WO2022164208A1 - Sars-coronavirus-2 fusion protein and immunogenic composition comprising same - Google Patents

Abstract

The present invention relates to a SARS-coronavirus-2 fusion protein and an immunogenic composition comprising same and, more specifically, to a fusion protein comprising a spike protein (S protein) of SARS-coronavirus-2 (SARS-CoV-2) and a constant region of an immunoglobulin molecule, and an immunogenic composition comprising same. The present invention can induce the production of an SARS-coronavirus-2-specific neutralizing antibody as well as remarkably increasing cell-mediated immune responses. Accordingly, the present invention can not only give rise to an immune response to mutant viruses, but also exhibit a long-term sustainable effect, thus finding advantageous applications in preventing SARS-coronavirus infections (COVID-19).

Description

SARS-coronavirus-2 fusion protein and immunogenic composition comprising same

The present invention relates to a SARS-coronavirus-2 fusion protein and an immunogenic composition comprising the same, and more particularly, to a SARS-coronavirus-2 (SARS-CoV-2) spike protein (S protein) and It relates to a fusion protein comprising the constant region of an immunoglobulin molecule and an immunogenic composition comprising the same.

Coronaviruses phylogeneically belong to the family Coronaviridae of the order Nidovirales, and are divided into four genera: alpha, beta, gamma and delta. Among human coronaviruses (HCoV) that infect humans, HCoV-229E and HCoV-NL63 belong to alphaCoV and cause common respiratory infections. HCoV-OC43, SARS-CoV, and MERS-CoV belong to beta-coronavirus (betaCoV), which also induces upper respiratory infections such as colds and digestive disorders. In addition, SARS-CoV and MERS-CoV belonging to betaCoV cause severe respiratory infectious diseases characterized by acute respiratory symptoms. Mortality rates associated with SARS and MERS-CoV are up to 10% and 35%, respectively. SARS-Coronavirus-2 (Severe Acute Respiratory Syndrome Coronavirus-2, SARS-CoV-2) is the second SARS-coronavirus and belongs to betaCoV as a result of homology analysis of a virus isolated from atypical pneumonia patients in Wuhan, China. It has been found, and it has high infectivity and transmission ability, and causes acute respiratory symptoms, leading to death. About 90 million people have been infected worldwide and about 1.9 million have died. Although therapeutic agents such as remdesivir and bamlanivimab and mRNA vaccines have been developed and approved in a short period of time and started to be distributed, there is a need to develop and distribute safer and cheaper vaccines to end the pandemic.

The genome of SARS-coronavirus-2 consists of about 30,000 RNAs, and the SARS-coronavirus-2 spike protein binds to the cell's ACE2 receptor (angiotensin converting enzyme 2), resulting in SARS-coronavirus- The bivalent enters the cell and causes viral replication. Crown-shaped projections (spikes) are characteristically expressed on the surface of the coronavirus. The spike protein consists of about 1,200 amino acids, and consists of a spike 1 (S1) and a spike 2 domain (S2 domain). Functionally, S1 is known to be involved in cell receptor binding and S2 is involved in fusion. It is known that the SARS-coronavirus-2 genome encodes not only structural proteins such as spike proteins, but also proteins involved in viral replication and infection, such as viral polymerases and proteolytic enzymes.

Vaccines currently used to prevent COVID-19 are largely mRNA vaccines, and virus vectored vaccines are being used with emergency use approval. The mRNA vaccine is a vaccine in which the mRNA encoding the spike protein is mixed with lipids, etc., and is administered intramuscularly to induce an immune response by expressing the spike protein in the human body. The viral vector vaccine is a vaccine in which the adenovirus surface protrusion is replaced with the SARS-coronavirus-2 spike protein. However, these two vaccines are novel vaccines and their efficacy and safety have been verified through clinical trials. Meanwhile, the use of vaccines due to virus mutations is also emerging.

Therefore, it is an existing vaccine form that does not have the disadvantages of this new form of vaccine, and has more safety confirmed, and a novel SARS-coronavirus-2 having a response to virus mutation and continuity of vaccine efficacy by selectively increasing cell-mediated immunogenicity There is a need to develop vaccine compositions. Also, in the case of mRNA vaccine, there is a need for a vaccine that can be distributed at 2-8ºC because it has the disadvantage of having to be distributed at a low temperature.

The safety and continuity of efficacy of the above vaccines should be further verified, and since there are problems in response to mutations and distribution, the effectiveness of prevention may be somewhat limited in a pandemic situation, and problems such as inducing anaphylactic reactions in some patients are being discovered

An object of the present invention is to provide a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.

In addition, another problem to be solved by the present invention is to provide a nucleic acid molecule encoding the fusion protein.

In addition, another problem to be solved by the present invention is to provide an expression vector comprising the nucleic acid molecule.

In addition, another problem to be solved by the present invention is to provide a host cell transformed with the above expression vector.

In addition, another problem to be solved by the present invention is to provide a method for preparing the fusion protein.

In addition, another problem to be solved by the present invention is to provide an immunogenic composition comprising the fusion protein.

In addition, another problem to be solved by the present invention is to provide a kit comprising the above immunogenic composition.

In addition, another problem to be solved by the present invention is to provide a method of inducing an immune response by administering the immunogenic composition.

In addition, another problem to be solved by the present invention is to provide a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.

In order to solve the above problems, the present invention provides a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.

In the fusion protein according to the present invention, the spike protein comprises: i) a full length S protein or a fragment thereof; ii) S1 protein or fragment thereof; iii) S2 protein or fragment thereof; Or iv) a receptor binding domain (RBD) region or a fragment thereof, but is not limited thereto. Here, the full-length S protein or S1 protein may include a receptor binding domain (RBD) region.

In addition, in the fusion protein according to the present invention, the spike protein comprises: i) a fusion protein of a) a S1 protein or a fragment thereof and b) an S2 protein or a fragment thereof; or ii) a) a fusion protein of a receptor binding domain (RBD) region or a fragment thereof and b) an S2 protein or a fragment thereof, but is not limited thereto.

In one embodiment, the spike protein may include any one of SEQ ID NOs: 1 to 4, but is not limited thereto.

In the fusion protein according to the present invention, the constant region (eg, Fc region) of the immunoglobulin molecule may bind to an Fc receptor to enhance immunogenicity. The constant region of the immunoglobulin molecule of the fusion protein according to the present invention may be an immunoglobulin heavy chain constant region, but is not limited thereto. The immunoglobulin may be any one selected from the group consisting of IgG, IgM, IgA, IgD and IgA, but is not limited thereto. In one embodiment, the immunoglobulin may be IgG, but is not limited thereto. In one embodiment, the IgG may be any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, but is not limited thereto. In one embodiment, the constant region of the immunoglobulin molecule may be a constant region of an IgG1 heavy chain, but is not limited thereto. In one embodiment, the constant portion of the immunoglobulin molecule may include a hinge region, a CH2 domain, and a CH3 domain of IgG1, and in another embodiment, the constant portion of the immunoglobulin molecule includes a CH1 domain of IgG1 may include, but is not limited thereto. In one embodiment, the constant region of the immunoglobulin molecule may include an Fc region. In one embodiment, the Fc region can enhance immunogenicity by binding to an Fc receptor.

In one embodiment, the Fc region may include an Fc variant. In one embodiment, the Fc variant may include one or more Fc variants capable of enhancing binding to an Fc receptor. In one embodiment, the Fc variant may include one or more Fc variants, which may enhance immunogenicity by enhancing binding to Fc receptors. In one embodiment, the Fc variant may include one or more mutations selected from the group consisting of G236A, S239D, A330L, and I332E. In one embodiment, the Fc variant may include all of the G236A / S239D / A330L / I332E mutation, but is not limited thereto. In one embodiment, the Fc region may include the sequence of SEQ ID NO: 5, but is not limited thereto. In one embodiment, the Fc variant may include the sequence of SEQ ID NO: 6, but is not limited thereto.

In one embodiment, the fusion protein may include any one of SEQ ID NOs: 7 to 18, but is not limited thereto.

In one embodiment, in the fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule, the spike protein is a) S1 protein or In the case of a fusion protein of a fragment thereof and b) an S2 protein or a fragment thereof, the sequence of SEQ ID NO: 19 may be included between the S1 protein and the S2 protein, but the sequence is not limited thereto.

The present invention also provides a nucleic acid molecule encoding the fusion protein.

In addition, the present invention provides an expression vector comprising the nucleic acid molecule.

In addition, the present invention provides a host cell transformed with the expression vector.

In addition, the present invention,

i) introducing the expression vector into an animal cell expression system; and

ii) performing expression of the fusion protein;

It provides a method for producing a fusion protein comprising a.

In order to solve another object of the present invention, the present invention provides an immunogenic composition comprising the fusion protein.

In one embodiment, the fusion protein may be a monomer, but is not limited thereto. In another embodiment, the fusion protein may be a dimer, but is not limited thereto. Here, the dimer may be formed by a disulfide bond in the hinge region of the two fusion proteins, but is not limited thereto.

The immunogenic composition according to the present invention may further include an adjuvant, wherein the adjuvant is one selected from the group consisting of a Toll-like receptor 4 (TLR4) agonist, aluminum salt, saponin, and liposome. It may be above, but is not limited thereto.

The Toll-like receptor 4 (TLR4) agonist may be at least one selected from the group consisting of Monophosphoryl Lipid A (MPL) and 3D-MPL, but is not limited thereto.

The aluminum salt may be at least one selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum sulfate, but is not limited thereto.

The saponin may be any one or more selected from the group consisting of QS21, QS17 and QuilA, but is not limited thereto.

In addition, in order to solve another object of the present invention, the present invention provides a kit comprising the immunogenic composition.

In addition, in order to solve another object of the present invention, the present invention provides a method of inducing an immune response by administering the immunogenic composition.

In addition, in order to solve another problem of the present invention, the present invention provides a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.

According to the present invention, a fusion protein comprising a spike protein, S protein of SARS-CoV-2 and a constant region of an immunoglobulin molecule, and an immunogenic composition comprising the same, are SARS-coronavirus It can induce -2 specific neutralizing antibodies as well as significantly increase cell-mediated immune responses. Accordingly, the fusion protein according to the present invention and the immunogenic composition comprising the same can exhibit a long-term sustainable effect as well as a corresponding immune response to the mutant virus, thus preventing SARS-coronavirus infection (COVID-19). It can be useful for prevention.

1 shows the amount of SARS-coronavirus-2 specific neutralizing antibody produced.

Figure 2 shows the production amount of SARS-coronavirus-2 specific total IgG antibody.

3 shows the production amount of SARS-coronavirus-2 specific IgG1 antibody.

4 shows the production amount of SARS-coronavirus-2 specific IgG2a antibody.

Hereinafter, the present invention will be described in more detail. However, this description is only for helping the understanding of the present invention, and the scope of the present invention is not limited by the details described in the detailed description in any sense.

The scope of the present invention is not limited by the content described below, and in particular, may include anything that may vary depending on the experimental conditions described in the following examples and the like. Since the scope of the present invention will be limited by the appended claims, the terminology used herein for further understanding only is for the purpose of describing detailed embodiments of the present invention, and the scope of the present invention is limited thereto. should not do

Unless defined otherwise herein, all technical and scientific terms used herein are to be interpreted with the same meanings as those commonly understood by one of ordinary skill in the art. All references mentioned herein are incorporated by reference in their entirety as the content of the present specification for the purpose of describing the invention herein.

The present invention relates to a fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule. As a more specific example, SARS-corona Fc-fusion in which the amino terminus of an immunoglobulin heavy chain constant region (fragment crystalizable, Fc) is linked to the carboxyl terminus of the spike protein on the surface of virus-2 (SARS-CoV-2) via a peptide bond provide protein.

The spike protein is the spike protein

i) a full length S protein or fragment thereof;

ii) S1 protein or fragment thereof;

iii) S2 protein or fragment thereof; or

iv) RBD (Receptor binding domain) region or fragment thereof

may include, but is not limited thereto.

Here, the full-length S protein or S1 protein may include a receptor binding domain (RBD) region. In one embodiment, the spike protein may include any one of SEQ ID NOs: 1 to 4, but is not limited thereto.

The immunoglobulin may be any one selected from the group consisting of IgG, IgM, IgA, IgD and IgA, but is not limited thereto. In one embodiment, the immunoglobulin may be IgG, but is not limited thereto. In one embodiment, the IgG may be any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4, but is not limited thereto. In one embodiment, the constant region of the immunoglobulin molecule may be a constant region of an IgG1 heavy chain, but is not limited thereto. In one embodiment, if the constant portion of the immunoglobulin molecule may include a hinge region, a CH2 domain and a CH3 domain of IgG1, the constant portion of the immunoglobulin molecule may further include a CH1 domain of IgG1, However, the present invention is not limited thereto. In one embodiment, the constant region of the immunoglobulin molecule may include an Fc region, but is not limited thereto. In one embodiment, the Fc region may enhance immunogenicity by binding to an Fc receptor, but is not limited thereto. In one embodiment, the Fc region may include the sequence of SEQ ID NO: 5, but is not limited thereto.

In addition, the Fc region may include an Fc variant, but is not limited thereto. The Fc variant may include one or more mutations selected from the group consisting of G236A, S239D, A330L, and I332E. The Fc variant may include all G236A/S239D/A330L/I332E mutations, but is not limited thereto. The numbering of the Fc mutation position is according to EU numbering, but is not limited thereto. In one embodiment, the Fc variant may include the sequence of SEQ ID NO: 6, but is not limited thereto.

In one embodiment of the present invention, the fusion protein may include any one of SEQ ID NOs: 7 to 18, but is not limited thereto.

The present invention also provides a nucleic acid molecule encoding the fusion protein.

In addition, the present invention provides an expression vector comprising the nucleic acid molecule.

In addition, the present invention provides a host cell transformed with the expression vector.

In addition, the present invention,

i) introducing the expression vector into an animal cell expression system; and

ii) performing expression of the fusion protein;

It provides a method for producing a fusion protein comprising a.

In addition, the present invention provides an immunogenic composition comprising the above fusion protein.

In one embodiment, the fusion protein may be a monomer, but is not limited thereto. In another embodiment, the fusion protein may be a dimer, but is not limited thereto. Here, the dimer may be formed by a disulfide bond in the hinge region of the two fusion proteins, but is not limited thereto.

The immunogenic composition according to the present invention may further include an adjuvant, wherein the adjuvant is one selected from the group consisting of a Toll-like receptor 4 (TLR4) agonist, aluminum salt, saponin, and liposome. It may be above, but is not limited thereto.

The Toll-like receptor 4 (TLR4) agonist may be at least one selected from the group consisting of Monophosphoryl Lipid A (MPL) and 3D-MPL, but is not limited thereto.

The aluminum salt may be at least one selected from the group consisting of aluminum hydroxide, aluminum phosphate, and aluminum sulfate, but is not limited thereto.

The saponin is a tripertene glycoside substance widely distributed in plant and marine animal kingdoms, and is an adjuvant mainly accepted in the art. According to one embodiment of the present invention, the saponin may be any one or more selected from the group consisting of QS21, QS17 and QuilA, but is not limited thereto. According to another embodiment of the present invention, it may be preferably QS21, but is not limited thereto. QS21 uses an extract derived from the bark of Quillaja saponaria by HPLC purification, and is also denoted as acylated 3,28-bisdesmodic triterpene glycosides (1,3).

The liposome is a lipid-based oval structure formed of a phospholipid bilayer, and is typically included in a vaccine composition or a pharmaceutical composition to serve as a drug delivery vehicle. According to an embodiment of the present invention, the liposome is dimethyldioctadecylammonium bromide (DDA), 1,2-dioleoyl-3-trimethyl ammonium propane (DOTAP), 3β-[N-(N′,N) '-Dimethylaminoethane carbamoyl cholesterol (3β-[N-(N,N-dimethylaminoethane) carbamoyl cholesterol, DC-Chol), 1,2-dioleoyloxy-3-dimethylammonium propane (DODAP), 1, 2-di-O-octadecenyl-3-triethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA), 1,2-dimyristoreoyl-sn-glycero -3-ethylphosphocholine (1,2-dimyristoleoyl-sn-glycero-3-ethyl phosphocholine, 14:1 Etyle PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethyl phosphocholine Choline (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, 16:0-18:1 Ethyl PC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (1 ,2-dioleoyl-snglycero-3-ethylphosphocholine, 18:1 Ethyl PC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (1,2-distearoyl-sn-glycero-3 -ethylphosphocholine, 18:0 Ethyl PC), 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine, 16:0 Ethyl PC) , 1,2-dimyristoyl-sn-glycero-3-ethyl phosphocholine (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, 14:0 Ethyl PC), 1,2-dilauro 1-sn-glycero-3-ethylphosphocholine (1,2-dilauroyl-sn-glycero-3-ethylphosphocholin, 12:0 Ethyl PC), N1-[2-((1S)-1-[(3) -Aminopro Phil)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (N1-[2-((1S)- 1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl) amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide, MVL5), 1,2-dimyristo yl-3-dimethylammonium-propane (1,2-dimyristoyl-3-dimethylammonium-propane, 14:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (1,2-dipalmitoyl-propane) 3-dimethyl ammonium-propane, 16:0 DAP), 1,2-distearoyl-3-dimethylammonium-propane (1,2-distearoyl-3-dimethylammonium-propane, 18:0 DAP), N- (4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium(N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis( oleoyloxy)propan-1-aminium, DOBAQ), 1,2-stearoyl-3-trimethylammonium-propane (1,2-stearoyl-3-trimethylammonium-propane, 18:0 TAP), 1,2-di Palmitoyl-3-trimethylammonium-propane (1,2-dipalmitoyl-3-trimethylammonium-propane, 16:0 TA), 1,2-dimyristoyl-3-trimethylammonium-propane (1,2- dimyristoyl-3-trimethyl ammonium-propane, 14:0 TAP) and N4-cholesteryl-spermine (N4-Cholesteryl-Spermine, GL67) may be at least one selected from the group consisting of, but is not limited thereto.

In addition, the liposome is additionally 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (1,2-Dimyristoyl-snglycero-3-phosphorylcholine, DMPC), 1,2-dioleoyl- sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) , 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC), 1,2-dilinoleoyl-sn-glycerol Rho-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC), phosphatidylserine (PS), phosphoethanolamine (PE), phosphatidylglycerol (PG), phosphoric acid ( PA) and may include any one neutral lipid selected from the group consisting of phosphatidylcholine (PC), but is not limited thereto.

In addition, the present invention provides a kit comprising the above immunogenic composition.

In addition, in order to solve another object of the present invention, the present invention provides a method of inducing an immune response by administering the immunogenic composition.

In addition, in order to solve another problem of the present invention, the present invention provides a method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition.

Each of the features described herein may be used in combination, and the fact that each of the features is recited in different dependent claims of the claims does not indicate that they cannot be used in combination.

Example 1. Preparation of viral antigen and Fc region fusion protein

S1-Fc, S1-Fc', S2-Fc' fusion proteins were prepared by the following method.

The DNA of a fusion protein in which the S protein of SARS-coronavirus-2 and the Fc region of human IgG are bound was synthesized and cloned into an animal cell expression vector, pCT146 vector. The synthesized S1-Fc contains wild-type human IgG Fc, and S1-Fc' and S2-Fc' contain mutant Fc in which amino acids of the human IgG Fc region are mutated (G236A/S239D/A330L/I332E), have. The synthesized S1-Fc, S1-Fc', and S2-Fc' DNA was digested with two restriction enzymes, ie, Nhe I and Pme I, and the pCT146 vector was also digested with the same restriction enzyme. The cleaved S1-Fc, S1-Fc', and S2-Fc' DNA and vector DNA were ligated using T4 ligase. The cloned pCT146 DNA was transformed into E. coli , colony was analyzed, and the S1-Fc, S1-Fc', S2-Fc' genes were inserted into the pCT146 vector bacterial clone (bacteria). clone) was selected. These vectors were named pCT665, pCT666, and pCT667, respectively.

Next, the DNA was purified by culturing the selected colonies. Transient transfection was performed using purified high-purity DNA in ExpiCHO-S animal cells, one of the Chinese hamster ovary cell lines (CHO). These ExpiCHO-S cells were fed-batch cultured to produce S1-Fc, S1-Fc', and S2-Fc' fusion proteins. The S1-Fc, S1-Fc', and S2-Fc' proteins released out of the cell were purified using Protein A affinity chromatography.

Example 2. Preparation of MPL and Aluminum Hydroxide and Preparation of Immunogenic Compositions

Next, for the experiment of the immunogenic composition of the present invention, MPL and aluminum hydroxide as adjuvants (immune enhancers) were prepared in the following manner.

Monophosphoryl Lipid A, also known as MPL or MPLA, is obtained from InvivoGen's MPLA-SM VacciGrade ^TM (Catalog code: vac-mpla) product according to the manufacturer's method. DMSO), and then stored frozen at -20°C for use.

MPL is extracted from lipopolysaccharide (LPS) produced from Re mutant of Salmonella Minnesota R595 strain, lipid A is mainly involved in the endotoxic activity of LPS and MPL is one in which one phosphate group has been removed from lipid A, which refers to a substance that has reduced toxicity and maintained the immune-enhancing effect (Infect Immun. 71 (5): 2498-507, Int Immunol. 14(11): 1325-32, J. Biol. Chem. 257(19): 11808-15, Infect Immun. 79(9): 3576-3587). Like LPS, MPL functions as a Toll-like receptor 4 (TLR4 agonist) and is known to strongly induce a Th1 immune response (Science 316(5831):1628-32, Infect Immun. 75 (12): 5939-46).

Next, as aluminum hydroxide, InvivoGen's alhydrogel adjuvant 2% (Alhydrogel® adjuvant 2%, catlog# vac-alu-250) product was dispensed and used.

It is known that an aluminum-based adjuvant increases antigen uptake of antigen presenting cells (APCs), induces a Th2 immune response, but does not induce a Th1 immune response (Immunity 33(4): 492-503) .

The antigen and fusion protein prepared in Example 1 were mixed with MPL and aluminum hydroxide at a specific concentration using a PBS solution (phosphate-buffered saline) to prepare the immunogenic composition of the present invention.

Experimental Example 1. Neutralizing antibody experiment

In order to evaluate the efficacy of the immunogenic composition prepared in Example 2, an experiment was conducted as follows.

SARS-Coronavirus-2 surface spike (Spike, S) protein domain (S1 or S2 domain) and a fusion protein antigen in which the Fc region of a human IgG1 antibody is bound. It was immunized by intramuscular injection. In order to measure the SARS-coronavirus-2 specific humoral and cellular immunity by the vaccine composition, orbital blood was collected for serum isolation from mice 2 weeks (14 days) after the second immunization. .

Experimental designs for evaluating the composition's neutralization response and humoral/cellular immune response are shown in Table 1 below. S1-Fc in Table 1 is an antigen in which the surface protein S1 domain of SARS-Coronavirus-2 and Fc of a human IgG1 antibody are fused, MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant), alum is aluminum hydroxyl seed (see Examples 1 and 2). In addition, S1-Fc' is an antigen in which the surface glycoprotein S1 domain of SARS-coronavirus-2 and Fc of an engineered human IgG1 antibody are fused, MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant) , alum means aluminum hydroxide. In addition, S2-Fc' is an antigen in which the surface protein S2 domain of SARS-coronavirus-2 and Fc of an engineered human IgG1 antibody are fused, MPL is monophosphoryl lipid A as an adjuvant (immune adjuvant), alum means aluminum hydroxide. In addition, the final volume of each composition in groups 1 to 9 was adjusted and administered by 100 μl.

group	vaccine composition	1st immunization (2 weeks)	2nd immunization (4 weeks)	Sampling (6 weeks)
One	PBS	IM	IM		Serum
2	S1 (5 μg) + alum	IM	IM	Serum
3	S1-Fc (5 μg) + alum	IM	IM	Serum
4	S1-Fc' (5 μg) + alum	IM	IM	Serum
5	S2-Fc' (5 μg) + alum	IM	IM	Serum
6	S1 (5 μg) + MPL + alum	IM	IM	Serum
7	S1-Fc (5 μg) + MPL + alum	IM	IM	Serum
8	S1-Fc' (5 μg) + MPL + alum	IM	IM	Serum
9	S2-Fc' (5 μg) + MPL + alum	IM	IM	Serum

In order to evaluate the neutralizing immune response of the present composition, FRNT (Focus Reduction Neutralization Test) analysis was performed using the isolated serum after 2 weeks from the second immunization to 5-week-old C57BL/6 female mouse (Koatech). To this end, the mouse blood samples were centrifuged (40 minutes, 4ºC) to separate the serum from the blood and stored at -80ºC.

Vero cells (Vero cells) were dispensed in 96-well plates and cell culture was performed for 16-18 hours. Using DMEM medium containing 2% FBS, mouse serum was serially diluted two-fold, and SARS-Coronavirus-2 (3.51 x 10 ⁶ PFU/ml, BetaCoV/Korea/KCDC03/2020, National Resources Bank of Korea) was prepared by diluting to 300 PFU using the same medium. A serum sample prepared by dilution and virus were mixed and reacted for 30 minutes at 37° C. in a CO ₂ cell incubator. The serum-virus mixture reacted in this way was added to Vero cells cultured in a 96-well plate, and reacted for 4 hours at 37° C. in a CO ₂ cell incubator. After the reaction, PBS was washed and formaldehyde was added to fix the cells at 4ºC for 16-18 hours.

After washing with PBS, blocking buffer (1% BSA, 0.5% goat serum, 0.5% tween-20 in PBS) was added and reacted at room temperature for 30 minutes. SARS-CoV-2 NP rabbit mAb (Sino Biological, 40143-R001) diluted at 1:3000 was added to the plate, and then reacted at 37°C for 1 hour. After washing 3 times with PBS washing solution containing 0.1% tween-20, goat anti rabbit IgG-HRP (Bio-Rad, 1721019) diluted 1:2000 was added and reacted at 37°C for 1 hour. After washing 3 times with PBS washing solution containing 0.1% tween-20, it was washed once with PBS. After KPL TrueBlue Peroxidase Substrate (Seracare, Cat:5510-0030)) was added to the plate, it was reacted for 30 minutes under dark conditions and room temperature, washed with distilled water, and dried. Foci stained with TrueBlue TS were measured using an immunospot reader (C.T.L.). Percentages were evaluated based on viral infection without serum.

As a result, compared to the PBS group and the alum adjuvant group, the composition containing the S1, S1-Fc, and S1-Fc' antigens and MPL/alum adjuvant showed a significant neutralizing immune response (Table 2, FIG. 1). The composition containing the S1 protein and MPL/alum adjuvant showed the highest neutralizing antibody titer, but it was about 2-3 times different from the compositions containing the S1-Fc or S1-Fc' fusion protein and MPL/alum adjuvant. did not show any significant difference.

	FRNT result
Group	PBS	S1 + alum	S1-Fc + alum	S1-Fc' + alum	S2-Fc' + alum	S1 +MPL + alum	S1-Fc +MPL + alum	S1-Fc' +MPL + alum	S2-Fc' +MPL + alum
FRNT 50 (individual values)	20	80	40	10	60	960	1280	640	40
	10	30	160	40	40	240	30	60	40
	20	320	80	160	60	240	240	320	40
	20	80	20	40	60	1280	320	40	20
	15	40	60	320	80	480	10	30	30
mean	17	110	72	114	60	640	376	218	34

Experimental Example 2. ELISA experiment

In order to evaluate the composition efficacy of the Experimental Example 1 combination (Groups 1 to 9), additional experiments were carried out in the following manner.

About 4 weeks after the second immunization, total IgG production, IgG1 antibody and IgG2a antibody production amount induced by the vaccine composition using serum isolated from mouse blood was measured by ELISA assay.

SARS-CoV-2 S protein (Acro Biosystem, SPN-C52H8) at a concentration of 0.5 μg/ml and SARS S1 protein (Acro Biosystem, S1N-852H5) at a concentration of 0.5 μg/ml were mixed with a coating buffer (coating buffer, carbonate/bicarbonate buffer) , Sigma, catalog number C3041-100CAP) was applied to a 96-well plate at 4° C. for 16 to 18 hours. Wash three times using washing solution (0.05 % tween 20 in 1x PBS, PBS-Tween tablet; Calbiochem, catalog# 524653 with 1L PW), and diluent (Diluent, Teknova, catalog# 2D5120-1L; 1% BSA, 0.05) % Tween-20, consisting of 1X PBS), the plate was blocked at room temperature for 1 hour using a dilution solution. After washing three times, serum samples of groups 1,2,3,4,6,7,8 were put on the plate coated with the S1 protein and reacted at room temperature for 1 hour 30 minutes to 2 hours. In addition, serum samples from groups 1, 5, and 9 were put on the SARS-CoV-2 S protein-coated plate and reacted at room temperature for 1 hour 30 minutes to 2 hours. After 3 washes, detection antibody: i) Goat anti-mouse IgG conjugated to HRP (SOUTHERN BIOTECH, Catalog# 1030-05, ii) horseradish peroxidase-conjugated goat anti-mouse IgG1 (BioRad, Catalog# STAR132P) or iii) horseradish Peroxidase-conjugated goat anti-mouse IgG2a (BioRad, Catalog# STAR133P) was added and reacted at room temperature for 1 hour. After washing 6 times, TMB (3,3', 5,5'-tetramethylbenzidine, Sigma, Catalog# T0440) was put on the plate and reacted for 5 minutes, and sulfuric acid solution (H ₂ SO ₄ , Merck, Catalog# 109072) was added The reaction was stopped. The absorbance (optical density) of each sample was measured using a plate reader. And the SARS-coronavirus-2 fusion protein, which is the result of the test, the total amount of IgG specific for coronavirus, and the production amount of IgG1 and IgG2a antibodies were measured. The amount of IgG1 produced refers to the product of a humoral immune response, whereas the amount of IgG2a produced refers to the product of a cellular immune response. Total IgG refers to all IgG antibodies such as IgG1, IgG2a, etc.

As a result, as shown in FIG. 2, total IgG production was significantly increased in all groups compared to PBS. It was confirmed that the combination of S2-Fc'+alum and S2-Fc'+MPL+alum was effective in generating IgG antibodies, indicating an increase in the humoral immune response. This trend was similarly confirmed in the IgG1 ELISA experiment ( FIG. 3 ). On the other hand, IgG2a production, an indicator of cellular immunogenicity, was observed only in the S1-Fc'+MPL+alum group. This means an increase in cellular immunogenicity by the effect of Fc engineering ( FIG. 4 ).

Taken together, the IgG1 antibody was generated in all experimental groups compared to the PBS group (negative control group), and a significant neutralizing antibody was generated in the composition including the S1 domain and MPL/alum adjuvant, especially S1 including the engineered Fc. -Fc' and the MPL/alum mixed composition induced a cellular immune response significantly.

Tables 3, 4, and 5 below correspond to FIGS. 2, 3, and 4, respectively.

group	vaccine composition	Total IgG antibody					Average	log2 Average	log2 Standard Deviation
One	PBS	0	0	0	0	0	n/a	n/a	n/a
2	S1 (5 μg) + alum	0.00	2500.00	500.00	0.00	0.00	600.0	9.2	5.6
3	S1-Fc (5 μg)+ alum	500	2500	500	2500	500	1300.0	10.3	1.3
4	S1-Fc' (5 μg) + alum	0	500	2500	0	500	700.0	9.5	5.4
5	S2-Fc' (5 μg)+ alum	62500	62500	62500	62500	62500	62500.0	15.9	0.0
6	S1 (5 μg) + MPL + alum	12500	2500	2500	2500	0	4000.0	12.0	5.4
7	S1-Fc (5 μg) + MPL + alum	500	2500	0	0	2500	1100.0	10.1	5.8
8	S1-Fc' (5 μg) + MPL + alum	500	500	2500	12500	500	3300.0	11.7	2.1
9	S2-Fc' (5 μg) + MPL + alum	1,562,500	62,500	312500	62,500	62,500	412500.0	18.7	2.1

group	vaccine composition	IgG1 antibody					Average	log2 Average	log2 Standard Deviation
One	PBS	0	0	0	0	0	n/a	n/a	n/a
2	S1 (5 μg) + alum	0.00	2500.00	500.00	0.00	0.00	600.0	9.2	5.6
3	S1-Fc (5 μg)+ alum	12500	12500	2500	12500	12500	10500.0	13.4	1.0
4	S1-Fc' (5 μg) + alum	0	500	12500	500	2500	3200.0	11.6	5.2
5	S2-Fc' (5 μg)+ alum	62500	62500	62500	62500	62500	62500.0	15.9	7.1
6	S1 (5 μg) + MPL + alum	12500	12500	2500	12500	0	8000.0	13.0	7.1
7	S1-Fc (5 μg) + MPL + alum	500	2500	0	0	2500	1100.0	10.1	5.8
8	S1-Fc' (5 μg) + MPL + alum	500	500	2500	12500	500	3300.0	11.7	2.1
9	S2-Fc' (5 μg) + MPL + alum	1,562,500	62,500	62500	12,500	62,500	352500.0	18.4	2.5

group	vaccine composition	IgG2a antibody					Average	log2 Average	log2 Standard Deviation
One	PBS	0	0	0	0	0	n/a	n/a	n/a
2	S1 (5 μg) + alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
3	S1-Fc (5 μg)+ alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
4	S1-Fc' (5 μg)+ alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
5	S2-Fc' (5 μg)+ alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
6	S1 (5 μg) + MPL + alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
7	S1-Fc (5 μg) + MPL + alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0
8	S1-Fc' (5 μg) + MPL + alum	0	0	500	0	0	100.0	6.6	4.0
9	S2-Fc' (5 μg) + MPL + alum	0.00	0.00	0.00	0.00	0.00	0.0	0	0

Claims (35)

1. A fusion protein comprising a spike protein (S protein) of SARS-CoV-2 and a constant region of an immunoglobulin molecule.
2. According to claim 1,

   The spike protein is

   i) a full length S protein or fragment thereof;

   ii) S1 protein or fragment thereof;

   iii) S2 protein or a fragment thereof; or

   iv) RBD (Receptor binding domain) region or fragment thereof

   A fusion protein comprising a.
3. According to claim 1,

   The spike protein is

   i) a fusion protein of a) S1 protein or fragment thereof and b) S2 protein or fragment thereof; or

   ii) a) a fusion protein of a receptor binding domain (RBD) region or a fragment thereof and b) an S2 protein or a fragment thereof

   A fusion protein comprising a.
4. According to claim 1,

   The spike protein is a fusion protein, characterized in that it comprises the sequence of any one of SEQ ID NOs: 1 to 4.
5. According to claim 1,

   The immunoglobulin is a fusion protein, characterized in that any one selected from the group consisting of IgG, IgM, IgA, IgD and IgA.
6. According to claim 1,

   The fusion protein, characterized in that the immunoglobulin is IgG.
7. According to claim 1,

   The IgG is a fusion protein, characterized in that any one selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.
8. According to claim 1,

   The fusion protein, characterized in that the constant region of the immunoglobulin molecule is the constant region of an IgG1 heavy chain.
9. According to claim 1,

   The fusion protein, characterized in that the constant region of the immunoglobulin molecule comprises a hinge region, a CH2 domain and a CH3 domain of IgG1.
10. According to claim 1,

    The fusion protein, characterized in that the constant region of the immunoglobulin molecule further comprises a CH1 domain of IgG1.
11. According to claim 1,

    The fusion protein, characterized in that the constant region of the immunoglobulin molecule comprises an Fc region.
12. 12. The method of claim 11,

    The Fc region is a fusion protein, characterized in that it binds to an Fc receptor to enhance immunogenicity.
13. 12. The method of claim 11,

    Wherein the Fc region comprises an Fc variant.
14. 14. The method of claim 13,

    The Fc variant is a fusion protein, characterized in that it comprises one or more mutations selected from the group consisting of G236A, S239D, A330L, and I332E.
15. 14. The method of claim 13,

    The Fc variant is a fusion protein, characterized in that it comprises a G236A / S239D / A330L / I332E mutation.
16. 12. The method of claim 11,

    The Fc region is a fusion protein, characterized in that it comprises the sequence of SEQ ID NO: 5.
17. 14. The method of claim 13,

    The Fc variant is a fusion protein, characterized in that it comprises the sequence of SEQ ID NO: 6.
18. According to claim 1,

    The fusion protein is characterized in that it comprises the sequence of any one of SEQ ID NOs: 7 to 18.
19. 4. The method of claim 3,

    A fusion protein comprising the sequence of SEQ ID NO: 19 between the S1 protein and the S2 protein.
20. 20. A nucleic acid molecule encoding the fusion protein of any one of claims 1-19.
21. An expression vector comprising the nucleic acid molecule of claim 20 .
22. A host cell transformed with the expression vector of claim 21 .
23. i) introducing the expression vector of claim 21 into an animal cell expression system; and

    ii) performing expression of the fusion protein

    A method for producing a fusion protein comprising a.
24. 20. An immunogenic composition comprising the fusion protein of any one of claims 1-19.
25. 25. The method of claim 24,

    The fusion protein is an immunogenic composition, characterized in that the monomer (monomer).
26. 25. The method of claim 24,

    The fusion protein is an immunogenic composition, characterized in that the dimer (dimer).
27. 27. The method of claim 26,

    The dimer is an immunogenic composition, characterized in that made by a disulfide bond (disulfide bond) in the hinge region of the two fusion proteins.
28. 25. The method of claim 24,

    An immunogenic composition, characterized in that it further comprises an adjuvant.
29. 29. The method of claim 28,

    The adjuvant is an immunogenic composition, characterized in that at least one selected from the group consisting of TLR4 (Toll-like receptor 4) agonist, aluminum salt, saponin and liposome.
30. 30. The method of claim 29,

    The TLR4 (Toll-like receptor 4) agonist is an immunogenic composition, characterized in that at least one selected from the group consisting of MPL (Monophosphoryl Lipid A) and 3D-MPL.
31. 30. The method of claim 29,

    The aluminum salt is an immunogenic composition, characterized in that any one or more selected from the group consisting of aluminum hydroxide (Aluminum hydroxide), aluminum phosphate (phosphate) and aluminum sulfate (sulphate).
32. 30. The method of claim 29,

    The saponin (saponin) is an immunogenic composition, characterized in that any one or more selected from the group consisting of QS21, QS17 and QuilA.
33. 33. A kit comprising the immunogenic composition of any one of claims 24-32.
34. 33. A method of inducing an immune response by administering the immunogenic composition of any one of claims 24-32.
35. 33. A method for preventing SARS-coronavirus infection (COVID-19) by administering the immunogenic composition of any one of claims 24-32.

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