A structured subunit vaccine based on framework nucleic acids, its preparation method and application

Abstract

This invention discloses a structured subunit vaccine based on framework nucleic acids, its preparation method, and its applications. The structured subunit vaccine comprises: framework nucleic acid, antigen, and adjuvant, wherein the antigen and adjuvant are coupled to the framework nucleic acid; the distance between the antigen and adjuvant is 5-400 nm. This structured subunit vaccine, by precisely controlling the spatial spacing between the antigen molecule and the TLR-like adjuvant at the nanoscale based on framework nucleic acid, achieves synergistic amplification of the B-cell antigen receptor and Toll-like receptor signal axes, thereby significantly improving the humoral immunogenicity of the subunit vaccine. Furthermore, the framework nucleic acid can be modified in topology and size as needed to construct multivalent, composite, and tandem structured vaccine systems, which is beneficial for wide application in various scenarios, including SARS-CoV-2, influenza, and tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a structured subunit vaccine based on framework nucleic acids, its preparation method, and its application. Background Technology

[0002] Subunit vaccines, due to their high safety and good controllability, have been widely used in the prevention and treatment of various diseases. However, subunit antigens themselves have weak immunogenicity and often need to be used in conjunction with highly effective molecular adjuvants to obtain sufficient humoral or cellular immune responses. Although traditionally used aluminum adjuvants have a certain promoting effect, their mechanism of action is limited, and the types of immune responses they induce are limited, making it difficult to meet the requirements of next-generation vaccines for strong immune activation. Therefore, Toll-like receptor (TLR) agonists (such as CpG ODN, MPLA, Telratolimod, etc.) with clear molecular targets and the ability to induce a wide range of immune pathways are gradually becoming an important direction in the development of subunit vaccines.

[0003] Combining antigens with TLR agonists through physical mixing (such as aluminum adjuvants) or randomly coupling them onto nanocarriers can improve adjuvant utilization and the strength of the immune response. While these strategies can enhance the immune effect to some extent, they cannot precisely control the nanoscale spatial relationship between antigens and adjuvants. Summary of the Invention

[0004] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a structured subunit vaccine based on framework nucleic acids.

[0005] The present invention also provides a method for preparing the above-mentioned structured subunit vaccine.

[0006] The present invention also provides an immunogenic composition.

[0007] The present invention also provides applications of the above-described structured subunit vaccines.

[0008] A structured subunit vaccine based on a framework nucleic acid, according to a first aspect of the present invention, comprises: a framework nucleic acid, an antigen, and an adjuvant, wherein the antigen and adjuvant are coupled to the framework nucleic acid; the distance between the antigen and the adjuvant is 5-400 nm. Optionally, the interval between the antigen and the adjuvant is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77. 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 19 8, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 29 3, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 3 31, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400nm, or any two of these values, are used as the range between the endpoint values.

[0009] This invention is the first to propose using framework nucleic acids to construct a structural carrier that links antigen molecules and TLR agonists at a strictly defined nanoscale spacing, thereby achieving "spatial coupling" of two types of immune stimulation signals at the subcellular structural level during B cell endocytosis. Through continuously adjustable FNA design, this invention sets the spacing between the antigen ligand and the TLR agonist to an arbitrary range of approximately 5-400 nm. The effects of different spacings on the BCR–TLR synergistic effect are systematically verified using B cell imaging and quantitative analysis of B cell activation stimulating factors, thus obtaining an "optimal spatial pattern" that can be used for vaccine development.

[0010] The antigen-adjuvant complex based on framework nucleic acids of this invention, after being endocytosed into endosomes by B cells, maintains a stable and controllable distance between the antigen and TLR agonist within the same intracellular compartment, thereby producing the following effects: It achieves synergistic amplification of BCR and TLR signals. The framework nucleic acid structure ensures that BCR and TLR are stably carried into the same intracellular compartment, enhancing endosome localization overlap. Appropriate spatial distribution of the antigen and adjuvant enhances the synergistic amplification effect of BCR and TLR, guiding the expression of higher levels of activation markers such as CD69 and CD86, enhancing B cell proliferation, promoting antibody affinity maturation, and generating a stronger humoral immune response.

[0011] Meanwhile, framework nucleic acids have good stability in vivo, with no significant toxicity or organ damage, and can achieve strong immune effects without the use of strong cytotoxic adjuvants, making them significantly safer than many existing vaccine enhancement technologies.

[0012] According to some embodiments of the present invention, the spacing between the antigen and the adjuvant refers to the straight-line distance between two sites linked to the framework nucleic acid.

[0013] According to some embodiments of the present invention, the spacing is achieved by changing the number of segments of the DNA double helix of the framework nucleic acid. The error in adjusting the spacing can be controlled within 1-2 nm.

[0014] According to some embodiments of the present invention, the DNA double helix structure of the framework nucleic acid has a defined pitch (approximately 10.5 bp per 3.4 nm), that is, the theoretical distance between the antigen and adjuvant is approximately 5 nm for approximately 15-16 bp, approximately 14 nm for 40-45 bp, approximately 20 nm for 60 bp, approximately 50 nm for 150 bp, and approximately 100 nm for 300 bp.

[0015] According to some embodiments of the present invention, the spacing can be further refined using PEG-DNA adapters, spacer arms, branched DNA, etc.

[0016] According to some embodiments of the present invention, the framework nucleic acid is constructed by base complementation of a long single-stranded DNA (usually bacteriophage DNA as the backbone sequence) with a series of designed short-stranded DNA (Staple) fragments to controllably construct the desired pattern or structure.

[0017] According to some embodiments of the present invention, the framework nucleic acid can be any framework nucleic acid.

[0018] According to some embodiments of the present invention, the framework nucleic acid is highly programmable.

[0019] According to some embodiments of the present invention, the surface of the framework nucleic acid extends an oligonucleotide single strand of 15-30 bases, and an antigen or adjuvant is pre-linked with DNA complementary to the single strand. The antigen or adjuvant hybridizes to the framework nucleic acid through base complementary pairing.

[0020] According to some embodiments of the present invention, the framework nucleic acid has a structure with a length of 20-400 nm.

[0021] According to some embodiments of the present invention, the framework nucleic acid may be in the shape of a columnar, cuboid, or hexagonal prism structure.

[0022] According to some embodiments of the present invention, the framework nucleic acid includes a backbone sequence and a Staple short chain.

[0023] According to some embodiments of the present invention, the sequence of the Staple short chain further includes thiophosphorylation modification. Thiophosphorylation modification ensures the stability of DNA in the in vivo environment.

[0024] According to some embodiments of the present invention, the backbone sequence of the framework nucleic acid includes a nucleotide sequence as shown in SEQ ID NO:1.

[0025] According to some embodiments of the present invention, the sequence of the Staple short chain includes one or more of sequences SEQ ID NO:2-SEQ ID NO:309.

[0026] According to some embodiments of the present invention, the framework nucleic acid is prepared by the following method: adding the backbone sequence and the short chain of Staple to a buffer solution for reaction to prepare the framework nucleic acid.

[0027] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:(8-12).

[0028] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:(9-11).

[0029] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:10.

[0030] According to some embodiments of the present invention, the buffer solution comprises TAE-Mg buffer solution.

[0031] According to some embodiments of the present invention, the TAE-Mg buffer comprises 4-6 mM Tris, 0.5-2 mM EDTA, and 14-18 mM MgCl2.

[0032] According to some embodiments of the present invention, the pH of the TAE-Mg buffer solution is 7-9.

[0033] According to some embodiments of the present invention, the reaction conditions include: heating at 75-85°C for 12-18 minutes, followed by annealing at 80-65°C decreasing by 1°C every 5 minutes; and at 65-30°C decreasing by 1°C every 30 minutes.

[0034] According to some embodiments of the present invention, the reaction further includes a step of centrifuging and ultrafiltration to remove excess staple short chains from the prepared reaction product.

[0035] According to some embodiments of the present invention, the centrifugation conditions are 2000-5000g, 8-12min.

[0036] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 10-100 kDa.

[0037] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 100 kDa.

[0038] According to some embodiments of the present invention, the antigen is linked to a designated location on a single-stranded DNA using His-tag, Cys-tag, or Click chemical modification.

[0039] According to some embodiments of the present invention, the antigen can be linked to the framework nucleic acid by covalent or non-covalent bonds, or by molecular adapters.

[0040] According to some embodiments of the present invention, the antigen can be linked to the surface of the framework nucleic acid through complementary base pairing or through molecular linkers.

[0041] According to some embodiments of the present invention, the antigen can be fused with a linker peptide, and then the antigen is linked to the framework nucleic acid by specifically linking the linker peptide. The linker peptide that can be used for antigen fusion can be any suitable peptide, such as those with specific nucleic acid aptamers.

[0042] According to some embodiments of the present invention, the antigen may be directly (e.g., covalently or non-covalently) fused with a linker peptide (i.e., there are no other molecular sequences between them, forming an antigen-linker peptide or a fusion protein with the reverse sequence), or indirectly fused through a spacer molecule (i.e., forming an antigen peptide-spacer-linker peptide or a fusion protein with the reverse sequence).

[0043] According to some embodiments of the present invention, the antigen is linked to nucleotide residues of the framework nucleic acid via molecular linkers.

[0044] According to some embodiments of the present invention, the molecular connector includes SMCC.

[0045] According to some embodiments of the present invention, the antigen includes a BCR activating ligand. The BCR activating ligand is used to induce antigen-specific BCR aggregation and activation.

[0046] According to some embodiments of the present invention, the BCR activating ligand comprises an antigenic peptide.

[0047] According to some embodiments of the present invention, the antigenic peptide may be any protein or polypeptide antigen.

[0048] According to some embodiments of the present invention, an antigenic peptide refers to a peptide that has antigenicity or immunogenicity. The antigenic peptide is derived from pathogens such as viruses, including but not limited to coronaviruses. Alternatively, the antigenic peptide may be a peptide fragment known in the art that can be used as a subunit vaccine.

[0049] According to some embodiments of the present invention, the antigenic peptide includes the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, anti-IgM, HA, OVA, and tumor antigen peptide.

[0050] According to some embodiments of the present invention, the adjuvant includes a TLR agonist.

[0051] According to some embodiments of the present invention, the TLR agonist includes at least one of a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist.

[0052] According to some embodiments of the present invention, the TLR agonist includes at least one of CpG oligodeoxynucleotide (ODN), ssRNA, and MPLA.

[0053] According to some embodiments of the present invention, the TLR agonist comprises CpG ODN 1018 and ssRNA 40.

[0054] According to some embodiments of the present invention, the TLR agonist hybridizes with the framework nucleic acid by extending a complementary sequence at the end.

[0055] According to some embodiments of the present invention, the vaccine is a dosage form for intramuscular, intradermal, or subcutaneous administration.

[0056] According to a second aspect of the present invention, the method for preparing the above-described structured subunit vaccine includes the following steps: coupling an antigen and a TLR agonist to a framework nucleic acid, and adjusting the distance between the antigen and the TLR agonist to 5-400 nm.

[0057] According to some embodiments of the present invention, the framework nucleic acid may be in the shape of a columnar, cuboid, or hexagonal prism structure.

[0058] According to some embodiments of the present invention, the framework nucleic acid includes a backbone sequence and a Staple short chain.

[0059] According to some embodiments of the present invention, the backbone sequence of the framework nucleic acid is shown in SEQ ID NO:1.

[0060] According to some embodiments of the present invention, the sequence of the Staple short chain includes one or more of sequences SEQ ID NO:2-SEQ ID NO:309.

[0061] According to some embodiments of the present invention, the framework nucleic acid is prepared by the following method: adding the backbone sequence and the short chain of Staple to a buffer containing magnesium ions for reaction to prepare the framework nucleic acid.

[0062] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:(8-12).

[0063] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:(9-11).

[0064] According to some embodiments of the present invention, the molar ratio of the backbone sequence to the Staple short chain is 1:10.

[0065] According to some embodiments of the present invention, the concentration of magnesium ions in the magnesium-containing buffer solution is 14-18 mM.

[0066] According to some embodiments of the present invention, the magnesium-containing buffer solution includes TAE-Mg buffer solution.

[0067] According to some embodiments of the present invention, the TAE-Mg buffer comprises 4-6 mM Tris, 0.5-2 mM EDTA, and 14-18 mM MgCl2.

[0068] According to some embodiments of the present invention, the pH of the TAE-Mg buffer solution is 7-9.

[0069] According to some embodiments of the present invention, the reaction conditions include: heating at 75-85°C for 12-18 minutes, followed by annealing at 80-65°C decreasing by 1°C every 5 minutes; and at 65-30°C decreasing by 1°C every 30 minutes.

[0070] According to some embodiments of the present invention, the reaction further includes a step of centrifuging and ultrafiltration to remove excess staple short chains from the prepared reaction product.

[0071] According to some embodiments of the present invention, the centrifugation conditions are 2000-5000g, 8-12min.

[0072] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 10-100 kDa.

[0073] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 100 kDa.

[0074] According to some embodiments of the present invention, prior to conjugation, the method further includes a step of functionalizing the antigen, wherein the functionalization step is as follows: The antigen is reacted with a bifunctional cross-linking agent to prepare an antigen activated by the cross-linking agent; the antigen activated by the cross-linking agent is reacted with a BCR ligand coupling sequence to prepare an antigen with complementary DNA functionalization; the BCR ligand coupling sequence is shown in SEQ ID NO:310.

[0075] According to some embodiments of the present invention, the bifunctional crosslinking agent includes SMCC, NHS, and Halota.

[0076] According to some embodiments of the present invention, the molar ratio of the antigen to the bifunctional cross-linking agent is 1:(4-6).

[0077] According to some embodiments of the present invention, the reaction time of the antigen with the bifunctional cross-linking agent is 25-35 min.

[0078] According to some embodiments of the present invention, after the antigen reacts with the bifunctional crosslinking agent, the reaction product is further subjected to centrifugation and ultrafiltration to remove excess SMCC.

[0079] According to some embodiments of the present invention, the centrifugation conditions are 2800-3200g, 3-7min.

[0080] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 10-50 kDa.

[0081] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 25-35 kDa.

[0082] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 30 kDa.

[0083] According to some embodiments of the present invention, the molar ratio of the antigen activated by the cross-linking agent to the BCR ligand coupling sequence is 1:(5-10).

[0084] According to some embodiments of the present invention, the reaction time between the antigen activated by the cross-linking agent and the BCR ligand coupling sequence is 7-9 hours.

[0085] According to some embodiments of the present invention, the reaction temperature of the antigen activated by the cross-linking agent with the BCR ligand coupling sequence is 2-6°C.

[0086] According to some embodiments of the present invention, after the antigen activated by the cross-linking agent reacts with the BCR ligand coupling sequence, the reaction product is further subjected to centrifugation and ultrafiltration to remove excess DNA sequence.

[0087] According to some embodiments of the present invention, the centrifugation conditions are 2000-4000g, 3-7min.

[0088] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 25-35 kDa.

[0089] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 30 kDa.

[0090] According to some embodiments of the present invention, the conjugation of antigen and TLR agonist to framework nucleic acid specifically includes the following steps: placing the antigen, TLR agonist and framework nucleic acid in a magnesium-containing buffer solution for hybridization reaction to obtain the desired result.

[0091] According to some embodiments of the present invention, the molar ratio of the antigen, TLR agonist and framework nucleic acid is (8-15):(8-15):1.

[0092] According to some embodiments of the present invention, the molar ratio of the antigen, TLR agonist and framework nucleic acid is (10-13):(10-13):1.

[0093] According to some embodiments of the present invention, the hybridization reaction is carried out through complementary DNA base pairing.

[0094] According to some embodiments of the present invention, the hybridization reaction takes 25-35 minutes.

[0095] According to some embodiments of the present invention, the temperature of the hybridization reaction is 35-38°C.

[0096] According to some embodiments of the present invention, the concentration of magnesium ions in the magnesium-containing buffer solution is 10-15 mM.

[0097] According to some embodiments of the present invention, the concentration of magnesium ions in the magnesium-containing buffer solution is 12-13 mM.

[0098] According to some embodiments of the present invention, the magnesium-containing buffer solution includes TAE-Mg buffer solution.

[0099] According to some embodiments of the present invention, the TAE-Mg buffer comprises 4-6 mM Tris, 0.5-2 mM EDTA, and 10-15 mM MgCl2.

[0100] According to some embodiments of the present invention, the pH of the TAE-Mg buffer solution is 7-9.

[0101] According to some embodiments of the present invention, the reaction further includes a step of centrifuging and ultrafiltration to remove excess antigen and TLR agonist from the prepared reaction product.

[0102] According to some embodiments of the present invention, the centrifugation conditions are 3800-4200g, 8-12min.

[0103] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 80-120 kDa.

[0104] According to some embodiments of the present invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 100 kDa.

[0105] An immunogenic composition according to a third aspect of the present invention comprises the above-described structured subunit vaccine and a pharmaceutically acceptable carrier.

[0106] According to some embodiments of the present invention, non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions, alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and colorants. Those skilled in the art will understand that other drug carriers can be used in this invention.

[0107] According to some embodiments of the present invention, the immunogenic composition is a dosage form for intramuscular, intradermal, or subcutaneous administration.

[0108] The application of the above-described structured subunit vaccine and immunogenic composition according to a fourth aspect embodiment of the present invention, wherein the application is in the preparation of a medicament for generating an immune response in a subject.

[0109] According to some embodiments of the present invention, the medicine includes an anti-novel coronavirus vaccine.

[0110] The term "subject" as used herein refers to an organism that suffers from or is susceptible to a disease or condition that can be treated with the structured subunit vaccine provided herein. Non-limiting examples include humans, other mammals such as cattle, rats, mice, dogs, monkeys, goats, sheep, cows, deer, and other non-mammals.

[0111] According to some embodiments of the present invention, the patient or object is a person.

[0112] The application of the above-described structured subunit vaccine and immunogenic composition according to the fifth aspect of the present invention is in the preparation of a medicament for the prevention or treatment of pathogen infection or tumor.

[0113] According to some embodiments of the present invention, the pathogen is selected from viruses, such as influenza virus, coronavirus, respiratory syncytial virus, herpes zoster virus, African swine fever virus, malaria parasite, and cytomegalovirus.

[0114] According to some embodiments of the present invention, the coronavirus includes the novel coronavirus.

[0115] According to some embodiments of the present invention, for a given pathogen, such as a virus, a subunit vaccine containing a corresponding antigen from the pathogen or an antigen that is immunogenic to the pathogen, as described herein, can be administered to a subject to prevent or treat infection with the pathogen or related diseases or conditions.

[0116] The present invention has at least the following beneficial effects: This invention provides a structured subunit vaccine based on framework nucleic acids. By precisely controlling the spatial spacing between antigen molecules and TLR-like adjuvant molecules at the nanoscale using framework nucleic acids, synergistic amplification of the B-cell antigen receptor (BCR) and Toll-like receptor (TLR) signaling axes is achieved, thereby significantly improving the humoral immunogenicity of the subunit vaccine. This solves the problems of uncontrollable antigen-adjuvant spacing, lack of optimal spacing rules, and inability to precisely enhance the BCR-TLR synergistic effect in existing technologies. Furthermore, the topology and size of framework nucleic acids can be modified as needed to construct multivalent, complex, and tandem structured vaccine systems, which is beneficial for wide application in various scenarios, including SARS-CoV-2, influenza, and tumor vaccines.

[0117] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0118] Figure 1 An atomic force microscope image of a columnar framework nucleic acid. Figure 2A schematic diagram illustrating the synthesis of a structured subunit vaccine; Figure 3 Electrophoretic characterization of complementary DNA functionalized Anti-IgM, where the left image shows the gel-red staining result and the right image shows the Coomassie brilliant blue staining result; Figure 4 The electrophoretic characterization results of BCR ligand Anti-IgM and TLR agonist CpG ODN 1018 assembled for framework nucleic acids are shown in the figure. Anti-IgM labeling was performed using Alexa 647 fluorescence, and CpG ODN 1018 labeling was performed using Cy3 fluorescence. Figure 5 The image shows the detection results of framework nucleic acids and framework nucleic acids coupled with Anti-IgM characterized by atomic force microscopy. Figure 6 Electrophoretic characterization results of BCR ligand Anti-IgM and TLR agonist ssRNA assembled from framework nucleic acids; Figure 7 This is a graph showing the detection results of RBD-DNA coupling analysis by electrophoresis. Figure 8 To quantify the number of RBDs in the framework nucleic acid load, where "**" indicates... p <0.01; Figure 9 This is a quantitative analysis of the CpG count map of nucleic acid load in the framework, where "***" indicates... p <0.001; Figure 10 This image shows the results of confocal imaging and flow cytometry analysis to evaluate the affinity of subunit vaccines for B cells. FNA-5 to FNA-100 represent subunit vaccines co-assembled with BCR ligands and TLR agonists at different intervals. A represents confocal imaging, and B represents flow cytometry analysis. "**" indicates... p <0.01; Figure 11 This is a flow cytometry quantitative analysis result evaluation of the subunit vaccine's uptake capacity by B cells, where "*" indicates... p <0.05, "***" indicates p <0.001; Figure 12 This is a graph showing the results of flow cytometry analysis of the positive rates of CD80, CD86, and CD69 in B cells. "*" indicates... p <0.05, "**" indicates p <0.01; Figure 13 This is a graph showing the flow cytometry results of the detection of the positive rates of CD80, CD86, and CD69 in B cells using ssRNA as a TLR agonist. "*" indicates...p <0.05, "**" indicates p <0.01, "***" indicates p <0.001; Figure 14 These are graphs showing the results of flow cytometry analysis of B cell proliferation and ELISA analysis of IL-6 cytokine secretion. In graph A, B represents the results of B cell proliferation detection, and in graph B, ELISA analysis of IL-6 cytokine secretion is shown. "*" indicates... p <0.05, "**" indicates p <0.01, "***" indicates p <0.001; Figure 15 This is a graph showing the detection results comparing antibody titers of framework nucleic acid subunit vaccines and aluminum adjuvant-coated vaccines, where "***" indicates... p <0.001.

[0119] Figure 16 A representative fluorescence micrograph of GFP expression in infected cells; Figure 17 Flow cytometry plots and statistical results of memory B cells for a framework nucleic acid subunit vaccine combined with an aluminum adjuvant vaccine, where "*" indicates... p <0.05, "****" indicates p <0.0001. Detailed Implementation

[0120] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0121] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0122] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0123] Example 1 This embodiment provides a structured subunit vaccine using a columnar framework nucleic acid as a vector. The columnar framework nucleic acid is coupled with an antigen (which can be a BCR ligand anti-IgM or a receptor binding domain (RBD)) and a TLR agonist (which can be CpG ODN 1018 or ssRNA 40 (sequence: GCCGUCUGUUGUGUGACUC (SEQ ID NO: 315), synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The distance between the antigen and the TLR agonist is 5-400 nm. A schematic diagram of the synthesis is shown below. Figure 1 As shown.

[0124] Example 2 This embodiment provides a structured subunit vaccine using a columnar framework nucleic acid as a carrier. The columnar framework nucleic acid is coupled with an antigen (BCR ligand anti-IgM) and a TLR agonist (CpG ODN 1018). The specific preparation method is as follows: 1. Preparation of Columnar Framework Nucleic Acids (FNAs) Experimental materials: The long scaffold DNA M13 ssDNA p8064 was purchased from Rudong Bio-Bailu Biotechnology Co., Ltd., with a length of 8064bp, and the sequence is shown in SEQ ID NO:1. Staple short-chain DNA consists of 272 oligonucleotides, and the corresponding staple strands (synthesized by Sangon Biotech) can be replaced according to different spacing requirements. The sequence of the unmodified staple single strand is shown in Table 1, and the sequences of the staple strand used for hybridization of BCR ligands and TLR agonists, as well as the BCR ligand sequences, are shown in Table 2.

[0125] p8064 backbone chain sequence:

[0126] Table 1. Unmodified staple chain sequences

[0127] The label indicates the position of this DNA molecule on the framework nucleic acid.

[0128] Table 2. DNA sequences of hybrid BCR ligand (arm-1) and TLR agonist (arm-2)

[0129] The asterisk (*) represents a phosphate dithioester bond modification, used to improve the stability of the DNA chain.

[0130] Experimental methods: Columnar framework nucleic acids were formed by mixing modified staple chains (with a molar ratio of 1:1 between each staple chain, as shown in Table 2) with backbone DNA chains at a molar ratio of 10:1 in TAE-Mg (5mM Tris, 1mM EDTA, 16mM MgCl2, pH 8.0) buffer, heating at 80°C for 15 min, and then annealing at 80-65°C every 5 minutes and then at 65-30°C every 30 minutes. Excess staple chains were removed by ultrafiltration three times using 100kDa ultrafiltration tubes at a speed of 4000g for 10 minutes each time.

[0131] A 2 nM sample was placed in the above-mentioned synthesis buffer TAE-Mg, dropped onto the surface of a mica sheet, and allowed to stand for 5 minutes before AFM imaging. Imaging was performed in a liquid environment using a Bruker MultiMode 8 atomic force microscope and a ScanAsyst-Fluid+ probe (Bruker).

[0132] AFM image as Figure 2 As shown in the figure, a columnar structure with a diameter of about 14 nm and a length of up to 220 nm was observed using an atomic force microscope, indicating that the FNA was successfully assembled.

[0133] 2. Functionalization of antigen (anti-IgM) Materials and Methods: Anti-IgM (anti-mouse IgM) was purchased from BioLegend at a concentration of 0.5 mg / mL. 400 μL of the stock solution was concentrated to approximately 2 mg / mL through a 30 kDa ultrafiltration tube, resulting in a volume of approximately 100 μL, and then replaced with phosphate buffer (10 mM Na2HPO4, 150 mM NaCl, pH 7.2).

[0134] The concentrated anti-IgM was coupled to the complementary DNA strand of arm 1 (i.e., the BCR ligand coupling sequence, as shown in Table 3) via a chemical ligation strategy, as follows: (1) The bifunctional crosslinking agent sulfo-SMCC (Merck, M6035) and anti-IgM were mixed in the above phosphate buffer at a molar ratio of 5:1. After reacting at room temperature for 30 min, the excess SMCC was removed by centrifugation three times through a 30 kDa ultrafiltration tube. The centrifugation conditions were 3000 g, 4 °C, and 5 min to obtain SMCC-activated anti-IgM.

[0135] (2) The SMCC-activated anti-IgM was reacted with the complementary DNA strand of Arm 1 in the above phosphate buffer at a molar ratio of 1:10 at 4°C for 8 hours. After the reaction, excess DNA was removed by centrifugation four times through a 30kDa ultrafiltration tube at 3000g, 4°C, and 5 minutes to obtain the complementary DNA-functionalized anti-IgM. The protein was verified by 6% polyacrylamide gel electrophoresis (PAGE) at 120V for 1 hour on ice, and then stained with Cologne blue.

[0136] Table 3. DNA sequences of BCR ligands coupled with TLR agonists

[0137] The asterisk (*) represents a phosphate dithioester bond modification, used to improve the stability of the DNA chain.

[0138] The results are as follows Figure 3 As shown in the figure, complementary DNA functionalized anti-IgM was successfully constructed.

[0139] 3. Construction and characterization of columnar framework nucleic acids loaded with BCR ligands and TLR agonists The purified (1 nM) FNA carrying specific extension sites of arm chain 1 and arm chain 2 was conjugated with 12 molar excess of anti-IgM-DNA1 product and 12 molar excess of anti-IgM-DNA1 TLR agonist CpG ODN 1018 or ssRNA (conjugation sequences are detailed in Table 3; in this example, it is CpG ODN 1018, sequence as shown in SEQ ID NO:311) in TAE-Mg²+ Hybridization was performed by mixing in a buffer system (5 mM Tris, 1 mM EDTA, 12.5 mM MgCl2, pH 8.0) at 37°C for 30 minutes, using DNA base complementarity pairing. Subsequently, the mixture was centrifuged at 4,000g for 10 minutes using a 100 kDa ultrafiltration tube, repeated three times, to remove excess anti-IgM-DNA1 conjugate and TLR agonist.

[0140] The construction and characterization of columnar framework nucleic acids loaded with BCR ligands and TLR agonists were analyzed by 1.5% agarose gel electrophoresis. The electrophoresis conditions were ice bath, and TAE-Mg²⁺ was used. + The electrophoresis was performed at 120 V for 1 hour using a buffer solution (5 mM Tris, 1 mM EDTA, 12.5 mM MgCl2, pH 8.0). The gels were pre-stained with GelRed and imaged using a ChemiDoc MP imaging system (Bio-Rad). Atomic force imaging conditions were the same as above.

[0141] The results are as follows Figure 4-5 As shown in the figure, the results of 1.5% agarose gel electrophoresis ( Figure 4 The results showed that the BCR ligand anti-IgM and the TLR agonist CpG ODN 1018 were successfully loaded into the FNA, with the spacing between the antigen anti-IgM and CpG ODN 1018 set to 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm, respectively. Atomic force microscopy imaging results (listed in...) Figure 5 The results showed that anti-IgM could indeed assemble near the top of the 12-helix FNA, and the position of CpG on the 12-helix FNA was also in line with the experimental expectations. The structured subunit vaccine with the spacing between the antigen anti-IgM and CpG ODN 1018 was successfully prepared at 5nm, 14nm, 20nm, 50nm and 100nm, respectively.

[0142] Example 3 This embodiment provides a structured subunit vaccine using a columnar framework nucleic acid as a carrier. The columnar framework nucleic acid is coupled with an antigen (specifically, a BCR ligand anti-IgM) and a TLR agonist (ssRNA). The specific preparation method is the same as in Example 2, except that step 3, the construction and characterization of the columnar framework nucleic acid loaded with the BCR ligand and TLR agonist, differs. The method is as follows: The purified (1 nM) FNA carrying specific extension sites of arm chain 1 and arm chain 2 was conjugated with 12 molar excess of anti-IgM-DNA1 product and 12 molar excess of anti-IgM-DNA1 TLR agonist TLR7 agonist (ssRNA) (sequence details are shown in Table 3; in this example, it is TLR7 agonist (ssRNA), sequence as shown in SEQ ID NO:312) in TAE-Mg² + Hybridization was performed by mixing in a buffer system (5 mM Tris, 1 mM EDTA, 12.5 mM MgCl2, pH 8.0) at 37°C for 30 minutes, using DNA base complementarity pairing. Subsequently, the mixture was centrifuged at 4,000g for 10 minutes using a 100 kDa ultrafiltration tube, repeated three times, to remove excess anti-IgM-DNA1 conjugate and TLR agonist. Structured subunit vaccines with distances of approximately 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm between the antigen and TLR agonist were prepared.

[0143] Electrophoretic characterization of structured subunit vaccines with distances of approximately 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm between the antigen and TLR agonist is shown below. Figure 6 As shown in the figure, the FNA band co-assembled with anti-IgM and ssRNA significantly lags behind the FNA, indicating that the structured subunit vaccine was successfully prepared.

[0144] Example 4 This embodiment provides a structured RBD vaccine, using the SARS-CoV-2 spike protein receptor-binding domain (RBD) as the BCR ligand, and demonstrates the immunostimulatory effect induced in vivo by the subunit vaccine constructed in this invention. The specific preparation method of the structured RBD vaccine is as follows: 1. Preparation of Columnar Framework Nucleic Acids (FNAs) The preparation method is the same as in Example 2.

[0145] 2. Preparation of model antigens The coding sequence of the SARS-CoV-2 spike protein receptor-binding domain (RBD, GenBank accession number: YP_009724390.1) (codon optimized for mammalian cell expression, the optimized sequence is RVQPTESIVRFP NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO:314)) was synthesized by a commercial company (Shanghai Tianxi Biotechnology Co., Ltd.).

[0146] (1) Preparation of receptor-binding domain (RBD-Halo tag) of SARS-CoV-2 spike protein carrying Halo tag as a model antigen 1) The N-terminal tissue plasminogen activator signal sequence (TPA signal peptide) with secretion signal peptide was cloned into the pcDNA3.1(+) vector.

[0147] 2) Following standard molecular cloning procedures, the RBD sequence, Halo tag sequence (GenBank accession number: ADN27525.1), and C-terminal 8×His tag were sequentially cloned downstream of the TPA signal peptide to construct the RBD-Halo tag expression vector.

[0148] 3) Expi293F suspension cells (Thermo Fisher Scientific) were cultured in OPM-293 CD05 medium (OPM Biosciences) at 37°C, 120 rpm, and 8% CO2. To prepare the RBD-Halo tag protein, the cell concentration was adjusted to 2 × 10⁻⁶ cells / year. 6 Cells / mL. In 100 μL OPM-293 CD05 medium, 1 μg of expression plasmid was mixed with 3 μg of polyethyleneimine (PEI, Polysciences), incubated at room temperature for 20 min, and then added to cell suspension for transfection. Approximately 20 hours after transfection, to promote protein expression, ProFeed (OPM Biosciences) transfection enhancement medium was added, and the cells were cultured for another 3–6 days. After culture, the cell suspension was centrifuged at 1000 g for 3 min, and the supernatant was collected for subsequent protein purification.

[0149] 4) Inject the culture supernatant into a Ni-NTA column (Smart-Lifesciences) at 4°C. Elute the His8-tagged protein stepwise with PBS buffer supplemented with 20mM, 50mM, and 250mM imidazole (pH 8). Collect the corresponding imidazole eluents and concentrate them using a 30kDa molecular weight cutoff centrifuge (MilliporeSigma). Load the concentrated protein onto a Superdex 200 Increase 16 / 600 GL column (GE Healthcare) and elute with PBS buffer to obtain high-purity RBD-HaloTag protein. Collect the corresponding peak fractions, concentrate, sterilely filter, and store at -80°C.

[0150] (2) Precise quantitative coupling of RBD-HaloTag to DNA employs a specific covalent binding strategy between HaloTag and chloroalkanes: Preparation of chloroalkane-modified BCR ligand-coupled chains: The amino-modified chain was reacted with chloroalkane modified with succinimide (NHS) ester in 1×PBS at a 1:50 molar ratio for 6 hours at room temperature; unreacted chloroalkane was removed by two ethanol precipitations; finally, the successful labeling of chloroalkane was confirmed by mass spectrometry. Coupling of DNA-chloroalkane with RBD-HaloTag: DNAX-chloroalkane was reacted with RBD-HaloTag protein at a 1:1 ratio at 37°C for 5 hours, forming RBD-DNA through the specific chemical bond of HaloTag; finally, the successful coupling of RBD and DNA was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE results are shown below. Figure 7 As shown in the figure, the 1:1 coupling of HaloTag with chloroalkane-labeled ssDNA successfully yielded RBD-DNA.

[0151] RBD-DNA and CpG ODN 1018 were co-assembled onto FNA via DNA base complementarity pairing (same as in Example 3). The purified FNA carrying specific arm chain 1 and arm chain 2 extension sites was then coupled with 12-fold molar excess of RBD-DNAX and 12-fold molar excess of CpG ODN 1018 in a TAE-Mg... 2+The mixture was placed in a buffer system (5 mM Tris, 1 mM EDTA, 16 mM MgCl2, pH 8.0) at 37°C for 30 minutes for hybridization via DNA base pairing. Excess RBD-DNA and CpG were then removed by centrifugation at 4000g for 10 minutes using a 100 kDa ultrafiltration tube, repeated three times. The final vaccine was analyzed and characterized by 1.5% agarose gel electrophoresis on ice. The construction and characterization of the columnar framework nucleic acid loaded with antigen and TLR agonist were performed, and the average number of RBDs and CpGs loaded on each FNA particle was determined using a quantitative real-time method based on strand displacement reaction.

[0152] The characterization results of the number of RBD loads are as follows: Figure 8 As shown, according to statistical results, approximately 50% of the six Cy3 fluorescent molecules assembled from each framework nucleic acid were replaced, indicating that the number of loaded RBDs was three. The characterization results of the CpG loading number are as follows... Figure 9 As shown, almost all of the Cy3 fluorescent molecules were replaced, indicating that approximately 6 CpG molecules were loaded.

[0153] Example 5: Cell binding and uptake analysis of structured subunit vaccines This embodiment tested the ability of the structured subunit vaccine prepared in Example 2 to recognize B cells.

[0154] Experimental methods: One spleen from a female BALB / c mouse was harvested and mechanically dispersed under aseptic conditions. Following the instructions of the PrecisionBioMedicals B cell isolation kit, approximately 30 million high-purity primary B cells were obtained. The sorted B cells were then processed at a concentration of 1×10⁻⁶ cells / mL. 6 Cells were seeded at a density of 10 cells / well in RPMI 1640 medium containing 10% FBS and 1×Penicillin-Streptomycin (P / S) for subsequent cell binding and uptake experiments. Columnar framework nucleic acids loaded with BCR ligands and TLR agonists are referred to as structured subunit vaccines.

[0155] To assess the recognition ability of this vaccine with B cells, a Cy5-labeled staple chain (TGCAATAAC(Cy5) GATGCATTTGACTTTCTCCGTGGCGCGGTTG (SEQ ID NO:313)) was used to synthesize an FNA. The preparation method was the same as in Example 2, except that a Cy5-labeled staple chain was added to the staple chain combination used in Example 2 for FNA synthesis. Approximately 1 million B cells were mixed with a subunit vaccine (structured subunit vaccine with antigen anti-IgM and CpG ODN 1018 spacing of 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm, respectively) at a final concentration of 2 nM and incubated at 4°C for 30 minutes in 300 μL of the above-mentioned culture medium. After incubation, the cells were thoroughly washed with PBS at 4°C, and then imaged using a confocal microscope. Fluorescence intensity data were collected using a flow cytometer (CytoFLEX S, Beckman Coulter).

[0156] To assess the uptake of structured immune adapters in B cells, 1 million B cells were incubated with a Cy5-labeled subunit vaccine under the same conditions at 37°C and 5% CO2 for 3 hours. After incubation, excess vaccine was removed by washing with cold PBS, and fluorescence intensity data were collected again using flow cytometry (CytoFLEX S). A control was prepared by incubating cells with an FNA containing only the TLR agonist CpG ODN 1018 at the same concentration. All experiments were performed in triplicate, and data were analyzed using FlowJo software.

[0157] The results are as follows Figure 10-11 As shown in the figure, confocal imaging and flow cytometry (…) Figure 10 The results showed significant fluorescence on the surface of B cells at 4°C, demonstrating that FNA could clearly bind to the B cell surface. The adsorption amount of vaccines at each interval subunit was significantly better than that of CpG-loaded and unloaded FNA, proving the promoting effect of BCR ligands on B cell recognition. Incubation at 37°C for 3 hours, followed by quantitative analysis by flow cytometry, further confirmed (…). Figure 11 Compared with the control group, the vaccine loaded with BCR ligand and TLR agonist showed significantly higher intracellular fluorescence intensity, demonstrating the promoting effect of BCR ligand on B cell uptake.

[0158] Example 6 Detection of B cell activation markers This embodiment tests the B cell activation ability of the structured subunit vaccines prepared in Examples 2 and 3.

[0159] (1) Detection of the B cell activation ability of the structured subunit vaccine prepared in Example 2 Spleens from two BALB / c female mice were harvested and mechanically dispersed under aseptic conditions. Following the instructions of the PrecisionBioMedicals B cell isolation kit, approximately 60 million high-purity primary B cells were obtained. The sorted B cells were then processed at a concentration of 1×10⁻⁶ cells / mL. 6 Cells were seeded at a density per well in RPMI 1640 medium containing 10% FBS and 1×Penicillin-Streptomycin (P / S) for subsequent B cell activation experiments.

[0160] The activation efficacy of FNAs co-assembled with anti-IgM and CpG ODN 1018 (FNA-5, FNA-14, FNA-20, FNA-50, and FNA-100, i.e., structured subunit vaccines with spacings of 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm between the antigen anti-IgM and CpG ODN 1018, respectively) on B cells was assessed. Control constructs included FNAs assembled only with anti-IgM (FNA-anti-IgM), FNAs assembled only with CpG ODN 1018 (FNA-CpG), and a mixture of both (Mix). 24-well plates were prepared, with 3 million B cells seeded in each well. 1 mL of culture medium containing the different structures was added to each well to achieve a final concentration of 2 nM for each group of structured subunit vaccines. The culture conditions were 37 ℃ and 5% CO2. After incubation for 24 hours in each group, excess vaccine was removed by washing with PBS, and B cells were collected and dispersed in 100 μL of PBS buffer. Then, 1 µL of APC anti-mouse CD80 and APC anti-mouse CD69 were mixed at 4°C for 20 minutes for staining. After staining, the cells were washed three times with PBS, and the positive rates of CD69 and CD86 in each treatment group were analyzed by flow cytometry. After incubation for 72 hours, PE anti-mouse CD80 (BioLegend) was stained using the same method, and the positive rate was analyzed by flow cytometry.

[0161] Flow cytometry analysis graph as follows Figure 12 As shown in the figure, the stimulation effect of the subunit vaccine with BCR ligand and TLR agonist loaded at various intervals is significantly better than that of the physical mixture of BCR ligand and TLR, proving that the co-assembled structure is significantly better than the simple mixture, and the effect is best with an interval of 20 nm.

[0162] (2) Detection of the B cell activation ability of the structured subunit vaccine prepared in Example 3 The experimental protocol was the same as in (1) above, except that the structured subunit vaccine prepared in Example 2 was replaced with the structured subunit vaccine prepared in Example 3, with the distance between the TLR7 agonist and the antigen being 5 nm, 14 nm, 20 nm, 50 nm, and 100 nm. Flow cytometry was used to analyze the expression of CD69, a marker of early B cell activation, and co-stimulatory molecules CD86 and CD80.

[0163] The results are as follows Figure 13 As shown, the flow cytometry and ELISA results indicate that stimulation with all different subunit vaccines can induce upregulation of the expression of CD69, a marker of early B cell activation, and co-stimulatory molecules CD86 and CD80, and is significantly better than that of the physical mixture group, indicating that the co-assembled structure has cross-platform universality of the TLR type.

[0164] Example 7 Analysis of B cell proliferation and cytokine secretion This embodiment tests the ability of the structured subunit vaccine prepared in Example 2 to induce B cell functional activation.

[0165] Test method: Freshly isolated B cells were stained according to the CFSE staining kit instructions: approximately ten million B cells were dispersed in PBS buffer (1 mL), and 0.5 μL of 5 mM CFDA SE stock solution was added. The cells were incubated at 37°C in the dark for 10 minutes. Excess staining agent was washed away with culture medium to obtain uniformly labeled B cells with CFSE fluorescent signals. These B cells were then cultured at 1 × 10⁻⁶ cells per well. 6 Cells were seeded into 24-well plates with 1 mL of culture medium and stimulated with equal volumes of subunit vaccines prepared in Example 2 at different spacings, and control groups (FNAs assembled only with anti-IgM (FNA-anti-IgM), FNAs assembled only with CpG ODN 1018 (FNA-CpG), and a mixture of both (Mix)). Incubation time was 72 hours. After incubation, cells were collected by centrifugation (1500 rpm, 5 min) and washed with PBS. The cell supernatant was collected for subsequent analysis of cytokine secretion. The serial dilution of CFSE signaling in B cells was then detected by flow cytometry to assess the promoting effect of the subunit vaccine on B cell proliferation. The IL-6 content in the culture supernatant was measured according to the ELISA kit instructions for quantitative analysis to assess the ability of the subunit vaccine to induce B cell functional activation.

[0166] The results are as follows Figure 14As shown in the figure, the subunit vaccine co-loaded with BCR ligand and TLR agonist induced significantly better B cell proliferation than the physical mixture group. Simultaneously, ELISA results also showed that the immune adapter loaded with BCR ligand and TLR agonist induced significantly better cytokine secretion than the control group, collectively demonstrating that the co-assembled structure was significantly superior to simple mixing, with a spacing of 20 nm yielding the best results.

[0167] Example 8: Evaluation of mouse immunization and vaccine efficacy This embodiment tested the immunization effect of the structured subunit vaccine prepared in Example 4.

[0168] Experimental methods: (1) Laboratory animals and grouping Six-week-old female Balb / c mice were selected, with n=6 in each group. The animals were randomly divided into three groups: 1) PBS control group; 2) Conventional vaccine group: a simple physical mixture of RBD antigen (5 μg), CpG ODN 1018 (1 μg) and aluminum adjuvant; 3) Structured vaccine group: The structured subunit vaccine RBD-CpG-20 prepared in Example 4 (the distance between RBD antigen and CpG ODN 1018 was set to 20nm) contained 5μg RBD and an equimolar amount of CpG.

[0169] (2) Immunoassay 1) Mice in each group were subcutaneously (sc) injected with 100 μL of the corresponding preparation on day 0 and day 14, while the PBS control group was injected with an equal volume of PBS solution.

[0170] 2) Blood was collected retroorbitally on days 21 and 28 after the first immunization, and the serum was separated for subsequent IgG titer testing.

[0171] (3) Vaccine efficacy testing: 1) The titer of RBD-specific IgG antibodies in serum was detected by ELISA to compare the differences between structured vaccines and conventional formulations in inducing humoral immunity.

[0172] ELISA test results as follows Figure 15 As shown in the figure, the serum IgG titer of mice in the RBD-CpG-20 immunization group was significantly higher than that of mice in the RBD@CpG1018@aluminum adjuvant immunization group at both 21 and 28 days post-immunization, confirming the superiority of the structured subunit vaccine.

[0173] 2) Combine neutralization experiments with memory B cell analysis to comprehensively evaluate the immune effect.

[0174] Methods: HEK-293T-ACE2 cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. 1×10⁶ cells were used... 5 HEK-293T-ACE2 cells were seeded into two glass-bottomed cell cultures. Subsequently, 1 µL of pseudovirus-SARS-CoV-2 and 100 µL of 10-fold diluted serum samples from days 21 and 28 were pre-incubated at 37°C for 1 h. After co-culturing the mixture with the cells for 12 h, the mixture was discarded, and fresh high-glucose DMEM medium was added for further culture for 48 h. Finally, the treated cells were resuspended in high-glucose DMEM medium, and GFP expression was detected by confocal imaging.

[0175] Figure 16 The image shows the results of confocal microscopy detection of the ability of mouse serum to neutralize SARS-CoV-2 pseudovirus. The results indicate that the serum from the framework nucleic acid subunit vaccine group was more effective in preventing pseudovirus infection of ACE2-overexpressing 293T cells on both day 21 and day 28 post-immunization.

[0176] After euthanizing the mice, the spleens of each immunized mouse were collected to isolate spleen cells. The cells were then stained with anti-mouse CD27 and CD19 antibodies and analyzed by flow cytometry. The results of the proportion of memory B cells in the spleen of mice 30 days post-immunization are shown below. Figure 17 As shown in the figure, the proportion of memory B cells in spleen B cells was significantly higher in the framework nucleic acid subunit vaccine group than in the aluminum adjuvant group.

[0177] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. A structured subunit vaccine based on framework nucleic acids, characterized in that, include: A framework nucleic acid, an antigen, and an adjuvant, wherein the antigen and adjuvant are coupled to the framework nucleic acid; The distance between the antigen and the adjuvant is 5-400 nm.

2. The structured subunit vaccine according to claim 1, characterized in that, The spacing is achieved by changing the number of segments in the DNA double helix of the framework nucleic acid; And / or, the shape of the framework nucleic acid includes one of columnar, cuboid, and hexagonal prism structures; Preferably, the framework nucleic acid includes a backbone sequence and a Staple short chain; More preferably, the backbone sequence of the framework nucleic acid includes the nucleotide sequence shown in SEQ ID NO:1; More preferably, the sequence of the Staple short chain includes one or more of sequences SEQ ID NO:2-SEQ ID NO:

309.

3. The structured subunit vaccine according to claim 1, characterized in that, The antigen can be linked to the framework nucleic acid via covalent or non-covalent bonds, or via molecular adapters; And / or, the antigen includes a BCR-activated ligand; Preferably, the BCR activating ligand comprises an antigenic peptide; More preferably, the antigenic peptide includes the SARS-CoV-2 S protein receptor-binding region, anti-IgM, HA, OVA, and tumor antigen peptide.

4. The structured subunit vaccine according to claim 1, characterized in that, The adjuvant includes a TLR agonist; Preferably, the TLR agonist includes at least one of a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR8 agonist, and a TLR9 agonist; More preferably, the TLR agonist includes at least one of CpG ODN, ssRNA, and MPLA.

5. A method for preparing a structured subunit vaccine as described in any one of claims 1-4, characterized in that, The method includes the following steps: conjugating the antigen and TLR agonist to the framework nucleic acid, and adjusting the spacing between the antigen and TLR agonist to 5-400 nm.

6. The preparation method according to claim 5, characterized in that, The framework nucleic acid was prepared by the following method: the backbone sequence and the short chain of Staple were added to a buffer containing magnesium ions and reacted to prepare the framework nucleic acid; Preferably, the concentration of magnesium ions in the magnesium-containing buffer solution is 14-18 mM; Preferably, the reaction further includes a step of centrifuging and ultrafiltration to remove excess staple short chains from the prepared reaction product; More preferably, the centrifugation conditions are 3800-4200g for 8-12min; More preferably, the ultrafiltration tube used in the ultrafiltration process has a molecular weight cutoff of 10-100 kDa.

7. The preparation method according to claim 5, characterized in that, Prior to conjugation, the process also includes a step of functionalizing the antigen, the steps of which are as follows: The antigen is reacted with a bifunctional cross-linking agent to prepare an antigen activated by the cross-linking agent; the antigen activated by the cross-linking agent is reacted with a BCR ligand coupling sequence to prepare an antigen with complementary DNA functionalization; the BCR ligand coupling sequence is shown in SEQ ID NO:

310. Preferably, the bifunctional crosslinking agent includes SMCC, NHS, and Halota; Preferably, the molar ratio of the antigen to the bifunctional cross-linking agent is 1:(4-6). Preferably, the reaction time between the antigen and the bifunctional cross-linking agent is 25-35 minutes; Preferably, the molar ratio of the antigen activated by the cross-linking agent to the BCR ligand coupling sequence is 1:(8-12). Preferably, the reaction time between the antigen activated by the cross-linking agent and the BCR ligand coupling sequence is 7-9 hours; Preferably, the reaction temperature between the antigen activated by the cross-linking agent and the BCR ligand coupling sequence is 2-6°C.

8. The preparation method according to claim 5, characterized in that, The specific steps for conjugating antigens and TLR agonists to framework nucleic acids include: placing the antigen, TLR agonist, and framework nucleic acid in a magnesium-containing buffer solution for hybridization reaction; Preferably, the molar ratio of the antigen, TLR agonist and framework nucleic acid is (8-15):(8-15):

1. Preferably, the hybridization reaction takes 25-35 minutes; Preferably, the temperature of the hybridization reaction is 35-38°C; Preferably, the hybridization reaction further includes a step of centrifuging and ultrafiltration to remove excess antigen and TLR agonist from the prepared reaction product.

9. An immunogenic composition, characterized in that, The immunogenic composition includes the structured subunit vaccine as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

10. The use of the structured subunit vaccine according to any one of claims 1-4, or the immunogenic composition according to claim 9, in any of the following: (1) Preparation of drugs for generating an immune response in subjects; (2) Prepare drugs for the prevention or treatment of pathogen infection and tumors.

Images