A vaccine adjuvant, its preparation method and application

Abstract

A vaccine adjuvant, its preparation method and application. The vaccine adjuvant is a MA105 immune adjuvant, which comprises: (1) QS-21, 50-300 μg / ml; (2) Poly I:C, 400-3000 μg / ml; and (3) a lipid molecule constituting a carrier; the carrier is a mixture of cationic liposomes and neutral liposomes.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical biotechnology, specifically relating to a vaccine adjuvant, its preparation method, and its application. Background Technology

[0002] 1. Immune adjuvants

[0003] In immunology, an adjuvant is a component that enhances and / or modulates an immune response to an antigen. An immune adjuvant is defined as any substance that, when bound to a specific vaccine antigen, accelerates, prolongs, or enhances an antigen-specific immune response. Many known adjuvants are widely used, including oils, aluminum salts, and viroids. It is generally believed that adjuvants that activate cellular immunity (especially CD4+ T cells) produce more effective therapeutic effects.

[0004] 1.1, QS21 adjuvant

[0005] The structural formula of saponin 21 (QS21) is as follows: molecular formula: C92H148O46, molecular weight: 1990.14. It is generally a colorless and transparent liquid.

[0006]

[0007] In cancer patients, QS-21 exhibits optimal activity and good tolerability at doses ranging from 100 to 200 μg. Toxicity within this dose range is characterized by erythema and induration of 2–10 cm at the injection site in most patients, and occasionally by mild, low-grade flu-like symptoms. No dose-limiting toxicities have been reported within this dose range. QS-21 is a novel pharmaceutical adjuvant with a high safety profile and good tolerability.

[0008] 1.2 Liposomes

[0009] Liposomes are artificial cell membrane-like spheres composed of lipids. They have a single- or multi-layered bilayer vesicle structure mainly composed of phosphate lipids, cholesterol, stearylamine, etc., and can encapsulate various vaccines, effectively introducing them into cells. In vivo, they tend to deposit within the reticuloendothelial-macrophage system, including the liver, spleen, and lymph nodes, prolonging their residence time and thus reducing vaccine dosage, minimizing toxicity and side effects, and enhancing immune function. They exhibit significant adjuvant and carrier effects for various antigens and antibodies, making them one of the more ideal vaccine adjuvant substances.

[0010] 1.3 Immune adjuvants and tumor immunotherapy

[0011] Immunotherapy (including immune checkpoint inhibitor antibodies, immunotherapy, and anti-tumor vaccines) is currently a trend and a hot topic in clinical cancer treatment. However, current cancer immunotherapies still have many limitations. These include low tumor vaccine release efficiency, impaired cross-presentation of tumor-specific antigens, and the effects of a series of immunosuppressive cytokines.

[0012] In the development of tumor vaccine products, immune adjuvants play a crucial role in enhancing the immunogenicity of tumor antigens and activating the body's adaptive immune response. As substances that enhance the body's adaptive immune response to antigens, adjuvants can compensate for the weak immunogenicity of tumor antigens, strengthen the immune response to tumor antigens, and effectively prevent immune tolerance induced by immature antigen-presenting cells (APCs) upon contact with antigens, thereby improving the immunotherapeutic efficiency of tumor vaccines.

[0013] Therefore, research on adjuvants in tumor immunotherapy has developed rapidly. In addition to traditional aluminum salt adjuvants, novel adjuvants such as pattern recognition receptor agonists (such as Toll-like receptor agonists and C-type agglutinating receptor agonists), polymer materials, and peptides have been found to have good immune activation effects.

[0014] 2. Shingles vaccine

[0015] In 2017, the Advisory Committee on Immunization Practice (ACIP) of the U.S. Centers for Disease Control and Prevention (CDC) issued an opinion recommending GlaxoSmithKline's recombinant subunit vaccine. Replacement of Merck's live attenuated vaccine For immunization of people aged 50 and above.

[0016] Merck's varicella-zoster live attenuated vaccine The protective efficacy of this vaccine gradually decreases with age. Phase III clinical trial results showed that the overall protection rate against shingles was approximately 64% in individuals aged 60-69, but dropped to 38% in those over 70 years of age. Furthermore, this vaccine cannot be used in individuals with compromised immune systems and will not provide effective protection. (GSK manufactures this vaccine.) It is a recombinant subunit vaccine, and its core technology originates from AS01. B The adjuvant system and specific antigens, along with Phase III clinical trial results, showed that the vaccine's overall efficacy was 97.2%, and its protection rate reached 97.5% in individuals aged ≥70 years. Simultaneously, it was found... The number of antigen-specific CD4+ T cells induced is approximately It induces 10 times the number of cells.

[0017] While aluminum adjuvant is a commonly used vaccine adjuvant, studies have shown that it primarily induces Th2 cell-mediated humoral immune responses, but not Th1 cell-mediated cellular immune responses (it cannot induce the expression of cytokines such as IL-2 and INF-γ associated with cellular immunity). Merck's inactivated varicella-zoster vaccine formulated with aluminum adjuvant failed to achieve the expected immunogenicity and was ultimately not marketed. This further illustrates that adjuvants capable of inducing cellular immune responses (especially CD4+ T cells) are a key factor in the high protective efficacy of varicella-zoster vaccines.

[0018] Based on this, the present invention relates to a novel MA105 adjuvant system, its preparation method, and the application of the adjuvant in the preparation of therapeutic products, wherein the therapeutic products include:

[0019] (1) Recombinant herpes zoster vaccine (CHO cell) preparation;

[0020] (2) Immune adjuvants that activate tumor immunity. Summary of the Invention

[0021] This invention first relates to an MA105 immune adjuvant, comprising:

[0022] (1) QS-21, 50-300μg / ml;

[0023] (2) Poly I:C, 400-3000 μg / ml; and

[0024] (3) Lipid molecules that constitute the common carrier of adjuvants and antigens;

[0025] The carrier is a liposome carrier, which can be a cationic liposome, a neutral liposome, or a mixture of both.

[0026] Preferably, the carrier is a mixture of cationic liposomes and neutral liposomes;

[0027] Preferably, the MA105 adjuvant comprises:

[0028] (1) QS-21, 50-200 μg / ml;

[0029] (1)Poly I:C, 400-3000μg / ml;

[0030] The lipid molecules are:

[0031] (3) DOTAP, 70-560 μg / ml;

[0032] (4) DOPC, 500-4000 μg / ml;

[0033] (5) Cholesterol, 175-1400 μg / ml.

[0034] More preferably, the MA105 adjuvant comprises:

[0035] (1) QS-21, 80-120 μg / ml;

[0036] (2)Poly I:C, 780-1600μg / ml;

[0037] The lipid molecules are:

[0038] (3) DOTAP, 140-280 μg / ml;

[0039] (4) DOPC, 1200-2500 μg / ml;

[0040] (5) Cholesterol, 350-600 μg / ml.

[0041] This invention also relates to a method for preparing the aforementioned MA105 adjuvant, comprising the following steps:

[0042] (1) Prepare cationic liposomes and neutral liposomes respectively;

[0043] (2) QS-21, Poly I:C, cationic liposomes and neutral liposomes were mixed and stirred evenly to prepare MA105 adjuvant.

[0044] The method for preparing the cationic liposomes is as follows:

[0045] (1) It is prepared by mixing 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC) and cholesterol in a mass ratio of 2:2:1;

[0046] (2) After the three lipid components are evenly dispersed, liposomes are prepared by ethanol injection method, thin film dispersion method, ultrasonic dispersion method and reverse evaporation method.

[0047] Preferably, the preparation is carried out using the ethanol injection method. The preparation method is as follows: after the three lipid components are evenly dispersed, the colostrum is prepared in the aqueous phase. After ultrafiltration to replace the buffer, the colostrum is extruded and shaped using extrusion membranes with pore sizes of 200 nm and 100 nm in sequence. Finally, the colostrum is filtered through a sterile filter to remove bacteria.

[0048] More preferably, the preparation steps of the cationic liposomes are as follows:

[0049] (1) The three lipid components are dissolved in ethanol and sheared online 1 to 3 times with the aqueous phase;

[0050] (2) Use a sucrose-containing buffer solution for ultrafiltration replacement, with a concentration factor of 2 to 4 times and a washing factor of 6 to 8 times;

[0051] (3) Use a filter membrane with a pore size of 200nm for extrusion, and perform multiple cycles of extrusion to control particle size uniformity;

[0052] (4) Use a filter membrane with a pore size of 100 nm for extrusion, and perform multiple cycles of extrusion to control particle size uniformity;

[0053] (5) Use a sterile filter to filter and sterilize.

[0054] The physicochemical parameters of the cationic liposomes are as follows:

[0055] Average particle size (D50): 30–90 nm;

[0056] Solution pH: 4.8–7.0; preferably 4.8–6.0

[0057] Zeta potential: 40–72 mV (pH 6.5).

[0058] The method for preparing the neutral liposomes is as follows:

[0059] (1) It is prepared by mixing dioleoylphosphatidylcholine (DOPC) and cholesterol in a mass ratio of 4:1;

[0060] (2) After the lipid components are evenly dispersed, liposomes are prepared using ethanol injection method, thin film dispersion method, ultrasonic dispersion method and reverse evaporation method;

[0061] Preferably, the preparation is carried out using the ethanol injection method. The preparation method is as follows: after uniform dispersion, a primary emulsion is prepared in an aqueous phase, and then extruded and shaped using extrusion membranes with pore sizes of 200 nm and 100 nm in sequence. Subsequently, ultrafiltration is performed to replace the buffer and filter for sterilization to obtain the final product.

[0062] More preferably, the preparation steps of the neutral liposomes are as follows:

[0063] (1) The two lipid components are dissolved in ethanol and sheared online with the aqueous phase 1 to 3 times;

[0064] (2) Use a filter membrane with a pore size of 200nm for filtration and perform multiple cyclic extrusions to control particle size uniformity;

[0065] (3) Use a filter membrane with a pore size of 100nm for filtration and perform multiple cyclic extrusions to control particle size uniformity;

[0066] (4) Use a sucrose-containing buffer solution for ultrafiltration replacement, with a concentration factor of 3 to 6 times and a washing factor of 6 to 8 times;

[0067] (5) Use a sterile filter to filter and sterilize.

[0068] The physicochemical parameters of the neutral liposomes are as follows:

[0069] Average particle size (D50): 80–140 nm;

[0070] Solution pH: 5.5-7.0, preferably 6.4-6.6.

[0071] The MA105 adjuvant system contains two immunostimulants (QS-21 and Poly I:C). To reduce the toxicity of QS-21 and Poly I:C and improve immunogenicity, a mixture of cationic and neutral liposomes is used to adsorb QS-21 and Poly I:C, or a mixture of cationic liposomes and neutral liposomes adsorbed with QS-21 is used to further adsorb Poly I:C. Poly I:C signaling primarily relies on Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA-5), strongly driving cellular immunity and effective type I interferon responses. It can also induce strong antigen-specific CD4+ T and CD8+ T cell responses through type I interferon (IFN-α / β) signaling and antigen cross-presentation. We use cationic liposomes (composed of DOTAP, DOPC, and cholesterol) as carriers to adsorb Poly I:C via electrostatic interactions.

[0072] This invention also relates to the application of the MA105 adjuvant in the preparation of vaccine formulations, wherein the vaccine formulation contains a therapeutically effective amount of antigen and the MA105 adjuvant, as well as necessary pharmaceutical excipients; preferably, the vaccine formulation is a recombinant herpes zoster vaccine formulation.

[0073] The pharmaceutical excipients mentioned include, but are not limited to, polysorbate 80, histidine, sodium dihydrogen phosphate, sucrose, and sodium chloride.

[0074] The present invention also relates to a method for preparing a recombinant herpes zoster vaccine formulation using the aforementioned MA105 adjuvant system, the method comprising the following steps:

[0075] (1) Add sucrose, histidine and Tween-80 to the purified antigen solution according to the formulation of the antigen buffer, and adjust the pH to obtain the antigen stock solution;

[0076] (2) The antigen stock solution and MA105 adjuvant are mixed and stirred evenly to prepare a semi-finished product;

[0077] (3) After repackaging, the recombinant herpes zoster vaccine formulation is obtained.

[0078] The antigen stock solution contains: gE protein antigen with the sequence shown in SEQ ID NO.1 (molecular weight 63530.6, isoelectric point 4.8-5.8);

[0079] SEQ ID NO.1:

[0080]

[0081] Preferably, per milliliter of vaccine formulation, the recombinant herpes zoster vaccine formulation prepared with the MA105 adjuvant system comprises:

[0082] (1) gE protein antigen with the sequence shown in SEQ ID NO.1, in a concentration of 95-110 ug / ml;

[0083] (2) A pharmaceutical excipient assembly for maintaining the stability of gE protein antigen and liposomes, said pharmaceutical excipient assembly comprising:

[0084] 1) Polysorbate 80, 450-550 μg / ml;

[0085] 2) Histidine, 200-950 μg / ml;

[0086] 3) Sucrose, 52-55 mg / ml;

[0087] (3) The MA105 immune adjuvant system, which includes:

[0088] 1) DOTAP, 140-215 μg / ml;

[0089] 2) DOPC, 1200-2200 μg / ml;

[0090] 2) Cholesterol, 320-550 μg / ml;

[0091] 3) QS-21, 80-110 μg / ml;

[0092] 4) Poly I:C, 780-830 μg / ml;

[0093] (4) Other pharmaceutical excipients, including:

[0094] 1) Sodium dihydrogen phosphate, 0-800 μg / ml;

[0095] 2) Sodium chloride, 2.5-6 mg / ml.

[0096] Furthermore,

[0097] The osmotic pressure of the vaccine formulation is 210–350 mOsmol / kg;

[0098] The average particle size of the vaccine formulation is 100-300 nm, preferably 130-200 nm;

[0099] The pH value of the vaccine preparation is 6.0 to 7.0.

[0100] The present invention also relates to a recombinant herpes zoster vaccine formulation prepared by the method described above.

[0101] The present invention also relates to the following applications of the recombinant herpes zoster vaccine formulation: treatment or prevention of disease caused by varicella-zoster virus (VZV), wherein the treatment is alone or in combination with other drugs or vaccines.

[0102] This invention also relates to an anti-tumor vaccine product, wherein the anti-tumor vaccine product comprises:

[0103] (1) An effective amount of tumor antigen, wherein the tumor antigen is a recombinant polypeptide antigen;

[0104] (2) MA105 adjuvant;

[0105] (3) Necessary pharmaceutical excipients.

[0106] Preferably, the antitumor vaccine product is a subcutaneous or intramuscular injection vaccine;

[0107] Preferably, the tumor antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.2, or an HPV antigen with an amino acid sequence as shown in SEQ ID NO.3.

[0108] SEQ ID NO.2:

[0109]

[0110] SEQ ID NO.3:

[0111]

[0112] Preferably, the pharmaceutical excipients include: stabilizers, lyophilization protectants, and osmotic pressure regulators.

[0113] More preferably, the pharmaceutical excipients include: polysorbate 80, sucrose, and sodium chloride.

[0114] This invention also relates to an antitumor agent comprising:

[0115] (1) An effective amount of MA105 adjuvant;

[0116] (2) Necessary pharmaceutical excipients.

[0117] Preferably, the antitumor agent is an intratumoral injection preparation or an orthotopic tumor vaccine.

[0118] Preferably, the tumor is melanoma, colorectal cancer, or lung cancer.

[0119] Preferably, the pharmaceutical excipients include: stabilizers, lyophilization protectants, and osmotic pressure regulators.

[0120] More preferably, the pharmaceutical excipients include: polysorbate 80, sucrose, sodium chloride, histidine, and sodium dihydrogen phosphate.

[0121] This invention also relates to the use of the aforementioned MA105 immune adjuvant or a formulation containing MA105 immune adjuvant in the preparation of an antitumor combination formulation, wherein the combination formulation comprises a therapeutically effective amount of MA105 immune adjuvant, and other drugs selected from any of the following:

[0122] (1) Cytotoxic chemotherapy drugs;

[0123] (2) Antibody-based antitumor drugs;

[0124] (3) Oncolytic peptides and oncolytic virus drugs;

[0125] (4) Cell therapy preparations, preferably CAR-T cell therapy preparations.

[0126] When used as a therapeutic agent, the MA105 adjuvant of the present invention or a vaccine composition containing the MA105 adjuvant can be administered orally or parenterally. In the case of parenteral administration, the adjuvant or vaccine composition can be administered by means of in situ injection, in situ injection at the lesion site, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, percutaneous administration, etc.

[0127] When administering the MA105 adjuvant of the present invention without antigen or an in situ tumor vaccine prepared using the MA105 adjuvant, intratumoral injection is used to activate the subject's immune protection.

[0128] This invention also relates to a method for preparing saponin 21 (QS21) immune adjuvant using semi-purified commercial-grade or food additive-grade saponins as raw materials, the method comprising the following steps:

[0129] (1) Saponin dissolution and filtration

[0130] The saponin raw material is dissolved in a diluent, and then the insoluble matter is filtered out using a filter.

[0131] (2) Reverse phase crude and pure,

[0132] Take the saponin filter sample solution obtained in step (1) and perform reverse-phase crude purification according to the following steps:

[0133] 1) Sample loading: Load the sample onto UniPSN 30-300 chromatographic medium;

[0134] 2) Elution: Elute the target peak with a linear gradient from 2.5% B to 21.25±5.00% B, and collect the high-purity sample in segments, and combine the crude saponin solution.

[0135] (3) Reverse phase purification,

[0136] Take the crude saponin solution obtained in step (2) and perform reverse-phase purification according to the following steps:

[0137] 1) Sample loading: Dilute the sample solution with water to an acetonitrile concentration of 22%–28%, and load the sample onto UniPS 10-300 chromatographic medium;

[0138] 2) Elution: Elute the target peak using a linear gradient from 10% B to 25±5% B, collect high-purity samples in segments and combine them;

[0139] 3) Repeat steps 1 to 2) above 1 to 3 times for the merged samples, preferably repeating 2 times;

[0140] (4) Precipitation and redissolution step.

[0141] 1) Take the purified saponin sample solution prepared in step (3), add 2 times the volume of water, mix well, place at room temperature, and then centrifuge to collect the precipitate;

[0142] 2) Add the dissolving solution to the precipitate, stir to dissolve, and then remove the organic solvent sample. Preferably, the amount of dissolving solution added is the same as the amount of purified saponin sample solution prepared in step (3).

[0143] (5) Ultrafiltration replacement step,

[0144] 1) Take the saponin sample solution obtained after redissolving the precipitate in step (4), and use the ultrafiltration replacement solution to ultrafiltration concentrate to obtain the concentrated QS21 adjuvant solution; preferably, ultrafiltration concentrate to 30% to 50% of the original volume;

[0145] In steps (1) to (5) above,

[0146] The diluent is an aqueous solution containing 30% acetonitrile, 5 mM citric acid, and pH 5.0.

[0147] Solution B is an aqueous solution containing 99% acetonitrile and 0.1% TFA;

[0148] The solution was an aqueous solution containing 5 mM histidine at pH 6.0;

[0149] The ultrafiltration replacement fluid was an aqueous solution containing 5 mM histidine at pH 5.0.

[0150] The saponin raw material has a QS21 content of not less than 8%, a moisture content of not more than 10%, and an ash content of not more than 3%.

[0151] The method described above achieves a purification yield of QS21 of no less than 50%, and the final purity of QS21 obtained is no less than 94%.

[0152] Furthermore,

[0153] In step (1), the saponin raw material is dissolved at a ratio of 1:25 (W / V) and then filtered through a 1.0+0.45μm filter;

[0154] In step (2), the sample loading capacity is 30-60 mg / ml, the linear flow rate is 90-360 cm / h, after the sample loading is completed, rinse with solution A1 and rinse with 2.5% solution B to remove impurities; during gradient elution, collect samples with a purity higher than 30% for merging.

[0155] In step (3), the sample loading capacity is 10-50 mg / ml. After the sample loading is completed, rinse with solution A2 and rinse with 10% B to remove impurities. Collect samples with a purity higher than 60% after the first reverse phase purification, collect samples with a purity higher than 80% after the second reverse phase purification, collect samples with a purity higher than 90% after the third reverse phase purification, and collect samples with a purity higher than 95% after the fourth reverse phase purification.

[0156] In step (4), the mixture should be placed at room temperature for at least 30 minutes, and the centrifugation parameters should be: 9000~11500g, 2~8℃, 10min;

[0157] In step (5), the ultrafiltration parameters are: using a 5kD membrane pack, transmembrane pressure of 0.2 to 0.4 bar, replacement times of 10, and washing with ultrafiltration replacement solution of not less than 8 times the volume;

[0158] in,

[0159] Solution A1 is an aqueous solution containing 30% acetonitrile and 0.1% TFA;

[0160] Solution A2 is an aqueous solution containing 20% ​​acetonitrile and 0.1% TFA.

[0161] The beneficial effects of this invention are as follows:

[0162] 1. The MA105 adjuvant system comprises a mixture of liposomes and two immune enhancers: Poly I:C and QS-21; it has a good safety profile and can induce high levels of antibodies and a high frequency of CD4+ T cell responses.

[0163] 1. When MA105 adjuvant is used alone, it is administered via intratumoral injection. It can significantly stimulate immune cells in the tumor, especially tumor-killing immune cells, and activate tumor immunity, thereby achieving the effect of treating tumors.

[0164] 2. When MA105 adjuvant is used in conjunction with tumor antigen, only a low dose of antigen is needed to stimulate the body to produce specific cellular immunity and generate memory-type immune protection.

[0165] Terminology Definitions and Explanations

[0166] Unless otherwise stated, the terms used herein have the meanings commonly understood by one of ordinary skill in the art. For terms explicitly defined herein, their meanings shall be as defined herein.

[0167] The term "effective dose" refers to the amount of a therapeutic agent, when administered alone or in combination with another therapeutic agent to cells, tissues, or subjects, that is effective in preventing or alleviating symptoms of a disease or the progression of that disease. "Effective dose" also refers to the amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing, or alleviating an associated medical condition, or increasing the rate at which such symptoms are treated, cured, prevented, or alleviated. When an active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, the therapeutically effective dose refers to the combined amount of active ingredients that produce the therapeutic effect, regardless of whether they are administered in combination, consecutively, or simultaneously. Effective doses can be variably defined based on factors such as formulation, method of administration, and the patient's age, weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and responsiveness.

[0168] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when used in this article.

[0169] The term "antitumor agent" refers to antitumor drugs, which are a class of drugs used to treat tumor diseases, including chemotherapy drugs and biological agents.

[0170] The terms "pharmaceutical formulation" or "pharmaceutical composition" refer to a formulation in which the biological activity of the active ingredient contained therein is permitted and which does not contain any additional components that would have unacceptable toxicity to a subject to whom the formulation will be administered. Vaccine adjuvants or vaccine compositions containing vaccine adjuvants according to exemplary embodiments of the present invention can be formulated using methods known in the art to enable rapid, sustained, or delayed release of the active ingredient upon administration to mammals. These formulations may include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powders.

[0171] The term "pharmaceutically acceptable" means a composition that is physiologically acceptable for human use and generally does not cause allergic reactions (e.g., gastrointestinal disturbances, dizziness, etc.) or similar reactions. Examples of carriers, excipients, and diluents may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, sodium alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, propyl methylparaben, talc, magnesium stearate, and mineral oil. Furthermore, fillers, anticoagulants, lubricants, humectants, fragrances, emulsifiers, preservatives, etc., may also be included.

[0172] The term "adjuvant" or "vaccine adjuvant" refers to a drug or immunizing agent administered to improve the immune response to a vaccine. In addition to the vaccine adjuvants described in this application, the vaccine composition may further include conventionally used vaccine adjuvants. Conventionally used vaccine adjuvants may include aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, MF59, virions, AS04 [a mixture of aluminum hydroxide and monophosphate A (MPLA)], AS03 (a mixture of DL-α-tocopherol, squalene, and polysorbate 80), CpG, flagellin, polyI:C, AS01, AS02, ISCOMs, etc.

[0173] The term "antigen" refers to a substance that induces an immune response. Therefore, in this invention, any substance exhibiting such activity as inducing an immune response can be used without limitation. Antigens can be peptides, proteins, nucleic acids, sugars, pathogens, attenuated pathogens, inactivated pathogens, viruses, virus-like particles (VLPs), cells, or cell fragments. Antigens can be selected from the following group: antigens of Japanese encephalitis virus, antigens of Haemophilus influenzae type B (HIB), antigens of Middle East Respiratory Syndrome (MERS) virus, antigens of Zika virus, antigens of Pseudomonas aeruginosa, antigens of pertussis, antigens of Mycobacterium tuberculosis, antigens of Bacillus anthracis, antigens of hepatitis A virus (HAV), antigens of hepatitis B virus (HBV), antigens of hepatitis C virus (HCV), antigens of human immunodeficiency virus (HIV), antigens of herpes simplex virus (HSV), antigens of Neisseria meningitidis, antigens of Corynebacterium diphtheriae, and antigens of Bordetella pertussis. Antigens of bacteria, Clostridium tetani, human papillomavirus (HPV), varicella-zoster virus, enterococcus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter spp., Helicobacter pylori, malaria, dengue virus, Orientia scrub typhus, Benjamin Butylitis virus (SFTS), SARS-CoV, influenza virus, Ebola virus, and Streptococcus pneumoniae. Attached Figure Description

[0174] Figure 1 Turbidity analysis of antigen preparation solutions with different formulations after shaking for 4 hours (turbidity was detected using a turbidimeter, and the vertical axis in the figure is in NTU).

[0175] Figure 2 The changes in polymer peak area after different freeze-thaw cycles of antigen preparation solutions with different formulations are shown in the figure. The peak area changes were detected by SEC-HPLC (the vertical axis is a percentage, for example, a change of 0.25 on the vertical axis indicates a change of 0.25% in peak area). The data for 0 freeze-thaw cycles show slight changes because there will be a certain detection bias when the protein is prepared into different formulations and tested before freeze-thaw.

[0176] Figure 3 Analysis of serum antibody subtype IgG2a titers 14 days after secondary immunization with the vaccine in each experimental group.

[0177] Figure 4 Cellular immunity analysis (CD4+ T cell response) 14 days after secondary immunization with the vaccine in each experimental group.

[0178] Figure 5 Cellular immunity analysis (CD4+ T cell response) 14 days after secondary immunization with the vaccine in each experimental group.

[0179] Figure 6 Flowchart for the preparation of MA105 adjuvant.

[0180] Figure 7 Transmission electron micrograph of recombinant herpes zoster vaccine.

[0181] Figure 8 Particle size distribution diagram of recombinant herpes zoster vaccine.

[0182] Figure 9 Tumor growth curves in TC-1 tumor-bearing mice after immunization with MA105 adjuvant and antigen.

[0183] Figure 10 The curve of weight change in TC-1 tumor-bearing mice after immunization with MA105 adjuvant and antigen.

[0184] Figure 11 The level of IFN-γ secretion in TC-1 tumor-bearing mice after immunization with MA105 adjuvant and antigen.

[0185] Figure 12 Antitumor activity of MA105 adjuvant alone (B16F10 tumor model, melanoma).

[0186] Figure 13 Antitumor activity of MA105 adjuvant alone (MC38 tumor model, colorectal cancer).

[0187] Figure 14 Antitumor activity of MA105 adjuvant alone (LLC tumor model, lung cancer).

[0188] Figure 15 MA105 adjuvant can significantly improve the therapeutic effect of anti-tumor antigens. Figure 5 A, Tumor volume after immunization Figure 5 B, body weight of mice after immunization.

[0189] Figure 16 After immunization with MA105 adjuvant and antigen, mice developed significant immune protection.

[0190] Figure 17 HPLC chromatogram of the QS21 sample after the third step of reverse-phase purification (after the fourth step of purification) during the QS21 purification process.

[0191] Figure 18 Analysis of antibodies conjugated to recombinant zoster vaccines produced using different processes

[0192] Figure 19 Cellular immune analysis (CD4+ T cell response) 14 days after secondary immunization with recombinant zoster vaccines produced by different processes. Detailed Implementation

[0193] 1. Herpes zoster antigen

[0194] The gE protein is the most abundant protein on the surface of the VZV virus. It is a transmembrane glycoprotein composed of 623 amino acid residues. Its N-terminal 1-30 amino acids form a signal peptide, amino acids 31-538 form an extramembrane segment, amino acids 539-559 form a transmembrane segment, and amino acids 560-623 form an intramembrane region. The gE protein dimers with the glycoprotein gI on the viral surface and plays an important role in viral infection of host cells. Natural infection or vaccination with a VZV attenuated vaccine can induce the production of gE-specific neutralizing antibodies and CD4+. + T-cell immune responses make gE an important target of the human immune response. The most important site for gE protein antibody response is located in the 50-135 amino acid segment at its N-terminus, containing at least one linear epitope that can be recognized by neutralizing antibodies. T-cell recognition sites for gE protein are distributed throughout its 31-538 amino acid extracellular region. The transmembrane region of gE protein does not contain any known antigenic epitopes or T-cell recognition sites.

[0195] The gE protein sequence used in this application's antigen is the extracellular region of the gE protein of common human varicella-zoster viruses (Strain NH29_3 (ABH08489.1), Dumas (NP_040190.1), DE10-2480 (AKG56356.1), and VZV-VSD (AAG48520.1). The amino acid sequence is 31-538, excluding the transmembrane and intramembrane regions. .

[0196] Antigen production: Each time, add 35-55L of basal culture medium, inoculate with 1.5-3L of cell suspension, and supplement with 8-10L of feed. The cell harvest volume is approximately 45-65L. The final yield is 5-10g gE antigen.

[0197] Host cells expressing antigens: The host cells, CHO-K1 cells, were purchased from Nanjing Genscript Biotech Co., Ltd., and the cell line was numbered CCL-61.

[0198] 2. Tumor antigens

[0199] The tumor antigens used in this invention are recombinant tumor antigens, which can be tumor-specific antigens / unique tumor antigens, tumor-associated antigens (TAAs), or neoantigens. Specifically, the tumor antigens include, but are not limited to, HPV antigens, PSA antigens, HER2 antigens, CEA antigens, VEGF antigens, EGFR antigens, and MUC1 antigens. Preferably, the antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.1, or an HPV antigen with an amino acid sequence as shown in SEQ ID NO.2.

[0200] SEQ ID NO.2:

[0201]

[0202] SEQ ID NO.3:

[0203]

[0204] 3. Other raw materials

[0205] Any commercially available product that meets pharmaceutical standards is acceptable.

[0206] DOPC: Dioleoylphosphatidylcholine;

[0207] DOTAP: 1,2-Dioleoyl-3-trimethylammonium chloride propane;

[0208] cholesterol;

[0209] Poly I:C: (Polyinosinic cytidine diphosphate).

[0210] Example 1: Screening of excipients for antigen stability

[0211] To ensure the long-term stability of gE protein at -70℃, we screened various excipients and studied their protective effects on the protein under heating conditions at 50℃ and room temperature shaking conditions. We also evaluated the differences in OD350 value, turbidity, main peak area detected by IEC-HPLC, and SDS-PAGE before and after sample treatment.

[0212] 1. Experiments have shown that Tween-80 can effectively reduce the aggregation of antigenic proteins in the formulation under oscillation conditions, and glycerol, histidine, sorbitol and sucrose can effectively reduce protein degradation under high temperature conditions.

[0213] 2. Since glycerol is not typically used for stock solution preservation, sorbitol, histidine, and other options were compared, with histidine chosen as the buffer system. Considering that sucrose is the preferred excipient in most recombinant biological products, 10% sucrose was selected as the heat stabilizer. The final stock solution formulation was 10% sucrose + 10mM histidine + 0.025% polysorbate 80 (Tween-80), pH 6.5.

[0214] Multiple formulations were screened, and the turbidity of the solutions under different formulations was examined. The results are shown in Table 1 below. Figure 1 As shown in Table 1, the antigen polymerization of different solvent systems after repeated freeze-thaw cycles was further investigated. Figure 2 As shown

[0215] Table 1. Screening of Antigen Solution Formulations

[0216] Buffer system formulation Shaking for 4 hours makes it turbid Test after 0 freeze-thaw cycles Test after 5 freeze-thaw cycles

[0217] 10% sucrose + 5mM PB, pH 6.5 7 0.27 0.7 10% sucrose + 5mM PB + 0.025% Tween-80 pH 6.5 2 0.26 0.3 10% sucrose + 10mM histidine, pH 6.5 7 0.29 0.71 10% sucrose + 10mM histidine + 0.025% Tween-80 pH 6.5 1 0.25 0.29 5mM PB pH 6.5 27 0.34 0.75

[0218] Sample Y202009-15 was prepared according to the above formula and subjected to stability testing. The specific stability testing parameters and steps are as follows.

[0219] Long-term conditions (-70℃), sampling points: 0 days, 3 months, 6 months

[0220] Acceleration conditions (2-8℃), sampling points: day 0, day 3, day 7, day 14

[0221] The test results showed that the sample was stable, with acceptable appearance, protein content, antigen activity, electrophoretic purity, HPLC purity, and sterility. Isoelectric point, peptide mapping, and N-terminal sequencing results indicated that the properties of the antigen protein remained unchanged. This indicates that the recombinant herpes zoster vaccine stock solution can withstand at least four freeze-thaw cycles.

[0222] Example 2: Formulation screening of shingles vaccine preparations

[0223] The shingles vaccine formulation of this invention is a suspension composed of recombinant varicella-zoster virus glycoprotein E (gE antigen) and the MA105 adjuvant system. The MA105 adjuvant system is a liposomal formulation containing two immune-enhancing components: saponin (QS-21) and polyinosinic-polycytidylic acid (Poly I:C). We determined the formulation and effective antigen content of the MA105 adjuvant system through formulation screening, and determined the preparation process of the semi-finished product through production process development.

[0224] The adjuvant system contains two immunostimulants (QS-21 and Poly I:C). To reduce the toxicity of QS-21 and Poly I:C and improve immunogenicity, we used a mixture of cationic and neutral liposomes to adsorb QS-21 and Poly I:C.

[0225] Poly I:C signaling primarily relies on Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA-5), strongly driving cellular immunity and effective type I interferon responses. It can also induce strong antigen-specific CD4+ T and CD8+ T cell responses through type I interferon (IFN-α / β) signaling and antigen cross-presentation. Since the receptor for Poly I:C is located on the intracellular membrane, and to ensure that Poly I:C targets lymph nodes after intramuscular injection and reduces adverse reactions from its diffusion into the bloodstream, we use cationic liposomes (composed of DOTAP, DOPC, and cholesterol) as carriers, which adsorb Poly I:C via electrostatic interactions.

[0226] The study found that Large flocculents or precipitates appeared after cationic liposomes adsorbed Poly I:C. To maintain the stability of the product's physical properties, this invention selects liposomes comprising cationic liposomes and neutral liposomes (composed of DOTAP, cholesterol, etc.). Adsorption of Poly I:C by mixed liposomes (composed of sterols) Poly I:C, adsorbed onto mixed liposomes after intramuscular injection, can be passively or actively delivered to antigen-presenting cells (APCs), limiting their interaction with non-specific phagocytes and modulating the resulting cytokine secretion characteristics. Toxicological studies of this product have confirmed that adsorbing Poly I:C onto mixed liposomes effectively improves product safety.

[0227] Free QS-21 can bind to cholesterol on cell membranes to form an irreversible complex, which can cause hemolysis when it binds to red blood cells. When QS-21 adsorbs onto cholesterol in the mixed liposomes, it prevents cholesterol from binding to the cell membrane. This combination significantly reduces the hemolytic effect. Experiments have shown that the amount of cholesterol added to the adjuvant system described in this invention can adsorb at least three times the dose of QS-21 in the finished product without causing hemolysis. This indicates that the cholesterol content is excessive, which can reduce the potential risks caused by the hemolytic activity of QS-21 and improve the safety boundary of the process.

[0228] Vaccines formulated using a single immune enhancer (Poly I:C or QS-21) in combination with a liposome mixture were able to induce high levels of binding antibodies and a high frequency of CD4+ T cell responses. When two immune enhancers are simultaneously mixed The vaccine formulated with liposomes induced higher levels of binding antibodies and a higher frequency of CD4+ T responses, indicating that... Poly I:C and QS-21 have a synergistic effect. Therefore, the combined use of two immune enhancers is more conducive to improving the immunogenicity of this product and the number of CD4+ T cells, as detailed in Example 4.

[0229] Polysorbate 80 (Tween-80) was chosen to avoid flocculent aggregates in the finished product. Sodium chloride was used to adjust the osmotic pressure of the vaccine and maintain the system's osmotic pressure (210–350 mOsmol / kg). Sucrose helps maintain the stability of the liposomes.

[0230] Specifically, the details of the active ingredients and excipients in each 1 ml of formulation are shown in Table 2 below:

[0231] Table 2. List of the content of major components in vaccine formulations

[0232]

[0233] DOPC: Dioleoylphosphatidylcholine;

[0234] DOTAP: 1,2-Dioleoyl-3-trimethylammonium chloride propane;

[0235] cholesterol;

[0236] QS-21: Saponin 21;

[0237] Poly I:C: Poly I:C

[0238] Example 3: Preparation and property testing of shingles vaccine formulation

[0239] The preparation steps for the vaccine formulation are as follows:

[0240] 3.1 Preparation of cationic liposomes;

[0241] The mixture was prepared by ethanol injection method using a formulation of 1,2-dioleoyl-3-trimethylammonium chloride propane (DOTAP), dioleoylphosphatidylcholine (DOPC), and cholesterol in a mass ratio of 2:2:1. The specific preparation steps are as follows:

[0242] (1) The three lipid components are dissolved in ethanol and sheared in the aqueous phase 1 to 3 times.

[0243] (2) Use sucrose-containing buffer for ultrafiltration replacement, with a concentration factor of 2 to 4 times and a washing factor of 6 to 8 times;

[0244] (3) 200nm liposome extrusion: Extrusion is performed using a filter membrane with a pore size of 200nm, and multiple cycles of extrusion are carried out to control particle size uniformity.

[0245] (4) 100nm liposome extrusion: Extrusion is performed using a filter membrane with a pore size of 100nm, and multiple cycles of extrusion are carried out to control particle size uniformity.

[0246] (5) Use a sterile filter to filter and sterilize.

[0247] The physicochemical parameters of the prepared cationic liposomes are as follows:

[0248] Average particle size (D50): 60–120 nm;

[0249] Solution pH: 4.8–7.0;

[0250] Zeta potential: 40–72 mV (pH 6.5).

[0251] After storage at 2-8℃ for 12 months, the morphology and composition of the cationic liposomes remained stable.

[0252] 3.2 Preparation of neutral liposomes;

[0253] Neutral liposomes are globular structures composed of a lipid bilayer of dioleoylphosphatidylcholine (DOPC) and cholesterol, with a DOPC to cholesterol mass ratio of 4:1. The specific preparation steps are as follows:

[0254] (1) The two lipid components are dissolved in ethanol and added to the aqueous phase by online shearing 1 to 3 times;

[0255] (2) 200nm liposome extrusion: filter with a 200nm pore size filter membrane and perform multiple cycles of extrusion to control particle size uniformity.

[0256] (3) 100nm liposome extrusion: filter with a 100nm pore size filter membrane and perform multiple cycles of extrusion to control particle size uniformity.

[0257] (4) Use sucrose-containing buffer for ultrafiltration replacement, with a concentration factor of 3 to 6 times and a washing factor of 6 to 8 times;

[0258] (5) Use a sterile filter to filter and sterilize.

[0259] The physicochemical parameters of the prepared neutral liposomes are as follows:

[0260] Average particle size (D50): 80–140 nm;

[0261] Solution pH: 5.5-7.0, preferably 6.4-6.6;

[0262] After storage at 2-8℃ for 12 months, the morphology and composition of the cationic liposomes remained stable.

[0263] 3.3 Preparation of vaccine formulations

[0264] The final content of each component of the vaccine formulation is prepared according to the proportions described in Table 2 of Example 2:

[0265] Cationic liposomes, neutral liposomes, and excipients were added according to the concentrations calculated in Table 2. Then, QS-21, Poly I:C, and gE antigen solutions were added separately, and the pH was adjusted to 6.0-7.0.

[0266] The complete vaccine preparation process is as follows: Figure 6 As shown.

[0267] 3.4 Quality testing of vaccine preparations

[0268] Multiple batches of vaccine preparations were prepared according to the above method, as shown in Table 3 below.

[0269] Table 3. Vaccine Formulation Production Batch Records

[0270]

[0271] The product underwent quality testing, which included checking the content of each major component. The test results are shown in Table 4 below.

[0272] Table 4. Results of vaccine preparation quality testing

[0273]

[0274]

[0275] Example 4: Property testing of shingles vaccine formulations and comparison of immunogenicity of different shingles vaccine formulations

[0276] The vaccine formulation of this invention was selected, with the aluminum hydroxide adjuvant formulation (gE / nano-type aluminum hydroxide adjuvant / QS-21 / Poly I:C) serving as control group 1. The (Shingrix) formulation served as control group 2, and the neutral liposome formulation (gE / neutral liposome / QS-21 / Poly I:C) served as control group 3. The effects on immunized mice were examined, and the results showed that...

[0277] (1) Mice immunized with gE antigen vaccines formulated with four different methods were all able to stimulate the production of high titers of binding antibodies. The mixed liposome formulation of this invention is superior to other formulations; detailed test results can be found in [link to test results]. Figure 3 In the picture:

[0278] The gE antigen concentration was consistent across all reference formulation groups (100 ug / ml);

[0279] MPL, 3M-52 (TLR7 / 8 agonist), aluminum hydroxide, and Poly I:C are immune adjuvants;

[0280] Group gE consists of a pure antigen solution without liposome preparations;

[0281] The nanoemulsion group is an oil-in-water formulation;

[0282] The formulation and preparation method of neutral liposomes are as described in Section 3.2 of Example 3. The amount of neutral liposomes used in the reference formulation of neutral liposomes is the same as that in the nanoemulsion group.

[0283] The buffer, solvent, QS-21, and Poly I:C used in all groups (except for the high and low dose Poly I:C groups) were kept consistent, and the dosage of each component is shown in Table 2.

[0284] (2) The mixed liposome formulation, neutral liposome formulation and of the present invention All formulas can induce irritation The stimulation of CD4+ T cell responses showed no significant differences among the groups, but all were superior to the aluminum hydroxide formulation group. See details for the test results. Figure 4 In the picture:

[0285] The gE antigen concentration was consistent across all reference formulation groups (100 ug / ml);

[0286] The blank control group contained no antigen, but the vaccine formulation was the complete formula;

[0287] The viral control consisted of only the antigen, but not of mixed liposomes, Poly I:C, and QS-21.

[0288] Xin Anli Shi is a commercially available vaccine product group;

[0289] The mixed liposome group is the mixed formulation of cationic liposomes and neutral liposomes described in Section 3.3 of Example 3;

[0290] The formulation and preparation method of neutral liposomes are as described in Section 3.2 of Example 3. The amount of neutral liposomes used in the reference formulation of neutral liposomes is the same as that in the nanoemulsion group.

[0291] The buffer, solvent, QS-21, and Poly I:C used in all groups were kept consistent, and the amounts of each component are shown in Table 2.

[0292] To investigate the immunomodulatory effects of different preparation methods and formulations, we conducted the experiments shown in the table below. Two formulations are listed in Table 2, and two preparation methods for MA105 adjuvant are shown in Table 3. Figure 6As shown in the table below, the sample information is as follows.

[0293] Sample 1 Formula 1 Preparation process 1 Sample 2 Formula 1 Preparation process 2 Sample 3 Formula 2 Preparation process 1 Sample 4 Formula 2 Preparation process 2

[0294] The results showed that different formulations and preparation methods could induce high levels of binding antibodies, and there were no significant differences between groups. Figure 18 However, sample 4 induced a higher frequency of (IL-2 / IFN-γ) CD4+ T cell responses. Figure 19 ).

[0295] Example 5: Comparison of the properties of different vaccine formulations

[0296] 5.1 Synergistic effect of immune enhancers

[0297] To investigate the effect of a single immunostimulant (Poly I:C or QS-21) on vaccine immunogenicity, and whether there is a synergistic effect between Poly I:C and QS-21 in the formulation on vaccine immunogenicity, the immunogenicity of a vaccine formulation using a single immunostimulant (Poly I:C or QS-21) and a mixture of liposomes was compared with that of a vaccine formulation containing Poly I:C, QS-21, and a mixture of liposomes.

[0298] Both immunostimulants and liposomes can induce high levels of binding antibodies and high frequencies of (IL-2 / IFN-γ) CD4+ T cell responses in vaccines formulated with these combinations. However, gE / combined adjuvant candidate vaccines induce even higher levels of binding antibodies and even higher frequencies of (IL-2 / IFN-γ) CD4+ T cell responses. Prove that Poly I:C and QS-21 have a synergistic effect. For detailed comparison results, please see Figure 5 In the picture:

[0299] The preparation methods for each reference group formulation are shown in Example 3. The specific differences in formulation components are as follows:

[0300] Blank control group: Contains no antigen, but the vaccine formulation is complete;

[0301] gE buffer group: without mixed liposomes, Poly I:C and QS-21;

[0302] gE mixed liposome group: mixed liposome formulation, but without Poly I:C and QS-21;

[0303] gE Mixed Liposomes / QS-21 Group: Mixed liposome formulation, without Poly I:C;

[0304] gE mixed liposomes / Poly I:C group: Mixed liposome formulation, QS-21 free;

[0305] gE mixed adjuvant group: the complete formulation prepared in Example 3.

[0306] 5.2 Dosage optimization of Poly I:C and QS-21

[0307] 5.2.1 Dosage optimization of Poly I:C

[0308] The immunogenicity of gE / combined adjuvant candidate vaccines formulated with different contents of Poly I:C (400 μg / ml, 800 μg / ml, 1600 μg / ml) was compared to determine the Poly I:C content in the vaccine formulation.

[0309] Candidate vaccines containing different doses of Poly I:C and gE / combined adjuvants all produced high levels of binding antibodies, with no significant differences between groups, and all induced a high frequency of (IL-2 / IFN-γ) CD4+ T cell responses. Considering that using mixed liposomes can improve the stability and delivery efficiency of Poly I:C, the Poly I:C content in the vaccine was tentatively set at 800 μg / ml.

[0310] 5.2.2 Dosage optimization of QS-21

[0311] The immunogenicity of gE / combined adjuvant candidate vaccines formulated with different contents of QS-21 (100 μg / dose, 50 μg / dose, 25 μg / dose, 12.5 μg / dose) was compared to determine the QS-21 content in the vaccine formulation (0.5 ml per dose).

[0312] Immunization with candidate vaccines containing different doses of QS-21 and gE / combined adjuvants all stimulated the production of high levels of binding antibodies; all experimental groups induced (IL-2 / IFN-γ) CD4+ T cell responses. (Reference) The formulation tentatively sets the concentration of QS-21 in this product at 50 μg / dose (100 μg / ml).

[0313] 5.3 Antigen Content Study

[0314] This vaccine is a liquid formulation containing a novel adjuvant. In accordance with the requirements of the "Technical Guidelines for Preclinical Research of Preventive Vaccines", four groups of gE vaccines prepared with different contents of gE antigen (100μg / dose; 50μg / dose; 25μg / dose; 5μg / dose; 0ug / dose, each dose is 0.5ml) and a pure adjuvant control were compared to study the effect of gE antigen content on vaccine immunogenicity.

[0315] Within the antigen content range studied in this experiment, gE vaccines formulated with different gE antigen concentrations all induced high levels of binding antibodies and a high frequency of (IL-2 / IFN-γ) CD4+ T cell responses. Humoral and cellular immunity results showed that gE antigen concentrations of 50 μg / dose and above induced a good immune response. The final antigen concentration was determined to be 50 μg / dose (concentration of 100 ug / ml).

[0316] Example 6: Preparation of antitumor vaccine formulation

[0317] Cationic and neutral liposomes for the MA105 adjuvant were prepared according to sections 3.1-3.2 of Example 3.

[0318] 6.1 Preparation of vaccine formulations

[0319] The final content of each component of the vaccine preparation is as described in Example 2, and the formulation is carried out according to the proportions shown in Table 5:

[0320] Table 5. List of the content of each component in antitumor vaccine formulations (per dose)

[0321]

[0322] The antigen is an HPV antigen with an amino acid sequence as shown in SEQ ID NO.2.

[0323] Example 7: Activity assay of antitumor vaccine formulation (TC-1 tumor model)

[0324] 7.1 Experimental Design

[0325] TC-1 tumor-bearing mice (C57BL / 6) were randomly divided into groups of 7 mice each, and the groups were immunized and subsequently observed and tested.

[0326] Immunization schedule: On day 7 after tumor transplantation (using tumor fragments excised from previous generation TC-1 tumor-bearing mice, inoculated on the lower part of one scapula), when the short diameter has grown to approximately 9 mm, the first immunization was performed via intramuscular injection in the hind limb, 200 μL per mouse (the vaccine formulation is shown in Table 6, 200 μL of each formulation was administered for each immunization). The second and third immunizations were performed on days 14 and 21 after tumor transplantation.

[0327] Testing sites:

[0328] (1) TC-1 tumor-bearing mice were observed every 3 days after immunization, and the long and short diameters of the tumor, the weight of the mice, and the general condition of the mice were recorded.

[0329] (2) Isolation of splenic lymphocytes from TC-1 tumor-bearing mice and detection of IFN-γ secretion by ELISPOT.

[0330] Table 6. Formulations of antitumor vaccine preparations

[0331]

[0332]

[0333] The antigen used is the HPV antigen with the amino acid sequence shown in SEQ ID NO.2.

[0334] 7.2 Experimental Results

[0335] (1) Tumor growth curves in TC-1 tumor-bearing mice

[0336] At the time of initial immunization, the average tumor volume in each group was 0.59 cm. 3 0.62cm 3 0.68cm 3 0.77cm 3 0.68cm 3 There was no statistically significant difference. Tumor growth curves of TC-1 tumor-bearing mice during the study period are shown in the figure. Figure 9 The results showed that, compared with the NC group, the D group exhibited significant tumor inhibition, with tumor inhibition rates of 82.16% (24d) and 73.30% (28d).

[0337] The formula for calculating tumor volume in the diagram is: V = 4 / 3 * π * major axis * minor axis 2

[0338] The formula for calculating tumor inhibition rate is: Inhibition rate = (average volume of xenograft in NC group - average volume of xenograft in experimental group) / average volume of xenograft in NC group * 100%.

[0339] (2) Body weight change curve of TC-1 tumor-bearing mice

[0340] Tumor growth curves of TC-1 tumor-bearing mice during the study period are shown in the figure. Figure 10 The results showed that after administration, the mice did not experience a significant decrease in body weight (the increase in body weight was due to tumor growth), indicating that the vaccines in each group were safe.

[0341] (3) IFN-γ secretion level in TC-1 tumor-bearing mice after triple immunization

[0342] Ten days after three immunizations, three mice from each group were randomly selected, and their spleens were harvested for splenic lymphocyte isolation and culture. The cells were stimulated with 5 μg and 50 μg of antigen, respectively. The IFN-γ secretion level in TC-1 tumor-bearing mice was detected using the DaYou ELISPOT-IFN-γ kit. The results are as follows: Figure 11 As shown, group D exhibited significantly higher immune activation efficiency compared to group NC.

[0343] The above results indicate that the MA105 adjuvant can significantly enhance the immunogenicity of tumor antigens and improve the therapeutic activity of vaccines.

[0344] Example 8 Antigen-free MA105 adjuvant Antitumor activity assay (B16F10 tumor model)

[0345] Experimental protocol

[0346] B16F10 tumor-bearing mice (C57BL / 6) were randomly divided into groups of 8 mice each. Group immunization and subsequent observation and testing were conducted.

[0347] Immunization schedule: After tumor inoculation (50,000 B16F10 tumor cells per mouse), immunizations were administered on days 9, 12, and 15. The administration method is intratumoral injection. Immunotherapy is only given No antigen adjuvant Each mouse was given 100 μL of the drug (formulation shown in Table 7). On day 18, mice that did not develop tumors underwent a second tumor cell inoculation (50,000 B16F10 tumor cells).

[0348] Monitoring points: After the initial treatment, observe every 2 days and record the long and short diameters of the tumor, the mouse's weight, and the mouse's general condition.

[0349] Table 7. Formulations without antigen adjuvants

[0350]

[0351] Tumor growth curves of B16F10 tumor-bearing mice during the study period are shown in the figure. Figure 12 .

[0352] The results showed that, compared with other groups, the MA105 adjuvant group exhibited significant tumor inhibition.

[0353] Example 9: Detection of antitumor activity of antigen-free MA105 adjuvant (allogeneic xenograft model of colon cancer MC38 cell line)

[0354] Experimental protocol

[0355] MC38 tumor-bearing mice (C57BL / 6) were randomly divided into groups of 10 mice each, and the groups were immunized and subsequently observed and tested.

[0356] Immunization program: Tumor inoculation (1×10⁻⁶ mice per inoculated) 5 After immunization with MC38 tumor cells, immunization was performed on days 11, 14, and 17. Intratumoral injection Immunotherapy Only administer without antigen adjuvant Each mouse was given 100 μL of the drug per dose (formulation as shown in Table 7 above).

[0357] Monitoring points: After the initial treatment, observe every 3 days and record the long and short diameters of the tumor, the weight of the mouse, and the general condition of the mouse; at the end of the experiment, dissect the mouse and weigh the tumor tissue.

[0358] Tumor growth curves of MC38 tumor-bearing mice during the study period and tumor weight analysis of each group at the end of the experiment are shown in the table below. Figure 13 .

[0359] The results showed that, compared with the saline control group, the MA105 adjuvant group exhibited significant tumor inhibition.

[0360] Example 10: Detection of antitumor activity of antigen-free MA105 adjuvant (lung cancer LLC cell allogeneic xenograft model)

[0361] Experimental protocol

[0362] LLC tumor-bearing mice (C57BL / 6) were randomly divided into groups of 10 mice each. Group immunization and subsequent observation and testing were conducted.

[0363] Immunization program: Tumor inoculation (6 × 10⁶ mice per mouse) 5 After (one LLC tumor cell), immunization was performed on days 11, 14, and 17. Intratumoral injection Immunotherapy Only administer without antigen adjuvant Each mouse was given 100 μL of the drug per dose (formulation as shown in Table 7 above).

[0364] Monitoring points: After the initial treatment, observe the mice every 3 days, record the weight and general condition of the mice; at the end of the experiment, dissect the mice, remove the tumor tissue and weigh it.

[0365] During the study, LLC tumors exhibited flattened growth, making it impossible to calculate growth curves. Tumor weight analysis for each group at the end of the experiment is shown below. Figure 14 .

[0366] The results showed that, compared with the saline control group, the MA105 adjuvant group exhibited significant tumor inhibition.

[0367] Example 11: Detection of the immunoprotective efficacy of a vaccine formulation (antigen + MA105 adjuvant) (TC-1 tumor model)

[0368] Experimental protocol

[0369] (1) A tumor-bearing mouse model was constructed according to the method in Example 2. TC-1 tumor-bearing mice (C57BL / 6) were randomly divided into groups of 5 mice each. When the short diameter of the transplanted tumor grew to about 5 mm, the mice were immunized for the first time (7 days). The second and third immunizations were performed on 14 days and 21 days, respectively.

[0370] (2) Each time, each mouse's left and right hind legs Intramuscular injection Administer 200μL of the following vaccine groups

[0371] 1) NC group (PBS 200μL);

[0372] 2) MKK700+MA105 group: MKK700 antigen is HPV antigen with the amino acid sequence shown in SEQ ID NO.1 + MA105 group (PBS buffer 20μL + MKK700 antigen 80μL + MA105 adjuvant 100μL, the concentration and dosage of antigen and adjuvant are the same as in Example 2);

[0373] 3) MA105 group (PBS buffer 100μL + MA105 adjuvant 100μL);

[0374] The formulations of the antigen solution and MA105 adjuvant are shown in Tables 6 and 7 above.

[0375] After administration, the tumor growth curve of mice is as follows: Figure 15 As shown, in the group of mice treated with antigen + MA105 adjuvant, 60% of the mice (3 / 5) achieved complete remission (CR) and 40% (2 / 5) achieved partial remission (PR).

[0376] Subsequently, TC-1 tumor blocks were reimplanted into other sites (contralateral subscapular region) in three mice that achieved complete remission after treatment. The results showed that the antigen + MA105 adjuvant treatment had a sustained immunoprotective effect, and the mice in complete remission developed strong immunity to the reimplanted TC-1 tumors, resulting in tumor cessation. Figure 16 ).

[0377] Example 12: Purification and Preparation of QS21

[0378] Main raw materials, equipment and reagents

[0379] Saponins (derived from dried bark extract of Quilla): purchased from Desert King (name: VET-SAP, CAS No.: 8047-15-2).

[0380] According to high performance liquid chromatography analysis, the raw material contains no less than 8% QS21, no more than 10% moisture, no more than 3% ash, and the content of heavy metals and pesticide residues meets pharmaceutical standards.

[0381] UniPSN 30-300 chromatographic media: Suzhou Nanomicro Technology Co., Ltd.

[0382] UniPS 10-300 chromatographic media: Suzhou Nanomicro Technology Co., Ltd.

[0383] Main equipment

[0384] High-speed refrigerated centrifuge H5-25KR Hunan Kecheng Instrument Equipment Co., Ltd. Membrane-encapsulated ultrafiltration system <![CDATA[0.5m 2 ]]> Hangzhou Kebote Filter Material Co., Ltd. High Performance Liquid Chromatography DAC150 Jiangsu Hanbang Technology Co., Ltd.

[0385] main buffer

[0386] Cleaning solution 1 (0.5M NaOH) Preservative solution 1 (100mM NaOH) Diluent (30% acetonitrile ~ 5mM citric acid, pH 5.0) Solution A1 (30% acetonitrile, 0.1% TFA) Solution A2 (20% acetonitrile, 0.1% TFA) Solution B (99% acetonitrile, 0.1% TFA) Solution (5mM histidine, pH 6.0) Ultrafiltration replacement fluid (5mM histidine, pH 5.0)

[0387] Step 1: Dissolving, filtering, and crude purifying saponins

[0388] 1.1 Saponin Dissolution and Filtration

[0389] Feeding amount: 200g saponin, 5L diluent;

[0390] Saponin was dissolved in a diluent (30% acetonitrile ~ 5 mM citric acid, pH 5.0) at a ratio of 1:25 (w / v), and filtered through a 1.0 ± 0.45 μm filter. The resulting product was... Saponin filtration sample 5.11~5.15L.

[0391] 1.2. Reversed-phase crude purification of saponin solution

[0392] (1) Pretreatment: Connect the chromatography column and the liquid chromatography system, and control the A1 solution to enter the A pump port and the B solution to enter the B pump port respectively. The linear flow rate of the system is 100-140 cm / h. After 100% B washing, the system is equilibrated with 100% A.

[0393] (2) Sample loading: Take the saponin filter sample (about 5L) obtained in step 1 and load it onto UniPSN 30-300 chromatographic medium with a loading of 30-60 mg / ml and a linear flow rate of 90-360 cm / h. After the sample loading is completed, rinse with A1 solution (4.5L) and then rinse with 2.5% B to remove impurities.

[0394] (3) Elution: The target peak was eluted with a linear gradient from 2.5% B to 21.25 ± 5.00% B, and the samples were collected in segments for purity testing. Samples with a purity higher than 30% were combined to obtain 4.7–5.5 L of the product. crude saponin solution ,

[0395] The sample loading and elution material quantities for the reverse-phase crude purification step are shown in Table 8 below.

[0396] Table 8. Reverse phase roughing and purification steps

[0397]

[0398] Step 2: Purification of saponins

[0399] 2.1 The first step in the reverse-phase purification of saponin solution

[0400] (1) Pretreatment: Connect the chromatography column and the liquid chromatography system, control A2 solution to enter pump A and B solution to enter pump B respectively, the linear flow rate of the system is 100-140 cm / h, and after 100% B washing, 100% A equilibration is performed.

[0401] (2) Sample loading: Dilute the crude saponin solution with water and control the acetonitrile concentration to 22%–28%. Load the sample into UniPS 10-300 chromatographic medium (load all the diluted crude saponin solution into the medium, with a loading capacity of about 10–50 mg / ml). Linear flow rate: 100–140 cm / h. After the sample loading is completed, rinse with solution A2 and then rinse with 10% solution B to remove impurities.

[0402] (3) Elution: Elute the target peak with a linear gradient from 10% B to 25±5% B, collect the samples in segments and test their purity. Combine the samples with a purity higher than 60% to obtain 4.94 to 5.30 L of two-step purified saponin sample solution.

[0403] The material quantities for the first step of reverse phase purification, including sample loading and elution, are shown in Table 9 below.

[0404] Table 9. Steps for the first step of reverse phase purification

[0405]

[0406] 2.2 Second step of saponin solution reverse-phase purification

[0407] (1) Pretreatment: Connect the chromatography column and the liquid chromatography system, control A2 solution to enter pump A and B solution to enter pump B respectively, the linear flow rate of the system is 100-140 cm / h, and after 100% B washing, 100% A equilibration is performed.

[0408] (2) Sample loading: Dilute the saponin sample solution purified in two steps with water, control the acetonitrile concentration to 22% to 28%, and load the sample onto UniPS 10-300 chromatographic medium (load all the diluted sample, with a loading of about 10 to 50 mg / ml). Linear flow rate: 100 to 140 cm / h. After loading, rinse with solution A2, and then rinse with 10% solution B to remove impurities.

[0409] (3) Elution: The target peak was eluted with a linear gradient from 10% B to 25±5% B, and the samples were collected in segments for purity testing. Samples with a purity higher than 80% were combined to obtain 1.76 to 2.30 L as the three-step purified saponin sample solution.

[0410] The material quantities used in the second step of reverse phase purification, including sample loading and elution, are shown in Table 10 below.

[0411] Table 10. Steps for the second step of reverse phase purification

[0412]

[0413] 2.3, Step 3 of Reverse-Phase Purification of Saponin Solution

[0414] (1) Pretreatment: Connect the chromatography column and the liquid chromatography system, and control the A2 solution to enter the A pump port and the B solution to enter the B pump port respectively. The linear flow rate of the system is 100-140 cm / h. After 100% B washing, 100% A equilibration is performed.

[0415] (2) Sample loading: Dilute the saponin sample solution purified in three steps with water, control the acetonitrile concentration to 22% to 28%, and load the sample onto UniPS 10-300 chromatographic medium (load all the diluted sample, with a loading of about 10 to 50 mg / ml). Linear flow rate: 100 to 140 cm / h. After loading, rinse with solution A2, and then rinse with 10% solution B to remove impurities.

[0416] (3) Elution: The target peak was eluted with a linear gradient from 10% B to 25±5% B, and the samples were collected in segments for purity testing. Samples with a purity higher than 95% were combined to obtain 1.41 to 1.81 L, which is the four-step purified saponin sample solution.

[0417] The material quantities used for sample loading and elution in the third step of reverse phase purification are shown in Table 11 below.

[0418] Table 11. Steps for the third step of reverse phase purification

[0419]

[0420] Step 3: Saponin precipitation, redissolution, ultrafiltration replacement, and dispensing.

[0421] 3.1 Precipitation and Redissolution Steps

[0422] (1) Take 1.4-1.8 L of the four-step purified saponin sample solution prepared in Example 2, add twice the volume of water and mix well. Let it stand at room temperature for at least 30 min, then centrifuge at 9000-11500 g and 2-8 °C for 10 min, and collect the precipitate.

[0423] (2) Add 1.5 to 3.0 L of dissolving solution to the precipitate, stir to dissolve, and obtain 1.5 to 3.0 L of organic solvent-free sample.

[0424] 3.2 Ultrafiltration Replacement Steps

[0425] (1) Rinse the ultrafiltration membrane with ultrafiltration replacement solution (5mM histidine, pH 5.0); take the saponin sample (about 1.5-3.0L) obtained after redissolving the precipitate in step 1 of this embodiment and concentrate it to 0.91-0.93L by ultrafiltration; 5kD membrane pack, transmembrane pressure: 0.2-0.4 bar, replacement times 10 times.

[0426] (2) Wash the filter with ultrafiltration replacement solution for a volume of not less than 8 times to obtain 0.91-0.93L of saponin ultrafiltration sample.

[0427] Using a 5kD membrane pack, the yield is no less than 70%, which is a significant improvement compared to the conventional 10kD ultrafiltration replacement membrane pack.

[0428] 3.3 Repackaging

[0429] The saponin ultrafiltration sample after sterilization and replacement was filtered through a 0.45 ± 0.2 μm filter to obtain the QS21 adjuvant solution described in this application, which was then aliquoted and stored at a temperature below -70°C.

[0430] Step 4: Key process quality inspection

[0431] 4.1 Detection Method

[0432] Intermediate samples from each step of the three batches of pilot-scale production were subjected to RP-HPLC analysis to compare and analyze the impurity peak removal effect of each chromatographic step. The chromatographic conditions are shown in Table 12 below.

[0433] Table 12. Chromatographic Procedure

[0434]

[0435] 4.2 Removal efficiency of major impurities

[0436] The first step of reversed-phase crude purification and reversed-phase fine purification yielded a number of HPLC impurity peaks in the sample. Among them, the main peak of QS-21 had a retention time of 22.622 min, and the three main impurity peaks had retention times of 20.376 min (impurity 1), 20.734 min (impurity 2), and 23.109 min (impurity 3).

[0437] The detection chromatogram of the QS21 sample after the third step of reverse-phase purification (after the fourth step of purification) is shown below. Figure 17 As shown;

[0438] Since the samples obtained in the first step of reversed-phase crude purification and reversed-phase fine purification have many impurity peaks, this study only calculates the chromatographic impurity removal effect based on the results of the second step of reversed-phase fine purification (after three-step purification) and the third and fourth steps of reversed-phase fine purification. The main impurity removal effects of the four-step purification compared to the three-step purification are shown in Table 13 below.

[0439] Table 13. Comparison of main impurity removal efficiency between four-step purification and three-step purification.

[0440]

[0441] Removal rate = chromatographically harvested sample Total amount of impurity peaks The total amount of impurity peaks in the sample loaded for chromatography, of which...

[0442] Total impurity peaks = (peak area of ​​UV peak detected by RP-HPLC / injection volume) * sample volume of chromatography step

[0443] 4.3. QS21 Purity and Yield Testing

[0444] The purity of QS21 was determined using the same RP-HPLC method, and the results are shown in Table 15 below.

[0445] Table 15. Purity test results of QS21

[0446]

[0447] The yield of QS21 was determined using the same RP-HPLC method, and the results are shown in Table 16 below.

[0448] Table 16. Yield test results of QS21

[0449] Inverse crude pure 76.77％ 75.37％ 80.60％ Phase refining step one 82.64％ 83.21％ 71.87％ Inverse purification step two 37.87％ 51.13％ 60.10％ Phase refining step 3 80.46％ 53.78％ 63.17％

[0450] Step 5: QS-21 Functional Verification

[0451] The QS21 samples prepared using the aforementioned steps were used to prepare MA105 adjuvant, shingles vaccine, and antitumor vaccine products according to the methods described in Examples 1-11 above, and their activities were compared with those of existing commercial QS21 products.

[0452] 5.1 Shingles vaccine products

[0453] The self-made QS-21 and the purchased QS-21 were formulated into gE / combined adjuvant candidate vaccines respectively, and the differences in immunogenicity between the two were compared.

[0454] Experimental methods: At the beginning of the study, pre-immunization was performed, and then the animals were divided into 3 groups: 10 animals in each experimental group and 5 animals in each gE / buffered buffer control group.

[0455] Pre-immunization method: 50 attenuated VZV viruses were administered subcutaneously to 5-6 week old female C57BL / 6 mice at 10⁴-5 TCID.

[0456] Five weeks after pre-immunization, mice were intramuscularly injected on days 0 and 28, with each injection containing 0.05 ml / 0.1 dose / mouse.

[0457] Blood samples were collected from mice 14 days after the second immunization, and the binding antibodies in individual serum were detected by ELISA.

[0458] Simultaneously, the spleen was isolated, and the frequency of CD4+ T cells producing cytokines (IL-2 / IFN-γ) was assessed by intracellular cytokine staining (flow cytometry).

[0459] The experimental results show that:

[0460] (1) There was no significant difference in the titer of binding antibodies after immunization with two different QS-21 vaccines formulated from different sources;

[0461] (2) Both vaccine formulations can induce stimulation to produce (IL-2 / IFN-γ)CD4+ T cell responses;

[0462] It is evident that the QS-21 prepared using the method and process of this embodiment and the gE / combination adjuvant candidate vaccine formulated with purchased QS-21 show no significant difference in inducing and activating immunity.

[0463] Finally, the same therapeutic activity results were also verified in antitumor vaccines based on MA105 adjuvant and antigen-free therapeutic agents (Examples 8-10).

[0464] Finally, it should be noted that the above embodiments are only used to help those skilled in the art understand the essence of the present invention, and are not intended to limit the scope of protection of the present invention.

Claims

1. A recombinant herpes zoster vaccine formulation, comprising: the vaccine formulation containing: A therapeutically effective amount of antigen, wherein the antigen is a gE protein antigen with the sequence shown in SEQ ID NO.1; MA105 adjuvant; And necessary pharmaceutical excipients; in, MA105 adjuvant contains: (1) QS-21, 50-300μg / ml; (2) Poly I:C, 400-3000 μg / ml; and (3) Lipid molecules that constitute the carrier; The carrier is a mixture of cationic liposomes and neutral liposomes; The lipid molecules are: DOTAP, 70-560 μg / ml; DOPC, 500-4000 μg / ml; and cholesterol, 175-1400 μg / ml.

2. The recombinant herpes zoster vaccine formulation according to claim 1, characterized in that, The adjuvant contains: QS-21, 50-200 μg / ml; Poly I:C, 400-3000μg / ml.

3. The recombinant herpes zoster vaccine formulation according to claim 2, characterized in that, The adjuvant contains: QS-21, 80-120μg / ml; Poly I:C, 780-1600μg / ml.

4. The recombinant herpes zoster vaccine formulation according to claim 1, characterized in that, The lipid molecules mentioned are: DOTAP, 140-280 μg / ml; DOPC, 1200-2500 μg / ml; and Cholesterol, 350-600 μg / ml.

5. The recombinant herpes zoster vaccine formulation according to any one of claims 1 to 4, wherein the preparation method of the MA105 adjuvant comprises the following steps: (1) Prepare cationic liposomes and neutral liposomes respectively; (2) QS-21, Poly I:C, cationic liposomes and neutral liposomes were mixed and stirred evenly to prepare MA105 adjuvant; The method for preparing the cationic liposomes is as follows: (1) It is prepared by mixing DOTAP, DOPC and cholesterol in a mass ratio of 2:2:1; (2) After the three lipid components are evenly dispersed, liposomes are prepared using ethanol injection method, thin film dispersion method, ultrasonic dispersion method and reverse evaporation method; The method for preparing the neutral liposomes is as follows: (1) Prepared by mixing DOPC and cholesterol in a mass ratio of 4:1; (2) After the lipid components are evenly dispersed, liposomes are prepared by ethanol injection method, thin film dispersion method, ultrasonic dispersion method and reverse evaporation method.

6. The recombinant herpes zoster vaccine formulation according to claim 5, characterized in that, The preparation steps of the cationic liposomes are as follows: (1) The three lipid components are dissolved in ethanol and sheared online 1 to 3 times with the aqueous phase; (2) Use a sucrose-containing buffer solution for ultrafiltration replacement, with a concentration factor of 2 to 4 times and a washing factor of 6 to 8 times; (3) Use a filter membrane with a pore size of 200 nm for extrusion, and perform multiple cycles of extrusion to control particle size uniformity; (4) Use a filter membrane with a pore size of 100 nm for extrusion, and perform multiple cycles of extrusion to control the uniformity of particle size.

7. The recombinant herpes zoster vaccine formulation according to claim 6, characterized in that, The preparation steps of the cationic liposomes also include step (5): using a sterile filter for sterilization.

8. The recombinant herpes zoster vaccine formulation according to claim 5, characterized in that, The physicochemical parameters of the cationic liposomes are as follows: Average particle size D50: 30–90 nm; Solution pH: 4.8–7.0; Zeta potential: 40–72 mV, pH 6.

5.

9. The recombinant herpes zoster vaccine formulation according to claim 8, characterized in that, The physicochemical parameters of the cationic liposomes include a solution pH of 4.8–6.

0.

10. The recombinant herpes zoster vaccine formulation according to claim 5, characterized in that, The preparation steps of the neutral liposomes are as follows: (1) The two lipid components are dissolved in ethanol and sheared online with the aqueous phase 1 to 3 times; (2) Use a filter membrane with a pore size of 200 nm for filtration and perform multiple cyclic extrusions to control particle size uniformity; (3) Use a filter membrane with a pore size of 100 nm for filtration and perform multiple cyclic extrusions to control particle size uniformity; (4) Use a sucrose-containing buffer solution for ultrafiltration replacement, with a concentration factor of 3 to 6 times and a washing factor of 6 to 8 times.

11. The recombinant herpes zoster vaccine formulation according to claim 10, characterized in that, The preparation steps of the neutral liposomes further include: step (5) using a sterile filter for sterilization.

12. The recombinant herpes zoster vaccine formulation according to claim 5, characterized in that, The physicochemical parameters of the neutral liposomes are as follows: Average particle size D50: 80–140 nm; Solution pH: 5.5-7.

0.

13. The recombinant herpes zoster vaccine formulation according to claim 12, characterized in that, Among the physicochemical parameters of the neutral liposomes, the solution pH value is 6.4-6.

6.

14. The recombinant herpes zoster vaccine formulation according to claim 1, characterized in that, The pharmaceutical excipients include: polysorbate 80, histidine, sodium dihydrogen phosphate, sucrose, and sodium chloride.

15. The method for preparing the recombinant herpes zoster vaccine formulation according to any one of claims 1 to 14, characterized in that, It includes the following steps: (1) Add sucrose, histidine and Tween-80 to the purified antigen solution, adjust the pH and prepare the antigen stock solution. The antigen in the antigen stock solution is the gE protein antigen with the sequence shown in SEQ ID NO.

1. (2) The antigen stock solution and the MA105 adjuvant described in any one of claims 1 to 13 are mixed and stirred evenly to prepare a semi-finished product; (3) After repackaging, the recombinant herpes zoster vaccine formulation is obtained.

16. The method according to claim 15, characterized in that, The recombinant herpes zoster vaccine formulation comprises, per milliliter: (1) The gE protein antigen with the sequence shown in SEQ ID NO.1, in a concentration of 95-110 μg / ml; (2) A pharmaceutical excipient combination for maintaining the stability of gE protein antigen, wherein the pharmaceutical excipient combination comprises: polysorbate 80, 450-550 μg / ml; histidine, 200-950 μg / ml; sucrose, 52-55 mg / ml; (3) MA105 immune adjuvant, which includes: DOTAP, 140-215 μg / ml; DOPC, 1200-2200 μg / ml; Cholesterol, 320-550 μg / ml; QS-21, 80-110 μg / ml; Poly I:C, 780-830 μg / ml; (4) Other pharmaceutical excipients, including: Sodium dihydrogen phosphate, 0-800 μg / ml; Sodium chloride, 2.5-6 mg / ml.

17. The recombinant herpes zoster vaccine formulation prepared by the method described in claim 15 or 16.

18. The recombinant herpes zoster vaccine formulation according to claim 17, characterized in that, The osmotic pressure of the vaccine formulation is 210–350 mOsmol / kg; The average particle size of the vaccine formulation is 100–300 nm; The pH value of the vaccine preparation is 6.0 to 7.

0.

19. The recombinant herpes zoster vaccine formulation according to claim 17, characterized in that, The average particle size of the vaccine formulation is 130~200nm.

20. The use of the recombinant herpes zoster vaccine formulation according to any one of claims 1 to 14, 17 and 18 in the preparation of a medicament for the prevention of disease caused by varicella-zoster virus (VZV).

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