Nano composite adjuvant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and discloses a nano composite adjuvant as well as a preparation method and application thereof. The composite adjuvant mainly comprises a TLR9 agonist CpG ODN, a nanoscale liposome, a cationic lipid DOTAP and a high-efficiency immunologic stimulant QS-21, several adjuvant components are prepared into the stable and uniform nanoscale liposome by adopting a microfluidic synthesis method, CpG is wrapped inside the nanoscale liposome, QS-21 is adsorbed outside the nanoscale liposome, a stable immunologic stimulation compound is formed, and the immunologic stimulant can be used for immunologic detection of the TLR9 agonist CpG ODN, the cationic lipid DOTAP and the high-efficiency immunologic stimulant QS-21. All the components have a remarkable synergistic effect, and are matched with corresponding antigens to generate strong and lasting cellular immune and humoral immune responses.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a nano-composite adjuvant and a preparation method and application thereof. BACKGROUND

[0002] The statements in this section merely provide background information related to the present disclosure and can not constitute the prior art.

[0003] To improve the immune effect of antigens, reduce the amount of antigens, and achieve more balanced cellular and humoral immune response levels, the development of new adjuvants has attracted more and more attention and has become a research hotspot in the field of vaccines in recent years. Due to the differences in the mechanisms of different types of adjuvants, the immune stimulation effects produced when they are combined with different antigens are quite different. For example, the commonly used aluminum adjuvant mainly stimulates the body to produce humoral immune response, CpG adjuvant can significantly improve the level of cellular immune response of the body, and liposomes can improve the phagocytosis of immune cells. To make the vaccine achieve better immune effect and improve the success rate of vaccine research and development, the use of composite adjuvant has become a difficulty and hotspot in vaccine development. For example, the AS series adjuvants (AS01, AS04, etc.) of GSK company mostly use two or more adjuvants to improve the immunogenicity of antigens. The patent WO2006094756A2 uses neutral liposomes, QS-21 and MPL as components of the composite adjuvant to improve the immunogenicity of the herpes zoster vaccine, but MPL is difficult to dissolve in water, which makes the preparation process of the vaccine complex and the production cost high. The patent CN105920599A describes a vaccine preparation method using cationic liposomes DOTAP as adjuvant. The method uses a film extrusion method to prepare liposomes, and the obtained liposomes have poor uniformity, large batch-to-batch difference, low antigen encapsulation rate, and long preparation process, which is not suitable for large-scale industrial production. The patent CN116350770B provides a herpes zoster vaccine preparation method. To improve the CD4+ cell response level, a composite adjuvant system is used. The adjuvant system uses a film extrusion method to prepare neutral liposomes and positive liposomes at the same time, and the liposomes have large particle size distribution, poor uniformity, complex preparation process, and the safety tolerance of the used adjuvant component PolyI:C needs further study. In the field of adjuvant preparation, the microfluidic method has not yet disclosed beneficial effects. SUMMARY

[0004] The present application aims at the problems of the preparation process of the current composite adjuvant, and provides a nano composite adjuvant, a preparation method and application thereof, so as to develop a more safe, efficient and self-owned intellectual property right composite adjuvant system. The present application uses a microflow preparation process, and innovatively encapsulates a self-designed CpG adjuvant molecule in a positive liposome to form stable and uniform nanoparticles, adsorbs an immunostimulant QS-21 to form an efficient immunostimulant complex, and has better stability and better uniformity. The CpG encapsulation rate is close to 100%, and the process flow is simpler. Animal experiments prove that the composite adjuvant can significantly improve the cellular immune and humoral immune response levels of the antigen, and has potential application value in the field of preventive vaccines or tumor treatment.

[0005] The technical scheme of the present application is as follows: The present application provides a nano composite adjuvant, which encapsulates a CpG ODN molecule in a positive liposome, and the outer side of the liposome adsorbs QS-21; the content of the QS-21 is 1-2.5 times the mass concentration of the CpG molecule.

[0006] Preferably, the molecular concentration of the encapsulated CpG molecule is between 200-1000 μg / ml.

[0007] The CpG ODN is a TLR9 agonist; the QS-21 is a high-efficiency immunostimulant; and the liposome can form an efficient immunostimulant complex, and simultaneously improve the cellular immune and humoral immune response levels of the body to the antigen, and has potential application value in the field of preventive vaccines or tumor treatment.

[0008] According to a preferred embodiment, the outer liposome is composed of DOTAP, DOPC and CHO, and the mass ratio concentration is DOTAP:DOPC:CHO=10-12 mg / ml:3-5 mg / ml:8-10 mg / ml; or, DOTMA and CHO are combined in a mass ratio concentration of DOTMA:CHO=10-15 mg / ml:10-15 mg / ml.

[0009] Preferably, DOTAP:DOPC:CHO=10 mg / ml:5 mg / ml:10 mg / ml or DOTAP:DOPC:CHO=12 mg / ml:5 mg / ml:8 mg / ml; DOTMA:CHO=12 mg / ml:13 mg / ml or DOTMA:CHO=10 mg / ml:15 mg / ml.

[0010] According to a preferred embodiment, the CpG molecule is CpGKHS6, CpGKHS3, CpG2006 or a combination of CpGKHS6 and CpGKHS3.

[0011] According to a preferred embodiment, the CpG molecule is CpGKHS6 or CpGKHS6 in combination with CpGKHS3.

[0012] Another aspect of the present application provides a preparation method of the nano-composite adjuvant, comprising the following steps: Step S1: Liposome components are dissolved in anhydrous ethanol as an oil phase according to a ratio, and the concentration is 15 mg / ml-50 mg / ml; preferably, the concentration of the liposome components is 20 mg / ml-40 mg / ml.

[0013] Step S2: The synthesized CpG dry powder is dissolved in PBS as an aqueous phase, and the concentration is 200 μg / ml-1000 μg / ml; preferably, the concentration of the CpG is 200 μg / ml-550 μg / ml; or more preferably, 400 μg / ml-550 μg / ml.

[0014] Step S3: The oil phase and the aqueous phase are obtained by a microfluidic synthesis chip at a flow rate ratio of 1:2-5, and the total flow rate is set to 12 ml / min-25 ml / min; preferably, the total flow rate is set to 15 ml / min-25 ml / min.

[0015] Preferably, the flow rate ratio of the oil phase and the aqueous phase is 1:2 or 1:3 or 1:5.

[0016] Preferably, the microfluidic synthesis chip is a CD01 prescription screening chip.

[0017] Step S4: Dilution, removal of anhydrous ethanol, and concentration to obtain a high-purity liposome solution.

[0018] Another aspect of the present application provides the use of the nano-composite adjuvant as described above in the preparation of a vaccine.

[0019] According to a preferred embodiment, the vaccine is an ovalbumin OVA vaccine, a norovirus-like particle VLP vaccine, a rabies vaccine, a pertussis vaccine, a diphtheria vaccine, a tetanus vaccine, a herpes simplex vaccine, a herpes zoster vaccine, a tuberculosis vaccine, a rotavirus vaccine, a meningitis vaccine, a human papillomavirus vaccine, a respiratory syncytial virus vaccine, a hand-foot-mouth disease virus vaccine, or a metapneumovirus vaccine.

[0020] Preferably, the norovirus vaccine subtypes involved include but are not limited to: GⅠ.1, GⅡ.2, GⅡ.3, GⅡ.4, GⅡ.6, GⅡ.17, etc.

[0021] Compared with the prior art, the present application has the following beneficial effects: 1. A nano-composite adjuvant, the application uses liposomes to encapsulate CpG adjuvant molecules, the immune stimulation effect is significantly improved compared with traditional aluminum adjuvant; at the same time, it overcomes the defect that traditional aluminum adjuvant can only produce single humoral immune response, and the cell immune response is weak, and can stimulate the body to produce strong and persistent cellular immune and humoral immune response when used with antigen; It can realize a relatively balanced Th1 / Th2 type cell reaction level, promote the body to secrete specific antibody IgG2a, improve the titer of specific blocking antibody in serum, and significantly improve the level of specific T cell response; 2. A nano-composite adjuvant, a stable and uniform nano-composite adjuvant system is formed by adsorbing immune enhancer QS-21 in the liposome cholesterol component, further improving the immune effect of the adjuvant; by using liposome to adsorb QS-21, compared with directly adding QS-21 alone, it can promote the absorption of immune stimulator and improve the immune stimulation effect; at the same time, the cholesterol component in the liposome of the application and the saponin component QS-21 form a stable and irreversible combination, which successfully eliminates its hemolytic property, the LNP-CpG molecule formed by the liposome encapsulation of the application can adsorb 2.5 times QS-21 without hemolytic reaction, which improves the safety of QS-21 injection; 3. A nano-composite adjuvant, by selecting suitable specific liposomes and combining them according to a specific mass concentration ratio, about 100% encapsulation rate of CpG ODN is successfully realized, and the encapsulated liposome molecules have excellent thermal stability and storage stability, which is beneficial to long-term storage and transportation; the liposome molecules also have good uniformity, preparation stability, particle size, PDI and potential, excellent use safety and immunogenicity; the synthesized liposome has good freeze-thaw and thermal acceleration stability, the particle size is about 110 nm, the Zeta potential is about 17 mV, the surface of the nanoparticle has a lower positive potential, which is easy to be captured and phagocytosed by immune cells, and also reduces the toxicity of cationic lipids, which is suitable for immune injection of the body. After 2 years of exploration, a large number of failed attempts, the above comprehensive effect significantly excellent formula technical scheme is finally obtained, which has outstanding progress; 4. The use of a nano-composite adjuvant, animal experiments show that the composite adjuvant can significantly improve the humoral immune level of antigen ovalbumin and norovirus-like particles VLP; improve the secretion level of norovirus blocking antibodies, IgG2a and specific total antibody IgG in serum; compared with the aluminum adjuvant group alone, improve the IgG1 / IgG2a ratio, balance the Th2 / Th1 type immune response; improve the cellular immune response level of the antigen, stimulate specific T cell response and cytokine IFN-γ secretion; the comprehensive immune stimulation effect is better than that of the composite adjuvant of Al(OH)3+CpG. It has obvious clinical application value in the field of cancer treatment and new vaccines; and for the first time, the composite adjuvant is used to improve the humoral immune and cellular immune response level of norovirus-like particles VLP, which provides an important reference for clinical trials.

[0022] 5. A preparation method of a nano-composite adjuvant, the preparation method of the present application is simple to operate, the liposome particles prepared have good uniformity, high stability in multiple preparations, suitable particle size, PDI and potential, and long-term storage and transportation stability. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 TEM picture of LNP-CpG nano-liposome prepared by formula 093 (uranyl acetate staining, scale = 200 nm); Figure 2 Cytokine IFN-α secretion in supernatant after in vitro stimulation of LNP-CpG on human PBMC cells; Figure 3 Stability investigation of LNP-CpG nano-particles for six months at 4°C by using formula 093 to encapsulate several different CpG molecules; due to the different structures and surface charges of CpG molecules, the particle sizes of the synthesized nano-particles are different; Figure 4 Cytokine IFN-γ and IL-4 secretion in supernatant after in vitro stimulation of ovalbumin OVA on mouse spleen lymphocytes; Figure 5 Cytokine IL-2 secretion in supernatant after in vitro antigen restimulation of ovalbumin OVA on mouse spleen lymphocytes; Figure 6 Tumor necrosis factor TNF-α secretion in supernatant after in vitro antigen restimulation of ovalbumin OVA on mouse spleen lymphocytes; Figure 7 Specific antibody IgG secretion level in serum of mice after immunization with ovalbumin OVA; Figure 8 Specific antibody IgG subtype analysis in serum of mice after immunization with ovalbumin OVA; Figure 9To investigate the serum antibody persistence of mice immunized with OVA, the specific antibody IgG secretion level of serum of mice immunized for 120 days was detected; Figure 10 To analyze the specific antibody IgG subtype of serum of mice immunized for 120 days after immunization with OVA; Figure 11 To analyze the secretion of cytokine IFN-γ and IL-4 in supernatant of spleen lymphocytes of mice immunized for 120 days after immunization with OVA in vitro restimulation; Figure 12 To analyze the secretion of cytokine IL-2 in supernatant of spleen lymphocytes of mice immunized for 120 days after immunization with OVA in vitro restimulation; Figure 13 To analyze the secretion of tumor necrosis factor TNF-α in supernatant of spleen lymphocytes of mice immunized for 120 days after immunization with OVA in vitro restimulation; Figure 14 To analyze the secretion of cytokine IFN-γ in supernatant of spleen lymphocytes of mice immunized for 28 days after immunization with Norovirus VLP in vitro restimulation; Figure 15 To detect the specific antibody IgG level of serum of mice immunized with Norovirus VLP containing different adjuvants; Figure 16 To analyze the specific antibody IgG subtype of serum of mice immunized with Norovirus VLP containing different adjuvants; Figure 17 To analyze the serum HBGA blocking antibody results of mice immunized with Norovirus VLP containing different adjuvants for 28 days; Figure 18 To analyze the particle size distribution of LNP-CpG nanoparticles prepared by 093 formula by dynamic light scattering method (DLS); Figure 19 To analyze the process characterization data of LNP-CpG nanoparticles prepared by 093 formula; Figure 20 To analyze the change of hemolysis rate of red blood cells after LNP-CpG nanoparticles adsorbed QS-21. DETAILED DESCRIPTION

[0024] The specific examples listed in the present application are only as examples of the present application, and the present application is not limited to the specific examples described below. Any equivalent modifications and alternatives to the examples described below are also within the scope of the present application for those skilled in the art. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be included in the scope of the present application. The specific conditions are not specified in the examples, and the conventional conditions or the conditions recommended by the manufacturer are used. All reagents or instruments are not specified by the manufacturer, and are conventional products that can be purchased on the market. In order to better illustrate the present application, numerous specific details are given in the following specific embodiments. Those skilled in the art should understand that the present application can also be implemented without certain specific details. In some embodiments, methods, means, apparatus and steps that are well known to those skilled in the art are not described in detail, in order to highlight the main idea of the present application.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in the specification are international standard units, and the numerical values and numerical ranges appearing in the present application should be understood to include the systematic errors that are inevitable in industrial production.

[0026] The features and properties of the present application are further described in detail below in conjunction with the examples.

[0027] Example 1 Preparation of LNP-CpG liposome 1.1 Liposome formula screening and stability investigation (1) Main experimental reagents: DOTAP, Avitormed (Shanghai) Pharmaceutical Technology Co., Ltd., batch number CG1086; Cholesterol CHO, Avitormed (Shanghai) Pharmaceutical Technology Co., Ltd., batch number C40110; DOPC, Avitormed (Shanghai) Pharmaceutical Technology Co., Ltd., batch number C40462; Phosphate buffer PBS, from Wuhan Sunriselife Biotechnology Co., Ltd., item number PB180327; DOTMA, Avitormed (Shanghai) Pharmaceutical Technology Co., Ltd., batch number A23T06K12; DMG-PEG2000, MCE company, item number HY-112764; CpG was synthesized by Anhui General Bio, dissolved in PBS and diluted to a concentration of 500 μg / ml as the water phase; anhydrous ethanol, Chengdu Kolon Chemicals, batch number 2022051901.

[0028] (2) Main equipment: Fast nano drug preparation system, Shanghai Maiannai Instrument Co., Ltd., model INano L; Multifunctional refrigerated centrifuge, Thermo Fisher Corporation, model ST4R Plus; Microspectrophotometer, Germany IMPLEN, Model NP80 Touch; Liposome synthesis chip, Shanghai Maianna Instrument Technology Co., Ltd., Item No. CD01; Nanoparticle size analyzer, Malvern Instruments, Model ZSU3305; 100KDa ultrafiltration centrifuge tube, Millipore, Item No. UFC910024; (3) Experimental method: In order to improve the uniformity of liposomes and the CpG encapsulation rate, and improve the utilization efficiency of raw materials, the microfluidic method is used to prepare nanoliposomes. According to the previous groping of liposome synthesis conditions, the components (DOTAP / DOPC / CHO, etc.) are dissolved in anhydrous ethanol as the oil phase, and the concentration is 25 mg / ml; the synthesized CpG dry powder is dissolved in PBS as the water phase, and the concentration is 500 μg / ml. The oil phase and the water phase are passed through the CD01 prescription screening microfluidic chip at a flow rate ratio of 1:3 to obtain nanoscale liposomes, and the total flow rate is set to 18 ml / min. After the synthesis of the liposomes, 0.05% Tween 80 is added to PBS to dilute 50 times, and the anhydrous ethanol is removed by centrifugation through a 100KDa pore size ultrafiltration tube. The centrifugal speed is 3000g, and after concentration, high-purity liposome solution is obtained. The ssDNA content, i.e. CpG concentration, is detected by sampling the centrifugal permeate and the concentrated solution, and the encapsulation rate is calculated. After sampling the liposomes, the particle size and potential are detected by the nanoparticle size analyzer.

[0029] Design 091-095 formula, as follows: The 091 formula is DOTMA:DOPC:CHO=2:2:1 molar ratio concentration; The 092 formula is DOTAP:DOPC:CHO=2:2:1 molar ratio concentration; The 093 formula is DOTAP:DOPC:CHO=12 mg / ml: 3 mg / ml: 10 mg / ml mass ratio concentration; molar ratio concentration = 9:2:14.

[0030] The 094 formula is DOTAP:DOPC:CHO:DMG-PEG2000=14.21 mg / ml: 3.2 mg / ml: 6.05 mg / ml: 1.54 mg / ml mass ratio concentration; The 095 formula is DOTMA:CHO=15 mg / ml: 10 mg / ml mass ratio concentration.

[0031] (4) Experimental results: The CpG encapsulation rate, surface potential, and liposome stability were investigated after the liposomes were synthesized according to the above-mentioned formula, and the results are shown in Tables 1, 2, and 3. The 093 formula and the 095 formula have the highest CpG synthesis encapsulation rate, and the particle size changes little after 6 freeze-thaw cycles. The particle size polydispersity coefficient PDI is still less than 0.15 after 4 weeks of thermal acceleration destruction at 37°C, indicating that the liposome stability is good and there is no obvious rupture.

[0032] Table 1, Stability investigation of CpG-KHS6 encapsulated by different liposome formulas (freeze-thaw experiment)

[0033] Table 2, Stability investigation of CpG-KHS6 encapsulated by different liposome formulas (37°C thermal acceleration)

[0034] As can be seen from the above table, different liposome formulas have different encapsulation rates and stability for CpG molecules. Among them, even if the same type of liposome, such as formula 092 and 093, it can be seen that the type of liposome is the same, but the proportions are different, and the encapsulation effects of the two are different. Formula 093 can achieve 100% encapsulation rate, while formula 092 only has 86.7%. At the same time, the particle size of formula 093 is significantly smaller than that of formula 092, and during repeated freeze-thawing, formula 093 can maintain significantly higher stability, while the particle size of formula 092 reaches 510.8 nm after 6 freeze-thaw cycles, and the PDI increases significantly. It can be seen that the liposome formula and proportion of the present application have significant synergy, and have made significant unexpected progress.

[0035] The molar ratio concentration of formula 091 and formula 092 is the same, and only the selection of DOTMA and DOTAP substances is different, and the two have differences in encapsulation rate, freeze-thawing, and heating stability.

[0036] The encapsulation rate of formula 094 decreases when a type of liposome DMG-PEG2000 is added. It is not as good as formula 095 which only selects DOTMA and CHO.

[0037] The liposome type used in formula 095 is the simplest, although the encapsulation rate is slightly lower than that of formula 093, but the freeze-thawing and heating stability are better than those of formula 093.

[0038] Different CpG molecules are encapsulated by using formula 093, and the encapsulation rate is shown in Table 3: Table 3, Change of encapsulation rate of LNP-CpG nanoparticles after multiple freeze-thawing

[0039] As shown in Table 3 above, different CpG molecules can form stable liposome complexes when encapsulated with the 093 formulation, and there is no significant leakage of CpG molecules after repeated freezing and thawing 5 times.

[0040] The transmission electron microscope picture of LNP-CpG nanoparticles encapsulated by the formulation 093 is shown in Figure 1 .

[0041] The stability data graph of LNP-CpG nanoparticles encapsulating several different CpG molecules with the 093 formulation stored at 4°C for half a year is shown in Figure 3 . Due to the different structures and surface charges of CpG molecules, the particle sizes of the synthesized nanoparticles are different.

[0042] As shown in Figure 18 , the particle size distribution of LNP-CpG nanoparticles prepared by the 093 formulation was detected by dynamic light scattering method (DLS). As shown in Figure 19 , the process characterization data of LNP-CpG nanoparticles prepared by the 093 formulation were statistically analyzed.

[0043] 1.2 Hemolytic performance study of LNP-CpG complex adjuvant Saponin, as a new type of immune stimulator, can activate the immune system through caspase and other signaling pathways. The most clinically used saponin component QS-21 has hemolytic properties. To overcome its hemolytic properties, saponin is generally combined with cholesterol to form an irreversible complex, eliminating its hemolytic properties. In this experiment, mouse red blood cells were obtained by heparin sodium anticoagulation method, washed with PBS three times, and then adjusted to a red blood cell concentration of 10 7 μg / ml, added to each experimental group at a ratio of 1:2, incubated at 37°C for 30 min, centrifuged, and the supernatant was taken to read the absorbance value at 540 nm wavelength. The hemolysis rate% = (experimental group - negative group) / (positive group - negative group) was calculated. According to the biological material compatibility judgment method, hemolysis rate less than 5% is considered non-hemolytic.

[0044] Since we used cholesterol and choline, which are biocompatible, as the formulation components of the liposome, the LNP-CpG nanoparticles alone are not hemolytic. The results shown in the figure above show that 10 μg / ml of LNP-CpG (concentration calculated based on CpG) can adsorb 25 μg / ml of QS-21 without causing red blood cell hemolysis. The results are shown in Figure 20 .

[0045] Example 2: In vitro stimulation effect of LNP-CpG on human PBMC cells (1) Main experimental reagents: Human lymphocyte separation medium, Tianjin Haoyang Bioproducts Co., Ltd., Catalog No. LTS10771; RPMI 1640 medium from Shanghai Yixing Biological Technology Co., Ltd., Catalog No. 41402ES76; Fetal bovine serum from Gibco, Catalog No. A5669801; Ampicillin-streptomycin double antibody from Gibco, Catalog No. 15140-122; Phosphate buffer PBS from Wuhan Sunrise Biotechnology Co., Ltd., Catalog No. PB180327; Trypan blue solution from Beijing Solabio Technology Co., Ltd., Catalog No. C0040; Human IFN-α cytokine detection kit from Shenzhen Dakewei Biotechnology Co., Ltd., Catalog No. 1110013.

[0046] The cationic formula encapsulating CpG uses the above-mentioned 093 formula, and the CpG sequence used is as shown in Table 4 below, all of which are artificially synthesized full-thio modified fragments: Table 4, sequence used in LNP-CpG stimulation of human PBMC cell experiment

[0047] (2) Experimental method: Human peripheral blood mononuclear cells PBMC mainly contain lymphocytes, dendritic cells, monocytes, etc., and are the main source of peripheral immune cells. CpG adjuvant as an immune stimulator can not only stimulate the proliferation of immune cells, but also promote the release of immune-related cytokines and improve the immune effect. The CpG molecule encapsulated by liposome can resist the decomposition of nuclease in serum and improve the phagocytosis efficiency of immune cells to CpG molecules.

[0048] The experiment uses heparin sodium anticoagulant tube to collect 40-60ml peripheral blood of healthy volunteers, and 5ml of fresh anticoagulated blood is slowly added into a 15ml centrifuge tube containing 4ml of lymphocyte separation medium. The centrifuge parameters are set as follows: temperature 20℃, rotation speed 600g, centrifugation time 25min. After centrifugation, the middle white lymphocyte layer is sucked into a new centrifuge tube, washed once with PBS, and the centrifugation parameters are the same as before. After centrifugation, the PBS is discarded and the cells are resuspended in 1640 culture medium, and the cell density is adjusted to 2-5*10 6 6 / ml, 1000ul / well is plated in a 24-well plate, and encapsulated and unencapsulated CpG stimulants (CpG concentration 10ug / ml) are added according to the scheme, and a control group is set, with PBS as the negative control group and CpG2006 as the positive control group. After incubation of the lymphocytes in a 37℃ incubator for 48h, the supernatant is taken to detect the secretion of cytokine IFN-α.

[0049] (3) Experimental results: For example Figure 2As shown, the release of type 1 interferon IFN-α was significantly promoted after CpG KHS6, CpG KHS3 or CpG 2006 were wrapped inside the liposomes, and the expression level was higher than that of the corresponding unencapsulated group. The release of cytokine IFN-α after stimulation of human PBMC by LNP-CpG-KHS6 was also significantly higher than that of all unencapsulated groups and positive sequence groups. It is indicated that the liposome encapsulation technology can promote the uptake of CpG by immune cells and improve the immune stimulation effect.

[0050] Example 3 Ovalbumin OVA verifies the adjuvant effect of LNP-CpG adjuvant system 3.1 Main experimental reagents: RPMI 1640 medium from Shanghai Yixing Biotechnology Co., Ltd., item number 41402ES76; fetal bovine serum from Gibco, item number A5669801; penicillin-streptomycin double antibody from Gibco, item number 15140-122; red blood cell lysis solution from Shanghai Yixing Biotechnology Co., Ltd., item number 40401ES76; sterile phosphate buffer PBS from Wuhan Sunrise Life Science and Technology Co., Ltd., item number PB180327; trypan blue solution from Beijing Solabio Technology Co., Ltd., item number C0040; ovalbumin OVA from Sigma, item number A5503; aluminum hydroxide adjuvant from CRODA company, item number 21645-51-2; CpG-KHS6 and CpG-KHS3, self-designed, synthesized by Anhui General Biotechnology Co., Ltd.; LNP-CpG, self-synthesized, batch number 20240501; QS-21 from MCE, batch number 235717; MF59, self-made according to the adjuvant formula published in the relevant literature (containing 4.1% squalene, 0.5% Tween 80, 0.5% Span 85); mouse cytokine IL-2 ELISA detection kit from Shenzhen Dakewo Biotechnology Co., Ltd., item number 1210202; mouse cytokine IFN-γ ELISA detection kit from Shenzhen Dakewo Biotechnology Co., Ltd., item number 121002; mouse cytokine IL-4 detection kit from Shenzhen Dakewo, item number 1210402; mouse TNF-α detection kit from Shenzhen Dakewo, item number 1110013; HRP-labeled goat anti-mouse IgG from Abeam company, item number Ab6789; HRP-labeled goat anti-mouse IgG1 from Abeam company, item number Ab97240; HRP-labeled goat anti-mouse IgG2a from Abeam company, item number Ab97245; ELISA enzyme-labeled plate from Corning company, item number 9018; TMB color developing solution from Seracare company, item number 5120-0074.

[0051] 3.2 Experimental animals: 6-8 weeks old SPF level Balb / c female mice, body weight 18-22 g, a total of 64, purchased from Beijing Vito Lihua Experimental Animal Technology Co., Ltd., license number: 511215600003615. After grouping according to the scheme, 4 mice per cage were raised in a constant temperature and humidity standard test animal room, with free water and food.

[0052] 3.3 Experimental method: (1) Experimental design: To verify the adjuvant effect of the independently designed nanoparticle LNP-CpG, the model antigen ovalbumin OVA was used as the immunogen to compare the humoral and cellular response levels of mice in each group after immunization. Each mouse was injected with 0.1 ml, 50 μl in the left and right hind legs for muscle injection. The details of the test grouping are shown in Table 5: Table 5, animal test grouping and immunization

[0053] The immunization route was hind leg muscle injection, and each group was immunized twice, once for basic immunization and once for booster immunization, with an interval of 14 days. According to the antigen, LNP-CpG mother liquor and aluminum adjuvant concentration, the amount of each component required was calculated, and each group of vaccines 1 ml was prepared in a clean bench, and then mixed gently with a vortex oscillator and stored at 4℃ for standby, and the vaccine was transported to the animal room within 1h for animal immunization. The single immunization amount of each component in the experimental group was: antigen 5 μg per mouse, CpG 20 μg per mouse, aluminum hydroxide adjuvant 100 μg per mouse, QS-21 10 μg per mouse.

[0054] At different time points after immunization, 5 mice were taken from each group for blood collection by jugular vein or orbit and serum separation, and the specific antibody IgG and antibody subtype (IgG1, IgG2a) serum expression level was detected by ELISA method; 28 days after immunization, three mice were taken from each group to separate splenic lymphocytes for in vitro culture and re-stimulation with antigen OVA, and the expression level of cytokines such as IFN-γ in the supernatant after stimulation was detected to analyze the antigen-specific T cell response level and verify the adjuvant effect.

[0055] (2) Cytokine detection method (cellular immune level) Two to two days after immunization, three mice from each group were euthanized by cervical dislocation and their entire bodies were disinfected by immersion in alcohol. Under sterile conditions, the spleens were isolated and thoroughly ground and broken up using a syringe plunger and a cell sieve (40 μm pore size). 10 ml of sterile PBS was added for washing, allowing lymphocytes to be washed into a 50 ml centrifuge tube at the bottom. The lymphocytes were centrifuged at 2000 rpm for 5 minutes using a low-temperature centrifuge. The supernatant PBS was discarded, and 10 ml of erythrocyte lysis buffer was added. The mixture was gently vortexed for 1 minute to ensure complete lysis of the erythrocytes. The cells were centrifuged again using the same parameters. The erythrocyte lysis buffer was discarded, and PBS was added to gently resuspend the cells at the bottom. The lymphocytes were washed once more, centrifuged, and the PBS was discarded. Finally, 10 ml of 1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrin antibodies was added to resuspend the cells. After cell counting, the cell concentration was adjusted to 5 x 10⁻⁶ cells / mL. 6 per ml.

[0056] Lymphocytes were seeded at 1000 μl / well in 24-well plates, with 3 replicates per group. Cells in each group were restimulated with antigen at a concentration of 20 μg / ml. After culturing the lymphocytes at 37°C for 5 days, the supernatant was collected and centrifuged at 5000 rpm for 15 min. Cell debris was discarded, and the supernatant was used for cytokine detection. The procedure was briefly described below according to the instructions of the Shenzhen Dactech ELISA kit: a. Prepare standards with concentrations of 0, 7.8, 15.6, 31.3, 62.5, 125, 250 and 500 pg / ml using sample dilution buffer. Add 100 μl of sample and standard to each well, then add 50 μl of biotin-labeled antibody to each well. Cover with sealing film and incubate at 37°C for 90 min. b. Washing the plate. Remove the liquid from the wells, add 300 μl of washing buffer per well, remove the washing buffer and blot dry on filter paper. Repeat washing 4 times. c. Add enzyme. Add 100 μl of HRP-labeled streptavidin-HRP to each well, cover with a sealing film, and incubate at 37°C for 30 min. d. Wash the plate. Repeat step 2, washing 4 times. e. Color development. Add 100 μl of TMB color development solution to each well and develop the color at 37℃ for 5-30 min. Based on the color, add 100 μl of stop solution per well to terminate the reaction. f. Plate reading. Immediately after termination, use a microplate reader to read the absorbance of each well at a wavelength of 450 nm, and calculate the cytokine concentration of each group according to the standard curve.

[0057] (3) Detection methods for specific antibody IgG and antibody subtype (humoral immunity level) Different time points after immunization, 100-200 μl of blood was collected from the jugular vein of the mice with a syringe, and serum was separated after centrifugation at 5000 rpm for 15 min, which was used for specific antibody IgG and antibody subtypes IgG1 and IgG2a detection. The antibody detection method is as follows: a. The antigen OVA was diluted to 1 μg / ml with PBS, and 100 μl / well was added to the enzyme-labeled plate, which was coated at 4°C overnight; b. The coated enzyme-labeled plate was taken out, and the plate was washed 3 times with PBS containing 0.05% Tween-20, 300 μl / well; the washing solution was discarded, and 100 μl / well of PBS containing 5% skimmed milk powder was added, and the plate was sealed at 37°C for 1.5 h; c. Discard the blocking solution, wash the plate 3 times, dilute the mouse serum 1:800, and add it to the enzyme-labeled plate, 100 μl / well, cover the plate with a sealing plate, and incubate at 37°C for 2 h; d. Discard the serum diluent, wash the plate 3 times, and pat dry on the water-absorbing paper, add HRP-labeled IgG, IgG1 or IgG2a (1:10000 dilution), cover the plate with a sealing plate, and incubate at 37°C for 1 h; e. Discard the HRP-labeled secondary antibody, wash the plate 5 times, add TMB color developing solution, 100 μl / well, and develop for 8 min; f. Add 2M sulfuric acid stop solution, and read the absorbance value at 450 nm wavelength after termination.

[0058] 3.4 Experimental results: (1) As shown in Figure 4 , Figure 5 and Figure 6 , the mouse spleen lymphocytes were isolated 28 days after immunization with antigen OVA for in vitro culture and antigen restimulation, and the results showed that the cell immune response level of the complex adjuvant group LNP-CpG-KHS6+QS-21 (LNP-CpG-S6+QS-21) and LNP-KHS3-KHS6+QS-21 (LNP-S3-S6+QS-21) was significantly higher than that of the aluminum adjuvant+CpG and MF59+CpG complex adjuvant groups, the cytokines IFN-γ and IL-2 representing Th1 type cell immune response were higher than those of other complex adjuvant groups and the control group, and the ratio of cytokines IFN-γ / IL-4 reflected the bias of Th1 / Th2 type immune response.

[0059] (2) Figure 7 and Figure 8The results of the antibody levels produced by the complex adjuvant groups LNP-CpG-KHS6+QS-21 (LNP-CpG-S6+QS-21) and LNP-KHS3-KHS6+QS-21 (LNP-S3-S6+QS-21) were higher than those of the traditional aluminum adjuvant group 35 days and 56 days after immunization, the IgG2a expression level was significantly higher than that of the aluminum adjuvant and control groups, the ratio of IgG1 / IgG2a was lower than that of the aluminum adjuvant group, indicating that the complex adjuvant group achieved a more balanced Th2 / Th1 type immune response.

[0060] (3) Figures 9-13 The results of the cell immune response and humoral immune response produced by the complex adjuvant groups LNP-CpG-KHS6+QS-21 (LNP-CpG-S6+QS-21) and LNP-KHS3-KHS6+QS-21 (LNP-S3-S6+QS-21) were significantly better than those of the conventional aluminum adjuvant group and other control groups 120 days after immunization, indicating that the complex adjuvant system stimulated the body to produce a relatively long-lasting immune memory effect.

[0061] 3.5 Experimental conclusion: The adjuvant effect of the LNP-CpG complex adjuvant was preliminarily verified by using the model antigen ovalbumin OVA, and it was found that the complex adjuvant can stimulate the body to produce strong and long-lasting humoral and cellular immune responses, significantly improve the level of specific T cell response, increase the expression level of specific antibody IgG and subtype antibody IgG2a, achieve a more balanced Th1 / Th2 type immune response, and play an excellent adjuvant effect, which has obvious clinical application value.

[0062] Example 4 Norovirus-like particles VLP as an antigen to verify the adjuvant effect of the LNP-CpG adjuvant system 4.1 Main experimental reagents RPMI 1640 medium from Shanghai Yikesheng Biotechnology Co., Ltd., item number 41402ES76; fetal bovine serum from Gibco, item number A5669801; penicillin-streptomycin double antibody from Gibco, item number 15140-122; red blood cell lysis solution from Shanghai Yikesheng Biotechnology Co., Ltd., item number 40401ES76; sterile phosphate buffer PBS from Wuhan Sunriser Biotechnology Co., Ltd., item number PB180327; trypan blue solution from Beijing Solabio Technology Co., Ltd., item number C0040; antigen Norovirus-like particles VLP GⅡ.3 from Chengdu Kanghua Biological Norovirus Project Team; aluminum hydroxide adjuvant from CRODA Company, item number 21645-51-2; CpG-KHS6, self-designed, synthesized by Anhui General Biotechnology Co., Ltd.; LNP-CpG, self-synthesized, batch number 20250427; QS-21 from MCE, batch number 235717; mouse cytokine IFN-γ ELISA detection kit from Shenzhen Dakewe Biotechnology Co., Ltd., item number 121002; HRP-labeled goat anti-mouse IgG from Abeam Company, item number Ab6789; HRP-labeled goat anti-mouse IgG1 from Abeam Company, item number Ab97240; HRP-labeled goat anti-mouse IgG2a from Abeam Company, item number Ab97245; ELISA enzyme-labeled plate from Corning Company, item number 9018; TMB color developing solution from Seracare Company, item number 5120-0074.

[0063] 4.2 Experimental animals: SPF level Balb / c female mice aged 6-8 weeks, weighing 18-22 g, a total of 35, from Beijing Vantoll Life Experimental Animal Technology Co., Ltd., qualified certificate number: 511215600003615. After grouping according to the scheme, 5 mice per cage were raised in a constant temperature and humidity standard animal room, with free access to water and food.

[0064] 4.3 Experimental method: (1) Experimental design: In this experiment, Norovirus-like particles VLP GⅡ.3 were used as antigens to verify the adjuvant effect of the nano-complex adjuvant system LNP-CpG, and the humoral and cellular response levels of the mice after immunization were investigated. Each mouse was injected with 0.1 ml, 50 μl of muscle injection in the left and right hind legs, and the test grouping details are shown in Table 6: Table 6, animal test grouping and immunization

[0065] Each group was immunized twice, once for basic immunization and once for booster immunization, with an interval of 14 days. The required amount of each component was calculated according to the antigen, LNP-CpG mother liquor and aluminum adjuvant concentration, and each group of vaccines 1 ml was prepared in a clean bench, and then mixed gently with a vortex shaker and stored at 4℃ for standby, and the vaccines were transported to the animal house within 1 h for animal immunization. The single immunization amount of each component in the experimental group was: antigen 2 μg per mouse, CpG 10 μg per mouse, aluminum hydroxide adjuvant 100 μg per mouse, QS-21 10 μg per mouse.

[0066] At different time points after immunization, 5 mice per group were collected by neck vein or orbital blood and serum was separated, and the expression levels of specific antibody IgG, serum blocking antibody and antibody subtype (IgG1, IgG2a) were detected by ELISA method; 28 days after immunization, four mice per group were taken to separate splenic lymphocytes for in vitro culture and re-stimulation with antigen VLP, and the expression level of cytokine IFN-γ in the supernatant after stimulation was detected to analyze the level of antigen-specific T cell response.

[0067] (2) Cellular immune response The experimental method is the same as part 3.3 of Example 3.

[0068] (3) Humoral immune response The experimental method is the same as part 3.3 of Example 3.

[0069] (4) Mouse serum blocking antibody titer determination a, coating. The mucin (Source Leaf Biological, Catalog No. S12066) was diluted with PBS to a final concentration of 20 μg / ml of coating working solution, 100 μl was added to each well of the 96-well plate, and the plate was sealed and placed in a 2-8℃ refrigerator for coating for 16-22 h.

[0070] b, washing the plate. Discard the liquid in the plate wells, add 300 μl of washing solution to each well, and wash 4 times. Add 200 μl of blocking solution to each well, and incubate at 25℃ for 60±10 min. After blocking, discard the liquid in the plate wells and wash the plate 4 times.

[0071] c, serum dilution. The mouse serum to be tested was diluted with PBS, and the dilution was in a ratio of 1:2 (the sample dilution factor can be adjusted according to the actual situation), and at least 4 gradients were diluted (serum reference dilution factor: 100, 200, 400, 800, 1600, 3200).

[0072] d. VLP dilution. Dilute VLP with PBS to a concentration of 0.3 μg / ml, mix 60 μl of diluted serum with an equal volume of corresponding VLP dilution to be detected (after mixing, the serum reference dilution ratio is: 200, 400, 800, 1600, 3200, 6400), seal the plate with a sealing film, and incubate at 25°C for 90±10 min. Add 100 μl of mixed and incubated sample to the blocked 96-well plate. Incubate at 25°C for 90±10 min with a sealing film.

[0073] e. Wash the plate. Remove the sealing film, discard the liquid in the plate well, add 300 μl of washing solution to each well, and wash 4 times. Tap the plate on absorbent paper to completely remove the residual liquid in the plate well.

[0074] f. Incubate rabbit polyclonal antibody. Dilute rabbit polyclonal antibody 1000 times with PBS, add 100 μl of prepared rabbit polyclonal antibody working solution to the corresponding well plate, seal with a sealing film, and incubate at 25°C for 60±5 min. Then wash the plate, discard the liquid in the plate well, add 300 μl of washing solution to each well, and wash 4 times.

[0075] g. Preparation of HRP-labeled goat anti-rabbit IgG working solution. Dilute HRP-labeled goat anti-rabbit IgG 5000 times with PBS. Add 100 μl of prepared IgG working solution to the corresponding well plate, seal with a sealing film, and incubate at 25°C for 60±5 min. After incubation, wash the plate 4 times.

[0076] h. Color development. Add 100 μl of TMB color developing agent to each well, and incubate in the dark for 5-20 min.

[0077] i. Stop color development. Add 50 μl of stop solution to each well to stop color development, and detect the absorbance value at a wavelength of 450 nm using a microplate reader. Use 1 / 2 of the mean value of the negative control as the Y value for calculation. The calculated value is the blocking titer BT50, which is defined as the maximum serum dilution that can block 50% of VLP binding to the receptor.

[0078] 4.4 Experimental results: (1) According to the results Figure 14 , the LNP-CpG complex adjuvant system can stimulate stronger cellular immune responses in mice, and higher levels of IFN-γ are produced after in vitro antigen restimulation of lymphocytes, which is significantly different from the aluminum adjuvant group alone.

[0079] (2) Figure 15 and Figure 16 The results show that the LNP-CpG complex adjuvant system can stimulate stronger humoral immune responses, and the level of specific antibody expressed in serum 28 days after immunization is significantly higher than that of other groups; The LNP-CpG complex adjuvant group also has higher IgG2a expression level, and the IgG2a / IgG1 ratio shows that the complex adjuvant stimulates more balanced Th1 / Th2 type immune response.

[0080] (3) Figure 17 The results show that the LNP-CpG complex adjuvant system can stimulate higher levels of blocking antibody titers and improve the immune protection effect of the vaccine.

[0081] 4.5 Experimental conclusion: The LNP-CpG complex adjuvant has strong immune stimulation effect, can improve the cellular immune and humoral immune response level of the antigen Norovirus VLP, promote the secretion of specific antibody IgG and antibody subtype IgG2a, improve the serum blocking antibody titer, achieve more balanced Th1 / Th2 type immune response, stimulate specific T cell response, and the comprehensive immune stimulation effect is better than that of the Al(OH)3+CpG complex adjuvant, and the Norovirus VLP is used in combination to play an excellent adjuvant effect.

[0082] The above-described embodiments only express the specific implementation of the present application, and the description is more specific and detailed, but it cannot be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the technical concept of the present application, a number of modifications and improvements can be made, which are within the protection scope of the present application.

Claims

1. A nanocomposite adjuvant, characterized in that, CpG molecules are encapsulated inside positive liposomes, and QS-21 is adsorbed on the outside of the liposomes; the content of QS-21 is 1-2.5 times the mass concentration of CpG molecules.

2. The nanocomposite adjuvant according to claim 1, characterized in that, The positive liposomes are composed of DOTAP, DOPC, and CHO in a mass ratio (mg / ml) of DOTAP:DOPC:CHO = 10⁻¹²: 3⁻⁵: 8⁻¹⁰; or DOTMA and CHO in a mass ratio (mg / ml) of DOTMA:CHO = 10⁻¹⁵: 10⁻¹⁵.

3. The nanocomposite adjuvant according to claim 1, characterized in that, The CpG molecule is CpGKHS6, CpGKHS3, CpG2006, or a combination of CpGKHS6 and CpGKHS3.

4. The nanocomposite adjuvant according to claim 3, characterized in that, The CpG molecule is CpGKHS6 or a combination of CpGKHS6 and CpGKHS3.

5. The nanocomposite adjuvant as described in claim 4, characterized in that, The molecular concentration of the CpG molecules is 200-1000 μg / ml.

6. A method for preparing a nanocomposite adjuvant according to any one of claims 1-5, characterized in that, Includes the following steps: Step S1: Dissolve the liposome components together in anhydrous ethanol according to the specified ratio to form the oil phase, with a concentration of 15-50 mg / ml; Step S2: Dissolve the synthesized CpG dry powder in PBS as the aqueous phase, with a concentration of 200-1000 μg / ml; Step S3: Obtain nanoscale liposomes by passing the oil phase and aqueous phase through a microfluidic synthesis chip at a flow rate ratio of 1:2-5, with the total flow rate set to 12-25 ml / min; Step S4: Dilute and remove anhydrous ethanol, then concentrate to obtain a high-purity liposome solution.

7. The method for preparing a nanocomposite adjuvant as described in claim 6, characterized in that, In step S3, the flow rate ratio of the oil phase to the water phase is 1:2, 1:3, or 1:

5.

8. The use of a nanocomposite adjuvant as described in any one of claims 1-5 in the preparation of vaccines.

9. The application according to claim 8, characterized in that, The vaccines mentioned are ovalbumin OVA vaccine, norovirus-like particle VLP vaccine, rabies vaccine, pertussis vaccine, diphtheria vaccine, tetanus vaccine, herpes simplex vaccine, herpes zoster vaccine, tuberculosis vaccine, rotavirus vaccine, meningococcal vaccine, human papillomavirus vaccine, respiratory syncytial virus vaccine, hand-foot-and-mouth disease virus vaccine, or metapneumovirus vaccine.

10. The application according to claim 9, characterized in that, Norovirus-like particle (VLP) vaccine subtypes include, but are not limited to: GⅠ.1, GⅡ.2, GⅡ.3, GⅡ.4, GⅡ.6, or GⅡ.17.

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