Method for preparing conformational polypeptide vaccine and use thereof in treatment of protein conformational disease

Abstract

A method for preparing a conformational polypeptide vaccine and the use thereof in the treatment of a protein conformational disease. Provided is a novel idea of using a conformational antigen as an active ingredient of a vaccine. The conformational antigen is a polypeptide assembly, is formed by self-assembly of an artificially designed polypeptide, has a conformation similar to that of a pathological protein aggregate having a β structure, and is a cross antigen for the pathological protein aggregate having the β structure. The conformational antigen is used as an active ingredient to prepare a vaccine. After the vaccine is injected, an antibody generated by the body can target the pathological protein aggregate having the β structure, thereby preventing and / or treating a protein conformational disease.

Description

A method for preparing a polypeptide conformation vaccine and its application in the treatment of protein conformation diseases. Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a method for preparing a polypeptide conformation vaccine and its application in the treatment of protein conformation diseases. Background Technology

[0002] Protein molecules need to fold into specific three-dimensional structures to perform their physiological functions, and conformational changes can lead to protein conformation disorders. A typical characteristic of protein conformation disorders is the self-assembly of one or more disease-related proteins accompanied by conformational inversion, resulting in loss of physiological function or the formation of toxic aggregates that induce other pathological changes. Typical protein conformation disorders include transmissible spongiform encephalopathy, Alzheimer's disease (AD), Parkinson's disease, and other neurodegenerative diseases, as well as non-neurological diseases such as type II diabetes and medullary thyroid carcinoma. These are zoonotic diseases that seriously threaten human health and property. In protein conformation disorders, conformational inversion manifests as α-helix-rich globulins or the formation of β-structure-dominant fibers from randomly structured polypeptides. The morphology of protein / polypeptide self-assembled fibers is closely related to the pathological classification of protein conformation disorders.

[0003] Antigen cross-reactivity refers to the reaction of antibodies or sensitized lymphocytes to different antigens that share a common epitope. Different species and origins of biological macromolecules can also complement the same antibody if their epitopes have partially similar spatial structures. Protein / peptide self-assembled fibers have advantages such as excellent mechanical properties and readily available raw materials; through rational design, peptides with a tendency to form specific structures (such as β-hairpins, β-sheets, etc.) can be obtained, which can then self-assemble into well-defined self-assembled fibers. By controlling the amino acid sequence of the peptide, peptide fibers can maintain the β-structure while avoiding toxicity and pathogenicity.

[0004] Invention Overview

[0005] Firstly, the inventors of this invention have pioneered a novel approach by introducing conformational antigens as active ingredients in vaccines. Conformational antigens are polypeptide assemblies, formed by the self-assembly of artificially designed polypeptides. They possess a conformational similarity to pathological protein aggregates with β-structures, thus serving as cross-antigens for these aggregates. Furthermore, the inventors have prepared vaccines using conformational antigens as active ingredients. After vaccination, the antibodies produced by the body can target pathological protein aggregates with β-structures, thereby preventing and / or treating protein conformation disorders. Technical issues

[0006] The technical problem solved by this invention is to provide a conformational vaccine with a novel design concept, which can be used for the prevention and / or treatment of protein conformational diseases. Technical solutions

[0007] This invention provides a polypeptide, named T1 polypeptide, whose amino acid sequence is shown in SEQ ID NO: 1 (RGYFWAGDYNYF), and all chiral amino acids in the polypeptide are L-type amino acids.

[0008] The T1 polypeptide is an artificially designed polypeptide.

[0009] This invention also protects a polypeptide, named the T1 truncated form, whose amino acid sequence is shown in a portion of SEQ ID NO: 1, and all chiral amino acids in the polypeptide are L-type amino acids. Exemplarily, the T1 truncated form is obtained by removing 1-5 amino acid residues from the N-terminus and / or C-terminus of the T1 polypeptide. Exemplarily, the T1 truncated form is obtained by removing 1-2 amino acid residues from the N-terminus and / or C-terminus of the T1 polypeptide. Exemplarily, the T1 truncated form is shown at positions 3 to 12 in SEQ ID NO: 1.

[0010] The present invention also protects derivatives of the T1 polypeptide, the molecular structure of which includes the T1 polypeptide.

[0011] The present invention also protects derivatives of the T1 truncated form, the molecular structure of which includes the T1 truncated form.

[0012] The present invention also protects a polypeptide assembly (named polypeptide assembly I), which is an oligomer, protofibril, fiber or fiber aggregate having a β structure assembled from the T1 polypeptide, a derivative of the T1 polypeptide, a truncated T1 polypeptide or a derivative of the truncated T1 polypeptide.

[0013] The present invention also protects a method for preparing polypeptide assembly I, comprising the following steps: dissolving the T1 polypeptide, a derivative of the T1 polypeptide, a truncated T1 polypeptide or a derivative of the truncated T1 polypeptide in a solvent, and the T1 polypeptide, the derivative of the T1 polypeptide, the truncated T1 polypeptide or a derivative of the truncated T1 polypeptide self-assembling to form polypeptide assembly I.

[0014] In the above preparation method, the solvent is an inorganic solvent (e.g., water) or an organic solvent.

[0015] In the above preparation method, commonly used methods in the prior art can be used to promote the dissolution of peptides in solvents, such as ultrasonic dissolution and / or heating.

[0016] In the above preparation method: under static or oscillating conditions, the polypeptide aggregates and self-assembles to form a polypeptide assembly.

[0017] As one specific embodiment, the preparation method of the polypeptide assembly is as follows: the T1 polypeptide, the derivative of the T1 polypeptide, the T1 truncated form or the derivative of the T1 truncated form are dissolved in an aqueous solvent to a concentration of 0.02-100 mg / mL, and incubated at 4-90℃.

[0018] As one specific implementation method, the preparation method of the polypeptide assembly is as follows: the T1 polypeptide, the derivative of the T1 polypeptide, the T1 truncated form or the derivative of the T1 truncated form are dissolved in water to a concentration of 1 mg / mL, and incubated at 37°C for 3 days.

[0019] The present invention also protects the use of polypeptides or polypeptide derivatives or polypeptide assemblies or compositions containing polypeptide assemblies in the preparation of vaccines for the prevention and / or treatment of protein conformation disorders;

[0020] The polypeptide is the T1 polypeptide or the T1 truncated form;

[0021] The derivative of the polypeptide is a derivative of the T1 polypeptide or a derivative of the T1 truncated polypeptide.

[0022] The polypeptide assembly is polypeptide assembly I;

[0023] The composition containing the polypeptide assembly consists of the polypeptide assembly I and a vaccine adjuvant.

[0024] The present invention also protects vaccines for the prevention and / or treatment of protein conformation disorders, the active ingredient of which includes said polypeptide assembly I or a composition containing said polypeptide assembly (composed of said polypeptide assembly I and a vaccine adjuvant).

[0025] This invention also protects a polypeptide assembly (named polypeptide assembly II), which is an oligomer, protofibril, filament, or filament aggregate having a β structure assembled from a Tx polypeptide or a Tx polypeptide derivative. The Tx polypeptide is a polypeptide capable of self-assembling to form a target structure (usually an artificially designed polypeptide). The amino acid sequence of the Tx polypeptide is not limited. The target structure is: an oligomer, protofibril, filament, or filament aggregate having a β structure. The target structure is: a structure identical or similar to that of polypeptide assembly I. The Tx polypeptide derivative is a substance whose molecular structure includes the Tx polypeptide.

[0026] The present invention also protects a method for preparing polypeptide assembly II, comprising the following steps: dissolving the Tx polypeptide or Tx polypeptide derivative in a solvent, wherein the Tx polypeptide or the Tx polypeptide derivative self-assembles to form polypeptide assembly II.

[0027] In the above preparation method, the solvent is an inorganic solvent (e.g., water) or an organic solvent.

[0028] In the above preparation method, commonly used methods in the prior art can be used to promote the dissolution of peptides in solvents, such as ultrasonic dissolution and / or heating.

[0029] In the above preparation method: under static or oscillating conditions, the polypeptide aggregates and self-assembles to form a polypeptide assembly.

[0030] The present invention also protects the use of polypeptides or polypeptide derivatives or polypeptide assemblies or compositions containing polypeptide assemblies in the preparation of vaccines for the prevention and / or treatment of protein conformation disorders;

[0031] The polypeptide is the Tx polypeptide;

[0032] The derivative of the polypeptide is the Tx polypeptide derivative;

[0033] The polypeptide assembly is polypeptide assembly II;

[0034] The composition containing the polypeptide assembly consists of polypeptide assembly II and a vaccine adjuvant.

[0035] The present invention also protects vaccines for the prevention and / or treatment of protein conformation disorders, the active ingredient of which includes said polypeptide assembly II or a composition containing said polypeptide assembly (composed of said polypeptide assembly II and a vaccine adjuvant).

[0036] The present invention also provides a composition comprising any of the polypeptide assemblies described above and a vaccine adjuvant.

[0037] In the composition, the mass ratio of the polypeptide assembly to the vaccine adjuvant is 1:1.

[0038] The present invention also provides a method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps: using any of the above-described polypeptide assemblies or the above-described compositions as the active ingredient of the vaccine.

[0039] As one specific implementation method, the vaccine preparation method is as follows:

[0040] (1) The T1 polypeptide, the derivative of the T1 polypeptide, the T1 truncated form or the derivative of the T1 truncated form are dissolved in water to a concentration of 0.02-100 mg / mL and incubated at 4-90℃ to obtain a polypeptide assembly solution.

[0041] (2) Dissolve QS-21 in DMSO, then dilute with buffer solution to obtain QS-21 solution;

[0042] (3) Mix the polypeptide assembly solution with the QS-21 solution and then add buffer solution to obtain the vaccine.

[0043] As one specific implementation method, the vaccine preparation method is as follows:

[0044] (1) The T1 polypeptide, the derivative of the T1 polypeptide, the T1 truncated form or the derivative of the T1 truncated form are dissolved in water to a concentration of 1 mg / mL and incubated at 37°C for 3 days to obtain the polypeptide assembly solution.

[0045] (2) Dissolve QS-21 in DMSO, and then dilute with buffer to 1 mg / mL to obtain the QS-21 solution;

[0046] (3) Mix 1 volume of polypeptide assembly solution with 1 volume of QS-21 solution in equal volume, and then add 1 volume of buffer solution to make the vaccine.

[0047] For example, the buffer solution is PBS buffer (pH 7.2).

[0048] The present invention also protects methods for preventing and / or treating protein conformation disorders, comprising the steps of administering to a subject a therapeutically effective amount of a polypeptide assembly (any of the above) or a composition (any of the above) or a vaccine (any of the above) to prevent and / or treat protein conformation disorders.

[0049] This invention also protects a pharmaceutical compound, characterized in that: the pharmaceutical compound is any of the polypeptide assemblies described above.

[0050] The pharmaceutical compound is used for the prevention and / or treatment of protein conformation disorders.

[0051] The present invention also provides a method for designing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps:

[0052] (1) Design the amino acid sequence of the polypeptide according to the following target: the polypeptide can self-assemble to form a polypeptide assembly with the target structure; the target structure is: an oligomer, protofibril, filament or filament aggregate with a β structure;

[0053] (2) Use polypeptide assemblies as the active ingredient of vaccines.

[0054] The present invention also provides a method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps:

[0055] (1) Design the amino acid sequence of the polypeptide according to the following target: the polypeptide can self-assemble to form a polypeptide assembly with the target structure; the target structure is: an oligomer, protofibril, filament or filament aggregate with a β structure;

[0056] (2) Synthesize the polypeptide according to the amino acid sequence designed in step (1), and then assemble it into a polypeptide assembly;

[0057] (3) The polypeptide assembly obtained in step (2) is used as the active ingredient of the vaccine.

[0058] Vaccine adjuvants are auxiliary substances that, when injected into the body together with or before the antigen, can enhance the body's immune response to the antigen and / or alter the type of immune response.

[0059] The vaccine adjuvants described above may be biological adjuvants and / or inorganic adjuvants and / or synthetic adjuvants and / or oils.

[0060] The vaccine adjuvants mentioned above include, but are not limited to: QS-21, Freund's adjuvant, incomplete Freund's adjuvant, alumina, aluminum phosphate, aluminum sulfate, 3-de-O-acylated monophosphoryl lipid A (MPL), TQL1055, QS-18, QS-17, QS-7, CpG, polyglutamic acid, polylysine, and AddaVax. TM and Any one or any combination of them.

[0061] The vaccines described above also include additives. These additives include, but are not limited to: carriers and / or excipients and / or pH buffers and / or osmotic pressure regulators and / or stabilizers and / or solubilizers. If desired, the additives may also include: colorants and / or preservatives and / or flavorings and / or taste agents and / or sweeteners, etc. These additives are well known and can be selected by those skilled in the art. Those skilled in the art select appropriate additives to formulate suitable dosage forms for use via selected routes of administration.

[0062] In some embodiments, the stabilizers and solubilizers include, but are not limited to, surfactants such as Tween and lauryl sarcosine. Other stabilizers and solubilizers include arginine, sucrose, trehalose, cyclodextrin, etc. The excipients include, but are not limited to, glycerol and polyethylene glycol. The osmotic pressure regulators include, but are not limited to, phosphates containing glucose, calcium, magnesium, aluminum, potassium, sodium, etc.

[0063] Other substances may be added to any of the vaccines described above, including neuroprotective agents, neuroregenerative substances, or neuroactive substances.

[0064] Any of the vaccines described above can be administered via injection, including subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, intracerebrospinal injection, or infusion; via cavity administration, such as rectal, vaginal, and sublingual administration; via respiratory tract administration, such as nasal administration; or via mucosal administration. The preferred route of administration is injection, and the preferred route of injection is intramuscular injection.

[0065] Any of the above-described assemblies is a self-assembly.

[0066] The vaccines described above for the prevention and / or treatment of protein conformational diseases are conformational vaccines for the prevention and / or treatment of protein conformational diseases.

[0067] In this application, "subject" includes a person who is being treated or prevented as a patient. The methods described herein can be used to treat animal subjects belonging to any classification. Examples of such animals include mammals. Mammals include, but are not limited to, rodents such as mice and hamsters, and lagomorphs such as rabbits. Mammals can be carnivores, including felines (cats) and canines (dogs). Mammals can be artiodactyla, including bovines (cattle, sheep) and suidae (pigs), or perissodactyla, including equines (horses). Mammals can be primates, ceboids, or simoids (monkeys) or anthropoids (humans and apes). In some embodiments, the mammal is a human.

[0068] The term "effective" when applied to dosage or amount refers to the amount of a compound or composition sufficient to produce the desired activity when administered to a subject in need. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary depending on the subject, including their species, age and general condition, the severity of the condition being treated, one or more specific medications being used, the mode of administration, etc.

[0069] The medicinal compound has the use of preventing and / or treating protein conformation disorders.

[0070] Any of the β structures described above can be β-hairpins and / or β-sheets and / or β-turns.

[0071] The synthesis methods of any of the above-mentioned polypeptides include, but are not limited to, total chemical synthesis, solid-phase polypeptide synthesis, expression in eukaryotic or prokaryotic cells by editing recombinant plasmids through genetic engineering, and purification and extraction from biological tissues.

[0072] The protein conformational diseases mentioned above include, but are not limited to: transmissible spongiform encephalopathy (including but not limited to mad cow disease, scrapie, Creutzfeldt-Jakob disease), Alzheimer's disease, Parkinson's disease, multiple sclerosis, type II diabetes, Gerstmann syndrome, Huntington's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy (oculocytopathy), Hallewarden-Schpatrick disease, auricula-jugular syndrome, chronic traumatic encephalopathy, ganglioglioma, meningeal angiomatosis, dialysis-associated amyloidosis, renal amyloidosis, subacute sclerosing panencephalitis, cardiac amyloidosis, injection-induced focal amyloidosis, pulmonary alveolar proteinosis, lead poisoning encephalopathy, tuberous sclerosis, lipofuscinosis, familial dementia in England, familial dementia in Denmark, light chain amyloidosis, (renal) Heavy chain amyloidosis (Herpes amyloidosis), pia mater amyloidosis, senile systemic amyloidosis, hereditary transthyretin amyloidosis, familial Finnish amyloidosis, hereditary fibrinogen Aα chain renal amyloidosis, Icelandic hereditary cerebral hemorrhage with amyloidosis, aortic medial amyloidosis, AA (secondary) amyloidosis, ApoAI amyloidosis, ApoAI / ApoAII / ApoAIV / ApoCII / ApoCIII amyloidosis, subepithelial corneal amyloidosis, odontogenic calcified epithelial tumors, lichenoid amyloidosis, patchy amyloidosis, nodular amyloidosis, simple hypotrichosis of the scalp, lattice corneal dystrophy, seminal vesicle amyloidosis, pituitary prolactinoma, medullary thyroid carcinoma, prostate cancer.

[0073] As a specific example, the protein conformational disease is Alzheimer's disease. Beneficial effects

[0074] The conformational antigen provided by this invention has the advantages of strong immunogenicity and high biocompatibility. Furthermore, the inventors of this invention use the conformational antigen as an active ingredient to prepare a vaccine. After vaccination, the antibodies produced by the body can target pathological protein aggregates in the β-structure, thereby preventing and / or treating protein conformational diseases. Attached Figure Description

[0075] Figure 1 is a schematic diagram of the molecular structure of the T1 polypeptide.

[0076] Figure 2 shows the high-resolution mass spectrum of the T1 polypeptide prepared in Example 1.

[0077] Figure 3 shows the structural characterization of the polypeptide assembly in Example 2.

[0078] Figure 4 shows the structural model of the polypeptide assembly in Example 2.

[0079] Figure 5 shows the antibody verification in the serum of animals after the immunization conformation vaccine in Example 4.

[0080] Figure 6 shows the results of the improvement of spatial memory in AD model animals by the conformational vaccine in Example 5.

[0081] Figure 7 shows the results of the conformational vaccine in Example 5 reducing the accumulation of amyloid protein in the brains of AD mice.

[0082] Figure 8 shows the results of the conformational vaccine in Example 5 clearing phosphorylated tau protein aggregates in the brains of AD mice.

[0083] Figure 9 shows the HE staining results of neurons in the brains of AD model mice protected by the conformational vaccine in Example 5.

[0084] Figure 10 shows a schematic diagram of sample selection in Example 5, as well as the UMAP visualization results of cell nuclei and the molecular characteristics of cell clustering.

[0085] Figure 11 shows the UMAP representation of cell clusters for all test samples in Example 5.

[0086] Figure 12 shows the Lrrc7 samples in all test samples of Example 5. + UMAP distribution map of neurons in the brain.

[0087] Figure 13 shows the Grin2b values ​​in all test samples from Example 5. + UMAP distribution map of neurons in the brain.

[0088] Figure 14 is a violin diagram showing the expression of marker genes of brain neurons in various samples in Example 5 and the proportion of brain neurons in the brain.

[0089] Figure 15 shows the relevant results of the safety evaluation of conformational vaccine protection in Example 5.

[0090] Implementation methods of the invention

[0091] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0092] Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. Except for glycine, all amino acids are chiral amino acids (classified as L-type and D-type amino acids). Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are performed in triplicate, and the results are averaged.

[0093] The terminology is defined as follows:

[0094] In this application, the term "β-sheet" means: a secondary structure of a protein, where two or more β-sheets are arranged in parallel and linked by hydrogen bonds to form a β-sheet.

[0095] In this application, the term "β-hairpin structure" means: a secondary structure of a protein consisting of parallel β-sheets that form a ring structure through interactions such as hydrogen bonds.

[0096] In this application, the term "β structure" means that the conformation of a protein or polypeptide is predominantly β-sheet.

[0097] In this application, the term "self-assembly" means: the spontaneous formation of supramolecular aggregates by polypeptide or protein molecules driven by non-covalent bonds (including but not limited to hydrogen bonds, hydrophobic interactions, π-π stacking, ionic bonds, etc.), wherein the conformation of polypeptide or protein molecules in supramolecular aggregates is predominantly β-structure. The morphology of supramolecular aggregates includes, but is not limited to: oligomers, protofibrils, fibers, or fiber aggregates.

[0098] In this application, the term "amyloid protein" means: a supramolecular aggregate formed by the self-assembly of polypeptide or protein molecules, with a β-structure as the predominant conformation.

[0099] In this application, the term "protein conformational disease" refers to a disease caused by conformational changes in protein molecules that require folding into specific three-dimensional structures to perform their physiological functions. A typical characteristic of protein conformational diseases is the self-assembly of one or more disease-related proteins accompanied by conformational inversion, resulting in loss of physiological function or the formation of toxic aggregates that induce other pathological changes.

[0100] In this application, the term "conformation vaccine" means: a composition in which a self-assembled product of a β-structured polypeptide (artificial or natural) or protein molecule is the active ingredient. This self-assembled product can act as a cross-antigen for pathological aggregates of the β-structure (the self-assembled product acts as an immunogen to induce the production of antibodies in the organism, which target the pathological aggregates of the β-structure), thereby preventing and / or treating protein conformation diseases.

[0101] AD model mouse (APP / PS1 mouse): VSM40043, VSM-40043, manufactured by VSM-40043. The APP / PS1 mouse is a commonly used Alzheimer's disease (AD) model mouse. It is bred by introducing human amyloid precursor protein (APP) and human presenilin 1 (PS1) gene into the mouse genome using transgenic technology. The APP / PS1 mouse mimics the pathological characteristics of AD patients, such as amyloid plaque formation and cognitive decline, and is widely used in AD research.

[0102] Example 1: Artificial Design and Solid-Phase Synthesis of Peptides

[0103] The amino acid sequence of the T1 polypeptide (SEQ ID NO: 1): RGYFWAGDYNYF.

[0104] Except for glycine, all amino acids in the T1 polypeptide are L-type amino acids.

[0105] Structural formula of T1 polypeptide: R 1 G 2 Y 3 F 4 W 5 A 6 G 7 D 8 Y 9 N 10 Y 11 F 12 G 7 D 8 It originates from the natural turn of the β-lactamase inhibitor BLIP, forming a type II β-turn. Two sets of "amino acid pairs" (Y-T1) are introduced into the T1 polypeptide. 3 -F 12 and F 4 -Y 11 ), and arranged six hydrophobic amino acids (Y). 3 F 4 W 5 A 6 and Y 9 N10 Y 11 F 12 This drives the polypeptide molecule to fold into a β-hairpin structure. In the T1 polypeptide, an arginine residue (R) is introduced at the N-terminus. 1 ), to increase the water solubility of peptides, and through glycine (G 2 It is linked to a β-hairpin structure to reduce the effect of arginine on the structure.

[0106] A schematic diagram of the molecular structure of the T1 polypeptide is shown in Figure 1.

[0107] T1 polypeptide (powder) was prepared by solid-phase synthesis using glycine and other amino acids (all L-type amino acids). The mass spectrometry structure confirmation data is shown in Figure 2 (Calculated: 1558.67, found: 1558.45 [M+H]+).

[0108] Example 2: Peptide self-assembly and conformation characterization

[0109] 1. Take T1 polypeptide powder, add ultrapure water to prepare an aqueous solution of 1 mg / mL, and then incubate at 37°C in the dark for 3 days.

[0110] 2. After completing step 1, the conformation was characterized by circular dichroism spectroscopy. The CD spectrum is shown in Figure 3a. The broad negative peak near 218 nm is a characteristic peak of the β-sheet, and the positive peak near 230 nm indicates the chiral arrangement of aromatic residues.

[0111] 3. After completing step 1, the morphology was characterized using atomic force microscopy. See Figure 3b and Figure 3c (the right image is a magnified view of a portion of the left image). It can be observed that the T1 polypeptide self-assembles into nanofibers, which are left-handed helices.

[0112] The above structural studies indicate that the T1 polypeptide self-assembles into an amyloid fibrous structure with β-sheets as the main component. A schematic diagram of the polypeptide assembly structure is shown in Figure 4.

[0113] Example 3: Preparation of Conformation Vaccine

[0114] QS-21 (CAS No.: 141256-04-4): Shanghai Yuanye Biotechnology Co., Ltd. QS-21 is dissolved in DMSO to a concentration of 10 mg / ml, which is the QS-21 stock solution. Take the QS-21 stock solution and dilute it 10 times with pH 7.2 PBS buffer to obtain the QS-21 solution.

[0115] Take T1 polypeptide powder, add ultrapure water to prepare an aqueous solution of 1 mg / ml, and then incubate at 37°C in the dark for 3 days to obtain T1 polypeptide fiber solution.

[0116] Mix 1 volume of T1 polypeptide fiber solution with 1 volume of QS-21 solution, and then add 1 volume of 2×PBS buffer (pH 7.2) to obtain the conformational vaccine.

[0117] Mix 1 volume of ultrapure water with 1 volume of QS-21 solution, and then add 1 volume of 2×PBS buffer (pH 7.2) to obtain the vaccine control.

[0118] Example 4: Detection of amyloid-binding antibodies using immunofluorescence technique

[0119] I. Animal Immunization

[0120] Four-month-old AD model mice were randomly divided into two groups (control group Q and experimental group L), with 12 mice in each group (6 males and 6 females). Female mice in the control group were denoted as QF, female mice in the experimental group as LF, male mice in the control group as QM, and male mice in the experimental group as LM. The experimental group was immunized monthly by intramuscular injection of the conformational vaccine prepared in Example 3 (40 μL per mouse per injection), for three consecutive months. The control group was immunized monthly by intramuscular injection of the vaccine control prepared in Example 3 (40 μL per mouse per injection), for three consecutive months.

[0121] II. Detection of amyloid-binding antibodies using immunofluorescence technology

[0122] Ten days after the mice in each group completed their third immunization in step one, blood was collected from the inner canthal vein, and serum was obtained. The serum obtained from the experimental group mice was designated as the experimental group serum, and the serum obtained from the control group mice was designated as the control group serum.

[0123] Sixteen-month-old AD model mice (normally fed, without any special treatment) were euthanized with carbon dioxide, and their brains were quickly harvested and paraffin sections were prepared (serial sections of the brain along the sagittal plane at the hippocampus, with a section thickness of 3 micrometers). The paraffin sections were dewaxed and antigen-retrieval was performed. Then, serum from the experimental group and the control group were added to different locations on the same section, incubated overnight, washed with PBST solution, and then incubated with Alexa Fluor 488-labeled goat anti-mouse secondary antibody. Cell nuclei were stained with DAPI, and then observed using an immunofluorescence microscope.

[0124] See Figure 5 for an example photograph. The control group serum staining was very weak, indicating that the control group was antibody negative. The experimental group serum staining showed the binding of diffuse plaques, fibrillary and globular amyloid aggregates, indicating that the experimental group was antibody positive, that is, the antibodies in the experimental group serum can recognize amyloid aggregates in the brain.

[0125] Example 5: Therapeutic effect of conformational vaccine on AD model mice

[0126] I. Animal Immunization

[0127] Four-month-old AD model mice were randomly divided into two groups (control group Q and experimental group L), with 12 mice in each group (6 males and 6 females). Female mice in the control group were denoted as QF, female mice in the experimental group as LF, male mice in the control group as QM, and male mice in the experimental group as LM. The experimental group was immunized monthly by intramuscular injection of the conformational vaccine prepared in Example 3 (40 μL per mouse per injection), for 12 consecutive months. The control group was immunized monthly by intramuscular injection of the vaccine control prepared in Example 3 (40 μL per mouse per injection), for 12 consecutive months.

[0128] II. Characterizing the improvement of spatial memory of conformational vaccines in AD model animals using the Morris water maze test After completing step one, 16-month-old mice in each group were subjected to the water maze test.

[0129] Specific testing steps: Training was conducted from day 1 to day 5 (twice a day). During the training, the escape platform was fixed 1 cm underwater. The time it took for the mouse to find the platform and its swimming trajectory were recorded and analyzed using software. If the mouse still could not find the escape platform within 2 minutes, the timer was stopped manually and the mouse was guided to the escape platform and left for 30 seconds to fully learn and memorize the spatial location of the platform. On day 6, a spatial memory test was conducted. During the test, the escape platform was removed and the mouse was placed in a water maze to swim freely. The mouse's swimming trajectory was recorded. The timer was stopped after 1 minute, and the mouse was removed from the water maze. The characteristics of the mouse's swimming path, the number of times the mouse passed the platform location, and the duration of the mouse in the target quadrant were analyzed using software.

[0130] The swimming path of the mice is shown in Figure 6a. The control group mice swam along the inner wall of the water maze and rarely searched for platforms, reflecting impaired spatial memory and a lack of memory of escape platforms. The experimental group mice searched for escape platforms near the correct locations, reflecting the protective effect of the conformational vaccine on spatial memory.

[0131] The statistical analysis results of the time mice spent in the target quadrant are shown in Figure 6b. Compared with the control group, the experimental group mice spent a significantly longer time in the target quadrant.

[0132] III. Conformation vaccines reduce the accumulation of amyloid protein (Aβ) in the brains of AD mice.

[0133] This step uses immunofluorescence assays to detect the effect of conformational vaccine immunization on pathological aggregates of amyloid protein (Aβ) in the brains of AD mice.

[0134] After completing the water maze test in step two, the mice were euthanized with carbon dioxide, and the brains were quickly harvested. Paraffin sections of the right brain were prepared (continuous sections were taken along the sagittal plane at the hippocampus, with a section thickness of 3 micrometers). The paraffin sections were dewaxed and antigens were retrieved, then primary antibodies were added, and the sections were incubated overnight. The sections were then washed with PBST solution, followed by secondary antibodies and incubation. The cell nuclei were stained with DAPI, and the cells were observed using an immunofluorescence microscope.

[0135] Primary antibody: Recombinant Anti-beta Amyloid 1-42 antibody (Abcam, mOC64, ab201060); used after 1:1000 dilution. Secondary antibody: Cy3-labeled goat anti-mouse IgG.

[0136] See Figure 7 for an example photograph. In the brains of control mice, typical Aβ1-42 aggregates were observed, appearing as diffusely distributed amyloid plaques (reflecting the aggressiveness of the disease) and globular or fibrous aggregates. In the brains of experimental mice, the Aβ1-42 aggregates had clearly defined edges (reflecting limited invasiveness), and the scattered globular or fibrous aggregates disappeared, reflecting that the antibodies produced by the conformational vaccine stimulation functioned to surround the Aβ1-42 aggregates and limit their growth.

[0137] IV. Conformation vaccines clear aggregates of phosphorylated tau protein in the brains of AD mice.

[0138] This step uses immunofluorescence assays to detect the effect of conformational vaccine immunization on pathological aggregates of phosphorylated tau protein in the brains of AD mice.

[0139] Take the paraffin sections obtained in step 3, dewax and retrieval antigen, then add primary antibody, incubate overnight, wash with PBST solution, add secondary antibody, incubate, stain cell nuclei with DAPI, and then observe using an immunofluorescence microscope.

[0140] Primary antibody: Phospho-Tau (Ser202, Thr205) Monoclonal Antibody (Thermofisher, AT8); used after 1:2000 dilution. Secondary antibody: Alexa Fluor 488-labeled goat anti-mouse IgG.

[0141] See Figure 8 for an example photograph. Globular or fibrillary aggregates of phosphorylated tau protein could be detected in the brains of control group mice. These aggregates were not detected in the brains of experimental group mice, reflecting that antibodies stimulated by the conformational vaccine could effectively clear phosphorylated tau protein aggregates.

[0142] V. Conformation-based vaccines protect neurons in the brains of AD model mice

[0143] Because the incidence of Alzheimer's disease is three times higher in women than in men, and because the disease is more severe in age-matched female APP / PS1 mice than in males, female mice were used in steps five and six. This step used HE staining to analyze the protective effect of the conformational vaccine on neurons in the brains of AD model mice.

[0144] Take the paraffin sections of the female mice obtained in step three and perform HE staining.

[0145] The results are shown in Figure 9. In the control group mice, neurons in the frontotemporal cortex were shrunken and deeply stained, with vacuolated cells, exhibiting the staining characteristics of apoptotic neurons. This indicates that aggregates of toxic Aβ and phosphorylated tau protein induced widespread apoptosis in the AD mouse brain. In the experimental group mice, neurons maintained normal morphology and structure, reflecting the protective effect of the conformational vaccine on brain neurons.

[0146] VI. Conformation-based vaccines protect neurons in the brains of AD model mice

[0147] This step uses single-cell sequencing to verify the protective effect of the conformational vaccine on neurons in the brains of AD model mice.

[0148] After completing the water maze test in step two, three female mice in the experimental group (L_1, L_2, L_3) and two female mice in the control group (Q_1, Q_2) were randomly selected and euthanized with carbon dioxide. The brains were quickly removed, the left brain was removed, cell nuclei were extracted, and single-cell transcriptome sequencing analysis was performed.

[0149] The UMAP visualization results of the cell nucleus are shown in Figure 10a. It can be seen that, compared with the brains of the experimental group mice, the brains of the control group mice lack a certain number of cells. The molecular characteristics of cell clustering are shown in Figure 10b (the size of the circle represents the percentage of cells expressing the gene, and the color scale represents the average gene expression level).

[0150] The UMAP representation of cell clusters for all test samples is shown in Figure 11 (color indicates cell type). Neurons in the lower right corner of the brain of the control group mice disappeared, while these neurons were present in the brains of the experimental group mice, indicating that these neurons were protected after the conformational vaccination.

[0151] Lrrc7 in all test samples + The UMAP distribution of neurons in the brain is shown in Figure 12. The brain neuron-specific gene Lrrc7 was highly expressed in the target region (i.e., the region corresponding to the missing neurons in the control group mice) of the experimental group mice.

[0152] Grin2b in all test samples +The UMAP distribution map of brain neurons is shown in Figure 13. The brain neuron-specific gene Grin2b was highly expressed in the target region (i.e., the region corresponding to the missing neurons in the control group mice) of the experimental group mice.

[0153] A violin diagram showing the expression of marker genes for brain neurons in various samples is shown in Figure 14a. The proportion of brain neurons in the brain is shown in Figure 14b. Compared with the control group mouse brains, the experimental group mouse brains showed a significantly increased proportion of brain neurons, indicating that the conformational vaccine has the effect of alleviating neuronal apoptosis in the AD pathogenesis process.

[0154] VII. Safety evaluation of conformational vaccine protection

[0155] After completing the water maze test in step two, the mice were euthanized with carbon dioxide, and the major organs (heart, liver, spleen, kidneys, and testes) were quickly removed and paraffin sections were prepared for HE pathological staining.

[0156] The results are shown in Figure 15. No inflammation or necrosis was observed in the major organs of the metabolic and circulatory systems of the mice, and no inflammation or necrosis was observed in the reproductive organs of the male mice, indicating that the conformational vaccine has excellent biocompatibility and no side effects.

[0157] The present invention has been described in detail above. For those skilled in the art, the invention can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application. Some basic features can be applied according to the scope of the appended claims. Any modifications, equivalent substitutions, improvements, etc., made within the principles and spirit of the invention, including changes or uses made using conventional techniques known in the art that depart from the scope disclosed in this application, should be included within the protection scope of the invention. Industrial applicability

[0158] The present invention discloses the following functions: First, the inventors of the present invention have pioneered a new approach of using conformational antigens as active ingredients in vaccines. The conformational antigens are polypeptide assemblies formed by the self-assembly of artificially designed polypeptides, which have a similar conformation to pathological protein aggregates of the β structure, and are cross-antigens of pathological protein aggregates of the β structure. Furthermore, the inventors of the present invention use conformational antigens as active ingredients to prepare vaccines. After the vaccine is injected, the antibodies produced by the body can target pathological protein aggregates of the β structure, thereby preventing and / or treating protein conformation diseases.

Claims

1. The use of polypeptides or polypeptide derivatives or polypeptide assemblies or compositions containing polypeptide assemblies in the preparation of vaccines for the prevention and / or treatment of protein conformation disorders; The polypeptide is a T1 polypeptide or a T1 truncated form; the T1 polypeptide is a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1, and all chiral amino acids in the polypeptide are L-type amino acids; the T1 truncated form is a polypeptide whose amino acid sequence is shown in a partial segment of SEQ ID NO: 1, and all chiral amino acids in the polypeptide are L-type amino acids. The polypeptide derivative is a substance whose molecular structure includes the polypeptide; The polypeptide assembly is an oligomer, protofibril, fiber, or fiber aggregate with a β structure assembled from the polypeptide or the polypeptide derivative. The composition containing the polypeptide assembly consists of the polypeptide assembly and a vaccine adjuvant.

2. A vaccine for the prevention and / or treatment of protein conformation disorders, wherein the active ingredient comprises the polypeptide assembly of claim 1 or the composition containing the polypeptide assembly.

3. A polypeptide whose amino acid sequence is shown in SEQ ID NO: 1, and all chiral amino acids in the polypeptide are L-type amino acids.

4. A polypeptide whose amino acid sequence is shown in a portion of SEQ ID NO: 1, and all chiral amino acids in the polypeptide are L-type amino acids.

5. A polypeptide derivative having a molecular structure comprising the polypeptide of claim 3 or 4.

6. A polypeptide assembly is an oligomer, protofibril, filament, or filament aggregate having a β structure assembled from a polypeptide or polypeptide derivative; wherein the polypeptide is the polypeptide of claim 3 or 4; and the polypeptide derivative is the polypeptide derivative of claim 5.

7. A method for preparing the polypeptide assembly of claim 6, comprising the following steps: dissolving the polypeptide or the polypeptide derivative in a solvent, wherein the polypeptide or the polypeptide derivative self-assembles to form a polypeptide assembly.

8. The use of polypeptides or polypeptide derivatives or polypeptide assemblies or compositions containing polypeptide assemblies in the preparation of vaccines for the prevention and / or treatment of protein conformation disorders; The polypeptide is a Tx polypeptide; the Tx polypeptide is an artificially designed polypeptide that can self-assemble into a polypeptide assembly with a target structure; the target structure is: an oligomer, fibril, filament, or filament aggregate with a β structure. The polypeptide derivative is a substance whose molecular structure includes the polypeptide; The polypeptide assembly is an oligomer, protofibril, fiber, or fiber aggregate with a β structure assembled from the polypeptide or the polypeptide derivative. The composition containing the polypeptide assembly consists of the polypeptide assembly and a vaccine adjuvant.

9. A vaccine for the prevention and / or treatment of protein conformation disorders, wherein the active ingredient comprises the polypeptide assembly of claim 8 or the composition containing the polypeptide assembly.

10. A polypeptide assembly is an oligomer, protofibril, filament, or filament aggregate having a β structure assembled from a polypeptide or polypeptide derivative; wherein the polypeptide is the polypeptide described in claim 8; and the polypeptide derivative is the polypeptide derivative described in claim 8.

11. A method for preparing the polypeptide assembly of claim 10, comprising the following steps: dissolving the polypeptide or the polypeptide derivative in a solvent, wherein the polypeptide or the polypeptide derivative self-assembles to form a polypeptide assembly.

12. A composition comprising a polypeptide assembly and a vaccine adjuvant; said polypeptide assembly being the polypeptide assembly of claim 6 or 10.

13. A method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the steps of: using a polypeptide assembly or composition as the active ingredient of the vaccine; wherein the polypeptide assembly is the polypeptide assembly of claim 6 or 10; and the composition is the composition of claim 12.

14. A method for preventing and / or treating protein conformation disorders, comprising the steps of administering a therapeutically effective amount of a polypeptide assembly, composition, or vaccine to a subject for the prevention and / or treatment of a protein conformation disorder; wherein the polypeptide assembly is the polypeptide assembly of claim 6 or 10; the composition is the composition of claim 12; and the vaccine is the vaccine of claim 2 or 9.

15. A pharmaceutical compound, characterized in that: The pharmaceutical compound is the polypeptide assembly described in claim 6 or 10.

16. The pharmaceutical compound according to claim 15, characterized in that: The medicinal compound has the use of preventing and / or treating protein conformation disorders.

17. A method for designing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps: (1) Design the amino acid sequence of the polypeptide according to the following target: the polypeptide can self-assemble to form a polypeptide assembly with the target structure; the target structure is: an oligomer, protofibril, filament or filament aggregate with a β structure; (2) Use polypeptide assemblies as active ingredients in vaccines.

18. A method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps: (1) Design the amino acid sequence of the polypeptide according to the following target: the polypeptide can self-assemble to form a polypeptide assembly with the target structure; the target structure is: an oligomer, protofibril, filament or filament aggregate with a β structure; (2) Synthesize the polypeptide according to the amino acid sequence designed in step (1), and then assemble it into a polypeptide assembly; (3) The polypeptide assembly obtained in step (2) is used as the active ingredient of the vaccine.

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