A method for preparing and use of a nano-adjuvant targeting antigen presenting cells

Abstract

The application provides a preparation method and application of a nano-adjuvant targeting antigen-presenting cells, and relates to the technical field of biological medicine. The nano-adjuvant targeting antigen-presenting cells is formed by compounding a mannose and / or a derivative thereof with a metal ion compound to form nanoparticles, and the average particle size of the nanoparticles is 50-500 nm. The novel vaccine adjuvant designed and developed by the application has the advantages of nano-adjuvant and antigen-presenting cell targeting performance. The antigen delivery efficiency and cross-presentation ability can be significantly improved while maintaining good safety, the humoral and cellular immune responses are comprehensively stimulated, and a key solution is provided for breaking through the research and development bottleneck of complex infectious diseases and tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing and applying a nanoadjuvant that targets antigen-presenting cells. Background Technology

[0002] Vaccines, as one of the greatest achievements in the history of human medicine, have made milestone breakthroughs in the prevention and treatment of many major diseases. However, effective vaccines still have not been developed for many diseases, such as infectious diseases caused by pathogens with high mutation rates like influenza viruses and SARS-CoV-2, and diseases caused by malignant tumors that lead to severe immunosuppression and immune escape. Vaccines for these diseases usually require inducing a strong immune response, especially a cytotoxic T lymphocyte (CTL)-mediated response, to completely eliminate pathogens, infected host cells, or tumor cells. Adjuvants play a crucial role in enhancing the strength and modulating the type of immune response; therefore, developing novel and highly efficient adjuvants and delivery systems is considered an effective way to overcome the current vaccine bottlenecks.

[0003] Traditional aluminum adjuvants, such as aluminum hydroxide and aluminum phosphate, have been widely used in various human vaccines. They effectively enhance humoral immune responses and increase antibody levels through mechanisms such as forming antigen reservoirs, promoting antigen uptake, and activating inflammasomes. However, aluminum adjuvants have limited efficiency in inducing cellular immunity, making it difficult to meet the immunization requirements of novel vaccines (especially tumor vaccines and multimutant virus vaccines). Furthermore, traditional aluminum adjuvant particles are generally micron-sized, often remaining locally after injection, potentially causing inflammatory reactions and adverse tissue reactions. Therefore, improving their physicochemical properties or using them in rational combination with other adjuvants to enhance cellular immune responses has become an urgent problem to be solved.

[0004] In recent years, with the development of nanotechnology, various nanoparticles such as liposomes, nanoemulsions, and nanoparticles have been widely explored as novel adjuvants and delivery systems. These nanoadjuvants have shown significant advantages in terms of controllable particle size, flexible surface functionalization, high delivery efficiency, and controlled release, which can partially compensate for the shortcomings of traditional aluminum adjuvants. However, most emerging nanoadjuvants still lack specific targeting ability to antigen-presenting cells and often rely on passive uptake, resulting in insufficient antigen utilization and low presentation efficiency.

[0005] In summary, there is an urgent need to design and develop novel vaccine adjuvants that combine the advantages of nano-adjuvants with antigen-presenting cell targeting capabilities. Summary of the Invention

[0006] This invention provides a method for preparing and applying a nano-adjuvant that targets antigen-presenting cells, thereby at least partially solving the above-mentioned problems.

[0007] The first aspect of this invention provides a nanovaccine adjuvant that targets antigen-presenting cells, wherein mannan and / or its derivatives are compounded with metal ion compounds to form nanoparticles, the nanoparticles having an average particle size of 50-500 nm.

[0008] Optionally, the mannan derivatives refer to natural endogenous, semi-synthetic, or fully synthetic derivatives with mannan as the backbone.

[0009] Optionally, the mannan derivative includes one or more of the following: mannan oligosaccharides (MOS), sulfated mannan, carboxymethyl mannan, phosphorylated mannan, mannan peptides, sulfated mannan peptides, carboxymethyl mannan peptides, and phosphorylated mannan peptides.

[0010] Optionally, the nanovaccine adjuvant further comprises one or more of the following immunostimulatory molecules: sodium aescinate, poly I:C, CpG, 2'3'-cGAMP, ginsenosides, and QuilA.

[0011] Optionally, the metal ion in the metal ion compound is selected from one or more of aluminum, iron, zirconium, calcium, manganese, cadmium, magnesium, cerium, cadmium, cobalt, gallium, or zinc, and the metal ion compound is in the form of an inorganic salt or an organic complex.

[0012] Optionally, the metal ion compound is an oxide, hydroxide, or phosphate of a metal ion; Optionally, the metal ion compound is one or more of iron hydroxide, manganese oxide, aluminum hydroxide, zinc hydroxide, aluminum phosphate, calcium phosphate, or manganese oxide.

[0013] Optionally, the mannan and its derivatives are derived from yeast and hemolytic streptococci; the mannan derivatives are... - Mannan peptides derived from hemolytic streptococci; the molecular weight range of the mannan and its derivatives is 16-90 kDa.

[0014] Optionally, the nanoparticles are mixed with antigens or the antigens are encapsulated within the nanoparticles, wherein the antigens are selected from: inactivated pathogens; protein antigens: malaria antigen, hepatitis A, hepatitis B or hepatitis C antigen, tetanus toxoid, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, SARS-CoV-2, herpes simplex virus, human papillomavirus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, and respiratory syncytial virus. Viruses, West Nile virus, cytomegalovirus, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, Group A Streptococcus antigen, or other recombinant protein antigens; protein antigens with weak immunogenicity, including: bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoprotein, soy albumin, wheat albumin, myoglobin, collagen, laminin; polypeptide antigens, including: TRP2, HGP100, p15E, HPV E6, HPV E7, OVA epitope peptide, hepatitis B epitope peptide, SARS-CoV-2 epitope peptide, MC38 antigen, or other synthetic polypeptide antigens and long polypeptide antigens containing several polypeptide sequences; one or more of the following: viral or bacterial lysate antigens, viral or bacterial outer membrane vesicle antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, and tumor cell exosome antigens.

[0015] Optionally, when the metal ion compound is aluminum hydroxide, the mass ratio of the metal ion compound to mannan or its derivative is 1-20:10-100.

[0016] Optionally, the nano-vaccine adjuvant is a lyophilized formulation.

[0017] A second aspect of this invention provides a method for preparing the nano-vaccine adjuvant described in the first aspect, the method comprising the following steps: Step 1: Dissolve a certain amount of mannan and / or its derivatives in HEPES buffer solution or sodium phosphate to obtain a first solution; Step 2: Mix the first solution with the metal ion solution to obtain a mixed solution; Step 3: The mixed solution is treated using ultrasound, vortexing, stirring, syringe pump or microfluidics to obtain nanoparticles; In cases where co-carrying of antigen is required, the antigen is dissolved in the HEPES buffer solution.

[0018] Optionally, in step 3, the ultrasonic power is 30-300W, the time is 0.5-30min, the vortex time is 10-60s, the infusion pump flow rate is 5-50ml / min, the microfluidic flow rate ratio is 1:1-1:5, and the flow rate is 5-40ml / min.

[0019] The third aspect of this invention provides the use of the nano-vaccine adjuvant described in the first aspect in the preparation of vaccines for the prevention or treatment of infectious diseases.

[0020] Optionally, the infectious diseases include influenza, hepatitis B, hepatitis C, SARS-CoV-2, and bacterial infections.

[0021] The fourth aspect of this invention provides the use of the nanovaccine adjuvant described in the first aspect in the preparation of vaccines for the prevention or treatment of tumors.

[0022] Optionally, the tumor includes melanoma, lung cancer, and colorectal cancer.

[0023] The fourth aspect of this invention provides the use of the nanovaccine adjuvant described in the first aspect in the preparation of a medicament that helps enhance the efficacy of immune checkpoint therapy.

[0024] As can be seen, the embodiments of the present invention propose an innovative strategy that combines the advantages of nano-adjuvants with the targeting performance of APC: 1. Targeting: Mannan and its derivatives can achieve specific delivery by binding to mannose receptors on the surface of APCs; 2. Adjuvant properties: Metal ions and mannan and its derivatives possess certain adjuvant properties, which can stabilize antigens and enhance immune responses; 3. Synergistic effect: The additional introduction of immunostimulatory molecules can further activate the innate immune pathway and enhance cellular immunity; 4. Broad spectrum: Applicable to a variety of antigens, including viral antigens, bacterial antigens, tumor antigens and synthetic polypeptide antigens.

[0025] Therefore, the nano-vaccine adjuvant provided in this embodiment of the invention can significantly improve antigen utilization and cross-presentation efficiency, while taking into account both safety and immune enhancement, providing a powerful adjuvant platform for the development of novel vaccines. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is the average fluorescence intensity result of FITC-labeled OVA in Example 44 of this invention.

[0028] Figure 2This is a schematic diagram showing the relative inhibition rates of blocked mannose receptors (Mannan) and unblocked mannose receptors (Noinhibitor) in Example 45 of the present invention; Figure 3 The H-2K antigen peptide carrying SIINFEKL on the surface of bone marrow-derived dendritic cells (BMDCs) in each treatment group of Example 46 of this invention is... b A schematic diagram showing the percentage of positive MHC-I molecules; Figure 4 This is a schematic diagram illustrating the differences in the positive rates of maturation molecular markers on the surface of mouse bone marrow-derived dendritic cells among different experimental treatment groups in Example 47 of the present invention; Figure 5 This is a schematic diagram of the mouse in vivo imaging experiment results in Example 48 of the present invention; Figure 6 This is a schematic diagram showing the distribution of cell subsets in the inguinal and popliteal lymph nodes of mice in different treatment groups in Example 49 of the present invention. Figure 7 This is a graph showing the detection results of the in vivo immune-induced CTL effect of the nano-vaccine in mice in Example 50 of this invention; Figure 8 This is a graph showing the results of the in vivo immune-induced anti-tumor immune response in mice using the nano-vaccine in Example 51 of this invention; Figure 9 This is a diagram showing the results of inducing antigen-specific immune responses in mice using nano-adjuvants loaded with antigens or mixed with antigens in Example 52 of this invention. Figure 10 This is a graph showing the freeze-drying stability results of the nano-vaccine in Example 53 of this invention; Figure 11 This is a diagram showing the results of promoting BMDC maturation by encapsulating different adjuvant molecules in aluminum hydroxide-mannan peptide nanoparticles in Example 54 of this invention. Figure 12 This is a graph showing the anti-tumor results of the nano-vaccine combined with an immune checkpoint inhibitor in a mouse subcutaneous cervical cancer tumor model in Example 55. Detailed Implementation

[0029] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0030] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents and other instruments whose manufacturers are not specified are all commercially available products.

[0031] Example 1 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg of mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 7.5, and add 400 μL of 10 mmol / L aluminum sulfate solution under vortex conditions to obtain the nanoadjuvant.

[0032] Example 2 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 1 mg of mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 7.5, and add 400 μL of 10 mmol / L aluminum sulfate solution under vortex conditions to obtain the nanoadjuvant.

[0033] Example 3 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 3 mg of mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 7.5, and add 400 μL of 10 mmol / L aluminum sulfate solution under vortex conditions to obtain the nanoadjuvant.

[0034] Example 4 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg of mannan in 400 μL of 100 mmol / L HEPES buffer solution with pH 7.5, and add 500 μL of 10 mmol / L aluminum sulfate solution under vortex conditions to obtain the nanoadjuvant.

[0035] Example 5 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 8, add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution, and sonicate for 3 min at 150 W to obtain the nanoadjuvant.

[0036] Example 6 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 8, add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution, and sonicate for 5 min at 100 W to obtain the nanoadjuvant.

[0037] Example 7 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg of mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 8 and place it in syringe A. Place 400 μL of 10 mmol / L aluminum sulfate solution in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1.25 and a flow rate of 10 mL / min. Mix the two phases to obtain the final product.

[0038] Example 8 Preparation of aluminum hydroxide-mannan nanoadjuvant: Dissolve 2 mg of mannan in 320 μL of 100 mmol / L HEPES buffer solution with pH 8 and place it in syringe A. Place 400 μL of 10 mmol / L aluminum sulfate solution in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0039] Example 9 Preparation of aluminum hydroxide-mannan peptide nanoadjuvant: 2 mg of mannan peptide was fully dissolved in 320 μL of 100 mmol / L HEPES buffer solution with a pH of 8 and placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. The two were placed on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were mixed to obtain the final product.

[0040] Example 10 Preparation of aluminum hydroxide-mannan peptide nanoadjuvant: Dissolve 2 mg of mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution with pH 8, and add 400 μL of 10 mmol / L aluminum sulfate solution under vortex conditions to obtain the nanoadjuvant.

[0041] Example 11 Preparation of aluminum hydroxide-mannan peptide-OVA nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution with pH 8 and mix well with 40 μL of 10 mg / mL OVA solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0042] Example 12 Preparation of aluminum hydroxide-mannan-RBD nanovaccine: Dissolve 2 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution (pH 8) and mix thoroughly with 30 μL of 10 mg / mL RBD solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0043] Example 13 Preparation of aluminum hydroxide-mannan-gE nanovaccine: Dissolve 3 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution (pH 8) and mix thoroughly with 40 μL of 10 mg / mL gE solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0044] Example 14 Preparation of aluminum hydroxide-mannan-HPV E744-62 peptide nanovaccine: Dissolve 3 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution (pH 8) and mix thoroughly with 20 μL of 10 mg / mL HPV E7 peptide solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0045] Example 15 Preparation of aluminum hydroxide-mannan-HPV E7 protein nanovaccine: Dissolve 2 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution (pH 8) and mix thoroughly with 20 μL of 10 mg / mL HPV E7 solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0046] Example 16 Preparation of aluminum hydroxide-mannan-HBsAg nanovaccine: Dissolve 3 mg mannan in 320 μL of 100 mmol / L HEPES buffer solution (pH 8) and mix thoroughly with 30 μL of 10 mg / mL HBsAg solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0047] Example 17 Preparation of aluminum hydroxide-mannan peptide-OVA nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mix well with 30 μL of 10 mg / mL OVA solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0048] Example 18 Preparation of aluminum hydroxide-mannan peptide-RBD nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mix well with 30 μL of 10 mg / mL RBD solution. Add 400 μL of 10 mmol / L aluminum sulfate solution to the above solution under vortex conditions to obtain the final product.

[0049] Example 19 Preparation of aluminum hydroxide-mannan peptide-gE nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L pH 7.5 HEPES buffer solution and mix well with 80 μL of 5 mg / mL gE solution. Place the mixture in syringe A. Take 400 μL of 10 mmol / L aluminum sulfate solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. Mix the two phases to obtain the final product.

[0050] Example 20 Preparation of aluminum hydroxide-mannan peptide-HBsAg nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mix well with 80 μL of 5 mg / mL HBsAg solution. Place the mixture in syringe A. Take 400 μL of 10 mmol / L aluminum sulfate solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. Mix the two phases to obtain the final product.

[0051] Example 21 Preparation of aluminum hydroxide-mannan peptide-gE nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L pH 7.5 HEPES buffer solution and mix well with 80 μL of 5 mg / mL gE solution. Place the mixture in syringe A. Take 400 μL of 10 mmol / L aluminum sulfate solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. Mix the two phases to obtain the final product.

[0052] Example 22 Preparation of aluminum hydroxide-mannan peptide-HBsAg nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed evenly with 80 μL of 5 mg / mL HBsAg solution. The mixture was placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both syringes were simultaneously passed through a microinfusion pump at a rate of 50 mL / min through a three-channel microfluidic device. The mixed liquid was collected to obtain the final product.

[0053] Example 23 Preparation of aluminum hydroxide-mannan peptide-ply nanovaccine: Dissolve 2 mg mannan peptide thoroughly in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mix thoroughly with 40 μL of 10 mg / mL ply solution. Place the mixture into syringe A. Place 400 μL of 10 mmol / L aluminum sulfate solution into syringe B. Simultaneously pass both syringes through a microinjection pump at a rate of 50 mL / min through a three-channel microfluidic device. Collect the mixed liquid to obtain the final product.

[0054] Example 24 Preparation of aluminum hydroxide-mannan peptide-ply-sodium aescin nanovaccine: 2 mg mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed evenly with 40 μL of 10 mg / mL ply solution and 40 μL of 5 mg / mL sodium aescin solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both syringes were simultaneously passed through a microinjection pump at a rate of 50 mL / min through a three-channel microfluidic device. The mixed liquid was collected to obtain the final product.

[0055] Example 25 Preparation of aluminum hydroxide-mannan peptide-OVA-CpG nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 10 mg / mL OVA solution and 40 μL of 1 mg / mL CpG solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both syringes were simultaneously passed through a microinjection pump at a rate of 50 mL / min through a three-channel microfluidic device. The mixed liquid was collected to obtain the final product.

[0056] Example 26 Preparation of aluminum hydroxide-mannan peptide-OVA-2'3'-cGAMP nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 10 mg / mL OVA solution and 40 μL of 1 mg / mL 2'3'-cGAMP solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0057] Example 27 Preparation of aluminum hydroxide-mannan peptide-OVA-poly(I:C) nanovaccine: 2 mg mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 10 mg / mL OVA solution and 40 μL of 1 mg / mL poly(I:C) solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of 1:1 (A:B) and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0058] Example 28 Preparation of aluminum hydroxide-mannan peptide-OVA-ginsenoside nanovaccine: 2 mg mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed evenly with 40 μL of 10 mg / ml OVA solution and 40 μL of 1 mg / ml ginsenoside solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. The two phases were then mixed to obtain the final product.

[0059] Example 29 Preparation of aluminum hydroxide-mannan peptide-OVA-QuilA nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 10 mg / mL OVA solution and 30 μL of 1 mg / mL QuilA solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0060] Example 30 Preparation of aluminum hydroxide-mannan peptide-PcrV-CpG nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 10 mg / mL PcrV solution and 40 μL of 1 mg / mL CpG solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0061] Example 31 Preparation of aluminum hydroxide-mannan peptide-OVA antigen peptide-CpG nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 40 μL of 50 mg / ml OVA antigen peptide solution and 40 μL of 1 mg / ml CpG solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0062] Example 32 Preparation of aluminum phosphate-mannan peptide-OVA antigen peptide-CpG nanovaccine: Dissolve 6 mg of mannan peptide thoroughly in 320 μL of 10 mmol / L sodium phosphate buffer solution and mix it evenly with 40 μL of 50 mg / ml OVA antigen peptide solution and 40 μL of 1 mg / ml CpG solution. Place the mixture in syringe A. Take 400 μL of 10 mmol / L aluminum sulfate solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0063] Example 33 Preparation of ferric hydroxide-mannan peptide-OVA antigen peptide-CpG nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 300 μL of 100 mmol / L HEPES buffer solution (pH 8) and mixed thoroughly with 40 μL of 50 mg / ml OVA antigen peptide solution and 40 μL of 1 mg / ml CpG solution. The mixture was then placed in syringe A. 200 μL of 5 mmol / L ferric chloride solution was placed in syringe B. Both solutions were placed on a microfluidic system with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. The two phases were then mixed to obtain the final product.

[0064] Example 34 Preparation of ferric hydroxide-mannan nanoadjuvant: Dissolve 3 mg of mannan solution thoroughly in 300 μL of 100 mmol / L HEPES buffer solution with a pH of 8, mix well, and place in syringe A. Take 200 μL of 5 mmol / L ferric chloride solution and place in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0065] Example 35 Preparation of ferric hydroxide-mannan nanoadjuvant: Dissolve 3 mg of mannan solution thoroughly in 300 μL of 100 mmol / L HEPES buffer solution with a pH of 8, mix well, and place in syringe A. Take 200 μL of 5 mmol / L ferric chloride solution and place in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0066] Example 36 Preparation of ferric hydroxide-mannan peptide-HPV E7 antigen peptide-CpG nanovaccine: Dissolve 3 mg of mannan peptide thoroughly in 300 μL of 100 mmol / L HEPES buffer solution (pH 8), and mix thoroughly with 40 μL of 10 mg / ml HPV E7 antigen peptide solution and 40 μL of 1 mg / ml CpG solution. Place the mixture in syringe A. Take 200 μL of 5 mmol / L ferric chloride solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0067] Example 37 Preparation of calcium phosphate-mannan peptide nanoadjuvant: Dissolve 4 mg of mannan peptide thoroughly in 400 μL of 6 mmol / L sodium phosphate buffer solution and place it in syringe A. Take 400 μL of 8 mmol / L calcium chloride solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 ml / min. Mix the two phases to obtain the final product.

[0068] Example 38 Preparation of calcium phosphate-mannan peptide-OVA nanovaccine: Dissolve 4 mg mannan peptide thoroughly in 400 μL of 6 mmol / L sodium phosphate buffer solution and mix well with 40 μL of 1 mg / mL OVA solution. Place the mixture in syringe A. Take 400 μL of 8 mmol / L calcium chloride solution and place it in syringe B. Place both on a microfluidic device with a flow rate ratio of A:B of 1:1 and a flow rate of 20 mL / min. Mix the two phases to obtain the final product.

[0069] Example 39 Preparation of manganese oxide-mannan nanoadjuvant: 20 mg of mannan was fully dissolved in 2 ml of 10 mM HEPES buffer. 500 μL of 10 mM MnCl2 solution was slowly added dropwise under stirring to adjust the pH of the system to 9. The mixture was stirred at room temperature for 60 min. The obtained product was dialyzed at 4 degrees Celsius using a 10 kDa dialysis bag for 48 h. The nanoparticles were then collected.

[0070] Example 40 Preparation of manganese oxide-mannan peptide-OVA nanovaccine: 20 mg mannan peptide and 200 μg OVA antigen were fully dissolved in 2 ml of 10 mM HEPES buffer. 500 μl of 10 mM MnCl2 solution was slowly added dropwise under stirring to adjust the pH of the system to 9. The mixture was stirred at room temperature for 60 min. The obtained product was dialyzed at 4 degrees Celsius using a 10 kDa dialysis bag for 48 h. The nanoparticles were then collected.

[0071] Example 41 Preparation of aluminum hydroxide-mannan peptide-OVA-sodium aescin nanovaccine: 2 mg of mannan peptide was thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed thoroughly with 100 μL of 10 mg / mL OVA antigen peptide solution and 40 μL of 5 mg / mL sodium aescin solution. The mixture was then placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both syringes were simultaneously passed through a microinjection pump at a rate of 50 mL / min through a three-channel microfluidic device. The mixed liquid was collected to obtain the final product.

[0072] Example 42 Preparation of aluminum hydroxide-mannan peptide-sodium aescin nanovaccine: 0.1 mg mannan peptide solution and 40 μL sodium aescin solution (5 mg / mL) were thoroughly dissolved in 320 μL of 100 mmol / L HEPES buffer solution (pH 7.5) and mixed evenly. The mixture was placed in syringe A. 400 μL of 10 mmol / L aluminum sulfate solution was placed in syringe B. Both syringes were simultaneously passed through a microinjection pump at a rate of 30 mL / min through a three-channel microfluidic device. The mixed liquid was collected to obtain the final product.

[0073] Example 43 Nanoparticle size determination: The particle size was determined using a Zetasizer Nano ZS90 laser particle size analyzer. (Example 1) The particle size distribution of 40 nano-adjuvants or nano-vaccines was determined by taking 200 μL of each sample from Example 1. A 40% solution of nanoparticles was prepared, and the sample was placed in the sample cell. The measurement temperature was set to 25°C. C, the results are shown in the table below. The results show that the nanoparticle size is about 50-500 nm and the particle size distribution is uniform.

[0074]

[0075] Example 44 Uptake of metal ion-mannan and its derivative nanovaccines on dendritic cells Plant 1×10⁻⁶ seeds per hole 5 After incubating DC2.4 cells for 8 hours, 50 μl of FITC-labeled OVA (FO) was added to each well, or the aluminum hydroxide-mannan-OVA nanovaccine (A) of Example 11, the aluminum hydroxide-mannan peptide-OVA nanovaccine (B) of Example 17, the calcium phosphate-mannan peptide-OVA nanovaccine (C) of Example 38, and the manganese oxide-mannan peptide-OVA nanovaccine (D) of Example 40 were prepared using FITC-labeled OVA (characters in parentheses indicate the group). Figure 1 (Identification in the text). After 1 hour of ingestion at 37°C, the supernatant was discarded, and the cell surface was gently washed twice with PBS. Cells were then centrifuged twice at 2000 rpm for 3 min, and finally resuspended in 400 μl PBS for flow cytometry analysis. Results are as follows: Figure 1 As shown, Figure 1 The average fluorescence intensity of FITC-labeled OVA is shown. It can be seen that aluminum hydroxide-mannan-OVA nanovaccines, aluminum hydroxide-mannan peptide-OVA nanovaccines, calcium phosphate-mannan peptide-OVA nanovaccines, and manganese oxide-mannan peptide-OVA nanovaccines can be efficiently taken up by antigen-presenting cells. The uptake rate on DC2.4 cells is much higher than that of free OVA.

[0076] Example 45 Uptake pathway of aluminum hydroxide-mannan peptide nanovaccine on dendritic cells Plant 1×10⁻⁶ seeds per hole 5DC2.4 cells were incubated for 8 hours. Then, mannose and mannan (final concentration 5 mg / mL) were added to block the mannose receptors in the cells. One hour later, 50 μL of FITC-labeled OVA (aluminum hydroxide-mannan peptide-OVA nanovaccine prepared in Example 17) was added. After 1 hour of uptake at 37°C, the supernatant was discarded, and the cell surface was gently washed twice with PBS. The cells were then centrifuged twice at 2000 rpm for 3 min, and finally resuspended in 400 μL PBS for flow cytometry analysis. Results are as follows: Figure 2 As shown, Figure 2 The relative inhibition rates of blocked mannose receptor (Mannan) and unblocked mannose receptor (No inhibitor) are shown. It can be seen that the uptake efficiency of aluminum hydroxide-mannan peptide nanovaccine is significantly reduced after blocking the mannose receptor, proving that the cell entry of aluminum hydroxide-mannan peptide nanovaccine mainly depends on the mannose receptor.

[0077] Example 46 Nanoparticle vaccines promote antigen cross-presentation at BMDC Inoculate 1×10⁻⁶ cells into 12-well plates 6 / well BMDC cells were added to each cell, along with free OVA antigen peptide (FO), a mixture of free antigen peptide and sodium aescinate (FOE), and 50 μl of nanoparticles (Example 17: Aluminum hydroxide-mannan peptide-OVA nanovaccine (AlMN-O), Example 41: Aluminum hydroxide-mannan peptide-OVA-sodium aescinate nanovaccine (AlMN-OE)) (characters in parentheses indicate the groups in which each group was added). Figure 3 (Identification in the text). Cells were cultured at 37℃ and 5% CO2 for 20 h. After washing, anti-mouse H2Kb-SIINFEKL-PE flow cytometry antibody was added, and the cells were incubated at 4℃ for 40 min for staining. After washing three times, the cells were detected by flow cytometry. Results are as follows: Figure 3 . Figure 3 The H-2K cells carrying the SIINFEKL antigen peptide on the surface of bone marrow-derived dendritic cells (BMDCs) in each group are shown. b The positive percentage of MHC-I molecules was determined. The experimental results indicate that the nanovaccine based on mannan-metal ion compound complex can effectively enhance the efficiency of BMDC antigen cross-presentation, which is beneficial for subsequent CD8 activation. + T-cell immune response.

[0078] Example 47 Nanoparticle vaccines promote BMDC maturation Inoculate 1×10⁻⁶ cells into 12-well plates 6 / well BMDC cells, 4 replicates, each containing 50 μl of nanoparticles (Example 11 Aluminum hydroxide-mannan peptide-OVA nanovaccine (A), Example 25 Aluminum hydroxide-mannan peptide-OVA-CpG nanovaccine (B), Example 26 Aluminum hydroxide-mannan peptide-OVA-2'3'-cGAMP nanovaccine (C), Example 27 Aluminum hydroxide-mannan peptide-OVA-poly(I:C) nanovaccine (D), Example 29 Aluminum hydroxide-mannan peptide-OVA-QuilA nanovaccine (E)), commercial aluminum gel (purchased from InvivoGene) and a physical mixture of OVA and mannan peptide (F), (where the amounts of aluminum and mannan peptide are the same as those of the nanoparticles) (characters in parentheses indicate the group in each case). Figure 4 (Identified in the text), LPS and PBS were added to a final concentration of 2 μg / mL. Cells were cultured at 37℃ and 5% CO2 for 24 h. After washing, anti-mouse CD40 / CD80 / CD86 antibody was added, and the cells were incubated at 4℃ for 40 min for staining. After washing three times, the cells were analyzed by flow cytometry. Results are as follows: Figure 4 . Figure 4 The study showed differences in the positive rates of maturation marker expression in mouse bone marrow-derived dendritic cells (BMDCs) from different experimental treatment groups. The results indicate that the nanovaccine based on mannan-metal ion compound can effectively induce BMDC maturation and is beneficial for activating the immune response for protection.

[0079] Example 48 Nanoparticle vaccine lymph node targeted delivery C57BL / 6 mice were intramuscularly injected with 50 μl of free Cy5-OVA solution, or a nanovaccine containing aluminum hydroxide-mannan peptides and Cy5-OVA, or a Cy5-OVA solution adsorbed on aluminum gel. Mice were sacrificed at 6 h, 12 h, and 24 h post-administration. Popliteal or inguinal lymph nodes were isolated, and the fluorescence intensity of Cy5-OVA in the lymph nodes was detected using small animal in vivo imaging. Results are shown below. Figure 5 . Figure 5The results of in vivo imaging experiments in mice are shown. Part A presents in vivo fluorescence imaging images of mouse lymph nodes. The top row shows fluorescence imaging images of inguinal lymph nodes from FO (free Cy5-OVA solution group), AIMN-O (aluminum hydroxide-mannan peptide nanovaccine group loaded with Cy5-OVA), and Algel (Cy5-OVA solution group adsorbed by aluminum gel). The bottom row shows fluorescence imaging images of popliteal lymph nodes from each experimental group. Part B presents the quantitative analysis results of lymph node fluorescence signals. The top row shows the quantitative analysis results of inguinal lymph node fluorescence signals, and the bottom row shows the quantitative analysis results of popliteal lymph node fluorescence signals. The experimental results show that the mannan-based nanovaccine can be rapidly and effectively delivered to draining lymph nodes in vivo, providing a possibility for rapid and effective immune response.

[0080] Example 49 The nano-vaccine was taken up by antigen-presenting cells in the lymph nodes. C57BL / 6 mice were intramuscularly injected with 50 μL of free Cy5-OVA solution (FO), or a nanovaccine containing Cy5-OVA in the form of aluminum hydroxide-mannan peptide (AlMN-O), a nanovaccine containing Cy5-OVA in the form of aluminum hydroxide-mannan peptide-sodium aescinate (AlMN-OE), or a Cy5-OVA solution adsorbed on aluminum gel (Algel-O). Mice were sacrificed 12 h after administration, and popliteal or inguinal lymph nodes were isolated and prepared into single-cell suspensions. Follicular dendritic cells (FDCs) and their subtypes (tissue-resident and migratory cDC1 and cDC2) were labeled using flow cytometry. The efficiency of Cy5-OVA uptake by each cell type was detected by flow cytometry. Results are shown in 6. Figure 6 The results of the subset distribution analysis of cells from different treatment groups in the inguinal and popliteal lymph nodes of mice are shown. The left figure shows the proportion of Cy5-positive cells among various immune cell subsets in the inguinal lymph nodes in different treatment groups, and the right figure shows the proportion of Cy5-positive cells among various immune cell subsets in the inguinal lymph nodes in different treatment groups. The experimental results show that the mannan-based nanovaccine can be effectively taken up by antigen-presenting cells in the lymph nodes, significantly better than traditional aluminum gel.

[0081] Example 50 Detection of CTL effect induced by nano-vaccine in mice Seven-week-old female C57BL / 6 mice were intramuscularly injected on days 0, 14, and 21 with the following formulations: PBS, free OVA antigen, a mixture of free OVA antigen and sodium aescinate (OVA + Esc), aluminum hydroxide-mannan peptide-OVA (AlMN-O), aluminum hydroxide-mannan peptide-OVA-sodium aescinate (AlMN-OE), and aluminum gel-adsorbed OVA + mannan peptide + sodium aescinate (Algel-MOE) (dosage per mouse: OVA 10 μg, mannan or mannan peptide 200 μg, sodium aescinate 20 μg, aluminum 20 μg). Serum was collected on day 28 to detect serum antibody levels. Mice were sacrificed on day 33 to assess CTL efficacy (each mouse was injected with 1 × 10⁶ CFSE-labeled target cells). 7 Cells were killed and tested 19 hours later. Results are as follows: Figure 7 As shown. Figure 7 The results of the detection of the immune-induced CTL effect of the nanovaccine in mice are shown in the figure. The left figure shows the schematic diagram of the in vivo specific killing experiment, and the right figure shows the statistical results of the antigen-specific cleavage rate of mice in different treatment groups.

[0082] It can be seen that nanovaccines based on mannan or mannan peptides can effectively improve the level of OVA-specific antibodies and induce a strong CTL effect, effectively killing target cells, with significantly better results than mannan peptides adsorbed by aluminum gel.

[0083] Example 51 Nanoparticle vaccines induce anti-tumor immune responses in mice. C57BL / 6 mice were subcutaneously inoculated with TC-1 tumor cells transfected with HPV 16 E7 on day 0, and treated on days 4, 10, and 14 with the following formulations: PBS, free HPV E744-62 peptide (FH), a mixture of free HPV E744-62 peptide and sodium aescinate (FHE), aluminum hydroxide-mannan-HPV E744-62 peptide nanovaccine (AlMN-H), aluminum hydroxide-mannan-HPV E744-62 peptide-sodium aescinate nanovaccine (AlMN-HE), and aluminum gel-adsorbed mannan peptide + HPV E744-62 peptide + sodium aescinate (dosage per mouse: 20 μg HPV E744-62 peptide, 200 μg mannan or mannan peptide, 20 μg sodium aescinate, 20 μg aluminum). Tumor volume was measured every two days, and mouse survival was recorded. Results are shown below. Figure 8 , Figure 8The diagram shows the results of the in vivo immune-induced anti-tumor immune response in mice using the nanovaccine. Part a shows a schematic timeline of the anti-tumor experiment in mice; part b shows the tumor growth curves of mice in different treatment groups; part c shows the survival curves of mice in each group; and part d shows the individual tumor growth curves of mice in each group. It is evident that the mannan-based nanovaccine can effectively delay tumor growth and improve mouse survival rates.

[0084] Example 52 Nanoparticle adjuvants encapsulate antigens or mix them with antigens to induce antigen-specific immune responses in mice. Seven-week-old female C57BL / 6 mice were intramuscularly injected on days 0, 14, and 21 with the following formulations: a mixture of free OVA antigen and sodium aescinate (FOE); aluminum hydroxide-mannan peptide nanoparticles co-loaded with OVA and sodium aescinate (AlMN-OE); an aluminum hydroxide-mannan peptide-sodium aescinate nanoparticle adjuvant mixed with free OVA (AlMNE+O); and aluminum gel adsorbed OVA + mannan peptide + sodium aescinate (Algel-MOE) (dosage per mouse: OVA 10 μg, mannan or mannan peptide 200 μg, sodium aescinate 20 μg, aluminum 20 μg). Serum was collected on day 28 to detect serum antibody levels. Figure 9 The diagram shows the results of inducing antigen-specific immune responses in mice using nano-adjuvants loaded with or mixed with antigens. Part a represents the titer of OVA-specific IgG antibodies; part b represents the titer of OVA-specific IgG1 antibodies; and part c represents the titer of OVA-specific IgG2a antibodies. The results demonstrate that whether the metal ion-mannan peptide nanoparticles co-load antigens and adjuvants, or are mixed with antigen proteins, they can induce antigen-specific immune responses in mice and increase the level of specific antibodies.

[0085] Example 53 Lyophilization stability of mannan-based nanovaccines Aluminum hydroxide-mannan peptide-OVA nanovaccine was prepared according to Example 11. After pre-freezing at -20°C for 4 hours, the nanovaccine was lyophilized. The lyophilized nanovaccine powder was reconstituted with water for injection and co-incubated with mouse BMDCs (indicated by After in the figure). A group of un-lyophilized nanovaccines co-incubated with BMDCs was also set up (indicated by Before in the figure). After 24 hours, BMDCs were collected, and the cell surface maturation molecules CD40 / CD80 were stained by flow cytometry. Flow cytometry was used to detect whether there was a difference in the promotion of BMDC maturation before and after lyophilization. The results showed that the nanovaccine could be lyophilized without a lyophilization protectant, and its stability was good; lyophilization did not affect its activity.

[0086] Example 54 Aluminum hydroxide-mannan peptide nanocarriers encapsulate different adjuvant molecules to improve BMDC maturation efficiency. Aluminum hydroxide-mannan peptide-OVA-CpG nanovaccine (AlMN-C) was prepared according to Example 25; aluminum hydroxide-mannan peptide-OVA-2'3'-cGAMP nanovaccine (AlMN-cGAMP) was prepared according to Example 26; aluminum hydroxide-mannan peptide-OVA-poly(I:C) nanovaccine (AlMN-P) was prepared according to Example 27; and aluminum hydroxide-mannan peptide-OVA-aescin sodium nanovaccine (AlMN-E) was prepared according to Example 41. Free solutions of these adjuvant molecules were prepared for each. BMDCs were co-incubated with either the free adjuvant or the adjuvant loaded in nanoparticles for 24 h. Cells were then collected, and the CD40 / CD80 maturation molecules on the cell surface were stained using flow cytometry. The expression levels of maturation molecules in BMDCs were detected by flow cytometry. The results showed that encapsulating other adjuvant molecules in the aluminum hydroxide-mannan peptide nanocarrier significantly improved the maturation efficiency of BMDCs.

[0087] Example 55 Antitumor effects of nano-vaccines combined with immune checkpoint inhibitors in a mouse model of cervical cancer subcutaneous tumors C57BL / 6 mice were subcutaneously inoculated with TC-1 tumor cells transfected with HPV 16 E7 on day 0, and treated on days 4, 10, and 14 with the following formulations: PBS, aluminum hydroxide-mannan-HPV E744-62 peptide-aescin sodium nanovaccine (AlMN-HE) (dosage per mouse: HPV E744-62 peptide 20ug, mannan or mannan peptide 200ug, aescin sodium 20ug, aluminum 20ug). Mice were intraperitoneally injected on days 7, 10, 13, 16, and 19. PD-1 monoclonal antibody (100 μg per mouse per dose), tumor volume was measured every two days, and mouse survival time was recorded. Results are shown below. Figure 12 , Figure 12 The administration regimen and antitumor effect of the nanovaccine in mice are illustrated. Part a shows a schematic diagram of the drug administration timeline in the in vivo antitumor experiment in mice; part b shows the tumor growth curves of mice in different treatment groups; and part c shows the survival curves of mice in each group. The results show that the mannan-based nanovaccine combined with PD-1 can significantly improve the efficacy of PD-1 monoclonal antibody in cervical cancer models and effectively increase the survival rate of mice. Therefore, the mannan-based nanovaccine provided in this embodiment of the invention can be applied to the preparation of drugs that adjuvantly enhance the efficacy of immune checkpoint therapy.

[0088] Mannans and their derivatives can achieve highly efficient targeted delivery and significantly improve antigen uptake efficiency by specifically binding to mannose receptors (MRs) on the surface of antigen-presenting cells (such as dendritic cells and macrophages). Simultaneously, mannans themselves possess certain immunostimulatory activity, which can further enhance the intensity of the immune response. More importantly, the mannose receptor-mediated endocytosis process can bypass the classic endosome-lysosome degradation pathway, allowing exogenous antigens to enter the MHC-I antigen presentation pathway, promoting cross-presentation and effectively activating CD8. + T cell responses enhance cellular immunity. However, currently, only mannan peptides are used as immune enhancers in other fields, and their mature application in vaccine adjuvants is still lacking.

[0089] In summary, the novel vaccine adjuvant designed and developed in this embodiment of the invention possesses the advantages of nano-adjuvants and exhibits antigen-presenting cell-targeting properties. It can significantly improve antigen delivery efficiency and cross-presentation capability while maintaining good safety, comprehensively stimulating humoral and cellular immune responses, and providing a key solution for overcoming the development bottlenecks of vaccines for complex infectious diseases and tumors.

[0090] Based on the above exploration and analysis, this invention provides a nanovaccine adjuvant that targets antigen-presenting cells, which is formed by combining mannan and / or its derivatives with metal ion compounds to form nanoparticles, wherein the average particle size of the nanoparticles is 50-500 nm.

[0091] Optionally, the mannan derivatives refer to natural endogenous, semi-synthetic, or fully synthetic derivatives with mannan as the backbone.

[0092] Optionally, the mannan derivative includes one or more of the following: mannan oligosaccharides (MOS), sulfated mannan, carboxymethyl mannan, phosphorylated mannan, mannan peptides, sulfated mannan peptides, carboxymethyl mannan peptides, and phosphorylated mannan peptides.

[0093] Optionally, the nanovaccine adjuvant further comprises one or more of the following immunostimulatory molecules: sodium aescinate, poly I:C, CpG, 2'3'-cGAMP, ginsenosides, and QuilA.

[0094] Optionally, the metal ion in the metal ion compound is selected from one or more of aluminum, iron, zirconium, calcium, manganese, cadmium, magnesium, cerium, cadmium, cobalt, gallium, or zinc, and the metal ion compound is in the form of an inorganic salt or an organic complex.

[0095] Optionally, the metal ion compound is an oxide, hydroxide, or phosphate of a metal ion; Optionally, the metal ion compound is one or more of iron hydroxide, aluminum hydroxide, zinc hydroxide, aluminum phosphate, calcium phosphate, or manganese oxide.

[0096] Optionally, the mannan and its derivatives are derived from yeast and hemolytic streptococci; the mannan derivatives are... - Mannan peptides derived from hemolytic streptococci; the molecular weight range of the mannan and its derivatives is 16-90 kDa.

[0097] Optionally, the nanoparticles are mixed with antigens or the antigens are encapsulated within the nanoparticles, wherein the antigens are selected from: inactivated pathogens; protein antigens: malaria antigen, hepatitis A, hepatitis B or hepatitis C antigen, tetanus toxoid, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, SARS-CoV-2, herpes simplex virus, human papillomavirus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, and respiratory syncytial virus. Virus, West Nile virus, cytomegalovirus, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, Group A Streptococcus antigen, or other recombinant protein antigens; protein antigens with weak immunogenicity, including: bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoprotein, soy albumin, wheat albumin, myoglobin, collagen, laminin; polypeptide antigens, including: TRP2, HGP100, p15E, HPV E6, HPVE7, OVA epitope peptide, hepatitis B epitope peptide, SARS-CoV-2 epitope peptide, MC38 antigen, or other synthetic polypeptide antigens and long polypeptide antigens containing several polypeptide sequences; one or more of the following: viral or bacterial lysate antigens, viral or bacterial outer membrane vesicle antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, and tumor cell exosome antigens.

[0098] Optionally, when the metal ion compound is aluminum hydroxide, the mass ratio of the metal ion compound to mannan or its derivative is 1-20:10-100.

[0099] Optionally, the nano-vaccine adjuvant is a lyophilized formulation.

[0100] This invention also provides a method for preparing the nano-vaccine adjuvant described in any of the above embodiments, the method comprising the following steps: Step 1: Dissolve a certain amount of mannan and / or its derivatives in HEPES buffer solution or sodium phosphate to obtain a first solution; Step 2: Mix the first solution with the metal ion solution to obtain a mixed solution; Step 3: The mixed solution is treated using ultrasound, vortexing, stirring, syringe pump or microfluidics to obtain nanoparticles; In cases where co-carrying of antigen is required, the antigen is dissolved in the HEPES buffer solution.

[0101] Optionally, in step 3, the ultrasonic power is 30-300W, the time is 0.5-30min, the vortex time is 10-60s, the infusion pump flow rate is 5-50ml / min, the microfluidic flow rate ratio is 1:1-1:5, and the flow rate is 5-40ml / min.

[0102] The present invention also provides the use of the nano-vaccine adjuvant described in any of the above embodiments in the preparation of vaccines for the prevention or treatment of infectious diseases.

[0103] Optionally, the infectious diseases include influenza, hepatitis B, hepatitis C, SARS-CoV-2, and bacterial infections.

[0104] The present invention also provides the use of the nanovaccine adjuvant described in any of the above embodiments in the preparation of vaccines for the prevention or treatment of tumors.

[0105] This invention also provides the use of the nanovaccine adjuvant described in any of the above embodiments in the preparation of medicaments that assist in enhancing the efficacy of immune checkpoint therapy.

[0106] Although preferred embodiments of the present invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present invention.

[0107] The preparation method and application of a nano-adjuvant targeting antigen-presenting cells provided by the present invention have been described in detail above. Specific examples have been used to illustrate the principle and implementation of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A nanovaccine adjuvant targeting antigen-presenting cells, characterized in that, Nanoparticles are formed by combining mannan and / or its derivatives with metal ionic compounds, wherein the average particle size of the nanoparticles is 50-500 nm.

2. The nano-vaccine adjuvant according to claim 1, characterized in that, The nanovaccine adjuvant also contains one or more of the following immunostimulatory molecules: sodium aescinate, poly I:C, CpG, 2'3'-cGAMP, ginsenosides, and QuilA.

3. The nano-vaccine adjuvant according to claim 1, characterized in that, The metal ion in the metal ion compound is selected from one or more of aluminum, iron, zirconium, calcium, manganese, cadmium, magnesium, cerium, cobalt, gallium, or zinc, and the metal ion compound is in the form of an inorganic salt or an organic complex.

4. The nano-vaccine adjuvant according to claim 1, characterized in that, The mannan and its derivatives are derived from yeast and hemolytic streptococci; the mannan derivatives are... - Mannan peptides derived from hemolytic streptococci; the molecular weight range of the mannan and its derivatives is 16-90 kDa.

5. The nano-vaccine adjuvant according to any one of claims 1-4, characterized in that, The nanoparticles are mixed with antigens or the antigens are encapsulated within the nanoparticles. The antigens are selected from: inactivated pathogens; protein antigens such as malaria antigen, hepatitis A, hepatitis B, or hepatitis C antigen, tetanus toxoid, diphtheria toxin, cholera toxin, pertussis toxin, Japanese encephalitis virus, influenza virus, tuberculosis, SARS-CoV-2, herpes simplex virus, human papillomavirus, measles virus, rubella virus, mumps virus, Ebola virus, rabies virus, and respiratory syncytial virus. The following antigens are included: West Nile virus, cytomegalovirus, Streptococcus pneumoniae, Legionella pneumophila, Neisseria meningitidis, Pseudomonas aeruginosa, Vibrio cholerae, Group A Streptococcus antigen, or other recombinant protein antigens; protein antigens with weak immunogenicity, including: bovine serum albumin, lysozyme, transferrin, insulin, lactalbumin, myoprotein, soy albumin, wheat albumin, myoglobin, collagen, laminin; polypeptide antigens, including: TRP2, HGP100, p15E, HPV E6, HPVE7, OVA epitope peptide, hepatitis B epitope peptide, SARS-CoV-2 epitope peptide, MC38 antigen, or other synthetic polypeptide antigens and long polypeptide antigens containing several polypeptide sequences; one or more of the following: viral or bacterial lysate antigens, viral or bacterial outer membrane vesicle antigens, tumor cell lysate antigens, tumor cell membrane vesicle antigens, and tumor cell exosome antigens.

6. The nano-vaccine adjuvant according to claim 1, characterized in that, When the metal ion compound is aluminum hydroxide, the mass ratio of the metal ion compound to mannan or its derivative is 1-20:10-100.

7. The nano-vaccine adjuvant according to claim 1, characterized in that, The nano-vaccine adjuvant is a lyophilized formulation.

8. A method for preparing the nano-vaccine adjuvant according to any one of claims 1-7, characterized in that, The method includes the following steps: Step 1: Dissolve a certain amount of mannan and / or its derivatives in HEPES buffer solution or sodium phosphate to obtain a first solution; Step 2: Mix the first solution with the metal ion solution to obtain a mixed solution; Step 3: The mixed solution is treated using ultrasound, vortexing, stirring, syringe pump or microfluidics to obtain nanoparticles; In cases where co-carrying of antigen is required, the antigen is dissolved in the HEPES buffer solution.

9. The method according to claim 8, characterized in that, In step 3, the ultrasonic power is 30-300W, the time is 0.5-30min, the vortex time is 10-60s, the infusion pump flow rate is 5-50ml / min, the microfluidic flow rate ratio is 1:1-1:5, and the flow rate is 5-40ml / min.

10. The application of the nano-vaccine adjuvant according to any one of claims 1-7, characterized in that, It can be used to prepare vaccines for the prevention or treatment of infectious diseases, or to prepare vaccines for the prevention or treatment of tumors, or to prepare drugs that can help enhance the effects of immune checkpoint therapy.

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