A vaccine carrier based on aluminum hydroxide nanoparticles

Abstract

A vaccine carrier based on aluminum hydroxide nanoparticles, which is formed by compounding PEG derivative materials with aluminum salts to form nanoparticles. It not only retains the powerful Th2 humoral immune adjuvant properties of aluminum salts, but can also be efficiently delivered to the draining lymph nodes in the body, and is easily taken up by dendritic cells (DCs), effectively cross-presents, induces cellular immune responses, and has a strong Th1 immune response.

Description

[0001] This application is a divisional application of an application filed on January 23, 2015, with the invention name “A vaccine carrier based on aluminum hydroxide nanoparticles”. The application was accepted by the State Intellectual Property Office on January 26, 2015, and the application number is 201510034381.X. Technical Field

[0002] The invention relates to a vaccine carrier based on an aluminum salt adjuvant, in particular to a vaccine carrier based on humoral immune response and cellular immune response of aluminum hydroxide nanoparticles and a preparation method thereof. Background Art

[0003] The emergence of vaccines has changed the passive situation of human beings in fighting against diseases and is one of the greatest discoveries in the history of human public health (Wayne C. Koff, Dennis R. Burton, et al. Accelerating next-generation vaccine development for global disease prevention. Science. 2013(340):1232910). Most of the vaccines that have been marketed protect the body by producing neutralizing antibodies through humoral immunity, but many global prevalent diseases such as malaria, tuberculosis, AIDS, etc. cannot provide good protection only by neutralizing antibodies. The pathogens that invade the body in these diseases need to rely on cellular immune responses to be effectively eliminated (Wayne C.Koff, Dennis R. Burton, et al. Accelerating next-generation vaccine development for global disease prevention. Science. 2013(340):1232910.). Therefore, vaccines that can induce strong humoral immunity and cellular immunity at the same time need to be studied urgently.

[0004] Traditional vaccines are prepared by attenuating or inactivating pathogens, and have been successful in treating a large number of diseases, such as smallpox, polio, tetanus, diphtheria, etc. (Darrell J. Irvine, Melody A. Swartz, Gregory L. Szeto. Engineering synthetic vaccines using cues from natural immunity. Nat materials. 2013(12):978-990.). However, the potential toxicity of traditional vaccines poses a safety hazard in their use. For this reason, people have begun to turn their attention to subunit vaccines. Subunit vaccines are made from a certain antigen (protein or a polypeptide) on the surface of pathogenic microorganisms, and are safer than traditional attenuated and inactivated vaccines. However, while subunit vaccines solve the safety problem, they also bring the trouble of reduced immunogenicity, that is, the body cannot produce an effective immune response after vaccination, so they need to be used in combination with adjuvants.

[0005] Among all adjuvants, aluminum salt adjuvant is the most widely used in human body. Since the immune effect of aluminum salt adjuvant was first described by Glenny in 1926 (Glenny AT, Pope CG, Waddington H, Wallace U. Immunological notes XVII to XXIV. J Pathol . 1926(29):31–40.), it has a history of more than 80 years of application and has been used clinically for more than 30 years. Its effectiveness and safety have been fully verified.

[0006] However, aluminum salt adjuvants also have obvious shortcomings, that is, although they can induce a strong Th2 humoral immune response, they lack a Th1 cellular immune response. This limits the application of aluminum adjuvants to vaccines targeting bacteria or relying mainly on the production of neutralizing antibodies, and fails to achieve satisfactory results in the application of vaccines for diseases that rely mainly on Th1 responses, such as HIV, tuberculosis, malaria, and tumors. In addition, traditional aluminum salt adjuvants have a high aluminum content when used, which is not easily metabolized at the injection site, and are often accompanied by severe irritation of local tissues, and side effects such as long-term inflammation, redness, swelling, and granulomas at the injection site. (Erik B. Lindblad. Aluminium adjuvants in retrospect and prospect. Vaccine . 2004(22):3658–68.)

[0007] CD8α is required for the production of Th1 cell immune response when exogenous antigens enter the body. +DC cells can cross-present and form antigen-MHC-I complexes to effectively activate CD8 + T cells, thereby generating a cellular immune response (Megan KL MacLeod, Amy S. McKee, et al. Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells. PANS . 2011(108):7914-7919.). CD8α + DCs are present in immune organs such as lymph nodes, but are present in very low amounts in the skin. Therefore, it is difficult to achieve this cross-presentation through traditional subcutaneous, intradermal, and intramuscular injections. + DC uptake is a very potential way to achieve cross-presentation and induce effective cellular immune responses.

[0008] Therefore, it is an urgent clinical need to improve the deficiency of aluminum adjuvant in Th1 immune response while making full use of its safety and potent Th2 immune response. Summary of the invention

[0009] One of the purposes of the present invention is to provide an aluminum salt adjuvant used as a vaccine carrier, which not only retains the powerful Th2 humoral immune adjuvant properties of aluminum salts, but can also be efficiently delivered to draining lymph nodes in the body, and is easily taken up by dendritic cells (DCs), effectively cross-presents, induces cellular immune responses, and has a strong Th1 immune response.

[0010] In vaccine research, lymph nodes are important sites for immune responses and play a very important role in the entire immune response process. Antigens that do not reach the lymph nodes cannot induce immune responses in the body (Rolf M. Zinkernagel, et al. Antigen localisation regμlates immune responses in adose- and time-dependent fashion: a geographical view of immune reactivity. Immunol.Rev.1997(156):199–209.). Small-sized nanoparticles can be effectively delivered to lymph nodes in vivo and are potential vaccine carriers (Theodora Fifis, Anita Gamvrellis, et al. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol . 2004(173):3148-3154.) (Sai ​​T Reddy, et al. Exploiting lymphatic transport and complement activation in nanoparticlevaccines. Nature biotechnology .2007(25):1159-1164.). However, cellular uptake increases with increasing particle size (Hyungjin Kim, Tomofumi Uto, Mitsuru Akashi, et al. Amphiphilic Poly (Amino Acid) Nanoparticles Induce Size-Dependent Dendritic Cell Maturation. Adv Funct Mater .2010(10):3925-3931.), so a good vaccine vector needs to find a balance between lymph node delivery and cellular uptake.

[0011] One of the purposes of the present invention is to provide an aluminum salt adjuvant used as a vaccine carrier, which is preferably aluminum salt nanoparticles, and more preferably aluminum hydroxide nanoparticles.

[0012] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles used as vaccine carriers, which can induce both humoral immune response (Th2 immune response) and cellular immune response (Th1 immune response).

[0013] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles used as vaccine carriers, characterized in that the nanoparticles are mainly formed by a composite of a PEG derivative material and aluminum hydroxide; wherein the aluminum hydroxide is preferably prepared from aluminum salts such as Al2(SO4)3 during the preparation process, so that the interaction between the PEG derivative material and the aluminum hydroxide can be optimized to obtain nanoparticles with a suitable particle size.

[0014] As one of the preferred embodiments of the present invention, an aluminum hydroxide nanoparticle used as a vaccine carrier is characterized in that the nanoparticle is mainly composed of a PEG derivative material and aluminum hydroxide, wherein the ratio of the PEG derivative material to the aluminum hydroxide is 0.05~1.85:0.042~0.866, preferably 0.75~1.11:0.125~0.173, based on weight.

[0015] The aluminum hydroxide nanoparticles used as vaccine carriers of the present invention have extremely low aluminum content and no side effects such as inflammation at the injection site; preferably, they can be specifically taken up by antigen presenting cells (such as DC), which is beneficial to the generation of immune response while reducing side effects caused by being taken up by other cells.

[0016] The PEG derivative material of the present invention is, but is not limited to, PEG-poly(AGE-Suc) (previously synthesized by our laboratory, Hanmei Li, Xun Sun, et al. A Cell-Specific Poly(ethylene glycol)Derivative with a Wheat-like Structure for Efficient Gene Delivery. Mol. Pharmaceutics , 2012, 9 (11): 2974–2985.).

[0017] In the study of Th1 immune response adjuvants, a series of adjuvants related to the TLR-like receptor family have shown good potential, such as double-stranded RNA (Poly I:C) (TLR3), LPS, MPLA (TLR4), Imiquiod (TLR7), CpG-OND (TLR9), etc. These adjuvants have reported positive results in laboratory, preclinical and clinical trials (Derek T. O'Hagan, Nicholas M. Valiante. Recent advances in the discovery and delivery of vaccine adjuvants .Nat Rev Immunol. 2003(2):727-735.). The present inventors therefore used a Th1 type immune adjuvant to combine with aluminum hydroxide nanoparticles. This complex can simultaneously induce a strong Th1 response and a Th2 response in vivo.

[0018] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles used as vaccine carriers, characterized in that the nanoparticles are mainly composed of a Th1 type immune adjuvant, a PEG derivative material and aluminum hydroxide to form a composite nanoparticle; wherein the aluminum hydroxide is preferably prepared from aluminum salts such as Al2(SO4)3 during the preparation process.

[0019] As one of the preferred embodiments of the present invention, the aluminum hydroxide nanoparticles used as a vaccine carrier described in the present invention are characterized in that the nanoparticles are formed by compounding a Th1 type immune adjuvant, a PEG derivative material and aluminum hydroxide, wherein based on the weight ratio of the PEG derivative material: aluminum hydroxide: Th1 type immune adjuvant is 0.05~1.85:0.042~0.866:0.001~0.5, preferably 0.75~1.11:0.125~0.173:0.008~0.03.

[0020] The vaccine carrier based on aluminum hydroxide nanoparticles described in the present invention is preferably an antigen carrier such as a polypeptide, protein or polysaccharide.

[0021] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles linked to (or encapsulating) OVA (model antigen), wherein the aluminum hydroxide nanoparticles are linked to OVA (model antigen) via a disulfide bond.

[0022] One of the purposes of the present invention is to provide an aluminum hydroxide nanoparticle having a Th1 type immune adjuvant and linked (or encapsulated) with OVA (model antigen), wherein the aluminum hydroxide nanoparticle is linked to OVA (model antigen) through a disulfide bond, an amide bond, an ester bond, etc., and its structure is as follows Figure 1 shown.

[0023] In the PEG-poly(AGE-Suc) of the present invention, the PEG unit length is between 20 and 120, preferably 50 to 90 units, the poly(AGE-Suc) unit length is between 1 and 50, preferably 5 to 15 units, and the molecular weight of the polymer is preferably about 3000 to 7000 Daltons. Its structural formula is as follows:

[0024]

[0025] The Th1 adjuvants of the present invention include: RNA (Poly I:C), MPLA, CpG-OND, Imiquiod, etc., preferably CpG-OND.

[0026] The linkage between OVA and PEG-poly(AGE-Suc) of the present invention includes disulfide bond, amide bond, ester bond, etc., preferably disulfide bond.

[0027] One of the purposes of the present invention is to provide a method for preparing aluminum hydroxide nanoparticles, which is characterized by comprising the following steps:

[0028] (1) Add PEG-poly(AGE-Suc) solution to Hepes buffer and stir evenly;

[0029] (2) Absorb Al2(SO4)3 solution and add it into the above mixed solution;

[0030] (3) Stir for 20-50 seconds, preferably 30 seconds.

[0031] As a preferred embodiment, when the final nanoparticle volume is 1 ml, the following is calculated:

[0032] Wherein, the amount of PEG-poly(AGE-Suc) in step (1) is 0.05~1.85 mg.

[0033] Wherein, the HEPES buffer in step (1) is 18.5~185 μmol.

[0034] The Al2(SO4)3 solution in step (2) is 0.27-5.55 μmol; the aluminum content in the Al2(SO4)3 solution converted to the final Al(OH)3 amount is 0.042-0.866 mg.

[0035] As one of the preferred embodiments of the present invention, the amount of PEG-poly(AGE-Suc) described in the present invention is preferably 0.75~1.11 mg.

[0036] As one of the preferred embodiments of the present invention, the HEPES buffer of the present invention is preferably 30-45 μmol.

[0037] As one of the preferred embodiments of the present invention, the Al2(SO4)3 solution of the present invention is preferably 0.8~1.11 μmol; the amount converted to Al(OH)3 is 0.125~0.173 mg.

[0038] One of the purposes of the present invention is to provide a method for preparing aluminum hydroxide nanoparticles having a Th1 type immune adjuvant and OVA, preferably comprising the following steps:

[0039] (1) Add PEG-poly(AGE-Suc)-OVA solution (or a mixture of PEG-poly(AGE-Suc) solution and OVA solution) and Th1 adjuvant solution to Hepes buffer and stir evenly;

[0040] (2) Absorb Al2(SO4)3 solution and add it into the above mixed solution;

[0041] (3) Stir for 20-50 seconds, preferably 30 seconds.

[0042] As a preferred embodiment, when the final nanoparticle volume is 1 ml, the following is calculated:

[0043] Wherein, the amount of PEG-poly(AGE-Suc)-OVA in step (1) is 0.05-1.85 mg; or, in the mixture of the PEG-poly(AGE-Suc) solution and the OVA solution in step (1), the amount of PEG-poly(AGE-Suc) is 0.05-1.85 mg, and the amount of OVA is 20-400 μg.

[0044] Wherein, the amount of Th1 adjuvant in step (1) is 1-500 μg.

[0045] Wherein, the HEPES buffer in step (1) is 18.5~185 μmol.

[0046] The Al2(SO4)3 solution in step (2) is 0.27-5.55 μmol; the aluminum content in the Al2(SO4)3 solution converted to the final Al(OH)3 amount is 0.042-0.866 mg.

[0047] As one of the preferred embodiments of the present invention, the dosage of PEG-poly(AGE-Suc)-OVA in the present invention is preferably 0.75~1.11 mg.

[0048] As one of the preferred embodiments of the present invention, the amount of PEG-poly(AGE-Suc) in the mixture of the PEG-poly(AGE-Suc) solution and the OVA solution of the present invention is preferably 0.75-1.11 mg, and the amount of OVA is 40-80 μg.

[0049] As one of the preferred embodiments of the present invention, the dosage of CpG-ODN in the present invention is preferably 8-30 μg.

[0050] As one of the preferred embodiments of the present invention, the HEPES buffer of the present invention is preferably 30-45 μmol.

[0051] As one of the preferred embodiments of the present invention, the Al2(SO4)3 solution of the present invention is preferably 0.8~1.11 μmol, which is converted into an amount of Al(OH)3 of 0.125~0.173 mg.

[0052] According to the present invention, the aluminum hydroxide nanoparticles prepared according to the above steps are about 50-140 nm and have a potential of -1 mV to -15 mV as measured by a Malvern Nano-ZS 90 particle size analyzer.

[0053] One of the purposes of the present invention is to provide a vaccine carrier of aluminum hydroxide nanoparticles linked to or encapsulating OVA (model antigen).

[0054] One of the purposes of the present invention is to provide a method for linking or encapsulating OVA (model antigen) using the aluminum hydroxide nanoparticles (with or without Th1 adjuvant).

[0055] One of the purposes of the present invention is also to provide a use of an aluminum salt in preparing an adjuvant that effectively induces both humoral immune response and cellular immune response.

[0056] One of the purposes of the present invention is also to provide a use of an aluminum salt adjuvant in preparing a vaccine vector, wherein the aluminum salt adjuvant effectively induces both humoral immune response and cellular immune response.

[0057] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles linked to (or encapsulated with) OVA (model antigen), wherein the OVA is linked to (or encapsulated with) the outside (inside) of the aluminum hydroxide nanoparticles.

[0058] One of the purposes of the present invention is to provide aluminum hydroxide nanoparticles having a Th1 type immune adjuvant and linked to (or encapsulated with) OVA (model antigen), wherein the OVA is linked to (or encapsulated with) the outside (inside) of the aluminum hydroxide nanoparticles.

[0059] The preparation method of the invention is simple, rapid and has mild conditions.

[0060] The present invention selects OVA as a model antigen, and proves through in vivo and in vitro experiments that aluminum hydroxide nanoparticles can effectively induce humoral immune response and cellular immune response in vivo.

[0061] As one of the preferred embodiments of the present invention, the prepared aluminum hydroxide nanoparticles are easily taken up by DC cells, but not by other cells such as B16 and L929, thereby reducing other side effects while ensuring the immune effect.

[0062] As one of the preferred embodiments of the present invention, when the aluminum content of the prepared sample is 1.25 μg, the IgG antibody response generated is equivalent to or better than that of the commercial aluminum gel with an aluminum content of 125 μg.

[0063] As one of the preferred embodiments of the present invention, when the aluminum content of the prepared sample was 1.25 μg, the CTL reaction generated was extremely significant (P<0.001), which was better than the commercial aluminum gel with an aluminum content of 125 μg.

[0064] As one of the preferred embodiments of the present invention, the prepared sample can be applied to dosage forms such as freeze-dried powder injection, injection, nasal drops, sprays, microneedles, patches, etc.

[0065] Beneficial Effects

[0066] The aluminum hydroxide nanoparticles used as vaccine carriers of the present invention can simultaneously induce humoral immune response (Th2 immune response) and cellular immune response (Th1 immune response). More importantly, the aluminum content is extremely low when used, and there is no side effect such as inflammation at the injection site.

[0067] The aluminum hydroxide nanoparticles of the present invention can be specifically taken up by antigen presenting cells (such as DC), which is beneficial to the generation of immune response and reduces the side effects caused by being taken up by other cells.

[0068] The aluminum hydroxide nanoparticles of the present invention retain the properties of aluminum salt adjuvant as a potent Th2 immune adjuvant even though the aluminum content is much lower than that of traditional aluminum gel, and the antibody titer induced by the nanoparticles is the same as or better than that of traditional aluminum gel.

[0069] One of the purposes of the present invention is to provide a new method for preparing aluminum hydroxide nanoparticles, which has simple preparation method, good repeatability and high stability.

[0070] The present invention places the Th1 type immune adjuvant and the Th2 type aluminum salt immune adjuvant in the same nanoparticle, which can be delivered to the same immune cells together with the antigen in the body, which is conducive to the rapid and effective generation of immune response; compared with other methods that physically mix the antigen and the adjuvant, the ability of this nanoparticle to generate immune response is stronger and more effective, and can reduce the systemic side effects caused by the systemic distribution of adjuvants such as CpG-ODN. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0072] Figure 1 This is a structural diagram of an aluminum hydroxide nanoparticle vaccine carrier containing a Th1-type immune adjuvant linked to OVA (model antigen).

[0073] Figure 2 This is the particle size data of aluminum hydroxide nanoparticles-OVA-CpG nanoparticles.

[0074] Figure 3 This is an electron micrograph of aluminum hydroxide nanoparticles-OVA-CpG nanoparticles.

[0075] Figure 4 This is the uptake of aluminum hydroxide nanoparticles.

[0076] Figure 5 Induce BMDC maturation.

[0077] Figure 6 This is the lymph node distribution of aluminum hydroxide nanoparticles.

[0078] Figure 7 For in vivo CTL experiments.

[0079] Figure 8 It is the IgG titer in the blood. DETAILED DESCRIPTION

[0080] The following examples are further explanations of the present invention, but are by no means limitations of the scope of the present invention. The present invention will be further described in detail below with reference to the examples, but it should be understood by those skilled in the art that the present invention is not limited to these examples and the preparation methods used. Moreover, those skilled in the art may carry out equivalent substitutions, combinations, improvements or modifications to the present invention based on the description of the present invention, but these will all be included within the scope of the present invention.

[0081] Example 1

[0082] Linkage between PEG-poly(AGE-Suc) and OVA

[0083] 1 mg OVA (purchased from Sigma) was dissolved in 2 ml PBS containing 25 mmol / L DTT (dithiothreitol), stirred at room temperature for two hours, and unreacted DTT and other small molecules were removed using a PD-10 desalting column (purchased from GE Healthcare) to obtain OVA with broken disulfide bonds, which was lyophilized for later use. PEG-poly (AGE-Suc) and linker SPDP (purchased from Pierce Biotechnology) were dissolved in 1.5 ml PBS-EDTA buffer (components: 100 mmol / L Na3PO4, 150 mmol / L NaCl, 1 mmol / L EDTA, pH=7.5) at a molar ratio of PEG-poly (AGE-Suc): SPDP=1:1.2, stirred at room temperature for 45 minutes, and unreacted SPDP and the like were removed using a PD-10 desalting column. Dissolve the disulfide-bond-broken OVA in 1 ml PBS-EDTA, then add it to the desalted PEG-poly(AGE-Suc) solution (molar ratio PEG-poly(AGE-Suc):OVA=1.2:1), stir overnight. Use double distilled water as the mobile phase, pass through a PD-10 desalting column to remove small molecules and PBS-EDTA solution, and freeze-dry.

[0084] Example 2

[0085] PEG-poly(AGE-Suc) linked to FITC (fluorescein isothiocyanate)

[0086] PEG-poly(AGE-Suc) and FITC (purchased from Sigma) were dissolved in 10 ml Na2CO3-NaHCO3 buffer (pH = 9.6) at a molar ratio of 1:1.8, stirred for 16 h in the dark, dialyzed for 3 days in a 1000 Da dialysis bag in the dark, and freeze-dried.

[0087] Example 3

[0088] Preparation of aluminum hydroxide-OVA-CpG nanoparticles

[0089] Add 185 μl 5 mg / ml PEG-poly(AGE-Suc)-OVA solution (Example 1, the same below) (0.925 mg) and 10 μl 1782 μg / ml CpG-ODN (purchased from Shanghai Biotech) (17.82 μg) to 370 μl 100 mmol / L pH=7.6 HEPES buffer (37 μmol), stir and mix; draw 555 μl 1.67 mmol / L Al2(SO4)3 solution (0.927 μmol), add to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide-OVA-CpG nanoparticles. The particle size was measured by Malvern Nano-ZS 90 particle size analyzer. The results are shown in Figure 2 Take a small amount of sample and examine it with transmission electron microscope. The results are shown in Figure 3 .

[0090] Example 4

[0091] Preparation of OVA-encapsulated aluminum hydroxide-CpG nanoparticles

[0092] To 370μl 100mmol / L pH=7.6 HEPES buffer (37μmol), add 185μl 5mg / ml PEG-poly(AGE-Suc) solution (0.925mg), 20μl 4mg / ml OVA solution (80μg), and 10μl 1782μg / ml CpG-ODN (17.82μg), and stir to mix; absorb 555μl 1.67mmol / L Al2(SO4)3 solution (0.927μmol), add to the above mixed solution, stir for 30s, and use a gel column to remove the unencapsulated OVA to obtain aluminum hydroxide-CpG nanoparticles encapsulating OVA.

[0093] Example 5

[0094] Preparation of aluminum hydroxide-CpG nanoparticles

[0095] Add 185μl 5mg / ml PEG-poly(AGE-Suc) solution (0.925mg) and 10μl 1782μg / ml CpG-ODN (17.82μg) to 370μl 100mmol / L pH=7.6 HEPES buffer (37μmol), and stir to mix; draw 555μl 1.67mmol / L Al2(SO4)3 solution (0.927μmol), add to the above mixed solution, stir for 30s, and obtain aluminum hydroxide-CpG nanoparticles.

[0096] Example 6

[0097] Preparation of aluminum hydroxide-FITC nanoparticles without Th1 adjuvant

[0098] To 370 μl of 100 mmol / L pH=7.6 HEPES buffer (37 μmol), add 185 μl of 5 mg / ml FITC-PEG-poly(AGE-Suc) solution (Example 2) (0.925 mg), and stir to mix; draw 555 μl of 1.67 mmol / L Al2(SO4)3 solution (0.927 μmol), add to the above mixed solution, and stir for 30 seconds to obtain aluminum hydroxide-FITC nanoparticles without Th1 adjuvant.

[0099] Example 7

[0100] Add 185 μl 6 mg / ml PEG-poly(AGE-Suc)-OVA solution (1.11 mg) and 10 μl 3500 μg / ml CpG-ODN (35 μg) to 370 μl 100 mmol / L HEPES buffer (37 μmol), and stir to mix; draw 555 μl 1.67 mmol / L Al2(SO4)3 solution (0.927 μmol), add to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0101] Example 8

[0102] Add 185 μl 6 mg / ml PEG-poly(AGE-Suc)-OVA solution (1.11 mg) and 10 μl 20 mg / ml CpG-ODN (200 μg) to 400 μl 100 mmol / L HEPES buffer (40 μmol), and stir to mix; absorb 500 μl 2 mmol / LAl2(SO4)3 solution (1 μmol), add it to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0103] Example 9

[0104] Add 185 μl 4 mg / ml PEG-poly(AGE-Suc)-OVA solution (0.74 mg) and 10 μl 500 μg / ml CpG-ODN (5 μg) to 300 μl 100 mmol / L HEPES buffer (30 μmol), and stir to mix; draw 300 μl 2 mmol / LAl2(SO4)3 solution (0.6 μmol), add it to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0105] Example 10

[0106] Add 185 μl 1 mg / ml PEG-poly(AGE-Suc)-OVA solution (0.185 mg) and 10 μl 1000 μg / ml CpG-ODN (10 μg) to 370 μl 50 mmol / L HEPES buffer (18.5 μmol), and stir to mix; draw 500 μl 0.5 mmol / L Al2(SO4)3 solution (0.25 μmol), add it to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0107] Embodiment 11

[0108] To 370 μl 50 mmol / L HEPES buffer (18.5 μmol), add 185 μl 0.5 mg / ml PEG-poly(AGE-Suc)-OVA solution (0.09 mg) and 10 μl 10 mg / ml Poly I:C (purchased from InvivoGen) (100 μg), and stir to mix; draw 500 μl 0.5 mmol / L Al2(SO4)3 solution (0.25 μmol), add it to the above mixed solution, and stir for 30 seconds to obtain aluminum hydroxide nanoparticles.

[0109] Example 12

[0110] Add 185 μl 8 mg / ml PEG-poly(AGE-Suc)-OVA solution (1.48 mg) and 10 μl 1000 μg / ml Poly I:C (10 μg) to 370 μl 150 mmol / L HEPES buffer (55.5 μmol), and stir to mix; absorb 555 μl 0.5 mmol / L Al2(SO4)3 solution (0.28 μmol), add it to the above mixed solution, and stir for 30 seconds to obtain aluminum hydroxide nanoparticles.

[0111] Embodiment 13

[0112] To 370 μl 300 mmol / L HEPES buffer (100 μmol), add 185 μl 6 mg / ml PEG-poly(AGE-Suc)-OVA solution (1.11 mg) and 10 μl 20 mg / ml MPLA (purchased from InvivoGen) (200 μg), and stir to mix; draw 500 μl 6 mmol / L Al2(SO4)3 solution (3 μmol), add it to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0113] Embodiment 14

[0114] Add 185 μl 10 mg / ml PEG-poly(AGE-Suc)-OVA solution (1.85 mg) and 10 μl 40 mg / ml MPLA (400 μg) to 370 μl 200 mmol / L HEPES buffer (74 μmol), and stir to mix; absorb 400 μl 10 mmol / L Al2(SO4)3 solution (4 μmol), add it to the above mixed solution, stir for 30 seconds, and obtain aluminum hydroxide nanoparticles.

[0115] Embodiment 15

[0116] Uptake of aluminum hydroxide nanoparticles by DC2.4, BMDC, B16, and L929

[0117] 5 × 10 5 / well DC2.4, B16 or L929 cells, 1×10 6 BMDC cells were cultured in 1 ml 1640 complete medium per well, and 100 μl aluminum hydroxide nanoparticles (Example 6) prepared by FITC-PEG-poly(AGE-Suc) (Example 2) were added thereto. The cells were cultured at 37°C and 5% CO2 for 1 hour. After washing with PBS three times, the cells were detected by flow cytometry. The results are shown in Table 1. Figure 4 The experimental results show that the prepared aluminum hydroxide nanoparticles are easily taken up by DC cells, which is beneficial to the generation of immune response; at the same time, they are almost not taken up by other cells, which is beneficial to reduce toxic side effects.

[0118] Example 16

[0119] Aluminum hydroxide nanoparticles induce BMDC maturation in vitro

[0120] 1 × 10 6 / well BMDC cells, 4 replicate wells, added 50μl aluminum hydroxide nanoparticles (Example 5), commercial aluminum gel (purchased from InvivoGene) and PEG-poly (AGE-Suc) physical mixture (wherein the amount of aluminum and PEG derivative materials is the same as that of aluminum hydroxide nanoparticles), final concentration 2μg / ml LPS, PBS. 37℃, 5% CO2 culture environment for 24h, cells washed, added anti-mouse CD40 / CD80 / CD86 antibody, incubated and stained at 4℃ for 1h, washed 3 times, and detected by flow cytometry. The results are shown in Figure 5 The experimental results show that aluminum hydroxide nanoparticles have a strong adjuvant effect, can effectively induce BMDC maturation, and are conducive to the generation of immune response.

[0121] Embodiment 17

[0122] Lymph node distribution of aluminum hydroxide nanoparticles

[0123] 25 μl of aluminum hydroxide nanoparticles prepared by FITC-PEG-poly(AGE-Suc) (Example 6) were injected into the paws of C57BL / 6 mice. The mice were killed at 12h, 24h, and 36h, respectively. Lymph nodes at the popliteal flexure were obtained, frozen sections were made, and cell nuclei were stained with DAPI (purchased from Sigma). The results were observed under a laser confocal microscope. Figure 6 The experimental results show that aluminum hydroxide nanoparticles can be quickly and effectively delivered to the draining lymph nodes in the body, providing the possibility for a rapid and effective immune response.

[0124] Embodiment 18

[0125] In vivo immunization experiment in C57BL / 6 mice

[0126] The content of OVA on aluminum hydroxide-OVA-CpG nanoparticles (Example 3) was detected using a BCA kit (purchased from Pierce), and the content of CpG was detected using Quant-iT™ OliGreen® (purchased from Life Technologies). On day 0, 25 μl of aluminum hydroxide-OVA-CpG nanoparticles (OVA content of 1.5 μg, CpG content of 445 ng, aluminum content of 1.25 μg) and 25 μl of commercial aluminum gel adsorbed with equal amounts of OVA and CpG (OVA content of 1.5 μg, CpG content of 445 ng, aluminum content of 125 μg) were injected into the paws of C57BL / 6 mice; on day 14, 25 μl of nanoparticles or aluminum gel were injected into the paws of mice to boost immunization. On day 28, blood was collected from the eye sockets to detect the content of OVA-specific IgG in the blood, and the in vivo cytotoxic T cell (CTL) response was detected by CFSE staining. The results are shown in Figure 7 and Figure 8 The experimental results show that the nanoparticles retain or even enhance the Th2 adjuvant effect of traditional aluminum salt adjuvants, and produce a strong CTL response, making up for the deficiency of traditional aluminum salt adjuvants in Th1 immune response.

[0127] The above conclusions show that the aluminum hydroxide nanoparticles in the present invention can be effectively delivered to the draining lymph nodes in vivo, and are quickly taken up by dendritic cells, inducing strong humoral immune responses and cellular immune responses. It effectively solves the disadvantage of weak cellular immune response of traditional aluminum salt adjuvants, and reduces the side effects of aluminum salts at a much lower aluminum content than traditional aluminum gels, retaining its characteristics as a strong humoral immune response adjuvant.

Claims

1. Aluminum hydroxide nanoparticles used as vaccine carriers, characterized in that The nanoparticles are mainly formed by compounding PEG derivative materials with aluminum hydroxide and Th1 type immune adjuvants, wherein the PEG derivative is PEG-poly (AGE-Suc).

2. The aluminum hydroxide nanoparticles used as a vaccine carrier according to claim 1, characterized in that The nanoparticles are mainly composed of PEG derivative materials, aluminum hydroxide and Th1 type immune adjuvants, wherein the PEG derivative is PEG-poly (AGE-Suc). When the antigen is encapsulated, the PEG derivative can be linked to the antigen through a disulfide bond, an amide group or an ester bond, and the Th1 type immune adjuvant is contained in the nanoparticles.

3. Use of the nanoparticles according to any one of claims 1 to 2 in the preparation of vaccine carriers.

4. The nanoparticles according to any one of claims 1-2, characterized in that The methods of encapsulating antigens include linking antigens through disulfide bonds, amide bonds or ester bonds, or directly encapsulating antigens in nanoparticles.

5. A method for preparing aluminum hydroxide nanoparticles as claimed in any one of claims 1 to 2, comprising the following steps: (1) Add PEG derivative material PEG-poly (AGE-Suc) and Th1 adjuvant to Hepes buffer and stir evenly; (2) Absorbing Al2(SO4)3 solution and adding it into the above mixed solution; (3) Stir for 20-50 seconds to obtain the product; When the aluminum hydroxide nanoparticle carrier directly encapsulates the antigen, the antigen can be added in step (1).

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