WO2022127236A1 - 蛋白质类抗原的单次注射疫苗及其制备方法 - Google Patents

蛋白质类抗原的单次注射疫苗及其制备方法 Download PDF

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WO2022127236A1
WO2022127236A1 PCT/CN2021/119029 CN2021119029W WO2022127236A1 WO 2022127236 A1 WO2022127236 A1 WO 2022127236A1 CN 2021119029 W CN2021119029 W CN 2021119029W WO 2022127236 A1 WO2022127236 A1 WO 2022127236A1
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antigen
protein
single injection
carrier
injection vaccine
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张拥军
王皓正
关英
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南开大学
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    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
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    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
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    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
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    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32311—Enterovirus
    • C12N2770/32334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a single injection vaccine of protein antigens and a preparation method thereof.
  • Subunit vaccines based on protein antigens are widely used in vaccine immunization programs because of their high safety.
  • vaccine adjuvants and multiple immunizations are required. Get the desired immune effect.
  • vaccine-preventable infectious diseases still claim the lives of approximately 1.5 million children each year due to inadequate vaccine distribution and management.
  • a significant number of these children received only one dose of the vaccine due to limited access to health care or other socioeconomic factors.
  • a single bolus administration is often insufficient to ensure strong and long-lasting immunity.
  • researchers have worked to create delivery systems that enable pulsed release of antigens, providing full immunity with just a single injection.
  • Degradable polymer microspheres encapsulated with protein antigens can achieve slow release of antigens, which is mainly due to the different degradation rates of microspheres of different sizes.
  • this immunization method cannot give a specific antigen release time, due to high
  • the degradation mechanism of molecular polymers is very complex and is affected by a variety of factors, so it is impossible to achieve immunity according to the vaccination plan.
  • the use of organic solvents in the preparation process will adversely affect protein antigens. More importantly, the immunization effect of the pulsed antigen release mode is better than that of the slow release antigen delivery mode.
  • the patent (application number KR: 20160160247) encapsulates inactivated microbial cells in polylactic-co-glycolic acid (PLGA) microspheres to compensate for the shortcomings of the existing formalin microbial inactivated vaccines.
  • the vaccine composition of the invention has significantly improved immunity and duration of immunity, and can more effectively improve immunity of individuals to whom the vaccine is administered.
  • This scheme is suitable for enhancing the effect of antigen immunity, but it cannot achieve complete immunity with a single injection of the vaccine. Therefore, it is still a difficult problem to develop a single-injection vaccine that simultaneously meets the requirements of simple preparation, precise pulse release, and no safety risks.
  • the purpose of the present invention is to provide a single-injection vaccine of protein antigens and a preparation method thereof, which can achieve ideal immune effect after a single injection.
  • the invention provides a single injection vaccine for protein antigens, which is composed of protein antigen carriers coated with tannic acid-high molecular polymer erodible coatings of different thicknesses on the surface.
  • the erosion coating dissociates at a constant rate and releases protein antigens in the form of multiple pulses, achieving the immune effect of multiple injections of conventional vaccines in a single injection.
  • the protein antigens include ovalbumin (OVA), novel coronavirus spike protein (His-Tag), hepatitis B surface antigen (HBsAg), human diphtheria antigen (Diphtheria-Ag), tetanus toxoid, spinal cord Poliovirus antigen.
  • OVA ovalbumin
  • His-Tag novel coronavirus spike protein
  • HBsAg hepatitis B surface antigen
  • human diphtheria antigen Diphtheria-Ag
  • tetanus toxoid spinal cord Poliovirus antigen.
  • the material of the carrier includes one or more of calcium carbonate, mesoporous silica, polymethyl methacrylate, polylactic acid and polylactic acid-glycolic acid copolymer.
  • the high molecular polymer comprises polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyethylene oxide-polypropylene oxide-polyethylene oxide copolymer (PEO-PPO-PEO ), one or more of hydroxypropyl methyl cellulose (HPMC) and hydroxymethyl cellulose (CMC).
  • PEG polyethylene glycol
  • PVP polyvinylpyrrolidone
  • PEO-PPO-PEO polyethylene oxide-polypropylene oxide-polyethylene oxide copolymer
  • HPMC hydroxypropyl methyl cellulose
  • CMC hydroxymethyl cellulose
  • the present invention also provides a method for preparing a single injection vaccine of the protein antigen described in the above technical solution, comprising the following steps:
  • step 2) Alternately soak the antigen carrier obtained in step 1) in the solution of tannic acid and high molecular polymer, so that the surface of the antigen carrier forms a tannic acid-high molecular polymer erodible coating, which is controlled by controlling the number of soaks. Erosable coating thickness.
  • step 2) Mixing the antigen carriers of different thicknesses obtained in step 2) can erode the coating-coated antigen carriers to obtain a single-shot vaccine of protein antigens.
  • the tannic acid-polymer erodible coating can dissociate at a constant rate after subcutaneous injection.
  • the pH of the solution of tannic acid and high molecular polymer is ⁇ 6.
  • the solution concentration of the tannic acid and the high molecular polymer should be 10 ⁇ g/mL-1 g/mL.
  • the soaking temperature is 1-37° C.
  • the single soaking time is 1 min to 60 min.
  • the alternating soaking times are 1-200 times.
  • the term “comprising” means including the stated elements, integers or steps, but not excluding any other elements, integers or steps.
  • the term “comprising” is used, unless otherwise indicated, it also encompasses the situation consisting of the stated elements, integers or steps.
  • antibody is intended to refer to immunoglobulin molecules having "variable region” antigen recognition sites.
  • antigen-binding fragment of an antibody refers to one or more portions of an antibody that contain antibody complementarity determining regions ("CDRs") and optionally include antibody “variable regions” antigen recognition sites framework residues and exhibit the ability to immunospecifically bind to antigens.
  • CDRs complementarity determining regions
  • Such fragments include Fab', F(ab') 2 , Fv, single chain (ScFv) and mutants thereof, naturally occurring variants, and include antibody "variable region” antigen recognition sites and heterologous proteins (eg, , toxins, antigen recognition sites of different antigens, enzymes, receptors or receptor ligands, etc.) fusion proteins.
  • the tannic acid-polymer erodible coatings with different thicknesses dissociate at a constant rate, so that the encapsulated protein antigens are dissociated at different time points. Achieve precise pulse release.
  • the immune effect produced is equivalent to that of multiple injections of the antigen carrier vaccine.
  • the single injection of the protein antigen vaccine provided by the present invention can achieve the immune effect of multiple injections of the antigen carrier vaccine by one injection, and improve the compliance of the people who need to be immunized.
  • the preparation method of the single injection vaccine provided by the present invention does not use an organic solvent, thereby avoiding the denaturation and inactivation of protein drugs.
  • the immunization effect is obviously better than the immunization mode of the common subcutaneous injection of the antigen.
  • Fig. 1 is a schematic diagram of a single injection vaccine of protein antigen prepared by the present invention.
  • a in Figure 1 is an acidic environment where tannic acid interacts with high molecular polymers through hydrogen bonds to form an erodible coating on the surface of the protein antigen carrier;
  • B in Figure 1 is a single shot of protein antigen After the vaccine is injected subcutaneously, the erodible coatings of different thicknesses are completely dissociated at different time points at a constant rate, so that the protein antigens can be accurately released at the corresponding time points, achieving multiple pulse release.
  • Figure 2 is a protein antigen carrier and a protein antigen whose surface is coated with an erodible coating ("tannic acid/polyethylene oxide-polypropylene oxide-polyethylene oxide copolymer" erodible coating)
  • an erodible coating tantadium oxide-polypropylene oxide-polyethylene oxide copolymer
  • a in Figure 2 is the surface morphology of the protein antigen carrier after 5000 times magnification
  • B in Figure 2 is the surface morphology of the protein antigen carrier after 15000 times magnification
  • C in Figure 2 is coated with an erodible coating
  • D in Figure 2 is the surface morphology of the protein antigen carrier coated with the erodible coating after 15000 times magnification.
  • Figure 3 Cumulative release curve of the antigen ovalbumin released in vitro from a single-injection vaccine using ovalbumin as an antigen model.
  • Figure 4 Comparison of changes in serum antibody concentration of mice after subcutaneous injection of single-injection vaccine and multiple-injection antigen carrier vaccine using ovalbumin as an antigen model into C57/BL/6 mice. A single-injection vaccine without protein antigen and a vaccine without adjuvant antigen were used as controls.
  • FIG. 1 Comparison of changes in serum antibody concentrations in mice after subcutaneous injection of single-injection vaccine and multiple-injection antigen vector vaccine of novel coronavirus into C57/BL/6 mice. A single-injection vaccine without protein antigen and a vaccine without adjuvant antigen were used as controls.
  • the invention provides a single injection vaccine for protein antigens, which is composed of protein antigen carriers coated with tannic acid-high molecular polymer erodible coatings of different thicknesses on the surface, and is characterized in that the surface erodible coatings It dissociates at a constant speed and releases protein antigens in the form of multiple pulses, so that a single injection can achieve the immune effect of multiple injections of conventional vaccines.
  • the protein antigens include ovalbumin (OVA), novel coronavirus spike protein (His-Tag), hepatitis B surface antigen (HBsAg), human diphtheria antigen (Diphtheria-Ag), tetanus toxoid , Poliovirus antigens.
  • OVA ovalbumin
  • His-Tag novel coronavirus spike protein
  • HBsAg hepatitis B surface antigen
  • HBsAg hepatitis B surface antigen
  • human diphtheria antigen Diphtheria-Ag
  • tetanus toxoid Poliovirus antigens.
  • the material of the carrier includes one or more of calcium carbonate, mesoporous silica, polymethyl methacrylate, polylactic acid and polylactic acid-glycolic acid copolymer.
  • the high molecular polymer includes polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), polyethylene oxide-polypropylene oxide-polyethylene oxide copolymer (PEO-PPO- One or more of PEO), hydroxypropyl methyl cellulose (HPMC) and hydroxymethyl cellulose (CMC).
  • PEG polyethylene glycol
  • PVP polyvinylpyrrolidone
  • PEO-PPO- One or more of PEO polyethylene oxide-polypropylene oxide-polyethylene oxide copolymer
  • HPMC hydroxypropyl methyl cellulose
  • CMC hydroxymethyl cellulose
  • the present invention also provides a method for preparing a single-injection vaccine for protein antigens according to the above technical solution, comprising: 1) encapsulating the protein antigens into a carrier to obtain an antigen carrier; 2) using the antigen carrier obtained in step 1). Alternate immersion in the solution of tannic acid and high molecular polymer forms a tannic acid-high molecular polymer erodible coating on the surface of the antigen carrier, and the thickness of the erodible coating is adjusted by controlling the number of immersion times. 3) The antigen carriers with different thicknesses obtained in the mixing step 2) can erode the coating-coated antigen carrier, and a single injection vaccine of protein antigens can be obtained.
  • the solution of the tannic acid and the high molecular polymer is preferably pH ⁇ 6; the tannic acid-high molecular polymer erodible coating is preferably injected subcutaneously, and can dissociate at a constant rate; the The solution concentration of protein drugs and macromolecular polymers is preferably 10 ⁇ g/mL-1g/mL; the soaking temperature is preferably 1-37°C; the single soaking time is preferably 1min-60min; the alternating soaking times is preferably 1-200 times.
  • the calcium carbonate carrier loaded with ovalbumin was soaked alternately in tannic acid and polyethylene glycol solution, each soaking time was 1 min, and one cycle was one layer.
  • the control system temperature was 37°C. Repeat the cycle 20 or 40 times to obtain antigen carriers coated with erodible coatings of different thicknesses, mix with antigen carriers without erodible coatings, and dry to obtain a single-injection vaccine of ovalbumin antigen.
  • the specific preparation steps are as follows: 9.8 g of cetyltrimethylammonium bromide and 5 g of sodium silicate are dissolved in 175 mL of ultrapure water, mixed and stirred, 17.5 mL of ethyl acetate is added, and the mixture is allowed to stand at room temperature for 5 hours. After 48h aging in a 90°C water bath, the product was collected by centrifugation. Finally, the product was calcined at 550 °C for 10 h in an air atmosphere to obtain mesoporous silica spheres.
  • the mesoporous silica sphere carrier loaded with the novel coronavirus characteristic protein was soaked alternately in the above two solutions, each soaking time was 15 min, and one cycle was one layer.
  • the temperature of the control system is 25°C. Repeat the cycle 10 or 20 times to obtain antigen carriers coated with erodible coatings of different thicknesses, mix with antigen carriers without erodible coatings, and dry to obtain a single injection vaccine of novel coronavirus antigen.
  • the hepatitis B surface antigen is dissolved into a 1% concentration of polyvinyl alcohol aqueous solution as an aqueous phase, and the antigen concentration is 0.5 mg/mL.
  • the polylactic acid was dissolved in dichloromethane to obtain a solution with a concentration of 8% as the oil phase.
  • 10 mL of the oil phase was added dropwise to 100 mL of the water phase while stirring to form an emulsion.
  • the emulsion was introduced into a membrane emulsifier and passed through a membrane to obtain the final emulsion.
  • the polylactic acid microsphere carrier loaded with hepatitis B surface antigen was soaked alternately in the two solutions, each soaking time was 30min, and one cycle was one layer.
  • the temperature of the control system is 5°C. Repeat the cycle 5 or 10 times to obtain antigen carriers coated with erodible coatings of different thicknesses, mix with antigen carriers without erodible coatings, and dry to obtain a single injection vaccine of hepatitis B surface antigen.
  • the specific preparation steps are: dissolving human diphtheria antigen protein into calcium chloride solution, the concentration is 1 mg/mL, adding an equal volume of sodium carbonate solution, stirring to form a calcium carbonate carrier loaded with human diphtheria antigen protein, and centrifuging to collect human diphtheria antigen protein carrier .
  • the calcium carbonate carrier loaded with human diphtheria antigen was soaked alternately in the two solutions, each soaking time was 8 min, and one cycle was one layer.
  • the temperature of the control system is 10°C. Repeat the cycle for 15 or 30 times to obtain antigen carriers coated with erodible coatings of different thicknesses, mix with antigen carriers without erodible coatings, and dry to obtain a single injection vaccine of human diphtheria antigen.
  • the calcium carbonate carrier loaded with the poliovirus antigen protein was soaked alternately in the two solutions, each soaking time was 20 min, and one cycle was one layer.
  • the temperature of the control system is 15°C. Repeat the cycle 10 or 20 times to obtain antigen carriers coated with erodible coatings of different thicknesses, mix with antigen carriers without erodible coatings, and dry to obtain a single-injection vaccine of poliovirus antigen.
  • Example 6 Morphological characterization of a single injection vaccine using ovalbumin as an antigen model
  • Example 2 Using the same method as Example 1, a single injection vaccine loaded with ovalbumin as an antigen model was prepared.
  • the antigen vaccine particles were dried and sprayed with gold for scanning electron microscope observation.
  • the surface morphology of the vaccine particles was shown in Figure 2.
  • the surface of the particles coated with the erodible coating appeared in the form of a film, indicating that the erodible coating had been successfully coated on the surface.
  • the surface of the ovalbumin antigen carrier The surface of the ovalbumin antigen carrier.
  • Example 7 In vitro release of antigens from a single injection of a new coronavirus vaccine
  • the new coronavirus vaccine prepared by the same method as Example 2 was soaked in phosphate buffer (pH 7.4) for a specified time, the content of the new coronavirus antigen in the release medium was detected by high performance liquid chromatography.
  • the cumulative release of 2019-nCoV antigens is shown in Figure 3. At a fixed time point, the antigen can be pulsed from the carrier for multiple times, simulating the time point of the vaccination plan.
  • Example 8 In vivo immune protection assessment of a single injection vaccine using ovalbumin as an antigen model
  • Example 2 Using the same method as Example 1, a single injection vaccine of ovalbumin was prepared. Using C57/BL/6 mice as an animal model to evaluate the immune effect of a single injection of the vaccine with ovalbumin as an antigen model. Twenty-four mice were randomly divided into four groups for the following immunization schedules:
  • the experimental group was a single injection of the vaccine with ovalbumin as the antigen model, subcutaneously injected only once on the 0th day, abbreviated as OVA@CaCO 3 /(TA/PEG) n .
  • the blank group is a single-injection vaccine without antigenic ovalbumin. Its preparation method is basically the same as that in Example 1. The only difference is that it is not loaded with ovalbumin. 3 /(TA/PEG) n .
  • Control group 1 is a carrier vaccine with ovalbumin as an antigen model.
  • the preparation method is basically the same as that of Example 1. The only difference is that there is no erodible coating on the surface of the antigen carrier.
  • the control group 2 is an antigen vaccine of ovalbumin, obtained by preparing a physiological saline solution of ovalbumin, and the concentration of the antigen protein is 0.5 mg/ml. It was injected subcutaneously three times on the 0th day, 14th day and 28th day, respectively. is OVA*3.
  • the total amount of antigen immunization was 30 ⁇ g/mouse.
  • 70 ⁇ L of orbital venous blood of mice was collected every week. After the blood samples were centrifuged at 3000 rpm for 10 min, the upper serum samples were taken, and the concentration of antigen and antibody in the serum was detected by enzyme-linked immunosorbent assay.
  • the changes in serum antibody concentration of mice after vaccination are shown in Figure 4.
  • the antibody content of a single subcutaneous injection of ovalbumin vaccine was not significantly different from that of three subcutaneous injections of ovalbumin carrier vaccine, indicating that a single injection
  • the immune effect of the vaccine is comparable to that of multiple injections of antigen carrier vaccines, which reduces the number of injections and improves the compliance of the population.
  • the antibody content of single injection vaccine is much higher than that of antigen vaccine, indicating that the immune effect of single injection vaccine is significantly better than that of ordinary subcutaneous injection of antigen vaccine.
  • Example 9 In vivo immune protection assessment of a single injection of novel coronavirus vaccine
  • Example 2 Using the same method as Example 2, a single injection vaccine of the novel coronavirus was prepared. The same method as Example 8 was used to evaluate the immune protection of a single injection of the new coronavirus vaccine. Four immunization schedules are implemented:
  • the experimental group was a single injection of the new coronavirus vaccine, which was subcutaneously injected only once on day 0, abbreviated as His-tag@SiO 2 /(TA/PEG) n .
  • the blank group is a single injection vaccine without loading the novel coronavirus antigen, and its preparation method is basically the same as that in Example 2. The only difference is that it is not loaded with the novel coronavirus antigen protein, and it is only injected subcutaneously once on day 0, referred to as is His-tag@SiO 2 /(TA/PEG) n .
  • the preparation method of the vector vaccine for the novel coronavirus in control group 1 is basically the same as that in Example 2, the only difference is that there is no erodible coating on the surface of the antigen carrier. is His-tag@SiO 2 *3.
  • the control group 2 is the antigen vaccine of the new coronavirus, obtained by preparing the physiological saline solution of the antigen protein of the new coronavirus, and the concentration of the antigen protein is 1 mg/mL. It was injected subcutaneously three times on the 0th, 14th, and 28th days, respectively. It is His-tag*3.

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Abstract

本发明提供了一种蛋白质类抗原的单次注射疫苗及其制备方法，该疫苗由表面涂覆有不同厚度单宁酸-高分子聚合物可侵蚀涂层的蛋白质类抗原载体组成，其特征在于，蛋白质类抗原载体表面的可侵蚀涂层以恒速解离，通过多次脉冲形式释放蛋白质类抗原，实现单次注射达到常规疫苗多次注射的免疫效果。

Description

蛋白质类抗原的单次注射疫苗及其制备方法
优先权和相关申请
本申请要求2020年12月16日提交的名称为“蛋白质类抗原的单次注射疫苗及其制备方法”的中国专利申请202011489968.7的优先权，该申请包括附录在内的全部内容作为参考并入本申请。
技术领域
本发明涉及生物医药技术领域，具体涉及蛋白质类抗原的单次注射疫苗及其制备方法。
背景技术
基于蛋白质类抗原的亚单位疫苗因其安全性高而被广泛应用到疫苗免疫接种计划中，但是因为蛋白质类抗原低免疫原性和半衰期短的特点，需要疫苗佐剂和多次免疫注射才可以获得理想的免疫效果。尽管在过去的四十年中，全球疫苗覆盖率大幅增长，但是由于疫苗分配和管理不充分，可疫苗预防的传染病仍然每年夺走约150万儿童的生命。在这些儿童中，有相当数量的人由于有限的医疗保健途径或其他社会经济因素只接受了一剂疫苗。不幸的是，单次推注给药通常不足以确保强大而持久的免疫力。在过去的35年中，研究人员致力于创建能够实现抗原脉冲释放的递送系统，仅需一次注射即可提供完全免疫力。
封装有蛋白质类抗原的可降解聚合物微球可以实现抗原的缓慢释放，这主要归因于不同尺寸的微球降解速度的不同，然而这种免疫方法无法给出具体的抗原释放时间，由于高分子聚合物的降解机理非常复杂，受多种因素的综合影响，因此无法准确按照接种计划来实现免疫。同时，制备过程中使用了有机溶剂会对蛋白质类抗原产生不良影响。更为重要的是，脉冲式抗原释放模式的免疫效果要好于缓慢释放抗原的递送模式。专利(申请号KR：20160160247)将灭活微生物细胞封装在聚乳酸-羟基乙酸共聚物(PLGA)微球中，以补偿现有福尔马林微生物灭活疫苗的缺点。与现有的福尔马林微生物灭活疫苗相比，该发明的疫苗组合物具有显着改善的免疫能力和免疫持续时 间，并且可以更有效地改善对其施用疫苗的个体的免疫力。该方案适用于抗原免疫效果的增强，却无法实现疫苗单次注射即可获得完全免疫力。因此，开发出一种同时满足制备简单、精确脉冲释放、无安全风险等要求的单次注射疫苗依然是一个需要解决的难题。
发明内容
有鉴于此，本发明的目的在于提供蛋白质类抗原的单次注射疫苗及其制备方法，能够实现单次注射即获得理想免疫效果。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了蛋白质类抗原的单次注射疫苗，由表面涂覆有不同厚度单宁酸-高分子聚合物可侵蚀涂层的蛋白质类抗原载体组成，其特征在于，蛋白质类抗原载体表面的可侵蚀涂层以恒速解离，通过多次脉冲形式释放蛋白质类抗原，实现单次注射达到常规疫苗多次注射的免疫效果。
优选地，所述蛋白质类抗原包括卵清蛋白(OVA)、新型冠状病毒棘突蛋白(His-Tag)、乙肝表面抗原(HBsAg)、人白喉抗原(Diphtheria-Ag)、破伤风类毒素、脊髓灰质炎病毒抗原。
优选地，所述载体的材料包括碳酸钙、介孔二氧化硅、聚甲基丙烯酸甲酯、聚乳酸和聚乳酸-羟基乙酸共聚物的一种或几种。
优选地，所述高分子聚合物包括聚聚乙二醇(PEG)、聚乙烯吡咯烷酮(PVP)、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷共聚物(PEO-PPO-PEO)、羟丙基甲基纤维素(HPMC)和羟甲基纤维素(CMC)的一种或几种。
本发明还提供了上述技术方案所述蛋白质类抗原的单次注射疫苗的制备方法，包括以下步骤：
1)将蛋白质类抗原封装到载体中，获得抗原载体；
2)将步骤1)中获得的抗原载体在单宁酸和高分子聚合物的溶液中交替浸泡，使抗原载体表面形成单宁酸-高分子聚合物可侵蚀涂层，通过控制浸泡次数来控制可侵蚀涂层厚度。
3)混合步骤2)获得的不同厚度可侵蚀涂层涂覆的抗原载体，获得蛋白质类抗原的单次注射疫苗。
所述单宁酸-高分子聚合物可侵蚀涂层注射到皮下后，可以恒定速率解 离。
优选地，所述单宁酸与高分子聚合物溶液的pH＜6。
优选地，所述单宁酸与高分子聚合物的溶液浓度应为10μg/mL-1g/mL。
优选地，所述浸泡的温度为1-37℃，单次浸泡时间为1min～60min。
优选地，所述交替浸泡次数为1-200次。
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
如本文中所用，术语“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。
如本文中所用，属于“抗体”旨在表示具有“可变区”抗原识别位点的免疫球蛋白分子。术语抗体的“抗原结合片段”是指抗体的一个或多个部分，所述一个或多个部分含有抗体互补决定区(“CDR”)和任选地包括抗体“可变区”抗原识别位点的构架残基，并且表现出免疫特异性结合抗原的能力。此类片段包括Fab′、F(ab′) 2、Fv、单链(ScFv)及其突变体、天然存在的变体，及包括抗体“可变区”抗原识别位点和异源蛋白(例如，毒素、不同抗原的抗原识别位点、酶、受体或受体配体等)的融合蛋白。
以下将详细说明本发明的各种示例性实施例、特征和方面。在这里专用的词“示例性”意为“用作例子、实施例或说明性”。这里作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
另外，为了更好地说明本发明，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本发明同样可以实施。在另外一些实例中，对于本领域技术人员熟知的方法、手段、器材和步骤未作详细描述，以便于凸显本发明的主旨。
如无特殊声明，本说明书中所使用的单位均为国际标准单位，并且本发明中出现的数值、数值范围，均应当理解为包含了工业生产中所不可避免的 系统性误差。
本发明具有如下的有益效果：
本发明提供的蛋白质类抗原的单次注射疫苗注射到皮下后，由于不同厚度单宁酸-高分子聚合物可侵蚀涂层均以恒速解离，使被封装的蛋白质类抗原在不同时间点实现精准脉冲释放。
本发明提供的蛋白质类抗原的单次注射疫苗注射到皮下后，产生的免疫效果与多次注射抗原载体疫苗相当。
本发明提供的蛋白质类抗原的单次注射疫苗，注射一次，即可达到抗原载体疫苗多次注射的免疫效果，改善了需要免疫人群的依从性。
本发明提供的单次注射疫苗的制备方法，没有使用有机溶剂，避免了蛋白质类药物的变性和失活。
利用本发明制备的蛋白质类抗原的单次注射疫苗皮下注射免疫后，免疫效果明显优于普通皮下注射抗原的免疫方式。
附图说明
图1为本发明制备的蛋白质类抗原的单次注射疫苗的示意图。图1中的A是在酸性环境下，单宁酸与高分子聚合物通过氢键相互作用，在蛋白质类抗原载体表面上形成可侵蚀涂层；图1中的B是蛋白质类抗原的单次注射疫苗注射到皮下后，不同厚度的可侵蚀涂层以恒定速率在不同时间点完全解离，使蛋白质类抗原在对应时间点准确释放，实现多次脉冲释放。
图2为蛋白质类抗原载体与表面涂覆有可侵蚀涂层(“单宁酸/聚环氧乙烷-聚环氧丙烷-聚环氧乙烷共聚物”可侵蚀涂层)的蛋白质类抗原载体在扫描电镜观察下的形态差异。图2中的A是蛋白质类抗原载体5000倍放大后的表面形态；图2中的B是蛋白质类抗原载体15000倍放大后的表面形态；图2中的C是涂覆有可侵蚀涂层的蛋白质类抗原载体5000倍放大后的表面形态；图2中的D是涂覆有可侵蚀涂层的蛋白质类抗原载体15000倍放大后的表面形态。
图3.以卵清蛋白为抗原模型的单次注射疫苗体外释放抗原卵清蛋白的累计释放曲线。
图4.以卵清蛋白为抗原模型的单次注射疫苗和多次注射抗原载体疫苗皮下注射到C57/BL/6小鼠体内后，小鼠血清抗体浓度的变化曲线对比图。以 未负载蛋白质类抗原的单次注射疫苗和无佐剂抗原疫苗作为对照。
图5.新型冠状病毒的单次注射疫苗和多次注射抗原载体疫苗皮下注射到C57/BL/6小鼠体内后，小鼠血清抗体浓度的变化曲线对比图。以未负载蛋白质类抗原的单次注射疫苗和无佐剂抗原疫苗作为对照。
具体实施方式
本发明提供了一种蛋白质类抗原的单次注射疫苗，由表面涂覆有不同厚度单宁酸-高分子聚合物可侵蚀涂层的蛋白质类抗原载体组成，其特征在于，表面可侵蚀涂层以恒速解离，通过多次脉冲形式释放蛋白质类抗原，实现单次注射达到常规疫苗多次注射的免疫效果。
在本发明中，所述蛋白质类抗原包括卵清蛋白(OVA)、新型冠状病毒棘突蛋白(His-Tag)、乙肝表面抗原(HBsAg)、人白喉抗原(Diphtheria-Ag)、破伤风类毒素、脊髓灰质炎病毒抗原。
在本发明中，所述载体的材料包括碳酸钙、介孔二氧化硅、聚甲基丙烯酸甲酯、聚乳酸和聚乳酸-羟基乙酸共聚物的一种或几种。
在本发明中，所述高分子聚合物包括聚乙二醇(PEG)、聚乙烯吡咯烷酮(PVP)、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷共聚物(PEO-PPO-PEO)、羟丙基甲基纤维素(HPMC)和羟甲基纤维素(CMC)的一种或几种。
本发明还提供了上述技术方案所述蛋白质类抗原的单次注射疫苗的制备方法，包括：1)将蛋白质类抗原封装到载体中，获得抗原载体；2)将步骤1)中获得的抗原载体在单宁酸和高分子聚合物的溶液交替浸泡，使抗原载体表面形成单宁酸-高分子聚合物可侵蚀涂层，通过控制浸泡次数调节可侵蚀涂层厚度。3)混合步骤2)获得的不同厚度可侵蚀涂层涂覆的抗原载体，即可获得蛋白质类抗原的单次注射疫苗。
在本发明中，所述单宁酸与高分子聚合物的溶液优选pH＜6；所述单宁酸-高分子聚合物可侵蚀涂层优选注射到皮下后，可以恒定速率解离；所述蛋白质类药物与高分子聚合物的溶液浓度优选为10μg/mL-1g/mL；所述浸泡的温度优选为1～37℃；单次浸泡时间优选为1min～60min；所述交替浸泡次数优选为1-200次。
下面将结合实施例对本发明的实施方案进行详细说明。以下实施例将有 助于本领域的技术人员进一步理解本发明，但不应视为限定本发明的范围。
实施例1 制备以卵清蛋白为抗原模型的单次注射疫苗
具体制备步骤为：将卵清蛋白溶解到氯化钙溶液中，浓度为1mg/mL，加入等体积碳酸钠溶液，搅拌形成负载有卵清蛋白的碳酸钙载体，离心收集蛋白质类抗原载体。将单宁酸与聚乙二醇分别溶于磷酸盐缓冲液中(pH＝6)，浓度均为1mg/mL。将负载有卵清蛋白的碳酸钙载体在单宁酸和聚乙二醇溶液中交替浸泡，每次浸泡时间为1min，一个循环为一层。控制系统温度为37℃。重复循环20或40次得到涂覆有不同厚度可侵蚀涂层的抗原载体，与没有可侵蚀涂层的抗原载体混合，干燥即得卵清蛋白抗原的单次注射疫苗。
实施例2 制备新型冠状病毒的单次注射疫苗
具体制备步骤为：将9.8g十六烷基三甲基溴化铵和5g硅酸钠溶于175mL超纯水中混合搅拌，加入17.5mL的乙酸乙酯，在室温下静置5h。经过90℃水浴中48h老化，离心收集产物。最后，将产物在空气气氛中550℃煅烧10h得到介孔二氧化硅球。将新型冠状病毒棘突蛋白(His-Tag)溶于磷酸缓冲液中(pH＝5)，浓度为2mg/mL，然后加入8mg介孔二氧化硅球，超声处理至分散，离心分离得到负载有新型冠状病毒特征蛋白的介孔二氧化硅球载体。将单宁酸和聚环氧乙烷-局环氧丙烷-聚环氧乙烷共聚物(PEO-PPO-PEO)分别溶于盐酸溶液中(pH＝3)，浓度均为10mg/mL。将负载有新型冠状病毒特征蛋白的介孔二氧化硅球载体在上述两种溶液中交替浸泡，每次浸泡时间为15min，一个循环为一层。控制系统温度为25℃。重复循环10或20次得到涂覆有不同厚度可侵蚀涂层的抗原载体，与没有可侵蚀涂层的抗原载体混合，干燥即得新型冠状病毒抗原的单次注射疫苗。
实施例3 制备乙肝表面抗原的单次注射疫苗
具体制备步骤为：将乙肝表面抗原溶解到1％浓度的聚乙烯醇水溶液中作为水相，抗原浓度为0.5mg/mL。将聚乳酸溶于二氯甲烷中获得浓度为8％的溶液作为油相。将10mL的油相逐滴滴入到100mL水相中，同时进行搅拌形成乳液。将乳液导入膜乳化器中过膜得到最终的乳液。将所制备出的乳液通风固化5小时，离心收集得到负载有乙肝表面抗原的聚乳酸微球载体。将单宁酸和聚乙烯吡咯烷酮分别溶于盐酸溶液中(pH＝2)，浓度均为5mg/mL。将负载有乙肝表面抗原的聚乳酸微球载体在两种溶液中交替浸泡，每次浸泡 时间为30min，一个循环为一层。控制系统温度为5℃。重复循环5或10次得到涂覆有不同厚度可侵蚀涂层的抗原载体，与没有可侵蚀涂层的抗原载体混合，干燥即得乙肝表面抗原的单次注射疫苗。
实施例4 制备人白喉抗原的单次注射疫苗
具体制备步骤为：将人白喉抗原蛋白溶解到氯化钙溶液中，浓度为1mg/mL，加入等体积碳酸钠溶液，搅拌形成负载有人白喉抗原蛋白的碳酸钙载体，离心收集人白喉抗原蛋白载体。将单宁酸与聚乙二醇分别溶于磷酸盐缓冲液中(pH＝5.5)，浓度均为7mg/mL。将负载有人白喉抗原的碳酸钙载体在两种溶液中交替浸泡，每次浸泡时间为8min，一个循环为一层。控制系统温度为10℃。重复循环15或30次得到涂覆有不同厚度可侵蚀涂层的抗原载体，与没有可侵蚀涂层的抗原载体混合，干燥即得人白喉抗原的单次注射疫苗。
实施例5 制备脊髓灰质炎病毒抗原的单次注射疫苗
具体制备步骤为：将脊髓灰质炎病毒抗原蛋白溶解到氯化钙溶液中，浓度为0.5mg/mL，加入等体积碳酸钠溶液，搅拌形成负载有脊髓灰质炎病毒抗原蛋白的碳酸钙载体，离心弃上清收集。将单宁酸与羟甲基纤维素分别溶于磷酸盐缓冲液中(pH＝3.5)，浓度均为8mg/mL。将负载有脊髓灰质炎病毒抗原蛋白的碳酸钙载体在两种溶液中交替浸泡，每次浸泡时间为20min，一个循环为一层。控制系统温度为15℃。重复循环10或20次得到涂覆有不同厚度可侵蚀涂层的抗原载体，与没有可侵蚀涂层的抗原载体混合，干燥即得脊髓灰质炎病毒抗原的单次注射疫苗。
实施例6 以卵清蛋白为抗原模型的单次注射疫苗形态表征
采用实施例1相同的方法，制备负载了以卵清蛋白为抗原模型的单次注射疫苗。将抗原疫苗微粒干燥后喷金进行扫描电镜观察，可见如图2所示的疫苗微粒表面形态，涂覆了可侵蚀涂层的微粒表面出现膜的形态，说明可侵蚀涂层已经成功涂敷在卵清蛋白抗原载体的表面。
实施例7 新型冠状病毒的单次注射疫苗体外释放抗原
新型冠状病毒的单次注射疫苗(采用实施例2相同的方法制备)在磷酸盐缓冲液(pH 7.4)中浸泡指定的时间后，以高效液相色谱检测释放介质中新型冠状病毒抗原的含量。新型冠状病毒抗原的累计释放量如图3所示，在 固定的时间点，抗原可以定时从载体中脉冲释放多次，模拟了疫苗接种计划的时间点。
实施例8 以卵清蛋白为抗原模型的单次注射疫苗体内免疫防护评估
采用实施例1相同的方法，制备了卵清蛋白的单次注射疫苗。使用C57/BL/6小鼠作为动物模型评估以卵清蛋白为抗原模型的单次注射疫苗的免疫效果。24只小鼠随机分为四组，分别进行以下免疫接种计划：
1)实验组为以卵清蛋白为抗原模型的单次注射疫苗，仅在第0天皮下注射一次，简称为OVA@CaCO 3/(TA/PEG) n。
2)空白组为没有负载抗原卵清蛋白的单次注射疫苗，其制备方法与实施例1方法基本相同，唯一不同点是没有负载卵清蛋白，仅在第0天皮下注射一次，简称为CaCO 3/(TA/PEG) n。
3)对照组1为以卵清蛋白为抗原模型的载体疫苗制备方法与实例1方法基本相同，唯一不同点是抗原载体表面没有可侵蚀涂层，分别在第0天，14天，28天皮下注射三次，简称为OVA@CaCO 3*3。
4)对照组2为卵清蛋白的抗原疫苗，通过配制卵清蛋白的生理盐水溶液获得，抗原蛋白浓度为0.5mg/ml，分别在在第0天，14天，28天皮下注射三次，记为OVA*3。
总抗原免疫量为30μg/只，注射免疫以后，每周取小鼠眼眶静脉血～70μL，血样品3000rpm离心10min后，取上层血清样品，以酶联免疫吸附法检测血清中抗原抗体的浓度。接种后小鼠血清抗体浓度变化如图4所示，单次皮下注射卵清蛋白的单次注射疫苗的抗体含量与分三次皮下注射卵清蛋白载体疫苗的抗体含量没有明显差异，说明单次注射疫苗的免疫效果与多次注射抗原载体疫苗相当，减少了注射次数，提高了人群的依从性。而与注射抗原疫苗相比，单次注射疫苗的抗体含量远高于抗原疫苗，说明单次注射疫苗的免疫效果明显优于普通皮下注射抗原疫苗的免疫方式。
实施例9 新型冠状病毒的单次注射疫苗的体内免疫防护评估
采用实施例2相同的方法，制备了新型冠状病毒的单次注射疫苗。采用实施例8相同的方法，对新型冠状病毒单次注射疫苗进行免疫防护评估。四种免疫接种计划被实施：
1)实验组为新型冠状病毒的单次注射疫苗，仅在第0天皮下注射一次， 简称为His-tag@SiO 2/(TA/PEG) n。
2)空白组为没有负载新型冠状病毒抗原的单次注射疫苗，其制备方法与实施例2方法基本相同，唯一不同点是没有负载新型冠状病毒抗原蛋白，仅在第0天皮下注射一次，简称为His-tag@SiO 2/(TA/PEG) n。
3)对照组1为新型冠状病毒的载体疫苗制备方法与实例2方法基本相同，唯一不同点是抗原载体表面没有可侵蚀涂层，分别在第0天，14天，28天皮下注射三次，简称为His-tag@SiO 2*3。
4)对照组2为新冠病毒的抗原疫苗，通过配制新型冠状病毒抗原蛋白的生理盐水溶液获得，抗原蛋白浓度为1mg/mL，分别在在第0天，14天，28天皮下注射三次，记为His-tag*3。
如图5所示，血清中单次皮下注射新型冠状病毒的单次注射疫苗的抗体含量与分三次皮下注射新型冠状病毒载体疫苗的抗体含量没有显著性差异，说明单次注射疫苗的免疫效果与多次注射抗原载体疫苗相当，减少了注射次数，提高了人群的依从性。而与注射抗原疫苗相比，单次注射疫苗的抗体含量远高于抗原疫苗，说明单次注射疫苗的免疫效果明显优于普通皮下注射抗原疫苗的免疫方式。
以上所述仅是本发明的优选实施例方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应该视为本发明的保护范围。

Claims (15)

  1. 一种单次注射疫苗，由表面涂覆有不同厚度单宁酸-高分子聚合物可侵蚀涂层的蛋白质类抗原载体组成，其特征在于，蛋白质类抗原载体表面的可侵蚀涂层以恒速解离，通过多次脉冲形式释放蛋白质类抗原，实现单次注射达到常规疫苗多次注射的免疫效果。
  2. 根据权利要求1所述的单次注射疫苗，其中所述蛋白质类抗原包括卵清蛋白(OVA)、新型冠状病毒棘突蛋白(His-tag)、乙肝表面抗原(HBsAg)、人白喉抗原(Diphtheria-Ag)、破伤风类毒素、脊髓灰质炎病毒抗原。
  3. 根据权利要求1所述的单次注射疫苗，其中所述载体包括碳酸钙、介孔二氧化硅、聚甲基丙烯酸甲酯、聚乳酸和聚乳酸-羟基乙酸共聚物的一种或几种。
  4. 根据权利要求1所述的单次注射疫苗，其中所述高分子聚合物包括聚乙二醇(PEG)、聚乙烯吡咯烷酮(PVP)、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷共聚物(PEO-PPO-PEO)、羟丙基甲基纤维素(HPMC)和羟甲基纤维素(CMC)的一种或几种。
  5. 权利要求1-4任一项所述的单次注射疫苗的制备方法，包括以下步骤：
    1)将蛋白质类抗原封装到载体中，获得抗原载体；
    2)将步骤1)中获得的抗原载体在单宁酸和高分子聚合物的溶液中交替浸泡，使抗原载体表面形成单宁酸-高分子聚合物可侵蚀涂层，通过控制浸泡次数来调节可侵蚀涂层厚度；
    3)混合步骤2)获得的不同厚度可侵蚀涂层涂覆的抗原载体，得到蛋白质类抗原的单次注射疫苗。
  6. 根据权利要求5所述的单次注射疫苗的制备方法，其特征在于，所述单宁酸和高分子聚合物的溶液pH＜6。
  7. 根据权利要求5所述的单次注射疫苗的制备方法，其特征在于，所述单宁酸和高分子聚合物的溶液浓度为10μg/mL-1g/mL。
  8. 根据权利要求5所述的单次注射疫苗的制备方法，其特征在于，所述制备过程的温度为1-37℃。
  9. 根据权利要求5所述的单次注射疫苗的制备方法，其特征在于，单次浸泡时间为1min-60min。
  10. 根根权利要求5所述的单次注射疫苗的制备方法，其特征在于，其中 所述交替浸泡次数为1-200次。
  11. 一种单次注射疫苗，其特征在于，所述单次注射疫苗包括：(1)蛋白质类抗原，(2)负载有蛋白质类抗原的载体，和(3)涂覆在所述负载有蛋白质类抗原的载体表面的、具有不同厚度的单宁酸-高分子聚合物可侵蚀涂层。
  12. 根据权利要求11所述的单次注射疫苗，其中所述蛋白质类抗原选自由以下抗原组成的组中的任一种：卵清蛋白(OVA)、新型冠状病毒棘突蛋白(His-tag)、乙肝表面抗原(HBsAg)、人白喉抗原(Diphtheria-Ag)、破伤风类毒素、脊髓灰质炎病毒抗原。
  13. 根据权利要求11或12所述的单次注射疫苗，其中所述负载有蛋白质类抗原的载体选自由碳酸钙、介孔二氧化硅、聚甲基丙烯酸甲酯、聚乳酸和聚乳酸-羟基乙酸共聚物组成的组中的一种以上。
  14. 根据权利要求11～13任一项所述的单次注射疫苗，其中所述高分子聚合物选自由聚乙二醇(PEG)、聚乙烯吡咯烷酮(PVP)、聚环氧乙烷-聚环氧丙烷-聚环氧乙烷共聚物(PEO-PPO-PEO)、羟丙基甲基纤维素(HPMC)和羟甲基纤维素(CMC)组成的组中的一种以上。
  15. 根据权利要求11～14任一项所述的单次注射疫苗，其中所述单宁酸-高分子聚合物可侵蚀涂层以恒速解离，通过多次脉冲形式释放所述蛋白质类抗原。
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