Lysosome-targeted organic polymer nanoparticle vaccine and preparation method and application thereof

Abstract

The application provides a kind of organic high molecular nanoparticle vaccine targeting lysosome and its preparation method and application.The organic high molecular nanoparticle vaccine can target lysosome, promote antigen presenting cell maturation and proinflammatory cytokine secretion, improve serum antibody titer, and enhance the immune protection effect on pathogen infection.The application also provides an organic high molecular polymer for preparing organic high molecular nanoparticle vaccine, which can accurately co-deliver adjuvant and antigen, and has good biocompatibility, degradability, and can reduce immunogenicity and side effects.

Description

Technical Field

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

[0002] TLR7 agonists, exemplified by 3M-052, possess numerous advantages and have become a hot topic in vaccine development. They can significantly activate innate immune cells, inducing strong immune responses, including promoting the generation of effector B cells and durable humoral immunity. For example, in non-human primates, 3M-052 has been shown to induce durable HIV-1 envelope-specific plasma cell and humoral immune responses, with antibody responses lasting up to approximately one year. However, TLR7 is primarily distributed within intracellular membrane structures such as lysosomes, making it difficult for TLR7 agonists to enter cells and target TLR7 in lysosomes. This results in low TLR7 activation efficiency, limiting their immune activation effects.

[0003] Organic polymer materials have broad application prospects in the field of vaccine carriers. However, current research both domestically and internationally still lacks organic polymer materials that can effectively co-deliver TLR7 agonists and antigen proteins. Summary of the Invention

[0004] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0005] The first aspect of this invention provides an organic polymer nanoparticle vaccine, which includes an organic polymer, an immune adjuvant, and an antigen.

[0006] The structure of the organic polymer is shown in formula (1):

[0007]

[0008] Equation (1);

[0009] In formula (1), R1 is a group with an amide bond;

[0010] In formula (1), R2 is a group with a pyrrole ring.

[0011] In some embodiments, the mass ratio of the organic polymer to the immune adjuvant is 10:1 to 50:1.

[0012] In one specific embodiment, the mass ratio of the organic polymer to the immune adjuvant is 30:1.

[0013] In some embodiments, the mass ratio of the organic polymer to the antigen is 10:1 to 50:1.

[0014] In one specific embodiment, the mass ratio of the organic polymer to the antigen is 30:1.

[0015] In some implementations, n in equation (1) is an integer from 2 to 500.

[0016] In some implementations, n in equation (1) is an integer between 50 and 200.

[0017] In one specific implementation, n is 113 in equation (1).

[0018] In some implementations, X in equation (1) is an integer from 1 to 500.

[0019] In one specific implementation, X in equation (1) is 113.

[0020] In one specific implementation, the structure of R1 in equation (1) is as shown in equation (2):

[0021]

[0022] Equation (2).

[0023] In one specific implementation, the structure of R2 in equation (1) is as shown in equation (3):

[0024]

[0025] Equation (3).

[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 Toll-like receptor agonists.

[0028] In some embodiments, the Toll-like receptor agonists include, but are not limited to, TLR1 receptor agonists, TLR2 receptor agonists, TLR3 receptor agonists, TLR4 receptor agonists, TLR5 receptor agonists, TLR6 receptor agonists, TLR7 receptor agonists, TLR8 receptor agonists, TLR9 receptor agonists, and TLR10 receptor agonists.

[0029] In some embodiments, the Toll-like receptor agonist is selected from TLR7 receptor agonists.

[0030] In some embodiments, the TLR7 receptor agonist includes, but is not limited to, 3M-052, Resiquimod, and Imiquimod.

[0031] In one specific embodiment, the TLR7 receptor agonist is selected from 3M-052.

[0032] In some implementations, the antigen includes, but is not limited to, nucleic acids, proteins, polypeptides, bacteria, fungi, viruses, or toxoids.

[0033] In some implementations, the antigen is selected from proteins.

[0034] In one specific embodiment, the protein is a Pseudomonas aeruginosa surface protein antigen, which includes, but is not limited to, outer membrane proteins, flagellin proteins, and fimbriae proteins.

[0035] A second aspect of the present invention provides an organic polymer, the structure of which is shown in formula (1):

[0036]

[0037] Equation (1);

[0038] In formula (1), R1 is a group with an amide bond;

[0039] In formula (1), R2 is a group with a pyrrole ring.

[0040] In some implementations, n in equation (1) is an integer from 2 to 500.

[0041] In some implementations, n in equation (1) is an integer between 50 and 200.

[0042] In some implementations, n is 113 in equation (1).

[0043] In some implementations, X in equation (1) is an integer from 1 to 500.

[0044] In one specific implementation, X in equation (1) is 113.

[0045] In one specific implementation, the structure of R1 in equation (1) is as shown in equation (2):

[0046]

[0047] Equation (2).

[0048] In one specific implementation, the structure of R2 in equation (1) is as shown in equation (3):

[0049]

[0050] Equation (3).

[0051] 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.

[0052] In some embodiments, the mass ratio of the organic polymer to the immune adjuvant is 10:1 to 50:1.

[0053] In one specific embodiment, the mass ratio of the organic polymer to the immune adjuvant is 30:1.

[0054] 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.

[0055] In some embodiments, the immune adjuvant is selected from Toll-like receptor agonists.

[0056] In some embodiments, the Toll-like receptor agonists include, but are not limited to, TLR1 receptor agonists, TLR2 receptor agonists, TLR3 receptor agonists, TLR4 receptor agonists, TLR5 receptor agonists, TLR6 receptor agonists, TLR7 receptor agonists, TLR8 receptor agonists, TLR9 receptor agonists, and TLR10 receptor agonists.

[0057] In some embodiments, the Toll-like receptor agonist is selected from TLR7 receptor agonists.

[0058] In some embodiments, the TLR7 receptor agonist includes, but is not limited to, 3M-052, Resiquimod, and Imiquimod.

[0059] In one specific embodiment, the TLR7 receptor agonist is selected from 3M-052.

[0060] 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.

[0061] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or excipient.

[0062] The fifth aspect of the present invention provides a method for preparing the organic polymer nanoparticle vaccine described in the first aspect of the present invention, the method comprising mixing the immune composition described in the third aspect of the present invention with an antigen.

[0063] In some implementations, the concentration of the antigen used is 1-50 μg / μL.

[0064] In one specific implementation, the concentration of the antigen used is 2.5 μg / μL.

[0065] 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:

[0066] Acid-sensitive dihydroxy monomer and L-lysine diisocyanate were dissolved in anhydrous N,N-dimethylformamide and reacted.

[0067] After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, it was added to the above reaction system, and the reaction continued.

[0068] After removing N,N-dimethylformamide and drying, an organic polymer is obtained.

[0069] In this invention, acid-sensitive dihydroxy monomers are a class of organic monomer molecules that can undergo specific chemical changes under acidic conditions and contain two hydroxyl groups (-OH).

[0070] In some embodiments, the acid-sensitive dihydroxy monomer has the structural formula shown in formula (2);

[0071]

[0072] Equation (2).

[0073] In some embodiments, the mass ratio of the acid-sensitive dihydroxy monomer, L-lysine diisocyanate and polyethylene glycol is (5-15):(5-15):(1-5).

[0074] In one specific embodiment, the mass ratio of the acid-sensitive dihydroxy monomer, L-lysine diisocyanate, and polyethylene glycol is 10:11:2.

[0075] In some embodiments, the concentration of the acid-sensitive dihydroxy monomer dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 g / mL.

[0076] In one specific embodiment, the concentration of the acid-sensitive dihydroxy monomer dissolved in anhydrous N,N-dimethylformamide is 0.1 g / mL.

[0077] In some embodiments, the concentration of the L-lysine diisocyanate dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 g / mL.

[0078] In one specific embodiment, the concentration of the L-lysine diisocyanate dissolved in anhydrous N,N-dimethylformamide is 0.11 g / mL.

[0079] In some embodiments, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 g / mL.

[0080] In one specific embodiment, the concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.02 g / mL.

[0081] In one specific embodiment, the polyethylene glycol is HO-PEG 5000-NHS.

[0082] In one specific implementation, the removal method includes dialysis.

[0083] In one specific implementation, the drying includes freeze drying.

[0084] In some embodiments, the method further includes a method for synthesizing acid-sensitive dihydroxy monomers, comprising:

[0085] Terephthalaldehyde and 2-hydroxyethylamine were dissolved in anhydrous methanol and reacted to obtain a precipitate. The precipitate was washed with anhydrous methanol and dried to obtain an acid-sensitive dihydroxy monomer.

[0086] In some embodiments, the mass ratio of terephthalaldehyde to 2-hydroxyethylamine is 1:1 to 1:5.

[0087] In one specific implementation, the mass ratio of terephthalaldehyde to 2-hydroxyethylamine is 1:2.

[0088] In some embodiments, the concentration of terephthalaldehyde dissolved in anhydrous methanol is 0.01-0.2 g / mL.

[0089] In one specific implementation, the concentration of terephthalaldehyde after dissolving in anhydrous methanol is 0.067 g / mL.

[0090] In some embodiments, the concentration of the 2-hydroxyethylamine dissolved in anhydrous methanol is 0.02-0.4 g / mL.

[0091] In one specific embodiment, the concentration of 2-hydroxyethylamine dissolved in anhydrous methanol is 0.13 g / mL.

[0092] 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.

[0093] In one specific embodiment, the organic solvent is selected from dimethyl sulfoxide.

[0094] In some embodiments, the concentration of the organic polymer dissolved in the organic solvent is 100-200 mg / mL.

[0095] In one specific embodiment, the concentration of the organic polymer dissolved in the organic solvent is 150 mg / mL.

[0096] The eighth aspect of the present invention provides any of the following methods:

[0097] (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;

[0098] (2) A method for activating the TLR7 signaling pathway, 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;

[0099] (3) A method for targeting lysosomes, 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.

[0100] (4) A method for promoting cytokine secretion, the method comprising administering an organic polymer nanoparticle vaccine as described in the first aspect of the present invention, or an organic polymer as described in the second aspect of the present invention, or an immune composition as described in the third aspect of the present invention, or a pharmaceutical composition as described in the fourth aspect of the present invention.

[0101] In one specific implementation, the cytokines are IL-12 and TNF-α.

[0102] The ninth aspect of the present invention provides any of the following applications:

[0103] (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;

[0104] In some implementations, the pathogens include, but are not limited to, bacteria, viruses, fungi, parasites, and prions.

[0105] In some implementations, the pathogen is selected from bacteria.

[0106] In one specific implementation, the bacteria include Pseudomonas aeruginosa.

[0107] (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;

[0108] (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;

[0109] 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.

[0110] (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;

[0111] Organic polymers or immune compositions can be used as medicated dressings to accelerate wound healing.

[0112] (5) The application of the organic polymer described in the second aspect of the present invention in loaded immune adjuvants;

[0113] 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.

[0114] In some embodiments, the immune adjuvant is selected from Toll-like receptor agonists.

[0115] In some embodiments, the Toll-like receptor agonists include, but are not limited to, TLR1 receptor agonists, TLR2 receptor agonists, TLR3 receptor agonists, TLR4 receptor agonists, TLR5 receptor agonists, TLR6 receptor agonists, TLR7 receptor agonists, TLR8 receptor agonists, TLR9 receptor agonists, and TLR10 receptor agonists.

[0116] In some embodiments, the Toll-like receptor agonist is selected from TLR7 receptor agonists.

[0117] In some embodiments, the TLR7 receptor agonist includes, but is not limited to, 3M-052, Resiquimod, and Imiquimod.

[0118] In one specific embodiment, the TLR7 receptor agonist is selected from 3M-052.

[0119] (6) 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 the maturation of dendritic cells;

[0120] (7) 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 activating the TLR7 signaling pathway;

[0121] (7) 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 targeted lysosomes;

[0122] (8) 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;

[0123] In some implementations, the cytokines are IL-12 and TNF-α.

[0124] The advantages and beneficial effects of this invention are as follows:

[0125] This invention provides an organic polymer nanoparticle vaccine targeting lysosomes, its preparation method, and its applications. The organic polymer nanoparticle vaccine targets lysosomes, promoting the maturation of antigen-presenting cells and the secretion of pro-inflammatory cytokines, increasing serum antibody titers, and enhancing the immune protection against pathogen infection. This invention also provides an organic polymer for preparing the organic polymer nanoparticle vaccine. This organic polymer can precisely co-deliver adjuvants and antigens and possesses good biocompatibility and degradability, reducing immunogenicity and side effects. Attached Figure Description

[0126] Figure 1 It is the 1H NMR spectrum of an acid-sensitive hydroxyl monomer.

[0127] Figure 2 This is the hydrogen nuclear magnetic spectrum of the polymer YAXA.

[0128] Figure 3Zeta potential maps of nanoparticles YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 prepared in Example 1 of this invention.

[0129] Figure 4 The particle size distribution of YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 prepared in Example 1 of this invention.

[0130] Figure 5 The image shows the Western blot and Coomassie brilliant blue staining verification results of YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 prepared in Example 1 of this invention.

[0131] Figure 6 The image shows the in vitro biocompatibility evaluation results of YIMIAO-3.4 prepared in Example 1 of this invention.

[0132] Figure 7 This is a graph showing the in vivo biosafety evaluation results of YIMIAO-3.7 prepared in Example 1 of this invention. Scale bar: 200 μm.

[0133] Figure 8 The image shows the effect of YIMIAO-3.7 prepared in Example 1 on promoting the maturation of isolated dendritic cells in mouse bone marrow.

[0134] Figure 9 The image shows the effect of YIMIAO-3.7 activating the TLR7 signaling pathway prepared in Example 1.

[0135] Figure 10 The image shows the effect of YIMIAO-3.7 prepared in Example 1 on promoting the secretion of IL-12 and TNF-α.

[0136] Figure 11 The image shows the effect of co-localization of YIMIAO-3.7 prepared in Example 1 with lysosomes.

[0137] Figure 12 The image shows the effect of YIMIAO-3.7 prepared in Example 1 on promoting the production of IgG antibodies in mice. Red represents YIMIAO-3.7, blue represents YIMIAO-3.3 (3M-052+VacPAE1), gray represents YIMIAO-3.2 (VacPAE1), and black represents Mock.

[0138] Figure 13 2.5×LD 50Survival curves of mice immunized with YIMIAO-3.7 after Pseudomonas aeruginosa challenge. Red represents YIMIAO-3.7, blue represents YIMIAO-3.3 (3M-052+VacPAE1), gray represents YIMIAO-3.2 (VacPAE1), and black represents Mock.

[0139] Figure 14 2.5×LD 50 Figure showing the weight changes of mice immunized with YIMIAO-3.7 after challenge with Pseudomonas aeruginosa. Red represents YIMIAO-3.7, blue represents YIMIAO-3.3 (3M-052+VacPAE1), gray represents YIMIAO-3.2 (VacPAE1), and black represents Mock.

[0140] Figure 15 2.5×LD 50 Organ bacterial load of mice immunized with YIMIAO-3.7 after challenge with Pseudomonas aeruginosa. Red represents YIMIAO-3.7, blue represents YIMIAO-3.3 (3M-052+VacPAE1), gray represents YIMIAO-3.2 (VacPAE1), and black represents Mock.

[0141] Figure 16 2.5×LD 50 Pathological changes in organs of mice immunized with YIMIAO-3.7 after challenge with Pseudomonas aeruginosa. Scale bar: 200 μm. Red represents YIMIAO-3.7, and blue represents YIMIAO-3.3 (3M-052+VacPAE1). Detailed Implementation

[0142] 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.

[0143] 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.

[0144] In this invention, the term "nanoparticle" refers to a substance with a particle size of 1 nm-100 nm or less than 1000 nm that exhibits physical, chemical, and biological effects related to its size.

[0145] 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.

[0146] 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.

[0147] 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 still 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.

[0148] Inactivated vaccines are a type of vaccine in which pathogenic microorganisms are completely deactivated by physical or chemical treatment 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.

[0149] 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.

[0150] 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.

[0151] Based on disease classification, vaccines can be divided into vaccines against tumors, vaccines against infectious diseases, and vaccines against chronic diseases.

[0152] 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.

[0153] 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, and Yersinia; 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.

[0154] Vaccines targeting chronic diseases can be used to treat chronic allergic diseases, diabetes, hypertension, obesity, Alzheimer's disease, rheumatoid arthritis, and other diseases.

[0155] In some implementations, the vaccine is selected from vaccines against infectious diseases.

[0156] In this invention, the infectious disease is a disease caused by bacteria.

[0157] In some embodiments, the bacteria are selected from the phylum Proteobacteria.

[0158] In some implementations, the bacteria are selected from the genus *Pseudomonas*.

[0159] In one specific implementation, the bacteria are selected from Pseudomonas aeruginosa.

[0160] In this invention, *Pseudomonas aeruginosa* is a Gram-negative opportunistic pathogen widely distributed in hospital environments and on the surface of organisms, and is one of the important pathogens causing nosocomial infections. This bacterium is known for its strong environmental adaptability and multidrug resistance, and can evade antibiotics through mechanisms such as efflux pump systems, biofilm formation, and horizontal transfer of resistance genes. It has been listed by the World Health Organization as a "high-risk resistant pathogen requiring the development of new antibiotics." *Pseudomonas aeruginosa* pneumonia is one of the main pathogens causing hospital-acquired pneumonia and ventilator-associated pneumonia, exhibiting high morbidity and mortality rates in intensive care units, immunocompromised patients, and patients with chronic lung disease. Its pathogenic mechanism is complex; it can damage host cells through type III secretory system toxins and secrete virulence factors such as elastase, exacerbating tissue damage.

[0161] 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.

[0162] In this invention, the pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, lubricants, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water. The binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginate and alginates, xanthan gum, and hydroxypropyl cellulose. The surfactants include, but are not limited to, sodium dodecyl sulfate, glyceryl monostearate, and hexadecyl alcohol. The lubricants include, but are not limited to, zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric acid sucrose ester, and magnesium dodecyl sulfate. The fillers include, but are not limited to, mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, and starch. The disintegrants include, but are not limited to, crosylvinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosylcarboxymethyl cellulose sodium, and soybean polysaccharides.

[0163] Example 1: Preparation and characterization of organic polymer nanoparticle vaccines targeting lysosomes

[0164] The first step is to prepare the polymer YAXA, which includes the following two steps:

[0165] (1) Synthesis of acid-sensitive dihydroxy monomer. Terephthalaldehyde (1 g, 134.13 mol) and 2-hydroxyethylamine (2 g, 122.16 mol) were dissolved in 15 mL of anhydrous methanol (8001AR0500, Concord). The mixture was magnetically stirred at room temperature for 3 h. After centrifugation, a white precipitate was obtained, which was washed with anhydrous methanol and finally dried to obtain acid-sensitive dihydroxy monomer M1.

[0166] (2) Synthesis of YAXA. Acid-sensitive dihydroxy monomer M1 (1 g) and L-lysine diisocyanate M2 (1.1 g, 248.85 mol) were dissolved in 10 mL of anhydrous N,N-dimethylformamide (8059AR0500, Concord), and the mixture was magnetically stirred at room temperature for 24 h. HO-PEG5k-NHS M3 (0.2 g) was dissolved in 10 mL of anhydrous N,N-dimethylformamide (8059AR0500, Concord) and added to the above reaction system, and the reaction was continued for another 24 h. After the reaction was complete, the solution was transferred to a dialysis bag (molecular weight cutoff 8-14 kDa, MD10 (8000-14000), Viskase) and dialyzed for 48 h. After the anhydrous N,N-dimethylformamide was completely removed from the dialysis bag, the solution was lyophilized to obtain the white solid product YAXA.

[0167] Then, the following nanoparticles were prepared, in which YIMIAO-3.7 is the final nanoparticle vaccine.

[0168] (1) YIMIAO-3.4: YAXA (60 mg) was dissolved in 0.4 mL of dimethyl sulfoxide (8068AR0500, Concord) by water bath sonication. The solution was then added dropwise to PBS (3.6 mL) and dialyzed (molecular weight cutoff 3.5 kDa, MD44-3-5, Viskase) for 3 h to remove DMSO from the solution to obtain YIMIAO-3.4.

[0169] (2) YIMIAO-3.5: YAXA (60 mg) and 3M-052 (2 mg, T17032, MedChemExpress) were dissolved in 0.4 mL of dimethyl sulfoxide (8068AR0500, Concord) by water bath sonication. The solution was then added dropwise to PBS (3.6 mL) and dialyzed (molecular weight cutoff 3.5 kDa, MD44-3-5, Viskase) for 3 h to remove DMSO from the solution, thus obtaining YIMIAO-3.5.

[0170] (3) YIMIAO-3.6: Take 160 μL of Pseudomonas aeruginosa surface protein antigen, abbreviated as VacPAE1 (2.5 μg / μL), 800 μL of YIMIAO-3.4 (YAXA 15 mg / mL) and 40 μL of PBS, mix them, stir magnetically at room temperature for 3 h, dialyze (molecular weight cutoff 100 kDa, MD10 (100000), Viskase) for 3 h to obtain YIMIAO-3.6.

[0171] (4) YIMIAO-3.7: Take 160 μL of protein VacPAE1 (2.5 μg / μL), 800 μL of YIMIAO-3.5 (YAXA 15 mg / mL, 3M-052 500 μg / μL) and 40 μL of PBS, mix them, stir magnetically at room temperature for 3 h, dialyze (molecular weight cutoff 100 kDa, MD10 (100000), Viskase) for 3 h to obtain YIMIAO-3.7.

[0172] Acid-sensitive hydroxyl monomers ( Figure 1 ) and YAXA ( Figure 2 The 1H NMR spectrum of YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 indicates the successful synthesis of the polymer framework. Zeta potential results show that YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 are all negatively charged (…). Figure 3 DLS results showed that the sizes of YIMIAO-3.4, YIMIAO-3.5, YIMIAO-3.6, and YIMIAO-3.7 gradually increased, ranging from 100 to 250 nm. Figure 4 Coomassie Brilliant Blue staining results showed successful loading of VacPAE1 protein onto YIMIAO-3.7 ( ); Figure 5 In summary, the organic polymer nanoparticle vaccine YIMIAO-3.7, which targets lysosomes, was successfully prepared.

[0173] All experimental groups in this invention are as follows:

[0174] All groups of this invention are:

[0175] (1) Mock (PBS)

[0176] (2) YIMIAO-3.1 (YM3.1): 3M-052 (free immune adjuvant)

[0177] (3) YIMIAO-3.2 (YM3.2): VacPAE1 (free antigen protein)

[0178] (4) YIMIAO-3.3 (YM3.3): VacPAE1+3M-052 (a mixture of free immune adjuvant and antigen protein)

[0179] (5) YIMIAO-3.4 (YM3.4): YAXA (simple polymer framework nanoparticles)

[0180] (6) YIMIAO-3.5 (YM3.5): YAXA+3M-052 (nanoparticles loaded with immune adjuvants)

[0181] (7) YIMIAO-3.6 (YM3.6): VacPAE1+YAXA (nanoparticles loaded with antigen protein)

[0182] (8) YIMIAO-3.7 (YM3.7): VacPAE1+YAXA+3M-052 (nanoparticles loaded with immune adjuvants and antigen proteins)

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

[0184] To evaluate the cellular safety of the nanoparticle vaccine vector YIMIAO-3.4, erythrocytes from C57BL / 6J mice were used in the experiment. YIMIAO-3.4 was dispersed in PBS to prepare solutions of different concentrations (50, 100, 250, and 500 μg / mL), which were then added to the erythrocytes. Erythrocytes dispersed in deionized water served as a positive control, and erythrocytes dispersed in PBS served as a negative control. All samples were then incubated at room temperature for 1 hour, centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected. The absorbance of the supernatant was measured at 570 nm. The hemolysis rate was calculated using the formula: Hemolysis rate (%) = (Sample absorbance - Negative control absorbance) / (Positive control absorbance - Negative control absorbance) × 100%.

[0185] In addition, the in vivo biosafety of YIMIAO-3.7 was evaluated using a C57BL / 6J mouse model. YIMIAO-3.7 was administered via lung delivery at a dose of 50 μL. On day 7 post-immunization, four C57BL / 6J mice were sacrificed (using CO2 lethality), and tissues from the heart, liver, spleen, and kidneys were dissected and fixed in 4% paraformaldehyde solution for at least 48 hours. Subsequently, the tissues were processed by Wuhan Sewell Biotechnology Co., Ltd., including washing, dehydration, clearing, paraffin embedding, sectioning, and HE staining, to prepare pathological sections. Finally, the pathological sections were observed under an optical microscope and histopathologically scored.

[0186] Experimental results showed that, even at a high concentration of 500 μg / mL, the hemolysis rate of YIMIAO-3.4 in the in vitro hemolysis experiment was still less than 5% (see [link to experiment]). Figure 6 This demonstrates good in vitro biocompatibility. In in vivo experiments, pathological examination of tissue sections revealed no significant histopathological changes at any time point after immunization (see...). Figure 7 This indicates that YIMIAO-3.7 also has good biocompatibility in vivo.

[0187] Example 3: Determination of TLR7 signaling pathway activation and lysosomal targeting efficacy of the YIMIAO-3.7 nanoparticle vaccine prepared in Example 1 of this invention.

[0188] This study used TLR7 deletion (Tlr7) - / - Bone marrow-derived dendritic cells (BMDCs) from C57BL / 6J mice and wild-type (WT) C57BL / 6J mice were used in five experimental groups: 1) Mock group; 2) YIMIAO-3.1 group; 3) YIMIAO-3.4 group; 4) YIMIAO-3.5 group; and 5) YIMIAO-3.7 group. Each group had three replicates. The samples from each group were mixed with BMDCs and incubated at 37°C with 5% CO2. Subsequently, the maturity of BMDCs was detected by flow cytometry, the protein expression levels of TLR7, IκBα, pIκBα, p65, and pp65 were analyzed by Western blotting, and the secretion levels of cytokines IL-12 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA). Specific detection methods are described below.

[0189] 1. Flow cytometry was used to detect the maturity of BMDCs.

[0190] (1) Take 2 mL of solution with a concentration of 1×10 6 BMDCs at a concentration of cells / mL were added to the stimuli of each of the above experimental groups and incubated for 12 hours.

[0191] (2) Transfer the cells to flow cytometry tubes, centrifuge at 400×g for 5 minutes, discard the supernatant, add 1 mL of DPBS, centrifuge again at 400×g for 5 minutes, and discard the supernatant. Repeat this step twice.

[0192] (3) In the flow cytometry assay for BMDC maturity, the following antibody combination was used for cell staining:

[0193] • Use 0.1 μL of FVD-ef780 antibody (labeled as FVD-ef780, fluorescent dye as APC-Cy7);

[0194] • Use 0.8 μL of CD11c antibody (fluorescent dye: FITC);

[0195] • Use 1.0 μL of MHC II antibody (fluorescent dye: PE-Cy7);

[0196] • Use 1.0 μL of CD86 antibody (fluorescent dye is PE);

[0197] • Use 1.0 μL of CD40 antibody (fluorescent dye: PerCP-Cy5.5).

[0198] The total antibody volume required per cell tube is 3.9 μL. In addition, each antibody is used in a separate single-staining tube for flow cytometry compensation adjustment.

[0199] (4) Staining steps:

[0200] 1) Add 3.9 μL of mixed antibody to each flow cytometry tube and incubate at room temperature in the dark for 25 minutes.

[0201] 2) Add 2 mL of DPBS, centrifuge at 400×g for 5 minutes, and repeat the washing twice.

[0202] 3) Discard the supernatant, add 50 μL of DPBS to resuspend the cells, and use a BD Verse flow cytometer to analyze the sample.

[0203] 2. Western blot detection of TLR7, IκBα, pIκBα, p65, and pp65 protein expression

[0204] (1) Sample preparation

[0205] 2 mL of a concentration of 1×10 6 BMDCs at concentrations of [number] cells / mL were added to the stimulants used in the above experimental groups. After stimulation for 3 hours, the supernatant was discarded, and 200 μL of cell lysis buffer was added and the cells were placed on ice for lysis for 30 minutes. Cells were then scraped off with a cell scraper, transferred to EP tubes, and centrifuged at 12000 rpm and 4°C for 10 minutes. The supernatant was discarded, and the precipitate was collected. An appropriate amount of 5× protein loading buffer was added, and the sample was heated in a boiling water bath for 10 minutes. After cooling, the sample was placed on ice and finally stored at -80°C for later use.

[0206] (2) Protein gel preparation

[0207] Prepare 12.5% ​​SDS-PAGE protein gels using the PAGE gel rapid preparation kit according to the corresponding formula.

[0208] (3) Electrophoresis

[0209] Add the prepared sample and protein marker to the gel wells, connect the electrophoresis tank to the power supply, set the voltage to 80V, and after the sample enters the stacking gel, adjust the voltage to 120V to continue electrophoresis.

[0210] (4) Transfer

[0211] Activate the cut PVDF membrane by immersing it in methanol solution, then place it in transfer solution for later use. Remove the protein gel, cut it according to the size of the target protein band, and place it on a transfer holder lined with a sponge pad and filter paper. Place the PVDF membrane, filter paper, and sponge pad on the gel in sequence, close the transfer holder, and place it in the transfer tank. Add pre-cooled transfer solution and an ice pack, and begin the transfer process. Adjust the transfer time according to the protein molecular weight.

[0212] (5) Closed

[0213] After the transfer is completed, the PVDF film is immersed in a sealing solution containing 5% skim milk powder and placed on a shaker at room temperature for 1 hour.

[0214] (6) Primary antibody incubation

[0215] After blocking, the primary antibodies (TLR7, IκBα, pIκBα, p65, pp65) were diluted at a ratio of 1:1000. The PVDF membrane was placed in a box containing the primary antibody and incubated at room temperature for 1 hour.

[0216] (7) Secondary antibody incubation

[0217] After primary antibody incubation, wash the PVDF membrane in 1×TBST solution for 5 minutes each time, for a total of 3 washes. After washing, place the membrane in a box containing secondary antibody and incubate at room temperature for 30 minutes.

[0218] (8) Exposure

[0219] After the secondary antibody incubation, the membrane was washed three times in 1×TBST solution for 5 minutes each time. The developing solution was prepared by mixing the luminescent solution A and solution B in a 1:1 ratio. After the membrane was drained on absorbent paper, it was immersed in the developing solution. Then, the membrane was placed in a chemiluminescence imaging system for automatic exposure.

[0220] 3. ELISA detection of cytokine secretion

[0221] (1) Sample preparation

[0222] Add BMDCs to the corresponding experimental group stimuli. After 6 hours of stimulation, aspirate the supernatant, centrifuge at 2200 rpm for 5 minutes, collect the supernatant and store it at -80℃ for later use.

[0223] (2) Preparation of ELISA kit

[0224] 1) Reagent warming: 30 minutes before the experiment, allow the reagent kit and test samples to warm to room temperature. If crystals appear in the concentrated washing solution, dissolve them in a 37°C water bath until the crystals completely disappear.

[0225] 2) Dissolving the standard: Add 1 mL of standard / sample diluent to the lyophilized standard tube, let stand for 15 minutes, and mix well after it is fully dissolved.

[0226] 3) Dilution of standard: Take 6 EP tubes and label them, and dilute the standard by 2 times in sequence. Take the standard / sample dilution solution as a blank control.

[0227] 4) Washing buffer preparation: Dilute 20× Washing Buffer with double-distilled water to 1×.

[0228] 5) Biotinylated antibody dilution: Calculate the required amount according to experimental needs, and dilute the 100× antibody concentrate with the detection diluent to 1× working solution (mix thoroughly before dilution).

[0229] 6) Preparation of streptavidin-enzyme conjugate: According to experimental requirements, dilute the 100× concentrated enzyme conjugate to 1× working solution with enzyme conjugate diluent (centrifuge before dilution).

[0230] (3) Detection steps

[0231] 1) Prepare reaction strips: Take out the required reaction strips that have been equilibrated to room temperature from the sealed bag, wash them 3 times with washing solution and shake dry before use.

[0232] 2) Add standard and sample: Add 100 μL of standard and test sample to the reaction well, seal the plate and incubate at 37°C for 90 minutes.

[0233] 3) Washing: Discard the liquid in the well, add 300 μL of washing solution to each well, let stand for 30 seconds, then shake off the liquid. Repeat 5 times, and finally pat dry on filter paper or absorbent paper.

[0234] 4) Add biotinylated antibody: Add 100 μL of biotinylated antibody working solution to each well, seal the membrane, and incubate at 37°C for 60 minutes.

[0235] 5) Washing: Repeat step 3.

[0236] 6) Add streptavidin-enzyme conjugate: Add 100 μL of enzyme conjugate working solution to each well, seal the membrane, and incubate at 37°C in the dark for 30 minutes.

[0237] 7) Washing: Repeat step 3.

[0238] 8) Add chromogenic substrate (TMB): Add 100 μL of TMB chromogenic substrate to each well and incubate at 37°C in the dark for 15 minutes.

[0239] 9) Add stop solution: Add 50 μL of stop solution to each well.

[0240] 10) Detection results: After the reaction was terminated, the OD value was measured using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450-630 nm, and the data was analyzed.

[0241] 4. Laser confocal microscopy for detecting colocalization of YIMIAO-3.7 nanoparticle vaccine with lysosomes

[0242] (1) Cell plating

[0243] Use confocal culture dishes, inoculating 4 × 10⁴ cells per dish. 5 Five BMDC cells were added to 2 mL of complete culture medium (1640 medium + 10% FBS). The experimental design included two time points, two experimental groups, and one negative control group, for a total of five culture dishes.

[0244] (2) Preparation of nuclear dyes and lysosomal dyes

[0245] 1) Lysosomal dye (LysoTracker): Stock solution concentration 1 mM, working concentration 100 nM, requires 10000-fold dilution. Preparation method: Add 1 μL of LysoTracker to 10 mL of 1640 medium.

[0246] 2) Nuclear dye (Hoechst 33342): Stock solution concentration 20 mM (12.3 μg / mL), working concentration 2 μg / mL, requires dilution 6150 times. Preparation method: Take 6150 μL of the above solution and add 1 μL of Hoechst 33342.

[0247] 3) Experimental volume: This experiment requires a total of 10 mL of dye solution, therefore 12.3 mL was actually prepared. The specific preparation method is as follows:

[0248] 4) Lysosomal dye: Add 2 μL of LysoTracker to 20 mL of 1640 medium.

[0249] 5) Nuclear dye: Take 20 mL of the above solution and add 3.25 μL of Hoechst 33342.

[0250] (3) Pre-staining with nuclear dyes and lysosomal dyes

[0251] Aspirate the culture medium from the culture dish, wash twice with DPBS, and then add 2 mL of culture medium containing nuclear dye and lysosomal dye to each culture dish and incubate for 0.5 hours.

[0252] (4) Administration

[0253] After aspirating the dye culture medium, rinse the wells and the bottom of the dishes with 2 mL of DPBS. After aspirating the DPBS, add 300 μL of culture medium containing YIMIAO-3.7 nanoparticles (1640 medium + 10% FBS, phenol red-free) to each culture dish. Place the culture dishes in a 37°C incubator and incubate for 10 minutes.

[0254] (5) Cell imaging

[0255] Imaging was performed using a laser confocal microscope, with magnifications set to 40× and 100×, to observe the co-localization of YIMIAO-3.7 nanoparticles and lysosomes.

[0256] Experimental results showed that the expression of cell surface maturation markers CD80 and CD86 was significantly upregulated in wild-type BMDCs after stimulation with YIMIAO-3.5 and YIMIAO-3.7. Figure 8 Furthermore, the protein expression levels of key molecules in the TLR7 signaling pathway, pIκBα and pp65, were also significantly increased. Figure 9 Simultaneously, the secretion levels of cytokines IL-12 and TNF-α significantly increased, indicating a synergistic effect between the immune adjuvant and the polymeric framework nanoparticles. Figure 10 Laser confocal imaging results showed that YIMIAO-3.7 had good co-localization with lysosomes. Figure 11 These results indicate that YIMIAO-3.7 can effectively enter lysosomes, activate the TLR7 signaling pathway, and thus promote the maturation of BMDCs and the secretion of cytokines.

[0257] Example 4: Determination of the immunoprotective effect of the YIMIAO-3.7 nanoparticle vaccine prepared in Example 1 of this invention.

[0258] The experiment was set up with 4 experimental groups: 1) Mock group (control group); 2) YIMIAO-3.2 group; 3) YIMIAO-3.3 group; 4) YIMIAO-3.7 group.

[0259] 1. Immunization and challenge protocols

[0260] Mice were immunized via lung delivery, with each group receiving three immunizations at 14-day intervals. On day 14 after the third immunization, mice were challenged with a 2.5×LD50 virus. 50The bacterial suspension of Pseudomonas aeruginosa strain F291007 was delivered via liquid aerosol lung delivery. The specific operation was as follows: (1) Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital at a dose of 100 mg / kg. (2) After successful anesthesia, the mice were fixed supine on the operating table, and the angle of the operating table was adjusted to make it at a suitable tilt angle with the horizontal direction. (3) The tracheal opening of the mice was fully exposed using a laryngoscope, and the generator head of the handheld liquid aerosol lung delivery device was inserted about 2 cm parallel to the trachea. (4) The push handle of the generator was quickly pushed to deliver 2.5×LD 50 The bacterial culture of Pseudomonas aeruginosa strain F291007 was delivered to the lungs of mice in the form of liquid aerosol. Evaluation of efficacy: (1) Antibody titer detection: The change in antibody titer in mice after immunization was detected by ELISA. (2) Evaluation of immunoprotective efficacy: The immunoprotective efficacy of the vaccine was comprehensively evaluated by survival analysis, histopathological analysis and organ bacterial load determination.

[0261] 2. Serum antibody level detection

[0262] (1) Sample collection and preparation: Six mice were randomly selected and labeled from each immunization group. Blood was collected from the labeled mice by tail sampling on days 7 and 14 after each immunization, for a total of six blood collections. The blood samples collected each time were placed in clean EP tubes and allowed to stand at room temperature for 4 hours. The serum was then separated by centrifugation at 3000 rpm and 4℃ for 10 minutes. The separated serum was aliquoted into new sterile EP tubes and stored at -80℃ for later use. The level of specific IgG antibodies in the serum was subsequently detected by ELISA.

[0263] (2) Coating: Dilute VacPAE1 protein to 1 μg / mL with coating buffer. Use a pipette to aspirate the diluted VacPAE1 and add 100 μL to each well of a 96-well ELISA plate. Coat overnight at 4°C.

[0264] (3) Blocking: Discard the coating solution, gently pat the 96-well ELISA plate dry, add 200 μL of blocking solution to each well, and incubate at 37°C for 2 hours.

[0265] (4) Primary antibody incubation: Discard the blocking solution and pat dry the wells. Dilute the serum from each immunization group serially with diluent, ranging from 1:200 to 1:6553600. After dilution, use a multi-channel pipette to add 100 μL of diluted serum to the coated 96-well ELISA plate in order from low to high concentration. At the same time, take the serum from 6 blank mice, dilute it 1:10, and add 100 μL to the control wells. Incubate at 37°C for 30 minutes.

[0266] (5) Washing the plate: Discard the primary antibody incubation solution, add 200 μL of washing solution to each well, shake for 1 minute and then discard. Repeat the washing process 5 times, and finally gently pat the plate dry.

[0267] (6) Secondary antibody incubation: Add 100 μL of HRP-labeled goat anti-mouse IgG and IgG1 antibodies diluted 1:10000 to each well of a 96-well ELISA plate using a multi-channel pipette, and incubate at 37°C for 20 minutes.

[0268] (7) Washing the plate: Discard the secondary antibody incubation solution, add 200 μL of washing solution to each well, shake for 1 minute and then discard. Repeat the washing process 5 times, and finally gently pat the plate dry.

[0269] (8) Color development: Add 100 μL of TMB color development solution to the 96-well ELISA plate using a pipette and incubate at 37°C in the dark for 5-10 minutes.

[0270] (9) Termination: Use a syringe to add 100 μL of termination solution to each well to terminate the reaction.

[0271] (10) Detection: The absorbance of each well of the 96-well ELISA plate was measured using an ELISA reader at wavelengths of 450 nm and 630 nm. A positive result was defined as the ratio of the absorbance of the sample well to the absorbance of the normal mouse serum well to a value greater than 2. The highest dilution gradient corresponding to the positive result was taken as the serum IgG antibody level.

[0272] 3. Culture of Pseudomonas aeruginosa strains

[0273] (1) Activation: Take 20 μL of Pseudomonas aeruginosa glycerol bacteria from the -80℃ freezer, thaw and inoculate it into 20 mL of BHI liquid medium containing meropenem antibiotic (10 μL), and culture at 37℃ and 200 rpm for 15 hours to allow the strain to grow to the plateau phase. This is the first generation of bacteria.

[0274] (2) Pre-culture: The first-generation bacterial culture was diluted 100 times and inoculated into 20 mL of BHI liquid medium containing meropenem (10 μL), and cultured at 37℃ and 200 rpm until OD. 600 When the concentration reaches 1.5, it is considered a second-generation bacterium.

[0275] (3) Formal culture: The second-generation bacterial culture was diluted 100 times and inoculated into 20 mL of antibiotic-free BHI liquid medium, and cultured at 37°C and 200 rpm until OD. 600 When the concentration reaches 1.5, it is considered a third-generation bacterium.

[0276] (4) Centrifugation to collect bacteria: Take 500 μL of third-generation bacterial solution, centrifuge at 3000×g for 10 minutes, and collect the bacterial cells.

[0277] (5) Bacterial resuspension: Discard the supernatant and resuspend the bacterial cells in an equal volume of physiological saline (containing 0.05% poloxamer), washing twice. Finally, adjust the OD of the bacterial culture with physiological saline containing 0.05% poloxamer. 600 The concentration of the bacterial culture was increased to 1.0, at which point the theoretical concentration was 4 × 10⁻⁶. 8 CFU / mL. Further dilute to the challenge concentration (3.5 × 10⁻⁶). 7 (CFU / mL), used for subsequent challenge experiments.

[0278] (6) Droplet count: The bacterial solution was serially diluted 5 times, and 10 μL of the diluted bacterial solution was dropped into a meropenem resistant culture dish. After incubation at 37°C for 15 hours, colony count was performed to calculate the actual bacterial concentration.

[0279] 4. Clinical symptom observation after challenge with Pseudomonas aeruginosa via liquid aerosol delivery to the lungs.

[0280] On day 14 after the third immunization, use 2.5×LD. 50 The bacterial solution was used to challenge mice in all immunized groups via liquid aerosol lung delivery, and the survival and weight of the mice were observed within 14 days after challenge. Survival curves and weight curves were plotted.

[0281] 5. Detection of bacterial load in organs after challenge with *Pseudomonas aeruginosa* via liquid aerosol delivery.

[0282] Twenty-four hours after lung challenge with *Pseudomonas aeruginosa* via liquid aerosol delivery, four mice from each challenge group were randomly selected and euthanized with CO2. Whole blood was collected by removing the eyeballs of the mice in a biosafety cabinet, and the lungs, spleen, and liver were dissected and placed in sterile petri dishes. Appropriately sized tissue samples were then cut, weighed, and placed into homogenization tubes containing 800 μL of sterile PBS. The homogenization was performed at 5200 rpm for 1 minute. -1 Homogenize for 90 seconds, then serially dilute the homogenate with PBS by 5 times. Take 10 μL of each dilution and drop it onto blood agar plates. Incubate the plates upside down in a 37 ℃ incubator for 15 hours and count the colonies of each organ at different dilution levels.

[0283] 6. Pathological examination of organ tissues after challenge with Pseudomonas aeruginosa via liquid aerosol delivery to the lungs.

[0284] (1) Tissue collection and fixation: On the second day after challenge with Pseudomonas aeruginosa via liquid aerosol delivery, three mice were randomly selected from each group and sacrificed using CO2. After dissection, the liver, spleen, and lungs were removed and fixed in 4% paraformaldehyde solution.

[0285] (2) Dehydration and transparency: After 48 hours of fixation, each organ was removed and dehydrated in 80%, 90%, 95% and 100% ethanol solutions, respectively, for 2 hours each time. The organs were then placed in xylene for 1 hour to complete the transparency treatment.

[0286] (3) Impregnation and embedding: The transparent organ is placed in liquid paraffin at 55°C and the paraffin solidifies naturally to form a wax block.

[0287] (4) Sectioning and staining: Cut the paraffin block into 4-6 μm thick sections. Gently pick up the sections with tweezers and place them on the surface of water at 40℃~45℃. After the sections have fully unfolded, gently lift them out and place them on a clean glass slide. After removing excess water from the glass slide, place it in a constant temperature oven at 60℃~65℃ for 30 minutes. Then perform HE staining and observe the changes in tissue cells under a microscope.

[0288] Experimental results showed that mice immunized with the YIMIAO-3.7 nanoparticle vaccine produced a strong humoral immune response, and the serum IgG antibody titer increased significantly with the progression of immunization, and was higher than that of other experimental groups. Figure 12 Under a 2.5×LD50 challenge condition, mice immunized with YIMIAO-3.7 showed the highest survival rate, significantly higher than the survival rates of the YIMIAO-3.2 (VacPAE1) and YIMIAO-3.3 (3M-052+VacPAE1) groups. Figure 13 , Figure 14 On day 2 post-challenge, the bacterial load in the lung tissue, spleen, and liver of mice in the YIMIAO-3.7 group was significantly reduced. Figure 15 ), and the pathological scores of these tissues were also significantly reduced ( Figure 16 In conclusion, YIMIAO-3.7 immunization significantly enhanced the anti-infective efficacy against Pseudomonas aeruginosa in mice.

[0289] 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 immune adjuvant was selected from 3M-052; The antigen was selected from the surface protein antigen of Pseudomonas aeruginosa. The structure of the organic polymer is shown in formula (1): Equation (1); The structure of R1 in equation (1) is shown in equation (2): Equation (2); The structure of R2 in equation (1) is shown in equation (3): Equation (3); In the above formula (1), n ​​is 113; In the above formula (1), X is 113.

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 10:1-50:

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 30:

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 10:1-50:

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 30:

1.

6. An organic polymer, characterized in that, The structure of the organic polymer is shown in formula (1): Equation (1); The structure of R1 in equation (1) is shown in equation (2): Equation (2); The structure of R2 in equation (1) is shown in equation (3): Equation (3); In the above formula (1), n ​​is 113; In the above formula (1), X is 113.

7. An immune composition, characterized in that, The immune composition comprises the organic polymer and immune adjuvant as described in claim 6; The immune adjuvant is selected from 3M-052.

8. The immune composition according to claim 7, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 10:1-50:

1.

9. The immune composition according to claim 8, characterized in that, The mass ratio of the organic polymer to the immune adjuvant is 30:

1.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the organic polymer nanoparticle vaccine according to any one of claims 1-5.

11. The pharmaceutical composition according to claim 10, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

12. A method for preparing the organic polymer nanoparticle vaccine according to any one of claims 1-5, characterized in that, The method includes mixing the immune composition according to any one of claims 7-9 with the antigen; The antigen is selected from the surface protein antigen of Pseudomonas aeruginosa.

13. The method according to claim 12, characterized in that, The concentration of the antigen used is 1-50 μg / μL.

14. The method according to claim 13, characterized in that, The concentration of the antigen used is 2.5 μg / μL.

15. A method for preparing the organic polymer of claim 6, characterized in that, The method includes: Acid-sensitive dihydroxy monomer and L-lysine diisocyanate were dissolved in anhydrous N,N-dimethylformamide and reacted. After dissolving polyethylene glycol in anhydrous N,N-dimethylformamide, it was added to the above reaction system, and the reaction continued. After removing N,N-dimethylformamide and drying, an organic polymer is obtained.

16. The method according to claim 15, characterized in that, The structural formula of the acid-sensitive dihydroxy monomer is shown in formula (2); Equation (2).

17. The method according to claim 15, characterized in that, The mass ratio of the acid-sensitive dihydroxy monomer, L-lysine diisocyanate and polyethylene glycol is (5-15):(5-15):(1-5).

18. The method according to claim 17, characterized in that, The mass ratio of the acid-sensitive dihydroxy monomer, L-lysine diisocyanate, and polyethylene glycol is 10:11:

2.

19. The method according to claim 15, characterized in that, The concentration of the acid-sensitive dihydroxy monomer dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 g / mL.

20. The method according to claim 19, characterized in that, The concentration of the acid-sensitive dihydroxy monomer dissolved in anhydrous N,N-dimethylformamide is 0.1 g / mL.

21. The method according to claim 15, characterized in that, The concentration of the L-lysine diisocyanate dissolved in anhydrous N,N-dimethylformamide is 0.05-0.15 g / mL.

22. The method according to claim 21, characterized in that, The concentration of the L-lysine diisocyanate dissolved in anhydrous N,N-dimethylformamide was 0.11 g / mL.

23. The method according to claim 15, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.01-0.05 g / mL.

24. The method according to claim 23, characterized in that, The concentration of the polyethylene glycol dissolved in anhydrous N,N-dimethylformamide is 0.02 g / mL.

25. The method according to claim 15, characterized in that, The polyethylene glycol is HO-PEG 5000-NHS.

26. The method according to claim 15, characterized in that, The removal method includes dialysis.

27. The method according to claim 15, characterized in that, The drying process includes freeze drying.

28. The method according to claim 15, characterized in that, The method also includes a method for synthesizing acid-sensitive dihydroxy monomers, including: Terephthalaldehyde and 2-hydroxyethylamine were dissolved in anhydrous methanol and reacted to obtain a precipitate. The precipitate was washed with anhydrous methanol and dried to obtain an acid-sensitive dihydroxy monomer.

29. The method according to claim 28, characterized in that, The mass ratio of terephthalaldehyde to 2-hydroxyethylamine is 1:1 to 1:

5.

30. The method according to claim 29, characterized in that, The mass ratio of terephthalaldehyde to 2-hydroxyethylamine is 1:

2.

31. The method according to claim 28, characterized in that, The concentration of terephthalaldehyde dissolved in anhydrous methanol is 0.01-0.2 g / mL.

32. The method according to claim 31, characterized in that, The concentration of terephthalaldehyde dissolved in anhydrous methanol is 0.067 g / mL.

33. The method according to claim 28, characterized in that, The concentration of the 2-hydroxyethylamine dissolved in anhydrous methanol is 0.02-0.4 g / mL.

34. The method according to claim 33, characterized in that, The concentration of the 2-hydroxyethylamine dissolved in anhydrous methanol is 0.13 g / mL.

35. A method for preparing the immune composition according to any one of claims 7-9, characterized in that, The method includes dissolving the organic polymer of claim 6 and the immune adjuvant in an organic solvent, and then removing the organic solvent by dialysis; the immune adjuvant is selected from 3M-052.

36. The method according to claim 35, characterized in that, The organic solvent is selected from dimethyl sulfoxide.

37. The method according to claim 35, characterized in that, The concentration of the organic polymer dissolved in the organic solvent is 100-200 mg / mL.

38. The method according to claim 37, characterized in that, The concentration of the organic polymer dissolved in the organic solvent is 150 mg / mL.

39. The use of the organic polymer nanoparticle vaccine of any one of claims 1-5, or the organic polymer and Pseudomonas aeruginosa surface protein antigen of claim 6, or the immune composition and Pseudomonas aeruginosa surface protein antigen of any one of claims 7-9, or the pharmaceutical composition of any one of claims 10-11, in the preparation of a drug for preventing pathogen infection; wherein the pathogen is selected from bacteria; and the bacteria include Pseudomonas aeruginosa.

40. The use of the organic polymer of claim 6 and the Pseudomonas aeruginosa surface protein antigen, or the immune composition of any one of claims 7-9 and the Pseudomonas aeruginosa surface protein antigen, or the pharmaceutical composition of any one of claims 10-11, in the preparation of a vaccine.

41. The use of the organic polymer of claim 6 in the preparation of a drug carrier loaded with the immune adjuvant 3M-052 and the surface protein antigen of Pseudomonas aeruginosa.

42. The use of the organic polymer of claim 6 in the preparation of a product loaded with an immune adjuvant; wherein the immune adjuvant is selected from 3M-052.

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