Preparation and use of aluminum hydroxide oxide nano-adjuvant based on calcium or silicon doping

Abstract

A preparation method for and the use of an aluminum hydroxide oxide nano-adjuvant based on calcium and silicon doping. The particle morphology of the AlOOH adjuvant is a 50-800 nm nano-particle, wherein silicon or calcium is doped in AlOOH crystal lattices, and the molar ratio of calcium or silicon to aluminum is 0.01-100:1. The preparation method comprises: synthesizing AlOOH by using an Al source, doping Ca or Si in a reaction solution by using a calcium source or a silicon source, precipitating Al(OH)3 and a dopant by adding an alkaline solution, and preparing a Ca- or Si-doped AlOOH adjuvant by means of a hydrothermal synthesis method. In a hepatitis B surface antigen and varicella-zoster virus glycoprotein E antigen model, the calcium- or silicon-doped AlOOH nano-adjuvant can simultaneously induce efficient humoral immunity and cellular immunity by means of in-vivo experiment verification of mice.

Description

Preparation and application of a calcium or silicon-doped aluminum oxyhydroxide nanoadjuvant Technical Field

[0001] The present invention relates to the preparation and application of an AlOOH nanoadjuvant doped with calcium and silicon elements, and specifically to a method of doping calcium and silicon elements into the AlOOH nanoadjuvant, which can be used in the development of vaccines and the prevention and treatment of various infectious diseases. Background Art

[0002] Infectious diseases have always been a major threat to human health and safety, resulting in a large number of deaths each year. For example, according to the World Health Organization (WHO), 296 million people were infected with chronic hepatitis B in 2019, resulting in an estimated 820,000 deaths, the majority of which were due to cirrhosis and hepatocellular carcinoma (primary liver cancer), with 1.5 million new cases of infection each year.

[0003] Vaccines, the most important weapon in preventing infectious diseases, have undergone several stages of development. In 1798, British physician Jenner developed the world's first cowpox vaccine, ushering in the history of vaccine application. Vaccine development entered its golden age in the mid-to-late 20th century. Adjuvants, a key component of vaccines, play a crucial role in enhancing the immune response to antigens. The development of adjuvants has evolved from natural ingredients to synthetic, engineered vaccine adjuvants. The discovery of the adjuvant effect of aluminum salts in 1926 was groundbreaking. To date, six types of adjuvants have been approved for use in vaccines by the US FDA: aluminum salts, MF59, AS03, AS04, CpG ODN, and AS01B. Aluminum salts are crucial in adjuvanted vaccines. They are widely used in vaccines for tetanus, diphtheria, pertussis, polio, hepatitis A, and hepatitis B.

[0004] However, aluminum salt adjuvants alone generally only enhance humoral immunity, not cellular immunity, and thus fail to provide a balanced immune response. Furthermore, to improve the interaction between the antigen and the adjuvant, excipients are often added to the vaccine formulation, a process that complicates vaccine production. Therefore, designing an infectious vaccine that, based on the vaccine adjuvant itself, can be simple to manufacture and formulate, while eliciting a robust and balanced immune response, is of great significance in the prevention and treatment of infectious diseases.

[0005] Summary of the Invention

[0006] To address the problems of the prior art, the present invention provides a method for preparing and applying a calcium- and silicon-doped aluminum oxyhydroxide (AlOOH) nanoadjuvant capable of eliciting a balanced immune response. This method utilizes a synthetic method for AlOOH adjuvants and incorporates calcium and silicon during the preparation process. The prepared AlOOH adjuvant exhibits a well-balanced immune response, inducing a more balanced humoral and cellular immune response, enabling the design of preventive or therapeutic vaccines. This method provides a new platform for the design of aluminum-adjuvanted vaccines.

[0007] A calcium- and silicon-doped AlOOH nanoadjuvant comprises calcium- and silicon-doped AlOOH adjuvants. Calcium or silicon atoms are present in the AlOOH lattice, and the adjuvant is primarily composed of AlOOH nanoparticles. AlOOH is synthesized using an Al source. A calcium or silicon source is simultaneously used in the reaction solution to dope the Ca or Si element. Al(OH)3 and the dopant are precipitated by adding an alkaline solution. The Ca- or Si-doped AlOOH adjuvant is prepared using a hydrothermal synthesis method.

[0008] Furthermore, the Al source is various inorganic or organic aluminum salts such as aluminum nitrate, aluminum chloride, and aluminum ethoxide. The aluminum element exists in the reaction solution in the form of aluminum ions, and the concentration of aluminum ions in the reaction system is 0.01-10 mol / L, preferably 0.01-2 mol / L.

[0009] Furthermore, the calcium source is an inorganic or organic soluble calcium salt such as calcium chloride, calcium nitrate, or tricalcium citrate, and the molar ratio of calcium to aluminum is 0.01-100:1, preferably 0.01-40:1; the calcium element exists in the reaction solution in the form of calcium ions with a concentration of 0.01-10 mol / L, and the preferred concentration of calcium element is 0.01-2 mol / L.

[0010] Furthermore, the silicon source is an organic silane such as tetraethoxysilane, a soluble silicate such as silicic acid or sodium silicate, potassium silicate, and the molar ratio of silicon element to aluminum element is: 0.01-100:1, preferably 0.01-40:1; the silicon element exists in the reaction liquid in the form of ions or organic matter containing silicon element, and the concentration of silicon element in the reaction liquid is 0.01-10 mol / L, preferably 0.01-2 mol / L.

[0011] Furthermore, the alkaline solution is an aqueous solution such as NaOH, KOH, ammonia water, or various alkaline buffer solutions, and the concentration of the alkaline solution is 0.01-10 mol / L, preferably 0.01-2 mol / L.

[0012] Furthermore, the calcium or silicon doped AlOOH nano-adjuvant is a nanoparticle, and the nanoparticle has a morphology of 20nm-1000nm (preferably 50-800nm).

[0013] The preparation method of the above-mentioned calcium- or silicon-doped AlOOH nanoadjuvant comprises the following steps:

[0014] ① For the synthesis of calcium-doped aluminum oxyhydroxide: at a temperature of 20-60°C, add an alkaline solution dropwise to an aluminum source solution containing a calcium source until the pH is 4-13, stirring throughout the process to obtain a dispersed system (reaction solution);

[0015] For the synthesis of silicon-doped aluminum oxyhydroxide: at a temperature of 20-60° C., an alkaline solution containing a silicon source is added dropwise to an aluminum source solution, and then the alkaline solution is added dropwise until the pH reaches 4-13, with stirring throughout the process to obtain a dispersed system (reaction solution);

[0016] ② Add the dispersed system of step ① into the reactor and prepare the reaction product by hydrothermal synthesis at a temperature of 50-300° C., preferably 90-250° C., and a hydrothermal time of 2-48 h, preferably 4-36 h;

[0017] ③ Centrifuge the reaction product of step ②, remove the supernatant, wash it with ultrapure water, and then store it or dry it.

[0018] Furthermore, in step ①, the concentration of the silicon source in the alkaline solution containing the silicon source is 0.01-10 mol / L, preferably 0.02-5 mol / L; the preparation method of the alkaline solution containing the silicon source is: adding the silicon source to the alkaline solution to obtain the alkaline solution containing the silicon source.

[0019] Furthermore, in step ①, the stirring rate is 300-1500 rpm, preferably 300-900 rpm, and the stirring time is 3 min-3 h.

[0020] Furthermore, in step ③, the centrifuge speed during washing is 8000-15000 rpm, preferably 11000 rpm; and the washing time is 10-30 min, preferably 25 min.

[0021] The present invention also provides the use of the above-mentioned calcium- or silicon-doped AlOOH nanoadjuvant in various preventive and therapeutic adjuvant vaccines.

[0022] Furthermore, the vaccine adjuvant is a calcium- or silicon-doped AlOOH nanoadjuvant, and the corresponding vaccine antigens include but are not limited to hepatitis B surface antigen, hepatitis B core antigen, human papillomavirus-like particles, the novel coronavirus spike protein receptor binding domain, human papillomavirus antigen, varicella-zoster virus recombinant glycoprotein, etc. Based on the above antigens, corresponding hepatitis B virus vaccines, human papillomavirus vaccines, novel coronavirus vaccines, herpes zoster virus vaccines, etc. are constructed.

[0023] The beneficial effects of the present invention are:

[0024] The calcium- or silicon-doped AlOOH nanoadjuvants of the present invention have been verified by in vivo experiments in mice to be able to induce efficient humoral immunity and cellular immunity at the same time.

[0025] The preparation method of AlOOH, which can simultaneously induce efficient humoral immunity and cellular immunity, is simple, easy to operate, has good reproducibility, and mild reaction conditions. The resulting vaccine nanoparticles are evenly dispersed and have a uniform particle size, and have good application prospects in the prevention and treatment of infectious viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The present invention is accompanied by 9 drawings,

[0027] Figure 1 shows transmission electron micrographs of AlOOH adjuvants doped with different concentrations of calcium and silicon (Si / Ca-AlOOH-1 / 2 / 3 / 4). Scale bar: 200 nm.

[0028] Figure 2 shows the X-ray diffraction (XRD) patterns of AlOOH adjuvants doped with different concentrations of calcium and silicon (Si / Ca-AlOOH-1 / 2 / 3 / 4).

[0029] Figure 3 shows the X-ray photoelectron spectra (XPS) of AlOOH adjuvant (Si / Ca-AlOOH-1 / 4) doped with different concentrations of calcium and silicon elements.

[0030] Figure 4 compares the elemental content in different parts of AlOOH adjuvants (Si / Ca-AlOOH-1 / 4) doped with varying concentrations of calcium and silicon. Inductively coupled plasma atomic emission spectrometry (ICP) was used to measure the bulk doping element content (orange), while XPS was used to measure the surface element content (green). The results demonstrate uniform doping of silicon and calcium within the AlOOH.

[0031] Figure 5 shows the in vitro immune evaluation of AlOOH adjuvants (Si / Ca-AlOOH-1 / 2 / 3 / 4) doped with different concentrations of calcium (A) and silicon (B).

[0032] Figure 6 shows the results of BMDC cell surface CD86 (A) and MHC II (B) expression and IL-1β (C), IL-6 (D), IL-12 (E) and TNF-α (F) cytokine release induced by different concentrations of calcium and silicon doped AlOOH adjuvant (Si / Ca-AlOOH-1 / 4).

[0033] Figure 7 shows the hepatitis B antibody levels induced by the Si / Ca-AlOOH-1 / 4 vaccine using 6-8 week old C57BL / 6 mice as a model; the specific hepatitis B antigen immunization strategy is to intramuscularly inject Si / Ca-AlOOH-1 / 4 containing 2 μg of hepatitis B surface antigen on day 0, inject an equal amount of Si / Ca-AlOOH-1 / 4 again on day 21, and collect serum and spleen on day 42 to detect humoral immunity and cellular immunity levels. Figures 7A and 7B show the specific antibody levels in the serum, which are total IgG and IgG1 levels, respectively. Among them, HBsAg is a simple hepatitis B surface antigen, Alum is a mixture of HBsAg and commercial aluminum hydroxide adjuvant, and the injection amount of all adjuvants is: the total mass of aluminum, calcium, and silicon elements is 50 μg. Figures 7C-7I are CD4 + Expression of CD69 on T cell surface (C), CD8 + Expression of CD69 on T cell surface (D), CD8 + Expression of FasL on T cell surface (E), CD4 + CD44 on T cells high CD62 low Expression of (F), CD8 + CD44 on T cells high CD62 low The expression of (G), the expression of CD69 on the surface of B cells (H) and the expression of CD27 on the surface of B cells (I).

[0034] Figure 8 shows the immunopathological analysis of the main organs of mice.

[0035] Figures 9A-9C show the expression of total IgG (A), IgG1 (B), and IgG specific in serum of female C57BL / 6 mice (8 weeks, n=6) after intramuscular inoculation of a mixture of doped AlOOH and varicella-zoster virus glycoprotein E antigen (VZV gE) on days 0 and 21. 2c Figure 9D is a radar chart showing the activation of functional T cells and B cells. Figures 9E-9F are flow cytometric analysis of CD69 in splenocytes of immunized mice restimulated with gE (2 μg / mL) for 120 h. + / CD4 + 、CD69 + / CD8 + 、FasL + / CD8 + 、CD44 high CD62 low / CD4 + 、CD44 high CD62L low / CD8 + 、CD69+ / CD19 + and CD27 + / CD19 + Figure 9E shows the cell activation spectrum of CD4 + Radar charts of Th1 (TNF-α, IFN-γ, IL-2) and Th2 (IL-4) cytokine production in T cells are shown in Figure 9F. + Radar charts show the production of Th1 (TNF-α, IFN-γ, IL-2) and Th2 (IL-4) cytokines in T cells. Figures 9G and 9H show the number of gE-specific IFN-γ (G) and IL-2-producing spot-forming cells (H) detected by enzyme-linked immunospot (ELISPOT) assay after 24 hours of restimulation of splenocytes from immunized mice with gE (2 μg / mL). Figures 9I and 9J show the levels of IL-4 (I) and IFN-γ (J) secretion by splenocytes from immunized mice 5 days after restimulation with gE (2 μg / mL). DETAILED DESCRIPTION

[0036] The following non-limiting examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0037] Example 1

[0038] A method for preparing a calcium-doped AlOOH nanoadjuvant, comprising the following steps:

[0039] ① Prepare 1.6 mol / L AlCl3·6H2O solution, 0.8 mol / L anhydrous CaCl2 solution, and 1 mol / L NaOH solution.

[0040] ② The above solutions were mixed and diluted to obtain calcium-doped aluminum solutions of different proportions, each 25 mL: 1.6 mol / L AlCl3·6H2O solution and 0.8 mol / L anhydrous CaCl2 solution were mixed and diluted in different volumes to prepare mixed solutions with calcium to aluminum element molar ratios (Ca / Al) of 0, 0.05, 0.1, 0.15 and 0.2, wherein the aluminum ion concentration was 0.5 mol / L. The prepared calcium-doped AlOOH nanoadjuvants (Ca-AlOOH-1 / 2 / 3 / 4) products were respectively recorded as AlOOH, Ca-AlOOH-1 (calcium to aluminum element molar ratio of 0.05), Ca-AlOOH-2 (calcium to aluminum element molar ratio of 0.1), Ca-AlOOH-3 (calcium to aluminum element molar ratio of 0.15), and Ca-AlOOH-4 (calcium to aluminum element molar ratio of 0.2).

[0041] ③ Add NaOH solution dropwise to the mixed solution in step ② until the pH is 7, stir at 600 rpm throughout the process, and the reaction temperature is 25°C to obtain a dispersed system;

[0042] ④ Add the dispersed system of step ③ into the reactor and hydrothermally prepare it at a temperature of 120°C for 4 hours to obtain a reaction product;

[0043] ⑤ Centrifuge the reaction product of step ④, remove the supernatant, wash it with ultrapure water, and then store it or dry it. The centrifuge speed during washing is 11000 rpm and the time is 25 minutes.

[0044] Example 2

[0045] A method for preparing a silicon-doped AlOOH nanoadjuvant, comprising the following steps:

[0046] ① Prepare 1.6 mol / L AlCl3·6H2O solution and 1 mol / L NaOH solution; take tetraethyl orthosilicate (TEOS) and add it to the 1 mol / L NaOH solution to obtain a NaOH solution containing 0.5 mol / L TEOS.

[0047] ② Dilute aluminum solution: dilute AlCl3·6H2O solution to 25mL, with a concentration of 0.5mol / L.

[0048] ③ To the aluminum solution diluted in step ②, different volumes of NaOH solution containing TEOS (sodium orthosilicate solution) were added dropwise, and then NaOH solution was added dropwise until the pH was 7, with stirring at 600 rpm throughout the process, and the reaction temperature was 25°C; according to the amount of NaOH solution containing TEOS added, solutions with a silicon to aluminum molar ratio (Si / Al) of 0, 0.05, 0.1, 0.15 and 0.2 were prepared, and the prepared silicon-doped AlOOH nanoadjuvants (Si-AlOOH-1 / 2 / 3 / 4) products were respectively recorded as AlOOH, Si-AlOOH-1 (silicon to aluminum molar ratio of 0.05), Si-AlOOH-2 (silicon to aluminum molar ratio of 0.1), Si-AlOOH-3 (silicon to aluminum molar ratio of 0.15), and Si-AlOOH-4 (silicon to aluminum molar ratio of 0.2).

[0049] ④ The subsequent processes such as hydrothermal treatment and washing are the same as those of calcium doping in Example 1.

[0050] Example 3

[0051] Detection of the physicochemical properties of the calcium and silicon-doped AlOOH nanoadjuvant prepared in Examples 1 and 2:

[0052] The hydrated particle size (Hydrodynamic size) and Zeta potential (Zeta potential) of AlOOH, Si-AlOOH-1 / 2 / 3 / 4 and Ca-AlOOH-1 / 2 / 3 / 4 in water and pH = 7.4, 10mM KNO3 are shown in Table 1.

[0053] Table 1

[0054] Transmission electron microscopy (TEM) examination of the morphology of the products in Examples 1 and 2 (as shown in FIG1 ) shows that as the silicon-aluminum ratio increases, the morphology of the material changes from rod-like to leaf-like. As the calcium-aluminum ratio increases, the morphology changes from rod-like to granular.

[0055] XRD analysis (as shown in Figure 2) shows that with the increase of Si and Ca elements, the XRD spectrum of doped AlOOH does not show any impurity peaks other than AlOOH, indicating that Si and Ca are evenly distributed in the AlOOH structure.

[0056] XPS analysis (Figure 3) further confirmed the successful doping of silicon and calcium. Quantification of silicon and calcium using XPS and ICP (Figure 4) showed similar doping levels on the surface and in the bulk, indicating uniform doping of silicon and aluminum in the AlOOH particles.

[0057] Example 4

[0058] The MTS assay was used to determine the cell viability of bone marrow-derived dendritic cells (BMDCs) treated with nanomaterials at different concentrations (0, 100, 200, 300, 400, and 500 μg / mL). The experimental results (as shown in FIG5 ) showed that the adjuvant had good biocompatibility. BMDC cell activation and cytokine production were analyzed (as shown in FIG6 ). The results showed that with increasing silicon content, the doped adjuvant induced higher production of IL-12 and IL-6, indicating that doped silicon induced the release of Th1 and Th2 type cytokines. With increasing calcium content, the doped adjuvant induced an increase in IL-12, while the IL-6 cytokine remained unchanged, suggesting that calcium doping induced enhanced release of Th1 type cytokines, but not Th2 type cytokines.

[0059] Example 5

[0060] 6-8 week old C57BL / 6 mice were used as animal models to detect the humoral immunity and cellular immunity induced by Si-AlOOH-1 / 2 / 3 / 4 and Ca-AlOOH-1 / 2 / 3 / 4 prepared in Examples 1 and 2. The method comprises the following steps: on day 0, mice were intramuscularly injected with 50 μL of vaccine, which contained 2 μg of hepatitis B surface antigen (HBV), 50 μg of aluminum, silicon and calcium in total, and the vaccine was dispersed in a physiological saline system. On day 21, the same amount of the above vaccine was injected again. On day 42, serum and spleen were collected to detect the levels of total IgG and IgG1 in the serum and the maturation and differentiation of spleen cells. Among them, control groups were added: a physiological saline group (each mouse was injected with 50 μL of physiological saline), an HBV group (each mouse was injected with only 2 μg of hepatitis B surface antigen), and an HBV+Alum group (each mouse was injected with a commercial aluminum oxyhydroxide adjuvant containing 2 μg HBV and 50 μg of aluminum element ( adjuvant 2%, InvivoGen) mixture, abbreviated as Alum group), the number of experimental mice in each group was 6.

[0061] Total IgG and IgG1 in serum are shown in Figures 7A-7B.

[0062] The maturation and differentiation of splenocytes are shown in Figures 7C-7I.

[0063] As shown in Figure 7, the characterization results of Example 5 showed that the antibody titer experimental results showed that the doped aluminum adjuvant could produce higher IgG and IgG1 antibody titers, proving that the doped AlOOH could produce higher humoral immunity levels. The results of spleen cell maturation and differentiation showed that the doped aluminum adjuvant could promote CD4 + 、CD8 + Cell activation and CTL cell killing mediators are highly expressed.

[0064] In addition, the immunopathological analysis of major organs (as shown in Figure 8) indicated that the doped aluminum adjuvant had good biosafety and strong application potential.

[0065] Example 6

[0066] Female C57BL / 6 mice (8 weeks, n=6) were inoculated intramuscularly on days 0 and 21 with a mixture of AlOOH-doped varicella-zoster virus glycoprotein E antigen (VZV gE). Each dose contained 5 μg gE and either 50 μg equivalent Al (Si-AlOOH-4 prepared in Example 1 or Ca-AlOOH-4 prepared in Example 2) or 100 μg equivalent Al (Si-AlOOH-4 prepared in Example 1, designated Si-AlOOH-4-H, or Ca-AlOOH-4 prepared in Example 2, designated Ca-AlOOH-4-H). gE (5 μg) served as a control. Serum specific for gE was measured on day 42.

[0067] Serum gE-specific total IgG, IgG1 and IgG 2c The titers are shown in Figures 9A to 9C. The results showed that both silicon- and calcium-doped AlOOH nanoadjuvants could induce an adjuvant-dose-dependent increase in gE-specific antibody responses.

[0068] The maturation and differentiation of splenocytes are shown in Figure 9D. Compared with the gE group, Si doping induced a higher CD69 + / CD4 + 、CD69 + / CD8 + 、FasL + / CD8 + 、CD62L low CD44 high / CD4 + 、CD69 + / CD19 + and CD27 + / CD19 + The percentage of cells, and with the increase of adjuvant content, FasL + / CD8 + and CD27 + / CD19 + The results showed that Si-AlOOH-4 induced a dose-dependent enhancement of CTL cell and memory B cell activation, as well as a dose-independent activation of other functional T and B cells. Compared with the gE group, Ca doping induced CD69 + / CD4 + 、CD69 + / CD8 + 、FasL + / CD8 + 、CD62L low CD44 high / CD4 + 、CD62L low CD44 high / CD8 + 、CD69 + / CD19 + 、CD27 + / CD19 + The proportion of cells was higher, and with the increase of adjuvant content, CD69 + / CD4 + 、CD69 + / CD8 + 、FasL + / CD8 + 、CD69 + / CD19 + This suggests that Ca-AlOOH-4 induces CD4 + T cells, CD8 + The activation of T cells, CTL cells and B cells was enhanced in a dose-dependent manner, while the activation of memory T and B cells was not dose-dependent.

[0069] The spleen cells of immunized mice were restimulated with gE (2 μg / mL) for 120 h, and CD69 was analyzed by flow cytometry. + / CD4 + 、CD69 + / CD8 + 、FasL + / CD8 + 、CD44 high CD62 low / CD4 + 、CD44 high CD62L low / CD8 + 、CD69 + / CD19 + and CD27 + / CD19 + Cell activation spectrum.

[0070] CD4 + The production of Th1 (TNF-α, IFN-γ, IL-2) and Th2 (IL-4) cytokines in T cells is shown in Figure 9E. Intracellular cytokine analysis showed that Si-AlOOH-4 and Si-AlOOH-4-H induced CD4 + The ratios of IL-4, IFN-γ, IL-2 and TNF-α in T cells were higher, indicating that Si-doped AlOOH nanoadjuvant induced CD4 + T cells produced Th2 and Th1 type cytokines, which was independent of the adjuvant dose. Compared with the gE group, Ca-AlOOH-4 induced CD4 +The ratio of IFN-γ and TNF-α in T cells was higher; compared with the Ca-AlOOH-4 group, Ca-AlOOH-4-H induced CD4 + The ratio of IL-4 and IL-2 in T cells was higher. The results showed that Ca-AlOOH-4 had the effect of inducing CD4 + The potential of T cells to produce Th2 and Th1 cytokines is determined by the adjuvant dose.

[0071] CD8 + The production of Th1 (TNF-α, IFN-γ, IL-2) and Th2 (IL-4) cytokines in T cells is shown in Figure 9F. + Compared with Si-AlOOH-4, Si-AlOOH-4-H induced the production of intracellular IFN-γ, IL-2, and TNF-α in CD8 T cells. + Ca-AlOOH-4 nanoadjuvants induce the expression of IFN-γ and TNF-α in CD8 T cells. + The proportion of T cells increased, and Ca-AlOOH-4-H induced CD8 + These results indicate that the proportion of CD8 T cells increased. + The production of Th2 and Th1 type cytokines in T cells was determined by the dose of Ca-AlOOH-4 nanoadjuvant.

[0072] After 24 hours of in vitro restimulation of splenocytes from immunized mice with VZV gE antigen (2 μg / mL), gE-specific T cell responses were detected by IFN-γ and IL-2 enzyme-linked immunosorbent assay (ELISPOT) assays, as shown in Figures 9G-9H. The results showed that compared with the gE group, the high-dose Ca-AlOOH-4 group had more IFN-γ and IL-2 spot-forming cells, indicating that Ca doping can promote a dose-dependent increase in IFN-γ and IL-2-producing cells. However, regardless of the adjuvant dose, Si-AlOOH-4 did not cause a significant increase in cytokine release, indicating that the T cell response induced by Si-AlOOH-4 nanoadjuvant was not significant.

[0073] Figures 9I-9J show the levels of extracellular IL-4 and IFN-γ secreted from splenocytes of immunized mice 5 days after restimulation with gE (2 μg / mL). The results show that Si-AlOOH-4 induced a 6.0-fold and 1.7-fold increase in IL-4 and IFN-γ secretion compared with the gE group, whereas increasing Si-AlOOH-4 doses decreased IL-4 and IFN-γ secretion to 2.1-fold and 1.0-fold, respectively. Compared with the gE group, Ca-AlOOH-4 and Ca-AlOOH-4-H induced an 11.6-fold and 7.1-fold increase in IL-4 secretion, respectively, with the magnitude of this increase decreasing with increasing Ca-AlOOH-4 dose. Furthermore, Ca-AlOOH-4 and Ca-AlOOH-4-H induced a 3.3-fold and 7.6-fold increase in IFN-γ secretion, respectively, indicating that the Ca-AlOOH-4-induced increase in IFN-γ release is adjuvant-dose dependent.

[0074] In the hepatitis B surface antigen and varicella-zoster virus glycoprotein E antigen models, the calcium or silicon-doped AlOOH nanoadjuvant of the present invention was verified by in vivo experiments in mice to be able to simultaneously induce efficient humoral immunity and cellular immunity.

Claims

1. A calcium or silicon-doped aluminum oxyhydroxide nanoadjuvant, characterized in that: Hydroxy aluminum oxide is synthesized using an Al source, and Ca or Si elements are doped in the reaction solution using a calcium source or a silicon source. Al(OH)3 and dopants are precipitated by adding an alkaline solution, and a Ca or Si-doped hydroxy aluminum oxide adjuvant is prepared using a hydrothermal synthesis method.

2. The calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to claim 1, characterized in that: The calcium source is an inorganic or organic soluble calcium salt, which includes but is not limited to calcium chloride, calcium nitrate or tricalcium citrate. The molar ratio of calcium to aluminum is 0.01-100:1, and the concentration of calcium in the reaction solution is 0.01-100 mol / L.

3. The calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to claim 1, characterized in that: The silicon source is organic silane, silicic acid or soluble silicate, the organic silane includes tetraethoxysilane, the soluble silicate includes sodium silicate or potassium silicate, and the molar ratio of silicon element to aluminum element is 0.01-100:

1.

4. The calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to claim 1, characterized in that: The alkaline solution includes an aqueous solution or buffer solution of NaOH, KOH or ammonia with a concentration of 0.01-10 mol / L.

5. The calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to claim 1, characterized in that: The Al source is an inorganic or organic aluminum salt, which includes aluminum nitrate, aluminum chloride or aluminum ethoxide. The concentration of aluminum in the reaction solution is 0.01-10 mol / L.

6. The calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to claim 1, characterized in that: The AlOOH nano-adjuvant doped with calcium or silicon is a nano-particle with a size of 20nm-1000nm.

7. A method for preparing a calcium or silicon doped aluminum oxyhydroxide nanoadjuvant according to any one of claims 1 to 6, characterized in that: The steps include: ① At a temperature of 20-60°C, add an alkaline solution to an aluminum source solution containing a calcium source until the pH is 4-13, or add an alkaline solution containing a silicon source to an aluminum source solution, then add an alkaline solution to the pH of 4-13, stirring throughout the process to obtain a dispersed system; ② hydrothermally synthesizing the dispersed system of step ① to prepare aluminum oxyhydroxide adjuvant; the hydrothermal temperature is 50-300° C., and the hydrothermal time is 2-48 hours to obtain a reaction product; ③ Centrifuge the reaction product of step ②, remove the supernatant, wash it with ultrapure water by centrifugation, and then store it or store it after drying.

8. The preparation method according to claim 7, characterized in that: In step ①, the concentration of the silicon source in the alkaline solution containing the silicon source is 0.01-10 mol / L; the stirring rate is 300-1500 rpm, and the stirring time is 3 min-3 h; in step ③, the centrifuge speed during the washing is 8000-15000 rpm, and the time is 10-30 min.

9. Use of a calcium or silicon-doped aluminum oxyhydroxide nanoadjuvant as claimed in any one of claims 1 to 8 in preventive and therapeutic adjuvant vaccines.

10. The use according to claim 9, characterized in that: The vaccines include hepatitis B virus vaccine, human papillomavirus vaccine, new coronavirus vaccine, and shingles virus vaccine.

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