A recombinant herpes zoster vaccine and a method for preparing the same

Abstract

The application belongs to the technical field of biological medicine, and discloses a recombinant herpes zoster vaccine and a preparation method thereof.The vaccine is composed of gE-gD protein and a liposome adjuvant.The liposome adjuvant is formed by co-dissolving 3D-MLA, phospholipid-containing lipids and steroidal components in an organic solvent, and then adding QS-21 at the end after PBS hydration and homogenization, and then being diluted with PBS.The process makes the enhancing components uniformly and stably anchored in the micro-domain of the membrane phase, and the particle structure is controllable.Under the same antigen dose and detection caliber, the D28 GMC of the vaccine is significantly improved, and a more sufficient and stronger initial humoral immune response is embodied.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to a recombinant herpes zoster vaccine and a preparation method thereof. BACKGROUND

[0002] Herpes zoster is caused by the reactivation of varicella-zoster virus after latency in the body. The virus can enter the body through the upper respiratory tract or conjunctiva and cause systemic infection. Primary infection is more common in children, presenting as chickenpox; in adulthood, it can be latent and enter a latent state. After recovery from primary infection, the virus can persist in the posterior root ganglion of the spinal cord for a long time; when the immune function of the body decreases, it is easily reactivated, and a unilateral rash and pain along the ganglion segment appear. Postherpetic neuralgia is the most common complication, which can be continuous or intermittent and repeated, significantly reducing the quality of life and increasing the social burden of disease. Epidemiological data shows that about one-fourth of the population has a risk of getting sick in their lifetime, with a higher proportion of the elderly.

[0003] Although clinical treatment methods have been continuously developed, from the perspective of public health, active immunization is still an economical and effective strategy to reduce the incidence of herpes zoster and related complications. Recombinant subunit vaccines use key surface glycoproteins of the virus as antigens, have the advantages of safety and production consistency; the immunogenicity is usually evaluated by the level of humoral immunity at a specific time point after vaccination, and the IgG antibody titer is used as the main endpoint, which has become a common practice. The binding level of IgG to viral epitopes can reflect the recognition and amplification of the body to the target antigen; for reactivation-related diseases, higher and longer-lasting IgG levels usually indicate better protection against re-exposure and lower risk of postherpetic neuralgia. Based on the characteristics of mature detection methods, unified statistical indicators and strong cross-study comparability, IgG titer has become a core indicator for evaluating the immunogenicity of such vaccines.

[0004] However, the existing recombinant herpes zoster vaccine still has room for improvement in the peak and persistence of IgG titer, especially in achieving high levels, low dispersion, and amplification in different populations and different batches. Without changing the original body, improving the immunization enhancement from the formulation level is generally considered an effective way to improve IgG. Among them, the optimization of adjuvants around the delivery and enhancement path has the potential to amplify the humoral immune response without increasing the dosage. However, existing public technologies still lack sufficient and reusable solutions on how to significantly and stably improve IgG titer through systematic design at the formulation level. Therefore, there is an urgent need in the field to propose a new and engineer-able technical solution to improve IgG antibody titer explicitly and stably while ensuring safety and manufacturability, thereby further improving the overall immune protection level of the recombinant herpes zoster vaccine. SUMMARY

[0005] In view of the above-mentioned defects of the prior art, the present application provides a recombinant herpes zoster vaccine and a preparation method thereof.

[0006] The specific technical solutions are as follows:

[0007] A recombinant herpes zoster vaccine, comprising gE-gD protein and a liposome adjuvant; the liposome adjuvant is prepared by co-dissolving 3D-MLA, phospholipid-containing lipid and steroidal components in an organic solvent to form a film, hydrating and homogenizing the film with PBS, adding QS-21, and then diluting to the volume with PBS.

[0008] Preferably, a recombinant herpes zoster vaccine comprises, per 0.50 mL / dose:

[0009] 30-90 μg / dose (in terms of active antigen mass) of gE-gD protein;

[0010] a liposome adjuvant comprising, per 0.50 mL / dose: 20-80 μg / dose of 3D-MLA, 1500-3600 μg / dose of phospholipid-containing lipid, 500-1800 μg / dose of steroidal component, and 30-90 μg / dose of QS-21; and the total volume is diluted to 0.50 mL with PBS buffer.

[0011] The phospholipid-containing lipid is an amphiphilic membrane lipid with a phospho group head, including phosphatidylcholine and sphingomyelin.

[0012] The phosphatidylcholine is selected from one or more of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, CAS No. 816-94-4), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC, CAS No. 63-89-8), hydrogenated phosphatidylcholine (HSPC, CAS No. 97281-48-6), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC, CAS No. 26853-31-6), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, CAS No. 4235-95-4), and pharmaceutically acceptable forms thereof.

[0013] The sphingomyelin is selected from one or more of N-palmitoyl-sphingosine-phosphocholine (C16:0-SM, CAS No. 6254-89-3), N-stearoyl-sphingosine-phosphocholine (C18:0-SM, CAS No. 2304-81-6), N-eicosanoyl-sphingosine-phosphocholine (C20:0-SM, CAS No. 7344-02-7), N-behenoyl-sphingosine-phosphocholine (C22:0-SM), and N-lignoceroyl-sphingosine-phosphocholine (C24:0-SM).

[0014] Further, the phospholipid-containing lipid consists of hydrogenated phosphatidylcholine and N-palmitoyl-sphingosine-phosphocholine, and the mass ratio of the two is (1-3): 1.

[0015] The steroidal component is selected from one or more of cholesterol, cholesteryl hemisuccinate (CAS No. 1510-21-0), cholesteryl sulfate, cholesteryl hemiglutarate, 7-dehydrocholesterol, beta-sitosterol, stigmasterol, campesterol, or a pharmaceutically acceptable derivative thereof.

[0016] The preparation method of the liposome adjuvant comprises: dissolving 3D-MLA, phospholipid-containing lipid and steroidal component in an organic solvent, evaporating and drying to form a liposome membrane; adding PBS buffer to hydrate and homogenize; and adding QS-21, and then adding PBS buffer to a predetermined working concentration.

[0017] Further, the preparation method of the liposome adjuvant comprises:

[0018] (a) dissolving 3D-MLA, phospholipid-containing lipid and steroidal component in isopropyl alcohol, and evaporating and drying to form a liposome membrane;

[0019] (b) adding PBS buffer to hydrate and homogenize to a particle size (Z-avg) of 90-110 nm and a PDI of less than or equal to 0.20;

[0020] (c) adding QS-21 under the conditions of 2-8°C and light shielding, and then adding PBS buffer to a predetermined working concentration;

[0021] (d) obtaining the liposome adjuvant through sterile filtration.

[0022] The application further provides a preparation method of a recombinant herpes zoster vaccine, comprising: mixing gE-gD protein and the liposome adjuvant, and then adding PBS buffer to a predetermined volume to obtain the recombinant herpes zoster vaccine.

[0023] The application cooperatively introduces 3D-MLA and QS-21 in a liposome carrier, dissolves the phospholipid-containing lipid and the steroidal component to form a membrane, then hydrates and homogenizes the membrane with PBS, and finally adds QS-21 and PBS to a predetermined working concentration. This method uniformly and stably anchors the enhanced components in the membrane phase microdomain with order and moderate fluidity, strengthens antigen exposure and uptake and presentation of dendritic cells. As a result, the D28 GMC is significantly improved under the same antigen dose and detection caliber, and a more sufficient and stronger primary humoral immune response is achieved. DETAILED DESCRIPTION

[0024] For the convenience of understanding and implementation, unless otherwise expressly limited, each of the numerical values and intervals described herein includes its endpoints, and the use of approximations within a reasonable error range of the endpoints is allowed. The upper and lower limits of any numerical range, the endpoints of different ranges, and the single-point data listed herein can be combined with each other to form new sub-ranges, which are also considered to be clearly disclosed and can be implemented accordingly.

[0025] Unless otherwise specified, the raw materials, reagents and equipment used in the present application are conventionally available in the art, can be purchased from multiple qualified suppliers or prepared according to the disclosed methods; the brand, purity and source difference do not affect the implementation and technical effects of the present application, and all belong to the reasonable scope of equivalent replacement.

[0026] The technical solutions and implementation paths of the present application are further illustrated below in combination with embodiments. The embodiments are only used to illustrate but not to limit the protection scope of the present application. Equivalent changes, replacements or combinations of parameters, processes or compositions made by those of ordinary skill in the art without departing from the core idea and essence of the present application should be covered within the scope defined by the claims of the present application.

[0027] A recombinant herpes zoster vaccine, which comprises, per 0.50 mL / dose:

[0028] gE-gD protein 30-90 μg / dose; liposome adjuvant, comprising the following components per 0.50 mL / dose: 3D-MLA 20-80 μg / dose, phospholipid-containing 1500-3600 μg / dose, sterol component 500-1800 μg / dose, QS-21 30-90 μg / dose; and supplemented with PBS buffer to a total volume of 0.50 mL of the dose. gE-gD determines specificity, 3D-MLA and QS-21 provide innate stimulation and humoral amplification, phospholipid-containing and sterol component constitute an engineered membrane phase carrier for anchoring and slow-release of immune-enhancing components.

[0029] The phospholipid-containing is an amphiphilic membrane lipid with a phosphate group head, including phosphatidylcholine and sphingomyelin. The amphiphilic membrane lipid with a phosphate group head is used to build the liposome skeleton, which not only provides the interfacial tension for forming closed vesicles and the stability of the bilayer, but also sets the order, fluidity and content barrier performance of the particles under the administration conditions by adjusting the saturation and chain length distribution, thereby affecting the residence of the enhancing components, the antigen surface exposure and the cellular uptake pathway.

[0030] The steroidal component is selected from one or more of cholesterol, cholestanol hemisuccinate, cholesterol sulfate, cholesterol hemiglutarate, cholestanol, 7-dehydrocholesterol, beta-sitosterol, stigmasterol, campesterol, or a pharmaceutically acceptable derivative thereof. By adjusting the compactness, surface charge and microdomain organization of the membrane through the hydrophobic backbone and polar modification of different steroids, the anchoring strength and release kinetics of components such as QS-21 are affected.

[0031] The preparation method of the liposome adjuvant comprises: (a) dissolving 3D-MLA, phospholipid-containing lipids and steroidal components with isopropyl alcohol, evaporating and drying to form a liposome membrane; (b) adding PBS buffer to hydrate and homogenize to a particle size (Z-avg) of 90-110 nm and a PDI of ≤0.20; (c) adding QS-21 under low-temperature and light-avoiding conditions, and then adding PBS buffer to a predetermined working concentration; and (d) sterilizing to obtain the liposome adjuvant. The components are co-dissolved and membrane-formed to ensure uniform distribution of the components at a molecular scale, then hydrated and homogenized with PBS buffer, and the particle size is controlled in a narrow range that is beneficial to lymphatic drainage and also takes into account stability, and the PDI is limited to reduce polydispersity to improve the predictability of release; then QS-21 is added under low-temperature and light-avoiding conditions, and then PBS buffer is added to a predetermined working concentration, thereby reducing the risk of degradation and aggregation caused by heat and shearing; and finally, sterilization is performed to remove particles and potential biological load, thereby forming an adjuvant mother liquor that can be directly used to prepare a vaccine.

[0032] The gE-gD protein is mixed with the liposome adjuvant, and PBS buffer is added to a predetermined volume to obtain the recombinant herpes zoster vaccine.

[0033] The parameters and sources of some raw materials in the examples are as follows: the gE-gD protein stock solution is provided by Jiangsu Huano Tai Biomedicine Technology Co., Ltd., and can also be prepared by the method of Example 2 in Chinese Invention Patent CN 119386170 A.

[0034] PBS refers to PBS buffer, NaCl 137 mM, KCl 2.7 mM, Na2HPO4 / NaH2PO4 (total inorganic phosphate 10 mM), adjusted to pH 7.20±0.05 at 25°C. Example 1:

[0035] Step 1: Membrane formation

[0036] In a 250 mL round-bottom flask, DOPC 250.0 mg, cholesterol 100.0 mg, 3D-MLA 6.00 mg were sequentially added with isopropyl alcohol 20.0 mL, and stirred at room temperature for 10 min until clear.

[0037] Rotary evaporation was performed at 42°C, 120 rpm, -0.09 MPa for 25 min to remove the solvent and form a uniform lipid film; then vacuum drying was performed at room temperature for 30 min.

[0038] The inner wall of the bottle forms a uniform dry film, and the isopropyl alcohol residue is controlled to be less than 500 ppm, which is used for the next step of hydration.

[0039] Step two: hydration and homogenization

[0040] Directly add 18.0 mL of PBS preheated to 60°C to the dry film bottle obtained in step one, hydrate at 60°C for 30 min, and obtain a crude dispersion; then transfer the crude dispersion into a high-pressure homogenizer, and circulate 5 times at 1000 bar, controlling the outlet temperature to be not higher than 30°C; after homogenization, cool at 5°C in the dark for 15 min, and obtain a liposome dispersion.

[0041] Step three: post-addition of QS-21 and constant volume

[0042] Take 6.00 mg of QS-21 and dissolve it in 2.00 mL of cold PBS (5°C); then add it dropwise to the liposome dispersion obtained in step two (5°C, dark), stand for 10 min, and constant volume to 25.0 mL with PBS, mix gently, collect by 0.22 μm PES sterile filtration, and obtain a liposome adjuvant stock solution;

[0043] Quality point detection (DLS): Z-avg 100±5 nm; PDI≤0.20. At this point, the working concentration of the adjuvant stock solution is fixed: 0.240 mg / mL 3D-MLA; 0.240 mg / mL QS-21; 14.0 mg / mL membrane material.

[0044] Step four: preparation of antigen stock solution

[0045] Take the gE-gD protein stock solution, determine the total protein concentration by BCA, and convert the active antigen concentration by purity, adjust to an active concentration of 240 μg / mL with PBS, constant volume to 25.0 mL; 0.22 μm PES final filtration, sterile storage at 5°C, and obtain an antigen stock solution with an active concentration of 240 μg / mL.

[0046] Step five: vaccine preparation and subpackaging

[0047] Mix 25.0 mL of the antigen stock solution and 25.0 mL of the adjuvant stock solution in a sterile condition according to the volume ratio of 1:1, and obtain a finished vaccine of 50.0 mL. Mix gently for 10 times, measure pH 7.20±0.05, sterilely subpackage 0.50 mL per branch, and store at 2-8°C in the dark.

[0048] Finally, the quantitative composition of the finished vaccine in Example 1 was as follows: per 0.50 mL / dose: gE-gD protein (based on activity) 60 μg; 3D-MLA 60 μg; QS-21 60 μg; DOPC 2.50 mg; cholesterol 1.00 mg; PBS to the fill volume. The physicochemical characteristics were: Z-avg 100±5 nm, PDI ≤0.20, and uniform milky white appearance. Example 2:

[0049] The only difference between step one and Example 1 is that steps two through five are the same. Specifically, step one is as follows:

[0050] Add 250.0 mg DSPC, 100.0 mg cholesterol, and 6.00 mg 3D-MLA to a 250 mL round-bottom flask in sequence, then add 20.0 mL isopropanol and stir magnetically at room temperature for 10 min until clear.

[0051] Rotary evaporation at 42°C, 120 rpm, and -0.09 MPa for 25 min was used to remove the solvent and form a uniform lipid film; subsequently, vacuum drying at room temperature and -0.09 MPa was performed for 30 min.

[0052] A uniform dry film forms on the inner wall of the bottle, controlling isopropanol residue to <500ppm, which is then used for the next step of hydration. Example 3:

[0053] The only difference between step one and Example 1 is that steps two through five are the same. Specifically, step one is as follows:

[0054] Add 250.0 mg DSPC, 100.0 mg cholesterol hemisuccinate, and 6.00 mg 3D-MLA to a 250 mL round-bottom flask in sequence, then add 20.0 mL isopropanol and stir magnetically at room temperature for 10 min until clear.

[0055] Rotary evaporation at 42°C, 120 rpm, and -0.09 MPa for 25 min was used to remove the solvent and form a uniform lipid film; subsequently, vacuum drying at room temperature and -0.09 MPa was performed for 30 min.

[0056] A uniform dry film forms on the inner wall of the bottle, controlling isopropanol residue to <500ppm, which is then used for the next step of hydration. Example 4:

[0057] The only difference between step one and Example 1 is that steps two through five are the same. Specifically, step one is as follows:

[0058] In a 250 mL round-bottom flask, add 250.0 mg of N-palmitoyl-sphingosine-phosphocholine, 100.0 mg of cholesterol hemisuccinate, and 6.00 mg of 3D-MLA in sequence, then add 20.0 mL of isopropanol and stir magnetically at room temperature for 10 min until clear.

[0059] Rotary evaporation 42°C, 120 rpm, -0.09 MPa, 25 min to remove solvent to form a uniform lipid film; followed by vacuum drying at room temperature -0.09 MPa for 30 min.

[0060] A uniform dry film formed on the inner wall of the bottle, with isopropanol residue controlled to be < 500 ppm, for the next step of hydration. Example 5:

[0061] Only step one is different from Example 1, steps two to five are the same. Specifically, step one is as follows:

[0062] Into a 250 mL round bottom flask, add DSPC 150.0 mg, N-palmitoyl-sphingosine- phosphocholine 100.0 mg, cholestanol hemisuccinate 100.0 mg, 3D-MLA 6.00 mg, add isopropanol 20.0 mL, magnetic stirring at room temperature for 10 min to clarify.

[0063] Rotary evaporation 42°C, 120 rpm, -0.09 MPa, 25 min to remove solvent to form a uniform lipid film; followed by vacuum drying at room temperature -0.09 MPa for 30 min.

[0064] A uniform dry film formed on the inner wall of the bottle, with isopropanol residue controlled to be < 500 ppm, for the next step of hydration. Example 6:

[0065] Only step one is different from Example 1, steps two to five are the same. Specifically, step one is as follows:

[0066] Into a 250 mL round bottom flask, add HSPC 250.0 mg, cholestanol hemisuccinate 100.0 mg, 3D-MLA 6.00 mg, add isopropanol 20.0 mL, magnetic stirring at room temperature for 10 min to clarify.

[0067] Rotary evaporation 42°C, 120 rpm, -0.09 MPa, 25 min to remove solvent to form a uniform lipid film; followed by vacuum drying at room temperature -0.09 MPa for 30 min.

[0068] A uniform dry film formed on the inner wall of the bottle, with isopropanol residue controlled to be < 500 ppm, for the next step of hydration. Example 7:

[0069] Only step one is different from Example 1, steps two to five are the same. Specifically, step one is as follows:

[0070] Into a 250 mL round bottom flask, add DOPC 250.0 mg, Cholesterol hemisuccinate 100.0 mg, 3D-MLA 6.00 mg, add isopropanol 20.0 mL, magnetic stirring at room temperature for 10 min until clear.

[0071] Rotary evaporation at 42°C, 120 rpm, -0.09 MPa for 25 min to remove solvent to form a uniform lipid film; then vacuum drying at room temperature -0.09 MPa for 30 min.

[0072] A uniform dry film is formed on the inner wall of the bottle, and the residual isopropanol is controlled to be less than 500 ppm, which is used for the next step of hydration. Example 8:

[0073] Only step one is different from example 1, steps two to five are the same. Specifically, step one is as follows:

[0074] Into a 250 mL round bottom flask, add DOPC 250.0 mg, Cholesterol hemisuccinate 100.0 mg, 3D-MLA 6.00 mg, add isopropanol 20.0 mL, magnetic stirring at room temperature for 10 min until clear.

[0075] Rotary evaporation at 42°C, 120 rpm, -0.09 MPa for 25 min to remove solvent to form a uniform lipid film; then vacuum drying at room temperature -0.09 MPa for 30 min.

[0076] A uniform dry film is formed on the inner wall of the bottle, and the residual isopropanol is controlled to be less than 500 ppm, which is used for the next step of hydration. Example 9:

[0077] Only step one is different from example 1, steps two to five are the same. Specifically, step one is as follows:

[0078] Into a 250 mL round bottom flask, add DOPC 250.0 mg, Cholesterol hemisuccinate 100.0 mg, 3D-MLA 6.00 mg, add isopropanol 20.0 mL, magnetic stirring at room temperature for 10 min until clear.

[0079] Rotary evaporation at 42°C, 120 rpm, -0.09 MPa for 25 min to remove solvent to form a uniform lipid film; then vacuum drying at room temperature -0.09 MPa for 30 min.

[0080] A uniform dry film is formed on the inner wall of the bottle, and the residual isopropanol is controlled to be less than 500 ppm, which is used for the next step of hydration.

[0081] Blank example:

[0082] gE-gD 6 μg; PBS made up to 0.05 mL. The difference from Example 1 is that it does not contain an adjuvant.

[0083] Test Example 1:

[0084] The test animals were BALB / c mice (6-8 weeks old, 18-22 g in weight), with n=10 in each sample group, and they were randomly grouped and double-blind coded in the blood sampling and testing stages. The mice were injected intramuscularly in the biceps femoris with a single needle, and the volume of each dose was 0.05 mL, i.e. 1 / 10 of the volume of the finished product.

[0085] Blood was sampled at two time points, D0 and D28: 100-150 μL of whole blood was taken from the retro-orbital or tail vein of each mouse, which was left to clot at room temperature for 30 minutes; then it was centrifuged at 4°C and a relative centrifugal force of 3000 x g for 10 minutes, and the supernatant serum was separated and carefully transferred to low-adsorption tubes, which were immediately frozen at -80°C to avoid repeated freeze-thawing; after all the samples were tested and the statistics were completed, the blinding was removed.

[0086] 96-well Nunc MaxiSorp high-binding plates were selected. Recombinant gE antigen was prepared at 1.0 μg / mL in carbonate coating buffer (pH 9.6), and 100 μL was added to each well, which was coated at 4°C overnight, and the liquid was poured out the next day. After the plates were washed with PBST (PBS + 0.05% Tween-20) 3 times, 1% BSA was added at 200 μL / well, which was incubated at room temperature for 1 h to complete the blocking, and the plates were washed with PBST 3 times and tapped to dry.

[0087] The test serum was serially diluted at a 3-fold gradient from 1:100, and 8 dilution points were prepared, with double wells for each dilution, and 100 μL was added to each well. The plates were incubated at room temperature for 1 h to complete the binding with the coated antigen, and then the plates were washed with PBST 4 times to completely remove the unbound components and avoid background elevation.

[0088] HRP-labeled anti-mouse IgG was diluted at 1:5000, and 100 μL was added to each well, which was incubated at room temperature for 1 h, and then the plates were washed with PBST 4 times. TMB color developing liquid was added at 100 μL / well, which was color developed at room temperature for about 10 min, and the OD was close to 1.0 for the medium control wells, and then 2 mol / L sulfuric acid was added at 100 μL / well to stop the reaction, and the absorbance was read within 10 min after stopping at a main wavelength of 450 nm and a reference wavelength of 630 nm.

[0089] High-titer reference serum standard was prepared in the same plate with 8 points in 3-fold gradient from 1000 AU / mL, and a 4-parameter logistic regression was used to establish a standard curve; negative and low / medium / high three levels of quality control were set and determined to be in control, with the requirements of plate CV≤10% and inter-plate CV≤20%, and the whole plate was discarded and retested if out of range. The sample OD was converted to AU / mL according to the curve of the plate; the LLOQ was set to 20 AU / mL, and the results below the LLOQ were counted as LLOQ / 2 (i.e. 10 AU / mL). The geometric mean concentration (GMC), serum conversion rate (more than 4 times of D0) and geometric multiplication rate (GMFR=D28 / D0) of each group were calculated; the log10 of AU / mL was taken for statistical t-test between groups, with a significant threshold of p<0.05, and the group difference (G1 / G2) and its 95% confidence interval were reported. The test data are shown in Table 1.

[0090] Table 1: Test table of vaccine geometric mean concentration

[0091]

[0092] Note: LLOQ=20 AU / mL; D0 below LLOQ is counted as 10 AU / mL; D28 is the main endpoint.

[0093] All groups were detected under the same ELISA caliber, and the serum AU / mL was first log 10 The geometric mean concentration GMC and 95% confidence interval were calculated after conversion, and the geometric mean ratio GMR was used for comparison between groups. D0 was only used to prove the consistency of the baseline, and those below the LLOQ were counted as 10 AU / mL (LLOQ / 2) and did not participate in the determination of the advantages and disadvantages; D28 was the main endpoint. Compared with the blank control, the D28 GMC of Example 1 was 3780 AU / mL (95% CI 2480-5760), and the GMR=5.40; on this basis, the D28 GMC of Examples 2-7 increased to 9020 AU / mL (95% CI 5900-13600), the relative GMR to the blank was between 6.37-12.89, and the GMR relative to Example 1 was also >1 (1.18-2.39). Accordingly, under the same antigen dose and administration conditions, the IgG antibody titer of the improved prescription at D28 was significantly higher than that of the blank control, and was better than the baseline prescription of Example 1, and the conclusion was robust.

[0094] Compared with Example 2, after replacing the steroid with cholestanol hemisuccinate in Example 3, the ordered membrane phase of saturated long-chain phospholipids obtained a moderate interfacial electrical property and a stronger hydrogen bond network, and the liquid ordered microdomains were more easily nucleated and remained connected, with reduced boundary line tension and leakage rate. Therefore, the anchoring of QS-21 and the steroid skeleton is more stable and longer, and the uptake and antigen presentation efficiency of dendritic cells are simultaneously improved, resulting in a higher D28 IgG geometric mean concentration under the same antigen dose.

[0095] Comparing Example 3, Example 6 and Example 8, the difference in nature is the saturation and chain length distribution of the membrane material. Hydrogenated phosphatidylcholine is composed of multiple saturated chain lengths, has a wider melting range, and can form a liquid-ordered background with both order and necessary fluidity at body temperature, which is more conducive to the synergistic organization of stable and connected microdomains with cholesterols hemisuccinate; distearoylphosphatidylcholine has a single chain length and a high melting point, and the membrane phase is biased hard and the boundary remodeling is insufficient; dioleoylphosphatidylcholine has a loose packing due to double bonds, and the membrane phase is biased soft and the connectivity is insufficient. Therefore, the hydrogenated phosphatidylcholine system gives QS-21 and steroid complex higher anchoring density and more stable residence, and the delivery and presentation kinetics are more controllable, so the D28 IgG peak and the compliance ratio of Example 6 are better than those of Example 3 and Example 8 at the same caliber.

[0096] Example 7 uses hydrogenated phosphatidylcholine and sphingomyelin as membrane materials, and cooperates with cholesterols hemisuccinate, QS-21 and 3D-MLA to form and maintain connected liquid-ordered microdomains at this formulation ratio; the microdomains provide high-density and stable anchoring sites, making it easier for steroids and sphingomyelin to complex and stably reside, and QS-21 and 3D-MLA achieve more sufficient local enrichment and membrane rearrangement at the boundary of the microdomains, thereby improving antigen presentation and endocytosis efficiency, and showing clear synergy. Example 5 uses distearoylphosphatidylcholine and sphingomyelin, the membrane phase is biased hard, the microdomains can nucleate but the boundary fluidity is insufficient, which limits the membrane rearrangement of QS-21 participation, and the overall only shows an additive effect without forming synergy. Example 9 uses dioleoylphosphatidylcholine and sphingomyelin, the membrane phase is biased soft and it is difficult to maintain microdomain connectivity, the anchoring sites are sparse and easy to disintegrate, and the spatial coupling of steroids and immunostimulatory components is difficult to sustain, so no synergy is produced.

[0097] The description only illustrates the preferred embodiments of the present application, and does not exhaust all technical forms of the present application. It should be understood that under the guidance of the general concept and purpose of the present application, those skilled in the art can make equivalent changes, substitutions or adaptive optimizations to materials, ratios, process sequences, equipment conditions and their combinations according to the disclosed technical means and parameter selection without creative labor; through conventional analysis, deduction or a small amount of verification tests, the same technical solutions as the present application can also be obtained. Any implementation form that follows the technical idea of the present application and achieves the same technical effect shall be subject to the claims, and falls within the protection scope of the present application.

Claims

1. A recombinant herpes zoster vaccine, characterized in that, Each 0.50 mL / dose contains: 30-90 μg / dose of gE-gD protein; liposome adjuvant, comprising the following components per 0.50 mL / dose: 20-80 μg / dose of 3D-MLA, 1500-3600 μg / dose of phospholipids, 500-1800 μg / dose of cholesterol hemisuccinate, and 30-90 μg / dose of QS-21; and is made up to a total volume of 0.50 mL with PBS buffer. The phospholipids consist of hydrogenated phosphatidylcholine and N-palmitoyl-sphingosine-phosphocholine.

2. The recombinant herpes zoster vaccine as described in claim 1, characterized in that, The mass ratio of hydrogenated phosphatidylcholine to N-palmitoyl-sphingosine-phosphocholine is (1-3):

1.

3. The recombinant herpes zoster vaccine as described in claim 2, characterized in that, The preparation of the liposome adjuvant The preparation method includes: dissolving 3D-MLA, phospholipids and cholesterol hemisuccinate in an organic solvent, evaporating and drying to form a liposome membrane; adding PBS buffer for hydration and homogenization; then adding QS-21 and adjusting the volume with PBS buffer.

4. The recombinant herpes zoster vaccine as described in claim 3, characterized in that, The method for preparing the liposome adjuvant includes: (a) Dissolve 3D-MLA, phospholipids and cholesterol hemisuccinate in isopropanol, and evaporate and dry to form a liposome membrane; (b) Add PBS buffer to hydrate and homogenize until the particle size is 90-110 nm and PDI ≤ 0.20; (c) Add QS-21 after being kept at 2-8℃ in the dark, and bring the volume up to the predetermined working concentration with PBS buffer. (d) The liposome adjuvant is obtained by sterile filtration.

5. A method for preparing a recombinant herpes zoster vaccine as described in claim 1, characterized in that, include: The gE-gD protein was mixed with liposome adjuvant and then replenished to the predetermined volume with PBS buffer to obtain the recombinant herpes zoster vaccine.

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