A composite water-soluble vaccine adjuvant and a preparation method thereof

Abstract

The application discloses a kind of composite water-soluble vaccine adjuvant and preparation method thereof, belong to biological medicine technical field.The adjuvant is based on the composite technology of water-soluble component and targeted delivery carrier, through the dual targeting effect of mannose modified beta-glucan, the uptake efficiency of dendritic cells to antigen is significantly improved, and the regulation of immune response is realized by synergistic levamisole.The adjuvant has the characteristics of uniform particle size, excellent water solubility, good storage stability, etc., and the immune enhancement effect is significantly better than traditional adjuvant, and can be widely used in the research and development of influenza, tuberculosis and tumor vaccines, has important clinical value and industrialization potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a composite water-soluble vaccine adjuvant and its preparation method. Background Technology

[0002] Vaccine adjuvants are key substances that enhance the body's specific immune response to antigens, and their performance directly determines the protective efficacy of vaccines. Traditional water-soluble adjuvants have attracted much attention due to their good biocompatibility, ease of large-scale production, and quality control.

[0003] β-glucan, as a natural immunomodulator, effectively initiates an immune response by activating the Dectin-1 receptor on the surface of dendritic cells. Existing technology CN111467488A discloses a water-soluble composite adjuvant that utilizes multiple stabilizers such as trehalose, raffinose, and glucan for synergistic effects, primarily designed for foot-and-mouth disease inactivated vaccines. It has shown some immunogenicity in freeze-dried vaccines against a few bacteria, including Brucella, Clostridium perfringens, and Streptococcus suis. However, research on its mechanism of action in synergistically activating immune pathways among its components is still insufficient, hindering subsequent formulation optimization and upgrades. Existing technology CN102600467A utilizes insoluble β-glucan particles loaded with antigenic peptides, which, while possessing some immunomodulatory capabilities, struggles to cover all cellular antigens, limiting its application effectiveness. Therefore, the development of a novel water-soluble vaccine adjuvant that combines physical stability, active targeting capability, and multi-pathway synergistic immune activation efficacy has become an urgent direction to meet current clinical needs in vaccine development. Summary of the Invention

[0004] This addresses the technical problems of existing water-soluble vaccine adjuvants, such as lack of active targeting ability, limited activation effect on a single immune pathway, and insufficient physical stability.

[0005] This invention provides a composite water-soluble vaccine adjuvant and its preparation method, which achieves simultaneous improvement in immunogenicity, targeting and stability through chemical modification, targeted modification and multi-component synergistic design.

[0006] To achieve the above objectives, the present invention provides a method for preparing a composite water-soluble vaccine adjuvant, as detailed below:

[0007] S1, Preparation of Base Fluid

[0008] Add 5-20g trehalose, 5-20g raffinose, 5-20g β-glucan / modified β-glucan, 1-10g xylitol, 1-10g polyethylene glycol, and 0.1-5g levamisole to 800-1000mL of phosphate buffer solution, and stir at 200-500rpm for 5-20min; let stand at 0-4℃ for later use within 8-24 hours after preparation.

[0009] S2, Preparation of the delivery carrier

[0010] S2.1 Preparation of Single-Component Solutions

[0011] Chitosan solution: Add 0.1-0.5g of chitosan to 5-10mL of 0.1mol / L hydrochloric acid, stir at 200-500rpm for 0.5-3h at room temperature, filter and store at 4℃.

[0012] Polyvinyl alcohol solution: Take 0.5-3g of polyvinyl alcohol and add 5-10mL of water. Stir in a water bath at 50-80℃ at 200-500rpm for 1-5 hours. After cooling to room temperature, filter to obtain the solution. Store in a sealed container at room temperature.

[0013] Preparation of S2.2 Composite Polymer Solution

[0014] Take the chitosan solution and polyvinyl alcohol solution prepared in S2.1 above, add 0.05-1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200-500 rpm for 1-5 h at room temperature; then continue stirring in a water bath at 50-80℃ for 1-5 h, and cool to room temperature to obtain the final product; seal and store at 4℃ for later use.

[0015] S3. Preparation of compound water-soluble vaccine adjuvant

[0016] Take 800-1000 mL of the base solution prepared in step S1 and 5-10 mL of the composite polymer solution, stir at 200-500 rpm for 0.5-3 h at room temperature, and then filter to obtain the final product.

[0017] The modified β-glucan is any one of octenyl succinic anhydride esterification modification, internal cyclization grafting modification, carboxymethylation modification, or mannose targeted modification.

[0018] The phosphate buffer solution has a concentration of 0.02 mol / L and a pH of 7.4.

[0019] The modified β-glucan described above is octenyl succinic anhydride esterified modification, and its preparation method is as follows:

[0020] Take 0.1-1g of β-glucan and add it to 100-200mL of water. Stir at 50-80℃ for 1-2h. After cooling to room temperature, centrifuge at 3000-5000rpm for 5-10min and collect the supernatant. Add 0.1-0.6mL of 2-octenylsuccinic anhydride and react at 50-60℃ for 1-5h. After cooling to room temperature, adjust the pH and continue stirring for 6-12h. After alcohol precipitation, centrifuge at 3000-5000rpm for 5-10min, collect the precipitate, dissolve it in water, and obtain the final product after water dialyze and vacuum freeze-drying.

[0021] The modified β-glucan described is an internal cyclization graft modified β-glucan, and its preparation method is as follows: Take 0.1-1g of β-glucan and add it to 100-200mL of water, stir at 50-80℃ for 1-2h; after cooling to room temperature, centrifuge at 3000-5000rpm for 5-10min, collect the supernatant β-glucan solution; then add 0.1-0.6mL of 2-octenylsuccinic anhydride and react at 50-60℃ for 1-5h; after cooling to room temperature, adjust the pH and continue stirring for 6-12h; after alcohol precipitation, centrifuge at 3000-5000rpm for 5-10min, collect the precipitate and dissolve it in water, dialyze it with water for later use, and obtain the prepared dialysate; then take 0.1-3g of β-glucan... 4-Amino-1-butanol and 0.1-3 g hexanediol diacrylate are dissolved in 0.1-5 g dimethyl sulfoxide and stirred at 50-90 °C and 200-500 rpm for 6-24 h under a nitrogen atmosphere; then mixed with 80-100 mL of the prepared dialysate, and after adjusting the pH, reacted at 30-80 °C for 1-6 h; the reaction solution is then dialyzed with water and freeze-dried under vacuum to obtain the final product.

[0022] The modified β-glucan is carboxymethylated and is prepared as follows: 0.1-1g of β-glucan is added to 30-100mL of sodium hydroxide aqueous solution and stirred at 200-500rpm for 1-2h at room temperature; then cooled to 0-4℃, 5-10mL of chloroacetic acid aqueous solution is added, the pH of the system is maintained, and the temperature is adjusted to 30-80℃ for 1-5h; after adjusting the pH, alcohol precipitation is performed at room temperature, the precipitate is collected and dissolved in water, and then obtained by water dialyze and vacuum freeze-drying.

[0023] The modified β-glucan described is a mannose-targeted modification, and its preparation method is as follows: 0.1-1 g of β-glucan is placed in 100-200 mL of water and stirred at 50-80℃ for 1-2 h; after cooling to room temperature, it is centrifuged at 3000-5000 rpm for 5-10 min, and the supernatant is collected; then 0.1-0.6 mL of 2-octenylsuccinic anhydride is added, and the mixture is reacted at 50-60℃ for 1-5 h; after cooling to room temperature, the pH is adjusted, and stirring is continued for 6-12 h; after alcohol precipitation, it is centrifuged at 3000-5000 rpm for 5-10 min, and the precipitate is collected and dissolved in water. After water dialysis and vacuum freeze-drying, it is dissolved in 10-30 mL of 2-morpholine ethanesulfonic acid buffer, and 0.1-1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-1 g of... N-hydroxysuccinimide was stirred at room temperature in the dark for 1-2 hours; 0.1-1 g of p-aminophenyl-α-D-mannoside was added, the pH was adjusted, and the mixture was stirred at room temperature in the dark for 4 hours; the product was obtained after water dialysis and vacuum freeze-drying.

[0024] The 2-morpholine ethanesulfonic acid buffer solution is a morpholine ethanesulfonic acid buffer solution with a pH of 6.0.

[0025] The alcohol precipitation process is as follows: add 1-4 times the amount of isopropanol to the reaction system and precipitate at room temperature for 6-12 hours.

[0026] The dialysis described above uses water as the dialysate, has a molecular weight cutoff of 14,000 Da, and a dialysis time of 24-48 hours, with the water being changed every 6 hours.

[0027] The vacuum freeze-drying conditions are set as follows: temperature -60 to -20℃, vacuum degree 80-100Pa, and drying time 6-12h.

[0028] A composite water-soluble vaccine adjuvant is prepared using the method described above.

[0029] The beneficial effects of this invention are:

[0030] 1. This invention uses a multi-element stabilizer system. Through the synergistic effect of multi-element stabilizers such as trehalose, raffinose, and xylitol with β-glucan / modified β-glucan, it has good physical stability, which is beneficial for long-term storage and transportation in subsequent vaccine research and development.

[0031] 2. This invention, through specific modification of β-glucan and mannoside, endows the adjuvant with the ability to actively target dendritic cells; and with the synergistic effect of levamisole, it achieves precise regulation of Th1 / Th2 response. It can be selected and combined according to the vaccine type that emphasizes humoral immunity or cellular immunity, thereby adapting to the actual needs of different vaccines.

[0032] 3. The modified β-glucan involved in this invention serves as a molecular backbone, interacting with chitosan and polyvinyl alcohol polymer networks through multi-point hydrogen bonds and electrostatic interactions. Together with the carrier network, they construct a multi-level stable structure. This structure helps maintain the nanoparticle size and good dispersibility of the adjuvant system, thereby promoting the synergistic enrichment and efficient antigen presentation of antigens or adjuvants, and potentially reducing the risk of non-targeted exposure. Furthermore, the preparation method of this invention is simple, mild, and easy to control, making it highly suitable for large-scale production and applicable to the development of various vaccines, including novel vaccines for influenza and tuberculosis. Detailed Implementation

[0033] The parameters and sources of some substances in the examples are as follows:

[0034] β-glucan: CAS No.: 9041-22-9; sourced from MedChemExpress; catalog number: HY-139413.

[0035] Polyethylene glycol, CAS No.: 25322-68-3; molecular weight: 200-1000 Da; sourced from Xi'an Xinfengda.

[0036] Chitosan: Deacetylation degree ≥90%, molecular weight 100kDa.

[0037] Levamisole: CAS: 16595-80-5; sourced from Qihui Pharmaceutical.

[0038] Polyvinyl alcohol: Model: PVA1788; Pharmaceutical grade; Brand: Jinyang.

[0039] 2-Octenylsuccinic anhydride: CAS No.: 26680-54-6; pharmaceutical grade.

[0040] 4-Amino-1-Butanol: CAS No.: 13325-10-5; Pharmaceutical grade.

[0041] Hexanediol diacrylate: CAS No.: 13048-33-4.

[0042] p-Aminophenyl-α-D-mannoside: CAS No.: 34213-86-0; Code: S46164; Derived from source leaf.

[0043] Example 1

[0044] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0045] S1, Preparation of Base Fluid

[0046] Add 12g trehalose, 10g raffinose, 10g β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0047] S2, Preparation of the delivery carrier

[0048] S2.1 Preparation of Single-Component Solutions

[0049] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0050] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0051] Preparation of S2.2 Composite Polymer Solution

[0052] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0053] S3. Preparation of compound water-soluble vaccine adjuvant

[0054] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0055] Example 2

[0056] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0057] S1, Preparation of Base Fluid

[0058] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0059] S2, Preparation of the delivery carrier

[0060] S2.1 Preparation of Single-Component Solutions

[0061] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0062] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0063] Preparation of S2.2 Composite Polymer Solution

[0064] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0065] S3. Preparation of compound water-soluble vaccine adjuvant

[0066] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0067] The preparation method of the modified β-glucan is as follows:

[0068] 0.5 g of β-glucan was added to 200 mL of water and stirred at 80 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant β-glucan solution was collected. 0.35 mL of 2-octenyl succinic anhydride was added, and the mixture was reacted at 50 °C for 4 h. After cooling to room temperature, the pH was adjusted to 8.5 with 3 wt% sodium hydroxide aqueous solution, and stirring was continued for 12 h. Isopropanol, four times the volume of the reaction system, was added, and the mixture was precipitated at room temperature for 12 h. The mixture was then centrifuged at 3000 rpm for 10 min, and the precipitate was collected and dissolved in 150 mL of water. The solution was then dialyzed with water for 48 h using a 14000 Da dialysis bag. The dialyzed solution was dried at -60 °C and 100 Pa vacuum for 6 h to obtain modified β-glucan.

[0069] Example 3

[0070] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0071] S1, Preparation of Base Fluid

[0072] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0073] S2, Preparation of the delivery carrier

[0074] S2.1 Preparation of Single-Component Solutions

[0075] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0076] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0077] Preparation of S2.2 Composite Polymer Solution

[0078] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0079] S3. Preparation of compound water-soluble vaccine adjuvant

[0080] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0081] The preparation method of the modified β-glucan is as follows:

[0082] 0.5 g of β-glucan was added to 200 mL of water and stirred at 80 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant β-glucan solution was collected. 0.35 mL of 2-octenyl succinic anhydride was added, and the mixture was reacted at 50 °C for 4 h. After cooling to room temperature, the pH was adjusted to 8.5 with 3 wt% sodium hydroxide aqueous solution, and stirring was continued for 12 h. Isopropanol, four times the volume of the reaction system, was added, and the mixture was precipitated at room temperature for 12 h. Subsequently, the mixture was centrifuged at 3000 rpm for 10 min, and the precipitate was collected and dissolved in 150 mL of water. The solution was then dialyzed with water for 48 h using a 14000 Da dialysis bag. The dialysate was then prepared for use.

[0083] Dissolve 0.9 g of 4-amino-1-butanol and 1.48 g of hexanediol diacrylate in 2.38 g of dimethyl sulfoxide, and stir at 70 °C and 200 rpm for 12 h under a nitrogen atmosphere. Then mix with 100 mL of the prepared 0.5 wt% dialysis solution, add 2 mol / L hydrochloric acid to adjust the pH to 7.5, and react at 50 °C for 6 h. Transfer the reaction solution to a dialysis bag with a molecular weight cutoff of 14000 Da and dialyze with water for 48 h. Dry at -60 °C and 100 Pa for 24 h to obtain the final product.

[0084] Example 4

[0085] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0086] S1, Preparation of Base Fluid

[0087] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0088] S2, Preparation of the delivery carrier

[0089] S2.1 Preparation of Single-Component Solutions

[0090] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0091] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0092] Preparation of S2.2 Composite Polymer Solution

[0093] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0094] S3. Preparation of compound water-soluble vaccine adjuvant

[0095] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0096] The preparation method of the modified β-glucan is as follows:

[0097] Add 5g of β-glucan to 50mL of 20wt% sodium hydroxide aqueous solution and stir at 200rpm for 2h at room temperature; then cool to 4℃ in an ice bath, add 10mL of 50wt% chloroacetic acid aqueous solution, and maintain the pH of the system at 8.5 with 20wt% sodium hydroxide aqueous solution during the process, and heat to 50℃ for 4h; adjust the pH to 7.5 with 1mol / L hydrochloric acid, add 4 times the amount of isopropanol in the reaction system, precipitate with alcohol at room temperature for 12h, collect the precipitate and dissolve it in 150mL of water, then dialyze with water for 48h through a 14000Da dialysis bag; dry the dialysate at -60℃ and 100Pa vacuum for 6h to obtain modified β-glucan.

[0098] Example 5

[0099] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0100] S1, Preparation of Base Fluid

[0101] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0102] S2, Preparation of the delivery carrier

[0103] S2.1 Preparation of Single-Component Solutions

[0104] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0105] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0106] Preparation of S2.2 Composite Polymer Solution

[0107] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0108] S3. Preparation of compound water-soluble vaccine adjuvant

[0109] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0110] The preparation method of the modified β-glucan is as follows:

[0111] 0.5 g of β-glucan was added to 200 mL of water and stirred at 80 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant β-glucan solution was collected. 0.35 mL of 2-octenyl succinic anhydride and 3 wt% sodium hydroxide aqueous solution were added to maintain pH 8.5, and the reaction was carried out at 55 °C for 3 h. 2 mol / L hydrochloric acid was added dropwise to adjust the pH to 6.5, and stirring was continued for 12 h. Four times the volume of isopropanol was added to the reaction mixture, and the mixture was precipitated at room temperature for 12 h. The precipitate was collected and dissolved in 150 mL of water, then dialyzed with water for 48 h using a 14000 Da dialysis bag. The dialysate was dried at -60 °C and 100 Pa for 6 h, then dissolved in 20 mL of 0.05 mol / L pH 6.0 2-morpholine ethanesulfonic acid buffer. 0.3 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.18 g of... N-hydroxysuccinimide was stirred at room temperature in the dark for 1 hour; 0.18 g of p-aminophenyl-α-D-mannoside was added, and the pH was adjusted to 7.4 with 3 wt% sodium hydroxide aqueous solution. The mixture was stirred at room temperature in the dark for 4 hours; the mixture was then dialyzed with water for 24 hours through a 14000 Da dialysis bag. The dialysate was dried at -60℃ and 100 Pa for 6 hours to obtain the final product.

[0112] Comparative Example 1

[0113] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0114] S1, Preparation of Base Fluid

[0115] Add 12g trehalose, 10g raffinose, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0116] S2, Preparation of the delivery carrier

[0117] S2.1 Preparation of Single-Component Solutions

[0118] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0119] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0120] Preparation of S2.2 Composite Polymer Solution

[0121] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0122] S3. Preparation of compound water-soluble vaccine adjuvant

[0123] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0124] Comparative Example 2:

[0125] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0126] S1, Preparation of Base Fluid

[0127] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0128] S2, Preparation of the delivery carrier

[0129] S2.1 Preparation of Single-Component Solutions

[0130] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0131] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0132] Preparation of S2.2 Composite Polymer Solution

[0133] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0134] S3. Preparation of compound water-soluble vaccine adjuvant

[0135] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0136] The preparation method of the modified β-glucan is as follows:

[0137] 0.5 g of β-glucan was added to 200 mL of water and stirred at 80 °C for 2 h. After cooling to room temperature, the mixture was centrifuged at 3000 rpm for 10 min, and the supernatant β-glucan solution was collected. 0.35 mL of 2-octenyl succinic anhydride was added, and the mixture was reacted at 45 °C for 3 h, maintaining the pH at 8.5 with 3 wt% sodium hydroxide aqueous solution. After cooling to room temperature, stirring was continued for another 2 h. The pH was adjusted to 6.5 by adding 2 mol / L hydrochloric acid, and four times the volume of isopropanol was added to precipitate the mixture at room temperature for 12 h. The precipitate was collected and dissolved in 150 mL of water. The mixture was then dialyzed with water for 48 h using a 14000 Da dialysis bag. The dialysate was dried at -60 °C and 100 Pa for 6 h, and then dissolved in 20 mL of 0.05 mol / L pH 6.0 solution. 0.18 g of p-aminophenyl-α-D-mannoside was added to 2-morpholine ethanesulfonic acid buffer, and the pH was adjusted to 7.4 with 3 wt% sodium hydroxide aqueous solution. The mixture was stirred at room temperature in the dark for 4 h. After dialyzing with water for 24 h through a 14000 Da dialysis bag, the dialysate was dried at -60℃ and 100 Pa for 6 h to obtain the final product.

[0138] Comparative Example 3:

[0139] A method for preparing a composite water-soluble vaccine adjuvant is as follows:

[0140] S1, Preparation of Base Fluid

[0141] Add 12g trehalose, 10g raffinose, 10g modified β-glucan, 7g xylitol, 4.5g polyethylene glycol, and 1.8g levamisole to 1L of 0.02mol / L pH7.4 phosphate buffer solution, stir at 500rpm for 15min; let stand at 4℃ for later use within 24 hours after preparation;

[0142] S2, Preparation of the delivery carrier

[0143] S2.1 Preparation of Single-Component Solutions

[0144] Chitosan solution: Add 0.2g of chitosan to 10mL of 0.1mol / L hydrochloric acid, stir at 300rpm for 1h at room temperature, then filter through a 0.22μm polyethersulfone filter membrane, and let stand at 4℃ for 30min for later use.

[0145] Polyvinyl alcohol solution: Add 1g of polyvinyl alcohol to 10mL of water, stir in an 80℃ water bath at 200rpm for 3h, and after cooling to room temperature, filter through a 0.22μm filter membrane and store in a sealed container at room temperature.

[0146] Preparation of S2.2 Composite Polymer Solution

[0147] Take 5 mL of the prepared chitosan solution and 3 mL of polyvinyl alcohol solution, add 0.1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200 rpm for 3 h at room temperature; then continue stirring in a 60 °C water bath for 1.5 h, cool to room temperature to obtain the final product; seal and store at 4 °C for later use.

[0148] S3. Preparation of compound water-soluble vaccine adjuvant

[0149] Take 1L of the prepared base solution and 10mL of the composite polymer solution, stir at 200rpm for 30min at room temperature, and then filter aseptically through a 0.22μm filter membrane to obtain the final product; store frozen at -20℃.

[0150] The preparation method of the modified β-glucan is as follows:

[0151] Take 2g of β-glucan and add it to 20mL of anhydrous pyridine. Stir at 300rpm for 15min at 50℃. Cool to 0℃ in an ice bath and slowly add 0.8mL of chlorosulfonic acid. React at 4℃ for 1h, then heat to 25℃ and react for 2h. Pour into 200mL of ice water and adjust the pH to 7.0 with 10wt% sodium hydroxide aqueous solution. Then dialyze with water for 24h through a 14000Da dialysis bag. Dry the dialysate at -60℃ and 100Pa vacuum for 6h to obtain the final product.

[0152] Test Example 1

[0153] Particle size distribution test

[0154] Dynamic light scattering technology was used to detect the adjuvant samples prepared in Examples 1-5 and Comparative Examples 1-3 at 25°C using a Malvern ZetaSizer Nano ZS-90 instrument. The samples were reconstituted with ultrapure water to 1 mg / mL, filtered through a 0.45 μm filter membrane, and the particle size distribution was measured. The results were repeated three times and the average value was taken.

[0155] Stability test

[0156] The above adjuvant samples were stored at 4°C for 14 days; their appearance was observed, and samples were taken for testing on days 0 and 14; the appearance was observed again, and the results were measured again. The results are shown in Table 1.

[0157] Table 1. Results of particle size distribution and stability tests

[0158]

[0159] Compared with Comparative Example 1, Example 1 uses unmodified β-glucan to construct the basic system, which not only shows that β-glucan is the core framework for maintaining adjuvant dispersibility; its intermolecular hydrogen bonds and weak hydrophobic effects maintain particle dispersibility, but the low surface charge density of β-glucan leads to limited interparticle repulsion.

[0160] Example 2 uses octenyl succinate-modified β-glucan, which forms an intramolecular hydrophobic core through its hydrophobic segments, driving self-assembly into smaller nanoparticles. Simultaneously, the introduced ester groups enhance electrostatic repulsion between particles, thereby improving uniformity and stability. Example 3 uses intracyclization grafting modification, where the amino groups impart a strong positive charge to the particles, allowing them to bind to negatively charged cell membranes via electrostatic adsorption, thus improving cellular uptake efficiency. However, a moderate positive charge is beneficial for interaction with negatively charged cell membranes, while the polymer network stabilizes the structure. Example 4 introduces carboxymethyl groups, whose strong hydrophilic anionic groups enhance intermolecular hydrogen bonds, leading to slight particle aggregation. While the high negative potential increases repulsion, it cannot completely counteract the aggregation tendency.

[0161] Example 5 describes a targeted system constructed using mannose modification. This retains the hydrophobic properties of octenyl succinate while maintaining the small particle size and uniformity of the adjuvant. Simultaneously, mannose molecules reduce surface charge, preventing aggregation caused by excessive negative charge. Furthermore, it forms a stable molecular network with levamisole, achieving an optimal balance between physical stability and subsequent targeting. In contrast, the physical mixing in Comparative Example 2 failed to form a stable bond, highlighting the necessity of chemical coupling. The sulfation modification in Comparative Example 3 introduced strong anionic groups, leading to charge repulsion imbalance; improper surface modification can compromise the stability of the adjuvant system.

[0162] Test Example 2

[0163] Humoral immune response assay

[0164] S1. Laboratory Animals and Grouping

[0165] Female BALB / c mice aged 6-8 weeks, SPF grade, weighing 18-22g, were selected and randomly divided into 10 groups of 6 mice each. They were kept at a temperature of 22±2℃ and a humidity of 50±10%, with a 12 / 12h light / dark cycle and free access to food and water. They were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and approved by the company's animal ethics committee.

[0166] A positive control group was set up using MF59 influenza vaccine adjuvant, as directed in the instructions; product number: C01001; sourced from Avetrol.

[0167] S2, Immune Procedure

[0168] Primary immunization: On day 0, all mice were injected intramuscularly into their hind legs with 0.1 mL of the vaccine formulation, which contained 100 μg of the compound water-soluble vaccine adjuvant prepared in Examples 1-5 and Comparative Examples 1-3; and 3 μg of purified influenza virus H1N1 (A / PuertoRico / 8 / 1934) hemagglutinin protein.

[0169] Booster immunization: On day 14, administer booster immunization at the same dose and route as the initial immunization.

[0170] Weight monitoring: Starting from day 0, weigh each mouse twice a week and record the weight of each mouse; if a single mouse's weight decreases by ≥10% from its initial weight, or if it shows abnormalities such as lethargy or ruffled fur, isolate it promptly and record the incident. If necessary, euthanize it according to ethical requirements and supplement the group with mice from the same batch to 6 mice per group and administer the immunization program.

[0171] S3, Sample Collection

[0172] On day 28, serum and spleen were collected from mice. Mice were anesthetized by isoflurane inhalation, and blood was collected through the retroocular venous plexus. The blood was allowed to stand at room temperature for 2 hours, centrifuged at 3000 rpm for 15 minutes, and the supernatant serum was collected, aliquoted and stored at -80℃ for antibody detection.

[0173] S4, Immune Detection

[0174] Coating: Dilute the purified influenza virus particles to 2 μg / mL with 0.05 mol / L pH 9.6 carbonate buffer, add 100 μL to each well of a 96-well microplate, and incubate overnight at 4°C; the purified influenza virus H1N1 particles (A / Puerto Rico / 8 / 1934) with a titer ≥10 8 TCID 50 / mL, sourced from Beijing Tiantan Biological Products Co., Ltd.;

[0175] Blocking solution: 0.02 mol / L pH 7.4 phosphate buffer solution containing 5 vol% skim milk;

[0176] Washing solution: 0.01 mol / L pH 7.4 phosphate buffer solution containing 0.05 vol% Tween-20;

[0177] Sealing: Discard the coating solution, wash the plate 3 times with washing solution; add sealing solution to each well, and seal at 37°C for 2 hours;

[0178] Serum addition: Wash the plate 3 times again; dilute mouse serum with 1% BSA-PBS starting at 1:100 and serially dilute 3-fold to 1:8100, adding 100 μL of diluted serum to each well and incubating at 37°C for 1.5 h; set 100 μL of unimmunized mouse serum as a negative control well, and 100 μL of 1% BSA-PBS dilution in the blank wells without any serum, and then add secondary antibody and perform color development as usual;

[0179] Add secondary antibody: Wash the plate 5 times with washing buffer; add 100 μL of horseradish peroxidase-labeled goat anti-mouse IgG, IgG1 or IgG2a secondary antibody to each well and incubate at 37°C for 1 h; the horseradish peroxidase is derived from the source leaf, labeled antibody grade, and has an enzyme activity ≥300 u / mg.

[0180] Color development and termination: Wash the plate 5 times with PBST; add 100 μL of TMB substrate solution to each well and develop color for 15 min in the dark.

[0181] S5, Data Recording

[0182] The reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid aqueous solution to each well; the absorbance of each well was immediately measured at 450 nm using an ELISA reader; the cut-off value was taken as 2.1 times the OD value of the negative serum control well. The geometric mean titer of mouse serum in each group was calculated. The results are shown in Table 2.

[0183] Table 2 Results of humoral immune response test in mice

[0184]

[0185] The immune response of the groups in Examples 1-5 was better than that of the comparative examples 1-3, and the humoral immune response of Examples 3 and 5 was significantly better than that of the positive control group.

[0186] Example 1 did not modify and activate downstream signaling pathways, but due to the lack of targeting and structural optimization, receptor binding efficiency was limited, resulting in a moderate response. Comparative Example 1, lacking β-glucan, verified that it is a core component for immune enhancement. Example 2 introduced a hydrophobic chain through octenyl succinic anhydride, driving β-glucan self-assembly to form small particles; it also promoted the immune response through endocytosis. Example 3, through internal cyclization grafting modification, its amino groups can activate the NF-κB signaling pathway, bind to β-glucan to regulate the immune response, and thus improve cellular uptake efficiency. Example 4, through carboxymethylation modification, its strongly hydrophilic anionic groups enhance intermolecular hydrogen bonding and hydrophilic interactions, leading to mild particle aggregation; although it can protect antigen integrity through a hydrophilic gel environment, excessive hydrophilic modification may weaken immune activation.

[0187] Furthermore, in Comparative Example 2, the physical mixing of mannosides failed to form a stable structure, resulting in low targeting efficiency and particle aggregation. In Comparative Example 3, the introduction of strong anionic groups and excessive negative charge hindered lectin receptor binding, and improper surface modification of β-glucan impaired adjuvant activity.

[0188] In summary, Example 5 achieves active targeting of dendritic cells by chemically coupling mannoside to anchor the β-glucan backbone; the dual binding of mannoside receptor and lectin receptor produces a synergistic effect, while the physically co-soluble levamisole in the system can further activate the recognition receptor signaling pathway, enhance its antigen presentation ability and pro-inflammatory factor secretion, thereby amplifying the immune response.

Claims

1. A method for preparing a composite water-soluble vaccine adjuvant, characterized in that, Includes the following steps: S1, Preparation of Base Fluid Add 5-20g trehalose, 5-20g raffinose, 5-20g modified β-glucan, 1-10g xylitol, 1-10g polyethylene glycol, and 0.1-5g levamisole to 800-1000mL of phosphate buffer solution, and stir at 200-500rpm for 5-20min; let stand at 0-4℃ for later use within 8-24 hours after preparation. S2, Preparation of the delivery carrier S2.1 Preparation of Single-Component Solutions Chitosan solution: Add 0.1-0.5g of chitosan to 5-10mL of 0.1mol / L hydrochloric acid, stir at 200-500rpm for 0.5-3h at room temperature, filter and store at 4℃. Polyvinyl alcohol solution: Take 0.5-3g of polyvinyl alcohol and add 5-10mL of water. Stir in a water bath at 50-80℃ at 200-500rpm for 1-5 hours. After cooling to room temperature, filter to obtain the solution. Store in a sealed container at room temperature. Preparation of S2.2 Composite Polymer Solution Take the chitosan solution and polyvinyl alcohol solution prepared in step S2.1, add 0.05-1 mL of 10 vol% glycidyl methacrylate aqueous solution, stir at 200-500 rpm for 1-5 h at room temperature; then continue stirring in a water bath at 50-80℃ for 1-5 h, and cool to room temperature to obtain the final product; seal and store at 4℃ for later use. S3. Preparation of compound water-soluble vaccine adjuvant Take 800-1000 mL of the base solution prepared in step S1 and 5-10 mL of the composite polymer solution, stir at 200-500 rpm for 0.5-3 h at room temperature, filter, and store frozen at -20℃. The modified β-glucan is a mannose-targeted modification; The modified β-glucan is a mannosyl-targeted modification, and its preparation method is as follows: Dissolve 0.1-1 g of β-glucan in 100-200 mL of water and stir at 50-80 °C for 1-2 h. After cooling to room temperature, centrifuge at 3000-5000 rpm for 5-10 min and collect the supernatant. Add 0.1-0.6 mL of 2-octenylsuccinic anhydride and react at 50-60 °C for 1-5 h. After cooling to room temperature, adjust the pH and continue stirring for 6-12 h. After alcohol precipitation, centrifuge at 3000-5000 rpm for 5-10 min, collect the precipitate, dissolve it in water, dialyze with water, freeze-dry under vacuum, and then dissolve it in 10-30 mL of 2-morpholine ethanesulfonic acid buffer. Add 0.1-1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.1-1 g of... N-hydroxysuccinimide was stirred at room temperature in the dark for 1-2 hours; 0.1-1 g of p-aminophenyl-α-D-mannoside was added, the pH was adjusted, and the mixture was stirred at room temperature in the dark for 4 hours; the product was obtained after water dialysis and vacuum freeze-drying.

2. The method for preparing the composite water-soluble vaccine adjuvant according to claim 1, characterized in that, The phosphate buffer solution has a concentration of 0.02 mol / L and a pH of 7.

4.

3. The method for preparing the composite water-soluble vaccine adjuvant according to claim 1, characterized in that, The alcohol precipitation process involves adding 1-4 times the amount of isopropanol to the reaction system and precipitating at room temperature for 6-12 hours.

4. The method for preparing the composite water-soluble vaccine adjuvant according to claim 1, characterized in that, The dialysis was performed using water as the dialysate, with a molecular weight cutoff of 14,000 Da, and a dialysis time of 24-48 hours, with the water being changed every 6 hours.

5. The method for preparing the composite water-soluble vaccine adjuvant according to claim 1, characterized in that, The vacuum freeze drying process involves setting the temperature to -60 to -20°C, the vacuum degree to 80-100 Pa, and the drying time to 6-12 hours.

6. A compound water-soluble vaccine adjuvant, characterized in that, Prepared by the method described in any one of claims 1-5.

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