Antigen double burst release self-boosting immune vaccine and application thereof

Abstract

The invention relates to the technical field of medicine, in particular to an antigen double burst release self-boosting immune vaccine and application thereof.The antigen double burst release self-boosting immune vaccine is characterized in that antigens are loaded inside and outside complex coacervation microcapsules in the vaccine, nanoparticles are prepared through a nanoprecipitation method, the nanoparticles and cell membranes jointly entrap the antigens to form functional nanoparticles, and the functional nanoparticles are used as functional nanoparticles; the preparation method comprises the following steps: coating functionalized nanoparticles by using a micro-fluidic technology and a complex coagulation method to prepare microcapsules, coating the surface of the microcapsules by using dopamine hydrochloride auto-polymerization reaction to form coating microcapsules, and finally blending with the functionalized nanoparticles, thereby dividing the antigen into an inner part and an outer part. After entering a body, the nano-microparticle carrier double burst release vaccine is subjected to antigen double burst release with time lag intervals in different time periods; the prepared vaccine can release antigens firstly, then effectively collect and activate antigen presenting cells, the antigen presenting cells uptake the antigen presenting cells to cause immune response, and the generation of specific antibodies can be effectively induced by two times of antigen release through the time-delay coating, so that a traditional natural immune procedure is simulated.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to an antigen double-splastic self-boosting immunization vaccine and its application. Background Technology

[0002] Infectious diseases have long been one of the main threats to global public health and human life. The outbreak of COVID-19 has further highlighted the importance of infectious disease prevention systems. As the most effective means of responding to biosecurity threats and blocking the spread of infectious diseases, the technological innovation and optimization of vaccination has always been a research focus in the medical field.

[0003] According to the World Health Organization's classification, vaccines currently used clinically mainly include six categories: live attenuated vaccines, inactivated vaccines, antitoxins, subunit vaccines, vector vaccines, and nucleic acid vaccines. Among them, subunit vaccines, because they contain only the specific antigenic components of the pathogen and do not contain nucleic acid substances, have significant advantages such as no risk of infection, high safety, and strong immunogenicity, showing broad application prospects in infectious disease prevention. However, the core drawback of subunit vaccines lies in their relatively weak immunogenicity, making it difficult to stimulate a sustained and effective immune protective response on its own. Typically, booster immunizations are needed after the primary immunization to enhance the immune effect (i.e., a multi-dose immunization program) in order to establish long-term immune memory.

[0004] Although multiple-dose immunization programs are currently the main way to compensate for the immunogenicity deficiencies of subunit vaccines, they have many insurmountable limitations in practical application: ① They are prone to dropout; multiple-dose immunization programs are time-consuming, costly, and require a robust vaccination management system. In economically underdeveloped areas or scenarios with limited vaccination resources, some populations often cannot complete the full course of vaccination on time, thus affecting the coverage of immunization protection; ② Vaccination compliance is poor among special populations; for highly mobile groups such as border guards and field workers, or those working in special environments, it is difficult to strictly follow the pre-set vaccination schedule, leading to interruptions in the immunization program; ③ They are not adaptable enough to sudden major epidemics; multiple-dose immunization programs require mobilizing a large amount of social resources for vaccine production, transportation, and vaccination services, which is not only costly but may also prevent the rapid construction of herd immunity due to vaccine shortages. Especially in developing countries, too many vaccinations can significantly limit vaccine accessibility.

[0005] Therefore, developing a vaccine formulation that can achieve similar immunization effects with a single dose has become key to solving the aforementioned problems. Such a single-dose vaccine not only needs to possess long-lasting immune protection but also needs to be convenient to administer, in order to increase vaccination coverage, reduce vaccination costs, and meet the needs of rapid prevention and control in the event of a sudden outbreak. Taking into account the necessity of booster immunization and preventing immune loss, developing a single-dose double-release vaccine that self-boosts immunization in vivo, achieves pulsed antigen release, and simulates the natural immunization process, has significant practical significance and application value for overcoming the application bottlenecks of existing subunit vaccines and improving the efficiency of infectious disease prevention and control. Summary of the Invention

[0006] The purpose of this invention is to provide a nanoparticle vaccine that achieves self-enhancing immunization in vivo through antigen pulse release. By loading antigen proteins into microcapsules, a time lag interval can be achieved, effectively solving the core problem of traditional subunit vaccines requiring multiple injections. It can be administered to the body via various methods such as intramuscular injection, subcutaneous injection, and pulmonary inhalation to induce an immune response in the body.

[0007] The objective of this invention is achieved through the following technical solution: The present invention provides a dual burst-release pulsed release vaccine, wherein the vaccine is a polydopamine-coated complex aggregated microcapsule, and both the interior and exterior of the complex aggregated microcapsule are loaded with antigens.

[0008] Furthermore, the antigen is selected from Spytag-OVA antigen, chicken ovalbumin antigen, recombinant antigen, novel coronavirus antigen, respiratory syncytial virus antigen, influenza virus antigen, or hepatitis B virus antigen; the nucleotide sequence of the Spytag-OVA antigen is shown in SEQ ID NO.1.

[0009] Furthermore, it also includes adjuvants selected from aluminum adjuvants or small molecule adjuvants; the aluminum adjuvants are selected from aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum; the small molecule adjuvants are selected from CpG, Toll-like receptor agonists or squalene.

[0010] Furthermore, the particle size of the complex condensed microcapsules is 10-150 μm, and the particle size of the nanoparticles is 20-500 nm.

[0011] The present invention also provides a method for preparing the aforementioned dual burst-release pulsed release vaccine, comprising the following steps: (1) Nanoparticles were prepared by nanoprecipitation method; (2) The nanoparticles are co-encapsulated with the cell membrane to obtain functionalized nanoparticles; (3) Using microfluidic technology, the functionalized nanoparticles and adjuvants are encapsulated by complex condensation method to prepare complex condensation microcapsules; (4) A polydopamine coating is applied to the surface of the complex coagulated microcapsules, and then the coating is blended with the functionalized nanoparticles to obtain the dual burst release pulse release vaccine.

[0012] Furthermore, in step (1), the nanoprecipitation method uses an organic solvent to dissolve the polymer to prepare the oil phase, and a surfactant to prepare the solution as the aqueous phase; The polymer is selected from polylactic acid, polyanhydride, lactide-glycolic acid copolymer, or polycaprolactone; the organic solvent is selected from dichloromethane, ethyl acetate, chloroform, acetone, diethyl ether, or ethanol; the surfactant is selected from polyethylene glycol octylphenyl ether, Tween, polyoxyethylene, polyvinyl alcohol, Span, poloxamer, polyethylene glycol, or sodium dodecyl sulfate.

[0013] Furthermore, in step (3), the three-phase gas pressure of the microfluidic technology is 10-2000 MPa; the inner phase of the complex coagulated microcapsule is an antigen and adjuvant solution, the intermediate phase is a capsule material solution, the capsule material is selected from gelatin, gum arabic or sodium carboxymethyl cellulose, and the outer phase is fluorinated oil with added surfactant.

[0014] Furthermore, in step (4), the polydopamine coating is carried out in a neutral buffer solution with a pH of 6-8 for a coating time of 1-240 min; the neutral buffer solution is selected from PBS, sodium citrate aqueous solution or sodium dihydrogen phosphate aqueous solution.

[0015] The present invention also provides the application of the aforementioned dual burst-release pulse-release vaccine in the preparation of preventive or therapeutic vaccines.

[0016] Furthermore, the preventive or therapeutic vaccine is used to prevent or treat novel coronavirus infection, respiratory syncytial virus infection, influenza, or hepatitis B; the vaccine is administered via intramuscular injection, subcutaneous injection, or inhalation.

[0017] Beneficial effects: This invention's dual-burst-release pulsed-release vaccine utilizes a structured design of polydopamine-coated complex aggregated microcapsules to achieve two time-delayed burst releases of antigen, effectively addressing the core challenge of traditional subunit vaccines requiring multiple immunizations. Functionalized nanoparticles loaded onto the vaccine's exterior rapidly release antigen for primary immunization; after the polydopamine coating degrades, the complex aggregated microcapsules rupture, releasing the internal antigen for booster immunization, precisely mimicking the enhanced stimulation of the natural immune program. This design efficiently recruits and activates antigen-presenting cells, promoting the aggregation of dendritic cells and macrophages at the injection site, significantly improving antigen uptake and presentation efficiency. Simultaneously, the time-delayed release effect effectively induces the proliferation and differentiation of germinal center B cells, promoting the continuous production of specific antibodies and activating CD4+. + and CD8 +T cells enhance cellular immune responses and form stable immune memory. Compared with the traditional two-dose immunization regimen, a single dose can achieve or even surpass the antibody levels and cellular immune effects of two-dose immunization, avoiding the problems of immune dropout, inconvenience of vaccination, and high cost caused by multiple-dose immunization. It is especially suitable for special populations such as border guards who have difficulty getting vaccinated on time, as well as for building herd immunity during sudden major epidemics. It can significantly improve vaccination rates and reduce vaccination costs and social resource consumption.

[0018] The vaccine preparation process of this invention has significant advantages. It employs a nanoprecipitation method combined with microfluidic technology, resulting in mild preparation conditions and precise control over the particle size of nanoparticles (20-500 nm) and complex aggregated microcapsules (10-150 μm), ensuring product uniformity and stability. Covalent linkage between antigen and nanoparticles is achieved through cell membrane encapsulation and bioclick chemistry, improving antigen loading efficiency and delivery stability. The polydopamine coating process is simple and controllable; the time lag interval between two antigen releases (3-5 weeks) can be flexibly adjusted by regulating the coating time to suit different immune needs. The vaccine exhibits good safety; as a subunit vaccine, it contains no nucleic acid components and poses no risk of infection. Animal experiments have shown that mice vaccinated showed no abnormalities in body weight or temperature, no toxic damage to major organs, and blood biochemical indicators remained within normal ranges. Furthermore, the vaccine supports multiple administration methods, including intramuscular injection, subcutaneous injection, and pulmonary administration, adapting to various application scenarios. Its antigen has a wide range of applicability, capable of loading antigens for various pathogens such as the novel coronavirus, influenza virus, and hepatitis B virus, providing an efficient, safe, and convenient technical solution for the prevention and treatment of various infectious diseases, and possessing significant practical application value. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. 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.

[0020] Figure 1 These are morphological images of nanoparticles, cell membranes, and functionalized nanoparticles in Example 1 of this invention.

[0021] Figure 2 This is a diagram showing the particle size distribution and potential distribution of nanoparticles, cell membranes, and functionalized nanoparticles in Example 2 of the present invention.

[0022] Figure 3 This is a diagram showing the preparation, morphology, and particle size distribution of microcapsules in Example 3 of the present invention.

[0023] Figure 4 This is a morphology diagram of the polydopamine-coated microcapsules in Example 4 of the present invention.

[0024] Figure 5 This is a graph showing the in vivo imaging and fluorescence changes of the microcapsule carrier in small animals in Experiment Example 1 of this invention.

[0025] Figure 6 This is a graph showing the antibody titer and its changes in polydopamine-coated microcapsules in Experiment Example 2 of this invention.

[0026] Figure 7 This is a diagram showing the activation of antigen-presenting cells in different immune groups in Experiment Example 3 of this invention.

[0027] Figure 8 This is a diagram showing the proportion of germinal center B cells in spleen tissue of different immune groups in Experiment Example 4 of this invention.

[0028] Figure 9 This is a diagram showing the in vitro antigen stimulation and proliferation of spleen cells in mice immunized with different immunization groups in Experiment Example 5 of this invention.

[0029] Figure 10 This is a diagram showing the activation status of specific T cells in different groups in Experiment Example 6 of this invention.

[0030] Figure 11 This is a graph showing the long-term specific antibody secretion in mice of different groups after immunization with the vaccine in Experiment Example 7 of this invention.

[0031] Figure 12 This is a graph showing the changes in body weight and body temperature of mice after immunization in different groups in Experiment Example 8 of this invention.

[0032] Figure 13 This is a graph showing the main blood biochemical indicators of mice after immunization in different groups in Experiment Example 8 of this invention.

[0033] Figure 14 This is a diagram showing the H&E staining analysis of the major organs of mice after immunization in different groups in Experiment Example 8 of this invention. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Unless otherwise specified, all chemical reagents, biochemical reagents and materials used in this invention are commercially available.

[0040] The present invention will now be described in detail with reference to the accompanying drawings, so that the advantages and features of the invention can be more easily understood by those skilled in the art, thereby providing a clearer and more definite definition of the scope of protection of the present invention. However, this does not limit the present invention to the scope of the described embodiments. The reagents and raw materials used in the following embodiments are all commercially available, and the test methods without specific conditions are generally performed under conventional conditions or according to the conditions recommended by the respective manufacturers. Furthermore, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0041] The preparation method of the dual burst-release pulsed release vaccine in this invention includes the following steps: (1) Nanoparticles are prepared by nanoprecipitation method. The polymer material is polylactic acid, polyanhydride, lactide-glycolic acid copolymer (PLGA) or polycaprolactone. The intrinsic viscosity of PLGA is 0.1-2.0 gL / g and the weight average molecular weight is 30,000-100,000.

[0042] Oil phase preparation: The polymer is dissolved in an organic solvent (dichloromethane, ethyl acetate, chloroform, acetone, diethyl ether or ethanol) at a concentration of 0.2-20 mg / ml.

[0043] Aqueous phase preparation: A solution is prepared using surfactants (polyethylene glycol octylphenyl ether, Tween, polyoxyethylene, polyvinyl alcohol, Span, poloxamer, polyethylene glycol or sodium dodecyl sulfate), with a surfactant mass-volume ratio concentration of 0.1%-5%.

[0044] Preparation conditions: the volume ratio of aqueous phase to organic phase is 1%-40%, the magnetic stirring speed is 400-1200 rpm, and nanoparticles are formed by high-speed shearing. The nanoparticle size is 20-500 nm, preferably 50-200 nm.

[0045] (2) The nanoparticles are co-encapsulated with the fusion protein antigen by the cell membrane to obtain functionalized nanoparticles: Cell membrane origin: from 10 5 -10 9 Membrane fragments were extracted from cells expressing the Spycatcher protein, thawed on ice, and resuspended for later use.

[0046] Cell membrane encapsulation: Nanoparticles at a mass concentration of 5-30 mg / ml are mixed with cell membrane fragments and then subjected to sonication under ice bath conditions. The sonication power is 50-600 W and the sonication time is 1-20 min to promote fusion encapsulation.

[0047] Antigen covalent linkage: The antigens include proteins with the sequence SEQ ID No. 1, chicken ovalbumin antigen, recombinant antigen, novel coronavirus antigen, etc., with an antigen solution concentration of 0.01-50 mg / ml. Cell membrane-encapsulated nanoparticles are co-incubated with the antigen solution at 0-8℃ for 4-96 h, and covalent linkage between the antigen and the cell membrane is achieved through Spydisplay bioclick chemistry.

[0048] (3) Using microfluidic technology to encapsulate functionalized nanoparticles and adjuvants via complex condensation, complex condensation microcapsules were prepared: Microcapsule structure: It consists of three phases: the inner phase is an aqueous solution or suspension of antigen and adjuvant, the middle phase is a capsule material solution, and the outer phase is fluorinated oil with added surfactant.

[0049] Phase parameters: Inner phase: The antigen concentration is 0.01-50 mg / ml. The adjuvant is selected from aluminum adjuvants such as aluminum hydroxide gel and aluminum phosphate, or small molecule adjuvants such as CpG, Toll-like receptor agonists, and squalene. The adjuvant needs to be dispersed in the antigen solution.

[0050] Intermediate phase: The capsule material is selected from gelatin, gum arabic or sodium carboxymethyl cellulose, with a solution concentration of 1-20% (w / v) and a preparation temperature of 40-60℃.

[0051] External phase: The concentration of surfactant added to fluorinated oil is 0.1%-5%, and the microfluidic three-phase gas pressure is 10-2000 MPa.

[0052] Volume ratio: The volume ratio of oil phase to mesophase is 1:1-1:50, and the volume ratio of mesophase to internal phase is 1:1-40:1.

[0053] Microcapsule particle size: The blank microcapsule particle size is 10-150μm, preferably 10-70μm.

[0054] (4) A polydopamine coating is applied to the surface of the complex aggregated microcapsules, which are then blended with the functionalized nanoparticles to obtain the dual burst-release pulse-release vaccine: Coating buffer: Use a neutral buffer (PBS, sodium citrate aqueous solution, sodium dihydrogen phosphate aqueous solution or tris(hydroxymethyl)aminomethane buffer) with a molar concentration of 0.1-2M and a pH of 6-8.

[0055] Coating system: Microcapsules were added to the buffer solution, followed by dopamine hydrochloride (concentration 1-10 mg / ml) and ammonium persulfate initiator (concentration 1-10 mg / ml). The coating reaction was carried out by stirring at room temperature.

[0056] Coating time: 1-240 min. The time lag between two antigen releases can be flexibly adjusted by adjusting the coating time to meet the immune requirements.

[0057] Example 1: Preparation of PLGA nanoparticles (functionalized nanoparticle precursors) In this embodiment, polylactic acid-glycolic acid copolymer (PLGA) nanoparticles were prepared using a nanoprecipitation method, serving as the core carrier for functionalized nanoparticles. The specific steps are as follows: Material selection: PLGA (model 7525 2CA) was selected, with an intrinsic viscosity of 0.12-0.20 dL / g and a weight-average molecular weight of approximately 15 kDa; the surfactant was polyoxyethylene-polyoxypropylene block copolymer (P188); and the organic solvent was acetone.

[0058] Oil phase preparation: Weigh 10 mg PLGA, add 3 mL acetone to dissolve it completely, and prepare a PLGA polymer oil phase solution.

[0059] Aqueous phase preparation: Prepare a 1% P188 aqueous solution as the aqueous phase, and take 20 mL for later use.

[0060] Nanoparticle formation: The oil phase is added dropwise to the aqueous phase using a constant flow pump, and the magnetic stirring speed is maintained at 800 rpm to form a primary emulsion through high-speed shear dispersion.

[0061] Solvent evaporation: Stir continuously for 6-8 hours to allow acetone to evaporate completely, resulting in a PLGA nanoparticle suspension.

[0062] Example 2: Preparation of Functionalized Nanoparticles (CNPs) In this embodiment, functionalized nanoparticles are prepared by covalently linking antigens to PLGA nanoparticles through cell membrane encapsulation and bioclick chemistry. The specific steps are as follows: Cell membrane preparation: Take 1×10 8 HEK293 cells expressing the Spycatcher protein were used to extract cell membrane fragments, which were then thawed on ice and resuspended for later use.

[0063] Cell membrane encapsulation of nanoparticles: The above-mentioned cell membrane fragments were added to 10 mg of PLGA nanoparticles prepared in Example 1. Under ice bath conditions, a No. 6 amplitude transformer was used, the ultrasonic power was set to 300 W, and the ultrasonic treatment was carried out for 10 min to promote the fusion of cell membrane and nanoparticles.

[0064] Antigen covalent linkage: Nanoparticles encapsulating cell membranes were mixed with 10 mg / mL Spytag-OVA antigen (nucleotide sequence as shown in SEQ ID NO.1) solution at a volume ratio of 1:1 and incubated on a horizontal shaker at 4°C for 24 h. Covalent linkage between antigen and cell membrane was achieved through Spydisplay bioclick chemistry.

[0065] Characterization and Detection: The particle size and potential of the functionalized nanoparticles were measured by sampling (see transmission electron microscopy images). Figure 1 Particle size and potential distribution are shown in Figure 2 ), and calculate the antigen loading.

[0066] In this embodiment, the amount of antigen carried inside and outside the microparticle can be adjusted by the cell membrane concentration and the concentration of antigen protein in the antigen mixture. There is a positive proportional trend between the concentration of the antigen solution and the amount of antigen in the microparticle.

[0067] Transmission electron microscopy images of the nanoparticles, cell membrane fragments, and functionalized nanoparticles prepared in Example 1 and Example 2 are shown below. Figure 1 As shown, Figure 1 A represents the morphology of the nanoparticles, which are round and well-formed. Figure 1 B represents the morphology of cell membrane fragments; Figure 1 C represents the morphology of the functionalized nanoparticles, which are visible as being encapsulated by the cell membrane.

[0068] The particle size distribution and potential distribution of nanoparticles, cell membrane fragments, and functionalized nanoparticles are as follows: Figure 2 As shown, Figure 2 A represents the particle size distribution, with nanoparticles having a diameter of approximately 100 nm, cell membrane fragments approximately 200 nm, and functionalized nanoparticles approximately 105-110 nm. Figure 2B represents the potential distribution. The potential of the nanoparticles is approximately -30 mV, the cell membrane potential is approximately -22 mV, and the potential of the functionalized nanoparticles is approximately -19 mV. It can be observed that the nanoparticles are encapsulated by the cell membrane, and the particle size is slightly increased, with the potential tending towards the cell membrane potential.

[0069] Example 3: Preparation of antigen / adjuvant loaded microcapsules using microfluidic technology This embodiment employs microfluidic technology, using gelatin-gum arabic composite encapsulation material, to prepare microcapsules encapsulating functionalized nanoparticles, antigens, and adjuvants. The specific steps are as follows: Preparation of solutions for each phase: Oil phase: Take 15 mL of 7500 fluorinated oil, add 1% surfactant by weight / volume, mix well and set aside; Intermediate phase: Weigh 2g gum arabic and 2g gelatin, add 20mL deionized water, heat in a 50℃ water bath until completely dissolved, and prepare a 10% (w / v) composite capsule solution. Inner phase: The antigen solution from Example 2 is mixed evenly with the adjuvant (aluminum adjuvant or CpG) to serve as the core substance of the inner phase.

[0070] Preparation of receiving solution: Take 50 mL of deionized water, adjust the pH to 3 with acetic acid, and keep it in an ice bath environment for later use.

[0071] Microcapsule preparation: The oil phase, intermediate phase and internal phase were sequentially connected to the PDMS microfluidic chip. The gas pressure of each phase was adjusted to control the microcapsule particle size at about 50 μm. After stabilization, the microcapsules were collected at the outlet using an ice bath receiving liquid.

[0072] Figure 3 For the preparation and characterization of microcapsules, Figure 3 A represents the shearing pattern of microcapsules passing through a microfluidic PDMS chip, where the inner phase is encapsulated by the intermediate phase and forms microdroplets under the shearing action of the outer phase; Figure 3 B is a microcapsule morphology diagram. The microcapsules are spherical and have a uniform particle size. Figure 3 C represents the microcapsule size distribution, where the microcapsule size is relatively concentrated, ranging from 50 to 52 μm.

[0073] Example 4: Preparation of polydopamine (PDA) coated microcapsules In this embodiment, a polydopamine coating is formed on the surface of the microcapsules through the self-polymerization reaction of dopamine hydrochloride, thereby regulating the antigen release lag. The specific steps are as follows: Buffer preparation: Weigh 25.81g of trisodium citrate and 11.99g of sodium dihydrogen phosphate, dissolve them in 100mL of deionized water, adjust the pH to 7.0 with sodium hydroxide, and prepare a neutral buffer solution for later use.

[0074] Coating reaction: Add the microcapsules prepared in Example 3 to the above buffer solution, mix well, and then add 40 mg of dopamine hydrochloride and 40 mg of ammonium persulfate (initiator) in sequence. Place on a magnetic stirrer and stir at 200 r / min at room temperature. Set the coating time to 10 min, 30 min and 60 min respectively.

[0075] In this embodiment, the experimental results are as follows: Figure 4 As shown, when microcapsules with different coating times are observed under a light microscope, the overall particle size of the microcapsules does not change, but the color of the microcapsules gradually deepens with increasing coating time.

[0076] Experimental Example 1: Investigation of In Vivo Antigen Burst Release Characteristics in Microcapsules This experiment used small animal in vivo imaging technology to investigate the antigen release pattern of the microcapsules in vivo. The specific method is as follows: Experimental animals: Male BALB / c mice aged 6-8 weeks and weighing 18-22g were randomly divided into two groups (Cy5-OVA group and OVA-MC group), with 3 mice in each group.

[0077] Sample preparation: Cy5-OVA-labeled microcapsules (Cy5-OVA-MC) were prepared according to the method in Example 3, with each group containing 35 μg of antigen; free Cy5-OVA was used as a control.

[0078] Immunization method: Mice in each group were injected intramuscularly with the corresponding sample in their thighs. The Cy5-OVA group was injected with free antigen, and the OVA-MC group was injected with microcapsule-loaded antigen.

[0079] Detection method: In vivo imaging of small animals was performed at 0d, 1d, 3d, 7d and 14d after immunization, and the changes in fluorescence intensity at the injection site were recorded.

[0080] In this embodiment, the experimental results are as follows: Figure 5 As shown, the fluorescence intensity at the injection site was highest in the Cy5-OVA group mice on day 0, gradually disappearing over time and essentially disappearing by day 3. In contrast, the fluorescence intensity of the Cy5-OVA-MC group mice was the highest on day 1, reaching its peak, and essentially disappeared by day 7. There was no statistically significant difference in fluorescence intensity between the two groups, indicating that the microcapsules ruptured and released the antigen on day 1 and were cleared by day 7, demonstrating that the microcapsules successfully delivered the antigen and achieved a burst release effect.

[0081] Experimental Example 2: In vivo time lag effect and specific antibody detection of polydopamine-coated microcapsules This experiment aims to verify the regulatory effect of polydopamine coating on the release lag of vaccine antigens. The immunization effect is reflected by detecting the specific antibody titer. Microcapsules were prepared according to Example 3, using physiological saline and CNPs as the internal phase. After the microcapsules were prepared, polydopamine coating was applied for 10 min according to Example 5. The design table of the coating microcapsule time lag carrier is shown in Table 1 below.

[0082] Table 1. Design of Coated Microcapsule Time-Delay Carriers

[0083] Female BALB / c mice aged 6-8 weeks were randomly divided into three groups of six each. Each group was inoculated with saline, Inside microcapsules, or Outside microcapsules, respectively. Immunization was performed only once. The immunization dosage and vector design are shown in Table 1. After immunization, blood was collected from the fundus venous plexus of the mice. Serum was analyzed using ELISA to detect the titer of Spytag-OVA-specific antibodies. The assay was performed over a period of two months.

[0084] The specific steps of the ELISA experimental method are as follows: Coating: Prepare a Spytag-OVA solution with a concentration of 20 μg / mL using 0.05 mol / L, pH 9.6 CBS buffer. Add 100 μL to each well and incubate overnight at 4°C. Blocking: Wash three times with PBST (0.01 mol / L, pH 7.4 PBS with 0.05% Tween 20), then add 150 μL of blocking buffer (2% BSA, PBST) to each well and block at 37°C for 1 h; Add serum: Add 300 μL of PBST washing buffer to each well of a 96-well plate and wash the plate three times. Dilute the serum sample with diluent (0.1% BSA, PBST), 1 / 100 for the first well, and then perform serial dilutions, 100 μL per well. The last column is the control group. Incubate at 37°C for 30 min. Add enzyme-labeled secondary antibody: Add 200 μL of PBST washing buffer to each well of a 96-well plate and wash the plate 5 times. Add 100 μL of HRP-labeled secondary antibody diluted with diluent to each well and incubate at 37°C for 30 min. Dilute with IgG (1:10000) diluent; wash the plate 5 times with PBST; add 200 μL of chromogenic reagent TMB to each well and incubate at room temperature in the dark for 5-10 min. Termination: Add 50 μL of 2M H2SO4 stop solution to each well. Serum titer determination: The absorbance (OD value) at 450 nm is measured using an ELISA reader. A positive result is defined as an OD value that is more than twice the OD value of negative serum. The maximum dilution factor is the titer of antigen-specific antibodies in the serum sample.

[0085] In this embodiment, the experimental results are as follows: Figure 6As shown, from 1 to 3 weeks post-immunization, no specific antibodies were produced in the Inside-coated microcapsule group (containing antigen internally but not externally), until 4 weeks, indicating that the microcapsules ruptured and released the internal antigen at 3 weeks. The antibody levels at 4 weeks were essentially the same as those at 1 week in the Outside-coated microcapsule group (containing antigen externally but not internally). Significant differences in antibody levels were observed between the two groups at 4-5 weeks post-immunization, which is related to the inconsistent antigen release time. From 6 to 8 weeks, there were no significant differences in antibody levels between the Inside and Outside groups. These results validate that the coating can effectively deliver antigens and produces a time-lag effect.

[0086] Example 3: Evaluation of antigen-presenting cell recruitment capacity at the injection site This experiment aimed to investigate the ability of the vaccine to recruit antigen-presenting cells (dendritic cells and macrophages) after vaccination. Six- to eight-week-old female BALB / c mice were randomly divided into seven groups of 35 mice each. The grouping and vector design are shown in Table 2. Table 2 Grouping and Immunization Regimens of Different Vaccine Vectors

[0087] The preparation method of the carrier used above is as follows: ①Saline group (blank control): Sterile saline was used directly without additional preparation. 50 μL / animal was injected intramuscularly during immunization.

[0088] ②Spytag-OVA group: Prepare a physiological saline solution containing 70 μg / mL Spytag-OVA antigen. Microencapsulation is not required. The immunization regimen is 50 μL intramuscularly on days 0 and 21 (each dose contains 35 μg Spytag-OVA).

[0089] ③MC group: Spytag-OVA (35 μg / dose) and CpG adjuvant (10 μg / dose) were used as the inner phase to prepare complex coagulation microcapsules according to the microfluidic complex coagulation method in Example 3. After the microcapsules were prepared, they were diluted with physiological saline, and Spytag-OVA (final concentration 700 μg / mL) and aluminum hydroxide adjuvant (final concentration 1 mg / mL) were added to the diluent and mixed evenly to obtain the microcapsules (the inner and outer parts of the microcapsules were Spytag-OVA, without polydopamine coating). 50 μL / animal was injected intramuscularly during immunization.

[0090] ④CNPs group: Functionalized nanoparticles (CNPs, each dose containing 35 μg Spytag-OVA) were prepared according to Example 2; during the first immunization (day 0), CNPs were mixed with aluminum hydroxide adjuvant (50 μg / dose) to prepare an immunization solution; during the second immunization (day 21), CNPs were mixed with CpG adjuvant (10 μg / dose) to prepare an immunization solution, and both were administered intramuscularly at a dose of 50 μL / animal.

[0091] ⑤ PDA 10min group: Using CNPs (each dose containing 35 μg Spytag-OVA) and CpG adjuvant (10 μg / dose) prepared in Example 2 as the inner phase, complex coagulation microcapsules were prepared according to Example 3; polydopamine coating was performed in pH 7.0 neutral buffer for 10 min according to the method in Example 4; after the microcapsule coating was completed, it was diluted with physiological saline, and CNPs (final concentration corresponding to 700 μg / mL Spytag-OVA) and aluminum hydroxide adjuvant (final concentration 1 mg / mL) prepared in Example 2 were added to the diluent and mixed evenly to obtain the microcapsule (both inside and outside the microcapsule are CNPs), and 50 μL / animal was injected intramuscularly.

[0092] ⑥ PDA 30min group: The preparation process is the same as ⑤, except that the polydopamine coating time is adjusted to 30min (both inside and outside the microcapsule are CNPs).

[0093] ⑦ PDA 60min group: The preparation process is the same as ⑤, except that the polydopamine coating time is adjusted to 60min (both inside and outside the microcapsule are CNPs).

[0094] At 3, 7, 28, 35, and 42 days post-immunization, tissues were collected from three mice in each group for injection site isolation. The tissues were washed with PBS, cut into fragments, and then digested in PBS containing 0.2% collagenase D and 10 U / ml DNase I at 37°C for 2 hours. The resulting single-cell suspension was prepared by filtration through a 70µm cell filter. Ten... 5 Cells were centrifuged to remove the supernatant, resuspended in 200 μL PBS, and then incubated on ice for 20 min in the dark with 1 μL APC anti-mouse CD11c antibody, 2 μL PE anti-mouse CD80 antibody, 1 μL FITC anti-mouse CD1lb antibody, and 2 μL BV421 anti-mouse F4 / 80 antibody. After incubation, the cells were washed twice with pre-cooled PBS, and finally resuspended in 210 μL PBS and 70 μL 4% paraformaldehyde. The difference in the number of dendritic cells and giant cells was detected by flow cytometry.

[0095] In this embodiment, the experimental results are as follows: Figure 7 As shown, Figure 7 A represents the recruitment of dendritic cells (DCs). The early functionalized nanoparticle-coated microcapsule group showed superior recruitment of antigen-presenting cells (APCs) compared to the other groups. Subsequently, the release lag time of each group resulted in varying degrees of recruitment. Figure 7B represents macrophage recruitment. Compared to dendritic cells (DCs), microcapsules and coated time-delayed microcapsules showed a greater tendency to recruit macrophages, which is related to the microcapsule size. At 3 weeks, the microcapsules in the PDA 10-min group released antigens after rupture, and at 4 weeks, macrophage recruitment in the PDA 10-min group was significantly higher than in the other groups. Similarly, at 4 weeks, the microcapsules in the PDA 30-min group released antigens after rupture, and at 5 weeks, macrophage recruitment in the PDA 10-min group significantly increased. At 5 weeks, the microcapsules in the PDA 60-min group released antigens after rupture, and at 6 weeks, macrophage recruitment in the PDA 60-min group significantly increased.

[0096] Experimental Example 4: Detection of B-cell proliferation capacity in germinal centers This experiment tested the induction effect of the vaccine on B cells in the germinal center. Six- to eight-week-old female BALB / c mice were randomly divided into seven groups of 35 mice each. The grouping and vector design are shown in Table 2. Lymph nodes from three mice in each group were collected at 3, 7, 28, 35, and 42 days post-immunization and placed in PBS. The cells were then milled through a 70 μm cell sieve to prepare a single-cell suspension. After washing 2-3 times with PBS, PE-Cy7 anti-mouse CD19 antibody, PerCP anti-mouse CD45R antibody, and PE anti-mousegL7 antibody were added, and the cells were incubated on ice in the dark for 30 min. After incubation, the cells were washed 2-3 times with PBS, resuspended in 1% paraformaldehyde, and analyzed by flow cytometry.

[0097] In this embodiment, the experimental results are as follows: Figure 8 As shown, Figure 8 A shows the production of GCBs in the lymph nodes of mice at 3 and 7 days. At 7 days, the number of germinal center B cells in each group was significantly increased compared to 3 days. Figure 8 B was 4 weeks. In the PDA 10 min group, due to the release of antigen from the ruptured microcapsules, the proportion of germinal center B cells was the highest, followed by the CNPs group. Both were superior to the Spytag-OVA group and the MC group. Figure 8 C was 5 weeks. The PDA 30 min group had the highest proportion of germinal center B cells due to the release of antigen from the ruptured microcapsules, which was superior to other groups. Figure 8 At 6 weeks (D), the PDA 60-minute group showed a significant increase in germinal center B cell levels due to the release of antigens from microcapsules. The levels of CNPs in all three groups of coated microcapsules were consistent with those in the secondary immunoimmune vaccine. This indicates that the time-delayed release of the coated microcapsules can effectively induce the production of germinal center B cells. These B cells further differentiate into plasma cells that secrete specific antibodies, thereby producing antibodies. A small number differentiate into memory B cells. Therefore, this microcapsule-based vaccine formulation is beneficial in promoting the production of specific antibodies and the subsequent formation of immune memory cells.

[0098] Example 5: Evaluation of the in vitro antigen-stimulated proliferation response of spleen cells This experiment aims to examine the strength of the vaccine-induced cellular immune response through a spleen cell proliferation assay. The specific method is as follows: Female BALB / c mice aged 6-8 weeks were randomly divided into 7 groups, with 35 mice in each group. The grouping and vector design are shown in Table 2. Spleens were collected from three mice in each group at 3, 7, 28, 35, and 42 days post-immunization, and single-cell suspensions were prepared. Under aseptic conditions, the spleens were removed, placed in a 70 μm cell sieve, and ground using a tissue homogenizer. Single-cell suspensions were prepared using RPMI 1640 medium. After washing once with RPMI 1640, 3 mL of erythrocyte lysis buffer was added, and lysis was performed for 3 min. After lysis, 12 mL of PBS was added to terminate lysis. After washing twice with RPMI 1640, the cells were resuspended in RPMI 1640 complete medium, and cell counts were performed. Trypan blue staining was added in 10 μL, and cell viability was assessed. The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 6 100 μL per well in a 96-well plate, i.e., 1 × 10⁶ cells. 5 20 μg Spytag OVA antigen was added to each well for in vitro stimulation of cells. A negative control (cells added only, no stimulant) and a blank control (RPMI 1640 complete medium added only) were included. Cells were cultured at 37°C in a 5% incubator. 1 / 10 volume of CCK-8 solution was added to each well. Incubation was performed for 1-4 hours. Cell proliferation was measured at 450 nm using a microplate reader. The formula for spleen cell proliferation rate is: Cell proliferation rate = (ODg) / (Spleen cell proliferation rate / (Spleen cell proliferation rate / (Spleen cell proliferation rate)) 疫苗 -OD 空白 ) / (OD 阴性 -OD 空白 ) × 100%.

[0099] In this embodiment, the experimental results are as follows: Figure 9 As shown, Figure 9 The results of spleen lymphocyte proliferation at week 5 showed that the PDA 10min group had the highest proliferation level, followed by the secondary immune CNPs group and the Spytag-OVA group. Figure 9 The results of spleen lymphocyte proliferation at week 6 showed that the PDA 30min group had the highest proliferation level, followed by the PDA 10min group, the secondary immune CNPs group, and the Spytag-OVA group. Figure 9The results of spleen lymphocyte proliferation at week 6 showed that the PDA 60min group had the highest proliferation level, followed by the PDA 30min group, the PDA 10min group, the secondary immunization CNPs group, and the Spytag-OVA group. This indicates that the coated microcapsules can effectively induce spleen proliferation and the proliferation will last for a period of time. When the antigen is encountered again, the proliferation response is better than that of other groups, indicating that the coated microcapsule vaccine can induce a rapid immune response when the body encounters the same antigen again.

[0100] Experimental Example 6: Detection of Specific T Cell Activation Levels This study aimed to investigate the effect of a vaccine on specific T cell activation. The specific methods are as follows: Female BALB / c mice aged 6-8 weeks were randomly divided into 7 groups, with 35 mice in each group. The grouping and vector design are shown in Table 2. Spleens were collected from three mice in each group at 3, 7, 28, 35, and 42 days post-immunization, and single-cell suspensions were prepared. Under aseptic conditions, the spleens were removed, placed in a 70 μm cell sieve, and ground using a tissue homogenizer. Single-cell suspensions were prepared using RPMI 1640 medium. After washing once with RPMI 1640, 3 mL of erythrocyte lysis buffer was added, and lysis was performed for 3 min. After lysis, 12 mL of PBS was added to terminate the lysis. Cells were washed twice with RPMI 1640, resuspended in RPMI 1640 complete medium, and cell counts were performed. Trypan blue staining was added in 10 μL, and cell viability was assessed. The cell concentration was adjusted to 5 × 10⁶ cells / mL. 6 100 μL per well in a 24-well plate, i.e., 5 × 10⁶ cells. 5 Cells were added to each well at a concentration of 10 cells per well, with 20 μg of Spytag-OVA antigen added per well for in vitro stimulation. A negative control was included (cells added without stimulant). Cells were incubated at 37°C for 48 h in a 5% incubator, and the cell suspension was collected. The cells were centrifuged (500g, 5 min) to remove the supernatant, washed twice with PBS, and then incubated with BV421 anti-mouse CD3 antibody, APC-anti-mouse CD4 antibody, PerCP anti-mouse CD8a antibody, and PE anti-mouse CD69 antibody on ice in the dark for 30 min. After centrifugation, the cells were resuspended in PBS and washed twice. 210 μL of PBS and 70 μL of 4% paraformaldehyde were added to the cells, and the T cell activation level was detected by flow cytometry.

[0101] In this embodiment, the experimental results are as follows: Figure 10 As shown, Figure 10 A is CD4 + T activation status, Figure 10 B is CD8+ T cell activation was observed, with overall activation levels correlated with time lag. T cell activation significantly increased after microcapsule rupture, especially CD8 activation. + The T-cell activity was significantly better than that of the two-treatment group, indicating that the single-needle time-delay microcapsule achieved and was superior to the T-cell activation and immune effect of the double-needle group.

[0102] Experimental Example 7: Detection of Long-Term Specific Antibody Secretion Levels This trial aims to investigate the long-term maintenance effect of vaccine-induced specific antibodies. The specific methods are as follows: Female BALB / c mice aged 6-8 weeks were randomly divided into 7 groups of 6 mice each. The grouping and vector design are shown in Table 2. After immunization, blood was collected from the fundus venous plexus of the mice, and the mouse serum was separated for testing for 3 months, once a week. The ELISA experimental method was the same as in Example 6.

[0103] In this embodiment, the experimental results are as follows: Figure 11 As shown, antibodies were measured in mice over a total of 12 weeks. Green represents the antibody immune response triggered by the burst antigen, pink represents the antibody immune response triggered by the secondary burst antigen, and blue represents the antibody titer maintenance phase. As shown in the figure, both the Spytag-OVA group and the CNPs group showed significant increases in antibody titer levels at 3 weeks after the second immunization and at 3-4 weeks. The MC group showed overlap between the first and second burst antigen releases, indicating that the microcapsules rupture rapidly and the two antigen stimulation times overlapped. The PDA 10min group also showed a significant increase in antibody titer levels at 3-4 weeks (with a lag of 3 weeks). The PDA 30min group showed a significant increase in antibody titer levels at 4-5 weeks (with a lag of 4 weeks). The PDA 60min group showed a significant increase in antibody titer levels at 5-6 weeks (with a lag of 5 weeks). In the later maintenance phase, with the same antigen dosage as the secondary immunization, the PDA 10min group achieved the same antibody titer level. In the microcapsule groups with longer time lags, the antibody titer levels in the maintenance phase were superior to those in the secondary immunization group. Time-delay microcapsules can not only achieve a dual burst release effect and realize self-enhanced secondary immunity, but also achieve antibody immunity levels superior to secondary immunity in the later maintenance phase.

[0104] Example 8: General Safety Evaluation of Vaccine Vector This study evaluated the safety of the vaccine vector from three dimensions: body weight, body temperature, blood biochemistry, and organ toxicity. The specific methods are as follows: Mouse weight and body temperature monitoring: After immunization, the weight and body temperature of the mice in Example 11 were monitored for three months after immunization, once every 3 days.

[0105] Blood biochemistry tests: 6-8 week old female BALB / c mice were randomly divided into 7 groups of 3 mice each. The grouping and vector design are shown in Table 2. Blood biochemistry tests were performed on the mice 3 months after immunization. The main indicators detected were aspartate aminotransferase (AST), alanine aminotransferase (ALT), aminophosphatase (ALP), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), total protein (TP), and albumin (ALB).

[0106] H&E toxicity assay of major organs: 6-8 week old female BALB / c mice were randomly divided into 7 groups of 3 mice each. The grouping and vector design are shown in Table 2. Eight weeks after immunization, the major organs (heart, liver, spleen, lung, and kidney) of the mice were collected for H&E staining analysis.

[0107] In this embodiment, the experimental results are as follows: Figure 12 As shown, in the monitoring of body weight and body temperature of immunized mice, no abnormal changes occurred in body weight and body temperature after vaccination with this vaccine.

[0108] Blood biochemistry test results as follows Figure 13 As shown, after immunization of mice with this formulation, the levels of AST, ALT, ALP, LDH, BUN, TP, and ALB were all within the normal range. The IgG content was largely consistent with the antibody titer results. There was no significant difference in IgG content between the Spytag-OVA and CNPs secondary immunization groups and the PDA 10-min group, which were released at the same time interval. The PDA 60-min group, with the longest release interval, had the highest IgG content, indicating that antibody content is related to the time lag interval. H&E analysis showed that immunization did not cause toxic reactions in major organs such as the heart, liver, spleen, lungs, and kidneys. In conclusion, this dual-release microcapsule vaccine delivery vector has good immunization efficacy and safety.

[0109] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A dual burst-release pulse-release vaccine, characterized in that, The vaccine is a polydopamine-coated complex coagulated microcapsule, with antigens loaded both inside and outside the complex coagulated microcapsule.

2. The dual burst-release pulse-release vaccine according to claim 1, characterized in that, The antigen is selected from Spytag-OVA antigen, chicken ovalbumin antigen, recombinant antigen, novel coronavirus antigen, respiratory syncytial virus antigen, influenza virus antigen, or hepatitis B virus antigen; the nucleotide sequence of the Spytag-OVA antigen is shown in SEQ ID NO.

1.

3. The dual burst-release pulse-release vaccine according to claim 1, characterized in that, It also includes adjuvants selected from aluminum adjuvants or small molecule adjuvants; the aluminum adjuvants are selected from aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum; the small molecule adjuvants are selected from CpG, Toll-like receptor agonists or squalene.

4. The dual burst-release pulse-release vaccine according to claim 1, characterized in that, The particle size of the complex condensed microcapsules is 10-150 μm, and the particle size of the nanoparticles is 20-500 nm.

5. A method for preparing a dual burst-release pulsed-release vaccine as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Nanoparticles were prepared by nanoprecipitation method; (2) The nanoparticles are co-encapsulated with the cell membrane to obtain functionalized nanoparticles; (3) Using microfluidic technology, the functionalized nanoparticles and adjuvants are encapsulated by complex condensation method to prepare complex condensation microcapsules; (4) A polydopamine coating is applied to the surface of the complex coagulated microcapsules, and then the coating is blended with the functionalized nanoparticles to obtain the dual burst release pulse release vaccine.

6. The preparation method according to claim 5, characterized in that, In step (1), the nanoprecipitation method uses an organic solvent to dissolve the polymer to prepare the oil phase and a surfactant to prepare the solution as the aqueous phase. The polymer is selected from polylactic acid, polyanhydride, lactide-glycolic acid copolymer, or polycaprolactone; the organic solvent is selected from dichloromethane, ethyl acetate, chloroform, acetone, diethyl ether, or ethanol; the surfactant is selected from polyethylene glycol octylphenyl ether, Tween, polyoxyethylene, polyvinyl alcohol, Span, poloxamer, polyethylene glycol, or sodium dodecyl sulfate.

7. The preparation method according to claim 5, characterized in that, In step (3), the three-phase gas pressure of the microfluidic technology is 10-2000 MPa; the inner phase of the complex coagulated microcapsule is an antigen and adjuvant solution, the intermediate phase is a capsule material solution, the capsule material is selected from gelatin, gum arabic or sodium carboxymethyl cellulose, and the outer phase is fluorinated oil with added surfactant.

8. The preparation method according to claim 5, characterized in that, In step (4), the polydopamine coating is carried out in a neutral buffer solution with pH 6-8 for a coating time of 1-240 min; the neutral buffer solution is selected from PBS, sodium citrate aqueous solution or sodium dihydrogen phosphate aqueous solution.

9. The use of a dual burst-release pulse-release vaccine as described in any one of claims 1-4 in the preparation of a preventive or therapeutic vaccine.

10. The application according to claim 9, characterized in that, The preventive or therapeutic vaccine is used to prevent or treat novel coronavirus infection, respiratory syncytial virus infection, influenza, or hepatitis B; the vaccine is administered via intramuscular injection, subcutaneous injection, or pulmonary administration.

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