A universal bacterial outer membrane vesicle delivery system GMMA

Abstract

The application discloses a general bacterial outer membrane vesicle delivery system GMMA, and relates to the technical field of synthetic biology vaccine delivery. The application first extracts outer membrane vesicles OMV with a diameter of 50-100 nm from gram-negative bacteria by a deoxycholic acid sodium method, and then couples tris (hydroxymethyl) aminomethane biotin to the surface of the vesicles by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The monomer avidin which is mutated at six sites A67D, G71K, A82N, G84Q, S101R and A105K and is truncated into 114 aa is fused and expressed with target antigens through a pentapeptide GGGGS, the obtained avidin-antigen fusion protein is self-assembled with the biotinylated vesicles to form the GMMA, and the binding affinity is KD=6.6*10 ‑8 The system does not need to be genetically modified with multiple genes of a bacterial strain, can load one or more antigens, has a reversible plug-in mode in the loading process, has an intact vesicle structure, is simple to prepare, has good safety, and is suitable for the fields of vaccine and other antigen delivery.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology vaccine delivery technology, and in particular to a universal bacterial outer membrane vesicle delivery system GMMA. Background Technology

[0002] OMVs are outer membrane vesicles released by Gram-negative bacteria, primarily containing bacterial outer membrane and periplasmic components. The outer membrane is composed of phospholipids and lipopolysaccharides (LPS), with the LPS located on the outer side interspersed with membrane proteins. The vesicle lumen may contain various substances derived from the bacterial periplasm or cytoplasm, such as proteins, RNA or DNA, and peptidoglycan. Because OMVs have been found to induce T-cell immune responses, numerous studies have investigated the potential immunogenicity of OMV components in vivo. Vesicles enter cells via receptor-mediated endocytosis. The common component OmpA in vesicles also facilitates cell entry and comprehensively activates macrophage proliferation. OMVs can extract surface proteins into APC cells, and their inherent characteristics give them an adjuvant effect, making them an ideal vaccine carrier. To date, the first-generation OMV vaccine Bexsero (Novartis) has been approved for marketing to prevent meningococcal meningitis (SMB) infection. Several other OMV-based products are in clinical trials focusing on infectious diseases, including pneumonia, meningitis, and pertussis. Furthermore, OMVs have demonstrated a range of functions in assisting bacterial infection of host cells. Most uniquely, OMVs serve as a delivery system for pathogen-associated molecular patterns (PAMPs). PAMP-loaded OMVs, along with other membrane components influencing infection-related host responses, can be delivered to distant cells. The immunomodulatory properties of OMVs point to the generation of protective mucosal immunity and bactericidal antibody responses, potentially providing a platform for vaccine development. The use of OMVs derived from *Escherichia coli*, *Salmonella*, and *Neisseria meningitidis* as vaccine antigen delivery systems to elicit effective B-cell responses has been extensively studied in numerous publications.

[0003] Neisseria meningitidis is a Gram-negative bacterium, morphologically a kidney-shaped or bean-shaped diplococcus with a flat or slightly concave contact surface, and a diameter of 0.6–0.8 μm. In artificial culture, it can form oval or spherical shapes, arranged irregularly, singly, in pairs, or in groups of four. In the cerebrospinal fluid of patients, it is mostly located within neutrophils, exhibiting a typical morphology; newly isolated strains mostly have capsules and pili. Group B meningococcal bacteria can secrete outer membrane vesicles (OMV), and the induced immunity primarily targets the highly variable outer membrane protein PorA. Randomized, double-blind, controlled clinical trials of two OMV vaccines (Cuban OMV and Norwegian OMV) conducted during the Chilean Group B meningococcal epidemic season showed that the OMV vaccine only provides serum subtype-specific protection, offering no protection against strains with heterologous PorA, and even showing no response in infants under one year old. However, it provides a good immune response against circulating strains with homologous PorA. Therefore, OMV is effective in eradicating epidemics caused by clonal strains within a certain area.

[0004] Studies have shown that covering 80% of the circulating strains in the United States requires a vaccine containing 20 different PorA antigens. Clearly, the diversity of PorA in many countries makes developing an unmodified OMV vaccine impractical. Furthermore, the duration of immunity is unclear; in large-scale vaccination campaigns in Zealand, infants were required to receive up to four doses. To address the limited coverage of a single PorA protein, another strategy has been developed: ensuring that an OMV contains more than two PorA proteins. The Netherlands Vaccine Institute has developed a hexavalent group B OMV vaccine, requiring a fourth booster dose between 12 and 18 months of age.

[0005] OMV vaccines have been used for decades, with more than 30 million doses administered. Their safety and efficacy have been verified. However, the most important limitation is that the immune response is mainly directed against homologous serum subtypes, which restricts the widespread use of OMV.

[0006] In summary, OMV is a spherical particle secreted during the growth of Gram-negative bacteria, with various antigens distributed on its surface. OMV has been used in vaccines and is also being developed as an antigen delivery vector. Much research has been conducted in the field of universal OMV development, with the most rapid progress being in the meningococcal OMV system, as well as in Salmonella, etc. This system is known in English as the GMMA antigen delivery system.

[0007] There are still many challenges in the development of OMV as a vaccine or vector. Taking meningococcal OMV as an example, firstly, the traditional OMV preparation process is complex and inefficient, with uneven particle size and unstable quality; secondly, using OMV to deliver porA protein of multiple serotypes requires complex gene editing to integrate porA from different strains into one bacterium to achieve the goal of multivalent antigen delivery; thirdly, OMV antigen delivery is limited to the delivery of antigens from the bacteria themselves and cannot be extended to any antigen; fourthly, as a GMMA system, the methods in the literature still lack flexibility. Summary of the Invention

[0008] In view of the aforementioned existing problems, the present invention is proposed.

[0009] This invention provides a universal bacterial outer membrane vesicle delivery system (GMMA) that solves the key technical problems of OMV multivalent antigen loading and safe and efficient delivery.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, embodiments of the present invention provide a universal bacterial outer membrane vesicle delivery system GMMA, comprising, (1) Biotinylated bacterial outer membrane vesicles (OMVs) with a diameter of 50 nm to 100 nm and uniform particle size distribution; and (2) At least one avidin-antigen fusion protein, said fusion protein containing the 114aa reconstructed monomeric avidin shown in SEQ ID NO:1, in: a) The carboxyl group of the OMV is biotinylated by coupling 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide with triethylene glycolamine biotin; b) The avidin in the avidin-antigen fusion protein reversibly and specifically binds to the biotin on the surface of the OMV to achieve antigen loading and delivery.

[0011] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the OMV is prepared by sodium deoxycholate extraction, specifically including the following steps: ① Extract Gram-negative bacterial cells with 0.5% sodium deoxycholate solution by shaking at 42 ℃; ② After centrifugation to remove cell debris, the cells are washed and concentrated using ≥300 kDa tangential flow ultrafiltration; ③ Add nuclease to remove nucleic acid impurities; ④ The OMV was obtained by purification by Sepharose 4 Fast Flow gel chromatography.

[0012] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the OMV biotinylation conditions are as follows: OMV and melamine biotin were mixed at a mass ratio of 1:0.06-0.08, the pH was adjusted to 6.8-7.4, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was added, and the mixture was reacted at 20-30 °C for 0.5-2 h. Uncoupled small molecules were then removed by centrifugation and gel chromatography.

[0013] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, wherein: reconstituted avidin is used relative to wild-type Rhizavidin: Remove 9 aa from the N-terminus and 12 aa from the C-terminus; It contains mutations at six sites: A67D, G71K, A82N, G84Q, S101R, and A105K. And it exists in the form of a single entity.

[0014] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the avidin and the antigen protein are sequentially fused via a pentapeptide GGGGS linker to form a single-chain avidin-antigen fusion protein.

[0015] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the antigen protein is fHbp of group B Neisseria meningitidis, and its amino acid sequence is SEQ ID NO:3.

[0016] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the GMMA is prepared by mixing OMV: avidin-antigen fusion protein at a ratio of 1:2, reacting at 20–30 °C for 1–2 h, and then collecting the coupling product by ultracentrifugation at ≥100,000 g.

[0017] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the same biotinylated OMV can be coupled with two or more different avidin-antigen fusion proteins to obtain a multivalent antigen delivery system.

[0018] As a preferred embodiment of the universal bacterial outer membrane vesicle delivery system GMMA described in this invention, the OMV is derived from at least one Gram-negative bacterium among Neisseria meningitidis, Salmonella, or Escherichia coli.

[0019] Secondly, the present invention provides the use of a universal bacterial outer membrane vesicle delivery system GMMA in the preparation of a vaccine composition for in vivo delivery of protein antigens, wherein intraperitoneal inoculation of BALB / c mice causes no visible pathological damage to the liver and spleen tissues.

[0020] The beneficial effects of this invention are as follows: This invention employs a one-step extraction-chromatographic purification process using sodium deoxycholate, yielding outer membrane vesicles with a diameter of 50 nm to 100 nm and uniform particle size distribution. The membrane structure is intact with minimal batch-to-batch variation, laying the foundation for subsequent chemical modification and large-scale production. By utilizing EDC-activated carboxyl groups and coupling them with polyethylene glycolamine biotin, modification can be completed at 25 °C and neutral pH conditions. This avoids protein denaturation caused by strong oxidation / strong acid / alkali conditions and provides sufficient space arms through short-chain PEG3, ensuring biotin exposure. SEQ ID NO:1 is obtained by truncating the N / C ends of Rhizavidin and mutating six hydrophobic sites to hydrophilic residues. This 114aa monomeric avidin retains high affinity (KD = 6.6 × 10⁻⁶). -8 Furthermore, it eliminates the spatial obstruction caused by natural dimers, resulting in higher insertion and extraction efficiency. By simply fusing SEQ ID NO:1 with the target antigen using the pentapeptide GGGGS as a flexible linker, homologous or heterologous proteins, membrane protein fragments, and even peptide epitopes can be loaded onto the same vesicle surface, avoiding the cumbersome steps of traditional GMMA that require the integration or tandem expression of multiple genes.

[0021] One biotinylated OMV of the present invention can simultaneously bind to two or more different avidin-antigen fusion proteins to form a multivalent complex, significantly expanding the antigen spectrum and potentially reducing the number of booster immunizations. Seven days after intraperitoneal injection of GMMA into BALB / c mice, no inflammation or necrosis was observed in liver and spleen HE sections, consistent with the negative control, indicating that the vesicle endogenous lipopolysaccharide has low residue and minimal toxicity after DOC treatment, meeting the safety requirements for vaccine adjuvants. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is an electron microscope image of the meningococcal OMV of the present invention.

[0024] Figure 2 The structure of biotin triethylene glycolamine is shown in this invention.

[0025] Figure 3 This is a schematic diagram of the Rhizavidin avidin dimer structure of the present invention.

[0026] Figure 4 This is an electrophoresis diagram of the biotinylated OMV and AVI-fHbp coupling product of the present invention.

[0027] Figure 5 This is a force map of the interaction between the biotinylated OMV and the AVI-fHbp molecule of the present invention.

[0028] Figure 6 These are pathological sections of the liver and spleen from the peritoneal immunoassay of BALB / c mice, as described in this invention. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0030] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0031] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0032] This invention relates to a GMMA antigen delivery system based on meningococcal OMV, specifically a universal antigen delivery system for Gram-negative bacteria OMV, also known as the GMMA system (Generalized Modules for Membrane Antigens). It employs a novel process to isolate bacterial OMV, using centrifugation for OMV extraction. The isolated OMV surface is rich in carboxyl groups; when mixed with PEG biotin containing amino groups at the ends, the OMV is biotinylated under the action of an EDAC coupling agent. Another important innovation of this invention is the reconstruction of wild-type avidin, mutating six hydrophobic amino acids, so that avidin no longer dimers but exists in solution as a monomer. Furthermore, the wild-type avidin is truncated, retaining the shortest non-antigenic amino acid sequence, shortening it from 135 amino acids in the wild-type to 114 amino acids. Experiments show that the truncated avidin maintains a high SPR value for biotin binding, indicating specific binding. The modified avidin can be fused with any antigen sequence for expression, thereby achieving antigen-OMV linkage. This invention uses the meningococcal outer membrane protein fHbp as an example to illustrate the avidin fusion expression process. The coupling of avidin fused with the fHbp antigen to biotin in the OMV is a simple process, requiring only mixing, similar to an insertion and removal. Therefore, the constructed GMMA system is versatile. The key points of this system are: first, a stable and homogeneous biotinylated OMV was prepared; second, an avidin fusion scheme with non-interfering domains was designed; third, the binding between biotin and avidin is specific; and fourth, the constructed GMMA system has excellent safety.

[0033] Example 1, referring to Figure 1 This is the first embodiment of the present invention, which provides the extraction, preparation, and characterization of OMV, including: outer membrane vesicles having a double membrane structure with a diameter of 50-100 nm. The outer membrane OMV is extracted using a deoxycholate process, followed by removal of cell debris and further purification to remove impurities. Taking meningococcal bacteria as an example, the process is as follows.

[0034] The seed culture was inoculated into the fermentation basal medium (composition: 20 g / L plant peptone, 20 g / L yeast extract, 5 g / L glucose•H2O, 3 g / L dipotassium hydrogen phosphate, 2 g / L potassium dihydrogen phosphate, 2 g / L anhydrous magnesium sulfate, and 14 g / L sodium chloride) at a ratio of 1:100 (volume ratio). Fermentation parameters were set as follows: pH 7.0, rotation speed 150 rpm, temperature 37℃, tank pressure 0.02 MPa, and aeration rate 60 L / min.

[0035] During fermentation, OD was measured every hour. 600Values; examine microscopically every 2 hours; adjust pH with ammonia and 6M hydrochloric acid respectively, and control it between 6.8 and 7.2; control dissolved oxygen >30%. At the 6th hour of fermentation, the bacteria enter the logarithmic growth phase, and the cells are harvested. Add 0.01M PBS to a centrifuge tube containing the bacterial precipitate, stir to resuspend and wash the cells, wash twice, centrifuge to collect the precipitate, and freeze at -20℃. Freeze for at least 24 hours, remove the cells from -20℃, and thaw at 2-8℃ for 18-24 hours. Resuspend the cells at a 1:10 ratio, add 10ml of sodium deoxycholate extraction solution (0.5% concentration) per gram of wet cells, and extract OMV by shaking at 42℃. Then centrifuge at 8000g to remove cell debris, wash repeatedly with a 300kDa tangential flow ultrafiltration system for 10 volumes, concentrate and collect the supernatant. Add nuclease to remove nucleic acid impurities, and collect the pure OMV vesicles by column chromatography using Sepharose 4FF. Samples were taken for testing for abnormal toxicity, bacterial endotoxins, and pyrogens (see Table 1), and characterized by electron microscopy. Figure 1 As shown, the spherical particles have a diameter of 50–100 nm, and the OMV particles are uniform in size, which allows for subsequent biotinylation operations.

[0036]

[0037] Only after all indicators are qualified can the process proceed to the next step.

[0038] Example 2, refer to Figure 2 This is a second embodiment of the present invention, which provides biotinylation of OMV, including the following steps: The selected biotin is biotin-melamine, such as... Figure 2 The structure shown has an amino group at one end for coupling OMV and a biotin-active group at the other end. The PEG linker provides the spatial space; this invention uses a PEG trimer, but a longer PEG polymer can also be chosen. Take 20 mg of OMV solution (3% sucrose system), add 1.2 mL of biotin-triethylene glycolamine (20 mg / mL), adjust the pH to 7.20, and then add 120 μL of carbodiimide. React at 25°C, 50 rpm for 1 h at room temperature. Monitor the pH change continuously during the reaction, recording the pH value every 10 min. After the reaction, centrifuge at 10000g for 10 min, discard the precipitate, and take the supernatant. Ultracentrifuge at 100000g for 1 h to separate the supernatant and precipitate. Redissolve the precipitate with the original volume of 3% sucrose solution. Ultrafiltration was performed using a 100KD membrane, followed by repeated washing with 0.10mol / L sodium chloride solution for 10 volumes. The solution was then loaded onto a Sepharose 4 Fast Flow gel chromatography column for purification. The monitoring wavelengths were 206nm and 280nm. The purified solution was collected and sterilized by 0.2μm filtration to obtain biotinylated OMV.

[0039] Example 3, referring to Figure 3 This is the third embodiment of the invention, which provides a reconstructed design for avidin, a biotin-binding protein with multiple sources and amino acid sequences. The present invention modifies the wild-type Rhizavidin sequence SEQ No. 2: First, the Rhizavidin amino acid sequence was shortened, with 9 amino acids removed from the N-terminus and 12 amino acids removed from the C-terminus, reducing the sequence from 135 amino acids to 114 amino acids. Then, the hydrophobic amino acid residues at the six dimer interfaces were mutated to hydrophilic amino acids: A67D, G71K, A82N, G84Q, S101R, and A105K, yielding sequence AVI SEQ No. 1. To clearly illustrate the difference between sequences SEQ No. 1 and SEQ No. 2, this invention used a Swiss-model to generate an online avidin structure diagram, as detailed in [link to documentation]. Figure 3 As shown in the figure, the two avidin molecules are linked together through hydrophobic interactions to form a dimer. The monomer formation method of this invention is to... Figure 3 The amino acids at the contact surfaces of the dimer molecules that are close to each other have been mutated, specifically involving six amino acids: A67, G71, A82, G84, S101, and A105.

[0040] Example 4 is the fourth embodiment of the present invention. This embodiment provides the fusion expression of the reconstructed avidin and the target antigen. SEQ No. 1 from Example 3 is fused and linked with the antigen. The present invention uses the fHbp sequence derived from group B meningococcal meningitis as an example, and the selected fHbp amino acid sequence is MenB20 SEQ No. 3. Using GGGGS as a linker, the sequence AVI-fHbp SEQ No. 4 is constructed.

[0041] The amino acid AVI-fHbp SEQ No. 4 was converted into a DNA sequence and optimized for synthesis by Nanjing GenScript Biotech Co., Ltd. It was then inserted into the pET-30a(+) vector, with an HIS tag added to the C-terminus to obtain the recombinant plasmid. 4 μg of the lyophilized recombinant plasmid was centrifuged at 6000g for 1 min, and then dissolved in 200 μl of water to a final plasmid concentration of 20 ng / μL. BL21(DE3) competent cells were thawed on ice and gently mixed (avoiding vigorous shaking). 1 μl (20 ng) of plasmid was added to 50 μL of competent cells and gently mixed. The cells were incubated on ice for 30 min, followed by a heat shock at 42℃ for 30 s, and then on ice for 2 min. 250 μl of SOC medium was added, and the cells were cultured at 37℃ with shaking for 1 h (225 rpm). 20 μl, 40 μl, 80 μl, and 100 μl of the bacterial culture were plated onto LB agar plates and incubated overnight at 37℃ (preheated in a 37℃ incubator before plating).

[0042] To observe colony growth on agar plates, pick a single colony from a plate with clearly defined colony boundaries and inoculate it into 5 ml of LB broth (containing 50 μg / ml kanamycin). Incubate overnight at 37°C with a shaker. Take 500 μl of the overnight culture and inoculate it into 50 ml (1% inoculation ratio) of LB broth. Incubate at 37°C with a shaker until OD (out of growth) is reached. 600 When the OD value is 0.6~0.8, take 0.9 ml of bacterial culture and add it to a 2 ml cryovial containing 0.1 ml of 80% glycerol. Mix well and store at -70℃. Take 2.5 ml of seed culture and inoculate it into 250 ml of LB liquid medium (containing 50 μg / mL kanamycin) at a ratio of 1:100 (v / v). Incubate at 37±1℃ and 200 rpm for 6 h on a shaker. 600 The pH should be between 1.0 and 3.0 before inoculating into the fermentation tank for fermentation.

[0043] During fermentation, the pH was adjusted using ammonia and 6 mol / L hydrochloric acid to maintain it within the range of 6.8–7.2; an antifoaming agent was used to eliminate foam; and dissolved oxygen was controlled to be >30%. At 6 hours of fermentation, the bacteria entered the logarithmic growth phase, and expression was induced. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.4 mmol / L, and fermentation was stopped after 6 hours of induction culture. The fermentation broth was centrifuged at 6800g for 10 min at 2–8℃, and the supernatant was discarded after centrifugation, collecting the precipitate. After collecting the bacterial cells, 0.01 mol / L PBS was added to the centrifuge tube containing the bacterial precipitate, and the cells were washed by stirring and resuspending. The mixture was then centrifuged at 7200g for 30 min at 2–8℃. After centrifugation, the supernatant was discarded, collecting the precipitate. This process was repeated twice, for a total of three washings.

[0044] Bacterial resuspension was performed at a ratio of 1:10 (m / v), and the volume of water for injection was calculated based on the wet weight of the bacterial cells. A high-pressure homogenizer was set to 800 bar, and homogenization was performed three times. The homogenate was centrifuged at 9600g for 30 min at 2–8℃, the precipitate was collected, inclusion bodies were dissolved using standard methods, diluted and refolded, and ultrafiltration was performed with buffer changes. Samples were loaded fractionally onto a Ni chromatography column, washed with equilibration buffer, and eluted with imidazole buffer. The target protein was eluted, the elution peak was collected, and stored at 2–8℃ for later use.

[0045] The recombinant AVI-fHbp protein prepared and purified through the above steps has a purity of over 95%, which can meet the requirements for subsequent conjugation.

[0046] Example 5, refer to Figure 4 and Figure 5This is the fifth embodiment of the present invention, which provides the conjugation of biotinylated OMV and AVI-fHBP proteins and SPR detection. The concentrations of OMV and AVI-fHBP are detected by reacting at a mass ratio of OMV:fHBP of 1:2 at room temperature for 2 hours. The product is then centrifuged at 100,000g for 1 hour to separate the supernatant and precipitate. The precipitate is dissolved in 5 ml of 3% sucrose solution. The concentrations of the products before and after conjugation are measured, and the conjugation is detected by SDS-PAGE. See details in [link to relevant documentation]. Figure 4 Electrophoresis diagram of the conjugation products (lane 1: GMMA conjugation product; lane 2: Marker; lane 3: Biotinylated OMV; lane 4: AVI-fHbp protein; marker molecular weights from top to bottom are 97.2 kDa, 66.4 kDa, 44.3 kDa, 29.0 kDa, 20.1 kDa, and 14.3 kDa). As shown in the figure, lane 1 contains the conjugation product, lane 2 the marker, lane 3 the biotinylated OMV, and lane 4 the AVI-fHbp protein. The marker molecular weights from top to bottom are 97.2 kDa, 66.4 kDa, 44.3 kDa, 29.0 kDa, 20.1 kDa, and 14.3 kDa. The results indicate that AVI-fHbp binds to OMV.

[0047] Biotinylated OMV was coupled to the chip, and the KD value of the interaction force between it and AVI-fHbp was determined to be 6.6 × 10⁻⁶ according to the standard method in the pharmacopoeia. -8 .See Figure 5 Force mapping between biotinylated OMV and AVI-fHbp molecules. The results show that the interaction between them is strong and belongs to a specific linkage.

[0048] Example 6, refer to Figure 6 This is the sixth embodiment of the present invention. This embodiment provides a pathological examination of major immune organ damage in BALB / c mice. The conjugate (GMMA) prepared in Example 5 and PBS were intraperitoneally inoculated into BALB / c mice, respectively. Seven days later, the mice were dissected, and major immune organs were harvested, stained with hematoxylin and eosin (HE), and pathologically observed. (See attached image.) Figure 6 As can be seen, the liver and spleen showed no significant pathological damage compared to the PBS control group. Therefore, GMMA delivery meets the safety requirements for vaccine adjuvant and antigen delivery systems, and is a safe and universal delivery system.

[0049] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A universal bacterial outer membrane vesicle delivery system GMMA, characterized in that, include: (1) Biotinylated bacterial outer membrane vesicles (OMV) with a diameter of 50 nm to 100 nm and uniform particle size distribution; as well as (2) At least one avidin-antigen fusion protein, said fusion protein containing the 114 aa reconstructed monomeric avidin shown in SEQ ID NO:1, in: a) The carboxyl group of the OMV is biotinylated by coupling 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide with triethylene glycolamine biotin; b) The avidin in the avidin-antigen fusion protein reversibly and specifically binds to the biotin on the surface of the OMV.

2. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 1, characterized in that, The OMV was prepared by sodium deoxycholate extraction, and the specific steps included: ① Extract Gram-negative bacterial cells with 0.5% sodium deoxycholate solution by shaking at 42 ℃; ② After centrifugation to remove cell debris, the cells were washed and concentrated using ≥300 kDa tangential flow ultrafiltration. ③ Add nuclease to remove nucleic acid impurities; ④ The OMV was obtained by purification by Sepharose 4 Fast Flow gel chromatography.

3. The universal bacterial outer membrane vesicle delivery system GMMA as described in any one of claims 1 to 2, characterized in that, The OMV biotinylation conditions are as follows: OMV and melamine biotin were mixed at a mass ratio of 1:0.06-0.08, the pH was adjusted to 6.8-7.4, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) was added, and the mixture was reacted at 20-30 °C for 0.5-2 h. Uncoupled small molecules were then removed by centrifugation and gel chromatography.

4. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 1, characterized in that, Reconstructed avidin relative to wild-type Rhizavidin: Remove 9 aa from the N-terminus and 12 aa from the C-terminus; It contains mutations at six sites: A67D, G71K, A82N, G84Q, S101R, and A105K. And it exists in the form of a single entity.

5. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 1, characterized in that, The avidin and the antigen protein are sequentially fused via a pentapeptide GGGGS linker to form a single-chain avidin-antigen fusion protein.

6. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 5, characterized in that, The antigen protein is fHbp of group B Neisseria meningitidis, and its amino acid sequence is SEQ ID NO:

3.

7. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 1, characterized in that, When preparing the GMMA, the OMV:avidin-antigen fusion protein was mixed at a ratio of 1:2, and reacted at 20–30 °C for 1–2 h. The coupling product was then collected by ultracentrifugation at ≥100,000 g.

8. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 1, characterized in that, The same biotinylated OMV can be coupled with two or more different avidin-antigen fusion proteins to obtain a multivalent antigen delivery system.

9. The universal bacterial outer membrane vesicle delivery system GMMA as described in claim 2, characterized in that, The OMV is derived from at least one Gram-negative bacterium among Neisseria meningitidis, Salmonella, or Escherichia coli.

10. Use of the universal bacterial outer membrane vesicle delivery system GMMA as described in any one of claims 1 to 9 in the preparation of vaccine compositions for in vivo delivery of protein antigens.

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