Herpes zoster vaccine containing novel adjuvant and preparation method thereof

Abstract

The invention discloses a herpes zoster vaccine containing a novel adjuvant and a preparation method of the herpes zoster vaccine, and relates to the technical field of vaccines. According to the vaccine, gE protein is used as an antigen, liposome and PLGA nanoparticles are compounded, phospholipid, cholesterol and cholesterol succinic acid monolipid are used for preparing the liposome, the liposome and an antigen solution containing a film-forming agent are mixed and subjected to ultrasonic treatment, then pre-freezing and freeze-drying are conducted, and a resoluble liposome freeze-dried preparation is obtained; the preparation method comprises the following steps: by taking PLGA as a carrier, emulsifying, dialyzing and homogenizing at high pressure to prepare a nano-particle dispersion liquid entrapped with the immunological enhancement factor; and mixing the two components, stabilizing with polyvinyl alcohol, and freeze-drying again to obtain the vaccine preparation. The problems of aggregation, particle size fluctuation and antigen conformation damage caused by easy oxidation instability of a liposome-antigen freeze-drying preparation in freeze-drying, redissolution and storage are solved, and the redissolution stability and consistency are improved through an internal and external synergistic anti-oxidation stable system.

Description

Technical Field

[0001] This invention relates to the field of vaccine technology, and in particular to a shingles vaccine containing a novel adjuvant and its preparation method. Background Technology

[0002] Herpes zoster is caused by the reactivation of the varicella-zoster virus (VZV), and the risk of developing the disease increases with age and a decline in immune function. Current prevention methods focus on enhancing VZV-specific immunity through vaccination in adults to reduce the risk of developing the disease and complications such as postherpetic neuralgia.

[0003] The existing technical routes for shingles vaccines mainly include two categories: one is live attenuated vaccines, which induce immunization through preparations containing live attenuated viruses; the other is recombinant subunit vaccines, which use VZV glycoprotein E (gE) as the key antigen and are combined with a strong adjuvant system to enhance the strength and duration of the immune response in the elderly and other populations.

[0004] CN108992667A discloses a shingles vaccine, its preparation method, and its application. Addressing the high safety risks associated with existing shingles vaccines, this vaccine uses Poly(I:C) as an adjuvant. Poly(I:C), a double-stranded RNA, has had its safety confirmed in large-scale clinical use. Poly(I:C) is a highly effective interferon inducer that can produce a viral infection-like immune response in vivo and can induce CD4+. + and CD8 + T cells enhance cellular and humoral immune responses. However, they are prone to oxidative instability during storage, leading to aggregation and particle size fluctuations. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem to be solved by the present invention is to improve the problem of oxidative instability of liposome-antigen lyophilized formulations during lyophilization, reconstitution and storage, which leads to aggregation, particle size fluctuation and antigen conformation damage. The present invention improves the reconstitution stability and consistency through an internal and external synergistic antioxidant stabilization system.

[0006] Liposome-nanoparticle complexes can be used to deliver a variety of antigens, especially in vaccines. Liposomes, as antigen carriers, can effectively encapsulate antigens and provide slow release. Nanoparticles, on the other hand, act as adjuvants, promoting uptake by antigen-presenting cells and enhancing the immune response.

[0007] To achieve the above objectives, the present invention provides a shingles vaccine containing a novel adjuvant and a method for preparing the same.

[0008] A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add phospholipids, cholesterol, and cholesterol succinate monoester to a mixed solvent and stir until homogeneous. Then transfer the mixture to a rotary evaporator to remove the solvent and obtain a thin film. Add the thin film to water, mix until homogeneous, and heat to obtain a liposome suspension. Add gE protein and film-forming agent to water and mix until homogeneous to obtain an antigen solution. Then add the antigen solution to the above liposome suspension and mix until homogeneous. Sonicate the mixture and finally place the mixture in a pre-freeze and freeze-dry to obtain a lyophilized liposome formulation. Step 2: Add PLGA to dichloromethane and mix evenly to obtain the oil phase; add immune-enhancing factor and PVA to water and mix evenly to obtain the aqueous phase; add the oil phase to the aqueous phase and stir to obtain the emulsion; then transfer to a rotary evaporator to remove the solvent, then transfer to a dialysis bag for dialysis, and finally homogenize using a high-pressure homogenizer to obtain the nanoparticle dispersion; Step 3: Add the lyophilized liposome preparation obtained above to PBS buffer solution and mix evenly to adjust the gE protein concentration in the liposomes; adjust the above nanoparticle dispersion according to the PLGA mass concentration; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 evenly, then add PVA and continue stirring evenly, then pre-freeze and freeze-dry to obtain a herpes zoster vaccine containing a novel adjuvant.

[0009] The phospholipids mentioned therein are at least one of soybean lecithin, hydrogenated lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylserine, distearate phosphatidylcholine (DSPC), dipalmitoyl phosphatidylcholine (DPPC), and myristoyl phosphatidylcholine (DMPC). The mixed solvent is composed of chloroform and methanol in a volume ratio of (1-3):1; The film-forming agent is at least one of proline, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, hydroxypropyl methylcellulose, dextran, sodium caseinate, and gelatin.

[0010] The immune-enhancing factor mentioned therein is at least one of TLR agonists, STING agonists, oligodeoxynucleotide immunomodulators, saponin immunomodulators, polysaccharide immunomodulators, and cytokine immunomodulators.

[0011] A further preferred method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 100-150 mg of phospholipids, 30-50 mg of cholesterol, 15-25 mg of cholesterol monosuccinate, and 5-12 mg of excipients to 20-50 mL of mixed solvent and stir until homogeneous. Then transfer the mixture to a rotary evaporator and treat it at 35-50℃, 100-300 rpm, and a vacuum degree ≤0.08 MPa for 40-60 min to obtain a thin film. Add the thin film to 20-50 mL of water and mix until homogeneous. Treat the mixture at 40-50℃ and 100-300 rpm for 30-60 min to obtain a liposome suspension. Add gE protein and film-forming agent to water and mix until homogeneous to obtain an antigen solution with a gE protein concentration of 0.1-0.5 mg / mL and a film-forming agent concentration of 3-6% (w / v). Then add 3-5 mL of the antigen solution to the above liposome suspension and mix until homogeneous. Sonicate the mixture and finally place the mixture in a pre-freeze environment and freeze-dry it to obtain a lyophilized liposome formulation. Step 2: Add 280-320 mg PLGA to 10-20 mL of dichloromethane and stir at 100-300 rpm for 10-30 min to obtain an oil phase; add immune-enhancing factor and PVA to water and mix well to obtain an aqueous phase with an immune-enhancing factor concentration of 0.5-1 mg / mL and a PVA concentration of 0.01-0.1% (w / v); add the oil phase to 5-10 mL of the aqueous phase and stir at 10000-20000 rpm for 5-10 min to obtain an emulsion; then transfer it to a rotary evaporator and treat it at 35-50℃, 100-300 rpm, and a vacuum degree ≤0.08 MPa for 60-120 min; then transfer it to a dialysis bag for dialyzing; finally, use a high-pressure homogenizer to treat it at 800-1000 bar 10-20 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposomes prepared above to a PBS buffer solution with pH 7.2-7.4 and mix thoroughly. Adjust the gE protein concentration in the liposomes to 30-100 µg / mL. Adjust the PLGA concentration of the above nanoparticle dispersion to 1-5 mg / mL. Mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1. Then add PVA and continue stirring until the final PVA concentration is 1-3% (w / v). Pre-freeze and freeze-dry to obtain a shingles vaccine containing a novel adjuvant.

[0012] The excipients mentioned therein are at least one of PEGylated lipids and tocopherols; The PEGylated lipid is at least one of DSPE-PEG2000, DSPE-PEG-Maleimide, DSPE-PEG-Biotin, DSPE-PEG-Mannose, and DSPE-PEG-Glutathione.

[0013] The tocopherols mentioned above are tocopherol succinates; More preferably, the excipient is a mixture of tocopherol succinate and DSPE-PEG-glutathione in a mass ratio of (1-3):(1-3); The conditions for ultrasonic treatment in step 1 are as follows: ultrasonic power of 80-100W, working time of 1-5s, interval of 1-5s, and total ultrasonic time of 10-20min. The pre-freezing conditions are -60℃ to -80℃ and the time is 1-3 hours; The freeze-drying conditions are -20°C to -40°C, 0.1-0.3 mbar, for 12-24 hours.

[0014] The PVA added in step 3 is used to improve the dispersion stability of the composite system after freeze-drying and reconstitution, and to reduce the fluctuation of particle size and PDI after reconstitution.

[0015] This invention reduces oxidative stress and interfacial damage during lyophilization, reconstitution, and storage by introducing the synergistic effect of tocopherol succinate and DSPE-PEG-glutathione into a liposome system. Tocopherol succinate is embedded in the lipid bilayer, scavenging lipid free radicals, inhibiting peroxidation, and maintaining membrane integrity; DSPE-PEG stabilizes particle dispersion, while glutathione scavenges reactive oxygen species in the aqueous phase and at the interface, providing a reducing buffer. Together, they significantly reduce particle aggregation and antigenic epitope damage, improving the stability and immunogenicity of the formulation.

[0016] The beneficial effects of this invention are: 1. Compared with the prior art, the present invention combines liposomes and nanoparticles and freeze-dries them together to enable antigen carriers and adjuvant carriers to achieve synergistic co-delivery within the same formulation unit: liposomes focus on the encapsulation, coexistence and delivery of antigens, while PLGA nanoparticles focus on the carrying and release of immune-enhancing factors. The composite dispersion system formed after reconstitution can form spatial coupling of isotopic delivery in the cellular uptake and intracellular processing stages, thereby enhancing the spatiotemporal matching of antigen and adjuvant in terms of mechanism.

[0017] 2. Compared with existing technologies, by combining the herpes zoster antigen gE protein with a liposome carrier and using a pre-freeze-freeze drying method to obtain a resolvable liposome lyophilized formulation, the antigen is placed in a protective system formed by the lipid bilayer, the aqueous microenvironment, and the film-forming agent. This reduces the risk of protein conformational disturbance and interfacial instability during preparation and storage, and facilitates the formation of a stable and resolvable antigen delivery carrier system.

[0018] 3. Compared with existing technologies, this invention introduces immune-enhancing factors into PLGA nanoparticles and prepares them in a dispersion system. The polymer network of PLGA provides a microenvironment for encapsulating and sustaining the release of these factors. Combined with the fact that nanoscale particles are more easily taken up by antigen-presenting cells, this allows for more effective delivery and aggregation of immune-enhancing factors at their sites of action during the in vivo presentation process. Mechanistically, this is beneficial for improving the activation efficiency of innate immune pathways and promoting antigen presentation. Detailed Implementation

[0019] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0020] DSPE-PEG-glutathione was prepared according to the specific method disclosed in "Optimisation of glutathione conjugation toliposomes quantified with a validated HPLC assay" (Joy N. Reginald-Opara, Darren Svirskis, Simon J. O'Carroll, Sreevalsan Sreebhavan, Justin M. Dean, Zimei Wu, International Journal of Pharmaceutics 567 (2019) 118451).

[0021] gE protein, His-tagged purified human recombinant VZV gE, provided by Jiangsu Huanotai Biopharmaceutical Technology Co., Ltd.

[0022] PLGA stands for poly(D,L-lactic acid-co-hydroxyacetic acid), model number RESERMER® RG 502 H, manufactured by Evonik, Germany. QS-21, Desert King, USA.

[0023] Other raw materials not mentioned are all common raw materials. The above content is only for the purpose of illustrating the present invention and should not be construed as a strict limitation of the present invention. Those skilled in the art can directly purchase the same / similar raw materials from the market or prepare them themselves.

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: A method for preparing a shingles vaccine containing a novel adjuvant, comprising the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, and 20 mg cholesterol succinate monoester to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film-like substance. Add the thin film-like substance to 20 mL of water and mix well. Treat at 40 °C and 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl methylcellulose to water and mix well to obtain... An antigen solution containing 0.5 mg / mL gE protein and 5% (w / v) hydroxypropyl methylcellulose was prepared. 5 mL of the antigen solution was then added to the liposome suspension and mixed thoroughly. The mixture was sonicated for 10 min (80 W, 5 s on, 5 s off), followed by incubation at 4°C for 30 min. The mixture was then pre-frozen at -80°C for 2 h and then freeze-dried at -40°C under 0.1 mBar vacuum for 12 h to obtain the lyophilized liposome formulation. The solvent was a mixture of chloroform and methanol in a 2:1 volume ratio. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0026] Example 2 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Combine 84mg egg yolk lecithin, 24mg phosphatidylserine, 12mg myristoyl phosphatidylcholine, 40mg cholesterol, 20mg cholesterol monosuccinate, and 10mg... DSPE-PEG-glutathione was added to 20 mL of mixed solvent and stirred for 20 min. The mixture was then transferred to a rotary evaporator and treated for 40 min at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa to obtain a thin film. The thin film was added to 20 mL of water and mixed thoroughly, then treated at 40 °C and 150 rpm for 30 min to obtain a liposome suspension. gE protein and hydroxypropyl methylcellulose were added to water and mixed thoroughly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). 5 mL of the antigen solution was then added to the liposome suspension and mixed thoroughly. The mixture was sonicated for 10 min at a power of 80 W, with a 5-second interval between sonication sessions. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C and a vacuum degree of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0027] Example 3 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, and 10 mg tocopheryl succinate to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C and 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl methylcellulose to water. An antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v) was obtained by mixing thoroughly. Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed thoroughly. The mixture was then sonicated for 10 min, with a sonication power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0028] Example 4 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, 7.5 mg tocopheryl succinate, and 2.5 mg DSPE-PEG-glutathione to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C, 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl... Hydroxypropyl methylcellulose was added to water and mixed evenly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed evenly. The mixture was then sonicated for 10 min, with an ultrasonic power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of water and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0029] Example 5 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Combine 84mg egg yolk lecithin, 24mg phosphatidylserine, 12mg myristoyl phosphatidylcholine, 40mg cholesterol, 20mg cholesterol monosuccinate, and 10mg... DSPE-PEG2000 was added to 20 mL of mixed solvent and stirred for 20 min. The mixture was then transferred to a rotary evaporator and treated for 40 min at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa to obtain a thin film. The thin film was added to 20 mL of water and mixed thoroughly, then treated at 40 °C and 150 rpm for 30 min to obtain a liposome suspension. gE protein and hydroxypropyl methylcellulose were added to water and mixed thoroughly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). 5 mL of the antigen solution was then added to the liposome suspension and mixed thoroughly. The mixture was sonicated for 10 min at a power of 80 W, with a 5-second interval between sonication sessions. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C and a vacuum degree of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0030] Example 6 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, 7.5 mg tocopheryl succinate, and 2.5 mg DSPE-PEG2000 to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C, 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl... Methylcellulose was added to water and mixed evenly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed evenly. The mixture was sonicated for 10 min, with a sonication power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add QS-21 and PVA1000 to water and mix well to obtain an aqueous phase with QS-21 concentration of 1 mg / mL and PVA1000 concentration of 0.1% (w / v); add the oil phase to 5 mL of water and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0031] Comparative Example 1 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, 7.5 mg tocopheryl succinate, and 2.5 mg DSPE-PEG-glutathione to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C, 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl... Hydroxypropyl methylcellulose was added to water and mixed evenly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed evenly. The mixture was then sonicated for 10 min, with an ultrasonic power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add PVA1000 to water and mix well to obtain a 0.1% (w / v) PVA1000 aqueous solution; add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and a vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L of pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0032] Comparative Example 2 A method for preparing a shingles vaccine containing a novel adjuvant includes the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, 7.5 mg tocopheryl succinate, and 2.5 mg DSPE-PEG-glutathione to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C, 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl... Hydroxypropyl methylcellulose was added to water and mixed evenly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed evenly. The mixture was then sonicated for 10 min, with an ultrasonic power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add 300 mg PLGA to 10 mL of dichloromethane and stir at 150 rpm for 15 min to obtain an oil phase; add PVA1000 to water and mix well to obtain a 0.1% (w / v) PVA1000 aqueous solution; add the oil phase to 5 mL of the aqueous phase and stir at 15000 rpm for 5 min to obtain an emulsion; then transfer to a rotary evaporator and treat at 40℃, 150 rpm, and a vacuum degree ≤0.08 MPa for 60 min; then transfer to a dialysis bag with a molecular weight cutoff of 10 kDa and dialyze in 2 L of pH 7.4 PBS at 4℃ for 48 h, changing the dialysate every 5 h; finally, use a high-pressure homogenizer to treat at 800 bar 15 times to obtain a nanoparticle dispersion. Step 3: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 50 µg / mL; adjust the above nanoparticle dispersion to a PLGA concentration of 3 mg / mL; mix the above liposome solution and nanoparticle dispersion at a volume ratio of 1:1, and then add 5 mg QS-21 to maintain the gE concentration in the composite system at 50 µg / mL; at this point, the final PLGA concentration in the composite system is 1.5 mg / mL; then add PVA1000 and continue stirring for 30 min to make the final PVA1000 concentration 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain a shingles vaccine containing a novel adjuvant.

[0033] Comparative Example 3 A method for preparing a shingles vaccine, comprising the following steps: Step 1: Add 84 mg egg yolk lecithin, 24 mg phosphatidylserine, 12 mg myristoyl phosphatidylcholine, 40 mg cholesterol, 20 mg cholesterol monosuccinate, 7.5 mg tocopheryl succinate, and 2.5 mg DSPE-PEG-glutathione to 20 mL of mixed solvent and stir for 20 min. Then transfer to a rotary evaporator and treat at 40 °C, 150 rpm, and a vacuum degree ≤0.08 MPa for 40 min to obtain a thin film. Add the thin film to 20 mL of water, mix well, and treat at 40 °C, 150 rpm for 30 min to obtain a liposome suspension. Add gE protein and hydroxypropyl... Hydroxypropyl methylcellulose was added to water and mixed evenly to obtain an antigen solution with a gE protein concentration of 0.5 mg / mL and a hydroxypropyl methylcellulose concentration of 5% (w / v). Then, 5 mL of the antigen solution was added to the above liposome suspension and mixed evenly. The mixture was then sonicated for 10 min, with an ultrasonic power of 80 W, a working time of 5 s, and a pause of 5 s. The mixture was then incubated at 4 °C for 30 min. Finally, the mixture was pre-frozen at -80 °C for 2 h and then freeze-dried at -40 °C under a vacuum of 0.1 mBar for 12 h to obtain the lyophilized liposome formulation. The mixed solvent was a mixture of chloroform and methanol in a volume ratio of 2:1. Step 2: Add the lyophilized liposome preparation obtained above to a PBS buffer solution at pH 7.2 and mix thoroughly to adjust the gE protein concentration in the liposomes to 100 µg / mL; mix the above liposome solution and the PBS buffer solution at pH 7.2 at a volume ratio of 1:1 to maintain the gE concentration in the composite system at 50 µg / mL; then add PVA1000 and continue stirring for 30 min to make the final concentration of PVA1000 2% (w / v); then pre-freeze at -80℃ for 2 h, and then freeze-dry at -40℃ and 0.1 mBar vacuum for 12 hours to obtain the shingles vaccine.

[0034] Test Example 1 (1) Reconstitution method: After the herpes zoster vaccine containing the novel adjuvant prepared in the examples and comparative examples was equilibrated at room temperature for 30 min, an equal volume of sterile pH 7.2 PBS solution was added. The solution was slowly added along the wall of the bottle, and the mixture was gently inverted to mix. Vigorous shaking and foaming were avoided. The 0-day reconstitution sample was obtained after the lyophilized cake had completely disintegrated and formed a homogeneous emulsion. If there were any visible aggregates or undisintegrated clumps, the phenomenon should be recorded and the bottle should be discarded.

[0035] (2) After reconstitution, the samples should be stored in groups according to the following conditions: Room temperature group: 25℃±2℃, placed in the dark; Acceleration group: 40℃±2℃, placed in the dark.

[0036] Samples were taken at 0d, 7d, and 28d after reconstitution (the time points can be adjusted as needed) for particle size / dispersion stability testing.

[0037] In addition, a portion of the reconstituted samples were subjected to freeze-thaw cycle tests: after the samples were frozen at -20℃ for 12 hours, they were rewarmed in a water bath at 25℃ until they were completely thawed, which was recorded as one freeze-thaw cycle. This was repeated 3 and 5 times. The particle size, PDI and ζ potential were measured immediately after the 3rd and 5th freeze-thaw cycles, respectively.

[0038] The Z-mean particle size and polydispersity index (PDI) of the samples were determined using a dynamic light scattering (DLS) particle size analyzer, and the zeta potential was measured using the same instrument or a matching electrophoretic light scattering module. The detection temperature was set to 25℃ and the scattering angle to 90°.

[0039] Table 1. Appearance test results of shingles vaccines containing novel adjuvants. As shown in Table 1, the particle size and PDI of Example 4 increased only slightly after being placed at 25°C and 40°C and after freeze-thaw cycles, and no visible flocculent precipitates were observed. Its stability was better than that of Examples 1-3. This is because it simultaneously reduced instability from both the membrane interior and the interface. Tocopheryl succinate inhibited membrane phase peroxidation and maintained membrane integrity, while DSPE-PEG-glutathione provided steric hindrance, a hydration layer, and an interface reduction buffer on the membrane surface. The combination of these two factors significantly reduced aggregation and precipitation.

[0040] Compared to Example 4, Comparative Example 1 was more prone to fine flocs and a small amount of precipitation. When there were only carrier particles and no effective immune-enhancing factor encapsulation, the system interface composition and interparticle interactions were more likely to shift towards flocculation. Comparative Example 2 showed the most significant particle size increase, stratification and a large amount of precipitation under accelerated and freeze-thaw conditions, indicating that free amphiphilic saponins are more likely to directly disturb liposomes and interface structures and induce bridging aggregation. Comparative Example 3 was also more prone to turbidity, flocculation and sedimentation under accelerated and freeze-thaw conditions, reflecting that the liposome system alone is more difficult to resist the fusion and aggregation caused by freeze concentration and particle collision.

[0041] Example 5: Introducing DSPE-PEG2000 into the liposome system alone resulted in significant increases in particle size and PDI under conditions of 25°C, 40°C, and freeze-thaw cycles. After 28 days at 40°C, the particle size increased to 222 nm and the PDI to 0.35. After five freeze-thaw cycles, the particle size increased to 236 nm and the PDI to 0.38, with the appearance of suspended flocculent matter. This indicates that while introducing PEGylated lipids alone can provide some steric hindrance and hydration protection on the particle surface, the lack of intramembrane antioxidant components limits its ability to inhibit lipid membrane oxidation and interfacial instability. 6. Simultaneously introducing tocopherol succinate and DSPE-PEG2000, the particle size was 170 nm and the PDI was 0.22 after being placed at 40°C for 28 days. After 5 freeze-thaw cycles, the particle size was 176 nm and the PDI was 0.24, which was significantly better than that of Example 5. This indicates that the intramembrane antioxidant effect and the PEGylation interface stabilization effect can synergistically reduce the risk of aggregation during freeze-drying reconstitution and storage. However, its overall stability is still slightly inferior to that of Example 4, indicating that the interface reduction buffer provided by DSPE-PEG-glutathione has a positive effect on further improving the stability of the system.

[0042] Test Example 2 Female BALB / c mice aged 6-8 weeks were selected, with 10 mice in each group. Immunization schedule: initial immunization on day 0, booster immunization on day 21; each dose contained 5µg of gE antigen, administered intramuscularly. Detection time: blood samples were collected on days 14, 28, and 42 for ELISA.

[0043] Table 2. Mouse gE-specific IgG antibody titers (GMT) As shown in Table 2, all groups were able to induce gE-specific IgG antibody responses after the first immunization. Furthermore, the antibody titers on days 28 and 42 after the booster immunization were generally higher than those on day 14, indicating that the vaccine formulation constructed in this application can effectively induce and maintain humoral immune responses against the gE antigen.

[0044] The differences in composition between the examples and comparative examples reveal that the groups with higher antibody titers and better persistence correspond to the synergistic effect of the liposome / PLGA nanoparticle composite system and the stabilizing excipients. This indicates that while improving the stability of lyophilized reconstitution, the system did not weaken the immunogenicity of the gE antigen. Conversely, groups lacking PLGA-encapsulated immunostimulants, using free immunostimulants, or using only liposome systems exhibited relatively weaker immune response strength or persistence. This suggests that the synergistic combination of antigen carrier, immunostimulant carrier, and stabilizing excipients is beneficial for enhancing the humoral immune response level to the gE antigen.

[0045] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A shingles vaccine containing a novel adjuvant, characterized in that: The vaccine is a freeze-dried formulation comprising: a liposome freeze-dried formulation containing varicella-zoster virus antigen, and a PLGA nanoparticle dispersion loaded with an immune-enhancing factor; wherein the liposome freeze-dried formulation is reconstituted and mixed with the PLGA nanoparticle dispersion, and then freeze-dried to obtain the varicella-zoster vaccine freeze-dried formulation containing the novel adjuvant.

2. The shingles vaccine containing a novel adjuvant as described in claim 1, characterized in that: The liposome lyophilized formulation containing varicella-zoster virus antigen is composed of varicella-zoster virus antigen, phospholipids, cholesterol, and cholesterol succinate monoester.

3. The shingles vaccine containing a novel adjuvant as described in claim 2, characterized in that: The phospholipid is at least one of soybean lecithin, hydrogenated lecithin, egg yolk lecithin, phosphatidylcholine, phosphatidylserine, distearyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or myristoyl phosphatidylcholine.

4. The shingles vaccine containing a novel adjuvant as described in claim 1, characterized in that: The liposome lyophilized formulation containing herpes zoster virus antigen also includes excipients, which are at least one of PEGylated lipids and tocopherols.

5. The shingles vaccine containing a novel adjuvant as described in claim 4, characterized in that: The PEGylated lipid is at least one of DSPE-PEG2000, DSPE-PEG-Maleimide, DSPE-PEG-Biotin, DSPE-PEG-Mannose, or DSPE-PEG-Glutathione; the tocopherol is tocopherol succinate.

6. The shingles vaccine containing a novel adjuvant as described in claim 1, characterized in that: The lyophilized PLGA nanoparticle formulation loaded with immune-enhancing factors contains at least one of the following: TLR agonists, STING agonists, oligodeoxynucleotide immunomodulators, saponin immunomodulators, polysaccharide immunomodulators, or cytokine immunomodulators.

7. A method for preparing a herpes zoster vaccine containing a novel adjuvant as described in any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Add phospholipids, cholesterol, and cholesterol succinate monoester to a mixed solvent and stir until homogeneous. Then transfer the mixture to a rotary evaporator to remove the solvent and obtain a thin film. Add the thin film to water, mix until homogeneous, and heat to obtain a liposome suspension. Add gE protein and film-forming agent to water and mix until homogeneous to obtain an antigen solution. Then add the antigen solution to the above liposome suspension and mix until homogeneous. Sonicate the mixture and finally place the mixture in a pre-freeze and freeze-dry to obtain a lyophilized liposome formulation. Step 2: Add PLGA to dichloromethane and mix evenly to obtain the oil phase; add immune-enhancing factor and PVA to water and mix evenly to obtain the aqueous phase; add the oil phase to the aqueous phase and stir to obtain the emulsion; then transfer to a rotary evaporator to remove the solvent, then transfer to a dialysis bag for dialysis, and finally homogenize using a high-pressure homogenizer to obtain the nanoparticle dispersion; Step 3: Add the lyophilized liposome preparation obtained above to PBS buffer solution and mix evenly to adjust the gE protein concentration in the liposomes; adjust the above nanoparticle dispersion according to the PLGA mass concentration; mix the above liposome solution and nanoparticle dispersion evenly, then add PVA and continue stirring evenly, then pre-freeze and freeze-dry to obtain a shingles vaccine containing a novel adjuvant.

8. The method for preparing the herpes zoster vaccine containing the novel adjuvant as described in claim 7, characterized in that, The mixed solvent is composed of chloroform and methanol in a volume ratio of (1-3):

1.

9. The method for preparing the herpes zoster vaccine containing the novel adjuvant as described in claim 7, characterized in that, The film-forming agent is at least one of proline, hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin, hydroxypropyl methylcellulose, dextran, sodium caseinate, or gelatin.

10. The method for preparing the herpes zoster vaccine containing the novel adjuvant as described in claim 7, characterized in that, The ultrasonic treatment conditions in step (1) are: ultrasonic power 80-100W, working time 1-5s, intermittent time 1-5s, total ultrasonic time 10-20min; the pre-freezing conditions are -60℃ to -80℃, time 1-3h; the freeze-drying conditions are -20℃ to -40℃, 0.1-0.3mbar, time 12-24h.

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