Vaccines against the novel coronavirus and their application

Abstract

This invention provides a vaccine against the novel coronavirus and its application. This invention discloses for the first time a novel method for modifying the S1 subunit of the coronavirus spike glycoprotein to enhance its immunogenicity, comprising: linking the S1 subunit of the coronavirus spike glycoprotein to a site-directed mutated IgG Fc; this invention also discloses a fusion polypeptide obtained by fusing the two and a vaccine obtained by mixing the fusion polypeptide with an aluminum adjuvant. The fusion polypeptide of this invention can be effectively expressed, and after expression, it can form the correct protein spatial structure; the vaccine obtained by mixing it with an aluminum adjuvant exhibits good immunogenicity.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and virology, and more specifically, this invention relates to vaccines against the novel coronavirus and their applications. Background Technology

[0002] The pneumonia caused by the 2019 novel coronavirus (2019-nCoV) has clinical manifestations very similar to viral pneumonia; the main clinical manifestations are fever, fatigue, and dry cough, and in severe cases, shock, sepsis, respiratory failure, and death can occur. It is characterized by its insidious nature and high infectivity, leading to a certain number of severe cases or deaths. Currently, there are no effective drugs for pneumonia caused by this virus, posing significant challenges to clinical diagnosis, treatment, and epidemic control. In addition to causing physical illness, according to the latest disclosures from the WHO, hundreds of millions of people worldwide are affected by varying degrees of mental health problems, and only a small minority have access to high-quality treatment.

[0003] Coronavirus particles are irregular in shape, with a diameter of approximately 60-220 nm. The nucleic acid of coronaviruses is positive-sense single-stranded RNA, which can use itself as a template to guide the synthesis of virus-related proteins. After entering a host cell, the virus first expresses RNA polymerase using viral RNA as a template. Subsequently, the RNA polymerase completes the transcription and synthesis of negative-sense RNA, the synthesis of various structural protein mRNAs, and the replication of the viral genomic RNA.

[0004] Coronaviruses have an envelope structure containing three proteins: spike protein (S), envelope protein (E), and membrane protein (M). A few species also contain hemagglutinin-esterase (HE). The spike protein plays a crucial role in recognizing and binding to host cell surface receptors and mediating the fusion of the viral envelope with the cell membrane. The M protein participates in the formation and budding of the viral envelope. The HE protein forms short protrusions on the envelope and may be related to early adsorption by coronaviruses; in some coronaviruses, the HE protein can induce erythrocyte agglutination and adsorption. The S protein is located on the viral surface, forming a rod-like structure; the N protein encapsulates the viral genome.

[0005] Currently, there is still a lack of effective vaccines against 2019-nCoV. Under these severe circumstances, it is of great significance to protect people's health and national security to develop safe and effective vaccines against 2019-nCoV as soon as possible to protect susceptible populations. Summary of the Invention

[0006] The purpose of this invention is to provide a vaccine against the novel coronavirus and its application.

[0007] In a first aspect of the invention, a method is provided for modifying the coronavirus spike glycoprotein (Spike) S1 subunit to enhance its immunogenicity (or immune effect or immune activity), comprising: linking the coronavirus spike glycoprotein S1 subunit to a site-directed mutated IgG Fc.

[0008] In a first aspect of the invention, a fusion polypeptide is provided comprising the following proteins interconnected: a coronavirus spike glycoprotein S1 subunit and a site-directed mutated IgG Fc.

[0009] In a preferred embodiment, the amino acid sequence of the site-directed mutated IgG Fc is shown as positions 686 to 917 in SEQ ID NO:1.

[0010] In another preferred embodiment, the amino acid sequence of the coronavirus spike glycoprotein S1 subunit is as shown in positions 16-685 or 1-685 of SEQ ID NO:1; or as shown in positions 16-685 or 1-685 of SEQ ID NO:5.

[0011] In another preferred embodiment, it comprises, from the amino terminus to the carboxyl terminus, the following components in sequence: the coronavirus spike glycoprotein S1 subunit and the site-directed mutated IgG Fc.

[0012] In another preferred embodiment, a linker may or may not be present between the coronavirus spike glycoprotein S1 subunit and the site-directed mutated IgG Fc.

[0013] In another aspect of the invention, a nucleic acid is provided that encodes the aforementioned fusion polypeptide.

[0014] In another aspect of the invention, a carrier is provided, the carrier containing the nucleic acid described above.

[0015] In another aspect of the invention, a host cell is provided, wherein the host cell contains the vector or the nucleic acid integrated into its genome.

[0016] In a preferred embodiment, the cell is a eukaryotic cell.

[0017] In another preferred embodiment, the cells include: 293 cells, CHO cells.

[0018] In another aspect of the invention, the use of the fusion polypeptide is provided for the preparation of a vaccine against the 2019-nCoV (COVID) virus.

[0019] In another aspect of the invention, a vaccine against the 2019-nCoV (COVID) virus is provided, comprising: the fusion polypeptide; and an aluminum adjuvant (alum).

[0020] In another aspect of the invention, a method for preparing a vaccine against the 2019-nCoV (COVID) virus is provided, comprising mixing the fusion polypeptide with an aluminum adjuvant.

[0021] In a preferred embodiment, the fusion peptide and aluminum adjuvant are in a weight ratio of 1 to 20:1000 (e.g., 2:1000, 3:1000, 5:1000, 8:1000, 15:1000); more preferably, it is 1 to 10:1000.

[0022] In another aspect of the invention, a kit is provided for immunization to produce antibodies against the 2019-nCoV (COVID) virus, comprising: a container, and the vaccine contained in the container; a container, and an aluminum adjuvant contained in the container.

[0023] In another aspect of the invention, a kit is provided for immunization to generate antibodies against the 2019-nCoV (COVID) virus, comprising: a container, and the said fusion polypeptide contained in the container; a container, and an aluminum adjuvant contained in the container.

[0024] In another aspect of the invention, a site-directed mutated IgG Fc or a nucleic acid encoding the same is provided, the site-directed mutated IgG Fc having an amino acid sequence as shown in positions 686 to 917 of SEQ ID NO:1.

[0025] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0026] Figure 1 A representative image of cells transiently transfected with 293 cells.

[0027] Figure 2 The expression level of S1-Fc protein in Expi293F cells (DAY6), where DAY represents the number of days.

[0028] Figure 3 Figure 1. Changes in S1-Fc protein expression and cell viability in Expi293F cells over culture time. VCD: Number of viable Expi293F cells; DAY1-6: Time for collecting culture supernatant after plasmid transfection. VCD: Viable cell density.

[0029] Figure 4 Flowchart of mouse immunization experiment.

[0030] Figure 5Results of IgM detection in immunized mice; Adjuvant control: only adjuvant was injected during immunization; RBD-Fc+ adjuvant: recombinant protein RBD-Fc+ adjuvant was injected; S1-Fc+ adjuvant: recombinant protein S1-Fc+ adjuvant was injected; HOPE-V+ adjuvant: recombinant protein HOPE-V+ adjuvant was injected; The vertical axis represents the A450 OD reading of IgM detection; The horizontal axis represents the mouse serum dilution concentration (n=3-4, average value was used for plotting).

[0031] Figure 6 Results of IgG detection in immunized mice; Adjuvant control: only adjuvant was injected during immunization; RBD-Fc+ adjuvant: recombinant protein RBD-Fc+ adjuvant was injected; S1-Fc+ adjuvant: recombinant protein S1-Fc+ adjuvant was injected; HOPE-V+ adjuvant: recombinant protein HOPE-V+ adjuvant was injected; The vertical axis represents the A450 OD reading of IgM detection; the horizontal axis represents the mouse serum dilution concentration (n=3-4, average value was used for plotting).

[0032] Figure 7 Results of the pseudovirus neutralization experiment in immunized mouse serum, including RLU readings (top figure) and neutralization activity (bottom figure); Adjuvant control: only adjuvant was injected during immunization; RBD-Fc+ adjuvant: recombinant protein RBD-Fc+ adjuvant was injected; RBD+ adjuvant: recombinant protein RBD+ adjuvant was injected; S1-Fc+ adjuvant: recombinant protein S1-Fc+ adjuvant was injected; S1-D614-Fc+ adjuvant: recombinant protein S1-D614-Fc+ adjuvant was injected; HOPE-V+ adjuvant: recombinant protein HOPE-V+ adjuvant was injected; The vertical axis represents the A450 OD reading of IgM detection; the horizontal axis represents the dilution concentration of mouse serum.

[0033] Figure 8 Results of SARS-CoV-2 neutralization assay in immunized mouse serum. Top figure: Neutralization activity; Bottom figure: Neutralization percentage.

[0034] Figure 9 Comparison of adjuvant processes in the neutralization experiment of SARS-CoV-2 eukaryotic virus in immunized mouse serum; Adjuvant control: only adjuvant was injected during immunization; S1+ adjuvant: recombinant protein S1+ adjuvant was injected; S1-Fc+ adjuvant: recombinant protein S1-Fc+ adjuvant was injected (n=4).

[0035] Figure 10 Comparison of Fc process results in SARS-CoV-2 neutralization assay of immunized mouse serum. Positive control: positive control serum from P3 laboratory; Adjuvant control: adjuvant only injected during immunization; S1+adjuvant: recombinant protein S1 (without Fc fragment) injected + adjuvant; S1-Fc+adjuvant: recombinant protein S1-Fc injected + adjuvant (n=4 per group).

[0036] In multiple figures, Fc can be selected from one of two variant forms, specifically the Fc sequence defined in the protein sequence in Table 1. Detailed Implementation

[0037] Through in-depth research, the inventors have for the first time disclosed a novel method for modifying the S1 subunit of the coronavirus spike glycoprotein to enhance its immunogenicity, comprising: linking the S1 subunit of the coronavirus spike glycoprotein with a site-directed mutated IgG Fc. This invention also discloses a fusion polypeptide obtained by fusing the two and a vaccine obtained by mixing the fusion polypeptide with an aluminum adjuvant. The fusion polypeptide of this invention can be effectively expressed, and after expression, it forms the correct protein spatial structure. The vaccine obtained by mixing the fusion polypeptide with an aluminum adjuvant exhibits good immunogenicity.

[0038] the term

[0039] As used herein, "operationally linked" or "operationally coupled to" refers to a situation where certain parts of a linear DNA sequence can influence the activity of other parts of the same linear DNA sequence. For example, if a promoter controls transcription of a coding sequence, then it is operationally coupled to the coding sequence.

[0040] As used herein, “containing,” “having,” or “including” includes “comprising,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0041] As used herein, a "pharmaceutically or immunologically acceptable" ingredient is a substance suitable for human use without excessive adverse side effects (such as toxicity), i.e., a reasonable benefit / risk ratio. The term "pharmaceutically or immunologically acceptable carrier" refers to a carrier used for the administration of immunotherapeutic agents, including various excipients and diluents. This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity after administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in a composition may contain liquids such as water, saline, glycerin, and sorbitol. Additionally, these carriers may contain auxiliary substances such as lubricants, flow aids, wetting agents or emulsifiers, pH buffers, and stabilizers such as albumin. Preferably, the adjuvant of the present invention serves as a pharmaceutically or immunologically acceptable carrier.

[0042] As used herein, “effective amount” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0043] Fusion Peptides

[0044] This invention provides a fusion polypeptide comprising a coronavirus spike glycoprotein S1 subunit and an IgG Fc fragment mutant, operatively linked together. Preferably, the fusion polypeptide is an isolated protein, a purified product of recombinant host cell culture, or a purified extract; the fusion polypeptide may also be present in a mixture, such as in a cell lysate or crude extract.

[0045] This invention includes derivatives and analogs of the coronavirus spike glycoprotein S1 subunit or IgG Fc fragment mutant. As used herein, the terms "derivative" and "analyte" refer to polypeptides that substantially retain the same biological function or activity as the coronavirus spike glycoprotein S1 subunit or IgG Fc fragment mutant of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a proteogen sequence, or a fusion protein formed with an antigen IgG fragment). It should be understood that certain positions in the IgG Fc fragment mutant derivatives and analogs are conserved, including “C” at position 690 and “EEM” at positions 826-828 of SEQ ID NO:1.

[0046] Spike protein is a trimeric transmembrane glycoprotein that forms a distinctive crown-like structure on the viral surface. It first binds to receptors on the cell surface, then undergoes metamorphosis, fusing the viral envelope with the cell membrane, thereby injecting viral genetic material into the cell and infecting it. Spike protein contains two subunits, S1 and S2. S1 primarily contains the receptor-binding domain (RBD), responsible for recognizing cellular receptors; S2 contains the essential elements required for membrane fusion. Spike protein is responsible for viral binding to host cell membrane receptors and membrane fusion.

[0047] In this invention, the term "coronavirus spike glycoprotein S1 subunit" refers to a polypeptide comprising the amino acid sequence shown at positions 16-685 or 1-685 of SEQ ID NO:1. This term also includes variations of the polypeptide having the same function as the coronavirus spike glycoprotein S1 subunit, comprising the amino acid sequence shown at positions 16-685 or 1-685 of SEQ ID NO:1. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-20, preferably 1-15, more preferably 1-10, more preferably 1-5, more preferably 1-2); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties often does not alter the function of the polypeptide. For example, adding or deleting one or more amino acids at the C-terminus and / or N-terminus often does not alter the function of the polypeptide. Therefore, the term also includes active fragments and active derivatives of the coronavirus spike glycoprotein S1 subunit.

[0048] In this invention, the term "IgG Fc mutant" refers to a polypeptide comprising the amino acid sequence shown in positions 686-917 of SEQ ID NO:1. This term also includes variants of the polypeptide having the same function as the IgG Fc mutant, comprising the amino acid sequence shown in positions 686-917 of SEQ ID NO:1. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-20, preferably 1-15, more preferably 1-10, more preferably 1-5, more preferably 1-2); and additions or deletions of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties often does not alter the function of the polypeptide. For instance, adding or deleting one or more amino acids at the C-terminus and / or N-terminus often does not alter the function of the polypeptide. It should be understood that some specific positions in the variant forms of the IgG Fc segment mutant are conserved, including "C" at position 690 and "EEM" at positions 826-828 of SEQ ID NO:1.

[0049] It should be understood that the present invention also includes proteins with high homology to the S1 subunit of the coronavirus spike glycoprotein or the IgG Fc segment mutant, such as having more than 90%, such as 95%, 98%, or 99% sequence identity. Methods and tools for comparing sequence identity are also well known in the art, such as BLAST. It should be understood that certain positions of the proteins with high homology to the IgG Fc segment mutant are conserved, including “C” at position 690 and “EEM” at positions 826–828 of SEQ ID NO:1.

[0050] The coronavirus spike glycoprotein S1 subunit and the IgG Fc mutant described in this invention are linked or coupled to each other by chemical bonds; the chemical bonds may be covalent or non-covalent. As a preferred embodiment of this invention, the coronavirus spike glycoprotein S1 subunit and the IgG Fc mutant are linked by chemical bonds; more preferably, the chemical bonds are peptide bonds.

[0051] As a preferred embodiment of the present invention, the fusion polypeptide comprises, from the amino terminus to the carboxyl terminus, the following components in sequence: the S1 subunit of the coronavirus spike glycoprotein and the IgG Fc fragment mutant.

[0052] The coronavirus spike glycoprotein S1 subunit and the IgG Fc mutant can be directly linked or linked via a polypeptide linker (linking peptide). The linker, for example, comprises 1-50 amino acids; preferably 1-30 or 2-20 amino acids. As a preferred embodiment of the invention, the coronavirus spike glycoprotein S1 subunit and the IgG Fc mutant are directly linked. The inventors have discovered that even without the assistance of flexible linking peptides, the coronavirus spike glycoprotein S1 subunit and the IgG Fc mutant can achieve compatibility and exhibit good biological activity.

[0053] On the other hand, the present invention also provides isolated nucleic acids encoding the fusion polypeptide, which may also be its complementary strand. Any nucleic acid encoding the fusion polypeptide is applicable to the present invention. The sequences mentioned in the examples below are applicable to the methods of the present invention.

[0054] The DNA sequence encoding the fusion polypeptide of the present invention can be synthesized artificially in its entirety, or the DNA sequences encoding the S1 subunit of the coronavirus spike glycoprotein and the amino acids of the IgG Fc segment mutant can be obtained separately by PCR amplification and then spliced ​​together to form the DNA sequence encoding the fusion polypeptide of the present invention.

[0055] The present invention also provides a vector comprising a nucleic acid molecule encoding the fusion polypeptide. The vector may further comprise an expression regulatory sequence operatively linked to the sequence of the nucleic acid molecule to facilitate the expression of the fusion polypeptide.

[0056] Various suitable vectors can be used in this invention, such as vectors for cloning and expression in bacteria, fungi, yeast, and mammalian cells, as described in, for example, Pouwels et al., Cloning Vectors: A Laboratory Manual. Preferably, the expression vector is an expression vector suitable for yeast cells.

[0057] In addition, recombinant cells containing nucleic acid sequences encoding the fusion polypeptide are also included in this invention.

[0058] In this invention, the term "host cell" includes prokaryotic cells and eukaryotic cells, preferably eukaryotic cells. Commonly used eukaryotic host cells include mammalian cells, yeast cells, and insect cells, preferably mammalian cells. In a preferred embodiment of this invention, the eukaryotic cells used are 293 cells or CHO cells; more preferably, Expi293F cells.

[0059] A method for producing the fusion polypeptide of the present invention is also included in the present invention. The method includes culturing recombinant cells containing nucleic acids encoded by the fusion polypeptide. The method may further include the isolation and / or purification of the fusion polypeptide.

[0060] The fusion peptides obtained in the above preparation can be purified to have essentially uniform properties, for example, appearing as a single band on SDS-PAGE electrophoresis.

[0061] Vaccine composition / formulation / reagent kit

[0062] The fusion peptides obtained in this invention exhibit ideal immunogenicity when mixed with a suitable adjuvant (such as aluminum adjuvant).

[0063] Therefore, the present invention provides an immunogenic vaccine composition, which is a preventive or therapeutic vaccine, said composition comprising: an effective amount of the fusion polypeptide described in this invention, and a pharmaceutically or immunologically acceptable carrier. In this invention, the vaccine composition may also be referred to as an immunogenic composition.

[0064] In a preferred embodiment of the present invention, aluminum adjuvant is used as an adjuvant in the vaccine composition. The inventors have found that when the aluminum adjuvant is combined with the fusion polypeptide, the fusion polypeptide exhibits excellent immunogenicity.

[0065] In a preferred embodiment of the present invention, the fusion polypeptide and aluminum adjuvant in the vaccine composition are in a weight ratio of 1 to 20:1000; more preferably, the ratio is 1 to 10:1000.

[0066] The composition can be formulated into various dosage forms suitable for administration to mammals, including but not limited to: solvents, emulsions, suspensions, and lyophilized formulations. Preferably, the dosage form is suitable for injection.

[0067] The fusion peptide or vaccine composition of the present invention can be tested in a suitable animal model system before being used in humans. Such animal model systems include, but are not limited to, mice, rabbits, and monkeys.

[0068] The vaccine compositions of the present invention can be used to protect or treat susceptible mammals, particularly humans, preferably administered via systemic or mucosal routes. Administration may include intramuscular, intraperitoneal, intradermal, or subcutaneous injection. Therefore, one aspect of the invention is to immunize a human host against disease caused by the 2019-nCoV (COVID) virus, a method comprising administering to the host an immune-protective dose (effective amount) of the vaccine composition of the present invention.

[0069] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). A suitable dose range may be, for example, 0.00001–100 mg / kg body weight. The optimal dosage for a particular vaccine can be determined through standard studies that include observing an appropriate immune response in subjects. Following primary vaccination, subjects may receive one or more booster immunizations at appropriate intervals.

[0070] Furthermore, for example, a single bolus injection can be administered, multiple separate doses can be given over time, or the dose can be proportionally reduced or increased depending on the urgency of the treatment situation. The fusion peptide or vaccine composition of the present invention is preferably sterile. Methods for sterilizing these fusion peptide or vaccine compositions are well known in the art. Precise dosing regimens for the target population are typically selected during clinical trials.

[0071] The vaccine composition of the present invention is immunoprotective and non-toxic, and is suitable for use by people of all ages.

[0072] The present invention also provides a medicine box containing the vaccine composition described above; or the medicine box contains both the fusion polypeptide and an adjuvant, which are mixed in a certain proportion during use. Preferably, the medicine box may also include an instruction manual explaining the method of using the vaccine composition.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Science Press, 2002, or according to the manufacturer's recommendations.

[0074] Example 1: Protein Sequence Modification and Plasmid Construction

[0075] 1. Protein sequence modification

[0076] To obtain proteins that can be efficiently expressed and are suitable for vaccine preparation, the inventors conducted in-depth research and comparisons on the novel coronavirus 2019-nCoV, modified the protein sequences, and selected proteins through recombinant expression. Based on repeated experimental analysis, the inventors established a series of recombinant protein modification schemes, and Table 1 shows some of the recombinant protein sequence information.

[0077] Table 1. Recombinant protein sequence information

[0078]

[0079]

[0080] In Table 1, amino acids MFVFLVLLPLVSSQC at positions 1-15 represent the signal peptide, the bolded black amino acid sequence represents the S1 functional protein region, and the underlined amino acid represents the Fc protein region.

[0081] 2. Construction of recombinant plasmids

[0082] (1) Experimental materials and reagents

[0083] Taq polymerase & pBO enzyme, dNTPs, competent cells, experimental water, T4 DNA ligase, recombinase (Clone EZEnzyme), endonuclease (NEB), pcDNA3.4 plasmid, all obtained from Genscript.

[0084] (2) Plasmid construction

[0085] The gene encoding the above protein sequence underwent codon optimization based on the human expression host. The optimized host was Human, and EcoRI and HindIII were filtered out in the optimized sequence.

[0086] The coding gene of the codon-optimized protein sequence was inserted into the multiple cloning site of the pcDNA3.4 plasmid to construct a recombinant plasmid.

[0087] Example 2: Expression of recombinant proteins

[0088] 1. Resuscitation of Expi293F cells

[0089] (1) Sterilize the biosafety cabinet with ultraviolet light for 30 minutes, rewarm the culture medium, and prepare 15ml centrifuge tubes;

[0090] (2) Remove Expi293F cells from liquid nitrogen and thaw them in a 37°C water bath;

[0091] (3) After thawing completely, wipe the cryovials with alcohol and place them in a biosafety cabinet;

[0092] (4) Take 10ml of culture medium into a centrifuge tube, put the cells into the centrifuge tube, and mix gently;

[0093] (5) Centrifuge at 1000 rpm for 5 min;

[0094] (6) After centrifugation, discard the supernatant, add 5 ml of fresh culture medium, and mix gently.

[0095] (7) Add 25ml of culture medium to a 125ml shake flask, then add the cell resuspension to it, for a final volume of 30ml;

[0096] (8) Place the shake flask in a shaker and incubate at 37°C, 125 rpm, 8% carbon dioxide concentration, and 25 mm diameter track conditions.

[0097] 2. Culture of Expi293F cells

[0098] (1) The seeding density of Expi293F cells was 0.4–0.6 × 10⁻⁶. 6 At this time, it is necessary to culture for 3 days to a density of 3-5 × 10⁻⁶. 6 ;

[0099] (2) The seeding density of Expi293F cells was 0.2–0.4 × 10⁻⁶. 6 At this time, it needs to be cultured for 4 days to a density of 3-5 × 10⁻⁶. 6 ;

[0100] 2.3 When the cell density is 3–5 × 10⁻⁶ 6 At this time, cell counting is performed, and fresh culture medium is added to adjust the cell density to 0.4–0.6 × 10⁻⁶. 6 Then you can continue training.

[0101] 3. Cryopreservation of Expi293F cells

[0102] (1) Prepare cell cryovials, a programmed cooling box, and prepare cryopreservation solution by adding 10% DMSO to 90% culture medium;

[0103] (2) When the cell density is 3-5 × 10 6 When the survival rate is greater than 95%, it can be frozen.

[0104] (3) Place the cells in a centrifuge tube and centrifuge at 1000 rpm for 5 min;

[0105] (4) Discard the supernatant and add cryopreservation solution to adjust the cell density to 1.0 × 10⁻⁶. 7 Dispense into 1ml cryovials;

[0106] (5) Place it in a programmable cooling box, put it in a -80℃ refrigerator overnight, and then put it in liquid nitrogen for long-term storage.

[0107] 4. Transfection conditions

[0108] Protein expression process:

[0109] DAY-1 (Day -1, the day before Day 0): Adjust cell density to 2.5–3.0 × 10⁻¹⁰. 6 ;

[0110] The cell density on DAY 0 was 4.5–5.5 × 10⁻⁶. 6 With a viability greater than 95%, the cell density was adjusted to 3.0 × 10⁻⁶. 6 Add the diluted transfection reagent to the diluted DNA, incubate for 10-20 minutes, and then add it to the cells;

[0111] Days 4-7 (Harvesting secretory proteins)

[0112] Taking a 125ml shake flask and a 30ml culture system as an example, the selection of transfection reagents, the amount of DNA used, and the optimization of the transfection reagent volume are as follows:

[0113] (1) When using the Thermo Expi293F expression system, the procedure and experimental conditions are as follows:

[0114] (2) Day 1, adjust cell density to 2.5–3.0 × 10⁻⁶. 6 , cultivate overnight stay;

[0115] (3) On Day 0, count a small number of cells, ensuring a viability greater than 95%. Add fresh culture medium and adjust the cell density to 3.0 × 10⁻⁶ cells / year. 6 25ml volume, put into a 125ml shake flask;

[0116] (4) Take 1.5 ml of Opti-MEM to dilute the plasmid DNA. The amount of DNA used is 3 ug / ml. When the DNA concentration is 1 ug / ul, the total amount is 30ul. After adding, mix gently.

[0117] (5) Take 1.4 ml of Opti-MEM to dilute the transfection reagent. The amount of transfection reagent used is 9 ul / ml, and the total amount is 90 ul. After adding, mix gently.

[0118] (6) Add the diluted transfection reagent to the diluted DNA, mix gently, and let stand at room temperature for 10 minutes.

[0119] (7) Add the mixture to the culture flask and place it on a shaker for incubation;

[0120] (8) DAY6 (Day 6) Harvest protein.

[0121] 5. Results of protein expression in shake flasks

[0122] To achieve good protein expression, the inventors first performed sequence optimization as described above; furthermore, they selected suitable host cells and determined Expi293F as the host cell for recombinant expression.

[0123] Cell image of Expi293F cells transiently transfected as shown below Figure 1 As shown. According to Figure 1 It is evident that Expi293F cells were in excellent condition after transfection and were able to effectively express recombinant proteins.

[0124] On day 6 post-transfection, the expression level of the S1-Fc recombinant protein was determined in the cell culture supernatant collected from Expi293F cells after transfection with the Expi293F protein expression plasmid. (DAY 6) Figure 2 The blank control consisted of the supernatant of untransfected Expi293F cell culture medium. Figure 2 It can be seen that, using Expi293F cells, the highest expression level of recombinant protein can reach 2000 ug / L, or 2 mg / L.

[0125] The changes in protein expression levels and cell viability of Expi293F cells with culture time at different time points after the initiation of S1-Fc recombinant expression are shown in the figure below. Figure 3 As shown in the figure, the results indicate that after transfection with Expi293F cells, the expression level of recombinant protein gradually increased with the extension of culture time, and the protein expression level reached 2 mg / L in the cell culture supernatant collected on day 6.

[0126] Through large-scale expression process optimization (transfection plasmid amount, PEI content, plasmid to PEI ratio, cell density during transfection, feeding process, etc.), the expression level of Expi293F cells can be significantly increased, reaching more than 20 mg / L.

[0127] Example 4: Protein chromatography purification

[0128] 1. Experimental materials and reagents

[0129] Chromatography column (GE), Tris (sigma), Nacl (sigma), imidazole (MACKLIN), AKTA pure (GE), CaptoQ (GE), SP-HP (GE), Superdex 200 (GE),

[0130] 2. Experimental Procedure

[0131] A. His affinity purification methods (suitable for proteins without Fc) include:

[0132] (1) Clarification and filtration of the sample: The prepared cell suspension supernatant was clarified using a 50ml syringe and a 0.22μm filter membrane;

[0133] (2) Protein capture and purification were performed using an AKTA protein chromatography column;

[0134] (3) Perform system flushing, then flush AKTA's A1 pump with equilibration solution, and flush B1 pump with eluent.

[0135] (4) Set the system flow rate to 1 ml / min, select the corresponding column position 1 to connect the protein chromatography column, use equilibration buffer to equilibrate AKTA and the column, and zero the UV after equilibration.

[0136] (5) Start loading the sample by transferring pump A1 into the loading centrifuge tube;

[0137] (6) After the sample loading is completed, transfer the A1 pump to the equilibration buffer, rinse the equilibration solution until the detection wavelength is stable, then distribute and elute, and collect the eluent;

[0138] (7) Rinse with equilibrium solution A, and finally rinse with 20% ethanol and store.

[0139] As a result, high-purity proteins were obtained; the above purification process is applicable to the protein sequences in Table 1.

[0140] B. Fc purification step (Protein A purification process)

[0141] Equilibration buffer A: 20mM PB, 150mM NaCl, pH 7.0;

[0142] Elution buffer B: 100 mM Gly, pH 3.0;

[0143] Elution buffer C: 100 mM Gly, pH 2.7;

[0144] (1) Clarification, filtration and concentration of samples: The cell supernatant volume was concentrated 10 times using tangential flow;

[0145] (2) Protein capture and purification were performed using an AKTA protein chromatography column;

[0146] (3) Perform system flushing. First, flush AKTA's A1 pump with equilibration solution A, then flush B1 pump with eluent B, and finally flush B2 pump with eluent C.

[0147] (4) Set the system flow rate to 1 ml / min, select the corresponding column position 2 to connect the protein chromatography column, and use equilibration buffer to equilibrate AKTA and the column. After equilibration, zero the UV filter.

[0148] (5) Start loading the sample by transferring pump A1 into the loading centrifuge tube;

[0149] (6) After the sample loading is completed, transfer pump A1 to equilibration buffer A, rinse the equilibration solution until the detection wavelength is stable, and then elute with elution buffer B and elution buffer C in one step, and collect the eluent;

[0150] (7) Finally, wash with equilibration buffer A. After use, replace equilibration buffer A with pure water and then store with 20% ethanol.

[0151] Example 5: Animal Immunization Experiment Procedure and Methods

[0152] Animal immunization experiments were conducted using the proteins expressed above. The flowchart of the immunization experiment is shown below. Figure 4 As shown; the immunization strategy is as follows:

[0153] d-5: 5 days before the first immunization;

[0154] d-0: Time of first immunization;

[0155] d13, d15, d22, d25, d32: These are the days following the first immunization. The arrows indicate the experimental procedures performed on those days. "Blood collection" refers to in vivo blood collection, and "immunization" refers to the injection of the recombinant protein immunogen expressed in this invention. The second immunization was performed on day 15 (d15), and the third immunization was performed on day 25 (d25).

[0156] The specific steps of animal immunization experiments include:

[0157] 1. C57 mice with the same weight and age were randomly divided into 10 groups.

[0158] 2. Before the experiment, pre-immune serum was collected from each mouse (pre-immune serum was collected on DAY-5 by blood collection through the eyeball, with an appropriate amount of blood collected to ensure the mice were in normal condition), and the collected serum was stored at -80℃.

[0159] 3. Preparation of aluminum adjuvant (aluminum hydroxide adjuvant) group: Before immunization, each antigen was diluted in 75 μL PBS to the corresponding dose (5 μg / mouse) and mixed with alum adjuvant (1 mg / mouse) at a volume ratio of antigen:adjuvant = 3:1 (i.e., 25 μl adjuvant was added to 75 μl of immunogen dilution); the adjuvant was shaken well before use, and the 25 μl adjuvant was slowly added dropwise to the immunogen solution; after the adjuvant and immunogen dilution were thoroughly mixed, they were mixed for 30 minutes to allow the adjuvant to effectively adsorb the antigen; subsequent procedures were performed according to the animal immunization experiment procedures.

[0160] 4. Group without aluminum adjuvant: The antigen was diluted in 100 μL of PBS to the corresponding doses in the table above, and 100 μL of immunogen was added. Subsequent procedures were carried out according to the procedures for immunizing animal experiments.

[0161] 5. Subcutaneous injection at 2-week intervals: The experiment was designed as a 3-immunization method, but blood was collected from the eyeballs 7 days after each immunization injection. Part of the mouse supernatant was obtained by centrifugation and the serum titer was first tested. 7 days after the last immunization, the maximum blood volume was collected from the heart, and the supernatant was obtained by centrifugation and stored at -80℃.

[0162] 6. Throughout the experiment, the mice were observed once a day starting from day 5 until the end of the experiment.

[0163] 7. Observation content: Observe the animal's death or near death, mental state, behavior, fecal characteristics, and the supply of feed and water at the cage.

[0164] 8. Physical appearance: Observation includes whether there are any abnormalities in the eyes, nose, mouth, urethral opening, perianal area, and genital area, and whether the animal is emaciated; abnormal changes such as redness, swelling, and scab formation at the injection site; behavioral activities: whether there is reduced activity, motor incoordination, lethargy or excessive excitement, restlessness and startling, muscle paralysis or tremors, abnormal gait, etc.

[0165] 9. Respiration: Observe for any difficulty in breathing.

[0166] 10. Excretion: Observe for hematuria, loose stools, bloody stools, and any abnormal bowel movements.

[0167] 11. Mortality rate: If any animals are near death or die, record the number of deaths and perform an autopsy promptly; euthanize near-death animals as soon as possible and collect the maximum amount of serum.

[0168] Example 6: Enzyme-linked reaction for detecting antibody titers in animals

[0169] 1. IgM antibody titer detection method

[0170] (1) Base plate coating: Dilute the antigen to 3ug / ml with coating diluent, add 100μl of the prepared coating solution to each well, and place in a 4℃ refrigerator for 24h.

[0171] (2) After 24 hours, remove it from the refrigerator and place it at 37°C for 30 minutes to equilibrate. Then discard the liquid in the hole; wash the hole with washing solution 3 times, 3 minutes each time.

[0172] (3) Blocking the enzyme-labeled reaction wells: Add 200 μL of 5% fetal bovine serum to each well and incubate at 37°C for 90 min. After blocking, wash the wells three times with washing buffer for 3 min each time.

[0173] (4) Add the sample to be tested: Dilute the sample according to the required ratio, add 100 μl of the diluted sample to each enzyme-labeled reaction well, place at 37℃ for 90 min; wash the well with washing solution 3 times, 3 min each time.

[0174] (5) Add enzyme-labeled antibody: Add the appropriate concentration of secondary antibody according to the instructions; wash 100 μl per well at 37℃ for 90 min as before.

[0175] (6) Add substrate solution: Add 100 μl of substrate per well and place at 37°C in the dark for 15 to 30 minutes.

[0176] (7) Termination of reaction: Add 50 μl of termination solution to each well to terminate the reaction, and measure the experimental results within 20 min.

[0177] 2. IgM antibody titer detection results

[0178] After immunizing mice, the IgM detection data are shown in Table 2 and Figure 5 .

[0179] Table 2

[0180]

[0181]

[0182] Table 2 and Figure 5 The results showed that all three vaccine + adjuvant groups could produce certain antibody titers; among them, the "S1-Fc + adjuvant" group had the highest antibody titer: after one routine immunization, the IgM antibody titer was as high as 1:12800 (compared with PBS control); after 2-3 routine booster immunizations, this recombinant protein vaccine could subsequently produce very high levels of immune antibodies.

[0183] 3. IgG antibody titer detection method

[0184] Test Procedure

[0185] Coating → Washing → Blocking → Washing → Adding sample → Incubation → Washing → Secondary antibody → Incubation → Washing → Developing solution → Stop solution → Reading.

[0186] Reagent preparation

[0187] Sample dilution buffer (PBS, pH 7.4): NaCl 0.8g, KHPO4 0.02g, Na2HPO4·12H2O 0.29g, KCl 0.02g, sodium azide 0.01g, add double-distilled water to 1000mL, and adjust pH to 7.4.

[0188] Washing solution (0.5% PBST): Tween-20 2.5 mL, 1×PBS (pH 7.4) 500 mL.

[0189] Coating dilution buffer (0.05 mol / L sodium carbonate-sodium bicarbonate buffer, pH=9.6): 0.15 g sodium carbonate, 0.29 g sodium bicarbonate, 0.02 g sodium azide, add double-distilled water to 100 mL, and adjust pH to 9.6.

[0190] Blocking solution (5% fetal bovine serum / PBS solution): 5 mL fetal bovine serum, 95 mL 1*PBS (pH 7.4).

[0191] The detailed steps are as follows:

[0192] (1) Bottom plate wrapping

[0193] Dilute the antigen to 0.5 ug / ml with coating diluent, add 100 μl of antigen to each well, incubate at 4℃ for 24 h; discard the liquid in the well.

[0194] (2) Seal the enzyme-labeled reaction wells

[0195] Block with 5% fetal bovine serum at room temperature for 90 minutes. After blocking, wash the wells three times with washing buffer for 1 minute each time.

[0196] (3) Add the sample to be tested

[0197] Dilute the sample according to the table below. Add 100 μl of the diluted sample to each well of the enzyme-labeled reaction and incubate at room temperature for 90 min. Wash the wells three times with washing buffer for 1 min each time.

[0198] (4) Add enzyme-labeled antibody

[0199] Add the appropriate concentration of secondary antibody at a 1:4000 dilution according to the instructions, incubate at room temperature for 60 minutes, add 100 μl to each well, and wash as before.

[0200] (5) Add substrate solution

[0201] (6) Substrate addition: 100 μl per well, place at room temperature in the dark for no more than 20 minutes, determine the incubation time based on the color, and add stop solution for color development.

[0202] (7) Termination of reaction

[0203] Add 50 μl of stop solution to each well to terminate the reaction, and measure the experimental results within 20 min. The measurement time should not be too long.

[0204] 4. IgG antibody titer test results

[0205] After immunizing mice, the IgG detection data are shown in Table 3 and Figure 6 As shown.

[0206] Table 3

[0207] Dilution factor 1∶200 1∶400 1∶800 1∶1600 1∶3200 1∶6400 1∶12800 1∶25600 1∶51200 adjuvant 0.144 0.100 0.075 0.066 0.055 0.066 0.065 0.059 0.066 RBD-FC-Adjuvant 2.841 1.822 1.069 0.602 0.328 0.198 0.124 0.096 0.124 RBD + adjuvant 0.397 0.242 0.161 0.116 0.127 0.084 0.072 0.055 0.088 HOPE-V+ adjuvant 1.441 0.905 0.576 0.295 0.160 0.118 0.104 0.081 0.075 S1-FC+Adjuvant 3.741 3.665 3.214 2.263 1.323 0.763 0.422 0.205 0.156

[0208] Table 3 and Figure 6 The results showed that some groups were able to produce high antibody titers, with the highest IgG antibody titer reaching 1:12800 after one booster immunization. Among them, the antibody titer of the "S1-Fc + adjuvant" group was significantly the highest, much higher than that of other groups.

[0209] Example 7: Experimental Method for Neutralizing Pseudoviruses

[0210] The procedure for the pseudovirus neutralization experiment is as follows:

[0211] 1. Cell seeding in 96-well plates

[0212] Cells: 293T-ACE2 cells;

[0213] Culture medium: DMEM + 10% FBS, DMEM stock solution;

[0214] Plate density: 30,000 cells / well;

[0215] After plating, the cells were placed in a 37°C, 5% CO2 incubator for further culture.

[0216] 2. Neutralization process of samples and pseudoviruses

[0217] Dilution: The sample was diluted at a concentration of 1:5.

[0218] 3. Incubation and sample addition

[0219] Sample group: Each drug was diluted to the above detection concentration, and 25 μL was mixed with pseudovirus. 25 μL was added to 293T-ACE2 cells that had been pre-coated and incubated at 37°C for 1 hour. Then the drug and virus were added to the cells. Each sample was in 5 replicates.

[0220] Positive wells: Equal volumes of pseudovirus and DMEM stock solution were mixed and incubated at 37°C for 1 hour. The mixture was then added to pre-coated 293T-ACE2 cells, with 5 replicates.

[0221] Negative wells: DMEM stock solution was incubated at 37°C for 1 hour and added to pre-coated 293T-ACE2 cells, 5 replicates.

[0222] After adding the samples and positive and negative controls to the cells, continue culturing at 37°C in a 5% CO2 incubator for 48-72 hours.

[0223] 4. Results Observation

[0224] The Luciferase emission value (RLU) of the samples in the 96-well plate was detected using a chemiluminescence analyzer; the lower the emission value (RLU reading), the better the neutralization effect.

[0225] The results of the pseudovirus neutralization experiment in the serum of immunized mice 22 days after the first immunization (d22) are shown in Table 4 and Figure 7 Including RLU reads ( Figure 7 (See above) and neutralizing activity ( Figure 7 (See the image below). S-ECD represents the numerical value of the conditional immunization result of the full-length COVID-19 S-ECD protein.

[0226] Table 4

[0227]

[0228] From Table 4 and Figure 7 The results show that, compared with the same recombinant protein, the addition of adjuvant did not produce a high level of neutralization ability for pseudoviruses, while the application of adjuvant significantly improved the neutralization ability. The neutralization percentage in the S-Fc group reached as high as 75.5%.

[0229] This result demonstrates that the application of adjuvants is crucial in the vaccine preparation method of this invention.

[0230] After the second immunization (d32), a pseudovirus experiment was performed, and the results of the neutralizing activity test are shown in Table 5.

[0231] Table 5

[0232]

[0233] The results of the third immunization test for pseudoviruses show that the S1-Fc+adjuvant group achieved an optimal neutralization rate of 97.41% for pseudoviruses.

[0234] Example 8: SARS-CoV-2 True Virus Challenge Experiment in P3 Laboratory

[0235] The procedure for the true virus neutralization experiment is as follows:

[0236] 1. This experiment was conducted in a P3 laboratory, using the SARS-CoV-2 strain and the 2019n-CoV true virus strain (from the Shenzhen P3 laboratory).

[0237] 2. The virus is passaged in designated production cells, and the serum diluent is mixed with 10... 2 One SARS-CoV-2 was incubated at 37°C for 1 hour.

[0238] 3. The serum-virus complex was added to the cell monolayer shown in the diagram in a 96-well plate and incubated at 37°C for 1 hour. Subsequently, the cells were coated with 1% (w / v) methylcellulose in MEM supplemented with 2% FBS.

[0239] 4. After 30 hours, remove the covering and fix it with 4% paraformaldehyde.

[0240] 5. Fix with paraformaldehyde in PBS at room temperature for 20 minutes. Wash the culture plate and incubate sequentially with 1 μg / mL CR3022 anti-S antibody and HRP-bound goat anti-human IgG in PBS. Observe SARS-CoV-2 infected cellular lesions using true blue peroxidase substrate (KPL) and perform quantitative analysis on an immunospot microanalyzer (cells).

[0241] On day 22 (d22) after the first immunization, blood was collected to test viral neutralization activity. The results are shown in Table 6. Figure 8 .

[0242] Table 6

[0243]

[0244] According to Table 6, on day 22 after the first immunization, "S1-FC+adjuvant" can produce a large number of neutralizing antibodies (n=3).

[0245] On day 32 after the first immunization, blood was collected to test the virus neutralization activity. The results are shown in Table 7 (where the numbers represent the ability to neutralize the true virus, i.e., the percentage of neutralizing antibodies (%)).

[0246] Table 7

[0247]

[0248]

[0249] On day 32 after the first immunization, the results of the real virus neutralization experiment showed that the S1-Fc+ adjuvant group had a higher neutralization percentage compared with the adjuvant control and other experimental groups, indicating that the S1-Fc+ adjuvant group could produce higher neutralizing antibodies to neutralize the SARS-CoV-2 virus after immunizing mice.

[0250] The above in vitro SARS-CoV-2 virus neutralization experiments demonstrate that the S1-FC+ adjuvant of the present invention produces significant neutralizing antibodies against the virus (n=4).

[0251] Summarize

[0252] 1. Regarding adjuvants

[0253] Based on the above SARS-CoV-2 neutralization experiments, a comparison regarding the use of adjuvants or the absence of adjuvants is shown in Table 8. Figure 9 (Values ​​are from Table 6) Figure 8 (See above image).

[0254] Table 8

[0255]

[0256] The results showed that, comparing the adjuvanted and unadjuvanted groups, mice immunized under the same conditions using the S1-Fc recombinant protein sequence selected in this invention underwent in vitro evovirus neutralization experiments. The unadjuvanted group showed the same results as the control group, both being negative and showing no neutralizing antibodies. However, in the S1-Fc + adjuvant group, all mice produced neutralizing antibodies. This result demonstrates that the application of adjuvants is indispensable in the vaccine preparation scheme of this invention.

[0257] 2. Fc segment process

[0258] Fc recombinant proteins are biologically active functional protein molecules fused with Fc fragments. The inventors have discovered that they require certain modifications to achieve better results.

[0259] Based on the above SARS-CoV-2 neutralization experiments, the results regarding different Fc mutants are summarized in Table 9 and... Figure 10 .

[0260] Table 9

[0261]

[0262] This result demonstrates that the fusion of Fc with the protein of this invention plays an important role in the vaccine preparation scheme of this invention.

[0263] Different Fc mutants have significantly different effects. The main difference between the "S1-Fc+adjuvant" group and the "S1-D614-FC" group, apart from one random mutation of aa (a natural viral mutation, which the inventors did not find to be substantially different in terms of its immune function), is that different Fc mutants were used in the Fc group.

[0264] according to Figure 7 The results of the pseudovirus neutralization experiment show that the "S1-Fc + adjuvant" group clearly had better efficacy; according to Figure 8 The results of the true virus neutralization experiment also show that the "S1-Fc + adjuvant" group obviously has a better effect.

[0265] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Houpu Biotechnology (Suzhou) Co., Ltd. <120> Vaccines against the novel coronavirus and their application <130> 207011 <160> 5 <170> SIPOSequenceListing 1.0 <210> 1 <211> 917 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(917) <223> Fusion Peptides <400> 1 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Glu Pro Lys 675 680 685 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 690 695 700 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 705 710 715 720 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 725 730 735 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 740 745 750 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 755 760 765 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 770 775 780 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 785 790 795 800 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 805 810 815 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln 820 825 830 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 835 840 845 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 850 855 860 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 865 870 875 880 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 885 890 895 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 900 905 910 Leu Ser Pro Gly Lys 915 <210> 2 <211> 476 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(476) <223> Fusion polypeptide <400> 2 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg 1 5 10 15 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 20 25 30 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 35 40 45 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 50 55 60 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 65 70 75 80 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 85 90 95 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 100 105 110 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 115 120 125 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 130 135 140 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 145 150 155 160 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 165 170 175 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 180 185 190 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 195 200 205 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 210 215 220 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ala Asp 225 230 235 240 Asp Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 245 250 255 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 260 265 270 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 275 280 285 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 290 295 300 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 305 310 315 320 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 325 330 335 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 340 345 350 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 355 360 365 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 370 375 380 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 385 390 395 400 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 405 410 415 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 420 425 430 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 435 440 445 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 450 455 460 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 3 <211> 685 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(685) [[ID=!39]]<223> Fusion polypeptide <400> 3 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg 675 680 685 <210> 4 <211> 238 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(238) <223> RBD domain <400> 4 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg 1 5 10 15 Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu 20 25 30 Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr 35 40 45 Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val 50 55 60 Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser 65 70 75 80 Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser 85 90 95 Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr 100 105 110 Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly 115 120 125 Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly 130 135 140 Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro 145 150 155 160 Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro 165 170 175 Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr 180 185 190 Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val 195 200 205 Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro 210 215 220 Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe 225 230 235 <210> 5 <211> 923 <212> PRT <213> Artificial Sequence <220> <221> PEPTIDE <222> (1)..(923) <223> Fusion polypeptide <400> 5 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ala Asp Asp 675 680 685 Asp Asp Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 690 695 700 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 705 710 715 720 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 725 730 735 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 740 745 750 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 755 760 765 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 770 775 780 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 785 790 795 800 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 805 810 815 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 820 825 830 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 835 840 845 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 850 855 860 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 865 870 875 880 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 885 890 895 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 900 905 910 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 915 920

Claims

1. Use of a fusion polypeptide in the preparation of a vaccine against 2019-nCoV virus, the vaccine comprising the fusion polypeptide and an aluminum adjuvant; The fusion polypeptide comprises, from the amino terminus to the carboxyl terminus, the following interconnected proteins: a signal peptide, a coronavirus spike glycoprotein S1 subunit, and a site-directed mutated IgG Fc; the amino acid sequence of the signal peptide is shown in positions 1-15 of SEQ ID NO:1; the amino acid sequence of the coronavirus spike glycoprotein S1 subunit is shown in positions 16-685 of SEQ ID NO:1; and the amino acid sequence of the site-directed mutated IgG Fc is shown in positions 686-917 of SEQ ID NO:

1. in, The fusion peptide and aluminum adjuvant are in a weight ratio of 1-20:1000.

2. The use as described in claim 1, characterized in that, The fusion peptide and aluminum adjuvant are in a weight ratio of 1 to 10:1000.

3. The use as described in claim 2, characterized in that, The fusion peptide and aluminum adjuvant are in a weight ratio of 5:1000.

4. The use as described in claim 1, characterized in that, The nucleic acid encoding the fusion polypeptide is contained in the pcDNA3.4 vector for expressing the fusion polypeptide.

5. The use as described in claim 4, characterized in that, The pcDNA3.4 vector is contained in host cells Expi293F to express the fusion polypeptide.

6. A method for preparing a vaccine against the 2019-nCoV virus, comprising mixing a fusion peptide with an aluminum adjuvant; The fusion polypeptide comprises, from the amino terminus to the carboxyl terminus, the following interconnected proteins: a signal peptide, a coronavirus spike glycoprotein S1 subunit, and a site-directed mutated IgG Fc; the amino acid sequence of the signal peptide is shown in positions 1-15 of SEQ ID NO:1; the amino acid sequence of the coronavirus spike glycoprotein S1 subunit is shown in positions 16-685 of SEQ ID NO:1; and the amino acid sequence of the site-directed mutated IgG Fc is shown in positions 686-917 of SEQ ID NO:

1. The fusion peptide and aluminum adjuvant are in a weight ratio of 1-20:1000.

7. The method as described in claim 6, characterized in that, The fusion peptide and aluminum adjuvant are in a weight ratio of 1 to 10:1000.

8. The method as described in claim 7, characterized in that, The fusion peptide and aluminum adjuvant are in a weight ratio of 5:1000.

9. Use of a vaccine in the preparation of a kit for immunization to produce antibodies against the 2019-nCoV virus, said kit comprising: The container, and the vaccine contained in the container; The vaccine is as defined in claim 1.

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