Novel broad-spectrum coronavirus vaccines, vaccine compositions and their applications

Abstract

This application relates to novel broad-spectrum coronavirus vaccines, vaccine compositions, and their applications. This application screens and constructs several epitope scaffold proteins and ferritin nanoparticles displaying these epitope scaffold proteins. These epitope scaffold proteins can specifically induce antibodies targeting the highly conserved stem-helix domain of the coronavirus S protein S2 subunit and antibodies targeting the highly conserved fusion peptide domain of the coronavirus S protein S2 subunit. Experiments have demonstrated that the vaccine composition provided by this invention has good immunogenicity, can induce the production of broad-spectrum antibodies binding to the coronavirus Spike protein in vivo, and provides broad-spectrum protection against infections including SARS-CoV-related coronaviruses and MERS-CoV. The broad-spectrum coronavirus vaccine provided by this invention has the potential for clinical application and can effectively prevent infection against multiple coronaviruses.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to novel broad-spectrum coronavirus vaccines, vaccine compositions and applications. Specifically, it relates to the design of epitope scaffold proteins and the formation of vaccine compositions by displaying them on nanoparticles, and their application in the prevention of multiple coronavirus infections in vivo. Background Technology

[0002] Coronaviruses have repeatedly triggered global public health crises. Based on their genetic characteristics, they can be divided into four distinct genera: α, β, γ, and δ coronaviruses. α and β coronaviruses primarily infect mammals; for example, the human seasonal coronaviruses HCoV-229E and HCoV-NL63, which cause the common cold, belong to the α genus, while SARS-CoV-2, SARS-CoV, and MERS-CoV, which cause high mortality rates, belong to the β genus. β coronaviruses are further divided into four subgenera: Embecovirus, Sarbecovirus, Merbecovirus, and Nobecovirus, also known as subgroups A, B, C, and D. Some typical coronaviruses exist within the β genus: HCoV-HKU1 and HCoV-OC43 belong to subgroup A, SARS-CoV-1 to subgroup B, and MERS-CoV to subgroup C. In addition to known coronaviruses that have already spread in humans, researchers are continuously discovering more coronaviruses in nature that can infect humans through mutation or natural host evolution.

[0003] Existing vaccines are mainly designed to target the spike protein of specific strains. When faced with rapidly mutating viruses (such as Omeprone and its subvariants) and the novel coronavirus, the neutralizing antibody titer decreases significantly or becomes ineffective, leading to immune escape and failing to provide broad-spectrum protection.

[0004] Therefore, developing a vaccine capable of inducing broad-spectrum neutralizing antibodies against conserved epitopes of multiple coronaviruses is a pressing technical challenge in this field. Traditional vaccine design methods struggle to accurately and stably represent conserved epitopes. Summary of the Invention

[0005] Technical Purpose One of the technical objectives of this application is to provide several epitope scaffold proteins that can be used to prevent various coronavirus infections.

[0006] Another technical objective of this application is to provide a fusion protein comprising the aforementioned epitope scaffold protein.

[0007] Another technical objective of this application is to provide ferritin nanoparticles containing the aforementioned fusion protein.

[0008] Another technical objective of this application is to provide a composition containing the aforementioned ferritin nanoparticles.

[0009] Another technical objective of this application is to provide the application of the above-mentioned epitope scaffold proteins, fusion proteins, ferritin nanoparticles, and compositions in the prevention of coronavirus infection (such as SARS-CoV-related coronaviruses and MERS-CoV).

[0010] Technical solution On one hand, the present invention provides an epitope scaffold protein comprising an amino acid sequence selected from SEQ ID NO:1, 2, 6, 8 and 16.

[0011] In some embodiments, the amino acid sequence of the epitope scaffold protein of the present invention is shown in SEQ ID NO:1, 2, 6, 8 or 16.

[0012] The epitope scaffold proteins provided in this application are a class of novel, non-natural proteins. They have an N-terminal epitope peptide (stem-helix domain DSFKEELDKYFKNH or fusion peptide domain PSKPSKRSFIEDLLFNKVTLADAGF) and a C-terminal supporting epitope scaffold structure. They all exhibit highly conserved epitopes in the coronavirus S protein. Experiments in this application have confirmed that they can induce antibodies against the coronavirus S protein in vivo.

[0013] On the other hand, the present invention provides a fusion protein of the above-mentioned epitope scaffold protein and SpyCatcher.

[0014] In a specific implementation, the epitope scaffold protein is fused with SpyCatcher via GSGGGGS (SEQ ID NO:53) for expression.

[0015] In a specific embodiment, the amino acid sequence of the fusion protein of the epitope scaffold protein and SpyCatcher is selected from SEQ ID NO:46-50.

[0016] In another aspect, the present invention provides a fusion protein of the above-mentioned epitope scaffold protein and ferritin.

[0017] On the other hand, the present invention provides a polynucleotide for encoding the above-mentioned epitope scaffold protein or the above-mentioned fusion protein.

[0018] On the other hand, the present invention also provides a carrier comprising the above-mentioned polynucleotides.

[0019] In another aspect, the present invention provides ferritin nanoparticles that display the above-mentioned epitope scaffold proteins on their surface.

[0020] In a specific embodiment, the ferritin nanoparticle is a 24-mer, which is formed by the self-assembly of a protein constructed by covalently binding a fusion protein of Ferritin-N96 and SpyTag and a fusion protein of the epitope scaffold protein and SpyCatcher.

[0021] In a specific embodiment, the ferritin nanoparticles are 24-mers, which are formed by the self-assembly of the aforementioned epitope scaffold protein and ferritin fusion protein.

[0022] In another aspect, the present invention provides a composition comprising the above-mentioned ferritin nanoparticles, and optionally an adjuvant.

[0023] In a specific embodiment, the adjuvant is selected from AddaVAX adjuvant.

[0024] In a specific embodiment, the composition comprises two or more types of ferritin nanoparticles, wherein the amino acid sequences of the corresponding epitope scaffold proteins and SpyCatcher fusion proteins in these ferritin nanoparticles are selected from SEQ ID NO:46-50.

[0025] In a specific embodiment, the composition comprises two types of ferritin nanoparticles, and the amino acid sequences of the corresponding epitope scaffold proteins and SpyCatcher fusion proteins in these two types of ferritin nanoparticles are SEQ ID NO:46 and 47, respectively.

[0026] In a specific embodiment, the composition comprises three types of ferritin nanoparticles, and the amino acid sequences of the corresponding epitope scaffold proteins and SpyCatcher fusion proteins in these three ferritin nanoparticles are SEQ ID NO:48, 49 and 50, respectively.

[0027] In a specific embodiment, the composition comprises five types of ferritin nanoparticles, wherein the amino acid sequences of the corresponding epitope scaffold proteins and the SpyCatcher fusion protein among the five types of ferritin nanoparticles are SEQ ID NO:46-50, respectively.

[0028] In a specific embodiment, the adjuvant is AddaVAX adjuvant.

[0029] In another aspect, the present invention provides the use of the above-mentioned epitope scaffold protein, the fusion protein of the above-mentioned epitope scaffold protein and SpyCatcher, the above-mentioned ferritin nanoparticles, and the above-mentioned vaccine composition in the preparation of a vaccine for the prevention of coronavirus infection.

[0030] In a specific implementation, the coronaviruses are α- and β-coronaviruses.

[0031] In a specific implementation, the coronavirus is selected from SARS-CoV-2, SARS-CoV and SARS-CoV-like coronaviruses, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and HCoV-229E.

[0032] Beneficial effects This invention provides a novel vaccine with broad-spectrum protection against coronaviruses, comprising five epitope scaffold proteins—ASH1, ASH2, AFP1, AFP3, and AFP11—and ferritin nanoparticles displaying these five epitope scaffold proteins. The ASH series proteins specifically induce antibodies targeting the highly conserved stem-helix domain of the coronavirus S protein S2 subunit; the AFP series proteins specifically induce antibodies targeting the highly conserved fusion peptide domain of the coronavirus S protein S2 subunit. The vaccine mixture provided by this invention exhibits good immunogenicity, inducing broad-spectrum antibodies that bind to the coronavirus Spike protein in vivo, and providing broad-spectrum protection against coronavirus infections including SARS-CoV-related coronaviruses and MERS-CoV. The broad-spectrum coronavirus vaccine provided by this invention has the potential for clinical application and can effectively prevent infection against multiple coronaviruses. Attached Figure Description

[0033] Figure 1 For the expression and purification of ASH series and AFP series proteins.

[0034] Figure 2 The titer of antibodies binding to the SARS-CoV-2 S protein in the serum of mice after immunization.

[0035] Figure 3 This is a molecular sieve chromatography chromatogram of Ferritin-N96SpyTag protein.

[0036] Figure 4 Molecular sieve chromatography chromatograms of ASH1-SpyCatcher, ASH2-SpyCatcher, AFP1-SpyCatcher, AFP3-SpyCatcher and AFP11-SpyCatcher proteins.

[0037] Figure 5 Molecular sieve chromatography chromatograms of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 proteins.

[0038] Figure 6Negative staining identification of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles.

[0039] Figure 7 The titers of antibodies against coronavirus S protein in mouse serum were determined by the binding of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles.

[0040] Figure 8 The titers of antibodies binding to the coronavirus S protein in mouse serum were ASH-NP, AFP-NP, and ASH-NP+AFP-NP.

[0041] Figure 9 The titer of antibodies binding to the coronavirus S protein in rhesus monkey serum.

[0042] Figure 10 The titers of antibodies binding to the coronavirus S protein in the serum of ASH-NP, AFP-NP, and ASH-NP+AFP-NP mice are given.

[0043] Figure 11 The viral load of RsSHC014 in the nasal cavity and lungs of mice.

[0044] Figure 12 The study included changes in mouse body weight and MERS-CoV viral load. Detailed Implementation

[0045] The technical solutions of this application are described in detail below through specific embodiments to enable those skilled in the art to better understand this application. However, the provision of these embodiments is not intended to limit the scope of this application.

[0046] Materials and reagents 293F cells: Thermo Fisher Scientific, R79007.

[0047] PVRC8400 vector, see: Structure of HIV-1 gp120 V1 / V2 domain with broadly neutralizing antibody PG9.[J]. Nature, 2011.

[0048] Nanoparticles Ferritin-N96SpyTag, see: A platform incorporating trimericantigens into self-assembling nanoparticles reveals SARS-CoV-2-spikenanoparticles to elicit substantially higher neutralizing responses thanspike alone. Sci Rep 10, 18149 (2020).

[0049] In this study, two highly conserved broad-spectrum neutralizing antibody epitopes in the S2 subunit of the coronavirus S protein were selected as the basis for the design of a broad-spectrum vaccine immunogen: the stem-helix domain and the fusion peptide domain. Deep learning AI was used to design epitope scaffold proteins that visualize these highly conserved broad-spectrum neutralizing antibody epitopes in the S2 subunit of the coronavirus S protein. Nineteen candidate proteins (five ASH series proteins displaying the stem-helix domain and fourteen AFP series proteins displaying the fusion peptide domain) were screened through computer simulation for experimental expression and validation. The candidate proteins were then screened through expression purification, binding activity with epitope-recognition antibodies, and immunogenicity in BALB / c mice, resulting in the selection of five epitope scaffold proteins (ASH1, ASH2, AFP1, AFP3, and AFP11). A vaccine mixture of five epitope scaffold protein nanoparticles was constructed, and its immunogenicity was validated in BALB / c mouse and rhesus monkey models. The heterologous sequential immunization as a novel coronavirus mRNA vaccine was studied, and the efficacy of the vaccine against severe acute respiratory syndrome-associated coronavirus RsSHC014 and Middle East respiratory syndrome coronavirus MERS-CoV was validated in humanized ACE2 mouse and humanized DPP4 mouse models, respectively.

[0050] Example 1: Design and Screening of Immunogens I. Design of Epitope Scaffold Proteins Based on Deep Learning 1. Target epitope selection Two highly conserved broad-spectrum neutralizing antibody epitopes in the S2 subunit of the coronavirus S protein were selected as the basis for the design of a broad-spectrum vaccine immunogen: the stem-helix domain and the fusion peptide domain. The amino acid sequence of the stem-helix domain is DSFKEELDKYFKNH (SEQ ID NO. 51), and the PDB structural reference number is 7RNJ; the amino acid sequence of the fusion peptide domain is PSKPSKRSFIEDLLFNKVTLADAGF (SEQ ID NO. 52), and the PDB structural reference number is 7X9E.

[0051] 2. AI-assisted design The target epitope was extracted from the three-dimensional structure of the original antigen protein and input as an independent motif / fragment into a self-built protein structure generation model. Scaffold protein design was carried out independently for stem-helix domain epitopes and fusion peptide domain epitopes. A pure scaffold model was used in the design process, with the target epitope as the fixed structural unit. Constraints were simultaneously applied to both the backbone and side chain conformations of the epitope to preserve, as much as possible, the local spatial conformational characteristics of the target epitope in the natural antigen.

[0052] In the design constraints, the generated scaffold protein is further required to meet the following conditions: First, the target epitope must be located on the surface exposed area of ​​the designed protein to ensure that the epitope can be effectively recognized by the immune system during subsequent immunization; Second, the scaffold body must not significantly obstruct the target epitope to avoid affecting the antigen presentation effect due to burial or coverage by adjacent structures; Third, hotspot residues are set in the design process to guide the scaffold structure to form a reasonable local support environment around the target epitope, thereby improving the structural stability and foldability of the overall design while maintaining the natural conformation of the epitope.

[0053] Regarding the scaffold length, the length of candidate scaffold proteins is controlled within 150-200 amino acids. This length range is determined based on protein structure design experience: on the one hand, this range is generally conducive to forming a relatively stable, independently foldable protein scaffold and has good feasibility for recombinant expression; on the other hand, this length can also provide sufficient spatial support and conformational constraints for the target epitope, reducing structural instability caused by an excessively short scaffold or additional structural complexity and expression burden caused by an excessively long scaffold.

[0054] Under the aforementioned constraints, a self-built protein structure generation model was run for each target epitope, generating 10,000 candidate scaffold protein designs. These candidate designs refer to the overall set of candidate molecules after backbone generation and sequence filling. For each candidate backbone output by the self-built protein structure generation model, ProteinMPNN was further used for amino acid sequence design. During the sequence design stage, eight candidate amino acid sequences were generated for each backbone, and the relatively superior sequences were selected based on the model score for subsequent structure validation and screening. Through this method, a large number of novel artificial scaffold protein candidate molecules constructed around the target epitope can be obtained while preserving the key spatial conformation of the target epitope.

[0055] 3. Computer simulation screening For the candidate scaffold proteins generated above, a multi-step computer simulation screening strategy was used for selection to evaluate their foldability, epitope conformation retention ability, and spatial matching when complexed with the corresponding neutralizing antibody.

[0056] First, AlphaFold2 was used to predict the structural folding of candidate protein sequences to assess whether the designed sequences could stably fold into the expected structure. For the structural folding results, candidate molecules with a predicted average pLDDT (Predicted Local Distance Difference Test) value greater than 80 were preferred as candidates with higher folding confidence. Simultaneously, the folded structures predicted by AlphaFold2 were compared with the initial design model, with a focus on calculating the Backbone RMSD (Root Mean Square Deviation) of the target epitope region to assess whether the target epitope could still maintain a spatial conformation close to the design after folding. Candidate molecules with a Backbone RMSD of less than 2 Å in the target epitope region were preferred for the next round of screening. This step eliminated candidate designs that, although constructed from a generative model, could not stably support the target epitope conformation at the sequence level.

[0057] Secondly, spatial compatibility analysis at the complex level is further conducted on candidate molecules screened through structural folding. Specifically, the target epitope displayed by the candidate scaffold protein and the corresponding neutralizing antibody are used to predict the complex structure, and the complex structure is evaluated using the steric hindrance score and interface metrics. Candidate molecules that generate significant steric hindrance within the complex, have poor interface geometry matching, or whose scaffold structure interferes with the antibody's approach to and binding to the target epitope, are eliminated. This step further ensures that the designed scaffold not only stably displays the target epitope but also effectively mimics the antibody-recognizable state in the natural antigen in terms of spatial conformation.

[0058] Based on the above screening, a comprehensive analysis was conducted on the sequence composition, surface physicochemical properties, and overall structural rationality of the candidate scaffold proteins to select candidate molecules with high folding probability, moderate surface hydrophobicity, and suitability for further experimental expression and verification.

[0059] Following this screening, five candidate proteins displaying a stem-helix domain were obtained, named ASH1 to ASH5, with their amino acid sequences shown in SEQ ID NO. 1-5. Fourteen candidate proteins displaying a fusion peptide domain were obtained, named AFP1 to AFP14, with their amino acid sequences shown in SEQ ID NO. 6-19.

[0060] II. Preparation of Candidate Proteins 1. Construction of recombinant expression vectors The double-stranded DNA molecule shown in SEQ ID NO:20 was inserted into the NotI and BamHI restriction sites of the PVRC8400 vector (see: Structure of HIV-1gp120 V1 / V2 domain with broadly neutralizing antibody PG9.[J]. Nature, 2011.) to obtain the PVRC8400-His tag vector. In SEQ ID NO:20, nucleotides 1-57 encode a signal peptide, nucleotides 58-65 form the NotI restriction recognition sequence, nucleotides 81-86 form the NheI restriction recognition sequence, nucleotides 87-104 encode a His6 tag, and nucleotides 105-107 are the stop codon.

[0061] The small fragments in the NotI and NheI restriction enzyme recognition sequences of the PVRC8400-His tag vector were replaced with double-stranded DNA molecules as shown in SEQ ID NO:21-39, respectively, to obtain recombinant plasmids PVRC8400-ASH-1 to 5 and PVRC8400-AFP-1 to 14. The recombinant plasmids express the following fusion protein, which, from the N-terminus to the C-terminus, contains a signal peptide, the functional region shown in SEQ ID NO:1-19, and a His6 tag. In cells, the signal peptide is cleaved, leaving the following active protein, which, from the N-terminus to the C-terminus, contains the functional region shown in SEQ ID NO:1-19 and a His6 tag.

[0062] 2. Preparation of protein-containing supernatant Nineteen recombinant plasmids, PVRC8400-ASH-1 to 5 and PVRC8400-AFP-1 to 14, were transfected into 293F cells (293F cells: Thermo Fisher Scientific, R79007). The cells were cultured in OPM-293 CD05 medium (OPM, 81075-001) for 72 h, and then centrifuged at 4000 rpm for 30 min. The supernatant containing ASH and AFP series proteins was collected.

[0063] 3. Protein purification The protein solutions to be purified are: the supernatants of 19 monomeric proteins prepared in step 2.

[0064] Add Ni-NTA purification medium (Qiagen, 30230) to the supernatant, incubate at 4°C for 3 hours, centrifuge at 400 rpm for 5 minutes, and collect the precipitate. Wash the precipitate with 100 mL of PBS solution containing 20 mM imidazole (Seville, G4202-500 mL) to remove impurities. Wash the precipitate with 20 mL of PBS solution containing 300 mM imidazole and collect the solution. Concentrate the obtained solution using a 3 kDa concentration tube to obtain the protein concentrate.

[0065] Of the five ASH series proteins, ASH5 could not be purified normally. Of the AFP series proteins, AFP2, AFP12, and AFP14 could not be purified normally. The obtained ASH-1 to 4 and AFP-1, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13 proteins were subjected to reducing SDS-PAGE (treated with β-mercaptoethanol) and non-reducing SDS-PAGE, respectively. Electrophoresis images are shown below. Figure 1 .

[0066] III. Binding activity of candidate proteins with epitope recognition antibodies S2P6, DH1057, and WS6 are all SARS-CoV-2 stem helix domain antibodies known in the prior art. 76E1, COV44-62, COV44-79, and VN01H1 are all SARS-CoV-2 fusion peptide domain antibodies known in the prior art.

[0067] SPR assay: Antibodies were coated on the sensor of Protein A, and then the ASH series and AFP series protein solutions prepared above were flowed in. Single-cycle kinetics detection was used to detect the binding activity of the candidate proteins to each antibody. The results are shown in Table 1 and Table 2.

[0068] The KD value of the binding affinity between the ASH series proteins and the S2P6 antibody is between 0.1 and 1 nM, and the KD value of the affinity with other antibodies of the same epitope is between 0.01 and 10 nM. The KD value of the binding affinity between the AFP series proteins and the 76E1 antibody is between 0.01 and 1 nM, and the KD value of the affinity with other antibodies of the same epitope is between 0.001 and 10 nM. After comprehensive comparison, 10 candidate proteins, namely ASH1, ASH2, AFP1, AFP3, AFP4, AFP5, AFP8, AFP9, AFP10, and AFP11, were selected.

[0069] Table 1

[0070] Table 2

[0071] IV. Preliminary Screening of the Immunogenicity of Candidate Proteins in BALB / c Mice 1. Animal Immunization Female BALB / C mice at 6 weeks of age (purchased from Zhejiang Vital River Laboratory Animal Science Co., Ltd., license number: SYXK (Zhejiang) 2023-0024) were divided into 10 groups, with 5 mice in each group, and were immunized with ASH1, ASH2, AFP1, AFP3, AFP4, AFP5, AFP8, AFP9, AFP10, and AFP11 proteins respectively. Four immunizations were carried out by intramuscular injection, and the immunization dose each time was 10 μg protein / mouse (specifically, the protein solution was diluted to 50 μL and then mixed with 50 μL AddaVAX adjuvant (Invivogen, vac-adx-10) before use). 14 days after the fourth immunization, 100 μL of blood was collected from the cheek, and the serum was taken for detection.

[0072] 2. Binding Antibody Titer of SARS-CoV-2 S-2P Protein in Serum Enzyme-linked immunosorbent assay (ELISA): (1) Antigen coating: The antigen (coronavirus S-2P protein) was diluted with PBS buffer and coated with 96-well plates at a coating volume of 100 ng / 100 μL / well. The coating was carried out overnight at 4°C.

[0073] (2) After coating the 96-well plate overnight, discard the liquid in the wells, wash the plate 3 times with PBST, add 300 μL of PBST containing 3% BSA (Yisheng, 36101ES80) to each well, and let it stand at 37°C for 2 hours to block.

[0074] (3) After the blocking is completed, wash the plate 3 times with PBST buffer.

[0075] (4) Primary antibody incubation: Dilute the serum with PBS solution serially; incubate at 37°C for 1 hour.

[0076] (5) Wash the plate 3 times with PBST buffer, add 100 μL of anti-mouse HRP antibody (Promega, W4021) (diluted with PBS solution to 1:4000) as secondary antibody, and incubate at 37°C for 1 hour.

[0077] (6) Wash the plate 3 times with PBST buffer, thoroughly dry the liquid in the wells, add 100 μL of TMB colorimetric solution (Kangwei Century, CW0050) to each well, incubate at room temperature for 8 minutes, and add 50 μL of 1M sulfuric acid to stop the color development.

[0078] (7) Use an ELISA reader to read the value at a wavelength of 450 nm, and use GraphPad software to plot the binding curve and calculate the ED50 value of the serum. The ED50 value is the serum dilution at which half of the antigen is bound by the antibody.

[0079] See results Figure 2 Five epitope scaffold proteins with strong immunogenicity—ASH1, ASH2, AFP1, AFP3, and AFP11—were selected for further evaluation.

[0080] Example 2: Preparation of Nanoparticle Vaccine I. Preparation of Ferritin-N96SpyTag Nanoparticles 1. Replace the small fragments in the NotI and NheI restriction enzyme recognition sequences of the PVRC8400-His tag vector with the double-stranded DNA molecule shown in SEQ ID NO:40, respectively, to obtain the recombinant plasmid PVRC8400-Ferritin-N96SpyTag. The recombinant plasmid expresses the following fusion protein, which, from the N-terminus to the C-terminus, contains a signal peptide, the functional region shown in SEQ ID NO:40, and a His6 tag. In cells, the signal peptide is cleaved, leaving the following active protein, which, from the N-terminus to the C-terminus, contains the functional region shown in SEQ ID NO:40 and a His6 tag.

[0081] 2. The recombinant plasmid PVRC8400-Ferritin-N96SpyTag was transfected into 293F cells and cultured in OPM-293 CD05 medium for 72 h. Then, the cells were centrifuged at 4000 rpm for 30 min, and the supernatant containing Ferritin-N96SpyTag protein (SEQ ID No:54) was collected.

[0082] 3. Add Ni-NTA purification medium to the supernatant, incubate at 4°C for 3 hours, centrifuge at 400 rpm for 5 minutes, and collect the precipitate. Wash the precipitate with 100 mL of PBS solution containing 20 mM imidazole to remove impurities. Wash the precipitate with 20 mL of PBS solution containing 300 mM imidazole and collect the solution. Concentrate the obtained solution using a 30 kDa concentrator to obtain a 1 mL concentrate. Perform molecular sieve chromatography on a Superose 6 Increase column using PBS solution as the eluent.

[0083] The chromatogram of the elution process is shown in [reference needed]. Figure 3 (The horizontal axis represents the effluent volume), and the effluent volume corresponding to the target peak is 16.57 mL.

[0084] Collect the elution solution (1 mL) corresponding to the target peak, which is the Ferritin-N96SpyTag nanoparticle protein solution.

[0085] II. Preparation of ASH1, ASH2, AFP1, AFP3 and AFP11-SpyCatcher proteins 1. Replace the small fragments in the NotI and NheI restriction enzyme recognition sequences of the PVRC8400-His tag vector with the double-stranded DNA molecules shown in SEQ ID NO: 41-45, respectively, to obtain recombinant plasmids PVRC8400-ASH1, ASH2, AFP1, AFP3, and AFP11-SpyCatcher. The recombinant plasmids express the following fusion proteins, which, from the N-terminus to the C-terminus, contain a signal peptide, the functional region shown in SEQ ID NO: 41-45, and a His6 tag. In cells, the signal peptide is cleaved, leaving the following active proteins, which, from the N-terminus to the C-terminus, contain the functional region shown in SEQ ID NO: 41-45 and a His6 tag.

[0086] 2. Five recombinant plasmids were transfected into 293F cells and cultured in OPM-293 CD05 medium for 72 h. Then, the cells were centrifuged at 4000 rpm for 30 min and the supernatant containing ASH1, ASH2, AFP1, AFP3 and AFP11-SpyCatcher proteins (amino acid sequences corresponding to SEQ ID NO:46-50) was collected.

[0087] 3. Add Ni-NTA purification medium to the supernatant, incubate at 4°C for 3 hours, centrifuge at 400 rpm for 5 minutes, and collect the precipitate. Wash the precipitate with 100 mL of PBS solution containing 20 mM imidazole to remove impurities. Wash the precipitate with 20 mL of PBS solution containing 300 mM imidazole and collect the solution. Concentrate the obtained solution using a 10 kDa concentrator to obtain a 1 mL concentrate. Perform molecular sieve chromatography on the concentrate using a Superdex 200 Increase 10 / 300 GL column, using PBS solution as the eluent.

[0088] The chromatogram of the elution process is shown in [reference needed]. Figure 4 (The horizontal axis represents the effluent volume), and the effluent volumes corresponding to the target peaks are 14.05, 13.20, 12.92, 15.95, and 14.69 mL, respectively.

[0089] Collect the elution solution (1 mL) corresponding to the target peak, which is the protein solution of ASH1-SpyCatcher, ASH2-SpyCatcher, AFP1-SpyCatcher, AFP3-SpyCatcher and AFP11-SpyCatcher.

[0090] III. Preparation of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles 1. The Ferritin-N96SpyTag nanoparticles prepared above were mixed with ASH1-SpyCatcher, ASH2-SpyCatcher, AFP1-SpyCatcher, AFP3-SpyCatcher, and AFP11-SpyCatcher proteins at a molar ratio of 1:1 (where Ferritin-N96SpyTag protein is calculated in monomer form), and incubated overnight at 4°C.

[0091] 2. The obtained solution was concentrated using a 30kD concentrator to obtain a 1mL concentrate. The concentrate was then subjected to molecular sieve chromatography using a Superose 6 Increase column with PBS as the eluent.

[0092] The chromatogram of the elution process is shown in [reference needed]. Figure 5 (The horizontal axis represents the effluent volume), and the effluent volumes corresponding to the target peaks are 12.60, 12.99, 12.42, 12.99, and 12.66 mL, respectively.

[0093] Collect the elution solution (1 mL) corresponding to the target peak, which is a solution of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles.

[0094] IV. Identification of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles Transmission electron microscopy (TEM) samples were prepared using negative staining techniques to observe the morphology of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles. Figure 6 .

[0095] Example 3: Evaluation of the immunogenicity of the vaccine of this application in animal models. I. Immunogenicity of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 individually 1. Immunization of BALB / c mice Six-week-old female BALB / c mice were divided into six groups of five each and immunized with: (1) Ferritin-ASH1; (2) Ferritin-ASH2; (3) Ferritin-AFP1; (4) Ferritin-AFP3; (5) Ferritin-AFP11; and (6) Ferritin-N96SpyTag (control). Four immunizations were administered intramuscularly, with each dose being 10 μg protein per mouse (specifically, the protein solution was diluted to 50 μL and then mixed with 50 μL of AddaVAX adjuvant before use). Fourteen days after the third immunization, 100 μL of blood was collected from the cheek for serum analysis.

[0096] 2. Serum antibody titers binding to SARS-CoV-2 S-2P protein The antibody titer in mouse serum was determined using the same detection method as in step four of Example 1. The antigen protein was the S-2P protein from SARS-CoV-2.

[0097] See results Figure 7The results showed that mouse serum immunized with individual nanoparticle proteins (Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP1) could bind to the Spike protein of the coronavirus SARS-CoV-2.

[0098] II. Immunogenicity of the vaccine in the BALB / c mouse model 1. Immunization of BALB / c mice Six-week-old female BALB / c mice were divided into four groups of five each and immunized with: (1) a mixture of Ferritin-ASH1 and Ferritin-ASH2 nanoparticles (ASH-NP); (2) a mixture of Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles (AFP-NP); (3) a mixture of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles (ASH-NP+AFP-NP); and (4) Ferritin-N96SpyTag nanoparticles (NP). Four immunizations were administered intramuscularly, with each dose being 10 μg protein per mouse (specifically, the protein solution was diluted to 50 μL and then mixed with 50 μL of AddaVAX adjuvant before use). Fourteen days after the fourth immunization, 100 μL of blood was collected from the cheek for serum analysis.

[0099] 2. Serum antibody titers binding to SARS-CoV-2 S-2P protein The binding antibody titer of mouse serum was detected using the same detection method as in step four of Example 1. The antigen protein was the Spike protein from seven coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1, and HCoV-229E).

[0100] See results Figure 8 The results showed that mouse serum immunized with the broad-spectrum anti-coronavirus vaccine of the present invention was able to bind Spike proteins from seven coronaviruses capable of infecting humans.

[0101] III. Immunogenicity of the vaccine in rhesus monkey models 1. Rhesus monkey immunization Female rhesus monkeys of suitable age were divided into two groups: Group 1 (n=8) and Group 2 (n=4). Each group was immunized with: (1) a mixture of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles; and (2) Ferritin-N96SpyTag nanoparticles. Four immunizations were administered via intramuscular injection, with each dose being 50 μg protein per monkey (specifically, the protein solution was diluted to 1 mL and then mixed with 1 mL of AddaVAX adjuvant before use). Fourteen days after the fourth immunization, 2 mL of blood was collected for serum analysis.

[0102] 2. Serum antibody titers binding to SARS-CoV-2 S-2P protein The binding antibody titer of rhesus monkey serum was detected using the same detection method as in step four of Example 1. The antigen protein was the S-2P protein from seven coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, OC43, NL63, HKU1, and 229E).

[0103] See results Figure 9 The results showed that rhesus monkey serum immunized with the broad-spectrum anti-coronavirus vaccine of the present invention was able to bind to the Spike protein from seven coronaviruses capable of infecting humans.

[0104] IV. Research on Vaccines as Heterologous Sequential Immunization 1. Immunization of BALB / c mice Six-week-old female BALB / c mice were divided into four groups of five mice each and immunized four times via intramuscular injection. The first two immunizations used a novel coronavirus mRNA vaccine (Watson Biotech RQ3013 vaccine), with a dose of 2 μg protein per mouse per immunization (specifically, the mRNA vaccine solution was diluted to 100 μL before use). The latter two immunizations used the broad-spectrum anti-coronavirus vaccine of this application, and the four groups were immunized with: (1) a mixture of Ferritin-ASH1 and Ferritin-ASH2 nanoparticles (ASH-NP); (2) a mixture of Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles (AFP-NP); (3) a mixture of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3, and Ferritin-AFP11 nanoparticles (ASH-NP+AFP-NP); and (4) Ferritin-N96SpyTag nanoparticles (NP). Each immunization dose was 10 μg protein per animal (specifically, the protein solution was diluted to 50 μL and then mixed with 50 μL of AddaVAX adjuvant before use). Fourteen days after the fourth immunization, 100 μL of blood was collected from the cheek, and the serum was used for testing.

[0105] 2. Serum antibody titers binding to SARS-CoV-2 S-2P protein The binding antibody titer of mouse serum was detected using the same detection method as in step four of Example 1. The antigen protein was the S-2P protein from seven coronaviruses (SARS-CoV-2, SARS-CoV, MERS-CoV, OC43, NL63, HKU1, and 229E).

[0106] See results Figure 10 The results showed that mouse serum immunized with the broad-spectrum anti-coronavirus vaccine of the present invention was able to bind Spike proteins from seven coronaviruses capable of infecting humans.

[0107] Example 4: Evaluation of the efficacy of a broad-spectrum anti-coronavirus vaccine in animal models. I. Vaccine efficacy against Severe Acute Respiratory Syndrome-associated Coronavirus RsSHC014 1. Challenge protocol: The broad-spectrum anti-coronavirus vaccine was used as a heterologous sequential immunization. A total of 4 groups of humanized ACE2 mice were immunized 4 times via intramuscular injection. The first immunization of G1, G2 and G3 used the novel coronavirus mRNA vaccine (Moderna, mRNA-1273), with an immunization dose of 1 μg protein / mouse. The subsequent 3 immunizations were respectively immunized with: (1) a mixture of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles; (2) Ferritin-N96SpyTag nanoparticles (NP); (3) the novel coronavirus mRNA vaccine (Moderna, mRNA-1273). The immunization dose of the broad-spectrum anti-coronavirus vaccine of this application is 10 μg protein / mouse (AddaVAX adjuvant). G4 was a blank control mouse that had not been immunized. 10 mice were infected intranasally 2 weeks after the 4th immunization. 5 The RsSHC014 virus in PFU.

[0108] 2. Evaluation of protective efficacy: Mice were sacrificed on day 2 post-infection, and nasal turbinate and lung tissue homogenates were collected to detect viral titers. Results are as follows: Figure 11 As shown, the viral titers in the nasal cavity and lungs of mice in group 1 were significantly lower than those in control groups 2 and 4, achieving the same protective effect as group 3 of the mRNA vaccine.

[0109] II. Vaccine efficacy against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) 1. Challenge protocol: The broad-spectrum anti-coronavirus vaccine was used as a heterologous sequential immunization. A total of 4 groups of humanized DPP4 mice were immunized three times by intramuscular injection. The first immunization of G1 and G2 used the novel coronavirus mRNA vaccine (Moderna, mRNA-1273), with an immunization dose of 1 μg protein / mouse. The subsequent two immunizations were respectively immunized with: (1) a mixture of Ferritin-ASH1, Ferritin-ASH2, Ferritin-AFP1, Ferritin-AFP3 and Ferritin-AFP11 nanoparticles; (2) the novel coronavirus mRNA vaccine (Moderna, mRNA-1273). The immunization dose of the broad-spectrum anti-coronavirus vaccine of this application is 10 μg protein / mouse (AddaVAX adjuvant). G3 was a blank control mouse that had not been immunized. Two weeks after the third immunization, 10 mice were infected intranasally. 5 PFU contains the MERS-CoV virus.

[0110] 2. Evaluation of protective efficacy: Mice were sacrificed on day 2 post-infection, and nasal turbinate and lung tissue homogenates were collected to detect viral titers. Results are as follows: Figure 12As shown, the viral titer in the lungs of mice in group 1 was significantly lower than that in control groups 2 and 3, providing better protection against MERS-CoV infection in the lower respiratory tract. The viral titer in the nasal cavity was significantly lower than that in control group 3, demonstrating a better broad-spectrum protective effect.

Claims

1. An epitope scaffold protein comprising an amino acid sequence selected from: SEQ ID NO:1, 2, 6, 8 and 16.

2. The epitope scaffold protein of claim 1, wherein, The amino acid sequence of the epitope scaffold protein is shown in SEQ ID NO: 1, 2, 6, 8 or 16.

3. The fusion protein of epitope scaffold protein and SpyCatcher as described in claim 1 or 2.

4. The fusion protein of claim 3, wherein, The epitope scaffold protein is expressed by fusion with SpyCatcher via GSGGGGS (SEQ ID NO:53).

5. The fusion protein of claim 4, wherein, The amino acid sequence of the fusion protein of the epitope scaffold protein and SpyCatcher is selected from SEQ ID NO:46-50.

6. The fusion protein of epitope scaffold protein and ferritin as described in claim 1 or 2.

7. Ferritin nanoparticles displaying the epitope scaffold protein as described in claim 1 or 2 on their surface.

8. The ferritin nanoparticles according to claim 8 are 24-mers formed by self-assembly of proteins constructed by covalently binding a fusion protein of Ferritin-N96 and SpyTag and a fusion protein of an epitope scaffold protein according to any one of claims 3-5 and SpyCatcher.

9. A composition comprising ferritin nanoparticles as described in claim 7 or 8, and optionally an adjuvant. Preferably, the composition comprises two or more types of ferritin nanoparticles, wherein the amino acid sequences of the fusion protein of the corresponding epitope scaffold protein and SpyCatcher in these ferritin nanoparticles are selected from SEQ ID NO:46-50; or The composition comprises two types of ferritin nanoparticles, wherein the amino acid sequences of the corresponding epitope scaffold proteins and the fusion protein of SpyCatcher in these two ferritin nanoparticles are SEQ ID NO:46 and 47, respectively; or The composition comprises three types of ferritin nanoparticles, the amino acid sequences of the corresponding epitope scaffold proteins and the SpyCatcher fusion protein in these three ferritin nanoparticles being SEQ ID NO:48, 49, and 50, respectively; or The composition comprises 5 ferritin nanoparticles, wherein, The amino acid sequences of the epitope scaffold proteins corresponding to these five ferritin nanoparticles and the fusion protein of SpyCatcher are SEQ ID NO:46-50, respectively.

10. The use of the epitope scaffold protein of claim 1 or 2, the fusion protein of the epitope scaffold protein of any one of claims 3-5 and SpyCatcher, the ferritin nanoparticles of claim 7 or 8, or the composition of claim 9 in the preparation of a vaccine for the prevention of coronavirus infection. Preferably, the coronavirus is an α- or β-coronavirus. More preferably, the coronavirus is selected from SARS-CoV-2, SARS-CoV and SARS-CoV-like coronaviruses, MERS-CoV, HCoV-OC43, HCoV-NL63, HCoV-HKU1 and HCoV-229E.

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