PREPARATION OF ZINC RISEDRONATE MICRO/NANO ADJUVANT AND USE THEREOF AS VACCINE ADJUVANT

Abstract

The present invention pertains to the field of pharmaceutical technology. Specifically, the present invention relates to a zinc risedronate micro/nano adjuvant with sustained-release function formed by mineralization of zinc ions and risedronic acid as main components and its use as a vaccine adjuvant. The present invention also relates to a method for preparing zinc risedronate micro/nano adjuvant. The present invention also relates to a chemical composition, vaccine adjuvant and vaccine composition comprising zinc risedronate micro/nano adjuvant. The present invention also relates to a use of zinc risedronate micro/nano adjuvant as a vaccine adjuvant.

Claims

1-8. (canceled)

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

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

11. The zinc risedronate micro/nanoparticle adjuvant according to claim 9, characterized in any one of the following: (1) said zinc risedronate micro/nanoparticle adjuvant further comprises a phosphate at a molar concentration ratio of zinc:phosphate of 1-8:1; and (2) said zinc risedronate 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 risedronate micro/nanoparticle adjuvant according to claim 9, characterized in any one of the following: (1) said zinc risedronate micro/nanoparticle adjuvant further comprises aluminum at a molar concentration ratio of zinc:aluminum of 0.02-1:1; and (2) said zinc risedronate 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 risedronate micro/nanoparticle adjuvant according to claim 9, wherein the zinc risedronate 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 risedronic acid and a sodium hydroxide; or evenly mixing the soluble salt solution of step a) with a risedronic 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 risedronate adjuvant.

14. The zinc risedronate micro/nanoparticle adjuvant according to claim 13, characterized in one or more of the following: (1) said soluble 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 risedronate 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 risedronate micro/nanoparticle adjuvant according to claim 9, characterized in one or more of the following: (1) said zinc risedronate micro/nanoparticle adjuvant has a pH of 8.0-9.0 before sterilization; (2) said zinc risedronate micro/nanoparticle adjuvant has a pH of 6.0-8.0 after sterilization; (3) said zinc risedronate micro/nanoparticle adjuvant has a particle size of 1-10 μm; (4) said zinc risedronate micro/nanoparticle adjuvant has a particle zero charge point of 4.0-11.4; and (5) said zinc risedronate micro/nanoparticle adjuvant has a protein adsorption rate of >60%.

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

18. A method for preparing a zinc risedronate 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 risedronic acid and a sodium hydroxide; or evenly mixing the soluble salt solution of step a) with a risedronic 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 risedronate adjuvant, wherein the obtained zinc risedronate adjuvant has a molar concentration ratio of zinc:risedronic acid of 1-8:1.

19. The method according to claim 18, wherein the obtained zinc risedronate adjuvant has a molar concentration ratio of zinc:risedronic 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 soluble 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 risedronate micro/nanoparticle adjuvant has a pH of 8.0-9.0 before sterilization; (2) the obtained zinc risedronate micro/nanoparticle adjuvant has a pH of 6.0-8.0 after sterilization; (3) the obtained zinc risedronate micro/nanoparticle adjuvant has a particle size of 1-10 μm; (4) the obtained zinc risedronate micro/nanoparticle adjuvant has a particle zero charge point of 4.0-11.4; and (5) the obtained zinc risedronate micro/nanoparticle adjuvant has a protein adsorption rate of >60%.

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 risedronate micro/nanoparticle adjuvant according to claim 9.

25. A vaccine composition, comprising an antigen and the zinc risedronate 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 risedronate 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

[0078] FIG. 1: schematic diagrams of three preparation processes for preparing the adjuvants.

[0079] FIG. 2: electron microscopic view of the zinc risedronate adjuvant.

[0080] FIG. 3: particle size of the zinc risedronate adjuvant.

[0081] FIG. 4: determination of the adjuvant activity of zinc risedronate adjuvant combined with recombinant VZV gE protein, mean±SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0082] FIG. 5: effect of zinc risedronate 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.

[0083] FIG. 6: determination of adjuvant activities of sodium risedronate adjuvant and zinc risedronate adjuvant combined with hepatitis B therapeutic protein, mean±SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

[0084] FIG. 7: study on dose-effect relationship of zinc risedronate adjuvant activity, mean±SD: n=5, *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

SPECIFIC MODES FOR CARRYING OUT THE PRESENT INVENTION

[0085] 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.

Preparation Example 1: Preparation of Zinc Risedronate Adjuvant (Zn-Risedronic Acid (1/0.25))

[0086] Risedronate sodium (C.sub.7H.sub.10NNaO.sub.7P.sub.2): purchased from Hunan Huateng Pharmaceutical Co., Ltd.

[0087] Anhydrous zinc chloride (ZnCl.sub.2): purchased from Xilong Chemical

[0088] Disodium hydrogen phosphate dodecahydrate (Na.sub.2HPO.sub.4.12H.sub.2O): purchased from Xilong Chemical

[0089] Sodium hydroxide (NaOH): purchased from Xilong Chemical

[0090] Preparation of Solutions:

[0091] According to a molar concentration ratio of Zn/risedronic acid 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 risedronic acid+36 mM sodium hydroxide+15.55 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.

[0092] Preparation of Zn-Risedronic Acid (1/0.25) Adjuvant Suspension and Determination of its Physical and Chemical Properties:

[0093] Solution A and Solution B were used to form a zinc risedronate adjuvant suspension according to the scheme as shown in FIG. 1A, that was, the prepared Solution B was added dropwise to Solution A at a volume ratio of 1:1 until all was added, forming a suspension.

[0094] After the mixing step according to FIG. 1A, the obtained zinc risedronate adjuvant was sterilized once by high-pressure steam at 121° C. for 60 min, and its physical and chemical properties such as pH value, particle size and particle morphology after sterilization were measured.

Preparation Example 2: Preparation of Zinc Risedronate Adjuvant (Zn-Risedronic Acid (1/1))

[0095] The source of reagents can be found in Preparation Example 1.

[0096] Preparation of Solutions:

[0097] According to a molar concentration ratio of Zn/risedronic acid 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 risedronic acid+60 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.

[0098] Preparation of Zn-Risedronic Acid (1/1) Adjuvant Suspension and Determination of its Physical and Chemical Properties:

[0099] Please refer to Preparation Example 1 for details.

Preparation Example 3: Preparation of Zinc Risedronate Adjuvant (Zn-Risedronic Acid (1/0.125))

[0100] The source of reagents can be found in Preparation Example 1.

[0101] Preparation of Solutions:

[0102] According to the molar concentration ratio of Zn/risedronic acid of 1:0.125, 1 L of 124.44 mM zinc chloride solution was prepared and defined as Solution A; 1 L of solution (15.56 mM risedronic acid+60 mM sodium hydroxide+184 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.

[0103] Preparation of Zn-Risedronic Acid (1/0.125) Adjuvant Suspension and Determination of its Physical and Chemical Properties:

[0104] Please refer to Preparation Example 1 for details.

Preparation Example 4: Preparation of Aluminum Adjuvant Al-002

[0105] Preparation of solutions: the source of reagents can be found in Preparation Example 1.

[0106] According to the molar concentration ratio of phosphate radical/Al of 0.3:1, 50 mL of a 62.22 mM aluminum chloride solution was prepared and defined as Solution A; 50 mL of 9.33 mM disodium hydrogen phosphate solution (50 mM sodium hydroxide) was prepared and defined as Solution B. Solutions were filtered with 0.22 μm filter membrane for later use.

[0107] Preparation of Aluminum Adjuvant Al-002 Suspension

[0108] Solution A and Solution B were prepared in a volume ratio of 1:1. The preparation was carried out according to the scheme of FIG. 1A in which the prepared Solution B was added dropwise to Solution A at a volume ratio of 1:1 until all was added, forming a suspension. The suspension obtained after mixing was sterilized at 121° C. for 60 min.

Preparation Example 5: Preparation of Aluminum Adjuvant Al-001-840

[0109] Preparation of solutions: the source of reagents can be found in Preparation Example 1.

[0110] 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, which also contained 150 mM sodium hydroxide. Solutions were filtered with 0.22 μm membrane for later use.

[0111] Aluminum adjuvant Al-001-840 suspension was prepared by the same method referring to the preparation of aluminum adjuvant Al-002 suspension.

Example 1: Determination of Physical and Chemical Properties of Zinc Risedronate Adjuvant (Zn-Risedronic Acid (1/0.125))

[0112] The zinc risedronate suspension obtained after mixing was sterilized once at 121° C. for 60 minutes, and the physical and chemical properties such as pH value, particle size and particle morphology, metal ion precipitation rate and other physical and chemical properties after sterilization were measured.

[0113] The following detection methods were applicable to any Zn/risedronic acid molar ratio, that was, any zinc risedronate adjuvant, such as zinc risedronate adjuvant doped with inorganic phosphoric acid;

[0114] (1) Observation of Adjuvant Particle Morphology

[0115] After the risedronate zinc 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.

[0116] Experimental results: As shown in FIG. 2, Zn-risedronic acid (1/1) adjuvant had an amorphous cluster shape, while in the Zn-risedronic acid (1/0.25) and Zn-risedronic acid (1/0.125) adjuvants, spherical nano-core particles could be clearly seen.

[0117] (2) pH Measurement

[0118] The samples to be tested was taken, equilibrated at room temperature for at least 30 minutes, and measured with a Sartorius pH glass electrode.

[0119] Standard buffer (pH7.00), standard buffer (pH4.01) and standard buffer (pH10.01) were selected to calibrate the instrument according to the requirements of the instruction manual.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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).

[0124] 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.

[0125] 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%.

[0126] 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.

[0127] The above operations were repeated to complete the third point (pH 10.01) calibration.

[0128] 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.

[0129] 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.

[0130] Experimental Results: Zn-Risedronic Acid (1/0.125) Adjuvant had a pH of 6.9-7.3 Before Sterilization and 6.4-6.8 after Sterilization;

[0131] (3) Determination of Particle Size

[0132] Beckman LS 13320 laser particle size analyzer was turned on and warmed up for 15 minutes.

[0133] 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.

[0134] The sample cell was placed on the sample tank and the compartment door was closed.

[0135] “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.

[0136] 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.

[0137] 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.

[0138] The closed compartment of the sample cell was opened and the sample cell was taken out.

[0139] 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.

[0140] 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.

[0141] “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.

[0142] 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.

[0143] “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.

[0144] Experimental results: As shown in FIG. 3, taking the Zn-risedronic acid (1/0.125) adjuvant as an example, the particle size was between 0.4-30 μm, and most of the particles had size of 6-7 μm.

[0145] (4) PZC Detection:

[0146] Instrument for Measurement: Nanobrook Omni (Brookhaven)

[0147] Experimental operation: 0.1M NaOH/1% HNO.sub.3 were used to adjust the pH of Zn-risedronic acid (1/0.125) to: 6.00/5.50/5.00/4.50/4.00/3.50/3.00/2.50/2.00.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] Results: The PZC of Zn-risedronic acid (1/0.125) adjuvant was 4.11.

[0152] (5) Determination of Adsorption Rate of Zinc Risedronate Adjuvant

[0153] 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).

[0154] 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.

[0155] BSA was mixed with adjuvant at a ration of 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.

[0156] Determination of protein concentration: 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 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

[0157] Experimental results: Taking Zn-risedronic acid (1/0.125) as an example, firstly, the BSA standard curve was measured. The content of BSA in the adjuvant supernatant was calculated according to the standard curve, and the adsorption rate of Zn-risedronic acid (1/0.125) adjuvant to BSA was calculated according to the absorption formula, which could reach about 70% when BSA was 0.5 mg/mL.

TABLE-US-00001 TABLE 1 Adsorption rate of Zn-risedronic acid (1/0.125) adjuvant BSA concentration (mg/mL) 0.50 1.00 2.00 3.00 5.00 10.00 BSA absorption rate 66% 53% 39% 34% 19% 3%

Example 2: Determination of Metal Ion Precipitation Rate of Zinc Risedronate Adjuvant

[0158] Method: Flame atomic absorption spectrometry, by measuring the content of zinc element in the supernatant of adjuvant, its meal ion precipitation rate was calculated.

[0159] The flame method (D2 lamp background correction) was used to determine the zinc content in the risedronate zinc adjuvants and the determination procedure was standardized. The detection instrument was an atomic absorption spectrophotometer: Shimadzu AA6300C (P/N 206-52430).

[0160] 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.

[0161] The preparation of the solution to be tested: the sample was diluted 400 times with 0.1M hydrochloric acid solution and mixed well by a vortex mixer under vibration.

[0162] The Zn-risedronic 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 ultrapure water for measurement. The measurement process was as follows:

[0163] Operation Method of AA-6300C and Usage of WizAArd Software:

[0164] 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;

[0165] 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;

[0166] 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;

[0167] Experimental results: Taking Zn-risedronic acid (1/0.125) as an example.

TABLE-US-00002 TABLE 2 Drawing of standard curve of zinc concentration and Abs Zinc standard concentration (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-00003 TABLE 3 Test results of zinc precipitation rate in Zn-risedronic acid (1/0.125) adjuvant Zinc Total zinc content of content of Zinc supernatant adjuvant precipitation Sample Abs (μg/mL) (μg/mL) rate (%) Zn-risedronic 0.155 6.8 4067 99.83% acid (1/0.125)

Example 3: Determination of Risedronic Acid Precipitation Rate of Zinc Risedronate Adjuvant

[0168] Method: UV Spectrophotometry, Instrument: UV800 (Beckman Coulter).

[0169] Risedronic acid contained a pyridine ring, which had a maximum absorption peak at 260 nm. The specific process of detecting the supernatant of the adjuvant at 260 nm by an ultraviolet spectrophotometer was as follows:

[0170] First, 2.374 mg/mL sodium risedronate solution (the content of sodium risedronate added in Zn-risedronate adjuvant (1/0.125)) was prepared with physiological saline solution, diluted with physiological saline to 0.08 mg/mL, 0.06 mg/mL, 0.04 mg/mL, 0.03 mg/mL, 0.02 mg/mL, 0.015 mg/mL, 0.01 mg/mL, and measured at the wavelength of 260 nm to obtain OD.sub.260 values, respectively. At the same time, we centrifuged the Zn-risedronic acid (1/0.125) at 13000 rpm for 10 min and then took the supernatant. The supernatant of the adjuvant was in an isotonic environment, and detected at a wavelength of 260 nm to obtain the absorbance of sodium risedronate in the supernatant. The results were as follows:

TABLE-US-00004 TABLE 4 Risedronate sodium concentration and OD.sub.260 standard curve Risedronate sodium standard concentration (x, mg/mL) 0.01 0.015 0.02 0.03 0.04 0.06 0.08 OD.sub.260 (y) 0.1316 0.1946 0.2704 0.3872 0.5365 0.7879 1.0185 Standard curve plotting y = 12.777x + 0.0098, R.sup.2 = 0.9989

TABLE-US-00005 TABLE 5 Risedronic acid precipitation rate of Zn-risedronic acid (1/0.125) adjuvant Risedronic Total risedronic Risedronic acid content of acid content of acid supernatant adjuvant precipitation Sample OD.sub.260 (μg/mL) (μg/mL) rate (%) Zn-risedronic 0.0141 0.3 2373 99.3 acid (1/0.125)

Example 4: Determination of Adjuvant Activity of Zinc Risedronate Adjuvant Combined with Recombinant Protein VZV gE

[0171] The prepared Zn-risedronic acid adjuvants were used as adjuvants, in which the molar concentration ratio of Zn/risedronic acid were 1:1 and 1:0.25 respectively, and they were separately used as adjuvants and mixed with VZV gE antigen at volume ratio of 1:1 to form vaccines, and then the vaccines were administered by intramuscular injection to mice to determine the specific antibody titers produced. The specific method was as follows:

[0172] Experimental animals: Balb/C mice, 6-8 weeks, 5 mice/group, female.

[0173] Experimental groups: (1) aluminum adjuvant group (Al-002); (2) Zn-risedronic acid (1/0.25) group; (3) Zn-risedronic acid (1/1) group;

[0174] Immunization protocol: antigen 5 μg/mouse, adjuvant and VZV gE antigen were mixed at a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected mice, 100 μL per mouse, and 50 μL on each hind leg. The immunization was performed in the 0.sup.th, 2.sup.nd and 4.sup.th week, that was, 2 weeks after the first immunization of the animals according to the immunization grouping, blood samples were collected from eye socket to determine the specific antibody titers in serum.

[0175] Antibody titers were measured in the 2.sup.nd week after the first immunization, booster immunization was performed in the 2.sup.nd week, blood samples were taken from eye socket 2 weeks after the second immunization, the specific antibody titers in serum were measured, the third immunization was performed at the same time, blood samples were taken from eye socket 2 weeks later, and antibody titers in serum were measured by ELISA.

[0176] Determination of Antibody Binding Titer by Enzyme-Linked Immunosorbent Assay (ELISA):

[0177] 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).

[0178] 2. Washing solution: PBST, INNOVAX Co. ELISA kit

[0179] 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.

[0180] 4. Color development solution A: INNOVAX Co. ELISA kit.

[0181] 5. Color development solution B: INNOVAX Co. ELISA kit.

[0182] 6. Stop solution: INNOVAX Co. ELISA kit.

[0183] Experiment Procedure:

[0184] (1) Coating plate: VZV gE antigen was diluted with PB7.4 coating buffer solution to a certain concentration. It was added to a 96-well polystyrene plate by 100 μL/well, and the plate was coated overnight at 4° C.

[0185] (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.

[0186] (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.

[0187] (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.

[0188] (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.

[0189] (6) Stopping: 2 M sulfuric acid stop solution was added by 50 μL/well to stop the reaction.

[0190] (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.

[0191] The experimental results were shown in FIG. 4:

[0192] Two weeks after the one dose of immunization of mice, the antibody titers of the Zn-risedronic acid adjuvant groups (1/0.25, 1/1) were higher than that of the control group, that was, they were more than 10 times that of the aluminum adjuvant group, showing features of fast onset of action. After two doses of immunization, advantages of humoral immune response enhancement were still noticeable. At the 4.sup.th week, the antibody titer of the Zn-risedronic acid adjuvant group (1/0.25) was 7.5 times that of the aluminum adjuvant group, and the Zn-risedronic acid adjuvant group (1/1) was 18 times that of the aluminum adjuvant group. After three doses of immunization, at the 6th week, the antibody titer of the Zn-risedronic acid adjuvant group (1/0.25) was 10 times that of the control group, and the Zn-risedronic acid adjuvant group (1/1) was 7.5 times that of the control group.

Example 5: Effect of Immunization of Mice with Zinc Risedronate Adjuvant Combined with Recombinant Protein VZV gE on Specific Antibody Isotypes

[0193] The prepared Zn-risedronic acid adjuvant was used as an adjuvant, in which the Zn/risedronic acid molar concentration ratio was 1:0.25, it was used as an adjuvant in combination with VZV gE antigen to inject intramuscularly into mice, and the specific antibody titer produced was measured. The specific method was as follows:

[0194] Experimental animals: Balb/C mice, 6-8 weeks, 5 mice/group, female.

[0195] Experimental groups: (1) aluminum adjuvant group (Al-002); (2) Zn-risedronic acid (1/0.25) group;

[0196] Immunization protocol: antigen 5 μg/mouse, adjuvant and VZV gE antigen were mixed at a volume ratio of 1:1 to form a vaccine, and then intramuscularly injected mice, 100 μL per mouse, and 50 μL on each hind leg. Immunization was performed at the 0.sup.th 2.sup.nd and 4.sup.th week, after 3 injections of immunization was completed, blood samples were collected from eye socket after 2 weeks for testing, and ELISA was used to determine the levels of specific antibody isotypes in serum.

[0197] Experimental Materials:

[0198] 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).

[0199] 2. Washing solution: PBST, ELISA kit from Beijing Wantai Company.

[0200] 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.

[0201] 4. Color development solution A: ELISA kit from Beijing Wantai Company.

[0202] 5. Color development solution B: ELISA kit from Beijing Wantai Company.

[0203] 6. Stop solution: Beijing Wantai Company ELISA kit.

[0204] Experiment Procedure:

[0205] (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.

[0206] (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.

[0207] (3) Adding serum to be tested: 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.

[0208] (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.

[0209] (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, that was an equal volume mixed A and B diluted by 3 times, by 100 μL/well, reacted at 25° C. for 10 min.

[0210] (6) Stopping: 50 μL/well of 2 M sulfuric acid stop solution was added to stop the reaction.

[0211] (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.

[0212] The experimental results were shown in FIG. 5:

[0213] Using the experimental procedure described in Example 4, mice were immunized by intramuscular injection with zinc risedronate adjuvant or aluminum adjuvant in combination with recombinant protein VZV gE, and the immunization procedure was the same as that of Example 4. After the three injections, blood samples were collected after 2 weeks, and the results of level of each isotypes of mouse serum antibody were shown in FIG. 5. Compared with the aluminum adjuvant group, the zinc risedronate adjuvant group could stimulate more intense IgG2a and IgG2b isotypes antibody levels, and the ratios of IgG1 to IgG2a and IgG2b were lower than that of the aluminum adjuvant group, indicating that it had a certain stimulating effect on Th1 immune pathway.

Example 6: Determination of Adjuvant Activity of Zinc Risedronate Adjuvant Combined with Hepatitis B Therapeutic Protein

[0214] Using the experimental procedure described in Example 4, mice were immunized with zinc risedronate adjuvant in combination with hepatitis B therapeutic protein by intramuscular injection, and serum antibody titers were detected. The specific method was as follows:

[0215] Experimental animals: Balb/C mice, 6-8 weeks, 5 mice/group, female.

[0216] Experimental groups: (1) normal saline; (2) aluminum adjuvant group (Al-001-840); (3) sodium risedronate group; (4) Zn-risedronic acid (1/0.125) group; among them the content of sodium risedronate in the group (3) was the same as the zinc risedronate in the group (4). Please refer to Preparation Example 3 for details.

[0217] Immunization protocol: antigen 1.2 μg/mouse, adjuvant and hepatitis B therapeutic protein were mixed at a volume ratio of 1:1 to form a vaccine, and then injected intramuscularly to mice, 150 μL per mouse, and 75 μL on each hind leg. The immunization was performed on the 0.sup.th, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th and 6.sup.th week, 1.2 μg of antigen per mouse, blood samples were collected from eye socket after the 6th week, and ELISA was used to determine the levels of specific antibodies in serum. Please refer to Example 4 for details.

[0218] Experimental results: as shown in FIG. 6, compared with the aluminum adjuvant group, the zinc risedronate adjuvant group was featured with quick and strong onset after one injection of immunization in mice, and the difference was statistically significant; after 3 injections of immunization, that was, at the 4.sup.th week, its humoral immune response enhancement advantage was still significant compared with the aluminum adjuvant group; after 5 injections of immunization, it was slightly better than that of the aluminum adjuvant group, while sodium risedronate itself also had a certain humoral immune stimulation effect, and showed no statistically significant difference compared with aluminum adjuvant.

Example 7: Study on Dose-Effect Relationship of Zinc Risedronate Adjuvant Activity

[0219] Using the experimental procedure described in Example 4, mice were immunized with zinc risedronate adjuvant in combination with hepatitis B therapeutic protein by intramuscular injection, and serum antibody titers were detected. The specific method was as follows:

[0220] Experimental animals: Balb/C mice, 6-8 weeks, 5 mice/group, female.

[0221] Experimental grouping 1: (1) aluminum adjuvant group (Al-001-840); (2) 2× Zn-risedronic acid (1/0.125) group; (3) 0.75× Zn-risedronic acid (1/0.125) group; (4) 0.35× Zn-risedronic acid (1/0.125) group.

[0222] Experimental grouping 2: (1) aluminum adjuvant group (Al-001-840); (2) 2× Zn-risedronic acid (1/0.125) group; (3) 1× Zn-risedronic acid (1/0.125) group.

[0223] Immunization protocol: antigen 1.2 μg/mouse, adjuvant and hepatitis B therapeutic protein were mixed to form a vaccine at a volume ratio of 1:1, and then injected intramuscularly to mice, 100 μL per mouse, 50 μL on each hind leg. The immunization was performed on the 0.sup.th, 2.sup.nd and 4.sup.th week, blood samples were collected from eye socket at the 0.sup.th, 2.sup.nd, 3.sup.rd, 4.sup.th, 5.sup.th and 6.sup.th week, and ELISA was used to determine the levels of specific antibodies in serum. Please refer to Example 4 for details.

[0224] Experimental results: as shown in FIG. 7, compared the serum antibody titers 2 weeks after 2 injections of immunization, it was found that when Zn-risedronic acid (1/0.125) decreased from 2× (2× was the concentration of adjuvant in the vaccine formulation) to 0.75× and 0.35×, its humoral immune enhancement level and amount of adjuvant used showed a significant dose-effect relationship; meanwhile, when Zn-risedronic acid (1/0.125) decreased from 2× to 1×, its humoral immune response enhancement level showed no statistically significant difference. Taking all the data into consideration, it is confirmed that Zn-risedronic acid (1/0.125) had a significant humoral immune enhancement effect and could be used as an immune enhancer in vaccines in the future.