Bivalent covid vaccine and methods of making and using same

Abstract

The present application relates to a bivalent new coronavirus vaccine, a preparation method and uses thereof, the bivalent new coronavirus vaccine comprises a fusion protein, the fusion protein comprises a receptor binding domain (RBD) of S protein of a first new coronavirus mutant or a functional fragment thereof, an immunoglobulin Fc and a receptor binding domain (RBD) of S protein of a second new coronavirus mutant or a functional fragment thereof, the first new coronavirus mutant and the second new coronavirus mutant are different mutants. The vaccine can significantly improve the neutralizing antibody titer against the new coronavirus Delta mutant and the Omicron mutant.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a bivalent COVID-19 vaccine, its preparation method, and its uses. Background Technology

[0002] The novel coronavirus (2019-nCoV, SARS-CoV-2) is a beta coronavirus that was first discovered in 2019. It is the seventh known coronavirus that can infect humans. Infection with this virus can cause symptoms such as fever, dry cough, and fatigue.

[0003] The novel coronavirus is composed of four structural proteins (spike glycoprotein, envelope protein, membrane protein, and nucleocapsid protein) and an RNA nucleic acid chain. The spike glycoprotein (S protein) is a glycoprotein located on the surface of the SARS-CoV-2 membrane and primarily functions in cell adhesion and cell membrane fusion. The S protein consists of two subunits, S1 and S2. The S1 subunit contains a receptor-binding domain (RBD), which is responsible for recognizing the host cell receptor ACE2. This is a key factor in virus-receptor interaction and viral invasion of cells, and is also a key target for vaccine design. The S2 subunit contains essential components required for membrane fusion, promoting the fusion of the virus with the host cell membrane.

[0004] The novel coronavirus is constantly mutating, and several worrying variants have emerged and spread, such as the Alpha mutant (B.1.1.7), the Beta mutant (B.1.351), the Gamma mutant (P.1), the Delta mutant (B.1.617.2), and the Omicron mutant (B.1.1.529).

[0005] The neutralizing antibodies produced after immunization with commercially available / investigation-stage COVID-19 vaccines primarily target the RBD (reactive protein barrier), blocking the interaction between the RBD and ACE2. Most SARS-CoV-2 mutants have acquired mutations in the neutralizing antibody epitopes of the RBD, allowing them to evade neutralizing antibodies and reduce vaccine efficacy. Currently, most commercially available / investigation-stage COVID-19 vaccines are designed using the wild-type full-length S protein antigen, offering good protection against wild-type or early COVID-19 mutant strains, but showing varying degrees of decreased protective efficacy against the currently prevalent Delta and Omicron mutant strains.

[0006] Therefore, there is an urgent need to develop new vaccines against the novel coronavirus, especially variants, to combat infection by mutant strains. Summary of the Invention

[0007] To overcome the limitations of existing vaccines, this invention provides a fusion protein. Immunization of animals with the vaccine containing the fusion protein significantly increases the titers of neutralizing antibodies against the SARS-CoV-2 Delta mutant and Omicron mutant strains.

[0008] In one aspect, the present invention provides a fusion protein comprising, in sequence, a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a first COVID-19 mutant strain, immunoglobulin Fc, and a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a second COVID-19 mutant strain, wherein the first COVID-19 mutant strain and the second COVID-19 mutant strain are different mutant strains.

[0009] In another aspect, the present invention provides a nucleic acid encoding the aforementioned fusion protein.

[0010] In another aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0011] In another aspect, the present invention provides a host cell that expresses the aforementioned fusion protein, or contains the aforementioned nucleic acid and / or the aforementioned expression vector.

[0012] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell, and one or more pharmaceutically acceptable vectors, diluents or excipients.

[0013] In another aspect, the present invention provides a vaccine comprising the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell, and one or more adjuvants.

[0014] In another aspect, the present invention provides the use of the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell and / or the aforementioned pharmaceutical composition in the preparation of a vaccine for the treatment or prevention of diseases or symptoms associated with SARS-CoV-2.

[0015] In another aspect, the present invention provides a method for inducing an immune response against SARS-CoV-2 in subjects with appropriate need, the method comprising administering the aforementioned vaccine to the subject.

[0016] Beneficial effects:

[0017] This invention utilizes immunoglobulin Fc to connect the receptor-binding domains (RBDs) of the S protein from different SARS-CoV-2 mutant strains at both ends. This not only maximizes the exposure of the binding sites of the two SARS-CoV-2 mutant S protein RBDs, preventing the binding region from being obscured and maximizing their effectiveness, but also allows immunoglobulin Fc to form a stable dimer through its own disulfide bonds, improving antigen stability and increasing immunogenicity. This activates the antigen presentation function of dendritic cells (DCs), resulting in better efficacy and improved purification efficiency.

[0018] This invention mutates the receptor-binding domain (RBD) of the S protein of the SARS-CoV-2 mutant strain Omicron to Q493K, thereby significantly enhancing its activity. The bivalent SARS-CoV-2 vaccine of this invention exhibits higher IFN-γ and IL-2 responses than single-adjuvant and unadjuvanted combinations, demonstrating a higher level of cellular immunity. The bivalent SARS-CoV-2 vaccine can provide cross-protection against different SARS-CoV-2 strains, including the original strain, Delta strain, and Omicron strain. In particular, the dual-adjuvant bivalent SARS-CoV-2 vaccine can generate specific IgG antibody titers as high as 143,360 and 122,880 against Delta strain and Omicron strain, respectively. The bivalent SARS-CoV-2 vaccine can generate high levels of cellular and humoral immunity. Attached Figure Description

[0019] Figure 1 The image shows the restriction enzyme digestion patterns of the PUC57 plasmid containing the OFD gene and the GDCHO vector, where: A is the restriction enzyme digestion pattern of the PUC57 plasmid containing the OFD gene; B is the restriction enzyme digestion pattern of the GDCHO vector.

[0020] Figure 2 The image shows the expression vector GDCHOOFD pattern and its identification pattern, where A is the expression vector GDCHOOFD pattern and B is the expression vector GDCHOOFD restriction enzyme digestion identification pattern.

[0021] Figure 3 The results show the IFN-γ and IL-2 responses after a second immunization with the bivalent COVID-19 vaccine with different adjuvant combinations. In this paper, A represents the IFN-γ response results after a second immunization with the bivalent COVID-19 vaccine, and B represents the IL-2 response results after a second immunization with the bivalent COVID-19 vaccine.

[0022] Figure 4 The levels of neutralizing antibodies in serum after immunization with pseudoviruses are shown. Detailed Implementation

[0023] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0024] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0025] As used herein and unless otherwise stated, the terms “comprising,” “including,” “having,” “containing,” and their grammatical equivalents, including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0026] As used herein, the term "coronavirus" belongs to the family Coronaviridae, genus Coronavirus, and can infect mammals and birds, causing various respiratory, digestive, and central nervous system diseases. Coronaviruses can be divided into four distinct genera based on genomic and serological differences: α, β, γ, and δ. Currently, only α and β coronaviruses infect humans. To date, six human coronaviruses (HCoVs) from two genera (α and β) have been identified. α coronaviruses include NL63 and 229E, while β coronaviruses include OC43, HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2.

[0027] As used herein, the term “COVID-19” is a viral illness typically characterized by high fever, cough, difficulty breathing, chills, persistent tremors, muscle pain, headache, sore throat, and new loss of taste and / or smell. In severe cases, a host of coagulopathy-related symptoms associated with COVID-19 severity may occur (e.g., blood clotting, thrombosis, acute respiratory distress syndrome, seizures, heart attack, stroke, multiple cerebral infarctions, renal failure, diabetes insipidus, and / or disseminated intravascular coagulation). In younger patients, rare inflammatory syndromes are sometimes associated with COVID-19 (e.g., atypical Kawasaki syndrome, toxic shock syndrome, pediatric multisystem inflammatory disease, and cytokine storm syndrome). The beta-coronavirus SARS-CoV-2 is the causative agent.

[0028] As used herein, the term "fusion protein" refers to a natural or synthetic molecule composed of one or more molecules, wherein two or more peptide- or protein-based (including glycoprotein) molecules with different specificities are optionally fused together by a chemical or amino acid-based linker molecule. This connection can be achieved via CN fusion or NC fusion (in the 5′→3′ orientation), with CN fusion being preferred.

[0029] As used herein, the term "antibody" or "immunoglobulin" has the broadest meaning, and in particular includes complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) consisting of at least two complete antibodies, and antibody fragments, provided they exhibit the desired biological activity. This term generally includes hybrid antibodies consisting of two or more antibodies or antibody fragments linked together with different binding specificities.

[0030] As used herein, the term "Fc" or "Fc region" is used to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus of the heavy chain. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, compositions of complete antibodies can comprise antibody populations with all K447 residues removed, antibody populations without any K447 residues removed, and antibody populations containing a mixture of antibodies with and without K447 residues.

[0031] As used herein, sequence “identity” or “commonality” has the meaning generally accepted in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of the polynucleotide or polypeptide or along a region of the molecule. Although many methods exist for measuring the similarity between two polynucleotides or polypeptides, the term “identity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0032] As used herein, the terms “disease” or “symptom” refer to the state of life or health of a patient or individual who can be treated with the fusion protein, pharmaceutical composition or method provided herein.

[0033] The term "vaccine" refers to a purified antigen vaccine or immunogenic composition, a subunit vaccine or immunogenic composition, an inactivated whole virus vaccine or immunogenic composition, or an attenuated virus vaccine or immunogenic composition. In some embodiments, the vaccine or immunogenic composition is a purified fusion protein.

[0034] As used herein, the term "treating" or "treatment" refers to any indication of successfully treating or improving an injury, disease, pathology, or condition, including any objective or subjective parameters, such as elimination; relief; reduction of symptoms or making the injury, pathology, or condition more tolerable for the patient; slowing the rate of deterioration or decline; or reducing the final point of deterioration; or improving the patient's physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters; and may include the results of physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term "treatment" and its adjuncts may include prevention of injury, pathology, condition, or disease. In this embodiment, treatment is prevention. In this embodiment, treatment does not include prevention.

[0035] As used herein (and fully understood in the art), “treating” also broadly includes any method used to obtain a beneficial or desired outcome (including clinical outcomes) in a subject’s condition. Beneficial or desired clinical outcomes may include, but are not limited to: relief or improvement of one or more symptoms or conditions; reduction of the severity of the disease; stabilization (i.e., non-deterioration) of the disease state; prevention of the spread or diffusion of the disease; delay or slowing the progression of the disease; improvement or remission of the disease state; reduction of disease recurrence; and remission (whether partial or complete, and whether detectable or undetectable). In other words, as used herein, “treatment” includes any cure, improvement, or prevention of a disease. Treatment may prevent the onset of disease; suppress the spread of disease; relieve the symptoms of disease; completely or partially eliminate the root cause of the disease; shorten the duration of the disease; or a combination of these.

[0036] As used herein, “treating” includes preventative treatment. Treatment methods involve administering a therapeutically effective amount of an active agent to a subject. Administration may consist of a single administration or may comprise a series of administrations. The length of treatment depends on various factors, such as the severity of symptoms, the patient's age, the concentration of the active agent, the activity of the composition used in treatment, or a combination thereof. It should also be understood that the effective dose of a pharmaceutical agent used for treatment or prevention may be increased or decreased during a particular treatment or prevention regimen. Changes in dose can be produced and become apparent using standard diagnostic assays known in the art. In some cases, chronic administration may be necessary. For example, administering the composition to the subject in an amount sufficient to treat the patient for a sufficient duration.

[0037] As used herein, the term "prevention" refers to reducing the occurrence of disease symptoms in a patient. As mentioned above, prevention can be complete (without detectable symptoms) or partial, resulting in fewer symptoms being observed than would be possible without treatment.

[0038] As used herein, "patient" or "subject in need" means a living organism suffering from or susceptible to suffering from a disease or symptom that can be treated by administration of a pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, bovine animals, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammals. In some embodiments, the patient is a human.

[0039] The term "combined administration" refers to the "combined use" of the fusion protein or vaccine of the present invention with a known drug (or other compound, or other vaccine), such that both have therapeutic or diagnostic effects. Such combined administration may include the administration of the drug (or other compound, or other vaccine) in parallel (i.e., simultaneously), before, or sequentially with respect to the administration of the fusion protein or vaccine of the present invention. Those skilled in the art will be able to readily determine the appropriate timing, order, and dosage of administration of a particular drug (or other compound, or other vaccine) and the combination of the present invention.

[0040] As used herein, the term "effective amount" is an amount sufficient to achieve the stated purpose (e.g., to achieve the effect it is administered to accomplish, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce protein function, to alleviate one or more symptoms of a disease or condition). Examples of "effective amounts" are amounts sufficient to induce treatment, prevention, or reduction of one or more symptoms of a disease; such amounts may also be referred to as "therapeutic effective amounts." "Reduction" of one or more symptoms means a reduction in the severity or frequency of one or more symptoms, or the elimination of one or more symptoms. A "preventive effective amount" of a drug is an amount of drug that, when administered to a subject, will have the expected preventive effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology, or condition or its symptoms. A complete preventive effect does not necessarily occur with a single dose and can occur after only a series of doses. Therefore, preventive effective amounts can be administered in the form of a single or multiple doses.

[0041] As used herein, the term "therapeutic effective dose" refers to an amount of therapeutic agent sufficient to improve the condition, as described above. For example, for a given parameter, a therapeutic effective dose will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease in "multiples." For example, a therapeutic effective dose may have an effect of at least 1.2 times, 1.5 times, 2 times, 5 times, or more relative to a control.

[0042] Dosage can vary depending on the patient's needs and the fusion protein or vaccine used. In the context of this invention, the dose administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The magnitude of the dose will also be determined by the presence, nature, and extent of any adverse side effects. Determining the appropriate dose for a specific situation is within the skill of the practitioner. Typically, treatment begins with a smaller dose than the optimal dose of the fusion protein or vaccine. Thereafter, the dose is increased in small increments until the optimal effect is achieved in these situations. The amount and interval of administration can be individually adjusted to provide a level of efficacy of the administered fusion protein or vaccine for the specific clinical indication being treated. This will provide a treatment regimen commensurate with the severity of the individual's disease state.

[0043] As used herein, the term "administration" means oral administration to a subject, administration in suppository form, local contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intrathecal, intranasal, or subcutaneous administration, or administration via implantation of a slow-release device (e.g., a microosmotic pump). Administration via any route includes parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or percutaneous) administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intravenous, and intracranial administration. Other delivery modalities include, but are not limited to, the use of liposome formulations, intravenous infusion, percutaneous patches, etc. In the embodiments, administration does not include the administration of any active agent other than the described active agents.

[0044] In one aspect, the present invention provides a fusion protein comprising, in sequence, a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a first COVID-19 mutant strain, immunoglobulin Fc, and a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a second COVID-19 mutant strain, wherein the first COVID-19 mutant strain and the second COVID-19 mutant strain are different mutant strains.

[0045] In some embodiments, the SARS-CoV-2 mutant strain is selected from Alpha mutant strain (B.1.1.7), Beta mutant strain (B.1.351), Gamma mutant strain (P.1), Delta mutant strain (B.1.617.2), or Omicron mutant strain (B.1.1.529).

[0046] In some implementations, the first COVID-19 mutant strain is the COVID-19 Omicron mutant strain, and the second COVID-19 mutant strain is the COVID-19 Delta mutant strain, or the first COVID-19 mutant strain is the COVID-19 Delta mutant strain, and the second COVID-19 mutant strain is the COVID-19 Omicron mutant strain.

[0047] In some implementations, the first COVID-19 mutant strain is the COVID-19 Omicron mutant strain, and the second COVID-19 mutant strain is the COVID-19 Delta mutant strain.

[0048] In some embodiments, the RBD of the S protein of the SARS-CoV-2 Omicron mutant strain comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:3, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the RBD of the S protein of the SARS-CoV-2 Omicron mutant strain is as shown in SEQ ID NO:3.

[0049] In some embodiments, the RBD of the S protein of the COVID-19 Delta mutant strain comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:4, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the RBD of the S protein of the COVID-19 Delta mutant strain is as shown in SEQ ID NO:4.

[0050] In some embodiments, the fusion protein comprises an amino acid sequence having 80% or more identity with the sequence shown in amino acids 26-726 of SEQ ID NO:2, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the fusion protein is as shown in amino acids 26-726 of SEQ ID NO:2.

[0051] In some implementations, a linker is included between the receptor-binding domain (RBD) of the S protein of the first COVID-19 mutant strain or a functional fragment thereof, immunoglobulin Fc, and the receptor-binding domain of the S protein of the second COVID-19 mutant strain or a functional fragment thereof.

[0052] In some embodiments, the immunoglobulin Fc is selected from IgG1 Fc, IgG2 Fc, IgG3 Fc, and IgG4 Fc, preferably IgG1 Fc; more preferably, it is IgG1 Fc.

[0053] In some embodiments, the immunoglobulin Fc comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:5, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the amino acid sequence of the immunoglobulin Fc is as shown in SEQ ID NO:5.

[0054] In some implementations, the joint is a flexible joint.

[0055] In some embodiments, the flexible joint is selected from GSGGGSGGGGSGGGGS (SEQ ID NO:7), GGGGS (SEQ ID NO:8), and GGGGSGGGGS (SEQ ID NO:9).

[0056] In some embodiments, the flexible polypeptide is GSGGGSGGGGSGGGGS (SEQ ID NO:7).

[0057] In one aspect, the present invention provides a nucleic acid encoding the aforementioned fusion protein.

[0058] In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleotide sequence having 80% or more identity with the sequence shown in nucleotides 91-2193 of SEQ ID NO:1, preferably having 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more identity, more preferably having 98% or 99% or more identity; more preferably, the nucleic acid encoding the fusion protein is as shown in nucleotides 91-2193 of SEQ ID NO:1.

[0059] In some implementations, the nucleic acid is mRNA.

[0060] In one aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid.

[0061] In some implementations, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector, preferably a eukaryotic expression vector.

[0062] In some implementations, the eukaryotic expression vector is pCHO1.0.

[0063] In some implementations, the eukaryotic expression vector is an adenovirus vector.

[0064] In one aspect, the present invention provides a host cell that expresses the aforementioned fusion protein, or contains the aforementioned nucleic acid and / or the aforementioned expression vector.

[0065] In some implementations, the host cell is a prokaryotic cell or a eukaryotic cell.

[0066] In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the prokaryotic cell is an Escherichia coli cell.

[0067] In some embodiments, the eukaryotic cells are selected from yeast cells, insect cells, and mammalian cells. In some embodiments, the mammalian cells are selected from CHO, HEK293, SP2 / 0, BHK, C127, etc. In some embodiments, the eukaryotic cells are CHO cells.

[0068] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell, and one or more pharmaceutically acceptable vectors, diluents or excipients.

[0069] In another aspect, the present invention provides a vaccine comprising the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell, and one or more adjuvants.

[0070] In some embodiments, the adjuvant is selected from at least one of aluminum hydroxide, CpG, aluminum phosphate, saponins such as Quil A, QS-21, GPI-0100, water-in-oil emulsions, oil-in-water emulsions, and water-in-oil-in-water emulsions.

[0071] In another aspect, the present invention provides the use of the aforementioned fusion protein, nucleic acid, expression vector and / or the aforementioned host cell and / or the aforementioned pharmaceutical composition in the preparation of a vaccine for the treatment or prevention of diseases or symptoms associated with SARS-CoV-2.

[0072] In another aspect, the present invention provides a method for inducing an immune response against SARS-CoV-2 in subjects with appropriate need, the method comprising administering the aforementioned vaccine to the subject.

[0073] In some implementations, the subject is a mammal or a bird.

[0074] In some implementations, the subjects are humans, cattle, dogs, cats, goats, sheep, pigs, horses, turkeys, ducks, or chickens.

[0075] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0076] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0077] Example 1: Construction of a high-efficiency expression vector

[0078] 1. Materials:

[0079] (1) The bivalent COVID-19 vaccine sequence was designed by Ginobiliary Health Products Co., Ltd.

[0080] The target gene was formed by sequentially linking the receptor-binding domain, linker, IgG1 Fc, and the coding nucleic acid sequences of the modified SARS-CoV-2 mutant omicron S protein receptor-binding domain and the modified SARS-CoV-2 mutant delta S protein receptor-binding domain. The entire recombinant gene sequence (OFD, SEQ ID NO:1) was optimized and synthesized by Nanjing Genscript Biotech Co., Ltd. The synthesized gene included restriction enzyme sites (AvrII, BstZ17I), a Kozak sequence (GCCGCCACC), a signal peptide, the target gene, and a stop codon, with a total length of 2205 bp. Codon optimization was performed during the synthesis of this recombinant gene to facilitate expression in Chinese hamster ovary cells (Cricetulus griseus, CHO cells).

[0081] The amino acid sequence of the OFD recombinant gene sequence encoding the OFD protein of the bivalent COVID-19 vaccine is shown in SEQ ID NO:2, and it includes:

[0082] The receptor-binding domain (RBD) sequence of the SARS-CoV-2 mutant strain omicron, shown in SEQ ID NO:3;

[0083] The delta receptor-binding domain (RBD) sequence of the SARS-CoV-2 mutant strain shown in SEQ ID NO:4;

[0084] The IgG1 Fc sequence shown in SEQ ID NO:5;

[0085] The signal peptide sequence shown in SEQ ID NO:6;

[0086] The connector sequence shown in SEQ ID NO:7.

[0087] OFD nucleotide sequence (2205bp, SEQ ID NO:1):

[0088]

[0089]

[0090] OFD amino acid sequence (SEQ ID NO:2):

[0091]

[0092]

[0093] in:

[0094] The first 25 amino acids form the signal peptide sequence (SEQ ID NO:6);

[0095] Amino acids 26-244 are the receptor-binding domain sequence of the SARS-CoV-2 mutant strain omicron (SEQ ID NO:3);

[0096] Amino acids 245-260 and 492-507 form the linker sequence listing (SEQ ID NO:7);

[0097] Amino acids 508-726 are the delta receptor-binding domain sequence of the SARS-CoV-2 mutant strain (SEQ ID NO:4).

[0098] (2) The high-efficiency expression vector GDCHO is derived from the commercially available pCHO1.0 expression vector, which replaces the pac with the existing GS gene selection marker and deletes CMV pA and PcmvIEF1 (provided by Beijing Gino Biotechnology Co., Ltd.).

[0099] 2. Methods

[0100] The PUC57 plasmid containing the OFD gene, synthesized by GenScript, was digested with AvrII, BstZ17I, and BspHI on agarose gel to obtain the OFD gene fragment. Simultaneously, the vector GDCHO was double-digested with AvrII and BstZ17I, and the digested large fragment of the vector GDCHO was obtained on agarose gel. Agarose gel electrophoresis of the digestion products is shown below. Figure 1 As shown.

[0101] The OFD gene fragment recovered from enzyme digestion was ligated with the digested vector GDCHO large fragment using ligase, transformed into E. coli competent cells TOP10, clones were picked, plasmids were rapidly extracted, and identified by AvrII and Bstz17I enzyme digestion. Agarose gel electrophoresis results showed... Figure 2 The plasmid that was correctly identified through enzyme digestion was sent to Tianyi Huiyuan Sequencing Company for sequencing. The correctly sequenced plasmid was named GDCHOOFD.

[0102] The correctly sequenced plasmid GDCHOOFD was transformed into E. coli competent cells TOP10. Single clones were picked, scaled up, and cultured overnight in 200 ml LB liquid medium. The plasmid was then extracted using the Tiangen Endotoxin-Free Large Extraction Kit, and the plasmid concentration was determined to be 1.3 mg / ml. The plasmid was then frozen for later use.

[0103] Example 2: Construction of a cell line stably expressing the OFD gene

[0104] The cell electroporator from Yida Biotechnology was placed in a clean bench, and the electroporation parameters were set as follows: voltage 200V, duration 2000μS, 6 pulses, and interval 1000ms. 20μg of GDCHOOFD plasmid was added to 200μl of CHO-K1 cells, and the plasmid containing the OFD gene was electroporated into the CHO-K1 cells.

[0105] After electroporation, CHO-K1 cells were transferred to T25 flasks containing 10 ml of CHO CD02 medium and incubated at 37°C with 5% CO2 for 24–48 hours. This CHO CD02 medium was a selection medium containing 25 μM methionine sulfoximine (MSX) and 200 nM methotrexate (MTX). Cells were then centrifuged and transferred to 125 ml shake flasks containing 30 ml of CHO CD02 medium. Change the selection medium every 3-5 days. When the cell density reaches 1.5 million / ml and the cell viability is above 95%, increase the MTX concentration to 1000nM and inoculate the cells at a density of 500,000 / ml. Cryopreserve the remaining cells for later use. Change the selection medium every 3-5 days. When the cell density reaches 1.5 million / ml and the cell viability is above 95%, inoculate the cells again at a density of 500,000 / ml and increase the MTX concentration to 2000nM. Cryopreserve the remaining cells for later use. Change the selection medium every 3-5 days. When the cell density reaches 1.5 million / ml and the cell viability is above 95%, cryopreserve 4 cell lines for later use. Adjust the cell density of the remaining cells using the limiting dilution method. Single-clone the cells at a density of 0.5 cells / well into 20 96-well plates. Seed 200μl / well into each 96-well plate and incubate at 37°C with 5% CO2.

[0106] After approximately 14 days of static culture in 96-well plates, the cells were observed under an optical microscope, and wells with good cell condition and single-cell expression were marked. When the clones reached 50-70% confluence in the 96-well plates, single-cell clones with good cell condition were selected and seeded into 24-well plates. When the cells reached 95% confluence, the culture supernatant was aspirated in batches as test samples to identify single-cell lines that highly expressed 3F9, 7E6, 13H3, and 20B8 as candidate cell lines.

[0107] Based on the fed-batch culture results, 7E6 was selected as the backup cell line for subsequent use.

[0108] Example 3: Large-scale culture of 7E6 cell line and purification and recovery of OFD protein

[0109] After inoculating the obtained high-expression monoclonal cell line 7E6, the culture was gradually scaled up to 1L shake flasks with a culture medium volume of 300ml. Then, the culture was transferred to eight 1L shake flasks and fed-batch cultured until the cell viability reached 50%. The culture was then stopped and the culture supernatant was harvested.

[0110] Centrifuge at 8000 rpm to remove cells and cell debris, and collect the cell culture supernatant. Filter through a 0.45 μm filter membrane, and pass the filtrate through a Protein A gel chromatography column pre-equilibrated with 20 mM PBS (pH 7.4, 150 mM NaCl). Wash with 2–4 column volumes of 20 mM PBS until A280 returns to baseline. Elute the OFD protein bound to the Protein A column with pH 3.0 100 mM glycine-hydrochloric acid buffer. Adjust the pH to approximately 7.0 with 1 M Tris. Detect OFD protein using SDS-PAGE electrophoresis. Freeze correctly detected proteins for later use.

[0111] Example 4: Purified OFD-immunized mice

[0112] Twenty-five 6-8 week old female C57BL / 6 mice were randomly divided into 5 groups. The mice were immunized twice with the immunogen on days 0 and 21. Fourteen days after the second immunization, the expression of IFN-γ and IL-2 cytokines in spleen lymphocytes of groups 1, 2, 3, 4, and 5 was detected by ELISPOT to assess the cellular immunogenicity of the vaccine. The humoral immunogenicity of the vaccine was assessed by detecting the titer of specific IgG antibodies in serum using ELISA.

[0113] Table 1: Immunization Protocol Design

[0114] Group Immunogen adjuvant immune system Immunization methods Number of immunizations Animal numbers 1 PBS - 0.1ml im 2 5 2 OFD 10μg - 0.1ml im 2 5 3 OFD 10μg <![CDATA[0.2mg AL(OH)3]]> 0.1ml im 2 5 4 OFD 10μg 10μg CpG 0.1ml im 2 5 5 OFD 10μg <![CDATA[0.2mg AL(OH)3+10μgCpG]]> 0.1ml im 2 5

[0115] ELISPOT assays were performed to detect IFN-γ and IL-2 cytokine levels in splenic lymphocytes after immunization. Mouse splenic lymphocytes were detected according to the instructions of the Murine IFN-γ Single-Color Enzymatic ELISPOT Assay (CTL catalog number mIFNgp-2M / 2) and Murine IL-2 Single-Color Enzymatic ELISPOT Assay (CTL catalog number mIL2p-2M / 2) kits. The experimental results are shown below. Figure 3 .

[0116] The levels of binding antibodies in serum after immunization were detected by ELISA, and the results are shown in Table 3. The levels of neutralizing antibodies in serum after immunization were detected by pseudovirus, and the results are shown in [Table 3]. Figure 4 The fake virus detection kits used in the testing are shown in Table 2.

[0117] Table 2: Fake virus test kits were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0118] fake virus name reagent kit name Item number Original strain pseudovirus SARS-CoV-2-Fluc WT DD1746-01 / 02 / 03 Delta strain pseudovirus SARS-CoV-2-Fluc B.1.617.2 DD1754-01 / 02 / 03 Omicron strain pseudovirus SARS-CoV-2-Fluc B.1.1.529 DD1768-01 / 02 / 03

[0119] Table 3: IgG antibody titers specific to RBD against different SARS-CoV-2 strains

[0120]

[0121] in conclusion: Figure 3 The results show the IFN-γ and IL-2 responses after a second immunization with a bivalent COVID-19 vaccine using different adjuvant combinations. A represents the IFN-γ response after a second immunization with the bivalent COVID-19 vaccine; B represents the IL-2 response. The results show that the dual-adjuvant bivalent COVID-19 vaccine resulted in higher IFN-γ and IL-2 responses than the single-adjuvant and unadjuvanted combinations, demonstrating a higher level of cellular immunity. Table 3 shows the RBD-specific IgG antibody titers against different COVID-19 strains, indicating that the bivalent COVID-19 vaccine can provide cross-protection against different SARS-CoV-2 strains, including the original strain, the Delta strain, and the Omicron strain. In particular, the dual-adjuvant bivalent COVID-19 vaccine produced specific IgG antibody titers as high as 143,360 and 122,880 against the Delta and Omicron strains, respectively. Figure 4 The results showed that the vaccine produced pseudovirus neutralizing antibody titers exceeding 3000 against the original strain, Delta strain, and Omicron strain. In summary, the newly developed bivalent COVID-19 vaccine can induce high levels of cellular and humoral immunity and exhibits significant cross-protective effects against different COVID-19 mutant strains.

[0122] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fusion protein comprising a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a first SARS-CoV-2 mutant strain, immunoglobulin Fc, and a receptor-binding domain (RBD) or a functional fragment thereof of the S protein of a second SARS-CoV-2 mutant strain, wherein the first SARS-CoV-2 mutant strain is the SARS-CoV-2 Omicron mutant strain, and the second SARS-CoV-2 mutant strain is the SARS-CoV-2 Delta mutant strain, and the amino acid sequence of the fusion protein is shown as amino acids 26-726 of SEQ ID NO:

2.

2. The fusion protein of claim 1, wherein, The fusion protein further comprises a signal peptide, the amino acid sequence of which is shown in SEQ ID NO:

6.

3. The fusion protein of claim 2, wherein, The amino acid sequence of the fusion protein is shown in SEQ ID NO:

2.

4. A nucleic acid encoding the fusion protein of claim 1, wherein, The nucleic acid encoding the fusion protein is shown as nucleotides 91-2193 of SEQ ID NO:

1.

5. The nucleic acid according to claim 4, wherein, The fusion protein further comprises a signal peptide, and the nucleic acid encoding the fusion protein containing the signal peptide is shown as nucleotides 16-2193 of SEQ ID NO:

1.

6. The nucleic acid according to claim 4 or 5, wherein, The nucleic acid in question is mRNA.

7. An expression vector comprising the nucleic acid according to any one of claims 4-6.

8. The expression vector according to claim 7, wherein, The expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

9. The expression vector according to claim 8, wherein, The expression vector is a eukaryotic expression vector.

10. The expression vector according to claim 9, wherein, The eukaryotic expression vector is pCHO1.

0.

11. The expression vector according to claim 9, wherein, The eukaryotic expression vector is an adenovirus vector.

12. A host cell expressing the fusion protein of any one of claims 1-3, or comprising the nucleic acid of any one of claims 4-6, or comprising the expression vector of any one of claims 7-11.

13. The host cell of claim 12, wherein, The host cell is a prokaryotic cell or a eukaryotic cell.

14. The host cell of claim 13, wherein, The prokaryotic cells mentioned are bacterial cells.

15. The host cell of claim 13, wherein, The prokaryotic cells are Escherichia coli cells.

16. The host cell of claim 13, wherein, The eukaryotic cells are selected from yeast cells, insect cells, and mammalian cells.

17. The host cell of claim 16, wherein, The mammalian cells were selected from CHO, HEK293, SP2 / 0, BHK, and C127.

18. The host cell of claim 17, wherein, The mammalian cells mentioned are CHO cells.

19. A pharmaceutical composition comprising a fusion protein according to any one of claims 1-3, a nucleic acid according to any one of claims 4-6, an expression vector according to any one of claims 7-11 or a host cell according to any one of claims 12-18, and one or more pharmaceutically acceptable carriers, diluents or excipients.

20. A vaccine comprising a fusion protein according to any one of claims 1-3, a nucleic acid according to any one of claims 4-6, an expression vector according to any one of claims 7-11 or a host cell according to any one of claims 12-18, and one or more adjuvants, said adjuvant being selected from at least one of aluminum hydroxide or CpG.

21. Use of the fusion protein of any one of claims 1-3, the nucleic acid of any one of claims 4-6, the expression vector of any one of claims 7-11, the host cell of any one of claims 12-18, or the pharmaceutical composition of claim 19 in the preparation of a vaccine for the prevention of disease caused by SARS-CoV-2 virus infection.

22. Use of the fusion protein of any one of claims 1-3, the nucleic acid of any one of claims 4-6, the expression vector of any one of claims 7-11, the host cell of any one of claims 12-18, or the pharmaceutical composition of claim 19 in the preparation of a vaccine that induces an immune response against SARS-CoV-2 in subjects with appropriate need.

23. The use as described in claim 22, wherein, The subjects were either humans or rodents.

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