Use of beta-hordothionin, active fragments thereof or derivatives thereof as intramolecular adjuvant

Abstract

This invention discloses the application of β-Hordothionin, its active fragment, or derivatives as intramolecular adjuvants, specifically involving a polypeptide derived from β-Hordothionin, the amino acid sequence of which is shown in SEQ ID NO.1, and the polynucleotide sequence of which is shown in SEQ ID NO.2. The polypeptide is fused with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and expressed, significantly enhancing the immunogenicity of the antigen. Experiments show that immunization of mice with the RBD-β-Hordothionin fusion protein induces a 1000-fold increase in specific IgG antibody titers compared to the RBD antigen alone. This fusion protein can be industrially prepared and can be used to enhance the immunogenicity of vaccine antigens.

Description

Application of β-Hordothionin, its active fragments, or its derivatives as intramolecular adjuvants Technical Field

[0001] This invention discloses an intramolecular adjuvant, belonging to the field of vaccine / adjuvant technology. Background Technology

[0002] Adjuvants are a key component of vaccines, a class of molecules that are co-inoculated with antigens to significantly enhance the body's antigen-specific immune response and improve the vaccine's protective efficacy. Currently approved adjuvants include: aluminum hydroxide adjuvant, aluminum phosphate adjuvant, MF59, AS01, AS02, AS03, AS04, PolyI:C, PolyA:U, QS21, RC-529, Virosome, imiquimod, and CpG ODN (1018 ISS). Aluminum hydroxide and aluminum phosphate adjuvants primarily enhance the body's immune response to antigens through mechanisms such as antigen adsorption, sustained release, and activation of the NLRP3 inflammasome signaling pathway. PolyI:C and others mainly enhance the body's immune response to antigens by activating downstream signaling pathways such as MyD88 through pattern recognition receptors. However, in addition to classic pathways such as pattern recognition receptor activation, statins that alter lipid metabolism can also be used as novel adjuvants.

[0003] Intramolecular adjuvants differ from classical adjuvants in that their chemical nature is a polypeptide or protein that is directly fused with the antigen protein to form a single protein. Because intramolecular adjuvants directly combine with the antigen protein to form a single protein, their use in vaccine design and preparation offers several advantages, including: simplified preparation (requiring only antigen expression and purification as in traditional vaccine preparation); compatibility with other adjuvants; and direct application to novel vaccines such as mRNA and DNA vaccines without the need for special processes. Currently developed intramolecular adjuvants are primarily structural or toxin proteins of viruses or bacteria. For example, tetanus toxin peptides, when fused with antigens, can significantly increase serum antibody titers after antigen immunization.

[0004] Intramolecular adjuvants have become one of the ideal adjuvant forms due to their advantages such as ease of preparation, great application potential, and applicability to novel mRNA and DNA vaccines. Therefore, there is a huge technological and market demand for providing novel intramolecular adjuvants and their applications.

[0005] β-Hordothionin is a cationic thioprotein found in barley (Hordeum vulgare) seeds. It is an important member of the plant defensive antimicrobial peptide family. This small protein, with a molecular weight of approximately 5 kDa, contains 6-8 conserved cysteine ​​residues and forms a stable β-sheet structure through 3-4 disulfide bonds, exhibiting an amphiphilic spatial conformation (positively charged regions and a hydrophobic core). Its main biological function is to bind to phospholipids in microbial membranes through electrostatic interactions, forming pores that lead to leakage of contents, thus exerting antibacterial effects against Gram-positive bacteria / fungi. Currently, it is mainly used as a candidate molecule for anti-drug-resistant bacteria and as a carrier for anticancer drugs utilizing its membrane permeability.

[0006] The purpose of this invention is to provide an intramolecular adjuvant that can significantly increase serum antibody titers after antigen immunization. Summary of the Invention

[0007] Based on the above objectives, the present invention provides the application of β-hordothionin, its active fragment or derivative thereof as an intramolecular adjuvant.

[0008] The intramolecular adjuvant described in this invention refers to an adjuvant molecule that is directly integrated into the antigen structure through chemical or genetic engineering methods to enhance the immunogenicity of the antigen, rather than the traditional method of physically mixing exogenous adjuvants (such as aluminum salts, oil emulsions, etc.) with the antigen. This design aims to more precisely regulate the immune response and improve vaccine efficacy.

[0009] This invention aims to protect the application of β-Hordothionin as an intramolecular adjuvant, rather than β-Hordothionin itself. Therefore, any modification or variation of β-Hordothionin that retains the fragment that exerts its complete protein biological activity, such as membrane permeability, is considered an active fragment of β-Hordothionin and falls within the scope of this invention.

[0010] The β-Hordothionin derivatives described in this invention refer to protein molecules obtained by modifying the structure of β-Hordothionin through methods such as chemical modification, enzymatic digestion, genetic engineering, or physical treatment. Therefore, these derivatives retain fragments that exhibit complete protein biological activity, such as membrane permeability. All of these are derivatives of β-Hordothionin and fall within the scope of this invention.

[0011] In a preferred embodiment, the application is the use of β-Hordothionin, its active fragment or derivative thereof, in the form of a fusion protein or chemically conjugated with an immunogenic peptide.

[0012] The fusion protein described in this invention refers to a single protein molecule produced by linking the coding sequences of two or more different genes together using genetic engineering techniques. The chemical coupling described in this invention refers to the covalently linking of two or more molecules (such as proteins, nucleic acids, small molecule drugs, fluorescent dyes, etc.) through a chemical reaction to form a stable complex. The chemical coupling involved in this invention is the coupling between two proteins, and the coupled antigen is mediated by a chemical crosslinker.

[0013] In a preferred embodiment, the application is an immunogenic polypeptide that is a human or animal autoantigen or derived from bacteria, viruses, fungi, or parasites.

[0014] In a more preferred embodiment, the application is β-Hordothionin, its active fragment or derivative thereof, recombined with an immunogenic polypeptide by direct linking or by linking with a flexible linker peptide to form a fusion protein.

[0015] More preferably, the application involves β-Hordothionin, its active fragment, or a derivative thereof linked to an immunogenic polypeptide via a flexible linker peptide (G4S)n, where n is a natural number from 1 to 6. In one specific embodiment, n = 6.

[0016] In one specific embodiment of the present invention, the amino acid sequence of β-Hordothionin is shown in SEQ ID NO.1.

[0017] In one specific embodiment of the present invention, the immunogenic polypeptide is the novel coronavirus RBD protein, and the sequence of the fusion protein is shown in SEQ ID NO. 5. In this invention, the novel coronavirus RBD protein serves as a model immunogenic polypeptide to demonstrate the technical effect of β-barbiturin as an intramolecular adjuvant. Therefore, the implementation of the novel coronavirus RBD protein as an immunogenic polypeptide should not constitute a limitation on the scope of protection of this invention.

[0018] Secondly, this invention provides a polynucleotide encoding the fusion protein, the sequence of which is shown in SEQ ID NO. 6. In this invention, the sequence of the polynucleotide uses human-optimized codons, suitable for expression of β-hordothionin via a mammalian expression vector. In one specific embodiment of this invention, cloning the polynucleotide into a pcDNA3.1 vector and transfecting the pcDNA3.1 vector into 293F cells yields a fusion protein of β-hordothionin and the immunogenic polypeptide-novel coronavirus RBD protein.

[0019] Finally, the present invention provides a vaccine composition comprising a fusion protein and chemical conjugate of β-Hordothionin and an immunogenic polypeptide prepared according to the aforementioned application of β-Hordothionin, its active fragment or derivative thereof as an intramolecular adjuvant, and an immune adjuvant.

[0020] In a preferred embodiment, the immune adjuvant is an aluminum adjuvant. In the composition, the mass ratio of the fusion protein to the aluminum adjuvant is preferably (0.5-2):(4-6), and in a specific embodiment, the mass ratio of the fusion protein to the aluminum adjuvant is 1:5.

[0021] Based on the application of β-Hordothionin, its active fragment, or its derivatives as intramolecular adjuvants according to the present invention, β-Hordothionin was fused with the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to obtain the RBD-β-Hordothionin fusion protein. Immunization of mice with the RBD-β-Hordothionin fusion protein induced a 1000-fold increase in specific IgG antibody titers compared to the RBD antigen alone, demonstrating the excellent technical effect of β-Hordothionin as an intramolecular adjuvant. When the RBD-β-Hordothionin fusion protein was used in combination with aluminum adjuvant, the antibody titer in the combined group was more than 8 times higher than that in the aluminum adjuvant-only group, showing a significant synergistic effect and demonstrating the application prospect of the fusion protein as a vaccine protein containing an intramolecular adjuvant in combination with an adjuvant. The RBD-β-Hordothionin fusion protein provided by the present invention can be prepared industrially, with a simple process and industrial applicability. Attached Figure Description

[0022] Figure 1. Map of pcDNA3.1-RBD-β-Hordothionin plasmid;

[0023] Figure 2. SDS-PAGE image of RBD-β-Hordothionin after preliminary purification;

[0024] Figure 3. Statistical graph of RBD antibody titers after immunization in RBD-β-Hordothionin mice;

[0025] Figure 4. Statistical chart of adsorption rate of RBD-β-Hordothionin aluminum adjuvant;

[0026] Figure 5. Statistical graph of RBD antibody titers in mice after immunization with RBD-β-Hordothionin in combination with aluminum adjuvant. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0028] The experimental reagents involved in this invention include: 293F cells, Hi-exp 293F medium (Shanghai OPMA Biotechnology Co., Ltd.), SDS-PAGE gel (GenScript Biotechnology Co., Ltd.), protein marker, ampicillin sodium (Beijing Solarbio Science & Technology Co., Ltd.), yeast extract powder (Oxoid), tryptone, sodium chloride, agar (Novon), PEI 4K (Polyplus), Opti-MEM (Gibco), endotoxin-free plasmid large-scale extraction kit (Guangzhou Meiji Biotechnology Co., Ltd.), anhydrous ethanol, BSA, pcDNA3.1 vector, sodium bicarbonate, sodium carbonate, PBS, RBD-immunized mouse serum, High Affinity Ni-Charged Resin FF (GenScript Biotechnology Co., Ltd.), imidazole (Sigma-Aldrich (Shanghai) Trading Co., Ltd.), 3kDa ultrafiltration tubes (Pall China), RBD protein, HRP-labeled goat anti-mouse antibody (Langeco Technology Co., Ltd.), ready-to-use TMB (Beijing Solarbio Science & Technology Co., Ltd.), ELISA stop solution (Beijing Solarbio Science & Technology Co., Ltd.), and 2019-nCoV. Spike Protein RBD ELISA Kit (Shanghai Beyotime Biotechnology Co., Ltd.), Alhydrogel® adjuvant 2% aluminum adjuvant (InvivoGen), BCA kit (Perce), 96-well EIA / RIA transparent flat-bottom polystyrene microplate (Corning).

[0029] The experimental animal involved in this invention is the BALB / c mouse (Viotonil).

[0030] Example 1. Expression and purification of RBD-β-Hordothionin protein

[0031] 1. Construction of recombinant plasmids

[0032] Human codons were optimized for the β-Hordothionin amino acid sequence (SEQ ID NO.1), and the corresponding polynucleotide sequence (SEQ ID NO.2) was synthesized. The β-Hordothionin gene fragment was amplified by PCR using the following primer set:

[0033] 5'-CTAGTGGTGGTGGTGGTGGTGGTGGTGCTTAGGGAAGCTGGAGGGAC-3' (SEQ ID NO.3, forward primer), and,

[0034] 5'-CAGTCTTCGTTTCGCCCTCCGGATCCAGAGTGCAG-3' (SEQ ID NO.4, reverse primer).

[0035] The amplified fragment was inserted into the pcDNA3.1 vector between the RBD coding region and the 8×His tag using seamless cloning technology. The linker peptide was designed as (GGGGS)2, and the recombinant plasmid pcDNA3.1-RBD-β-Hordothionin was constructed. The amino acid sequence of the RBD-β-Hordothionin fusion protein is shown in SEQ ID NO.5, and the coding sequence is shown in SEQ ID NO.6. Among them, positions 1-66 are the signal peptide encoding nucleic acid. The mature RBD-β-Hordothionin fusion protein does not include this signal peptide. After transformation into competent cells, sequencing verification was performed. (See Figure 1)

[0036] 2. Protein Expression and Purification

[0037] The correct plasmid was transfected into 293F cells (density 1×10^6 cells / mL) using a polyethyleneimine (PEI)-mediated transfection method at a plasmid-to-PEI ratio of 1:3 (w / v). After 5 days of post-transfection culture, the cell supernatant was collected. The cells were purified by Ni-Charged Resin affinity chromatography, eluting sequentially with PBS buffer containing 20 mM and 250 mM imidazole. The purity of the purified protein was verified by SDS-PAGE; the molecular weight was approximately 37 kD, as expected (Figure 2). The protein was then replaced with PBS buffer and its concentration was determined.

[0038] Example 2. Evaluation of the adjuvant activity of β-Hordothionin

[0039] 1. Mouse immunization and serum collection

[0040] 10 μg each of the RBD-β-Hordothionin protein prepared in Example 1 and the control RBD protein were diluted in PBS to a final concentration of 0.1 mg / mL. BALB / c mice were purchased and divided into 3 groups of 5 mice each. 100 μL was injected into the quadriceps femoris muscle on days 0 and 14. Tail vein blood was collected on day 28.

[0041] Tail vein blood collected on day 28 was incubated at 37°C for 30 min, centrifuged at 3000×g for 15 min, and the supernatant was then frozen and stored at -80°C.

[0042] 2. Serum RBD antibody titer determination

[0043] The ELISA method for determining antibody titers is as follows: 2 μg / mL RBD protein (0.05M carbonate buffer, pH 9.6) was coated overnight at 4°C; blocked with 1% BSA / PBST solution at room temperature for 1 h; serially diluted serum (2-fold dilution) was incubated at room temperature for 1 h, followed by incubation with HRP-labeled secondary antibody (1:10000) at room temperature for 1 h; after reacting with TMB substrate, the OD at 450 nm was measured. Results showed that the antibody titer in the experimental group (log...)... 10 The antibody titers were approximately 1000-fold higher than the control group (Figure 3). The antibody titers shown in Figure 3 for each group were: PBS: 1.398±0.000, RBD: 2.120±0.1649, and RBD-β-Hordothionin: 5.131±0.4565.

[0044] Example 3. Evaluation of the adsorption compatibility of β-Hordothionin with aluminum adjuvant

[0045] RBD protein and RBD-β-Hordothionin (20 μg / mL) were mixed with equal volumes of 2% aluminum adjuvant (Alhydrogel®) and adsorbed at 4°C for 30 min. After centrifugation, the supernatant was collected, and the concentration of unadsorbed antigen was detected by ELISA.

[0046] Calculation formula: Adsorption rate = (PBS group concentration - aluminum adjuvant group concentration) / PBS group concentration × 100%.

[0047] The results showed that β-Hordothionin did not affect the antigen adsorption capacity of aluminum adjuvant (Figure 4), demonstrating the compatibility of the two in combination. The adsorption rates shown in Figure 4 for the two groups were: RBD: 37.60±5.85%, and RBD-β-Hordothionin: 97.33±2.939%.

[0048] Example 4. Synergistic effect of β-Hordothionin and aluminum adjuvant

[0049] The experimental group consisted of 10 μg of RBD-β-Hordothionin fusion protein combined with 50 μg of aluminum adjuvant, while the control groups consisted of RBD protein alone plus aluminum adjuvant and RBD-β-Hordothionin alone (dosage as in Example 2). The immunization procedure and ELISA detection method were the same as in Example 2. The results showed that the antibody titer in the combination group was more than 8-fold higher than that in the aluminum adjuvant-only group (Figure 5), demonstrating a significant synergistic effect. Figure 5 shows the antibody titers (log) for each group. 10 ) are: PBS: 1.398±0.000, RBD: 2.120±0.1649, RBD+Al: 4.167±0.1346, RBD-β-Hordothionin: 5.131±0.4565, RBD-β-Hordothionin+Al: 5.673±0.2519.

Claims

1. The application of β-Hordothionin in the preparation of intramolecular adjuvants, characterized in that, The application involves the use of β-Hordothionin and an immunogenic peptide in the form of a fusion protein. The amino acid sequence of the β-Hordothionin is shown in SEQ ID NO.1, the immunogenic peptide is the novel coronavirus RBD protein, and the sequence of the fusion protein is shown in SEQ ID NO.

5.

2. A polynucleotide encoding the fusion protein of claim 1, characterized in that, The sequence of the polynucleotide is shown in SEQ ID NO.

6.

3. A vaccine composition, characterized in that, The vaccine composition contains the fusion protein as described in claim 1 and an immune adjuvant, wherein the immune adjuvant is an aluminum adjuvant.

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