Inhalable organic nanoparticle vaccine and preparation method and application thereof

Abstract

The application provides an inhaled organic nanoparticle vaccine and a preparation method and application thereof. The inhaled organic nanoparticle vaccine comprises an organic high polymer, an immune adjuvant and an antigen, the organic nanoparticle vaccine has good appearance, uniform particles, simple preparation, good safety, convenient immunization scheme, high protection rate and other characteristics, and has a significant effect on preventing pathogen infection. The organic high polymer has positive charges on the surface, is conducive to loading the immune adjuvant through electrostatic adsorption, has a cyclic amino group with a proton sponge effect, can mediate lysosome escape of the immune adjuvant, can efficiently load the antigen and the adjuvant, and can realize target co-delivery of the antigen and the adjuvant.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an inhaled organic nanoparticle vaccine, its preparation method, and its application. Background Technology

[0002] Subunit vaccines, due to their inherent degradability and low immunogenicity, typically require adjuvants to enhance their protective effect. cGAMP, as a highly effective STING pathway agonist, can enhance vaccine immunogenicity, activate antigen-presenting cells, and strengthen specific T-cell responses, thus it is considered a potential next-generation vaccine adjuvant. However, its poor cell membrane permeability and rapid clearance after administration limit its application as a vaccine adjuvant. Organic nanoparticle vaccines can effectively deliver and protect adjuvants and antigens, achieving targeted delivery and thus improving immunogenicity. Furthermore, organic polymer nanoparticle vaccines exhibit good biocompatibility and degradability, contributing to improved vaccine safety. However, currently, there is a lack of organic polymers capable of effectively co-delivering cGAMP adjuvants and antigens, both domestically and internationally. Summary of the Invention

[0003] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0004] The first aspect of this invention provides an organic polymer nanoparticle vaccine, wherein the organic polymer nanoparticle vaccine comprises an organic polymer, an immune adjuvant, and an antigen;

[0005] The organic polymer is composed of CP1 and CP2;

[0006] The structure of CP1 is shown in equation (1):

[0007]

[0008] Equation (1);

[0009] The structure of CP2 is shown in equation (2):

[0010]

[0011] Equation (2).

[0012] In this invention, the term "vaccine" refers to a biological composition that can stimulate the immune system and enable it to fight infection or disease. The vaccine includes preventative vaccines and therapeutic vaccines. Preventative vaccines are used in healthy individuals or individuals who have not yet been infected with a specific pathogen to prevent future infections or diseases by activating the immune system in advance. Therapeutic vaccines are used in individuals who are already ill to help control disease progression and eliminate pathogens or abnormal cells (such as cancer cells) by regulating or enhancing the immune system.

[0013] In some implementations, the vaccine is selected from preventative vaccines.

[0014] In some implementations, the mass ratio of CP1 to CP2 is 1:2 to 5:1.

[0015] In one specific implementation, the mass ratio of CP1 to CP2 is 3:2.

[0016] In some embodiments, the mass ratio of the organic polymer to the immune adjuvant is 5:1 to 20:1.

[0017] In one specific embodiment, the mass ratio of the organic polymer to the immune adjuvant is 10:1.

[0018] In some embodiments, the mass ratio of the organic polymer to the antigen is 5:1 to 20:1.

[0019] In one specific embodiment, the mass ratio of the organic polymer to the antigen is 10:1.

[0020] In some implementations, m in equations (1)-(2) is an integer from 2 to 500.

[0021] In some implementations, m in equations (1)-(2) is an integer between 50 and 200.

[0022] In one specific implementation, m in equations (1)-(2) is 113.

[0023] In the equations (1) and (2), n is an integer between 2 and 20.

[0024] In one specific implementation, n is 10 in equations (1)-(2).

[0025] In this invention, the term "immune adjuvant" refers to a nonspecific immune enhancer, also known as a nonspecific immune proliferator, which, when injected into the body together with or beforehand with an antigen, can effectively enhance the strength of the immune response or alter the type of the immune response.

[0026] In some embodiments, the immune adjuvant includes, but is not limited to, aluminum salt adjuvants, STING agonists, water-in-oil emulsions, saponin adjuvants, Toll-like receptor agonists, liposome / nanoparticle adjuvants, and cytokine adjuvants.

[0027] In some embodiments, the immune adjuvant is selected from STING agonists.

[0028] In some embodiments, the STING agonist includes, but is not limited to, cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, and SNX281.

[0029] In one specific implementation, the STING agonist is selected from cGAMP.

[0030] In this invention, the term "antigen" refers to a substance that induces a specific immune response in a host animal. Types of antigens include, but are not limited to, nucleic acids, proteins, polypeptides, bacteria, fungi, viruses, or toxoids.

[0031] Based on the type of antigen, vaccines can be classified into, but are not limited to, live attenuated vaccines, inactivated vaccines, polysaccharide and polysaccharide conjugate vaccines, subunit vaccines, and nucleic acid vaccines.

[0032] In this invention, attenuated live vaccines refer to a type of live vaccine in which the structure of pathogens such as live viruses or live bacteria is changed after being treated by chemical or physical methods, resulting in a significant reduction in their toxicity while retaining their antigenicity. They generally have a long development cycle, but can induce strong immune efficacy and are of great significance for pandemics of highly lethal and highly pathogenic infectious diseases.

[0033] Inactivated vaccines are a type of vaccine in which pathogenic microorganisms are completely deactivated by physical or chemical treatments but retain their immunogenicity. Compared with live attenuated vaccines, inactivated vaccines are relatively safer but have lower immunogenicity. Considering their lower immunogenicity and immunogenicity, they are usually used together with immune adjuvants to help activate the immune system.

[0034] Subunit vaccines are composed of specific components of a pathogen that can elicit an immune response, typically including proteins and peptides. Compared to attenuated or inactivated vaccines, subunit vaccines retain only the pathogen antigens that trigger an immune response, thus effectively improving vaccine safety.

[0035] Nucleic acid vaccines are vaccines that use plasmids as a medium to inject a foreign gene sequence that encodes a specific protein into the body and express the corresponding protein antigen in the host cells, thereby inducing the host to produce a corresponding immune response and exerting the effect of a vaccine.

[0036] In some implementations, the antigen is selected from proteins.

[0037] In some implementations, the vaccine is selected from subunit vaccines.

[0038] Based on disease classification, vaccines can be divided into vaccines against tumors, vaccines against infectious diseases, and vaccines against chronic diseases.

[0039] Among these, tumor vaccines include, but are not limited to, messenger RNA tumor vaccines, viral vector tumor vaccines, and tumor peptide vaccines. Messenger RNA tumor vaccines are produced through in vitro transcription to obtain mRNA sequences encoding tumor-specific antigens or tumor-associated antigens. These sequences are then prepared as vaccines and injected into the human body, where they translate to produce antigen proteins, thereby inducing a specific immune response. Viral vector tumor vaccines utilize modified viruses with replication defects or attenuated viruses as vectors to deliver the genetic information encoding tumor antigens to host cells, prompting the expression of these antigens and activating a specific anti-tumor immune response. Tumor peptide vaccines are vaccines produced from peptides designed and chemically synthesized based on the amino acid sequences of tumor antigen epitopes.

[0040] Vaccines for infectious diseases include, but are not limited to, vaccines against diseases caused by pathogens such as bacteria and viruses. The bacteria include, but are not limited to, the phyla Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, Spirochetes, and Chlamydia. Proteobacteria include, but are not limited to, Enterobacteriaceae, Vibrioceae, Pseudomonas, Yersinia, and Klebsiella; Firmicutes include, but are not limited to, Bacillus, Staphylococcus, and Clostridium; Actinobacteria include, but are not limited to, Mycobacterium and Streptomyces; Bacteroidetes include, but are not limited to, Bacteroidetes and Prevotella; Spirochetes include, but are not limited to, Leptospira and Treponema; and Chlamydia include, but are not limited to, Chlamydia. The viruses include, but are not limited to, the following families of viruses: Retroviridae, Clonorviridae, Coronaviridae, Filoviridae, Rhabdoviridae, Bunyaviridae, Orthomyxoviridae, Paramyxoviridae, Arenaviridae, Hepatoviridae, Herpesviridae, Flaviviridae, Baculoviridae, Poxviridae, or Picornaviridae.

[0041] Vaccines targeting chronic diseases can be used to treat chronic allergic diseases, diabetes, hypertension, obesity, Alzheimer's disease, rheumatoid arthritis, and other diseases.

[0042] In some implementations, the vaccine is selected from vaccines against infectious diseases.

[0043] In this invention, the infectious disease is a disease caused by bacteria.

[0044] In some embodiments, the bacteria are selected from the phylum Proteobacteria.

[0045] In some embodiments, the bacteria are selected from the genus Klebsiella.

[0046] In one specific implementation, the bacteria are selected from Klebsiella pneumoniae.

[0047] In this invention, Klebsiella pneumoniae (KP) is a Gram-negative bacterium widely distributed in nature, typically colonizing human mucous membranes such as the gastrointestinal tract, genitourinary tract, and oropharynx. Highly virulent Klebsiella pneumoniae (hvKP) is a pathogenic form still evolving, with stronger virulence than classic Klebsiella pneumoniae (cKP). hvKP infection occurs globally, primarily infecting healthy individuals, leading to community-acquired infections including liver abscess, meningitis, necrotizing fasciitis, endophthalmitis, and severe pneumonia, which can be life-threatening.

[0048] In this invention, the protein can be a recombinant protein or a natural protein.

[0049] In some implementations, the protein is selected from recombinant proteins.

[0050] In one specific embodiment, the protein is a Klebsiella pneumoniae-associated protein, which includes, but is not limited to, fimbrial adhesion proteins, including, but not limited to, fimH protein.

[0051] In some implementations, the organic nanoparticle vaccine may be administered via methods including, but not limited to, inhalation, injection, oral administration, nasal spray, and transdermal administration.

[0052] In some implementations, the organic nanoparticle vaccine is administered via inhalation.

[0053] A second aspect of the present invention provides an organic polymer composed of CP1 and CP2;

[0054] The structure of CP1 is shown in equation (1):

[0055]

[0056] Equation (1);

[0057] The structure of CP2 is shown in equation (2):

[0058]

[0059] Equation (2).

[0060] In some implementations, the mass ratio of CP1 to CP2 is 1:2 to 5:1.

[0061] In one specific implementation, the mass ratio of CP1 to CP2 is 3:2.

[0062] In some implementations, m in equations (1)-(2) is an integer from 2 to 500.

[0063] In some implementations, m in equations (1)-(2) is an integer between 50 and 200.

[0064] In one specific implementation, m in equations (1)-(2) is 113.

[0065] In the equations (1) and (2), n is an integer between 2 and 20.

[0066] In one specific implementation, n is 10 in equations (1)-(2).

[0067] A third aspect of the present invention provides an immune composition comprising the organic polymer and an immune adjuvant described in the second aspect of the present invention.

[0068] In some embodiments, the mass ratio of the organic polymer to the immune adjuvant is 5:1 to 20:1.

[0069] In one specific embodiment, the mass ratio of the organic polymer to the immune adjuvant is 10:1.

[0070] In some embodiments, the immune adjuvant includes, but is not limited to, aluminum salt adjuvants, water-in-oil emulsions, saponin adjuvants, Toll-like receptor agonists, liposome / nanoparticle adjuvants, and cytokine adjuvants.

[0071] In some embodiments, the immune adjuvant is selected from STING agonists.

[0072] In some embodiments, the STING agonist includes, but is not limited to, cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, and SNX281.

[0073] In one specific implementation, the STING agonist is selected from cGAMP.

[0074] A fourth aspect of the present invention provides a pharmaceutical composition comprising the organic polymer nanoparticle vaccine of the first aspect of the present invention, the organic polymer of the second aspect of the present invention, or the immune composition of the third aspect of the present invention.

[0075] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0076] In some embodiments, the pharmaceutically acceptable carrier and / or excipients include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0077] In some embodiments, the diluent includes, but is not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binder includes, but is not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginates, xanthan gum, and hydroxypropylcellulose. The surfactant includes, but is not limited to, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol. The lubricant includes, but is not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolaurate, and magnesium lauryl sulfate. The filler includes, but is not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrant includes, but is not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropylmethyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0078] The fifth aspect of the present invention provides a method for preparing the organic polymer nanoparticle vaccine according to the first aspect of the present invention, the method comprising mixing the organic polymer according to the second aspect of the present invention with an immune adjuvant and an antigen.

[0079] In some embodiments, the concentration of the immune adjuvant used is 100-500 μg / mL.

[0080] In one specific implementation, the concentration of the immune adjuvant used is 200 μg / mL.

[0081] In some implementations, the concentration of the antigen used is 100-500 μg / mL.

[0082] In one specific implementation, the concentration of the antigen used is 200 μg / mL.

[0083] The sixth aspect of the present invention provides a method for preparing the organic polymer described in the second aspect of the present invention, the method comprising: dissolving CP1 and CP2 in an organic solvent and dialysis to remove the organic solvent.

[0084] In some embodiments, the organic solvent includes, but is not limited to, methanol, ethanol, isopropanol, formalin, chloroform, acetone, hydrogen sulfide, and dimethyl sulfoxide.

[0085] In some embodiments, the organic solvent is selected from dimethyl sulfoxide.

[0086] In some embodiments, the concentration of CP1 after dissolving in an organic solvent is 20-40 mg / mL.

[0087] In one specific embodiment, the concentration of CP1 after dissolving in an organic solvent is 30 mg / mL.

[0088] In some embodiments, the concentration of CP2 after dissolving in an organic solvent is 10-30 mg / mL.

[0089] In one specific embodiment, the concentration of CP2 after being dissolved in an organic solvent is 20 mg / mL.

[0090] In some embodiments, the method further includes a method for preparing CP1, comprising the following steps:

[0091] 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl) disulfide were dissolved in anhydrous N,N-dimethylformamide, and the reaction was carried out to obtain the reaction system;

[0092] After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, the solution was added to the reaction system, and the reaction continued.

[0093] Remove N,N-dimethylformamide and dry to obtain the intermediate product;

[0094] The intermediate was dissolved in N,N-dimethylformamide, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After the reaction, 2-(4-methylpiperidin-1-yl)ethanol was added and the reaction was continued.

[0095] Remove N,N-dimethylformamide and dry to obtain CP1.

[0096] In some embodiments, the equivalent ratio of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide and polyethylene glycol is (5-15):(5-15):(1-5).

[0097] In one specific embodiment, the equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is 11:10:2.

[0098] In some embodiments, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0099] In one specific embodiment, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.11 eq / mL.

[0100] In some embodiments, the concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0101] In one specific embodiment, the concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL.

[0102] In some embodiments, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

[0103] In one specific embodiment, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.02 eq / mL.

[0104] In some embodiments, the equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 2-(4-methylpiperidin-1-yl)ethanol is (0.5-5):(1-5):(1-5):(5-10).

[0105] In one specific embodiment, the equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 2-(4-methylpiperidin-1-yl)ethanol is 1:2:2:8.

[0106] In some embodiments, the concentration of the intermediate product after dissolving in N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0107] In one specific embodiment, the concentration of the intermediate product after dissolving in N,N-dimethylformamide is 0.1 eq / mL.

[0108] In some implementations, the drying process includes, but is not limited to, freeze drying, vacuum distillation, and spray drying.

[0109] In one specific implementation, the removal method includes dialysis.

[0110] In some implementations, the drying process includes freeze drying.

[0111] In some embodiments, the method further includes a method for preparing CP2, comprising the following steps:

[0112] 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl) disulfide were dissolved in anhydrous N,N-dimethylformamide, and the reaction was carried out to obtain the reaction system;

[0113] After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, the solution was added to the reaction system, and the reaction continued.

[0114] Remove N,N-dimethylformamide and dry to obtain the intermediate product;

[0115] The intermediate was dissolved in N,N-dimethylformamide, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After the reaction, 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid was added, and the reaction was continued.

[0116] Remove N,N-dimethylformamide and dry to obtain CP2.

[0117] In some embodiments, the equivalent ratio of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide and polyethylene glycol is (5-15):(5-15):(1-5).

[0118] In one specific embodiment, the equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is 11:10:2.

[0119] In some embodiments, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0120] In one specific embodiment, the concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.11 eq / mL.

[0121] In some embodiments, the concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0122] In one specific embodiment, the concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL.

[0123] In some embodiments, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

[0124] In one specific embodiment, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.02 eq / mL.

[0125] In some embodiments, the equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid is (0.5-5):(1-5):(1-5):(1-5).

[0126] In one specific embodiment, the equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 2-(4-methylpiperidin-1-yl)ethanol is 1:2:2:2.

[0127] In some embodiments, the concentration of the intermediate product after dissolving in N,N-dimethylformamide is 0.05-0.15 eq / mL.

[0128] In one specific embodiment, the concentration of the intermediate product after dissolving in N,N-dimethylformamide is 0.1 eq / mL.

[0129] In some implementations, the drying process includes, but is not limited to, freeze drying, vacuum distillation, and spray drying.

[0130] In one specific implementation, the removal method includes dialysis.

[0131] In some implementations, the drying process includes freeze drying.

[0132] The seventh aspect of the present invention provides a method for preparing the immune composition described in the third aspect of the present invention, the method comprising dissolving the organic polymer and the immune adjuvant described in the second aspect of the present invention in an organic solvent, and dialysis to remove the organic solvent.

[0133] In some embodiments, the organic solvent includes, but is not limited to, methanol, ethanol, isopropanol, formalin, chloroform, acetone, hydrogen sulfide, and dimethyl sulfoxide.

[0134] In some embodiments, the organic solvent is selected from dimethyl sulfoxide.

[0135] In some embodiments, the concentration of the organic polymer dissolved in the organic solvent is 30-70 mg / mL.

[0136] In one specific embodiment, the concentration of the organic polymer dissolved in the organic solvent is 50 mg / mL.

[0137] In some embodiments, the concentration of the immune adjuvant dissolved in the organic solvent is 1-10 mg / mL.

[0138] In one specific embodiment, the concentration of the immune adjuvant dissolved in the organic solvent is 5 mg / mL.

[0139] The eighth aspect of the present invention provides any of the following methods:

[0140] (1) A method for promoting the maturation of dendritic cells, the method comprising administering the organic polymer nanoparticle vaccine of the first aspect of the present invention or the organic polymer of the second aspect of the present invention or the immune composition of the third aspect of the present invention or the pharmaceutical composition of the fourth aspect of the present invention;

[0141] (2) A method for promoting cytokine secretion, the method comprising administering the organic polymer nanoparticle vaccine of the first aspect of the present invention, or the organic polymer of the second aspect of the present invention, or the immune composition of the third aspect of the present invention, or the pharmaceutical composition of the fourth aspect of the present invention.

[0142] In some implementations, the cytokine is IFN-β.

[0143] The ninth aspect of the present invention provides any of the following applications:

[0144] (1) The use of the organic polymer nanoparticle vaccine of the first aspect of the present invention, or the organic polymer of the second aspect of the present invention, or the immune composition of the third aspect of the present invention, or the pharmaceutical composition of the fourth aspect of the present invention in the preparation of a drug for preventing pathogen infection;

[0145] In some implementations, the pathogens include, but are not limited to, bacteria, viruses, fungi, parasites, and prions.

[0146] In some implementations, the pathogen is selected from bacteria.

[0147] In one specific implementation, the bacteria include Klebsiella pneumoniae.

[0148] (2) The use of the organic polymer described in the second aspect of the present invention, the immune composition described in the third aspect of the present invention, or the pharmaceutical composition described in the fourth aspect of the present invention in the preparation of vaccines;

[0149] (3) The use of the organic polymer described in the second aspect of the present invention or the immune composition described in the third aspect of the present invention as a drug carrier;

[0150] Organic polymers or immune compositions can serve as drug carriers to deliver antibacterial, antiviral, or anticancer drugs, exerting antibacterial, antiviral, or anticancer effects while enhancing immune stimulation.

[0151] (4) The use of the organic polymer described in the second aspect of the present invention or the immune composition described in the third aspect of the present invention as a pharmaceutical dressing;

[0152] Organic polymers or immune compositions can be used as medicated dressings to accelerate wound healing.

[0153] (5) The application of the organic polymer described in the second aspect of the present invention in loaded immune adjuvants;

[0154] In some embodiments, the immune adjuvant includes, but is not limited to, aluminum salt adjuvants, STING agonists, water-in-oil emulsions, saponin adjuvants, Toll-like receptor agonists, liposome / nanoparticle adjuvants, and cytokine adjuvants.

[0155] In some embodiments, the immune adjuvant is selected from STING agonists.

[0156] In some embodiments, the STING agonist includes, but is not limited to, cGAMP, MSA-2, ADU-S100, MK-1454, SR-717, diABZI, and SNX281.

[0157] In one specific implementation, the STING agonist is selected from cGAMP.

[0158] (6) The application of the organic polymer nanoparticle vaccine of the first aspect of the present invention, or the organic polymer of the second aspect of the present invention, or the immune composition of the third aspect of the present invention, or the pharmaceutical composition of the fourth aspect of the present invention in promoting the maturation of dendritic cells;

[0159] (7) The use of the organic polymer nanoparticle vaccine of the first aspect of the present invention, or the organic polymer of the second aspect of the present invention, or the immune composition of the third aspect of the present invention, or the pharmaceutical composition of the fourth aspect of the present invention in promoting cytokine secretion.

[0160] In some implementations, the cytokine is IFN-β.

[0161] The advantages and beneficial effects of this invention are as follows:

[0162] This invention provides an inhaled organic nanoparticle vaccine, its preparation method, and its application. The inhaled organic nanoparticle vaccine comprises an organic polymer, an immune adjuvant, and an antigen. The organic nanoparticle vaccine exhibits good morphology, uniform particle size, and simple preparation, and possesses characteristics such as good safety, convenient immunization protocol, and high protection rate, demonstrating significant effectiveness in preventing pathogen infection. The organic polymer carries a positively charged surface, which facilitates the loading of the immune adjuvant through electrostatic adsorption. Its cyclic amino groups possess a proton sponge effect, mediating the lysosomal escape of the immune adjuvant, enabling efficient loading of both antigen and adjuvant, and achieving targeted co-delivery of both. Attached Figure Description

[0163] Figure 1 The 1H NMR spectra of polymers CP1 and CP2 prepared in Example 1 of this invention are shown. a is the 1H NMR spectrum of CP1, and b is the 1H NMR spectrum of CP2.

[0164] Figure 2 Scanning electron microscope image of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention. Scale bar: 200 nm.

[0165] Figure 3 This is a Zeta potential diagram of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention and during the process of nanoparticle preparation.

[0166] Figure 4 This document describes the preparation of an organic nanoparticle vaccine against Klebsiella pneumoniae in Example 1 of the present invention, and the particle size distribution of the nanoparticles during the process.

[0167] Figure 5 The image shows the Coomassie brilliant blue staining verification results of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention.

[0168] Figure 6 This figure shows the cellular-level biosafety evaluation results of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention.

[0169] Figure 7 The figure shows the in vitro biocompatibility evaluation results of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention.

[0170] Figure 8 This image shows the in vivo biosafety evaluation results of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention. Magnification: 200x.

[0171] Figure 9 This image shows the effect of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention on promoting the maturation of isolated dendritic cells in mouse bone marrow.

[0172] Figure 10 This is a diagram showing the effect of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention on promoting the secretion of type I interferon.

[0173] Figure 11 The image shows the effect of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention in promoting the production of IgG antibodies in mice.

[0174] Figure 12 For 20×LD 50 Survival curves of mice immunized with organic nanoparticle vaccines after Klebsiella pneumoniae challenge.

[0175] Figure 13For 20×LD 50 Figure showing the weight changes in mice immunized with organic nanoparticle vaccines after Klebsiella pneumoniae challenge. Detailed Implementation

[0176] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0177] Example 1: Preparation and characterization of organic nanoparticle vaccine loaded with Klebsiella pneumoniae VacKPN9 protein

[0178] Synthesis of polymer CP1: 1,2,4,5-cyclohexanetetracarboxylic dianhydride (1.1 eq) and bis(2-hydroxyethyl) disulfide (1.0 eq) were dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF). The mixture was magnetically stirred at room temperature and reacted for 24 h. Then, polyethylene glycol (0.2 eq) was dissolved in 10 mL of anhydrous DMF and added to the reaction mixture, and the reaction was continued for another 24 h. After the reaction was complete, the reaction solution was transferred to a dialysis bag (8-14 kDa) and dialyzed for 48 h. After the DMF was completely removed from the dialysis bag, the solution was transferred to a lyophilizer and freeze-dried to obtain a white solid product. Weigh out the above-mentioned white polymer (1.0 eq) and dissolve it in 10 mL of DMF. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.0 eq) and 4-dimethylaminopyridine (2.0 eq). After activating the reaction for 30 min, add 2-(4-methylpiperidin-1-yl)ethanol (8.0 eq) and continue the reaction for 24 h before stopping the reaction. Transfer the reaction solution to a dialysis bag and dialyze for 48 h. After the DMF in the dialysis bag is completely removed, transfer the solution to a lyophilizer and freeze-dry to obtain the white solid product CP1.

[0179] Synthesis of polymer CP2: 1,2,4,5-cyclohexanetetracarboxylic dianhydride (1.1 eq) and bis(2-hydroxyethyl) disulfide (1.0 eq) were dissolved in 10 mL of DMF and reacted with magnetic stirring at room temperature for 24 h. Then, polyethylene glycol (0.2 eq) was dissolved in 10 mL of anhydrous DMF and added to the reaction mixture, and the reaction continued for another 24 h. After the reaction was complete, the reaction solution was transferred to a dialysis bag and dialyzed for 48 h. After the DMF was completely removed from the dialysis bag, the solution was transferred to a lyophilizer and freeze-dried to obtain a white solid product. Weigh out the above-mentioned white polymer (1.0 eq) and dissolve it in 10 mL of DMF. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (2.0 eq) and 4-dimethylaminopyridine (2.0 eq). After activating the reaction for 30 min, add 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid (2.0 eq) and continue the reaction for 24 h. Then stop the reaction. Transfer the reaction solution to a dialysis bag and dialyze for 48 h. After the DMF in the dialysis bag is completely removed, transfer the solution to a lyophilizer and freeze-dry to obtain the white solid product CP2.

[0180] CP1 and CP2 together constitute the polymeric backbone of the organic nanoparticle vaccine. The 1H NMR spectra of the organic polymers CP1 and CP2 are shown below. Figure 1 .

[0181] Preparation of organic nanoparticle vaccine: CP1 (3 mg), CP2 (2 mg), and cGAMP (0.5 mg) were weighed and dissolved in 0.1 mL of dimethyl sulfoxide (DMSO) using a water bath sonication method. The solution was then rapidly added to 0.9 mL of PBS and stirred. The solution was then dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 3.5 kDa to remove DMSO, and the concentration of cGAMP in the solution was measured. Subsequently, a certain volume of the above solution (to achieve a working concentration of cGAMP of 200 μg / mL), 125 μL of Klebsiella pneumoniae-associated protein, abbreviated as VacKPN9 protein (stock solution concentration of 1.6 μg / μL), and a certain volume of PBS were taken to a final volume of 1 mL. The reaction was carried out with magnetic stirring at room temperature for 3 h, followed by dialysis for 3 h to obtain an organic nanoparticle vaccine loaded with Klebsiella pneumoniae VacKPN9 protein and cGAMP adjuvant.

[0182] After preparation, transmission electron microscopy results showed that the nanoparticles were uniformly spherical with rough surfaces and had basically similar morphologies. Figure 2 Zeta potential results showed that the polymer backbone was positively charged, which was beneficial for loading cGAMP; after loading cGAMP and protein, it became negatively charged. Figure 3DLS results showed that the hydrodynamic size of the Klebsiella pneumoniae organic nanoparticle vaccine was approximately 160 nm. Figure 4 Coomassie brilliant blue staining results showed that VacKPN9 protein loading was successful. Figure 5 In summary, an organic nanoparticle vaccine loaded with Klebsiella pneumoniae VacKPN9 protein and cGAMP adjuvant was successfully prepared.

[0183] Example 2: Biosafety verification of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention.

[0184] BMDCs were used to evaluate the cytotoxicity of Klebsiella pneumoniae organic nanoparticle vaccines. The nanoparticle vaccines were dispersed in PBS at concentrations of 200, 300, 400, and 500 μg / mL, and BMDC cells were stimulated for 24 h. After collecting the supernatant, 100 μL CCK-8 working solution was added to each well, and the culture plates were incubated in an incubator for 4 h. The absorbance at 450 nm was measured using an ELISA reader.

[0185] The in vitro blood compatibility of a Klebsiella pneumoniae organic nanoparticle vaccine was assessed using mouse erythrocytes. First, the nanoparticle vaccine was prepared at different concentrations (200, 300, 400, and 500 μg / mL) in PBS solution. These nanoparticle solutions were then mixed with erythrocytes. As controls, deionized water-treated erythrocytes served as a positive control, and PBS-treated erythrocytes served as a negative control. All samples were incubated at room temperature for 1 h, then centrifuged at 3000 r / min for 10 min, and the supernatant was collected. Finally, the hemolysis rate was assessed by measuring the absorbance of the supernatant at 570 nm using the formula: Hemolysis rate (%) = (Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control absorbance) × 100%.

[0186] The in vivo safety of the Klebsiella pneumoniae organic nanoparticle vaccine was evaluated using a C57BL / 6J mouse model. The nanoparticle vaccine, containing 10 μg cGAMP and 10 μg VacKPN9, was administered via lung delivery in a 50 μL volume. On day 7 post-immunization, five mice were euthanized using carbon dioxide and dissected to collect the heart, liver, spleen, lungs, and kidneys. These tissues were then fixed in 4% paraformaldehyde for over 48 h for further processing. The fixed tissues were sent to Wuhan Sewell Biotechnology Co., Ltd. for a series of histological processing steps, including washing, dehydration, clearing, paraffin embedding, sectioning, and HE staining, to prepare pathological sections. Finally, these sections were examined under an optical microscope and histopathologically scored.

[0187] The CCK-8 assay results showed that when the working concentration of the nanoparticle vaccine was increased to 500 μg / mL, it had no significant toxicity to BMDC cells after 24 h of stimulation. Figure 6 This indicates that the nanoparticle vaccine has good safety at the cellular level. The in vitro safety of the vaccine was preliminarily verified using a hemolysis assay, which showed a hemolysis rate of less than 5% at a concentration of 500 μg / mL. Figure 7 This indicates that the vaccine has good biosafety in vitro. In vivo pathological sections showed no significant histopathological changes in any tissue after immunization. Figure 8 This indicates that the vaccine has good biocompatibility in vivo.

[0188] Example 3: Determination of the activation effect of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of the present invention on innate immunity.

[0189] Dendritic cells (BMDCs) isolated from the bone marrow of C57BL / 6J mice were used in two experimental groups and one negative control group. The experimental groups were Mock, cGAMP + VacKPN9, and polymeric backbone + cGAMP + VacKPN9, respectively, with three replicates per group. After being added to the BMDCs, they were incubated at 37 ℃ in a 5% CO2 incubator. BMDC maturity was assessed by flow cytometry, and the secretion of type I IFN was detected by ELISA. Specific detection methods are as follows.

[0190] 1. Flow cytometry was used to detect the maturity of BMDCs.

[0191] (1) At a concentration of 1×10 in 1 mL 6 The corresponding experimental group stimulants were added to BMDCs at a concentration of cells / mL and incubated for 24 h.

[0192] (2) Transfer the cells to a flow cytometer, centrifuge at 400×g for 5 min, and discard the supernatant. Then add 1 mL of DPBS, centrifuge again at 400×g for 5 min, discard the supernatant, and repeat this step twice.

[0193] (3) Staining

[0194] 1) Add 0.1 μL of FVD780 (APC-Cy7) live / dead dye to each flow cytometer. Incubate at room temperature for 10 min.

[0195] 2) Add 1 mL of FACS buffer (DPBS containing 1% FBS), mix well, centrifuge at 350×g for 5 min, discard the supernatant, and disperse the cell clumps.

[0196] 3) Add 1 µl of Fc receptor blocker (CD16 / 32) and incubate at room temperature in the dark for 10 min.

[0197] 4) Add the flow cytometry antibody (prepared in advance), mix well, and incubate at room temperature in the dark for 25 min.

[0198] 5) Add 2 mL of DPBS washing buffer, mix well, centrifuge (350×g, 5 min), discard the supernatant, and break up the cell clumps. Repeat this step twice.

[0199] 6) Add 0.2 mL of DPBS washing buffer and analyze the sample using a BD flow cytometer.

[0200] 2. ELISA detection of cytokine secretion

[0201] (1) Sample preparation: The corresponding stimulants for the experimental groups were added to the pre-coated BMDCs. After 24 h of stimulation, the cell supernatant was collected. Then, the supernatant was centrifuged at 1000 r / min for 5 min and the supernatant was aspirated.

[0202] (2) Preparation of ELISA kit:

[0203] Thirty minutes before the experiment, allow the samples to warm to room temperature. If crystals appear in the concentrated wash buffer, heat it in a 37°C water bath until the crystals are completely dissolved. Then, calculate the required volume of diluted wash buffer based on the experimental needs, and dilute the 20-fold concentrated wash buffer with deionized water to prepare the working solution. Store any unused concentrated wash buffer at 4°C. When washing the plate, first shake off all liquid from the wells, then pat dry on absorbent paper. Next, add 300 μL of wash buffer to each well using a wash bottle, let stand for 30 seconds, shake off all liquid from the wells again, and pat dry on absorbent paper. Repeat this process three times. Finally, prepare the standards: add 1 mL of standard / sample diluent to the lyophilized standard, let stand for 15 minutes to dissolve completely, then gently mix. Perform serial dilutions of 2-fold each time.

[0204] (3) Testing:

[0205] 1) Add 100 μL of standard and sample to each reaction well, with 3 replicates per group. After sealing, incubate the plate at 37 °C for 90 min, then wash the plate 4 times.

[0206] 2) Prepare the biotinylated antibody working solution by diluting the 100-fold antibody concentrate with the detection diluent (mix thoroughly before dilution) and adding 100 μL to each well within 30 min. After sealing the plate, incubate at 37 ℃ for 60 min, and then wash the plate 4 times.

[0207] 3) Prepare the enzyme conjugate working solution. Dilute the 100-fold concentrated enzyme conjugate with enzyme conjugate diluent to prepare the working solution (centrifuge before dilution), and add 100 μL to each well within 30 min. After sealing the plate, incubate at 37 ℃ for 30 min, and then wash the plate 5 times.

[0208] 4) Add 100 μL of chromogenic substrate to the reaction wells, seal the plate, and develop the color at 37 °C in the dark for 15 min.

[0209] 5) Add 50 μL of stop solution and immediately measure the OD value at 450 nm using a microplate reader (this must be completed within 5 minutes). Then perform data analysis.

[0210] The results showed that stimulation of C57BL / 6J mouse BMDCs with Klebsiella pneumoniae organic nanoparticle vaccine significantly increased the expression of the cell surface maturity marker CD86. Figure 9 The secretion level of the cytokine IFN-β was significantly increased. Figure 10 This indicates that the organic nanoparticle vaccine can promote cell maturation, activation of the STING signaling pathway, and secretion of cytokines, and has the effect of activating innate immune stimulation.

[0211] Example 4: Determination of the immunoprotective effect of the Klebsiella pneumoniae organic nanoparticle vaccine prepared in Example 1 of this invention.

[0212] Five experimental groups were set up: Mock, VacKPN9, cGAMP + VacKPN9, polymeric backbone + VacKPN9, and polymeric backbone + cGAMP + VacKPN9. Mice were immunized three times via lung delivery, 14 days apart. 20×LD20 was administered 14 days after the last immunization. 50 Klebsiella pneumoniae strain NTUH-K2044 was challenged via liquid aerosol delivery into the lungs. Changes in antibody titers in mice after immunization were evaluated using ELISA; the immunoprotective efficacy of the vaccine was assessed through survival analysis and weight change measurement. The specific implementation methods are as follows.

[0213] 1. Mouse immunization and challenge protocol

[0214] Immunization was administered via lung delivery, with immunizations given every 14 days for a total of three immunizations.

[0215] On day 14 after the completion of the third immunization, 2000 CFU (20×LD) was administered. 50Mice in all immunized groups were challenged with Klebsiella pneumoniae strain NTUH-K2044 via liquid aerosol lung delivery. First, mice were anesthetized via intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 mg / kg. The mice were then fixed to the operating table in a supine position. Next, the tilt angle of the operating table was adjusted to a suitable position, and the tracheal opening of the mouse was exposed using a laryngoscope. Then, the nozzle of a handheld liquid aerosol delivery device was inserted approximately 2 cm deep parallel to the trachea. The actuator was quickly operated to deliver Klebsiella pneumoniae strain NTUH-K2044 as a liquid aerosol into the mouse lungs.

[0216] 2. Serum antibody level detection

[0217] (1) Sample collection and preparation: Eight mice were randomly selected from each immunization group and marked. Blood was collected from the marked mice in each group on days 7 and 14 after each immunization using the tail vein sampling method. The mice were fixed in a restraint table with their tails exposed. The tip of the tail was cut obliquely with scissors, and blood was gently collected along the direction of the tail (about 50 μL each time). Hemostasis was achieved by applying pressure with a cotton ball, and then the sample was disinfected with alcohol. The collected serum was allowed to stand at room temperature for 2 h, then centrifuged at 3000 r / min for 10 min. The supernatant serum was aspirated into a new centrifuge tube, marked, and stored at -80 ℃ for later testing.

[0218] (2) Coating: Take 10 μg of antigen (VacKPN9) and add 10 mL of coating buffer to prepare a working solution of 1 μg / mL. Shake to mix well and add to a 96-well plate labeled with enzyme (100 μL / well, i.e. 0.1 μg / well). Seal and pack overnight at 4 ℃.

[0219] (3) Sealing: Shake off the coating liquid, add 200 μL of sealing liquid to each well, and seal the membrane at 37 ℃ for 2 h.

[0220] (4) Primary antibody incubation: Shake dry and tap to remove blocking solution. Take another 96-well plate, take dilution solution, and serially dilute the primary antibody (to be tested) 2 times. Use a multi-channel pipette to add 100 μL of the corresponding antibody at each dilution gradient to each enzyme-labeled 96-well plate, seal the plate and incubate at 37 ℃ for 30 min, then wash the plate.

[0221] (5) Secondary antibody incubation: Prepare secondary antibody (enzyme-labeled antibody), dilute 1:10000, add 100 μL to each well of each enzyme-labeled plate, seal the plate and incubate at 37 ℃ for 20 min, then wash the plate.

[0222] (6) Color development: Add 100 μL of color development reagent TMB to each well of each microplate and perform the color development reaction in the dark for 10 min.

[0223] (7) Termination: Add 100 μL of termination solution to each well of each microplate.

[0224] (8) Detection: The absorbance values ​​at wavelengths of 450 nm and 630 nm were detected using an enzyme-linked immunosorbent assay (ELISA) reader.

[0225] 3. Culture of Klebsiella pneumoniae strain

[0226] (1) Take out 20 μL of Klebsiella pneumoniae glycerol bacteria from the -80 ℃ freezer, thaw it, and transfer it to 20 mL of BHI liquid medium. Incubate at 37 ℃ and 200 r / min for 13 h to allow the bacteria to grow to the plateau phase. The absorbance at 600 nm measured by UV spectrophotometer should be between 2.2 and 2.8. This is the first generation of bacteria.

[0227] (2) Continue to dilute and culture the first generation bacteria at a ratio of 1:250 at 37 ℃ and 200 r / min for about 2.5 h to allow the bacteria to grow to the logarithmic phase. The absorbance at 600 nm measured by a UV spectrophotometer should be between 1.3 and 1.6, which indicates that this is the second generation bacteria.

[0228] (3) The second generation bacteria were passaged again at a ratio of 1:250 for about 3 hours to obtain the third generation bacteria in the logarithmic growth phase.

[0229] (4) Take 1 mL of the original third-generation bacterial culture, centrifuge at 4 ℃ and 3000 × g for 10 min, and discard the supernatant. Add 1 mL of physiological saline containing 0.05% poloxamer to resuspend the bacteria, centrifuge at 4 ℃ and 3000 × g for 10 min, and discard the supernatant.

[0230] (5) Resuspend the bacteria in physiological saline containing 0.05% poloxamer until the absorbance at 600 nm is between 1 and 1.05. The theoretical concentration of the bacterial suspension at this point is 4 × 10⁻⁶. 8 CFU / mL, and diluted to the challenge concentration for subsequent challenge experiments.

[0231] (6) Drop plate counting: After diluting the bacterial solution by a ratio of 5, take 10 μL of the bacterial solution and drop it onto a blood plate. After incubating overnight in a bacterial incubator at 37 ℃, count the colonies to calculate the actual bacterial concentration.

[0232] 4. Clinical symptom observation after Klebsiella pneumoniae challenge via liquid aerosol delivery to the lungs.

[0233] On day 14 after the third immunization, 2000 CFU (20×LD) was administered. 50Mice in all immunized groups were challenged with Klebsiella pneumoniae strain NTUH-K2044 via liquid aerosol lung delivery. The survival and weight of the mice were observed within 14 days after the challenge, and survival and weight curves were plotted.

[0234] The results showed that mice immunized with the Klebsiella pneumoniae organic nanoparticle vaccine produced a strong humoral immune response, and the serum antibody IgG titer increased with the progress of immunization and was higher than that of other immunized groups. Figure 11 ). In 20×LD 50 After challenge, the Klebsiella pneumoniae organic nanoparticle vaccine maintained a 90% survival rate in mice, significantly higher than other groups. Figure 12 , Figure 13 In summary, the Klebsiella pneumoniae organic nanoparticle vaccine enhanced the protection against Klebsiella pneumoniae infection in mice.

[0235] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An organic polymer nanoparticle vaccine, characterized in that, The organic polymer nanoparticle vaccine comprises organic polymer, immune adjuvant, and antigen; The organic polymer is composed of CP1 and CP2; The structure of CP1 is shown in equation (1): Equation (1); The structure of CP2 is shown in equation (2): Equation (2); In equations (1) and (2), m is 113; In equations (1) and (2), n is 10; The mass ratio of CP1 to CP2 is 3:2; The immune adjuvant is cGAMP; The antigen is a Klebsiella pneumoniae-associated protein.

2. The organic polymer nanoparticle vaccine according to claim 1, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 5:1 to 20:

1.

3. The organic polymer nanoparticle vaccine according to claim 2, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 10:

1.

4. The organic polymer nanoparticle vaccine according to claim 1, characterized in that, The mass ratio of the organic polymer to the antigen is 5:1 to 20:

1.

5. The organic polymer nanoparticle vaccine according to claim 4, characterized in that, The mass ratio of the organic polymer to the antigen is 10:

1.

6. The organic polymer nanoparticle vaccine according to claim 1, characterized in that, The organic nanoparticle vaccine is administered via inhalation.

7. An organic polymer, characterized in that, The organic polymer is composed of CP1 and CP2; The structure of CP1 is shown in equation (1): Equation (1); The structure of CP2 is shown in equation (2): Equation (2); The mass ratio of CP1 to CP2 is 3:2; In equations (1) and (2), m is 113; In equations (1) and (2), n is 10.

8. An immune composition, characterized in that, The immune composition comprises the organic polymer of claim 7 and the immune adjuvant; The immune adjuvant is cGAMP.

9. The immune composition according to claim 8, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 5:1 to 20:

1.

10. The immune composition according to claim 9, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 10:

1.

11. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the organic polymer nanoparticle vaccine according to any one of claims 1-6.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

13. A method for preparing the organic polymer nanoparticle vaccine according to any one of claims 1-6, characterized in that, The method includes mixing the organic polymer of claim 7 with an immune adjuvant and an antigen; The immune adjuvant is cGAMP; The antigen is a Klebsiella pneumoniae-associated protein.

14. The method according to claim 13, characterized in that, The concentration of the immune adjuvant used is 100-500 μg / mL.

15. The method according to claim 14, characterized in that, The concentration of the immune adjuvant used is 200 μg / mL.

16. The method according to claim 13, characterized in that, The concentration of the antigen used is 100-500 μg / mL.

17. The method according to claim 16, characterized in that, The concentration of the antigen used is 200 μg / mL.

18. A method for preparing the organic polymer of claim 7, characterized in that, The method includes: dissolving CP1 and CP2 in an organic solvent and dialysis to remove the organic solvent.

19. The method according to claim 18, characterized in that, The organic solvent is selected from dimethyl sulfoxide.

20. The method according to claim 18, characterized in that, The concentration of CP1 after dissolving in an organic solvent is 20-40 mg / mL.

21. The method according to claim 20, characterized in that, The concentration of CP1 after dissolving in an organic solvent is 30 mg / mL.

22. The method according to claim 18, characterized in that, The concentration of CP2 after dissolving in an organic solvent is 10-30 mg / mL.

23. The method according to claim 22, characterized in that, The concentration of CP2 after being dissolved in an organic solvent is 20 mg / mL.

24. The method according to claim 18, characterized in that, The method also includes a method for preparing CP1, comprising the following steps: 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl) disulfide were dissolved in anhydrous N,N-dimethylformamide, and the reaction was carried out to obtain the reaction system; After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, the solution was added to the reaction system, and the reaction continued. Remove N,N-dimethylformamide and dry to obtain the intermediate product; The intermediate was dissolved in N,N-dimethylformamide, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After the reaction, 2-(4-methylpiperidin-1-yl)ethanol was added and the reaction was continued. Remove N,N-dimethylformamide and dry to obtain CP1.

25. The method according to claim 24, characterized in that, The equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide and polyethylene glycol is (5-15):(5-15):(1-5).

26. The method according to claim 25, characterized in that, The equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is 11:10:

2.

27. The method according to claim 24, characterized in that, The concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

28. The method according to claim 27, characterized in that, The concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide was 0.11 eq / mL.

29. The method according to claim 24, characterized in that, The concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

30. The method according to claim 29, characterized in that, The concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL.

31. The method according to claim 24, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

32. The method according to claim 31, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide was 0.02 eq / mL.

33. The method according to claim 24, characterized in that, The equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 2-(4-methylpiperidin-1-yl)ethanol is (0.5-5):(1-5):(1-5):(5-10).

34. The method according to claim 23, characterized in that, The equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 2-(4-methylpiperidin-1-yl)ethanol is 1:2:2:

8.

35. The method according to claim 24, characterized in that, The concentration of the intermediate product dissolved in N,N-dimethylformamide is 0.05-0.15 eq / mL.

36. The method according to claim 35, characterized in that, The concentration of the intermediate product dissolved in N,N-dimethylformamide was 0.1 eq / mL.

37. The method according to claim 24, characterized in that, The removal method includes dialysis.

38. The method according to claim 24, characterized in that, The drying process includes freeze drying.

39. The method according to claim 18, characterized in that, The method also includes a method for preparing CP2, comprising the following steps: 1,2,4,5-cyclohexanetetracarboxylic dianhydride and bis(2-hydroxyethyl) disulfide were dissolved in anhydrous N,N-dimethylformamide, and the reaction was carried out to obtain the reaction system; After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, the solution was added to the reaction system, and the reaction continued. Remove N,N-dimethylformamide and dry to obtain the intermediate product; The intermediate was dissolved in N,N-dimethylformamide, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added. After the reaction, 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid was added, and the reaction was continued. Remove N,N-dimethylformamide and dry to obtain CP2.

40. The method according to claim 39, characterized in that, The equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide and polyethylene glycol is (5-15):(5-15):(1-5).

41. The method according to claim 40, characterized in that, The equivalent ratio of 1,2,4,5-cyclohexanetetracarboxylic dianhydride, bis(2-hydroxyethyl) disulfide, and polyethylene glycol is 11:10:

2.

42. The method according to claim 39, characterized in that, The concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

43. The method according to claim 42, characterized in that, The concentration of the 1,2,4,5-cyclohexanetetracarboxylic dianhydride dissolved in anhydrous N,N-dimethylformamide was 0.11 eq / mL.

44. The method according to claim 39, characterized in that, The concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 eq / mL.

45. The method according to claim 44, characterized in that, The concentration of the bis(2-hydroxyethyl) disulfide dissolved in anhydrous N,N-dimethylformamide is 0.1 eq / mL.

46. ​​The method according to claim 39, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 eq / mL.

47. The method according to claim 46, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide was 0.02 eq / mL.

48. The method according to claim 39, characterized in that, The equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid is (0.5-5):(1-5):(1-5):(1-5).

49. The method according to claim 48, characterized in that, The equivalent ratio of the intermediate product, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 3-(4-methyl-2,5-dioxo-2,5-dihydrofuran-3-yl)propionic acid is 1:2:2:

2.

50. The method according to claim 39, characterized in that, The concentration of the intermediate product dissolved in N,N-dimethylformamide is 0.05-0.15 eq / mL.

51. The method according to claim 50, characterized in that, The concentration of the intermediate product dissolved in N,N-dimethylformamide was 0.1 eq / mL.

52. The method according to claim 39, characterized in that, The removal method includes dialysis.

53. The method according to claim 39, characterized in that, The drying process includes freeze drying.

54. A method for preparing the immune composition according to any one of claims 8-10, characterized in that, The method includes dissolving the organic polymer and the immune adjuvant of claim 7 in an organic solvent, and then removing the organic solvent by dialysis. The immune adjuvant is cGAMP.

55. The method according to claim 54, characterized in that, The organic solvent is selected from dimethyl sulfoxide.

56. The method according to claim 54, characterized in that, The concentration of the organic polymer dissolved in the organic solvent is 30-70 mg / mL.

57. The method according to claim 56, characterized in that, The concentration of the organic polymer dissolved in the organic solvent is 50 mg / mL.

58. The method according to claim 54, characterized in that, The concentration of the immune adjuvant dissolved in the organic solvent is 1-10 mg / mL.

59. The method according to claim 58, characterized in that, The concentration of the immune adjuvant dissolved in the organic solvent is 5 mg / mL.

60. The use of the organic polymer nanoparticle vaccine according to any one of claims 1-6 or the pharmaceutical composition according to any one of claims 11-12 in the preparation of a drug for preventing pathogen infection; wherein the pathogen is selected from bacteria, and the bacteria is Klebsiella pneumoniae.

61. Use of the pharmaceutical composition according to any one of claims 11-12 in the preparation of a vaccine.

62. The use of the organic polymer of claim 7 in the preparation of drug carriers loaded with the immune adjuvant cGAMP and Klebsiella pneumoniae.

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