Metal aluminum nano-adjuvant, vaccine composition and preparation method therefor and use thereof
12133889 ยท 2024-11-05
Assignee
Inventors
- Kun LIU (Changchun, CN)
- Hua YU (Changchun, CN)
- Chenggong YANG (Changchun, CN)
- Tianmeng SUN (Changchun, CN)
- Ye WANG (Changchun, CN)
- Ge ZHU (Changchun, CN)
Cpc classification
A61K9/19
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K39/001157
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
International classification
A61K39/39
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
Abstract
Disclosed are a metal aluminum nano-adjuvant, a vaccine composition and a preparation method therefor and a use thereof. The vaccine adjuvant comprises metal aluminum nanoparticles, and can be used as a candidate adjuvant for preventive vaccines and therapeutic vaccines for various diseases such as infections, autoimmune diseases and tumors. The combined use of the vaccine adjuvant provided by the present disclosure and antigen can effectively enhance the humoral immune response and the cellular immune response of the vaccine, and the enhancement effect is significantly better than that of the commercially available aluminum hydroxide adjuvant.
Claims
1. A vaccine composition, wherein, the vaccine composition comprises the vaccine adjuvant, and an antigen or DNA encoding the antigen; wherein the vaccine adjuvant comprises metal aluminum nanoparticles, wherein the average particle size of the metal aluminum nanoparticles is 10-1000 nm, and wherein the metal aluminum nanoparticles are composed of a metal aluminum core with zero valent aluminum (Al.sup.0) and amorphous alumina layer with a thickness of 3-5 nm on the surface.
2. The vaccine composition as defined in claim 1, wherein the antigen is one or more of short peptides, polypeptides and proteins.
3. The vaccine composition as defined in claim 2, wherein, the short peptide is antigen peptide OT-1 of ovalbumin OVA, antigenic glycoprotein gp100 of melanoma cells or apoptosis inhibitory protein survivin/birc5-1; or, the polypeptide is telomerase activity catalytic unit TERT, or, T cell recognition melanoma antigen MART-1, MOG35-55, PADRE, Trp2 or survivin/birc5-2; or, the protein is ovalbumin OVA; or, the mass ratio of the metal aluminum nanoparticles to the antigen is 2:(0.1-10).
4. The vaccine composition as defined in claim 3, wherein, sequence of the antigenic peptide OT-1 is shown in SEQ ID NO: 1; molecular weight of the antigenic peptide OT-1 is 963.14 g/mol; or, sequence of the TERT is shown in SEQ ID NO: 2; molecular weight of the TERT is 3046.70 g/mol; or, molecular weight of the OVA is 298.4 g/mol; or, the mass ratio of the metal aluminum nanoparticles to the antigen is 2:3 or 1:1.
5. The vaccine composition as defined in claim 1, where the average particle size of the metal aluminum nanoparticles is 10-300 nm, or, the metal aluminum nanoparticles are metal aluminum nanoparticles with a dispersion coefficient of circumscribed circle diameter0.21.
6. The vaccine composition as defined in claim 1, wherein, the average particle size of the metal aluminum nanoparticles is 88.85 nm8.86 nm, 147.14 nm11.95 nm, 139.7642.81 nm or 287.82 nm24.13 nm.
7. The vaccine composition as defined in claim 1, wherein, the metal aluminum nanoparticles are prepared by electric explosion method or ligand regulation method.
8. The vaccine composition as defined in claim 7, wherein, the electric explosion method comprises the following steps: in an inert environment, vaporizing the aluminum wire by electric current to form aluminum vapor, and after condensation, obtaining the metal aluminum nanoparticles; or, the ligand regulation method comprises the following steps: in an atmosphere with a water content lower than 10 ppm and an oxygen content lower than 100 ppm, in the presence of titanium catalyst, reacting the ligand solution with the precursor solution; the ligand is a polymer with a functional group containing sulfur atom as a terminal group, and the polymerization degree of the ligand is 10-1000; the structural formula of the precursor is H.sub.3AlX, and the X is an organic molecule, and the organic molecule contains atoms that are able to coordinate with aluminum and have lone pair electrons.
9. The vaccine composition as defined in claim 7, wherein, in the ligand regulation method, the titanium catalyst is titanium tetraisopropanolate; or, in the ligand regulation method, the ligand is ##STR00016## wherein: R.sup.1 is C.sub.1-10 alkyl, C.sub.6-30 aryl, or C.sub.6-30 aryl substituted by R.sup.1a; R.sup.2 is ##STR00017## R.sup.a is C.sub.1-10 alkyl, or C.sub.1-10 alkyl substituted by R.sup.a1, R.sup.a1 is C.sub.6-30 aryl; R.sup.b is H or C.sub.1-10 alkyl; R.sup.C is C.sub.6-30 aryl, C.sub.6-30 aryl substituted by R.sup.c1, or ##STR00018## R.sup.1a and R.sup.c1 are each independently C.sub.1-10 alkyl or halogen; or, in the ligand regulation method, the polymerization degree of the ligand is 20-1000; or, in the ligand regulation method, the PDI of the ligand is 1-2; or, in the ligand regulation method, the structural formula of the ligand is shown in formula (1), wherein Mn is 4.5 kg/mol, n is 42, and PDI is 1.09; ##STR00019## or, in the ligand regulation method, in the precursor, X is an organic molecule containing N or O atoms; or, in the ligand regulation method, in the reaction solution, the concentration of the precursor is 15-500 mM; or, in the ligand regulation method, the molar ratio of the ligand to the precursor is 3:(500-800); or, in the ligand regulation method, the molar ratio of the titanium catalyst to the precursor is 1:(350-550); or, in the ligand regulation method, the molar ratio of the ligand, the precursor and the titanium catalyst is 1:(60-520):(0.04-1.6); or, in the ligand regulation method, the solvent in the ligand solution and the precursor solution is an aprotic solvent.
10. The vaccine composition as defined in claim 9, wherein, in the ligand regulation method, the polymerization degree of the ligand is 40-240; or, in the ligand regulation method, the PDI of the ligand is 1-1.51; or, in the ligand regulation method, in the precursor, X is tertiary amine or tetrahydrofuran; or, in the ligand regulation method, in the reaction solution, the concentration of the precursor is 20-100 mM; or, in the ligand regulation method, the molar ratio of the ligand to the precursor is 3:800; or, in the ligand regulation method, the molar ratio of the titanium catalyst to the precursor is 1:400; or, in the ligand regulation method, the molar ratio of the ligand, the precursor and the titanium catalyst is 1:(70-500):(0.05-1.5); or, in the ligand regulation method, the solvent in the ligand solution and the precursor solution is one or more of toluene, tetrahydrofuran and ether solvents.
11. The vaccine composition as defined in claim 1, wherein, a preparation method of the vaccine adjuvant comprises the following steps: mixing the metal aluminum nanoparticles with solvent A to prepare a vaccine adjuvant suspension.
12. The vaccine composition as defined in claim 11, wherein, the solvent A is one or more of alcohol solvents, ether solvents, ketone solvents, dimethyl sulfoxide, N,N-dimethylformamide and tetrahydrofuran; or, in the vaccine adjuvant suspension, the concentration of the metal aluminum nanoparticles is 0.1-100 mg/ml; or, the mixing method is ultrasonic dispersion.
13. A lyophilized vaccine, wherein, the lyophilized vaccine comprises metal aluminum nanoparticles and antigens, wherein the average particle size of the metal aluminum nanoparticles is 10-1000 nm, wherein the metal aluminum nanoparticles are composed of a metal aluminum core with zero valent aluminum (Al.sup.0) and amorphous alumina layer with a thickness of 3-5 nm on the surface, and wherein the antigens is one or more of short peptides, polypeptides and proteins.
14. A preparation method of the vaccine composition as defined in claim 1, wherein, the preparation method comprises the following steps: mixing the vaccine adjuvant with a solution containing the antigen, and carrying out an incubation reaction.
15. The preparation method of the vaccine composition as defined in claim 14, wherein, the solution containing the antigen is prepared by the following method: mixing the antigen with solvent B.
16. The preparation method of the vaccine composition as defined in claim 15, wherein, the solvent B is water, PBS buffer, DMF or alcohol solvent; or, in the solution containing the antigen, the concentration of the antigen is 10 mg/ml; or, when the vaccine adjuvant in the form of the vaccine adjuvant suspension is mixed with the solution containing the antigen, the solvent A and the solvent B are soluble; or, the incubation reaction is carried out according to the following steps: carrying out ultrasonic dispersion first, followed by incubation; or, the incubation reaction is carried out under the condition of 80-800 rpm; or, the time of the incubation reaction is 1-24 hours; or, after the incubation reaction, the vaccine composition is centrifuged to remove supernatant.
17. The lyophilized vaccine as defined in claim 13, wherein, the lyophilized vaccine is prepared by the following method: mixing the vaccine composition with water and lyophilizing; the vaccine composition comprises the vaccine adjuvant, and an antigen or DNA encoding the antigen, and the vaccine adjuvant comprises metal aluminum nanoparticles.
18. A method for enhancing a humoral or cellular immune response comprising administering to a subject in need thereof metal aluminum nanoparticles and a vaccine; wherein the average particle size of the metal aluminum nanoparticles is 10-1000 nm, and wherein the metal aluminum nanoparticles are composed of a metal aluminum core with zero valent aluminum (Al.sup.0) and amorphous alumina layer with a thickness of 3-5 nm on the surface.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(13) The following embodiments further illustrate the present disclosure, but the present disclosure is not limited thereto. The experimental methods without specific conditions in the following embodiments are selected according to conventional methods and conditions, or according to the product description.
(14) In the following embodiments and comparative embodiments:
(15) Ovalbumin OVA was purchased from sigma-aldrich, molecular weight was 298.4 g/mol;
(16) short peptides or polypeptides were purchased from Gill Biochemical (Shanghai) Co., Ltd. The specific information was as follows:
(17) polypeptide Tert (DI-27): sequence was DLQPYIVIGQFLKHLQDSDASALRN SVVI (SEQ ID NO: 2), molecular weight was 3046.70 g/mol;
(18) short peptide OT-1: sequence was SIINFEKL (SEQ ID NO: 1), molecular weight was 963.14 g/mol.
Embodiment 1 Preparation of Metal Aluminum Nanoparticles by Electric Explosion Method
(19) In argon atmosphere, an aluminum wire with a diameter of 0.2 mm and a purity greater than or equal to 95% was fixed between the high-voltage electrodes, continuously powered by a mechanical device, and the power supply capacitance was 96 F, and the electrode discharge voltage was 4.0-4.4 kV. Under a high-density current, the aluminum wire was heated and radially expanded to be vaporized to form aluminum vapor, and then condensed to form aluminum particles with an average particle size of 139.7642.81 nm, then aluminum particles were collected in an argon-filled collector. (See document: Passivation Process for Superfine Aluminum Powders Obtained by Electrical Explosion of Wires. Appl. Sur. Sci. 2003, 211, 57-67.)
Embodiment 2 Preparation of Metal Aluminum Nanoparticles by Ligand Regulation Method
(20) (1) Synthesis and Characterization of Precursor H.sub.3Al (1-MP)
(21) In a glove box (both oxygen and water content were lower than 1 ppm), 3.748 g of lithium aluminum hydride and 4.132 g of aluminum chloride were added to a flask containing 45 mL of anhydrous toluene. Under strong stirring (the stirring speed was 800 rpm), 11.65 mL of 1-methylpyrrolidine was added dropwise. Wherein, the molar ratio of lithium aluminum hydride, aluminum chloride, and 1-methylpyrrolidine was 3:1:1. After the reaction was carried out overnight at room temperature, the reaction mixture was filtered with a funnel to remove solid impurities. For further purification, the obtained filtrate was filtered again with an organic phase filter membrane with a pore size of 0.22 m, and the obtained filtrate was a toluene solution of H.sub.3Al (1-MP) (structural formula was
(22) ##STR00014##
the yield was 90%), and the solution was then stored in a glove box refrigerator at low temperature; the storage temperature was 10 C. The concentration of the solution can be calibrated by 1H nuclear magnetic resonance spectrum.
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(25) (2) Synthesis and characterization of ligand (2-phenyl-2-propyl benzodithioate)-terminated polystyrene (CDTB-PS)
(26) 90.90 g of styrene, 0.0576 g of 2,2-azobis(2-methylpropionitrile), and 0.4107 g of 2-phenyl-2-propyl benzodithioate (CDTB) were added to the Schelenk flask. After three times of liquid nitrogen freezing-vacuumizing-melting treatment, the mixed solution was reacted in an oil bath at 60 C. for 12 hours under stirring, and then the reaction solution was cooled to room temperature. Then, most of the unreacted styrene was removed with a rotary evaporator. Finally, the reaction solution was washed by sedimentation with methanol-ultrasonic centrifugation three times to remove the reactant. The product was placed in a vacuum oven at 120 C. for 1 day, and the final solid product was stored in a glove box refrigerator at low temperature. The molecular weight (Mn) of the product was about 4.5 kg/mol, and the dispersion index (PDI) was 1.09. 7.64 g of the product was obtained with a yield of 8.5%. The structure of the product was as follows:
(27) ##STR00015##
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(29)
(3) Synthesis and Characterization of Aluminum Nanoparticles
(30) Preparation of the Solution:
(31) An anhydrous THF solution of CDTB-PS with a concentration of 20 mM was prepared, and a little excess CDTB-PS was added roughly according to the molecular weight, and the accurate concentration was calibrated by ultraviolet quantitative method.
(32) An anhydrous THF solution of H.sub.3Al (1-MP) with a concentration of 1M was prepared. The specific preparation method was as follows: the concentration of H.sub.3Al (1-MP) solution (usually greater than 1M) was determined according to the integral area relationship of specific peak of liquid .sup.1H NMR. By calculation, the anhydrous THF solution of H.sub.3Al(1-MP) with a concentration of 1M was obtained by adding a specific volume of THF.
(33) An anhydrous THF solution of Ti(i-PrO).sub.4 with a concentration of 100 mM was prepared. The mass of Ti(i-PrO).sub.4 was weighed with a balance and a specific volume of THF was added.
(34) In a glove box (both oxygen and water content were lower than 1 ppm), 75 L of a solution of 20 mM CDTB-PS in anhydrous THF was added to 4.425 mL of anhydrous tetrahydrofuran (THF), and the reaction solution was heated and stabilizing to 50 C. Under strong stirring (500 rpm), 400 L of a solution of 1M H.sub.3Al(1-MP) in anhydrous THF and 100 L of a solution of 10 mM Ti(i-PrO).sub.4 in anhydrous THF were added successively. Wherein, the molar ratio of ligand, precursor and Ti(i-PrO).sub.4 was 3:800:2; after the feeding was completed, in the whole reaction solution (solvent 5.0 mL), the concentration of H.sub.3Al(1-MP) precursor was 80 mM, the concentration of titanium catalyst was 0.2 mM. The reaction was carried out at 50 C. under strong stirring (500 rpm), and the reaction solution was cooled to room temperature at 45 minutes, 1.5 hours, and 4 hours, respectively; the reaction solution was centrifuged at 8000 rpm for 10 minutes. The supernatant was removed, and an equal amount of anhydrous THF was added, then the precipitate was washed by shaking. After repeating three times, aluminum nanoparticles with good monodispersity with a size of 88.85 nm8.86 nm and a dispersion coefficient of 0.10; a size of 147.14 nm11.95 nm and a dispersion coefficient of 0.09; a size of 287.82 nm24.13 nm and a dispersion coefficient of 0.08 were obtained respectively.
Embodiment 3
(35) The vaccine AlNPs-OVA was prepared by mixing metal aluminum nanoparticles obtained by electric explosion with OVA. Specific steps were as follows: (1) The metal aluminum nanoparticles obtained by electric explosion and prepared in Embodiment 1 were prepared into a solution of 0.1-5.0 mg/mL (using DMF as solvent), and the concentration of the metal aluminum nanoparticles in the present embodiment was 2.0 mg/mL, and ultrasonic dispersion was performed for 20 minutes. (2) Ovalbumin OVA PBS solution with a concentration of 0.1-5.0 mg/mL was prepared. In the present embodiment, the concentration of ovalbumin OVA was 3.0 mg/mL. (3) A PBS solution of ovalbumin OVA and a DMF solution of aluminum nanoparticles were mixed according to the volume of 1:1, after ultrasonic dispersion for half an hour, the reaction solution was transferred to a shaker, and shaked at room temperature at 320 rpm for 12 hours. After stopping shaking, the reaction solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed and the samples was lyophilized to remove the remaining small amount of solvent.
Embodiment 4
(36) The vaccine 287.82 nm Al-OVA was prepared by mixing 287.82 nm metal aluminum nanoparticles with OVA. Specific steps were as follows:
(37) The aluminum particles with a size of 287.82 nm24.13 nm and a dispersion coefficient of 0.08 prepared in Embodiment 2 were used, and the remaining steps were the same as those in Embodiment 3.
Embodiment 5
(38) The vaccine 147.14 nm Al-OVA was prepared by mixing 147.14 nm metal aluminum nanoparticles with OVA. Specific steps were as follows:
(39) The aluminum particles with a size of 147.14 nm11.95 nm and a dispersion coefficient of 0.09 prepared in Embodiment 2 were used, and the remaining steps were the same as those in Embodiment 3.
Embodiment 6
(40) The vaccine 88.85 nm Al-OVA was prepared by mixing 88.85 nm metal aluminum nanoparticles with OVA. Specific steps were as follows:
(41) The aluminum particles with a size of 88.85 nm8.86 nm and a dispersion coefficient of 0.10 prepared in Embodiment 2 were used, and the remaining steps were the same as those in Embodiment 3.
Embodiment 7
(42) The vaccine 147.14 nm Al-OT-1 was prepared by mixing 147.14 nm metal aluminum nanoparticles with short peptide OT-1. Specific steps were as follows: (1) The metal aluminum nanoparticles with a size of 147.14 nm11.95 nm and a dispersion coefficient of 0.09 prepared in Embodiment 2 were prepared into a solution of 0.1-5.0 mg/mL (using DMF as solvent), and the concentration of the metal aluminum nanoparticles in the present embodiment was 2.0 mg/mL, and ultrasonic dispersion was performed for 20 minutes. (2) 0.1-5.0 mg/mL DMF solution of OT-1 was prepared. In the present embodiment, the concentration of OT-1 was 2.0 mg/mL. (3) The OT-1 solution and the metal aluminum nanoparticle solution were mixed according to the volume of 1:1, after ultrasonic dispersion for half an hour, the reaction solution was transferred to a shaker, and shaked at room temperature at 320 rpm for 12 hours. After stopping shaking, the reaction solution was centrifuged at 6000 rpm/min for 10 minutes, and the supernatant was removed and the samples was lyophilized to remove the remaining small amount of solvent.
Embodiment 8
(43) The vaccine 147.14 nm Al-Tert was prepared by mixing 147.14 nm metal aluminum nanoparticles with polypeptide Tert (DI-27).
(44) Except the following conditions, other operations and conditions in the present embodiment were the same as those in Embodiment 7:
(45) 2.0 mg/mL DMF solution of Tert (DI-27) was prepared and mixed with the metal aluminum nanoparticle solution.
Comparative Embodiment 1
(46) A vaccine (nano Al(OH).sub.3-OVA) was prepared by mixing aluminum hydroxide nanoparticles with OVA.
(47) The preparation method of aluminum hydroxide nanoparticles was as follows: in a 20 mL small white flask, 5 mL of 3.6 mg/mL aluminum chloride hexahydrate AlCl.sub.3.6H.sub.2O and 5 mL of 0.04 M NaOH solution were added successively, and then the pH of the reaction solution was adjusted with 0.01 M NaOH to 7. After the mixture was stirred at room temperature for 20 minutes, the mixture was centrifuged at 8000 rpm to remove the supernatant. After the residue was washed twice with ultrapure water, dried under reduced pressure, and DMF was added to prepare a solution with a concentration of 2.0 mg/mL. The average particle size measured by TEM was 150.3532.84 nm.
(48) The preparation method of the vaccine nano Al(OH).sub.3-OVA was as follows: the aluminum hydroxide nanoparticles were mixed with OVA to prepare the vaccine, and other conditions were the same as those in Embodiment 3.
Comparative Embodiment 2
(49) The vaccine (Al(OH).sub.3-OVA) was prepared by mixing commercial aluminum hydroxide gel adjuvant with OVA.
(50) Specifically: the vaccine was prepared by mixing aluminum hydroxide gel adjuvant (purchased from Thermo Scientific, particle size was 4-10 m) with OVA, and other conditions were the same as those in Embodiment 3.
Effect Embodiment 1 Humoral Immune Response
(51) The lyophilized vaccines prepared in Embodiment 3, Comparative Embodiment 1, and Comparative Embodiment 2 were taken and their activities of stimulating humoral immune response were detected.
(52) Experimental Conditions:
(53) 1. Animal Immunity:
(54) 7-week-old female C.sub.57BL/6 mice were randomly divided into 5 groups with 3 mice in each group. Taking OVA as the antigen, the groups were 2.5 mg/mL OVA group, 10 mg/mL Al(OH).sub.3-OVA group, 10 mg/mL nano Al(OH).sub.3-OVA group, 10 mg/mL AlNPs-OVA group and control PBS group, respectively. The mice were immunized by subcutaneous injection of 100 L each in the right inguinal area on the 0 th day and the 7 th day, respectively. On the 14 th day, the eyeballs were removed and serum was collected for IgG1 and IgG2a detection.
(55) 2. Preparation of Mouse Serum:
(56) 1) Removing the eyeball for blood collection: (1) the ears and the skin of the back of the neck of the mouse were grabbed with the thumb and index finger of the left hand, and the tail was fixed with the little finger; (2) the left forelimb of the mouse was pressed on the heart of the sternum with the middle finger, and the abdomen was pressed by the ring finger, then the thumb was twisted, and the eye skin on the blood side was gently pressed to make the eyeball congested and protrude; (3) the eyeball was taken with an elbow tweezer; (4) the thumb and index finger were twisted as needed to make the blood flow vertically into the centrifuge tube from the orbit at different speeds; (5) the left middle finger was simultaneously used to gently press the mouse heart to speed up the pumping speed of the heart; (6). when the blood was exhausted, the mouse was killed by dislocation.
(57) 2) Separation of serum: (1) the blood in the centrifuge tube was put at room temperature for 2 hours; (2) the blood was stored in a refrigerator at 4 C. for 3 hours; (3) after the blood had clotted and the clot had contracted, the mixture was centrifuged at 4000 rpm for 10 minutes; (4) the supernatant was taken in a clean EP tube and stored in a 80 C. refrigerator for later use.
(58) 3. Detection of Specific Antibody Subtype ELISA:
(59) Referred to Mouse Anti-Ovalbumin IgG2a ELISA Kit, 96 tests, Quantitative Kit and Mouse Anti-Ovalbumin IgG1 ELISA Kit, 96 tests, Quantitative Kit produced by Alpha Diagnostic International, and detection was performed according to their instructions.
(60) The specific experimental results could be found in Table 1,
(61) TABLE-US-00001 TABLE 1 Th2 antibody subtype Th1 antibody subtype Group Ig G1 titer (pg/mL) Ig G2a titer (pg/mL) PBS 20.062 2.73 ** 10.062 2.73 ** OVA 669.9742 84.47 ** 119.9742 13.76 * Al(OH).sub.3-OVA 987.2912 123.58 * 287.2912 17.84 * Nano Al(OH).sub.3-OVA 1107.307 77.11 * 332.8065 28.25 * AlNPs-OVA 1556.6 83.54 566.5995 69.39.sup. Note: * P < 0.05, ** P < 0.01, there were significant differences between the AlNPs-OVA group and the other four groups.
(62) It can be seen from Table 1,
(63) the ability of AINPs-OVA as a vaccine to induce the production of Th2 antibody subtype IgG1 and Th1 antibody subtype IgG2a is significantly higher than that of the other four groups.
Effect Embodiment 2 Cellular Immune Response
(64) The lyophilized vaccines prepared in Embodiment 3, Comparative Embodiment 1, and Comparative Embodiment 2 were taken and their activities of stimulating cellular immune response were detected.
(65) Experimental Conditions:
(66) 1. Animal Immunity:
(67) 7-week-old female C.sub.57BL/6 mice were randomly divided into 5 groups with 2 mice in each group. Taking OVA as the antigen, the groups were 2.5 mg/mL OVA group, 10 mg/mL Al(OH).sub.3-OVA group, 10 mg/mL nano Al(OH).sub.3-OVA group, 10 mg/mL AlNPs-OVA group and control PBS group. The mice were immunized by subcutaneous injection of 100 L each in the right inguinal area on the 0 th day and the 7 th day, respectively. On the 14 th day, the mice were sacrificed by cervical dislocation, and the spleen and draining lymph nodes were collected for flow cytometry.
(68) 2. Preparation of Mouse Spleen Cells
(69) 1) The mice were sacrificed by cervical dislocation, and the spleen was taken into a 60 mm dish containing 5 mL of PBS, then the spleen was ground to disperse cells, then filtered into a 15 mL centrifuge tube through a 200 mesh filter, then centrifuged at 1200 rpm at 4 C. for 5 min, and the supernatant was discarded;
(70) 2) 1 mL of red blood cell lysate was added, and the mixture was stood at 4 C. for 15 min and shaked every 5 min, then 10 mL of PBS was added to terminate, then the mixture was centrifuged at 1200 rpm at 4 C. for 5 min; the supernatant was discarded, and the spleen cells were resuspended in 1 mL of FACS and counted.
(71) 3. Preparation of Draining Lymph Nodes of Mice
(72) Mice were sacrificed by cervical dislocation, and the right inguinal lymph nodes were taken into a 60 mm dish containing 5 mL of PBS, grounded to disperse cells, then filtered into a 15 mL centrifuge tube through a 200 mesh filter, then centrifuged at 1200 rpm at 4 C. for 5 min, and the supernatant was discarded, then lymph node cells were resuspended in 1 mL of FACS (FACS referred to a PBS solution containing 0.1% bovine serum albumin) and counted.
(73) 4. Detection of Antigen-Specific T Lymph Node Cells by Flow Cytometry
(74) 110.sup.6 spleen/lymph node cells were taken into a flow tube, and CD45-Pacific Blue, CD3-PE-Dazzle-594, CD4-BV421, CD8-PE-Cy5, CD19-BV650, CD11c-APC-Cy7, CD11b-BV711, Anti-SIINFEKL-H-2kb-PE were flow antibody stained (the above antibodies were purchased from BD Company (Becton, Dickinson and Company) in the United States), stained at 4 C. for 30 min in the dark, and 3 mL of FACS (FACS referred to PBS solution containing 0.1% bovine serum albumin) was added, then the mixture was centrifuged at 1200 rpm for 5 min at 4 C.; the supernatant was discarded, and the cells were vortexed to resuspend, and detected by Cytek Aurora flow analyzer.
(75) The specific experimental results could be found in Table 2-3,
(76) TABLE-US-00002 TABLE 2 Counts of spleen cells and draining lymph node cells in mice Draining lymph node cells/ Spleen cells/ Group 1 10.sup.6 cells 1 10.sup.6 cells PBS 1.85 0.28 74.00 9.19 OVA 2.76 0.83 69.5 10.61 Al(OH).sub.3-OVA 8.61 2.25 151.75 34.29 Nano Al(OH).sub.3-OVA 1.70 0.78 133.5 45.25 AlNPs-OVA 13.65 2.65 109.75 8.84
(77) It can be seen from Table 2 that the ability of AlNPs-OVA as a vaccine to induce the production of spleen cells and draining lymph node cells is significantly higher than that of the other four groups.
(78) TABLE-US-00003 TABLE 3 Percentage of antigen-specific T cells in draining lymph node cells and spleen cells of mice Percentage of Percentage of antigen-specific antigen-specific T cells in draining T cells in Group lymph node cells/% spleen cells/% PBS 0 * 0.009 0 **.sup. OVA 0.3 0.03 * 0.075 0.02 * Al(OH).sub.3-OVA 0.225 0.09 * 0.145 0.06 * Nano Al(OH).sub.3-OVA 1.325 0.28 * 0.31 0.03 * AlNPs-OVA 5.21 1.17 0.559 0.07.sup. Note: * P < 0.05, ** P < 0.01, there were significant differences between the AlNPs-OVA group and the other four groups.
(79) It can be seen from Table 3,
Effect Embodiment 3
(80) The lyophilized vaccines prepared in Embodiment 4 and Embodiment 5 were taken and their activities of stimulating immune response were detected.
(81) Experimental Conditions:
(82) Female C.sub.57BL/6 mice, 8-week-old, were purchased from Beijing Vital River Company;
(83) the mice were randomly divided into 5 groups with 4 mice in each group, and the groups were PBS group, OVA protein group (160 g OVA/mouse), metal aluminum nanoparticle group (1 mg/mouse, wherein, 1 mg of metal aluminum nanoparticles were composed of 0.5 mg 287.82 nm Al and 0.5 mg 147.14 nm Al), 287.82 nm Al-OVA group (1 mg Al/160 g OVA/mouse) and 147.14 nm Al-OVA group (1 mg Al/160 g OVA/mouse), respectively.
(84) On the 0 th and 7 th day, mice were immunized by intradermal injection on the left back, 100 L/mouse/time.
(85) On the 14 th day, 5'10.sup.5 B16F10 cells/mouse were subcutaneously inoculated on the right back of the mice.
(86) The tumor was measured every 2 days from the 21 st day, and the tumor volume=lengthwidthwidth/2.
(87) The specific experimental results could be seen in Table 4 and
(88) TABLE-US-00004 TABLE 4 Days after tumor Tumor Group inoculation/d volume/mm.sup.3 PBS 26 974.15 351.57 OVA (Free-OVA) 26 886.91 431.86 Metal aluminum nanoparticles 26 551.36 481.76 (Free-Al) 287.82 nm Al-OVA 26 44.71 18.16* 147.14 nm Al-OVA 26 21.63 15.58* Note: *P < 0.05, there were significant difference between the 147.14 nm Al-OVA group, the PBS group and the Free-Al group, and there were significant difference between the 287.82 nm Al-OVA group, the PBS group and the Free-Al group.
(89) It can be seen from Table 4 and
Effect Embodiment 4
(90) The lyophilized vaccines prepared in Embodiment 5 and Comparative Embodiment 2 were taken and their activities of stimulating immune response were detected.
(91) Experimental conditions:
(92) Female C.sub.57BL/6 mice, 8-week-old, were purchased from Beijing Vital River Company;
(93) on the 0 th day, 110.sup.6 B16F10 cells/mouse were subcutaneously inoculated on the right back of the mice.
(94) The mice were randomly divided into 4 groups with 2 mice in each group, and the groups were PBS group, OVA protein group (100 g OVA/mouse), commercial aluminum hydroxide gel group (1.332 mg Al (OH).sub.3/100 g OVA/mouse) and 147.14 nm Al-OVA group (0.23 mg Al/100 g OVA/mouse), respectively.
(95) On the 7th day, the mice were immunized by subcutaneous injection on the right groin, once every 3 days, a total of 6 times, 100 L/mouse/time.
(96) The tumor was measured every 2 days from the 7 th day, and the tumor volume=lengthwidthwidth/2.
(97) The specific experimental results could be seen in Table 5 and
(98) TABLE-US-00005 TABLE 5 Days after tumor Tumor Group inoculation/d volume/mm.sup.3 PBS 16 727.47 132.65 OVA (Free-OVA) 16 467.39 88.51 Al(OH).sub.3-OVA 16 144.50 31.94** 147.14 nm Al-OVA 16 37.75 16.91** Note: **P < 0.01, there were significant difference between the 147.14 nm Al-OVA group, the PBS group and the Free-OVA group, and there were significant difference between the Al(OH).sub.3-OVA group and the PBS group.
(99) It can be seen from Table 5 and
Effect Embodiment 5
(100) The lyophilized vaccines prepared in Embodiment 7 and Comparative Embodiment 2 were taken and their activities of stimulating immune response were detected.
(101) Experimental Conditions:
(102) Female C57BL/6 mice, 8-week-old, were purchased from Beijing Vital River Company;
(103) The mice were randomly divided into 5 groups with 3 mice in each group, and the groups were PBS group, 147.14 nm Al group (0.8 mg/mouse), OT1 short peptide group (50 g OT1/mouse), and commercial aluminum hydroxide gel group (0.8 mg Al (OH).sub.3/50 g OT1/mouse), 147.14 nm Al-OVA group (0.8 mg Al/50 g OVA/mouse).
(104) On the 0 th and 7 th day, mice were immunized by subcutaneous injection in the right groin, 100 L/mouse/time.
(105) On the 14 th day, 110.sup.6 B16F10 cells/mouse were subcutaneously inoculated on the right back of the mice.
(106) The tumor was measured every 2 days from the 25 th day, and the tumor volume=lengthwidthwidth/2.
(107) The specific experimental results could be seen in Table 6 and
(108) TABLE-US-00006 TABLE 6 Days after tumor Tumor Group inoculation/d volume/mm.sup.3 PBS 15 1106.10 316.53 OT1 (Free- OT1) 15 719.39 129.69* Metal aluminum nanoparticles 15 634.86 114.94 (Free-Al) Al(OH).sub.3-OT1 15 603.98 154.92* 147.14 nm Al-OT1 15 255.50 73.32*** Note: *P < 0.05, ***P < 0.001, there were significant difference between the 147.14 nm Al-OT1 group, the PBS group and the Free-Al group, and there were significant difference between the Al(OH).sub.3-OT1 group and the PBS group, and there were significant difference between the OT1 (Free-OT1) group and the PBS group.
(109) It can be seen from Table 6 and
Effect Embodiment 6
(110) The lyophilized vaccine prepared in Embodiment 8 was taken and its activity of stimulating immune response was detected.
(111) Experimental Conditions:
(112) Female C.sub.57BL/6 mice, 8-week-old, were purchased from Beijing Vital River Company;
(113) the mice were randomly divided into 4 groups with 3 mice in each group, and the groups were PBS group, 147.14 nm Al group (2.5 mg Al/mouse), Tert (DI-27) polypeptide group (250 g Tert (DI-27)/mouse) and 147.14 nm Al-Tert (DI-27) group (2.5 mg Al/250 g Tert (DI-27)/mouse).
(114) On the 0 th and 7 th day, mice were immunized by subcutaneous injection in the right groin, 100 L/mouse/time.
(115) On the 14 th day, 510.sup.5 B16F10 cells/mouse were subcutaneously inoculated on the right back of the mice.
(116) The tumor was measured every 2 days from the 21 st day, and the tumor volume=lengthwidthwidth/2.
(117) The specific experimental results could be seen in Table 7 and
(118) TABLE-US-00007 TABLE 7 Days after tumor Tumor Group inoculation/d volume/mm.sup.3 PBS 21 748.43 159.59 Metal aluminum nanoparticles 21 722.58 97.72 (Free-Al) Tert (Free-Tert) 21 604.92 167.18 147.14 nm Al-Tert 21 134.49 38.63** Note: **P < 0.01, there were significant difference between the 147.14 nm Al-Tert group, the PBS group, the Free-Al group and the Tert (Free-Tert) group.
(119) It can be seen from Table 7 and