Polypeptide chiral vaccine, preparation method therefor and use thereof in treatment of protein conformational diseases

Abstract

Provided are a polypeptide chiral vaccine, a preparation method therefor and the use thereof in the treatment of protein conformational diseases. Firstly, a D-amino acid is introduced into a polypeptide to obtain a polypeptide having a D-amino acid, which is abbreviated to a D-polypeptide. Then, the D-polypeptide is assembled to obtain a polypeptide assembly. The prepared polypeptide assembly has a conformation similar to that of a pathological protein aggregate and is a cross antigen of the pathological protein aggregate. The polypeptide assembly is then used as an active ingredient to prepare a vaccine, i.e., the chiral vaccine. After injection of the chiral vaccine, antibodies produced by the body can target pathological protein aggregates, thereby preventing and / or treating protein conformational diseases.

Description

A chiral polypeptide vaccine, its preparation method, and its application in the treatment of protein conformation disorders. Technical Field

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

[0002] Chirality refers to the asymmetric characteristic of a substance that cannot be superimposed on its mirror image. The singularity of chirality is a prerequisite for life activities, meaning that the amino acids used by ribosomes to synthesize proteins are all L-type. The importance of molecular chirality in drug synthesis, biomaterials, and artificial materials is widely known. L-type amino acid residues that make up proteins, especially L-aspartic acid (Asp,D), are easily converted to D-type amino acids through racemization, leading to the collapse of the protein's spatial structure and the formation of misfolded, toxic protein assemblies, resulting in protein conformation disorders.

[0003] Protein molecules need to fold into specific three-dimensional structures to perform their physiological functions, and conformational changes can lead to protein conformation disorders. The typical characteristics of protein conformation disorders are: conformational flipping of α-helical globulins or random polypeptides to β-structures; self-assembly of one or more disease-related proteins; formation of toxic aggregates; and induction of other pathological changes. Typical protein conformation disorders include transmissible spongiform encephalopathy, Alzheimer's disease, Parkinson's disease, and other neurodegenerative diseases, as well as non-neurological diseases such as type II diabetes and medullary thyroid carcinoma. These are a group of zoonotic diseases that seriously threaten human health and property safety.

[0004] The racemic conversion of L-amino acids to D-amino acids plays an important role in the pathogenesis of protein conformation disorders. For example, D-isomerization of Asp7 or Asp23 in Aβ and D-isomerization of Asp314 in tau protein cause the protein to lose its physiological function and transform into an assembly of β structure, leading to neurotoxicity.

[0005] 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 biomolecules can also complement the same antibody if their epitopes have partially similar spatial structures. Protein / peptide self-assembled fibers possess advantages such as excellent mechanical properties and readily available raw materials; through rational design, peptides with specific structures (such as β-hairpins, β-sheets, etc.) can be obtained, which can then self-assemble into structurally 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.

[0006] Invention Overview

[0007] First, the inventors of this invention introduced D-type amino acids into the polypeptide, obtaining a polypeptide containing D-type amino acids, referred to as a D-type polypeptide. Then, the inventors assembled the D-type polypeptide to obtain a polypeptide assembly. The polypeptide assembly prepared by this invention has a similar conformation to pathological protein aggregates, serving as a cross-antigen for pathological protein aggregates. The inventors then used the polypeptide assembly as the active ingredient to prepare a vaccine, namely a chiral vaccine. After injection of the chiral vaccine, the antibodies produced by the body can target pathological protein aggregates, thereby preventing and / or treating protein conformation diseases. This invention pioneers a new approach to vaccines. Technical issues

[0008] The technical problem solved by this invention is to provide a chiral vaccine with an innovative design concept, which can be used for the prevention and / or treatment of protein conformation diseases. Technical solutions

[0009] The present invention provides a polypeptide named (d)-T1 polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1 (rgyfwagdynyf), and at least one chiral amino acid in the polypeptide is a D-type amino acid.

[0010] Specifically, all chiral amino acids in the (d)-T1 polypeptide are D-type amino acids.

[0011] The (d)-T1 polypeptide is an artificially designed polypeptide.

[0012] This invention also protects a polypeptide, named (d)-T1 truncated, whose amino acid sequence is shown in a portion of SEQ ID NO: 1, and at least one chiral amino acid in the polypeptide is a D-type amino acid. Specifically, all chiral amino acids in the (d)-T1 truncated polypeptide are D-type amino acids. Exemplarily, the (d)-T1 truncated polypeptide is obtained by removing 1-5 amino acid residues from the N-terminus and / or C-terminus of the (d)-T1 polypeptide. Exemplarily, the (d)-T1 truncated polypeptide is obtained by removing 1-2 amino acid residues from the N-terminus and / or C-terminus of the (d)-T1 polypeptide. Exemplarily, the (d)-T1 truncated polypeptide is shown at positions 3 to 12 in SEQ ID NO: 1.

[0013] The present invention also protects derivatives of the (d)-T1 polypeptide, the molecular structure of which includes the (d)-T1 polypeptide.

[0014] The present invention also protects derivatives of the (d)-T1 truncated form, the molecular structure of which includes the (d)-T1 truncated form.

[0015] This invention also protects a polypeptide, named (d)-Tx polypeptide, having any amino acid sequence, wherein at least one chiral amino acid is a D-type amino acid. As an example, all chiral amino acids in the (d)-Tx polypeptide are D-type amino acids. The (d)-Tx polypeptide is an artificially designed polypeptide (amino acid sequence not limited) capable of forming a target structure. The target structure is: an α-helix and / or a β-sheet and / or a random coil. The target structure is: an oligomer, fibril, fiber, or fiber aggregate having an α-helix and / or a β-sheet and / or a random coil. Exemplarily, the target structure is: an oligomer, fibril, fiber, or fiber aggregate having a β-structure.

[0016] The present invention also protects derivatives of the (d)-Tx polypeptide, the molecular structure of which includes the (d)-Tx polypeptide.

[0017] This invention also protects a polypeptide assembly (named polypeptide assembly I), which is assembled from the (d)-T1 polypeptide, a derivative of the (d)-T1 polypeptide, a truncated form of the (d)-T1 polypeptide, or a derivative of the truncated form of the (d)-T1 polypeptide. The polypeptide assembly I is an oligomer, protofibril, filament, or filament aggregate having a β-structure.

[0018] This invention also protects a polypeptide assembly (named polypeptide assembly II) assembled from the (d)-Tx polypeptide or a derivative of the (d)-Tx polypeptide. The polypeptide assembly II is an oligomer, protofibril, filament, or filament aggregate having α-helices and / or β-sheets and / or random coils. Exemplarily, the polypeptide assembly II is an oligomer, protofibril, filament, or filament aggregate having a β-structure. Exemplarily, the polypeptide assembly II has the same or similar structure as polypeptide assembly I.

[0019] Polypeptide assembly I and polypeptide assembly II are collectively referred to as polypeptide assemblies.

[0020] This invention also protects a method for preparing polypeptide assemblies, comprising the following steps: dissolving a polypeptide in a solvent, and the polypeptide self-assembling to form a polypeptide assembly. When the polypeptide is the T1 polypeptide, a derivative of the T1 polypeptide, a truncated form of the T1 polypeptide, or a derivative of the truncated form of the T1 polypeptide, polypeptide assembly I is prepared. When the polypeptide is the (d)-Tx polypeptide or a derivative of the (d)-Tx polypeptide, polypeptide assembly II is prepared.

[0021] In the above preparation method, the solvent is an inorganic solvent (e.g., water) or an organic solvent.

[0022] 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.

[0023] In the above preparation method: under static or oscillating conditions, the polypeptide aggregates and self-assembles to form a polypeptide assembly.

[0024] As one specific embodiment, the preparation method of the polypeptide assembly is as follows: the (d)-T1 polypeptide, the derivative of (d)-T1, the truncated form of (d)-T1 or the derivative of the truncated form of (d)-T1 are dissolved in an aqueous solvent to a concentration of 0.02-50 mg / mL, and incubated at 4-90°C.

[0025] As one specific implementation method, the preparation method of the polypeptide assembly is as follows: the (d)-T1 polypeptide, the derivative of the (d)-T1 polypeptide, the (d)-T1 truncated form or the derivative of the (d)-T1 truncated form are dissolved in water to a concentration of 1 mg / mL and incubated at 37°C for 3 days.

[0026] The present invention also provides a composition comprising any of the polypeptide assemblies described above and a vaccine adjuvant.

[0027] In the composition, the mass ratio of the polypeptide assembly to the vaccine adjuvant is 1:1.

[0028] 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.

[0029] The adjuvant may be a biological adjuvant and / or an inorganic adjuvant and / or a synthetic adjuvant and / or an oil.

[0030] The adjuvants 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, AddaVax TM and Any one or any combination of them.

[0031] The present invention also protects vaccines for the prevention and / or treatment of protein conformation disorders, the active ingredients of which include any of the polypeptide assemblies or compositions described above.

[0032] The vaccine also includes 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.

[0033] 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.

[0034] Other substances may also be added to the vaccine, including neuroprotective agents, neuroregenerative substances, or neuroactive substances.

[0035] The vaccine 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.

[0036] The present invention also protects a method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the steps of: using any of the above-described polypeptide assemblies or the compositions as the active ingredient of the vaccine.

[0037] As one specific implementation method, the vaccine preparation method is as follows:

[0038] (1) The (d)-T1 polypeptide, the derivative of the (d)-T1 polypeptide, the (d)-T1 truncated form or the derivative of the (d)-T1 truncated form are dissolved in water to a concentration of 0.02-50 mg / mL and incubated at 4-90℃ to obtain a polypeptide assembly solution.

[0039] (2) Dissolve QS-21 in DMSO, then dilute with buffer solution to obtain QS-21 solution;

[0040] (3) Mix the polypeptide assembly solution with the QS-21 solution and then add buffer solution to obtain the vaccine.

[0041] As one specific implementation method, the vaccine preparation method is as follows:

[0042] (1) The (d)-T1 polypeptide, the derivative of the (d)-T1 polypeptide, the (d)-T1 truncated form or the derivative of the (d)-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.

[0043] (2) Dissolve QS-21 in DMSO, and then dilute with buffer to 1 mg / mL to obtain the QS-21 solution;

[0044] (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.

[0045] For example, the buffer solution is PBS buffer (pH 7.2).

[0046] The present invention also protects the use of the (d)-T1 polypeptide, derivatives of the (d)-T1 polypeptide, truncated form of the (d)-T1 polypeptide, truncated form of the (d)-T1 polypeptide, (d)-Tx polypeptide, derivatives of the (d)-Tx polypeptide, any of the above polypeptide assemblies, or the composition thereof in the preparation of vaccines for the prevention and / or treatment of protein conformation disorders.

[0047] 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.

[0048] This invention also protects a pharmaceutical compound, characterized in that: the pharmaceutical compound is any of the polypeptide assemblies described above.

[0049] The medicinal compound has the use of preventing and / or treating protein conformation disorders.

[0050] This invention also protects a method for designing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps:

[0051] (1) Design D-type polypeptides according to the following objectives: the polypeptides form polypeptide assemblies with a target conformation; the target conformation is a characteristic conformation of protein conformational disorders; a D-type polypeptide refers to a polypeptide in which at least one chiral amino acid is a D-type amino acid;

[0052] (2) Peptide assemblies are the active ingredients of vaccines.

[0053] This invention also protects a method for preparing a vaccine for the prevention and / or treatment of protein conformation disorders, comprising the following steps:

[0054] (1) Design D-type polypeptides according to the following objectives: the polypeptides form polypeptide assemblies with a target conformation; the target conformation is a characteristic conformation of protein conformational disorders; a D-type polypeptide refers to a polypeptide in which at least one chiral amino acid is a D-type amino acid;

[0055] (2) Synthesize D-type polypeptides according to the amino acid sequence designed in step (1), and then assemble them into polypeptide assemblies;

[0056] (3) The polypeptide assembly obtained in step (2) is used as the active ingredient of the vaccine.

[0057] Any of the above-described assemblies can be self-assembled.

[0058] The vaccines described above for the prevention and / or treatment of protein conformation diseases are chiral vaccines for the prevention and / or treatment of protein conformation diseases.

[0059] 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 and 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.

[0060] 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.

[0061] Any of the above-described features may be α-helices and / or β-folds and / or random coils.

[0062] Any of the β structures described above can be β-hairpins and / or β-sheets and / or β-turns.

[0063] 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.

[0064] The protein conformational diseases mentioned above include, but are not limited to: disseminated spongiform encephalopathy (including but not limited to mad cow disease, scrapie, kuru), Alzheimer's disease, Parkinson's disease, multiple sclerosis, type II diabetes, Gerstmann syndrome, Huntington's disease, Pick's disease, corticobasal degeneration, progressive supranuclear palsy (oculocele), 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, meningeal 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.

[0065] As a specific example, the protein conformational disease is Alzheimer's disease. Beneficial effects

[0066] The polypeptide assemblies prepared from D-type polypeptides provided by this invention serve as cross-antigens for pathological protein aggregates, exhibiting advantages such as strong immunogenicity and high biocompatibility. Chiral vaccines prepared using these polypeptide assemblies as active ingredients, upon injection, induce antibodies produced by the body that can target pathological protein aggregates, thereby preventing and / or treating protein conformation disorders. Attached Figure Description

[0067] Figure 1 is a schematic diagram of the molecular structure of the T1 polypeptide.

[0068] Figure 2 shows the high-resolution mass spectrum of the T1 polypeptide prepared in Example 1 (Calculated: 1558.67, found: 779.80 [M+2H]2+).

[0069] Figure 3 shows the structural characterization of the polypeptide assembly in Example 2.

[0070] Figure 4 shows the structural model of the polypeptide assembly in Example 2.

[0071] Figure 5 shows the antibody verification in the serum of animals after immunization with the chiral vaccine in Example 4.

[0072] Figure 6 shows the results of the improvement of spatial memory in AD model animals by the chiral vaccine in Example 5.

[0073] Figure 7 shows the results of chiral vaccine reducing amyloid protein accumulation in the brains of AD mice in Example 5.

[0074] Figure 8 shows the results of the chiral vaccine clearing phosphorylated tau protein aggregates in the brains of AD mice in Example 5.

[0075] Figure 9 shows the HE staining results of neurons in the brains of AD model mice protected by the chiral vaccine in Example 5.

[0076] 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.

[0077] Figure 11 shows the UMAP representation of cell clusters for all test samples in Example 5.

[0078] Figure 12 shows the Lrrc7 samples in all test samples of Example 5. + UMAP distribution map of neurons in the brain.

[0079] Figure 13 shows the Grin2b values ​​in all test samples from Example 5. + UMAP distribution map of neurons in the brain.

[0080] 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.

[0081] Figure 15 shows the relevant results of the safety evaluation of chiral vaccine protection in Example 5.

[0082] Figure 16 shows the results of the effects of chiral vaccine immunization on AD-related genes and signaling pathways in microglia in Example 6.

[0083] Figure 17 shows the results of the effect of the chirality of the immunogen on the treatment mechanism in Example 7.

[0084] Implementation methods of the invention

[0085] 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.

[0086] 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.

[0087] The terminology is defined as follows:

[0088] In this application, the term "D-type amino acid" means: an amino acid in which the amino group is located to the right of the chiral carbon atom in the Fischer projection.

[0089] In this application, the term "D-type polypeptide" means a polypeptide sequence containing one or more D-type amino acids, or a polypeptide consisting entirely of D-type amino acids.

[0090] 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.

[0091] In this application, the term "β structure" means that the conformation of a protein or polypeptide is predominantly β-sheet.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] In this application, the term "chiral vaccine" means: a composition in which a self-assembled product of a D-type polypeptide or a protein molecule having D-type amino acids is the active ingredient. This self-assembled product can act as a cross-antigen for pathological aggregates (the self-assembled product acts as an immunogen to induce the production of antibodies in the organism, and the antibodies target the pathological aggregates), thereby preventing and / or treating protein conformation disorders.

[0096] 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.

[0097] Example 1: Artificial Design and Solid-Phase Synthesis of Peptides

[0098] The amino acid sequence of the (d)-T1 polypeptide (SEQ ID NO: 1): rgyfwagdynyf. Except for glycine, all amino acids in the (d)-T1 polypeptide are D-type amino acids. The structural formula of the (d)-T1 polypeptide is: (d)-r 1 g 2 y 3 f 4 w 5 a 6 g 7 d 8 y 9 n 10 y 11 f 12 The molecular structure of (d)-T1 polypeptide is shown in Figure 1. (d)-T1 polypeptide (powder) was prepared by solid-phase synthesis using glycine and other amino acids (all D-type amino acids). The mass spectrometry structure confirmation data is shown in Figure 2 (Calculated: 1558.67, found: 779.80 [M+2H]2+).

[0099] Note: The amino acid sequence of the (l)-T1 polypeptide is: RGYFWAGDYNYF. All amino acids in the (l)-T1 polypeptide, except for glycine, are L-type amino acids. The structural formula of the (l)-T1 polypeptide is: R 1 G 2 Y 3 F 4 W5 A 6 G 7 D 8 Y 9 N 10 Y 11 F 12 (l)-T1 polypeptide (powder) was prepared by solid-phase synthesis using glycine and other amino acids (all L-type amino acids).

[0100] The (d)-T1 polypeptide and the (l)-T1 polypeptide are mirror images of each other.

[0101] Example 2: Peptide self-assembly and conformation characterization

[0102] 1. Take (d)-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.

[0103] 2. After completing step 1, the morphology was characterized using atomic force microscopy. The photograph is shown in Figure 3a. It can be observed that the (d)-T1 polypeptide self-assembles into a fibrous structure with a right-handed helix.

[0104] 3. Take (l)-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.

[0105] 4. After completing step 1 or step 3, characterize the conformation using circular dichroism spectroscopy and broad-spectrum infrared spectroscopy (DT1 represents the product of step 1, LT1 represents the product of step 3). The CD spectrum is shown in Figure 3b (DT1 represents the product of step 1, LT1 represents the product of step 3). The CD spectrum of the (d)-T1 peptide is a mirror image of that of the (l)-T1 peptide; the characteristic peak at 218 nm indicates that the peptide conformation is predominantly β-sheet. The infrared spectrum is shown in Figure 3c, with peaks at 1626 and 1635 cm⁻¹. -1 These are absorption peaks of the β-sheet, at 1662, 1674, and 1685 cm⁻¹. -1 Indicating β-sheet turn angles. The results showed that step 1 yielded the (d)-T1 polypeptide assembly, and step 3 yielded the (l)-T1 polypeptide assembly.

[0106] A schematic diagram of the (d)-T1 polypeptide assembly structure model is shown in Figure 4.

[0107] Example 3: Preparation of chiral vaccines

[0108] 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.

[0109] Take (d)-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 (d)-T1 polypeptide fiber solution.

[0110] Mix 1 volume of (d)-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 chiral vaccine.

[0111] 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.

[0112] Example 4: Detection of amyloid-binding antibodies using immunofluorescence technique

[0113] I. Animal Immunization

[0114] Four-month-old AD model mice were randomly divided into two groups (control group Q and experimental group D), with 12 mice in each group (6 males and 6 females). Female mice in the control group were designated QF, female mice in the experimental group were designated DF, male mice in the control group were designated QM, and male mice in the experimental group were designated DM. The experimental group was immunized monthly by intramuscular injection of the chiral 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.

[0115] II. Detection of amyloid-binding antibodies using immunofluorescence technology

[0116] 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.

[0117] 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.

[0118] 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.

[0119] Example 5: Therapeutic effect of chiral vaccine on AD model mice

[0120] I. Animal Immunization

[0121] Four-month-old AD model mice were randomly divided into two groups (control group Q and experimental group D), with 12 mice in each group (6 males and 6 females). Female mice in the control group were designated QF, female mice in the experimental group were designated DF, male mice in the control group were designated QM, and male mice in the experimental group were designated DM. The experimental group was immunized monthly by intramuscular injection of the chiral 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.

[0122] II. Characterizing the improvement of spatial memory of chiral 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.

[0123] 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.

[0124] The swimming path of the mice is shown in the left image of Figure 6. 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 chiral vaccine on spatial memory.

[0125] The statistical analysis results of the time mice spent in the target quadrant are shown in the right panel of Figure 6. Compared with the control group, the experimental group mice spent a significantly longer time in the target quadrant.

[0126] III. Chiral vaccines reduce the accumulation of amyloid-β (Aβ) protein in the brains of AD mice.

[0127] This step uses immunofluorescence assays to detect the effect of chiral vaccine immunization on pathological aggregates of amyloid protein (Aβ) in the brains of AD mice.

[0128] 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.

[0129] 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.

[0130] See Figure 7 for an example photograph. Typical Aβ(1-42) aggregates were observed in the brains of control mice, 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 clear edges (reflecting limited invasiveness), and the scattered globular or fibrous aggregates disappeared, reflecting that the antibodies produced by the chiral vaccine stimulation functioned to surround the Aβ(1-42) aggregates and limit their growth.

[0131] IV. Chiral vaccines clear phosphorylated tau protein aggregates in the brains of AD mice.

[0132] This step uses immunofluorescence assays to detect the effect of chiral vaccine immunization on pathological aggregates of phosphorylated tau protein in the brains of AD mice.

[0133] 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.

[0134] 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.

[0135] 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 produced by chiral vaccine stimulation can effectively clear phosphorylated tau protein aggregates.

[0136] V. Chiral vaccines protect neurons in the brains of AD model mice

[0137] 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 chiral vaccine on neurons in the brains of AD model mice.

[0138] Take the paraffin sections of the female mice obtained in step three and perform HE staining.

[0139] 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 chiral vaccine on brain neurons.

[0140] VI. Chiral vaccines protect neurons in the brains of AD model mice

[0141] This step uses single-cell sequencing to verify the protective effect of the chiral vaccine on neurons in the brains of AD model mice.

[0142] After completing the water maze test in step two, three female mice in the experimental group (D_1, D_2, D_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, various cells were collected, and cell nuclei were extracted for single-cell transcriptome sequencing analysis.

[0143] 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).

[0144] 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 control group mice disappeared, while these neurons were present in the brains of experimental group mice, indicating that these neurons were protected after immunization with the chiral vaccine.

[0145] 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.

[0146] 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.

[0147] 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 chiral vaccine has the effect of alleviating neuronal apoptosis in the AD pathogenesis process.

[0148] VII. Safety evaluation of chiral vaccine protection

[0149] 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.

[0150] 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 chiral vaccine has excellent biocompatibility and no side effects.

[0151] Example 6: Effects of chiral vaccine immunization on AD-related genes and signaling pathways in microglia.

[0152] Microglia are immune cells in the central nervous system and are currently central to research on the pathogenesis of Alzheimer's disease. Microglia results were obtained from single-cell sequencing in Example 6.

[0153] The results are shown in Figure 16. Compared with the control group, the genes involved in Aβ clearance (Apoe, Trem2) in microglia of mice in the experimental group were significantly upregulated, as were the GO pathways related to neuronal structure and function, and the GO pathway related to autophagy. This indicates that the chiral vaccine improved the pathogenesis of AD.

[0154] Example 7: The Influence of Immunogen Chirality on the Mechanism of Treatment

[0155] Four-month-old AD model mice were randomly divided into two groups (experimental group D and experimental group L), with 10 female mice in each group. Experimental group D mice were immunized monthly by intramuscular injection of the chiral vaccine prepared in Example 3 (40 μL per mouse per injection), for 12 consecutive months. Experimental group L mice were immunized monthly by intramuscular injection of the L-type control vaccine (40 μL per mouse per injection), for 12 consecutive months.

[0156] Preparation method of L-type control vaccine: (l)-T1 polypeptide powder is used instead of (d)-T1 polypeptide powder, and the other preparation methods are the same as those of the chiral vaccine in Example 3.

[0157] At 16 months of age, three female mice (D1, D2, D3) were randomly selected from experimental group D and three female mice (L1, L2, L3) were randomly selected from experimental group L. The mice were euthanized with carbon dioxide, and the brains were quickly removed. The left brain was removed, and various cells were collected. The cell nuclei were extracted and single-cell transcriptome sequencing analysis was performed.

[0158] The results are shown in Figure 17. The results indicate that the chiral vaccine using the (d)-T1 assembly as the immunogen has a different therapeutic mechanism for AD compared to the control vaccine using the (l)-T1 assembly as the immunogen. Compared to the experimental group L mice, the genes involved in Aβ clearance (Apoe, Trem2), stem cell differentiation-related GO pathway, and neural regeneration-related GO pathway were significantly upregulated in the microglia of the experimental group D mice. These results suggest that the chiral vaccine treats AD through a more complex mechanism than the control vaccine.

[0159] 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

[0160] This invention discloses the following: First, the inventors introduced D-type amino acids into a polypeptide, obtaining a polypeptide containing D-type amino acids, referred to as a D-type polypeptide. Then, the inventors assembled the D-type polypeptide to obtain a polypeptide assembly. The polypeptide assembly prepared by this invention has a similar conformation to pathological protein aggregates and serves as a cross-antigen for pathological protein aggregates. Then, the inventors used the polypeptide assembly as an active ingredient to prepare a vaccine, namely a chiral vaccine. After injection of the chiral vaccine, the antibodies produced by the body can target pathological protein aggregates, thereby preventing and / or treating protein conformation disorders.

Claims

1. A polypeptide having the amino acid sequence shown in SEQ ID NO: 1, wherein at least one chiral amino acid in the polypeptide is a D-type amino acid.

2. A polypeptide whose amino acid sequence is shown in a portion of SEQ ID NO: 1, wherein at least one chiral amino acid in the polypeptide is a D-type amino acid.

3. A polypeptide derivative having a molecular structure comprising the polypeptide of claim 1 or 2.

4. A polypeptide assembly, which is assembled from a polypeptide or a polypeptide derivative; wherein the polypeptide is the polypeptide of claim 1 or 2; and the polypeptide derivative is the polypeptide derivative of claim 3.

5. A method for preparing the polypeptide assembly of claim 4, 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.

6. A polypeptide having any amino acid sequence, wherein at least one chiral amino acid is a D-type amino acid.

7. A polypeptide derivative having a molecular structure comprising the polypeptide of claim 6.

8. A polypeptide assembly, which is assembled from a polypeptide or a polypeptide derivative; wherein the polypeptide is the polypeptide of claim 6; and the polypeptide derivative is the polypeptide derivative of claim 7.

9. A method for preparing the polypeptide assembly of claim 8, 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.

10. A composition comprising a polypeptide assembly and a vaccine adjuvant; said polypeptide assembly being the polypeptide assembly of claim 4 or 8.

11. A vaccine for the prevention and / or treatment of protein conformation disorders, wherein the active ingredient comprises a polypeptide assembly or a composition containing a polypeptide assembly; said polypeptide assembly is the polypeptide assembly of claim 4 or 8; said composition containing a polypeptide assembly is the composition of claim 10.

12. 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 a composition containing a polypeptide assembly as the active ingredient of the vaccine; wherein the polypeptide assembly is the polypeptide assembly of claim 4 or 8; and the composition containing the polypeptide assembly is the composition of claim 10.

13. The use of a polypeptide or polypeptide derivative or polypeptide assembly or composition containing a polypeptide assembly in the preparation of a vaccine for the prevention and / or treatment of protein conformation disorders; wherein the polypeptide is the polypeptide of claim 1, 2 or 6; the polypeptide derivative is the polypeptide derivative of claim 3 or 7; the polypeptide assembly is the polypeptide assembly of claim 4 or 8; and the composition containing a polypeptide assembly is the composition of claim 10.

14. A method for preventing and / or treating protein conformation disorders, comprising the steps of administering to a subject a therapeutically effective amount of a polypeptide assembly, composition, or vaccine to prevent and / or treat the protein conformation disorder; wherein the polypeptide assembly is the polypeptide assembly of claim 4 or 8; the composition is the composition of claim 10; and the vaccine is the vaccine of claim 11.

15. A pharmaceutical compound, characterized in that: The pharmaceutical compound is the polypeptide assembly described in claim 4 or 8.

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 D-type polypeptides according to the following objectives: the polypeptides form polypeptide assemblies with a target conformation; the target conformation is a characteristic conformation of protein conformational disorders; a D-type polypeptide refers to a polypeptide in which at least one chiral amino acid is a D-type amino acid; (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 D-type polypeptides according to the following objectives: the polypeptides form polypeptide assemblies with a target conformation; the target conformation is a characteristic conformation of protein conformational disorders; a D-type polypeptide refers to a polypeptide in which at least one chiral amino acid is a D-type amino acid; (2) Synthesize D-type polypeptides according to the amino acid sequence designed in step (1), and then assemble them into polypeptide assemblies; (3) The polypeptide assembly obtained in step (2) is used as the active ingredient of the vaccine.

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