CN116041544A

CN116041544A - Bivalent new crown vaccine and its preparation method and use

Info

Publication number: CN116041544A
Application number: CN202211584733.5A
Authority: CN
Inventors: 王希良; 李世崇; 王莉; 王亚丽; 程晋霞; 司炳银
Original assignee: Beijing Jinuo Sanitary Products Technology Co ltd
Current assignee: Beijing Jinuo Sanitary Products Technology Co ltd
Priority date: 2022-12-09
Filing date: 2022-12-09
Publication date: 2023-05-02

Abstract

The invention relates to a bivalent new crown vaccine, a preparation method and application thereof, wherein the bivalent new crown vaccine comprises fusion protein, the fusion protein comprises a Receptor Binding Domain (RBD) of S protein of a first new crown mutant strain or a functional fragment thereof, immunoglobulin Fc and a Receptor Binding Domain (RBD) of S protein of a second new crown mutant strain or a functional fragment thereof, and the first new crown mutant strain and the second new crown mutant strain are different mutant strains. The vaccine provided by the invention can obviously improve the titer of neutralizing antibodies to the novel crown Delta mutant strain and the Omicron mutant strain.

Description

Bivalent new crown vaccine and its preparation method and use

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a bivalent new crown vaccine, a preparation method and application thereof.

Background

The novel coronavirus (2019-nCoV, SARS-CoV-2) is a kind of beta coronavirus, which is found for the first time in 2019 and is a seventh known coronavirus capable of infecting human, and after infection, the virus can cause symptoms such as fever, dry cough, hypodynamia and the like of patients.

The novel coronavirus consists of four structural proteins (spinous, envelope, membrane and nucleocapsid proteins) and RNA nucleic acid strands. Wherein the spinous process protein (Spike Glycoprotein, S protein) is a glycoprotein, which is located on the surface of a novel coronavirus membrane and mainly acts on cell adhesion and cell membrane fusion. The S protein consists of two subunits S1 and S2, wherein the S1 subunit contains a receptor binding domain (Receptor Binding Domain, RBD), which is responsible for recognizing the host cell receptor ACE2, is a key factor for virus and receptor interaction and virus invasion cells, and is also a key target for vaccine design. The S2 subunit contains the essential elements required for the membrane fusion process and is capable of facilitating fusion of the virus with the host cell membrane.

Among the constantly mutating novel coronaviruses, several alarming varieties have emerged and propagated, such as Alpha mutant (b.1.1.7), beta mutant (b.1.351), gamma mutant (p.1), delta mutant (b.1.617.2), omicron mutant (b.1.1.529), etc.

Neutralizing antibodies generated after immunization with commercial/developed new coronal vaccines are directed primarily against RBD to block the interaction between RBD and ACE 2. Most SARS-CoV-2 mutants acquire mutations in the neutralizing antibody epitopes of RBD, which can escape neutralizing antibodies, reducing vaccine efficacy. At present, the commercial/research new crown vaccines mostly adopt wild type full-length S protein antigen design, have better epidemic prevention capability on wild type or early new crown mutant strains, but have different degrees of protective force reduction phenomena on the mainstream Delta mutant strains and Omicron mutant strains at present.

Thus, there is an urgent need to develop new vaccines against novel coronaviruses, especially mutant novel coronaviruses, to cope with infection with mutant strains.

Disclosure of Invention

To avoid the limitations of existing vaccines, the present invention provides a fusion protein. After the vaccine containing the fusion protein is adopted to immunize animals, the neutralizing antibody titer of the novel crown Delta mutant strain and the Omicron mutant strain can be obviously improved.

In one aspect, the invention provides a fusion protein comprising, in order, the Receptor Binding Domain (RBD) of the S protein of a first novel corona-mutant, or a functional fragment thereof, the immunoglobulin Fc, and the Receptor Binding Domain (RBD) of the S protein of a second novel corona-mutant, or a functional fragment thereof, the first novel corona-mutant and the second novel corona-mutant being different mutants.

In another aspect, the invention provides a nucleic acid encoding the fusion protein described above.

In another aspect, the invention provides an expression vector comprising the aforementioned nucleic acid.

In another aspect, the invention provides a host cell expressing the fusion protein described above, or comprising the nucleic acid described above and/or the expression vector described above.

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

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

In another aspect, the 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 a disease or condition associated with SARS-CoV-2.

In another aspect, the invention provides a method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising: the vaccine described above is administered to a subject.

The beneficial effects are that:

according to the invention, the Receptor Binding Domains (RBDs) of the S proteins of different novel crown mutant strains are connected at two ends of the immunoglobulin Fc, so that the Receptor Binding Domains (RBDs) of the S proteins of the two novel crown mutant strains can expose binding sites to the greatest extent, the binding domains are prevented from being shielded and play a role to the greatest extent, meanwhile, the immunoglobulin Fc forms a stable dimer through disulfide bonds, the stability of antigens is improved, the immunogenicity is increased, the antigen presentation effect of Dendritic Cells (DCs) is activated, the effect is better, and the purification efficiency is also improved.

The invention makes mutation Q493K aiming at Receptor Binding Domain (RBD) of S protein of novel crown mutant omacron, thus the activity is greatly improved, the combination of Yu Bishan adjuvant and non-adjuvant for IFN-gamma and IL-2 response is higher by adopting the divalent novel crown vaccine provided by the invention, the higher cellular immunity level is embodied, the divalent novel crown vaccine can generate cross protection to different novel coronaviruses, including original strain, delta strain and omacron strain, especially the divalent novel crown vaccine with double adjuvant can generate specific IgG antibody titer up to 143360 and 122880 aiming at Delta strain and omacron strain, and the divalent novel crown vaccine can generate higher cellular immunity and humoral immunity.

Drawings

FIG. 1 shows the gene cleavage map of the PUC57 plasmid containing the OFD gene and the GDCHO vector cleavage map in which: a is a gene restriction map of a PUC57 plasmid containing an OFD gene; b is GDCHO vector restriction enzyme map.

FIG. 2 shows an expression vector GDCHOOFD map and an identification map for the expression vector, wherein A is the expression vector GDCHOOFD map; b is an expression vector GDCHOOFD enzyme digestion identification map.

FIG. 3 shows IFN-gamma and IL-2 responses after a new coronal bivalent vaccine in different adjuvant combinations, wherein A is the IFN-gamma response results after a new coronal bivalent vaccine is post-exempt; b is the IL-2 response result after the new crown bivalent vaccine is subjected to secondary immunity.

Figure 4 shows serum neutralizing antibody levels after pseudovirus detection immunization.

Detailed Description

In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein and unless otherwise indicated, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where integers are required, the term refers to rounding up or down to the nearest integer within plus or minus 10% of a given value or range.

As used herein and unless otherwise indicated, the terms "comprising," "including," "having," "containing," and their grammatical equivalents are generally understood to be open-ended and not to be limiting, e.g., not to exclude other, unrecited elements or steps.

As used herein, the term "Coronavirus" belongs to the family coronaviridae, genus Coronavirus, which can infect mammals and birds, causing various diseases of the respiratory system, digestive and central nervous. Coronaviruses can be divided into four different genera based on genomic and serological differences: alpha, beta, gamma and delta, only alpha and beta coronaviruses currently infect humans. Up to now 6 human coronaviruses (HCoV) from two genera (α and β) have been identified, including NL63 and 229E, and β coronaviruses including OC43, HKU1, acute respiratory syndrome coronavirus (SARS-CoV), middle east respiratory syndrome coronavirus (MERS-CoV) and novel coronaviruses (SARS-CoV-2).

As used herein, the term "covd-19" is a viral disease, typically characterized by symptoms of high fever, cough, dyspnea, chills, sustained tremors, muscle pain, headache, sore throat, new gustatory and/or olfactory loss. In severe cases, a number of coagulopathy-related symptoms (e.g., blood clotting, thrombosis, acute respiratory distress syndrome, seizures, heart attacks, stroke, multiple cerebral infarction, renal failure diabetes insipidus, and/or disseminated intravascular coagulation) may occur that are related to the severity of covd-19. In young patients, rare inflammatory syndromes are sometimes associated with covd-19 (e.g., atypical kawasaki syndrome, toxic shock syndrome, pediatric multisystemic inflammatory disease, and cytokine storm syndrome). The coronavirus SARS-CoV-2 of the genus beta is a causative agent.

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 (including glycoproteins) based molecules of different specificity are optionally fused together by chemical or amino acid based linker molecules. The ligation may be achieved by C-N fusion or N-C fusion (in the 5 '. Fwdarw.3' direction), preferably C-N fusion.

As used herein, the term "antibody" or "immunoglobulin" is intended to be in the broadest sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) made up of at least 2 intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. This term generally includes hybrid antibodies consisting of 2 or more antibodies or antibody fragments having different binding specificities linked together.

As used herein, the term "Fc" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as extending from position Cys226, or from an amino acid residue at Pro230 to the carboxy terminus of the heavy chain. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example during antibody production or purification, or by recombinant engineering of nucleic acids encoding the heavy chain of the antibody. Thus, a composition of an intact antibody may comprise a population of antibodies that have all K447 residues removed, a population of antibodies that have no K447 residues removed, and a population of antibodies that have a mixture of antibodies with and without K447 residues.

As used herein, sequence "identity" or "identity" has art-recognized meanings, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity may be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan (carrello, H. & Lipman, d.,. SIAM J Applied Math 48:1073 (1988)).

As used herein, the term "disease" or "condition" refers to the survival or health state of a patient or individual that can be treated with the fusion proteins, pharmaceutical compositions, or methods provided herein.

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

As used herein, the term "treatment" refers to any indication of successful treatment or amelioration of a lesion, disease, pathology, or condition, including any objective or subjective parameter, such as elimination; relief; alleviating symptoms or making lesions, pathologies or conditions more tolerable to the patient; slowing the rate of degradation or decay; or less degradation at the final point of degradation; improving physical or mental health of the patient. Treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of physical examination, neuropsychiatric examination, and/or psychiatric assessment. The term "treatment" and its conjugation may comprise preventing injury, pathology or disease. In embodiments, the treatment is prophylaxis. In embodiments, the treatment does not include prophylaxis.

As used herein (and as is well understood in the art), "treating" or "treatment" also broadly encompasses any method for achieving a beneficial or desired result (including clinical results) in a subject's condition. Beneficial or desired clinical results may include, but are not limited to: alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilization (i.e., not worsening) of the disease state, preventing spread or spread of disease, delaying or slowing of disease progression, amelioration or palliation of the disease state, reduction of disease recurrence, and remission (whether partial or total, and whether detectable or undetectable). In other words, as used herein, "treating" includes any cure, amelioration, or prevention of a disease. Treatment may prevent disease occurrence; inhibiting disease spread; alleviating symptoms of the disease, completely or partially removing the root cause of the disease, shortening the duration of the disease, or a combination of these.

As used herein, "treatment" includes prophylactic treatment. The method of treatment comprises administering to the subject a therapeutically effective amount of an active agent. The administering step may consist of a single administration or may comprise a series of administrations. The length of the treatment period depends on a variety of factors, such as the severity of the condition, the age of the patient, the concentration of the active agent, the activity of the composition used in the treatment, or a combination thereof. It will also be appreciated that the effective dosage of the agent for treatment or prevention may be increased or decreased during a particular treatment or prevention regimen. Variations in dosage may be produced and become apparent by standard diagnostic assays known in the art. In some cases, chronic administration may be required. For example, the composition is administered to the subject in an amount sufficient to treat the patient for a sufficient duration.

As used herein, the term "preventing" refers to reducing the occurrence of disease symptoms in a patient. As described above, prevention may be complete (no detectable symptoms) or partial, such that fewer symptoms are observed than would occur in the absence of treatment.

As used herein, "patient" or "subject in need thereof" refers to a living organism that is suffering from or susceptible to a disease or condition 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, cows, deer, and other non-mammals. In some embodiments, the patient is a human.

The term "co-administration" refers to the "co-administration" of a fusion protein or vaccine of the invention with a known drug (or other compound, or other vaccine) such that both have a therapeutic or diagnostic effect. Such combination administration may include concurrent (i.e., simultaneous), prior, or sequential administration of the agents (or other compounds, or other vaccines) relative to administration of the fusion proteins or vaccines of the present invention. One of ordinary skill in the art will be readily able to determine the appropriate timing, order, and dosage of administration of a particular drug (or other compound, or other vaccine) and the combination of the invention.

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 achieve, treat a disease, reduce enzyme activity, increase enzyme activity, reduce protein function, alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to cause treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount". "reducing" of one or more symptoms means reducing the severity or frequency of one or more symptoms, or eliminating one or more symptoms. A "prophylactically effective amount" of a drug is an amount of the drug that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset (or recurrence) of a lesion, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of a lesion, disease, pathology, or condition, or symptoms thereof. The complete prophylactic effect does not necessarily occur by administration of one dose, and may occur after administration of only a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic agent sufficient to ameliorate a condition as described above. For example, a therapeutically effective amount will exhibit an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90% or at least 100% for a given parameter. Treatment efficacy may also be expressed as a "fold" increase or decrease. For example, a therapeutically effective amount may have an effect of at least 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more relative to a control.

The dosage may vary depending on the needs of the patient and the fusion protein or vaccine employed. In the context of the present invention, the dose administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the presence, nature and extent of any adverse side effects. It is within the skill of the practitioner to determine the appropriate dosage for a particular situation. Typically, the 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 under these circumstances is reached. The amount and interval of administration can be individually adjusted to provide a level of fusion protein or vaccine administered that is effective for the particular clinical indication being treated. This will provide a treatment regimen commensurate with the severity of the individual's disease state.

As used herein, the term "administration" means oral administration to a subject, administration in suppository form, topical contact, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a slow release device (e.g., a micro osmotic pump). Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palate, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular and intracranial administration. Other modes of delivery include, but are not limited to, use of liposome formulations, intravenous infusion, transdermal patches, and the like. In embodiments, the administration does not comprise administration of any active agent other than the recited active agents.

In some embodiments, the novel crown mutant is selected from Alpha mutant (b.1.1.7), beta mutant (b.1.351), gamma mutant (p.1), delta mutant (b.1.617.2) or Omicron mutant (b.1.1.529).

In some embodiments, the first novel crown mutant is a novel crown Omicron mutant, the second novel crown mutant is a novel crown Delta mutant, or the first novel crown mutant is a novel crown Delta mutant, the second novel crown mutant is a novel crown Omicron mutant.

In some embodiments, the first novel crown mutant is a novel crown Omicron mutant and the second novel crown mutant is a novel crown Delta mutant.

In some embodiments, the RBD of the S protein of the novel crown Omacron mutant comprises an amino acid sequence having 80% or more identity to the amino acid sequence set forth in SEQ ID NO. 3, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of RBD of S protein of the novel crown Omicron mutant is shown as SEQ ID NO. 3.

In some embodiments, the RBD of the S protein of the novel crown Delta mutant strain comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or more identity to the amino acid sequence set forth in SEQ ID NO. 4; more preferably, the amino acid sequence of RBD of S protein of the novel crown Delta mutant strain is shown as SEQ ID NO. 4.

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

In some embodiments, the Receptor Binding Domain (RBD) of the S protein of the first novel corona-mutant or a functional fragment thereof, the immunoglobulin Fc, and the receptor binding domain of the S protein of the second novel corona-mutant or a functional fragment thereof comprise a linker therebetween.

In some embodiments, the immunoglobulin Fc is selected from the group consisting of IgG1 Fc, igG2 Fc, igG3 Fc, igG4 Fc, preferably IgG1 Fc; more preferably, the IgG1 Fc.

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

In some embodiments, the linker is a flexible linker.

In some embodiments, the flexible linker is selected from GSGGGSGGGGSGGGGS (SEQ ID NO: 7), GGGGS (SEQ ID NO: 8), GGGGSGGGGS (SEQ ID NO: 9).

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

In one aspect, the invention provides nucleic acids encoding the aforementioned fusion proteins

In some embodiments, the nucleic acid encoding the fusion protein comprises a nucleotide sequence having 80% or more identity to the sequence set forth in nucleotide 91-2193 of SEQ ID NO. 1, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence 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.

In some embodiments, the nucleic acid is mRNA.

In one aspect, the invention provides an expression vector comprising the aforementioned nucleic acid.

In some embodiments, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector, preferably the expression vector is a eukaryotic expression vector.

In some embodiments, the eukaryotic expression vector is pcho1.0.

In some embodiments, the eukaryotic expression vector is an adenovirus vector.

In one aspect, the invention provides a host cell expressing the fusion protein described above, or comprising the nucleic acid described above and/or the expression vector described above.

In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the prokaryotic cell is an e.

In some embodiments, the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell. In some embodiments, the mammalian cell is selected from CHO, HEK293, SP2/0, BHK, C127, and the like. In some embodiments, the eukaryotic cell is a CHO cell.

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, water-in-oil-in-water emulsions.

In some embodiments, the subject is a mammal or bird.

In some embodiments, the subject is a human, cow, dog, cat, goat, sheep, pig, horse, turkey, duck, or chicken.

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

EXAMPLE 1 construction of efficient expression vectors

1. Materials:

(1) The bivalent new crown vaccine sequence was designed by the product of Jino hygiene Co., ltd:

the modified novel crown mutant strain omacron S protein receptor binding domain, linker (linker), igG1 Fc, linker (linker) and the modified novel crown mutant strain delta S protein receptor binding domain are sequentially connected to form the target gene. The whole recombinant gene sequence (OFD for short, SEQ ID NO: 1) entrusts optimization synthesis of Nanjing Jinsri biotechnology Co., ltd, and the synthesized gene comprises an enzyme cutting site (AvrII, bstZ 17I), a Kozak sequence (GCCGCCACC), a signal peptide, a target gene and a stop codon, wherein the total length of the gene is 2205bp. The recombinant gene was synthesized with codon optimization to facilitate expression in chinese hamster ovary cells Cricetulus griseus (CHO cells).

The amino acid sequence of the bivalent new crown vaccine OFD protein coded by the OFD recombinant gene sequence is shown as SEQ ID NO. 2, and the bivalent new crown vaccine OFD protein comprises:

the novel crown mutant omacron Receptor Binding Domain (RBD) sequence set forth in SEQ ID NO. 3;

the novel crown mutant delta Receptor Binding Domain (RBD) sequence shown in SEQ ID NO. 4;

an IgG1 Fc sequence shown in SEQ ID NO. 5;

a signal peptide sequence shown in SEQ ID NO. 6;

the linker sequence shown in SEQ ID No. 7.

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

OFD amino acid sequence (SEQ ID NO: 2):

wherein:

amino acids 1-25 are the signal peptide sequence (SEQ ID NO: 6);

amino acids 26-244 are the novel crown mutant omicron receptor binding domain sequence (SEQ ID NO: 3);

amino acids 245 to 260 and 492 to 507 are the linker sequence list (SEQ ID NO: 7);

amino acids 508-726 are the delta receptor binding domain sequence of the novel crown mutant (SEQ ID NO: 4).

(2) The high-efficiency expression vector GDCHO is obtained by replacing pac with a GS gene screening mark in the prior art on the basis of commercializing the existing pCHO1.0 expression vector and deleting CMV pA and PcmvIEF1 (supplied by Beijing Jino health products technology Co., ltd.).

2. Method of

The PUC57 plasmid containing the OFD gene synthesized by Jinsri was recovered by three enzyme-digested agarose gels of AvrII, bstZ17I and BspHI to obtain a fragment containing the OFD gene. Meanwhile, the carrier GDCHO is digested with AvrII and BstZ17I, and the digested carrier GDCHO large fragment is recovered by agarose gel. Agarose gel electrophoresis of the digested products is shown in FIG. 1.

The OFD gene fragment obtained by digestion and recovery is connected with the large fragment of the vector GDCHO after digestion by ligase, the competent TOP10 of the escherichia coli is transformed, clones are selected, the plasmid is rapidly extracted, and the cut and identification are carried out by AvrII and Bstz17I, and the agarose electrophoresis identification result is shown (figure 2). The plasmid identified as correct by restriction enzyme was sent to the Tian Yi Hui Yuan sequencing Co for sequencing, and the plasmid sequenced correctly was named GDCHOOFD.

The plasmid GDCHOOFD with correct sequence is transformed into escherichia coli competent TOP10, monoclonal is selected and amplified into 200ml LB liquid medium for overnight culture, then plasmid is extracted by using a large endotoxin-free extraction kit of the root of the day, the concentration of the plasmid is measured to be 1.3mg/ml, and the plasmid is frozen for standby.

EXAMPLE 2 construction of cell lines stably expressing the OFD Gene

Placing a cell electrotransformation instrument of Yida living beings into an ultra-clean workbench, and setting electrotransformation parameters as follows: the voltage is 200V, the duration is 2000 mu S, the pulse is 6 times, and the interval is 1000ms. Mu.g of GDCHOOFD plasmid was added to 200. Mu.l of CHO-K1 cells, and the plasmid containing the OFD gene was electrotransferred into CHO-K1 cells.

Transferring the electrotransformed CHO-K1 cells into a T25 square bottle containing 10ml CHO CD02 medium, placing at 37 ℃ and 5% CO ₂ Culturing in an incubator for 24-48 hours. The CHO CD02 medium is a screening medium containing 25 μm methionine iminosulfone (methionine sulfoximine, MSX) and 200nM Methotrexate (MTX). The cells were then transferred to 125ml shake flasks containing 30ml CHO cd02 medium by centrifugation. Changing the screening culture medium once in 3-5 days, increasing MTX concentration to 1000nM when cell density reaches 150 ten thousand/ml and cell activity is above 95%, inoculating cellsThe density is 50 ten thousand/ml, and the residual cells are frozen for standby; changing the screening culture medium once in 3-5 days, inoculating with 50 ten thousand/ml cell density when the cell density reaches 150 ten thousand/ml and the cell activity is over 95%, increasing MTX concentration to 2000nM, and freezing the rest cells for later use; changing the screening medium once in 3-5 days, freezing 4 cells for standby when the cell density reaches 150 ten thousand/ml and the cell activity is more than 95%, regulating the cell density of the rest cells by a limiting dilution method, monoclonalizing to 20 96-well plates according to the density of 0.5 cells/well, inoculating 200 mu l/well into the 96-well plates, inoculating at 37 ℃ and 5% CO ₂ And (5) standing and culturing.

The 96-well plate is subjected to static culture for about 14 days, and the cell well plate is observed under an optical microscope, and the well plate is marked and is a monoclonal well. When the clone grows to 50-70% of the confluence in the 96-well plate, selecting single cell clone with good cell state, inoculating the single cell clone to the 24-well plate, when the cell grows to 95% of the confluence, sucking culture supernatant in batches to serve as a detection sample, and determining the single cell strain with high expression of 3F9, 7E6, 13H3 and 20B8 as a candidate cell strain.

And determining 7E6 as a subsequent standby cell strain according to the fed-batch culture result.

EXAMPLE 3 mass culture of 7E6 cell lines and purification recovery of OFD proteins

And (3) inoculating the obtained high-expression monoclonal cell strain 7E6, culturing, gradually amplifying to a volume of 300ml in 1L shake flasks, transferring to 8 1L shake flasks, and culturing by fed-batch feeding until the cell activity is 50%, and stopping culturing, harvesting and culturing supernatant.

Cells and cell debris were removed by centrifugation at 8000r/min and cell culture supernatants were collected. The filtrate was filtered through a 0.45 μm filter and passed through a Protein A gel column pre-equilibrated with 20mM PBS (pH 7.4, 150mM NaCl). Washing 2-4 column volumes by 20mM PBS until the A280 returns to the baseline level, eluting the OFD protein bound on the protein A column by using a glycine-hydrochloric acid buffer solution with the pH of 3.0 mM, adjusting the pH to about 7.0 by using 1M Tris, detecting the OFD protein by using SDS-PAGE electrophoresis, and freezing and storing the detected correct protein for later use.

EXAMPLE 4 OFD immunized mice after purification

25C 57BL/6 female mice of 6-8 weeks old were randomly divided into 5 groups, immunized twice with immunogen for 0d and 21d, the cellular immune efficacy of the vaccine was evaluated by detecting IFN-gamma and IL-2 cytokine expression by splenic lymphocytes of

groups

1, 2, 3, 4 and 5 of the second-immunized 14d after the two-immunization day by ELISPOT, and the humoral immune efficacy of the vaccine was evaluated by detecting specific IgG antibody titers in serum by ELISA method.

Table 1: immunization protocol design

Group of	Immunogens	Adjuvant	Immune body mass	Immunization mode	Number of immunizations	Number of animals
							1	PBS	-	0.1ml	i.m	2	5
2	OFD 10μg	-	0.1ml	i.m	2	5
							3	OFD 10μg	0.2mg AL(OH) ₃	0.1ml	i.m	2	5
4	OFD 10μg	10μg CpG	0.1ml	i.m	2	5
							5	OFD 10μg	0.2mg AL(OH) ₃ +10μgCpG	0.1ml	i.m	2	5

ELISPOT assay of spleen lymphocytes IFN-gamma and IL-2 cytokine levels following immunization, mice spleen lymphocytes were assayed according to the Murine IFN-gamma Single-Color Enzymatic ELISPOT Assay (CTL cat# mIFNgp-2M/2) kit and Murine IL-2Single-Color Enzymatic ELISPOT Assay (CTL cat# mIL2 p-2M/2) kit instructions, and the experimental results are shown in FIG. 3.

ELISA measures serum binding antibody levels after immunization, and the test results are shown in Table 3. The pseudovirus detected serum neutralizing antibody levels after immunization, and the test results are shown in FIG. 4. The pseudovirus detection kit used for detection is shown in Table 2.

Table 2: pseudovirus kit was purchased from Nanjinouzan biotechnology Co., ltd

Pseudovirus name	Kit name	Goods number
			Pseudovirus of original strain	SARS-CoV-2-Fluc WT	DD1746-01/02/03
Delta 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

Table 3: specific IgG antibody titers against RBD of different novel coronal strains

Conclusion: FIG. 3 shows IFN-gamma and IL-2 responses after a new coronal bivalent vaccine in different adjuvant combinations, wherein A is the IFN-gamma response results after a new coronal bivalent vaccine is post-exempt; b is the IL-2 response result after the new crown bivalent vaccine is subjected to secondary immunity. The results show that the bivalent new crown vaccine IFN-gamma and IL-2 with double adjuvant mode responds more than Yu Bishan adjuvant and no adjuvant combination, which shows higher cellular immunity level. From the results shown in table 3 for RBD specific IgG antibody titers against different new coronal strains, it can be seen that bivalent new coronal vaccines can cross-protect against different new coronaviruses, including the original strain, delta strain, omicron strain, and especially that double-adjuvant bivalent new coronal vaccines can produce specific IgG antibody titers against Delta strain, omicron strain as high as 143360 and 122880. Pseudovirus neutralizing antibody titers of up to 3000 or more were generated against the original strain, delta strain, omacron strain as shown in fig. 4. In conclusion, the newly developed bivalent novel crown vaccine can generate higher cellular immunity and humoral immunity, and has obvious cross protection effect on different novel crown mutant strains.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A fusion protein comprising a Receptor Binding Domain (RBD) of an S protein of a first novel corona-mutant or a functional fragment thereof, an immunoglobulin Fc, and a Receptor Binding Domain (RBD) of an S protein of a second novel corona-mutant or a functional fragment thereof, said first and second novel corona-mutant being different mutants.

2. The fusion protein of claim 1, wherein the novel crown mutant is selected from Alpha mutant (b.1.1.7), beta mutant (b.1.351), gamma mutant (p.1), delta mutant (b.1.617.2) or Omicron mutant (b.1.1.529);

preferably, the first new crown mutant is a new crown Omicron mutant, the second new crown mutant is a new crown Delta mutant, or the first new crown mutant is a new crown Delta mutant, the second new crown mutant is a new crown Omicron mutant;

more preferably, the first novel crown mutant is a novel crown omacron mutant and the second novel crown mutant is a novel crown Delta mutant;

preferably, the RBD of the S protein of the novel crown Omicron mutant comprises an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO. 3, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of RBD of S protein of the novel crown Omicron mutant is shown as SEQ ID NO. 3;

preferably, the RBD of the S protein of the novel crown Delta mutant comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or 99% or more identity to the amino acid sequence set forth in SEQ ID NO. 4; more preferably, the amino acid sequence of RBD of S protein of the novel crown Delta mutant strain is shown as SEQ ID NO. 4;

preferably, the fusion protein comprises an amino acid sequence having 80% or more identity, preferably 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably 98% or more identity to the sequence shown as amino acids 26-726 of SEQ ID NO. 2; more preferably, the amino acid sequence of the fusion protein is shown as amino acids 26-726 of SEQ ID NO. 2.

3. The fusion protein of claim 1 or 2, wherein the Receptor Binding Domain (RBD) of the S protein of the first novel corona-mutant strain or a functional fragment thereof, the immunoglobulin Fc, and the receptor binding domain of the S protein of the second novel corona-mutant strain or a functional fragment thereof comprise a linker therebetween;

preferably, the immunoglobulin Fc is selected from the group consisting of IgG1 Fc, igG2 Fc, igG3 Fc, igG4 Fc, preferably IgG1 Fc; more preferably, the IgG1 Fc;

preferably, the immunoglobulin Fc comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO. 5, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably an amino acid sequence having 98% or more identity; more preferably, the amino acid sequence of the immunoglobulin Fc is shown in SEQ ID NO. 5;

preferably, the joint is a flexible joint;

preferably, the flexible linker is selected from the group consisting of the sequences shown in SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9;

more preferably, the flexible polypeptide is the sequence shown in SEQ ID NO. 7.

4. A nucleic acid encoding the fusion protein of any one of claims 1-3;

preferably, the nucleic acid encoding the fusion protein comprises a nucleotide sequence having 80% or more identity to the sequence shown in nucleotide 91-2193 of SEQ ID NO. 1, preferably a nucleotide sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, more preferably a nucleotide sequence 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;

preferably, the nucleic acid is mRNA.

5. An expression vector comprising the nucleic acid of claim 4;

preferably, the expression vector is a prokaryotic expression vector or a eukaryotic expression vector; preferably, the expression vector is a eukaryotic expression vector;

preferably, the eukaryotic expression vector is pcho1.0;

preferably, the eukaryotic expression vector is an adenovirus vector.

6. A host cell expressing the fusion protein of any one of claims 1-3, or comprising the nucleic acid of claim 4 and/or comprising the expression vector of claim 5;

preferably, the host cell is a prokaryotic cell or a eukaryotic cell;

preferably, the prokaryotic cell is a bacterial cell; preferably, the prokaryotic cell is an E.coli cell;

preferably, the eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell; preferably, the mammalian cell is selected from CHO, HEK293, SP2/0, BHK, C127 and the like; more preferably, the eukaryotic cell is a CHO cell.

7. A pharmaceutical composition comprising the fusion protein of any one of claims 1-3, the nucleic acid of claim 4, the expression vector of claim 5, and/or the host cell of claim 6, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

8. A vaccine comprising the fusion protein of any one of claims 1-3, the nucleic acid of claim 4, the expression vector of claim 5 and/or the host cell of claim 6, and one or more adjuvants;

preferably, the adjuvant is selected from at least one of aluminium hydroxide, cpG, aluminium phosphate, saponins such as Quil A, QS-21, GPI-0100, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions.

9. Use of the fusion protein of any one of claims 1-3, the nucleic acid of claim 4, the expression vector of claim 5 and/or the host cell of claim 6 and/or the pharmaceutical composition of claim 7 in the manufacture of a vaccine for the treatment or prevention of a disease or condition associated with SARS-CoV-2.

10. A method of inducing an immune response against SARS-CoV-2 in a subject in need thereof, the method comprising: administering the vaccine of claim 8 to a subject;

preferably, the subject is a mammal or bird;

preferably, the subject is a human, cow, dog, cat, goat, sheep, pig, horse, turkey, duck or chicken.