Preparation of zinc zoledronate micro / nanoparticle adjuvant and its use as a vaccine adjuvant

Abstract

A zinc zoledronic acid micro-nanoparticle adjuvant containing zinc and zoledronic acid, optionally containing phosphate and aluminum, is disclosed. Its preparation method involves co-precipitation of a soluble salt solution containing zinc ions, zoledronic acid, and sodium hydroxide. The adjuvant can be used in the preparation of vaccines, etc.

Description

[Technical field]

[0001] The present invention relates to a vaccine adjuvant, particularly a zinc zoledronate micro / nanoparticle adjuvant with sustained release effect, which can significantly enhance humoral and cellular immune responses and can be used as a vaccine adjuvant in vaccine preparation.The present invention relates to the field of immunology. [Background technology]

[0002] Zinc is an important chemical element with unique biological functions. For example, trace levels of zinc are present in about 300 enzymes involved in cellular metabolism, and play important roles in cellular respiration, cell division, protein synthesis, DNA synthesis, damage repair, and immune function activation. Thus, zinc deficiency or disorders are associated with many diseases, especially those of the immune system (Non-Patent Document 1). Within immune cells, divalent cations including zinc play an important role in regulating intracellular signaling pathways. Zinc deficiency affects lymphocyte survival, proliferation, and maturation in innate and adaptive immunity. Studies have reported that zinc deficiency adversely affects immune cell cytokine production and function, as well as the number, proliferation, and differentiation of peripheral and thymic T cells. Studies have found that trace zinc deficiency causes immunosenescence in the elderly (Non-Patent Document 2). In brief, zinc plays an important role in the production, development, and function of T cells.

[0003] Bisphosphonates (BP), the most important drugs for the treatment of osteoporosis, are inorganic pyrophosphate analogs. Bisphosphonates are formed by replacing the oxygen atom in the POP bond of pyrophosphate with a carbon atom and have a unique PCP bond skeleton. Bisphosphonates are not easily hydrolyzed by enzymes in the body, are resistant to high temperatures, and are stable against many chemicals. Meanwhile, the PCP molecular configuration allows its three-dimensional structure to 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. Zoledronic acid is characterized by strong efficacy, low dosage, and ease of use. Clinical studies have shown that bisphosphonates, used as adjuvant therapy for diseases such as multiple myeloma, breast cancer, renal cancer, and prostate cancer, can reduce the incidence of bone-related diseases in patients, reduce the recurrence rate of cancer, and extend the survival time of patients, thereby improving clinical outcomes. Zoledronate is particularly advantageous in terms of antitumor effect, and is widely used in clinical studies of various malignant tumors.In addition, other studies have found that bisphosphonate drugs also have immunomodulatory effects, and can improve humoral immune response by acting on B cells (Non-Patent Document 3), and that zoledronic acid can enhance antigen-specific CD8+ T cell response (Non-Patent Document 4).

[0004] Dieter Berndardt et al. invented zinc hydroxide gel or iron hydroxide gel, its preparation process, and its application as a vaccine adjuvant (Patent Document 1, Patent Grant Date: October 12, 1993). Zinc hydroxide gel or iron hydroxide gel has good adsorption effect on viruses or proteins and has good adjuvant effect. Zinc hydroxide gel can stimulate both humoral and cellular immune responses of the body, but iron hydroxide gel mainly enhances humoral immune response, both have good local and systemic tolerance, and both have better immune stimulating effect when combined with lecithin.

[0005] Hu Yunzhang et al. invented a vaccine adjuvant (Patent Document 2, publication date: June 3, 2009). This invention relates to a vaccine adjuvant, which is a zinc hydroxide compound with a chemical formula of Zn(OH)2 and a formula weight of 99.4046 Daltons, used in the preparation of adjuvants for vaccines and used in the preparation of adjuvants for Hepatitis A vaccines. Zinc hydroxide used in combination with vaccines as an adjuvant can effectively enhance the humoral immune response of vaccines, and its immune enhancing effect is better than that of aluminum adjuvants. In addition, the results of animal experiments show that zinc hydroxide as a new vaccine adjuvant has better sensitization effect and better safety profile than aluminum adjuvants.

[0006] Wang Haixuan et al. (Non-Patent Document 5) studied the immune enhancement of heparan sulfate (HS) and zinc hydroxide as adjuvants for humoral immune response in mice induced by recombinant hepatitis C virus antigen epitope polypeptide vaccine, and the results showed that the composite adjuvant of HS and zinc hydroxide can effectively enhance the humoral immune response in mice immunized with recombinant HCV antigen epitope polypeptide vaccine, and can save the dose of antigen to a certain extent.

[0007] Hu Yunzhang et al. invented a zinc phosphate vaccine adjuvant (Patent Document 3, Publication date: September 26, 2012). This invention relates to a zinc phosphate vaccine adjuvant, in particular to the use of zinc phosphate as a vaccine adjuvant and the use of zinc phosphate vaccine adjuvant in the manufacture of vaccines. Zinc phosphate is used as a vaccine adjuvant in combination with an antigen, which can effectively enhance the humoral immune response of the vaccine. Its immune enhancing effect is better than aluminum adjuvant and zinc hydroxide adjuvant, and the results of animal experiments show that zinc phosphate exhibits better sensitizing effect and better safety profile than aluminum adjuvant and zinc hydroxide adjuvant when used as a vaccine adjuvant.

[0008] Hu Yunzhang et al. invented a composite vaccine adjuvant (Patent Document 4, Publication (Announcement) Date: December 26, 2012). This invention provides a composite vaccine adjuvant characterized by comprising the following components: sodium ferulate and zinc hydroxide in a mass ratio of 10:1 to 50:1. The combined use of the composite vaccine adjuvant with a vaccine can effectively enhance the humoral immune response of the vaccine. The enhancing effect is similar to that of aluminum adjuvant, but is better than sodium ferulate adjuvant alone and zinc hydroxide adjuvant alone. In addition, this adjuvant is non-toxic, has no side effects, and is safe and reliable within the range of dosage required for immunization. The raw materials of the composite vaccine adjuvant of this invention are easily available and are commercial products. The composite vaccine adjuvant has a simple preparation process, low cost and stable performance. The composite vaccine adjuvant can be used as an adjuvant for hepatitis B vaccine, genetically engineered vaccine, virus vaccine, etc.

[0009] Wang Haixuan et al. invented a zoledronic acid adjuvant and a vaccine containing the zoledronic acid adjuvant (Patent Document 5, Publication Date: March 2, 2016). This invention provides a zoledronic acid adjuvant and a vaccine containing the zoledronic acid adjuvant, each vaccine dose containing 10 μg to 20 μg of the zoledronic acid adjuvant. The zoledronic acid adjuvant provided by this invention has low toxic side effects, is safe and reliable to use within the range of dosage required for immunization, can effectively induce antigen-specific humoral immune response, has the same immune effect as aluminum adjuvant, its raw materials are easily available, has stable performance, and can be used as a vaccine adjuvant. Takuya SHISHIDO et al. invented a bisphosphonate-containing vaccine pharmaceutical composition that can generate an efficient humoral immune response in the body and can be administered transdermally or mucosally (Patent Document 6, Publication Date: October 5, 2017).

[0010] Cai Hongzhi et al. (Non-Patent Document 6) studied the effect of zinc hydroxide and heparan sulfate (HS) composite adjuvant on the humoral immune response of mice induced by rabies vaccine, and the results showed that the zinc hydroxide and HS composite adjuvant could enhance the humoral immune response of mice induced by rabies vaccine.

[0011] Chen Zhenpu et al. invented zinc glycinate adjuvant and vaccine containing zinc glycinate adjuvant (Patent Document 7, Publication (Announcement) Date: October 15, 2014). This invention provides zinc glycinate adjuvant and vaccine containing zinc glycinate adjuvant, where each vaccine dose contains 0.1mg-1mg zinc glycinate adjuvant dissolved in PBS with pH=6-8. The zinc glycinate adjuvant provided by this invention is both a pharmaceutical adjuvant and a zinc nutritional fortifier. This adjuvant has low toxic side effects and is safe and reliable to use within the dosage range required for immunization. This adjuvant can effectively induce antigen-specific humoral immune response, and the induced humoral immune response effect is better than the non-adjuvant group. The raw materials of zinc glycinate adjuvant are easily available and are commercial products. This adjuvant has a simple preparation process, low cost, stable performance, high biological efficacy, no toxic or harmful effects, and can be added to a variety of conventional vaccines and genetically engineered vaccines as a vaccine adjuvant.

[0012] Mahsa Afshari et al. (Non-Patent Document 7) used a BALB / c mouse model with Leishmania infection. They found that oral administration of zinc sulfate to mice could significantly reduce parasite burden and increase the expression level of Th1 cytokine mRNA. This indicates that zinc sulfate can induce a strong Th1 immune response in Leishmania-infected BALB / c mice. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] U.S. Pat. No. 5,252,327 [Patent Document 2] China Patent Application Publication No. 101444623 [Patent Document 3] China Patent Application Publication No. 101972477 [Patent Document 4] China Patent Application Publication No. 102085366 [Patent Document 5] China Patent Application Publication No. 103768595 [Patent Document 6] US Patent Application Publication No. 2017 / 0281759 [Patent Document 7] China Patent Application Publication No. 104096229 [Non-patent literature]

[0014] [Non-Patent Document 1] Autoimmunity Reviews, 2015, 14 (04) 277-285 [Non-Patent Document 2] Immunity & Ageing, 2009, 6(9): 1-17 [Non-Patent Document 3] Cell Rep., 2013, 5(2) [Non-Patent Document 4] Vaccine, 2016, 34(10):1275-81 [Non-Patent Document 5] Chinese Journal of Biological Sciences, 2011, 24(10): 1174-1176 [Non-Patent Document 6] Chinese Journal of Biological Sciences, 2013, 26 (05): 608-610 [Non-Patent Document 7] Cytokine, 2016, 81:71-76 Summary of the Invention

[0015] In order to overcome the shortcomings of existing vaccine aluminum adjuvants, namely, inability to stimulate Th1 immune response and inability to enhance the immunogenicity of polypeptide and nucleic acid vaccines, and also considering the role of zoledronic acid in immune enhancement, a zinc zoledronate micro / nanoparticle adjuvant is formed by co-precipitation of zoledronic acid with zinc, a trace element that also plays an important role in immune regulation. Through creative research, the inventors of the present invention have found that the zinc zoledronate micro / nanoparticle adjuvant can significantly improve humoral and cellular immune responses. As a result, a zinc zoledronate vaccine adjuvant with significant immune enhancement effect is provided.

[0016] Thus, in one aspect, the present invention relates to a zinc zoledronate micro / nanoparticle adjuvant prepared by co-precipitation of zinc ions with phosphonic acid groups, phosphate radicals and hydroxide radicals, abbreviated as zinc zoledronate adjuvant.

[0017] In certain embodiments, the zinc zoledronate adjuvant of the present invention can be obtained by homogeneously mixing a soluble salt solution containing zinc ions with zoledronic acid and sodium hydroxide solution, or homogeneously mixing a soluble salt solution containing zinc ions with zoledronic acid, sodium hydroxide and sodium phosphate solutions to obtain the zinc zoledronate adjuvant.

[0018] In certain embodiments, the mixing methods include, but are not limited to, sequential precipitation, separate precipitation followed by mixing, or co-precipitation.

[0019] In one embodiment, the molar ratio of zinc:zoledronic acid in the zoledronic acid zinc adjuvant is generally not limited. In a preferred embodiment, the molar ratio of zinc:zoledronic acid in the zoledronic acid zinc adjuvant can be (1-8): 1. The molar ratio of zinc:zoledronic acid is preferably selected from 1:1, 4:1 and 8:1.

[0020] In one embodiment, the zinc zoledronate adjuvant may contain phosphate. For example, the zinc zoledronate complex adjuvant is prepared by replacing zoledronic acid with phosphate in various molar ratios (but not completely replacing it) using various mixing methods (such as successive precipitation, mixing after separate precipitation, or co-precipitation, etc.). In such zinc zoledronate complex adjuvant, the molar concentration ratio of Zn:phosphate radical is generally not limited. In a preferred embodiment, the molar concentration ratio of Zn:phosphate radical can be (1-8):1. The molar concentration ratio of Zn:phosphate radical is preferably selected from 1.5:1 and 4:1, thereby forming an organic-inorganic hybrid zinc zoledronate adjuvant.

[0021] In another embodiment of the zinc zoledronate adjuvant, the zinc zoledronate adjuvant may further comprise aluminum (Al). For example, zinc zoledronate-aluminum adjuvants are prepared by replacing Zn with Al in different proportions (but not completely replacing) using various mixing methods (such as continuous precipitation, mixing after separate precipitation, or co-precipitation, etc.). In such zinc zoledronate-aluminum adjuvants, the molar concentration ratio of Zn:Al is generally not limited. In a preferred embodiment, the molar concentration ratio of Zn:Al can be (0.02-1):1. The molar concentration ratio of Zn:Al is preferably selected from 0.375:1, 0.5:1, and 0.8:1.

[0022] In one embodiment, the specific type of zinc compound is not limited, for example, zinc hydroxide, zinc phosphate, zinc sulfate, zinc carbonate, or other types of zinc adjuvants known in the art are acceptable, so long as the zinc zoledronate adjuvant is prepared by precipitation of zinc ions with phosphonate groups, phosphate radicals, and hydroxide radicals.

[0023] In one embodiment, the phosphate solution can be selected from, but is not limited to, sodium phosphate, disodium hydrogen phosphate (anhydrous, dihydrate, heptahydrate, dodecahydrate), sodium dihydrogen phosphate (anhydrous, dihydrate), potassium phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, pyrophosphate, polyphosphate, and any mixture thereof.

[0024] In one embodiment, the specific type of aluminum compound is not limited, for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate, or other aluminum adjuvant types known in the art are acceptable, so long as the zinc zoledronate-aluminum adjuvant is prepared by precipitation of zinc and aluminum ions with phosphonate groups, phosphate radicals, and hydroxide radicals.

[0025] In one aspect, the invention relates to a method of making a zinc zoledronate adjuvant comprising precipitating zinc ions by reacting the zinc ions with a phosphonate group, a phosphate radical and a hydroxide radical separately or simultaneously in a soluble salt solution, thereby preparing the zinc zoledronate adjuvant.

[0026] In certain embodiments, the method further comprises: a) providing a soluble salt solution containing zinc ions; b) homogeneously mixing the soluble salt solution of step a) with zoledronic acid and sodium hydroxide, or homogeneously mixing the soluble salt solution of step a) with zoledronic acid, sodium hydroxide and sodium phosphate solutions in the manner of continuous precipitation, mixing after separate precipitation, or co-precipitation to obtain zinc zoledronate adjuvant; Includes.

[0027] In embodiments of the present invention, the soluble salt solution is generally not limited and may be selected from, for example, a solution of hydrochloric acid.

[0028] In a preferred embodiment, the method further comprises sterilizing the zinc zoledronate adjuvant suspension formed after mixing in step b) by autoclaving under high temperature and pressure conditions at 121°C for 60 minutes, cooling to room temperature, and then allowing to stand at 2°C to 8°C, preferably storing at 4°C for later use. In one embodiment, the molar ratio of zinc:zoledronic acid in the zinc zoledronate adjuvant obtained by the method of the present invention can be 1 to 8:1. In a preferred embodiment, the molar ratio of zinc:zoledronic acid is selected from 1:1, 4:1, or 8:1.

[0029] In another embodiment, during the method of the invention for obtaining the zinc-aluminum zoledronate adjuvant, the zinc-aluminum zoledronate adjuvant is prepared by precipitating zinc ions and aluminum ions with phosphonic acid groups, phosphate radicals and hydroxide radicals by various mixing methods (such as sequential precipitation, separate precipitation followed by mixing, or co-precipitation), and the Zn / Al molar concentration ratios are set to 0.375:1, 0.5:1 and 0.8:1.

[0030] As used herein, a soluble salt solution of zinc ions can be any soluble salt solution of zinc ions, preferably a solution of zinc ions in hydrochloric acid.

[0031] The zoledronate solution used herein is preferably an alkaline solution of zoledronic acid and sodium hydroxide.

[0032] The means for precipitating zinc and phosphonic acid groups used herein can be any means in which a precipitation reaction occurs by thoroughly mixing a soluble salt solution of zinc ions with an alkaline solution of zoledronic acid and sodium hydroxide.Preferably, the zinc zoledronate adjuvant can be prepared by methods such as sequential precipitation, separate precipitation followed by mixing, or co-precipitation.

[0033] Sterilization as used herein can be any method suitable for sterilizing zinc zoledronate adjuvant. For example, high-temperature high-pressure steam sterilization technology can be applied. For example, sterilization is performed at 121°C for 30 to 60 minutes, preferably 60 minutes.

[0034] In one embodiment, the present invention also relates to a method for measuring the physical and chemical properties of the resulting zinc zoledronate adjuvant. In one embodiment, the pH value, particle size, zeta potential, particle point of zero charge (PZC), protein adsorption and dissociation rate, metal ion precipitation rate, organic phosphonic acid precipitation rate, precipitate dissolution rate in vivo and in vitro, etc. of the adjuvant are measured. The physical and chemical properties of the adjuvant can be measured by conventional methods (see, for example, U.S. Pat. No. 9,573,811; Ai Xulu et al., Analysis of physicochemical properties of three aluminum hydroxide adjuvant, "Chinese Journal of Biologicals", 2015, 28(1): 44-47) and as described in the Examples herein.

[0035] In one embodiment, the zinc zoledronate adjuvants described herein have one or more of the following properties: a pre-sterilization pH of 8.0-9.0, a post-sterilization pH of 6.0-8.0, a particle size of 1 μm-10 μm, a point of zero charge of the particle of 4.0-11.4, a metal ion precipitation rate of greater than 99%, and a protein adsorption rate of greater than 80%.

[0036] In one aspect, the invention relates to compositions, particularly pharmaceutical formulations or compositions, comprising the zinc zoledronate adjuvants described herein.

[0037] Methods of making a pharmaceutical formulation or composition include the step of combining the zinc zoledronate adjuvant with the carrier and, optionally, one or more accessory ingredients.

[0038] In general, the formulations are prepared by uniformly and intimately bringing into association a zinc zoledronate adjuvant with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0039] Liquid dosage forms for oral administration of active ingredients include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the zinc zoledronate adjuvant, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0040] Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0041] Suspension formulations may contain, in addition to the zinc zoledronate adjuvant, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0042] The pharmaceutical compositions of the present invention for rectal or vaginal administration can be provided as suppositories, which can be prepared by mixing the zinc zoledronate adjuvant with one or more suitable non-irritating excipients or carriers, including, for example, cocoa butter, polyethylene glycol, a suppository wax, or salicylates, which are solid at room temperature and liquid at body temperature, and thus melt in the rectum or vagina to release the zinc zoledronate adjuvant. Formulations of the present invention suitable for vaginal administration also include vaginal suppositories, tampons, creams, gels, pastes, foams or sprays containing suitable carriers known in the art.

[0043] Pharmaceutical compositions of the invention suitable for parenteral administration in combination with the zinc zoledronate adjuvant include solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions before use, comprising one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous carriers which may contain antioxidants, buffers, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0044] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size (in the case of dispersions), and by the use of surfactants.

[0045] These compositions may contain adjuvants such as wetting agents, emulsifying agents, and dispersing agents. It may also be desirable to add isotonic agents to the compositions, such as sugars, sodium chloride, etc. In addition, agents that delay absorption, such as aluminum monostearate and gelatin, can be added to bring about prolonged absorption of the injectable pharmaceutical form.

[0046] Injectable depot forms can be made by forming microencapsule matrices of the zinc zoledronate adjuvant in a biodegradable polymer, such as polylactide-polyglycolide. Depending on the ratio of the zinc zoledronate adjuvant to the polymer and the nature of the particular polymer used, the release rate of the zinc zoledronate adjuvant can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot 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 filtration through a bacteria-retaining filter.

[0047] The formulations may be presented in single-dose or multi-dose hermetically sealed containers, for example ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0048] In one aspect, the present invention also relates to an immunogenic composition comprising the zinc zoledronate adjuvant described herein and one or more antigens.

[0049] As used herein, an immunogenic composition stimulates a protective immune response against one or more of the antigens contained therein when administered to a subject or animal.

[0050] In one aspect, the present invention also relates to a vaccine composition comprising a zinc zoledronate adjuvant as described herein and one or more antigens.

[0051] As used herein, a vaccine composition, when administered to a subject or animal, stimulates a protective immune response against, for example, a microorganism, or effectively protects the subject or animal from infection.

[0052] The vaccine composition can be used to prevent or ameliorate a pathological condition that responds favorably to modulation of the immune response. Such a vaccine composition can be a prophylactic or therapeutic vaccine. Preferably, the vaccine composition comprises a genetically engineered vaccine, such as a protein vaccine, for example a varicella zoster virus recombinant protein vaccine.

[0053] In one aspect, the present invention also relates to a vaccine adjuvant comprising the zinc zoledronate adjuvant described herein. For example, such a vaccine adjuvant may also comprise a secondary adjuvant, as described below.

[0054] The term "adjuvant" or "vaccine adjuvant," as used herein, refers to a substance that can non-specifically accelerate, prolong, or enhance the immune response to an antigen.

[0055] Advantageously, adjuvants can also reduce the number of immunizations or the amount of antigen required for protective immunity.

[0056] It is known that adjuvants enhance immune responses to antigens, while adjuvants themselves do not stimulate any or little immune response. Thus, the zinc zoledronate adjuvant of the present invention can be combined with one or more antigens to create immunogenic compositions or vaccines that can be used to stimulate immune responses in individuals. A variety of substances can be used as antigens in immunogen- or vaccine-type complexes or formulations (e.g., attenuated and inactivated viral and bacterial pathogens, purified macromolecules, proteins, polysaccharides, toxoids, recombinant antigens, organisms with foreign genes derived from pathogens, synthetic peptides, polynucleotides, antibodies, and tumor cells, etc.). Antigens can be used in both prophylactic and therapeutic vaccines. Antigens include protein antigens, such as varicella zoster virus gE glycoprotein antigen (VZV gE).

[0057] Various immunomodulatory molecules can also be used in combination with the zinc zoledronate adjuvant of the present invention to change immune response in individuals.The immunomodulatory agent described herein refers to a type of preparation that can regulate, balance and restore immune function of the body.Commonly used immunomodulatory agents include three major categories: immunostimulants, immunosuppressants and immune interactive regulators.

[0058] The amounts of antigen and zinc zoledronate adjuvant in the vaccine composition of the present invention and the dosages thereof will be determined by techniques known to those skilled in the pharmaceutical arts and will take into account factors such as: the particular antigen, the age, sex, weight, species and condition of the particular animal or patient, and the route of administration.

[0059] In a preferred embodiment, 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 adjusters, preservatives, osmotic pressure adjusters, propellants, water, and any mixtures thereof.

[0060] The vaccine composition of the present invention may further comprise a pharma- ceutically acceptable carrier. The amount of carrier depends on the amount selected for the other components, the desired concentration of the antigen, the choice of the route of administration (oral or parenteral), etc. 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, before administration. Alternatively, the carrier can be used to manufacture the final product.

[0061] Examples of suitable carriers include, but are not limited to, sterile water, saline, buffered saline, phosphate buffered saline, buffered sodium chloride, vegetable oil, minimal essential medium (MEM), MEM containing HEPES buffer, and the like.

[0062] Optionally, the vaccine compositions of the invention may contain a conventional secondary adjuvant in varying amounts depending on the adjuvant and the desired result.

[0063] Examples of suitable secondary adjuvants include stabilizers; emulsifiers; pH adjusters such as sodium hydroxide, hydrochloric acid, and the like; surfactants such as Tween™ 80 (Polysorbate 80, commercially available from Sigma Chemical Co., St. Louis, Mo.); liposomes; iscom adjuvants; synthetic glycopeptides such as muramyl dipeptide; bulking agents such as dextran; carboxypolymethylene; bacterial cell walls such as mycobacterial cell wall extracts; their derivatives such as Corynebacterium parvum; Propionibacterium acnes; Mycobacterium bovis, e.g., Bovine Calmette-Guerin; Adjuvants that may be used include, but are not limited to, BCG (Cas9-Guerin); vaccinia or animal poxvirus proteins; subviral particle adjuvants such as orbivirus; cholera toxin; N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine (pyridine); monophosphoryl lipid A; dimethyldioctadecylammonium bromide (DDA, commercially available from Kodak, Rochester, NY); and combinations and mixtures thereof.

[0064] Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, digested protein extracts such as NZ-Amine or NZ-Amine AS, etc. Examples of emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil, and other standard metabolizable non-toxic oils useful in injectable or intranasal vaccine compositions.

[0065] For purposes of the present invention, these adjuvants are referred to herein as "secondary" in contrast to the zinc zoledronate adjuvant described above simply because the combination of the zinc zoledronate adjuvant with an antigenic agent can significantly enhance the humoral immune response to the antigenic agent. Secondary adjuvants are included in vaccine formulations primarily as processing aids, although certain adjuvants have some degree of immune enhancing properties and are dual purpose.

[0066] Conventional preservatives can be added to the vaccine composition in effective amounts ranging from about 0.0001% to about 0.1% by weight. Depending on the preservative used in the formulation, amounts below or above this range may be useful. Typical preservatives include, for example, potassium sorbate, sodium metabisulfite, phenol, methylparaben, propylparaben, thimerosal, and the like.

[0067] The selection of inactivated, modified or other types of vaccine compositions and the preparation of improved vaccine composition formulations of the present invention are known or readily determined by those of skill in the art.

[0068] In general, the vaccine composition of the present invention can be conveniently administered orally, parenterally (subcutaneously, intramuscularly, intravenously, intradermally or intraperitoneally), bucally, intranasally or transdermally. The route of administration contemplated by the present invention depends on the antigenic substance and formulation aids. For example, if the vaccine composition contains saponin, care must be taken not to inject the sapogenin glycoside into the bloodstream, since saponin is non-toxic when administered orally or intranasally, but acts as a strong hemolytic agent. Also, many antigens are not effective when taken orally. It is preferred to administer the vaccine composition intramuscularly or intraperitoneally.

[0069] The dosage of the vaccine composition will depend on the particular antigen selected, the route of administration, the species, and other standard factors. It is contemplated that one of skill in the art can easily and readily titrate the appropriate dosage to the immunogenic response for each antigen and determine the effective immunizing dose and route of administration.

[0070] As a vaccine adjuvant, the zinc zoledronate adjuvant of the present invention can improve the efficacy of a vaccine by enhancing the immunogenicity of weaker antigens (e.g., highly purified or recombinant antigens), reducing the amount of antigen required for an immune response, or reducing the number of immunizations required for protective immunity. The zinc zoledronate adjuvant of the present invention can improve the efficacy of a vaccine in individuals with reduced or weakened immune responses (e.g., neonates, elderly, and immunocompromised individuals) and can enhance the efficacy of a vaccine in target tissues. Alternatively, the zinc zoledronate adjuvant of the present invention can promote cellular and / or humoral immunity by inducing a specific cytokine profile.

[0071] Combinations of antigens and / or immunomodulatory molecules with the zinc zoledronate adjuvants of the present invention can be tested in a variety of preclinical toxicity and safety studies known in the art.

[0072] For example, such combinations can be evaluated in animal models in which the antigen is found to be immunogenic and can be reproducibly immunized by the same route as proposed for human clinical trials.

[0073] Combinations of antigens and / or immunomodulatory molecules with the zoledronic acid adjuvants of the invention can be tested, for example, by approaches defined by the Center for Bioproducts Evaluation and Research / Food and Drug Administration and the National Institute of Allergy and Infectious Diseases (Goldenthal, KL et al. AID Res Hum Retroviruses, 9: S45-9 (1993)).

[0074] For a particular combination of an antigen and / or immunomodulatory molecule with a conjugate adjuvant of the invention, one skilled in the art knows how to determine the appropriate antigen payload, route of immunization, dose, purity of antigen and vaccination regimen that will be useful for treating a particular pathological condition in a particular animal species.

[0075] The immunogenic composition or vaccine of the present invention for inducing an immune response can be administered as a solution or suspension with a pharma- ceutically acceptable vehicle.

[0076] Such pharma- ceutically acceptable vehicles can be, for example, water, phosphate buffered saline, saline or other buffered saline, or other solvents or vehicles, such as glycols, glycerol, and oils, such as olive oil, or injectable organic esters. Pharmaceutically acceptable vehicles may also contain liposomes or micelles, or immune stimulating complexes prepared by mixing a polypeptide or peptide antigen with a detergent and a glycoside, such as Quil A.

[0077] The immunogenic compositions or vaccines of the invention can be administered by a variety of routes to stimulate an immune response. For example, the immunogenic compositions or vaccines can be delivered subcutaneously, intradermally, intralymphatically, intramuscularly, intratumorally, intravesically, intraperitoneally, and intracerebrally.

[0078] Those of skill in the art will know how to select an appropriate delivery route for a particular formulation of the zoledronic acid adjuvant of the invention.

[0079] In a preferred embodiment of the invention, a vaccination method for the treatment or prevention of infections in mammals comprises the use of a vaccine according to the invention, which is administered in particular intramuscularly or intraperitoneally. The vaccine may be administered as a single dose or, preferably, following a prime / boost strategy, several times, for example 2, 3 or 4 times per week or month. The appropriate dose depends on various parameters.

[0080] In one aspect, the present invention also relates to the use of a zinc zoledronate adjuvant as described herein for the manufacture of a vaccine adjuvant, pharmaceutical composition, immunogenic composition or vaccine composition, preferably comprising a protein vaccine such as a varicella zoster virus protein recombinant vaccine. [Brief description of the drawings]

[0081] [Figure 1] FIG. 1 is a schematic diagram of three preparation processes for preparing adjuvants. [Diagram 2] FIG. 1 shows particle morphology of zinc zoledronate adjuvant, zinc aluminium zoledronate adjuvant and aluminium zoledronate adjuvant. [Diagram 3] Figure 1. Determination of adjuvant activity of zinc zoledronate adjuvant in VZV gE vaccine. Mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. [Figure 4] Figure 1 shows the effect of zinc zoledronate adjuvant in combination with recombinant VZV gE protein on mouse antibody isotypes. Mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. [Diagram 5] Figure 1 shows the determination of the adjuvant activity of zinc zoledronate adjuvant to Hepatitis B therapeutic protein. Mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. [Figure 6] Figure 1 shows the effect of other zinc bisphosphonate adjuvants (Zn / Zol=1 / 0.25) in combination with VZV gE on mouse serum antibody titers. Mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. [Figure 7] Figure 1 shows the effect of zoledronic acid and zoledronic acid zinc modified adjuvant combined with Hepatitis B therapeutic protein on mouse serum antibody titers. Mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] Specific Model for Implementing the Invention Hereinafter, the embodiments of the present invention will be described in detail with reference to the examples. If no specific conditions are given in the examples, they shall be carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment used without manufacturer's designation are conventional products that can be purchased commercially.

[0083] The materials used in the following preparation examples are as follows: Anhydrous zinc chloride (ZnCl2): purchased from Xilong Chemical; Aluminum chloride hexahydrate (AlCl3·6H2O): purchased from Xilong Chemical; Disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O): purchased from Xilong Chemical; Sodium hydroxide (NaOH): purchased from Xilong Chemical; Zoledronic acid (C5H 10 N2O7P2):Purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0084] Preparation Example 1: Preparation of Zn-zoledronic acid (1 / 0.25) adjuvant According to a Zn / zoledronic acid molar ratio of 1:0.25, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (7.78 mM zoledronic acid + 43 mM sodium hydroxide + 7.78 mM disodium hydrogen phosphate) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0085] Preparation of Zn-Zoledronic Acid (1 / 0.25) Adjuvant Suspension: Solutions A and B were used to form a zinc zoledronate adjuvant suspension according to the scheme depicted in Figure 1A, i.e., the prepared solution B was added dropwise to solution A according to a 1:1 volume ratio until all was added to form a suspension.

[0086] The zinc zoledronate adjuvant obtained after mixing according to FIG. 1A was subjected to autoclave steam sterilization at 121°C for 60 min, followed by cooling to room temperature and then storing at 2°C to 8°C.

[0087] Preparation Example 2: Preparation of Zn-Zoledronic Acid (1 / 1) Adjuvant Preparation of solutions: According to a 1:1 Zn / zoledronic acid molar ratio, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (31.11 mM zoledronic acid + 87 mM sodium hydroxide) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0088] With reference to Preparation Example 1, a Zn-zoledronic acid (1 / 1) adjuvant suspension was prepared.

[0089] Preparation Example 3: Preparation of zinc-aluminum zoledronic acid (FH-001-zoledronic acid) adjuvant Preparation of solutions: According to the Al / Zn molar concentration of 1:0.375, 50 mL of a mixed solution of aluminum chloride (122.44 mM) and zinc chloride (46.66 mM) was prepared as solution A, and 50 mL of a solution (18.66 mM zoledronic acid + 380 mM sodium hydroxide) was prepared as solution B. Solution A and solution B were filtered through 0.22 μm filter membrane for further use.

[0090] With reference to Preparation Example 1, an FH-001-zoledronic acid adjuvant suspension was prepared.

[0091] Preparation Example 4: Preparation of aluminum zoledronic acid (Al-001-zoledronic acid) adjuvant Preparation of solutions: According to an Al / Zol molar concentration of 1:0.075, 50 mL of 122.44 mM aluminum chloride solution was prepared, designated as solution A, and 50 mL of a solution (9.33 mM zoledronic acid + 9.33 mM disodium hydrogen phosphate dodecahydrate + 370 mM sodium hydroxide) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0092] An Al-001-zoledronic acid adjuvant suspension was prepared with reference to Preparation Example 1.

[0093] Preparation Example 5: Preparation of Zn-zoledronic acid (1 / 0.125) adjuvant Preparation of solutions: According to a Zn / zoledronic acid molar ratio of 1:0.125, 50 mL of 124.44 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (15.56 mM zoledronic acid + 65.7 mM sodium hydroxide) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0094] With reference to Preparation Example 1, a Zn-zoledronic acid (1 / 0.125) adjuvant suspension was prepared.

[0095] Preparation Example 6: Preparation of aluminum adjuvant (Al-002) Preparation of solutions: Aluminum chloride hexahydrate was added to water to prepare 0.5 L of 62.22 mM aluminum chloride solution, designated as solution A, and disodium hydrogen phosphate dihydrate and sodium hydroxide were added to water to prepare 0.5 L of a solution containing 18.68 mM phosphate and 0.12 M sodium hydroxide, designated as solution B. These were filtered through a 0.22 μm filter membrane for further use.

[0096] With reference to Preparation Example 1, aluminum adjuvant Al-002 was prepared.

[0097] Preparation Example 7: Preparation of aluminum adjuvant (Al-001-840) Preparation of solutions: According to a phosphate / Al molar ratio of 0.15, 0.5 L of 124 mM aluminum chloride solution was prepared, designated solution A, and 0.5 L of 18.6 mM disodium hydrogen phosphate solution was prepared, designated solution B. Solution B further contained 150 mM sodium hydroxide, and they were filtered through a 0.22 μm membrane filter for further use.

[0098] Referring to Preparation Example 1, a suspension of aluminum adjuvant Al-001-840 was prepared.

[0099] Preparation Example 8: Preparation of zinc-aluminum adjuvant (FH-001) Preparation of solutions: Aluminum chloride hexahydrate and anhydrous zinc chloride were added to water to prepare 0.25 L of a solution containing 124.44 mM aluminum chloride and 46.68 mM zinc chloride, designated solution A, and disodium hydrogen phosphate dihydrate and sodium hydroxide were added to water to prepare 0.25 L of a solution containing 18.68 mM phosphate and 0.425 M sodium hydroxide, designated solution B. These were filtered through a 0.22 μm filter membrane for further use.

[0100] Preparation of aluminum complex adjuvant: As shown in Figure 1A, 0.25 L of solution B was added dropwise to 0.25 L of solution A until all was added, thereby obtaining zinc-aluminum complex adjuvant FH-001. The obtained zinc-aluminum complex adjuvant was subjected to autoclave steam sterilization at 121°C for 60 minutes, then cooled to room temperature and stored at 2°C to 8°C.

[0101] Preparation Example 9: Preparation of Zn-etidronic acid (1 / 0.25) adjuvant According to a Zn / etidronic acid molar ratio of 1:0.25, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (7.78 mM etidronic acid + 34 mM sodium hydroxide + 7.78 mM disodium hydrogen phosphate) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0102] Referring to Preparation Example 1, a Zn-etidronic acid (1 / 0.25) adjuvant suspension was prepared.

[0103] Preparation Example 10: Preparation of Zn-clodronic acid (1 / 0.25) adjuvant According to a Zn / clodronic acid molar ratio of 1:0.25, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (7.78 mM clodronic acid + 36 mM sodium hydroxide + 7.78 mM disodium hydrogen phosphate) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0104] Referring to Preparation Example 1, a Zn-clodronic acid (1 / 0.25) adjuvant suspension was prepared.

[0105] Preparation Example 11: Preparation of Zn-alendronic acid (1 / 0.25) adjuvant According to a Zn / alendronic acid molar ratio of 1:0.25, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (7.78 mM alendronic acid + 36 mM sodium hydroxide + 7.78 mM disodium hydrogen phosphate) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0106] Referring to Preparation Example 1, a Zn-alendronic acid (1 / 0.25) adjuvant suspension was prepared.

[0107] Preparation Example 12: Preparation of Zn-pamidronic acid (1 / 0.25) adjuvant According to a Zn / pamidronic acid molar ratio of 1:0.25, 50 mL of 31.11 mM zinc chloride solution was prepared, designated as solution A, and 50 mL of a solution (7.78 mM pamidronic acid + 32 mM sodium hydroxide + 7.78 mM disodium hydrogen phosphate) was prepared, designated as solution B. Solution A and solution B were filtered through a 0.22 μm filter membrane for further use.

[0108] Referring to Preparation Example 1, a Zn-pamidronic acid (1 / 0.25) adjuvant suspension was prepared.

[0109] This specification discloses the following inventions. [1] 1. A zinc zoledronic acid micro / nanoparticle adjuvant comprising zinc and zoledronic acid, having a molar ratio of zinc:zoledronic acid selected from 1:1, 4:1 and 8:1, and optionally further comprising phosphate, having a molar ratio of zinc:phosphate selected from 1.5 and 4, and optionally further comprising aluminium, having a molar ratio of zinc:aluminium selected from 0.375:1, 0.5:1 and 0.8:1. [2] The zinc zoledronate micro / nanoparticle adjuvant a) providing a soluble salt solution containing zinc ions, preferably said soluble salt solution comprising a solution of hydrochloric acid; b) homogeneously mixing the soluble salt solution of step a) with zoledronic acid and sodium hydroxide, or homogeneously mixing the soluble salt solution of step a) with zoledronic acid, sodium hydroxide and sodium phosphate solutions by continuous precipitation, separate precipitation followed by mixing, or co-precipitation to obtain zinc zoledronate adjuvant; and preferably further comprising subjecting the mixed solution of step b) to sterilization and storing it at 2°C to 8°C for later use, preferably wherein the sterilization comprises sterilization using high temperature and high pressure sterilization techniques, for example sterilization at 121°C for 30 minutes to 60 minutes, preferably 60 minutes. [3] The following characteristics: Pre-sterilization pH of 8.0-9.0; pH after sterilization of 6.0 to 8.0; Particle size: 1μm~10μm Particle zero charge point of 4.0 to 11.4, A zinc zoledronate micro / nanoparticle adjuvant according to [1] or [2], having one or more protein adsorption rates of greater than 80%, preferably wherein the protein comprises bovine serum albumin. [4] 1. A method of making a zinc zoledronate micro / nanoparticle adjuvant comprising: a) providing a soluble salt solution containing zinc ions, preferably said soluble salt solution comprising a solution of hydrochloric acid; b) homogeneously mixing the soluble salt solution of step a) with zoledronic acid and sodium hydroxide, or homogeneously mixing the soluble salt solution of step a) with zoledronic acid, sodium hydroxide and sodium phosphate solutions by continuous precipitation, separate precipitation followed by mixing, or co-precipitation to obtain zinc zoledronate adjuvant; and preferably further comprising subjecting the mixed solution of step b) to sterilization and storing it at 2°C to 8°C for later use, preferably the sterilization comprises sterilization using high temperature and high pressure sterilization techniques, for example sterilization at 121°C for 30 minutes to 60 minutes, preferably 60 minutes; The method, wherein the resulting zinc zoledronic acid adjuvant has a molar ratio of zinc:zoledronic acid selected from 1:1, 4:1 and 8:1. [5] The resulting zinc zoledronate adjuvant has the following characteristics: Pre-sterilization pH of 8.0-9.0; pH after sterilization of 6.0 to 8.0; Particle size: 1μm~10μm Particle zero charge point of 4.0 to 11.4, The method according to [4], having one or more protein adsorption rates of more than 80%, preferably the protein comprising bovine serum albumin. [6] A vaccine adjuvant, pharmaceutical composition, or immunogenic composition comprising the zinc zoledronate micro / nanoparticle adjuvant according to any one of [1] to [3]. [7] A vaccine composition comprising an antigen and the zinc zoledronate micro / nanoparticle adjuvant according to any one of [1] to [3], wherein the antigen preferably comprises a protein antigen such as a varicella-zoster virus gE glycoprotein antigen. [8] Use of the zinc zoledronate micro / nanoparticle adjuvant according to any one of [1] to [3] in the manufacture of a vaccine adjuvant, a pharmaceutical composition, a drug delivery vehicle, an immunogenic composition or a vaccine composition, preferably wherein the vaccine comprises a protein vaccine such as a varicella zoster virus recombinant protein vaccine. The present invention will be further described below in conjunction with examples. EXAMPLES

[0110] Example 1: Determination of the physical and chemical properties of zinc zoledronate adjuvants The following determination method was applicable to any Zn / zoledronic acid molar ratio, i.e., any zinc zoledronic acid adjuvant, such as inorganic phosphate doped or Al doped zinc zoledronic acid adjuvant.

[0111] (1) Morphological Observation of Adjuvant Particles The zinc zoledronate adjuvant was diluted 50 times with deionized water and then observed using a JEM-2100 transmission electron microscope (TEM) from JEOL Ltd. The specific steps were as follows: the adjuvant sample was dropped onto a copper mesh coated with a carbon film, adsorbed for 10 minutes, the remaining liquid was wiped off with filter paper, and the sample was sent to the sample chamber of the transmission electron microscope to observe the morphology of the sample and take a picture for further analysis.

[0112] Experimental results: It was clearly confirmed that the Zn-zoledronic acid (1 / 0.25) adjuvant and the Al-001-zoledronic acid adjuvant had spherical nanocore particles, while the FH-001-zoledronic acid adjuvant showed a seaweed-like cluster shape (Figure 2).

[0113] (2) Determination of pH Samples to be tested were taken, equilibrated at room temperature for at least 30 minutes and measured using a Sartorius pH glass electrode.

[0114] Standard buffer (pH 7.00), standard buffer (pH 4.01) and standard buffer (pH 10.01) were selected and the instrument was calibrated according to the requirements in the instruction manual.

[0115] The "Mode" button could be used to switch between pH and mV modes. Usually, the pH mode was selected when determining the pH value of a solution.

[0116] Press the "SETUP" button until the display reads Clear buffer, then press the "ENTER" button to confirm and clear the previous calibration data.

[0117] Press the "SETUP" button until the buffer solution group "4.01, 7.00, 10.01" appears on the display, then press the "ENTER" button to confirm.

[0118] The electrodes were carefully removed from the electrode storage solution, the electrodes were thoroughly rinsed with deionized water, and the water on the surface was dried with filter paper after thorough rinsing (taking care not to wipe the electrode).

[0119] The instrument was automatically calibrated by immersing the electrode in the first buffer solution (pH 7.00) and pressing the "STANDARDIZE" button after the value stabilized and displayed "S." If the calibration was successful, "7.00" and the electrode slope were displayed.

[0120] The instrument was automatically calibrated by removing the electrode from the first buffer solution, rinsing the electrode thoroughly with deionized water, and immersing the electrode in the second buffer solution (pH 4.01) in sequence, and pressing the "STANDARDIZE" button after the value stabilized and "S" was displayed. If the calibration was successful, the messages "4.01 7.00" and "Slope" were displayed. Slope displayed the measured electrode slope value, which was acceptable within the range of 90% to 105%.

[0121] If there is a significant deviation from the theoretical value, an error message (Err) is displayed and the electrode needs to be cleaned and the above steps need to be repeated for calibration.

[0122] The above procedure was repeated to complete the third calibration point (pH 10.01).

[0123] After the calibration was completed, the electrode was thoroughly rinsed with deionized water, then lightly dried with filter paper. The sample solution was shaken evenly, the glass electrode was immersed in the sample solution, and the reading was confirmed after the pH value did not change more than ±0.05 in 1 minute.

[0124] The sample solution was shaken evenly and the measurement was taken again. The difference between the two pH values ​​should not exceed 0.1. The average of the two readings was considered as the pH value of the test article.

[0125] Experimental results (Table 1): The pH range of the Zn-zoledronic acid adjuvant was 8.0-9.0 before sterilization and 6.5-7.0 after sterilization.

[0126] (3) Determination of particle size The Beckman LS 13320 laser particle size analyzer was turned on and allowed to preheat for 15 minutes.

[0127] The control software of the analyzer was started, the closed compartment of the sample cell was opened, the sample cell was removed from the sample bath, and 12 mL of purified water was added.

[0128] The sample cell was placed onto the sample chamber and the compartment lid was closed.

[0129] Click on “start cycle”, select “Measure Offsets”, “Align”, “Measure Background”, and finally click on “start” and then on “OK” in the pop-up dialogue box to start the blank background calibration.

[0130] The sample cell was removed and a certain amount of standard sample (provided with the analyzer) was added. The standard sample was measured by clicking "start cycle", selecting "Measure Loading", "Enter Sample Info", "Enter run setting", "start runs", and finally clicking "start". The name of the standard sample was entered in the pop-up dialogue box, and when the "Obscuration" parameter in the software reached 8%-12%, clicking "OK".

[0131] In order to ensure the accuracy and reliability of the experimental data, the blank background needs to be calibrated, and the size of the standard sample needs to be measured before each measurement.

[0132] The closed compartment of the sample cell was opened and the sample cell was removed.

[0133] The aqueous solution containing the standard samples in the sample cell was discarded, deionized water was added to the sample cell, and the sample cell was washed three times.

[0134] After rinsing, 12 mL of deionized water was added, the sample cell was placed on the sample bath, and the compartment lid was closed.

[0135] Click on “start cycle”, select “Measure Offsets”, “Align”, “Measure Background”, and finally click on “start” and then on “OK” in the pop-up dialogue box to start the blank background calibration.

[0136] The sample cell was removed, a volume of test sample was added, the lid of the sample test compartment was opened, the sample cell was placed on the sample chamber, and the lid of the compartment was closed.

[0137] Click on “start cycle”, select “Enter Sample Info”, “Enter run setting”, “start runs”, and finally click on “start”, enter the name of the standard sample in the pop-up dialogue box, click on “OK” when the “Obscuration” parameter in the software is between 8% and 12%, and record the measured particle size of the sample.

[0138] Test Results: The particle size of the zinc zoledronate adjuvant ranged from 0.4 μm to 30 μm, with most of the particles being between 6 μm and 7 μm in size.

[0139] [Table 1]

[0140] (4) Determination of adsorption rate Standard curve plot of BSA standard: BSA standard (2 mg / mL) was serially diluted using 150 mM NaCl as dilution buffer, and the absorbance at 280 nm was detected by UV2100 pro. OD280 showed high precision at 0.2-0.8 (expanded to 0.2-1.5).

[0141] BSA Gradient Dilution (EP): Using 150 mM NaCl as the dilution buffer, a fixed amount of BSA sample was weighed and diluted to the concentration gradient specified in the EP: 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 5 mg / mL, 10 mg / mL for further use.

[0142] BSA was mixed with adjuvant at a volume ratio of 3:1 (BSA:adjuvant), which was set as the condition for measuring the adsorption rate. After shaking the adjuvant evenly, it was mixed with different concentrations of BSA according to the experimental conditions, and adsorption was carried out at room temperature for 1 hour, during which shaking was performed 5 times. After centrifugation at 13000 rpm / min for 3 minutes, the supernatant was collected for later use.

[0143] Determination of protein concentration: The protein concentration in EP was determined using the Lowry method. In this experiment, according to the actual situation, the absorbance of the supernatant at 280 nm was directly determined using UV2100pro, and the reading value was kept between 0.2 and 0.8, otherwise the supernatant needs to be diluted.

[0144] Calculation of the adsorption rate: Adsorption rate = [1-OD 280(上清の希釈係数X) / OD 280(希釈率Xの吸着率が0の場合) ] x 100

[0145] The experimental results were as follows:

[0146] [Table 2]

[0147] (5) Decision of PZC: Measurement equipment: Nanobrook Omni (Brookhaven)

[0148] Experimental procedure: The pH of Zn-zoledronic acid (1 / 0.125) was adjusted to 6.00 / 5.50 / 5.00 / 4.50 / 4.00 / 3.50 / 3.00 / 2.50 / 2.00 using 0.1M NaOH / 1% HNO3.

[0149] Passivation of the electrodes: 3 mL to 4 mL of adjuvant was added to the sample tube. After inserting the electrodes, the SOP cycle was set to 50 and the instrument was run to passivate the electrodes.

[0150] Sample measurement: After removing the electrode, rinse its lower end with deionized water, then add the corresponding sample, set the cycle in SOP to 15, set the measurement to 3, set the pH to the corresponding pH of each sample, and run the instrument.

[0151] Data processing: The corresponding zeta potentials were obtained at different pH values, and the PZC values ​​were obtained by running the software provided with the instrument.

[0152] RESULTS: The PZC of the Zn-zoledronic acid (1 / 0.125) adjuvant was 4.08.

[0153] Example 2: Determination of the zinc precipitation ratio of Zn-Zoledronic Acid (1 / 0.125) adjuvant The zinc content in the zoledronic acid zinc adjuvant was determined using the flame method (D2 lamp background correction) and the determination procedure was standardized. The detection equipment was an atomic absorption spectrophotometer: AA6300C (P / N 206-52430) manufactured by Shimadzu Corporation.

[0154] Preparation of standard solutions and samples to be determined: Preparation of standard curve: The original concentration of zinc standard was 500 μg / mL, which was diluted with 0.1 M hydrochloric acid solution to obtain standards of 500 ng / mL, 1000 ng / mL, 1500 ng / mL, 2000 ng / mL and 2500 ng / mL.

[0155] Zn-Zoledronic acid (1 / 0.125) adjuvant was centrifuged at 13000r / min for 10 minutes, and the supernatant was removed. The sample was diluted 5 times with 0.1M nitric acid solution for determination, and was shaken and mixed well using a vortex mixer. The determination process was as follows:

[0156] How to use AA-6300C and Wizard software: Turn on the power: turn on the computer, the power switch of the AA-6300C, the switch of the air compressor (pressure was set to 0.35MPa), and the switch of the ventilation system.

[0157] Acetylene release: The acetylene valve was slowly opened so that the primary pressure was 0.5 MPa and the secondary pressure was 0.1 MPa.

[0158] Basic operation procedure of WizAArd software: Log in to Wizard → Select an element → Click <Connect / Send parameters> on the "Unconnected device / Send parameters" page → Set up on the "Initialize device" page → Check each item in the "Frame analysis device check catalog" and check it. <ok>Click → Set the wavelength (213.86), slit width (0.7), and illumination method (light emission), set the (Lamp position setting) on ​​the "Optical parameters" page so that the actual position of the Zn hollow cathode lamp is the same as the preset position, and select (Lamp lighting) → Line search → Adjust the burner origin position → Select (Parameter) from the menu bar → (Edit parameters) → Change the illumination method <bgc-d2>Change to → Line search → Ignite: After confirming that C2H2 is turned on and the pressure meets the requirements, press the PURGE and IGNITE buttons on the host simultaneously until ignition occurs → Auto zero → Set the blank group (BLK), standard (STD) and test sample (UNK) on the MRT worksheet, enter the theoretical concentration of the standard and the name of the sample, manually add the sample through the sample tube extended from the nebulizer, set the sample volume to at least 1mL each time, select (Start) to test → Stop the frame → Save the data and disconnect the instrument from the computer → Shut down.

[0159] Test results:

[0160] [Table 3]

[0161] [Table 4]

[0162] Example 4: Determination of Zoledronic Acid Precipitation Rate of Zn-Zoledronic Acid (1 / 0.125) Adjuvant Method: UV spectrophotometry, equipment: UV800 (Beckman coulter).

[0163] Zoledronic acid has an imidazole ring and has a maximum absorption peak at 215 nm. The specific process of measuring the supernatant of adjuvant zoledronic acid at 215 nm by ultraviolet spectrophotometer was as follows:

[0164] First, standard zoledronate sodium solutions of 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, 0.015 mg / mL, 0.0075 mg / mL, and 0.00375 mg / mL were prepared in saline, and their OD 215 The values ​​were measured at a wavelength of 215 nm, and a standard curve was plotted. At the same time, Zn-zoledronic acid (1 / 0.125) was centrifuged at 13000 rpm for 10 minutes, and the supernatant was collected. The absorbance of sodium zoledronate in the supernatant was measured at a wavelength of 215 nm. The results were as follows:

[0165] [Table 5]

[0166] [Table 6]

[0167] Example 5: Determination of the adjuvant activity of Zn-zoledronic acid adjuvant in a recombinant protein VZV gE vaccine The prepared zoledronic acid zinc adjuvant (also known as Zn-zoledronic acid) was used as an adjuvant with a Zn / zoledronic acid molar concentration ratio of 1:0.25 or 1:1. These were used as adjuvants in combination with VZV gE antigen, and both were intramuscularly injected into mice, and the specific antibody titers produced were measured. The specific method was as follows: Experimental animals: Balb / C mice, 6-8 weeks old, 5 mice / group, female. Drug concentration: antigen: 50 μg / ml. Dosage: 5 μg / 50 μl / mouse; aluminum adjuvant: 50 μl / mouse; zinc zoledronate adjuvant: 50 μl / mouse. Experimental groups: (1) basic aluminum adjuvant group (Al-002); (2) Zn-zoledronic acid (1 / 0.25); (3) Zn-zoledronic acid (1 / 1).

[0168] Immunization protocol: Two weeks after the first immunization of animals according to the immunization group, blood samples were taken from the orbit to determine the antibody titers in the serum. Antibody titers were measured two weeks after the first immunization. A booster immunization was performed at week 2. Two weeks after the booster immunization, blood samples were taken from the orbit to determine the antibody titers in the serum, and a third immunization was performed. Two weeks later, the antibody titers in the serum were determined by ELISA.

[0169] Determination of antibody titers by enzyme-linked immunosorbent assay (ELISA): 1. Antigen coating solution: 1×PB 7.4 buffer solution (4.343 g Na2HPO4·7H2O; 0.456 g NaH2PO4). 2. Washing solution: PBST, ELISA kit from Beijing Wantai 3. Blocking solution: 2x ED (Enzyme Dilution): 1x PBS + 0.5% casein + 2% gelatin + 0.1% preservative (proclin-300) diluted 1x with ultrapure or distilled water for sealing and sample dilution. 4. Coloring solution A: ELISA kit from Beijing Wantai. 5. Color solution B: ELISA kit from Beijing Wantai. 6. Stop solution: ELISA kit from Beijing Wantai.

[0170] Testing Procedure: (1) Plate coating: VZV gE antigen was diluted to a certain concentration, usually 100 ng / well, in PB7.4 coating buffer solution and added to a 96-well polystyrene plate at 100 μL / well, and the plate was coated overnight at 4° C. (2) Blocking: The coating solution in the wells was discarded, the plate was washed once with PBST washing solution, and dried by centrifugation. Blocking solution was added at 200 μL / well, and blocking was carried out at room temperature for 4 hours. (3) Addition of serum at a certain dilution: discard the blocking solution in the wells, wash the plate once with PBST, and centrifuge dry. Add the serum to be tested to the first well at 150 μL / well, add ED diluent to each of the subsequent wells at 100 μL / well, dilute in a 3-fold gradient, and incubate at 25°C for 1 hour to react. (4) Addition of enzyme-labeled antibody (GAM-HRP): The serum diluent in the wells was discarded, the plate was washed five times with PBST, and dried by centrifugation. 100 μL / well of enzyme-labeled antibody (GAM-HRP, V:V=1:5000) was added, and the plate was incubated at 25° C. for 1 hour to allow the reaction to proceed. (5) Color development: The secondary antibody in the wells was discarded, the plate was washed five times with PBST, and spun dry. A color development solution containing equal amounts of A and B was added at 100 μL / well, and the reaction was allowed to proceed at 25° C. for 10 minutes. (6) Stopping: 50 μL / well of 2 M sulfuric acid stopping solution was added to stop the reaction. (7) Reading the plate: The measurement wavelengths of the microplate reader were set to 450 nm and 630 nm, and the OD value of each reaction well was measured.

[0171] The experimental results are shown in Figure 3.

[0172] The adjuvant was mixed with the same amount of antigen and the mice were immunized. Immunization, blood sampling, and serum antibody titer determination were performed according to the above-mentioned strategy. In the zoledronate zinc adjuvant group, the mice showed rapid and strong expression after one immune injection. After two immune injections, the benefit of humoral immunity enhancement showed statistically significant difference compared with the aluminum adjuvant group, and the difference remained significant after three immune injections.

[0173] Example 6: Effect of zoledronic acid zinc adjuvant in combination with recombinant protein VZV gE on mouse antibody isotypes The immunization strategy was as described in Example 5.

[0174] Test materials: 1. Antigen coating solution: 1×PB 7.4 buffer solution (4.343 g Na2HPO4·7H2O; 0.456 g NaH2PO4). 2. Washing solution: PBST, ELISA kit from Beijing Wantai 3. Blocking solution: 2x ED (Enzyme Dilution): 1x PBS + 0.5% casein + 2% gelatin + 0.1% preservative (proclin-300) diluted 1x with ultrapure or distilled water for sealing and sample dilution. 4. Coloring solution A: ELISA kit from Beijing Wantai. 5. Coloring solution B: ELISA kit from Beijing Wantai. 6. Stop solution: ELISA kit from Beijing Wantai.

[0175] Testing Procedure: (1) Plate coating: VZV gE antigen was diluted to a certain concentration in PB7.4 coating buffer solution and added to a 96-well polystyrene plate at 100 μL / well, and the plate was coated overnight at 4° C. (2) Blocking: The coating solution in the wells was discarded, the plate was washed once with PBST, and dried by centrifugation. Blocking solution was added at 200 μL / well, and blocking was carried out at room temperature for 4 hours. (3) Addition of test subject serum: Serum used for serum antibody isotype detection: serum from 2 weeks after the completion of the three immune injections, i.e., the 6th week; the blocking solution in the wells was discarded, the plate was washed once with PBST, and centrifuged and dried. A certain dilution of the test subject serum was added at 100 μL / well, and incubated at 25° C. for 1 hour to allow the reaction. (4) Addition of enzyme-labeled antibodies: The serum diluent in the wells was discarded, the plate was washed five times with PBST, and dried by centrifugation. 100 μL / well of enzyme-labeled antibodies that specifically recognize each antibody isotype (IgG1, V:V = 1:30000; IgG2a, V:V = 1:1000; IgG2b, V:V = 1:1000) were added, and the plate was incubated at 25°C for 1 hour to allow the reaction to proceed. (5) Color development: The enzyme-labeled antibody in the wells was discarded, the plate was washed five times with PBST, and dried by centrifugation. Equal amounts of A and B were mixed, and 100 μL / well of a 3-fold diluted color development solution was added and allowed to react at 25° C. for 10 minutes. (6) Stop: 50 μL / well of 2 M sulfuric acid stop solution was added to stop the reaction. (7) Reading the plate: The measurement wavelengths of the microplate reader were set to 450 nm and 630 nm, and the OD value of each reaction well was measured.

[0176] The experimental results are shown in Figure 4.

[0177] The experimental procedure described in Example 5 was used. Mice were immunized with zinc zoledronate adjuvant or aluminum adjuvant combined with recombinant VZV gE protein by intramuscular injection. The immunization procedure was the same as in Example 5. After three immunization injections, blood samples were collected two weeks later, and the results of the levels of each antibody isotype in mouse serum are shown in Figure 4. Compared with the aluminum adjuvant group, the zinc zoledronate adjuvant group can stimulate higher levels of IgG2a and IgG2b antibody isotypes, and the ratio of IgG1 to IgG2a and IgG2b is lower than that of the aluminum adjuvant group, indicating that it has a certain stimulating effect on Th1 immune pathway.

[0178] Example 7: Determination of the adjuvant activity of zinc zoledronate adjuvant in hepatitis B therapeutic protein The experimental procedure described in Example 5 was used. Mice were immunized with zoledronic acid zinc adjuvant combined with hepatitis B therapeutic protein by intramuscular injection, and serum antibody titers were determined. The results are shown in Figure 5. Compared with the aluminum adjuvant group, the zoledronic acid zinc adjuvant group showed rapid onset and strong immune response in mice after one immunization injection, and the difference was statistically significant. After two immunization injections, the advantage of humoral immunity enhancement was still significant compared with the aluminum adjuvant group, and after three immunization injections, it was slightly better than the aluminum adjuvant group.

[0179] Example 8: Effect of other zinc bisphosphonate adjuvants in combination with VZV gE on mouse serum antibody titers The experimental procedure described in Example 5 was used. Mice were immunized with zinc bisphosphonate adjuvant combined with recombinant VZV gE protein by intramuscular injection, and serum antibody titers were determined. The results are shown in Figure 6. After three immunization injections in mice, the zinc zoledronate adjuvant group showed significant differences in promoting humoral immune enhancement compared with the aluminum adjuvant group, but the effects of other zinc bisphosphonate adjuvants were not significantly different from aluminum adjuvant.

[0180] Example 9: Effect of Zinc Zoledronate Adjuvant in Combination with VZV gE on Mouse Serum Antibody Titers The experimental procedure described in Example 5 was used. Mice were immunized with zinc zoledronate adjuvant combined with VZV gE by intramuscular injection, and serum antibody titers were determined. As shown in Figure 7, aluminum adjuvant, complexed zinc-aluminum adjuvant, complexed zinc-aluminum adjuvant doped with zoledronic acid, and zinc zoledronate adjuvant showed a gradual trend in the level of antibody enhancement, with significant differences observed by comparing the zinc zoledronate adjuvant group with the aluminum adjuvant group. < / ok>

Claims

1. 1. A method of making a zinc zoledronate micro / nanoparticle adjuvant comprising: a) providing a soluble salt solution containing zinc ions; b) homogeneously mixing the soluble salt solution of step a) with zoledronic acid and sodium hydroxide, or homogeneously mixing the soluble salt solution of step a) with zoledronic acid, sodium hydroxide and sodium phosphate solutions in the manner of continuous precipitation, separate precipitation followed by mixing, or co-precipitation to obtain zinc zoledronate adjuvant; wherein the resulting zinc zoledronic acid adjuvant has a molar concentration ratio of zinc:zoledronic acid of 1-8:

1.

2. 2. The method of claim 1, wherein the resulting zinc zoledronic acid adjuvant has a molar concentration ratio of zinc:zoledronic acid selected from 1:1, 4:1 and 8:

1.

3. One or more of the following: (1) the soluble salt solution comprises a solution of hydrochloric acid; and (2) the method further comprises subjecting the mixture of step b) to sterilization and storing it at 2°C to 8°C for later use; 2. The method according to claim 1 .

4. One or more of the following: (1) The sterilization includes sterilization using high temperature and high pressure sterilization techniques; and (2) The sterilization includes sterilization at 121°C for 30 to 60 minutes; 4. The method according to claim 3 .

5. One or more of the following characteristics: (1) the resulting zinc zoledronate micro / nanoparticle adjuvant has a pre-sterilization pH of 8.0-9.0; (2) the resulting zinc zoledronate micro / nanoparticle adjuvant has a pH after sterilization of 6.0-8.0; (3) the resulting zinc zoledronate micro / nanoparticle adjuvant has a particle size between 1 μm and 10 μm; (4) the resulting zinc zoledronate micro / nanoparticle adjuvant has a particle zero charge point of 4.0 to 11.4; and (5) The resulting zinc zoledronate micro / nanoparticle adjuvant has a protein adsorption rate of greater than 80%; 2. The method according to claim 1 .

6. The method of claim 5 , wherein the protein comprises bovine serum albumin.

7. 10. A method of producing a vaccine adjuvant, pharmaceutical composition, drug delivery vehicle, immunogenic composition or vaccine composition comprising providing a zinc zoledronate micro / nanoparticle adjuvant according to the method of any one of claims 1 to 6.

8. One or more of the following: (1) the vaccine is a protein vaccine; and (2) The vaccine is a varicella-zoster virus recombinant protein vaccine; 8. The method according to claim 7 .

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