A sars-cov-2 epitope type vaccine multi-epitope combination and application

Abstract

The application discloses a SARS-CoV-2 epitope type vaccine multi-epitope combination and application, and belongs to the technical field of coronavirus vaccine research and development.The first aspect of the application relates to a fusion protein, which comprises in sequence: (a) a SARS-CoV-2 spike protein receptor binding domain or a functional fragment thereof; (b) a T cell epitope domain, comprising: a CTL epitope cluster, the CTL epitope cluster comprising at least one CD8+ T cell epitope polypeptide selected from SEQ ID NO: 1-15; and (c) an immunoglobulin Fc domain.The application adopts a tandem strategy of immunodominant epitopes + conserved epitopes, predicts high-affinity T cell epitopes by computational biology methods, evaluates the HLA restriction in different populations, introduces a flexible linker peptide for optimization design, evaluates the immune effect difference of different combinations through in vitro and animal models, analyzes the synergistic or competitive relationship between epitopes, and optimizes the vaccine design.Through systematic comparison of the immunological effect difference of different epitope combinations and the adaptability to various vaccine platforms, the application establishes an optimized safe, efficient, broad-spectrum and long-acting multi-epitope vaccine design strategy, and has significant application value and important transformation value.

Description

Technical Field

[0001] This invention relates to the field of coronavirus vaccine research and development technology, specifically to a multi-epitope combination of SARS-CoV-2 epitope-type vaccines and its application. Background Technology

[0002] The COVID-19 pandemic caused by SARS-CoV-2 has had a profound impact on global health and socioeconomic development. While existing vaccines (such as inactivated vaccines, mRNA vaccines, and adenovirus vector vaccines) have played a significant role in reducing severe illness and mortality rates, the efficacy of neutralizing antibodies induced by some vaccines has significantly decreased due to continuous viral mutations (such as Omicron and its subspecies), highlighting the limitations of relying solely on humoral immunity. Epitope-based vaccines, as an emerging precision immunization strategy, are rationally designed by screening key B-cell and T-cell epitopes, offering potential advantages such as high safety, ease of large-scale production, and rapid adaptation to variant strains, making them a key direction for next-generation vaccine development. T-cell immunity plays a crucial role in clearing virus-infected cells, providing long-term protection, and responding to variant strains; therefore, T-cell epitope-based vaccine design has become an important strategy for enhancing broad-spectrum immune protection.

[0003] Against the backdrop of SARS-CoV-2's continuous evolution leading to immune evasion, the development of vaccines with broad-spectrum protective efficacy urgently needs to meet two core requirements: firstly, it must target highly conserved antigenic epitopes; and secondly, it must possess the molecular basis for activating polyclonal T-cell immune responses. In this context, multi-epitope vaccines based on conserved epitopes have become a strategic direction for novel vaccine development due to their potential to overcome immune evasion by variant strains.

[0004] Compared with traditional vaccines, T-cell epitope vaccines have the following advantages: (1) they can target conserved viral regions, reducing the risk of immune escape; (2) they are not affected by antibody-dependent enhancement (ADE); and (3) they are easy to combine multiple epitopes to cover populations with different HLA (human leukocyte antigen) backgrounds. However, their development still faces key challenges, including optimizing the immunogenicity of epitopes, differences in population coverage due to HLA restrictions, and the immune interference effects that may be caused by the combination of multiple epitopes. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a multi-epitope combination for SARS-CoV-2 epitope-type vaccines and its application.

[0006] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a recombinant fusion protein, comprising, in sequence: (a) The receptor-binding domain of the SARS-CoV-2 spike protein or a functional fragment thereof; (b) A T cell epitope domain comprising: a CTL epitope cluster, wherein the CTL epitope cluster includes at least one CD8+ T cell epitope polypeptide selected from SEQ ID NO: 1-15; And, (c) the immunoglobulin Fc domain.

[0007] Optionally, the amino acid sequence of the SARS-CoV-2 spike protein receptor-binding domain is shown in SEQ ID NO: 16; and the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO: 19.

[0008] Optionally, the CTL epitope domain comprises all CD8+ T cell epitope peptides as shown in SEQ ID NO: 1-15.

[0009] Optionally, the domains and epitope peptides in the recombinant fusion protein are connected by linkers. The amino acid sequence of the linker is shown in SEQ ID NO: 28.

[0010] A second aspect of the invention relates to an isolated nucleic acid molecule that encodes the aforementioned recombinant fusion protein.

[0011] Optionally, the nucleic acid molecule is mRNA, comprising: derived from human... β - The 5' untranslated region of the globulin gene, the signal peptide sequence encoding the tissue plasminogen activator, the coding region encoding the recombinant fusion protein; and the poly-A tail.

[0012] A third aspect of the present invention relates to an immunogenic composition comprising: The aforementioned nucleic acid molecules; and Lipid nanoparticles encapsulating the nucleic acid molecules.

[0013] Optionally, the lipid nanoparticles have an average particle size of 140 nm to 60 nm.

[0014] A fourth aspect of the present invention relates to the use of the above-described recombinant fusion protein, or the above-described nucleic acid molecule, or the above-described immunogenic composition in the preparation of a medicament for the prevention or treatment of coronavirus infectious diseases.

[0015] Optionally, the coronavirus infectious disease is caused by the SARS-CoV-2 Omicron variant.

[0016] The beneficial effects of this invention are: This invention employs a tandem strategy of "immunodominant epitopes + conserved epitopes," using computational biology methods to predict high-affinity T-cell epitopes and assess their HLA restriction in different populations. Flexible linker peptides are introduced for optimized design, and the differences in immune effects of different combinations are evaluated using in vitro and animal models. The synergistic or competitive relationships between epitopes are analyzed to optimize vaccine design. By systematically comparing the differences in immunological effects of different epitope combinations and their compatibility with various vaccine platforms, this study establishes an optimized, safe, efficient, broad-spectrum, and long-lasting multi-epitope vaccine design strategy, demonstrating significant application and translational value. Attached Figure Description

[0017] The invention will now be further described with reference to the accompanying drawings.

[0018] Figure 1 A summary of dominant HLA gene frequencies in Chinese and Caucasian populations; Figure 2 and Figure 15 For the 117 CD8 entries of this application + Results of T-cell epitope peptide detection in 30 recovered patients; Figure 2 In the table, (a) represents a candidate epitope for the E protein, and (b) represents a candidate epitope for the M protein. Figure 15 In the table, (c) represents the candidate epitope of the N protein, and (d) represents the candidate epitope of the S protein; Figure 3 For the 15 CD8 entries of this application + MHC restriction and scoring of T cell epitope peptides; Figure 4 For the secondary structure analysis of the CTL combination in this application, H: α-helix, S: β-sheet, T: β-turn, and C: random coil. Figure 5 For the structural analysis of the combined form of this application, the RBD-CTL-Fc combination is used; Figure 6 For the analysis of the secondary structure in the form of a combination, the RBD-CTL-Fc combination is used. Figure 7 For the analysis of the three-level structure in the form of combination, the RBD-CTL-Fc combination is used. Figure 8 For combined Laplace chart analysis, the RBD-CTL-Fc combination is used. Figure 9 This is a schematic diagram of the mRNA assembly in this application; wherein, (a) is a schematic diagram of the ligation insertion fragment, and (b) is a schematic diagram of the mRNA construction; Figure 10The encapsulation rates of LNP-encapsulated mRNAs in different combinations of this application are shown; where (a) is the RBD-CTL-Fc combination, (b) is the RBD-Fc combination, and (c) is the Fc combination. Figure 11 The specific antibody levels of mice immunized with different combinations of multi-epitope candidate vaccines of this application were measured; among them, (a) is the ELISA result data of HLA-A2 / DR1 double transgenic mice, and (b) is the ELISA result data of HLA-A11 / DR1 double transgenic mice. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. P < 0.0001; ns indicates no statistically significant difference; Figure 12 For the purposes of this application HLA-A2 / DR1 and HLA-A11 / DR1 ELISpot results of double transgenic mice; (a) shows the ELISpot results of HLA-A2 / DR1 double transgenic mice stimulated with A2 mixed peptides, and (b) shows the ELISpot results of HLA-A11 / DR1 double transgenic mice stimulated with A11 mixed peptides. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. P < 0.0001; ns indicates no statistically significant difference; Figure 13 This is an evaluation of the immunoprotective effect of the mouse vaccine in this application; the image shows the qPCR results of lung tissue from HLA-A2 / DR1-HACE2 transgenic mice after challenge. This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001. P < 0.0001; ns indicates no statistically significant difference; Figure 14 The following images are used to evaluate the pathological damage of lung tissue in mice challenged with the virus in this application: (a) is a pathological image of RBD-CTL-Fc, (b) is a pathological image of RBD-Fc, and (c) is a pathological image of Fc. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1: SARS-CoV-2 viral protein sequence search and MHC restriction selection 1. Protein sequence retrieval After data analysis, 14 original antigenic protein sequences of SARS-CoV-2 (WuHan strain) were retrieved from the NCBI database. These included the structural proteins: envelope protein E (75 amino acids), membrane protein M (222 amino acids), nucleocapsid protein N (419 amino acids), and spike protein S (1273 amino acids). The precursor proteins ORF1a and ORF1b contained 4405 and 7096 amino acids, respectively. The remaining accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 contained 275, 61, 121, 43, 121, and 38 amino acids, respectively.

[0021] 2. MHC Restrictive Selection The high-frequency MHC restriction patterns identified in the Chinese population were summarized and a heatmap was created. Figure 1 The darker the color, the higher the frequency of MHC restriction. By comparing the high-frequency MHC restriction in the Chinese population with that in Caucasians, high-frequency MHC restriction in the Chinese population was identified.

[0022] 1923 CTL epitope peptides with complete and correct sequence information were obtained through IEDB screening. These T cell epitopes were then organized, and a preliminary database of high-frequency MHC-restricted T cell epitopes in the Chinese population was established.

[0023] Example 2: Establishment of a database of T cell epitopes and high MHC affinity 1. Screening of T-cell epitopes using intracellular cytokine detection method in SARS-CoV-2 recovered patients. Our research group collected peripheral blood samples from 30 recovered SARS-CoV-2 patients and used an intracellular cytokine detection method to analyze 117 CD8+ cells. + A systematic study of T-cell epitope peptides was conducted. Results were screened through visual analysis. Figure 2As can be observed from the two-dimensional and three-dimensional graphs, the higher the height of the block bar, the stronger the individual's response to the restriction; and the greater the number of similar peptides, the wider the number of individuals covered by the peptide segment.

[0024] Based on the viral protein structure: The S protein's RBD region is located at 319-514, the transmembrane region at 1213-1237, and the intracellular region at 1237-1273. E protein: Intramembrane region located at 1-11, transmembrane region located at 12-37, extramembrane region located at 38-75. M protein: Extramembrane region located at 1-19, 74-77; transmembrane region located at 20-39, 52-73, 79-100; intramembrane region located at 41-50, 104-222. N protein: entirely within the membrane. In T cell epitope selection, intramembrane regions should be preferred. However, since the M protein crosses the membrane multiple times, epitopes targeting the M protein should be selected primarily based on experimental results.

[0025] 2. Establishment of a candidate T cell epitope database The experimental results screened in Example 1 were integrated and analyzed with high-frequency MHC-restricted epitopes in the Chinese population reported in the IEDB database. Efforts were made to screen for T-cell epitope peptides that overlapped between the two methods. If the number of candidate epitope peptides was insufficient, Experiment 1 was used as the primary selection criterion. Ultimately, 15 CD8 epitopes were identified. + T cell epitope peptides (Table 1, ...) Figure 3 ).

[0026] Table 1. CD8 + T cell candidate epitope peptides Continued from Table 1. CD8 + T cell candidate epitope peptides Example 3: Epitope Combination Design Strategy 1. Combined Single-Unit Predictive Analysis CD8 +T cell epitopes were combined, and each peptide was linked together using a linker. After optimizing the linking results using relevant software, the combination was called the CTL combination. For linker selection, flexible linkers that have been validated and reported in the literature were selected: GGGGS, AAY, GGPPG, and GGSGGGGSGG. Different linkers were used to predict and analyze the new epitopes and coverage. The serine of GGGS combined with the epitope to form a new high-resolution epitope. The prediction results of AAY and GGPPG showed large differences between the Th combination and the CTL combination. Finally, GGSGGGGSGG (SEQ ID NO:17) was selected as the optimal linker.

[0027] AllerTop v2.0 was used to predict that both combinations were non-sensitizing. Immunogenicity was analyzed using Vaxijen, and the CTL combination showed an immunogenicity score of 0.5785, consistent with the viral model.

[0028] Predict its secondary structure using the SOPMA online tool ( Figure 4 In the diagram, blue represents α-helical structures, purple represents β-sheet structures, and yellow represents irregular coil structures.

[0029] 2. Combination Form Prediction Analysis The RBD and CTL segments were arranged and combined, with Fc tags added to each combination. A comprehensive analysis of the physicochemical properties, secondary structure, and tertiary structure of various combinations was performed. Each epitope was connected using the selected linker: GGSGGGGGSGG. RBD and CTL fragments were also connected using the GGSGGGGGSGG linker. The Fc tag was directly added after the combination, without adding linkers before or after it.

[0030] The following combinational form was constructed: RBD-CTL-Fc combination ( Figure 5 Table 2 shows the protein sequences (SEQ ID NO. 19) for the four combinations, which contain: SEQ ID NO.16 of the RBD sequence of BA.2.9.3: RVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFT GCVIAWNSNKLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF, Linker sequence SEQ ID NO.17: GGSGGGGGSGG; hIgG1 Fc tag sequence SEQ ID NO.18: DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK .

[0031] Table 2. Analysis of candidate epitope combination sequences AllerTop v2.0 was used to predict that the combination was non-sensitizing. Immunogenicity was analyzed using Vaxijen, and the RBD-CTL-Fc combination had an immunogenicity score of 0.5848, consistent with the viral model.

[0032] Predict its secondary structure using the SOPMA online tool ( Figure 6 In the diagram, blue represents α-helical structures, purple represents β-sheet structures, and yellow represents irregular coil structures.

[0033] In this embodiment, the AlphaFold 3 deep learning platform was used to systematically predict the three-dimensional spatial configuration of multi-epitope combinations. Figure 7 Molecular dynamics simulations and structural clustering analysis revealed that the linker peptide regions in the recombinant molecules exhibited distinct β-sheet secondary structure characteristics, but no cross-over phenomenon caused by non-specific interactions between epitopes was observed. Further analysis showed that the interface pTM (predicted TM-score) and overall pTM (predicted TM-score) scores, as evaluation parameters for novel composite structures, were both consistently higher than 0.62 (95% confidence interval).

[0034] To further analyze the reliability of various combinations, a Laplace plot of the combination structure was generated using procheck. Figure 8 This method visualizes the distribution of dihedral angles (φ and ψ) along the protein backbone to determine if the structure conforms to known stereochemical rules. Blue dots represent amino acid residues; the red area in the Laplace plot is the core region; ideally, over 90% of the most desirable φ / ψ combinations (such as α-helices and β-sheets) should fall within this region, indicating good conformational space. Brown represents allowed regions, which are suboptimal but acceptable dihedral angles; yellow represents unconventional conformations found in a few natural proteins; and beige represents disallowed regions. The results show that >80% of these four combinations fall within the core plus allowed regions, constituting a reasonable model, with no more than five outliers. This also reflects the reliability of our combined structures.

[0035] Example 4: Preparation of mRNA-based epitope vaccines 1. Plasmid preparation This embodiment uses molecular cloning technology to construct a recombinant expression vector system. Through reverse genetics system design, the core structure of the mRNA includes the following functional elements (… Figure 9 (1) Codon-optimized 5' untranslated region (5'UTR, derived from the human β-globulin gene); (2) Tissue plasminogen activator (tPA) signal peptide sequence (MDAMKRGLCCVLLLCGAVFVSPS), used to guide the secretion of recombinant proteins; (3) NheⅠ (GCTAGC) and KpnⅠ (4) A directed cloning framework consisting of two GGTACC restriction sites; (5) A length-optimized 110-base poly-A tail to enhance mRNA stability; BsaⅠ (GGTCTC) linearization sites are used for in vitro transcription template preparation.

[0036] 2. In vitro transcription and capping The linearized products were transcribed and capped in vitro. The theoretical length of the mRNA fragment of the RBD-CTL-Fc combination was 834 nucleotides (nt), and the theoretical length of the mRNA fragment of the RBD-Fc combination was 335 nucleotides (nt).

[0037] 3. In vitro validation of LNP-encapsulated mRNA Liposome solution and sodium citrate solution were used to encapsulate the capped and purified mRNA. To determine the encapsulation efficiency and drug release rate, the encapsulation efficiency and particle size were measured (Table 3). The results showed that the standard particle size range for mRNA vaccines is approximately 70-150 nm, and the particle size of the LNP combination in this experiment was all within the range of 150 nm. Figure 10 Ideally, a high-quality mRNA vaccine should have an encapsulation rate of over 80%. In this experiment, the encapsulation rates of all mRNA combinations were greater than 90%. The results confirm that the LNP encapsulation effect is good and suitable for use in subsequent immunization experiments.

[0038] Table 3. LNP Quality Control Indicators Example 5: Immunogenicity assay of multi-epitope candidate vaccines 1. Results of specific antibody level assay in immunized mice Mice were randomly assigned to different groups and immunized twice with different multi-epitope combination mRNA vaccines at 6-8 weeks of age. HLA-A2 / DR1 Double transgenic mice and HLA-A11 / DR1 Double transgenic mice. Serum was collected 14 days after the second immunization and the levels of the corresponding specific antibodies were detected by indirect ELISA.

[0039] Experimental results showed that all multi-epitope vaccines induced the production of specific IgG antibodies in mice after immunization. Comparison of different groups of single-strain mice revealed significant statistical differences between the RBD-CTL-Fc group and the RBD-Fc and Fc groups. P<0.05 () Figure 11 ).

[0040] 2. Determination of cellular immunity levels in immunized mice using enzyme-linked immunospot assay. Cellular immune responses were validated using the ELISpot assay, employing various combinations of HLA-restricted epitope mixtures to verify post-immunization responses. HLA-A2 / DR1 Double transgenic mice, HLA-A11 / DR1 T-cell immune response in double transgenic mice. After 14 days of immunization with different doses of the above-mentioned immunization groups, the mice were euthanized and dissected. The mouse spleen cells were stimulated with a selected HLA-restricted epitope mixed peptide, and the formation of IFN-γ spots was measured.

[0041] The results showed that after immunization with RBD-CTL-Fc HLA-A2 / DR1 Transgenic mice, HLA-A11 / DR1All transgenic mice exhibited significant antigen-specific T-cell responses. Specifically, ELISpot assays revealed that these mice had significantly higher numbers of spotted forming units (SFUs) than the RBD-Fc group and the Fc control group. P<0.05 () Figure 12 ).

[0042] Example 6: Immunoprotective efficacy assay of multi-epitope candidate vaccines 1. Changes in body weight and viral replication results in mice after challenge with the virus. Based on the above experimental results, select HLA-A2 / DR1-HACE2 Transgenic mice were used to determine the immunoprotective effect of the vaccine. Immunization was performed according to the above-described mRNA vaccine immunization protocol. Fourteen days after the last immunization, mice were intranasally injected with 1×10⁻⁶ mRNA vaccine. 4 TCID 50 Mice infected with the SARS-CoV-2 Omicron BA.5 strain were sacrificed on the third day after challenge, and their body weight was monitored daily. The results showed that the body weight of mice in the control group (Fc group) decreased significantly, the body weight of mice in the RBD-Fc group decreased slightly, and the body weight of other groups did not change significantly.

[0043] On the third day after challenge, mice were euthanized, and lung tissue was collected by dissection to detect viral load. Results showed that the viral load in the RBD-CTL-Fc group was significantly lower than that in the RBD-Fc and Fc groups, with statistically significant differences. P<0.05 () Figure 13 ).

[0044] 2. Evaluation of lung tissue pathological damage in mice after viral challenge To investigate the effects of different immune groups on SARS-CoV-2 infection HLA-A2 / DR1-HACE2 The immunogenicity of transgenic mice was assessed by infecting mice in each immunization group with the virus and evaluating their protective effect. In this example, the presence of pathological changes in the lung tissue of mice was observed, and histopathological examination of the lung tissue was performed on day 3 post-infection.

[0045] Pathological results showed that in the RBD-Fc group, perivascular inflammatory cell sheathing was formed, involving some blood vessels (2+), with a large number of neutrophils around some blood vessels, perivascular edema, fibrin exudation, and inflammatory exudate in the bronchioles; the Fc control group had large areas of pulmonary consolidation, increased lymphomonocytes, and multifocal inflammatory cell infiltration in the alveoli, some of which formed plaques, indicating substantial damage to the lung tissue. Figure 14 Compared to the Fc control group, the RBD-CTL-Fc group showed milder pathological damage, which is consistent with the viral load results.

[0046] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0047] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A recombinant fusion protein, characterized in that, In order, they include: (a) The receptor-binding domain of the SARS-CoV-2 spike protein or a functional fragment thereof; (b) A T cell epitope domain comprising: a CTL epitope cluster, said CTL epitope cluster including at least one CD8 group selected from SEQ ID NO: 1-15. + T-cell epitope polypeptides; And, (c) the immunoglobulin Fc domain.

2. The recombinant fusion protein according to claim 1, characterized in that, The amino acid sequence of the SARS-CoV-2 spike protein receptor-binding domain is shown in SEQ ID NO: 16; the amino acid sequence of the immunoglobulin Fc domain is shown in SEQ ID NO:

19.

3. The recombinant fusion protein according to claim 1, characterized in that, The CTL epitope structure domain contains all of the CD8 as shown in SEQ ID NO: 1-15. + T-cell epitope peptides.

4. The recombinant fusion protein according to claim 1, characterized in that, The domains and epitope peptides in the recombinant fusion protein are connected by linkers. The amino acid sequence of the linker is shown in SEQ ID NO:

17.

5. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the recombinant fusion protein as described in any one of claims 1 to 4.

6. The nucleic acid molecule according to claim 5, characterized in that, The nucleic acid molecule is mRNA, containing: derived from human... β - The 5' untranslated region of the globulin gene, the signal peptide sequence encoding the tissue plasminogen activator, the coding region encoding the recombinant fusion protein; and the poly-A tail.

7. An immunogenic composition, characterized in that, Include: The nucleic acid molecule as described in claim 5 or 6; and Lipid nanoparticles encapsulating the nucleic acid molecules.

8. The immunogenic composition according to claim 7, characterized in that, The lipid nanoparticles have an average particle size of 140 nm to 60 nm.

9. The use of the recombinant fusion protein according to any one of claims 1 to 4, or the nucleic acid molecule according to claim 5 or 6, or the immunogenic composition according to claim 7 or 8 in the preparation of a medicament for the prevention or treatment of coronavirus infectious diseases.

10. The application according to claim 9, characterized in that, The coronavirus infectious disease is caused by the SARS-CoV-2 Omicron variant.

Images