A varicella-zoster virus vaccine and uses thereof

Abstract

The application provides a varicella-zoster virus vaccine and application thereof, and belongs to the technical field of vaccines.The varicella-zoster virus vaccine comprises liposome nanoparticles, zoster virus glycoprotein E and an adjuvant encapsulated in the liposome nanoparticles.The adjuvant comprises triterpenoid saponins.In the application, the zoster virus glycoprotein E (gE) is wrapped by the liposome nanoparticles, which can effectively promote antigen-presenting cells to phagocytose and efficiently deliver the antigen, and realize sustained stimulation of the vaccine to the body to produce a specific cellular immune response against VZV-gE.The triterpenoid saponins used in the application can effectively realize cross-presentation of the antigen zoster virus glycoprotein E and induce an antigen-specific cellular immune response.In addition, the cholesterol rich in the liposome nanoparticles can effectively neutralize the cytotoxicity of the triterpenoid saponins, ensuring the safety of the vaccine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of vaccines, and particularly relates to a varicella-zoster virus vaccine and application thereof. BACKGROUND

[0002] Varicella-Zoster Virus (VZV) is widespread throughout the world, with a very strong infectivity, and only one serotype has been found so far. VZV only infects humans in nature. VZV can cause both chickenpox and herpes zoster (HZ). Chickenpox is usually seen in childhood, while herpes zoster occurs in adulthood. After the primary infection of chickenpox, the virus can be latent in the host's ganglion. With the increase of age, immune dysfunction or immunosuppression, VZV can be reactivated and cause herpes zoster. Globally, most adults are at risk of developing herpes zoster and its related complications.

[0003] The attenuated live vaccine of Oka strain developed by Michiaki Takahashi of Japan was approved by FDA in 1995 for vaccination of children and adults to prevent chickenpox (vaccination amount: 1000-10000 PFU (plaque forming unit)), and has been widely used in the world. Subsequent studies have found that the Oka strain, like the wild-type virus, can establish latent infection and thus may also lead to the occurrence of herpes zoster.

[0004] A single subcutaneous booster immunization of high-dose attenuated live vaccine (vaccination amount: about 19400 PFU) in people over 50 years old who have been infected with VZV virus can effectively prevent herpes zoster. The corresponding product Zostavax of Merck was launched in 2005, and the protection rates for people aged 50-59, 60-69 and over 70 years old were about 70%, 64% and 38%, respectively. The decrease of this protection rate with age is mainly due to the weakening of the cellular immune response that occurs with the aging of the immune system.

[0005] Shingrix, a zoster genetically engineered subunit vaccine launched by GSK in late 2017, uses the conserved viral glycoprotein E (gE) expressed by Chinese hamster ovary (CHO) cells as an antigen, and uses the adjuvant AS01B to effectively enhance the specific cellular immune response to VZV-gE, making the protection rate of the vaccine in healthy people over 50 years old as high as 97.2% (96.6%, 97.3% and 91.3 for people aged 50-59, 60-69 and over 70, respectively) and showing good safety and effectiveness in immunodeficient people including HIV carriers. The triterpene polysaccharide QS21 and the monophosphoryl lipid (MPL) A in the AS01B adjuvant system work synergistically based on the liposome carrier to induce CD4-positive T cells specific to gE, playing a key role in the effectiveness of the vaccine.

[0006] Encapsulating CpG motif-containing oligodeoxynucleotides (CpG ODN) into ionizable lipid nanoparticles (LNP) can enhance antigen-specific humoral and cellular immune responses (PMID: 33805880).

[0007] Due to the strong cytotoxicity of QS21, many studies have not been clinically applied. Therefore, how to enhance the VZV-gE-specific cellular immune response under the premise of ensuring the safety of the vaccine components and obtain an immune effect similar to that of the Shingrix zoster vaccine by using appropriate adjuvant components is a problem that needs to be solved in vaccine development. SUMMARY

[0008] Therefore, the purpose of the present application is to provide a varicella-zoster virus vaccine and its application. The varicella-zoster virus vaccine of the present application can effectively enhance the specific cellular immune response to VZV-gE and be used as a zoster vaccine. Moreover, the varicella-zoster virus vaccine of the present application has high safety.

[0009] The present application provides a varicella-zoster virus vaccine, comprising a liposome nanoparticle and a zoster virus glycoprotein E and an adjuvant encapsulated in the liposome nanoparticle; the adjuvant comprises a triterpenoid saponin.

[0010] Preferably, the content of the zoster virus glycoprotein E in the varicella-zoster virus vaccine is 5-100 μg / dose.

[0011] Preferably, the content of the triterpenoid saponin in the varicella-zoster virus vaccine is 1-100 μg / dose.

[0012] Preferably, the triterpenoid saponin comprises QS21.

[0013] Preferably, the adjuvant further comprises a GC-rich single-stranded oligodeoxynucleotide fragment.

[0014] Preferably, the content of the GC-rich single-stranded oligodeoxynucleotide fragment in the varicella-zoster virus vaccine is 5 μg to 2 mg per dose.

[0015] Preferably, the liposome nanoparticle comprises a cationic liposome and a polyethylene glycol derivative; and the molar ratio of the cationic liposome to the polyethylene glycol derivative is (46-50):(1.5-1.6).

[0016] Preferably, the particle size of the varicella-zoster virus vaccine is 20-400 nm.

[0017] Preferably, the dosage form of the varicella-zoster virus vaccine comprises an injection.

[0018] The application also provides the use of the varicella-zoster virus vaccine described in the above-mentioned scheme in the preparation of a medicament for preventing or improving herpes zoster and / or post-herpes zoster sequelae.

[0019] The application provides a varicella-zoster virus vaccine, comprising a liposome nanoparticle and a herpes zoster virus glycoprotein E and an adjuvant encapsulated in the liposome nanoparticle; the adjuvant comprises a triterpenoid saponin. In the application, the herpes zoster virus glycoprotein E (gE) is wrapped by the liposome nanoparticle, which can effectively promote the phagocytosis of antigen-presenting cells and efficiently deliver the antigen, and realize the sustained stimulation of the vaccine to the body to produce a specific cellular immune response against VZV-gE. The triterpenoid saponin used in the application can effectively realize the cross-presentation of the antigen herpes zoster virus glycoprotein E, and induce an antigen-specific cellular immune response. Moreover, the cholesterol rich in the liposome nanoparticle can effectively neutralize the cytotoxicity of the triterpenoid saponin, ensuring the safety of the vaccine. The varicella-zoster virus vaccine described in the application is proved to specifically enhance the cellular immune response against the herpes zoster virus glycoprotein E through animal experiments, and can be used as a herpes zoster vaccine. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 - shows the varicella-zoster virus vaccine prepared by the example, the antigen encapsulation rate detected by experimental example 1 (A in Figure 1 ), the nucleic acid encapsulation rate detected by experimental example 2 (B in Figure 1 ), the QS21 encapsulation rate detected by experimental example 3 (C in Figure 1 ), the particle size detected by experimental example 4 (D in Figure 1 ), and the polydispersity index detected by experimental example 4 (E in Figure 1 );

[0021] Figure 2- showing the varicella-zoster virus vaccine prepared by the example, the cytotoxicity of the obtained cells detected by experimental example 5;

[0022] Figure 3 - showing the varicella-zoster virus vaccine prepared by the example, the gE-specific IgG antibody titers of the obtained cells detected by experimental example 6, experimental example 7, experimental example 12;

[0023] Figure 4 - showing the varicella-zoster virus vaccine prepared by the example, the IL-2 concentrations of the obtained cells detected by experimental example 6, experimental example 8, experimental example 9, experimental example 12;

[0024] Figure 5 - showing the varicella-zoster virus vaccine prepared by the example, the IFN-γ concentrations of the obtained cells detected by experimental example 6, experimental example 8, experimental example 9, experimental example 12;

[0025] Figure 6 - showing the varicella-zoster virus vaccine prepared by the example, the number of spots of IL-2 secreted by 2x10 5

[0026] Figure 7 - showing the varicella-zoster virus vaccine prepared by the example, the number of spots of IFN-γ secreted by 2x10 5

[0027] Figure 8 - showing the varicella-zoster virus vaccine prepared by the example, the proportion of CD4+T cells secreting IL-2 detected by experimental example 6, experimental example 8, experimental example 11, experimental example 12;

[0028] Figure 9 - showing the varicella-zoster virus vaccine prepared by the example, the proportion of CD4+T cells secreting IFN-γ detected by experimental example 6, experimental example 8, experimental example 11, experimental example 12. DETAILED DESCRIPTION

[0029] The present application provides a varicella-zoster virus vaccine, comprising a liposome nanoparticle and a zoster virus glycoprotein E and an adjuvant encapsulated in the liposome nanoparticle; the adjuvant comprises a triterpenoid saponin.

[0030] In the present application, each component in the varicella-zoster virus vaccine is combined by physical electroadsorption or physical wrapping.

[0031] ​​In the present application, the content of the herpes zoster virus glycoprotein E in the varicella-zoster virus vaccine is preferably 5-100 μg / dose. In the present application, the herpes zoster virus glycoprotein E (gE) is wrapped by liposome nanoparticles, which can effectively promote the phagocytosis of antigen-presenting cells and high-efficiency delivery of antigens, and realize the sustained stimulation of the vaccine to continuously stimulate the body to produce specific cellular immune response against VZV-gE.

[0032] In the present application, the content of the triterpenoid saponin in the varicella-zoster virus vaccine is preferably 1-100 μg / dose. In the present application, the triterpenoid saponin preferably includes QS21 extracted from the bark of Quillaja saponaria in South America. The triterpenoid saponin used in the present application can effectively realize the cross-presentation of the antigen herpes zoster virus glycoprotein E, and induce antigen-specific cellular immune response. Moreover, the cholesterol rich in the liposome nanoparticles can effectively neutralize the cytotoxicity of the triterpenoid saponin, and ensure the safety of the vaccine.

[0033] In the present application, the adjuvant preferably further includes a GC-rich single-stranded oligodeoxynucleotide fragment (CpG ODN), and more preferably CpG ODN 1018. In the present application, the content of the GC-rich single-stranded oligodeoxynucleotide fragment in the varicella-zoster virus vaccine is preferably 5 μg-2 mg / dose. In the present application, the CpG ODN is wrapped by liposome nanoparticles, which can effectively avoid the degradation of nucleases on one hand, and the escaped CpG ODN before being phagocytosed by the presenting cells can be rapidly degraded by nucleases in the body, thereby effectively avoiding the systemic inflammatory side effects that may be caused by the non-specific diffusion of CpG ODN from the injection site of the vaccine, and making the adjuvant present "local" and "transient" characteristics, which meet the safety requirements. In addition, the varicella-zoster virus vaccine described in the present application uses CpG ODN that can be taken up by TLR9 in endosomes, induces interferon secretion, and effectively activates antigen-specific T cells by promoting the cross-presentation of antigens. Among them, the class A of CpG ODN can stimulate dendritic cells to produce type I interferon, activate natural killer cells, the class B can quickly transfer from early endosomes to late endosomes, stimulate B cell proliferation, stimulate plasmacytoid dendritic cell maturation and the production of TNF-α, IL-6 and IL-12, and the class C has the characteristics of both class A and class B, balancing the promotion of humoral and cellular immune responses. The type C CpG ODN that can form a local stem-loop structure in the cytoplasm may induce related acquired immune responses through the stimulator of IFN genes (STING) innate immune pathway by activating cyclic GMP-AMP synthase (cGAS). The present application simultaneously uses triterpenoid saponin and CpG ODN, which have good synergistic effect in inducing antigen-specific cellular immune response.

[0034] In the present application, the liposome nanoparticles comprise cationic liposome and polyethylene glycol derivative; the molar ratio of the cationic liposome and polyethylene glycol derivative is (46-50):(1.5-1.6). In the present application, the cationic liposome preferably comprises ((4-hydroxybutyl)azabicycloalkyl)bis(hexane-6,1-diyl)bis(2-hexyldecylazanediyl) (ALC-0315) and / or heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((undecyloxy)hexyl)azanediyl)octanoate) (SM-102). In the present application, the polyethylene glycol derivative preferably comprises methoxypolyethylene glycol dimyristyl acetamide (ALC-0159) and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG2000).

[0035] In the present application, the particle size of the varicella-zoster virus vaccine is preferably 20-400 nm.

[0036] In the present application, the dosage form of the varicella-zoster virus vaccine preferably comprises injection.

[0037] The present application does not have special restrictions on the preparation method of the varicella-zoster virus vaccine, and the conventional liposome nanoparticle coating method in the art can be used. In the specific implementation process of the present application, the microfluidic technology equipment is preferably used to prepare the varicella-zoster virus vaccine.

[0038] In the present application, the administration mode of the varicella-zoster virus vaccine is preferably injection administration; the injection preferably comprises subcutaneous injection or intramuscular injection.

[0039] The present application also provides the use of the varicella-zoster virus vaccine described in the above scheme in the preparation of a drug for preventing or improving herpes zoster and / or sequelae of herpes zoster. In the present application, the sequelae of herpes zoster preferably include postherpetic neuralgia.

[0040] The technical solutions in the present application will be described clearly and completely in combination with the examples in the present application.

[0041] According to the single-dose vaccine composition in Table 1 below, the required component feed of each vaccine group of 20 doses is calculated.

[0042] Table 1. Single-dose vaccine feed

[0043]

[0044] - Not added; √ Added.

[0045] Comparative Example 1 - 0.2 mg of CHO-expressed gE ectodomain glycoprotein (purchased from Kunming Diangong Technology Co., Ltd.), 0.2 mg of thio-oxidized CpG 1018 (purchased from InvivoGen Co., Ltd.), and 0.1 mg of QS21 (purchased from Alpha Diagnostic Co., Ltd.) were weighed and dissolved in 1 mL of PBS to obtain 20 doses of Comparative Example 1 vaccine.

[0046] Comparative Example 2 - 0.2 mg of CHO-expressed gE ectodomain glycoprotein, 0.2 mg of thio-oxidized CpG 1018, and 0.1 mg of QS21 were weighed and dissolved in 0.5 mL of PBS, and mixed with an equal volume of aluminum adjuvant (purchased from Thermo Fisher Co., Ltd.) to obtain 20 doses of Comparative Example 2 vaccine.

[0047] Example - ALC-0315 (purchased from Xiamen Senobangge Biotechnology Co., Ltd.), DSPC (purchased from Shanghai Aivetuo Pharmaceutical Technology Co., Ltd.), cholesterol (purchased from Shanghai Aivetuo Pharmaceutical Technology Co., Ltd.), and ALC-0159 (purchased from Xiamen Senobangge Biotechnology Co., Ltd.) were weighed in a molar ratio of 46.3:9.4:42.7:1.6 and dissolved in anhydrous ethanol. A microfluidic nanomedicine manufacturing system (Precision Nanosystems Co., Ltd.) was used to mix the solution with 100 mM, pH 4.0 citric acid buffer containing 0.24 mg of gE, 0.3 mg of CpG 1018, and 0.12 mg of QS21 at a ratio of 1:3 to obtain 20 doses of Example vaccine.

[0048] The vaccines prepared in the above example and Comparative Examples 1-2 were subjected to the following experiments for determination:

[0049] Experimental Example 1, gE concentration

[0050] The Example vaccine was lysed in 0.1 M sodium hydroxide and 0.1% (w / v) sodium dodecyl sulfate buffer at room temperature overnight. The gE concentration was detected using a BCA colorimetric protein detection kit (Shanghai Biyun Tian Biotechnology Co., Ltd.) and the protein loading efficiency was calculated.

[0051] Experimental Example 2, nucleic acid concentration

[0052] The Example vaccine was lysed in 0.1 M sodium hydroxide and 0.1% (w / v) sodium dodecyl sulfate buffer at room temperature overnight. The nucleic acid concentration was detected using a nucleic acid detection kit Quant-iT OliGreen ssDNA Regent Kit (purchased from Thermo Fisher Co., Ltd.) and the nucleic acid loading efficiency was calculated.

[0053] Experimental Example 3, QS21 concentration

[0054] Example vaccines were lysed in 0.1 M sodium hydroxide and 0.1% (w / v) sodium dodecyl sulfate buffer overnight at room temperature. The amount of encapsulated QS21 in the examples was determined using high performance liquid chromatography (HPLC, purchased from Waters Corporation) with a 4.6 x 250 mm C18 column (purchased from Waters Corporation) and calculated the QS21 loading efficiency using free QS21 as a standard.

[0055] Experimental Example 4, Particle size and polydispersity index

[0056] The particle size and polydispersity index of the LNP of the example vaccine were detected using a nanoparticle size detector (Malvern).

[0057] The results of the examples and experimental examples 1-4 are shown in Figure 1 Table 1. The encapsulation efficiency of the LNP lipid nanovaccine gE prepared by the example using the microfluidic nanomedicine manufacturing system was 49.57% (A in Table 1), about 5.95 μg / needle; the encapsulation efficiency of the CpG ODN nucleic acid was 41.85% (B in Table 1), about 6.28 μg / needle; the loading efficiency of QS21 was 57.15% (C in Table 1), about 3.43 μg / needle; the nanoparticle size was between 190.3-194.7 nm (D in Table 1), and the polydispersity index was between 0.269-0.322 (E in Table 1). Figure 1 Figure 1 Figure 1 Figure 1 Figure 1

[0058] Experimental Example 5, Cytotoxicity

[0059] The femur and tibia of a specific pathogen-free C57BL / 6J mouse (female, 6-8 weeks old, 16-18 g, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.) were taken, and the red blood cells were lysed to obtain bone marrow cells. The immature bone marrow-derived dendritic cells (BMDCs) were induced using 1640 complete medium (purchased from Thermo Fisher) containing 20 ng / mL GM-CSF (purchased from Suzhou Paitaike Biological Technology Co., Ltd.). 2 x 10 5 cells were inoculated in a 96-well plate per well, and then the sample was added for further culture for 24 h. The cell viability was detected using a CCK-8 kit (purchased from MedChemExpress).

[0060] The results of the examples and experimental example 5 are shown in Figure 2 Table 2. The BMDC cell activity in the presence of 10 μg / mL of the comparative free QS21 was only about 18%. When encapsulated by LNP, the same concentration of QS21 in the examples did not show obvious cytotoxicity. ​​​​​

[0061] Experimental Example 6, Animal Immunization

[0062] C57BL / 6 mice (6 / group, female, 6-8 weeks old, 16-18 g) were immunized twice with 50 μl of the vaccine prepared in Example, Comparative Example 1, and Comparative Example 2 by intramuscular injection at 4-week intervals. Two weeks after the final immunization, the spleen was removed, and the blood was collected from the heart, centrifuged at 3500 rpm for 30 min at 4°C, and the serum was prepared. The serum was used for subsequent immunological analysis.

[0063] Experimental Example 7, Antibody Titer Test

[0064] Capture antigen gE extracellular region glycoprotein 2 μg / mL in PBS was added to each well of a 96-well enzyme plate (purchased from Corning) at 100 μl, and the plate was coated at 4°C overnight. After washing the plate once with PBST (0.05% (v / v) Tween 20 (Sigma) in PBS), 200 μl of 5% (w / v) skim milk powder blocking solution in PBS was added to each well, and the plate was blocked at 37°C for 1 h. After discarding the blocking solution, the plate was washed 4 times with PBST. 100 μl of 1% blocking solution was added to each well, and the plate was incubated at 37°C for 1 h. After washing the plate 5 times with PBST, 100 μl of color developing solution (purchased from BD) was added to each well at a ratio of 1:1. After placing the plate in the dark at room temperature for 5 min, 100 μl of 1M sulfuric acid was added to stop the reaction, and the optical absorbance was measured at 450 nm. The serum dilution concentration at which OD450 was greater than 0.15 was used as the antibody titer, and the titer at which OD450 was less than 0.15 at a dilution of 1:2000 was defined as 100 for calculation.

[0065] The results of Comparative Examples 1 and 2, Example, and Experimental Examples 6 and 7 are shown in Table 1. Figure 3 The gE-specific IgG titer in the serum of the mouse immunized with Example was 170667, which was comparable to that of Comparative Example 2 and 1.3 times (IgG titer: 128000) that of Comparative Example 1.

[0066] Experimental Example 8, Isolation of Spleen Lymphocytes

[0067] The spleen was placed on a cell strainer (purchased from Wuxi Nai Si Life Science Technology Co., Ltd.), and ACK red blood cell lysis solution was added thereto. After placing the mixture at room temperature for 5 min, the cells were counted after centrifugation at 1800 rpm, and the cells were resuspended to 1×10 7 cells / mL using 1640 medium (purchased from Thermo Fisher) containing 10% fetal bovine serum (purchased from Thermo Fisher) and a secondary antibody.

[0068] Experimental Example 9, Cytokine analysis

[0069] 100 μΐ of 1 x 10 7 Splenocytes at 1 x 10 7 After 24 h incubation at 37 °C in a 5% CO2 environment, cell supernatants were collected and assayed for IL-2 and IFN-γ by ELISA. IL-2 (3 pg / mL) and IFN-γ (4 pg / mL) capture antibodies (purchased from Thermo Fisher) dissolved in PBS were used to coat 96-well plates at 4 °C for 16 h. After blocking with 5% skim milk blocking solution for 1 h at 37 °C, 50 μΐ of cell supernatant was added to each well and incubated for 3 h at room temperature. Mouse IL-2 and IFN-γ protein standards (purchased from Suzhou Pipers Tek Biological Technology Co., Ltd.) dissolved in PBS were used to generate standard curves. Biotin-conjugated antibodies specific for IL-2 or IFN-γ (2 pg / mL, purchased from Thermo Fisher) and HRP-conjugated streptavidin (1 pg / mL, purchased from BioLegend) were subsequently added and incubated for 1.5 h. The reaction was stopped and detected as described in the antibody titer assay.

[0070] The results of Comparative Examples 1-2, Example, Experimental Examples 6, 8, 9, 12 are shown in Table 1. Figures 4-5 The IL-2 level in the supernatant of Example was 2509 pg / mL (p<0.0001) by ELISA analysis. This level was 1.25 times that of Comparative Example 1 (2011 pg / mL, p=0.72) and 2.69 times that of Comparative Example 2 (934.3 pg / mL, p=0.02). Figure 4 The IFN-γ level in the supernatant of Example was 6722 pg / mL (p<0.0001). This level was 1.21 times that of Comparative Example 1 (5572 pg / mL, p=0.42) and 1.6 times that of Comparative Example 2 (4207 pg / mL, p=0.02). Figure 5

[0071] Experimental Example 10, Enzyme linked immunospot assay (ELISPOT)

[0072] The IL-2 and IFN-γ detection kits were purchased from BD and operated according to the instructions. The specific steps are as follows: After diluting the capture antibody with coating buffer, 100 μL / well was added to an ELISPOT plate. After coating overnight at 4°C, the coating buffer was discarded. The plate was washed once with 200 μL / well of blocking buffer. 200 μL of blocking buffer was added to each well and blocked at room temperature for 2 h. After discarding the blocking buffer, 100 μL of 1640 complete culture medium containing 20 μg / mL gE was added, along with spleen cells obtained from the spleen lymphocyte isolation process to bring the final concentration to 2 × 10⁻⁶. 5 Cells / well, incubated overnight at 37°C. Centrifuged at 800g for 5 min, supernatant discarded. Washed twice with 200 μL / well deionized water (5 min each time), then three times with 200 μL / well washing buffer 1. Added 100 μL / well of the detection antibody diluted with diluent and incubated at room temperature for 2 h. Washed three times with 200 μL / well washing buffer 1 (2 min each time). Added 100 μL / well of the enzyme conjugate Streptavidin-HRP diluted with diluent and incubated at room temperature for 1 h. Washed four times with 200 μL / well washing buffer 1 (2 min each time), then twice with 200 μL / well washing buffer 2. Added 100 μL of substrate solution and reacted for the appropriate time. Washed with deionized water to terminate the reaction. After drying, spots were counted using an ELISPOT plate reader (AID Diagnostika GmbH).

[0073] The results of Comparative Examples 1-2, Examples, and Experimental Examples 6, 8, 10, and 12 are as follows: Figures 6-7 As shown. ELISPOT analysis in this example showed that the number of cells secreting IL-2 after gE stimulation was 224.3 / 2×102. 5 One spleen cell ( Figure 6 This figure is 2.1 times that of scale 1 (per 2 × 10). 5 The number of spleen cells was 106.8 per 2 × 10⁸ (p < 0.001), which was 1.87 times that of control example 2 (per 2 × 10⁸). 5 119.8 splenocytes (p = 0.002). In this example, the number of IFN-γ secretory cells after gE stimulation was 2 × 10⁸ cells per 10⁸ cells. 5 293.7 spleen cells ( Figure 7 This figure is 1.67 times that of scale 1 (per 2 × 10). 5 The number of spleen cells was 175.5 per 2 × 10⁻⁶ (p = 0.008), which was 1.87 times that of control example 2 (per 2 × 10⁻⁶). 5 157 spleen cells were detected (p = 0.002).

[0074] Experiment 11: Flow Cytometry Analysis

[0075] All flow cytometry reagents were purchased from BioLegend. A total of 2 × 10⁻⁶ reagents were used. 6with 10 pg / mL of protein gE for 2 h at 37°C in 5% CO2, followed by the addition of 5 pg / mL of Brefeldin A. Spleen cells were incubated overnight in the same conditions to block cytokine release. After washing with staining buffer, 100 mΐ of Zombie NIR TM were added to each sample and incubated for 30 min. Then 5 pg / ml of anti-CD16 / CD32 antibody was added and spleen cells were incubated for 10 min at 4°C to block non-specific binding to Fc receptors. Subsequently, PerCP-Cy5.5-conjugated anti-mouse CD4 was added and incubated for 30 min at 4°C. PE-conjugated anti-mouse IFN-γ and APC-conjugated anti-mouse IL-2 antibodies were used for intracellular staining. After staining, cells were gated (forward and side scatter, FSC / SSC) and samples of over 20,000 CD4+ cell events were analyzed with a CytoFLEX flow cytometer (Beckman) and FlowJo_V10 software.

[0076] The results of Comparative Examples 1-2, Example, Experimental Examples 6, 8, 11, 12 are shown in Table 1. Figures 8-9 According to the flow cytometry analysis, the proportion of CD4+ T cells expressing IL-2 after gE stimulation in the Example was 0.6633% ( Figure 8 ). This level was 2.54 times that of Comparative Example 1 (0.2612%, p = 0.008) and 3.04 times that of Comparative Example 2 (0.2183%, p = 0.004). The proportion of CD4+ T cells expressing IFN-γ after gE stimulation in the Example was 0.7598% ( Figure 9 ). This level was 2.11 times that of Comparative Example 1 (0.3598%, p = 0.04) and 3.53 times that of Comparative Example 2 (0.2152%, p = 0.004).

[0077] Experimental Example 12, Statistical Analysis

[0078] Data were analyzed using GraphPad Prism 9.2 software and expressed as mean ± SD. Significant differences between experimental groups were analyzed by ordinary one-way ANOVA and Dunnett's multiple comparison test, with the Example as the reference. The asterisks represent the p-value classification: *p < 0.05, **p < 0.01 and ***p < 0.001.

[0079] The results of Experimental Example 12 are shown in Table 1. Figures 4-9

[0080] Although the above-described example makes a detailed description of the present application, it is only a part of the examples of the present application but not all the examples, and other examples can be obtained according to the present example without creativity, which all belong to the protection scope of the present application.​

Claims

1. A varicella-zoster virus vaccine, which is composed of a liposome nanoparticle and a zoster virus glycoprotein E and an adjuvant encapsulated in the liposome nanoparticle; the adjuvant is QS21 and a GC-rich single-stranded oligodeoxynucleotide fragment. The content of the zoster virus glycoprotein E in the varicella-zoster virus vaccine is 5-100 μg / dose. The content of QS21 in the varicella-zoster virus vaccine is 1-100 μg / dose. The content of the GC-rich single-stranded oligodeoxynucleotide fragment in the varicella-zoster virus vaccine is 5 μg-2 mg / dose.

2. The varicella-zoster virus vaccine according to claim 1, characterized in that, The liposome nanoparticle comprises a cationic liposome and a polyethylene glycol derivative; the molar ratio of the cationic liposome and the polyethylene glycol derivative is (46-50):(1.5-1.6).

3. The varicella-zoster virus vaccine of claim 1, wherein the varicella-zoster virus vaccine is a live attenuated varicella-zoster virus vaccine. The particle size of the varicella-zoster virus vaccine is 20-400 nm.

4. The varicella-zoster virus vaccine of claim 1, wherein the VZV vaccine is a live attenuated VZV vaccine. The dosage form of the varicella-zoster virus vaccine includes injection.

5. Use of the varicella-zoster virus vaccine according to any one of claims 1-4 in the preparation of a drug for preventing or improving zoster and / or post-zoster sequelae.

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