A herpes zoster vaccine composition and a method of preparing the same

Abstract

The application discloses a zoster vaccine composition and a preparation method, and belongs to the technical field of vaccines.The zoster vaccine composition comprises gE protein and an adjuvant; the adjuvant is at least one selected from aluminum salt adjuvants, saponin adjuvants, TLR pathway agonists, STING pathway agonists, emulsion adjuvants and liposome adjuvants.The application also provides a preparation method thereof.Compared with the prior art, the prepared gE protein is jointly constructed with a glassy sugar and a buffer system to form a protective matrix, hydrogen bonds are replaced and a fixed conformation is glassified during freezing and drying, interface-induced unfolding and aggregation are reduced, a small amount of surfactant weakens air-liquid and solid-liquid interfacial tension, terminal low adsorption filtration reduces non-specific adsorption loss, and a monomer state and immunogenicity are maintained after reconstitution.

Description

Technical Field

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

[0002] Varicella-zoster virus (VZV) is one of eight herpesviruses that infect humans, also known as human herpesvirus 3 (HHV-3). This virus is the causative agent of varicella-zoster virus disease and has only one serotype. Varicella-zoster virus is widely distributed and highly contagious. Initial infection in childhood triggers infection, which then remains latent in the body. When the body's immunity declines to a certain threshold, the varicella-zoster virus can be reactivated.

[0003] CN109200280A discloses a nanovaccine, a vaccine composition, its preparation method, and its application. The nanovaccine and two vaccine compositions are described. The nanovaccine is a DSPE-PEG2000-antigen peptide, and the vaccine composition consists of DSPE-PEG2000-CpG and the DSPE-PEG2000-antigen peptide from the aforementioned nanovaccine. The other vaccine composition consists of the DSPE-PEG2000-antigen peptide and the adjuvant Montandid ISA 51VG ST. The invention also provides a method for preparing the above-mentioned vaccine compositions and the application of the nanovaccine and vaccine compositions in the preparation of tumor immunotherapy drugs. After entering the body, the above-mentioned nanovaccine and the two vaccine compositions can effectively target and drain lymph nodes. The materials required for the preparation method are all FDA-approved medical materials, showing potential for clinical application. By co-loading the antigen and adjuvant into a single vaccine composition for co-delivery, integrating the carrier, antigen, and adjuvant into one unit, it can fully stimulate immune cells and further enhance various downstream immune responses.

[0004] Therefore, the present invention provides a shingles vaccine composition and a method for preparing the same. Summary of the Invention

[0005] In view of the above-mentioned deficiencies of the prior art, the technical problem actually solved by the present invention is to solve the structural stability and activity maintenance of antigens during preparation, storage and reconstitution; to avoid compatibility inactivation caused by long-term coexistence of antigens and liposomes, and to improve shelf-life stability; and to improve the uptake and delivery efficiency of antigen-presenting cells by optimizing the liposome membrane composition and surface modification.

[0006] To achieve the above objectives, the present invention provides a shingles vaccine composition, characterized in that it comprises gE protein and adjuvant.

[0007] The adjuvant is selected from at least one of aluminum salt adjuvants, saponin adjuvants, TLR pathway agonists, STING pathway agonists, emulsion adjuvants, and liposome adjuvants.

[0008] More preferably, a shingles vaccine composition may further include at least one of a buffer, stabilizer, surfactant, isotonicity modifier, preservative, chelating agent, and antioxidant.

[0009] The present invention also discloses a method for preparing a shingles vaccine composition.

[0010] A method for preparing a shingles vaccine composition includes the following steps:

[0011] Step 1: Sterols and lipids are added to a solvent and mixed evenly to obtain a sterol-lipid solution; phospholipids are added to a solvent and mixed evenly to obtain a phospholipid solution; the sterol-lipid solution, phospholipid solution, and immunostimulant solution are mixed evenly, and then DSPE-PEG-TD1 is added and mixed evenly to obtain a mixed solution. The mixed solution is added to a buffer solution using a continuous flow shear press to obtain a liposome dispersion. The liposome dispersion is passed through a high-pressure homogenizer, and the homogenized sample is dialyzed by ultrafiltration and filtered; the obtained filtrate is stored to obtain the liposome adjuvant.

[0012] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant until homogeneous to obtain the shingles vaccine composition.

[0013] Preferably, a method for preparing a shingles vaccine composition includes the following steps:

[0014] Step 1: Add 0.5-1.3g of sterol and 0.1-0.3g of lipid to 50-150mL of anhydrous ethanol and mix thoroughly to obtain a sterol-lipid solution; add 1-3.5g of phospholipid to 80-150mL of anhydrous ethanol and mix thoroughly to obtain a phospholipid solution; mix the sterol-lipid solution, phospholipid solution, and immunostimulant solution thoroughly, then add 0.01-0.06g of DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 800-1200mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion using a high-pressure homogenizer at 500-1000 bar 1-6 times, then homogenize at 1000-1200 bar 1-5 times, and then perform ultrafiltration dialysis (MWCO 100). The ethanol was removed and concentrated using kDa, and the final volume was adjusted with buffer to a concentration of 10-50 µg / mL for the immunostimulant. The solution was then filtered through a 0.22 μm filter. The filtrate was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0015] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant evenly to obtain the herpes zoster vaccine composition; wherein the reconstituted gE protein solution and the liposome adjuvant are mixed at a volume ratio of (1-3):(1-3), and the final volume of each dose is 0.50 mL, then the immunostimulant is 10-15 µg / dose, and the gE protein is 40-60 µg / dose.

[0016] The buffer solution is an aqueous solution containing 3-6 wt% sucrose, 8-12 mM histidine, and 50-70 mM NaCl, with a pH of 6.1-6.6.

[0017] The concentration of the immunostimulant solution is 20-30 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is at least one of GLA, QS-21, 3M-052, CpG ODN, and cGAMP.

[0018] The preparation method of the lyophilized formulation containing gE protein is as follows: Take 100-300 mL of water for injection and place it in an ice bath at 2-8℃. Add 6-10% (w / v) trehalose, 1-3% (w / v) mannitol, and 8-12 mM histidine buffer to adjust the pH to 6.1-6.8 (25℃). Add 0.001-0.006% (w / v) Tween-20 and bring the volume to 200-300 mL. Let it stand at 0-4℃ for 30-60 min. Add gE protein at 2-8℃ and mix to 0.1-0.3 mg / mL. Avoid vigorous stirring and foaming throughout the process. Use a low-protein adsorption PVDF / PES membrane for 0.22 μm terminal sterile filtration, discard the first filtrate, and collect the entire volume. Use 2 mL vials, aliquoting 0.25 mL into each vial. Insert a half-inserted lyophilization stopper and perform lyophilization to obtain the lyophilized formulation containing gE protein.

[0019] The freeze-drying process involves pre-freezing at -45°C to -40°C for 120-240 min; drying at 100-150 mTorr at -35°C to -30°C for 16-24 h; and then drying a second time by raising the temperature to 20-25°C at 0.1-0.3°C / min and holding for 6-10 h, so that the residual moisture content is ≤1.5%.

[0020] The sterol mentioned therein is at least one of cholesterol hemisuccinate and rapeseed sterol;

[0021] The lipids mentioned therein are at least one of archaea tetraether lipids and dialkyl glycerol tetraether lipids (GDGTs);

[0022] The phospholipids mentioned above are at least one of soybean lecithin and phosphatidylserine;

[0023] gE protein constructs a protective matrix with glassy sugars and a buffer system. During freezing and drying, it fixes its conformation through hydrogen bond replacement and glassization, reducing interface-induced unfolding and aggregation. A small amount of surfactant weakens the air-liquid and solid-liquid interfacial tension, and terminal low-adsorption filtration reduces non-specific adsorption loss. After resolvation, it maintains its monomeric state and immunogenicity.

[0024] The antigen is lyophilized and the adjuvant is preserved in liposome liquid to block adsorption and deactivation caused by long-term physical contact and avoid the influence of surfactants in the lipid phase on protein structure. It is mixed immediately before use and only spatial colocalizes in vivo, improving effective presentation without sacrificing shelf-life stability.

[0025] Sterols and cholesterol derivatives regulate membrane order and surface charge, promoting the insertion of amphiphilic GLA and its stable embedding in the bilayer; changes in lipid phase charge and fluidity in an acidic microenvironment facilitate endosome membrane fusion and content release, enhancing the accessibility of innate immune receptors.

[0026] Phosphatidylserine is internalized by recognition receptors of macrophages and dendritic cells; surface PEGylation provides steric hindrance to inhibit the formation of nonspecific protein crowns, reducing local aggregation, while retaining specific interactions with ligand peptides, promoting directional migration along the injection site-lymph node axis.

[0027] Dialkylglycerol tetraether esters possess a transmembrane monomolecular tetraether structure, which imparts higher membrane density, permeability resistance, antioxidant properties, and shear stability. When coexisting with phospholipids and sterols, dialkylglycerol tetraether esters tend to form relatively ordered skeletal domains, significantly inhibiting the permeate flux of water and small molecules.

[0028] The beneficial effects of this invention are:

[0029] gE protein constructs a protective matrix with glassy sugars and a buffer system. During freezing and drying, it fixes its conformation through hydrogen bond replacement and glassization, reducing interface-induced unfolding and aggregation. A small amount of surfactant weakens the air-liquid and solid-liquid interfacial tension, and terminal low-adsorption filtration reduces non-specific adsorption loss. After resolvation, it maintains its monomeric state and immunogenicity.

[0030] The antigen is lyophilized and the adjuvant is preserved in liposome liquid to block adsorption and deactivation caused by long-term physical contact and avoid the influence of surfactants in the lipid phase on protein structure. It is mixed immediately before use and only spatial colocalizes in vivo, improving effective presentation without sacrificing shelf-life stability. Detailed Implementation

[0031] 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.

[0032] Cholesterol hemisuccinate, product number CHO2003, was purchased from Shenzhen Botai Biotechnology Co., Ltd.

[0033] Rapeseed sterol, CAS: 474-67-9, purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0034] In this embodiment, DSPE-PEG-TD1 is DSPE-PEG(2000) Maleimide, catalog number 880126, purchased from Avanti Polar Lipids, Inc.

[0035] gE protein, His-tagged purified human recombinant VZV gE, provided by Jiangsu Huanotai Biopharmaceutical Technology Co., Ltd.

[0036] GLA is a pyranose glucolipid adjuvant, purchased from Avanti Polar Lipids, Inc. (Alabaster, AL); product number 699800.

[0037] Dialkylglycerol tetraether ester, Catalog#1303, purchased from Cayman Chemical;

[0038] 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.

[0039] 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.

[0040] Example 1

[0041] A method for preparing a shingles vaccine composition includes the following steps:

[0042] Step 1: Add 0.7g cholesterol hemisuccinate, 0.35g rapeseed sterol, and 0.2g dialkylglycerol tetraether to 100mL of anhydrous ethanol and mix thoroughly to obtain a cholesterol hemisuccinate-rapeseed sterol solution; add 2g soybean lecithin and 0.4g phosphatidylserine to 100mL of anhydrous ethanol and mix thoroughly to obtain a soybean lecithin-phosphatidylserine solution; mix the cholesterol hemisuccinate-rapeseed sterol solution, soybean lecithin-phosphatidylserine solution, and immunostimulant solution thoroughly, then add 0.05g DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 1000mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion using a high-pressure homogenizer at 800bar 5 times, then at 1100bar 3 times, and then perform ultrafiltration dialysis (MWCO 100). The solution was diluted with kDa to remove ethanol and concentrated. The final volume was adjusted with buffer to a concentration of 50 µg / mL for the immunostimulant, and then filtered through a 0.22 μm filter. The filtrate was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0043] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant until homogeneous to obtain the herpes zoster vaccine composition; wherein the reconstituted gE protein solution and the liposome adjuvant are mixed at a volume ratio of 1:1, and the final volume of each dose is 0.50 mL, then the immunostimulant is 12.5 µg / dose, and the gE protein is 50 µg / dose.

[0044] The buffer solution is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, with a pH of 6.5.

[0045] The concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is GLA.

[0046] The reconstituted solution containing gE protein was prepared by reconstituted the lyophilized formulation containing gE protein to 0.25 mL using a pH = 6.6, 10 mM histidine buffer, with gE protein concentration of 0.2 mg / mL.

[0047] The method for preparing the lyophilized formulation containing gE protein is as follows:

[0048] Take 200 mL of water for injection and place it in an ice bath at 2-8℃. Add 8% (w / v) trehalose, 2% (w / v) mannitol, and adjust the pH to 6.6 (25℃) with 10 mM histidine buffer. Add 0.005% (w / v) Tween-20 and bring the volume to 250 mL. Let it stand at 4℃ for 30 min. Add gE protein at 2-8℃ and mix well to 0.20 mg / mL. Avoid vigorous stirring and foaming throughout the process. Use a pre-wetted PVDF / PES membrane with low protein adsorption for 0.22 μm terminal sterile filtration. Discard the first filtrate and collect the entire volume. Use 2 mL vials and dispense 0.25 mL into each vial. Insert a half-inserted lyophilization stopper and freeze-dry to obtain a lyophilized formulation containing gE protein.

[0049] The freeze-drying process involves pre-freezing at -45°C for 120 min; drying at -35°C for 24 h at 120 mTorr; and then drying a second time by raising the temperature to 20°C at 0.3°C / min and holding for 8 h, so that the residual moisture content is ≤1.5%.

[0050] Example 2

[0051] A method for preparing a shingles vaccine composition includes the following steps:

[0052] Step 1: Add 1.25g of dialkylglycerol tetraether to 100mL of anhydrous ethanol and mix thoroughly to obtain a dialkylglycerol tetraether solution; add 2g of soybean lecithin and 0.4g of phosphatidylserine to 100mL of anhydrous ethanol and mix thoroughly to obtain a soybean lecithin-phosphatidylserine solution; mix the dialkylglycerol tetraether solution, soybean lecithin-phosphatidylserine solution, and immunostimulant solution thoroughly, then add 0.05g of DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 1000mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion using a high-pressure homogenizer at 800bar 5 times, then at 1100bar 3 times, and then perform ultrafiltration dialysis (MWCO 100). The solution was diluted with kDa to remove ethanol and concentrated. The final volume was adjusted with buffer to a concentration of 50 µg / mL for the immunostimulant, and then filtered through a 0.22 μm filter. The filtrate was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0053] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant until homogeneous to obtain the herpes zoster vaccine composition; wherein the reconstituted gE protein solution and the liposome adjuvant are mixed at a volume ratio of 1:1, and the final volume of each dose is 0.50 mL, then the immunostimulant is 12.5 µg / dose, and the gE protein is 50 µg / dose.

[0054] The buffer solution is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, with a pH of 6.5.

[0055] The concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is GLA.

[0056] The reconstituted solution containing gE protein was prepared by reconstituted the lyophilized formulation containing gE protein to 0.25 mL using a pH = 6.6, 10 mM histidine buffer, with gE protein concentration of 0.2 mg / mL.

[0057] The method for preparing the lyophilized formulation containing gE protein is as follows:

[0058] Take 200 mL of water for injection and place it in an ice bath at 2-8℃. Add 8% (w / v) trehalose, 2% (w / v) mannitol, and adjust the pH to 6.6 (25℃) with 10 mM histidine buffer. Add 0.005% (w / v) Tween-20 and bring the volume to 250 mL. Let it stand at 4℃ for 30 min. Add gE protein at 2-8℃ and mix well to 0.20 mg / mL. Avoid vigorous stirring and foaming throughout the process. Use a pre-wetted PVDF / PES membrane with low protein adsorption for 0.22 μm terminal sterile filtration. Discard the first filtrate and collect the entire volume. Use 2 mL vials and dispense 0.25 mL into each vial. Insert a half-inserted lyophilization stopper and freeze-dry to obtain a lyophilized formulation containing gE protein.

[0059] The freeze-drying process involves pre-freezing at -45°C for 120 min; drying at -35°C for 24 h at 120 mTorr; and then drying a second time by raising the temperature to 20°C at 0.3°C / min and holding for 8 h, so that the residual moisture content is ≤1.5%.

[0060] Example 3

[0061] A method for preparing a shingles vaccine composition includes the following steps:

[0062] Step 1: Add 0.8g cholesterol hemisuccinate and 0.45g rapeseed sterol to 100mL of anhydrous ethanol and mix thoroughly to obtain a cholesterol hemisuccinate-rapeseed sterol solution; add 2g soybean lecithin and 0.4g phosphatidylserine to 100mL of anhydrous ethanol and mix thoroughly to obtain a soybean lecithin-phosphatidylserine solution; mix the cholesterol hemisuccinate-rapeseed sterol solution, soybean lecithin-phosphatidylserine solution, and immunostimulant solution thoroughly, then add 0.05g DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 1000mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion using a high-pressure homogenizer at 800bar 5 times, then at 1100bar 3 times, and then perform ultrafiltration dialysis (MWCO 100). The solution was diluted with kDa to remove ethanol and concentrated. The final volume was adjusted with buffer to a concentration of 50 µg / mL for the immunostimulant, and then filtered through a 0.22 μm filter. The filtrate was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0063] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant until homogeneous to obtain the herpes zoster vaccine composition; wherein the reconstituted gE protein solution and the liposome adjuvant are mixed at a volume ratio of 1:1, and the final volume of each dose is 0.50 mL, then the immunostimulant is 12.5 µg / dose, and the gE protein is 50 µg / dose.

[0064] The buffer solution is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, with a pH of 6.5.

[0065] The concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is GLA.

[0066] The reconstituted solution containing gE protein was prepared by reconstituted the lyophilized formulation containing gE protein to 0.25 mL using a pH = 6.6, 10 mM histidine buffer, with gE protein concentration of 0.2 mg / mL.

[0067] The method for preparing the lyophilized formulation containing gE protein is as follows:

[0068] Take 200 mL of water for injection and place it in an ice bath at 2-8℃. Add 8% (w / v) trehalose, 2% (w / v) mannitol, and adjust the pH to 6.6 (25℃) with 10 mM histidine buffer. Add 0.005% (w / v) Tween-20 and bring the volume to 250 mL. Let it stand at 4℃ for 30 min. Add gE protein at 2-8℃ and mix well to 0.20 mg / mL. Avoid vigorous stirring and foaming throughout the process. Use a pre-wetted PVDF / PES membrane with low protein adsorption for 0.22 μm terminal sterile filtration. Discard the first filtrate and collect the entire volume. Use 2 mL vials and dispense 0.25 mL into each vial. Insert a half-inserted lyophilization stopper and freeze-dry to obtain a lyophilized formulation containing gE protein.

[0069] The freeze-drying process involves pre-freezing at -45°C for 120 min; drying at -35°C for 24 h at 120 mTorr; and then drying a second time by raising the temperature to 20°C at 0.3°C / min and holding for 8 h, so that the residual moisture content is ≤1.5%.

[0070] Example 4

[0071] It is basically the same as Example 1, except that Tween-20 is not added to the lyophilized formulation containing gE protein.

[0072] Example 5

[0073] It is basically the same as Example 1, except that: in the freeze-dried formulation containing gE protein, 10% sucrose is used to replace 8% trehalose and 2% mannitol.

[0074] Example 6

[0075] It is basically the same as Example 1, except that phosphatidylserine is not added.

[0076] Example 7

[0077] It is basically the same as Example 1, except that 1.05g of cholesterol is used instead of 0.7g of cholesterol hemisuccinate and 0.35g of campesterol.

[0078] Example 8

[0079] Essentially the same as Example 1, except that DSPE-PEG-TD1 is not added. Example 9

[0080] It is basically the same as Example 1, except that gE protein is used instead of the lyophilized formulation containing gE protein.

[0081] Example 10

[0082] A method for preparing a shingles vaccine composition includes the following steps:

[0083] Step 1: Add 0.7g cholesterol hemisuccinate, 0.35g rapeseed sterol, and 0.2g dialkylglycerol tetraether to 100mL of anhydrous ethanol and mix thoroughly to obtain a cholesterol hemisuccinate-rapeseed sterol solution; add 2g soybean lecithin and 0.4g phosphatidylserine to 100mL of anhydrous ethanol and mix thoroughly to obtain a soybean lecithin-phosphatidylserine solution; mix the cholesterol hemisuccinate-rapeseed sterol solution and the soybean lecithin-phosphatidylserine solution thoroughly, then add 0.05g DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 1000mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion through a high-pressure homogenizer at 800bar 5 times, then homogenize at 1100bar 3 times, and then perform ultrafiltration dialysis (MWCO 100). The solution was purified by removing ethanol and concentrating the solution, then filtered through a 0.22 μm filter. The filtrate was mixed with an immunostimulant solution to achieve an immunostimulant concentration of 50 µg / mL. The mixture was stirred and homogenized using a magnetic stirrer. The final product was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0084] Step 2: Mix the reconstituted gE protein solution with the liposome adjuvant until homogeneous to obtain the herpes zoster vaccine composition; wherein the reconstituted gE protein solution and the liposome adjuvant are mixed at a volume ratio of 1:1, and the final volume of each dose is 0.50 mL, then the immunostimulant is 12.5 µg / dose, and the gE protein is 50 µg / dose.

[0085] The buffer solution is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, with a pH of 6.5.

[0086] The concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is GLA.

[0087] The reconstituted solution containing gE protein was prepared by reconstituted the lyophilized formulation containing gE protein to 0.25 mL using a pH = 6.6, 10 mM histidine buffer, with gE protein concentration of 0.2 mg / mL.

[0088] The method for preparing the lyophilized formulation containing gE protein is as follows:

[0089] Take 200 mL of water for injection and place it in an ice bath at 2-8℃. Add 8% (w / v) trehalose, 2% (w / v) mannitol, and adjust the pH to 6.6 (25℃) with 10 mM histidine buffer. Add 0.005% (w / v) Tween-20 and bring the volume to 250 mL. Let it stand at 4℃ for 30 min. Add gE protein at 2-8℃ and mix well to 0.20 mg / mL. Avoid vigorous stirring and foaming throughout the process. Use a pre-wetted PVDF / PES membrane with low protein adsorption for 0.22 μm terminal sterile filtration. Discard the first filtrate and collect the entire volume. Use 2 mL vials and dispense 0.25 mL into each vial. Insert a half-inserted lyophilization stopper and freeze-dry to obtain a lyophilized formulation containing gE protein.

[0090] The freeze-drying process involves pre-freezing at -45°C for 120 min; drying at -35°C for 24 h at 120 mTorr; and then drying a second time by raising the temperature to 20°C at 0.3°C / min and holding for 8 h, so that the residual moisture content is ≤1.5%.

[0091] Example 11

[0092] A method for preparing a shingles vaccine composition includes the following steps:

[0093] Step 1: Add 0.7g cholesterol hemisuccinate, 0.35g rapeseed sterol, and 0.2g dialkylglycerol tetraether to 100mL of anhydrous ethanol and mix thoroughly to obtain a cholesterol hemisuccinate-rapeseed sterol solution; add 2g soybean lecithin and 0.4g phosphatidylserine to 100mL of anhydrous ethanol and mix thoroughly to obtain a soybean lecithin-phosphatidylserine solution; mix the cholesterol hemisuccinate-rapeseed sterol solution, soybean lecithin-phosphatidylserine solution, and immunostimulant solution thoroughly, then add 0.05g DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution; turn on a continuous flow shear press and add the mixed solution to 1000mL of buffer to obtain a liposome dispersion; homogenize the liposome dispersion using a high-pressure homogenizer at 800bar 5 times, then at 1100bar 3 times, and then perform ultrafiltration dialysis (MWCO 100). The solution was diluted with kDa to remove ethanol and concentrated. The final volume was adjusted with buffer to a concentration of 50 µg / mL for the immunostimulant, and then filtered through a 0.22 μm filter. The filtrate was stored in a pharmaceutical cooler at 2-8°C to obtain the liposome adjuvant.

[0094] Step 2: Mix gE protein and liposome adjuvant evenly, place in a semi-inserted freeze-drying plug for freeze-drying. The freeze-drying process is as follows: pre-freeze at -45℃ for 120 min; dry at -35℃ for 24 h at 120 mTorr; then dry a second time by heating to 20℃ at 0.3℃ / min and holding for 8 h, so that the residual moisture is ≤1.5%, to obtain the shingles vaccine composition. The final volume of each dose is 0.50 mL, so the immunostimulant is 12.5 µg / dose and the gE protein is 50 µg / dose.

[0095] The buffer solution is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, with a pH of 6.5.

[0096] The concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; the immunostimulant is GLA.

[0097] Test Example 1

[0098] The particle size and polydispersity index of liposome adjuvants were determined using a Zetasizer Nao ZS paricle size analyzer (Malvern, UK).

[0099] Table 1. Particle size and polydispersity index of liposome adjuvants

[0100]

[0101] As shown in Table 1, Example 1 exhibits the lowest particle size and polydispersity index (PDI), indicating the best dispersibility. Example 3, based on Example 1, removes dialkylglycerol tetraether ester, thus lacking the transmembrane tetraether monomolecular framework. This reduces the bilayer's shear and permeation resistance, decreases continuity, and weakens interfacial tension. Consequently, the particles are more susceptible to transient energy fluctuations during shearing, leading to an increase in PDI. Overall, the particle size is slightly larger, and the PDI is higher than in Example 1. Example 2, lacking cholesterol hemisuccinate and rapeseed sterol, results in a loss of rigid filling in the hydrophobic core, decreased continuity, and reduced membrane surface tension and flexural modulus. The granulation stage is more sensitive to transient shear energy, making secondary collisions and size increases more likely, leading to an increased PDI. Even with the presence of dialkylglycerol tetraether ester, it is difficult to replace the sterol's compaction of the hydrophobic core and its ordering effect on the head-base region. Therefore, the particle size and PDI are higher than in Example 1. Example 7 uses cholesterol instead of cholesterol hemisuccinate and rapeseed sterol. Cholesterol can compact the membrane phase, but the lack of the weakly acidic head group of cholesterol hemisuccinate and the stronger compaction brought by the C-24 side chain of rapeseed sterol results in insufficient hydration and surface charge regulation in the head group region, leading to a more heterogeneous microphase. After homogenization, the particle size and PDI are generally higher than in Example 1. Example 8 lacks DSPE-PEG-TD1, resulting in decreased steric hindrance and anti-protein ability, and an increased probability of short-range aggregation. The effect on particle size is a slight upward floating, while the effect on PDI is more significant. The difference between Example 10 and Example 1 is that the immunostimulant is added last. Post-loading causes post-insertion and rearrangement within the membrane, increasing short-term diffusion on the inner membrane surface and local domain reconstruction. This is generally manifested as a slight upward floating of particle size and a slight increase in PDI compared to Example 1, but still within the stable dispersion range.

[0102] Test Example 2

[0103] The lyophilized formulations containing gE protein in Examples 1 and 4-5 were tested, with 10 samples for each example.

[0104] 1. Appearance inspection

[0105] Visual inspection: Observe the cake by slowly rotating it 360° on the inspection table: integrity (judgment criteria are complete, slight shrinkage, collapse, cracks), color;

[0106] Taking photos: Photo archiving, with the same lighting and background;

[0107] 2. Residual water (coulometric method, KF)

[0108] System suitability: 95-105% recovery rate when calibrated with 1.0 mg / g pure water standard solution.

[0109] Blank: Use an empty crucible / empty sample boat as a blank; the reading should be ≤5 µg.

[0110] Determination: Weigh 50-100 mg of the freeze-dried block, place it in a heating furnace and heat it to 60-80℃. After the reading stabilizes, record the µg moisture content.

[0111] Calculation: Moisture (%) = (µg moisture / sample mass mg) × 100%;

[0112] 3. DSC test

[0113] Tg (lyophilized preparation): Take 5-8 mg of lyophilized block and seal it; heat to 120-140℃ at 10℃ / min (avoid sample decomposition); read the Tg.

[0114] 4. Reconstitution time:

[0115] (1) Slowly inject 0.25 mL of water for injection along the bottle wall, and start timing as soon as the liquid is added;

[0116] (2) Manually reverse the rotation, once every 2 seconds, with an amplitude of 180°;

[0117] (3) Endpoint determination: No visible particles or flocculent matter, and the liquid surface is stable without streaks.

[0118] (4) Record the stopwatch time (s).

[0119] Table 2 Appearance test results of lyophilized formulations containing gE protein

[0120]

[0121] Test Example 3

[0122] Using the shingles vaccine composition provided in the examples as the test object, the induction levels of IL-2 and IFN-γ by the vaccine composition were detected.

[0123] Control group: PBS buffer solution was used as control group 1.

[0124] The specific detection method is as follows: Six-week-old female Balb / c mice were randomly divided into groups of 10 each. The herpes zoster vaccine composition provided in the example and the sample of the control group were diluted 10 times, that is, 1 / 10 of the content of each composition was taken and made up to 0.1 mL with PBS. 0.1 mL was injected subcutaneously into each group of mice. Each group of mice was immunized twice, with an interval of 2 weeks.

[0125] Mice 14 days after secondary immunization were euthanized by cervical dislocation, immersed in 75% alcohol, and transferred to a biosafety cabinet. The spleens were isolated and placed on a 200-mesh cell sieve. 2 mL of PBS buffer was added, and the cells were ground on the sieve using a 5 mL syringe handle until no obvious tissue remained. After repeatedly and slowly rinsing the sieve, the suspension was collected and transferred to a 15 mL centrifuge tube, centrifuged at 300g for 10 min. The supernatant was discarded, 4 mL of ACK lysis buffer was added, the mixture was pipetted and incubated for 3-5 min, then centrifuged at 300g for 10 min. The supernatant was discarded, 4 mL of RPMI 1640 medium containing 10% FBS was added, the mixture was pipetted and incubated for 10 min, then centrifuged at 300g for 10 min. The supernatant was discarded again, 4 mL of RPMI 1640 medium containing 10% FBS was added, the mixture was pipetted and incubated, and the cells were counted using a cell counter. Each cell group was diluted to 2 × 10⁻⁶ cells / mL. 6 Cells / mL were added to pre-activated IFN-γ and IL-2 detection PVDF plates (Mabtech), 50 µL per well. A negative control, gE protein stimulation (10 μg / mL), and a positive control (PHA) were included. The negative control was brought to 100 μL / well with culture medium. For gE protein stimulation and the positive control, gE protein and PHA were diluted to 20 μg / mL with culture medium, and 50 μL was added to each well. Each sample was tested in triplicate. Cells were incubated at 37°C and 5% CO2 for 48 h. The liquid in the wells was discarded, and 200 μL of PBS was added to each well for washing five times. Then, 100 μL of diluted biotin-labeled antibody was added to each well, and the cells were incubated at room temperature for 2 h. The liquid in the wells was discarded, and 200 μL of PBS was added to each well for washing five times. Then, 100 μL of diluted ALP-labeled streptavidin was added to each well, and the cells were incubated at room temperature for 1 h. Pour out the liquid from the wells, add 200 μL of PBS to each well and wash 5 times. Then add 100 μL of filtered NCIP / NBT-plus solution to each well and develop for 5-30 min until obvious spots appear. Rinse with deionized water to stop the color development. IL-2 and IFN-γ levels in each group of experiments were detected using the ELISPOT method.

[0126] Table 3. IL-2 and IFN-γ test results of the shingles vaccine composition.

[0127]

[0128] As shown in Table 3, Example 1 used cholesterol hemisuccinate, rapeseed sterol, and dialkylglycerol tetraether to improve compaction and reduce basal leakage. Cholesterol hemisuccinate triggered membrane remodeling and fusion in a weakly acidic environment, enhancing the effective exposure of GLA in the endosome-lysosome pathway and promoting Th1-biased activation of the TLR4 pathway. Therefore, IL-2 and IFN-γ readings were significantly higher than those in the PBS control. In Example 10, the introduction of GLA caused post-insertion and rearrangement. The intracellular localization was slightly shallower than co-assembly, and the domain remodeling time was slightly longer. The storage period remained stable, but the timing of release after cell entry was slightly delayed compared to Example 1, and IFN-γ and IL-2 were slightly lower than the reasonable trend in Example 1.

[0129] 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 method of preparing a herpes zoster vaccine composition, characterized by, The method comprises the following steps: Step 1: uniformly mixing sterol and lipid in a solvent to obtain a sterol-lipid solution; uniformly mixing phospholipid in a solvent to obtain a phospholipid solution; uniformly mixing the sterol-lipid solution, the phospholipid solution and an immunostimulant solution, and then adding DSPE-PEG 2000 Maleimide to obtain a mixed solution; adding the mixed solution into a buffer solution to obtain a liposome dispersion by starting a continuous flow shear machine; passing the liposome dispersion through a high-pressure homogenizer; passing the homogenized sample through ultrafiltration dialysis and filtration; and storing the obtained filtrate to obtain a liposome adjuvant; Step 2: uniformly mixing the redissolved gE protein-containing solution and the liposome adjuvant to obtain a herpes zoster vaccine composition; The redissolved gE protein-containing solution is obtained by redissolving a gE protein-containing freeze-dried preparation in a histidine buffer; The preparation method of the gE protein-containing freeze-dried preparation comprises the following steps: taking water for injection in an ice bath at 2-8 DEG C, adding trehalose and mannitol, adjusting the pH with a histidine buffer, adding Tween-20, and then constant volume; standing at 0-4 DEG C for 30-60 min; adding gE protein under the condition of 2-8 DEG C, uniformly mixing to 0.1-0.3 mg / mL; filtering by using a low-protein adsorption PVDF / PES membrane, and freeze-drying to obtain the gE protein-containing freeze-dried preparation; The sterol is obtained by mixing cholestanol hemisuccinate and brassicasterol at a mass ratio of 2:1; The lipid is glyceryl dialkyl glyceryl tetraether; The phospholipid is obtained by mixing soybean lecithin and phosphatidylserine at a mass ratio of 2:0.4; The immunostimulant is GLA.

2. The method of preparing a zoster vaccine composition according to claim 1, wherein: The buffer is an aqueous solution containing 3-6 wt% sucrose, 8-12 mM histidine and 50-70 mM NaCl, and the pH is 6.1-6.

6.

3. The method of preparing a zoster vaccine composition according to claim 1, wherein: The immunostimulant solution is prepared by mixing an immunostimulant and ethanol.

4. The method of preparing a zoster vaccine composition according to claim 1, wherein The method comprises the following steps: Step 1: uniformly mixing 0.7 g cholestanol hemisuccinate, 0.35 g brassicasterol and 0.2 g glyceryl dialkyl glyceryl tetraether in 100 mL anhydrous ethanol to obtain a cholestanol hemisuccinate-brassicasterol solution; uniformly mixing 2 g soybean lecithin and 0.4 g phosphatidylserine in 100 mL anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; uniformly mixing the cholestanol hemisuccinate-brassicasterol solution, the soybean lecithin-phosphatidylserine solution and an immunostimulant solution, and then uniformly mixing 0.05 g DSPE-PEG 2000 Maleimide to obtain a mixed solution; adding the mixed solution into 1000 mL of a buffer solution by starting a continuous flow shear machine to obtain a liposome dispersion; passing the liposome dispersion through a high-pressure homogenizer, homogenizing 5 times at 800 bar, and then homogenizing 3 times at 1100 bar; performing ultrafiltration dialysis at an MWCO of 100 kDa to remove ethanol and concentrate; finally, constant volume with the buffer solution, the immunostimulant concentration is 50 µg / mL, and then 0.22 μm filtration; and storing the obtained filtrate in a 2-8 DEG C drug cool cabinet to obtain a liposome adjuvant. Step 2: mixing the reconstituted gE protein-containing solution with the liposome adjuvant to obtain a herpes zoster vaccine composition; wherein the reconstituted gE protein-containing solution and the liposome adjuvant are mixed at a volume ratio of 1:1, and the final volume of each dose is 0.50 mL, so that the immunostimulant is 12.5 µg / dose and the gE protein is 50 µg / dose; the buffer is an aqueous solution containing 5 wt% sucrose, 10 mM histidine, and 60 mM NaCl, and the pH is 6.5; the concentration of the immunostimulant solution is 25 mg / mL; the immunostimulant solution is prepared by mixing an immunostimulant and ethanol; and the immunostimulant is GLA; the reconstituted gE protein-containing solution is prepared by reconstituting a gE protein-containing lyophilized preparation with a 10 mM histidine buffer at pH 6.6 to a volume of 0.25 mL, and the gE protein is 0.2 mg / mL; the preparation method of the gE protein-containing lyophilized preparation is as follows: 200 mL of water for injection is placed in an ice bath at 2-8°C, 8% w / v trehalose, 2% w / v mannitol, and 10 mM histidine buffer are added to adjust the pH to 6.6, and 0.005% w / v Tween-20 is added to make up to 250 mL; 4°C for 30 min; under the condition of 2-8°C, gE protein is added and mixed to 0.20 mg / mL; avoid vigorous stirring and foaming throughout the process; 0.22 µm terminal sterile filtration is performed using a low-protein adsorption PVDF / PES membrane, and the full amount is collected after discarding the initial filtrate; 2 mL of a vial is selected, and 0.25 mL is dispensed per vial; a semi-inserted freeze-drying plug is used for freeze-drying treatment to obtain a gE protein-containing lyophilized preparation; the freeze-drying treatment process is pre-freezing at -45°C for 120 min; primary drying at -35°C for 24 h under 120 mTorr; and secondary drying at 20°C for 8 h at a temperature increase of 0.3°C / min, so that the residual moisture is ≤1.5%.

5. A zoster vaccine composition, characterized in that, The gE protein-containing lyophilized preparation is prepared by the preparation method of any one of claims 1-4.

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