Novel herpes zoster vaccine composition and preparation method thereof

Abstract

The invention discloses a novel herpes zoster vaccine composition and a preparation method, and belongs to the technical field of vaccines. The novel herpes zoster vaccine composition comprises gE protein and an adjuvant, the adjuvant is selected from at least one of an aluminum salt adjuvant, a saponin adjuvant, a TLR pathway agonist, an STING pathway agonist, an emulsion adjuvant and a liposome adjuvant. The invention also provides a preparation method of the composition. Compared with the prior art, the gE protein prepared by the method disclosed by the invention has the advantages that a protective matrix is jointly constructed by saccharides capable of forming a glassy state and a buffer system, and conformation is fixed through hydrogen bond replacement and vitrification in freezing and drying processes, so that interface-induced folding 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 the monomer state and immunogenicity are maintained after redissolution.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of vaccine technology, in particular to a novel zoster vaccine composition and a preparation method thereof. BACKGROUND

[0002] Varicella zoster virus (VZV) is one of the eight herpes viruses that infect humans, also known as human herpes virus-3 (HHV-3). The virus is the pathogen that causes varicella-zoster virus, and there is only one serotype. Varicella-zoster virus is widely distributed and highly contagious, and is induced by initial infection in childhood, and then latent in the human body, and can be reactivated when the human body immunity decreases to a certain threshold.

[0003] CN109200280A discloses a nano vaccine, a vaccine composition, a preparation method and application thereof. The nano vaccine and two vaccine compositions, the nano vaccine is DSPE-PEG2000-antigen peptide, the vaccine composition is composed of DSPE-PEG2000-CpG and the DSPE-PEG2000-antigen peptide in the above nano vaccine; and another vaccine composition is composed of DSPE-PEG2000-antigen peptide and adjuvant Montanid ISA 51VG ST. The application also provides a preparation method of the above vaccine composition and application of the nano vaccine and the vaccine composition in preparing tumor immunological drugs. The above nano vaccine and two vaccine compositions can effectively target the draining lymph nodes after entering the body, the materials required in the preparation method are all FDA-approved medical materials, and have clinical application potential; by loading the antigen and the adjuvant in one vaccine composition for common delivery, the carrier, the antigen and the adjuvant are integrated, the immune cells can be fully stimulated, and various downstream immune responses can be further enhanced.

[0004] Therefore, the present application provides a novel zoster vaccine composition and a preparation method thereof. SUMMARY

[0005] In view of the above defects of the prior art, the present application actually solves the technical problems of solving the structural stability and activity maintenance of the antigen in preparation, storage and reconstitution, avoiding the compatibility inactivation caused by the long-term coexistence of the antigen and the liposome, and improving the shelf life stability. Through optimizing the composition of the liposome membrane and the surface modification, the uptake and delivery efficiency of the antigen presenting cells are improved.

[0006] To achieve the above-mentioned purpose, the present application provides a novel zoster vaccine composition, characterized in that: comprising gE protein and adjuvant.

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

[0008] Further preferably, the novel herpes zoster vaccine composition can further comprise at least one of a buffer, a stabilizer, a surfactant, an isotonicity adjusting agent, a preservative, a chelating agent, and an antioxidant.

[0009] The present application also discloses a preparation method of the novel herpes zoster vaccine composition.

[0010] The preparation method of the novel herpes zoster vaccine composition comprises the following steps:

[0011] Step 1: uniformly mix sterol and lipid in a solvent to obtain a sterol-lipid solution; uniformly mix phospholipid in a solvent to obtain a phospholipid solution; uniformly mix the sterol-lipid solution, the phospholipid solution, and an immunostimulant solution, and then uniformly mix DSPE-PEG-TD1 to obtain a mixed solution; open a continuous flow shear machine to add the mixed solution into a buffer to obtain a liposome dispersion; pass the liposome dispersion through a high-pressure homogenizer; pass the homogenized sample through ultrafiltration dialysis and filtration; store the obtained filtrate to obtain a liposome adjuvant;

[0012] Step 2: uniformly mix the reconstituted gE protein-containing solution and the liposome adjuvant to obtain the novel herpes zoster vaccine composition.

[0013] Preferably, the preparation method of the novel herpes zoster vaccine composition comprises the following steps:

[0014] Step 1: uniformly mix 0.5-1.3 g of sterol and 0.1-0.3 g of lipid in 50-150 mL of anhydrous ethanol to obtain a sterol-lipid solution; uniformly mix 1-3.5 g of phospholipid in 80-150 mL of anhydrous ethanol to obtain a phospholipid solution; uniformly mix the sterol-lipid solution, the phospholipid solution, and an immunostimulant solution, and then uniformly mix 0.01-0.06 g of DSPE-PEG-TD1 to obtain a mixed solution; open a continuous flow shear machine to add the mixed solution into 800-1200 mL of a buffer to obtain a liposome dispersion; pass the liposome dispersion through a high-pressure homogenizer, homogenize 1-6 times at 500-1000 bar, and then homogenize 1-5 times at 1000-1200 bar; perform ultrafiltration dialysis at an MWCO of 100 kDa to remove ethanol and concentrate; finally, use a buffer to dilute the immunostimulant to a concentration of 10-50 µg / mL, and then filter through a 0.22 μm filter; store the obtained filtrate in a 2-8℃ pharmaceutical refrigerator to obtain a liposome adjuvant;

[0015] Step 2: mixing the gE protein-containing solution after reconstitution with the liposome adjuvant uniformly to obtain a novel herpes zoster vaccine composition; wherein the gE protein-containing solution after reconstitution and the liposome adjuvant are mixed according to a volume ratio of (1-3):(1-3), and the final volume per dose is 0.50 mL, so that the immunostimulant is 10-15 µg / dose, and the gE protein is 40-60 µg / dose.

[0016] The buffer is an aqueous solution containing 3-6 wt% sucrose, 8-12 mM histidine, and 50-70 mM NaCl, and has 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; and the immunostimulant is at least one of GLA, QS-21, 3M-052, CpG ODN, and cGAMP.

[0018] The gE protein-containing lyophilized preparation is prepared by the following method: taking 100-300 mL of water for injection in an ice bath at 2-8°C, adding 6-10% (w / v) trehalose, 1-3% (w / v) mannitol, adjusting the pH to 6.1-6.8 (25°C) with 8-12 mM histidine buffer, adding 0.001-0.006% (w / v) Tween-20, and making up to 200-300 mL; standing at 0-4°C for 30-60 min; adding gE protein under the condition of 2-8°C, mixing uniformly to 0.1-0.3 mg / mL; avoiding vigorous stirring and foaming throughout the process; using a low-protein adsorption PVDF / PES membrane for 0.22 µm terminal sterile filtration, collecting the whole amount of filtrate after discarding the first section; selecting a 2 mL vial, and dispensing 0.25 mL per vial; placing a half-inserted freeze-drying plug for freeze-drying treatment to obtain the gE protein-containing lyophilized preparation;

[0019] The freeze-drying treatment process is pre-freezing at -45°C to -40°C for 120-240 min; primary drying at 100-150 mTorr at -35 to -30°C for 16-24 h; and secondary drying at 0.1-0.3°C / min to 20-25°C for 6-10 h, so that the residual moisture is ≤1.5%.

[0020] The sterol is at least one of cholesterol hemisuccinate and brassicasterol;

[0021] The lipid is at least one of archaeal tetraether lipids and glycerol dialkyl glycerol tetraether lipids (GDGTs);

[0022] The phospholipid is at least one of soybean lecithin and phosphatidylserine;

[0023] The gE protein constructs a protective matrix with a sugar that can form a glass and a buffer system, freezes and fixes the conformation by hydrogen bond replacement and glassification during the freezing and drying process, reduces 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, and maintains the monomer state and immunogenicity after reconstitution.

[0024] The antigen is freeze-dried and shaped, and the adjuvant is stored in a liposome liquid, which blocks adsorption and deactivation caused by long-term physical contact, and avoids the influence of interfacial active substances in the lipid phase on the protein structure; instant mixing is used when using, only spatial co-localization occurs in vivo, which improves effective presentation without sacrificing shelf life stability.

[0025] Sterols and cholesterol derivatives regulate membrane order and surface electrical properties, prompting the insertion of amphiphilic GLA and its stable embedding by the bilayer; the charge and fluidity of the lipid phase change in an acidic microenvironment, which is beneficial to endosome membrane fusion and content release, and improves the accessibility of innate immune receptors.

[0026] Phosphatidylserine is endocytosed by macrophages and dendritic cells through recognition receptors; surface PEGylation provides steric hindrance to inhibit the formation of non-specific protein crown, reduces local aggregation, while retaining specific interaction with ligand peptides, and promotes directional migration along the injection site-lymph node axis.

[0027] Glycerol dialkyl glycerol tetraether lipids have a transmembrane monomolecular tetraether structure, which gives the membrane phase higher density, permeability resistance and oxidation resistance, and shear stability. When coexisting with phospholipids and sterols, glycerol dialkyl glycerol tetraether lipids tend to form a relatively ordered skeletal domain, which significantly inhibits the flux of water and small molecules across the membrane.

[0028] The beneficial effects of the present application are:

[0029] The gE protein constructs a protective matrix with a sugar that can form a glass and a buffer system, freezes and fixes the conformation by hydrogen bond replacement and glassification during the freezing and drying process, reduces 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, and maintains the monomer state and immunogenicity after reconstitution.

[0030] The antigen is freeze-dried and shaped, and the adjuvant is stored in a liposome liquid, which blocks adsorption and deactivation caused by long-term physical contact, and avoids the influence of interfacial active substances in the lipid phase on the protein structure; instant mixing is used when using, only spatial co-localization occurs in vivo, which improves effective presentation without sacrificing shelf life stability. DETAILED DESCRIPTION

[0031] The endpoints of the ranges and any values specified in the disclosure are not limited to the precise values recited. The ranges and values should be interpreted as being approximate, meaning they are intended to be a close approximation of the values and are equivalent to the values they approximate within normal experimental variability. For numerical ranges, the end points of the ranges are included within the range. The numerical ranges are intended to include any and all values within the range, including the end points. Any numerical range can be combined with any other numerical range to form a new numerical range.

[0032] Cholesterol hemisuccinate, Cat. No. CHO2003, purchased from Shenzhen Botai Biological Technology Co., Ltd.

[0033] Brassicasterol, CAS: 474-67-9, purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0034] DSPE-PEG-TD1 in the examples is DSPE-PEG(2000) Maleimide, Cat. No. 880126, purchased from Avanti Polar Lipids, Inc.

[0035] gE protein is His-tag purified human recombinant VZV gE, provided by Jiangsu Huano Tai Biological Medicine Technology Co., Ltd.

[0036] GLA is a glucopyranose lipid adjuvant, purchased from Avanti Polar Lipids, Inc. (Alabaster, AL); Product No. 699800.

[0037] Glycerol dialkyl glycerol tetraether lipid, Catalog #1303, purchased from Cayman Chemical.

[0038] The other raw materials not mentioned are common raw materials, and the above content is only for the purpose of helping to illustrate the present application, and should not be understood as a strict limitation of the present application. Those skilled in the art can directly purchase or prepare the same / similar raw materials.

[0039] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with specific embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0040] Example 1

[0041] A preparation method of a new herpes zoster vaccine composition, comprising the following steps:

[0042] Step 1: 0.7 g of cholestanol hemisuccinate, 0.35 g of brassicasterol, and 0.2 g of glycerol dialkyl glycerol tetraether were mixed uniformly in 100 mL of anhydrous ethanol to obtain a cholestanol hemisuccinate-brassicasterol solution; 2 g of soybean lecithin and 0.4 g of phosphatidylserine were mixed uniformly in 100 mL of anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; the cholestanol hemisuccinate-brassicasterol solution, the soybean lecithin-phosphatidylserine solution, and the immunostimulant solution were mixed uniformly, and then 0.05 g of DSPE-PEG-TD1 was added and mixed uniformly to obtain a mixed solution; the mixed solution was added to 1000 mL of a buffer solution by starting a continuous flow shear machine to obtain a liposome dispersion; the liposome dispersion was subjected to high-pressure homogenization by using 800 bar for 5 times and then 1100 bar for 3 times, and then ultrafiltration dialysis was performed with a MWCO of 100 kDa to remove ethanol and concentrate; finally, the buffer solution was used for constant volume, the concentration of the immunostimulant was 50 μg / mL, and then 0.22 μm filtration was performed; the obtained filtrate was stored in a 2-8℃ pharmaceutical refrigerator, and a liposome adjuvant was obtained;

[0043] Step 2: The reconstituted gE protein-containing solution was mixed uniformly with the liposome adjuvant to obtain a novel herpes zoster vaccine composition; the reconstituted gE protein-containing solution and the liposome adjuvant were mixed according to a volume ratio of 1:1, the final volume of each dose was 0.50 mL, the immunostimulant was 12.5 μg / dose, and the gE protein was 50 μg / dose.

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

[0045] 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.

[0046] The reconstituted gE protein-containing solution is prepared by reconstituting a gE protein-containing freeze-dried preparation to 0.25 mL with a pH = 6.6, 10 mM histidine buffer, and the gE protein is 0.2 mg / mL.

[0047] The preparation method of the gE protein-containing freeze-dried preparation is as follows:

[0048] Take 200 mL of water for injection in a 2-8℃ ice bath, add 8% (w / v) trehalose, 2% (w / v) mannitol, add 10 mM histidine buffer to adjust pH to 6.6 (25℃), add 0.005% (w / v) Tween-20, constant volume to 250 mL; 4℃ for 30 min; under the condition of 2-8℃, add gE protein, mix to 0.20 mg / mL; avoid vigorous stirring and foaming throughout the process; use low protein adsorption PVDF / PES membrane (pre-wet), perform 0.22 μm terminal sterile filtration, discard the first filtrate and collect the full amount; choose 2 mL of a syringe, each bottle is 0.25 mL; put into a half-inserted freeze-drying plug for freeze-drying treatment, get gE protein-containing freeze-dried preparation;

[0049] The freeze-drying process is pre-cooling at -45℃ for 120 min; one-time drying at -35℃ for 24 h under 120 mTorr; then secondary drying at 0.3℃ / min to 20℃ for 8 h, residual moisture ≤1.5%.

[0050] Example 2

[0051] A preparation method of a new herpes zoster vaccine composition, comprising the following steps:

[0052] Step 1: Mix 1.25 g of glyceryl dialkyl glyceryl tetraether fat in 100 mL of anhydrous ethanol to obtain a glyceryl dialkyl glyceryl tetraether fat solution; mix 2 g of soybean lecithin and 0.4 g of phosphatidylserine in 100 mL of anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; mix the glyceryl dialkyl glyceryl tetraether fat solution, the soybean lecithin-phosphatidylserine solution, and the immunostimulant solution uniformly, then add 0.05 g of DSPE-PEG-TD1 to obtain a mixed solution; open the continuous flow shear machine and add the mixed solution to 1000 mL of buffer to obtain a liposome dispersion; pass the liposome dispersion through a high-pressure homogenizer, homogenize 5 times at 800 bar, and then homogenize 3 times at 1100 bar; then perform ultrafiltration dialysis with a MWCO of 100 kDa to remove ethanol and concentrate; finally, use the buffer to constant volume to an immunostimulant concentration of 50 μg / mL, and then filter through a 0.22 μm filter; store the obtained filtrate in a 2-8℃ drug cool cabinet to obtain a liposome adjuvant;

[0053] Step 2: Mix the reconstituted gE protein-containing solution and the liposome adjuvant uniformly to obtain a new herpes zoster vaccine composition; 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 the immunostimulant is 12.5 μg / dose and the gE protein is 50 μg / dose.

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

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

[0056] The gE protein-containing solution after reconstitution is prepared by reconstituting a gE protein-containing lyophilized preparation with a pH = 6.6, 10 mM histidine buffer to 0.25mL, and the gE protein is 0.2mg / mL.

[0057] The preparation method of the gE protein-containing lyophilized preparation is as follows:

[0058] Take 200 mL of water for injection in a 2-8℃ ice bath, add 8% (w / v) trehalose, 2% (w / v) mannitol, adjust the pH to 6.6 (25℃) with 10mM histidine buffer, add 0.005% (w / v) Tween-20, and dilute to 250 mL; 4℃ for 30min; add gE protein under 2-8℃ conditions, mix to 0.20 mg / mL; avoid vigorous stirring and foaming throughout the process; use low protein adsorption PVDF / PES membrane (pre-wet) for 0.22μm terminal sterile filtration, discard the first filtrate and collect the full amount; choose 2mL of a syringe, each containing 0.25 mL; insert a semi-inserted freeze-drying plug for freeze-drying treatment, and obtain a gE protein-containing lyophilized preparation;

[0059] The freeze-drying process is as follows: pre-freeze at -45℃ for 120 min; primary drying at -35℃ for 24 h under 120 mTorr; secondary drying at 20℃ for 8 h with a temperature rise of 0.3℃ / min, so that the residual moisture is ≤1.5%.

[0060] Example 3

[0061] A preparation method of a novel herpes zoster vaccine composition, comprising the following steps:

[0062] Step 1: 0.8 g of cholestanol hemisuccinate and 0.45 g of brassicasterol were mixed uniformly in 100 mL of anhydrous ethanol to obtain a cholestanol hemisuccinate-brassicasterol solution; 2 g of soybean lecithin and 0.4 g of phosphatidylserine were mixed uniformly in 100 mL of anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; the cholestanol hemisuccinate-brassicasterol solution, the soybean lecithin-phosphatidylserine solution, and the immunostimulant solution were mixed uniformly, and then 0.05 g of DSPE-PEG-TD1 was added and mixed uniformly to obtain a mixed solution; the mixed solution was added to 1000 mL of a buffer solution by starting a continuous flow shear machine to obtain a liposome dispersion; the liposome dispersion was subjected to high-pressure homogenization by using a high-pressure homogenizer, 800 bar for 5 times, and 1100 bar for 3 times, and then ultrafiltration dialysis MWCO 100 kDa was performed to remove ethanol and concentrate, and finally the buffer solution was used for constant volume, the immunostimulant concentration was 50 μg / mL, and then 0.22 μm filtration was performed; the obtained filtrate was stored in a 2-8℃ pharmaceutical refrigerator, and a liposome adjuvant was obtained;

[0063] Step 2: The reconstituted gE protein-containing solution was mixed uniformly with the liposome adjuvant to obtain a novel herpes zoster vaccine composition; the reconstituted gE protein-containing solution and the liposome adjuvant were mixed according to a volume ratio of 1:1, the final volume of each dose was 0.50 mL, the immunostimulant was 12.5 μg / dose, and the gE protein was 50 μg / dose.

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

[0065] 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.

[0066] The reconstituted gE protein-containing solution is prepared by reconstituting a gE protein-containing freeze-dried preparation to 0.25 mL with a pH = 6.6, 10 mM histidine buffer solution, and the gE protein is 0.2 mg / mL.

[0067] The preparation method of the gE protein-containing freeze-dried preparation is as follows:

[0068] Take 200 mL of water for injection in a 2-8℃ ice bath, add 8% (w / v) trehalose, 2% (w / v) mannitol, add 10 mM histidine buffer to adjust pH to 6.6 (25℃), add 0.005% (w / v) Tween-20, constant volume to 250 mL; 4℃ for 30 min; under the condition of 2-8℃, add gE protein, mix to 0.20 mg / mL; avoid vigorous stirring and foaming throughout the process; use low protein adsorption PVDF / PES membrane (pre-wet), perform 0.22 μm terminal sterile filtration, discard the first filtrate and collect the full amount; choose 2 mL of a test tube, each containing 0.25 mL; insert a half-insert freeze-drying plug for freeze-drying treatment, and obtain a gE protein-containing freeze-dried preparation;

[0069] The freeze-drying treatment process is pre-freezing at -45℃ for 120 min; one-time drying at -35℃ for 24 h under 120 mTorr; then secondary drying at 0.3℃ / min to 20℃ for 8 h, with residual moisture ≤1.5%.

[0070] Example 4

[0071] Basically the same as Example 1, the only difference is that no Tween-20 is added to the gE protein-containing freeze-dried preparation.

[0072] Example 5

[0073] Basically the same as Example 1, the only difference is that 10% sucrose is used to replace trehalose 8% and mannitol 2% in the gE protein-containing freeze-dried preparation.

[0074] Example 6

[0075] Basically the same as Example 1, the only difference is that no phosphatidylserine is added.

[0076] Example 7

[0077] Basically the same as Example 1, the only difference is that 1.05 g of cholesterol is used to replace 0.7 g of cholesterol hemisuccinate and 0.35 g of campesterol.

[0078] Example 8

[0079] Basically the same as Example 1, the only difference is that no DSPE-PEG-TD1 is added.

[0080] Example 9

[0081] Basically the same as Example 1, the only difference is that gE protein is used instead of gE protein-containing freeze-dried preparation.

[0082] Example 10

[0083] A method for preparing a novel herpes zoster vaccine composition, comprising the following steps:

[0084] Step 1: 0.7 g of cholestanol hemisuccinate, 0.35 g of brassicasterol, and 0.2 g of glycerol dialkyl glycerol tetraether lipid are mixed in 100 mL of anhydrous ethanol to obtain a cholestanol hemisuccinate-brassicasterol solution; 2 g of soybean lecithin and 0.4 g of phosphatidylserine are mixed in 100 mL of anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; the cholestanol hemisuccinate-brassicasterol solution and the soybean lecithin-phosphatidylserine solution are mixed uniformly, and then 0.05 g of DSPE-PEG-TD1 is added and mixed uniformly to obtain a mixed solution; the mixed solution is added to 1000 mL of a buffer solution by starting a continuous flow shear machine to obtain a liposome dispersion; the liposome dispersion is filtered through a high-pressure homogenizer, and is subjected to 800 bar homogenization for 5 times and 1100 bar pressure homogenization for 3 times; the ethanol is removed by ultrafiltration dialysis with a MWCO of 100 kDa, and then concentrated; and the obtained filtrate is filtered through a 0.22 μm filter; the obtained filtrate is mixed with an immunostimulant solution to make the concentration of the immunostimulant 50 μg / mL, and the mixture is stirred uniformly by a magnetic stirrer; and the final product is stored in a 2-8℃ medicine cool cabinet to obtain a liposome adjuvant.

[0085] Step 2: The reconstituted gE protein-containing solution is mixed with the liposome adjuvant uniformly to obtain a novel herpes zoster vaccine composition; the reconstituted gE protein-containing solution and the liposome adjuvant are mixed according to a volume ratio of 1:1, the final volume of each dose is 0.50 mL, the immunostimulant is 12.5 μg / dose, and the gE protein is 50 μg / dose.

[0086] The buffer is an aqueous solution containing 5wt% sucrose, 10 mM histidine, and 60 mM NaCl, and has a pH of 6.5.

[0087] 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.

[0088] The reconstituted gE protein-containing solution is prepared by reconstituting a gE protein-containing freeze-dried preparation in 0.25 mL of a pH = 6.6, 10 mM histidine buffer, and the gE protein is 0.2 mg / mL.

[0089] The preparation method of the gE protein-containing freeze-dried preparation is as follows:

[0090] Take 200 mL of water for injection in a 2-8℃ ice bath, add 8% (w / v) trehalose, 2% (w / v) mannitol, add 10 mM histidine buffer to adjust the pH to 6.6 (25℃), add 0.005% (w / v) Tween-20, and make up to 250 mL; 4℃ for 30 min; under the condition of 2-8℃, add gE protein, mix to 0.20 mg / mL; avoid vigorous stirring and foaming throughout the process; use low protein adsorption PVDF / PES membrane (pre-wet), perform 0.22 μm terminal sterile filtration, discard the first filtrate and collect the full amount; choose 2 mL of a syringe, each containing 0.25 mL; insert into a semi-insert freeze-drying plug for freeze-drying treatment, and obtain a gE protein-containing freeze-dried preparation;

[0091] The freeze-drying treatment process is pre-freezing at -45℃ for 120 min; one-time drying at -35℃ for 24 h under 120 mTorr; and then secondary drying at 0.3℃ / min to 20℃ for 8 h, with residual moisture ≤1.5%.

[0092] Example 11

[0093] A preparation method of a new herpes zoster vaccine composition, comprising the following steps:

[0094] Step 1: 0.7 g of cholesteryl hemisuccinate, 0.35 g of brassicasterol, and 0.2 g of glycerol dialkyl glycerol tetraether fat are mixed uniformly in 100 mL of anhydrous ethanol to obtain a cholesteryl hemisuccinate-brassicasterol solution; 2 g of soybean lecithin and 0.4 g of phosphatidylserine are mixed uniformly in 100 mL of anhydrous ethanol to obtain a soybean lecithin-phosphatidylserine solution; the cholesteryl hemisuccinate-brassicasterol solution, the soybean lecithin-phosphatidylserine solution, and the immunostimulant solution are mixed uniformly, and then 0.05 g of DSPE-PEG-TD1 is added and mixed uniformly to obtain a mixed solution; the mixed solution is added to 1000 mL of buffer solution by starting a continuous flow shear machine to obtain a liposome dispersion; the liposome dispersion is subjected to high-pressure homogenization by a high-pressure homogenizer at 800 bar for 5 times and at 1100 bar for 3 times, and then ultrafiltration dialysis is performed at a MWCO of 100 kDa to remove ethanol and concentrate; finally, the buffer solution is used to make up the immunostimulant concentration to 50 μg / mL, and then 0.22 μm filtration is performed; the obtained filtrate is stored in a 2-8℃ drug cool cabinet to obtain a liposome adjuvant;

[0095] Step 2: The gE protein is mixed with the liposome adjuvant uniformly, and is placed into a semi-inserted freeze-drying plug for freeze-drying treatment, wherein the freeze-drying treatment process is pre-cooling at -45°C for 120 min; one-time drying at -35°C for 24 h under 120 mTorr; and then secondary drying at 0.3°C / min to 20°C for 8 h, so that the residual moisture is ≤1.5%, to obtain a novel herpes zoster vaccine composition; wherein the final volume of each dose is 0.50 mL, and the immunostimulant is 12.5 μg / dose, and the gE protein is 50 μg / dose.

[0096] The buffer is an aqueous solution containing 5wt% sucrose, 10 mM histidine, and 60 mM NaCl, and the pH is 6.5.

[0097] 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.

[0098] Test Example 1

[0099] The particle size and polydispersity index of the liposome adjuvant are detected by using a nano particle size detector Zetasizer Nano ZS paricle size analyzer (Malvern, UK).

[0100] Table 1 Particle size and polydispersity index of the liposome adjuvant

[0101]

[0102] From Table 1, the particle size and its polydispersity index in Example 1 are the lowest, and the dispersity is the best. Example 3 removes glyceryl dialkyl glyceryl tetraether fat on the basis of Example 1, that is, lacks a transmembrane tetraether monomolecular skeleton, and the shear resistance and permeation resistance of the bilayer are reduced, the continuity is reduced, and the interfacial line tension is weakened, resulting in that the particles are more easily affected by transient energy fluctuations during shear forming, the PDI is increased, and the overall performance is that the particle size is slightly larger and the PDI is higher than that of Example 1. In Example 2, no cholestanol hemisuccinate or brassicasterol is added, the hydrophobic core loses rigid filling and continuity is reduced, the membrane surface line tension and bending modulus are reduced; the particles are more sensitive to transient shear energy during granulation, and are prone to secondary coalescence and size floating, the PDI is increased, and even if the glyceryl dialkyl glyceryl tetraether fat exists, it is difficult to replace the compaction of the sterol on the hydrophobic core and the ordering effect of the head group region, so the particle size and PDI are higher than those of Example 1. Example 7 uses cholestanol to replace cholestanol hemisuccinate and brassicasterol, and cholestanol can densify the membrane phase, but the weak acid head group of cholestanol hemisuccinate and the stronger compaction brought by the C-24 side chain of brassicasterol are missing, the hydration and surface electrical property regulation of the head group region are insufficient, and the microphase is more heterogeneous; after homogenization, the particle size and PDI are usually higher than those of Example 1. Example 8 lacks DSPE-PEG-TD1, the steric hindrance and protein resistance are reduced, the probability of short-range coalescence is increased, the influence on the particle size is slight floating, and the influence on the PDI is more significant; the difference between Example 10 and Example 1 is that the immunostimulant is finally added, and the post-loading causes post-insertion and rearrangement in the membrane, the diffusion and local domain reconstruction of the membrane surface increase in a short time, and generally, the particle size is slightly larger than that of Example 1, and the PDI is slightly increased, but it is still in the stable dispersion interval.

[0103] Test Example 2

[0104] Test the freeze-dried preparation containing gE protein in Example 1, Example 4-5, 10 samples for each example.

[0105] 1. Appearance test

[0106] Visual inspection: observe on the light inspection table along 360° slowly: cake integrity (determination criteria are complete, slight shrinkage, collapse, crack), color;

[0107] Photograph: photograph for archiving, same light and background;

[0108] 2. Residual water (Karl Fischer method, KF)

[0109] System suitability: calibrate with 1.0 mg / g pure water standard solution, and the recovery is 95-105%.

[0110] Blank: blank crucible / empty sample boat for blank, the reading should be ≤5 µg;

[0111] Determination: Weigh 50-100 mg of the lyophilized block, put it into a heating furnace and heat to 60-80℃, record the µg of moisture after the reading is stable;

[0112] Calculation: Moisture (%) = (µg of moisture / sample mass mg) x 100%;

[0113] 3. DSC test

[0114] Tg (lyophilized preparation): Take 5-8 mg of the lyophilized block and seal it; heat it to 120-140℃ at a rate of 10℃ / min (to avoid sample decomposition); read the Tg.

[0115] 4. Resuspension time:

[0116] (1) Slowly inject 0.25 mL of water for injection along the bottle wall, and start timing as soon as the liquid is injected; (2) Manually invert every 2 s, with an amplitude of 180°; (3) End point determination: no visible particles or floccules, and stable liquid surface without streaks; (4) Record the stopwatch time (s).

[0117] Table 2. Results of appearance test of lyophilized preparations containing gE protein

[0118]

[0119] Test Example 3

[0120] The new herpes zoster vaccine composition provided in the examples was taken as the detection object, and the induction levels of IL-2 and IFN-γ of the vaccine composition were detected.

[0121] Control group: PBS buffer solution was used as control group 1.

[0122] The detection method is as follows: 6-week-old female Balb / c mice were randomly divided into groups, 10 in each group, and the new herpes zoster vaccine composition provided in the examples and the samples of the control group were diluted 10 times, i.e. 1 / 10 of the content of each composition was taken and supplemented with PBS to 0.1 mL, and each group of mice was subcutaneously injected with 0.1 mL. Each group of mice was immunized twice with an interval of 2 weeks.

[0123] The mice were sacrificed by cervical dislocation 14 days after the second immunization, and transferred to a biological safety cabinet by 75% alcohol immersion. The mouse spleen was isolated and placed on a 200 mesh cell screen, 2 mL of PBS buffer was added dropwise, and a 5 mL syringe handle was used to grind on the 200 mesh cell screen until there was no obvious tissue. After repeatedly washing the screen, the suspension was collected and transferred to a 15 mL centrifuge tube, and centrifuged at 300g for 10 min. The supernatant was discarded, 4 mL of ACK lysis buffer was added, mixed by blowing and placed for 3-5 min, and centrifuged at 300g for 10 min. The supernatant was discarded, 4 mL of RPMI 1640 medium containing 10% FBS was added, mixed by blowing, and centrifuged at 300g for 10 min. The supernatant was discarded, 4 mL of RPMI 1640 medium containing 10% FBS was added, mixed by blowing and counted by a cell counter. Each group of cells was diluted to 2x10 6 cells / mL, and pre-activated IFN- γ and IL-2 were added to the PVDF plate for detection (Mabtech), 50 μL per well. Negative control, gE protein stimulation (10 μg / mL) and positive control (PHA) were set. The negative control was supplemented with medium to 100 μL / well, and the gE protein stimulation and positive control were diluted to 20 μg / mL with medium, then 50 μL was added to each well, and 3 replicate wells were made for each sample. Incubate the cells at 37°C, 5% CO2 for 48h. Pour the liquid in the hole, wash 5 times with 200 μL PBS per well, then add diluted biotin-labeled antibody, 100 μL per well, and incubate at room temperature for 2h. Pour the liquid in the hole, wash 5 times with 200 μL PBS per well, then add diluted ALP-labeled streptavidin, 100 μL per well, and incubate at room temperature for 1h. Pour the liquid in the hole, wash 5 times with 200 μL PBS per well, then add 100 μL of filtered NCIP / NBT-plus solution per well, and develop for 5-30 min until obvious spots appear, and rinse with deionized water to stop color development. The IL-2 and IFN- γ levels in each test group were detected by ELISPOT method.

[0124] Table 3 IL-2 and IFN- γ test results of new shingles vaccine compositions

[0125]

[0126] As can be seen from Table 3, Example 1 uses cholesterol hemisuccinate, brassicasterol, glycerol dialkyl glycerol tetraether lipid to improve compactness and reduce basal leakage, and cholesterol hemisuccinate triggers membrane remodeling and fusion in a weak acid environment, so that the effective exposure of GLA in the endosome-lysosome pathway is enhanced, and the Th1 biased activation of the TLR4 pathway is promoted, so that the readings of IL-2 and IFN-γ are significantly higher than those of the PBS control. The introduction of GLA in Example 10 causes post-insertion and rearrangement, and the in-membrane positioning is slightly shallower than co-assembly, and the domain remodeling time is slightly longer; the storage period is still stable, but the timing of triggering release after entering the cell is slightly delayed, and IFN-γ and IL-2 are slightly lower than those of Example 1, which is a reasonable trend.

[0127] The preferred embodiments of the present application are described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and changes without creative labor based on the concept of the present application. Therefore, any technical solutions obtained by logical analysis, reasoning or limited experiments based on the prior art within the concept of the present application shall be within the protection scope defined by the claims.

Claims

1. A method for preparing a novel herpes zoster vaccine composition, characterized in that, Includes the following steps: Step 1: Add sterols and lipids to a solvent and mix thoroughly to obtain a sterol-lipid solution; add phospholipids to a solvent and mix thoroughly to obtain a phospholipid solution; After thoroughly mixing the sterol-lipid solution, phospholipid solution, and immunostimulant solution, add DSPE-PEG-TD1 and mix thoroughly to obtain a mixed solution. Turn on the continuous flow shear machine to add the mixed solution to the buffer to obtain a liposome dispersion. Pass the liposome dispersion through a high-pressure homogenizer. The homogenized sample is then subjected to ultrafiltration dialysis and filtered. The obtained filtrate is stored to obtain the liposome adjuvant. Step 2: Mix the reconstituted gE protein solution with liposome adjuvant until homogeneous to obtain a novel herpes zoster vaccine composition.

2. The method for preparing the novel herpes zoster vaccine composition as described in claim 1, characterized in that: 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.

3. The method for preparing the novel herpes zoster vaccine composition as described in claim 1, characterized in that: The immunostimulant solution is prepared by mixing an immunostimulant and ethanol.

4. The method for preparing the novel herpes zoster vaccine composition as described in claim 3, characterized in that: The immunostimulant is at least one of GLA, QS-21, 3M-052, CpG ODN, and cGAMP.

5. The method for preparing the novel herpes zoster vaccine composition as described in claim 4, characterized in that: The reconstituted solution containing gE protein was prepared by reconstituted a lyophilized formulation containing gE protein using histidine buffer.

6. The method for preparing the novel herpes zoster vaccine composition as described in claim 5, characterized in that: The preparation method of the lyophilized formulation containing gE protein is as follows: Take water for injection and place it in an ice bath at 2-8℃, add trehalose and mannitol, adjust the pH with histidine buffer, add Tween-20, and make up to volume; 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; filter using a low protein adsorption PVDF / PES membrane, and lyophilize to obtain the lyophilized formulation containing gE protein.

7. The method for preparing the novel herpes zoster vaccine composition according to claim 1, characterized in that: The sterol is at least one of cholesterol hemisuccinate and rapeseed sterol.

8. The method for preparing the novel herpes zoster vaccine composition as described in claim 1, characterized in that: The lipid is at least one of archaeal tetraether lipids and dialkyl glycerol tetraether lipids (GDGTs).

9. A novel shingles vaccine composition, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

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