PREPARATION OF ZINC ZOLEDRONATE MICRO-NANOPARTICLE ADJUVANT AND USE THEREOF AS VACCINE ADJUVANT

Abstract

Disclosed is a zinc zoledronate micro-nanoparticle adjuvant, which contains zinc and zoledronic acid and optionally contains a phosphate and aluminum. The preparation method therefor comprises performing mixed precipitation on a soluble salt solution containing zinc ions, zoledronic acid, and sodium hydroxide. The adjuvant can be used to prepare vaccines, etc.

Claims

1-8. (canceled)

9. A zinc zoledronate micro/nanoparticle adjuvant, comprising zinc and zoledronic acid at a molar concentration ratio of zinc:zoledronic acid of 1-8:1.

10. The zinc zoledronate micro/nanoparticle adjuvant according to claim 9, wherein said zinc zoledronate micro/nanoparticle adjuvant has a molar concentration ratio of zinc:zoledronic acid selected from 1:1, 4:1 and 8:1.

11. The zinc zoledronate micro/nanoparticle adjuvant according to claim 9, characterized in any one of the following: (1) said zinc zoledronate micro/nanoparticle adjuvant further comprises a phosphate at a molar concentration ratio of zinc:phosphate of 1-8:1; and (2) said zinc zoledronate micro/nanoparticle adjuvant further comprises a phosphate at a molar concentration ratio of zinc:phosphate selected from 1.5:1 and 4:1.

12. The zinc zoledronate micro/nanoparticle adjuvant according to claim 9, characterized in any one of the following: (1) said zinc zoledronate micro/nanoparticle adjuvant further comprises aluminum at a molar concentration ratio of zinc:aluminum of 0.02-1:1; and (2) said zinc zoledronate micro/nanoparticle adjuvant further comprises aluminum at a molar concentration ratio of zinc:aluminum selected from 0.375:1, 0.5:1 and 0.8:1.

13. The zinc zoledronate micro/nanoparticle adjuvant according to claim 9, wherein the zinc zoledronate micro/nanoparticle adjuvant is prepared by a method comprising the following steps: a) providing a soluble salt solution containing zinc ions; b) evenly mixing the soluble salt solution of step a) with a zoledronic acid and a sodium hydroxide; or evenly mixing the soluble salt solution of step a) with a zoledronic acid, a sodium hydroxide, and a sodium phosphate solution, in a manner of sequential precipitation, separated precipitation followed by mixing, or co-precipitation, to obtain the zinc zoledronate adjuvant.

14. The zinc zoledronate micro/nanoparticle adjuvant according to claim 13, characterized in one or more of the following: (1) said salt solution comprises a solution of hydrochloric acid; and (2) said method further comprises subjecting the mixture of step b) to sterilization, and storing at 2° C. to 8° C. for later use.

15. The zinc zoledronate micro/nanoparticle adjuvant according to claim 14, characterized in any one of the following: (1) said sterilization comprises a sterilization using a high temperature and high pressure sterilization technique; and (2) said sterilization comprises a sterilization performed at 121° C. for 30 to 60 minutes.

16. The zinc zoledronate micro/nanoparticle adjuvant according to claim 9, characterized in one or more of the following: (1) said zinc zoledronate micro/nanoparticle adjuvant has a pH of 8.0-9.0 before sterilization; (2) said zinc zoledronate micro/nanoparticle adjuvant has a pH of 6.0-8.0 after sterilization; (3) said zinc zoledronate micro/nanoparticle adjuvant has a particle size of 1-10 μm; (4) said zinc zoledronate micro/nanoparticle adjuvant has a particle zero charge point of 4.0-11.4; and (5) said zinc zoledronate micro/nanoparticle adjuvant has a protein adsorption rate of >80%.

17. The zinc zoledronate micro/nanoparticle adjuvant according to claim 16, wherein said protein comprises bovine serum albumin.

18. A method for preparing a zinc zoledronate micro/nanoparticle adjuvant, comprising: a) providing a soluble salt solution containing zinc ions; b) evenly mixing the soluble salt solution of step a) with a zoledronic acid and a sodium hydroxide; or evenly mixing the soluble salt solution of step a) with a zoledronic acid, a sodium hydroxide, and a sodium phosphate solution, in a manner of sequential precipitation, separated precipitation followed by mixing, or co-precipitation, to obtain the zinc zoledronate adjuvant, wherein the obtained zinc zoledronate adjuvant has a molar concentration ratio of zinc:zoledronic acid of 1-8:1.

19. The method according to claim 18, wherein the obtained zinc zoledronate adjuvant has a molar concentration ratio of zinc:zoledronic acid selected from 1:1, 4:1 and 8:1.

20. The method according to claim 18, characterized in one or more of the following: (1) said salt solution comprises a solution of hydrochloric acid; and (2) said method further comprises subjecting the mixture of step b) to sterilization, and storing at 2° C. to 8° C. for later use.

21. The method according to claim 20, characterized in one or more of the following: (1) said sterilization comprises a sterilization using a high temperature and high pressure sterilization technique; and (2) said sterilization comprises a sterilization performed at 121° C. for 30 to 60 minutes.

22. The method according to claim 18, characterized in one or more of the following: (1) the obtained zinc zoledronate micro/nanoparticle adjuvant has a pH of 8.0-9.0 before sterilization; (2) the obtained zinc zoledronate micro/nanoparticle adjuvant has a pH of 6.0-8.0 after sterilization; (3) the obtained zinc zoledronate micro/nanoparticle adjuvant has a particle size of 1-10 μm; (4) the obtained zinc zoledronate micro/nanoparticle adjuvant has a particle zero charge point of 4.0-11.4; and (5) the obtained zinc zoledronate micro/nanoparticle adjuvant has a protein adsorption rate of >80%.

23. The method according to claim 22, wherein the protein comprises bovine serum albumin.

24. A vaccine adjuvant, a pharmaceutical composition or an immunogenic composition, comprising the zinc zoledronate micro/nanoparticle adjuvant according to claim 9.

25. A vaccine composition, comprising an antigen and the zinc zoledronate micro/nanoparticle adjuvant according to claim 9.

26. The vaccine composition according to claim 25, characterized in one or more of the following: (1) said antigen comprises a protein antigen; and (2) said antigen comprises a varicella-zoster virus gE glycoprotein antigen.

27. A method of preparing a vaccine adjuvant, a pharmaceutical composition, a drug delivery vehicle, an immunogenic composition or a vaccine composition, comprising providing the zinc zoledronate micro/nanoparticle adjuvant according to claim 9 in the vaccine adjuvant, the pharmaceutical composition, the drug delivery vehicle, the immunogenic composition or the vaccine composition.

28. The method according to claim 27, characterized in one or more of the following: (1) said vaccine is a protein vaccine; and (2) said vaccine is a varicella-zoster virus recombinant protein vaccine.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] FIG. 1: schematic diagrams of three preparation processes for preparing the adjuvants.

[0083] FIG. 2: morphologies of particles of zinc zoledronate adjuvant, zinc-aluminum zoledronate adjuvant, and aluminum zoledronate adjuvant.

[0084] FIG. 3: determination of the 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.

[0085] FIG. 4: effect of zinc zoledronate adjuvant combined 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.

[0086] FIG. 5: determination of the adjuvant activity of zinc zoledronate adjuvant for hepatitis B therapeutic protein, mean±SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0087] FIG. 6: effect of other zinc bisphosphonate adjuvants (Zn/Zol=1/0.25) combined with VZV gE on mouse serum antibody titer, mean±SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0088] FIG. 7: effect of zoledronic acid and zinc zoledronate modified adjuvants combined with hepatitis B 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

[0089] The following describes the embodiments of the present invention in detail in conjunction with the examples. For those not indicating specific conditions in the examples, they shall be carried out in accordance with the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are all conventional products that can be purchased commercially.

[0090] The materials used in the following preparation examples are as follows:

[0091] Anhydrous zinc chloride (ZnCl.sub.2): purchased from Xilong Chemical;

[0092] Aluminum chloride hexahydrate (AlCl.sub.3.6H.sub.2O): purchased from Xilong Chemical;

[0093] Disodium hydrogen phosphate dodecahydrate (Na.sub.2HPO.sub.4.12H.sub.2O): purchased from Xilong Chemical;

[0094] Sodium hydroxide (NaOH): purchased from Xilong Chemical;

[0095] Zoledronic acid (C.sub.5H.sub.10N.sub.2O.sub.7P.sub.2): purchased from Hunan Huateng Pharmaceutical Co., Ltd.

PREPARATION EXAMPLE 1

Preparation of Zn-Zoledronic Acid (1/0.25) Adjuvant

[0096] According to a Zn/zoledronic acid molar concentration ratio of 1:0.25, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (7.78 mM zoledronic acid+43 mM sodium hydroxide+7.78 mM disodium hydrogen phosphate) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0097] Preparation of Zn-zoledronic acid (1/0.25) adjuvant suspension:

[0098] Solution A and B were used to form a zinc zoledronate adjuvant suspension according to the scheme as shown in FIG. 1A, that was, the prepared Solution B was added dropwise according to a volume ratio of 1:1 to Solution A to form a suspension until all was added.

[0099] The zinc zoledronate adjuvant obtained after mixing according to FIG. 1A was subjected to an autoclave steam sterilization at 121° C. for 60 min, and then was cooled to room temperature, and then stored at 2° C. to 8° C.

PREPARATION EXAMPLE 2

Preparation of Zn-Zoledronic Acid (1/1) Adjuvant

[0100] Preparation of solutions:

[0101] According to a Zn/zoledronic acid molar concentration ratio of 1:1, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (31.11 mM zoledronic acid+87 mM sodium hydroxide) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

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

PREPARATION EXAMPLE 3

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

[0103] Preparation of solutions:

[0104] According to an Al/Zn molar concentration of 1:0.375, 50 mL of a mixture solution of aluminum chloride (122.44 mM) and zinc chloride (46.66 mM) was prepared and defined as Solution A; 50 mL of a solution (18.66 mM zoledronic acid+380 mM sodium hydroxide) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0105] Referring to Preparation Example 1, the FH-001-zoledronic acid adjuvant suspension was prepared.

PREPARATION EXAMPLE 4

Preparation of Aluminum Zoledronate (Al-001-Zoledronic Acid) Adjuvant

[0106] Preparation of solutions:

[0107] According to an Al/Zol molar concentration of 1:0.075, 50 mL of 122.44 mM aluminum chloride solution was prepared and defined as Solution A; 50 mL of a solution (9.33 mM zoledronic acid+9.33 mM disodium hydrogen phosphate dodecahydrate+370 mM sodium hydroxide) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0108] Referring to Preparation Example 1, the Al-001-zoledronic acid adjuvant suspension was prepared.

PREPARATION EXAMPLE 5

Preparation of Zn-Zoledronic Acid (1/0.125)

[0109] Preparation of solutions:

[0110] According to a Zn/zoledronic acid molar concentration ratio of 1:0.125, 50 mL of 124.44 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (15.56 mM zoledronic acid+65.7 mM sodium hydroxide) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0111] Referring to Preparation Example 1, the Zn-zoledronic acid (1/0.125) adjuvant suspension was prepared.

PREPARATION EXAMPLE 6

Preparation of Aluminum Adjuvant (A1-002)

[0112] Preparation of solutions:

[0113] Aluminum chloride hexahydrate was added to water to prepare 0.5 L of a 62.22 mM aluminum chloride solution and defined as Solution A; 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 and defined as Solution B. They were filtered with 0.22 μm filter membrane for later use.

[0114] Referring to Preparation Example 1, the aluminum adjuvant Al-002 was prepared.

PREPARATION EXAMPLE 7

Preparation of Aluminum Adjuvant (Al-001-840)

[0115] Preparation of solutions:

[0116] According to a phosphate/Al molar concentration ratio of 0.15, 0.5 L of a 124 mM aluminum chloride solution was prepared and defined as Solution A; 0.5 L of an 18.6 mM disodium hydrogen phosphate solution was prepared and defined as Solution B. Solution B further contained 150 mM sodium hydroxide. They were filtered with 0.22 μm membrane filter for later use.

[0117] Referring to Preparation Example 1, the aluminum adjuvant Al-001-840 suspension was prepared.

PREPARATION EXAMPLE 8

Preparation of Zinc-Aluminum Adjuvant (FH-001)

[0118] Preparation of solutions:

[0119] 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, which was defined as Solution A; 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, which defined as solution B. They were filtered with 0.22 μm filter membrane for later use.

[0120] Preparation of aluminum composite adjuvant:

[0121] As shown in FIG. 1A, 0.25 L of Solution B was added dropwise to 0.25 L of Solution A until all was added, thereby obtaining the zinc-aluminum composite adjuvant FH-001. The obtained zinc-aluminum composite adjuvant was subjected to an autoclave steam sterilization at 121° C. for 60 min, and then was cooled to room temperature, and then stored at 2° C. to 8° C.

PREPARATION EXAMPLE 9

Preparation of Zn-Etidronic Acid (1/0.25) Adjuvant

[0122] According to the Zn/etidronic acid molar concentration ratio of 1:0.25, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (7.78 mM etidronic acid+34 mM sodium hydroxide+7.78 mM disodium hydrogen phosphate) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0123] Referring to Preparation Example 1, the Zn-etidronic acid (1/0.25) adjuvant suspension was prepared.

PREPARATION EXAMPLE 10

Preparation of Zn-Clodronic Acid (1/0.25) Adjuvant

[0124] According to a Zn/clodronic acid molar concentration ratio of 1:0.25, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (7.78 mM clodronic acid+36 mM sodium hydroxide+7.78 mM disodium hydrogen phosphate) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0125] Referring to Preparation Example 1, the Zn-clodronic acid (1/0.25) adjuvant suspension was prepared.

PREPARATION EXAMPLE 11

Preparation of Zn-Alendronic Acid (1/0.25) Adjuvant

[0126] According to a Zn/alendronic acid molar concentration ratio of 1:0.25, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (7.78 mM alendronic acid+36 mM sodium hydroxide+7.78 mM disodium hydrogen phosphate) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0127] Referring to Preparation Example 1, the Zn-alendronic acid (1/0.25) adjuvant suspension was prepared.

PREPARATION EXAMPLE 12

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

[0128] According to a Zn/pamidronic acid molar concentration ratio of 1:0.25, 50 mL of a 31.11 mM zinc chloride solution was prepared and defined as Solution A; 50 mL of a solution (7.78 mM pamidronic acid+32 mM sodium hydroxide+7.78 mM disodium hydrogen phosphate) was prepared and defined as Solution B. Solution A and Solution B were filtered with 0.22 μm filter membrane for later use.

[0129] Referring to Preparation Example 1, the Zn-pamidronic acid (1/0.25) adjuvant suspension was prepared.

[0130] The present invention will be further described below in conjunction with examples.

EXAMPLE 1

Determinations of Physical and Chemical Properties of Zinc Zoledronate Adjuvant

[0131] The following determination methods were applicable to any Zn/zoledronic acid molar ratio, that was, any zinc zoledronate adjuvant, such as zinc zoledronate adjuvant doped with inorganic phosphoric acid or doped with Al.

[0132] (1) Observation of Morphology of the Adjuvant Particles

[0133] After the zinc zoledronate adjuvant was diluted 50 times with deionized water, the observation was performed with a JEM-2100 transmission electron microscope (TEM) of Japan Electronics. The specific steps were as follows: dropping the adjuvant sample on a copper mesh coated with carbon film, allowing 10 minutes for absorption, wiping off the residual liquid with filter paper, sending samples to the sample chamber of the transmission electron microscope to observe the morphology of the sample, and taking photos for further analysis.

[0134] Experimental results: it could be clearly seen that Zn-zoledronic acid (1/0.25) adjuvant and Al-001-zoledronic acid adjuvant had spherical nano-core particles, while FH-001-zoledronic acid adjuvant showed a laver cluster shape (FIG. 2).

[0135] (2) Determination of pH

[0136] The samples to be tested was taken, equilibrated at room temperature for at least 30 minutes, and measured with a Sartorius pH glass electrode.

[0137] Standard buffer (pH 7.00), standard buffer (pH 4.01) and standard buffer (pH 10.01) were selected to calibrate the instrument according to the requirements of the instruction manual.

[0138] The “Mode” button could be pressed to switch between pH and mV modes. Usually, the pH mode was selected when determining the pH value of a solution.

[0139] The “SETUP” button was pressed until the display showed Clear buffer, and then the “ENTER” button was pressed to confirm and clear the previous calibration data.

[0140] The “SETUP” button was pressed until the display showed the buffer solution group “4.01, 7.00, 10.01”, and then the “ENTER” button was pressed to confirm.

[0141] The electrode was taken out of the electrode storage solution carefully, the electrode was rinsed thoroughly with deionized water, and the water on the surface was dried with filter paper after well rinsed (be careful not to wipe the electrode).

[0142] The electrode was immersed in the first buffer solution (pH 7.00), until the value was stable and “S” appeared, then the “STANDARDIZE” button was pressed, until the instrument was automatically calibrated. After the calibration was successful, “7.00” and the electrode slope were displayed.

[0143] The electrode was taken out from the first buffer solution, and the electrode was rinsed thoroughly with deionized water, and the electrode was immersed in the second buffer solution (pH 4.01) in sequence, until the value was stable and “S” appeared, then the “STANDARDIZE” button was pressed, until the instrument was calibrated automatically. After the calibration was successful, “4.01 7.00” and the message “Slope” displayed. Slope displayed the measured electrode slope value, which was acceptable in the range of 90-105%.

[0144] If there was a big deviation from the theoretical value, an error message (Err) would be displayed, then the electrode should be cleaned, and the above steps should be repeated for calibration.

[0145] The above operations were repeated to complete the third point (pH 10.01) calibration.

[0146] After the calibration was completed, the electrode was rinsed thoroughly with deionized water, and then dried gently with filter paper. The sample solution was shaken evenly, the glass electrode was immersed in the sample solution, until the pH value change no more than ±0.05 within 1 minute, and then the reading was confirmed.

[0147] The sample solution was shaken evenly and the measurement was carried out again. The difference between the two pH values should not exceed 0.1. The average of the two readings was taken as the pH value of the test product.

[0148] Experimental results (Table 1):

[0149] The pH range of Zn-zoledronic acid adjuvants were 8.0 to 9.0 before sterilization, and were 6.5 to 7.0 after sterilization.

[0150] (3) Determination of Particle Size

[0151] Beckman LS 13320 laser particle size analyzer was turned on and warmed up for 15 minutes.

[0152] The analyzer control software were started and the closed compartment of the sample cell were opened, the sample cell was taken from the sample tank and added with 12 mL of purified water.

[0153] The sample cell was placed on the sample tank and the compartment door was closed.

[0154] “start cycle” was clicked, “Measure Offsets”, “Align”, “Measure Background” were selected in turn, and finally “start” was clicked, “OK” in the pop-up dialog box was clicked to start the calibration of blank background.

[0155] The sample cell was taken out, and added with a certain amount of standard sample (come with the analyzer); “start cycle” was clicked, “Measure Loading”, “Enter Sample Info”, “Enter run setting”, “start runs” were selected in turn, and finally “start” was clicked, the name of the standard sample was input in the pop-up dialog box, “OK” was clicked when “Obscuration” parameter in the software was 8% to 12%, so the measurement of the standard sample was carried out.

[0156] In order to ensure the accuracy and reliability of the experimental data, the blank background should be calibrated and the size of the standard sample should be measured before each measurement was performed.

[0157] The closed compartment of the sample cell was opened and the sample cell was taken out.

[0158] The aqueous solution containing the standard sample in the sample cell was discarded, and deionized water was added into the sample cell to clean the sample cell 3 times.

[0159] After cleaning, 12 mL of deionized water was added, and the sample cell was placed on the sample tank, and the compartment door was closed.

[0160] “start cycle” was clicked, “Measure Offsets”, “Align”, “Measure Background” were selected in turn, and finally “start” was clicked, “OK” in the pop-up dialog box was clicked to start the calibration of blank background.

[0161] The sample cell was taken out, added with a certain volume of a test sample, the sample test compartment door was opened, the sample cell was placed on the sample tank and the compartment door was closed.

[0162] “start cycle” was clicked, “Enter Sample Info”, “Enter run setting”, “start runs” were selected in turn, and finally “start” was clicked, the name of the standard sample was input in the pop-up dialog box, “OK” was clicked when “Obscuration” parameter in the software was 8% to 12%, the particle sizes of samples that were measured were recorded.

[0163] Experimental results:

[0164] The particle size of the zinc zoledronate adjuvants was between 0.4 to 30 μm, and most of the particles were 6 to 7 μm in size.

TABLE-US-00001 TABLE 1 pH/particle size of several batches of Zn-zoledronic acid (1/0.25) adjuvant Adjuvant type Batch pH Particle size (μm) Zn-zoledronic 2018041802 6.73 6.6 acid 2018041803 6.78 6.6 (1/0.25) 2018042501 6.62 6.4

[0165] (4) Determination of Adsorption Rate

[0166] Plotting standard curve for BSA standard: 150 mM NaCl was used as dilution buffer, the BSA standard (2 mg/mL) was serially diluted, and the absorbance at 280 nm was detected with UV2100 pro. The OD280 showed a high accuracy at 0.2-0.8 (broaden to 0.2-1.5).

[0167] BSA gradient dilution (EP): 150 mM NaCl was used as dilution buffer, a certain amount of BSA sample was weighed and diluted to the concentration gradient specified in EP: 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 3 mg/mL, 5 mg/mL, 10 mg/mL, for later use.

[0168] BSA was mixed with adjuvant under BSA: adjuvant=3:1 (volume ratio), which was set as the usage condition for the measurement of adsorption rate; after the adjuvant was shaken evenly, it was mixed with different concentrations of BSA according to the experimental conditions, and the adsorption at room temperature was performed for 1 hour, and shaking was performed 5 times during the period; a centrifugation was performed at 13000 rpm/min for 3 minutes, the supernatant was taken afterwards, for later use.

[0169] Determination of protein concentration: Lowry method was used to determine the protein concentration in EP. In this experiment, according to the practical situation, UV2100 pro was used to directly determine the absorbance of the supernatant at 280 nm, the reading was kept between 0.2-0.8, otherwise the supernatant should be diluted.

Calculation of adsorption rate: adsorption rate=[1−OD.sub.280(supernatant dilution factor X)/OD.sub.280(when the adsorption rate of dilution X is 0)]*100

[0170] The experimental results were as follows:

TABLE-US-00002 TABLE 2 Protein adsorption capacity of several batches of Zn-zoledronic acid (1/0.25) adjuvant Batch Absorption capacity (%) BSA(mg/mL) 0.5 1 2 3 5 10 Lot 2018041802 97 92 59 42 25 20 Lot 2018041803 99 90 55 42 26 17 Lot 2018041804 96 78 50 48 27 19

[0171] (5) Determination of PZC:

[0172] Instrument for measurement: Nanobrook Omni (Brookhaven)

[0173] Experimental operation: 0.1M NaOH/1% HNO.sub.3 were used to adjust the pH of 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.

[0174] Passivated electrode: 3-4 mL of the 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.

[0175] Sample measurement: After the electrode was taken out, its lower end was rinsed with deionized water, and then the corresponding sample was added, the cycle was set to 15 in the SOP, the measure was set to 3, the pH was set to the corresponding pH of each sample, and the instrument was run.

[0176] Data processing: the corresponding Zeta potentials under different pH values were got, the software that came with the instrument was run to get the PZC values.

[0177] Results: The PZC of Zn-zoledronic acid (1/0.125) adjuvant was 4.08.

EXAMPLE 2

Determination of Zinc Precipitation Rate of Zn-Zoledronic (1/0.125) Adjuvant

[0178] The flame method (D2 lamp background correction) was used to determine the zinc content in the zinc zoledronate adjuvants and the determination procedure was standardized. The detection instrument was an atomic absorption spectrophotometer: Shimadzu AA6300C (P/N 206-52430).

[0179] Preparation of standard solution and sample to be determined: preparation of standard curve: the original concentration of zinc standard was 500 μg/mL, and it was diluted with 0.1 M hydrochloric acid solution to obtain 500 ng/mL, 1000 ng/mL, 1500 ng/mL, 2000 ng/mL and 2500 ng/mL standards.

[0180] The Zn-zoledronic acid (1/0.125) adjuvant was centrifuged at 13000 r/min for 10 min and the supernatant was removed. The sample was diluted 5 times with 0.1M nitric acid solution for determination, and a vortex mixer was used for shaking and mixing well. The determination process was as follows:

[0181] Operation method of AA-6300C and usage of WizAArd software:

[0182] Power turning on: the computer, AA-6300C power switch, air compressor switch (the pressure was set at 0.35 MPa), and ventilation system switch were turned on;

[0183] Acetylene opening: the acetylene valve was slowly opened to ensure that the primary pressure was 0.5 MPa and the secondary pressure was 0.1 MPa;

[0184] Basic operation procedure of WizAArd software: log in WizAArd.fwdarw.select element.fwdarw.at “unconnected instrument/send parameter” page, click <connect/send parameter>.fwdarw.set in “instrument initialization” page.fwdarw.check and tick each of items in “flame analysis instrument check catalog”, click <OK>.fwdarw.set wavelength [213.86], slit width [0.7], lighting method [emission], [lamp position setting] in “optical parameters” page to ensure the actual position and preset position of the Zn hollow cathode lamp be identical, select [lamp on].fwdarw.line search.fwdarw.burner origin position adjustment.fwdarw.select [parameter] in the menu bar.fwdarw.[edit parameter].fwdarw.change the lighting method to <BGC-D2>.fwdarw.line search.fwdarw.ignition: after ensuring that C2H2 was turned on and the pressure met the requirements, press the PURGE and IGNITE buttons on the host at the same time until ignition.fwdarw.auto zero.fwdarw.set the blank group (BLK), standard product (STD), and test sample (UNK) on the MRT worksheet, input the theoretical concentration of the standard and the name of the sample, and manually load the sample through the sample tube extended from the nebulizer, set the sample volume at least 1 mL each time, select [start] to test.fwdarw.stop the flame.fwdarw.save the data and disconnect the instrument from the computer.fwdarw.shut down;

[0185] Test results:

TABLE-US-00003 TABLE 3 Drawing of standard curve of zinc concentration and Abs Concentration of Zinc standard (X, ng/mL) 0 500 1000 1500 2000 2500 Abs (y) 0.017 0.093 0.161 0.248 0.314 0.379 Standard curve formula y = 0.0001x + 0.0191, R.sup.2 = 0.9984

TABLE-US-00004 TABLE 4 Test results of zinc precipitation rate in Zn-zoledronic acid (1/0.125) adjuvant Zinc content Total zinc content Zinc of supernatant of adjuvant precipitation Sample Abs (μg/mL) (μg/mL) rate (%) Zn-zoledronic 0.165 7.3 4067 99.82 acid (1/0.25)

EXAMPLE 4

Determination of Zoledronic Acid Precipitation Rate of Zn-Zoledronic Acid (1/0.125) Adjuvant

[0186] Method: UV spectrophotometry, instrument: UV800 (Beckman coulter).

[0187] Zoledronic acid has an imidazole ring and has a maximum absorption peak at 215 nm. The specific process for measuring the supernatant of the zoledronic acid of the adjuvant at 215 nm by an ultraviolet spectrophotometer was as follows:

[0188] First, 0.04 mg/mL, 0.03 mg/mL, 0.02 mg/mL, 0.015 mg/mL, 0.0075 mg/mL, 0.00375 mg/mL of standard zoledronate sodium solutions were prepared with physiological saline, and their OD.sub.215 values were measured at a wavelength of 215 nm, and a standard curve was plotted. At the same time, we centrifuged Zn-zoledronic acid (1/0.125) at 13000 rpm for 10 minutes and took the supernatant afterward. The absorbance of the zoledronate sodium in the supernatant was measured at a wavelength of 215 nm. The results were as follows:

TABLE-US-00005 TABLE 5 Sodium zoledronate concentration and OD.sub.215 standard curve concentration of zoledronate sodium standard (x, mg/mL) 0.00375 0.0075 0.015 0.02 0.03 0.04 OD.sub.215 (y) 0.0517 0.1039 0.1944 0.2669 0.3931 0.5183 Standard curve y = 12.876x + 0.0053, R.sup.2 = 0.9997 plotting

TABLE-US-00006 TABLE 6 Measurement results of zoledronic acid precipitation rate of Zn-zoledronic acid (1/0.125) adjuvant Content of Content of Zoledronic Zoledronic acid total zoledronic acid in supernatant acid in adjuvant precipitation Sample OD.sub.215 (μg/mL) (μg/mL) rate (%) Zn-zoledronic 0.0359 2.4 2373 99.89% acid (1/0.125)

EXAMPLE 5

Determination of Adjuvant Activity of Zn-Zoledronic Adjuvant in Recombinant Protein VZV gE Vaccine

[0189] The prepared zinc zoledronate adjuvants (also known as Zn-zoledronic acid) were used as an adjuvant, in which the Zn/zoledronic acid molar concentration ratio was 1:0.25 or 1:1. As adjuvants, they were used in combination with VZV gE antigen and together they were injected intramuscularly into mice, and the specific antibody titers produced were measured. The specific method was as follows:

[0190] Experimental animals: Balb/C mice, 6-8 weeks, 5 mice/group, female.

[0191] Drug concentration: antigen: 50 μg/ml;

[0192] Dosage: 5 μg/50 μl/mouse; aluminum adjuvant: 50 μl/mouse; zinc zoledronate adjuvant: 50 μl/mouse;

[0193] Experimental groups: (1) basic aluminum adjuvant group (A1-002); (2) Zn-zoledronic acid (1/0.25); (3) Zn-zoledronic acid (1/1);

[0194] Immunization protocol: At 2 weeks after the first immunization of the animals according to the immunization grouping, blood samples were collected from the eye socket to determine the antibody titer in serum. The antibody titer was measured in the second week after the first immunization. A booster immunization was given in the second week. At 2 weeks after the booster immunization, blood samples were collected from the eye socket to determine the antibody titer in serum, and a third immunization was given. After 2 weeks, the antibody titer in serum was determined by ELISA.

[0195] Determination of antibody binding titer by enzyme-linked immunosorbent assay (ELISA):

[0196] 1. Antigen coating solution: 1× PB 7.4 buffer solution (4.343 g of Na.sub.2HPO.sub.4.7H.sub.2O; 0.456 g of NaH.sub.2PO.sub.4).

[0197] 2. Washing solution: PBST, Beijing Wantai ELISA kit

[0198] 3. Blocking solution: 2× ED (Enzyme Dilution): 1× PBS+0.5% casein+2% gelatin+0.1% preservative (proclin-300), diluted to 1× with ultrapure water or distilled water for sealing and sample dilution.

[0199] 4. Color development solution A: Beijing Wantai ELISA kit.

[0200] 5. Color development solution B: Beijing Wantai ELISA kit.

[0201] 6. Stop solution: Beijing Wantai ELISA kit.

[0202] Experiment procedure:

[0203] (1) Coating plate: VZV gE antigen was diluted with PB7.4 coating buffer solution to a certain concentration, usually 100 ng/well, added to a 96-well polystyrene plate by 100 μL/well, and the plate was coated overnight at 4° C.

[0204] (2) Blocking: the coating solution in the well was discarded, the plate was washed with PBST washing solution once, spin-dried, added with the blocking solution by 200 μL/well, and blocking was performed for 4 hours at room temperature.

[0205] (3) Adding serum of a certain degree of dilution: the blocking solution in the well was discarded, the plate was washed with PBST once, spin-dried, the first well was added with the serum to be tested by 150 μL/well, each of the following well was added with ED diluent by 100 μL/well, diluted at a gradient of 3 times, incubated and reacted at 25° C. for 1 h.

[0206] (4) Adding enzyme-labeled antibody (GAM-HRP): the serum diluent in the well was discarded, the plate was washed 5 times with PBST, spin-dried, added with enzyme-labeled antibody (GAM-HRP, V:V=1:5000) by 100 μL/well, incubated and reacted at 25° C. for 1 h.

[0207] (5) Color development: the secondary antibody in the well was discarded, the plate was washed 5 times with PBST, spin-dried, added with 100 μL/well of a color development solution, an equal volume mixed A and B, and reacted at 25° C. for 10 min.

[0208] (6) Stopping: 2 M sulfuric acid stop solution was added by 50 μL/well to stop the reaction.

[0209] (7) Reading plate: dual wavelengths of 450 nm and 630 nm were set as the measuring wavelengths on microplate reader and the OD value of each reaction well was measured.

[0210] The experimental results were shown in FIG. 3:

[0211] The adjuvant was mixed with the antigen in equal volume to immunize the mice. The immunization, the blood sampling and the determination of serum antibody titer were performed according to the above-mentioned strategy. In the zinc zoledronate adjuvant group, the mice showed a rapid and strong onset after one immunization injection; after two immunization injections, the humoral immune enhancement advantage showed a statistically significant difference as compared with the aluminum adjuvant group, and the difference was still significant after three immunization injections.

EXAMPLE 6

Effect of Zinc Zoledronate Adjuvant Combined with Recombinant Protein VZV gE on Mouse Antibody Isotypes

[0212] The immunization strategy referred to Example 5.

[0213] Experimental Materials:

[0214] 1. Antigen coating solution: 1× PB 7.4 buffer solution (4.343 g of Na.sub.2HPO.sub.4.7H.sub.2O; 0.456 g of NaH.sub.2PO.sub.4).

[0215] 2. Washing solution: PBST, ELISA kit from Beijing Wantai Company.

[0216] 3. Blocking solution: 2× ED (Enzyme Dilution): 1× PBS+0.5% casein+2% gelatin+0.1% preservative (proclin-300), diluted to 1× with ultrapure water or distilled water for sealing and sample dilution.

[0217] 4. Color development solution A: ELISA kit from Beijing Wantai Company.

[0218] 5. Color development solution B: ELISA kit from Beijing Wantai Company.

[0219] 6. Stop solution: Beijing Wantai Company ELISA kit.

[0220] Experiment Procedure:

[0221] (1) Coating plate: the VZV gE antigen was diluted with PB7.4 coating buffer solution to a certain concentration, added to a 96-well polystyrene plate by 100 μL/well, and the plate was coated overnight at 4° C.

[0222] (2) Blocking: the coating solution in the well was discarded, the plate was washed once with PBST, spin-dried, added with blocking solution by 200 μL/well, and blocking was performed for 4 hours at room temperature.

[0223] (3) Adding serum to be tested: Serum used for serum antibody isotype detection: 2 weeks post the completion of 3 immunization injections, that was, the serum of the 6th week; the blocking solution in the well was discarded, the plate was washed once with PBST, spin-dried, added with the serum to be tested at a certain degree of dilution by 100 μL/well, incubated and reacted for 1 h at 25° C.

[0224] (4) Adding enzyme-labeled antibody: the serum diluent in the well was discarded, the plate was washed 5 times with PBST, spin-dried, added with enzyme-labeled antibody that specifically recognized each antibody isotype (IgG1, V:V=1:30000; IgG2a, V:V=1:1000; IgG2b, V:V=1:1000) by 100 μL/well, incubated and reacted at 25° C. for 1 h.

[0225] (5) Color development: the enzyme-labeled antibody in the well was discarded, the plate was washed 5 times with PBST, spin-dried, added with a color development solution, a equal volume mixed A and B diluted by 3 times, by 100 μL/well, reacted at 25° C. for 10 min.

[0226] (6) Stopping: 50 μL/well of 2 M sulfuric acid stop solution was added to stop the reaction.

[0227] (7) Reading plate: dual wavelengths of 450 nm and 630 nm were set as the measuring wavelengths on microplate reader, and the OD value of each reaction well was measured.

[0228] The experimental results were shown in FIG. 4:

[0229] The experimental procedure described in Example 5 was used. The mice were immunized with zinc zoledronate adjuvant or aluminum adjuvant in combination with recombinant VZV gE protein by intramuscular injection. The immunization procedure was the same as that in Example 5. After 3 immunization injections, blood samples were collected after 2 weeks, and the results of levels of each antibody isotype in mouse serum were shown in FIG. 4. Compared with the aluminum adjuvant group, the zinc zoledronate adjuvant group could stimulate higher levels of IgG2a and IgG2b antibody isotypes, and the ratios of IgG1 to IgG2a and IgG2b was lower than that of the aluminum adjuvant group, indicating that it had a certain stimulating effect on the Th1 immune pathway.

EXAMPLE 7

Determination of Adjuvant Activity of Zinc Zoledronate Adjuvant in Hepatitis B Therapeutic Protein

[0230] The experimental procedure as described in Example 5 was used. The 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 were shown in FIG. 5: compared with the aluminum adjuvant group, the zinc zoledronate adjuvant group showed rapid onset and strong immune response after one injection of immunization in mice, and the difference was statistically significant; after 2 immunization injections, the humoral immunity enhancement advantage was still significant as compared with the aluminum adjuvant group, and was slightly better than the aluminum adjuvant group after 3 immunization injections.

EXAMPLE 8

Effect of Other Zinc Bisphosphonate Adjuvants Combined with VZV gE on Mouse Serum Antibody Titers

[0231] The experimental procedure as described in Example 5 was used. The mice were immunized with a zinc bisphosphonate adjuvant in combination with recombinant VZV gE protein by intramuscular injection, and serum antibody titers were determined. The results were shown in FIG. 6: after 3 immunization injections in mice, the zinc zoledronate adjuvant group showed a significant difference in advancement of humoral immunity enhancement as compared with the aluminum adjuvant group, while the effects of other zinc bisphosphonate adjuvants were not significantly different from that of aluminum adjuvant.

EXAMPLE 9

Effect of Zinc Zoledronate Adjuvant Combined with VZV gE on Mouse Serum Antibody Titers

[0232] The experimental procedure as described in Example 5 was used. The 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, the aluminum adjuvant, composite zinc-aluminum adjuvant, composite zinc-aluminum adjuvant doped with zoledronic acid, and zinc zoledronate adjuvant showed a gradually increased trend in the level of antibody enhancement, and there was a significant difference by comparing the zinc zoledronate adjuvant group with the aluminum adjuvant group.