Fusion proteins and their applications

Abstract

This application relates to one or more fusion proteins comprising a domain of the SARS-CoV-2 S (Spike) protein. This application also relates to immunogenic compositions comprising the fusion protein and their applications.

Description

Technical Field

[0001] This application relates to the biomedical field, specifically to fusion proteins, compositions, and applications of SARS-CoV-2 spike protein (S protein) containing the receptor-binding domain (RBD) or its functionally active fragment and / or the N-terminal domain (NTD) or its functionally active fragment. Background Technology

[0002] Currently, the methods for controlling the COVID-19 pandemic are implementing quarantine, isolation, and maintaining physical distancing. Therefore, there is an urgent need for an effective vaccine against COVID-19 to alleviate the enormous burden of mortality and morbidity associated with SARS-CoV-2 infection.

[0003] The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) is considered the primary antigenic target region for inducing the production of neutralizing antibodies. Using the RBD as a vaccine can focus the neutralizing antibodies produced by the body more precisely on the receptor binding to the virus, thereby improving the immunogenicity and immunization efficiency of the vaccine.

[0004] The N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) is a sequence at the N-terminus of the viral S protein. It can bind to proteins or glycoproteins of the host cell, mediating viral invasion of the host cell. Therefore, this region may contain epitopes that induce the production of neutralizing antibodies. Summary of the Invention

[0005] This application provides a fusion protein, composition, and application thereof comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof and / or an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof, which has one or more of the following properties: 1) the fusion protein is immunogenic; 2) the COVID-19 subunit vaccine containing the fusion protein is more immunogenic and can induce the production of more neutralizing antibodies.

[0006] On one hand, this application provides a fusion protein comprising: 1) a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0007] In some implementations, the RBD is derived from SARS-CoV-2 wild-type or a mutant thereof.

[0008] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0009] In some embodiments, the RBD has mutations at one or more amino acid sites selected from the group consisting of K417, L452, T478, E484, and N501, compared to the RBD of wild-type SAR-CoV-2.

[0010] In some embodiments, the RBD comprises one or more of the amino acid mutations K417T / N, L452R, T478K, E484K, and N501Y.

[0011] In some embodiments, the RBD contains amino acid mutations of K417T, E484K, and N501Y.

[0012] In some embodiments, the RBD contains amino acid mutations of K417N, E484K, and N501Y.

[0013] In some embodiments, the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0014] In some embodiments, the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0015] In some embodiments, the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0016] In some embodiments, the P2 or its functional active fragment is directly or indirectly connected to the N-terminus or C-terminus of the RBD or its functional active fragment.

[0017] In some embodiments, the RBD or its functionally active fragment is fused with the P2 or its functionally active fragment within the frame.

[0018] In some embodiments, the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibrin.

[0019] In some embodiments, the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0020] In some embodiments, the foldon domain or its functionally active segment is directly or indirectly connected to the N-terminus or C-terminus of the RBD or its functionally active segment.

[0021] In some implementations, the RBD or its functionally active fragment is fused within the frame with the foldon domain or its functionally active fragment.

[0022] In some embodiments, the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0023] In some embodiments, the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin.

[0024] In some embodiments, the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0025] In some embodiments, the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0026] In some embodiments, the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0027] In some embodiments, the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0028] In some embodiments, the ferritin or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0029] In some embodiments, the RBD or its functionally active fragment is fused with the ferritin or its functionally active fragment within the frame.

[0030] In some embodiments, the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0031] In some embodiments, the hepatitis B surface antigen or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0032] In some embodiments, the RBD or its functionally active fragment is fused with the hepatitis B surface antigen or its functionally active fragment within the frame.

[0033] In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 1-17, 80, and 86.

[0034] On the other hand, this application provides a fusion protein comprising: 1) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, the foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0035] In some implementations, the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0036] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0037] In some embodiments, the NTD includes mutations at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0038] In some embodiments, the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0039] In some implementations, the NTD contains L18F, D80A, D215G, L242-244del, and R246I mutations.

[0040] In some embodiments, the NTD contains amino acid mutations in L18F, T20N, P26S, D138Y, and R246I.

[0041] In some embodiments, the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0042] In some embodiments, the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0043] In some embodiments, the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0044] In some embodiments, the P2 or its functional active fragment is directly or indirectly connected to the N-terminus or C-terminus of the NTD or its functional active fragment.

[0045] In some embodiments, the NTD or its functionally active fragment is fused with the P2 or its functionally active fragment within the frame.

[0046] In some embodiments, the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibrin.

[0047] In some embodiments, the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0048] In some embodiments, the foldon domain or its functionally active segment is directly or indirectly connected to the N-terminus or C-terminus of the NTD or its functionally active segment.

[0049] In some embodiments, the NTD or its functionally active fragment is fused within the frame with the foldon domain or its functionally active fragment.

[0050] In some embodiments, the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0051] In some embodiments, the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin.

[0052] In some embodiments, the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0053] In some embodiments, the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0054] In some embodiments, the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0055] In some embodiments, the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0056] In some embodiments, the ferritin or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0057] In some embodiments, the NTD or its functionally active fragment is fused with the ferritin or its functionally active fragment within the frame.

[0058] In some embodiments, the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0059] In some embodiments, the hepatitis B surface antigen or its functionally active fragment is directly or indirectly connected to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0060] In some embodiments, the NTD or its functionally active fragment is fused with the hepatitis B surface antigen or its functionally active fragment within the frame.

[0061] In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 20-36, 81 and 87.

[0062] On the other hand, this application provides a fusion protein comprising: 1) a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0063] In some embodiments, the fusion protein further includes one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0064] In some implementations, the RBD is derived from SARS-CoV-2 wild-type or a mutant thereof.

[0065] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0066] In some embodiments, the RBD has mutations at one or more amino acid sites selected from the group consisting of K417, L452, T478, E484, and N501, compared to the RBD of wild-type SAR-CoV-2.

[0067] In some embodiments, the RBD comprises one or more of the amino acid mutations K417T / N, L452R, T478K, E484K, and N501Y.

[0068] In some embodiments, the RBD contains amino acid mutations of K417T, E484K, and N501Y.

[0069] In some embodiments, the RBD contains amino acid mutations of K417N, E484K, and N501Y.

[0070] In some embodiments, the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0071] In some implementations, the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0072] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0073] In some embodiments, the NTD includes mutations at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0074] In some embodiments, the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0075] In some implementations, the NTD contains L18F, D80A, D215G, L242-244del, and R246I mutations.

[0076] In some embodiments, the NTD contains amino acid mutations in L18F, T20N, P26S, D138Y, and R246I.

[0077] In some embodiments, the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0078] In some embodiments, the RBD or its functionally active fragment is directly or indirectly connected to the NTD or its functionally active fragment.

[0079] In some embodiments, the RBD or its functionally active fragment is fused with the NTD or its functionally active fragment within the frame.

[0080] In some embodiments, the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0081] In some embodiments, the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0082] In some embodiments, the P2 or its functionally active fragment is directly or indirectly connected to the RBD or its functional fragment and / or the NTD or its functionally active fragment.

[0083] In some embodiments, the P2 or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0084] In some embodiments, the N-terminus of the P2 or its functional active segment is directly or indirectly connected to the C-terminus of the RBD or its functional active segment, and the N-terminus of the RBD or its functional active segment is directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0085] In some embodiments, the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibrin.

[0086] In some embodiments, the amino acid residues at the C-terminus of the phage T4 fibrin contain the amino acid sequence shown in SEQ ID NO:78.

[0087] In some embodiments, the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0088] In some embodiments, the foldon domain or its functionally active fragment is directly or indirectly connected to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0089] In some embodiments, the foldon domain or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0090] In some embodiments, the N-terminus of the foldon domain or its functionally active segment is directly or indirectly connected to the C-terminus of the RBD or its functionally active segment, and the N-terminus of the RBD or its functionally active segment is directly or indirectly connected to the C-terminus of the NTD or its functionally active segment.

[0091] In some embodiments, the N-terminus of the foldon domain or its functional active segment is directly or indirectly connected to the C-terminus of the P2 or its functional active segment, the N-terminus of the P2 or its functional active segment is directly or indirectly connected to the C-terminus of the RBD or its functional active segment, and the N-terminus of the RBD or its functional active segment is directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0092] In some embodiments, the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0093] In some embodiments, the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin.

[0094] In some embodiments, the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0095] In some embodiments, the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0096] In some embodiments, the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0097] In some embodiments, the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0098] In some embodiments, the ferritin or its functionally active fragment is directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0099] In some embodiments, the ferritin or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0100] In some embodiments, the N-terminus of the ferritin or its functionally active fragment is directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the N-terminus of the RBD or its functionally active fragment is directly or indirectly connected to the C-terminus of the NTD or its functionally active fragment.

[0101] In some embodiments, the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0102] In some embodiments, the hepatitis B surface antigen or its functionally active fragment is directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0103] In some embodiments, the hepatitis B surface antigen or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0104] In some embodiments, the N-terminus of the hepatitis B surface antigen or its functionally active fragment is directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the N-terminus of the RBD or its functionally active fragment is directly or indirectly connected to the C-terminus of the NTD or its functionally active fragment.

[0105] In some embodiments, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 39-44, 79, 85 and 96.

[0106] On the other hand, this application provides an immunogenic composition comprising the fusion protein described above.

[0107] In some embodiments, the immunogenic composition comprises a first component comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising a fusion protein.

[0108] In some embodiments, the immunogenic composition comprises a first component comprising a fusion protein; and a second component comprising the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0109] In some embodiments, the immunogenic composition comprises a first component comprising the fusion protein; and a second component comprising the fusion protein.

[0110] In some embodiments, the immunogenic composition is formulated in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0111] In some embodiments, the immunogenic composition comprises: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0112] On the other hand, this application provides an immunogenic composition comprising a first component comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0113] In some implementations, the RBD is derived from SARS-CoV-2 wild-type or a mutant thereof.

[0114] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0115] In some embodiments, the RBD has mutations at one or more amino acid sites selected from the group consisting of K417, L452, T478, E484, and N501, compared to the RBD of wild-type SAR-CoV-2.

[0116] In some embodiments, the RBD comprises one or more of the amino acid mutations K417T / N, L452R, T478K, E484K, and N501Y.

[0117] In some embodiments, the RBD contains amino acid mutations of K417T, E484K, and N501Y.

[0118] In some embodiments, the RBD contains amino acid mutations of K417N, E484K, and N501Y.

[0119] In some embodiments, the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0120] In some implementations, the RBD is derived from SARS-CoV-2 wild-type or a mutant thereof.

[0121] In some embodiments, the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0122] In some embodiments, the NTD includes mutations at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0123] In some embodiments, the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0124] In some implementations, the NTD contains L18F, D80A, D215G, L242-244del, and R246I mutations.

[0125] In some embodiments, the NTD contains amino acid mutations in L18F, T20N, P26S, D138Y, and R246I.

[0126] In some embodiments, the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0127] In some embodiments, the immunogenic composition is formulated in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0128] In some embodiments, the immunogenic composition comprises: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0129] In some embodiments, the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18-19, 76, 83, 97-108.

[0130] In some embodiments, the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37-38, 77, 84 and 109-111.

[0131] On the other hand, this application provides a pharmaceutical composition comprising the fusion protein or the immunogenic composition described above, and optionally a pharmaceutically acceptable excipient.

[0132] On the other hand, this application provides the use of the fusion protein or the immunogenic composition described herein in the preparation of a vaccine.

[0133] In some implementations, the vaccine is used to prevent and / or treat COVID-19.

[0134] On the other hand, this application provides the aforementioned fusion protein or the aforementioned immunogenic composition for the treatment and / or prevention of COVID-19.

[0135] On the other hand, this application provides a method for preparing a COVID-19 subunit vaccine, comprising:

[0136] 1) Provide the fusion protein or the immunogenic composition described above; and

[0137] 2) Mix the fusion protein or immunogenic composition described in 1) with a pharmaceutically acceptable adjuvant.

[0138] On the other hand, this application provides a method for detecting SARS-CoV-2 neutralizing antibodies, comprising:

[0139] 1) Administer the described COVID-19 subunit vaccine to the subjects; and

[0140] 2) Detect neutralizing antibodies produced in the subjects described in 1) after receiving the COVID-19 subunit vaccine.

[0141] On the other hand, this application provides a method for treating and / or preventing COVID-19, comprising administering the fusion protein, the immunogenic composition, or the COVID-19 subunit vaccine to a subject.

[0142] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0143] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0144] Figure 1 The image shows the results of SDS-PAGE electrophoresis and WB verification of the RBD-P2-6*HIS protein.

[0145] Figure 2 The image shows the results of SDS-PAGE electrophoresis and WB verification of the NTD-P2-6*HIS (GP101150-8) protein.

[0146] Figure 3 The image shows the results of SDS-PAGE electrophoresis and WB verification of the NTD-RBD-foldon-8*HIS protein.

[0147] Figure 4 The image shows the amino acid sequence alignment of the NTD-RBD-foldon-6×His (Gamma mutant) sequencing results.

[0148] Figure 5 The image shown is a size exclusion chromatography pattern of the NTD-RBD-foldon (Gamma mutant) protein.

[0149] Figure 6 The image shows the results of SDS-PAGE electrophoresis and Western blot verification of NTD-RBD-foldon (Gamma mutant) protein.

[0150] Figure 7 The image shows the amino acid sequence alignment results of NTD-RBD-foldon-6×His (Beta mutant strain).

[0151] Figure 8 The image shown is a size exclusion chromatography pattern of the NTD-RBD-foldon (Beta mutant) protein.

[0152] Figure 9 The image shows the results of SDS-PAGE electrophoresis and Western blot verification of NTD-RBD-foldon (Beta mutant) protein. Detailed Implementation

[0153] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0154] Terminology Definition

[0155] In this application, the term "fusion protein" generally refers to a biologically functional protein molecule obtained through genetic engineering technology. In this application, the fusion protein may be a fusion protein composed of RBD or its functionally active fragment and any of the following sequences: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, or hepatitis B surface antigen HBsAg or its functionally active fragment. In this application, the fusion protein may be a fusion protein composed of NTD or its functionally active fragment and any of the following sequences: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, or hepatitis B surface antigen HBsAg or its functionally active fragment. In this application, the fusion protein may be a fusion protein composed of NTD or its functionally active fragment and RBD or its functionally active fragment. In this application, the fusion protein may be a fusion protein composed of NTD or its functionally active fragment, RBD or its functionally active fragment, and any of the following fragments: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, or hepatitis B surface antigen HBsAg or its functionally active fragment.

[0156] GISAID uses the complete genome sequence (EPI_ISL_402124) of strain hCoV-19 / WIV04 / 2019 (WIV04) as the official reference sequence. The WIV04 strain is generally defined as a wild-type or original strain. In this application, the term "SARS-CoV-2 wild-type" usually refers to the WIV04 strain.

[0157] In this application, the term "P2" generally refers to an epitope peptide of tetanus toxin. For example, P2 can be a peptide of epitope peptides 830 to 844 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide of epitope peptides 830 to 845 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide of epitope peptides 829 to 844 of tetanus toxin or a mutant thereof. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids from the N-terminus or C-terminus of epitope peptides 829 to 844 of tetanus toxin. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids from the N-terminus or C-terminus of epitope peptides 830 to 845 of tetanus toxin. For example, P2 can be a peptide obtained by truncating or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 844. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids.

[0158] In this application, the term "foldon domain" generally refers to residues at the C-terminus of phage T4 fibrin. In this application, the foldon domain can be the 27 residues at the C-terminus of phage T4 fibrin or a mutant. In this application, the foldon domain can be a truncated or extended form obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of phage T4 fibrin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference can be a substitution, deletion, or insertion of one or more amino acids.

[0159] In this application, the terms "ferritin" generally refer to *Spodoptera litura* ferritin or *Helicobacter pylori* ferritin. In this application, *Spodoptera litura* ferritin has a heavy chain and a light chain (ferritin LC and ferritin HC). In this application, *Helicobacter pylori* ferritin has a single-chain structure. In this application, ferritin can be a mutant of *Spodoptera litura* or *Helicobacter pylori* ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In this application, "light chain of *Spodoptera litura* ferritin" and "LC" are used interchangeably with "ferritin LC". In this application, "heavy chain of *Spodoptera litura* ferritin" and "HC" are used interchangeably with "ferritin HC".

[0160] In this application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, or it can be suitably truncated or augmented with 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus, or it can have protein mutations, such as deletions, substitutions, or insertions of one or more amino acids.

[0161] In this application, the term "functionally active fragment" generally refers to a fragment that has similar biological activity to RBD, NTD, P2, foldon domain, ferritin, and hepatitis B surface antigen HBsAg.

[0162] In this application, the term "immunogenic composition" generally refers to a subunit composition. In this application, a subunit composition is a composition in which the components have been isolated and purified to a purity of at least 50%, 60%, 70%, 80%, or 90% before being mixed to form an antigenic composition. For example, the subunit composition may be an aqueous solution of a water-soluble protein. For example, the subunit composition may contain a detergent. For example, the subunit composition may contain a non-ionic, zwitterionic, or ionic detergent. For example, the subunit composition may contain lipids. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and a fusion protein containing an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein containing an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein containing an RBD or a functionally active fragment thereof and a fusion protein containing an NTD or a functionally active fragment thereof. In this application, the immunogenic composition may further include an adjuvant. For example, the adjuvant may include an aluminum salt (e.g., aluminum hydroxide gel or aluminum phosphate), but may also be a calcium salt, iron salt, or zinc salt, or may be an insoluble suspension of acylated tyrosine or acylated sugar, cationic or anionic derivatized polysaccharide, or polyphosphazene. For example, the immunogenic composition may also be selected as a Th1-type response preferential inducer. For example, a Th1-type response preferential inducer may include monophospholipid A or a derivative thereof. For example, the adjuvant may be a combination of monophospholipid A (e.g., 3-de-O-acylated monophospholipid A (3D-MPL)) and an aluminum salt. An adjuvant enhancement system may include a combination of monophospholipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL disclosed in WO94 / 00153, or a composition as disclosed in WO96 / 33739 that quenches QS21 with cholesterol, thereby reducing reactivity. For example, the adjuvant may also be the adjuvant described in WO95 / 17210, which contains QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion. For example, the adjuvant may be a homogeneous droplet emulsion formed by microfluidization of a mixture of Tween 80, sorbitol trioleate, and squalene under high pressure. For example, the adjuvant may contain unmethylated CpG of oligonucleotides (WO96 / 02555).

[0163] In this application, the term "parts by weight" generally refers to the weight ratio of the first component and the second component of the immunogenic composition. In this application, each dose of the immunogenic composition may contain 5-60 μg of the first component and 5-60 μg of the second component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the first component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the second component.

[0164] In this application, the term "RBD" refers to the receptor-binding domain of the SARS-CoV-2 spike protein (S protein). In this application, the RBD may be a peptide segment of the SARS-CoV-2 spike protein (S protein) between 310 and 560 amino acids, or a mutant thereof, or may be truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. In this application, the RBD may be a peptide segment of the SARS-CoV-2 spike protein (S protein) between 319 and 541 amino acids, or a mutant thereof, or may be suitably truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. In this application, the RBD may be a peptide segment of the SARS-CoV-2 spike protein (S protein) between amino acids 331 and 524, or a mutant thereof, or may suitably truncate 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. In this application, a "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences may be substitutions, deletions, or insertions of one or more amino acids.

[0165] In some implementations, the RBD may comprise an RBD of SARS-CoV-2 (Gamma mutant).

[0166] In some implementations, the RBD may comprise an RBD of SARS-CoV-2 (Beta mutant).

[0167] In some implementations, the RBD may comprise an RBD of SARS-CoV-2 (WIV04-1).

[0168] In this application, the term "NTD" refers to the N-terminal domain of the SARS-CoV-2 spike protein (S protein). In this application, the NTD can be a peptide of 13-353 amino acids from the SARS-CoV-2 spike protein (S protein) or a mutant thereof, or suitably truncated to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25, or 30 amino acids from the N-terminus or C-terminus. For example, the NTD can be a peptide of 13-303 amino acids from the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In this application, the NTD can be a peptide of 14-304 amino acids from the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In this application, the NTD can be a peptide of 18-353 amino acids from the SARS-CoV-2 spike protein (S protein) or a mutant thereof. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference can be a substitution, deletion, or insertion of one or more amino acids. In some embodiments, the NTD may comprise an NTD of SARS-CoV-2 (Gamma mutant). In some embodiments, the NTD may comprise an NTD of SARS-CoV-2 (Beta mutant). In some embodiments, the NTD may comprise an NTD of SARS-CoV-2 (WIV04-1).

[0169] In this application, the term "S protein," also known as "Spike protein" or "spike protein," generally refers to the capsid surface glycoprotein of coronaviruses. SARS-CoV-2 binds to the ACE2 receptor and invades cells via the S protein. The S protein consists of 1213 amino acids and contains a transmembrane region comprising a segment from the N-terminus or from amino acid 14 of a coronavirus capsid surface glycoprotein up to amino acid 1213, or a corresponding region from other SARS viruses. The S1 protein is subunit 1 of the S protein, primarily referring to the segment from the N-terminus or amino acid 14 up to amino acid 685.

[0170] In this application, the mutant may comprise any one or more currently known SAR-CoV-2 mutant strains (mutants). For example, the Gamma mutant strain may refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1.1.28 lineage with GenBank accession number QRN46961.1. For example, the Gamma mutant strain may refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1.1.28 lineage with GenBank accession number QLF80256.1. For example, the Gamma mutant strain may refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the P.1 lineage with GenBank accession number QVE55289.1. For example, the Gamma mutant strain may be Gamma mutant strain 501Y.V3, whose surface glycoprotein amino acid sequence may be as shown in SEQ ID NO: 88.

[0171] For example, the Beta mutant strain can refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1.351 lineage, GenBank accession number QUA12570.1. For example, the Beta mutant strain can refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1 lineage, GenBank accession number QIZ15537.1. For example, the Beta mutant strain can refer to the amino acid sequence of the SARS-CoV-2 surface glycoprotein of the B.1.351 lineage, GenBank accession number QVI03430.1. For example, the Beta mutant strain can be the 501Y.V2 Beta mutant strain, whose surface glycoprotein amino acid sequence is shown in SEQ ID NO:82.

[0172] As used in this article, the term "hemagglutinin / hemagglutinin protein / HA" generally refers to antibodies or other substances that can cause red blood cell agglutination. These substances can be found on the surfaces of influenza viruses, measles viruses (and many other bacteria and viruses), and can attach to the red blood cells of various animals, causing agglutination, which can be fatal in severe cases. In this article, it refers to the surface glycoprotein hemagglutinin (HA) of the influenza virus. Every year, the WHO website publishes predictions of the influenza sequence for that year. This article refers to the influenza sequence for the Northern Hemisphere in 2021-2022, specifically the influenza virus strains available on the GISAID website: influenza A (H1N1) EPI1661231|HA|A / Wisconsin / 588 / 2019|EPI_ISL_404460, influenza A (H3N2) EPI1843589|HA|A / Cambodia / e0826360 / 2020|EPI_ISL_944639, influenza B EPI1394970|HA|B / Washington / 02 / 2019|EPI_ISL_347829, and influenza B EPI529345|HA|B / PHUKET / 3073 / 2013|EPI_ISL_161843.

[0173] In this application, the term "comprising" generally means including, encompassing, containing, or including. In some cases, it also means "to be" or "composed of".

[0174] In this application, the term "around" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0175] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which inserts...

[0176] The inserted nucleic acid molecules are transferred to host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for DNA or RNA replication.

[0177] The vector is a vector for transcription and / or translation of expression. The vector also includes vectors having a variety of the functions described above. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.

[0178] In this application, the term "host cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector including the nucleic acid molecules described in this application, or that is capable of expressing the antibodies or antigen-binding fragments described in this application. The host cell may include progeny of a single host cell. Due to natural, accidental, or intentional mutations, progeny cells may not necessarily be morphologically or genomically identical to the original parent cell, but they need to be capable of expressing the fusion protein described in this application. The host cell can be obtained by in vitro transfection of cells using the vector described in this application. The host cell can be a prokaryotic cell (e.g., *Escherichia coli*) or a eukaryotic cell (e.g., yeast cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell may be a CHO cell. Invention Details

[0180] Fusion protein

[0181] On one hand, this application provides a fusion protein comprising: 1) a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0182] In this application, the RBD or its functionally active fragment may be derived from the RBD of the wild-type S protein of SARS-CoV-2 or from the RBD of the mutant S protein of SARS-CoV-2. In some embodiments, the RBD or its functionally active fragment may contain an amino acid mutation at one or more amino acid sites among K417, L452, T478, E484, and N501.

[0183] In this application, the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations selected from K417T, L452R, T478K, E484K, and N501Y. For example, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 83. In this application, the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations selected from K417N, E484K, and N501Y. For example, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 76.

[0184] In this application, the P2 or its functionally active fragment may contain an epitope peptide of tetanus toxin.

[0185] For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 844 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 845 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 829 to 844 or a mutant thereof. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 829 to 844. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 845. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 844. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids. For example, the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0186] In this application, the P2 or its functional active fragment may be directly or indirectly connected to the N-terminus or C-terminus of the RBD or its functional active fragment.

[0187] In this application, the P2 or its functional active fragment may be directly or indirectly connected to the N-terminus of the RBD or its functional active fragment.

[0188] In this application, the P2 or its functional active fragment may be directly or indirectly connected to the C-terminus of the RBD or its functional active fragment.

[0189] In this application, the RBD or its functionally active fragment may be fused with the P2 or its functionally active fragment within the frame.

[0190] In this application, the foldon domain or its functionally active fragment may contain amino acid residues at the C-terminus of phage T4 fibrin.

[0191] In this application, the foldon domain or its functionally active fragment may comprise the 27 amino acid residues at the C-terminus of phage T4 fibrin or a mutant. In this application, the foldon domain may be a truncated or extended form obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of the 27 residues at the C-terminus of phage T4 fibrin. In this application, a "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids.

[0192] For example, the foldon domain or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0193] In this application, the foldon domain or its functionally active segment can be directly or indirectly connected to the N-terminus or C-terminus of the RBD or its functionally active segment.

[0194] In this application, the foldon domain or its functionally active segment can be directly or indirectly connected to the N-terminus of the RBD or its functionally active segment.

[0195] In this application, the foldon domain or its functionally active fragment can be directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment.

[0196] In this application, the RBD or its functionally active fragment can be fused with the foldon domain or its functionally active fragment within the frame.

[0197] In this application, the ferritin or its functionally active fragment may include Spodoptera litura ferritin or its functionally active fragment.

[0198] In this application, the ferritin of *Spodoptera litura* or its functionally active fragment may include the heavy chain or light chain of *Spodoptera litura* ferritin.

[0199] In this application, ferritin can be a mutant of *Spodoptera litura* or *Helicobacter pylori* ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In this application, "light chain of *Spodoptera litura* ferritin" and "LC" can be used interchangeably with "ferritin LC". In this application, "heavy chain of *Spodoptera litura* ferritin" and "HC" can be used interchangeably with "ferritin HC". In this application, "Helicobacter pylori ferritin" can be used interchangeably with "HP ferritin".

[0200] In this application, the heavy chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 70.

[0201] In this application, the light chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 71.

[0202] In this application, the ferritin or its functionally active fragment may include Helicobacter pylori ferritin or its functionally active fragment.

[0203] In this application, the Helicobacter pylori ferritin or its functionally active fragment may contain the amino acid sequence shown in SEQ ID NO: 72.

[0204] In this application, the ferritin or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0205] In this application, the ferritin or its functionally active fragment can be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0206] For example, the ferritin or its functionally active fragment can be directly or indirectly linked to the N-terminus of the RBD or its functionally active fragment.

[0207] For example, the ferritin or its functionally active fragment can be directly or indirectly linked to the C-terminus of the RBD or its functionally active fragment.

[0208] In this application, the RBD or its functionally active fragment may be fused with the ferritin or its functionally active fragment within the frame.

[0209] In this application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, or it can be suitably truncated or augmented with 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus, or it can have protein mutations, such as deletions, substitutions, or insertions of one or more amino acids.

[0210] For example, the hepatitis B surface antigen (HBsAg) or its functionally active fragment may contain the amino acid sequence shown in SEQ ID NO: 73.

[0211] In this application, the hepatitis B surface antigen (HBsAg) or its functional active fragment can be directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functional active fragment.

[0212] In this application, the hepatitis B surface antigen (HBsAg) or its functionally active fragment can be directly or indirectly linked to the N-terminus of the RBD or its functionally active fragment.

[0213] In this application, the hepatitis B surface antigen (HBsAg) or its functional active fragment can be directly or indirectly linked to the C-terminus of the RBD or its functional active fragment.

[0214] In this application, the RBD or its functionally active fragment may be fused with the hepatitis B surface antigen (HBsAg) or its functionally active fragment within the frame.

[0215] In this application, "direct or indirect linking" generally refers to two different linking methods: direct linking and indirect linking of two sequences. Direct linking means that the connection between the two sequences does not involve any artificially added sequences, such as flexible linkers, rigid linkers, or cuttable linkers. Indirect linking refers to the connection of two sequences achieved by artificially adding a linker sequence, such as a flexible linker, rigid linker, or cuttable linker. In some embodiments, the flexible linker may contain the amino acid sequence GGSSG (SEQ ID NO: 89). In some embodiments, the linker sequence may contain the amino acid sequence SFTVEKGIYQTSNF (SEQ ID NO: 90).

[0216] In this application, the fusion protein may further comprise a signal peptide. In some embodiments, a signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) may be added to the N segment of the fusion protein.

[0217] In this application, the fusion protein may contain the amino acid sequence shown in any one of SEQ ID NO: 1-17, 80 and 86.

[0218] On the other hand, this application provides a fusion protein comprising: 1) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, the foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0219] In this application, the NTD or its functionally active fragment may be derived from the NTD of the wild-type S protein of SARS-CoV-2 or from the NTD of the mutant S protein of SARS-CoV-2. In some embodiments, the NTD or its functionally active fragment may contain an amino acid mutation at one or more amino acid sites selected from L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246. In some embodiments, the NTD or its functionally active fragment may contain one or more amino acid mutations selected from L18F, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0220] In this application, the NTD or its functionally active fragment may comprise an NTD of the Gamma mutant S protein. In some embodiments, the NTD may comprise amino acid mutations of L18F, T20N, P26S, D138Y, and R190S. For example, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 84.

[0221] In this application, the NTD or its functionally active fragment may comprise, for example, an NTD of a Beta mutant S protein. In some embodiments, the NTD or its functionally active fragment may comprise L18F, D80A, D215G, L242-244del, and R246I mutations. In some embodiments, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 77.

[0222] In this application, the P2 or its functionally active fragment may contain an epitope peptide of tetanus toxin.

[0223] For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 844 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 845 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 829 to 844 or a mutant thereof. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 829 to 844. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 845. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 844. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids. For example, the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0224] In this application, the P2 or its functional active fragment can be directly or indirectly connected to the N-terminus or C-terminus of the NTD or its functional active fragment.

[0225] In this application, the P2 or its functional active fragment can be directly or indirectly connected to the N-terminus of the NTD or its functional active fragment.

[0226] In this application, the P2 or its functional active fragment can be directly or indirectly connected to the C-terminus of the NTD or its functional active fragment.

[0227] In this application, the NTD or its functional active fragment may be fused with the P2 or its functional active fragment within the frame.

[0228] In this application, the foldon domain or its functionally active fragment may contain amino acid residues at the C-terminus of phage T4 fibrin. In some embodiments, the amino acid residues at the C-terminus of phage T4 fibrin may contain the amino acid sequence shown in SEQ ID NO: 78.

[0229] In this application, the foldon domain or its functionally active fragment may comprise the 27 amino acid residues at the C-terminus of phage T4 fibrin or a mutant. In this application, the foldon domain may be a truncated or extended form obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of the 27 residues at the C-terminus of phage T4 fibrin. In this application, a "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids.

[0230] For example, the foldon domain or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0231] In this application, the foldon domain or its functional active segment can be directly or indirectly connected to the N-terminus or C-terminus of the NTD or its functional active segment.

[0232] In this application, the foldon domain or its functional active segment can be directly or indirectly connected to the N-terminus of the NTD or its functional active segment.

[0233] In this application, the foldon domain or its functional active segment can be directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0234] In this application, the NTD or its functionally active fragment can be fused with the foldon domain or its functionally active fragment within the frame.

[0235] In this application, the ferritin or its functionally active fragment may include Spodoptera litura ferritin or its functionally active fragment.

[0236] In this application, the ferritin of *Spodoptera litura* or its functionally active fragment may include the heavy chain or light chain of *Spodoptera litura* ferritin.

[0237] In this application, ferritin can be a mutant of *Spodoptera litura* or *Helicobacter pylori* ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In this application, "light chain of *Spodoptera litura* ferritin" and "LC" can be used interchangeably with "ferritin LC". In this application, "heavy chain of *Spodoptera litura* ferritin" and "HC" can be used interchangeably with "ferritin HC". In this application, "Helicobacter pylori ferritin" can be used interchangeably with "HP ferritin".

[0238] In this application, the heavy chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 70.

[0239] In this application, the light chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 71.

[0240] In this application, the ferritin or its functionally active fragment may include Helicobacter pylori ferritin or its functionally active fragment.

[0241] In this application, the Helicobacter pylori ferritin or its functionally active fragment may contain the amino acid sequence shown in SEQ ID NO: 72.

[0242] In this application, the ferritin or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0243] In this application, the ferritin or its functionally active fragment can be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0244] For example, the ferritin or its functionally active fragment can be directly or indirectly linked to the N-terminus of the NTD or its functionally active fragment.

[0245] For example, the ferritin or its functionally active fragment can be directly or indirectly linked to the C-terminus of the NTD or its functionally active fragment.

[0246] In this application, the NTD or its functionally active fragment may be fused with the ferritin or its functionally active fragment within the frame.

[0247] In this application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, or it can be suitably truncated or augmented with 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus, or it can have protein mutations, such as deletions, substitutions, or insertions of one or more amino acids.

[0248] For example, the hepatitis B surface antigen (HBsAg) or its functionally active fragment may contain the amino acid sequence shown in SEQ ID NO: 73.

[0249] In this application, the hepatitis B surface antigen (HBsAg) or its functional active fragment can be directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functional active fragment.

[0250] In this application, the hepatitis B surface antigen (HBsAg) or its functional active fragment can be directly or indirectly linked to the N-terminus of the NTD or its functional active fragment.

[0251] In this application, the hepatitis B surface antigen (HBsAg) or its functional active fragment can be directly or indirectly linked to the C-terminus of the NTD or its functional active fragment.

[0252] In this application, the NTD or its functionally active fragment may be fused with the hepatitis B surface antigen (HBsAg) or its functionally active fragment within the frame.

[0253] In this application, "direct or indirect linking" generally refers to two different linking methods: direct linking and indirect linking of two sequences. Direct linking means that the connection between the two sequences does not involve any artificially added sequences, such as flexible linkers, rigid linkers, or cuttable linkers. Indirect linking refers to the connection of two sequences achieved by artificially adding a linker sequence, such as a flexible linker, rigid linker, or cuttable linker. In some embodiments, the flexible linker may contain the amino acid sequence GGSSG (SEQ ID NO: 89). In some embodiments, the linker sequence may contain the amino acid sequence SFTVEKGIYQTSNF (SEQ ID NO: 90).

[0254] In this application, the fusion protein may further comprise a signal peptide. In some embodiments, a signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) may be added to the N segment of the fusion protein.

[0255] In this application, the fusion protein may comprise the amino acid sequence shown in any one of SEQ ID NO: 20-36, 81 and 87.

[0256] On the other hand, this application provides a fusion protein comprising: 1) a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0257] In this application, the RBD or its functionally active fragment may be derived from the RBD of the wild-type S protein of SARS-CoV-2 or from the RBD of the mutant S protein of SARS-CoV-2. In some embodiments, the RBD or its functionally active fragment may contain amino acid mutations at one or more amino acid sites among K417, L452, T478, E484, and N501.

[0258] In this application, the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations among K417T, E484K, and N501Y. For example, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 83. In this application, the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations among K417N, E484K, and N501Y. For example, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 76.

[0259] In this application, the NTD or its functionally active fragment may be derived from the NTD of the wild-type S protein of SARS-CoV-2 or from the NTD of the mutant S protein of SARS-CoV-2. In some embodiments, the NTD or its functionally active fragment may contain an amino acid mutation at one or more amino acid sites selected from L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246. In some embodiments, the NTD or its functionally active fragment may contain one or more amino acid mutations selected from L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0260] In this application, the NTD or its functionally active fragment may comprise an NTD of the Gamma mutant S protein. In some embodiments, the NTD may comprise amino acid mutations of L18F, T20N, P26S, D138Y, and R190S. For example, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 84.

[0261] In this application, the NTD or its functionally active fragment may comprise the NTD of the Beta mutant S protein. In some embodiments, the NTD or its functionally active fragment may comprise the L18F, D80A, D215G, L242-244del, and R246I mutations. For example, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 77.

[0262] In this application, the RBD or its functional active fragment is directly or indirectly connected to the NTD or its functional active fragment.

[0263] In this application, the N-terminus of the RBD or its functional active segment is directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0264] In this application, the C-terminus of the RBD or its functional active segment is directly or indirectly connected to the N-terminus of the NTD or its functional active segment.

[0265] In this application, the RBD or its functionally active fragment is fused with the NTD or its functionally active fragment within the frame.

[0266] In this application, the fusion protein may further include one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0267] In this application, the P2 or its functionally active fragment may contain an epitope peptide of tetanus toxin.

[0268] For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 844 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 830 to 845 or a mutant thereof. For example, P2 can be a peptide of tetanus toxin epitope peptides 829 to 844 or a mutant thereof. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 829 to 844. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 845. For example, P2 can be a peptide obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of tetanus toxin epitope peptides 830 to 844. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences may be substitutions, deletions, or insertions of one or more amino acids.

[0269] For example, the epitope peptide of the tetanus toxin may contain the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0270] In this application, the P2 or its functional active fragment may be directly or indirectly connected to the RBD or its functional fragment and / or the NTD or its functional active fragment.

[0271] For example, the N-terminus of the RBD or its functional active segment can be directly or indirectly connected to the C-terminus of the NTD or its functional active segment, and the N-terminus of the P2 or its functional active segment can be directly or indirectly connected to the C-terminus of the RBD or its functional active segment.

[0272] In this application, the P2 or its functional active fragment can be fused within the frame with the RBD or its functional active fragment and the NTD or its functional active fragment.

[0273] In this application, the foldon domain or its functionally active fragment may contain amino acid residues at the C-terminus of phage T4 fibrin. In some embodiments, the amino acid residues at the C-terminus of the phage T4 fibrin contain the amino acid sequence shown in SEQ ID NO: 78.

[0274] In this application, the foldon domain or its functionally active fragment may comprise the 27 amino acid residues at the C-terminus of phage T4 fibrin or a mutant. In this application, the foldon domain may be a truncated or extended form obtained by shortening or adding 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus of the 27 residues at the C-terminus of phage T4 fibrin. In this application, a "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). This difference may be a substitution, deletion, or insertion of one or more amino acids.

[0275] For example, the foldon domain or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0276] In this application, the foldon domain or its functionally active fragment is directly or indirectly connected to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0277] For example, the N-terminus of the foldon domain or its functional active segment can be directly or indirectly connected to the C-terminus of the RBD or its functional active segment, and the N-terminus of the RBD or its functional active segment can be directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0278] In this application, the foldon domain or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and the NTD or its functionally active fragment.

[0279] In this application, the N-terminus of the foldon domain or its functional active segment is directly or indirectly connected to the C-terminus of the P2 or its functional active segment, the N-terminus of the P2 or its functional active segment is directly or indirectly connected to the C-terminus of the RBD or its functional active segment, and the N-terminus of the RBD or its functional active segment is directly or indirectly connected to the C-terminus of the NTD or its functional active segment.

[0280] In this application, the P2 or its functional active fragment, the foldon domain or its functional active fragment, the RBD or its functional active fragment, and the NTD or its functional active fragment are fused within the frame.

[0281] In this application, the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0282] In this application, the ferritin of *Spodoptera litura* or its functionally active fragments include the heavy chain or light chain of *Spodoptera litura* ferritin.

[0283] In this application, ferritin can be a mutant of *Spodoptera litura* or *Helicobacter pylori* ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence due to the presence of one or more differences (mutations). These differences can be substitutions, deletions, or insertions of one or more amino acids. In this application, "light chain of *Spodoptera litura* ferritin" and "LC" can be used interchangeably with "ferritin LC". In this application, "heavy chain of *Spodoptera litura* ferritin" and "HC" can be used interchangeably with "ferritin HC". In this application, "Helicobacter pylori ferritin" can be used interchangeably with "HP ferritin".

[0284] In this application, the heavy chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 70.

[0285] In this application, the light chain of the ferritin from *Spodoptera litura* may contain the amino acid sequence shown in SEQ ID NO: 71.

[0286] In this application, the ferritin or its functionally active fragment may include Helicobacter pylori ferritin or its functionally active fragment.

[0287] In this application, the ferritin or its functionally active fragment may contain the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0288] In this application, the ferritin or its functionally active fragment may be directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0289] For example, the N-terminus of the ferritin or its functional active fragment can be directly or indirectly connected to the C-terminus of the RBD or its functional active fragment, and the N-terminus of the RBD or its functional active fragment can be directly or indirectly connected to the C-terminus of the NTD or its functional active fragment.

[0290] For example, the N-terminus of the Helicobacter pylori ferritin or its functional active fragment can be directly or indirectly connected to the C-terminus of the RBD or its functional active fragment, and the N-terminus of the RBD or its functional active fragment can be directly or indirectly connected to the C-terminus of the NTD or its functional active fragment.

[0291] In this application, the Helicobacter pylori ferritin or its functionally active fragment can be fused within the frame with the RBD or its functionally active fragment and the NTD or its functionally active fragment.

[0292] In this application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of hepatitis B surface antigen can be the sequence corresponding to protein sequence accession numbers AAA45524, ANJ76941, CAA24234, or AAC34729 in NCBI, or it can be suitably truncated or augmented with 1, 2, 3, 4, 5, 6, or 10 amino acids from the N-terminus or C-terminus, or it can have protein mutations, such as deletions, substitutions, or insertions of one or more amino acids.

[0293] In this application, the hepatitis B surface antigen or its functionally active fragment may contain the amino acid sequence shown in SEQ ID NO: 73.

[0294] In this application, the hepatitis B surface antigen or its functionally active fragment may be directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0295] In this application, the N-terminus of the hepatitis B surface antigen or its functional active fragment can be directly or indirectly connected to the C-terminus of the RBD or its functional active fragment, and the N-terminus of the RBD or its functional active fragment can be directly or indirectly connected to the C-terminus of the NTD or its functional active fragment.

[0296] In this application, the hepatitis B surface antigen or its functionally active fragment is fused with the RBD or its functionally active fragment and the NTD or its functionally active fragment within the frame.

[0297] In this application, "direct or indirect linking" generally refers to two different linking methods: direct linking and indirect linking of two sequences. Direct linking means that the connection between the two sequences does not involve any artificially added sequences, such as flexible linkers, rigid linkers, or cuttable linkers. Indirect linking refers to the connection of two sequences achieved by artificially adding a linker sequence, such as a flexible linker, rigid linker, or cuttable linker. In some embodiments, the flexible linker may contain the amino acid sequence GGSSG (SEQ ID NO: 89). In some embodiments, the linker sequence may contain the amino acid sequence SFTVEKGIYQTSNF (SEQ ID NO: 90).

[0298] In this application, the fusion protein may further comprise a signal peptide. In some embodiments, a signal peptide sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 91) may be added to the N segment of the fusion protein.

[0299] In this application, the fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO: 39-44, 79, 85 and 96.

[0300] Immunogenic Compositions

[0301] On the other hand, this application provides an immunogenic composition comprising the fusion protein described above.

[0302] In this application, the term "immunogenic composition" generally refers to a subunit composition. In this application, a subunit composition is a composition in which the components have been isolated and purified to a purity of at least 50%, 60%, 70%, 80%, or 90% before being mixed to form an antigenic composition. For example, the subunit composition may be an aqueous solution of a water-soluble protein. For example, the subunit composition may contain a detergent. For example, the subunit composition may contain a non-ionic, zwitterionic, or ionic detergent. For example, the subunit composition may contain lipids. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include an RBD or a functionally active fragment thereof and a fusion protein containing an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein containing an RBD or a functionally active fragment thereof and an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may include a fusion protein containing an RBD or a functionally active fragment thereof and a fusion protein containing an NTD or a functionally active fragment thereof. In some cases, the immunogenic composition may also contain one or more human influenza virus hemagglutinin proteins (HA). In this application, the HA may contain the amino acid sequence of any one of SEQ ID NO: 92-95.

[0303] In some embodiments, the influenza hemagglutinin is selected from influenza strains that are prevalent strains published annually on the WHO website. For example, the prevalent strains published on the WHO website in 2021 are: type A (H1N1) EPI1661231|HA|A / Wisconsin / 588 / 2019|EPI_ISL_404460, type A (H3N2) EPI1843589|HA|A / Cambodia / e0826360 / 2020|EPI_ISL_944639, type B EPI1394970|HA|B / Washington / 02 / 2019|EPI_ISL_347829 and type B EPI529345|HA|B / PHUKET / 3073 / 2013|EPI_ISL_161843. In one embodiment, the preparation of a vaccine composition for preventing influenza and COVID-19 includes 50 μg of each of the above-mentioned HA protein types, 50 μg of the mutant NTD-RBD-foldon protein, and adjuvants commonly used in vaccine compositions, such as AS03 adjuvant.

[0304] For example, the immunogenic composition may comprise a first component and a second component. The first component may comprise a fusion protein of the RBD or its functionally active fragment and the NTD or its functionally active fragment as described in this application. The fusion protein may also comprise one or more polypeptides selected from the group consisting of: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment. The second component may comprise the RBD or its functionally active fragment, the NTD or its functionally active fragment, or optionally one or more human influenza virus hemagglutinin proteins (HA).

[0305] In this application, the RBD or its functionally active fragment may be derived from the RBD of the wild-type S protein of SARS-CoV-2 or from the RBD of the mutant S protein of SARS-CoV-2. In some embodiments, the RBD or its functionally active fragment may contain an amino acid mutation at one or more amino acid sites among K417, L452, T478, E484, and N501.

[0306] In this application, the RBD or its functionally active fragment may comprise the RBD of the Gamma mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations among K417T, E484K, and N501Y. For example, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 83. In this application, the RBD or its functionally active fragment may comprise the RBD of the Beta mutant S protein. In some embodiments, the RBD or its functionally active fragment may comprise one or more amino acid mutations among K417N, E484K, and N501Y. In some embodiments, the RBD may comprise the amino acid sequence shown in SEQ ID NO: 76.

[0307] In this application, the NTD or its functionally active fragment may be derived from the NTD of the wild-type S protein of SARS-CoV-2 or from the NTD of the mutant S protein of SARS-CoV-2. In some embodiments, the NTD or its functionally active fragment may contain an amino acid mutation at one or more amino acid sites selected from L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246. In some embodiments, the NTD or its functionally active fragment may contain one or more amino acid mutations selected from L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0308] In this application, the NTD or its functionally active fragment may comprise an NTD of the Gamma mutant S protein. In some embodiments, the NTD may comprise amino acid mutations of L18F, T20N, P26S, D138Y, and R190S. For example, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 84.

[0309] In this application, the NTD or its functionally active fragment may comprise the NTD of the Beta mutant S protein. In some embodiments, the NTD or its functionally active fragment may comprise the L18F, D80A, D215G, L242-244del, and R246I mutations. For example, the NTD may comprise the amino acid sequence shown in SEQ ID NO: 77.

[0310] In this application, the immunogenic composition may further include an adjuvant. For example, the adjuvant may include an aluminum salt (e.g., aluminum hydroxide gel or aluminum phosphate), but may also be a calcium salt, iron salt, or zinc salt, or may be an insoluble suspension of acylated tyrosine or acylated sugar, cationic or anionic derivatized polysaccharide, or polyphosphazene. For example, the immunogenic composition may also be selected as a Th1-type response preferential inducer. For example, a Th1-type response preferential inducer may include monophospholipid A or a derivative thereof. For example, the adjuvant may be a combination of monophospholipid A (e.g., 3-de-O-acylated monophospholipid A (3D-MPL)) and an aluminum salt. An adjuvant enhancement system may include a combination of monophospholipid A and a saponin derivative, particularly the combination of QS21 and 3D-MPL disclosed in WO94 / 00153, or a composition as disclosed in WO96 / 33739 that quenches QS21 with cholesterol, thereby reducing reactivity. For example, the adjuvant may also be the adjuvant described in WO95 / 17210, which contains QS21, 3D-MPL, and tocopherol in an oil-in-water emulsion. For example, the adjuvant may be a homogeneous droplet emulsion formed by microfluidization of a mixture of Tween 80, sorbitol trioleate, and squalene under high pressure. For example, the adjuvant may contain unmethylated CpG of oligonucleotides (WO96 / 02555).

[0311] In this application, the immunogenic composition comprises a first component comprising the fusion protein; and a second component comprising the fusion protein.

[0312] In this application, the immunogenic composition is prepared in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0313] In this application, the immunogenic composition may comprise: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0314] In this application, the term "parts by weight" generally refers to the weight ratio of the first component and the second component of the immunogenic composition. In this application, each dose of the immunogenic composition may contain 5-60 μg of the first component and 5-60 μg of the second component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the first component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the second component.

[0315] On the other hand, this application provides an immunogenic composition comprising a first component comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0316] In this application, the immunogenic composition is prepared in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0317] In this application, the immunogenic composition may comprise: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0318] In this application, the RBD may contain the amino acid sequence shown in any one of SEQ ID NO: 18-19.

[0319] In this application, the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37-38.

[0320] In this application, the term "parts by weight" generally refers to the weight ratio of the first component and the second component of the immunogenic composition. In this application, each dose of the immunogenic composition may contain 5-60 μg of the first component and 5-60 μg of the second component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the first component. For example, the immunogenic composition may contain 5 μg, 10 μg, 20 μg, 30 μg, or 40 μg of the second component.

[0321] In some cases, the immunogenic composition may comprise a first component, NTD-RBD-foldon protein, 20-100 μg; and a second component, NTD-foldon protein, 20-100 μg, or RBD-foldon protein, or influenza virus hemagglutinin protein, 15-80 μg.

[0322] In some embodiments, the immunogenic composition comprises 20-100 μg of NTD-RBD-foldon (Gamma mutant) protein, 20-100 μg of NTD-foldon (Gamma mutant) protein, and a lyophilization protectant formulated according to the following formula: 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5.

[0323] In some embodiments, the immunogenic composition comprises 20-100 μg of NTD-RBD-foldon (Gamma mutant) protein, 20-100 μg of RBD-foldon (Gamma mutant) protein, and a lyophilization protectant formulated according to the following formula: 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5.

[0324] In some embodiments, the immunogenic composition comprises 20-100 μg of NTD-RBD-foldon (Gamma mutant) protein, 15-180 μg of influenza virus hemagglutinin protein, and a lyophilization protectant formulated according to the following formula: 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5.

[0325] On the other hand, the present invention provides a vaccine comprising the fusion protein or the immunogenic composition described herein, and optionally a pharmaceutically acceptable adjuvant.

[0326] In some embodiments, the vaccine comprises 0.5 mL of a fusion protein or immunogenic composition and 0.5 mL of optionally pharmaceutically acceptable adjuvant.

[0327] The adjuvants described in this invention are selected from one or more small molecules chosen from squalene, MF59, ASO3, monophosphatidyllipid A, flagellin, CpG-ODN, muramyl dipeptide, and aluminum or calcium salts. These adjuvants are all well known in the art and are available through several commercial channels.

[0328] In some embodiments, the adjuvant is aluminum hydroxide adjuvant.

[0329] In some embodiments, the adjuvant is MF59 adjuvant.

[0330] In some embodiments, the adjuvant is preferably an AS03 adjuvant.

[0331] The components of the AS03 adjuvant include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, and 0.09 mg of potassium dihydrogen phosphate.

[0332] The MF59 adjuvant comprises 4.5% squalene, 0.5% Tween 80, and 0.5% Span 85.

[0333] In some embodiments, the vaccine comprises 20-100 μg of NTD-RBD-foldon protein, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, and AS03 adjuvant.

[0334] In some embodiments, the vaccine comprises 20-100 μg of NTD-RBD-foldon protein, 15-45 μg of HA from the H1N1 strain, 15-45 μg of HA from the H3N2 strain, and 15-45 μg of HA from the B / Washington / 02 / 2019 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, as well as AS03 adjuvant.

[0335] In some embodiments, the vaccine comprises 20-100 μg of NTD-RBD-foldon protein, 15-45 μg of HA from the H1N1 strain, 15-45 μg of HA from the H3N2 strain, 15-45 μg of HA from the B / Washington / 02 / 2019 strain, and 15-45 μg of HA from the B / PHUKET / 3073 / 2013 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, and AS03 adjuvant.

[0336] In some embodiments, the vaccine comprises 20-100 μg of NTD-RBD-foldon protein, 15-45 μg of HA from the H1N1 strain, 15-45 μg of HA from the H3N2 strain, and 15-45 μg of HA from the B / Washington / 02 / 2019 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, as well as MF59 adjuvant.

[0337] In some embodiments, the vaccine comprises 20-100 μg of NTD-RBD-foldon protein, 15-45 μg of HA from the H1N1 strain, 15-45 μg of HA from the H3N2 strain, 15-45 μg of HA from the B / Washington / 02 / 2019 strain, and 15-45 μg of HA from the B / PHUKET / 3073 / 2013 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, as well as MF59 adjuvant.

[0338] In some preferred embodiments, the vaccine comprises 50 μg of NTD-RBD-foldon protein, 45 μg of HA from the H1N1 strain, 45 μg of HA from the H3N2 strain, 45 μg of HA from the B / Washington / 02 / 2019 strain, and 45 μg of HA from the B / PHUKET / 3073 / 2013 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, and AS03 adjuvant.

[0339] In some preferred embodiments, the vaccine comprises 50 μg of NTD-RBD-foldon protein, 45 μg of HA from the H1N1 strain, 45 μg of HA from the H3N2 strain, 45 μg of HA from the B / Washington / 02 / 2019 strain, and 45 μg of HA from the B / PHUKET / 3073 / 2013 strain, 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80, and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5, and MF59 adjuvant.

[0340] In this application, the RBD-NTD-foldon (Beta mutant strain) may contain the amino acid sequence shown in SEQ ID NO: 79. In this application, the RBD-NTD-foldon (Gamma mutant strain) may contain the amino acid sequence shown in SEQ ID NO: 85. In this application, the RBD-NTD-foldon (WIV04-1) may contain the amino acid sequence shown in SEQ ID NO: 96.

[0341] In this application, the immunogenic composition may further comprise one or more human influenza virus hemagglutinin proteins (HA). In this application, the HA may comprise the amino acid sequence of any one of SEQ ID NO: 92-95.

[0342] In this application, the RBD may also contain the amino acid sequence shown in any one of SEQ ID NO: 97-108.

[0343] In this application, the NTD may also contain the amino acid sequence shown in any one of SEQ ID NO: 109-111.

[0344] Nucleic acid molecules, pharmaceutical compositions, applications

[0345] On the other hand, this application also provides one or more isolated nucleic acid molecules that can encode the fusion protein described in this application. In some embodiments, the nucleic acid molecule may comprise mRNA.

[0346] On the other hand, this application provides a pharmaceutical composition comprising the fusion protein or the immunogenic composition described above, and optionally a pharmaceutically acceptable excipient.

[0347] The pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or nonionic surfactants, etc.

[0348] In this application, the pharmaceutical composition may be formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir.

[0349] The pharmaceutical composition may be used to inhibit or delay the development or progression of a disease or condition, and / or to alleviate and / or stabilize the state of a disease or condition.

[0350] The pharmaceutical composition described in this application may contain a preventive and / or therapeutically effective amount of the fusion protein, or the immunogenic composition described herein.

[0351] The effective dose for prevention and / or treatment is the dose required to prevent and / or treat (at least partially treat) a disease or condition and / or any of its complications in a subject who has or is at risk of developing it.

[0352] In this application, the term "effective amount" generally refers to the amount of medicine that can alleviate or eliminate a subject's disease or symptoms, or that can preventively inhibit or prevent the occurrence of a disease or symptoms. Typically, the specific effective amount can be determined based on the subject's weight, age, sex, diet, excretion rate, medical history, current treatment, time of administration, dosage form, method of administration, route of administration, combination of drugs, the subject's health status and potential for cross-infection, allergies, hypersensitivity and side effects, and / or the degree of epithelial (or endothelial) tissue disease development, etc. Those skilled in the art (e.g., physicians or veterinarians) can proportionally reduce or increase the dosage based on these or other conditions or requirements.

[0353] In this application, the subject may include humans or non-human animals. For example, the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice, and rats. Furthermore, non-human animals may also include any animal species other than humans, such as livestock, rodents, primates, domesticated animals, or poultry. The human may be Caucasian, African, Asian, Semitic, or of other races, or a hybrid of various races. For example, the human may be elderly, adult, adolescent, child, or infant.

[0354] The effective dose in humans can be inferred from the effective dose in laboratory animals. For example, Freireich et al. described the dose-response relationship between animals and humans (based on milligrams per square meter of body surface area) (Freireich et al., Cancer Chemother. Rep. 50, 219 (1966)). Body surface area can be approximated from the patient's height and weight. See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, NY, 537 (1970).

[0355] On the other hand, this application provides the use of the fusion protein or the immunogenic composition described herein in the preparation of a vaccine.

[0356] In this application, the vaccine may be used to prevent and / or treat COVID-19.

[0357] On the other hand, this application provides the aforementioned fusion protein or the aforementioned immunogenic composition for the treatment and / or prevention of COVID-19.

[0358] On the other hand, this application provides a method for preparing a COVID-19 subunit vaccine, comprising:

[0359] 1) Provide the fusion protein or the immunogenic composition described above; and

[0360] 2) Mix the fusion protein or immunogenic composition described in 1) with a pharmaceutically acceptable adjuvant.

[0361] On the other hand, this application provides a method for detecting SARS-CoV-2 neutralizing antibodies, comprising:

[0362] 1) Administer the described COVID-19 subunit vaccine to the subjects; and

[0363] 2) Detect neutralizing antibodies produced in the subjects described in 1) after receiving the COVID-19 subunit vaccine.

[0364] On the other hand, this application provides a method for treating and / or preventing COVID-19, comprising administering the fusion protein, the immunogenic composition, or the COVID-19 subunit vaccine to a subject.

[0365] This application also relates to the following specific implementation methods:

[0366] 1. A fusion protein comprising: 1) a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0367] 2. The fusion protein according to Embodiment 1, wherein the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0368] 3. The fusion protein according to Embodiment 2, wherein the SAR-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant and Alpha mutant.

[0369] 4. The fusion protein according to any one of embodiments 1-3, wherein the RBD has a mutation at one or more amino acid sites selected from the group consisting of wild-type SARS-CoV-2 compared with the RBD of wild-type SARS-CoV-2: K417, L452, T478, E484 and N501.

[0370] 5. The fusion protein according to any one of embodiments 1-4, wherein the RBD comprises one or more amino acid mutations K417T / N, L452R, T478K, E484K and N501Y.

[0371] 6. The fusion protein according to any one of embodiments 1-5, wherein the RBD comprises amino acid mutations of K417T, E484K, and N501Y.

[0372] 7. The fusion protein according to any one of embodiments 1-6, wherein the RBD comprises amino acid mutations of K417N, E484K, and N501Y.

[0373] 8. The fusion protein according to any one of embodiments 1-7, wherein the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0374] 9. The fusion protein according to any one of embodiments 1-8, wherein the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0375] 10. The fusion protein according to embodiment 9, wherein the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0376] 11. The fusion protein according to any one of embodiments 1-10, wherein the P2 or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0377] 12. The fusion protein according to any one of embodiments 1-11, wherein the RBD or its functionally active fragment is fused with the P2 or its functionally active fragment within the frame.

[0378] 13. The fusion protein according to any one of embodiments 1-12, wherein the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibrin.

[0379] 14. The fusion protein according to any one of embodiments 1-13, wherein the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0380] 15. The fusion protein according to any one of embodiments 1-14, wherein the foldon domain or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0381] 16. The fusion protein according to any one of embodiments 1-15, wherein the RBD or its functionally active fragment is fused with the foldon domain or its functionally active fragment within the frame.

[0382] 17. The fusion protein according to any one of embodiments 1-16, wherein the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0383] 18. The fusion protein according to embodiment 17, wherein the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin of *Spodoptera litura*.

[0384] 19. The fusion protein according to embodiment 18, wherein the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0385] 20. The fusion protein according to any one of embodiments 18-19, wherein the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0386] 21. The fusion protein according to any one of embodiments 1-20, wherein the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0387] 22. The fusion protein according to any one of embodiments 1-21, wherein the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0388] 23. The fusion protein according to any one of embodiments 1-22, wherein the ferritin or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0389] 24. The fusion protein according to any one of embodiments 1-23, wherein the RBD or its functionally active fragment is fused with the ferritin or its functionally active fragment within the frame.

[0390] 25. The fusion protein according to any one of embodiments 1-24, wherein the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0391] 26. The fusion protein according to any one of embodiments 1-25, wherein the hepatitis B surface antigen or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the RBD or its functionally active fragment.

[0392] 27. The fusion protein according to any one of embodiments 1-26, wherein the RBD or its functionally active fragment is fused with the hepatitis B surface antigen or its functionally active fragment within the frame.

[0393] 28. The fusion protein according to any one of embodiments 11-27, wherein the direct or indirect linkage may include linkage via a linker.

[0394] 29. The fusion protein according to embodiment 28, wherein the linker may comprise a rigid linker, a flexible linker, or other sequences.

[0395] 30. The fusion protein according to any one of embodiments 28-29, wherein the linker may comprise the amino acid sequence shown in any one of SEQ ID NO: 89-90.

[0396] 31. The fusion protein according to any one of embodiments 1-30, comprising the amino acid sequence shown in any one of SEQ ID NO: 1-17, 80 and 86.

[0397] 32. A fusion protein comprising: 1) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, the foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0398] 33. The fusion protein according to embodiment 32, wherein the NTD is derived from the NTD of the wild-type or mutant S protein of SARS-CoV-2.

[0399] 34. The fusion protein according to embodiment 33, wherein the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant and Alpha mutant.

[0400] 35. The fusion protein according to any one of embodiments 32-34, wherein the NTD comprises a mutation at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0401] 36. The fusion protein according to any one of embodiments 32-35, wherein the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0402] 37. The fusion protein according to any one of embodiments 32-36, wherein the NTD comprises L18F, D80A, D215G, L242-244del and R246I mutations.

[0403] 38. The fusion protein according to any one of embodiments 32-37, wherein the NTD comprises amino acid mutations of L18F, D80A, D215G, L242-244del and R190S.

[0404] 39. The fusion protein according to any one of embodiments 32-38, wherein the NTD comprises amino acid mutations of L18F, T20N, P26S, D138Y, and R246I.

[0405] 40. The fusion protein according to any one of embodiments 32-39, wherein the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0406] 41. The fusion protein according to any one of embodiments 32-40, wherein the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0407] 42. The fusion protein according to embodiment 41, wherein the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0408] 43. The fusion protein according to any one of embodiments 32-42, wherein the P2 or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0409] 44. The fusion protein according to any one of embodiments 32-43, wherein the NTD or its functionally active fragment is fused with the P2 or its functionally active fragment within the frame.

[0410] 45. The fusion protein according to any one of embodiments 32-44, wherein the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibroin.

[0411] 46. ​​The fusion protein according to any one of embodiments 32-45, wherein the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0412] 47. The fusion protein according to any one of embodiments 32-46, wherein the foldon domain or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0413] 48. The fusion protein according to any one of embodiments 32-47, wherein the NTD or its functionally active fragment is fused with the foldon domain or its functionally active fragment within the frame.

[0414] 49. The fusion protein according to any one of embodiments 32-48, wherein the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0415] 50. The fusion protein according to embodiment 49, wherein the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin of *Spodoptera litura*.

[0416] 51. The fusion protein according to embodiment 50, wherein the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0417] 52. The fusion protein according to any one of embodiments 50-51, wherein the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0418] 53. The fusion protein according to any one of embodiments 32-52, wherein the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0419] 54. The fusion protein according to any one of embodiments 32-53, wherein the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0420] 55. The fusion protein according to any one of embodiments 32-54, wherein the ferritin or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0421] 56. The fusion protein according to any one of embodiments 32-55, wherein the NTD or its functionally active fragment is fused with the ferritin or its functionally active fragment within a frame.

[0422] 57. The fusion protein according to any one of embodiments 32-56, wherein the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0423] 58. The fusion protein according to any one of embodiments 32-57, wherein the hepatitis B surface antigen or its functionally active fragment is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0424] 59. The fusion protein according to any one of embodiments 32-58, wherein the NTD or its functionally active fragment is fused with the hepatitis B surface antigen or its functionally active fragment within the frame.

[0425] 60. The fusion protein according to any one of embodiments 43-59, wherein the direct or indirect linkage may include linkage via a linker.

[0426] 61. The fusion protein according to embodiment 60, wherein the linker may comprise a rigid linker, a flexible linker, or other sequences.

[0427] 62. The fusion protein according to any one of embodiments 60-61, wherein the linker may comprise the amino acid sequence shown in any one of SEQ ID NO: 89-90.

[0428] 63. The fusion protein according to any one of embodiments 32-62, comprising the amino acid sequence shown in any one of SEQ ID NO: 20-36, 81 and 87.

[0429] 64. A fusion protein comprising: 1) the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0430] 65. The fusion protein according to embodiment 64, wherein the RBD is derived from wild-type SARS-CoV-2 or a mutant thereof.

[0431] 66. The fusion protein according to embodiment 65, wherein the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0432] 67. The fusion protein according to any one of embodiments 64-66, wherein the RBD has a mutation at one or more amino acid sites selected from the group consisting of K417, L452, T478, E484 and N501.

[0433] 68. The fusion protein according to any one of embodiments 64-67, wherein the RBD comprises one or more amino acid mutations K417T / N, L452R, T478K, E484K and N501Y.

[0434] 69. The fusion protein according to any one of embodiments 64-68, wherein the RBD comprises amino acid mutations of K417T, E484K, and N501Y.

[0435] 70. The fusion protein according to any one of embodiments 64-69, wherein the RBD comprises amino acid mutations of K417N, E484K, and N501Y.

[0436] 71. The fusion protein according to any one of embodiments 64-70, wherein the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0437] 72. The fusion protein according to any one of embodiments 64-71, wherein the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0438] 73. The fusion protein according to embodiment 72, wherein the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant and Alpha mutant.

[0439] 74. The fusion protein according to any one of embodiments 64-73, wherein the NTD comprises a mutation at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0440] 75. The fusion protein according to any one of embodiments 64-74, wherein the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0441] 76. The fusion protein according to any one of embodiments 64-75, wherein the NTD comprises L18F, D80A, D215G, L242-244del and R246I mutations.

[0442] 77. The fusion protein according to any one of embodiments 64-76, wherein the NTD comprises amino acid mutations of L18F, D80A, D215G, L242-244del, and R190S.

[0443] 78. The fusion protein according to any one of embodiments 64-77, wherein the NTD comprises amino acid mutations of L18F, T20N, P26S, D138Y, and R246I.

[0444] 79. The fusion protein according to any one of embodiments 64-78, wherein the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0445] 80. The fusion protein according to any one of embodiments 64-79 further comprises one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0446] 81. The fusion protein according to any one of embodiments 64-80, wherein the RBD or its functionally active fragment is directly or indirectly linked to the NTD or its functionally active fragment.

[0447] 82. The fusion protein according to any one of embodiments 64-81, wherein the RBD or its functionally active fragment is fused with the NTD or its functionally active fragment within the frame.

[0448] 83. The fusion protein according to any one of embodiments 64-82, wherein the P2 or its functionally active fragment comprises an epitope peptide of tetanus toxin.

[0449] 84. The fusion protein according to embodiment 83, wherein the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0450] 85. The fusion protein according to any one of embodiments 64-84, wherein the P2 or its functionally active fragment is directly or indirectly linked to the RBD or its functional fragment and / or the NTD or its functionally active fragment.

[0451] 86. The fusion protein according to any one of embodiments 64-85, wherein the P2 or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0452] 87. The fusion protein according to any one of embodiments 64-86, wherein the N-terminus of P2 or its functional active fragment is directly or indirectly connected to the C-terminus of RBD or its functional active fragment, and the N-terminus of RBD or its functional active fragment is directly or indirectly connected to the C-terminus of NTD or its functional active fragment.

[0453] 88. The fusion protein according to any one of embodiments 64-87, wherein the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibroin.

[0454] 89. The fusion protein according to any one of embodiments 64-88, wherein the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0455] 90. The fusion protein according to any one of embodiments 64-89, wherein the foldon domain or its functionally active fragment is directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0456] 91. The fusion protein according to any one of embodiments 64-90, wherein the foldon domain or its functionally active fragment is fused within the frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0457] 92. The fusion protein according to any one of embodiments 64-91, wherein the N-terminus of the foldon domain or its functionally active fragment is directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the N-terminus of the RBD or its functionally active fragment is directly or indirectly connected to the C-terminus of the NTD or its functionally active fragment.

[0458] 93. The fusion protein according to any one of embodiments 64-92, wherein the N-terminus of the foldon domain or its functional active fragment is directly or indirectly connected to the C-terminus of the P2 or its functional active fragment, the N-terminus of the P2 or its functional active fragment is directly or indirectly connected to the C-terminus of the RBD or its functional active fragment, and the N-terminus of the RBD or its functional active fragment is directly or indirectly connected to the C-terminus of the NTD or its functional active fragment.

[0459] 94. The fusion protein according to any one of embodiments 64-93, wherein the ferritin or its functionally active fragment comprises Spodoptera litura ferritin or its functionally active fragment.

[0460] 95. The fusion protein according to embodiment 94, wherein the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin of *Spodoptera litura*.

[0461] 96. The fusion protein according to embodiment 95, wherein the heavy chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 70.

[0462] 97. The fusion protein according to any one of embodiments 95-96, wherein the light chain of the ferritin from *Spodoptera litura* comprises the amino acid sequence shown in SEQ ID NO: 71.

[0463] 98. The fusion protein according to any one of embodiments 64-97, wherein the ferritin or its functionally active fragment comprises Helicobacter pylori ferritin or its functionally active fragment.

[0464] 99. The fusion protein according to any one of embodiments 64-98, wherein the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0465] 100. The fusion protein according to any one of embodiments 64-99, wherein the ferritin or its functionally active fragment is directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0466] 101. The fusion protein according to any one of embodiments 64-100, wherein the ferritin or its functionally active fragment is fused within a frame with the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0467] 102. The fusion protein according to any one of embodiments 64-101, wherein the N-terminus of the ferritin or its functionally active fragment is directly or indirectly connected to the C-terminus of the RBD or its functionally active fragment, and the N-terminus of the RBD or its functionally active fragment is directly or indirectly connected to the C-terminus of the NTD or its functionally active fragment.

[0468] 103. The fusion protein according to any one of embodiments 64-102, wherein the hepatitis B surface antigen or its functionally active fragment comprises the amino acid sequence shown in SEQ ID NO: 73.

[0469] 104. The fusion protein according to any one of embodiments 64-103, wherein the hepatitis B surface antigen or its functionally active fragment is directly or indirectly linked to the RBD or its functionally active fragment and / or the NTD or its functionally active fragment.

[0470] 105. The fusion protein according to any one of embodiments 64-104, wherein the hepatitis B surface antigen or a functionally active fragment thereof is fused within a frame with the RBD or a functionally active fragment thereof and / or the NTD or a functionally active fragment thereof.

[0471] 106. The fusion protein according to any one of embodiments 81-105, wherein the direct or indirect linkage may include linkage via a linker.

[0472] 107. The fusion protein according to embodiment 106, wherein the linker may comprise a rigid linker, a flexible linker, or other sequences.

[0473] 108. The fusion protein according to embodiments 81-107, wherein the linker may comprise the amino acid sequence shown in any one of SEQ ID NO: 89-90.

[0474] 109. The fusion protein according to any one of embodiments 64-108, comprising the amino acid sequence shown in any one of SEQ ID NO: 39-44, 79, 85 and 96.

[0475] 110. An immunogenic composition comprising the fusion protein described in any one of embodiments 1-109.

[0476] 111. The immunogenic composition according to embodiment 110, comprising a first component comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising the fusion protein of any one of embodiments 1-109.

[0477] 112. The immunogenic composition according to embodiment 110, comprising a first component comprising the fusion protein of any one of embodiments 1-109; and a second component comprising the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0478] 113. The immunogenic composition according to embodiment 110, comprising a first component comprising the fusion protein of any one of embodiments 1-109; and a second component comprising the fusion protein of any one of embodiments 1-109.

[0479] 114. An immunogenic composition according to any one of embodiments 110-113, which is formulated in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0480] 115. An immunogenic composition according to any one of embodiments 110-114, comprising: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0481] 116. An immunogenic composition comprising a first component comprising a receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and a second component comprising an N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0482] 117. The immunogenic composition according to embodiment 116, wherein the RBD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0483] 118. The immunogenic composition according to embodiment 117, wherein the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0484] 119. The immunogenic composition according to any one of embodiments 116-118, wherein the RBD has a mutation at one or more amino acid sites selected from the group consisting of K417, L452, T478, E484 and N501.

[0485] 120. The immunogenic composition according to any one of embodiments 116-119, wherein the RBD comprises one or more of the amino acid mutations K417T / N, L452R, T478K, E484K and N501Y.

[0486] 121. The immunogenic composition according to any one of embodiments 116-120, wherein the RBD comprises amino acid mutations of K417T, E484K, and N501Y.

[0487] 122. The immunogenic composition according to any one of embodiments 116-121, wherein the RBD comprises amino acid mutations of K417N, E484K, and N501Y.

[0488] 123. The immunogenic composition according to any one of embodiments 116-122, wherein the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18, 19, 76, 83, 97-108.

[0489] 124. The immunogenic composition according to any one of embodiments 116-123, wherein the NTD is derived from SARS-CoV-2 wild type or a mutant thereof.

[0490] 125. The immunogenic composition according to embodiment 124, wherein the SARS-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant, and Alpha mutant.

[0491] 126. The immunogenic composition according to any one of embodiments 116-125, wherein the NTD comprises a mutation at one or more amino acid sites selected from the group consisting of: L18, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0492] 127. The immunogenic composition according to any one of embodiments 116-126, wherein the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0493] 128. The immunogenic composition according to any one of embodiments 116-127, wherein the NTD comprises L18F, D80A, D215G, L242-244del and R246I mutations.

[0494] 129. The immunogenic composition according to any one of embodiments 116-128, wherein the NTD comprises amino acid mutations of L18F, D80A, D215G, L242-244del, and R190S.

[0495] 130. The immunogenic composition according to any one of embodiments 116-129, wherein the NTD comprises amino acid mutations of L18F, T20N, P26S, D138Y, and R246I.

[0496] 131. The immunogenic composition according to any one of embodiments 116-130, wherein the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0497] 132. An immunogenic composition according to any one of embodiments 116-131, which is formulated in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0498] 133. An immunogenic composition according to any one of embodiments 111-132, comprising: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0499] 134. The immunogenic composition according to any one of embodiments 116-133, wherein the RBD comprises the amino acid sequence shown in any one of SEQ ID NO: 18-19.

[0500] 135. The immunogenic composition according to any one of embodiments 116-134, wherein the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37-38.

[0501] 136. One or more isolated nucleic acid molecules encoding the fusion protein of any one of claims 1-109.

[0502] 137. The nucleic acid molecule of claim 136, comprising mRNA.

[0503] 138. A pharmaceutical composition comprising the fusion protein of any one of embodiments 1-109, or the immunogenic composition of any one of embodiments 110-135, or the nucleic acid molecule of any one of embodiments 136-137, and optionally a pharmaceutically acceptable excipient.

[0504] 139. Use of the fusion protein of any one of embodiments 1-109 or the immunogenic composition of any one of embodiments 110-135 in the preparation of a vaccine.

[0505] 140. The use according to embodiment 139, wherein the vaccine is used for the prevention and / or treatment of COVID-19.

[0506] 141. The fusion protein of any one of embodiments 1-109, or the immunogenic composition of any one of embodiments 110-135, or the nucleic acid molecule of any one of embodiments 136-137, for the treatment and / or prevention of COVID-19.

[0507] 142. A method for preparing a COVID-19 subunit vaccine, comprising:

[0508] 1) Provide the fusion protein according to any one of embodiments 1-109 or the immunogenic composition according to any one of embodiments 110-135; and

[0509] 2) Mix the fusion protein or immunogenic composition described in 1) with a pharmaceutically acceptable adjuvant.

[0510] 143. A method for detecting SARS-CoV-2 neutralizing antibodies, comprising:

[0511] 1) Administering the COVID-19 subunit vaccine as described in Implementation Method 142 to the subjects; and

[0512] 2) Detect neutralizing antibodies produced in the subjects described in 1) after receiving the COVID-19 subunit vaccine.

[0513] 144. A method of treating and / or preventing COVID-19, comprising administering to a subject the fusion protein of any one of embodiments 1-109, or the immunogenic composition of any one of embodiments 110-135, or the nucleic acid molecule of any one of embodiments 136-137, or the COVID-19 subunit vaccine of embodiment 142.

[0514] This application also includes the following implementation schemes:

[0515] 1. A fusion protein comprising: 1) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof; and 2) one or more polypeptides selected from the group consisting of: P2 or a functionally active fragment thereof, the foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0516] 2. The fusion protein according to embodiment 1, wherein the NTD is derived from the NTD of the wild-type SARS-CoV-2 or its mutant S protein.

[0517] 3. The fusion protein according to embodiment 2, wherein the SAR-CoV-2 mutant is selected from any one of the following groups: Gamma mutant, Beta mutant, Delta mutant and Alpha mutant.

[0518] 4. The fusion protein according to any one of embodiments 1-3, wherein the NTD comprises a mutation at one or more amino acid sites selected from the group consisting of: L18, T19, T20, P26, D80, D138, R190, D215, L242-244, and R246.

[0519] 5. The fusion protein according to any one of embodiments 1-4, wherein the NTD comprises one or more amino acid mutations selected from the group consisting of: L18F, T19R, T20N, P26S, D80A, D138Y, R190S, D215G, L242-244del, and R246I.

[0520] 6. The fusion protein according to any one of embodiments 1-5, wherein the NTD comprises L18F, D80A, D215G, L242-244del and R246I mutations.

[0521] 7. The fusion protein according to any one of embodiments 1-6, wherein the NTD comprises amino acid mutations of L18F, T20N, P26S, D138Y, and R190S.

[0522] 8. The fusion protein according to any one of embodiments 1-7, wherein the NTD comprises the amino acid sequence shown in any one of SEQ ID NO: 37, 38, 77, 84 and 109-111.

[0523] 9. The fusion protein according to any one of embodiments 1-8, wherein the polypeptide is directly or indirectly linked to the N-terminus or C-terminus of the NTD or its functionally active fragment.

[0524] 10. The fusion protein according to any one of embodiments 1-9, wherein the NTD or its functionally active fragment is fused to the polypeptide within the frame.

[0525] 11. The fusion protein according to any one of embodiments 1-0, wherein the P2 or its functionally active fragment comprises a tetanus toxin epitope peptide.

[0526] 12. The fusion protein according to embodiment 11, wherein the epitope peptide of the tetanus toxin comprises the amino acid sequence shown in any one of SEQ ID NO: 64-66.

[0527] 13. The fusion protein according to any one of embodiments 1-12, wherein the foldon domain or its functionally active fragment comprises amino acid residues at the C-terminus of phage T4 fibroin.

[0528] 14. The fusion protein according to any one of embodiments 1-13, wherein the foldon domain or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 67-69 and 78.

[0529] 15. The fusion protein according to any one of embodiments 1-14, wherein the ferritin or its functionally active fragment comprises *Spodoptera litura* ferritin, *Helicobacter pylori* ferritin, or its functionally active fragment.

[0530] 16. The fusion protein according to embodiment 15, wherein the ferritin of *Spodoptera litura* or its functionally active fragment comprises the heavy or light chain of ferritin of *Spodoptera litura*.

[0531] 17. The fusion protein according to embodiment 16, wherein the heavy chain of the *Spodoptera litura* ferritin comprises the amino acid sequence shown in SEQ ID NO: 70, and the light chain of the *Spodoptera litura* ferritin comprises the amino acid sequence shown in SEQ ID NO: 71.

[0532] 18. The fusion protein according to any one of embodiments 1-17, wherein the ferritin or its functionally active fragment comprises the amino acid sequence shown in any one of SEQ ID NO: 70-72.

[0533] 19. The fusion protein according to any one of embodiments 1-18, wherein the hepatitis B surface antigen or a functionally active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 73.

[0534] 20. The fusion protein according to any one of embodiments 9-19, wherein the direct or indirect linkage comprises linkage via a linker.

[0535] 21. The fusion protein according to embodiment 20, wherein the linker comprises a rigid linker, a flexible linker, or other sequences.

[0536] 22. The fusion protein according to any one of embodiments 20-21, wherein the linker comprises the amino acid sequence shown in any one of SEQ ID NO: 89-90.

[0537] 23. The fusion protein according to any one of embodiments 1-22, comprising the amino acid sequence shown in any one of SEQ ID NO: 20-36, 81 and 87.

[0538] 24. An immunogenic composition comprising the fusion protein described in any one of embodiments 1-23.

[0539] 25. The immunogenic composition according to embodiment 24, comprising a first component comprising the fusion protein of any one of claims 1-23, and a second component comprising the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S protein) or a functionally active fragment thereof.

[0540] 26. The immunogenic composition according to embodiment 25 is formulated in the following weight ratios: 1) a first component (1-15 parts by weight); and / or 2) a second component (1-15 parts by weight).

[0541] 27. The immunogenic composition according to any one of embodiments 25-26, comprising: 1) 5-60 μg of a first component; and / or 2) 5-60 μg of a second component.

[0542] 28. The immunogenic composition according to any one of embodiments 24-27, further comprising at least one human influenza virus hemagglutinin protein HA.

[0543] 29. The immunogenic composition according to embodiment 28, wherein the HA comprises the amino acid sequence of any one of SEQ ID NO: 92-95.

[0544] 30. One or more isolated nucleic acid molecules encoding a fusion protein according to any one of embodiments 1-23.

[0545] 31. The nucleic acid molecule according to embodiment 30, comprising mRNA.

[0546] 32. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 1-23, an immunogenic composition according to any one of embodiments 24-29, or a nucleic acid molecule according to any one of embodiments 30-31, and optionally a pharmaceutically acceptable excipient.

[0547] 33. Use of the fusion protein of any one of embodiments 1-23, the immunogenic composition of any one of embodiments 24-29, the nucleic acid molecule of any one of embodiments 30-31, or the pharmaceutical composition of embodiment 32 in the preparation of a vaccine.

[0548] 34. The use as described in embodiment 33, wherein the vaccine is used for the prevention and / or treatment of COVID-19.

[0549] 35. A method for preparing a COVID-19 subunit vaccine, comprising:

[0550] 36. Provides the fusion protein of any one of embodiments 1-23, the immunogenic composition of any one of embodiments 24-29, or the nucleic acid molecule of any one of embodiments 30-31; and

[0551] 37. To mix the immunogenic composition described in 1) with a pharmaceutically acceptable adjuvant.

[0552] 38. A method for detecting SARS-CoV-2 neutralizing antibodies, comprising:

[0553] 1) Administer the COVID-19 subunit vaccine as described in Implementation Plan 35 to the subjects; and

[0554] 2) Detect neutralizing antibodies produced in the subjects described in 1) after receiving the COVID-19 subunit vaccine.

[0555] The embodiments described below are not intended to be limited by any theory; they are merely for illustrating the fusion protein, immunogenic composition, preparation method, and uses of this application, and are not intended to limit the scope of the invention. Furthermore, the presence or absence of 6 or 8 HIS tags or N-terminal signal peptides in the embodiments does not affect the scope of protection. Non-essential modifications and adjustments made to the embodiments by those skilled in the art based on the content of this invention still fall within the scope of protection of this invention.

[0556] Example

[0557] Example 1: Construction of RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid

[0558] Signal peptide addition: MGVPAVPEASSPRWGTLLLAIFLAASRGLVAA (SEQ ID NO: 74). Vector selection: pcdna3.1 (+), codon optimized according to host CHO cells.

[0559] Cloning of the fragment. The RBD-P2-6*HIS fragment was amplified by PCR using primers, and then recombined into the target vector pcdna3.1(+) (BamHI-XhoI digested vector) using a multi-segment recombination method to obtain the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid. The reaction system is shown in Table 1.

[0560] Table 1. Reaction system for linking the treated target fragment with the carrier

[0561]

[0562] The above ligation solution was ligated at a constant temperature of 52℃ for 30 min to obtain the recombinant expression plasmid RBD-P2-6*HIS (GP101150-7).

[0563] Transformation method: (1) Add 1-3 μl of the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid at a concentration of 100 ng / μl to 100 μl of competent cells, gently shake and rotate to mix, and place on ice for 3 minutes. (2) Incubate in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μl of LB medium pre-warmed at 37℃ to each tube, and gently shake at 200 rpm on a 37℃ shaker for 40 minutes.

[0564] Verification of the recombinant expression plasmid RBD-P2-6*HIS (GP101150-7): (1) Prepare agar plates containing the corresponding antibiotics. (2) Take 100 μl of bacterial culture and spread it evenly on the agar plate containing the corresponding antibiotics. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick bacteria from the plate, shake the plate at 37°C at 250 rpm for 14 hours, and perform PCR identification using the bacterial culture. Send the positive clones for sequencing.

[0565] Identification method of cloned plasmid: PCR amplification of the RBD-P2-6*HIS fragment. The primer sequences were synthesized by the company's internal primer department. The expected fragment length was 861 bp. The PCR reaction used a 20 μL system: 0.5 μL primer, 2 μL template bacterial culture, 0.5 μL polymerase buffer, 3 μL buffer, and 14 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, 23 cycles, and a final extension at 72℃ for 1 min. Positive clones were screened by bacterial culture PCR. The obtained positive bacterial cultures were shaken at 37℃ to extract plasmids, which were then sequenced. Plasmids that matched the sequencing results were double-digested with BamHI-XhoI to obtain two fragments, 861 bp and 5372 bp.

[0566] RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid extraction: *E. coli* cells (stbl3) containing 1% of the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid were inoculated into 2 ml LB medium and cultured overnight at 37°C with shaking. After cell treatment, 100 μg of plasmid was extracted using a plasmid extraction kit. The correctly aligned plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 861 bp and 5372 bp.

[0567] Example 2: Cell transfection and purification of RBD-P2-6*HIS protein

[0568] During all cell manipulations, gently rotate the host cells to mix, avoiding vigorous mixing / pipetting. Subculture and expand CHO cells (EXPICHO from Thermo) until a cell density of 4 × 10⁶ cells / year is reached. 6 –6×10 6 Cells / mL. Day -1: Cell expansion, expanding cultured cells to 3 × 10⁻⁶. 6 –4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 –10×10 6 Cells were transfected at a density of 95–99% viability using fresh expression medium preheated to 37°C, and then diluted to a final density of 6 × 10⁶ cells / mL. 6Cells were cultured at 37°C (90 rpm) in a 50 mm amplitude incubator with 8% CO2. Transfection reagent and the RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid complex were prepared using OPti-PRO SFM medium (4°C). For example, for 1 ml of CHO cells, 40 μl of OPti-PRO SFM was added with 0.8 μg of RBD-P2-6*HIS (GP101150-7) recombinant expression plasmid, mixed, and incubated for 5 min; 40 μl of OPti-PRO SFM was added with 3 μl of Expi Fectamine CHO reagent, mixed, and incubated for 5 min at room temperature for 1–5 minutes. The solution was then slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were cultured at 37°C (90 rpm) in a 50 mm amplitude incubator with 8% CO2. 18–22 hours post-transfection, Enhancer and ExpiCHO Feed were added, and standard experimental protocols were followed. Example: Add 6 μl of Enhancer and 0.24 ml of ExpiCHO Feed to 1 ml of cells, and incubate the cells at 37°C with a 50 mm amplitude incubator at 90 rpm and 8% CO2. Collect the cells 8 days after transfection for further purification.

[0569] Protein purification. The culture medium was centrifuged, and the supernatant was added to a Ni column. The column was incubated with shaking for 2 hours, followed by affinity chromatography purification using a gravity-fed empty column. Equilibration buffer: PBS, pH 7.4, 10 CV wash; Washing buffer: PBS, pH 7.4 with 20 mM imidazole, 10 CV wash; Elution buffer: PBS, pH 7.4 with 500 mM imidazole, 1 CV elution, repeated 5 times, yielding the purified RBD-P2-6*HIS protein, as shown in Table 2.

[0570] Table 2. Concentration and volume of purified protein

[0571]

[0572] SDS-PAGE electrophoresis and Western blotting verification. A. 1.0 mm PAGE gel, 8 μl sample loading, gel running sequence: M. Molecular weight marker. Me. Culture medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix RBD-P2-6*HIS purified protein with loading buffer (RBD-P2-6*HIS purified protein: 5x loading buffer = 4:1), boil for 5 min, and then load the sample. First, perform electrophoresis at a constant voltage of 100V. You can see the marker gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25 min). C. Transfer membrane: Pry open the glass plate and carefully remove the gel. Activate the PVDF membrane with methanol for 30 s, cut it and the filter paper to the same size as the gel block, and soak them in transfer buffer. From bottom to top: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between the layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 50kDa protein requires 20 minutes. Larger molecular weight proteins require longer transfer times. D. Antibody incubation: After transfer, remove the PVDF membrane; you will see the marker transferred onto the membrane. Block with 5% skim milk (1g skim milk powder to 100ml PBST) for 1 hour, then wash with PBST 2 x 5 minutes each time. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder to 100ml TBS), incubate in a small container, and react at 4°C on a decolorizing shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4 x 10 minutes each time. Dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder and incubate for 1 hour. Wash with PBST 4 x 10 minutes each time. Use a shaker for all reactions. E. Color development, ECL color development: For each PBST membrane, mix 1 mL of solution A and 10 μL of solution B, drop the mixture onto the membrane, and record the image using a chemiluminescence imaging system after 5 minutes. Figure 1 As shown.

[0573] Example 3: Construction of NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid

[0574] Signal peptide addition: MFVFLVLLPLVS (SEQ ID NO: 75), vector selection: pcdna3.1(+). Codon optimization was performed according to the host CHO cells. The NTD-P2-6*HIS primers were amplified by PCR to obtain the fragment PCR product, which was then recombined into the target vector pcdna3.1(+) (BamHI-XhoI digested vector) using a multi-segment recombination method to obtain the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid. The reaction system is shown in Table 3.

[0575] Table 3. Reaction system for linking the treated target fragment with the carrier

[0576]

[0577] Transformation method: Add 1-3 μl of the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid at a concentration of approximately 100 ng / μl to approximately 100 μl of competent cells. Gently shake and rotate to mix, then place on ice for 3 minutes. Incubate at 42°C for 90 seconds without shaking; then incubate on ice for approximately 3 minutes. Add 500-800 μl of pre-warmed LB medium at 37°C to each tube, and gently shake at 200 rpm on a 37°C shaker for 40 minutes.

[0578] Verification of the recombinant expression plasmid NTD-P2-6*HIS (GP101150-8): (1) Prepare agar plates containing the corresponding antibiotics. (2) Take 100 μl of bacterial culture and spread it evenly on the agar plate containing the corresponding antibiotics. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick bacteria from the plate, shake the bacteria at 37°C and 250 rpm for 14 hours, perform PCR identification with the bacterial culture, and send the positive clones for sequencing.

[0579] Identification method of cloned plasmid: PCR amplification of the NTD-P2-6*HIS fragment. The primer sequences were synthesized by the company's in-house primer department. The expected fragment length was 1005 bp. The PCR reaction used a 20 μL system: 0.5 μL primer, 2 μL template bacterial culture, 0.5 μL polymerase buffer, 3 μL buffer, and 14 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, 23 cycles, and a final extension at 72℃ for 1 min. Positive clones were screened by bacterial culture PCR. The obtained positive bacterial cultures were shaken at 37℃ to extract plasmids, which were then sequenced. Plasmids that matched the sequencing results were double-digested with BamHI-XhoI to obtain two fragments of 1005 bp and 5372 bp.

[0580] Extraction of the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid. *E. coli* cells (stbl3) containing 1% of the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid were inoculated into 2 ml LB medium and cultured overnight at 37°C with shaking. After cell treatment, 100 μg of plasmid was extracted using a plasmid extraction kit. The correctly aligned plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 1005 bp and 5372 bp.

[0581] Example 4 Cell transfection and purification of NTD-P2-6*HIS (GP101150-8) protein

[0582] During all cell manipulations, gently rotate to mix the host cells; avoid vigorous mixing / pipetting. Subculture and expand CHO cells until a cell density of 4 × 10⁶ cells / year is reached. 6 –6×10 6 Cells / mL. Day -1: CHO cell expansion, expanding cultured cells to 3 × 10⁻⁶. 6 –4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 –10×10 6 Transfect cells at a density of 95–99% viability using fresh cell expression medium preheated to 37°C, and dilute to a final density of 6 × 10⁶ cells / mL. 6 Cells were cultured at 37°C (90 rpm) in a 50 mm amplitude incubator with 8% CO2. Transfection reagent and the NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid complex were prepared using OPti-PRO SFM medium (4°C). For example, for 1 ml of cells, 40 μl of OPti-PRO SFM was added with 0.8 μg of NTD-P2-6*HIS (GP101150-8) recombinant expression plasmid, mixed, and incubated for 5 min; 40 μl of OPti-PRO SFM was added with 3 μl of Expi Fectamine CHO reagent, mixed, and incubated for 5 min at room temperature for 1–5 minutes. The solution was then slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were then cultured at 37°C (90 rpm) in a 50 mm amplitude incubator with 8% CO2. 18–22 hours post-transfection, Enhancer and ExpiCHO feed were added, and standard experimental protocols were followed. Example: Add 6 μl of Enhancer and 0.24 ml of ExpiCHO Feed to 1 ml of cells, and incubate the cells at 37°C with a 50 mm amplitude incubator at 90 rpm and 8% CO2. Collect the cells 8 days after transfection for further purification.

[0583] Protein purification was performed by centrifugation of the culture medium, adding the supernatant to a Ni column, incubating with shaking for 2 hours, and then performing affinity chromatography using a gravity-fed empty column. Equilibration buffer: PBS, pH 7.4, 10 CV wash; Washing buffer: PBS, pH 7.4 with 20 mM Mimidazole, 10 CV wash; Elution buffer: PBS, pH 7.4 with 500 mM Mimidazole, 1 CV elution. This was repeated 5 times to obtain NTD-P2-6*HIS (GP101150-8) purified protein. The results are shown in Table 4.

[0584] Table 4. Concentration and volume of purified protein from NTD-P2-6*HIS (GP101150-8)

[0585]

[0586] SDS-PAGE electrophoresis and Western blotting verification. A. 1.0 mm PAGE gel, 8 μl sample volume, gel running sequence: M. Molecule weight marker. Me. Cylinder medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix NTD-P2-6*HIS (GP101150-8) purified protein with loading buffer (NTD-P2-6*HIS (GP101150-8) purified protein: 5x loading buffer = 4:1), boil for 5 min, and then load the sample. First, perform electrophoresis at a constant voltage of 100V. You will see the marker gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25 min). C. Transfer. Pry open the glass plate and carefully remove the gel. After activating the PVDF membrane with methanol for 30 seconds, cut it and the filter paper to the same size as the gel block, and soak it in transfer buffer. The order from bottom to top is: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between the layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 50kDa protein requires 20 minutes. Larger molecular weight proteins require longer transfer times. D. Antibody incubation. After transfer, remove the PVDF membrane; the marker will be visible transferred to the membrane. Block with 5% skim milk (1g skim milk powder to 100ml PBST) for 1 hour, then wash with PBST 2 x 5 minutes each time. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder to 100ml TBS), incubate in a small container, and react at 4°C on a decolorizing shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4 x 10 minutes each time. The secondary antibody (goat anti-mouse) was also diluted with 5% skim milk powder and incubated for 1 hour. Wash with PBST 4 x 10 min. Shake on a shaker for all steps. E. Color development. ECL color development: For each PBST membrane, mix 1 mL of solution A and 10 μL of solution B, drop the mixture onto the membrane, and record the image using a chemiluminescence imaging system after 5 min. Figure 2 As shown.

[0587] Example 5: Construction of NTD-RBD-foldon-8*HIS recombinant expression plasmid

[0588] Signal peptide addition: MFVFLVLLPLVS (SEQ ID NO: 75), vector selection: pcdna3.1(+). Codon optimization was performed according to the host CHO cells. The primers for NTD-RBD-foldon-8*HIS were amplified by PCR to obtain the fragment PCR product, which was then recombined into the target vector pcdna3.1(+) (BamHI-XhoI digested vector) using a multi-segment recombination method to obtain the NTD-RBD-foldon-8*HIS recombinant expression plasmid. The reaction system is shown in Table 5.

[0589] Table 5. Reaction system for linking the treated target fragment with the carrier

[0590]

[0591] Transformation method: Add 1-3 μl of the NTD-RBD-foldon-8*HIS recombinant expression plasmid (at a concentration of approximately 100 ng / μl) to approximately 100 μl of competent cells, gently shake and rotate to mix, and incubate on ice for 3 minutes. Incubate at 42°C for 90 seconds without shaking; incubate on ice for approximately 3 minutes; add 500-800 μl of pre-warmed LB medium at 37°C to each tube, and gently shake at 200 rpm on a 37°C shaker for 40 minutes.

[0592] Verification of the NTD-RBD-foldon-8*HIS recombinant expression plasmid: (1) Prepare agar plates containing the corresponding antibiotics. (2) Take 100 μl of bacterial culture and spread it evenly on the agar plate containing the corresponding antibiotics. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick bacteria from the plate, shake the plate at 37°C for 250 rpm for 14 hours, and perform PCR identification using the bacterial culture. Send the positive clones for sequencing.

[0593] Identification method of cloned plasmid: PCR amplification of the NTD-RBD-foldon-8*HIS fragment. The primer sequences were synthesized by the company's internal primer department. The expected fragment length was 1761 bp. The PCR reaction used a 20 μL system: 0.5 μL primer, 2 μL template bacterial culture, 0.5 μL polymerase buffer, 3 μL buffer, and 14 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 20 s, 23 cycles, and a final extension at 72℃ for 1 min. Positive clones were screened by bacterial culture PCR. The obtained positive bacterial cultures were shaken at 37℃ to extract plasmids, which were then sequenced. Plasmids that matched the sequencing results were double-digested with BamHI-XhoI to obtain two fragments, 1761 bp and 5372 bp.

[0594] Extraction of the NTD-RBD-foldon-8*HIS recombinant expression plasmid. *E. coli* cells (stbl3) containing 1% of the NTD-RBD-foldon-8*HIS recombinant expression plasmid were inoculated into 2 ml LB medium and cultured overnight at 37°C with shaking. After cell treatment, 100 μg of plasmid was extracted using a plasmid extraction kit. The correctly aligned plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 1761 bp and 5372 bp.

[0595] Example 6 Cell transfection and protein purification

[0596] During all cell manipulations, gently rotate to mix the host cells; avoid vigorous mixing / pipetting. Subculture and expand CHO cells until a cell density of 4 × 10⁶ cells / year is reached. 6 –6×10 6 Cells / mL. Day -1: Cell expansion, expanding cultured cells to 3 × 10⁻⁶. 6 –4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 –10×10 6 Cells were transfected at a density of 95–99% viability using fresh expression medium preheated to 37°C, and then diluted to a final density of 6 × 10⁶ cells / mL. 6 Cells / mL, cultured at 37°C in a 50mm amplitude incubator at 90rpm with 8% CO2.

[0597] The transfection reagent and NTD-RBD-foldon-8*HIS recombinant expression plasmid complex were prepared using OPti-PRO SFM medium (4°C). For example, for 1 ml of cells, 40 μL of OPti-PRO SFM was added, followed by 0.8 μg of NTD-RBD-foldon-8*HIS recombinant expression plasmid. The mixture was incubated for 5 min. Then, 40 μL of OPti-PRO SFM was added, followed by 3 μL of Expi Fectamine CHO reagent. The mixture was incubated for 5 min at room temperature for 1–5 minutes, then the solution was slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were incubated at 37°C with 90 rpm in a 50 mm amplitude incubator at 8% CO2. 18–22 hours after transfection, Enhancer and ExpiCHO feed were added, following standard experimental protocols. For example, 6 μL of Enhancer and 0.24 ml of ExpiCHO feed were added to 1 ml of cells. Cells were incubated at 37°C with 90 rpm in a 50 mm amplitude incubator at 8% CO2. Cells were collected 8 days after transfection for further purification.

[0598] Protein purification: After centrifugation of the culture medium, the supernatant was added to a Ni column, and the mixture was incubated with shaking for 2 hours. Affinity chromatography was then performed using a gravity-fed empty column for purification. Equilibration buffer: PBS, pH 7.4, 10 CV wash; Washing buffer: PBS, pH 7.4 with 20 mM Mimidazole, 10 CV wash; Elution buffer: PBS, pH 7.4 with 500 mM Mimidazole, 1 CV elution. This was repeated 5 times to obtain the purified NTD-RBD-foldon-8*HIS protein. The results are shown in Table 6.

[0599] Table 6 Concentration and volume of NTD-RBD-foldon-8*HIS purified protein

[0600]

[0601] SDS-PAGE electrophoresis and Western blotting verification. A. 1.0 mm PAGE gel, 8 μL sample volume, gel running sequence: M. Molecular weight marker. Me. Culture medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix NTD-RBD-foldon-8*HIS purified protein with loading buffer (NR-foldon-8*HIS purified protein: 5x loading buffer = 4:1), boil for 5 min, and then load the sample. First, perform electrophoresis at a constant voltage of 100V. You can see the marker gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (about 25 min). C. Transfer membrane: Pry open the glass plate and carefully remove the gel. Activate the PVDF membrane with methanol for 30 s, cut it and the filter paper to the same size as the gel block, and soak them in transfer buffer. From bottom to top: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between the layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 50kDa protein requires 20 minutes. Larger molecular weight proteins require longer transfer times. D. After antibody incubation and transfer, remove the PVDF membrane; you will see the marker transferred onto the membrane. Block with 5% skim milk (1g skim milk powder to 100ml PBST) for 1 hour, then wash with PBST 2 x 5 minutes each time. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder to 100ml TBS), incubate in a small container, and react at 4°C on a shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4 x 10 minutes each time. Dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder and incubate for 1 hour. Wash with PBST 4 x 10 minutes each time, using a shaker. E. Color development, ECL color development: For each PBST membrane, mix 1 mL of solution A and 10 μL of solution B, drop the mixture onto the membrane, and record the image using a chemiluminescence imaging system after 5 minutes. Figure 3 As shown.

[0602] Example 7: Construction of vector and protein expression

[0603] Referring to the above embodiments, the target fragment to be expressed was constructed as follows, and the protein was expressed and purified, as shown in Table 7.

[0604] Table 7 Different target segments

[0605]

[0606] Example 8 Preparation of vaccine composition

[0607] 0.04 mg / mL of RBD protein, 0.04 mg / mL of NTD protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM histidine buffer at pH 5.0 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0608] Example 9 Preparation of vaccine composition

[0609] 0.04 mg / mL of RBD-P2 protein, 0.04 mg / mL of NTD-P2 protein; 2% to 15% (w / v) sorbitol; 0.01% to 0.05% (w / v) polysorbate 20; and 5 mM to 20 mM histidine buffer at pH 5.5 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0610] Example 10 Preparation of vaccine composition

[0611] 0.04 mg / mL of RBD protein, 0.04 mg / mL of NTD-P2 protein; 2% to 15% (w / v) sucrose; 0.01% to 0.05% (w / v) Tween 80; and 5 mM to 25 mM succinate buffer at pH 4.5 to 5.5 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0612] Example 11 Preparation of vaccine composition

[0613] 0.04 mg / mL of RBD-P2 protein, 0.04 mg / mL of NTD protein; 2% to 15% (w / v) sorbitol; 0.01% to 0.05% (w / v) polysorbate 20; and 5 mM to 20 mM histidine buffer at pH 5.5 to 7.0 were aliquoted into 2 ml vials, 0.5 ml per vial, and lyophilized.

[0614] Example 12 Preparation of vaccine composition

[0615] The immunogenic composition of any one of Examples 8-11, wherein the composition further comprises aluminum hydroxide adjuvant at a concentration of 1 mg / mL.

[0616] CpG1018 adjuvant was added to the immunogenic composition described in any one of Examples 8-11 at a concentration of 6 mg / mL. The CpG adjuvant (CpG was synthesized by Shanghai Sangon Biotech Co., Ltd. according to the CpG1018 sequence)

[0617] Example 13 Preparation of vaccine composition

[0618] The immunogenic composition of any one of Examples 8-11, the composition further comprising an adjuvant, for example, in an adjuvant vial of 0.5 ml, 50 μg of MPL, 500 μg of aluminum hydroxide, 150 mM of NaCl, 8 mM of disodium hydrogen phosphate dihydrate, and water for injection to a final volume of 0.5 ml.

[0619] Example 14 Preparation of vaccine composition

[0620] The immunogenic composition described in any one of Examples 8-11, further comprising an adjuvant, for example, in a 0.25 ml adjuvant vial containing 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na₂HPO₄, 0.09 mg of KH₂PO₄, and water for injection.

[0621] Example 15 Preparation of vaccine composition

[0622] The immunogenic composition of any one of Examples 8-11, further comprising an adjuvant, for example, in a 0.5 ml adjuvant vial containing 50 μg of MPL, 50 μg of QS-21, 1 mg of DOPC dioleoylphosphatidylcholine, 0.25 mg of cholesterol, 0.15 mg of anhydrous disodium phosphate, 0.54 mg of potassium dihydrogen phosphate, 4.385 mg of sodium chloride, and water for injection.

[0623] Example 16 Preparation of vaccine composition

[0624] The immunogenic composition of any one of Examples 8-11, further comprising an adjuvant, for example, in a 0.5 ml adjuvant vial containing 9.75 mg squalene, 1.175 mg Span 85, 1.175 mg Tween 80, 0.66 mg trisodium citrate dihydrate and 0.04 mg citric acid monohydrate, and water for injection.

[0625] Example 17 Mouse Immunization Experiment with Different Antigens and Adjuvants

[0626] The antigen protein was combined with different adjuvants to immunize mice, and the level of protein-induced neutralizing antibodies was determined by ELISA. Mouse grouping and immunization regimens are shown in Table 8.

[0627] Table 8. Mouse Grouping Table

[0628]

[0629] The NTD protein sequence is amino acids 1-291 of SEQ ID NO: 20, and the RBD sequence is amino acids 1-223 of SEQ ID NO: 1. The concentrations of Al(OH)3 adjuvant, CpG adjuvant, and CpG+Al(OH)3 adjuvant are as described in Example 12. The composition of AS01 is as described in Example 15, and the composition of AS04 is as described in Example 13. The results of the mouse immunization 28 days later are shown in Table 9. The results show that RBD+NTD induces a stronger level of neutralizing antibodies.

[0630] Table 9 Results of mouse immunization 28 days later

[0631]

[0632] Example 18 Immunogenicity Evaluation Experiment of Different Antigens and Adjuvants

[0633] The antigen protein and MF59 adjuvant were used to immunize Japanese white rabbits. The level of neutralizing antibodies induced by the protein was determined by ELISA. The grouping and immunization regimen of the Japanese white rabbits are shown in Table 10.

[0634] Table 10 Immunization Grouping Table for Japanese White Rabbits

[0635]

[0636] The sequence of NTD-P2 is shown in SEQ ID NO: 20, the sequence of RBD-P2 is shown in SEQ ID NO: 1, the sequence of NTD-foldon is shown in SEQ ID NO: 22, the sequence of RBD-foldon is shown in SEQ ID NO: 3, the sequence of NTD-ferritin LC is shown in SEQ ID NO: 23, the sequence of RBD-ferritin is shown in SEQ ID NO: 5, the sequence of NTD-HBsAg is shown in SEQ ID NO: 25, the sequence of RBD-HBsAg is shown in SEQ ID NO: 6, and the adjuvant MF59 is shown in Example 16.

[0637] The sequences of NTD-RBD-P2 are as shown in SEQ ID NO: 40, but without a HIS tag at the C-terminus; the sequences of NTD-RBD-foldon are as shown in SEQ ID NO: 41, but without a HIS tag at the C-terminus; the sequences of NTD-RBD-P2-foldon are as shown in SEQ ID NO: 42, but without a HIS tag at the C-terminus; the sequences of NTD-RBD-ferritin are as shown in SEQ ID NO: 43; and the sequences of NTD-RBD-HBsAg are as shown in SEQ ID NO: 44. The detection results of the fusion proteins 28 days after immunization with Japanese White rabbits are shown in Table 11, indicating that the fusion proteins are immunogenic.

[0638] Table 11 Results of Japanese White Rabbit Immunization 28 Days Later

[0639]

[0640] Example 19 Immunogenicity Evaluation Experiment of Different Antigens and Adjuvants

[0641] The antigen protein was used in combination with different adjuvants to immunize rhesus monkeys, and the level of protein-induced neutralizing antibodies was determined by ELISA. The rhesus monkeys were grouped and immunization regimens are as follows, with an inactivated COVID-19 vaccine as the positive control. The test results of the rhesus monkeys 28 days after immunization are shown in Table 12.

[0642] Table 12 Immunization Results of Rhesus Monkeys

[0643]

[0644] Example 20: Detection of the effect of recombinant COVID-19 vaccine candidate antigen inducing neutralizing antibody production in mice

[0645] Based on the WIV04-1 sequence, NTD and RBD were selected as candidate antigens. The immunogenicity of NTD alone, RBD alone, co-immunization with NTD and RBD, the NTD-RBD fusion protein (hereinafter referred to as NR), and the NTD-RBD-foldon fusion protein (hereinafter referred to as NR-foldon) was detected, and the results are shown in Table 14. The results showed that immunization of BALB / c mice with NR-foldon antigen and BFA03 adjuvant significantly and effectively induced the production of high-titer neutralizing antibodies (based on the SARS-CoV-2 virus strain and VSV pseudovirus detection system) and antigen-specific IgG antibodies. The neutralizing effect against the WIV04 prototype strain and the D614G and Gamma mutant strains was significantly better than that against other antigens.

[0646] Table 13 Overview of Experimental Design for Recombinant SARS-CoV-2 Vaccine Candidate Antigens in BALB / c Mice

[0647]

[0648] Table 14 Screening results of recombinant COVID-19 vaccine candidate antigens in BALB / c mice

[0649]

[0650] Note: 1. Detection of neutralizing antibodies against the novel coronavirus based on the SARS-CoV-2 strain, the same applies below.

[0651] 2. Pseudovirus types: Prototype strain (WIV04-1), major circulating strain (D614G); VOC variant strains: UK mutant strain (Alpha, B.1.1.7), South African mutant strain (Beta, B.1.351), Brazilian mutant strain (Gamma, P.1), Indian mutant strain (Delta, B.1.617.2); VOI variant strains: Peruvian mutant strain (Lambda, C.37), the same below.

[0652] Example 21 Immunological Effect Detection of Different Adjuvants

[0653] BALB / c mice were immunized with Al(OH)3, BFA01, BFA02, BFA03, BFA04, and CpG / Al(OH)3 in combination with NR-foldon antigen, and the levels of neutralizing antibodies and antigen-specific IgG antibodies in the serum were detected. The results showed that among the candidate adjuvants, BFA03 adjuvant exhibited the best immunogenicity and was significantly superior to traditional aluminum adjuvants (aluminum hydroxide adjuvants), as well as to composite adjuvant systems based on aluminum salt adjuvants.

[0654] Table 15 Overview of Experimental Design for Recombinant COVID-19 Vaccine Candidate Adjuvants in BALB / c Mice

[0655]

[0656] Note: 1. The human dose (0.5ml) contains the content of the main component in the adjuvant. The immunization dose in BALB / c mice is 1 / 5HD.

[0657] Table 16 Experimental results of recombinant COVID-19 vaccine candidate adjuvants in BALB / c mice

[0658]

[0659] Example 22: Detection of the Immunogenic Effect of Different Doses in Mouse Experiments

[0660] BALB / c mice were immunized with 1 / 10 and 1 / 5 human doses of NR-foldon antigen (i.e., 4 μg and 8 μg), respectively, in combination with 1 / 5 or 1 / 10 human doses of BFA03 adjuvant. The results showed that, with a fixed antigen dose, immunization with 1 / 10 HD adjuvant produced a lower titer of neutralizing antibodies than immunization with 1 / 5 HD adjuvant. However, there was no statistically significant difference between the groups. This suggests that a proper ratio of antigen to adjuvant can induce a stronger immune response.

[0661] Table 17 Results of dose evaluation of recombinant COVID-19 vaccine in BALB / c mice

[0662]

[0663] Example 23: Detection of the Immunogenic Effect of Different Doses in Rabbit Experiments

[0664] Japanese white rabbits were immunized with one human dose of BFA03 adjuvant (0.5 ml) in combination with two different doses of NR-foldon antigen, namely 20 μg and 40 μg. The results are shown in Table 18. The antibody titer produced by immunization with 40 μg antigen was higher.

[0665] Table 18 Results of immunization of Japanese White rabbits with different doses of adjuvant

[0666]

[0667] Japanese white rabbits were immunized with 1 HD (0.5 ml), 1 / 2 HD, and 1 / 4 HD BFA03 adjuvants in combination with 1 HD NR-foldon antigen. The results are shown in Table 19. The 1 HD adjuvant immunized the rabbits with the 1 HD antigen and induced the highest levels of neutralizing and binding antibodies. Reducing the adjuvant dose would reduce the immunization effect.

[0668] Table 19 Results of immunization of Japanese White rabbits with different doses of adjuvant

[0669]

[0670] Example 24: Detection of the neutralizing effect of antigen adjuvant combination on different variants of SARS-CoV-2

[0671] The following are the GMT results of neutralizing antibody titers produced by ReCOV vaccine (NR-foldon antigen / BFA03 adjuvant) immunized in different species of animals. As shown in the table, the determined antigen-adjuvant combination has good neutralizing effect against SARS-CoV-2 virus and different variants.

[0672] Table 20 Results of neutralizing antibody titers produced by different animal species

[0673]

[0674] Effects of using S protein NTD and RBD in Gamma mutant strains and S protein NTD and RBD in Beta mutant strains.

[0675] Example 25 Construction of recombinant expression plasmids for NTD-RBD-foldon-6×His (Gamma mutant) and NTD-RBD-foldon-6×His (Beta mutant)

[0676] As shown in SEQ ID NO: 85, NTD-RBD-foldon (Gamma mutant) was supplemented with the signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 91) and 6×His; as shown in SEQ ID NO: 79, NTD-RBD-foldon (Beta mutant) was supplemented with the signal peptide MGWSCIILFLVATATGVHS (SEQ ID NO: 91) and 6×His. The full-length amino acid sequences were codon-optimized by Wuxi WuXi Biologics Co., Ltd., and the DNA fragments of NTD-RBD-foldon-6×His (Gamma mutant) and NTD-RBD-foldon-6×His (Beta mutant) were synthesized by PCR. After gel purification, the purified DNA fragments were recombined into the target vector pcDNA3.1(+) to obtain the recombinant expression plasmids of NTD-RBD-foldon-6×His (Gamma mutant) and NTD-RBD-foldon-6×His (Beta mutant). And the sequence was verified by Sanger sequencing, which showed that it was 100% correct. See the sequencing results below. Figure 1 and Figure 7 .

[0677] Transformation of host bacteria: (1) Add 1-3 μL of the recombinant expression plasmid NTD-RBD-foldon-6×His (Gamma mutant) or NTD-RBD-foldon-6×His (Beta mutant) at a concentration of 100 ng / μL to 100 μL of E. coli Top10 competent cells, gently shake and rotate to mix, and place on ice for 3 minutes. (2) Incubate in a 42℃ water bath for 90 s without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μL of LB medium pre-warmed at 37℃ to each tube, and gently shake at 200 rpm on a 37℃ shaker for 40 minutes.

[0678] Recombinant expression plasmid extraction and sequencing: (1) Prepare an agar plate containing 100 µg / mL ampicillin. (2) After culturing 100 μL of bacterial culture, gently spread the mixture onto an LB agar plate (containing 100 µg / mL ampicillin) using a sterile glass spreader, and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick a single colony from the plate and inoculate it into 300 mL of LB medium for expansion culture. Prepare a large number of plasmids using the NucleoBond Xtra Maxi EF kit according to the instructions. Sequencing was performed to verify the target gene, and the sequencing results were consistent with the designed genome sequence.

[0679] Example 26 Cell transfection and protein purification

[0680] Cell transfection:

[0681] During all cell manipulations, gently rotate the host cells to mix, avoiding vigorous mixing / pipetting. Subculture and expand CHO cells (ExpiCHO from Thermo) until a cell density of 4 × 10⁶ cells / year is reached. 6 ~6×10 6 Cells / mL. Day -1 after transfection: Adjust cell density to 3 × 10⁻⁶. 6 ~4×10 6 Cells / mL, and allowed to grow overnight. Day 0: Transfect cells, determine viable cell density and survival rate; cell density should reach 7 × 10⁶ cells / mL. 6 ~10×10 6 Transfection was performed when the cell viability reached 95-99% at a density of 6 × 10⁶ cells / mL. Fresh cell expression medium was preheated to 37°C, and cells were diluted to a final density of 6 × 10⁶ cells / mL. Cells were then cultured at 37°C with 90 rpm in a 50 mm amplitude incubator under 8% CO₂. Transfection reagent and the NTD-RBD-foldon-6×His (Gamma mutant / Beta mutant) recombinant expression plasmid complex were prepared using OptiPRO™ SFM medium (4°C). For example, for 1 mL CHO cells, 40 μL of OptiPRO™ SFM was added to 0.8 μg of NTD-RBD-foldon-6×His (Gamma mutant / Beta mutant) recombinant expression plasmid, mixed, and incubated for 5 minutes. 40 μL of OptiPRO™ SFM was then added to 3 μL of LexpiFectamine™ CHO reagent, mixed, and incubated for 5 minutes at room temperature for 1-5 minutes. The solution was then slowly transferred to a shaker flask with gentle shaking during the addition process. Cells were cultured at 37°C with a 50mm amplitude incubator at 90 rpm and 8% CO2. 18-22 hours post-transfection, Enhancer and ExpiCHO Feed were added, and standard experimental protocols were followed. For example, 1 mL of cells was incubated with 6 μL Enhancer and 0.24 mL ExpiCHO Feed, and the cells were then cultured at 37°C with a 50mm amplitude incubator at 90 rpm and 8% CO2. Cells were collected 6 days post-transfection for further purification.

[0682] Protein purification:

[0683] Purification was performed using a combination of immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC). A Hi Trap 5ml chelating HP column (GE Healthcare, USA) loaded with NiSO4 was used. After pretreatment with chelating chromatography media, the column was packed and washed with 50 mmol / L EDTA, 0.2 mol / L NaOH, and ultrapure water, respectively. After equilibration with at least 5 times the volume of lysis buffer, the cell lysis supernatant was directly loaded onto the column. Gradient elution was performed using solution A (20 mmol / L PB, pH 7.4, 0.15 mol / L NaCl) and solution B (20 mmol / L PB, pH 7.4, 0.15 mol / L NaCl, 0.5 mol / L imidazole) as the mobile phase (the concentration of solution B was increased from 4% (v / v) to 100% (v / v)). The target fraction was collected and further separated using a HiLoad 26 / 60 Superdex-200 pre-grade gel column (GE Healthcare, USA). Elution was performed using solution C (20 mmol / L PB, pH 7.4, 0.15 mol / L NaCl) as the mobile phase for one column volume at an elution rate of 2.5 mL / min. The chromatogram of the purified SEC is shown below. Figure 5 and Figure 8 .

[0684] The purified protein NTD-RBD-foldon-6×His (Gamma mutant / Beta mutant) was obtained. The protein purification results are shown in Table 21. The purity of the purified target protein can reach 93.6% (Gamma mutant) and 95% (Beta mutant).

[0685] Table 21 Protein purification results

[0686]

[0687] SDS-PAGE electrophoresis and Western blot verification:

[0688] A. 1.0 mm PAGE gel, 8 μL sample volume, gel running sequence: M. Molecular weight marker. Me. Culture medium. FT. Flow through. W. Wash. E. Eluted fractions. B. Electrophoresis: Mix NTD-RBD-foldon (Gamma mutant / Beta mutant) purified protein with loading buffer (NTD-RBD-foldon (Gamma mutant / Beta mutant) purified protein: 5 × loading buffer = 4:1), boil for 5 minutes, and then load the sample. First, perform electrophoresis at a constant voltage of 100V; the marker will gradually become a thin line. After the marker enters the separating gel, adjust the voltage to 300V and continue electrophoresis until the blue bromophenol blue band reaches the bottom of the gel (approximately 25 minutes). C. Transfer: Pry open the glass plate and carefully remove the gel. Activate the PVDF membrane with methanol for 30 seconds, cut it and filter paper to the same size as the gel block, and soak them in transfer buffer. From bottom to top: filter paper - PVDF membrane - gel - filter paper. Ensure there are no air bubbles between layers, especially between the gel and the membrane. Pour all the transfer buffer from the petri dish into the transfer container. Transfer at a constant voltage of 20V; 20 minutes is required for a 50kDa protein. Larger molecular weight proteins require longer transfer times. D. Antibody incubation: After transfer, remove the PVDF membrane; you will see the marker transferred onto the membrane. Block with 5% skim milk (1g skim milk powder to 100mL PBST) for 1 hour, then wash with PBST 2×5min. Dilute the primary antibody (anti-His Mab) with 5% skim milk powder (1g skim milk powder, 100mL TBS), incubate in a small container, and react at 4℃ on a decolorizing shaker for 2 hours. Remove the PVDF membrane and wash with PBST 4×10min. Dilute the secondary antibody (goat anti-mouse) with 5% skim milk powder and incubate for 1 hour. Wash with PBST 4×10min. Use a shaker for all reactions. E. Color development, ECL color development: For each PBST membrane, mix 1 mL of solution A and 10 μL of solution B, drop the mixture onto the membrane, and record the image using a chemiluminescence imaging system after 5 minutes. Figure 6 and Figure 9 As shown in the SDS-PAGE electrophoresis diagram and the Western blot results, it can be seen that the largest band on the electrophoretic lane of the NTD-RBD-foldon (Beta mutant) solution after SEC separation is NTD-RBD-foldon (Beta mutant) and the largest band on the electrophoretic lane of the NTD-RBD-foldon (Gamma mutant) solution is NTD-RBD-foldon (Gamma mutant), and it mainly exists in the form of trimer. The corresponding target bands can be seen in the Western blot results.

[0689] Example 27 Construction of vector and protein expression

[0690] Referring to the above embodiments, construct the target fragment to be expressed as shown in Table 22, and express and purify the protein.

[0691] Table 22 Different target segments

[0692]

[0693] Example 28 Preparation of Immunogenic Composition

[0694] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein, 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL vials, 0.5 mL per vial, and lyophilized.

[0695] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein, 50 μg of purified RBD-foldon (Beta mutant / Gamma mutant) protein; 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL pipettes, 0.5 mL per tube, and lyophilized.

[0696] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein; 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL vials, 0.5 mL per vial, and lyophilized.

[0697] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein, 50 μg of purified NTD-RBD-foldon (WIV04-1) protein; 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL tubes (0.5 mL per tube) and lyophilized.

[0698] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein; 135 μg of HA protein (45 μg of H1N1 HA protein, 45 μg of H3N2 HA protein, and 45 μg of B / Washington / 02 / 2019 HA protein); 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL pipettes (0.5 mL per tube) and lyophilized. The selected HA proteins are shown in Table 23.

[0699] 50 μg of purified NTD-RBD-foldon (Gamma mutant / Beta mutant) protein; 180 μg of HA protein (45 μg of H1N1 HA protein, 45 μg of H3N2 HA protein, 45 μg of B / Washington / 02 / 2019 HA protein, 45 μg of B / Phuket / 3073 / 2013 HA protein); 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80; and 10 mM PB buffer (Na2HPO4, NaH2PO4) at pH 7.5 were aliquoted into 2 mL pipettes (0.5 mL per tube) and lyophilized. The selected HA proteins are shown in Table 23.

[0700] Table 23 Influenza strains used in combination

[0701]

[0702] Example 29 Vaccine Preparation

[0703] The immunogenic composition described in any of the foregoing embodiments further comprises aluminum hydroxide adjuvant at a concentration of 1 mg / mL.

[0704] The immunogenic composition described in any of the foregoing embodiments further comprises adjuvant AS03, the adjuvant vial being 0.5 mL, and the components including 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of polysorbate 80, 3.53 mg of sodium chloride, 0.09 mg of potassium chloride, 0.51 mg of disodium hydrogen phosphate, 0.09 mg of potassium dihydrogen phosphate, and water for injection.

[0705] The immunogenic composition described in any of the foregoing embodiments further comprises adjuvant MF59, the adjuvant vial being 0.5 mL, and the components including 4.5% squalene, 0.5% Tween 80, 0.5% Span 85, and water for injection.

[0706] Example 30 Mouse Immunization Experiment of Vaccine (Beta Mutant Strain)

[0707] Mice were immunized with the immunogenic composition described in Example 28 combined with AS03 adjuvant at a dose of 2 / 25 of the human dose. An immunogenic composition prepared from NTD-RBD-foldon (WIV04-1) protein combined with AS03 adjuvant served as a control. The level of induced neutralizing antibodies was determined using a SARS-CoV-2 pseudovirus neutralizing antibody detection method based on the VSV (vesicular stomatitis virus) system. Mouse grouping and immunization protocols are shown in Table 24.

[0708] Table 24 Mouse Immunization Grouping Table

[0709]

[0710] The AS03 adjuvant was prepared according to Example 29. On day 28 after mouse immunization, serum was obtained by collecting blood from the orbital vein. The neutralizing antibody titer was detected using a VSV-based pseudovirus detection system. Neutralization experiments were conducted using pseudoviruses of the main circulating strain of SARS-CoV-2 (D614G), the Alpha mutant strain of SARS-CoV-2 (B.1.1.7), the Beta mutant strain of SARS-CoV-2 (501Y.V2), and the Gamma mutant strain of SARS-CoV-2 (501Y.V3) to investigate the neutralizing effect of the vaccine on different SARS-CoV-2 variants. The test results are shown in Table 25. The results showed that the neutralizing antibodies induced by NTD-RBD-foldon (WIV04-1) as antigen and AS03 adjuvant had a good neutralizing effect on the main circulating pseudovirus and Gamma mutant pseudovirus (GMT much higher than 10,000), but a poor neutralizing effect on Alpha mutant pseudovirus and Beta mutant pseudovirus (GMT much lower than 10,000). The neutralizing antibodies induced by NTD-RBD-foldon (Beta mutant) as antigen and AS03 adjuvant had a good neutralizing effect on both Beta mutant pseudovirus and Gamma mutant pseudovirus (GMT much higher than 10,000). More surprisingly, the neutralizing antibody GMT for Gamma mutant pseudovirus was as high as 20,944, but its neutralizing effect on the main circulating pseudovirus and Alpha mutant pseudovirus was slightly weaker. It is evident that the monovalent vaccine using NTD-RBD-foldon (Beta mutant strain) as the immunogen has good immunizing effects against all four SARS-CoV-2 mutant strains. In particular, it can serve as a new COVID-19 candidate vaccine against SARS-CoV-2 mutant strains (especially the South African and Gamma mutant strains), and more efficiently induce the production of protective neutralizing antibodies to combat immune escape from SARS-CoV-2 mutant strains.

[0711] The bivalent vaccine, using NTD-RBD-foldon (Beta mutant strain) combined with NTD-RBD-foldon (WIV04-1) as the immunogen, induced an overall increase in neutralizing antibody levels compared to both the NTD-RBD-foldon (Beta mutant strain) and NTD-RBD-foldon (WIV04-1) monovalent vaccines. This enhanced the efficacy of the NTD-RBD-foldon (WIV04-1) monovalent vaccine against both the Beta mutant strain and the major circulating strains of the UK. This bivalent vaccine, with its broader scope and stronger efficacy, is more suitable as a candidate vaccine for combating COVID-19.

[0712] Table 25 Results of mouse immunization 28 days later

[0713]

[0714] Example 31 Mouse Immunization Experiment of Vaccine (Gamma Mutant)

[0715] Mice were immunized with the immunogenic composition described in Example 28 combined with AS03 adjuvant at a dose of 2 / 25 of the human dose. The level of induced neutralizing antibodies was determined by a SARS-CoV-2 pseudovirus neutralizing antibody detection method based on the VSV (vesicular stomatitis virus) system. Mouse grouping and immunization regimens are shown in Table 26.

[0716] Table 26 Mouse Immunization Grouping Table

[0717]

[0718] The AS03 adjuvant was prepared according to Example 29. On day 28 after mouse immunization, serum was obtained by collecting blood from the orbital vein. The neutralizing antibody titer was detected using a VSV-based pseudovirus detection system. Neutralization experiments were conducted using pseudoviruses of the main circulating strain of SARS-CoV-2 (D614G), the Alpha mutant strain of SARS-CoV-2 (B.1.1.7), the Beta mutant strain of SARS-CoV-2 (501Y.V2), and the Gamma mutant strain of SARS-CoV-2 (501Y.V3) to investigate the neutralizing effect of the vaccine on different SARS-CoV-2 variants. The test results are shown in Table 27. The results showed that immunization of mice with NTD-RBD-foldon (Gamma mutant strain) as the antigen and AS03 adjuvant induced the highest antibody level to neutralize the Beta mutant strain (501Y.V2) pseudovirus, followed by the antibody level to neutralize the Gamma mutant strain (501Y.V3) pseudovirus. The antibody levels to neutralize the predominantly circulating strain (D614G) and the Alpha mutant strain (B.1.1.7) pseudovirus were relatively low. This indicates that the monovalent vaccine using NTD-RBD-foldon (Gamma mutant strain) as the immunogen is effective against all four SARS-CoV-2 mutant strains. It could serve as a novel COVID-19 vaccine candidate against SARS-CoV-2 mutant strains (especially the South African and Gamma mutant strains), more efficiently inducing protective neutralizing antibodies against immune evasion by SARS-CoV-2 mutant strains.

[0719] Table 27 Results of mouse immunization 28 days later

[0720]

[0721] Based on the above results, it can be determined that if a bivalent vaccine combining NTD-RBD-foldon (Gamma mutant) and NTD-RBD-foldon (Beta mutant), or a bivalent vaccine combining NTD-RBD-foldon (Gamma mutant) and NTD-RBD-foldon (WIV04-1), or a trivalent vaccine combining NTD-RBD-foldon (Gamma mutant) and NTD-RBD-foldon (Beta mutant) and NTD-RBD-foldon (WIV04-1), or a multivalent vaccine combining NTD-RBD-foldon (Gamma mutant) and NTD-RBD-foldon (Beta mutant), NTD-RBD-foldon (WIV04-1) and influenza hemagglutinin HA, etc., is prepared according to the above method, higher neutralizing antibody titers may be produced. sequence list <110> Jiangsu Provincial Center for Disease Control and Prevention (Jiangsu Provincial Institute of Public Health); Jiangsu Ruike Biotechnology Co., Ltd.; Beijing Anbaisheng Biotechnology Co., Ltd. <120> Fusion proteins and their applications <130> 0107-PA-005 <160> 111 <170> PatentIn version 3.5 <210> 1 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> RBD-P2 <400> 1 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 225 230 235 240 Thr Glu Leu <210> 2 <211> 243 <212> PRT <213> Artificial Sequence <220> <223> P2-RBD <400> 2 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe <210> 3 <211> 255 <212> PRT <213> Artificial Sequence <220> <223> RBD - foldon <400> 3 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 225 230 235 240 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly 245 250 255 <210> 4 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> RBD-ferritin LC <400> 4 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Glu Tyr Gly Ser His Gly Asn Val Ala Thr Glu Leu 225 230 235 240 Gln Ala Tyr Ala Lys Leu His Leu Glu Arg Ser Tyr Asp Tyr Leu Leu 245 250 255 Ser Ala Ala Tyr Phe Asn Asn Tyr Gln Thr Asn Arg Ala Gly Phe Ser 260 265 270 Lys Leu Phe Lys Lys Leu Ser Asp Glu Ala Trp Ser Lys Thr Ile Asp 275 280 285 Ile Ile Lys His Val Thr Lys Arg Gly Asp Lys Met Asn Phe Asp Gln 290 295 300 His Ser Thr Met Lys Thr Glu Arg Lys Asn Tyr Thr Ala Glu Asn His 305 310 315 320 Glu Leu Glu Ala Leu Ala Lys Ala Leu Asp Thr Gln Lys Glu Leu Ala 325 330 335 Glu Arg Ala Phe Tyr Ile His Arg Glu Ala Thr Arg Asn Ser Gln His 340 345 350 Leu His Asp Pro Glu Ile Ala Gln Tyr Leu Glu Glu Glu Phe Ile Glu 355 360 365 Asp His Ala Glu Lys Ile Arg Thr Leu Ala Gly His Thr Ser Asp Leu 370 375 380 Lys Lys Phe Ile Thr Ala Asn Asn Gly His Asp Leu Ser Leu Ala Leu 385 390 395 400 Tyr Val Phe Asp Glu Tyr Leu Gln Lys Thr Val 405 410 <210> 5 <211> 409 <212> PRT <213> Artificial Sequence <220> <223> RBD-ferritin HC <400> 5 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Arg Ser Cys Arg Asn Ser Met Arg Gln Gln Ile Gln 225 230 235 240 Met Glu Val Gly Ala Ser Leu Gln Tyr Leu Ala Met Gly Ala His Phe 245 250 255 Ser Lys Asp Val Val Asn Arg Pro Gly Phe Ala Gln Leu Phe Phe Asp 260 265 270 Ala Ala Ser Glu Glu Arg Glu His Ala Met Lys Leu Ile Glu Tyr Leu 275 280 285 Leu Met Arg Gly Glu Leu Thr Asn Asp Val Ser Ser Leu Leu Gln Val 290 295 300 Arg Pro Pro Thr Arg Ser Ser Trp Lys Gly Gly Val Glu Ala Leu Glu 305 310 315 320 His Ala Leu Ser Met Glu Ser Asp Val Thr Lys Ser Ile Arg Asn Val 325 330 335 Ile Lys Ala Cys Glu Asp Asp Ser Glu Phe Asn Asp Tyr His Leu Val 340 345 350 Asp Tyr Leu Thr Gly Asp Phe Leu Glu Glu Gln Tyr Lys Gly Gln Arg 355 360 365 Asp Leu Ala Gly Lys Ala Ser Thr Leu Lys Lys Leu Met Asp Arg His 370 375 380 Glu Ala Leu Gly Glu Phe Ile Phe Asp Lys Lys Leu Leu Gly Ile Asp 385 390 395 400 Val Asp Tyr Lys Asp Asp Asp Asp Lys 405 <210> 6 <211> 454 <212> PRT <213> Artificial Sequence <220> <223> RBD-HBsAg <400> 6 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu 225 230 235 240 Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile 245 250 255 Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly 260 265 270 Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His 275 280 285 Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys 290 295 300 Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile 305 310 315 320 Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro 325 330 335 Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys 340 345 350 Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr 355 360 365 Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp 370 375 380 Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp 385 390 395 400 Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro 405 410 415 Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser 420 425 430 Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe 435 440 445 Cys Leu Trp Val Tyr Ile 450 <210> 7 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> RBD‑P2‑foldon <400> 7 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 225 230 235 240 Thr Glu Leu Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp 245 250 255 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 260 265 270 Phe Leu Gly 275 <210> 8 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> P2-RBD-foldon <400> 8 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp 245 250 255 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 260 265 270 Phe Leu Gly 275 <210> 9 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> RBD‑foldon‑P2 <400> 9 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr 225 230 235 240 Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly 245 250 255 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 260 265 270 Thr Glu Leu 275 <210> 10 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> RBD‑P2‑ferritin LC <400> 10 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 225 230 235 240 Thr Glu Leu Gly Ser Gly Ser Gly Glu Tyr Gly Ser His Gly Asn Val 245 250 255 Ala Thr Glu Leu Gln Ala Tyr Ala Lys Leu His Leu Glu Arg Ser Tyr 260 265 270 Asp Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn Tyr Gln Thr Asn Arg 275 280 285 Ala Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser Asp Glu Ala Trp Ser 290 295 300 Lys Thr Ile Asp Ile Ile Lys His Val Thr Lys Arg Gly Asp Lys Met 305 310 315 320 Asn Phe Asp Gln His Ser Thr Met Lys Thr Glu Arg Lys Asn Tyr Thr 325 330 335 Ala Glu Asn His Glu Leu Glu Ala Leu Ala Lys Ala Leu Asp Thr Gln 340 345 350 Lys Glu Leu Ala Glu Arg Ala Phe Tyr Ile His Arg Glu Ala Thr Arg 355 360 365 Asn Ser Gln His Leu His Asp Pro Glu Ile Ala Gln Tyr Leu Glu Glu 370 375 380 Glu Phe Ile Glu Asp His Ala Glu Lys Ile Arg Thr Leu Ala Gly His 385 390 395 400 Thr Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn Asn Gly His Asp Leu 405 410 415 Ser Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu Gln Lys Thr Val 420 425 430 <210> 11 <211> 429 <212> PRT <213> Artificial Sequence <220> <223> RBD‑P2‑ferritin HC <400> 11 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 225 230 235 240 Thr Glu Leu Gly Ser Gly Ser Gly Arg Ser Cys Arg Asn Ser Met Arg 245 250 255 Gln Gln Ile Gln Met Glu Val Gly Ala Ser Leu Gln Tyr Leu Ala Met 260 265 270 Gly Ala His Phe Ser Lys Asp Val Val Asn Arg Pro Gly Phe Ala Gln 275 280 285 Leu Phe Phe Asp Ala Ala Ser Glu Glu Arg Glu His Ala Met Lys Leu 290 295 300 Ile Glu Tyr Leu Leu Met Arg Gly Glu Leu Thr Asn Asp Val Ser Ser 305 310 315 320 Leu Leu Gln Val Arg Pro Pro Thr Arg Ser Ser Trp Lys Gly Gly Val 325 330 335 Glu Ala Leu Glu His Ala Leu Ser Met Glu Ser Asp Val Thr Lys Ser 340 345 350 Ile Arg Asn Val Ile Lys Ala Cys Glu Asp Asp Ser Glu Phe Asn Asp 355 360 365 Tyr His Leu Val Asp Tyr Leu Thr Gly Asp Phe Leu Glu Glu Gln Tyr 370 375 380 Lys Gly Gln Arg Asp Leu Ala Gly Lys Ala Ser Thr Leu Lys Lys Leu 385 390 395 400 Met Asp Arg His Glu Ala Leu Gly Glu Phe Ile Phe Asp Lys Lys Leu 405 410 415 Leu Gly Ile Asp Val Asp Tyr Lys Asp Asp Asp Asp Lys 420 425 <210> 12 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> P2‑RBD‑ferritin LC <400> 12 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe Gly Ser Gly Ser Gly Glu Tyr Gly Ser His Gly Asn Val 245 250 255 Ala Thr Glu Leu Gln Ala Tyr Ala Lys Leu His Leu Glu Arg Ser Tyr 260 265 270 Asp Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn Tyr Gln Thr Asn Arg 275 280 285 Ala Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser Asp Glu Ala Trp Ser 290 295 300 Lys Thr Ile Asp Ile Ile Lys His Val Thr Lys Arg Gly Asp Lys Met 305 310 315 320 Asn Phe Asp Gln His Ser Thr Met Lys Thr Glu Arg Lys Asn Tyr Thr 325 330 335 Ala Glu Asn His Glu Leu Glu Ala Leu Ala Lys Ala Leu Asp Thr Gln 340 345 350 Lys Glu Leu Ala Glu Arg Ala Phe Tyr Ile His Arg Glu Ala Thr Arg 355 360 365 Asn Ser Gln His Leu His Asp Pro Glu Ile Ala Gln Tyr Leu Glu Glu 370 375 380 Glu Phe Ile Glu Asp His Ala Glu Lys Ile Arg Thr Leu Ala Gly His 385 390 395 400 Thr Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn Asn Gly His Asp Leu 405 410 415 Ser Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu Gln Lys Thr Val 420 425 430 <210> 13 <211> 429 <212> PRT <213> Artificial Sequence <220> <223> P2‑RBD‑ferritin HC <400> 13 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe Gly Ser Gly Ser Gly Arg Ser Cys Arg Asn Ser Met Arg 245 250 255 Gln Gln Ile Gln Met Glu Val Gly Ala Ser Leu Gln Tyr Leu Ala Met 260 265 270 Gly Ala His Phe Ser Lys Asp Val Val Asn Arg Pro Gly Phe Ala Gln 275 280 285 Leu Phe Phe Asp Ala Ala Ser Glu Glu Arg Glu His Ala Met Lys Leu 290 295 300 Ile Glu Tyr Leu Leu Met Arg Gly Glu Leu Thr Asn Asp Val Ser Ser 305 310 315 320 Leu Leu Gln Val Arg Pro Pro Thr Arg Ser Ser Trp Lys Gly Gly Val 325 330 335 Glu Ala Leu Glu His Ala Leu Ser Met Glu Ser Asp Val Thr Lys Ser 340 345 350 Ile Arg Asn Val Ile Lys Ala Cys Glu Asp Asp Ser Glu Phe Asn Asp 355 360 365 Tyr His Leu Val Asp Tyr Leu Thr Gly Asp Phe Leu Glu Glu Gln Tyr 370 375 380 Lys Gly Gln Arg Asp Leu Ala Gly Lys Ala Ser Thr Leu Lys Lys Leu 385 390 395 400 Met Asp Arg His Glu Ala Leu Gly Glu Phe Ile Phe Asp Lys Lys Leu 405 410 415 Leu Gly Ile Asp Val Asp Tyr Lys Asp Asp Asp Asp Lys 420 425 <210> 14 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> P2‑RBD‑ferritin <400> 14 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe Gly Ser Gly Ser Gly Asp Ile Ile Lys Leu Leu Asn Glu 245 250 255 Gln Val Asn Lys Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser 260 265 270 Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe 275 280 285 Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe 290 295 300 Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro 305 310 315 320 Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu 325 330 335 His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala 340 345 350 Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val 355 360 365 Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys 370 375 380 Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln 385 390 395 400 Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser 405 410 <210> 15 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> RBD‑P2‑HBsAg <400> 15 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 225 230 235 240 Thr Glu Leu Gly Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe 245 250 255 Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg 260 265 270 Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn 275 280 285 Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro 290 295 300 Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr 305 310 315 320 Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu 325 330 335 Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu 340 345 350 Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro 355 360 365 Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser 370 375 380 Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile 385 390 395 400 Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val 405 410 415 Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val 420 425 430 Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr 435 440 445 Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu 450 455 460 Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 465 470 <210> 16 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> P2-RBD-HBsAg <400> 16 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro 20 25 30 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 35 40 45 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 50 55 60 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 65 70 75 80 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 85 90 95 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 100 105 110 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 115 120 125 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 130 135 140 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 145 150 155 160 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 165 170 175 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 180 185 190 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 195 200 205 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 210 215 220 Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys 225 230 235 240 Val Asn Phe Gly Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe 245 250 255 Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg 260 265 270 Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn 275 280 285 Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro 290 295 300 Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr 305 310 315 320 Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu 325 330 335 Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu 340 345 350 Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro 355 360 365 Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser 370 375 380 Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile 385 390 395 400 Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val 405 410 415 Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val 420 425 430 Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr 435 440 445 Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu 450 455 460 Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 465 470 <210> 17 <211> 474 <212> PRT <213> Artificial Sequence <220> <223> RBD‑HBsAg‑P2 <400> 17 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly 210 215 220 Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro Leu 225 230 235 240 Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr Ile 245 250 255 Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly Gly 260 265 270 Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn His 275 280 285 Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met Cys 290 295 300 Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu Ile 305 310 315 320 Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys Pro 325 330 335 Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr Cys 340 345 350 Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys Thr 355 360 365 Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser Trp 370 375 380 Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser Trp 385 390 395 400 Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser Pro 405 410 415 Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro Ser 420 425 430 Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe Phe 435 440 445 Cys Leu Trp Val Tyr Ile Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala 450 455 460 Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 465 470 <210> 18 <211> 223 <212> PRT <213> Artificial Sequence <220> <223> RBD 319‑541 <400> 18 Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn 1 5 10 15 Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val 20 25 30 Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser 35 40 45 Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val 50 55 60 Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp 65 70 75 80 Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln 85 90 95 Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr 100 105 110 Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly 115 120 125 Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys 130 135 140 Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr 145 150 155 160 Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser 165 170 175 Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val 180 185 190 Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly 195 200 205 Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 210 215 220 <210> 19 <211> 194 <212> PRT <213> Artificial Sequence <220> <223> RBD 331-524 <400> 19 Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg 1 5 10 15 Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val 20 25 30 Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys 35 40 45 Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn 50 55 60 Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile 65 70 75 80 Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro 85 90 95 Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp 100 105 110 Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys 115 120 125 Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln 130 135 140 Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe 145 150 155 160 Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln 165 170 175 Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala 180 185 190 Thr Val <210> 20 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> NTD-P2 <400> 20 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys 290 295 300 Phe Ile Gly Ile Thr Glu Leu 305 310 <210> 21 <211> 311 <212> PRT <213> Artificial Sequence <220> <223> P2‑NTD <400> 21 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Lys 305 310 <210> 22 <211> 323 <212> PRT <213> Artificial Sequence <220> <223> NTD‑foldon <400> 22 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp 290 295 300 Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr 305 310 315 320 Phe Leu Gly <210> 23 <211> 478 <212> PRT <213> Artificial Sequence <220> <223> NTD-ferritin LC <400> 23 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Glu Tyr Gly Ser His Gly Asn Val Ala 290 295 300 Thr Glu Leu Gln Ala Tyr Ala Lys Leu His Leu Glu Arg Ser Tyr Asp 305 310 315 320 Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn Tyr Gln Thr Asn Arg Ala 325 330 335 Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser Asp Glu Ala Trp Ser Lys 340 345 350 Thr Ile Asp Ile Ile Lys His Val Thr Lys Arg Gly Asp Lys Met Asn 355 360 365 Phe Asp Gln His Ser Thr Met Lys Thr Glu Arg Lys Asn Tyr Thr Ala 370 375 380 Glu Asn His Glu Leu Glu Ala Leu Ala Lys Ala Leu Asp Thr Gln Lys 385 390 395 400 Glu Leu Ala Glu Arg Ala Phe Tyr Ile His Arg Glu Ala Thr Arg Asn 405 410 415 Ser Gln His Leu His Asp Pro Glu Ile Ala Gln Tyr Leu Glu Glu Glu 420 425 430 Phe Ile Glu Asp His Ala Glu Lys Ile Arg Thr Leu Ala Gly His Thr 435 440 445 Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn Asn Gly His Asp Leu Ser 450 455 460 Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu Gln Lys Thr Val 465 470 475 <210> 24 <211> 476 <212> PRT <213> Artificial Sequence <220> <223> NTD-ferritin HC <400> 24 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Arg Ser Cys Arg Asn Ser Met Arg Gln 290 295 300 Gln Ile Gln Met Glu Val Gly Ala Ser Leu Gln Tyr Leu Ala Met Gly 305 310 315 320 Ala His Phe Ser Lys Asp Val Val Asn Arg Pro Gly Phe Ala Gln Leu 325 330 335 Phe Phe Asp Ala Ala Ser Glu Glu Arg Glu His Ala Met Lys Leu Ile 340 345 350 Glu Tyr Leu Leu Met Arg Gly Glu Leu Thr Asn Asp Val Ser Ser Leu 355 360 365 Leu Gln Val Arg Pro Pro Thr Arg Ser Ser Trp Lys Gly Gly Val Glu 370 375 380 Ala Leu Glu His Ala Leu Ser Met Glu Ser Asp Val Thr Lys Ser Ile 385 390 395 400 Arg Asn Val Ile Lys Ala Cys Glu Asp Asp Ser Glu Phe Asn Asp Tyr 405 410 415 His Leu Val Asp Tyr Leu Thr Gly Asp Phe Leu Glu Glu Gln Tyr Lys 420 425 430 Gly Gln Arg Asp Leu Ala Gly Lys Ala Ser Thr Leu Lys Lys Leu Met 435 440 445 Asp Arg His Glu Ala Leu Gly Glu Phe Ile Phe Asp Lys Lys Leu Leu 450 455 460 Gly Ile Asp Val Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 475 <210> 25 <211> 521 <212> PRT <213> Artificial Sequence <220> <223> NTD‑HBsAg <400> 25 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe Leu 290 295 300 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile 305 310 315 320 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 325 330 335 Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 340 345 350 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg 355 360 365 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 370 375 380 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro 385 390 395 400 Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys 405 410 415 Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys 420 425 430 Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 435 440 445 Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg 450 455 460 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly 465 470 475 480 Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp 485 490 495 Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro 500 505 510 Ile Phe Phe Cys Leu Trp Val Tyr Ile 515 520 <210> 26 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> NTD‑P2‑foldon <400> 26 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30<(...)>Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Note: There seems to be an incomplete tag in line 39 which should be ` ` as per the pattern. I've translated it as `<(...)>` in the above output to maintain the integrity of the translation process. If it's a different tag, please correct it for a more accurate translation.Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 290 295 300 Ile Gly Ile Thr Glu Leu Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala 305 310 315 320 Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu 325 330 335 Leu Ser Thr Phe Leu Gly 340 <210> 27 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> P2‑NTD‑foldon <400> 27 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala 305 310 315 320 Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu 325 330 335 Leu Ser Thr Phe Leu Gly 340 <210> 28 <211> 342 <212> PRT <213> Artificial Sequence <220> <223> NTD-foldon-P2 <400> 28 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly 290 295 300 Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe 305 310 315 320 Leu Gly Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 325 330 335 Ile Gly Ile Thr Glu Leu 340 <210> 29 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> NTD‑P2‑ferritin LC <400> 29 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 290 295 300 Ile Gly Ile Thr Glu Leu Gly Ser Gly Ser Gly Glu Tyr Gly Ser His 305 310 315 320 Gly Asn Val Ala Thr Glu Leu Gln Ala Tyr Ala Lys Leu His Leu Glu 325 330 335 Arg Ser Tyr Asp Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn Tyr Gln 340 345 350 Thr Asn Arg Ala Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser Asp Glu 355 360 365 Ala Trp Ser Lys Thr Ile Asp Ile Ile Lys His Val Thr Lys Arg Gly 370 375 380 Asp Lys Met Asn Phe Asp Gln His Ser Thr Met Lys Thr Glu Arg Lys 385 390 395 400 Asn Tyr Thr Ala Glu Asn His Glu Leu Glu Ala Leu Ala Lys Ala Leu 405 410 415 Asp Thr Gln Lys Glu Leu Ala Glu Arg Ala Phe Tyr Ile His Arg Glu 420 425 430 Ala Thr Arg Asn Ser Gln His Leu His Asp Pro Glu Ile Ala Gln Tyr 435 440 445 Leu Glu Glu Glu Phe Ile Glu Asp His Ala Glu Lys Ile Arg Thr Leu 450 455 460 Ala Gly His Thr Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn Asn Gly 465 470 475 480 His Asp Leu Ser Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu Gln Lys 485 490 495 Thr Val <210> 30 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> NTD‑P2‑ferritin HC <400> 30 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 290 295 300 Ile Gly Ile Thr Glu Leu Gly Ser Gly Ser Gly Arg Ser Cys Arg Asn 305 310 315 320 Ser Met Arg Gln Gln Ile Gln Met Glu Val Gly Ala Ser Leu Gln Tyr 325 330 335 Leu Ala Met Gly Ala His Phe Ser Lys Asp Val Val Asn Arg Pro Gly 340 345 350 Phe Ala Gln Leu Phe Phe Asp Ala Ala Ser Glu Glu Arg Glu His Ala 355 360 365 Met Lys Leu Ile Glu Tyr Leu Leu Met Arg Gly Glu Leu Thr Asn Asp 370 375 380 Val Ser Ser Leu Leu Gln Val Arg Pro Pro Thr Arg Ser Ser Trp Lys 385 390 395 400 Gly Gly Val Glu Ala Leu Glu His Ala Leu Ser Met Glu Ser Asp Val 405 410 415 Thr Lys Ser Ile Arg Asn Val Ile Lys Ala Cys Glu Asp Asp Ser Glu 420 425 430 Phe Asn Asp Tyr His Leu Val Asp Tyr Leu Thr Gly Asp Phe Leu Glu 435 440 445 Glu Gln Tyr Lys Gly Gln Arg Asp Leu Ala Gly Lys Ala Ser Thr Leu 450 455 460 Lys Lys Leu Met Asp Arg His Glu Ala Leu Gly Glu Phe Ile Phe Asp 465 470 475 480 Lys Lys Leu Leu Gly Ile Asp Val 485 <210> 31 <211> 498 <212> PRT <213> Artificial Sequence <220> <223> P2-NTD-ferritin LC <400> 31 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Gly Ser Gly Ser Gly Glu Tyr Gly Ser His 305 310 315 320 Gly Asn Val Ala Thr Glu Leu Gln Ala Tyr Ala Lys Leu His Leu Glu 325 330 335 Arg Ser Tyr Asp Tyr Leu Leu Ser Ala Ala Tyr Phe Asn Asn Tyr Gln 340 345 350 Thr Asn Arg Ala Gly Phe Ser Lys Leu Phe Lys Lys Leu Ser Asp Glu 355 360 365 Ala Trp Ser Lys Thr Ile Asp Ile Ile Lys His Val Thr Lys Arg Gly 370 375 380 Asp Lys Met Asn Phe Asp Gln His Ser Thr Met Lys Thr Glu Arg Lys 385 390 395 400 Asn Tyr Thr Ala Glu Asn His Glu Leu Glu Ala Leu Ala Lys Ala Leu 405 410 415 Asp Thr Gln Lys Glu Leu Ala Glu Arg Ala Phe Tyr Ile His Arg Glu 420 425 430 Ala Thr Arg Asn Ser Gln His Leu His Asp Pro Glu Ile Ala Gln Tyr 435 440 445 Leu Glu Glu Glu Phe Ile Glu Asp His Ala Glu Lys Ile Arg Thr Leu 450 455 460 Ala Gly His Thr Ser Asp Leu Lys Lys Phe Ile Thr Ala Asn Asn Gly 465 470 475 480 His Asp Leu Ser Leu Ala Leu Tyr Val Phe Asp Glu Tyr Leu Gln Lys 485 490 495 Thr Val <210> 32 <211> 488 <212> PRT <213> Artificial Sequence <220> <223> P2‑NTD‑ferritin HC <400> 32 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Gly Ser Gly Ser Gly Arg Ser Cys Arg Asn 305 310 315 320 Ser Met Arg Gln Gln Ile Gln Met Glu Val Gly Ala Ser Leu Gln Tyr 325 330 335 Leu Ala Met Gly Ala His Phe Ser Lys Asp Val Val Asn Arg Pro Gly 340 345 350 Phe Ala Gln Leu Phe Phe Asp Ala Ala Ser Glu Glu Arg Glu His Ala 355 360 365 Met Lys Leu Ile Glu Tyr Leu Leu Met Arg Gly Glu Leu Thr Asn Asp 370 375 380 Val Ser Ser Leu Leu Gln Val Arg Pro Pro Thr Arg Ser Ser Trp Lys 385 390 395 400 Gly Gly Val Glu Ala Leu Glu His Ala Leu Ser Met Glu Ser Asp Val 405 410 415 Thr Lys Ser Ile Arg Asn Val Ile Lys Ala Cys Glu Asp Asp Ser Glu 420 425 430 Phe Asn Asp Tyr His Leu Val Asp Tyr Leu Thr Gly Asp Phe Leu Glu 435 440 445 Glu Gln Tyr Lys Gly Gln Arg Asp Leu Ala Gly Lys Ala Ser Thr Leu 450 455 460 Lys Lys Leu Met Asp Arg His Glu Ala Leu Gly Glu Phe Ile Phe Asp 465 470 475 480 Lys Lys Leu Leu Gly Ile Asp Val 485 <210> 33 <211> 478 <212> PRT <213> Artificial Sequence <220> <223> P2‑NTD‑ferritin <400> 33 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Gly Ser Gly Ser Gly Asp Ile Ile Lys Leu 305 310 315 320 Leu Asn Glu Gln Val Asn Lys Glu Met Asn Ser Ser Asn Leu Tyr Met 325 330 335 Ser Met Ser Ser Trp Cys Tyr Thr His Ser Leu Asp Gly Ala Gly Leu 340 345 350 Phe Leu Phe Asp His Ala Ala Glu Glu Tyr Glu His Ala Lys Lys Leu 355 360 365 Ile Ile Phe Leu Asn Glu Asn Asn Val Pro Val Gln Leu Thr Ser Ile 370 375 380 Ser Ala Pro Glu His Lys Phe Glu Gly Leu Thr Gln Ile Phe Gln Lys 385 390 395 400 Ala Tyr Glu His Glu Gln His Ile Ser Glu Ser Ile Asn Asn Ile Val 405 410 415 Asp His Ala Ile Lys Ser Lys Asp His Ala Thr Phe Asn Phe Leu Gln 420 425 430 Trp Tyr Val Ala Glu Gln His Glu Glu Glu Val Leu Phe Lys Asp Ile 435 440 445 Leu Asp Lys Ile Glu Leu Ile Gly Asn Glu Asn His Gly Leu Tyr Leu 450 455 460 Ala Asp Gln Tyr Val Lys Gly Ile Ala Lys Ser Arg Lys Ser 465 470 475 <210> 34 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> NTD‑P2‑HBsAg <400> 34 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe 290 295 300 Ile Gly Ile Thr Glu Leu Gly Ser Gly Ser Gly Met Glu Asn Ile Thr 305 310 315 320 Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu 325 330 335 Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr 340 345 350 Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser 355 360 365 Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys 370 375 380 Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe 385 390 395 400 Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln 405 410 415 Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser 420 425 430 Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met 435 440 445 Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys 450 455 460 Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp 465 470 475 480 Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln 485 490 495 Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met 500 505 510 Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile 515 520 525 Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 530 535 540 <210> 35 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> P2‑NTD‑HBsAg <400> 35 Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu Gly 1 5 10 15 Ser Gly Ser Gly Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Gly Ser Gly Ser Gly Met Glu Asn Ile Thr 305 310 315 320 Ser Gly Phe Leu Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu 325 330 335 Leu Thr Arg Ile Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr 340 345 350 Ser Leu Asn Phe Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser 355 360 365 Gln Ser Pro Thr Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys 370 375 380 Pro Gly Tyr Arg Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe 385 390 395 400 Ile Leu Leu Leu Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln 405 410 415 Gly Met Leu Pro Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser 420 425 430 Thr Gly Pro Cys Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met 435 440 445 Phe Pro Ser Cys Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys 450 455 460 Ile Pro Ile Pro Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp 465 470 475 480 Ala Ser Val Arg Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln 485 490 495 Trp Phe Val Gly Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met 500 505 510 Met Trp Tyr Trp Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile 515 520 525 Pro Leu Leu Pro Ile Phe Phe Cys Leu Trp Val Tyr Ile 530 535 540 <210> 36 <211> 541 <212> PRT <213> Artificial Sequence <220> <223> NTD-HBsAg-P2 <400> 36 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Gly Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe Leu 290 295 300 Gly Pro Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile 305 310 315 320 Leu Thr Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 325 330 335 Leu Gly Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr 340 345 350 Ser Asn His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg 355 360 365 Trp Met Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu 370 375 380 Cys Leu Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro 385 390 395 400 Val Cys Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys 405 410 415 Lys Thr Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys 420 425 430 Cys Cys Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro 435 440 445 Ser Ser Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg 450 455 460 Phe Ser Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly 465 470 475 480 Leu Ser Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp 485 490 495 Gly Pro Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro 500 505 510 Ile Phe Phe Cys Leu Trp Val Tyr Ile Gly Ser Gly Ser Gly Gln Tyr 515 520 525 Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu 530 535 540 <210> 37 <211> 291 <212> PRT <213> Artificial Sequence <220> <223> NTD 14‑304 <400> 37 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys 290 <210> 38 <211> 336 <212> PRT <213> Artificial Sequence <220> <223> NTD 18‑353 <400> 38 Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr 1 5 10 15 Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His 20 25 30 Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe 35 40 45 His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn 50 55 60 Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys 65 70 75 80 Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys 85 90 95 Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys 100 105 110 Val Cys Glu Phe Gln Phe Cys Asn Asp Gly Val Tyr Tyr 115 120 125 His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser 130 135 140 Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met 145 150 155 160 Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val 165 170 175 Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro 180 185 190 Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro 195 200 205 Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu 210 215 220 Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly 225 230 235 240 Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg 245 250 255 Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val 260 265 270 Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser 275 280 285 Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln 290 295 300 Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro 305 310 315 320 Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp 325 330 335 <210> 39 <211> 536 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑6His <400> 39 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Gly Ser His His His His His His 530 535 <210> 40 <211> 556 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑P2‑6His <400> 40 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 530 535 540 Ile Thr Glu Leu Gly Ser His His His His His His 545 550 555 <210> 41 <211> 588 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑foldon‑6His <400> 41 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr 305 310 315 320 Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 325 330 335 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 340 345 350 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 355 360 365 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 370 375 380 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 385 390 395 400 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 405 410 415 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 420 425 430 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 435 440 445 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 450 455 460 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 465 470 475 480 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 485 490 495 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 500 505 510 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 515 520 525 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 530 535 540 Cys Val Asn Phe Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg 545 550 555 560 Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser 565 570 575 Thr Phe Leu Gly Gly Ser His His His His His His 580 585 <210> 42 <211> 608 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑P2‑foldon‑6His <400> 42 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro 20 25 30 Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys 35 40 45 Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro 50 55 60 Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr 65 70 75 80 Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly 85 90 95 Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile 100 105 110 Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn 115 120 125 Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn 130 135 140 Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met 145 150 155 160 Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu 165 170 175 Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn 180 185 190 Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe 195 200 205 Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro 210 215 220 Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile 225 230 235 240 Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu 245 250 255 Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr 260 265 270 Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu 275 280 285 Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser 290 295 300 Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr 305 310 315 320 Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe 325 330 335 Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr 340 345 350 Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys 355 360 365 Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe 370 375 380 Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr 385 390 395 400 Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln 405 410 415 Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu 420 425 430 Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu 435 440 445 Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg 450 455 460 Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr 465 470 475 480 Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr 485 490 495 Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr 500 505 510 Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro 515 520 525 Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys 530 535 540 Cys Val Asn Phe Gly Ser Gly Ser Gly Gln Tyr Ile Lys Ala Asn Ser 545 550 555 560 Lys Phe Ile Gly Ile Thr Glu Leu Gly Ser Gly Ser Gly Tyr Ile Pro 565 570 575 Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp 580 585 590 Val Leu Leu Ser Thr Phe Leu Gly Gly Ser His His His His His His 595 600 605 <210> 43 <211> 695 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑ferritin <400> 43 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Met Leu Ser Lys Asp Ile Ile Lys Leu Leu Asn Glu Gln Val Asn Lys 530 535 540 Glu Met Asn Ser Ser Asn Leu Tyr Met Ser Met Ser Ser Trp Cys Tyr 545 550 555 560 Thr His Ser Leu Asp Gly Ala Gly Leu Phe Leu Phe Asp His Ala Ala 565 570 575 Glu Glu Tyr Glu His Ala Lys Lys Leu Ile Ile Phe Leu Asn Glu Asn 580 585 590 Asn Val Pro Val Gln Leu Thr Ser Ile Ser Ala Pro Glu His Lys Phe 595 600 605 Glu Gly Leu Thr Gln Ile Phe Gln Lys Ala Tyr Glu His Glu Gln His 610 615 620 Ile Ser Glu Ser Ile Asn Asn Ile Val Asp His Ala Ile Lys Ser Lys 625 630 635 640 Asp His Ala Thr Phe Asn Phe Leu Gln Trp Tyr Val Ala Glu Gln His 645 650 655 Glu Glu Glu Val Leu Phe Lys Asp Ile Leu Asp Lys Ile Glu Leu Ile 660 665 670 Gly Asn Glu Asn His Gly Leu Tyr Leu Ala Asp Gln Tyr Val Lys Gly 675 680 685 Ile Ala Lys Ser Arg Lys Ser 690 695 <210> 44 <211> 759 <212> PRT <213> Artificial Sequence <220> <223> NTD‑RBD‑HBsAg <400> 44 Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr 1 5 10 15 Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser 20 25 30 Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn 35 40 45 Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys 50 55 60 Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala 65 70 75 80 Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr 85 90 95 Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn 100 105 110 Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu 115 120 125 Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe 130 135 140 Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln 145 150 155 160 Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu 165 170 175 Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser 180 185 190 Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser 195 200 205 Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg 210 215 220 Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp 225 230 235 240 Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr 245 250 255 Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile 260 265 270 Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys 275 280 285 Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn 290 295 300 Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr 305 310 315 320 Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser 325 330 335 Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr 340 345 350 Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly 355 360 365 Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala 370 375 380 Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly 385 390 395 400 Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe 405 410 415 Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val 420 425 430 Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu 435 440 445 Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser 450 455 460 Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln 465 470 475 480 Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg 485 490 495 Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys 500 505 510 Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 515 520 525 Gly Ser Gly Ser Gly Met Glu Asn Ile Thr Ser Gly Phe Leu Gly Pro 530 535 540 Leu Leu Val Leu Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile Leu Thr 545 550 555 560 Ile Pro Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe Leu Gly 565 570 575 Gly Ser Pro Val Cys Leu Gly Gln Asn Ser Gln Ser Pro Thr Ser Asn 580 585 590 His Ser Pro Thr Ser Cys Pro Pro Ile Cys Pro Gly Tyr Arg Trp Met 595 600 605 Cys Leu Arg Arg Phe Ile Ile Phe Leu Phe Ile Leu Leu Leu Cys Leu 610 615 620 Ile Phe Leu Leu Val Leu Leu Asp Tyr Gln Gly Met Leu Pro Val Cys 625 630 635 640 Pro Leu Ile Pro Gly Ser Thr Thr Thr Ser Thr Gly Pro Cys Lys Thr 645 650 655 Cys Thr Thr Pro Ala Gln Gly Asn Ser Met Phe Pro Ser Cys Cys Cys 660 665 670 Thr Lys Pro Thr Asp Gly Asn Cys Thr Cys Ile Pro Ile Pro Ser Ser 675 680 685 Trp Ala Phe Ala Lys Tyr Leu Trp Glu Trp Ala Ser Val Arg Phe Ser 690 695 700 Trp Leu Ser Leu Leu Val Pro Phe Val Gln Trp Phe Val Gly Leu Ser 705 710 715 720 Pro Thr Val Trp Leu Ser Ala Ile Trp Met Met Trp Tyr Trp Gly Pro 725 730 735 Ser Leu Tyr Ser Ile Val Ser Pro Phe Ile Pro Leu Leu Pro Ile Phe 740 745 750 Phe Cys Leu Trp Val Tyr Ile 755 <210> 45 <211> 1500 <212> DNA <213> Artificial Sequence <220> <223> RBD-HBsAg-6*HIS <400> 45 ggatccgccg ccaccatggg cgtgcccgct gtgccagagg ccagctctcc taggtggggc 60 accctgctgc tggccatctt tctggccgcc agcagaggcc tggtggccgc tagagtgcag 120 cctaccgaga gcatcgtgcg gttcccaaac atcaccaacc tgtgtccttt cggcgaggtg 180 ttcaatgcca ccaggttcgc tagcgtgtat gcctggaaca ggaagaggat ctccaattgt 240 gtggctgact actccgtgct gtacaacagc gcttccttta gcaccttcaa gtgctacggc 300 gtgtccccca ccaagctgaa cgacttgtgc tttaccaacg tgtatgccga cagctttgtg 360 atcaggggcg acgaggtgcg gcagattgct cctggacaga ccggaaagat cgctgactat 420 aattacaagc tgcccgacga cttcaccggc tgcgttatcg cttggaatag caacaatctg 480 gattccaagg tgggcggcaa ttacaattat ctgtaccggc tgttcaggaa gtccaacctg 540 aagcccttcg agagggatat cagcaccgag atctatcagg ctggctccac cccctgtaac 600 ggcgttgaag gattcaattg ctacttcccc ctgcagagct acggctttca gcctaccaat 660 ggcgtgggct atcagcccta cagggtggtt gtgctgagct ttgagctgct gcacgctcct 720 gctaccgtgt gtggaccaaa gaagtccacc aacctggtga agaataagtg cgtgaatttt 780 ggatctggaa gcggaatgga gaacatcacc tccggcttcc tgggcccact gttggttctg 840 caggccggat tcttcctgct gaccagaatc ctgaccatcc cccagtccct ggacagctgg 900 tggacatctc tgaattttct gggcggctcc cccgtgtgcc tgggacagaa ctctcagagc 960 cccaccagca accactcccc aacaagctgc ccccctatct gtcccggata taggtggatg 1020 tgtctgaggc ggttcatcat cttcctgttt atcctgctgc tgtgcctgat cttcctgctg 1080 gtgctgctgg actaccaggg catgctgcca gtgtgccctc tgatcccagg ctctaccacc 1140 accagcaccg gaccttgtaa gacctgcacc acccctgccc agggaaatag catgtttcct 1200 tcctgttgct gcaccaagcc caccgatggc aactgcacct gtatccccat ccctagcagc 1260 tgggcctttg ctaagtacct gtgggagtgg gcttccgtgc ggtttagctg gctgtccctg 1320 ctggtgcctt tcgtgcagtg gttcgtgggc ctgagcccta cagtgtggct gtctgctatc 1380 tggatgatgt ggtattgggg ccctagcctg tacagcatcg tgtccccatt cattcctctg 1440 ctgcctatct ttttctgcct gtgggtgtat atccaccacc accaccatca ctgactcgag 1500 <210> 46 <211> 1644 <212> DNA <213> Artificial Sequence <220> <223> NTD-HBsAg-6*HIS <400> 46 ggatccgccg ccaccatgtt cgtgtttctg gtgctgctgc ccctggtgtc ctcccagtgt 60 gttaatctga ccaccaggac ccagctgcct cctgcttaca ccaattcctt taccaggggc 120 gtgtactacc ccgacaaggt gttcaggtcc tccgtgctgc acagcaccca ggatctgttc 180 ctgcctttct ttagcaacgt gacctggttc cacgctatcc acgtgagcgg caccaacgga 240 accaagagat tcgacaatcc cgtgctgccc ttcaacgatg gcgtgtactt cgccagcacc 300 gagaagagca acatcatccg gggctggatc ttcggcacca ccttggattc caagacccag 360 agcctgctga tcgtgaacaa cgctaccaac gtggtcatta aggtgtgtga gttccagttt 420 tgtaacgacc cctttctggg cgtgtattat cacaagaaca ataagtcctg gatggagagc 480 gagtttaggg tgtattcctc cgctaacaac tgcacctttg agtatgtgag ccagcctttt 540 ctgatggacc tggagggcaa gcagggcaat ttcaagaacc tgagggagtt cgtgtttaag 600 aacatcgatg gctactttaa gatctactcc aagcacaccc ctatcaacct ggtgcgggat 660 ctgcctcagg gctttagcgc tctggagcct ctggtggatc tgccaatcgg catcaatatc 720 acccggtttc agaccctgct ggctctgcac aggagctacc tgacccctgg agatagctcc 780 agcggctgga cagctggagc tgctgcttac tacgtgggct acctgcagcc cagaaccttt 840 ctgctgaagt acaatgagaa cggcaccatc accgacgccg tggattgcgc tctggaccct 900 ttgtccgaga ccaagtgtac cctgggatct ggaagcggaa tggagaacat cacctccggc 960 ttcctgggcc cactgttggt tctgcaggcc ggattcttcc tgctgaccag aatcctgacc 1020 atcccccagt ccctggacag ctggtggaca tctctgaatt t...

Claims

1. A fusion protein comprising: 1) the N-terminal domain (NTD) of the SARS-CoV-2 spike protein (S protein); 2) the receptor-binding domain (RBD) of the SARS-CoV-2 S protein; and 3) a foldon domain; wherein The NTD is derived from wild-type SARS-CoV-2, Gamma mutant strain, or Beta mutant strain; The RBD is derived from wild-type SARS-CoV-2, Gamma mutant strain, or Beta mutant strain; The NTD and the RBD are derived from the same strain; The amino acid sequence of the foldon domain is shown in any one of SEQ ID NO: 67-69 and 78; The C-terminus of the NTD is connected to the N-terminus of the RBD via the connector shown in SEQ ID NO:90, and the C-terminus of the RBD is connected to the foldon structural domain via the connector shown in SEQ ID NO:

89.

2. The fusion protein according to claim 1, wherein the amino acid sequence of the NTD is shown in any one of SEQ ID NO: 37, 77 and 84.

3. The fusion protein according to claim 1, wherein the amino acid sequence of the RBD is shown in any one of SEQ ID NO: 18, 76 and 83.

4. The fusion protein according to claim 1, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID NO: 41, 79, 85 and 96.

5. The fusion protein according to claim 1, wherein the fusion protein derived from wild-type SARS-CoV-2 further comprises a tetanus toxin epitope peptide P2, the N-terminus of P2 being linked to RBD via the linker shown in SEQ ID NO:89, and the C-terminus being linked to the foldon domain via the linker shown in SEQ ID NO:

89.

6. The fusion protein according to claim 5, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO:

42.

7. An immunogenic composition comprising the fusion protein of any one of claims 1-6.

8. An immunogenic composition comprising a first component and a second component, wherein the first component is a fusion protein comprising the NTD of the SARS-CoV-2S protein and a foldon domain, and the second component is a fusion protein comprising the RBD of the SARS-CoV-2S protein and a foldon domain; wherein The NTD and the RBD are derived from the wild type of SARS-CoV-2; The amino acid sequence of the foldon domain is shown in any one of SEQ ID NO: 67-69 and 78; The C-terminus of the NTD is connected to the foldon structural domain via the connector shown in SEQ ID NO:89; the C-terminus of the RBD is connected to the foldon structural domain via the connector shown in SEQ ID NO:

89.

9. The immunogenic composition according to claim 8, wherein the amino acid sequence of the NTD is as shown in SEQ ID NO:

37.

10. The immunogenic composition according to claim 8, wherein the amino acid sequence of the RBD is as shown in SEQ ID NO:

18.

11. The immunogenic composition of claim 8, wherein the amino acid sequence of the fusion protein in the first component is as shown in SEQ ID NO:22, and the amino acid sequence of the fusion protein in the second component is as shown in SEQ ID NO:

3.

12. The immunogenic composition according to claim 8, which is formulated in the following weight ratios: 1) 1-15 parts by weight of the first component; and 2) 1-15 parts by weight of the second component.

13. The immunogenic composition according to claim 8, wherein the contents of the first component and the second component are: 1) 5-60 μg of the first component; and 2) 5-60 μg of the second component.

14. The immunogenic composition according to any one of claims 8-13, further comprising at least one human influenza virus hemagglutinin protein HA, the amino acid sequence of said HA being shown in any one of SEQ ID NO: 92-95.

15. An isolated nucleic acid molecule encoding the fusion protein of any one of claims 1-6.

16. The nucleic acid molecule according to claim 15, wherein it is mRNA.

17. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-6, an immunogenic composition according to any one of claims 7-14, or a nucleic acid molecule according to any one of claims 15-16, and optionally a pharmaceutically acceptable excipient.

18. Use of the fusion protein of any one of claims 1-6, the immunogenic composition of any one of claims 7-14, the nucleic acid molecule of any one of claims 15-16, or the pharmaceutical composition of claim 17 in the preparation of a vaccine for the prevention and / or treatment of COVID-19.

19. A method for preparing a COVID-19 subunit vaccine, comprising: 1) Provide the fusion protein according to any one of claims 1-6, the immunogenic composition according to any one of claims 7-14, or the nucleic acid molecule according to any one of claims 15-16; as well as 2) Mix the fusion protein, immunogenic composition or nucleic acid molecule described in 1) with a pharmaceutically acceptable adjuvant.

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