RU2801598C2 - Obtaining adjuvant based on zinc zoledronate micro/nanoparticles and its use as a vaccine adjuvant - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention can be used in medical practice. The invention discloses a vaccine adjuvant based on zinc zoledronate microparticles, which contains zinc and zoledronic acid, respectively, as well as possible additives based on phosphate and aluminum. The method of its preparation includes carrying out mixed precipitation in a soluble salt solution containing zinc ions, zoledronic acid and sodium hydroxide.

EFFECT: invention can be used as an immunostimulating additive and adjuvant in the preparation of pharmaceutical compositions and formulations.

19 cl, 9 ex, 6 tbl, 7 dwg

Description

Technical field

The present invention relates to a vaccine adjuvant, in particular, a micro/nanoparticle zinc zoledronate adjuvant with a sustained release effect, which can significantly enhance the humoral immune response and the cellular immune response, and can be used as a vaccine adjuvant in the production of vaccines. The present invention relates to the field of immunology.

State of the art

Zinc is an important chemical element with specific biological functions. For example, trace amounts of zinc are present in approximately 300 enzymes involved in cellular metabolism, and they play an important role in cellular respiration, cell division, protein synthesis, DNA synthesis, damage repair, and activation of immune function. Thus, zinc deficiency or metabolic disorders are associated with many diseases, in particular, with diseases of the immune system (Autoimmunity Reviews, 2015, 14(04) 277-285). In immunocytes, divalent cations, including zinc, play an important role in the regulation of intracellular signaling pathways. Zinc deficiency affects survival, proliferation, and maturation of lymphocytes in the innate and adaptive immune systems; studies have shown that zinc deficiency adversely affects the production and function of cytokines synthesized by immunocytes and the number, proliferation and differentiation of peripheral and thymic T cells. Research has shown that a deficiency in trace amounts of zinc leads to "aging" of the immune system in the elderly (Immunity & Aging, 2009, 6(9): 1-17). In general, zinc plays an important role in the production, development, and function of T cells.

Bisphosphonates (BP), as the most important drugs for the treatment of osteoporosis, are analogues of inorganic pyrophosphate. They are formed by replacing the oxygen atom in the P-O-P pyrophosphate bond with a carbon atom and have a specific backbone of the P-C-P bond. They cannot easily be enzymatically hydrolyzed in the body, are resistant to high temperatures, and are stable to many chemicals. Meanwhile, due to the P-C-P molecular configuration, their 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. It has strong action, low dose and ease of use. Clinical studies have shown that bisphosphonates, used as an adjuvant treatment for diseases such as multiple myeloma, breast cancer, kidney cancer, prostate cancer, etc., can reduce the incidence of bone-related diseases in patients, reduce the recurrence rate cancer and prolong patient survival, thereby improving clinical outcomes. Zoledronate is particularly advantageous in terms of antitumor activity and is widely used in clinical trials with various cancers. In addition, other studies have shown that bisphosphonate-based drugs also have immunomodulatory effects and can increase the humoral immune response by targeting B cells (Cell Rep., 2013, 5(2)); and zoledronic acid can increase the antigen-specific response of CD8+ T cells (Vaccine, 2016, 34 (10): 1275-81).

Dieter Bernardt et al. developed a zinc hydroxide gel or an iron hydroxide gel, a method for producing the same, and its use as an adjuvant for a vaccine (U.S. Patent No. 5,252,327, issue date: October 12, 1993). Zinc hydroxide gel or iron hydroxide gel has a good adsorption effect on viruses or proteins, and has a good adjuvant effect; zinc hydroxide gel can stimulate both humoral and cellular immune response, while iron hydroxide gel mainly enhances humoral immune response, and both have good local and systemic tolerance; and both have a higher immunostimulatory effect when used in combination with lecithin.

Hu Yunzhang et al. developed a vaccine adjuvant (China Patent Publication (Notice) No. CN101444623A, Publication Date (Notice): June 3, 2009). The invention relates to a vaccine adjuvant, i.e. to a zinc hydroxide compound having the chemical formula Zn(OH) ₂ and a molecular weight according to the chemical formula 99.4046 daltons, for use in the production of an adjuvant for vaccines, and for use in the preparation of an adjuvant for hepatitis A vaccines. Zinc hydroxide used in combination with the vaccine as an adjuvant can effectively enhance the humoral immune response of the vaccine, and its immune enhancing effect is higher than that of aluminum adjuvant. And animal test results show that zinc hydroxide used as a new vaccine adjuvant has a better sensitizing effect and better safety performance than aluminum adjuvant.

Wang Haixuan et al. (Chinese Journal of Biologicals, 2011, 24 (10): 1174-1176) studied the immunopotentiation of heparan sulfate (HS) and zinc hydroxide as an adjuvant on the humoral immune response in mice induced by a recombinant polypeptide vaccine with an antigenic epitope of hepatitis C virus, and the results showed that the combined adjuvant of HS and zinc hydroxide can effectively enhance the humoral immune response in mice immunized with a recombinant polypeptide vaccine with an HCV antigenic epitope and can reduce the dose of antigen to some extent.

Hu Yunzhang et al. developed a zinc phosphate vaccine adjuvant (China Patent Publication (Notice) No. CN101972477B, Publication Date (Notice): September 26, 2012). The invention relates to a zinc phosphate vaccine adjuvant, in particular, to the use of zinc phosphate as a vaccine adjuvant and the use of a zinc phosphate vaccine adjuvant in the manufacture of vaccines. Zinc phosphate is used as a vaccine adjuvant in combination with an antigen that can effectively enhance the humoral immune response to a vaccine. Its immune enhancing effect is higher than that of aluminum adjuvant and zinc hydroxide adjuvant, and animal test results showed that when used as a vaccine adjuvant, zinc phosphate showed higher sensitizing effect and better safety properties than aluminum adjuvant and zinc hydroxide adjuvant.

Hu Yunzhang et al. developed a combined vaccine adjuvant (China Patent Publication (Notice) No. CN102085366B, Publication Date (Notice): December 26, 2012). The invention relates to a combined vaccine adjuvant, characterized in that it consists of the following components: sodium ferulate and zinc hydroxide in a mass ratio of 10:1 to 50:1. The combined use of a combined vaccine adjuvant and vaccine can effectively enhance the humoral immune response to the vaccine. The enhancement effect is similar to that of aluminum adjuvant, but superior to that of sodium ferulate adjuvant alone and zinc hydroxide adjuvant alone. And it is non-toxic, has no side effects, is safe and reliable at the dose required for immunization. The raw materials for the combined vaccine adjuvant of the invention are widely available and are commercially available products. The combined vaccine adjuvant has a simple preparation method, low cost and stable efficacy. The combined vaccine adjuvant can be used as an adjuvant for hepatitis B vaccine, genetically engineered vaccine, viral vaccine, and the like.

Wang Haixuan et al. developed a zoledronic acid adjuvant and a vaccine containing a zoledronic acid adjuvant (China Patent Publication (Notice) No. CN103768595 B, Publication Date (Notice): March 2, 2016). The invention relates to a zoledronic acid adjuvant and a vaccine containing a zoledronic acid adjuvant, i.e. each dose of the vaccine contains 10 to 20 μg of the zoledronic acid adjuvant. The zoledronic acid adjuvant provided by the invention has low toxicity and side effects, is safe and reliable when used at a dose required for immunization, can effectively induce an antigen-specific humoral immune response, and has an immune effect similar to that of an aluminum adjuvant, and its The raw material is widely available, and it has stable efficacy, and it can be used as an adjuvant for vaccines. Takuya SHISHIDO et al. developed a vaccine pharmaceutical composition containing a bisphosphonate that can induce an effective humoral immune response in the body and that can be administered transdermally or through the mucous membranes (US patent, publication number: 2017/0281759A1, publication date: October 5, 2017).

Cai Hongzhi et al. (Chinese Journal of Biologicals, 2013, 26 (05): 608-610) investigated the effect of a combined adjuvant based on zinc hydroxide and heparan sulfate (HS) on the rabies vaccine-induced humoral immune response in mice, and the results showed that the combined adjuvant on based on zinc hydroxide and HS can enhance the humoral immune response in mice induced by the rabies vaccine.

Chen Zhenpu et al. developed a zinc glycinate adjuvant and a vaccine containing a zinc glycinate adjuvant (China Patent Publication (Notice) No. CN104096229A, Publication Date (Notice): October 15, 2014). The invention relates to a zinc glycinate adjuvant and a vaccine containing a zinc glycinate adjuvant, i.e. each vaccine dose contains 0.1 to 1 mg of zinc glycinate adjuvant dissolved in PBS pH=6-8. The zinc glycinate adjuvant provided by the invention is an adjuvant for medical practice, and also a zinc nutritional enhancer. It has low toxicity and side effects and is safe and reliable at the dose required for immunization. It can effectively induce antigen-specific humoral immune responses, and the effect of the induced humoral immune response is higher than that of the group without adjuvant. Raw materials for zinc glycinate adjuvant are widely available, and are commercially available products. It has an easy production process, low cost, stable efficacy, high biological activity, no toxic or adverse effects, and can be added to various conventional vaccines and genetically engineered vaccines as a vaccine adjuvant.

Mahsa Afshari et al. (Cytokine, 2016, 81: 71-76) used a Leishmania infection model in BALB/c mice. It was found that oral feeding of zinc sulfate to mice can significantly reduce the parasitic load and increase the level of mRNA expression of the Th1-profile cytokine. This indicates that zinc sulfate can induce a strong Th1 immune response in BALB/c mice infected with Leishmania .

The essence of the invention

To overcome the shortcomings of existing vaccine aluminum adjuvants, i.e., the inability to stimulate a Th1-mediated immune response and the inability to enhance the immunogenicity of vaccines based on polypeptides and nucleic acids, and also taking into account the role of zoledronic acid in enhancing immunity, an adjuvant based on micro/nanoparticles of zoledronate was developed. zinc by co-precipitation of zoledronic acid and the trace element zinc, which also plays an important role in the regulation of the immune system. Through creative work, the inventors of the present invention found that the micro/nanoparticle zinc adjuvant zoledronate can significantly increase the humoral immune response and the cellular immune response. Thus, the present invention relates to a vaccine adjuvant based on zinc zoledronate with a significant immunostimulatory effect.

Therefore, in one aspect, the present invention relates to a micro/nanoparticulate zinc zoledronate adjuvant obtained by co-precipitating zinc ions together with phosphonic acid groups, phosphate radicals and hydroxide radicals, which is referred to as zinc zoledronate adjuvant for short.

In a specific embodiment, the zoledronate zinc adjuvant of the present invention can be prepared by uniformly mixing a solution of a soluble salt containing zinc ions with zoledronic acid and sodium hydroxide solution, or by uniformly mixing a solution of a soluble salt containing zinc ions with zoledronic acid, sodium hydroxide solution and sodium phosphate to obtain an adjuvant based on zinc zoledronate.

In a specific embodiment, the mixing method includes, but is not limited to, successive precipitation, separate precipitation followed by mixing or co-precipitation.

In one embodiment, the molar ratio of zinc:zoledronic acid in the zinc zoledronate adjuvant is generally not limited. In a preferred embodiment, the molar ratio of zinc:zoledronic acid in the zinc zoledronate adjuvant may be (1-8):1. Preferably, the molar ratio of zinc:zoledronic acid is selected from 1:1, 4:1 and 8:1.

In one embodiment, the zinc zoledronate adjuvant may also contain a phosphate. For example, a zinc zoledronate combination adjuvant is prepared by displacing zoledronic acid with various molar ratios of phosphate (incomplete substitution) using various mixing methods (eg sequential precipitation, separate precipitation followed by mixing or co-precipitation, etc.). In such a combined zinc zoledronate adjuvant, the molar ratio of Zn:phosphate radical is not generally limited. In a preferred embodiment, the ratio of molar concentrations of Zn:phosphate radical may be (1-8):1. Preferably the ratio of molar concentrations of Zn:phosphate radical is selected from 1.5:1 and 4:1, thereby forming an organic-inorganic hybrid adjuvant zinc zoledronate.

In another embodiment of the zinc zoledronate adjuvant, the zoledronate zinc adjuvant may further comprise aluminum (Al); for example, a zinc-aluminum zoledronate adjuvant is prepared by substituting Zn for Al in various ratios (incomplete substitution) and using various mixing methods (eg sequential precipitation, split precipitation followed by mixing or co-precipitation, etc.). In such an adjuvant, zinc-aluminum zoledronate, the Zn:Al molar ratio is generally not limited. In a preferred embodiment, the Zn:Al molar ratio may be (0.02-1):1, and preferably the Zn:Al molar ratio is selected from 0.375:1, 0.5:1 and 0.8:1.

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-containing adjuvants known in the art are acceptable, provided that the adjuvant zinc zoledronate is prepared by precipitation of zinc ions and phosphonoacid groups, phosphate radicals and hydroxide radicals.

In one embodiment, the phosphate solution can be selected from, but not limited to, sodium phosphate, sodium hydrogen phosphate (anhydrous, dihydrate, heptahydrate, dodecahydrate), sodium dihydrogen phosphate (anhydrous, dihydrate), potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, pyrophosphate, polyphosphoric acids and any mixtures thereof.

In one embodiment, the specific type of aluminum compound is not limited, for example, aluminum hydroxide, aluminum phosphate, aluminum sulfate, or other types of aluminum-based adjuvants known in the art are acceptable, provided that the zinc-aluminum zoledronate adjuvant is prepared by precipitation of zinc ions and aluminum with phosphonic acid groups, phosphate radicals and hydroxide radicals.

In one aspect, the present invention relates to a process for the preparation of a zinc zoledronate adjuvant, which comprises:

precipitation of zinc ions by reacting zinc ions with phosphonic acid groups, phosphate radicals and hydroxide radicals individually or simultaneously in a soluble salt solution, thereby obtaining a zinc zoledronate adjuvant.

In a specific embodiment, the method includes:

a) obtaining a soluble salt solution containing zinc ions,

b) Uniform mixing of the soluble salt solution from step a) with zoledronic acid, sodium hydroxide or uniform mixing of the soluble salt solution from step a) with zoledronic acid, sodium hydroxide solution and sodium phosphate by successive precipitation, separate precipitation followed by mixing or co-precipitation to obtain zinc zoledronate adjuvant.

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

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

In yet another embodiment, in the method of the present invention for preparing a 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 using various mixing methods (e.g. , sequential precipitation, separate precipitation followed by mixing or co-precipitation, etc.), where the ratio of molar concentrations of Zn/Al is set at 0.375:1, 0.5:1 and 0.8:1.

The soluble zinc ion salt solution used herein may be any soluble zinc ion salt solution, and is preferably a solution of zinc ions in hydrochloric acid.

Within the scope of the present invention, the zoledronate solution is preferably an alkaline solution of zoledronic acid and sodium hydroxide.

Within the scope of the present invention, methods for precipitating zinc and phosphonic acid groups can be any methods in which the precipitation reaction is carried out by thoroughly mixing a solution of a soluble salt of zinc ions and an alkaline solution of zoledronic acid and sodium hydroxide. Preferably, the zinc zoledronate adjuvant can be prepared in a manner such as sequential precipitation, separate precipitation followed by mixing or co-precipitation, etc.

Within the scope of the present invention, sterilization may be any method suitable for sterilizing the zinc zoledronate adjuvant. For example, steam sterilization at high temperature and high pressure can be used. For example, sterilization is carried out at 121°C for 30-60 minutes, preferably 60 minutes.

In one embodiment, the present invention also relates to a method for determining the physical and chemical properties of the resulting zinc zoledronate adjuvant. In one embodiment, the pH value, particle size, zeta potential, point of zero charge (PZC) of the particles, degree of adsorption and dissociation with proteins, degree of precipitation of metal ions, degree of precipitation of organic phosphonic acid, degree of dissolution in vivo and in vitro are determined for the adjuvant. draft, etc. A. The physical and chemical properties of the adjuvant can be determined by conventional methods, for example, see US 9573811; Ai Xulu et al., Analysis of physicochemical properties of three aluminum hydroxide adjuvant, Chinese Journal of Biologicals, 2015, 28(1): 44-477; and as described in the examples here.

In one embodiment, the zinc zoledronate adjuvant as described herein has one or more of the following properties: pH 8.0-9.0 before sterilization, pH 6.0-8.0 after sterilization, particle size 1-10 microns , the point of zero charge of particles 4.0-11.4, the degree of deposition of metal ions> 99% and the degree of protein adsorption> 80%.

In one aspect, the present invention relates to a composition, in particular a pharmaceutical composition or composition, which contains an adjuvant zinc zoledronate, as described here.

The method for preparing a pharmaceutical formulation or composition includes the step of combining the zinc zoledronate adjuvant with the carrier and, optionally, one or more accessory ingredients.

In essence, formulations are prepared by uniformly and uniformly combining the zinc zoledronate adjuvant with liquid carriers or particulate solid carriers, or both, and then shaping the product if necessary.

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

In addition to inert diluents, compositions for oral administration may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preservative agents.

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

The pharmaceutical composition of the present invention for rectal or vaginal administration may be presented in the form of a suppository which may 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, suppository wax, or salicylate), which are solid at room temperature and liquid at body temperature, thereby melting in the rectum or vagina to release the adjuvant zinc zoledronate. A composition suitable for vaginal administration of the present invention also includes a vaginal suppository, tampon, cream, gel, paste, foam or spray containing suitable carriers known in the art.

A pharmaceutical composition of the present invention suitable for parenteral administration comprises a zoledronate zinc adjuvant and one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous carriers in combination therewith, including a solution, dispersion, suspension or emulsion, or a sterile powder, which may be reconstituted into a sterile injectable solution or dispersion prior to use, which may contain antioxidants, buffers, solutes that render the composition isotonic with the blood of the intended recipient, or suspending agents or thickeners.

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

These compositions may also contain adjuvants such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be provided by the inclusion of absorption delaying agents such as aluminum monostearate and gelatin.

Depot injections can be prepared by matrixing microcapsules of zinc adjuvant zoledronate in biodegradable polymers (such as polylactide-polyglycolide). Depending on the ratio of zinc zoledronate adjuvant to polymer and the nature of the particular polymer used, the release rate of zinc zoledronate adjuvant can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectables are also prepared by incorporating the zinc zoledronate adjuvant into liposomes or microemulsions that are compatible with body tissues. Injectable materials can be sterilized, for example, by filtration through a bacteria-retaining filter.

Formulations may be presented in single-dose or multi-dose sealed containers (eg, ampoules and vials) and may be stored in a lyophilized state requiring only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Solutions and suspensions for the preparation of extemporo can be prepared from sterile powders, granules and tablets of the type described above.

In one aspect, the present invention also provides an immunogenic composition comprising a zinc zoledronate adjuvant as described herein and one or more antigens.

Within the scope of the present invention, an immunogenic composition, when administered to a subject or animal, induces a protective immune response against said one or more antigens contained therein.

In one aspect, the present invention also provides a vaccine composition comprising a zinc zoledronate adjuvant as described herein and one or more antigens.

Within the scope of the present invention, the vaccine composition, when administered to a subject or animal, induces a protective immune response, for example against a microorganism, or to effectively protect the subject or animal from infection.

The vaccine composition can be used to prevent or alleviate a pathological condition in which there will be a favorable response to the modulation of the immune response. Such a vaccine composition may be a prophylactic vaccine or a therapeutic vaccine. Preferably, the vaccine composition comprises a genetically engineered vaccine such as a protein vaccine such as a recombinant varicella zoster virus vaccine.

In one aspect, the present invention also relates to a vaccine adjuvant, including a zinc zoledronate adjuvant as described herein. For example, such a vaccine adjuvant may also include an additional adjuvant, as described below.

In the context of the present invention, the term "adjuvant" or "vaccine adjuvant" refers to a substance capable of non-specifically accelerating, prolonging or enhancing the immune response against an antigen.

Advantageously, adjuvants may also reduce the number of immunizations or the amount of antigen required to elicit protective immunity.

It is well known that the adjuvant itself does not stimulate or hardly stimulate the immune response, but the adjuvant enhances the immune response against the antigen. Therefore, the zoledronate zinc adjuvant of the present invention can be combined with one or more antigens to form an immunogenic composition or vaccine that can be used to stimulate an immune response in a subject. Various substances can be used as antigens in an immunogenic or vaccine type complex or formulation. For example, attenuated and inactivated viral and bacterial pathogens, purified macromolecules, proteins, polysaccharides, toxoids, recombinant antigens, microorganisms containing a foreign pathogen gene, synthetic peptides, polynucleotides, antibodies and tumor cells, etc. Antigens can be used both in preventive and therapeutic vaccines. Antigens include protein antigens such as the varicella zoster virus glycoprotein antigen gE (VZV gE).

Various immunomodulatory molecules can also be used in combination with the zinc zoledronate adjuvant of the present invention to alter the immune response in a subject. The immunomodulators described herein belong to a group of compounds that can regulate, balance and restore the body's immune function. Commonly used immunomodulators fall into three main categories, which are immune enhancers, immunosuppressors, and bidirectional immune modulators.

The amounts of antigen and adjuvant based on zinc zoledronate in the vaccine composition of the present invention and the dose to be administered are determined by methods well known to those skilled in the pharmaceutical art, where factors such as the following must be taken into account: specific antigen, age, sex, body weight, species and condition. specific animal or patient, as well as the route of administration.

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 promoters, water-absorbing polymers, substances that inhibit enzymatic degradation, alcohols, organic solvents, oils, agents, pH adjusters, preservatives, osmotic pressure adjusters, propellants, water, and any mixtures thereof.

The vaccine composition of the present invention may further include a pharmaceutically acceptable carrier. The amount of carrier will depend on the amounts chosen for the other ingredients, the concentration of antigen required, the choice of route of administration (oral or parenteral), etc. The carrier may be added to the vaccine at any suitable time. In the case of a lyophilized vaccine, the carrier may, for example, be added prior to administration. Alternatively, the final product may be made with a carrier.

Examples of suitable carriers include, but are not limited to, sterile water, saline, buffers, phosphate buffered saline, buffered sodium chloride, vegetable oils, minimal essential medium (MEM), HEPES buffered MEM, etc.

Optionally, the vaccine composition of the invention may contain conventional additional adjuvants in varying amounts depending on the adjuvant and the desired result.

Examples of suitable additional adjuvants include, but are not limited to, stabilizers; emulsifiers; pH adjusters such as sodium hydroxide, hydrochloric acid, etc.; surfactants such as ^Tween® 80 (polysorbate 80, commercially available from Sigma Chemical Co., St. Louis, Mo.); liposomes; iscom adjuvant; synthetic glycopeptides such as muramyl dipeptides; fillers such as dextran; carboxypolymethylene; bacterial cell walls such as mycobacterial cell wall extract; their derivatives such as Corynebacterium parvum ; Propionibacterium acne ; Mycobacterium bovis , such as bovine bacillus Calmette-Guérin (BCG); 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); synthetic agents and mixtures thereof.

Examples of suitable stabilizers include, but are not limited to, sucrose, gelatin, peptone, protein hydrolysate extracts such as NZ-amine or NZ-amine AS. Examples of emulsifiers include, but are not limited to, mineral oil, vegetable oil, peanut oil, and other standard, metabolizable, non-toxic oils suitable for injectable or intranasal vaccine compositions.

For the purposes of the present invention, these adjuvants are referred to herein as "additional" adjuvants, simply to contrast with the zinc zoledronate adjuvant described above, since the combination of the zinc zoledronate adjuvant and the antigenic substance can significantly enhance the humoral immune response to the antigenic substance. Additional adjuvants are primarily included in the vaccine as an aid to the manufacturing process, although some adjuvants do have some degree of immuno-enhancing properties and have a dual purpose.

Conventional preservatives may be added to the vaccine composition in effective amounts ranging from about 0.0001% to about 0.1% by weight. Depending on the particular preservative used in the formulation, amounts below or above the stated range may be suitable. Typical preservatives include, for example, potassium sorbate, sodium metabisulphite, phenol, methylparaben, propylparaben, thimerosal, etc.

The choice of an inactivated, modified, or other type of vaccine composition and obtaining an improved composition of the vaccine composition of the present invention is known or easily determined by experts in this field.

In general, the vaccine composition of the present invention can be conveniently administered orally, parenterally (subcutaneously, intramuscularly, intravenously, intradermally, or intraperitoneally), buccally, intranasally, or transdermally. The route of administration contemplated by the present invention will depend on the antigenic substance and associated excipients. For example, if the vaccine composition contains saponins that are non-toxic when administered orally or intranasally, then care must be taken when introducing sapogenin glycosides into the bloodstream, since they act as strong hemolytics. In addition, many antigens will not be effective when administered orally. Preferably, the vaccine composition is administered intramuscularly or intraperitoneally.

The dosage of the vaccine composition will depend, inter alia, on the selected antigen, route of administration, species and other usual factors. It is believed that a person skilled in the art can easily and without difficulty titrate the appropriate dose to induce an immunogenic response to each antigen to determine the effective immunizing amount and route of administration.

With respect to a vaccine adjuvant, the zoledronate zinc adjuvant of the present invention may increase vaccine efficacy by enhancing the immunogenicity of weaker antigens (e.g., highly purified or recombinant antigens), by reducing the amount of antigens needed to induce an immune response, or by reducing frequency of immunizations required for protective immunization. The zinc zoledronate adjuvant of the present invention may enhance vaccine efficacy in subjects (eg, neonates, elderly, and immunocompromised individuals) with reduced or compromised immune responses and may enhance vaccine efficacy in target tissues. Alternatively, the adjuvant zinc zoledronate of the present invention may enhance cellular immunity and/or humoral immunity by triggering a specific cytokine profile.

The combination of an antigen and/or immunomodulatory molecule and a zinc zoledronate adjuvant of the present invention can be tested in a variety of preclinical toxicology and safety studies well known in the art.

For example, such a combination can be evaluated in an animal model in which the antigen has been shown to be immunogenic and can be reproducibly immunized in the same way that is proposed for clinical trials in humans.

The combination of an antigen and/or immunomodulatory molecule and a zinc zoledronate adjuvant of the present invention can be tested, for example, using the approach established by the Center for Biological Evaluation and Research/Food and Drug Administration and the National Institute of Allergy and Infectious Diseases (Goldenthal K. L. et al. AID Res Hum Retroviruses, 9: S45-9 (1993)).

Those skilled in the art will know how to determine, for a particular combination of an antigen and/or immunomodulatory molecule with a combination adjuvant of the invention, the appropriate antigen load, immunization route, dose, antigen purity, and vaccination schedule suitable for the treatment of a particular pathological condition in a particular animal species.

The immunogenic composition or vaccine of the invention for inducing an immune response may be administered as a solution or suspension along with a pharmaceutically acceptable medium.

Such a pharmaceutically acceptable medium may be, for example, water, phosphate buffered saline, normal saline or other physiologically buffered saline, or another solvent or carrier such as glycol, glycerin, and an oil such as olive oil or an organic ester for injections. The pharmaceutically acceptable medium may also contain liposomes or micelles and may contain immunostimulatory complexes obtained by mixing polypeptide or peptide antigens with a detergent and a glycoside (such as Quil A).

The immunogenic composition or vaccine of the invention can be administered in a variety of ways to stimulate an immune response. For example, the immunogenic composition or vaccine can be delivered subcutaneously, intradermally, intralymphatic, intramuscularly, intratumorally, intravesically, intraperitoneally, and intracerebral.

Those skilled in the art will know how to select appropriate delivery routes for particular zoledronic acid adjuvant formulations of the invention.

In a preferred embodiment of the present invention, a vaccination method for treating or preventing an infection in a mammal comprises administering a vaccine of the present invention, wherein the vaccine will be specifically administered intramuscularly or intraperitoneally. The vaccine may be administered as a single dose, or preferably administered multiple times, eg two, three or four times per week or per month, using a prime/boost strategy. The appropriate dose depends on many different parameters.

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, the vaccine comprises a protein vaccine, such as a recombinant varicella-zoster virus protein vaccine.

Brief description of the figures

In FIG. 1: diagrams of three methods for preparing adjuvants are shown.

In FIG. 2: Particle morphology of Zinc Zoledronate Adjuvant, Zinc Aluminum Zoledronate Adjuvant and Aluminum Zoledronate Adjuvant is shown.

In FIG. 3: the results of evaluation of the adjuvant activity of the zinc zoledronate adjuvant in the VZV gE vaccine are shown, mean ± standard deviation: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

In FIG. 4: 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.

In FIG. 5: the results of evaluating the adjuvant activity of the adjuvant based on zinc zoledronate with a therapeutic protein against hepatitis B virus are shown, mean value ± standard deviation: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

In FIG. 6: shows the effect of other zinc bisphosphonate adjuvants (Zn/Zol=1/0.25) in combination with VZV gE on mouse serum antibody titer, mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

In FIG. 7: shows the effect of modified adjuvants based on zoledronic acid and zoledronate zinc, in combination with an anti-hepatitis B virus therapeutic protein, on mouse serum antibody titer, mean ± SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

Specific models for carrying out the present invention

Embodiments of the present invention are described in detail below along with examples. In the examples that do not specify specific conditions, it is understood that they should be carried out in accordance with the usual conditions or conditions proposed by the manufacturer. Reagents or instruments used without a manufacturer's name are common products that can be purchased commercially.

The materials used in the following production examples are as follows:

Anhydrous zinc chloride (ZnCl ₂ ): manufactured by Xilong Chemical;

Aluminum chloride hexahydrate (AlCl ₃ ·6H ₂ O): manufactured by Xilong Chemical;

Sodium hydrogen phosphate dodecahydrate (Na ₂ HPO ₄ ·12H ₂ O): manufactured by Xilong Chemical;

Sodium hydroxide (NaOH): manufactured by Xilong Chemical;

Zoledronic acid (C ₅ H ₁₀ N ₂ O ₇ P ₂ ): manufactured by Hunan Huateng Pharmaceutical Co., Ltd.

Preparation Example 1 Preparation of Zn-zoledronic acid adjuvant (1/0.25)

According to a Zn/zoledronic acid molar ratio of 1:0.25, 50 ml of a 31.11 mM zinc chloride solution were prepared, which was identified as solution A; 50 ml of a solution (7.78 mM zoledronic acid + 43 mM sodium hydroxide + 7.78 mM sodium hydrogen phosphate) was prepared, which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for further use.

Preparation of adjuvant suspension Zn-zoledronic acid (1/0.25):

Solutions A and B were used to prepare the zinc zoledronate adjuvant suspension according to the scheme shown in FIG. 1A, i.e. the prepared solution B was added dropwise in accordance with a 1:1 volume ratio to solution A to form a suspension until it was completely added.

The zinc zoledronate adjuvant obtained after mixing as shown in FIG. 1A was subjected to steam autoclave sterilization at 121°C for 60 min, and then cooled to room temperature and then stored at 2-8°C.

Production Example 2: Production of Zn-zoledronic acid adjuvant (1/1)

Preparation of solutions:

According to the Zn/zoledronic acid molar ratio of 1:1, 50 ml of a 31.11 mM zinc chloride solution were prepared, which was identified as solution A; 50 ml of a solution (31.11 mM zoledronic acid + 87 mM sodium hydroxide) was prepared, which was identified as solution B. Solution A and solution B were filtered through a 0.22 µm membrane filter for further use.

As described in Preparation Example 1, a suspension of Zn-zoledronic acid adjuvant (1/1) was prepared.

Preparation Example 3 Preparation of Zinc Aluminum Zoledronic Adjuvant (FH-001-Zoledronic Acid)

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 mmol) and zinc chloride (46.66 mmol) was prepared, which was determined as solution A; 50 ml of a solution (18.66 mM zoledronic acid + 380 mM sodium hydroxide) was prepared, which was identified as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for further use.

As described in Preparation Example 1, a suspension of FH-001-zoledronic acid adjuvant was prepared.

Preparation Example 4 Preparation of Aluminum Zoledronate Adjuvant (Al-001-zoledronic Acid)

Preparation of solutions:

According to a molar concentration of Al/Zol 1:0.075, 50 ml of a 122.44 mM aluminum chloride solution were prepared, which was determined as solution A; 50 ml of a solution (9.33 mM zoledronic acid + 9.33 mM sodium hydrogen phosphate dodecahydrate + 370 mM sodium hydroxide) was prepared, which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm to further use.

As described in Preparation Example 1, a suspension of Al-001-zoledronic acid adjuvant was prepared.

Production Example 5: Preparation of Zn-zoledronic acid (1/0.125)

Preparation of solutions:

According to a Zn/zoledronic acid molar ratio of 1:0.125, 50 ml of a 124.44 mM zinc chloride solution were prepared, which was identified as solution A; 50 ml of a solution (15.56 mM zoledronic acid+65.7 mM sodium hydroxide) was prepared, which was identified as solution B. Solution A and solution B were filtered through a 0.22 µm membrane filter for further use.

As described in Preparation Example 1, a suspension of Zn-zoledronic acid adjuvant (1/0.125) was prepared.

Production Example 6: Preparation of Aluminum Adjuvant (Al-002)

Preparation of solutions:

Aluminum chloride hexahydrate was added to water to give 0.5 L of a 62.22 mM aluminum chloride solution, which was identified as solution A; Sodium hydrogen phosphate dihydrate and sodium hydroxide were added to water to give 0.5 L of a solution containing 18.68 mM phosphate and 0.12 M sodium hydroxide, which was identified as solution B. The solutions were filtered through a 0.22 µm membrane filter for further usage.

As described in Preparation Example 1, aluminum adjuvant Al-002 was prepared.

Production Example 7: Production of Aluminum Adjuvant (Al-001-840)

Preparation of solutions:

According to a phosphate/Al molar ratio of 0.15, 0.5 L of a 124 mM aluminum chloride solution was prepared, which was identified as solution A; prepared 0.5 l of 18.6 mm solution of sodium hydrogen phosphate, which was identified as solution B. Solution B additionally contained 150 mm sodium hydroxide. The solutions were filtered through a membrane filter with a pore size of 0.22 μm for further use.

As described in Production Example 1, a suspension of aluminum adjuvant Al-001-840 was prepared.

Production Example 8: Preparation of Zinc-Aluminum Adjuvant (FH-001)

Preparation of solutions:

Aluminum chloride hexahydrate and anhydrous zinc chloride were added to water to give 0.25 L of a solution containing 124.44 mM aluminum chloride and 46.68 mM zinc chloride, which was identified as solution A; sodium hydrogen phosphate dihydrate and sodium hydroxide were added to water to obtain 0.25 L of a solution containing 18.68 mM phosphate and 0.425 M sodium hydroxide, which was determined as solution B. The solutions were filtered through a membrane filter with a pore size of 0.22 μm for further use.

Preparation of combined aluminum adjuvant:

As shown in FIG. 1A, 0.25 L of solution B was added dropwise to 0.25 L of solution A until it was completely added, thereby obtaining a combined zinc-aluminum adjuvant FH-001. The resulting combined zinc-aluminum adjuvant was sterilized in a steam autoclave at 121°C for 60 minutes, and then cooled to room temperature and then stored at 2-8°C.

Production Example 9: Production of Zinc Etidronic Acid Adjuvant (1/0.25)

According to the Zn/etidronic acid molar ratio of 1:0.25, 50 ml of a 31.11 mM zinc chloride solution were prepared, which was identified as solution A; prepared 50 ml of a solution (7.78 mM etidronic acid + 34 mM sodium hydroxide + 7.78 mM sodium hydrogen phosphate), which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for subsequent use.

As described in Preparation Example 1, a suspension of Zn-etidronic acid adjuvant (1/0.25) was prepared.

Preparation Example 10 Preparation of Zn-clodronic acid adjuvant (1/0.25)

According to the Zn/clodronic acid molar ratio of 1:0.25, 50 ml of a 31.11 mM zinc chloride solution were prepared, which was identified as solution A; prepared 50 ml of a solution (7.78 mM clodronic acid + 36 mM sodium hydroxide + 7.78 mM sodium hydrogen phosphate), which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for subsequent use.

As described in Preparation Example 1, a suspension of Zn-clodronic acid adjuvant (1/0.25) was prepared.

Production Example 11 Preparation of Zn-alendronic acid adjuvant (1/0.25)

In accordance with the ratio of the molar concentrations of Zn/alendronic acid 1:0.25 prepared 50 ml of 31.11 mm solution of zinc chloride, which was determined as solution A; prepared 50 ml of a solution (7.78 mM alendronic acid + 36 mM sodium hydroxide + 7.78 mM sodium hydrogen phosphate), which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for further use.

As described in Preparation Example 1, a suspension of Zn-alendronic acid adjuvant (1/0.25) was prepared.

Production Example 12 Preparation of Zn-Pamidronic Acid Adjuvant (1/0.25)

According to the Zn/pamidronic acid molar ratio of 1:0.25, 50 ml of a 31.11 mM zinc chloride solution were prepared, which was identified as solution A; prepared 50 ml of a solution (7.78 mM pamidronic acid + 32 mM sodium hydroxide + 7.78 mM sodium hydrogen phosphate), which was determined as solution B. Solution A and solution B were filtered through a membrane filter with a pore size of 0.22 μm for subsequent use.

As described in Preparation Example 1, a suspension of Zn-pamidronic acid adjuvant (1/0.25) was prepared.

Further, the present invention will be described below in conjunction with examples.

Example 1 Determination of Physical and Chemical Properties of Zinc Zoledronate Adjuvant

The following determination methods were applicable to any Zn/zoledronic acid molar ratio, i.e. any zinc zoledronate adjuvant such as zinc zoledronate adjuvant with inorganic phosphoric acid addition or Al addition.

(1) Determination of the morphology of adjuvant particles

After diluting zinc zoledronate adjuvant 50 times with deionized water, analysis was performed using a JEM-2100 transmission electron microscope (TEM) from Japan Electronics. Specific steps were as follows: applying the adjuvant sample to a copper mesh coated with carbon film, allowing 10 min for absorption, removing residual liquid with filter paper, placing the samples in a transmission electron microscope sample chamber to determine sample morphology, and taking photographs for further analysis.

Experimental results: It could be clearly seen that the adjuvant Zn-zoledronic acid (1/0.25) and the adjuvant Al-001-zoledronic acid had spherical particles with a nano-core, while the adjuvant FH-001-zoledronic acid had the shape of an algae bundle (Fig. 2).

(2) pH determination

Samples were taken for testing, equilibrated at room temperature for at least 30 minutes and pH measured using a Sartorius glass electrode pH meter.

Standard buffer (pH 7.00), standard buffer (pH 4.01) and standard buffer (pH 10.01) were selected to calibrate the instrument in accordance with the instructions for use of the instrument.

The "Mode" button was pressed to switch between pH and mV modes. Usually the pH mode was chosen when determining the pH value of the solution.

The "Setup" button was pressed until the display showed "clear buffer", and then the "Enter" button was pressed to confirm and delete the previous calibration data.

Press the "Set" button until the display shows "buffer group" "4.01, 7.00, 10.01", and then press the "Enter" button to confirm.

The electrode was carefully removed from the electrode storage solution, the electrode was rinsed thoroughly with deionized water, and water on the surface was removed by blotting with filter paper after rinsing thoroughly (care should be taken not to rub the electrode).

The electrode was immersed in the first buffer solution (pH 7.00) until the value stabilized and the letter “S” appeared, then the “Standardize” button was pressed until the instrument was automatically calibrated. After a successful calibration, the number "7.00" and the slope of the electrode were displayed.

The electrode was removed from the first buffer solution, rinsed thoroughly with deionized water and sequentially immersed the electrode in the second buffer solution (pH 4.01) until the value stabilized and the letter “S” appeared, then the “Standardize” button was pressed until the instrument will not automatically calibrate. After a successful calibration, "4.01 7.00" and the message "Tilt" are displayed. The slope displays the measured value of the electrode slope, which was acceptable in the range of 90-105%.

If there was a large deviation from the theoretical value, then an error message (Err) would be displayed, in which case you should clean the electrode and repeat the above steps to calibrate.

The above operations were repeated to complete the calibration of the third point (pH 10.01).

After calibration was completed, the electrode was thoroughly rinsed with deionized water, and then gently blotted with filter paper. The sample solution was shaken evenly, the glass electrode was immersed in the sample solution until the pH changed by no more than ± 0.05 for 1 min, after which the readings were confirmed.

The sample solution was evenly shaken and measured again. The difference between the two measured pH values must not exceed 0.1. The average of the two readings was taken as the pH value of the test product.

Experimental results (Table 1):

The pH range for Zn-zoledronic acid adjuvants was 8.0 to 9.0 before sterilization and 6.5 to 7.0 after sterilization.

(3) Particle Sizing

A Beckman LS 13320 laser particle size analyzer was turned on and warmed up for 15 min.

The analyzer control software was started, the closed sample cuvette compartment was opened, the sample cuvette was removed from the sample reservoir, and 12 ml of purified water was added.

The sample cuvette was placed in the sample tank and the compartment door was closed.

Clicked "start cycle", select "offset measurement", "alignment", "background measurement" in turn, and finally clicked "start", clicked "OK" in the pop-up dialog box to start blank background calibration.

The sample cuvette was removed and a certain amount of standard sample (supplied with the analyzer) was added to it; click "run cycle", select "load measurement", "sample information input", "input analysis setting", "start analysis" in turn, and finally click "start", the name of the standard sample is entered into the pop-up dialog box, "OK" was pressed when the "shading" parameter in the software was from 8% to 12%, thus measuring the standard sample.

To ensure the accuracy and reliability of the experimental data, a blank background should be calibrated and the standard sample size should be measured before each measurement.

The closed compartment of the sample cuvette was opened and the sample cuvette was removed.

The aqueous solution containing the standard sample in the sample cuvette was removed, and deionized water was added to the sample cuvette 3 times to clean the sample cuvette.

After cleaning, 12 ml of deionized water was added, the sample cuvette was placed in the sample reservoir, and the compartment door was closed.

Clicked "start cycle", select "offset measurement", "alignment", "background measurement" in turn, and finally clicked "start", clicked "OK" in the pop-up dialog box to start blank background calibration.

The sample cuvette was removed, a certain volume of the test sample was added thereto, the test sample compartment door was opened, the sample cuvette was placed in the sample reservoir, and the compartment door was closed.

Press "run cycle", select "input information by sample", "introduce analysis setting", "start analysis" in turn, and finally press "run", enter the name of the standard sample in the pop-up dialog box, press "OK", when the "shading" parameter in the software was from 8% to 12%, thus, the measurements of the particle size of the samples were recorded.

Experiment results:

The particle size of adjuvants based on zinc zoledronate ranged from 0.4 to 30 µm, and the majority of the particles ranged from 6 to 7 µm.

Table 1
pH/particle size of multiple batches of adjuvant Zn-zoledronic acid (1/0.25) adjuvant type The consignment pH Particle size (µm) Zn-zoledronic acid
(1/0.25) 2018041802 6.73 6.6 2018041803 6.78 6.6 2018042501 6.62 6.4

(4) Determination of the degree of adsorption

Building a standard curve for the BSA standard: 150 mM NaCl was used as a dilution buffer, prepared serial dilutions of the BSA standard (2 mg/ml) and determined the optical density at 280 nm using a UV2100 pro spectrophotometer. The OD280 value showed a high accuracy of 0.2-0.8 (limits up to 0.2-1.5).

BSA dilution gradient (EP): 150 mM NaCl was used as dilution buffer, a certain amount of BSA sample was weighed and diluted to the concentration gradient indicated in EP: 0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml, 5 mg/ml, 10 mg/ml, for further use.

BSA was mixed with the adjuvant at a ratio of BSA:adjuvant=3:1 (volume ratio), which was set as a condition for measuring the degree of adsorption; after evenly shaking the adjuvant, it was mixed with various concentrations of BSA according to the experimental conditions, and adsorption was carried out at room temperature for 1 hour, and shaking was performed 5 times during this period; centrifugation was performed at 13,000 rpm for 3 min, then the supernatant was collected for further use.

Protein Concentration Determination: The Lowry method was used to determine the protein concentration in EP. In this experiment, according to the practical situation, UV2100pro was used to directly determine the absorbance of the supernatant at 280 nm, the reading was maintained in the range of 0.2-0.8, otherwise the supernatant had to be diluted.

Adsorption rate calculation: Adsorption rate=[1 - OD _{280 (supernatant X dilution ratio)} /OD280 _{(when X dilution adsorption ratio is 0)} ] × 100

The results of the experiment were as follows:

table 2
Adsorption capacity of several batches of adjuvant Zn-zoledronic acid (1/0.25) for protein The consignment Adsorption capacity (%) BSA(mg/ml) 0.5 1 2 3 5 10 series 2018041802 97 92 59 42 25 20 series 2018041803 99 90 55 42 26 17 series 2018041804 96 78 50 48 27 19

(5) Definition of PZC:

Measurement device: Nanobrook Omni (Brookhaven)

Experimental procedure: used 0.1 M NaOH/1% HNO ₃ to adjust the pH of the adjuvant Zn-zoledronic acid (1/0.125) to: 6.00/5.50/5.00/4.50/4.00/3 .50/3.00/2.50/2.00.

Passivation of the electrode: 3-4 ml of adjuvant was added to the sample tube. After the electrode was inserted, the cycle in the SOP was set to 50 and the instrument was run to passivate the electrode.

Sample analysis: after removing the electrode, its lower end was rinsed with deionized water, and then the appropriate sample was added, the cycle was set to 15 in SOP, the index was set to 3, the pH was set to the corresponding pH of each sample, and the instrument was started.

Data processing: the corresponding zeta potentials were obtained at various pH values, the software supplied with the instrument was run to obtain the PZC values.

Results: The PZC of the adjuvant Zn-zoledronic acid (1/0.125) was 4.08.

Example 2 Determination of the Degree of Zinc Precipitation in Zn-Zoledronic Acid Adjuvant (1/0.125)

Flame atomic absorption spectrometry (D2 lamp background correction) was used to determine the zinc content of zinc zoledronate adjuvants, and the determination procedure was standardized. The instrument for analysis was an atomic absorption spectrophotometer: Shimadzu AA6300C (P/N 206-52430).

Preparation of standard solution and sample for determination: plotting a standard curve: the initial concentration of the zinc standard was 500 μg/ml, and it 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.

The adjuvant Zn-zoledronic acid (1/0.125) was centrifuged at 13,000 rpm for 10 min and the supernatant was removed. The sample was diluted 5 times with 0.1 M nitric acid for determination, and a vortex mixer was used for shaking and mixing. The determination process was as follows:

AA-6300C working principle and using WizAArd software:

Power on: turned on the computer, AA-6300C power switch, air compressor switch (pressure 0.35MPa), ventilation system switch;

Acetylene port: The acetylene valve was slowly opened to ensure that the primary pressure is 0.5 MPa and the secondary pressure is 0.1 MPa;

WizAArd software basic operation procedure: enter WizAArd → select an item → on the "unconnected instrument/send parameter" page, press <connect/send parameter> → set on the "instrument initialization" page → check and mark each item in the "flame analysis test directory" instrument", press <OK> → set wavelength [213.86], slit width [0.7], illumination method [emission], [lamp position setting] on the "optical parameters" page to ensure that the actual position and target position of the hollow cathode lamp for Zn ion detection are identical, select [turn on the lamp] → search by line → adjust the initial position of the burner → select [parameter] in the menu bar → [change parameter] → change the illumination method to <BGC-D2> → line search → ignition: after making sure that C2H2 is on and the pressure is correct, simultaneously press the “purge” and “ignition” buttons on the main device until ignition occurs → automatic zeroing → set blank control (BLK), standard product (STD) and test sample (UNK) on the MRT sheet, enter the theoretical concentration of the standard and the name of the sample, and manually load the sample through the sample tube coming out of the nebulizer, set the sample volume to at least 1 ml each time, select [start] for testing → stop the flame → save the data and disconnect the device from the computer → turn off;

Test results:

Table 3
Plotting a Standard Curve for Zinc and Ab Concentrations Concentration
zinc standard
(X, ng/ml) 0 500 1000 1500 2000 2500 Ab(y) 0.017 0.093 0.161 0.248 0.314 0.379 Formula for Standard Curve y=0.0001x+0.0191, R²=0.9984

Table 4
Results of determining the degree of zinc precipitation in the Zn-zoledronic acid adjuvant (1/0.125) Sample Ab Zinc content in supernatant (µg/ml) Total Zinc in Adjuvant (µg/mL) Degree of zinc precipitation (%) Zn-zoledronic acid (1/0.25) 0.165 7.3 4067 99.82

Example 4 Determination of the Precipitation Rate of Zoledronic Acid in Zn-Zoledronic Acid Adjuvant (1/0.125)

Method: UV spectrophotometry, instrument: UV800 (Beckman coulter).

Zoledronic acid contains an imidazole ring and has an absorption maximum at 215 nm. The specific procedure for measuring zoledronic acid in the adjuvant supernatant at 215 nm using an ultraviolet spectrophotometer was as follows:

First, standard solutions of zoledronate sodium were prepared in saline 0.04 mg/ml, 0.03 mg/ml, 0.02 mg/ml, 0.015 mg/ml, 0.0075 mg/ml, 0.00375 mg/ml, and their OD ₂₁₅ values were measured at a wavelength of 215 nm and a standard curve was drawn. At the same time, the adjuvant Zn-zoledronic acid (1/0.125) was centrifuged at 13,000 rpm for 10 minutes, and then 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:

Table 5
Sodium Zoledronate Concentration and OD ₂₁₅ Standard Curve The concentration of zoledronate sodium,
standard
(x, mg/ml) 0.00375 0.0075 0.015 0.02 0.03 0.04 OD ₂₁₅ (y) 0.0517 0.1039 0.1944 0.2669 0.3931 0.5183 Formula for Standard Curve y=12.876x+0.0053， R²=0.9997

Table 6
The results of determining the degree of precipitation of zoledronic acid in the adjuvant Zn-zoledronic acid (1/0.125) Sample OD ₂₁₅ Content
zoledronic acid
in supernatant (µg/ml) General content
zoledronic acid
in adjuvant (µg/ml) Degree of precipitation of zoledronic acid
(%) Zn-zoledronic acid
(1/0.125) 0.0359 2.4 2373 99.89%

Example 5: Determination of Adjuvant Activity of Zn-Zoledronate Adjuvant in VZV gE Recombinant Protein Vaccine

Prepared adjuvants based on zinc zoledronic acid (also referred to as Zn-zoledronic acid) were used as an adjuvant in which the Zn/zoledronic acid molar ratio was 1:0.25 or 1:1. As adjuvants, they were used in combination with the VZV gE antigen, and administered intramuscularly to mice together, and specific antibody titers were determined. The specific method was as follows:

Experimental animals: Balb/C mice, 6-8 weeks old, 5 mice/group, females.

Drug concentration: antigen: 50 µg/ml.

Dose: 5 µg/50 µl/mouse; aluminum adjuvant: 50 µl/mouse; adjuvant zinc zoledronate: 50 µl/mouse.

Experimental groups: (1) main group with aluminum adjuvant (Al-002); (2) Zn-zoledronic acid (1/0.25); (3) Zn-zoledronic acid (1/1).

Protocol of immunization: 2 weeks after the first immunization of animals, in accordance with the distribution into groups according to immunization options, blood samples were taken from the orbit to determine the titer of antibodies in serum. The antibody titer was measured in the second week after the first immunization. Boost immunization was performed in the second week. Two weeks after the boost immunization, blood samples were taken from the orbit to determine the serum antibody titer, and a third immunization was performed. After 2 weeks, the serum antibody titer was determined by ELISA.

Determination of binding antibody titer using enzyme immunoassay (ELISA):

1. Antigen coating solution: 1X PB buffer solution 7.4 (4.343 g Na ₂ HPO ₄ · 7H ₂ O; 0.456 g NaH ₂ PO ₄ ).

2. Wash 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 to 1X with ultrapure water or distilled water to seal and dilute the sample.

4. Stain development solution A: ELISA kit from Beijing Wantai.

5. Solution B for stain development: ELISA kit from Beijing Wantai.

6. Stop solution: ELISA kit from Beijing Wantai.

The order of the experiment:

(1) Plate coating: VZV gE antigen was diluted with PB7.4 coating buffer to a certain concentration, usually 100 ng/well, added to a 96-well polystyrene plate at 100 μl/well, and the plate was coated overnight at 4° WITH.

(2) Blocking: The coating solution in the well was removed, the plate was washed once with PBST washing solution, dried by centrifugation, blocking solution was added at 200 μl/well, and blocking was performed for 4 hours at room temperature.

(3) Addition of serum at a certain dilution: the blocking solution in the well was removed, the plate was washed once with PBST, centrifuged, serum to be tested was added to the first well at 150 µl/well, ED diluent was added to each of the following wells at the rate of 100 µl/well, diluted with a gradient 3 times, incubated and reacted at 25°C for 1 h.

(4) Addition of enzyme labeled antibody (GAM-HRP): the serum diluent in the well was removed, the plate was washed 5 times with PBST, dried by centrifugation, enzyme labeled antibody (GAM-HRP, V:V=1:5000) was added at a rate of 100 μl/ well, incubated and reacted at 25°C for 1 hour.

(5) Stain development: secondary antibodies in the well were removed, the plate was washed 5 times with PBST, dried by centrifugation, 100 µl/well of stain development solution, mixed solution of A and B in equal volumes was added, and the reaction was carried out at 25°C for 10 min.

(6) Reaction stop: 2M stop solution containing sulfuric acid was added at the rate of 50 µl/well to stop the reaction.

(7) Plate Reading: Two wavelengths of 450 nm and 630 nm were set as measurement wavelengths on the microplate reader, and the OD value of each reaction well was determined.

The results of the experiment are shown in Fig. 3:

The adjuvant was mixed with the antigen in equal volume to immunize mice. Immunization, blood sampling and determination of serum antibody titer were carried out in accordance with the strategy described above. In the adjuvanted zinc zoledronate group, mice showed a rapid and pronounced onset of effect after just one immunizing injection; after two immunizing injections, the benefit of enhanced humoral immunity showed a statistically significant difference compared to the aluminum adjuvant group, and the difference was still significant after three immunizing injections.

Example 6 Effect of Adjuvant Zinc Zoledronate in Combination with Recombinant VZV gE Protein on Murine Antibody Isotypes

The immunization strategy is described in Example 5.

Experimental materials:

1. Antigen coating solution: 1X PB buffer solution 7.4 (4.343 g Na ₂ HPO ₄ • 7H ₂ O; 0.456 g NaH ₂ PO ₄ ).

2. Wash 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 to 1X with ultrapure water or distilled water to seal and dilute the sample.

4. Stain development solution A: ELISA kit from Beijing Wantai.

5. Solution B for stain development: ELISA kit from Beijing Wantai.

6. Stop solution: ELISA kit from Beijing Wantai.

The order of the experiment:

(1) Plate coating: VZV gE antigen was diluted with PB7.4 coating buffer to a certain concentration, 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 well was removed, the plate was washed once with PBST, dried by centrifugation, blocking solution was added at 200 μl/well, and blocking was performed for 4 hours at room temperature.

(3) Addition of serum for testing: serum used to determine the isotype of antibodies in serum: 2 weeks after completion of 3 immunization injections, i.e., serum obtained at week 6; the blocking solution in the well was removed, the plate was washed once with PBST, dried by centrifugation, the test serum was added at a certain dilution rate at the rate of 100 μl/well, incubated and reacted for 1 h at 25°C.

(4) Enzyme-labeled antibody addition: the serum diluent in the well was removed, the plate was washed 5 times with PBST, dried by centrifugation, an enzyme-labeled antibody that specifically recognizes each antibody isotype (IgG1, V:V=1:30000; IgG2a, V:V) was added. =1:1000; IgG2b, V:V=1:1000) at 100 µl/well, incubated and the reaction was carried out at 25°C for 1 hour.

(5) Stain development: Enzyme-labeled antibody, removed from well, plate was washed 5 times with PBST, dried by centrifugation, stain development solution added, mixed solution A and B in equal volumes, diluted 3 times, added to the well at a rate of 100 μl /well, the reaction was carried out at 25°C for 10 min.

(6) Reaction stop: 50 µl/well of 2 M stop solution containing sulfuric acid was added to stop the reaction.

(7) Reading the plate: two wavelengths of 450 nm and 630 nm were set as measurement wavelengths on the microplate reader, and the OD value of each reaction well was measured.

The results of the experiment are shown in Fig. 4:

The experimental procedure described in Example 5 was used. Mice were immunized with zinc zoledronate adjuvant or aluminum adjuvant in combination with recombinant VZV gE protein by intramuscular injection. The immunization procedure was similar to that described in Example 5. After 3 immunization injections, blood samples were collected after 2 weeks, and the results of determining the serum levels of each antibody isotype in mice are shown in FIG. 4. Compared with the aluminum adjuvant group, stimulation of higher levels of IgG2a and IgG2b antibody isotypes was observed in the zinc zoledronate adjuvant group, and the ratio of IgG1 to IgG2a and IgG2b was lower than in the aluminum adjuvant group, indicating that it has a certain stimulatory effect on the Th1-mediated immune pathway.

Example 7: Determination of Adjuvant Activity of Zinc Zoledronate Adjuvant in Combination with Hepatitis B Virus Therapeutic Protein

The experimental procedure described in Example 5 was used. Mice were immunized with zinc zoledronate adjuvant in combination with hepatitis B therapeutic protein by intramuscular injection and serum antibody titers were determined. The results are shown in FIG. 5: Compared with the aluminum adjuvant group, the zinc zoledronate adjuvant group showed a rapid onset and strong immune response after just one immunization injection in mice, and the difference was statistically significant; after 2 immunizing injections, the advantage in enhancing humoral immunity was still significant compared to the aluminum adjuvanted group and slightly better than in the aluminum adjuvanted group after 3 immunizing injections.

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 in combination with recombinant VZV gE protein by intramuscular injection and serum antibody titers were determined. The results are shown in FIG. 6: After 3 immunization injections in mice, the zinc zoledronate adjuvant group had a significant difference in enhancement of humoral immunity compared to the aluminum adjuvant group, while the effects of other zinc bisphosphonate adjuvants were not significantly different compared to the aluminum adjuvant. .

Example 9 Effect of Zinc Adjuvant Zoledronate in Combination with VZV gE on Antibody Titers in Mouse Serum

The experimental procedure described in Example 5 was used. Mice were immunized with zinc zoledronate adjuvant in combination with VZV gE by intramuscular injection and serum antibody titers were determined. As shown in FIG. 7, aluminum adjuvant, zinc-aluminum combined adjuvant, combined zinc-aluminum adjuvant with addition of zoledronic acid, and zinc zoledronate adjuvant showed a gradually increasing trend in antibody titer increase, and when compared, a significant difference was observed between the zoledronate-based adjuvant group. zinc, with an aluminum adjuvanted group.

Claims (55)

1. Vaccine adjuvant containing an effective amount of zinc zoledronate microparticles; moreover, the specified vaccine adjuvant has a ratio of molar concentrations of zinc:zoledronic acid 1-8:1. 2. A vaccine adjuvant according to claim 1, wherein said vaccine adjuvant has a zinc:zoledronic acid molar concentration ratio selected from 1:1, 4:1, and 8:1. 3. A vaccine adjuvant according to claim 1, characterized by any of the following: (1) said vaccine adjuvant additionally contains phosphate at a zinc:phosphate molar ratio of 1-8:1; And (2) said vaccine adjuvant further contains phosphate at a zinc:phosphate molar ratio selected from 1.5:1 and 4:1. 4. A vaccine adjuvant according to claim 1, characterized by any of the following: (1) the specified vaccine adjuvant additionally contains aluminum at a ratio of molar concentrations of zinc: aluminum 0.02-1:1; And (2) said vaccine adjuvant further contains aluminum at a zinc:aluminum molar ratio selected from 0.375:1, 0.5:1, and 0.8:1. 5. The vaccine adjuvant according to claim 1, where the vaccine adjuvant is obtained by a method comprising the following steps: a) obtaining a soluble salt solution containing zinc ions; b) uniformly mixing the soluble salt solution from step a) with zoledronic acid and sodium hydroxide; or uniform mixing of the soluble salt solution from step a) with zoledronic acid, sodium hydroxide and sodium phosphate solution, by successive precipitation, separate precipitation followed by mixing or co-precipitation to obtain a vaccine adjuvant. 6. A vaccine adjuvant according to claim 5, characterized by one or more of the following: (1) said salt solution contains a hydrochloric acid solution; And (2) said method further comprises sterilizing the mixture from step b) and storing at 2-8°C for later use. 7. A vaccine adjuvant according to claim 6, characterized by any of the following: (1) said sterilization includes sterilization using a high temperature high pressure sterilization method; And (2) said sterilization includes sterilization performed at 121°C for 30-60 minutes. 8. A vaccine adjuvant according to claim 1, characterized by one or more of the following: (1) said vaccine adjuvant has a pH of 8.0-9.0 prior to sterilization; (2) said vaccine adjuvant has a pH of 6.0-8.0 after sterilization; (3) said vaccine adjuvant has a particle size of 1-10 microns; (4) said vaccine adjuvant has a zero particle charge point of 4.0-11.4; And (5) said vaccine adjuvant has a protein adsorption rate >80%. 9. The vaccine adjuvant of claim 8, wherein said protein is bovine serum albumin. 10. A method for obtaining a vaccine adjuvant, including: a) obtaining a soluble salt solution containing zinc ions; b) uniformly mixing the soluble salt solution from step a) with zoledronic acid and sodium hydroxide; or uniform mixing of the soluble salt solution from step a) with zoledronic acid, sodium hydroxide and sodium phosphate solution, sequential precipitation, separate precipitation followed by mixing or co-precipitation to obtain a vaccine adjuvant, where the resulting vaccine adjuvant has a ratio of molar concentrations of zinc:zoledronic acid 1-8:1. 11. The method of claim 10, wherein the resulting vaccine adjuvant has a zinc:zoledronic acid molar ratio selected from 1:1, 4:1, and 8:1. 12. The method of claim 10, characterized by one or more of the following: (1) said salt solution contains a hydrochloric acid solution; And (2) said method further comprises sterilizing the mixture from step b) and storing at 2-8°C for later use. 13. The method of claim 12, characterized by one or more of the following: (1) said sterilization includes sterilization using a high temperature high pressure sterilization method; And (2) said sterilization includes sterilization performed at 121°C for 30-60 minutes. 14. The method of claim 10, characterized by one or more of the following: (1) the resulting vaccine adjuvant has a pH of 8.0-9.0 before sterilization; (2) the resulting vaccine adjuvant has a pH of 6.0-8.0 after sterilization; (3) the resulting vaccine adjuvant particle size 1-10 microns; (4) the resulting vaccine adjuvant has a zero particle charge point of 4.0-11.4; And (5) the resulting vaccine adjuvant has a protein adsorption rate >80%. 15. The method of claim 14 wherein the protein comprises bovine serum albumin. 16. Vaccine composition containing an effective amount of antigen and vaccine adjuvant according to any one of paragraphs. 1-9, characterized by one or more of the following: (1) said antigen includes a protein antigen; And (2) said antigen includes the varicella zoster virus gE glycoprotein antigen. 17. The use of a vaccine adjuvant according to any one of paragraphs. 1-9 as an immunostimulating additive in the preparation of an immunogenic composition. 18. The use of a vaccine adjuvant according to any one of paragraphs. 1-9 as an immunostimulating additive in the preparation of a vaccine composition. 19. Use according to claim 18, characterized by one or more of the following: (1) said vaccine is a protein vaccine; And (2) said vaccine is a recombinant protein-based varicella-zoster virus vaccine.

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