Microcapsule vaccine delivery system based on nano-vesicles as well as preparation method and application of microcapsule vaccine delivery system

Abstract

The invention provides a microcapsule vaccine delivery system based on nano-vesicles and a preparation method and application thereof, and relates to the technical field of biological medicine, an inner core of the vaccine delivery system is a nano-compound, and the nano-compound is a compound of bacterial-derived nano-vesicles and antigens; the outer layer of the nano-composite is coated with a calcium alginate layer and a chitosan layer. The preparation method of the microcapsule vaccine delivery system based on the nano vesicles is simple and rapid, can be applied to preparation of various types of vaccines, and realizes co-delivery of antigens, adjuvants and multivalent antigens. The prepared micro-nano fusion system used as the oral vaccine carrier can induce humoral immune response, cellular immune response and mucosal immune response at the same time.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to a nanovesicle-based microcapsule vaccine delivery system and a preparation method and application thereof. Background Art

[0002] The application of vaccines has greatly reduced the morbidity and mortality of common epidemic infectious diseases. The immunization route of vaccines has a great influence on the type of immunity they induce. Traditional vaccination methods such as subcutaneous injection, intramuscular injection and intradermal injection have been repeatedly proven to be unable to activate mucosal immunity due to tissue restrictions. The most effective way to induce mucosal immune response is to administer the drug via the mucosal route. However, high concentrations of soluble antigens can easily induce immune tolerance when stimulated on the mucosal surface.

[0003] So far, the most widely used mucosal vaccines on the market are oral polio vaccine and oral rotavirus vaccine. However, both vaccines are live attenuated vaccines, which have the potential risk of reversion to virulence, instability, difficulty in storage and transportation, and are not suitable for people with immune deficiency and immunodeficiency. In addition, the use of vaccine delivery carriers, such as liposomes and nanoparticles, can significantly improve the stability of vaccines and increase the level of immune response. However, most of these vaccines are administered by injection, which cannot induce effective mucosal immune response and cannot achieve the secretion of antigen-specific SIgA.

[0004] Therefore, it is urgent to develop an effective and safe mucosal vaccine delivery system to achieve multiple protections of humoral immune response, cellular immune response and mucosal immune response. Summary of the invention

[0005] The present application provides a nanovesicle-based microcapsule vaccine delivery system and a preparation method and application thereof to solve the above-mentioned problems existing in the existing vaccine delivery systems.

[0006] In a first aspect of an embodiment of the present application, a nanovesicle-based microcapsule vaccine delivery system is provided. The core of the vaccine delivery system is a nanocomplex, which is a complex of bacterial-derived nanovesicles and antigens. The outer layer of the nanocomplex is coated with a calcium alginate layer and a chitosan layer.

[0007] In an optional embodiment of the present application, the antigen includes one or more of a protein antigen, a protein antigen with weak immunogenicity, a polypeptide antigen, a virus or bacteria lysate antigen, a virus or bacteria outer membrane vesicle antigen, a tumor cell lysate antigen, a tumor cell membrane vesicle antigen, a tumor cell exosome antigen, or a tumor pattern antigen chicken ovalbumin;

[0008] The protein antigens include: hepatitis A, hepatitis B or hepatitis C antigens, tetanus toxoid, human papillomavirus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, respiratory syncytial virus, West Nile virus, cytomegalovirus, malaria antigen, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, group A streptococcal antigens, or other recombinant protein antigens; the protein antigens with weak immunogenicity include: bovine serum albumin, lysozyme, The invention relates to a polypeptide antigen comprising: a tyrosinase-related protein 2 antigen fragment (TRP2), a human melanoma antigen fragment (HGP100), a retrovirus tumor antigen fragment (p15E) endogenously expressed in MC38 tumor cells, a chicken ovalbumin antigen fragment (SIINFEKL), a human papillomavirus oncoprotein (E6 / E7), a hepatitis B antigen epitope peptide S28-39, or other synthetic polypeptide antigens and a long polypeptide antigen containing several polypeptide sequences.

[0009] In an optional embodiment of the present application, the particle size of the nanocomplex is less than 500 nm; the nanovesicle-based microcapsule vaccine delivery system is a microspherical gel with a particle size of less than 500 μm.

[0010] In a second aspect of the embodiment of the present application, a method for preparing a nanovesicle-based microcapsule vaccine delivery system according to any one of the first aspects is provided, the method comprising:

[0011] A nanocomplex is prepared by using an aqueous solution of bacteria-derived nanovesicles and an antigen;

[0012] Based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution, calcium alginate-chitosan microcapsules are prepared as the nanovesicle-based microcapsule vaccine delivery system.

[0013] In an optional embodiment of the present application, the bacteria-derived nanovesicles in the bacteria-derived nanovesicle aqueous solution are prepared according to the following steps:

[0014] The cultured bacterial suspension was placed in a centrifuge and centrifuged at 6000 g / min for 10 min. The precipitate was collected and washed twice with a sterile PBS solution and resuspended in a PBS solution containing PMSF to obtain a bacterial suspension.

[0015] Using an ultrasonic probe, ultrasonically disrupting the bacteria at 20-60% ultrasonic power, and ultrasonicating until the bacterial suspension is clear to obtain a bacterial disruption solution;

[0016] The bacterial disruption solution was placed in a centrifuge and centrifuged at 4000 g / min for 10 min, and the supernatant was collected;

[0017] The supernatant was subjected to ultracentrifugation at 100,000 g / min for 40 min, and the precipitate was collected and centrifuged using a 0.2 × 10 -3 The resulting mixture was washed with M ethylenediaminetetraacetic acid 1-2 times to obtain the bacteria-derived nanovesicles.

[0018] In an optional embodiment of the present application, a nanocomplex is prepared using a bacterial-derived nanovesicle aqueous solution and an antigen, comprising:

[0019] Add 1 mg / mL-10 mg / mL of antigen to 30-200 mM Hepes buffer, mix well, and obtain an antigen solution;

[0020] Add 0.5 mg / mL-10 mg / mL poly-L-arginine membrane-penetrating peptide R8 solution to the antigen solution, vortex mix for 5-60 seconds, and stand at room temperature for 30-60 minutes to obtain an antigen complex;

[0021] The antigen complex is slowly added dropwise to the bacterial-derived nanovesicle aqueous solution for mixing to obtain the nanocomplex, wherein the mixing mass ratio is antigen: bacterial-derived nanovesicle = 1:0.1-2.0.

[0022] In an optional embodiment of the present application, the method of slowly dropping the antigen complex into the bacterial-derived nanovesicle aqueous solution for mixing includes: vortexing, ultrasound or mixing by a microinjection pump;

[0023] The vortex mixing time is 5-60S; the ultrasonic mixing power is 50-300W, and the ultrasonic time is 1-10min; the flow rate of the micro-injection pump mixing is 30mL / min-80mL / min.

[0024] In an optional embodiment of the present application, calcium alginate-chitosan microcapsules are prepared based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution, including:

[0025] Dispersing 0.5 mg / mL-10 mg / mL of the nanocomplex in 1%-2% (m / v) sodium alginate solution to obtain a sodium alginate suspension;

[0026] The sodium alginate suspension is placed in a syringe pump device, and the sodium alginate suspension is pushed downward at a speed of 0.1-1.0 mL / min. Under the action of the electric field force, the sodium alginate suspension falls into a measuring cup containing a 0.1 mol / L-0.2 mol / L calcium chloride solution in the form of droplets, thereby obtaining calcium alginate microgel beads encapsulating the nanocomposite;

[0027] The calcium alginate microgel beads carrying the nanocomposite are reacted with 0.4%-0.8% (m / v) chitosan solution at a volume ratio of 1:1 to form a film, thereby obtaining calcium alginate-chitosan microcapsules.

[0028] In a third aspect of an embodiment of the present application, a nanovesicle-based microcapsule vaccine delivery system is provided for use in the preparation of oral immune drugs. The nanovesicle-based microcapsule vaccine delivery system is prepared according to any one of the above-mentioned second aspects.

[0029] The present application includes the following advantages: The present application provides a nanovesicle-based microcapsule vaccine delivery system and its preparation method and application, wherein the core of the vaccine delivery system is a nanocomplex, and the nanocomplex is a complex of bacterial-derived nanovesicles and antigens; the outer layer of the nanocomplex is coated with a calcium alginate layer and a chitosan layer. The preparation method of the nanovesicle-based microcapsule vaccine delivery system is simple and rapid, and can be applied to the preparation of various types of vaccines to achieve co-delivery of antigens and adjuvants, and multivalent antigens. The prepared "micro-nano" fusion system used as an oral vaccine carrier can simultaneously induce humoral immune response, cellular immune response, and mucosal immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0031] Figure 1 This is a morphology characterization result diagram of a nanocomposite provided in an embodiment of the present application;

[0032] Figure 2 This is a schematic diagram of the characterization results of a calcium alginate-chitosan microcapsule provided in an example of the present application;

[0033] Figure 3 This is a morphological characterization diagram of BMV-RO-1@A / C and BMV-RO-2@A / C in artificial gastric juice AGF provided in the examples of the present application;

[0034] Figure 4This is a morphological characterization diagram of BMV-RO-1@A / C and BMV-RO-2@A / C in artificial intestinal fluid ASF provided in the examples of the present application;

[0035] Figure 5 This is a graph showing the uptake of nanocomplexes by different antigen presenting cells proposed in the present application;

[0036] Figure 6 This is a fluorescence microscope image of a Cy5.5 fluorescently labeled BMV nanocomplex provided in an example of the present application;

[0037] Figure 7 This is a fluorescence quantity characterization diagram of Peyer's patches and mesenteric lymph nodes in the intestine provided in an embodiment of the present application;

[0038] Figure 8 This is a flow cytometric percentage diagram of the maturation and differentiation of antigen presenting cells in Peyer's patches of the mouse intestine induced by an oral vaccine provided in an embodiment of the present application;

[0039] Fig. 9 This is a flow cytometric percentage diagram of the maturation and differentiation of antigen presenting cells in the mesenteric lymph nodes of mice induced by an oral vaccine provided in an example of the present application;

[0040] Fig.10 This is a flow cytometric percentage diagram of the production of B cells and plasma cells secreting IgA in the intestinal lamina propria of mice induced by an oral vaccine provided in an embodiment of the present application;

[0041] Fig.11 This is a flow cytometric percentage diagram of the production of B cells and plasma cells secreting IgA in the mesenteric lymph nodes of mice induced by an oral vaccine provided in an embodiment of the present application;

[0042] Fig.12 This is a graph showing the antigen-specific IgA antibody results produced on the 28th day after mice were immunized with an oral vaccine provided in an embodiment of the present application;

[0043] Fig.13 This is a graph showing the results of antigen-specific IgG and its subtype antibodies produced on the 28th day after mice were immunized with an oral vaccine provided in an example of the present application. DETAILED DESCRIPTION

[0044] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0045] The biological or chemical reagents used in the examples of this application are all commercially available.

[0046] The route of vaccination greatly affects the type of immunity it induces. Traditional vaccination methods such as subcutaneous injection, intramuscular injection and intradermal injection have been repeatedly proven to be unable to activate mucosal immunity due to tissue restrictions. The most effective way to induce mucosal immune response is to administer the drug via the mucosal route. However, high concentrations of soluble antigens can easily induce immune tolerance when stimulated on the mucosal surface. In addition, extreme pH, abundant proteases, epithelial barriers and mucosal barriers often result in unsatisfactory immune responses induced by mucosal administration. So far, the most widely used mucosal vaccines on the market are oral polio vaccine and oral rotavirus vaccine. However, both vaccines are live attenuated vaccines, which have the potential risk of virulence reversion, instability, difficulty in storage and transportation, and are not suitable for vaccination in immunodeficient and immunocompromised people. In addition, the use of vaccine delivery carriers, such as liposomes and nanoparticles, can significantly improve the stability of vaccines and increase the level of immune response. However, most of these vaccines are administered by injection, which cannot induce effective mucosal immune responses and cannot achieve the secretion of antigen-specific SIgA. Therefore, it is urgent to develop an effective and safe mucosal vaccine delivery system to achieve multiple protections of humoral immune response, cellular immune response and mucosal immune response.

[0047] In order to solve the above problems, considering that oral vaccines can eliminate the risk of infection by blood-borne pathogens, avoid the pain of needle puncture, and have good patient compliance, and do not require complete antigen purification, and have the advantages of high economic value, this application proposes to prepare a mucosal vaccine delivery system by oral delivery, but the various physiological barriers in the gastrointestinal tract seriously limit the successful delivery of oral vaccines, such as the antigens in the gastrointestinal tract are subject to the attack of low pH gastrointestinal fluid, mechanical movement process, degradation of digestive enzymes, and crossing of intestinal epithelial barriers; In addition, the immune tolerance of mucosal tissue may hinder the immune response of the vaccine, and the interference of intestinal flora will also affect the stability and effectiveness of the vaccine. Therefore, inducing immune responses through oral routes usually requires higher doses, but it also creates safety hazards. The above barriers can be overcome by appropriate adjuvants and delivery systems. The vaccine delivery system can protect the antigen from being degraded by gastric acid and proteases, thereby ensuring the efficacy of the vaccine. In order to further improve the efficacy, the oral vaccine delivery system should have both adjuvant and mucosal adhesion properties. More importantly, they must be safe. In order to achieve the above goals, this application proposes a nano-micro combination oral vaccine delivery system.

[0048] In the first aspect of the embodiment of the present application, a microcapsule vaccine delivery system based on nanovesicles is proposed, the core of the vaccine delivery system is a nanocomposite, and the nanocomposite is a complex of bacterial-derived nanovesicles and antigens; the outer layer of the nanocomposite is coated with a calcium alginate layer and a chitosan layer; the bacterial-derived nanovesicles (BMVs) are extracted from bacteria, containing a variety of pathogen-related molecular patterns such as bacterial membrane proteins, lipoproteins, carbohydrates and nucleic acids, etc., which can be used as natural carriers and adjuvants for cancer immunotherapy; it is due to its strong ability to activate innate immune signaling pathways, and can induce the maturation of dendritic cells (DCs) in intestinal-related lymph nodes and lamina propria after oral administration; at the same time, its preparation method is convenient, high in yield, and is conducive to production and application. The sources of the bacterial-derived nanovesicles used in the present application include Gram-positive bacteria and Gram-negative bacteria, and can be preferably Bifidobacterium and Akkermansia.

[0049] Antigen, as the most important component of a vaccine, is the key to determining the protective effect of a vaccine. It is necessary to consider selecting the best specific antigen for a certain disease. However, for a pathogen, the antigens of different subtypes may not be the same, that is, they have a certain heterogeneity, and some antigens may even mutate. Therefore, the vaccine vector that delivers a single antigen has limited effect and may even be disabled. In order to induce the body to produce a more comprehensive and stronger protective effect, it is necessary to develop a vaccine vector with the ability to deliver multivalent antigens. Preferably, a multivalent antigen delivery system that can deliver different properties (hydrophilicity) can induce the body to produce a comprehensive and strong protective effect.

[0050] In the present application, the antigens in the above-mentioned nanocomplex include one or more of protein antigens, protein antigens with weak immunogenicity, polypeptide antigens, virus or bacterial lysate antigens, virus or bacterial outer membrane vesicle antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, tumor cell exosome antigens or tumor pattern antigen chicken egg white albumin; the protein antigens include: hepatitis A, hepatitis B or hepatitis C antigens, tetanus toxoid, human papillomavirus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, respiratory syncytial virus, West Nile virus, cytomegalovirus, malaria antigens, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa The invention relates to an antigen of a monocytic organism, a vibrio cholerae, a group A streptococcus antigen, or other recombinant protein antigen; the protein antigen with weak immunogenicity includes: one of bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, soy albumin, wheat albumin, myoglobin, collagen, and lamin; the polypeptide antigen includes: a tyrosinase-related protein 2 antigen fragment (TRP2), a human melanoma antigen fragment (HGP100), a retrovirus tumor antigen fragment (p15E) endogenously expressed in MC38 tumor cells, a chicken ovalbumin antigen fragment (SIINFEKL), a cancer protein of human papillomavirus (E6 / E7), a hepatitis B antigen epitope peptide S28-39, or other synthetic polypeptide antigens and a long polypeptide antigen containing several polypeptide sequences.

[0051] Optionally, the particle size of the nanocomplex in the nanovesicle-based microcapsule vaccine delivery system is less than 500 nm, preferably less than 200 nm; the above-mentioned nanovesicle-based microcapsule vaccine delivery system is a microspherical gel with a particle size less than 500 μm, preferably less than 200 μm.

[0052] In a second aspect of the embodiment of the present application, a method for preparing a nanovesicle-based microcapsule vaccine delivery system according to the first aspect is provided, the method comprising the following steps:

[0053] Step A: Based on the principle of electrostatic adsorption self-assembly, a nanocomplex is prepared using a bacterial-derived nanovesicle aqueous solution and an antigen;

[0054] Step B: Using the principle of high voltage electrostatics, calcium alginate-chitosan microcapsules are prepared based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution as a microcapsule vaccine delivery system.

[0055] Among them, the present application adopts ultrasound-centrifugation method to extract and prepare bacterial-derived nanovesicles. The components of the bacterial shell (including structures such as the cell outer membrane, cell wall and cell membrane) will spontaneously self-assemble from tiny fragments into a vesicle-like particle structure during the ultrasonic fragmentation process, which is beneficial to the uptake and activation of immune cells.

[0056] Specifically, the bacteria-derived nanovesicles in the bacteria-derived nanovesicle aqueous solution are prepared according to the following steps:

[0057] Step a: placing the cultured bacterial suspension on a centrifuge, centrifuging at 6000 g / min for 10 min, collecting the precipitate, washing it twice with a sterile PBS solution, and resuspending it in a PBS solution containing a serine protease inhibitor (Phenylmethanesulfonyl fluoride, PMSF) to obtain a bacterial suspension;

[0058] Step b: using an ultrasonic probe to ultrasonically disrupt the bacteria at 20-60% ultrasonic power, and ultrasonicating until the bacterial suspension is clear to obtain a bacterial disruption solution;

[0059] Step c: placing the bacterial disruption solution on a centrifuge, centrifuging at a speed of 4000 g / min for 10 min, and collecting the supernatant;

[0060] Step d: After the supernatant was subjected to ultra-high speed centrifugation at 100,000 g / min for 40 min, the precipitate was collected and separated using a 0.2 × 10 -3 The resulting nanovesicles were washed with ethylenediaminetetraacetic acid (EDTA) for 1-2 times to obtain the bacteria-derived nanovesicles.

[0061] After obtaining the bacteria-derived nanovesicles, the bacteria-derived nanovesicles are placed in an aqueous solution according to a use concentration to obtain a negatively charged bacteria-derived nanovesicle aqueous solution.

[0062] In an optional embodiment of the present application, in step a, the culture process of the bacterial suspension used is as follows:

[0063] If the bacteria are anaerobic bacteria, the seed culture solution is added to the corresponding culture medium, and cultured in an anaerobic incubator at a culture temperature of 37° C. for 3-5 days to obtain the cultured bacterial suspension; the anaerobic bacteria may be Bifidobacterium or Akkermansia;

[0064] If the bacteria are facultative anaerobic bacteria, add 50 μL of seed culture solution to 10 mL of liquid LB medium and culture overnight at a culture temperature of 37° C. and a rotation speed of 200 r / min; the bacteria after overnight culture are expanded according to an inoculation volume of 1:50 and cultured again until the OD value of the bacterial suspension reaches 0.6-0.8 to obtain the cultured bacterial suspension.

[0065] In an optional embodiment of the present application, a nanocomplex is prepared using a bacterial-derived nanovesicle aqueous solution and an antigen, specifically comprising the following steps:

[0066] Step A-1: ​​Add 1 mg / mL-10 mg / mL of antigen to 30-200 mM Hepes buffer and mix well to obtain an antigen solution;

[0067] Step A-2: Add 0.5 mg / mL-10 mg / mL poly-L-arginine membrane-penetrating peptide R8 solution to the antigen protein solution, vortex mix for 5-60 seconds, and stand at room temperature for 30-60 minutes to obtain a positively charged antigen complex;

[0068] Step A-3: Slowly drop the antigen complex into the aqueous solution of bacteria-derived nanovesicles to mix them, thereby obtaining a nanocomplex.

[0069] Optionally, in step A-3, the antigen complex is slowly added dropwise to the bacterial-derived nanovesicle aqueous solution for mixing, including: vortexing, ultrasound or mixing by a microinjection pump; wherein the vortex mixing time is 5-60S; the ultrasound mixing power is 50-300W, and the ultrasound time is 1-10min; the flow rate of the microinjection pump mixing is 30mL / min-80mL / min.

[0070] In an optional embodiment of the present application, calcium alginate-chitosan microcapsules are prepared based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution, which specifically includes the following steps:

[0071] Step B-1: dispersing 0.5 mg / mL-10 mg / mL of the nanocomplex in 1%-2% (m / v) sodium alginate solution to obtain a sodium alginate suspension;

[0072] Step B-2: The sodium alginate suspension is placed in a syringe pump device, and the sodium alginate suspension is pushed downward at a speed of 0.1-1.0 mL / min. Under the action of the electric field force, the sodium alginate suspension falls into a measuring cup containing a 0.1 mol / L-0.2 mol / L calcium chloride solution in the form of droplets, thereby obtaining calcium alginate microgel beads encapsulating the nanocomposite;

[0073] Step B-3: The calcium alginate microgel beads encapsulating the nanocomplex are reacted with 0.4%-0.8% (m / v) chitosan solution at a volume ratio of 1:1 to form a film, thereby obtaining calcium alginate / chitosan (A / C) microcapsules.

[0074] The preparation method proposed in this application first adopts the principle of electrostatic adsorption self-assembly to prepare bacterial-derived nanovesicle-antigen complex (nanocomplex) to obtain a stable nano preparation. Then, the high-voltage electrostatic principle is adopted to disperse the nanocomplex in the sodium alginate solution to obtain a sodium alginate suspension, overcome the viscosity and surface tension, and the sodium alginate suspension rapidly undergoes a cross-linking reaction with the calcium chloride solution in the form of droplets, solidifies to form calcium alginate microgel beads, and coats a layer of chitosan on its surface. The microencapsulation process has good stability, and the size and shape of the formed calcium alginate microgel beads are uniform, and the surface is coated with chitosan to avoid the influence of the physiological barrier during oral administration; and it does not involve high temperature and covalent bond formation in thermal cross-linking and chemical cross-linking reactions, and is suitable for the encapsulation of antigens and adjuvants, providing a platform for building a safe, effective and easy-to-convert oral vaccine system.

[0075] In the third aspect of the embodiment of the present application, a nanovesicle-based microcapsule vaccine delivery system prepared by any of the preparation methods in the second aspect is provided for use in the preparation of oral immune drugs. The "micro-nano" fusion mucosal vaccine delivery system prepared by the above preparation method efficiently delivers oral vaccine drugs containing bacteria-derived nanovesicle-antigen nanocomplexes to intestinal mucosal tissues, and penetrates the epithelial barrier to reach intestinal-related lymph nodes; through oral immunization, it induces the body to produce humoral immunity, cellular immunity and mucosal immune responses, and has broad application prospects.

[0076] The present application provides a nanovesicle-based microcapsule vaccine delivery system and its preparation method and application. The core of the vaccine delivery system is a nanocomplex, which is a complex of bacterial-derived nanovesicles and antigens; the outer layer of the nanocomplex is coated with a calcium alginate layer and a chitosan layer. The preparation method of the nanovesicle-based microcapsule vaccine delivery system is simple and rapid, and can be applied to the preparation of various types of vaccines to achieve the co-delivery of antigens and adjuvants, and multivalent antigens. The prepared "micro-nano" fusion system used as an oral vaccine carrier can simultaneously induce humoral immune response, cellular immune response and mucosal immune response.

[0077] The nanovesicle-based microcapsule vaccine delivery system prepared by the above preparation method is used in the preparation of oral immune drugs. The technical solution provided by the present application is described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0078] Example 1: Preparation and characterization of nanocomposites

[0079] The bacteria were resuscitated and cultured, and bacterial-derived nanovesicles BMVs were prepared by ultrasound-centrifugation. The OVA complex (RO) modified with the positively charged transmembrane peptide R8 was introduced, and the BMV-R8-OVA (BMV-RO, BRO) nanocomplex was prepared by electrostatic adsorption self-assembly, where OVA refers to the tumor model antigen chicken ovalbumin.

[0080] Specifically, the preparation method is as follows:

[0081] Step 1: adding the seed-keeping bacterial solution to the corresponding culture medium, and culturing the culture in an anaerobic incubator at a culture temperature of 37° C. for 3-5 days to obtain the cultured bacterial suspension, wherein the bacteria in the seed-keeping bacterial solution include one of Bifidobacterium or Akkermansia;

[0082] Step 2: Place the cultured bacterial suspension on a centrifuge and centrifuge at 6000 g / min for 10 min, collect the precipitate, wash it twice with a sterile PBS solution, and resuspend it in a PBS solution containing PMSF to obtain a bacterial suspension;

[0083] Step 3: using an ultrasonic probe to ultrasonically disrupt the bacteria at 40% ultrasonic power, and ultrasonicating until the bacterial suspension is clear to obtain a bacterial disruption solution;

[0084] Step 4: Place the bacterial disruption solution in a centrifuge, centrifuge at 4000 g / min for 10 min, and collect the supernatant;

[0085] Step 5: After ultracentrifugation of the supernatant at 100,000 g / min for 40 min, the precipitate was collected and purified using a 0.2 × 10 -3 The resulting solution was washed with ethylenediaminetetraacetic acid 1-2 times to obtain bacteria-derived nanovesicles.

[0086] The bacterial-derived nanovesicles prepared from Bifidobacterium are used as Example 1, and the bacterial-derived nanovesicles prepared from Akkermansia are used as Example 2.

[0087] Based on Examples 1 and 2, a nanocomposite coated with antigen OVA was prepared, and Example 1 was used to illustrate the method for preparing the nanocomposite:

[0088] Add 1 mg / mL of OVA antigen to 100 mM Hepes buffer and mix well to obtain an antigen solution;

[0089] A 1 mg / mL poly-L-arginine membrane-penetrating peptide R8 solution was added to the antigen solution, vortexed for 60 seconds, and allowed to stand at room temperature for 60 minutes to obtain an antigen complex;

[0090] The antigen complex was slowly added dropwise to the BMVs aqueous solution of Example 1 and mixed to obtain a nanocomplex as Example 3.

[0091] The above method was repeated to compound with the aqueous solution of BMVs in Example 2, and the obtained nanocomposite was designated as Example 4. The nanocomposite of Example 3 was referred to as BRO-1, and the nanocomposite of Example 4 was referred to as BRO-2.

[0092] Nanoparticle tracking analyzer was used to analyze Examples 1 to 4. The analysis method was the same as the existing method and will not be repeated here. The results are shown in Table 1. The average particle size and potential of Examples 1 and 2 were 150.8nm / -22.47±0.20mV and 146.5nm / -26.77±0.66mV, respectively; the particle size of BRO-1 (Example 3) and BRO-2 (Example 4) formed after compounding with OVA was basically unchanged (152.3nm and 140.4nm, respectively), and the potential was reversed to positive charge, which was 20.63±0.12mV and 18.43±0.64mV, respectively, proving that the compounding process was effective.

[0093] Table 1 Particle size and potential characterization results of nanocomposites

[0094]

[0095]

[0096] The morphology of Examples 1 to 4 was characterized by transmission electron microscopy. The characterization method is the same as the steps of the existing characterization method and will not be repeated here. Figure 1 , Figure 1 This is a morphology characterization result diagram of a nanocomposite provided in an example of the present application. Figure 1 Part (a) is the morphology characterization result of Example 1. Figure 1 Part (b) is the morphology characterization result of Example 3. Figure 1 Part (c) is the morphology characterization result of Example 2. Figure 1 Part (d) is the morphology characterization result of Example 4.

[0097] It can be seen that the BMVs extracted from bacteria (Examples 1 and 2) are in the form of nanovesicles, and the surface of the nanocomplexes with OVA (Examples 3 and 4) is rougher, but it does not affect its vesicle shape, proving the rationality and reliability of the electrostatic self-assembly preparation process. From the results of the particle size, potential and morphology characterization of the above nanocomplexes, it can be seen that the method for preparing nanocomplexes is highly stable.

[0098] Example 2: Preparation and characterization of calcium alginate-chitosan microcapsules loaded with nanocomposites

[0099] The nanocomposite of Example 3 and Example 4 is used as the core, and a calcium alginate layer and a chitosan layer are coated on the surface to form a calcium alginate-chitosan microcapsule carrying the nanocomposite. The specific preparation method is as follows:

[0100] 1 mg / mL of the nanocomposite (BRO-1) was dispersed in a 1% (m / v) sodium alginate solution to obtain a sodium alginate suspension;

[0101] The sodium alginate suspension was placed in a syringe pump device, and the sodium alginate suspension was pushed downward at 0.2 mL / min. Under the action of the electric field force, the sodium alginate suspension fell into a measuring cup containing 0.2 mol / L calcium chloride solution in the form of droplets, thereby obtaining calcium alginate microgel beads encapsulating the nanocomposite.

[0102] The calcium alginate microgel beads containing the nanocomposite were reacted with 0.8% (m / v) chitosan solution at a volume ratio of 1:1 to form a film, thereby obtaining calcium alginate-chitosan microcapsules as Example 5 (hereinafter referred to as BMV-RO-1@A / C).

[0103] The nanocomposite BRO-1 of Example 3 was replaced with the nanocomposite BRO-2 of Example 4, and the calcium alginate-chitosan microcapsules prepared according to the above method were used as Example 6 (hereinafter referred to as BMV-RO-2@A / C). Blank calcium alginate microcapsules without nanocomposite loading were prepared by the same method as above.

[0104] The blank calcium alginate microgel beads without nanocomposites and Examples 5 and 6 were characterized for their morphology. Figure 2 , Figure 2 is a schematic diagram of the characterization results of a calcium alginate-chitosan microcapsule provided in the examples of the present application, Figure 2 Parts (a) and (b) are the actual schematic diagram and characterization diagram of blank calcium alginate microgel beads. Figure 2 Parts (c) and (d) are characterization images of calcium alginate microgel beads loaded with nanocomposites BRO-1 and BRO-2, respectively. Figure 2 Parts (e) and (f) are the characterization diagrams of Example 6 and Example 5, respectively.

[0105] It can be seen that under the action of the electric field force, the sodium alginate solution reacts with the calcium chloride solution and solidifies in situ into blank calcium alginate microgel beads with uniform shape and size and smooth surface ( Figure 2 (a) and (b)). The BMV-RO nanocomposite was dispersed in a sodium alginate solution and the same preparation method was used to successfully encapsulate the nanocomposite (the encapsulation rate of the antigen OVA was about 79.4%) without affecting the morphology and size of the calcium alginate microgel beads ( Figure 2(c) and (d)). After incubation with chitosan solution, a calcium alginate / chitosan (A / C) microcapsule preparation loaded with BMV-RO nanocomposite was finally formed, with a diameter of about 120 μm and very good uniformity ( Figure 2 (e) and (f) of the annex).

[0106] The calcium alginate-chitosan microcapsules of Examples 5 and 6 were incubated in artificial gastric juice and artificial intestinal juice, and their morphologies in the artificial gastric juice and artificial intestinal juice were observed. The characterization results are shown in Figure 3 and Figure 4 , Figure 3 This is a morphological characterization diagram of BMV-RO-1@A / C and BMV-RO-2@A / C in artificial gastric juice AGF provided in the examples of this application. Figure 4 This is a morphological characterization diagram of BMV-RO-1@A / C and BMV-RO-2@A / C in artificial intestinal fluid ASF provided in the examples of the present application. Figure 3 Parts (a), (b), and (c) are the morphological characterization images of BMV-RO-1@A / C incubated in artificial gastric fluid AGF for 1 h, 2 h, and 4 h, respectively. Figure 3 Parts (d), (e), and (f) are the morphological characterization images of BMV-RO-2@A / C incubated in artificial gastric fluid AGF for 1 h, 2 h, and 4 h, respectively. Figure 4 Parts (a), (b), (c) and (d) are the morphological characterization images of BMV-RO-1@A / C incubated in artificial intestinal fluid ASF for 1h, 3h, 12h and 24h, respectively. Figure 4 Parts (e), (f), (g) and (h) are morphological characterization pictures of BMV-RO-2@A / C incubated in artificial intestinal fluid ASF for 1h, 3h, 12h and 24h respectively.

[0107] First, BMV-RO-1@A / C and BMV-RO-2@A / C were incubated in artificial gastric juice AGF (pH 1.0) for 4 h. Figure 3 It can be seen that the morphology still maintains the microbead form; then BMV-RO-1@A / C and BMV-RO-2@A / C were further immersed in artificial intestinal fluid ASF (pH 6.8). Figure 4 As can be seen in parts (b) and (f), after 3 hours of incubation, BMV-RO-1@A / C and BMV-RO-2@A / C began to slowly disintegrate. Figure 4As can be seen in parts (d) and (h), BMV-RO-1@A / C and BMV-RO-2@A / C still have the basic morphology of microspheres until 24 hours of incubation. It can be seen that the calcium alginate-chitosan microcapsules prepared in this application (i.e., nanovesicle-based microcapsule vaccine delivery system) are beneficial to the gastrointestinal delivery and sustained release of oral vaccines.

[0108] Example 3: Targeted delivery of oral vaccine drugs to intestinal-associated lymph nodes

[0109] At the cellular level, it is verified whether the nanocomplex loaded with antigens OVA and BMVs can be highly taken up by antigen presenting cells. The method includes: using fluorescent molecules FITC and Cy5.5 to label the antigens OVA and BMVs respectively, and then using the fluorescently labeled antigens OVA and BMVs to obtain a fluorescently labeled BMV-RO nanocomplex according to the preparation method of Preparation Example 3, and then incubating the fluorescently labeled BMV-RO nanocomplex with DC2.4 cells or RAW264.7 cells for 2 hours, and using flow cytometry to quantitatively determine the uptake ratio of the nanocomplex by different cells.

[0110] The results can be found in Figure 5 , Figure 5 This is a graph showing the uptake of nanocomplexes by different antigen presenting cells proposed in the present application. Figure 5 Each part is the uptake result of FITC marker in RAW264.7 cells, the uptake result of Cy5.5 marker in RAW264.7 cells, the co-uptake result of FITC and Cy5.5 marker in RAW264.7 cells, the uptake result of FITC marker in DC2.4 cells, the uptake result of Cy5.5 marker in DC2.4 cells, and the co-uptake result of FITC and Cy5.5 marker in DC2.4 cells.

[0111] Depend on Figure 5 It can be seen that at the cellular level, BMVs can be highly taken up by antigen-presenting cells including macrophages and DC cells, especially can significantly improve the efficiency of DC cells in taking up antigens, laying the foundation for subsequent in vivo transmembrane transport and activation of antigen-presenting cells research.

[0112] In order to further characterize the encapsulation of the nanocomposite by the microcapsule vaccine system, the nanocomposite was replaced with a fluorescently labeled BMV-RO nanocomposite by the method of Preparation Example 5 to obtain a fluorescently labeled BMV-RO@A / C oral vaccine drug. The characterization results are shown in Figure 6 , Figure 6 This is a fluorescence microscope image of a Cy5.5 fluorescently labeled BMV nanocomplex provided in an example of the present application. Figure 6Part A is a schematic diagram of the method for preparing fluorescently labeled BMV-RO@A / C oral vaccine drugs. Figure 6 Part B shows the fluorescence images of the prepared fluorescently labeled BMV-RO@A / C oral vaccine drugs, which are FITC fluorescence labeling images, Cy5.5 fluorescence labeling images, and the combined images of FITC fluorescence labeling and Cy5.5 fluorescence labeling. The results of fluorescence microscopy observation show that the fluorescently labeled OVA and BMVs are co-loaded in the microcapsule vaccine delivery system, which is expected to achieve the oral co-delivery effect of vaccine antigens and adjuvants.

[0113] The BMV-RO@A / C microcapsule drug was administered orally to C57BL / 6 mice (the dosage of oral administration was 100 μg of antigen OVA and 100 μg of BMVs). The intestinal Peyer's patches and mesenteric lymph nodes of the mice were removed at different time points (2 hours, 4 hours, 6 hours, and 8 hours). The fluorescence amount of the preparation reaching the intestinal related lymph nodes and its changes over time were observed by in vivo imaging. The results are shown in Figure 7 , Figure 7 This is a fluorescence quantity characterization diagram of Peyer's patches and mesenteric lymph nodes in the intestine provided in the embodiment of the present application. Figure 7 Each figure is divided into two columns of characterization results, the right column of each figure is the fluorescence characterization results of Peyer's patches in the intestine, and the left column of each figure is the fluorescence characterization results of mesenteric lymph nodes. Figure 7 It can be seen that the microcapsule drug of BMV-RO@A / C reaches the Peyer's patches in the intestine within 4 hours and drains to the mesenteric lymph nodes within 6 hours, which preliminarily proves that the oral vaccine drug has the targeting ability to the intestinal-associated lymphoid tissue in the body.

[0114] Example 4: Oral vaccine drugs have significant activation ability on antigen presenting cells in gut-associated lymph nodes

[0115] In Example 3, it has been preliminarily demonstrated that oral vaccine drugs can quickly migrate to lymph nodes after oral administration, but it does not guarantee the successful induction of immune response. Therefore, by examining the activation of antigen presenting cells in intestinal immune induction sites such as Peyer's patches (PPs) and mesenteric lymph nodes (MLNs), it is further verified that they have the ability to initiate the body's immune response.

[0116] The specific method is as follows: After oral administration of the calcium alginate-chitosan microcapsule oral immune drug (BMV-RO-1@A / C or BMV-RO-2@A / C) prepared in Example 5 or Example 6 to C57BL / 6 mice (6-8 weeks old) four times (once a week for four consecutive weeks) (the oral dose is 100 μg of antigen OVA, 100 μg of BMVs), the intestinal Peyer's patches and mesenteric lymph nodes of the mice are dissected and separated, CD11c antibody is used to mark dendritic cells DCs, and flow cytometry is used to detect the expression levels of DCs surface molecules CD40, CD80, and CD86 to evaluate the ability of the oral vaccine to induce DCs maturation in the intestinal lymph nodes; in addition, F4 / 80+ antibody is used to mark macrophages, and flow cytometry is used to examine the typing of macrophages and changes in their proportions.

[0117] The results refer to Figure 8 and Fig. 9 , Figure 8 This is a flow cytometric percentage diagram of the maturation and differentiation of antigen presenting cells in Peyer's patches in the intestine of mice induced by an oral vaccine provided in an example of the present application. Fig. 9 This is a flow cytometric percentage diagram of the maturation and differentiation of antigen presenting cells in the mesenteric lymph nodes of mice induced by an oral vaccine provided in an example of the present application. Figure 8 Part A in the middle shows the percentage of macrophage surface molecules CD80+, CD86+, and CD86+CD80+ in Peyer's patches of the mouse intestine from left to right. The vertical axis is the percentage of different macrophage surface molecules. Figure 8 Part B in the middle shows the percentage of DC cell surface molecules CD11c+, CD86+, CD86+CD80+ in Peyer's patches of the mouse intestine from left to right. The vertical axis is the percentage of different DC cell surface molecules, and the horizontal axis is the control group, BRO-1@A / C group, BRO-2@A / C group, RO@A / C group and OVA@A / C group. Fig. 9 Part A in the middle shows the percentage of mouse mesenteric lymph node surface molecule CD11c+ and DC cell surface molecules CD80+, CD86+, and CD40+ from left to right. The vertical axis is the percentage of different DC cell surface molecules. Fig. 9 Part B in the middle shows the percentage of macrophage surface molecules F4 / 80+, CD80+, and CD86+ in the mesenteric lymph nodes of mice from left to right. The vertical axis is the percentage of different macrophage surface molecules, and the horizontal axis is the control group, BRO-1@A / C group, BRO-2@A / C group, RO@A / C group, and OVA@A / C group.

[0118] Among them, ctrl in the figure is the control group, which is a group not given any microcapsule preparation, RO@A / C is a calcium alginate-chitosan microcapsule prepared by the preparation method of Example 5 without containing BMVs (hereinafter referred to as RO@A / C), and OVA@A / C is a calcium alginate-chitosan microcapsule prepared by the preparation method of Example 5 without containing poly-L-arginine transmembrane peptide R8 solution and BMVs (hereinafter referred to as OVA@A / C). The above experimental groups are set up to verify the adjuvant effect of bacterial membrane vesicles BMVs.

[0119] The preparation method of RO@A / C is as follows: add 1 mg / mL of OVA antigen to 100 mM Hepes buffer, mix well, and obtain an antigen solution; add 1 mg / mL of poly-L-arginine transmembrane peptide R8 solution to the antigen solution, vortex mix for 60 seconds, and stand at room temperature for 60 minutes to obtain an antigen complex; then disperse the antigen complex (RO) in 1% (m / v) sodium alginate solution to obtain a sodium alginate suspension; and mix the sodium alginate suspension. The device is installed in a syringe pump device, and the sodium alginate suspension is pushed downward at 0.2 mL / min. Under the action of the electric field force, the sodium alginate suspension falls into a measuring cup containing a 0.2 mol / L calcium chloride solution in the form of droplets to obtain calcium alginate microgel beads encapsulating nanocomposites; the calcium alginate microgel beads encapsulating the nanocomposites and a 0.8% (m / v) chitosan solution are reacted in a volume ratio of 1:1 to form a film, and calcium alginate-chitosan microcapsules (hereinafter referred to as RO@A / C) are obtained.

[0120] Among them, the preparation method of OVA@A / C is as follows: add 1 mg / mL of OVA antigen to 100 mM Hepes buffer, mix evenly, and obtain an antigen solution; then disperse the antigen solution in 1% (m / v) sodium alginate solution to obtain a sodium alginate suspension; load the sodium alginate suspension into a syringe pump device, push the sodium alginate suspension downward at 0.2 mL / min, and under the action of the electric field force, the sodium alginate suspension falls into a measuring cup containing 0.2 mol / L calcium chloride solution in the form of droplets to obtain calcium alginate microgel beads encapsulating nanocomplexes; react the calcium alginate microgel beads encapsulating nanocomplexes with 0.8% (m / v) chitosan solution at a volume ratio of 1:1 to form a film, and obtain calcium alginate-chitosan microcapsules (hereinafter referred to as OVA@A / C).

[0121] In the in vivo flow cytometric study, compared with the simple OVA@A / C group, the microcapsule system loaded with BMV-RO nanocomplexes from different bacterial sources (BRO-1@A / C and BRO-2@A / C) showed a certain activation effect on DCs and macrophages in mouse PPs, but there was no significant difference compared with the RO@A / C group ( Figure 8As for MLNs, oral vaccine preparations can induce the maturation of DC cells, including significantly increasing the expression levels of CD40+, CD86+, and CD80+ on the surface of DC cells ( Fig. 9 Part A); it also has a significant activation effect on M1 macrophages ( Fig. 9 Part B of the study), proving that the oral vaccine system has the basic conditions to initiate the body's immune response.

[0122] Example 5: Potential of oral vaccine drugs to induce mucosal immune response

[0123] After being activated by antigens, mature B cells differentiate into antibody-secreting cells in germinal centers or follicles. IgA-secreting plasma cells are mainly produced in intestinal-associated lymphoid tissues such as mesenteric lymph nodes. Intestinal IgA+ plasma cells are one of the largest B cell pools in the body and can produce a large amount of IgA. Therefore, the applicant tested the induction of IgA plasma cells after immunizing mice with the preparation in order to preliminarily evaluate the potential of the oral vaccine to induce mucosal immune responses in the body.

[0124] The specific method is as follows: C57BL / 6 mice (6-8 weeks old) were given calcium alginate-chitosan microcapsule oral immune drugs (BMV-RO-1@A / C or BMV-RO-2@A / C) prepared in Example 5 or Example 6 by gavage four times (once a week for four consecutive weeks) (the dose of gavage was 100μg antigen OVA, 100μg BMVs), the mesenteric lymph nodes and intestinal segments of the mice were dissected, and the intestinal lamina propria immune cell suspensions of each group of mice were obtained by separation and digestion, mesh filtration, Percoll density gradient centrifugation and washing. The proportions of B cells (B220+IgA+) and plasma cells (B220-CD38+IgA+) secreting IgA in the mesenteric lymph nodes and lamina propria immune cells were detected by flow cytometry. The results refer to Fig.10 and Fig.11 , Fig.10 This is a flow cytometric percentage diagram of B cells and plasma cells secreting IgA in the intestinal lamina propria of mice induced by an oral vaccine provided in the examples of the present application. Fig.11 This is a flow cytometric percentage diagram of the production of IgA-secreting B cells and plasma cells in the mesenteric lymph nodes of mice induced by an oral vaccine provided in an example of the present application. Fig.10 and Fig.11 The figure on the left shows the results of B cells. Fig.10 and Fig.11 The figure on the right in the middle shows the results for plasma cells.

[0125] Depend on Fig.10 and Fig.11It can be seen that the flow cytometry results of the secretory IgA (SIgA) levels of B cells (B220+) and plasma cells (B220-CD38+) in the intestinal lamina propria (LP) and mesenteric lymph nodes (MLNs) of mice showed that oral vaccine drugs showed different degrees of promotion on SIgA. The BRO@A / C group (i.e., the corresponding groups of BMV-RO-1@A / C and BMV-RO-2@A / C, abbreviated as BRO@A / C group) can significantly increase the expression level of IgA in B cells and plasma cells, which is crucial for inducing effective mucosal immune responses.

[0126] In addition, the applicant collected mouse feces and intestinal segments, collected the supernatant by centrifugation after certain treatment, and detected the content of mucosal antigen-specific SIgA by ELISA. The experimental method is as follows:

[0127] 1. Coat the ELISA plate one day in advance: weigh OVA and prepare it into 1 mg mL -1 The concentration was then diluted to 10 μg mL -1 , add 100 μL per well to the ELISA plate with a pipette. After sealing, place it at 4°C for overnight coating.

[0128] 2. Blocking: Take out the coated ELISA plate, pour out the liquid in the plate, add 200μL of washing solution to each well of the ELISA plate with an eight-channel pipette, and shake it with an oscillator for 1 minute. After pouring out the liquid, tap it on the filter paper to clean it. Repeat the above operation twice. Use a pipette to add 200μL of blocking solution to each well of the ELISA plate and place it at 37℃ for 1 hour.

[0129] 3. Incubate with primary antibody: Take out the ELISA plate and pour out the liquid in it. Wash twice according to step 2, add the sample diluted with diluent, 100 μL per well, and incubate at 37°C for 2 hours.

[0130] 4. Incubate the secondary antibody: Take out the incubated ELISA plate, remove the ELISA plate and pour out the liquid in it, wash it 5 times according to step 2, dilute the HRP-coupled secondary antibody 5000 times with diluent, add 100 μL per well to the ELISA plate, and incubate at 37°C for 1-2 hours.

[0131] 5. Color development: Take out the ELISA plate and pour out the liquid in it. Wash it 5 times according to step 2, then add 100 μL of TMB (Tetramethylbenzidine, as substrate color development solution) to each well of the ELISA plate, incubate at 37°C for 20 minutes, and add 50 μL of stop solution to each well after the liquid turns blue. The color of the liquid will immediately turn from blue to yellow.

[0132] 6. Reading: After adding the stop solution, the absorbance at 450nm should be measured as soon as possible using a microplate reader and recorded as OD450.

[0133] Mouse feces and intestinal tissues were collected and processed to obtain the supernatant. The secretion level of mucosal antigen-specific SIgA was determined by ELISA. Fig.12 , Fig.12 This is a graph showing the results of antigen-specific IgA antibodies produced on the 28th day after mice were immunized with an oral vaccine provided in an example of the present application. Fig.12 The left picture shows the secretion level of SIgA in the intestine. Fig.12 The right figure shows the results of fecal SIgA secretion levels. Based on the absorbance values ​​of the feces and intestinal segments after 10-fold dilution, the BRO@A / C group can promote the significant secretion of SIgA, thereby activating the mucosal immune response.

[0134] Example 6: Oral vaccine drug induces the production of antigen OVA-specific antibodies in serum

[0135] Antigen-specific antibodies in serum are important indicators for evaluating the strength of humoral immune response induced by vaccines. They are produced in vivo by B cells that proliferate and divide into antibody-secreting cells, namely plasma cells. IgG is one of the most abundant antibodies in serum and is the main antibody for the body's humoral immunity. There are many subtypes of IgG, including IgG1, IgG2, IgG3, and IgG4. It is generally believed that IgG1 is mainly mediated by Th2 cells, reflecting the ability to induce humoral immune response; while IgG2a is believed to be mediated by Th1 cells. Since Th1 cells can secrete IFN-γ and promote CD8+T activation, the level of IgG2a can also reflect the strength of cellular immune response. Antibody detection is simple to operate and time-saving. It can be detected without killing mice, which is convenient for monitoring the level of immune response at different time periods. Therefore, antibody detection is usually an important step in evaluating the efficacy of vaccines.

[0136] The specific operation is as follows: The ELISA method for determining IgG and its subtypes (IgG1, IgG2a) in serum is consistent with the method for determining the content of mucosal antigen-specific SIgA. The only difference is that in step 4, the HRP-coupled antibody is diluted with diluent at 1:10000.

[0137] The above experimental results refer to Fig.13 , Fig.13 This is a graph showing the antigen-specific IgG and its subtype antibody results produced on the 28th day after mice were immunized with an oral vaccine provided in the examples of this application. Fig.13 Part A in the middle shows the IgG secretion level results of serum (from left to right, 100 and 1000 times dilution respectively). Fig.13Part B shows the secretion level results of IgG2a and IgG1 in serum (diluted 100 times).

[0138] Compared with the RO@A / C and OVA@A / C groups, the BRO@A / C group was able to simultaneously increase the levels of IgG, IgG1, and IgG2 antibodies in mouse serum, and IgG1 and IgG2a were associated with the activation of Th2 and Th1 cells, respectively, proving that the oral vaccine can induce humoral and cellular immune responses. From the results that the BRO-1 group was able to more significantly increase the secretion levels of IgG1 and IgG2a in mouse serum, BMVs from different bacterial sources may have personalized immune activation effects, showing different degrees of humoral and cellular immune response induction capabilities.

[0139] The above is a detailed introduction to a nanovesicle-based microcapsule vaccine delivery system, a preparation method and an application thereof provided by the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A nanovesicle-based microcapsule vaccine delivery system, characterized in that: The core of the vaccine delivery system is a nanocomplex, which is a complex of bacteria-derived nanovesicles and antigens; the outer layer of the nanocomplex is coated with a calcium alginate layer and a chitosan layer.

2. The nanovesicle-based microcapsule vaccine delivery system according to claim 1, characterized in that: The antigens include one or more of protein antigens, protein antigens with weak immunogenicity, polypeptide antigens, viral or bacterial lysate antigens, viral or bacterial outer membrane vesicle antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, tumor cell exosome antigens, or tumor pattern antigen chicken ovalbumin; The protein antigens include: hepatitis A, hepatitis B or hepatitis C antigens, tetanus toxoid, human papillomavirus, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, herpes simplex virus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, respiratory syncytial virus, West Nile virus, cytomegalovirus, malaria antigen, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, group A streptococcal antigens, or one of other recombinant protein antigens; the immunogen The weaker protein antigens include: one of bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoalbumin, soy albumin, wheat albumin, myoglobin, collagen, and lamin; the polypeptide antigens include: tyrosinase-related protein 2 antigen fragment, human melanoma antigen fragment, retroviral tumor antigen fragment endogenously expressed in MC38 tumor cells, chicken ovalbumin antigen fragment, human papilloma virus oncoprotein, hepatitis B antigen epitope peptide, or other synthetic polypeptide antigens and one of long polypeptide antigens containing several polypeptide sequences.

3. The nanovesicle-based microcapsule vaccine delivery system according to claim 1, characterized in that: The particle size of the nanocomplex is less than 500 nm; the microcapsule vaccine delivery system based on nanovesicles is a microspherical gel with a particle size of less than 500 μm.

4. A method for preparing a nanovesicle-based microcapsule vaccine delivery system according to any one of claims 1 to 3, characterized in that: The method comprises: A nanocomplex is prepared by using an aqueous solution of bacteria-derived nanovesicles and an antigen; Based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution, calcium alginate-chitosan microcapsules are prepared as the nanovesicle-based microcapsule vaccine delivery system.

5. The method for preparing a nanovesicle-based microcapsule vaccine delivery system according to claim 4, characterized in that: The bacteria-derived nanovesicles in the bacteria-derived nanovesicle aqueous solution are prepared according to the following steps: The cultured bacterial suspension was placed in a centrifuge and centrifuged at 6000 g / min for 10 min. The precipitate was collected and washed twice with a sterile PBS solution and resuspended in a PBS solution containing PMSF to obtain a bacterial suspension. Using an ultrasonic probe, ultrasonically disrupting the bacteria at 20-60% ultrasonic power, and ultrasonicating until the bacterial suspension is clear to obtain a bacterial disruption solution; The bacterial disruption solution was placed in a centrifuge and centrifuged at 4000 g / min for 10 min, and the supernatant was collected; The supernatant was subjected to ultracentrifugation at 100,000 g / min for 40 min, and the precipitate was collected and centrifuged using a 0.2 × 10 -3 The resulting mixture was washed with M ethylenediaminetetraacetic acid 1-2 times to obtain the bacteria-derived nanovesicles.

6. The method for preparing a nanovesicle-based microcapsule vaccine delivery system according to claim 4, characterized in that: The nanocomplex is prepared by using a bacterial-derived nanovesicle aqueous solution and an antigen, including: Add 1 mg / mL-10 mg / mL of antigen to 30-200 mM Hepes buffer, mix well, and obtain an antigen solution; Add 0.5 mg / mL-10 mg / mL poly-L-arginine membrane-penetrating peptide R8 solution to the antigen solution, vortex mix for 5-60 seconds, and stand at room temperature for 30-60 minutes to obtain an antigen complex; The antigen complex is slowly added dropwise to the bacterial-derived nanovesicle aqueous solution for mixing to obtain the nanocomplex, wherein the mixing mass ratio is antigen: bacterial-derived nanovesicle = 1:0.1-2.

0.

7. The method for preparing a nanovesicle-based microcapsule vaccine delivery system according to claim 6, characterized in that: The antigen complex is slowly added dropwise to the aqueous solution of bacteria-derived nanovesicles for mixing, including: vortexing, ultrasound or mixing by a microinjection pump; The vortex mixing time is 5-60S; the ultrasonic mixing power is 50-300W, and the ultrasonic time is 1-10min; the flow rate of the micro-injection pump mixing is 30mL / min-80mL / min.

8. The method for preparing a nanovesicle-based microcapsule vaccine delivery system according to claim 4, characterized in that: Based on the nanocomposite, sodium alginate solution, calcium chloride solution and chitosan solution, calcium alginate-chitosan microcapsules are prepared, including: Dispersing 0.5 mg / mL-10 mg / mL of the nanocomplex in 1%-2% (m / v) sodium alginate solution to obtain a sodium alginate suspension; The sodium alginate suspension is placed in a syringe pump device, and the sodium alginate suspension is pushed downward at a speed of 0.1-1.0 mL / min. Under the action of the electric field force, the sodium alginate suspension falls into a measuring cup containing a 0.1 mol / L-0.2 mol / L calcium chloride solution in the form of droplets, thereby obtaining calcium alginate microgel beads encapsulating the nanocomposite; The calcium alginate microgel beads carrying the nanocomposite are reacted with 0.4%-0.8% (m / v) chitosan solution at a volume ratio of 1:1 to form a film, thereby obtaining calcium alginate-chitosan microcapsules.

9. An application of a nanovesicle-based microcapsule vaccine delivery system, characterized in that: Applied to the preparation of oral immune drugs, the nanovesicle-based microcapsule vaccine delivery system is prepared according to any one of claims 4 to 8 above.

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