mixed vesicles, only soft mixed vesicles, or a mixture of hard mixed vesicles and soft mixed vesicles in different ratios can be used).

[0057] In this embodiment, these two types of liposomes jointly achieve precise control over the mechanical properties of the OMVs-liposome fusion, thereby modulating the intensity of the immune response induced by the vesicle system. The hard mixed vesicles exhibit stronger APC uptake capacity due to their mechanical stability and longer-term interaction with the cell surface. In contrast, the soft mixed vesicles are particularly effective in promoting endosome escape, thereby facilitating antigen cross-presentation and enhancing CD8. + T-cell response. The vesicle system provided in this embodiment (including hard mixed vesicles and soft mixed vesicles) is a dual-function platform that can both act as an adjuvant to activate the innate immune response and as a carrier to promote antigen delivery and presentation.

[0058] In some embodiments, the mass ratio of the OMVs to the hard liposomes in the hard mixed vesicles is (25-75):(25-75), for example, 25:25, 25:50, 25:75, 50:25, 50:75, 75:25, or 75:50; and the mass ratio of the OMVs to the soft liposomes in the soft mixed vesicles is (25-75):(25-75), for example, 25:25, 25:50, 25:75, 50:25, 50:75, 75:25, or 75:50.

[0059] In this embodiment, different mass ratios are used to fine-tune the physicochemical and immunological properties of hard mixed vesicles and soft mixed vesicles.

[0060] Despite the promising prospects of OMVs in vaccine development, researchers and companies studying OMVs still face the challenge of controlling their high reactivity. This is mainly due to the presence of lipopolysaccharide (LPS) in OMVs. LPS can effectively activate the Toll-like receptor 4 (TLR4)-MD-2 complex, leading to a strong release of pro-inflammatory cytokines, thus resulting in strong reactivity. Furthermore, the lipid A structure of LPS and its interaction with LPS-binding protein (LBP) and CD14 amplify immune signals. This strong immune activation can lead to systemic inflammation, making LPS a key endotoxin that triggers conditions such as septic shock. Therefore, a key challenge in OMV-based vaccine development is to minimize the reactivity of OMVs without compromising their immunogenicity. Currently available technical strategies for reducing the reactivity of OMVs include: (1) using bacterial mutants that produce detoxifying LPS variants, which modify strains such as Escherichia coli by removing specific acyl chains from the lipid A structure to produce LPS that is low in response to TLR4. This reduces the immune response triggered by OMVs to some extent; however, OMVs remain highly reactive even with this process alone. (2) Selective detergent treatment uses mild detergents (such as Triton X-100 or sodium deoxycholate) to extract and remove LPS from OMVs without damaging their overall structure. By carefully optimizing detergent concentration and incubation time, endotoxin levels can be reduced while maintaining the integrity of key antigenic components. However, overuse of detergents may result in the loss of important membrane proteins or other components crucial to the immunogenicity of OMVs. Furthermore, selective removal of LPS is not always thorough, and trace amounts of LPS may still remain in the final formulation. (3) Hydrostatic pressure filtration dialysis (HFD) is a scalable and cost-effective method that uses hydrostatic pressure to filter OMVs and remove soluble LPS contaminants. This technology achieves high recovery rates of OMVs with minimal processing pressure, making it an attractive option for large-scale production. However, hydrostatic pressure filtration dialysis is not effective in removing LPS that is tightly bound to the vesicle membrane, and this method needs further optimization to improve its efficiency and purity, especially in industrial-scale applications. In addition, there is limited data on the level of precision in LPS reduction achievable by this technique. (4) Affinity purification. LPS removal is achieved by adding affinity tags (such as His tags) to the surface proteins of OMVs. These labeled OMVs can be purified using techniques such as immobilized metal affinity chromatography (IMAC) to selectively capture vesicles while washing away LPS contaminants. However, although this method is effective in isolating OMVs, it requires genetic modification to introduce affinity tags, which limits its applicability to specific strains. In addition, the recovery rate of OMVs is low due to aggregation or steric hindrance, and the process is time-consuming and costly. (4) Gradient filtration.The process involves separating OMVs from smaller contaminants, including free LPS molecules, using membranes with gradually decreasing pore sizes. This method efficiently separates vesicles while maintaining structural integrity. However, gradient filtration can lead to reduced yields and the loss of smaller vesicle subpopulations during filtration. It also has limitations in removing highly bound or aggregated LPS, which may remain in the final product. In summary, despite these advancements in detoxification methods, no strategy currently perfectly addresses the challenge of LPS reactivity in OMV-based vaccines. While LPS is a key component in activating the immune system, excessive LPS, even after detoxification, can lead to an overly strong inflammatory response. Importantly, OMVs also contain other active components, such as nucleic acids (DNA and RNA), lipoproteins, and peptidoglycans, which collectively influence their overall reactivity. Therefore, a primary goal for OMV-based vaccines involves not only LPS detoxification but also reducing the amount of LPS to ensure a safer and more effective product. Thus, further improvements to these strategies and optimization of the application of OMVs in vaccines and treatments are needed. In summary, existing technologies for LPS detoxification or purification in OMVs offer a range of strategies for the safe application of OMVs in vaccines, but none are entirely satisfactory. More effective and scalable methods are still needed to comprehensively address the LPS reactivity issue while preserving the immunogenicity of OMVs.

[0061] Based on this, in some embodiments, the present invention employs a specific method for obtaining OMVs, which can reduce the reactivity of OMVs while retaining their immunogenicity. Specifically, the OMVs are obtained by culturing and centrifuging a mutant strain of E. coli;

[0062] The E. coli mutant strain was obtained by inactivating the pagP (lipid A palmitoyltransferase) and msbB (lipid A 3'-O-myristoyltransferase) genes based on the E. coli BL21(DE3) strain, or the E. coli mutant strain was obtained by inactivating the ompA (outer membrane protein A), pagP and msbB genes based on the E. coli BL21(DE3) strain.

[0063] In this embodiment, gene inactivation can be achieved by gene knockout, gene silencing, or insertion inactivation (i.e., inserting a foreign DNA fragment into the target gene).

[0064] In this embodiment, inactivation of the pagP gene prevents palmitoylation of lipid A, alters the lipid A structure of LPS, reduces the number of acyl chains, and thus reduces innate immune stimulation. Inactivation of the msbB gene (encoding a late acyltransferase responsible for hexaacylation of lipid A) further reduces the endotoxin content of LPS by producing a pentacylated lipid A form (i.e., it reduces hexaacylation of lipid A, thereby reducing endotoxin content). In other words, inactivation of the msbB and pagP genes leads to the production of hypoacylated lipid A within LPS, reducing LPS reactivity and significantly reducing the innate inflammatory response typically associated with Gram-negative bacterial OMVs. This allows the produced OMVs to maintain immunogenicity while exhibiting safer characteristics in terms of endotoxin activity. Inactivation of the ompA gene disrupts the outer membrane structure, induces hypervesicle formation, and increases OMV production. The OMVs produced using the *E. coli* mutant strain of this invention can increase OMV production and significantly improve its safety (reduced LPS reactivity), making it more suitable for human vaccine formulations.

[0065] In other words, in this embodiment, OMVs derived from a genetically engineered *E. coli* BL21(DE3) strain are fused with liposomes to obtain a vesicle system comprising hard mixed vesicles and / or soft mixed vesicles. This modified strain reduces LPS reactivity and enhances vesicle formation, while the liposomes impart the desired charge, rigidity, and membrane fusion properties. Compared to conventional OMVs, the resulting vesicle system exhibits improved APC uptake, enhanced immunomodulation, and higher safety, and can precisely modulate reactivity and immunogenicity to adapt to the specific needs of different immunization strategies. Furthermore, the decisive advantage of this system lies in its ability to influence and redirect cellular uptake pathways. Due to the mixed nature of hard and soft mixed vesicles (i.e., different liposomes are fused and mixed with OMVs respectively), the mechanism of APC internalization of OMVs can be selectively modulated. Hard and soft mixed vesicles can be internalized via the phagolysosome / endosome pathway (promoting MHC-II molecule processing and CD4). + T cell initiation), and delivery via a fusion-driven cytoplasmic pathway (facilitating cross-presentation via MHC-I molecules and activation of cytotoxic CD8). + (T cell response). This dual-pathway capability provides remarkable versatility for tailoring immune responses to a wide range of targets, including intracellular pathogens and tumors.

[0066] This invention also provides a method for preparing the vesicle system described above, wherein the method for preparing the vesicle system includes a method for preparing hard mixed vesicles and / or a method for preparing soft mixed vesicles;

[0067] The method for preparing the hard hybrid vesicles includes the following steps:

[0068] At room temperature, OMVs are mixed with hard liposomes and then hard hybrid vesicles are prepared by one of the following methods: passive incubation (passive incubation time is greater than or equal to 1 hour), mechanical extrusion (e.g., extrusion by a micro extruder), ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method.

[0069] The method for preparing the soft hybrid vesicles includes the following steps:

[0070] At room temperature, OMVs are mixed with soft liposomes, and then soft hybrid vesicles are prepared by one of the following methods: passive incubation (passive incubation time greater than or equal to 1 hour), mechanical extrusion (e.g., extrusion via a microextruder), ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method. In this embodiment, both hard and soft hybrid vesicles can be prepared using various methods. However, to develop a scalable and industrially feasible method for preparing hard and soft hybrid vesicles, this invention systematically evaluates various fusion methods between OMVs and liposomes. These mainly include mechanical extrusion using a microextruder, probe ultrasonic treatment, and passive incubation at room temperature. Quantitative flow cytometry, fluorescence resonance energy transfer assays of vesicles labeled with nitrobenzoxadiazole (NBD) / rhodamine, and dual-fluorescence labeled confocal microscopy measurements revealed comparable membrane fusion efficiencies across all methods, demonstrating the stability of the fusion process. However, significant differences emerged in process complexity, material preservation, and applicability to downstream manufacturing. While sonication achieves acceptable fusion efficiency, it leads to significant degradation and loss of internal components of OMVs, including nucleic acids with inherent immunostimulatory properties such as DNA and RNA. This loss of molecules can impair the complete adjuvant potential of the final vesicle system. Mechanical extrusion using microextruders, while effective, presents logistical and scalability challenges in manufacturing environments due to its equipment requirements, pressure demands, and the need for repetitive manual operation. These limitations make it unsuitable for large-scale, GMP-compliant vaccine production, especially under resource constraints. In contrast, passive incubation of OMVs and liposomes (hard or soft liposomes) at room temperature for just one hour achieves efficient and reproducible membrane fusion without specialized equipment or additional energy input (no mechanical shearing or external force required). This method preserves the integrity of OMVs (maintaining the structural and biochemical integrity of their components), is easily scalable and cost-effective, and avoids the complexities associated with sonication or extrusion. Furthermore, comparative analysis of 1-hour and 24-hour incubation showed no significant difference in fusion efficiency, making passive incubation a rapid, low-cost, mild, and practical method.

[0071] Meanwhile, to systematically optimize the physicochemical parameters affecting vesicle fusion efficiency and APC uptake, a Plackett-Burman experimental design was employed. Seven process variables were tested for the following 12 mixed vesicles (as shown in Table 1): fusion temperature, DMG-PEG2000 content (referred to as DMG-PEG in Table 1), DOTAP concentration, PE / PC (PE / DPPC or DOPE / DOPC) ratio, mechanical stress (passive vs. mechanical), fusion time, and fusion method. Analysis was performed using FACS (Fluorescence-Activated Cell Sorting), and data were statistically processed using Minitab software. Pareto analysis results are shown below. Figure 1 As shown, fusion time (factor F) and fusion method (factor G) are the most significant statistical factors contributing to the functional performance of hybrid vesicles (normalized effect > 2.776, α = 0.05). Notably, fusion time is positively correlated with uptake efficiency and membrane integration (i.e., uptake efficiency and membrane integration efficiency increase with increasing fusion time), and spontaneous fusion at room temperature yields comparable or even better results than energy-intensive methods (such as sonication or extrusion), thus simplifying the production process. Other influencing parameters include polyethylene glycolation (factor B) and DOTAP concentration (factor C), both of which significantly affect membrane fluidity and surface charge, thereby modulating vesicle-cell interactions. Interestingly, mechanofusion (factor E) shows a small but significant effect, further highlighting the practical advantages of non-mechanical room temperature incubation. The above tests show that passive incubation fusion at ambient temperature (only 1 hour) is both efficient and economical, requiring no special equipment. In addition, the PEG content and cationic lipid balance must be fine-tuned to optimize vesicle stability and immunogenicity (the specific optimized content is described above). The production process of passive incubation fusion is scalable, enabling rapid and low-cost manufacturing without significant infrastructure investment.

[0072] Table 1. Relevant information on 12 types of mixed vesicles

[0073]

[0074]

[0075] In some embodiments, the method for preparing the hard liposomes includes the following steps:

[0076] The components of the hard liposomes are mixed with a first organic solvent (such as chloroform) to obtain a first homogeneous lipid solution;

[0077] The first homogeneous lipid solution is transferred to a container, the first organic solvent is evaporated, and a lipid film is formed on the inner wall of the container.

[0078] Phosphate buffer was added to the container, followed by vortex hydration and sonication to obtain the hard liposomes.

[0079] In some embodiments, the method for preparing the soft liposomes includes the following steps:

[0080] The components of the soft liposomes are mixed with a second organic solvent (such as chloroform) to obtain a second homogeneous lipid solution;

[0081] The second homogeneous lipid solution is transferred to a container, the second organic solvent is evaporated, and a lipid film is formed on the inner wall of the container.

[0082] Phosphate buffer was added to the container, followed by vortex hydration and sonication to obtain the soft liposomes.

[0083] In some embodiments, the preparation method of the OMVs includes the following steps:

[0084] Mutant strains of Escherichia coli were obtained by inactivating the pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain, or by inactivating the ompA, pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain.

[0085] After culturing and centrifuging the Escherichia coli mutant strain, OMVs were obtained.

[0086] In this embodiment, the use of genetically engineered Escherichia coli BL21(DE3) strain for OMV preparation enhances OMV release while significantly reducing endotoxin content, minimizing adverse inflammatory responses while preserving the immunostimulatory capacity of OMVs. The method provided in this embodiment enables the preparation of high-yield, low-toxicity OMVs.

[0087] In some implementations, the pagP, msbB, and ompA genes are inactivated on Escherichia coli BL21(DE3) strains via the CRISPR-associated transposon system.

[0088] This invention also provides a vaccine adjuvant, wherein the vaccine adjuvant includes the vesicle system described above in this invention embodiment, or the vaccine adjuvant includes a vesicle system prepared using the preparation method described above in this invention embodiment.

[0089] In this embodiment, the vesicle system includes hard mixed vesicles and soft mixed vesicles, which can be internalized via the phagocytosolic / endosome pathway (promoting MHC-II molecule processing and CD4). + T cell initiation) can also be achieved through a fusion-driven cytoplasmic pathway (which facilitates cross-presentation via MHC-I molecules and activation of cytotoxic CD8).+ (T cell response). This dual-pathway capability provides significant versatility for tailoring immune responses to a wide range of targets, including intracellular pathogens and tumors. Furthermore, the differences in intracellular transport dynamics between mixed and soft mixed vesicles can influence the quality and intensity of the immune response; therefore, this vesicle system provides a platform that can be appropriately modulated according to desired immunological outcomes. The vesicle system exhibits improved APC uptake, enhanced immunomodulation, and greater safety, and allows for precise modulation of reactivity and immunogenicity to suit the specific needs of different immunization strategies.

[0090] The vesicle system in this embodiment of the invention has the following advantages:

[0091] Controllable reactivity: Reduced LPS content and acylation mode limit uncontrolled inflammation and enhance safety.

[0092] Optimized uptake: Adjustable biophysical properties that improve phagocytosis and / or membrane fusion based on composition and structure.

[0093] Enhanced immune activation: This vesicle system delivers immunogenic components via endosomal or cytoplasmic pathways, supporting the presentation of MHC-I and MHC-II antigens and promoting balanced Th1, Th2, Th17 and humoral responses.

[0094] Modular immune enhancement: The adjuvant effect can be enhanced or modulated through genetic or chemical addition (such as plasmids encoding cytokines or model antigens).

[0095] Manufacturing Scalability and Cost-Effectiveness: The production of this vesicle system relies on genetically engineered E. coli BL21(DE3) strains and standard commercially available liposome components. Manufacturing is highly reproducible and scalable through mature fermentation, offering a simple technology and significant economic advantages. For example, 1 liter of standard bacterial culture in a laboratory-scale fermenter can produce several milligrams of OMVs, sufficient to formulate hundreds to thousands of vaccine doses (depending on the final concentration). This low-cost, high-volume production strategy, coupled with the inherent stability of the mixed vesicles, makes this vesicle system a promising candidate for numerous applications.

[0096] In some embodiments, the vaccine adjuvant further includes at least one of an antigen (such as a protein, peptide, or nucleic acid), a TLR (Toll-like receptor) agonist, a cytokine, and a stabilizing excipient. At least one of the TLR agonist, cytokine, and stabilizing excipient may be physically mixed with the vesicle system.

[0097] In some embodiments, the vaccine adjuvant further includes a plasmid encapsulated in the hard and / or soft mixed vesicles, the plasmid encoding a cytokine including at least one of IL-2 (interleukin-2), IL-17 (interleukin-17), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ). This plasmid may be a human expression plasmid based on pIRES.

[0098] In this embodiment, although the vaccine adjuvant (i.e., the adjuvant platform) is fully functionalized, its immunostimulatory capacity can be further enhanced. Specifically, in this embodiment, hard mixed vesicles and / or soft mixed vesicles are electroporated to encapsulate plasmids (such as pIRES-based human expression plasmids) encoding immunomodulatory cytokines such as IL-2, TNF-α, IFN-γ, and IL-17. These cytokines play key roles in T cell proliferation, cytotoxic response activation, and regulation of the inflammatory environment, thereby amplifying cellular and humoral immunity. The adjuvant platform provided by this invention allows for precise immunological customization according to therapeutic purposes, positioning the platform as a highly adaptable and customizable system applicable to a variety of vaccine applications, including but not limited to vaccines for the prevention or treatment of infectious diseases and cancer.

[0099] This invention also provides the application of the vaccine adjuvant described above in the present invention in the preparation of preventive and therapeutic vaccines against infectious diseases, cancer, and chronic inflammatory diseases.

[0100] The present invention will be further described below through specific embodiments.

[0101] Unless otherwise specified, the materials used in the following embodiments are all commercially available products.

[0102] Example 1: Construction of Escherichia coli mutant strain

[0103] The construction of a three-gene-inactivated E. coli mutant strain using the CRISPR-related transposon system involves inserting exogenous genetic material sequences into three target genes, ompA, pagP, and msbB, to inactivate the target genes. The steps include:

[0104] (1) Construction and validation of mutant strains

[0105] The vector pSL1765 (Addgene#160734; 100 ng / μL, which is an INTEGRATE vector without crRNA) and the custom plasmid pUC57-OMVs (synthesized by Qingke Biotechnology; 100 ng / μL, obtained by inserting crRNA sequences targeting three target genes ompA, pagP and msbB into plasmid pUC57, the nucleotide sequence of crRNA is shown in SEQ ID NO: 1, plasmid pUC57 is a commercial product) were mixed at a molar ratio of 2:1 and assembled by the Golden Gate cloning method to obtain the vector pSL1765-OMVs.

[0106] The assembly process of the Kinmen cloning method is as follows:

[0107] The above assembly reaction system (containing BsaI-HFv2 and Hi-T4 DNA ligase) was digested (37°C, 2 minutes) and ligated (25°C, 2 minutes) for a total of 25 cycles. Then, it was incubated at 37°C for 30 minutes, 60°C for 5 minutes and 80°C for 10 minutes in sequence to complete the final digestion and enzyme inactivation. The total reaction time was about 2.5 hours.

[0108] Subsequently, 10 μL of the assembly product was transformed into 100 μL of chemocompetent E.coli DH10B cells. The specific steps were: incubation on ice for 30 minutes, heat shock at 42°C for 60–90 seconds (90 seconds in this example), and then recovery on ice for 2 minutes.

[0109] Cells were then revived in 900 μL of NEB 10-beta resuscitation medium (37°C, 1 h, 220 rpm) and spread on LB agar plates containing kanamycin (50 μg / mL). Colonies were obtained by incubation at 37°C for 24–48 h (36 h in this example).

[0110] Three single colonies were picked and inoculated into 3 mL of LB broth containing kanamycin (50 μg / mL) and cultured overnight (37°C, 220 rpm). After Sanger sequencing verification, the correct pSL1765-OMVs plasmid was extracted and transformed into E. coli BL21(DE3). The colonies were cultured on LB plates containing kanamycin (50 μg / mL) at 25°C for 48–72 hours (60 hours in this example).

[0111] (2) Gene integration verification

[0112] Colony PCR and Sanger sequencing were performed using target gene-specific primers to verify genome integration.

[0113] For incompletely integrated colonies, enrichment was achieved by three consecutive subcultures (1:100 dilution, 25°C) in selective LB-kanamycin medium, followed by rescreening. Completely integrated colonies were subcultured in antibiotic-free LB medium (1:1000 dilution, 37°C) to eliminate editing plasmids.

[0114] The resulting E. coli mutant strain was preserved by mixing the culture with a 50% glycerol solution at a 1:1 ratio, thus completing the inactivation of the three target genes of the strain.

[0115] The *E. coli* mutant strain was subjected to agarose gel electrophoresis (2%) to verify that the exogenous genetic material sequence had been inserted into the *ompA*, *msbB*, and *pagP* genes of *E. coli* Bl21(DE3), i.e., to verify whether the *ompA*, *msbB*, and *pagP* genes were inactivated (or destroyed). The results are as follows: Figure 2 As shown (using the molecular weight marker DL 5000 as a size reference), PCR amplification of the wild-type *E. coli* alleles produced products of approximately 325 bp, 365 bp, and 363 bp, respectively, while PCR amplification of the *E. coli* mutant strains prepared above produced mutant amplicones of 1302 bp, 1342 bp, and 1340 bp, respectively, consistent with the expected size increase due to the insertion of exogenous genetic material sequences. These results confirm the correct integration of all three target sites, and the ompA, msbB, and pagP genes were successfully inactivated. The primers used for gene inactivation verification are shown in Table 2, and the PCR program used a standard three-step method: 35 cycles, with an annealing temperature of 57°C.

[0116] Table 2. Primer sequences

[0117]

[0118] Example 2: Preparation of OMVs

[0119] The *E. coli* mutant strain prepared in Example 1 was cultured in LB broth to mid-log phase, and then bacterial cells were removed by centrifugation. The supernatant was filtered through a 0.22 μm membrane and then ultracentrifuged at 100,000 × g for 2 hours at 4 °C. The resulting precipitate (i.e., OMV particles) was washed in PBS, resuspended, and stored at 4 °C or -80 °C for later use.

[0120] Example 3: Preparation of hard liposomes and hard mixed vesicles

[0121] (1) Preparation of hard liposomes

[0122] The lipid components (DOPE, DOTAP, DOPC, and DMG-PEG2000) were lyophilized.

[0123] The lyophilized lipid fraction (composed of 54 parts by weight of DOPE, 33 parts by weight of DOTAP, 10 parts by weight of DOPC and 3 parts by weight of DMG-PEG2000) was dissolved in chloroform to obtain a homogeneous lipid solution with a lipid fraction concentration of 10 mg / mL.

[0124] The homogeneous lipid solution was transferred to a clean glass tube, and the solvent was evaporated under an argon atmosphere to form a lipid membrane along the inner side of the tube. Then, phosphate buffer was added to make the lipid membrane concentration 1 μg / μL to obtain a suspension.

[0125] Vigorously vortex the hydrated suspension for 1 minute to promote liposome formation, then place it in a 37°C water bath and sonicate for 30 minutes to enhance homogeneity. During the 30-minute sonication, after 15 minutes of sonication, briefly vortex for 30 seconds to resuspend any aggregates, and vortex again for 1 minute at the end of the 30-minute sonication to obtain bilayer spherical hard liposomes.

[0126] (2) Preparation of hard mixed vesicles

[0127] At room temperature (25°C), OMVs and hard liposomes were mixed in phosphate buffer at mass ratios of 75:25, 50:50 and 25:75, respectively, and incubated for one hour to obtain three types of hard mixed vesicles (referred to as hard mixed vesicle 1, hard mixed vesicle 2 and hard mixed vesicle 3, respectively).

[0128] Example 4: Preparation of soft liposomes and soft mixed vesicles

[0129] (1) Preparation of soft liposomes

[0130] The lipid components (DPPC, DOTAP, PE, and DMG-PEG2000) were lyophilized.

[0131] The lyophilized lipid fraction (composed of 54 parts by mass of DPPC, 33 parts by mass of DOTAP, 10 parts by mass of PE and 3 parts by mass of DMG-PEG2000) was dissolved in chloroform to obtain a homogeneous lipid solution with a lipid fraction concentration of 10 mg / mL.

[0132] The homogeneous lipid solution was transferred to a clean glass tube, and the solvent was evaporated under an argon flow to form a lipid membrane along the inner side of the tube. Then, phosphate buffer was added to hydrate the lipid membrane to a concentration of 1 μg / μL, thus obtaining a suspension.

[0133] The hydrated suspension was vigorously vortexed for 1 minute to promote liposome formation, and then placed in a 37°C water bath and sonicated for 30 minutes to enhance homogeneity. During the 30-minute sonication, after 15 minutes of sonication, the suspension was briefly vortexed for 30 seconds to resuspend any aggregates, and then vortexed again for 1 minute at the end of the 30-minute sonication to obtain bilayer spherical soft liposomes.

[0134] (2) Preparation of soft mixed vesicles

[0135] At room temperature (25°C), OMVs and soft liposomes were mixed in phosphate buffer at mass ratios of 75:25, 50:50 and 25:75, respectively, and incubated for one hour to obtain three soft mixed vesicles (referred to as soft mixed vesicle 1, soft mixed vesicle 2 and soft mixed vesicle 3, respectively).

[0136] test:

[0137] (1) The soft and hard liposomes prepared in Examples 3 and 4 were deposited and developed on mica using atomic force microscopy (AFM) and then subjected to nanomechanical analysis. The results are as follows: Figure 3 As shown in the figure (log) 10 (E / Pa) In this context, E represents Young's modulus. Compared to soft liposomes, hard liposomes exhibit greater thickness due to their hydrocarbon-tailed crystal conformation, along with higher delamination forces and a higher Young's modulus (180 MPa, compared to only 38 MPa for soft mixed vesicles). Furthermore, well-separated domains were found within individual liposomes, and this phase separation may play an important role in cellular uptake.

[0138] (2) Flow cytometry with dual fluorescent labeling was used to visualize colocalization and membrane integration. Specifically, different membrane intercalation dyes were used for flow cytometry analysis: PKH26 was used to label OMVs, and PKH67 was used to label soft liposomes. The results are as follows: Figure 4 As shown in A in the diagram.

[0139] Dual-fluorescent labeling confocal microscopy was used to visualize colocalization and membrane integration. Specifically, OMVs were labeled with PKH26 and soft liposomes with PKH67. Soft mixed vesicles were deposited on a confocal culture dish, and then confocal microscopy was used to display signal overlap and colocalization. The results are shown below. Figure 4 As shown in B, it can be seen that OMVs fused with liposomes.

[0140] Fluorescence resonance energy transfer (FRET) assays confirmed membrane fusion between OMVs and soft liposomes, as shown in the results. Figure 4As shown in C, the FRET effect (black curve) is activated by inserting dual fluorescent groups (i.e., nitrobenzoxadiazole fluorescent group (NBD) and rhodamine (RHOD)) into the liposome, while the fusion of OMVs with the liposome inactivates the effect (red curve).

[0141] Dynamic light scattering (DLS) was used to evaluate size distribution and stability, and the results... Figure 4 As shown in D, the particle size of OMVs is 50-100 nm, the particle size of soft liposomes is 120 nm, and the particle size of soft mixed vesicles 2 is 200 nm.

[0142] Therefore, it can be seen that OMVs and soft liposomes were successfully fused to a membrane, resulting in the preparation of soft hybrid vesicles 2. This invention employs a method of directly incubating OMVs with liposomes to prepare soft hybrid vesicles 2. This simplified fusion process not only promotes wider implementation and technology transfer but also enhances its applicability for rapid, low-cost production in industrial and decentralized environments.

[0143] (3) APC (macrophage) uptake test

[0144] (31) Using different substances to treat human macrophages (1×10 5 Cells / wells were processed and co-incubated for 3 hours. Images were then acquired using a confocal microscope (60x magnification, 3x zoom). The specific steps are as follows:

[0145] Human macrophages (1×10) 5 After incubating cells / well with 5 μg of hard-mixed vesicles for 1 hour, Hoechst33342 and LysoTracker were added. TM Deep Red was used to label the cell nuclei and acidic compartments, respectively. At the end of the 3-hour incubation period, the hardened mixed vesicles were removed and the wells were washed twice with PBS. Images were then acquired using a confocal microscope.

[0146] Repeat the above process, replacing the hard mixed vesicles with OVMs, hard liposomes, soft liposomes, and soft mixed vesicles respectively. After incubation for 3 hours, images are acquired using a confocal microscope.

[0147] The results are as follows Figure 5 As shown, cell nuclei are blue (Hoechst 33342 labeled), PKH26-labeled OMVs are red, PKH67-labeled liposomes are green, and phagolysosomes are magenta (LysoTracker). TM (Deep Red marker), the co-location area is yellow.

[0148] Figure 5In the diagram, A represents the negative control; B represents OMVs treatment; C represents hard liposome treatment; D represents soft liposome treatment; E represents hard mixed vesicle 1 (OMVs: hard liposomes = 75:25) treatment; F represents hard mixed vesicle 2 (OMVs: hard liposomes = 50:50); G represents hard mixed vesicle 3 (OMVs: hard liposomes = 25:75) treatment; H represents soft mixed vesicle 1 (OMVs: soft liposomes = 75:25) treatment; I represents soft mixed vesicle 2 (OMVs: soft liposomes = 50:50) treatment; and L represents soft mixed vesicle 3 (OMVs: soft liposomes = 25:75) treatment.

[0149] Depend on Figure 5 This demonstrates that the intracellular delocalization of OMVs and liposomes, and their spatial distribution within or on the cell surface after fusion to form mixed vesicles, differ from their intracellular distribution. Normally, liposomes are primarily localized to the cell membrane, while OMVs are internalized. This behavior indicates that mixed vesicles exhibit a mixed uptake mechanism upon contact with the cell: liposomes partially fuse with the plasma membrane while OMVs are internalized, or both components are internalized simultaneously. Confocal images also show colocalization and spatial separation between OMVs and phagolysosomes, further illustrating that mixed vesicles exhibit two distinct internalization pathways: direct membrane fusion and endocytic uptake.

[0150] The fusion process between OMVs and liposomes results in a structure with phase-separated domains, in which the native OMV membrane is dispersed within the lipid phase of the liposome. This asymmetric distribution stems from the inherent differences in membrane density, rigidity, and curvature between the OMVs and the liposome components. When macrophages encounter mixed vesicles rich in native OMV membranes, endocytosis is triggered, directing the mixed vesicles towards the phagocytosolic system. Conversely, when mixed vesicles contact the cell surface via the phospholipid domains of the liposome, membrane fusion occurs, allowing the OMVs to directly enter the cytoplasm.

[0151] In addition, hard mixed vesicles exhibited better uptake efficiency than soft mixed vesicles and were able to promote the maturation of phagolysosomes.

[0152] (32) Using different substances to treat human macrophages (1×10 5 Cells / well were processed and co-incubated for 4 hours. Images were then acquired using a confocal microscope (60x magnification, 3x zoom). The specific steps are as follows:

[0153] Human macrophages (1×10) 5 Cells / well) were incubated with 5 μg of hard-mixed vesicles, and Hoechst 33342 and LysoTracker were added after 1 hour. TM Deep Red was used to label the cell nuclei and acidic compartments, respectively. At the end of the 4-hour incubation period, the hardened mixed vesicles were removed and the wells were washed twice with PBS. Images were then acquired using a confocal microscope.

[0154] Repeat the above process, replacing the hard mixed vesicles with OVM, hard liposomes, soft liposomes, and soft mixed vesicles respectively. After incubation for 3 hours, images are acquired using a confocal microscope.

[0155] The results are as follows Figure 6 As shown, cell nuclei are blue (Hoechst 33342), PKH26-labeled OMVs are red, PKH67-labeled liposomes are green, and phagolysosomes are magenta (LysoTracker). TM (Deep Red), the co-location area is yellow.

[0156] In this diagram, A represents the negative control; B represents OMVs treatment alone; C represents hard liposome treatment; D represents soft liposome treatment; E represents hard mixed vesicle 1 (OMVs: hard liposomes = 75:25) treatment; F represents hard mixed vesicle 2 (OMVs: hard liposomes = 50:50) treatment; G represents hard mixed vesicle 3 (OMVs: hard liposomes = 25:75) treatment; H represents soft mixed vesicle 1 (OMVs: soft liposomes = 75:25) treatment; I represents soft mixed vesicle 2 (OMVs: soft liposomes = 50:50) treatment; and L represents soft mixed vesicle 3 (OMVs: soft liposomes = 25:75) treatment.

[0157] Figure 6 This can explain that hard mixed vesicles ( Figure 6 E, F, and G in the data are compared to OMVs ( Figure 6 B) or soft mixed vesicles ( Figure 6 Treatment with H, I, and L in macrophages induces greater maturation of phagolysosomes. Specifically, hard-mixed vesicles exhibit superior uptake kinetics and promote the maturation of larger phagolysosomes in human macrophages. Hard-mixed vesicles also trigger more efficient antigen presentation and co-stimulatory marker expression.

[0158] From the above Figure 5 and Figure 6 The results demonstrate that a significant characteristic of mixed vesicles is their mixing behavior upon uptake by APCs, reflecting the properties of both components (OMVs and liposomes). Confocal images show colocalization and spatial separation between OMVs and phagolysosomes, indicating two distinct internalization pathways for mixed vesicles: direct membrane fusion and endocytic uptake (the coexistence of these two pathways is due to the heterogeneous topology of the mixed vesicle membrane). This embodiment reveals, through confocal microscopy, two main and mechanistically different pathways for macrophage internalization of mixed vesicles, each influencing antigen presentation outcomes and adaptive immune activation:

[0159] (a) Phagolysosomal uptake pathway: Mixed vesicles are internalized via classical endocytosis, similar to natural OMVs. After recognition and phagocytosis, the mixed vesicles are transported to phagolysosomes, where they undergo enzymatic degradation. This compartmentalization facilitates antigen presentation via the MHC class II pathway, thereby initiating CD4 uptake. + T helper cells. This uptake pathway is particularly beneficial in bacterial infections or extracellular pathogen environments that require a strong Th1 / Th17 response.

[0160] (b) Direct membrane fusion and cytoplasmic delivery: In another pathway, the liposomal component of the mixed vesicle (rich in fusion phospholipids) fuses directly with the macrophage plasma membrane. This fusion results in the cytoplasmic release of the contents of the OMVs, bypassing lysosomal degradation. This intracellular delivery pathway supports cross-presentation via the MHC-I pathway and subsequent activation of CD8. + Cytotoxic T lymphocytes make them particularly suitable for antiviral and anticancer vaccine applications.

[0161] (33) Human macrophages were stimulated with 5 μg hard liposomes, 5 μg OMVs, and 5 μg hard mixed vesicle 2 (OMVs: hard liposomes = 50:50), respectively. Images were acquired using confocal microscopy at different time points (60-100 cells from 6 samples were used at each time point). Images were segmented to identify individual cells and the average signal intensity was quantified cell by cell, i.e., quantitative analysis was performed using confocal microscopy. The results are as follows: Figure 7 As shown, colocalization of OMV contents within cellular regions is clearly observed only after a considerable time. In contrast, hard mixed vesicles exhibit the rapid kinetic advantage derived from hard liposomes, delivering OMVs into cells within fifteen minutes. This indicates that the stiffness of mixed vesicles can modulate the rate and manner of internalization, further influencing downstream immunological characteristics. This underscores the importance of modulating the biophysical properties of mixed vesicles (such as membrane stiffness and surface charge), which directly affect mixed vesicle transport and tailor antigen presentation pathways for specific immune outcomes.

[0162] In summary, this invention provides a vesicle system and its preparation method, a vaccine adjuvant, and its application. Firstly, this invention achieves high-yield, low-toxicity OMV production. Specifically, it utilizes a genetically engineered *E. coli* mutant strain to enhance OMV release while significantly reducing endotoxin content, minimizing adverse inflammatory responses while preserving the immunostimulatory capacity of OMVs. Then, combining genetic engineering, vesicle biophysics, and modular delivery capabilities, it provides a modular vesicle system (vesicle platform) with dual functional characteristics, capable of improving safety and inducing a strong and controllable immune response. Specifically, two different types of hybrid vesicles—hard hybrid vesicles and soft hybrid vesicles—are developed by fusing OMVs with component-optimized liposomes. The ratio of OMVs to liposomes and the liposome components can be finely adjusted. Both hard and soft hybrid vesicles are internalized by APCs through a dual mechanism of phagolysosomal uptake and cytoplasmic delivery, delivering OMV components to the cytosol, supporting MHC-I cross-presentation, and simultaneously undergoing intracellular processing in the phagolysosomal region for MHC-II presentation. However, hard mixed vesicles tend to shift the focus of substance delivery mechanisms towards the lysosomal pathway, while soft mixed vesicles tend to fuse with the cell membrane, thereby delivering substances directly in the cytoplasm. Therefore, the balance between these two internalization mechanisms can be modulated and altered through hard and soft mixed vesicles, thereby regulating the immune response. Hard mixed vesicles, due to their higher membrane rigidity, more effectively promote the maturation of phagolysosomes. In contrast, soft mixed vesicles exhibit slower uptake kinetics and induce less phagolysosomal maturation. These differences in intracellular transport kinetics affect the quality and intensity of the immune response, making the vesicle system a platform that can be appropriately modulated according to desired immunological outcomes. This platform enables the development of a new class of adjuvants with improved efficacy and safety, adaptable to diverse prophylactic and therapeutic vaccination needs, by providing synthetic-biological hybrids with controllable physicochemical and immunological properties. Furthermore, the platform allows for the incorporation of different plasmids into mixed vesicles, thereby enabling customizable modulation of immune responses for different vaccine applications. This allows it to be used in the preparation of preventive or therapeutic vaccine formulations (including but not limited to vaccines against intracellular pathogens, emerging viruses, or tumor antigens).

[0163] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A vesicle system, characterized in that, The vesicle system includes hard hybrid vesicles and soft hybrid vesicles. The hard hybrid vesicles are obtained by membrane fusion of hard liposomes and outer membrane vesicles, and the soft hybrid vesicles are obtained by membrane fusion of soft liposomes and outer membrane vesicles. The hard liposomes comprise the following components in parts by weight: 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline 45.9~62.1 parts, (2,3-dioleoyl-propyl)-trimethylammonium chloride 28.1~38 parts, 1,2-distearate-sn-glycerol-3-phosphate ethanolamine 8.5~11.5 parts, and 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000 2.6~3.5 parts; The soft liposomes comprise the following components in parts by weight: 45.9~62.1 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine, 28.1~38 parts of (2,3-dioleoyl-propyl)-trimethylammonium chloride, 8.5~11.5 parts of 1,2-dioleoyl-sn-glycerol-3-phosphate choline, and 2.6~3.5 parts of 1,2-dimyristoyl-racemic-glycerol-3-methoxy polyethylene glycol 2000; The outer membrane vesicles were obtained by culturing and centrifuging a mutant strain of Escherichia coli. The Escherichia coli mutant strain was obtained by inactivating the pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain, or the Escherichia coli mutant strain was obtained by inactivating the ompA, pagP and msbB genes on the basis of Escherichia coli BL21(DE3) strain. The vesicle system is internalized via a phagocytosomal / endosome pathway and simultaneously delivered via a fusion-driven cytoplasmic pathway.

2. The vesicle system according to claim 1, characterized in that, In the hard hybrid vesicles, the mass ratio of the outer membrane vesicles to the hard liposomes is (25~75):(25~75). In the soft mixed vesicles, the mass ratio of the outer membrane vesicles to the soft liposomes is (25~75):(25~75).

3. A method for preparing the vesicle system according to any one of claims 1-2, characterized in that, The preparation method of the vesicle system includes a method for preparing hard hybrid vesicles and / or a method for preparing soft hybrid vesicles; The method for preparing the hard hybrid vesicles includes the following steps: At room temperature, outer membrane vesicles are mixed with hard liposomes, and then hard mixed vesicles are prepared by one of the following methods: passive incubation, mechanical extrusion, ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method. The method for preparing the soft hybrid vesicles includes the following steps: At room temperature, outer membrane vesicles are mixed with soft liposomes, and then soft mixed vesicles are prepared by one of the following methods: passive incubation, mechanical extrusion, ultrasonic treatment, cryogenic cycling, or microfluidic-assisted method.

4. The preparation method according to claim 3, characterized in that, The method for preparing the hard liposomes includes the following steps: The components of the hard liposomes are mixed with a first organic solvent to obtain a first homogeneous lipid solution; The first homogeneous lipid solution is transferred to a container, the first organic solvent is evaporated, and a lipid film is formed on the inner wall of the container. Phosphate buffer solution was added to the container, followed by vortex hydration and sonication to obtain the hard liposomes; The method for preparing the soft liposomes includes the following steps: The components of the soft liposomes were mixed with a second organic solvent to obtain a second homogeneous lipid solution; The second homogeneous lipid solution is transferred to a container, the second organic solvent is evaporated, and a lipid film is formed on the inner wall of the container. The soft liposomes were obtained by adding phosphate buffer to the container and then performing vortex hydration and sonication.

5. The preparation method according to claim 3, characterized in that, The method for preparing the outer membrane vesicles includes the following steps: Based on the Escherichia coli BL21(DE3) strain, the pagP and msbB genes were inactivated, or the ompA, pagP and msbB genes were inactivated on the Escherichia coli BL21(DE3) strain, to obtain Escherichia coli mutant strains; The Escherichia coli mutant strain was cultured and centrifuged to obtain outer membrane vesicles.

6. The preparation method according to claim 3, characterized in that, The passive incubation time is greater than or equal to 1 hour.

7. A vaccine adjuvant, characterized in that, The vaccine adjuvant includes the vesicle system according to any one of claims 1-2, or the vaccine adjuvant includes the vesicle system prepared by the preparation method according to any one of claims 3-6.

8. The vaccine adjuvant according to claim 7, characterized in that, The vaccine adjuvant also includes at least one of antigen, TLR agonist, cytokine, and stabilizing excipient.

9. The vaccine adjuvant according to claim 7, characterized in that, The vaccine adjuvant further includes a plasmid encapsulated in the hard mixed vesicles and / or soft mixed vesicles, the plasmid encoding a cytokine including at least one of IL-2, IL-17, TNF-α, and IFN-γ.

10. The use of a vaccine adjuvant according to any one of claims 7-9 in the preparation of preventive and therapeutic vaccines against infectious diseases, cancer, and chronic inflammatory diseases.

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