Composite nano aluminum phosphate adjuvant as well as preparation method and application thereof

Abstract

The invention relates to a composite nano aluminum phosphate adjuvant as well as a preparation method and application thereof, and belongs to the technical field of vaccine adjuvants. The gel is prepared from the following raw materials: ethylene glycol chitosan GCS with the molecular weight of 1-30 kDa and an aluminum phosphate adjuvant AP in a weight ratio of n: 1, wherein n is equal to 0.1-7. The invention has the advantages of excellent dispersibility and stability, high-pressure sterilization and freeze-drying treatment tolerance, and can be stored at normal temperature for more than 15 months. Animal experiments show that compared with a traditional aluminum phosphate adjuvant, the nano aluminum adjuvant disclosed by the invention can induce body fluid and cell immune response which is stronger in effect, safer and more balanced, can be used as a novel adjuvant platform to carry adjuvants such as ZMF59, C-di-AMP or Imiquimod, further improves the immune effect, and is suitable for being applied to vaccines needing Th1 / Th2 immune balance or strong cell immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of vaccine adjuvants and relates to a composite nano-aluminum phosphate adjuvant and a preparation method and application thereof. Background Art

[0002] Since aluminum adjuvants were licensed for use in human vaccines in the 1920s, they remain the most widely used adjuvant to date. However, aluminum adjuvants often induce a Th2-biased immune response, limiting their application in vaccines requiring cellular immunity and thus failing to meet the needs of vaccines requiring a cellular immune response. Furthermore, traditional aluminum adjuvants cannot withstand freezing and lyophilization, resulting in a relatively limited shelf life and requiring only cold chain transportation. Furthermore, aluminum adjuvants can cause significant adverse reactions at the injection site, including redness, swelling, and nodules. To overcome these limitations, attempts have been made to load aluminum adjuvants with other immunostimulants or to modify their physicochemical properties, such as size and surface characteristics. Among these, nano-aluminum adjuvants have been reported to more effectively promote antigen absorption and presentation and activate the NLRP3 inflammasome, thereby stimulating T and B cells and enhancing adaptive immune responses. Furthermore, nano-aluminum adjuvants can reduce injection site reactions. Various methods have been developed to prepare nano-aluminum adjuvants, including ultrasonic dispersion, hydrothermal synthesis, laser ablation, and high-pressure microfluidics. Nanoaluminum may reaggregate shortly after ultrasonic treatment. Compared with micronized aluminum adjuvants, nanoaluminum prepared by hydrothermal synthesis showed higher suspension stability after antigen adsorption. Nanoaluminum adjuvants prepared by laser ablation showed anticancer and antipathogenic activities, but their adjuvant activity was not tested. ® After microfluidization of a mixture of stabilizers and polyacrylic acid, a stable nano-aluminum adjuvant was obtained, which can be repeatedly frozen and thawed and enhance Th1 / Th2 immune responses. In summary, although the development of nano-aluminum adjuvants is still under exploration, these evidences suggest that nano-aluminum adjuvants may have advantages over traditional aluminum adjuvants in overcoming vaccine temperature stability and improving vaccine efficacy, and have good clinical application potential. In addition, microfluidization of the mixture of stabilizers and aluminum adjuvants is currently a relatively mature and stable method for preparing nano-aluminum adjuvants. More safe and effective nano-aluminum stabilizers are urgently needed to be developed.

[0003] Chitosan, a natural cationic polysaccharide, has been used in FDA-approved wound dressing products and exhibits excellent biocompatibility, antimicrobial activity, and biodegradability. Similar to chitosan, its derivative, GCS, also exhibits good biocompatibility, high biodegradability, and mucosal adhesion. Due to the hydrophilicity of its ethylene glycol side chain, GCS is soluble at all pH values, whereas chitosan is only soluble in acidic solutions below pH 6. Recent studies have shown that chitosan can promote type I interferon production, promote dendritic cell (DC) maturation, and enhance Th1-mediated cellular immune responses by activating the cGAS-STING pathway. Previous studies have shown that GCS, when used as an adjuvant in respiratory syncytial virus (RSV) F protein subunit vaccines, exhibited limited ability to enhance antigen-specific immune responses. Furthermore, low-molecular-weight chitosan has been reported to have greater penetration into antigen-presenting cells (APCs), resulting in stronger adjuvant activity.

[0004] Small molecule immunostimulants play an important role in enhancing the induction of desired antigen-specific immune responses by traditional adjuvants. Among them, Toll-like receptor (TLR) agonists are gaining increasing attention due to their well-defined structure-activity relationship and acceptable safety profile. For example, IMQ, a TLR7 / 8 agonist, activates NF-κB to produce type I interferons, proinflammatory cytokines, and chemokines, thereby generating a potent cellular immune response. CdA, a cGAS-STING agonist, activates innate immunity, initiates type I interferon production, and induces antigen-specific Th1 / Th2 / Th17 responses. However, when used alone, small molecule stimulants are easily disseminated systemically, degraded, and metabolized, leading to side effects such as inflammation. Therefore, they need to be combined with nanodelivery systems to enhance immune responses and prevent rapid clearance.

[0005] The squalene nanoemulsion adjuvant MF59, a squalene-based oil-in-water nanoemulsion, was the first non-aluminum adjuvant used in licensed human vaccines and continues to be used today. MF59 can create a transient immune microenvironment at the injection site by promoting the secretion of various cytokines and chemokines and the recruitment of immune cells, subsequently triggering the infiltration of antigen-presenting cells into the draining lymph nodes (dLNs). MF59 also induces high levels of germinal center (GC) development and the production of follicular helper T cells (TFH), leading to a Th1 / Th2-type response. However, MF59 has a weak interaction with antigens, and its combination with aluminum adjuvants enhances its antigen delivery efficiency and DC targeting.

[0006] In summary, a new nano-aluminum adjuvant system that has balanced, safe and efficient immune activation capabilities, can carry various immunostimulants, and can withstand freeze-drying and be stored at room temperature needs to be developed. Summary of the Invention

[0007] The present invention provides a composite nano-aluminum phosphate adjuvant and a preparation method and use thereof, so as to solve the problems that traditional aluminum adjuvants are intolerant to freezing, their storage conditions need to be optimized, and their cellular immune response and safety need to be improved.

[0008] The technical solution adopted by the present invention is: a composite nano-aluminum phosphate adjuvant is made of the following raw materials in the following weight ratios: Glycol chitosan (GCS) with a molecular weight of 1~30 kDa: aluminum phosphate adjuvant (AP) = n: 1, n = 0.1~7.

[0009] The present invention provides a method for preparing a composite nano-aluminum phosphate adjuvant, comprising the following steps: Step 1: Glycol chitosan (GCS) with a molecular weight of 1-30 kDa and aluminum phosphate adjuvant (AP) were mixed in a weight ratio of n:1 in deionized water and tumbled overnight at 4 °C; Step 2: Homogenize the mixture using a high shear homogenizer at 11,000-39,000 rpm for 5-10 minutes. Step 3: After homogenization, the suspension was microfluidized using an M-110L microfluidizer at an air pressure of 20-100 psi to obtain the composite nano-aluminum phosphate adjuvant Gn-A1.

[0010] The aluminum concentration in step 1 of the present invention is 0.5-5 mg / mL.

[0011] In step 3 of the present invention, the number of microfluidization times is preferably such that the nano-aluminum particle size is within the range of 170-178 nm without any significant decrease.

[0012] The present invention further comprises step 4, high pressure sterilization, wherein the nano-aluminum adjuvant is high pressure sterilized at 0.1 MPa and 121° C. for 20 minutes, and the particle size after sterilization is 172-4735 nm.

[0013] The present invention also includes freeze drying, and the freeze drying protective agent is trehalose.

[0014] The composite nano-aluminum phosphate adjuvant is obtained by the preparation method of the composite nano-aluminum phosphate adjuvant.

[0015] The composite nano-aluminum phosphate adjuvant of the present invention is carried with imiquimod (IMQ), cyclic diadenylic acid C-di-AMP (CdA) or ZMF59 emulsion.

[0016] The vaccine preparation of the composite nano-aluminum phosphate adjuvant described in the present invention also contains antigens, which are protein antigens, including human papillomavirus (HPV) type 16 L1 protein virus-like particles (VLPs), respiratory syncytial virus protein antigens, influenza antigens, and herpes zoster virus protein antigens.

[0017] The present invention has achieved the following beneficial effects: A series of positively charged nanoaluminum adjuvants were developed by microfluidics of glycol chitosan (GCS) and aluminum phosphate adjuvant (AP). When the GCS / AP ratio was 5-7, they exhibited excellent stability after autoclaving. Furthermore, they could be freeze-dried in the presence of trehalose and stored at room temperature for at least 15 months without changes in physicochemical properties, potentially offering greater transport and storage convenience compared to traditional aluminum adjuvants. The candidate adjuvant, G7-A1, outperformed traditional aluminum adjuvants in inducing potent immune responses, as demonstrated by significantly activated DCs leading to a Th1 / Th2 immune balance, robust CTL responses, and high levels of memory T cells. It also induced milder local inflammatory infiltration than traditional aluminum adjuvants, demonstrating a better safety profile. Combining G7-A1 with ZMF59 or CdA produced a synergistic effect, significantly enhancing the immune response compared to using either component alone, without increasing the local inflammatory response compared to the single adjuvants. Notably, the G7-A1 / CdA combination outperformed G7-A1 and the combination of aluminum phosphate adjuvant and monophosphatidic acid lipid A (ZAS04) (an adjuvant system similar to the AS04 developed by GSK) in three key indicators: DC maturation level, Th1 cell immune response, and durability of neutralizing antibody response. These results demonstrate that G7-A1 is a comprehensive, potent, and safe adjuvant platform with transformative potential for the development of a new generation of vaccines and is a candidate adjuvant for clinical translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Characterization diagram of the Gn-A1 nano-aluminum adjuvant of the present invention: appearance (A), particle size distribution (B) and potential (C). The data are shown as the mean ± SD of three independent experiments. ® and TEM images of Gn-A1 nanoaluminum adjuvant without staining (D), the scale bars represent 200 nm, 500 nm, or 100 nm, respectively, TEM images of G0-A1 and G7-A1 nanoaluminum adjuvants stained with 2% (w / v) phosphotungstic acid (E), the scale bar is 500 nm; Figure 2 Figure 3 is a graph showing the physical and chemical properties of the Gn-A1 nanoaluminum adjuvant after autoclaving of the present invention, including the appearance of G0-A1 to G7-A1 after autoclaving (A), the particle size distribution (B) and potential (C) of G4-A1 to G7-A1 after autoclaving, and the pH values of G4-A1 to G7-A1 before and after autoclaving (D). Data are the mean ± SD of three independent experiments. Figure 3Figure 2 shows the appearance, particle size, and potential of the reconstituted nanoaluminum adjuvant after freeze-drying and storage at room temperature for 193 days. The data represent the results of three repeated experiments and are expressed as mean ± SD. Figure 4 Figure 3 is the particle size (A) and potential (B) of the reconstituted nanoaluminum adjuvant after freeze-drying and storage at room temperature for 458 days. The data represent the results of three repeated experiments and are expressed as mean ± SD. Figure 5 Characterization of HPV16 VLP adsorption on Gn-A1, including (A) representative TEM images of HPV16 L1 VLP and G0-A1 and G7-A1 stained with 2% (w / v) phosphotungstic acid after antigen adsorption, scale bar, 200 nm, (B) binding capacity of different aluminum adjuvants to HPV16 L1 VLPs at a ratio of 1 / 20 (VLPs / aluminum, w / w), potential (C), particle size (D), and PDI (E) of antigen-adsorbed Gn-A1 adjuvant. Data are the mean ± SD of three independent experiments. Figure 6 Schematic diagram of mouse immunization and antibody level assessment, where (A) C57BL / 6 mice were immunized on days 0 and 14, and blood was collected on days 14 and 28, respectively. Antibody titers of HPV16 L1 VLP-specific IgG (B and C), IgG1 and IgG2c (D), IgG1 / IgG2c ratio (E), and neutralizing antibodies (F and G) were presented (n = 5). Significance was assessed using one-way and two-way analysis of variance (ANOVA), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; Figure 7 Characterization diagrams of G7-A1 / ZMF59 composite adjuvants with and without HPV16 L1 VLPs, (A) Appearance of G7-A1 / ZMF59 adjuvants with different ratios, particle size distribution (B and C) and potential (D) of G7-A1 / ZMF59 adjuvant, particle size (E), PDI (F) and potential (G) of HPV16 L1 VLP loaded with G7-A1 / ZMF59 adjuvant, the buffer of HPV16 L1 VLPs solution is 8 mM PB, containing 0.15 M NaCl and 0.05% Polysorbate 80, pH 6.0, data are the mean ± SD of three independent experiments; Figure 8Figure 3 Antibody responses to HPV16 L1 VLPs vaccines with different adjuvants. (A) Schematic diagram of vaccination and serum sampling. Mice (n = 4 or 5) were injected intramuscularly twice, 2 weeks apart. Serum samples were collected 2, 4, 8, and 12 weeks after the initial immunization. HPV16 L1 VLP-specific IgG (B), IgG1 (C), and IgG2c (D) antibody titers, IgG1 / IgG2c ratio (E), and neutralizing antibody titers (F) were shown. Significance was assessed using t-test and one-way analysis of variance (ANOVA). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns indicates not significant. Figure 9 Figure 1 is a schematic diagram of vaccination and cellular immune response assessment, where (A) C57BL / 6 mice were injected intramuscularly on days 0 and 14, and spleens were harvested on day 28. The levels of IFN-γ (B), TNF-α (C), IL-4 (D), and IL-10 (E) in splenocyte supernatants were measured by ELISA (n=3-5). The levels of HPV16 L1 VLP-specific IFN-γ-secreting splenocytes (F) and representative images of IFN-γ ELISPOT assays (G) are shown (n=4 or 5). The levels of HPV16 L1 VLP-specific CD4+ (H) and CD8+ T cells (I) expressing IFN-γ, IL-2, and TNF-α were measured by flow cytometry (n=4 or 5). Significance was assessed by t-test and one-way analysis of variance (ANOVA), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Figure 10 Figure 3 is a graph showing the levels of HPV16-specific memory T cells determined by flow cytometry; the levels of HPV-specific CD4+ Tcm (A), CD4+ TEM (B), and total memory T cells (C) in T cells, and the levels of CD8+ Tcm (D), CD8+ TEM (E), and total memory T cells (F) in T cells (n = 4 or 5), with significance assessed by t-test and one-way analysis of variance (ANOVA), *P < 0.05, **P < 0.01, ***P < 0.001; Figure 11 Figure 2 is an immunofluorescence analysis of activated DCs in the draining lymph nodes of mice 1, 3, and 7 days after immunization. Representative images of one mouse per group at each time point are shown. Paraffin sections of draining lymph nodes were stained with anti-CD11c antibody (green), anti-CD86 antibody (red), and DAPI (blue). The scale bar is 1 mm. Figure 12 Figure 3 shows the safety evaluation of mice after vaccination with G7-A1-based composite adjuvants, where (A) the body weight of mice was monitored within 28 days after immunization, (B) the body temperature of mice was monitored at 0, 0.5, 1, 2, and 4 hours after immunization, and (C) representative images of H&E staining of the muscles at the injection site. On day 0, C57BL / 6 mice were injected intramuscularly with various adjuvants adsorbed with HPV16 L1 VLPs. Sterile PBS or HPV16 L1 VLPs alone were used as negative controls, and ZAS04 was used as a positive control. On days 1, 3, and 7, the mice were euthanized, and muscle samples from the injection site were collected for H&E staining. The scale bar is 1 mm. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is not limited to the specific implementation methods listed below. Based on the examples in the implementation methods, other embodiments obtained by those skilled in the art without making any creative work shall fall within the scope of protection of the present invention.

[0020] The preparation method of ethylene glycol chitosan GCS with a molecular weight of 1 to 30 kDa used in the following examples is as follows: Step 1: Dissolve 0.8 g of GCS in 60 mL of 4 M HCl in a 50°C water bath with continuous magnetic stirring for 96–144 hours. Step 2: Place the acid hydrolyzate in a 1-14 kDa dialysis bag and dialyze against deionized water for 24 hours, changing the solution every 4-6 hours until the pH is neutral; Step 3: freeze-dry the dialyzed product and store it in a sealed drying dish; Step 4: The lyophilized product was dissolved in 0.3 M sodium acetate (2 mg / mL) and the molecular weight was determined by gel permeation chromatography.

[0021] The aluminum phosphate adjuvant AP used in the following examples is a commercial aluminum phosphate adjuvant Adju-Phos ® . Example 1

[0022] Step 1: Glycol chitosan (GCS) with a molecular weight of 20 kDa and aluminum phosphate adjuvant (AP) were mixed in deionized water at a weight ratio of 1:1, with an aluminum concentration of 0.5 mg / mL, and tumbled at 4 °C overnight. Step 2: Homogenize using a high shear homogenizer at 11,000 rpm for 5 minutes; Step 3: After homogenization, the suspension was microfluidized using an M-110L microfluidizer at an air pressure of 50 psi to obtain a nano-aluminum particle size of 170-178 nm without a significant decrease, thereby obtaining the composite nano-aluminum phosphate adjuvant G1-A1. Example 2

[0023] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 2:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G2-A1. Example 3

[0024] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 3:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G3-A1. Example 4

[0025] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 4:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G4-A1. Example 5

[0026] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 5:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G5-A1. Example 6

[0027] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 6:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G6-A1. Example 7

[0028] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 7:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G7-A1. Example 8

[0029] In step 1, glycol chitosan (GCS) and aluminum phosphate adjuvant (AP) are mixed in deionized water at a weight ratio of 0.1:1, and the remaining steps are the same as those in Example 1 to obtain composite nano-aluminum phosphate adjuvant G0.1-A1. Example 9

[0030] Step 1: Glycol chitosan (GCS) with a molecular weight of 1 kDa and aluminum phosphate adjuvant (AP) were mixed in deionized water at a weight ratio of 4:1, with an aluminum concentration of 2.5 mg / mL, and tumbled at 4 °C overnight. Step 2: Homogenize using a high shear homogenizer at 25,000 rpm for 7 minutes; Step 3: After homogenization, the suspension was microfluidized using an M-110L microfluidizer at an air pressure of 20 psi to obtain a nano-aluminum particle size of 170-178 nm without a significant decrease, thereby obtaining the composite nano-aluminum phosphate adjuvant G4-A1. Example 10

[0031] Step 1: Glycol chitosan (GCS) with a molecular weight of 30 kDa and aluminum phosphate adjuvant (AP) were mixed in deionized water at a weight ratio of 6:1, with an aluminum concentration of 5 mg / mL, and tumbled at 4 °C overnight. Step 2: Homogenize using a high shear homogenizer at 39,000 rpm for 10 minutes; Step 3: After homogenization, the suspension was microfluidized using an M-110L microfluidizer at an air pressure of 100 psi to obtain a nano-aluminum particle size of 170-178 nm without a significant decrease, thereby obtaining the composite nano-aluminum phosphate adjuvant G6-A1. Example 11

[0032] The composite nano-aluminophosphate adjuvant Gn-A1 obtained in Examples 1 to 10 was respectively sterilized under high pressure at 0.1 MPa and 121° C. for 20 minutes. The particle size after sterilization was 172 to 4735 nm. Example 12

[0033] The composite nano-aluminophosphate adjuvant Gn-A1 obtained in Examples 1 to 11 or the composite nano-aluminophosphate adjuvant after high-pressure sterilization was respectively taken, and a lyophilization protective agent was added, such as trehalose, with a concentration range of 2% to 10% (w / v), and the mixture was freeze-dried and sealed for long-term dry storage. Example 13

[0034] The composite nano-aluminophosphate adjuvants obtained in Examples 1 to 12 were respectively taken and loaded with Imiquimod (IMQ). Example 14

[0035] The dosage is 100 μL per dose, and 50 μg of the composite nano-aluminophosphate adjuvant, 7.5 μg of IMQ, and 4 μg of antigen obtained in Examples 1 to 12 are taken respectively, wherein the antigen is a protein antigen, such as human papillomavirus (HPV) type 16 L1 protein virus-like particles (VLPs), respiratory syncytial virus protein antigens, influenza antigens, herpes zoster virus protein antigens, etc. Example 15

[0036] The composite nano-aluminophosphate adjuvants obtained in Examples 1 to 12 were respectively taken and loaded with cyclic diadenylic acid C-di-AMP (CdA). Example 16

[0037] The dosage is 100 μL per dose, and 50 μg of the composite nano-aluminophosphate adjuvant, 7.5 μg of CdA, and 4 μg of the antigen obtained in Examples 1 to 12 are taken respectively, wherein the antigen is a protein antigen, such as human papillomavirus (HPV) type 16 L1 protein virus-like particles (VLPs), respiratory syncytial virus protein antigens, influenza antigens, herpes zoster virus protein antigens, etc. Example 17

[0038] The composite nano-aluminophosphate adjuvants obtained in Examples 1 to 12 were respectively taken and loaded with ZMF59 emulsion. Example 18

[0039] The dosage is 100 μL per dose, and 19.73 μg of the composite nano-aluminophosphate adjuvant obtained in Examples 1 to 12, 59.2 μL of ZMF59, and 4 μg of antigen are taken respectively, wherein the antigen is a protein antigen, such as human papillomavirus (HPV) type 16 L1 protein virus-like particles (VLPs), respiratory syncytial virus protein antigens, influenza antigens, herpes zoster virus protein antigens, etc.

[0040] The present invention is further described below through experimental examples.

[0041] Corresponding measurement results of the composite nano-aluminum phosphate adjuvant prepared in Experimental Example 1.

[0042] Aluminum phosphate adjuvant Adju-phos ® It is mainly composed of aggregated plate-like entities with a size of 15-50 nm ( Figure 1 D), the average size of the aggregates is about 1-3 μm. After the introduction of low molecular weight GCS as a stabilizer and microfluidization treatment with AP, a stable and uniform milky white nano-aluminum suspension was generated ( Figure 1 A). The average particle size of G0-A1 and G1-A1 ranged from 190 to 195 nm, while the particle size of the nanoaluminum adjuvants produced using a higher concentration of GCS was approximately 178 nm. The polydispersity index (PDI) of all nanoaluminum adjuvants ranged from 0.1 to 0.148, indicating a monodisperse system ( Figure 1 B). G0-A1 without GCS coating showed a potential of -21.8 mV. As the GCS / AP ratio increased, the potential of the nano-aluminum adjuvant increased from 21.17 mV to 33.1 mV ( Figure 1 C). At the same time, the pH value also decreased from 6.88 to 5.293 (Table 1). TEM examination showed that the Gn-A1 nanoaluminum adjuvant showed an irregular shape with an average size of 100-200 nm, and no significant difference was observed under different GCS ratios ( Figure 1 D). After staining G7-A1 with 2% phosphotungstic acid, a shell was observed around the nanoaluminum core, which was different from the appearance of G0-A1 ( Figure 1 E). It is speculated that the shell is composed of GCS, because only the hydroxyl groups and amide bonds in GCS can interact with phosphotungstic acid, leading to dye deposition.

[0043] Table 1 Particle size, potential and pH value of different nano-aluminum adjuvants name Particle size (nm) PDI Potential (mV) pH G0-A1 195.33±43.23 0.148±0.063 -21.8±0.61 6.88±0.079 G1-A1 190.80±9.49 0.118±0.009 21.17±2.86 6.32±0.139 G2-A1 178.67±5.19 0.106±0.015 26.37±1.62 5.847±0.006 G3-A1 177.93±2.06 0.102±0.007 25.87±4.98 5.680±0.075 G4-A1 175.57±2.10 0.111±0.013 31.47±0.15 5.590±0.218 G5-A1 178.03±1.66 0.107±0.008 31.80±0.70 5.463±0.175 G6-A1 179.60±5.56 0.113±0.011 33.07±0.50 5.397±0.197 G7-A1 178.50±5.56 0.100±0.007 33.10±0.79 5.293±0.18 The preparation method of G0-A1 in the table is as follows: in Example 1, the amount of glycol chitosan (GCS) used is 0, and the remaining steps are the same.

[0044] After autoclaving, nano-aluminum adjuvants G1-A1 to G3-A1 showed different degrees of precipitation at the bottom of the test tube ( Figure 2 A). When the GCS / AP weight ratio was higher than 4:1, the particle sizes of all nano-aluminum adjuvants were between 207 nm and 211 nm, and the PDI was between 0.131 and 0.136, indicating that they were monodisperse systems ( Figure 2 B and Table 2). After autoclaving, G1-A1-G3-A1 showed aggregation, while G5-A1 to G7-A1 could withstand standard autoclaving without significant changes in their physicochemical properties.

[0045] Table 2 Particle size, potential and pH value of different nano-aluminum adjuvants after sterilization name Particle size (nm) PDI Potential (mV) pH G0-A1 195.63±44.32 0.145±0.041 -27.17±4.77 7.097±1.075 G1-A1 2763.67±1972.18 0.253±0.111 3.58±5.73 7.16±0.382 G2-A1 998.33±261.39 0.226±0.029 9.37±9.62 7.017±0.709 G3-A1 1085.70±733.31 0.279±0.129 14.06±10.51 6.74±0.92 G4-A1 761.33±855.61 0.241±0.088 19.53±8.81 6.37±0.686 G5-A1 211.17±13.24 0.136±0.028 25.87±1.30 5.853±0.097 G6-A1 179.60±5.56 0.113±0.011 33.07±0.50 5.397±0.197 G7-A1 178.50±5.56 0.100±0.007 33.10±0.79 5.293±0.18 It is well known that traditional aluminum adjuvants can neither be frozen nor freeze-dried. Here, the present invention freeze-dried the above-mentioned nano-aluminum adjuvants in the presence of different concentrations of the cryoprotectant trehalose, and then re-dissolved and characterized. For all nano-aluminum adjuvants, when the trehalose concentration was maintained at 2%, different degrees of collapse occurred, especially G0-A1. As the GCS / AP weight ratio increased, the appearance became more stable. When the trehalose concentration was higher than 4%, all freeze-dried nano-aluminum adjuvants did not show phenomena such as collapse or shrinkage. After the nano-aluminum adjuvants with a GCS / AP ratio higher than 3:1 were freeze-dried in the presence of 6% or higher trehalose, their particle size, PDI and potential were comparable to those of freshly prepared nano-aluminum adjuvants ( Figure 3 and Table 3).

[0046] Table 3 Characterization of nano-aluminum adjuvants 193 days after freeze-drying Similar advantages were also observed in freeze-dried nano-aluminum adjuvants stored for 15 months ( Figure 4 These results indicate that high levels of GCS and trehalose can effectively prevent the aggregation of freeze-dried nano-aluminum adjuvant particles after reconstitution. GCS and trehalose give the nano-aluminum adjuvant excellent freeze-drying resistance, which will facilitate its future storage and transportation.

[0047] Table 4 Characterization of nano-aluminum adjuvants 458 days after freeze-drying Experimental Example 2 Antigen adsorption experiment with different nano-aluminum phosphate adjuvants, including the following steps: Step 1: Add HPV16 L1 VLPs with a final concentration of 20 μg / mL to the diluted nanoaluminum adjuvant with an aluminum content of 20, 200, or 400 mg / mL, and invert at 4 degrees for 1 hour.

[0048] Step 2: Centrifuge at 100,000 g for 16 minutes, collect the supernatant, concentrate 10-fold, and perform SDS-PAGE with Coomassie blue staining. Use a 25-300 μg / mL BSA standard as a control. Measure the OD700nm brightness of the bands using a far-infrared fluorescence imaging system. After obtaining a standard curve, calculate the residual HPV protein content in the supernatant and the adjuvant adsorption rate: Adsorption rate = (1-protein content in supernatant / total protein content) × 100% The results of the antigen adsorption experiments with different nano-aluminum phosphate adjuvants are as follows: To investigate the adsorption capacity of various Gn-A1 nanoaluminum adjuvants on HPV16 L1 VLPs, we prepared HPV16 L1 VLPs with a size of approximately 60 nm and a potential of 1.5 mV. Precipitation occurred when HPV16 L1 VLPs were added to G0-A1, which can be explained by the flocculation of nanoaluminum particles mediated by VLP adsorption. TEM analysis results ( Figure 5 A) As can be seen, the VLP is located within the GCS shell of G7-A1. The HPV16 L1 protein has both negatively and positively charged amino acids on its surface. Therefore, electrostatic interactions between the GCS and HPV16 L1 proteins may still exist.

[0049] In terms of adsorption rate, as the aluminum / antigen weight ratio increases, Gn-A1 adjuvant can adsorb higher concentrations of HPV16L1 VLPs. However, as GCS increases, the adsorption capacity of Gn-A1 nano-aluminum adjuvant decreases. This may be due to the shielding effect of the negatively charged particle surface of the nano-aluminum adjuvant and the limited loading capacity of the GCS shell. When the weight ratio of HPV to aluminum is 1 / 20, G5-A1 to G7-A1 adjuvants only adsorbed about 50% of the antigen ( Figure 5 B). When the antigen / aluminum weight ratio approaches 1:1, Gn-A1 nanoaluminum particles undergo antigen adsorption-mediated flocculation, as evidenced by the increase in particle size and PDI value ( Figure 5 D and E). In addition, their potential is also closer to that of HPV16 L1 VLPs ( Figure 5 C).

[0050] Experimental Example 3 Immunity experiments with different nano-aluminum phosphate adjuvants.

[0051] The following steps are involved: Step 1: Add HPV16 L1 VLPs to a final concentration of 20 μg / mL to the diluted adjuvant containing 0.5 mg / mL aluminum. Incubate at 4°C for 1 hour. Simultaneously, establish a PBS group, a GCS group alone (350 μg / dose), an antigen group alone, and a ZAS04 group (50 μg AP + 5 μg MPLA / dose) as controls.

[0052] Step 2: Five C57BL / 6 mice per group were immunized intramuscularly with a 0.1 mL dose. A second immunization was performed two weeks after the first immunization using the same method and dose. Blood was collected from the orbitals two weeks after the first and second immunizations, and serum was isolated and assayed for IgG, IgG1, IgG2c, and neutralizing antibody titers.

[0053] The results of the immune experiments with different nano-aluminum adjuvants are as follows: HPV16 L1 VLPs prepared with different adjuvants were used to immunize C57BL / 6 mice intramuscularly twice at intervals of two weeks. Figure 6 As shown, the antigen-specific IgG induced by nano-aluminum adjuvants with a GCS / aluminum ratio higher than 5:1 was significantly higher than that of the single antigen group two weeks after the initial immunization ( Figure 6 B). Similar phenomenon was observed in the neutralizing antibody titer, with only the G7-A1 group showing statistical difference ( Figure 6 F). After booster immunization, IgG and neutralizing antibody titers increased significantly ( Figure 6 C and G). In addition, the antibody levels of all adjuvanted vaccine groups, excluding the GCS group, were significantly higher than those of the antigen-only group, indicating that the adjuvant activity was strong. As the GCS / aluminum ratio increased, the IgG1 / IgG2c ratio decreased ( Figure 6 D and E), indicating a more balanced humoral and cellular immunity. ® Compared with ZAS04, the vaccine formulated with nano-aluminum adjuvant with a GCS / aluminum ratio higher than 5:1 induced slightly higher levels of neutralizing antibodies ( Figure 6 G). In conclusion, nano-aluminum adjuvants with high GCS ratios are superior to Adju-phos in inducing early antibody production, protective neutralizing antibodies, and balanced Th1 / Th2 immune responses. ® The effect is equivalent to or better than ZAS04.

[0054] Experimental Example 4 Screening experiment on the mixing ratio of nano-aluminum adjuvant and ZMF59 emulsion.

[0055] The following steps are involved: Step 1: Mix G7-A1 and ZMF59 emulsions in a volume ratio of 1 / 9 to 9 / 1, and then measure the particle size and potential.

[0056] Step 2: HPV16 L1 VLPs with a final concentration of 40 μg / mL were added to the emulsion mixture of G7-A1 and ZMF59 in a volume ratio of 1 / 9 to 9 / 1, and the particle size and potential were detected.

[0057] ZMF59 is a homemade squalene-based emulsion adjuvant similar to MF59, with a milky white appearance, a particle size of 152.8 nm, and a potential of -25.3 mV. All G7-A1 / ZMF59 composite adjuvants with different volume ratios have a similar appearance to ZMF59 ( Figure 7 A). With the increase of G7-A1 / ZMF59 ratio, the particle size, PDI and potential of the composite adjuvant increased ( Figure 7 C and D). Except for the 9 / 1 ratio, no obvious precipitation was observed in all the combinations ( Figure 7 A). HPV16 L1 VLP-loaded G7-A1 / ZMF59 composite adjuvants were further characterized. As the G7-A1 / ZMF59 ratio increased, antigen-loaded G7-A1 / ZMF59 showed similar trends to the antigen-free composite adjuvant in terms of particle size, PDI, and potential ( Figure 7 In subsequent experiments, a 4 / 6 ratio of G7-A1 / ZMF59 ( Figure 7 C and F).

[0058] Experimental Example 5 Immunization experiment in which G7-A1 was combined with IMQ / CdA / ZMF59 and loaded with HPV16 L1 VLPs.

[0059] The following steps are involved: Step 1: The aluminum content of G7-A1 and AP was adjusted to 0.5 mg / mL, and the experimental groups included PBS group, antigen alone group, G7-A1 group, AP group, IMQ group (7.5 μg / dose), G7-A1 / IMQ group (50 μg G7-A1 + 7.5 μg IMQ / dose), CdA group (7.5 μg / dose), G7-A1 / CdA group (50 μg G7-A1 + 7.5 μg CdA / dose), ZMF59 group (59.2 μL / dose), G7-A1 / ZMF59 group (19.73 μg G7-A1 + 59.2 μL ZMF59 / dose), and ZAS04 group (50 μg AP + 5 μg MPLA / dose). HPV16 L1 VLPs were added to all vaccine preparations except the PBS group at a final concentration of 40 μg / mL and adsorbed at 4°C for 1 hour.

[0060] Step 2: Each group of 5 C57BL / 6 mice received an intramuscular immunization of 0.1 mL of the vaccine. A second immunization was performed 2 weeks after the first immunization using the same immunization method and dose.

[0061] (1) Two weeks after the second immunization, the spleens of mice were harvested and single cell suspensions were prepared. The levels of IFN-γ secreting cells, memory T cells, Th1 / CTL cells, and IFN-γ, TNF-α, IL-4, and IL-10 cytokines in the spleen cells were determined.

[0062] (2) Blood was collected from the eye sockets 2, 4, 8, and 12 weeks after the initial immunization, and the serum was separated and the titers of IgG, IgG1, IgG2c, and neutralizing antibodies in the serum were measured.

[0063] (3) Two mice were killed in each group 1, 3, and 7 days after the initial immunization. The muscle tissue at the injection site was taken for H&E staining, and the inguinal draining lymph nodes were taken for immunofluorescence to observe the activation of DCs.

[0064] The results of the immune experiment of the composite nano-aluminum phosphate adjuvant are as follows: Humoral immune response such as Figure 8 As shown, 6 weeks after booster immunization, G7-A1 and Adju-phos ® The IgG antibody levels elicited were significantly higher than those in the antigen group, and the IgG, IgG1, and IgG2c titers induced by the G7-A1 composite adjuvant were higher than those induced by ZMF59 or a single immunostimulant ( Figure 8 BD). In addition, with Adju-phos ® Compared with the control group, G7-A1 and its co-adjuvants with CdA or ZMF59 induced a more balanced IgG1 / IgG2c immune response 2 weeks after booster immunization ( Figure 8 E). In terms of neutralizing antibodies, the G7-A1-based composite adjuvant induced significantly higher antibody titers after booster injections than ZMF59 or individual immunostimulants ( Figure 8 F). G7-A1 / CdA or G7-A1 / ZMF59 induced faster and more sustained antibody responses, and a more balanced Th1 and Th2 immune response.

[0065] Cellular immune response such as Figure 9 As shown, with Adju-phos ® Compared with the control group, G7-A1 induced significantly higher levels of TNF-α, IL-4, and IL-10, and the combined adjuvants of G7-A1 promoted the production of IFN-γ, TNF-α, IL-4, and IL-10 to varying degrees ( Figure 9BE). The number of IFN-γ splenocyte spots induced by G7-A1 was 2.73 times higher than that in the AP group, the number of IFN-γ splenocyte spots in the G7-A1 / IMQ group was 7.23 times higher than that in the IMQ group, and the number of IFN-γ splenocyte spots in the G7-A1 / ZMF59 group was 4.49 times higher than that in the ZMF59 group. The number of IFN-γ splenocyte spots in the G7-A1 / CdA group was 2.32 times higher than that in the CdA group ( Figure 9 FG). G7-A1 / CdA adjuvant induced the highest proportion of polyfunctional CD4+ T cells among all adjuvants, followed by G7-A1 / ZMF59. G7-A1 alone induced the highest proportion of antigen-specific polyfunctional CD8+ T cells, followed by G7-A1 / CdA ( Figure 9 In conclusion, the above results further confirmed that G7-A1 and G7-A1-based composite adjuvants (especially G7-A1 / CdA and G7-A1 / ZMF59) are more effective than Adju-Phos alone. ® and immunostimulants, and have advantages in inducing cellular immunity.

[0066] Antigen-specific memory T cell responses such as Figure 10 As shown, the G7-A1 / CdA group significantly enhanced the CD4+ Tcm response, followed by G7-A1 / ZMF59, and G7-A1 / IMQ triggered more CD8+ Tcm cells ( Figure 10 A and D). G7-A1 elicited more CD4+ and CD8+ TEM responses than Adju-Phos ® More strongly, G7-A1 / CdA significantly increased the number of CD4+ and CD8+ TEM cells, and the TEM level of G7-A1 / ZMF59 was also increased compared with ZMF59 alone ( Figure 10 B and E). Overall memory T cell responses as Figure 10 As shown in C and F, G7-A1 and G7-A1 / CdA may be more effective in maintaining long-term immune responses.

[0067] Immunofluorescence was used to evaluate the infiltration of CD11c+CD86+ cells in the lymph nodes of mice vaccinated with various vaccines. At all time points, the expression of CD11c+CD86+ cells in the G7-A1 group was significantly higher than that in the Adju-Phos ® Group( Figure 11 When G7-A1 is combined with IMQ, CdA or ZMF59, it can also bind to Adju-Phos ® Compared with G7-A1, IMQ, CdA, or ZMF59, the number of CD11c+CD86+ cells was significantly higher. These results suggest that G7-A1 can promote the proliferation and activation of dendritic cells in the abdominal lymph nodes and enhance the adaptive immune response.

[0068] To evaluate the safety of nano-aluminum adjuvants, we monitored the body temperature and body weight of mice after vaccination with different vaccines ( Figure 12 A and B). Within 28 days after the first vaccination, all groups of mice gained weight, and no significant differences were found. Within 4 hours after vaccination, the body temperature of mice in all groups did not fluctuate more than 0.9 °C. Inflammation at the injection site was as follows Figure 12 As shown in C, no inflammatory cell infiltration was found in the HPV16 L1 VLP group, and a mild inflammatory reaction was observed in the IMQ group on day 1. Transient inflammatory cell infiltration was observed in the G7-A1 and CdA groups on day 3 after inoculation, while MF59 caused a mild inflammatory reaction on days 3 and 7. ® The inflammatory response was more severe than that of the other adjuvants at all sampling time points, characterized by a significant accumulation of inflammatory cells at the inoculation site. The inflammatory response induced by ZAS04 was milder but persisted from day 1 to day 7, and similar phenomena were observed in the G7-A1 / IMQ, G7-A1 / CdA, and G7-A1 / ZMF59 groups. These findings suggest that G7-A1 and its co-adjuvants did not induce any significant systemic toxicity or severe local adverse reactions.

[0069] The new nano-aluminum adjuvant G7-A1 prepared by the present invention can be freeze-dried and stored at room temperature for more than one year, and induces safer, more efficient and more balanced Th1 / Th2 responses than traditional aluminum phosphate adjuvants, as well as activates stronger CD8+ T cell and memory T cell responses. When G7-A1 is used in combination with ZMF59 or CdA, the immune response can be significantly enhanced without increasing the local inflammatory response. The G7-A1 / CdA combination is superior to G7-A1 and ZAS04 in three key indicators: DC maturation level, Th1 immune response and persistence of neutralizing antibody response. This discovery is more applicable to antigens that require cellular immunity or humoral / cellular immunity balance in the future. It meets the urgent needs of the public health field and establishes a multifunctional platform for the global fight against various pathogens and cancers.

Claims

1. A composite nano-aluminum phosphate adjuvant, characterized in that It is made of the following raw materials in the following weight ratios: Glycol chitosan GCS with a molecular weight of 1~30 kDa: aluminum phosphate adjuvant AP=n:1, n=0.1~7.

2. The method for preparing the composite nano-aluminum phosphate adjuvant according to claim 1, characterized in that The following steps are involved: Step 1: Glycol chitosan (GCS) with a molecular weight of 1-30 kDa and aluminum phosphate adjuvant (AP) were mixed in a weight ratio of n:1 in deionized water and tumbled overnight at 4 °C. Step 2: Homogenize the mixture using a high shear homogenizer at 11,000-39,000 rpm for 5-10 minutes. Step 3: After homogenization, the suspension was microfluidized using an M-110L microfluidizer at an air pressure of 20-100 psi to obtain the composite nano-aluminum phosphate adjuvant Gn-A1.

3. The method for preparing the composite nano-aluminum phosphate adjuvant according to claim 2, wherein: The aluminum concentration in step 1 is 0.5-5 mg / mL.

4. The method for preparing the composite nano-aluminum phosphate adjuvant according to claim 2, wherein: In step 3, the number of microfluidization times is preferably such that the nano-aluminum particle size is within the range of 170-178 nm without any significant decrease.

5. The method for preparing the composite nano-aluminum phosphate adjuvant according to claim 2, wherein: The method further includes step 4, high-pressure sterilization, wherein the nano-aluminum adjuvant is high-pressure sterilized at 0.1 MPa and 121° C. for 20 minutes, and the particle size after sterilization is 172-4735 nm.

6. The method for preparing the composite nano-aluminum phosphate adjuvant according to claim 2 or 5, characterized in that: It also includes freeze-drying, and the freeze-drying protective agent is trehalose.

7. The composite nano-aluminum phosphate adjuvant obtained by the preparation method of the composite nano-aluminum phosphate adjuvant according to any one of claims 2 to 6.

8. A vaccine preparation containing the composite nano-aluminum phosphate adjuvant according to claim 1 or 7, characterized in that: It also contains antigens, which are protein antigens, including human papillomavirus HPV16 type L1 protein virus-like particles VLP, respiratory syncytial virus protein antigens, influenza antigens, and herpes zoster virus protein antigens.

9. The composite nano-aluminum phosphate adjuvant according to claim 1 or 7, characterized in that: Emulsions containing imiquimod IMQ, cyclic diadenylic acid CdA or ZMF59.

10. A vaccine preparation containing the composite nano-aluminum phosphate adjuvant according to claim 9, characterized in that: It also contains antigens, which are protein antigens, including human papillomavirus HPV16 type L1 protein virus-like particles VLP, respiratory syncytial virus protein antigens, influenza antigens, and herpes zoster virus protein antigens.

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