WO2020207472A1 - 唑来膦酸锌微纳米颗粒佐剂的制备及作为疫苗佐剂的用途 - Google Patents

唑来膦酸锌微纳米颗粒佐剂的制备及作为疫苗佐剂的用途 Download PDF

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WO2020207472A1
WO2020207472A1 PCT/CN2020/084190 CN2020084190W WO2020207472A1 WO 2020207472 A1 WO2020207472 A1 WO 2020207472A1 CN 2020084190 W CN2020084190 W CN 2020084190W WO 2020207472 A1 WO2020207472 A1 WO 2020207472A1
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zinc
adjuvant
zoledronate
solution
vaccine
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PCT/CN2020/084190
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English (en)
French (fr)
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赵勤俭
黄小芬
陈思伊
李宜珂
张彩
夏宁邵
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厦门万泰沧海生物技术有限公司
厦门大学
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Priority to AU2020256646A priority Critical patent/AU2020256646A1/en
Priority to EP20786788.8A priority patent/EP3954386A4/en
Priority to BR112021020377A priority patent/BR112021020377A2/pt
Priority to US17/602,214 priority patent/US20220193231A1/en
Priority to JP2021560015A priority patent/JP2022527606A/ja
Priority to CA3136495A priority patent/CA3136495A1/en
Priority to KR1020217036922A priority patent/KR20220004085A/ko
Publication of WO2020207472A1 publication Critical patent/WO2020207472A1/zh
Priority to ZA2021/08092A priority patent/ZA202108092B/en

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    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A61K39/25—Varicella-zoster virus
    • 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
    • A61K39/12—Viral antigens
    • A61K39/29—Hepatitis virus
    • A61K39/292—Serum hepatitis virus, hepatitis B virus, e.g. Australia antigen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • 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
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16734—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a vaccine adjuvant, in particular to a zinc zoledronate micronanoparticle adjuvant with slow-release effect, which can significantly enhance humoral immunity and cellular immune response, and can be used as a vaccine adjuvant in vaccine preparation , Belongs to the field of immunology.
  • Zinc is an important chemical element with special biological functions. For example, trace levels of zinc are present in about 300 enzymes involved in cell metabolism, and it plays an important role in cell respiration, cell division, protein synthesis, DNA synthesis, damage repair, and immune function activation. Therefore, zinc deficiency or disorder is related to many diseases, especially diseases of the immune system (Autoimmunity Reviews, 2015, 14(04)277-285). In immune cells, divalent cations include zinc, which plays an important role in the regulation of intracellular signaling pathways.
  • Zinc deficiency will affect the survival, proliferation, and maturation of lymphocytes in innate immunity and adaptive immunity; studies have reported that zinc deficiency will affect the production and function of immune cells, peripheral and thymic T cells The number, its proliferation and differentiation have adverse effects. Studies have found that the lack of trace zinc will lead to immune aging in the elderly (Immunity & Ageing, 2009, 6(9): 1-17). In short, zinc plays an important role in the production, development and function of T cells.
  • Bisphosphonates as the main anti-osteoporosis treatment drug, are inorganic pyrophosphate analogues. They are formed by replacing oxygen atoms in pyrophosphate POP bonds with carbon atoms for special PCP bond skeletons, which are not easily affected by the body. Enzymatic hydrolysis, high temperature resistance, stable to many chemicals. At the same time, the molecular configuration of P-C-P determines that its three-dimensional structure can be combined with hydroxyapatite or calcium, iron, and zinc metal ions.
  • Zoledronic acid (ZOL) is a nitrogen-containing bisphosphonic acid drug with a heterocyclic structure, which is powerful, low-dose, and convenient to use.
  • Zinc hydroxide colloid or ferric hydroxide colloid has a good adsorption effect on viruses or proteins and has a good adjuvant effect; zinc hydroxide colloid can stimulate the body's humoral and cellular immunity at the same time, while ferric hydroxide colloid mainly enhances humoral immune response , And the two have good local and systemic tolerance; the combination of the two with lecithin has better immune stimulation effect.
  • Hu Yunzhang and others invented a vaccine adjuvant (Chinese Patent, Publication (Announcement) No. CN101444623A, Publication (Announcement) Date June 3, 2009).
  • the invention relates to a vaccine adjuvant, that is, the application of zinc hydroxide, a compound with a chemical formula of Zn(OH) 2 and a chemical formula weight of 99.4046 Daltons, in the preparation of vaccine adjuvants, and the preparation of hepatitis A vaccine adjuvants application.
  • the combined application of zinc hydroxide as an adjuvant and vaccine can effectively enhance the humoral immune response of the vaccine, and its immune enhancement effect is better than that of aluminum adjuvants.
  • the results of animal experiments show that zinc hydroxide as a new vaccine adjuvant has both sensitization and The safety is better than aluminum adjuvant.
  • Recombinant epitope polypeptide vaccine induces the enhancement of mouse humoral immunity, and it is found that the composite adjuvant of HS and zinc hydroxide can effectively enhance the mouse humoral immune response induced by HCV recombinant epitope polypeptide vaccine, and it can to a certain extent Save the amount of antigen used.
  • Hu Yunzhang and others invented a zinc phosphate vaccine adjuvant (Chinese Patent, Publication (Announcement) No. CN101972477B, Publication (Announcement) Date September 26, 2012), the invention relates to a zinc phosphate vaccine adjuvant, especially zinc phosphate as a vaccine adjuvant And the application of zinc phosphate vaccine adjuvant in the preparation of vaccines.
  • the zinc phosphate is used as a vaccine adjuvant in combination with an antigen, which can effectively enhance the humoral immune response of the vaccine, and its immune enhancement effect is better than aluminum adjuvant and zinc hydroxide adjuvant, and the results of animal experiments show that zinc phosphate is used as a vaccine adjuvant Its sensitization and safety are better than aluminum adjuvant and zinc hydroxide adjuvant.
  • Hu Yunzhang et al. invented a compound vaccine adjuvant (Chinese Patent, Publication (Announcement) No. CN102085366B, published (Announcement) date December 26, 2012).
  • the invention provides a compound vaccine adjuvant characterized by the following components Composition: The mass ratio of sodium ferulate to zinc hydroxide is 10:1 to 50:1.
  • the combined application of the composite vaccine adjuvant and the vaccine can effectively enhance the humoral immune response of the vaccine, and the enhancement effect is similar to that of the aluminum adjuvant, and is superior to a single sodium ferulate adjuvant and a single zinc hydroxide adjuvant, and is non-toxic , Side effects, it is safe and reliable to use within the immune dose range.
  • the composite vaccine adjuvant of the invention has easy-to-obtain raw materials, is a commercially available product, has simple preparation process, low cost and stable performance, and can be used as an adjuvant for hepatitis B vaccine, genetic engineering vaccine, virus vaccine and the like.
  • the zoledronic acid adjuvant provided by the present invention has small toxic and side effects, is safe and reliable to use within an immune dose range, can effectively induce antigen-specific humoral immune responses, and has an immune effect similar to aluminum adjuvants, and raw materials are easily available It has stable performance and can be used as an adjuvant for vaccines.
  • Takuya SHISHIDO et al. invented a bisphosphonate-containing vaccine pharmaceutical composition that can induce the body to produce an efficient humoral immune response, and can be administered transdermally or mucosally (US Patent, Publication Number: US 2017/0281759A1, Publication Date: 2017 October 5).
  • the invention provides a zinc glycinate adjuvant and contains
  • the zinc glycinate adjuvant provided by the invention is a medicinal adjuvant, a zinc nutrient fortifier, with small toxic and side effects, is safe and reliable to use within an immune dose range, and can effectively induce antigen-specific humoral immune responses, and the induced humoral The immune response effect is better than that of the non-adjuvant group.
  • the raw material of zinc glycinate adjuvant is easily available. It is a commercially available product. The preparation process is simple, low cost, stable performance, high biological potency, non-toxic effect, and can be added to a variety of traditional Used as vaccine adjuvant in vaccines and genetic engineering vaccines.
  • the trace element zinc which plays an important role, forms a zinc zoledronate micronanoparticle adjuvant through co-precipitation.
  • the inventors discovered through creative research that the zinc zoledronate micronanoparticle adjuvant can significantly improve humoral immunity and cellular immune response, thereby providing a zinc zoledronate vaccine adjuvant with obvious immune enhancement effect.
  • the present invention relates to a zinc zoledronate micronanoparticle adjuvant prepared by co-precipitation of zinc ions and phosphonic acid groups, phosphate and hydroxide, referred to as zinc zoledronate adjuvant.
  • the zinc zoledronate adjuvant of the present invention can be prepared by combining a soluble salt solution containing zinc ions with zoledronic acid, sodium hydroxide solution, or combining a soluble salt solution containing zinc ions with zoledronic acid.
  • the acid, sodium hydroxide, and sodium phosphate solution are uniformly mixed to obtain a zinc zoledronate adjuvant.
  • the mixing method includes, but is not limited to, sequential precipitation, separate precipitation and remixing, or co-precipitation.
  • the zinc:zoledronic acid molar concentration ratio in the zinc zoledronate adjuvant is generally not limited.
  • the zinc: zoledronic acid molar concentration ratio in the zinc zoledronate adjuvant may be 1-8.
  • the zinc:zoledronic acid molar concentration ratio is selected from 1, 4, and 8.
  • the zinc zoledronate adjuvant may also contain phosphate.
  • phosphate can be used to replace zoledronic acid in different molar ratios (not all replacements) and through different mixing methods (such as sequence Precipitation, separate precipitation and then mixing or co-precipitation, etc.) to prepare a zinc zoledronate complex adjuvant.
  • the molar concentration ratio of Zn:phosphate is usually not limited.
  • the molar concentration ratio of Zn:phosphate radical can be 1-8.
  • the molar concentration ratio of Zn:phosphate radical is selected from 1.5 and 4 to form an organic-inorganic hybrid zinc zoledronate complex adjuvant. Agent.
  • the zinc zoledronate adjuvant may also contain aluminum (Al).
  • Al can be used to replace Zn in different proportions (not all replacement) and through different mixing methods (such as sequence precipitation, Separate precipitation and then mixing or co-precipitation, etc.) to prepare zoledronate zinc aluminum adjuvant.
  • the Zn:Al molar concentration ratio is generally not limited.
  • the Zn:Al molar concentration ratio may be 0.02-1, and preferably, the Zn:Al molar concentration ratio is selected from 0.375, 0.5 and 0.8.
  • the specific type of zinc compound is not limited, for example, it can be zinc hydroxide, zinc phosphate, zinc sulfate, zinc carbonate or other types of zinc adjuvants known in the art, as long as the zinc zoledronate adjuvant It is prepared by precipitation of zinc ions and phosphonic acid groups, phosphate and hydroxide.
  • the phosphate solution can be selected from but not limited to sodium phosphate, disodium hydrogen phosphate (no crystal water, dihydrate, heptahydrate, dodecahydrate), sodium dihydrogen phosphate (no crystal water, dihydrate) , Potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, pyrophosphate, polyphosphoric acid and any mixtures thereof.
  • the specific aluminum compound type is not limited, for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate or other aluminum adjuvant types known in the art, as long as the zoledronate zinc aluminum adjuvant is It is prepared by precipitation of zinc and aluminum ions with phosphonic acid groups, phosphate and hydroxide.
  • the present invention relates to a method of preparing a zinc zoledronate adjuvant, which comprises:
  • the zinc ion is reacted with the phosphonic acid group, the phosphate group and the hydroxide group separately or simultaneously in a soluble salt solution to precipitate the zinc ion, thereby preparing a zinc zoledronate adjuvant.
  • the method includes:
  • step a and zoledronic acid, sodium hydroxide or the soluble salt solution of step a and zoledronic acid, sodium hydroxide, sodium phosphate solution are precipitated in sequence, separately precipitated and then mixed or co-precipitated Mix uniformly to obtain zinc zoledronate adjuvant.
  • the soluble salt solution is generally not limited, and may be selected from hydrochloric acid solutions, for example.
  • the method further includes sterilizing the zinc zoledronate adjuvant suspension formed after mixing in step b at 121°C for 60 minutes at high temperature and autoclaving, and then placing it at 2-8°C after cooling to room temperature, It is preferably stored at 4°C for later use.
  • the molar concentration ratio of zinc: zoledronic acid in the zinc zoledronate adjuvant obtained by the method of the present invention may be 1-8.
  • the zinc:zoledronic acid molar concentration ratio is selected from 1, 4, and 8.
  • Al is used in different mixing methods (such as sequence precipitation, sequence precipitation, Zn/Al molar concentration ratio of 0.375, 0.5, and 0.8). Separate precipitation and then mixing or co-precipitation, etc.) to prepare zinc aluminum adjuvant of zoledronate by precipitation of zinc ion and aluminum ion with phosphonic acid group, phosphate and hydroxide.
  • the soluble salt solution of zinc ion as used herein may be any soluble salt solution of zinc ion, and is preferably a hydrochloric acid solution of zinc ion.
  • the zoledronate solution as used herein is preferably an alkaline solution of zoledronic acid sodium hydroxide.
  • the method of precipitation of zinc and phosphonic acid groups as used herein may be any method in which a soluble salt solution of zinc ions and an alkali solution of zoledronic acid sodium hydroxide are thoroughly mixed to cause a precipitation reaction.
  • the zinc zoledronate adjuvant can be prepared by sequential precipitation, separate precipitation and then mixing, or co-precipitation.
  • Sterilization as used herein may be any manner suitable for sterilizing the zinc zoledronate adjuvant.
  • high temperature and high pressure sterilization technology can be used for sterilization, such as sterilization at 121°C for 30-60 minutes, preferably 60 minutes.
  • the present invention also relates to the determination of the physical and chemical properties of the obtained zinc zoledronate adjuvant.
  • adjuvants can be determined by conventional techniques, for example, see US9573811; Ai Xulu et al., Analysis of the physical and chemical properties of three aluminum hydroxide adjuvants, "Chinese Journal of Biological Products", 2015, 28(1): 44-47; and as described in the Examples herein.
  • the zinc zoledronate adjuvant as described herein has one or more of the following properties: the pH before sterilization is 8.0-9.0, the pH after sterilization is 6.0-8.0, and the particle size is 1-10 ⁇ m, particle zero charge point is 4.0-11.4, metal ion precipitation rate>99%, protein adsorption rate>80%.
  • the present invention relates to a composition
  • a composition comprising a zinc zoledronate adjuvant as described herein, particularly a pharmaceutical formulation or composition.
  • the method for preparing the pharmaceutical preparation or composition includes the step of combining the zinc zoledronate adjuvant and the carrier and/or optionally one or more accessory ingredients together.
  • the preparation is prepared as follows: the zinc zoledronate adjuvant and the liquid carrier or the pulverized solid carrier or both are uniformly and tightly combined, and then the product is shaped if necessary.
  • Liquid dosage forms for oral administration of zinc zoledronate adjuvant include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage form can also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, and ethyl acetate.
  • Benzyl alcohol benzyl benzoate, propylene glycol, 1,3-butanediol, oil (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerin, tetrahydrofurfuryl alcohol , Fatty acid esters of polyethylene glycol, sorbitan, and mixtures thereof.
  • oil specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil
  • glycerin tetrahydrofurfuryl alcohol
  • Fatty acid esters of polyethylene glycol sorbitan, and mixtures thereof.
  • the oral composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, flavoring agents, and preservatives.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, flavoring agents, and preservatives.
  • the suspension formulation may also contain suspending agents, such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, and partial hydroxide Aluminum, bentonite, agar, tragacanth and their mixtures.
  • suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, and partial hydroxide Aluminum, bentonite, agar, tragacanth and their mixtures.
  • the formulation of the pharmaceutical composition of the present invention for rectal or vaginal administration can be provided as a suppository, which can be prepared by combining a zinc zoledronate adjuvant with one or more suitable non-irritating excipients or carriers (including, for example, cocoa butter , Polyethylene glycol, suppositories are prepared by mixing wax or salicylate), and are solid at room temperature and liquid at body temperature, which will melt in the rectum or vagina to release the zinc adjuvant of zoledronate .
  • suitable for vaginal administration of the present invention also include vaginal suppositories, tampons, creams, gels, pastes, foams or sprays containing suitable carriers known in the art.
  • the pharmaceutical composition of the present invention suitable for parenteral administration comprises a zinc zoledronate adjuvant and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous carriers in combination with it, including solutions, dispersions Preparations, suspensions or emulsions or sterile powders that can be reconstituted into sterile injectable solutions or dispersions before use, which may contain antioxidants, buffers, and solutes that make the preparations isotonic with the blood of the intended recipient Or suspending agent or thickening agent.
  • aqueous and non-aqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and Injectable organic esters such as ethyl oleate.
  • the proper fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size (in the case of dispersants), and by using surfactants.
  • compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, etc., in the composition.
  • isotonic agents such as sugars, sodium chloride, etc.
  • the prolonged absorption of the injectable drug form can be achieved by incorporating substances that delay absorption, such as aluminum monostearate and gelatin.
  • An injectable long-acting formulation can be prepared by forming a microcapsule matrix of a zinc zoledronate adjuvant in a biodegradable polymer (such as polylactide-polyglycolide). According to the ratio of the zinc zoledronate adjuvant to the polymer and the nature of the polymer used, the release rate of the zoledronic acid adjuvant can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Long-acting injectable formulations can also be prepared by encapsulating the zinc zoledronate adjuvant in liposomes or microemulsions that are compatible with body tissues. The injectable material can be sterilized, for example, by filtering with a bacteria retention filter.
  • the preparation or composition can be provided in a single dose, or in a multi-dose sealed container (such as ampoules and vials), and can be stored under lyophilized conditions, and only a sterile liquid carrier such as water for injection needs to be added before use. Injection solutions and suspensions can be prepared immediately from the above-mentioned types of sterile powders, granules and tablets.
  • the invention also relates to an immunogenic composition
  • an immunogenic composition comprising a zinc zoledronate adjuvant as described herein and one or more antigens.
  • the immunogenic composition as used herein when administered to a subject or animal, is a composition capable of eliciting a protective immune response against the one or more antigens contained therein.
  • the present invention also relates to a vaccine composition
  • a vaccine composition comprising a zinc zoledronate adjuvant as described herein and one or more antigens.
  • the vaccine composition as used herein when administered to a subject or animal, can elicit a protective immune response against, for example, microorganisms, or can effectively protect the subject or animal from infection.
  • the vaccine composition can be used to prevent or ameliorate pathological conditions that would respond favorably to immune response modulation.
  • Such vaccine composition may be a prophylactic vaccine or a therapeutic vaccine.
  • the vaccine composition includes a genetically engineered vaccine, such as a protein vaccine, such as a varicella-zoster virus recombinant protein vaccine.
  • the invention also relates to a vaccine adjuvant comprising a zinc zoledronate adjuvant as described herein.
  • a vaccine adjuvant comprising a zinc zoledronate adjuvant as described herein.
  • such vaccine adjuvants may also include secondary adjuvants as described below.
  • adjuvant means a substance capable of non-specifically accelerating, prolonging or enhancing an immune response to an antigen.
  • adjuvants can also reduce the number of immunizations or the number of antigens required for protective immunity.
  • the zinc zoledronate adjuvant of the present invention can be combined with one or more antigens to produce an immunogenic composition or vaccine that can be used to elicit an immune response in an individual.
  • antigens include protein antigens, such as varicella zoster virus gE glycoprotein antigen (VZV gE).
  • immunomodulatory molecules can also be used in combination with the zinc zoledronate adjuvant of the present invention to modify the immune response in an individual.
  • the immunomodulators described herein refer to a class of preparations that can regulate, balance and restore the body's immune function. Commonly used immunomodulators include immune boosters, immunosuppressants and immune two-way regulators.
  • the amount of the antigen and the zinc zoledronate adjuvant in the vaccine composition of the present invention and the dose to be administered are determined by techniques well known to those skilled in the pharmaceutical field, in which factors such as the following should be considered: specific antigen, specific The age, sex, weight, species and condition of the animal or patient, and the route of administration.
  • the vaccine composition of the present invention further comprises one or more components selected from the group consisting of surfactants, absorption enhancers, water-absorbing polymers, substances that inhibit enzymatic degradation, alcohols, organic Solvents, oils, pH control agents, preservatives, osmotic pressure control agents, propellants, water, and any mixtures thereof.
  • the vaccine composition of the present invention may also include a pharmaceutically acceptable carrier.
  • the amount of the carrier will depend on the selected amount of other ingredients, the desired concentration of the antigen, the choice of route of administration (oral or parenteral) and so on.
  • the carrier can be added to the vaccine at any convenient time. In the case of a lyophilized vaccine, the carrier can be added, for example, just before administration. Alternatively, the final product can be manufactured with a carrier.
  • Suitable carriers include, but are not limited to, sterile water, saline, buffer, phosphate buffered saline, buffered sodium chloride, vegetable oil, minimal essential medium (MEM), MEM with HEPES, and the like.
  • the vaccine composition of the present invention may contain different amounts of conventional secondary adjuvants, depending on the adjuvant and the desired result.
  • suitable secondary adjuvants include, but are not limited to, stabilizers; emulsifiers; pH adjusters, such as sodium hydroxide, hydrochloric acid, etc.; surfactants, such as Tween.RTM.80 (polysorbate 80, available from Sigma Chemical Co. (St.
  • liposomes are used as adjuvant; synthetic glycopeptides, such as muramyl dipeptide; extenders, such as dextran; carbopol; bacteria Cell walls, such as mycobacterial cell wall extracts; their derivatives, such as Corynebacterium parvum; Propionibacterium acnes (Propionibacterium acne); Mycobacterium bovis, such as Bovine Calmette Guerin, BCG); vaccinia or animal pox virus protein; subviral particle adjuvants, such as circovirus; cholera toxin; N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propane Diamine (pyridine); monophosphoryl lipid A; dimethyl dioctadecyl ammonium bromide (DDA, commercially available from Kodak Corporation (Rochester, NY)); their compositions and mixtures.
  • synthetic glycopeptides such as muramyl dipeptide
  • extenders such as dex
  • suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-amine or NZ-amine AS.
  • emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil, and other standard, metabolizable, non-toxic oils that can be used in injection or intranasal vaccine compositions.
  • these adjuvants are referred to herein as "minor” only to contrast with the above-mentioned zinc zoledronate adjuvant, because the combination of zoledronate zinc adjuvant and antigenic substance can significantly Improve the immune response to the antigenic substance so that it can be used as an essential ingredient in the vaccine composition.
  • Secondary adjuvants are mainly included in vaccine formulations as processing aids, although some adjuvants do have a certain degree of immune-enhancing properties and thus have a dual purpose.
  • preservatives can be added to the vaccine composition in an effective amount of about 0.0001% by weight to about 0.1% by weight. Depending on the preservative used in the formulation, amounts below or above this range may also be used.
  • Typical preservatives include, for example, potassium sorbate, sodium metabisulfite, phenol, methyl paraben, propyl paraben, thimerosal and the like.
  • the vaccine composition of the present invention is conveniently administered by oral, parenteral (subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal or intraperitoneal), intraoral, intranasal or transdermal routes.
  • the route of administration envisaged by the present invention will depend on the antigenic substance and adjuvant.
  • the vaccine composition contains saponins, although they are non-toxic when administered orally or intranasally, care must be taken not to inject sapogenin glycosides into the blood stream because they act as strong hemolytic agents. Also, many antigens will not be effective if taken orally.
  • the vaccine composition is administered by intramuscular or intraperitoneal route.
  • the dosage of the vaccine composition will significantly depend on the selected antigen, route of administration, species and other standard factors. It is envisaged that a person of ordinary skill in the art can easily titrate an appropriate dose for the immune response to each antigen, so as to know the effective immunization amount and administration method.
  • the zinc zoledronate adjuvant of the present invention can enhance the immunogenicity of weaker antigens (such as highly purified or recombinant antigens), reduce the amount of antigen required for immune response, and reduce protection
  • weaker antigens such as highly purified or recombinant antigens
  • the frequency of immunity required for sexual immunization can improve the effectiveness of the vaccine, which can improve the efficacy of the vaccine in individuals with reduced or weakened immune responses (such as neonates, the elderly, and immunocompromised individuals), and can enhance the effectiveness in target tissues. Or by triggering a specific cytokine profile to promote cell-mediated immunity and/or humoral immunity.
  • antigen and/or immunomodulatory molecule and the zinc zoledronate adjuvant of the present invention can be tested in a variety of preclinical toxicology and safety studies known in the art.
  • the combination can be evaluated in an animal model: the antigen has been found to be immunogenic in the animal model, and the animal model can be reproducibly reproducible through the same approach as proposed for human clinical trials Get immunized.
  • the combination of antigens and/or immunomodulatory molecules and the zoledronic acid adjuvant of the present invention can be obtained, for example, by the US Food and Drug Administration Center for Biological Products Evaluation and Research and the National Institute of Allergy and Infectious Diseases (Goldenthal, KL Et al. AID Res Hum Retroviruses, 9: S45-9 (1993)) proposed the experiment.
  • the immunogenic composition or vaccine of the present invention for inducing an immune response can be administered as a solution or suspension together with a pharmaceutically acceptable medium.
  • Such a pharmaceutically acceptable medium may be, for example, water, phosphate buffered saline, physiological saline or other physiological buffered saline, or other solvents or media, such as glycol, glycerol and oil (such as olive oil) or injectable organic esters.
  • the pharmaceutically acceptable medium may also contain liposomes or micelles, and may contain an immunostimulatory complex prepared by mixing a polypeptide or peptide antigen with a detergent and a glycoside (such as Quil A).
  • the immunogenic composition or vaccine of the present invention can be administered in various ways to stimulate an immune response.
  • the immunogenic composition or vaccine can be delivered by subcutaneous, intradermal, intralymphatic, intramuscular, intratumoral, intravesical, abdominal, intraperitoneal, and intracerebral routes.
  • the vaccination method for treating or preventing infection in a mammal includes the use of the vaccine of the present invention, which will specifically be administered intramuscularly or intraperitoneally.
  • the vaccine may be administered as a single dose, or preferably several times a week or a month according to the primary immunization/booster immunization pattern, for example two, three or four times. The appropriate dose depends on various parameters.
  • the present invention also relates to the use of the zinc zoledronate adjuvant as described herein for the preparation of vaccine adjuvants, pharmaceutical compositions, immunogenic compositions or vaccine compositions.
  • the vaccine comprises Protein vaccines, such as varicella-zoster virus protein recombinant vaccines.
  • FIG. 1 Schematic diagram of three adjuvant preparation processes.
  • Figure 2 Particle morphology of zinc zoledronate, zinc aluminum zoledronate, aluminum zoledronate adjuvant
  • Aluminum chloride hexahydrate (AlCl 3 ⁇ 6H 2 O): purchased from Xilong Chemical;
  • Disodium hydrogen phosphate dodecahydrate Na 2 HPO 4 ⁇ 12H 2 O: purchased from Xilong Chemical;
  • Zoledronic acid (C 5 H 10 N 2 O 7 P 2 ): purchased from Hunan Huateng Pharmaceutical Co., Ltd.
  • the zinc risedronate adjuvant obtained after mixing in Figure 1A was sterilized once by autoclaving at 121°C for 60 minutes, cooled to room temperature and stored at 2-8°C.
  • the aluminum adjuvant Al-002 was prepared.
  • the aluminum adjuvant Al-001-840 suspension was prepared.
  • solution A Use aluminum chloride hexahydrate and anhydrous zinc chloride with water to prepare 0.25L of a solution containing 124.44mM aluminum chloride and 46.68mM zinc chloride, defined as solution A; use disodium hydrogen phosphate dihydrate and sodium hydroxide to prepare with water 0.25L of solution containing 18.68mM phosphate and 0.425M sodium hydroxide, defined as B solution, filtered with 0.22 ⁇ m filter membrane for use;
  • Example 1 Determination of physical and chemical properties of zoledronate zinc adjuvant
  • any Zn/zoledronic acid molar ratio that is, any zinc zoledronate adjuvant, such as an inorganic phosphoric acid or Al-doped zinc zoledronate adjuvant;
  • test product Take the test product, put it at room temperature to equilibrate for at least 30 minutes, and measure it with Sartorius pH glass electrode.
  • Err error message
  • the pH range of Zn-zoledronic acid adjuvant before sterilization is 8.0-9.0, and after sterilization it is 6.5-7.0;
  • the blank background and the size of the test standard sample must be corrected before each start-up measurement.
  • the particle size of zinc zoledronate adjuvant is between 0.4-30 ⁇ m, and most of the particles are 6-7 ⁇ m.
  • BSA gradient dilution 150mM NaCl is the dilution buffer, weigh a certain amount of BSA sample, and dilute to the concentration gradient specified in EP: 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 5mg/mL , 10mg/mL, set aside.
  • BSA: adjuvant 3:1 (volume ratio) as the adsorption rate detection condition; after shaking the adjuvant, mix it with different concentrations of BSA according to the experimental conditions, and adsorb at room temperature for 1 hour, shaking during the period 5 times; centrifuge at 13000rpm/min for 3min and take the supernatant, set aside.
  • Protein concentration determination Lowry method is used to determine the protein concentration in EP. In this experiment, UV2100pro is used to directly measure the absorbance of the supernatant at 280nm, so that the reading is between 0.2-0.8, otherwise it needs to be diluted.
  • adsorption rate [1-OD 280 (supernatant dilution times X) )/OD 280 (when the dilution X times the adsorption rate is 0) ]*100
  • Passivated electrode Add 3-4mL adjuvant to the sample tube. After the electrode is inserted, set the cycle to 50 in SOP, and run the instrument to passivate the electrode.
  • Sample detection After the electrode is taken out, rinse the lower end with deionized water, add the corresponding sample, adjust the cycle setting in SOP to: 15, the measure setting to: 3, the pH setting to the corresponding pH of each sample, and run the instrument.
  • the flame method (D2 lamp background correction) was used to determine the zinc content in zoledronate zinc adjuvant and standardize the determination operation process.
  • the detection instrument is an atomic absorption spectrophotometer: Shimadzu AA6300C (P/N 206-52430);
  • Preparation of standard solution and sample to be tested Preparation of standard curve: original concentration of zinc standard 500 ⁇ g/mL, diluted with 0.1M hydrochloric acid solution to 500ng/mL, 1000ng/mL, 1500ng/mL, 2000ng/mL and 2500ng /mL standard product.
  • Open acetylene slowly open the acetylene valve to ensure that the primary pressure is 0.5MPa and the secondary pressure is 0.1MPa;
  • WizAArd Basic operation of WizAArd software: log in WizAArd ⁇ select element ⁇ "unconnected instrument/send parameter” page, click ⁇ connect/send parameter> ⁇ set in the "instrument initialization” page ⁇ in the "flame analysis instrument check directory" one by one Check and tick the items, click ⁇ OK> ⁇ set wavelength [213.86], slit width [0.7], lighting method [emission], [lamp position setting] in the "optical parameters” page to ensure the actual position of the Zn hollow cathode lamp Same as the setting position, select [Lighting] ⁇ Spectrum search ⁇ burner origin position adjustment ⁇ Select [Parameter] ⁇ [Edit parameter] in the menu bar ⁇ change the lighting method to ⁇ BGC-D2> ⁇ Spectrum search ⁇ Ignition: After ensuring that C2H2 is turned on and the pressure is up to the requirements, press the PURGE and IGNITE buttons on the host at the same time until ignition ⁇ auto zero ⁇ set the blank group (BLK), standard product (
  • Example 4 Determination of the precipitation rate of Zn-zoledronic acid (1/0.125) adjuvant zoledronic acid
  • Zoledronic acid contains an imidazole ring and has a maximum absorption peak at 215nm.
  • the specific process for detecting the supernatant zoledronic acid of the adjuvant at 215nm by an ultraviolet spectrophotometer is as follows:
  • Example 5 Determination of adjuvant activity of Zn-zoledronic acid adjuvant in recombinant protein VZV gE vaccine
  • the prepared zinc zoledronate adjuvant (also known as Zn-zoledronic acid) was used as an adjuvant, and the molar concentration ratio of Zn/zoledronic acid was 1:0.25 or 1:1, which was used as an adjuvant.
  • mice were injected intramuscularly to determine the specific antibody titers produced. The specific method is as follows:
  • mice Balb/C mice, 6-8 weeks, 5 mice/group, female.
  • Drug concentration Antigen: 50 ⁇ g/ml
  • Immunization protocol 2 weeks after the first immunization of the animals according to the immunization group, blood was collected from the orbit to determine the antibody titer in the serum. The antibody titer was measured in the second week after the first immunization, the second week was boosted, and 2 weeks after the second immunization, blood was taken from the orbit to determine the antibody titer in the serum, and the third immunization was performed. After 2 weeks, the serum was determined by ELISA Antibody titers.
  • Enzyme-linked immunosorbent assay detects antibody binding titer:
  • Antigen coating solution 1X PB 7.4 buffer (4.343g Na 2 HPO 4 ⁇ 7H 2 O; 0.456g NaH 2 PO 4 ).
  • Blocking solution 2X ED Enzyme Dilution: 1X PBS + 0.5% casein + 2% gelatin + 0.1% preservative (proclin-300), diluted to 1X with ultrapure water or distilled water, used for sealing and sample dilution.
  • Chromogenic Solution A ELISA kit from Beijing Wantai Company.
  • Chromogenic Solution B ELISA kit from Beijing Wantai Company.
  • Coating plate Dilute the VZV gE antigen to a certain concentration with PB7.4 coating buffer, generally 100ng/well, and add 100 ⁇ L/well to a 96-well polystyrene plate, and coat overnight at 4°C.
  • Blocking discard the coating solution in the well, wash the plate once with PBST washing solution, spin dry, add 200 ⁇ L/well of blocking solution, and block for 4 hours at room temperature.
  • Color development discard the secondary antibody in the well, wash the plate with PBST 5 times, spin dry, add 100 ⁇ L/well of the color development solution A and B in equal volumes, and react at 25°C for 10 min.
  • Reading the plate Set the detection dual wavelengths of 450nm and 630nm on the microplate reader, and measure the OD value of each reaction well.
  • the adjuvant was mixed with the antigen in equal volumes, and the mice were immunized according to the above-mentioned immunization strategy.
  • the immunization and blood sampling and serum antibody titers were tested; in the zoledronate zinc adjuvant group, the mice had a fast onset after immunization.
  • the humoral immunity enhancement advantage compared with aluminum adjuvant group the difference is statistically significant, and the difference is still significant after three injections.
  • Example 6 The effect of zoledronate zinc adjuvant combined with recombinant protein VZV gE on mouse antibody subtypes
  • Antigen coating solution 1X PB 7.4 buffer (4.343g Na 2 HPO 4 ⁇ 7H 2 O; 0.456g NaH 2 PO 4 ).
  • Blocking solution 2X ED Enzyme Dilution: 1X PBS + 0.5% casein + 2% gelatin + 0.1% preservative (proclin-300), diluted to 1X with ultrapure water or distilled water, used for sealing and sample dilution.
  • Chromogenic Solution A ELISA kit from Beijing Wantai Company.
  • Chromogenic Solution B ELISA kit from Beijing Wantai Company.
  • Coating plate Dilute the VZV gE antigen to a certain concentration with PB7.4 coating buffer, add 100 ⁇ L/well to a 96-well polystyrene plate, and coat overnight at 4°C.
  • Blocking discard the coating solution in the well, wash the plate once with PBST, spin dry, add 200 ⁇ L/well of blocking solution, and block for 4h at room temperature.
  • Serum used for serum antibody subtype detection 2 weeks after the completion of 3 injections of immunization, that is, the 6th week of serum, discard the blocking solution in the well, wash the plate once with PBST, spin dry, and add a certain dilution 100 ⁇ L/well of serum to be tested, incubate the reaction at 25°C for 1h.
  • Color development discard the enzyme-labeled antibody in the well, wash the plate with PBST 5 times, spin dry, mix the same volume of color development solution A and B and dilute it three times, 100 ⁇ L/well, and react at 25°C for 10 min.
  • Reading the plate Set the detection dual wavelengths of 450nm and 630nm on the microplate reader, and measure the OD value of each reaction well.
  • mice were immunized with zoledronate zinc adjuvant or aluminum adjuvant and recombinant VZV gE protein by intramuscular injection.
  • the immunization procedure was the same as that in Example 5.
  • blood was collected for 2 weeks.
  • the mouse serum antibody levels of each subclass the results are shown in Figure 4.
  • the zinc zoledronate adjuvant group can arouse more intense IgG2a and IgG2b subtype antibody levels, and IgG1 and IgG2a
  • the ratio of IgG2b to IgG2b is lower than that of the aluminum adjuvant group, indicating that it has a certain stimulating effect on the Th1 immune pathway.
  • Example 7 Determination of the adjuvant activity of zoledronate zinc adjuvant in hepatitis B therapeutic protein
  • mice were immunized with zoledronate zinc adjuvant and hepatitis B therapeutic protein by intramuscular injection, and serum antibody titers were detected.
  • Example 8 The effect of other zinc bisphosphonate adjuvants combined with VZV gE on mouse serum antibody titers
  • Example 5 Using the experimental procedure described in Example 5, the zinc bisphosphonate adjuvant was combined with recombinant VZV gE protein to immunize mice by intramuscular injection, and the serum antibody titer was detected.
  • the results shown in Figure 6 show that the humoral immunity enhancement advantage of the zoledronate zinc adjuvant group after three injections of mice was significantly different from that of the aluminum adjuvant group, while the other bisphosphonates The effect of zinc adjuvant is not significantly different from that of aluminum adjuvant.
  • Example 9 The effect of zoledronate zinc adjuvant with VZV gE on mouse serum antibody titers
  • mice were immunized with zoledronate zinc adjuvant and VZV gE by intramuscular injection, and serum antibody titers were detected.
  • aluminum adjuvant, composite zinc-aluminum adjuvant, composite zinc-aluminum adjuvant doped with zoledronic acid, and zinc zoledronate adjuvant showed a gradual increase in the level of antibody enhancement.
  • the zinc phosphonate adjuvant was significantly different.

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Abstract

一种唑来膦酸锌微纳米颗粒佐剂，包含锌及唑来膦酸，任选包含磷酸盐和铝。其制备方法包括将含锌离子的可溶性盐溶液与唑来膦酸、氢氧化钠混合沉淀。所述佐剂可用于制备疫苗等。

Description

唑来膦酸锌微纳米颗粒佐剂的制备及作为疫苗佐剂的用途 技术领域
本发明涉及一种疫苗佐剂，尤其涉及一种具有缓释作用的唑来膦酸锌微纳米颗粒佐剂，其可明显增强体液免疫及细胞免疫反应，可作为疫苗佐剂应用于疫苗制备中，属于免疫学领域。
技术背景
锌是一种重要的化学元素，具有特殊的生物学功能。例如，痕量级的锌存在于大约300种参与细胞代谢的酶中，并且其在细胞呼吸、细胞分裂、蛋白质合成、DNA合成、损伤修复以及免疫功能激活等中扮演重要角色。因此，锌的缺乏或紊乱与众多疾病相关，尤其是免疫系统方面的疾病(Autoimmunity Reviews，2015，14(04)277–285)。在免疫细胞内，二价阳离子包括锌，对于细胞内信号通路的调节具有重要的作用。锌的缺乏对先天性免疫及适应性免疫中淋巴细胞的存活、增殖、以及成熟都将产生影响；有研究报道称，锌缺乏会对免疫细胞的细胞因子的产生和功能、外周及胸腺T细胞的数量、其增殖和分化产生不利影响。有研究调查发现微量锌的缺乏将导致老年人免疫衰老(Immunity&Ageing，2009，6(9)：1-17)。简而言之，锌在T细胞生成、发育及其功能发挥具有重要作用。
双膦酸(Bisphosphonates,BPs)作为抗骨质疏松治疗最主要的药物，为无机焦磷酸类似物，由焦磷酸P-O-P键中氧原子被碳原子取代为特殊的P-C-P键骨架而形成，不易被体内酶水解，耐高温，对许多化学物质稳定。同时，P-C-P分子构型决定了其三维结构可与羟基磷灰石或钙、铁、锌金属离子结合。唑来膦酸(zoledronic acid，ZOL)，为具有杂环结构含氮双膦酸药物，其具有强效、低剂量、使用方便的特点。临床研究显示双膦酸盐用于多发性骨髓瘤、乳腺癌、肾癌、前列腺癌等疾病的辅助治疗可降低患者的骨相关疾病的发生率，癌症复发率以及提高患者的存活期，以改善临床结果，尤其是唑来膦酸盐的抗肿瘤效果更具优势，被广泛的运用于多种恶性肿瘤的临床研究；其次，另有研究发现，双膦酸药物亦具有免疫调节效果，可通过作用于B细胞提高体液免疫反应(Cell Rep.，2013，5(2))；唑来膦酸可提高抗原特异性CD8+T细胞反应(Vaccine，2016，34(10):1275-81)。
Dieter Berndardt等发明一种氢氧化锌胶体或氢氧化铁胶体，其制备过程及其 作为疫苗佐剂的应用(美国专利，专利号US 5252327，授权日期：1993年10月12号)。氢氧化锌胶体或者氢氧化铁胶体对病毒或蛋白具有良好的吸附作用并具有良好的佐剂效应；氢氧化锌胶体可同时激发机体的体液和细胞免疫，而氢氧化铁胶体主要增强体液免疫反应，并且二者具有良好的局部及全身耐受性；二者与卵磷脂联合使用具有更好的免疫刺激作用。
胡云章等发明一种疫苗佐剂(中国专利，公开(公告)号CN101444623A，公开(公告)日2009年6月3日)。该发明涉及一种疫苗佐剂，即化学式为Zn(OH) 2，化学式量为99.4046道尔顿的化合物氢氧化锌在制备疫苗的佐剂方面的应用，以及在制备甲肝疫苗的佐剂方面的应用。该氢氧化锌作为佐剂与疫苗联合应用能够有效增强疫苗的体液免疫应答，其免疫增强效果优于铝佐剂，并且动物试验结果显示，氢氧化锌作为新型的疫苗佐剂其致敏性和安全性均优于铝佐剂。
王海漩等(中国生物制品学杂志，2011，24(10)：1174-1176)研究硫酸乙酰肝素(Heparan sulfate，HS)与氢氧化锌复合佐剂对丙型肝炎病毒(Hepatitis C virus，HCV)重组抗原表位多肽疫苗诱导小鼠体液免疫的增强作用，结果发现HS与氢氧化锌的复合佐剂能有效增强HCV重组抗原表位多肽疫苗诱导的小鼠体液免疫应答，且能够在一定程度上节省抗原的使用量。
胡云章等发明磷酸锌疫苗佐剂(中国专利，公开(公告)号CN101972477B，公开(公告)日2012年9月26日)，该发明涉及一种磷酸锌疫苗佐剂，尤其涉及磷酸锌作为疫苗佐剂的应用，以及磷酸锌疫苗佐剂在制备疫苗中的应用。所述磷酸锌作为疫苗佐剂与抗原联合应用，能够有效增强疫苗的体液免疫应答，其免疫增强效果优于铝佐剂和氢氧化锌佐剂，并且经动物试验结果显示：磷酸锌作为疫苗佐剂其致敏性和安全性均优于铝佐剂和氢氧化锌佐剂。
胡云章等发明一种复合疫苗佐剂(中国专利，公开(公告)号CN102085366B，公开(公告)日2012年12月26日)，该发明提供一种复合疫苗佐剂，其特征在于由下列组分组成：阿魏酸钠与氢氧化锌的质量比为10:1～50:1。所述复合疫苗佐剂与疫苗联合应用能够有效增强疫苗的体液免疫应答，增强效果与铝佐剂相似，并优于单一的阿魏酸钠佐剂和单一的氢氧化锌佐剂，而且无毒、副作用，在免疫剂量范围内使用是安全可靠的。该发明的复合疫苗佐剂原料易得，均为市售产品，制备工艺简单，成本低，性能稳定，可用作乙肝疫苗、基因工程疫苗、病毒疫苗等的佐剂。
王海璇等发明唑来膦酸佐剂及含有唑来膦酸佐剂的疫苗(中国专利，公开(公告)号CN103768595B，公开(公告)日：2016年3月2日)，该发明提供一种唑来膦酸佐剂及含有唑来膦酸佐剂的疫苗，即每单份疫苗剂中含有10～20μg的唑来膦酸佐剂。本发明提供的唑来膦酸佐剂的毒副作用小，在免疫剂量范围内使用是安全可靠的，能够有效地诱导抗原特异性的体液免疫应答，且免疫效果与铝佐剂接近，原料易得，性能稳定，可作为疫苗的佐剂。Takuya SHISHIDO等发明一种含双膦酸盐疫苗药物组合物可诱导机体产生高效的体液免疫应答，并可经皮或者粘膜给药(美国专利，公开号：US 2017/0281759A1，公开日：2017年10月5日)。
蔡泓志等(中国生物制品学杂志，2013，26(05)：608-610)研究氢氧化锌与硫酸乙酰肝素(Heparan sulfate，HS)复合佐剂对狂犬病疫苗诱导的小鼠体液免疫应答的影响，结果发现氢氧化锌和HS复合佐剂能增强狂犬病疫苗诱导的小鼠体液免疫应答。
陈振普等发明甘氨酸锌佐剂及含甘氨酸锌佐剂的疫苗(中国专利，公开(公告)号CN104096229A，公开(公告)日2014年10月15日)，该发明提供一种甘氨酸锌佐剂及含有甘氨酸锌佐剂的疫苗，即每单份疫苗剂中含有用pH＝6～8的PBS溶解的0.1～1mg的甘氨酸锌佐剂。该发明提供的甘氨酸锌佐剂是药用辅料，是锌营养强化剂，毒副作用小，在免疫剂量范围内使用是安全可靠的，能够有效诱导抗原特异性的体液免疫应答，且所诱导的体液免疫应答效果优于无佐剂组，甘氨酸锌佐剂原料易得，为市售产品，制备工艺简单，成本低，性能稳定，生物学效价较高、无毒害作用，可添加于多种传统疫苗和基因工程疫苗中作为疫苗佐剂。
Mahsa Afshari等(Cytokine，2016，81:71-76)采用利什曼虫感染的BALB/c小鼠模型中，口服喂养小鼠硫酸锌，发现其可显著降低寄生虫载量，并且可提升Th1细胞因子mRNA的表达量。这说明，硫酸锌可诱导利什曼虫感染的BALB/c小鼠强的Th1免疫反应。
发明内容
为解决现有疫苗铝佐剂无法激发Th1型免疫反应及无法增强多肽、核酸类疫苗的免疫原性等方面的缺陷，结合唑来膦酸在免疫增强方面的作用，将其与同样在免疫调节方面具有重要作用的微量元素锌通过共沉淀形成唑来膦酸锌微纳米颗粒佐剂。本发明人通过创造性的研究发现，唑来膦酸锌微纳米颗粒佐剂可明显提高体液免疫及细胞免疫反应，从而提供了具有明显免疫增强作用的唑来膦酸锌疫苗佐剂。
因此，在一个方面，本发明涉及锌离子和膦酸基团、磷酸根和氢氧根共沉淀制备 的唑来膦酸锌微纳米颗粒佐剂，简称唑来膦酸锌佐剂。
在具体的实施方案中，本发明的唑来膦酸锌佐剂可通过使包含锌离子的可溶性盐溶液与唑来膦酸、氢氧化钠溶液或使包含锌离子的可溶性盐溶液与唑来膦酸、氢氧化钠、磷酸钠溶液均匀混合以获得唑来膦酸锌佐剂。
在具体的实施方案中，所述混合方式包括但不限于序列沉淀、分别沉淀再混合或共同沉淀等方式。
在一个实施方案中，唑来膦酸锌佐剂中的锌:唑来膦酸摩尔浓度比通常不受限制。在优选的实施方案中，唑来膦酸锌佐剂中的锌:唑来膦酸摩尔浓度比可以是1-8。优选地，锌:唑来膦酸摩尔浓度比选自1、4和8。
在一个实施方案中，唑来膦酸锌佐剂中还可包含磷酸盐，例如，可以使用磷酸盐以不同的摩尔比例替代唑来膦酸(不全部替代)并通过不同的混合方式(诸如序列沉淀、分别沉淀再混合或共同沉淀等方式)而制备成唑来膦酸锌复合型佐剂。在这样的唑来膦酸锌复合型佐剂中，Zn:磷酸根摩尔浓度比通常不受限制。在优选的实施方案中，Zn:磷酸根摩尔浓度比可以是1-8，优选地，Zn:磷酸根摩尔浓度比选自1.5、4，形成有机无机杂合的唑来膦酸锌复合型佐剂。
在又一个实施方案中，唑来膦酸锌佐剂中还可包含铝(Al)，例如，可以使用Al以不同的比例替代Zn(不全部替代)并通过不同的混合方式(诸如序列沉淀、分别沉淀再混合或共同沉淀等方式)而制备成唑来膦酸锌铝佐剂。在这样的唑来膦酸锌铝佐剂中，Zn:Al摩尔浓度比通常不受限制。在优选的实施方案中，Zn:Al摩尔浓度比可以是0.02-1，优选地，Zn:Al摩尔浓度比选自0.375、0.5和0.8。
在一个实施方案中，具体的锌化合物类型不受限制，例如可以是氢氧化锌、磷酸锌、硫酸锌、碳酸锌或其它本领域已知的锌佐剂类型，只要唑来膦酸锌佐剂是通过使得锌离子与膦酸基团、磷酸根和氢氧根产生沉淀制备而成的。
在一个实施方案中，磷酸盐溶液可以选自但不限于磷酸钠、磷酸氢二钠(无结晶水，二水，七水，十二水)、磷酸二氢钠(无结晶水，二水)、磷酸钾、磷酸氢二钾、磷酸二氢钾、焦磷酸、多聚磷酸及其任意混合物。
在一个实施方案中，具体的铝化合物类型不受限制，例如可以是氢氧化铝、磷酸铝、硫酸铝或其它本领域已知的铝佐剂类型，只要该唑来膦酸锌铝佐剂是通过使得锌铝离子与膦酸基团、磷酸根和氢氧根产生沉淀制备而成的。
在一个方面，本发明涉及制备唑来膦酸锌佐剂的方法，其包括：
使锌离子在可溶性盐溶液中分别或同时与膦酸基团、磷酸根和氢氧根反应，以沉淀所述锌离子，从而制备唑来膦酸锌佐剂。
在具体的实施方案中，该方法包括：
a.提供包含锌离子的可溶性盐溶液，
b.将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠或将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠、磷酸钠溶液以序列沉淀、分别沉淀再混合或共同沉淀方式均匀混合以获得唑来膦酸锌佐剂。
在本发明的实施方案中，所述可溶性盐溶液通常不受限制，例如可以选自盐酸溶液。
在优选的实施方案中，所述方法还包括将步骤b混合后形成的唑来膦酸锌佐剂悬浊液121℃高温高压灭菌60分钟，待冷却至常温后置于2-8℃，优选4℃储存备用。在一个实施方案中，通过本发明的方法获得的唑来膦酸锌佐剂中的锌:唑来膦酸摩尔浓度比可以是1-8。在优选的实施方案中，锌:唑来膦酸摩尔浓度比选自1、4和8。
在又一个实施方案中，在通过本发明的方法获得唑来膦酸锌铝佐剂的过程中，Al以Zn/Al摩尔浓度比为0.375、0.5和0.8通过不同的混合方式(诸如序列沉淀、分别沉淀再混合或共同沉淀等方式)使得锌离子和铝离子与膦酸基团、磷酸根和氢氧根沉淀而制备成唑来膦酸锌铝佐剂。
如本文所用的锌离子的可溶性盐溶液可以是锌离子任何可溶性盐溶液，优选为锌离子的盐酸溶液。
如本文所用的唑来膦酸盐溶液优选为唑来膦酸氢氧化钠碱溶液。
如本文所用的锌与膦酸基团沉淀方式可以是使锌离子的可溶性盐溶液与唑来膦酸氢氧化钠碱溶液充分混合以发生沉淀反应的任何方式。优选地可以通过序列沉淀、分别沉淀再混合或共同沉淀等方式制备成唑来膦酸锌佐剂。
如本文所用的灭菌可以是适合于对唑来膦酸锌佐剂进行灭菌的任何方式。例如，可以采用高温高压灭菌技术来灭菌，例如在121℃下灭菌30-60分钟，优选60分钟。
在一个实施方案中，本发明还涉及对获得的唑来膦酸锌佐剂进行理化性质的测定。在一个实施方案中，测定了佐剂的pH值、粒径、ZETA电位(Zeta potential)、颗粒零电荷点(PZC，potential of zero charg)、蛋白吸附解离速率、金属离子沉 淀率，有机膦酸的沉淀率、沉淀体内外溶解速率等。佐剂的理化性质可以采用常规技术来测定，例如可参见US9573811；艾绪露等人，3种氢氧化铝佐剂的理化性质分析，《中国生物制品学杂志》，2015，28(1):44-47；以及如本文实施例所述。
在一个实施方案中，如本文所述的唑来膦酸锌佐剂具有以下的一个或多个性质：在灭菌前pH为8.0-9.0，在灭菌后pH为6.0-8.0，粒径为1-10μm，颗粒零电荷点为4.0-11.4，金属离子沉淀率>99％,蛋白吸附率>80％。
在一个方面，本发明涉及包含如本文所述的唑来膦酸锌佐剂的组合物，特别是药物制剂或组合物。
制备药物制剂或组合物的方法包括使唑来膦酸锌佐剂与载体和/或任选的一种或多种辅助成分结合在一起的步骤。
一般而言如下制备制剂：将唑来膦酸锌佐剂与液体载体或粉碎的固体载体或两者均匀地和紧密的结合在一起，然后有必要的话将产品成形。
用于口服施用唑来膦酸锌佐剂的液体剂型包括药学上可接受的乳剂、微乳剂、溶液剂、混悬剂、糖浆剂和酏剂。除了唑来膦酸锌佐剂之外，液体剂型还可含有本领域常用的惰性稀释剂，例如水或其他溶剂、增溶剂和乳化剂，如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苄醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、油(具体而言为棉籽油、花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢糠醇、聚乙二醇、脱水山梨糖醇的脂肪酸酯，以及它们的混合物。
除了惰性稀释剂之外，口服组合物还可包含佐剂如湿润剂、乳化剂和悬浮剂、甜味剂、矫味剂、着色剂、香味剂和防腐剂。
混悬剂制剂除了含有唑来膦酸锌佐剂之外还可含有悬浮剂，例如乙氧基化异十八醇、聚氧乙烯山梨醇和脱水山梨糖醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和黄蓍胶以及它们的混合物。
供直肠或阴道施用的本发明药物组合物的制剂可作为栓剂提供，其可通过将唑来膦酸锌佐剂与一种或多种合适的非刺激性赋形剂或载体(包括例如可可脂、聚乙二醇、栓剂用蜡或水杨酸盐)混合来制备，且在室温下为固体而在体温下为液体，从而在直肠或阴道中会熔化而释放出唑来膦酸锌佐剂。本发明的适合于阴道施用的制剂还包括含有本领域已知适用的载体的阴道栓剂、棉塞、乳膏剂、凝胶剂、糊剂、泡沫剂或喷雾剂。
适合于胃肠外施用的本发明药物组合物包含唑来膦酸锌佐剂和与其相组合的一种或多种药学上可接受的无菌等渗含水或非水载体，包括溶液剂、分散剂、混悬剂或乳剂或者可在临用前重构成无菌可注射溶液剂或分散剂的无菌散剂，其可含有抗氧化剂、缓冲剂、使制剂与预定接受者的血液等渗的溶质或者悬浮剂或增稠剂。
可应用于本发明药物组合物的合适的含水和非水载体的例子包括水、乙醇、多元醇(如甘油、丙二醇、聚乙二醇等)和它们的合适混合物、植物油如橄榄油、以及可注射的有机酯如油酸乙酯。可例如通过使用包衣材料如卵磷脂，通过维持所需的颗粒大小(在分散剂的情况中)，和通过使用表面活性剂，来维持适当的流动性。
这些组合物还可含有辅剂如湿润剂、乳化剂和分散剂。还可能需要在组合物中包含等渗剂，如糖类、氯化钠等。另外，可通过掺入能延迟吸收的物质如单硬脂酸铝和明胶，实现可注射药物形式的延长吸收。
可通过形成唑来膦酸锌佐剂于生物可降解聚合物(如聚丙交酯-聚乙醇酸交酯)中的微囊基质，制备出可注射的长效制剂形式。根据唑来膦酸锌佐剂与聚合物的比例和具体采用的聚合物的性质，可控制唑来膦酸佐剂的释放速度。其他生物可降解聚合物的例子包括聚(原酸酯)和聚(酸酐)。长效可注射制剂也可通过将唑来膦酸锌佐剂包埋在与身体组织相容的脂质体或微乳中来制备。可注射材料可例如通过用细菌保留滤器过滤来进行除菌。
制剂或组合物可以单剂量提供，或者以多剂量密封容器(例如安瓿和小瓶)提供，且可在冻干条件下保存，在临用前仅需要加入无菌液体载体如注射用水。可从上述类型的无菌粉末、颗粒和片剂即时制备注射溶液和混悬剂。
在一个方面，本发明还涉及包含如本文所述的唑来膦酸锌佐剂以及一种或多种抗原的免疫原性组合物。
如本文所用的免疫原性组合物在被施用至受试者或动物时，能引起针对其中所包含的所述一种或多种抗原的保护性免疫应答的组合物。
在一个方面，本发明还涉及包含如本文所述的唑来膦酸锌佐剂以及一种或多种抗原的疫苗组合物。
如本文所用的疫苗组合物在被施用至受试者或动物时，能引起针对例如微生物的保护性免疫应答，或者能有效地保护受试者或动物免受感染。
疫苗组合物可用于防止或改善会对免疫应答调节作出有利响应的病理状况。这样 的疫苗组合物可以是预防性疫苗或治疗性疫苗。优选地，疫苗组合物包括基因工程疫苗，例如蛋白质疫苗，例如水痘带状疱疹病毒重组蛋白疫苗。
在一个方面，本发明还涉及包含如本文所述的唑来膦酸锌佐剂的疫苗佐剂。例如，这样的疫苗佐剂还可以包含如下文所述的次要佐剂。
如本文所用的术语“佐剂”或“疫苗佐剂”意在指能够非特异性地加速、延长或增强对抗原的免疫应答的物质。
有利地，佐剂还可减少进行保护性免疫所需的免疫次数或抗原数量。
众所周知，佐剂本身不会或几乎不会诱发免疫应答，但是佐剂会提高对抗原的免疫应答。因此，可将本发明的唑来膦酸锌佐剂与一种或多种抗原进行组合，以产生可用于在个体中引起免疫应答的免疫原性组合物或疫苗。有多种物质可用作免疫原性类型的或疫苗类型的化合物或制剂中的抗原，例如，减毒的和灭活的病毒和细菌病原体、纯化的大分子、蛋白质、多糖、类毒素、重组抗原、含有来自病原体的外来基因的生物体、合成的肽、聚核酸、抗体和肿瘤细胞等。抗原在预防性和治疗性疫苗中都可使用。抗原包括蛋白质抗原，例如水痘带状疱疹病毒gE糖蛋白抗原(VZV gE)。
多种免疫调节分子也可与本发明的唑来膦酸锌佐剂组合使用，以改变个体中的免疫应答。本文所述的免疫调节剂，是指能调节、平衡及恢复机体免疫功能的一类制剂，常用的免疫调节剂有免疫促进剂、免疫抑制剂及免疫双向调剂三大类。
抗原和唑来膦酸锌佐剂在本发明疫苗组合物中的量及所施用的剂量，通过制药领域技术人员公知的技术来确定，其中要考虑到诸如以下的因素：具体的抗原，具体的动物或患者的年龄、性别、体重、物种和状况，以及施用途径。
在一个优选的实施方案中，本发明的疫苗组合物还包含一种或多种选自以下的组分：表面活性剂、吸收促进剂、吸水聚合物、抑制酶促降解的物质、醇、有机溶剂、油、pH控制剂、防腐剂、渗透压控制剂、抛射剂、水以及它们的任意混合物。
本发明的疫苗组合物还可包含药学上可接受的载体。载体的量将取决于其他成分所选定的量、抗原的所需浓度、施用途径的选择(口服还是胃肠外途径)等。载体可在任何方便的时间加到疫苗。在冻干的疫苗的情况中，可例如在临施用前加入载体。或者，最终产品可制造成带有载体。
适当的载体的例子包括但不限于无菌水、盐水、缓冲液、磷酸缓冲盐水、缓冲氯化钠、植物油、最小必需培养基(MEM)、具有HEPES的MEM，等等。
任选地，本发明的疫苗组合物可含有不同数量的常规次要佐剂，数量取决于该佐剂和所需的结果。
合适的次要佐剂的例子包括但不限于稳定剂；乳化剂；pH调节剂，如氢氧化钠、盐酸等；表面活性剂，如Tween.RTM.80(聚山梨醇酯80，可从Sigma Chemical Co.(St.Louis,Mo.)市售获得)；脂质体；iscom佐剂；合成的糖肽，如胞壁酰二肽；增量剂，如右旋糖苷；聚羧乙烯；细菌细胞壁，如分枝杆菌细胞壁提取物；它们的衍生物，如短小棒状杆菌(Corynebacterium parvum)；痤疮丙酸杆菌(Propionibacterium acne)；牛分支杆菌(Mycobacterium bovis)，例如卡介菌(Bovine Calmette Guerin，BCG)；牛痘或动物痘病毒蛋白；亚病毒颗粒佐剂，如环状病毒；霍乱毒素；N,N-双十八烷基-N',N'-双(2-羟乙基)-丙二胺(吡啶)；单磷酰脂质A；二甲基双十八烷基溴化铵(DDA，可从Kodak公司(Rochester,N.Y.))市售获得；它们的合成物和混合物。
合适的稳定剂的例子包括但不限于蔗糖、明胶、蛋白胨、消化的蛋白质提取物如NZ-胺或NZ-胺AS。乳化剂的例子包括但不限于矿物油、植物油、花生油和其他可用于注射剂或鼻内疫苗组合物的标准、可代谢、无毒的油。
出于本发明的目的，这些佐剂在本文指称为“次要”仅仅是为了与上述的唑来膦酸锌佐剂形成对比，因为唑来磷膦酸锌佐剂与抗原物质相组合可显著提高针对该抗原物质的免疫应答从而其作为疫苗组合物中的必要成分。次要佐剂主要是作为加工助剂包括在疫苗制剂中，尽管某些佐剂的确具有一定程度的免疫增强性质从而具有双重目的。
常规的防腐剂可以以约0.0001％(重量)至约0.1％(重量)的有效量加到疫苗组合物。取决于制剂中所采用的防腐剂，这个范围以下或以上的数量也可使用。典型的防腐剂包括例如山梨酸钾、焦亚硫酸钠、苯酚、羟苯甲酯、羟苯丙酯、硫柳汞等。
失活的、修饰的或其他类型的疫苗组合物的选择以及本发明的改进的疫苗组合物制剂的制备方法，是本领域普通技术人员知道的或者容易确定的。
一般而言，本发明的疫苗组合物便利地通过口服、胃肠外(皮下、肌肉内、静脉内、真皮内、腹腔或腹膜内)、口腔内、鼻内或透皮途径施用。本发明所设想的施用途径将取决于抗原物质和辅助剂。例如，如果疫苗组合物含有皂苷类，虽然它们口服或鼻内施用时是无毒的，但必须小心不要将皂苷元糖苷注射到血流中，因为它们起到 强溶血剂的作用。还有，许多抗原如果口服的话将不会有效。优选地，疫苗组合物通过肌肉或腹腔途径施用。
疫苗组合物的剂量将显著取决于所选择的抗原、施用途径、物种和其他标准因素。设想到，本领域普通技术人员可很容易地滴定出用于针对每种抗原的免疫应答的适当剂量，以获知有效的免疫量和施用方法。
作为疫苗佐剂，本发明的唑来膦酸锌佐剂可通过增强较弱的抗原(如高度纯化的或重组的抗原)的免疫原性、减少免疫应答所需的抗原的量、减少提供保护性免疫所需的免疫频率来提高疫苗的有效性，可改进疫苗在免疫应答减低或弱化的个体(如新生儿、老年人和免疫受损的个体)中的功效，和可增强在靶标组织中的免疫性，或者通过引发特定的细胞因子谱来促进细胞介导的免疫和/或体液免疫。
抗原和/或免疫调节分子与本发明的唑来膦酸锌佐剂的组合，可在本领域公知的多种临床前毒理学和安全性研究中进行试验。
例如，可在这样的动物模型中评价这种组合：该抗原已被发现在该动物模型中具有免疫原性，且该动物模型可通过与提议用于人临床试验的途径相同的途径可再现地进行免疫。
抗原和/或免疫调节分子与本发明的唑来膦酸佐剂的组合，可例如通过由美国食品与药物管理局生物制品评价与研究中心与美国国家过敏症与传染病研究所(Goldenthal,KL等人.AID Res Hum Retroviruses,9:S45-9(1993))提出的方案进行试验。
本领域技术人员会知道如何为抗原和/或免疫调节分子与本发明的复合佐剂的具体组合确定适当的抗原负荷、免疫途径、剂量、抗原纯度和可用于在特定动物物种中治疗特定病理状况的接种方案。
用于诱导免疫应答的本发明免疫原性组合物或疫苗，可与药学上可接受的介质一起作为溶液或悬浮液施用。
这种药学上可接受的介质可为例如水、磷酸缓冲盐水、生理盐水或其他生理缓冲盐水、或者其他溶剂或介质，如二元醇、甘油和油(如橄榄油)或可注射有机酯。药学上可接受的介质还可含有脂质体或胶束，且可含有通过将多肽或肽抗原与去污剂和糖苷(如Quil A)混合在一起制备的免疫刺激复合物。
本发明的免疫原性组合物或疫苗可通过多种途径施用以刺激免疫应答。例如，免 疫原性组合物或疫苗可通过皮下、真皮内、淋巴内、肌肉内、肿瘤内、膀胱内、腹腔、腹膜内和脑内途径递送。
本领域技术人员会知道如何选择用于本发明唑来膦酸锌佐剂的具体制剂的适当递送途径。
在本发明的一个优选实施方案中，用于在哺乳动物中治疗或预防感染的接种方法包括使用本发明的疫苗，该疫苗具体而言将通过肌肉或腹腔施用。疫苗可作为单独剂量施用，或者优选地根据初次免疫/加强免疫模式每周或每月施用数次，例如两次、三次或四次。适当的剂量根据各种参数而定。
在一个方面，本发明还涉及如本文所述的唑来膦酸锌佐剂用于制备疫苗佐剂、药物组合物、免疫原性组合物或疫苗组合物的用途，优选地，所述疫苗包括蛋白质疫苗，例如水痘带状疱疹病毒蛋白重组疫苗。
附图说明
图1：三种制备佐剂工艺示意图。
图2：唑来膦酸锌、唑来膦酸锌铝、唑来膦酸铝佐剂颗粒形态
图3：唑来膦酸锌佐剂在VZV gE疫苗中的佐剂活性测定，平均值±SD：n＝5，*p<0.05；**p<0.01；***p<0.001，****p<0.0001。
图4：唑来膦酸锌佐剂联合重组VZV gE蛋白对小鼠抗体亚型的影响，平均值±SD：n＝5，*p<0.05；**p<0.01；***p<0.001，****p<0.0001。
图5：唑来膦酸锌佐剂在乙肝治疗性蛋白上的佐剂活性测定，平均值±SD：n＝5，*p<0.05；**p<0.01；***p<0.001，****p<0.0001。
图6：其他双膦膦酸锌佐剂(Zn/Zol＝1/0.25)配伍VZV gE对小鼠血清抗体滴度的影响，平均值±SD：n＝5，*p<0.05；**p<0.01；***p<0.001，****p<0.0001。
图7：唑来膦酸、唑来膦酸锌修饰佐剂配伍乙肝治疗性蛋白对小鼠血清抗体滴度的影响，平均值±SD：n＝5，*p<0.05；**p<0.01；***p<0.001，****p<0.0001。
具体实施方式
下面结合实施例对本发明实施方案进行详细描述，实施例中的未注明具体条件者，按照常规条件或制作商建议的条件进行。所用试剂或仪器未注明生产商者，均为 可以通过市购获得的常规产品。
下述制备例所使用的材料如下：
无水氯化锌(ZnCl 2)：购自西陇化工；
六水氯化铝(AlCl 3·6H 2O)：购自西陇化工；
十二水合磷酸氢二钠(Na 2HPO 4·12H 2O)：购自西陇化工；
氢氧化钠(NaOH)：购自西陇化工；
唑来膦酸(C 5H 10N 2O 7P 2)：购自湖南华腾制药有限公司
制备例1：Zn-唑来膦酸(1/0.25)佐剂的制备
按照1：0.25的Zn/唑来膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(7.78mM唑来膦酸+43mM氢氧化钠+7.78mM磷酸氢二钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
Zn-唑来膦酸(1/0.25)佐剂悬浊液的制备：
将A和B溶液按照图1A所示的方式形成唑来膦酸锌佐剂悬浊液，即将配制好的B溶液，按照体积比1：1的方式逐滴直至全部加入A溶液形成的悬浊液。
对图1A混合后获得的利塞膦酸锌佐剂进行一次121℃，60min高压蒸汽灭菌，冷却至室温后置于2-8℃中储存。
制备例2：Zn-唑来膦酸(1/1)佐剂的制备
溶液配制：
按照1：1的Zn/唑来膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(31.11mM唑来膦酸+87mM氢氧化钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-唑来膦酸(1/1)佐剂悬浊液的制备。
制备例3：唑来膦酸锌铝(FH-001-唑来膦酸)佐剂的制备
溶液配制：
按照1：0.375的Al/Zn的摩尔浓度，配制50mL氯化铝(122.44mM)氯化锌(46.66mM)混合溶液，定义为A溶液；配制50mL(18.66mM唑来膦酸+380mM氢 氧化钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行FH-001-唑来膦酸佐剂悬浊液的制备。
制备例4：唑来膦酸铝(Al-001-唑来膦酸)佐剂的制备
溶液配制：
按照1:0.075的Al/Zol的摩尔浓度，配制50mL122.44mM氯化铝溶液，定义为A溶液；配制50mL(9.33mM唑来膦酸+9.33mM十二水磷酸氢二钠+370mM氢氧化钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Al-001-唑来膦酸佐剂悬浊液的制备。
制备例5：Zn-唑来膦酸(1/0.125)的制备
溶液配制：
按照1：0.125的Zn/唑来膦酸摩尔浓度比，配制50mL 124.44mM的氯化锌溶液，定义为A溶液；配制50mL(15.56mM唑来膦酸+65.7mM氢氧化钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-唑来膦酸(1/0.125)佐剂悬浊液的制备。
制备例6：铝佐剂(Al-002)的制备
溶液的配制：
采用六水氯化铝加水配制成62.22mM的氯化铝溶液0.5L，定义为A溶液；采用二水合磷酸氢二钠和氢氧化钠加水配制成含18.68mM磷酸盐和0.12M氢氧化钠溶液0.5L，定义为B溶液，0.22μm滤膜过滤备用；
参考制备例1进行铝佐剂Al-002的制备。
制备例7：铝佐剂(Al-001-840)的制备
溶液配制：
按照0.15的磷酸根/Al摩尔浓度配比，配制0.5L 124mM的氯化铝溶液，定义为A溶液；配制0.5L 18.6mM浓度的磷酸氢二钠溶液，定义为B溶液，B溶液中含有150mM氢氧化钠，0.22μm滤膜过滤备用。
参考制备例1进行铝佐剂Al-001-840悬浊液的制备。
制备例8：锌铝佐剂(FH-001)的制备
溶液配制：
采用六水氯化铝和无水氯化锌加水配制成含124.44mM氯化铝和46.68mM氯化锌的溶液0.25L，定义为A溶液；采用二水合磷酸氢二钠和氢氧化钠加水配制成含18.68mM磷酸盐和0.425M氢氧化钠的溶液0.25L，定义为B溶液，采用0.22μm滤膜过滤备用；
铝复合佐剂的制备：
按照图1A所示，将0.25L B溶液往0.25L A溶液里逐滴滴加，直至完全加完，获得锌铝复合佐剂FH-001，对获得的锌铝复合佐剂进行一次121℃、60min的高压蒸汽灭菌，在室温冷却后储存在2-8℃中。
制备例9：Zn-依替膦酸(1/0.25)佐剂的制备
按照1：0.25的Zn/依替膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(7.78mM依替膦酸+34mM氢氧化钠+7.78mM磷酸氢二钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-依替膦酸(1/0.25)佐剂悬浊液的制备。
制备例10：Zn-氯屈膦酸(1/0.25)佐剂的制备
按照1：0.25的Zn/氯屈膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(7.78mM氯屈膦酸+36mM氢氧化钠+7.78mM磷酸氢二钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-氯屈膦酸(1/0.25)佐剂悬浊液的制备。
制备例11：Zn-阿伦膦酸(1/0.25)佐剂的制备
按照1：0.25的Zn/阿伦膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(7.78mM阿伦膦酸+36mM氢氧化钠+7.78mM磷酸氢二钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-阿伦膦酸(1/0.25)佐剂悬浊液的制备。
制备例12：Zn-帕米膦酸(1/0.25)佐剂的制备
按照1：0.25的Zn/帕米膦酸摩尔浓度比，配制50mL 31.11mM的氯化锌溶液，定义为A溶液；配制50mL(7.78mM帕米膦酸+32mM氢氧化钠+7.78mM磷酸氢二钠)溶液，定义为B溶液，A溶液和B溶液用0.22μm滤膜过滤备用。
参考制备例1进行Zn-帕米膦酸(1/0.25)佐剂悬浊液的制备。
下面结合实施例对本发明做进一步描述。
实施例1：唑来膦酸锌佐剂的理化性质测定
以下检测方法适用于任何一种Zn/唑来膦酸摩尔配比，即任何一种唑来膦酸锌佐剂，例如掺杂无机磷酸或掺杂Al的唑来膦酸锌佐剂；
(1)佐剂颗粒形态观察
将唑来膦酸锌佐剂用去离子水稀释50倍后，利用日本Electronics的JEM-2100透射电子显微镜(Transmission Electron Microscopy,TEM)进行观察。具体的步骤如下：将佐剂样品滴于渡炭的铜网上，令其吸附10min，用滤纸擦去残液，送入透射电镜的样品室里，观察样品形态，拍照以备进一步分析。
实验结果：Zn-唑来膦酸(1/0.25)与Al-001-唑来膦酸佐剂可以清晰看到球形纳米核心颗粒，而FH-001-唑来膦酸佐剂呈现紫菜团状(图2)。
(2)pH测定
取供试品，放置室温平衡至少30分钟，利用Sartorius pH玻璃电极进行测量。
选择标准缓冲液(pH7.00)、标准缓冲液(pH4.01)、标准缓冲液(pH10.01)按照使用说明书要求校正仪器。
按“Mode”(转换)键可以在pH和mV模式之间进行切换。通常测定溶液pH值将模式至于pH状态。
按“SETUP”键，显示屏显示Clear buffer，按“ENTER”键确认，清除以前的校准数据。
按“SETUP”键直至显示屏显示缓冲溶液组“4.01，7.00，10.01”，按“ENTER”键确认。
将电极小心从电极储存液中取出，用去离子水充分冲洗电极，冲洗干净后用滤纸吸干表面水(注意不要擦拭电极)。
将电极浸入第一种缓冲溶液(pH 7.00)，等到数值稳定并出现“S”时，按“STANDARDIZE”键，等待仪器自动校准。校准成功后，将显示“7.00”和电极斜率。
将电极从第一种缓冲溶液中取出，用去离子水充分淋洗电极，将电极依次浸入第二种缓冲溶液(pH 4.01)，等到数值稳定并出现“S”时，按“STANDARDIZE”键，等待仪器自动校准。校准成功后，将显示“4.01 7.00”和信息“Slope”。Slope显示测量的电极斜率值，该测量值在90-105％范围内可以接受。
如果与理论值有很大偏差，将显示错误信息(Err)，电极应清洗，并重复上述步骤校准。
重复以上操作完成第三点(pH 10.01)校准。
校准完成后，用去离子水充分淋洗电极，然后滤纸轻轻吸干。摇匀供试品溶液，将玻璃电极浸入供试品溶液中，至pH值的读数在1分钟内改变不超过±0.05时进行读数。
摇匀供试品溶液，重新测量，两次pH值的度数相差应不超过0.1。取两次读数的平均值作为供试品pH值。
实验结果(表1)：
Zn-唑来膦酸佐剂灭菌前pH的范围为8.0-9.0，灭菌后为6.5-7.0；
(3)粒径大小的测定
打开Beckman LS 13320激光粒径测试仪，预热15min。
打开仪器控制软件和样品池封闭舱，将样品池从样品槽中取出并加入12mL去离子水中。
将样品池置于样品槽上，关闭舱门。
点击“start cycle”，依次选择“Measure Offsets”、“Align”、“Measure Background”，最后点击“start”，在弹出的对话框中点击“OK”开始校正空白背景。
取出样品池，加入一定质量的标准样品(仪器自带)，点击“start cycle”，依次选择“Measure Loading”、“Enter Sample Info”、“Enter run setting”、“start runs”，最后点击“start”，在弹出的对话框中输入标准样品名称，待软 件中“Obscuration”参数为8％-12％时点击“OK”，进行标准样品的测试。
为保证实验数据的准确性和可靠性，每次开机测量之前均需校正空白背景和测试标准样品的大小。
打开样品池封闭舱，取出样品池。
将样品池内含有标准样品的水溶液倒弃，倒入去离子水清洗样品池3遍。
清洗完毕后加入12mL去离子水，将样品池置于样品槽上，关闭舱门。
点击“start cycle”，依次选择“Measure Offsets”、“Align”、“Measure Background”，最后点击“start”，在弹出的对话框中点击“OK”开始校正空白背景。
取出样品池，加入一定体积的检测样品，打开样品测试舱门，将
样品池置于样品槽上，关闭舱门。
点击“start cycle”，选择“Enter Sample Info”、“Enter run setting”、“start runs”，最后点击“start”，在弹出的对话框中输入样品名称，待软件中“Obscuration”参数为8％-12％时点击“OK”，记录样品测得的粒径大小；
实验结果
唑来膦酸锌佐剂颗粒大小在0.4-30μm之间，而大部分颗粒大小为6-7μm。
表1：几个批次的Zn-唑来膦酸(1/0.25)佐剂的pH/粒径大小
Figure PCTCN2020084190-appb-000001
(4)吸附率检测
BSA标准品绘制标准曲线：150mM NaCl为稀释缓冲液，将BSA标准品(2mg/mL)进行系列稀释，用UV2100pro检测其在280nm处的吸光值，OD280在0.2-0.8(放宽至0.2-1.5)之间准确度高。
BSA梯度稀释(EP)：150mM NaCl为稀释缓冲液，称取一定量BSA样品，稀释至EP中规定的浓度梯度：0.5mg/mL、1mg/mL、2mg/mL、3mg/mL、5mg/mL、10mg/mL，待用。
BSA与佐剂混合:BSA:佐剂＝3:1(体积比)做为吸附率检测使用条件；将佐剂摇匀后，按照实验条件与不同浓度BSA分别进行混合，室温吸附1h，期间摇晃5次；用13000rpm/min离心3min后取上清，待用。
蛋白浓度测定：EP中蛋白浓度测定采用Lowry法，结合实际情况，本实验中采用UV2100pro直接测定上清在280nm处的吸光值，使读值在0.2-0.8之间，否则需进行稀释。
吸附率计算：吸附率＝[1-OD 280(上清稀释倍数X))/OD 280(稀释X倍吸附率为0时)]*100
实验结果如下表：
表2：几个批次的Zn-唑来膦酸(1/0.25)佐剂的蛋白吸附能力
Figure PCTCN2020084190-appb-000002
(5)PZC检测
仪器检测：Nanobrook Omni(Brookhaven)
实验操作：使用0.1M NaOH/1％HNO 3将Zn-唑来膦酸(1/0.125)的pH调节至：6.00/5.50/5.00/4.50/4.00/3.50/3.00/2.50/2.00。
钝化电极：向样品管中加入3-4mL佐剂，电极插入后，SOP中将cycle设置为：50，运行仪器，使得电极钝化。
样品检测：电极取出后，去离子水冲洗下端，加入相应样品，SOP中调节cycle设置为：15，measure设置为：3，pH设置为各样品对应的pH，运行仪器。
数据处理：得到不同pH值下对应的Zeta电位，运行仪器自带软件，得到PZC值
结果：Zn-唑来膦酸(1/0.125)佐剂的PZC为4.08。
实施例2：Zn-唑来膦酸(1/0.125)佐剂锌沉淀率测定
采用火焰法(D2灯背景校正)测定唑来膦酸锌佐剂中锌含量并规范测定操作流程。检测仪器为原子吸收分光光度计：岛津AA6300C(P/N 206-52430)；
标准品溶液及待测样品的配制：标准曲线的配制：锌标准品原始浓度500μg/mL，利用0.1M盐酸溶液将其稀释成500ng/mL、1000ng/mL、1500ng/mL、2000ng/mL和2500ng/mL的标准品。
将Zn-唑来膦酸(1/0.125)佐剂13000r/min，离心10min后去上清用0.1M硝酸溶液将样品进行5倍稀释后进行检测，需用涡旋混和仪振荡混匀，检测过程如下：
AA-6300C操作方法及WizAArd软件使用方法：
打开电源：计算机开机、AA-6300C电源开关、空气压缩机开关(保证压力为0.35MPa)、通风系统开关；
打开乙炔：缓慢打开乙炔气阀，保证初级压力为0.5MPa，次级压力为0.1MPa；
WizAArd软件基本操作：登录WizAArd→选择元素→“未连接仪器/发送参数”页面，点击<连接/发送参数>→在“仪器初始化”页面中进行设置→在“火焰分析的仪器检查目录”中逐项检查并打钩，点击<OK>→在“光学参数”页面中设定波长[213.86]，狭缝宽[0.7]，点灯方式[发射]，[灯位设置]保证Zn空心阴极灯的实际位置与设定位置相同，选择[点灯]→谱线搜索→燃烧器原点位置调节→在菜单栏中选择[参数]→[编辑参数]→将点灯方式改为<BGC-D2>→谱线搜索→点火：确保C2H2打开且压力达到要求后，同时按下主机上的PURGE和IGNITE键，直至点火→自动调零→在MRT工作单上设置空白组(BLK)、标准品(STD)、待测样品(UNK)，输入标准品的理论浓度以及样品名称，通过雾化器延伸出来的上样管进行手动上样，每次上样量最少1mL，选择[开始]进行检测→熄火→保存数据并断开仪器与计算机的连接→关机；
实验结果：
表3：锌浓度与Abs标准曲线的绘制
Figure PCTCN2020084190-appb-000003
表4：Zn-唑来膦酸(1/0.125)佐剂中锌沉淀率检测结果
Figure PCTCN2020084190-appb-000004
实施例4：：Zn-唑来膦酸(1/0.125)佐剂唑来膦酸沉淀率测定
方法：紫外分光光度法，仪器：UV800(Beckman coulter)。
唑来膦酸内含咪唑环，在215nm处有最大吸收峰，通过紫外分光光度计，在215nm检测佐剂的上清唑来膦酸的具体过程如下：
首先，利用生理盐水配制0.04mg/mL，0.03mg/mL，0.02mg/mL,0.015mg/mL,0.0075mg/mL,0.00375mg/mL标准唑来磷酸钠溶液，并分别测定在215nm波长下的OD 215，绘制标准曲线。同时，我们将Zn-唑来膦酸(1/0.125)，13000rpm离心10min后取上清，在215nm波长处检测上清中唑来膦酸钠的吸光度，结果如下：
表5：唑来膦酸钠浓度与OD 215标准曲线
Figure PCTCN2020084190-appb-000005
表6：Zn-唑来膦酸(1/0.125)佐剂唑来膦酸沉淀率检测结果
Figure PCTCN2020084190-appb-000006
实施例5：Zn-唑来膦酸佐剂在重组蛋白VZV gE疫苗中的佐剂活性测定
将制备好的唑来膦酸锌佐剂(也被称为Zn-唑来膦酸)作为佐剂，其中Zn/唑来膦酸的摩尔浓度比1:0.25或1:1，将其作为佐剂与VZV gE抗原二者联用肌肉注射小 鼠，测定产生的特异性抗体滴度。具体方法如下：
实验动物：Balb/C小鼠，6-8周，5只/组，雌性。
药物使用浓度：抗原：50μg/ml；
给药剂量：5μg/50μl/鼠；铝佐剂：50μl/鼠；唑来膦酸锌佐剂：50μl/鼠；
实验组：(1)基础铝佐剂组(Al-002)；(2)Zn-唑来膦酸(1/0.25)；(3)Zn-唑来膦酸(1/1)；
免疫方案：动物按照免疫分组首次免疫后2周，眼眶采血，测定血清中抗体滴度。首次免疫注射后第二周检测抗体滴度，第2周加强免疫，二次免疫后2周，眼眶取血，测定血清中抗体滴度，并进行第3次免疫，2周后ELISA测定血清中的抗体滴度。
酶联免疫吸附法(ELISA)检测抗体结合滴度：
1.抗原包被液：1X PB 7.4缓冲液(4.343g Na 2HPO 4·7H 2O；0.456g NaH 2PO 4)。
2.洗涤液：PBST，北京万泰公司ELISA试剂盒
3.封闭液2X ED(Enzyme Dilution)：1X PBS+0.5％酪蛋白+2％明胶+0.1％防腐剂(proclin-300)，用超纯水或蒸馏水稀释至1X后用于封闭及样品稀释。
4.显色液A：北京万泰公司ELISA试剂盒。
5.显色液B：北京万泰公司ELISA试剂盒。
6.终止液：北京万泰公司ELISA试剂盒。
实验过程：
(1)包板：用PB7.4包被缓冲液将VZV gE抗原稀释至一定浓度，一般为100ng/孔，100μL/孔加至聚苯乙烯96孔板中，4℃包被过夜。
(2)封闭：弃去孔内包被液，PBST洗液洗板1次，甩干，加封闭液200μL/孔，室温封闭4h。
(3)加一定稀释度的血清：弃去孔内封闭液，PBST洗板1次，甩干，首孔加待检血清150μL/孔，后面的每孔加入100μl ED稀释液，3倍梯度稀释，25℃孵育反应1h。
(4)加酶标抗体(GAM-HRP)：弃去孔内血清稀释液，PBST洗板5次，甩干，加酶标抗体(GAM-HRP，V：V＝1：5000)100μL/孔，25℃孵育反应1h。
(5)显色：弃去孔内二抗，PBST洗板5次，甩干，以100μL/孔加入A、B等 体积混匀的显色液，25℃反应10min。
(6)终止：50μL/孔加2M硫酸终止液，终止反应。
(7)读板：在酶标仪上设置检测双波长为450nm和630nm，测各反应孔的OD值。
实验结果如图3所示：
佐剂与抗原等体积混合，免疫小鼠，按照上述的免疫策略进行，免疫和采血以及血清抗体滴度检测；唑来膦酸锌佐剂组，小鼠免疫一针后，其具有起效快且强的特点，免疫2针后，其体液免疫增强优势与铝佐剂组相比，差异具有统计学意义，三针免疫后差异仍然显著。
实施例6：唑来膦酸锌佐剂联合重组蛋白VZV gE对小鼠抗体亚型的影响
免疫策略参考实施例5。
实验材料：
1.抗原包被液：1X PB 7.4缓冲液(4.343g Na 2HPO 4·7H 2O；0.456g NaH 2PO 4)。
2.洗涤液：PBST，北京万泰公司ELISA试剂盒
3.封闭液2X ED(Enzyme Dilution)：1X PBS+0.5％酪蛋白+2％明胶+0.1％防腐剂(proclin-300)，用超纯水或蒸馏水稀释至1X后用于封闭及样品稀释。
4.显色液A：北京万泰公司ELISA试剂盒。
5.显色液B：北京万泰公司ELISA试剂盒。
6.终止液：北京万泰公司ELISA试剂盒。
实验过程：
(1)包板：用PB7.4包被缓冲液将VZV gE抗原稀释至一定浓度，100μL/孔加至聚苯乙烯96孔板中，4℃包被过夜。
(2)封闭：弃去孔内包被液，PBST洗板1次，甩干，加封闭液200μL/孔，室温封闭4h。
(3)加待检血清：血清抗体亚型检测所用血清：完成3针免疫2周后，即第6周的血清，弃去孔内封闭液，PBST洗板1次，甩干，加一定稀释度的待检血清100μL/孔， 25℃孵育反应1h。
(4)加酶标抗体：弃去孔内血清稀释液，PBST洗板5次，甩干，加特异性识别各抗体亚型的酶标抗体(IgG1，V:V＝1:30000；IgG2a，V:V＝1:1000；IgG2b，V:V＝1:1000)100μL/孔，25℃孵育反应1h。
(5)显色：弃去孔内酶标抗体，PBST洗板5次，甩干，将显色液A、B等体积混匀后稀释三倍，100μL/孔，25℃反应10min。
(6)终止：加2M硫酸终止液50μL/孔，终止反应。
(7)读板：在酶标仪上设置检测双波长为450nm和630nm，测各反应孔的OD值。
实验结果如图4所示：
采用实施例5所述的实验流程，将唑来膦酸锌佐剂或铝佐剂配伍重组VZV gE蛋白通过肌肉注射免疫小鼠，免疫程序同实施例5，免疫3针后再2周采血检测，小鼠血清抗体各亚类水平，结果如图4，与铝佐剂组相比，唑来膦酸锌佐剂组能激起更为强烈的IgG2a和IgG2b亚型抗体水平，且IgG1与IgG2a和IgG2b的比值较铝佐剂组更低，表明起在Th1免疫途径具有一定的刺激作用。
实施例7：唑来膦酸锌佐剂在乙肝治疗性蛋白中的佐剂活性测定
采用实施例5所述的实验流程，将唑来膦酸锌佐剂配伍乙肝治疗性蛋白通过肌肉注射来免疫小鼠，检测血清抗体滴度。如图5所示的结果：与铝佐剂组相比，唑来膦酸锌佐剂组，在小鼠免疫一针后，具有起效快且强的特点，差异具有统计学意义；免疫2针后，其体液免疫增强优势与铝佐剂组相比，差异仍然显著，三针免疫后略优于铝佐剂组。
实施例8：其他双膦膦酸锌佐剂配伍VZV gE对小鼠血清抗体滴度的影响
采用实施例5所述的实验流程，将双膦酸锌佐剂配伍重组VZV gE蛋白通过肌肉注射来免疫小鼠，检测血清抗体滴度。如图6所示的结果显示：唑来膦酸锌佐剂组，在小鼠免疫三针后，其体液免疫增强优势与铝佐剂组相比，差异具有显著性，而其他的双膦酸锌佐剂效果与铝佐剂相比无明显差别。
实施例9：唑来膦酸锌佐剂配伍VZV gE对小鼠血清抗体滴度的影响
采用实施例5所述的实验流程，将唑来膦酸锌佐剂配伍VZV gE通过肌肉注射来免疫小鼠，检测血清抗体滴度。如图7所示，铝佐剂、复合锌铝佐剂、掺杂唑来膦酸的复合锌铝佐剂、唑来膦酸锌佐剂在抗体增强水平上，呈现逐级递增趋势，唑来膦酸锌佐剂与铝佐组相比，差异显著。

Claims (8)

  1. 一种唑来膦酸锌微纳米颗粒佐剂，其包含锌及唑来膦酸，其中锌:唑来膦酸摩尔浓度比选自1、4和8；任选地，其还包含磷酸盐，其中锌:磷酸根摩尔浓度比选自1.5、4；任选地，其还包含铝，其中锌:铝摩尔浓度比选自0.375、0.5和0.8。
  2. 权利要求1的唑来膦酸锌微纳米颗粒佐剂，其中所述唑来膦酸锌微纳米颗粒佐剂通过包括以下步骤的方法来制备：
    a.提供包含锌离子的可溶性盐溶液，优选地，所述盐溶液包括盐酸溶液，
    b.将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠或将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠、磷酸钠溶液以序列沉淀、分别沉淀再混合或共同沉淀方式均匀混合以获得唑来膦酸锌佐剂，
    优选地，还包括将步骤b混合后的溶液灭菌，并于2-8℃储存备用，优选地，所述灭菌包括采用高温高压灭菌技术的灭菌，例如在121℃下灭菌30-60分钟，优选60分钟。
  3. 权利要求1-2中任一项的唑来膦酸锌微纳米颗粒佐剂，其具有以下的一个或多个性质：
    在灭菌前pH为8.0-9.0，
    在灭菌后pH为6.0-8.0，
    粒径为1-10μm，
    颗粒零电荷点为4.0-11.4，
    蛋白吸附率>80％，优选地，所述蛋白包括牛血清白蛋白。
  4. 一种制备唑来膦酸锌微纳米颗粒佐剂的方法，其包括：
    a.提供包含锌离子的可溶性盐溶液，优选地，所述盐溶液包括盐酸溶液，
    b.将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠或将步骤a的可溶性盐溶液与唑来膦酸、氢氧化钠、磷酸钠溶液以序列沉淀、分别沉淀再混合或共同沉淀方式均匀混合以获得唑来膦酸锌佐剂，
    优选地，还包括将步骤b混合后的溶液灭菌，并于2-8℃储存备用，优选地，所述灭菌包括采用高温高压灭菌技术的灭菌，例如在121℃下灭菌30-60分钟，优选60分钟，
    其中获得的唑来膦酸锌佐剂中的锌:唑来膦酸摩尔浓度比选自1、4和8。
  5. 权利要求4的方法，其中获得的唑来膦酸锌微纳米颗粒佐剂具有以下的一个或多个性质：
    在灭菌前pH为8.0-9.0，
    在灭菌后pH为6.0-8.0，
    粒径为1-10μm，
    颗粒零电荷点为4.0-11.4，
    蛋白吸附率>80％，优选地，所述蛋白包括牛血清白蛋白。
  6. 一种疫苗佐剂、药物组合物或免疫原性组合物，其包含权利要求1-3中任一项的唑来膦酸锌微纳米颗粒佐剂。
  7. 一种疫苗组合物，其包含抗原和权利要求1-3中任一项的唑来膦酸锌微纳米颗粒佐剂，
    优选地，所述抗原包括蛋白质抗原，例如水痘带状疱疹病毒gE糖蛋白抗原。
  8. 权利要求1-3中任一项的唑来膦酸锌微纳米颗粒佐剂用于制备疫苗佐剂、药物组合物、药物递送载体、免疫原性组合物或疫苗组合物的用途，
    优选地，所述疫苗包括蛋白质疫苗，例如水痘带状疱疹病毒重组蛋白疫苗。
PCT/CN2020/084190 2019-04-11 2020-04-10 唑来膦酸锌微纳米颗粒佐剂的制备及作为疫苗佐剂的用途 WO2020207472A1 (zh)

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BR112021020377A BR112021020377A2 (pt) 2019-04-11 2020-04-10 Preparação de adjuvante de micro/nanopartícula de zoledronato de zinco e uso do mesmo como adjuvante de vacina
US17/602,214 US20220193231A1 (en) 2019-04-11 2020-04-10 Preparation of zinc zoledronate micro-nanoparticle adjuvant and use thereof as vaccine adjuvant
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