Coronavirus vaccine composition

Abstract

The present invention relates to a conjugate in which a coronavirus receptor-binding domain (RBD) and a Hepatitis B surface antigen (HBsAg) are linked, a virus-like particle containing the conjugate, and a coronavirus vaccine composition containing the conjugate and / or the virus-like particle.

Description

[Technical Field]

[0001] Cross-reference to related application(s) This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0044328 dated April 5, 2021, and all contents disclosed in the documents of said Korean Patent Application are incorporated herein by reference.

[0002] The present invention relates to a conjugate in which a coronavirus receptor-binding domain (RBD) and a Hepatitis B surface antigen (HBsAg) are linked, a virus-like particle containing the conjugate, and a coronavirus vaccine composition containing the conjugate and / or the virus-like particle. [Background technology]

[0003] Coronaviruses are a species of virus belonging to the family Coronaviridae. They are positive-sense RNA viruses that infect humans and animals and cause respiratory, gastrointestinal, and / or neurological diseases. Examples of coronaviruses include severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 (COVID-19), which can cause serious infectious diseases in humans.

[0004] In particular, vaccines using mRNA and adenovirus have been approved for use against the novel coronavirus (SARS-CoV-2; COVID-19), which was newly discovered in 2019, but their effectiveness is reduced against mutated viruses.

[0005] There is a demand for the development of a vaccine that can effectively prevent infection with coronaviruses, particularly the novel coronavirus or its mutant viruses. Summary of the Invention [Problem to be solved by the invention]

[0006] A SARS-CoV-2 RBD-HBsAg conjugate is provided, in which the receptor binding domain (RBD) of the spike protein of a coronavirus, particularly SARS-CoV-2 (COVID-19) (hereinafter referred to as "SARS-CoV-2 RBD") and the Hepatitis B surface antigen (HBsAg) are linked with or without a linker.

[0007] The SARS-CoV-2 RBD can be wild-type or mutant.

[0008] The hepatitis B virus surface antigen (HBsAg) or the conjugate may be in the form of a virus-like particle (VLP).

[0009] In the conjugate, the SARS-CoV-2 RBD can be linked to a Hepatitis B virus surface antigen in a VLP form with or without a linker, or the Hepatitis B virus surface antigen linked to the SARS-CoV-2 RBD with or without a linker can self-assemble to form a VLP form in which the SARS-CoV-2 RBD is exposed on the particle surface. When the conjugate is in the form of a virus-like particle, the SARS-CoV-2 RBD can be located on the particle surface.

[0010] In one example, the SARS-CoV-2 RBD and the Hepatitis B virus surface antigen may be linked via a chemical linker. The chemical linker may be selected from cross-linking (coupling) compounds having a functional group capable of linking proteins. In one example, the chemical linker may be a compound having one or more functional groups selected from the group consisting of functional groups containing oxygen, nitrogen, or sulfur, such as aldehyde, imide, ester, thiol, sulfonyl, etc., such as glutaraldehyde, SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate), SMPB (succinimidyl 4-(p-maleimido-phenyl)butyrate), Sulfo-SMPB, Sulfo-EMCS [Sulfo-(N-ε-maleimidocaproyl-oxysulfosuccinimide ester)], Sulfo-GMBS [Sulfo-(N-γ-maleimidobutyryl-oxysuccinimide ester)], SM[PEG]n(NHS-PEGn-Maliemide), AMAS(N-(α-Maleimidoacetoxy)-succinimide ester), BMPS(N-(β-Maleimidopropyloxy)succinimide ester), EMCA(N-ε-Maleimidocaproic acid), EMCS(N-(ε-Maleimidocaproyloxy)succinimide ester), GMBS(N-(γ-Maleimidobutyryloxy)succinimide ester), LC-SMCC(Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate)), MBS(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), SBAP(Succinimdyl 3-(bromoacetamido)propionate), SIA(N-succinimidylThe sulfo- or hydroxybenzoic acid may be one or more selected from the group consisting of, but not limited to, sulfo- or hydroxybenzoic acid esters ...

[0011] The conjugate may form a virus-like particle (VLP). The VLP may have the SARS-CoV-2 RBD located (exposed) on the particle surface, and may be formed with a hepatitis B virus surface antigen as described above. Thus, another example provides a virus-like particle containing the conjugate.

[0012] The conjugates and / or virus-like particles may function as immunogens or antigenic substances in the organism.

[0013] Another example provides a nucleic acid molecule for producing the conjugate, which may comprise a combination of a SARS-CoV-2 RBD-encoding polynucleotide and a Hepatitis B virus surface antigen-encoding polynucleotide.

[0014] Another example provides a recombinant vector containing the nucleic acid molecule. The recombinant vector can be used as an expression vector in a host cell. The recombinant vector can contain a SARS-CoV-2 RBD-encoding polynucleotide and a Hepatitis B virus surface antigen-encoding polynucleotide together in one vector or in two vectors, respectively.

[0015] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector. The recombinant cell may be obtained by introducing the recombinant vector into a host cell.

[0016] Another example provides a vaccine composition against coronavirus comprising the conjugate and / or virus-like particle, which may additionally comprise an immune enhancer.

[0017] Other examples provide the use of the conjugates and / or virus-like particles for immunization against coronaviruses or for the manufacture of vaccine compositions against coronaviruses, which may additionally comprise an immune enhancer.

[0018] Another example provides a method of immunizing against coronavirus, comprising administering said conjugate and / or virus-like particle or said vaccine composition to an individual in need thereof.

[0019] Another example provides a composition for preventing coronavirus infection or diseases and / or symptoms associated with infection, comprising said conjugate and / or virus-like particle, or said vaccine composition.

[0020] Other examples provide the use of said conjugates and / or virus-like particles, or said vaccine compositions, for the prevention of coronavirus infection or diseases and / or symptoms associated with infection, or for the manufacture of a composition for the prevention of coronavirus infection or diseases and / or symptoms associated with infection.

[0021] Another example provides a method for preventing coronavirus infection or a disease and / or symptom associated with infection, comprising administering said conjugate and / or virus-like particle or said vaccine composition to an individual in need thereof.

[0022] Another example provides a pharmaceutical composition for treating coronavirus infection or a disease and / or symptom associated with infection, comprising an antibody that binds to the conjugate and / or virus-like particle, or blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered, wherein the antibody can be isolated from blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered.

[0023] Another example provides the use of antibodies that bind to the conjugates and / or virus-like particles, or blood or serum isolated from an individual to whom the conjugates and / or virus-like particles have been administered, for the treatment of coronavirus infection or diseases and / or symptoms associated with infection, or for the manufacture of a pharmaceutical composition for the treatment of coronavirus infection or diseases and / or symptoms associated with infection, where the antibodies may have been isolated from blood or serum isolated from an individual to whom the conjugates and / or virus-like particles have been administered.

[0024] Another example provides a method for treating coronavirus infection or a disease and / or symptom associated with infection, comprising administering to a patient in need of treatment an antibody that binds to the conjugate and / or virus-like particle, or blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered, wherein the antibody can be isolated from blood or serum isolated from the individual to whom the conjugate and / or virus-like particle has been administered.

[0025] Another example provides a method for producing a conjugate comprising SARS-CoV-2 RBD and Hepatitis B virus surface antigen, a virus-like particle comprising the conjugate, or a coronavirus vaccine composition comprising the same, comprising expressing a nucleic acid molecule encoding SARS-CoV-2 RBD and Hepatitis B virus surface antigen in a host cell. The method may additionally comprise a step of adding a chemical linker before, after, or simultaneously with the expressing step.

[0026] The coronavirus may be the SARS-CoV-2 virus. [Means for solving the problem]

[0027] Definition of Terms As used herein, the term "vaccine" refers to a biological preparation containing an antigen that induces immunity in a living body, and refers to an immunogen or antigenic substance that confers immunity to a living body when administered to humans or animals for the prevention of infectious diseases.

[0028] As used herein, a coronavirus vaccine composition or a vaccine composition against a coronavirus refers to a pharmaceutical composition that induces an immune response against a coronavirus, and unless otherwise specified, can be used equivalently and interchangeably with a coronavirus immunogenic composition.

[0029] As used herein, the expressions "comprise (a specified component)" or "consist (of a specified component)" mean that the specified component is essentially included, and may not exclude the inclusion of additional and / or auxiliary components as long as they exert an effect equivalent to the intended effect.

[0030] As used herein, the phrase "a nucleic acid molecule (which may be used in conjunction with "gene") or a polypeptide (which may be used in conjunction with "protein") that "comprises a specific nucleic acid sequence or amino acid sequence, consists of a specific nucleic acid sequence or amino acid sequence, or is expressed by a specific nucleic acid sequence or amino acid sequence" is an equivalent and interchangeable expression, and may mean that the nucleic acid molecule or polypeptide essentially contains the specific nucleic acid sequence or amino acid sequence, and may be interpreted as including "substantially equivalent sequences" in which mutations (deletions, substitutions, modifications, and / or additions) have been made to the specific nucleic acid sequence or amino acid sequence to the extent that the original function and / or intended function of the nucleic acid molecule or polypeptide is maintained (or as not excluding the mutations).

[0031] The present invention will now be described in more detail.

[0032] SARS-CoV-2 RBD-HBsAg conjugate One example is a SARS-CoV-2 RBD-HBsAg conjugate comprising a receptor-binding domain of a coronavirus spike protein (e.g., SARS-CoV-2 RBD) and a hepatitis B virus surface antigen (HBsAg). In the conjugate, the receptor-binding domain of the coronavirus spike protein and the hepatitis B virus surface antigen can be linked with or without a linker. In one example, the conjugate can additionally include a chemical linker. In this case, the receptor-binding domain of the coronavirus spike protein and the hepatitis B virus surface antigen can be linked via the chemical linker.

[0033] In one embodiment, the coronavirus can be SARS-CoV-2 (COVID-19).

[0034] The SARS-CoV-2 RBD and Hepatitis B virus surface antigen may be produced by recombinant or chemical synthesis, and when recombinantly produced, they may be produced alone or together in a conjugate form in a suitable host cell, but are not limited thereto.

[0035] SARS-CoV-2 (COVID-19) has a spike protein (Spike protein; GenBank: QHD43416.1; 1273aa; SEQ ID NO: 1) on the surface of the virus, and the RBD (Receptor Binding Domain) of the spike protein binds to a receptor on the host cell (human cell) to enter the cell. Therefore, the SARS-CoV-2 (COVID-19) RBD can function as an antigen for SARS-CoV-2 vaccines.

[0036] The SARS-CoV-2 RBD may be the amino acid sequence from the 319th (R319) to the 541st (F541) amino acid from the N-terminus of the spike protein of SEQ ID NO: 1, or a part or extension thereof.

[0037] In one example, the SARS-CoV-2 RBD is based on the spike protein of SEQ ID NO: 1 and is the amino acid sequence from positions 319 to 541 from the N-terminus (wild type or original; SEQ ID NO: 2); a polypeptide fragment in which a portion thereof (for example, 10 to 30, 10 to 25, or 10 to 20 (for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) consecutive or non-consecutive amino acids are deleted independently from the N-terminus, C-terminus, or both termini of SEQ ID NO: 2 (when all amino acids are deleted from both termini, the number of amino acids deleted from each terminus may be the same or different); for example, a polypeptide fragment containing 190 or more consecutive amino acids within SEQ ID NO: 2, including at least SEQ ID NO: 5), extensions (e.g., polypeptide extensions in which 10 to 30, 10 to 25, or 10 to 20 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids have been added (extended) to the N-terminus, C-terminus, or both termini of SEQ ID NO: 2 or the aforementioned polypeptide fragments); or These amino acid sequences may be expressed as mutant amino acid sequences in which mutations have been added.

[0038] In one specific example, when the SARS-CoV-2 RBD is conjugated to the hepatitis B virus surface antigen through a free cysteine ​​(e.g., using a linker such as SMPH or SMPB), the polypeptide fragment or extension may contain one or more cysteines selected from the group consisting of C301, C336, C361, C379, C432, C391, C480, C488, C525 and C538, for example, C301, C538, or all of these, in free form, but is not limited thereto.

[0039] The SARS-CoV-2 RBD mutations may include, from the N-terminus of the spike protein of SEQ ID NO: 1, one or more (e.g., 1, 2, 3, 4, or 5) selected from the group consisting of: a substitution of lysine (K417) at amino acid residue 417 or a corresponding amino acid residue with another amino acid (e.g., K417N or K417T); a substitution of leucine (L452) at amino acid residue 452 or a corresponding amino acid residue with another amino acid (e.g., L452R); a substitution of serine (S477) at amino acid residue 477 or a corresponding amino acid residue with another amino acid (e.g., S477N); a substitution of glutamic acid (E484) at amino acid residue 484 or a corresponding amino acid residue with another amino acid (e.g., E484K); and a substitution of asparagine (N501) at amino acid residue 501 or a corresponding amino acid residue with another amino acid (e.g., N501Y).

[0040] More specifically, the mutation of the SARS-CoV-2 RBD is based on the spike protein of SEQ ID NO: 1, from the N-terminus: Substitution of the 501st amino acid residue, asparagine (N501) or a corresponding amino acid residue with another amino acid (e.g., N501Y) (e.g., UK variant SARS-CoV-2 virus, type B.1.1.7); one or more (e.g., one, two, or three) amino acid residues selected from the group consisting of substitution of lysine at amino acid residue 417 (K417) or a corresponding amino acid residue with another amino acid (e.g., K417N), substitution of glutamic acid at amino acid residue 484 (E484) or a corresponding amino acid residue with another amino acid (e.g., E484K), and substitution of asparagine at amino acid residue 501 (N501) or a corresponding amino acid residue with another amino acid (e.g., N501Y) (e.g., South African variant SARS-CoV-2 virus, type B.1.351); one or more (e.g., one, two, or three) amino acid residues selected from the group consisting of substitution of lysine (K417) at amino acid residue 417 or a corresponding amino acid residue with another amino acid (e.g., K417T), substitution of glutamic acid (E484) at amino acid residue 484 or a corresponding amino acid residue with another amino acid (e.g., E484K), and substitution of asparagine (N501) at amino acid residue 501 or a corresponding amino acid residue with another amino acid (e.g., N501Y) (e.g., the Brazilian variant SARS-CoV-2 virus, type P.1); A substitution of the 452nd amino acid residue, leucine (L452) or a corresponding amino acid residue with another amino acid (e.g., L452R) (e.g., California variant SARS-CoV-2 virus, B.1.427 / B.1.429); or One or more (e.g., one or two) amino acid residues selected from the group consisting of a substitution of the 477th amino acid residue, serine (S477) or a corresponding amino acid residue, with another amino acid (e.g., S477N) and a substitution of the 484th amino acid residue, glutamic acid (E484) or a corresponding amino acid residue, with another amino acid (e.g., E484K) (e.g., New York variant SARS-CoV-2 virus, B.1.526 type) It may include:

[0041] In one embodiment, the mutation occurs at the 417th amino acid residue from the N-terminus of SEQ ID NO: 1 (spike protein) (SEQ ID NO: 2 (wild-type SARS-CoV-2 The amino acid residues may be substituted with other amino acids at one or more (e.g., two, three, four, or five) selected from the group consisting of lysine (K417) at the 99th amino acid residue from the N-terminus of the RBD or an amino acid residue corresponding thereto, leucine (L452) at the 452nd amino acid residue or an amino acid residue corresponding thereto (the 134th amino acid residue from the N-terminus of SEQ ID NO: 2), serine (S477) at the 477th amino acid residue or an amino acid residue corresponding thereto (the 159th amino acid residue from the N-terminus of SEQ ID NO: 2), glutamic acid (E484) at the 484th amino acid residue (the 166th amino acid residue from the N-terminus of SEQ ID NO: 2) or an amino acid residue corresponding thereto, and asparagine (N501) at the 501st amino acid residue (the 183rd amino acid residue from the N-terminus of SEQ ID NO: 2) or an amino acid residue corresponding thereto. In one example, the mutation may include, but is not limited to, one or more (e.g., 1, 2, 3, 4, or 5) mutations selected from the group consisting of: a substitution of lysine at the 417th amino acid residue from the N-terminus with threonine (K417T) or asparagine (K417N); a substitution of leucine at the 452nd amino acid residue (L452) with arginine (L452R); a substitution of serine at the 477th amino acid residue (S477) with asparagine (S477N); a substitution of glutamic acid at the 484th amino acid residue with lysine (E484K); and a substitution of asparagine at the 501st amino acid residue with tyrosine (N501Y).

[0042] The wild-type and mutant amino acid sequences of the SARS-CoV-2 RBD are exemplified in Table 1 below. [Table 1-1] [Table 1-2]

[0043] The SARS-CoV-2 RBD contained in the conjugates provided herein may be selected from, but is not limited to, SEQ ID NOs: 2 to 11.

[0044] The hepatitis B virus surface antigen is a C-terminal fragment of the hepatitis B virus (HBV) middle S protein (UniprotKB:D0ESR0) (SEQ ID NO: 14), and can be expressed as a recombinant protein in a suitable host cell (e.g., yeast, E. coli, animal cells including CHO, etc.) and used in the production of a hepatitis prophylactic vaccine in the form of a virus-like particle (VLP). In one example, the hepatitis B virus surface antigen can be selected from SEQ ID NOs: 12 to 14 (Table 2). [Table 2]

[0045] The conjugates provided herein may comprise a combination of the Hepatitis B virus surface antigen and SARS-CoV-2 RBD described above at 1:0.1 to 100, 1:0.1 to 70, 1:0.1 to 50, 1:0.1 to 30, 1:0.1 to 10, 1:0.1 to 7, 1:0.1 to 5, 1:0.1 to 3.5, 1:0.1 to 3, 1:0.1 to 2.5, 1:0.1 to 2.1, 1:0.1 to 2, 1:0.1 to 1.5, 1:0.1 to 1, 1:0.1 to 0.7, 1:0.1 to 0.5, 1:0.3 to 100, 1:0.3 to 70, 1:0.3 to 5 0, 1:0.3-30, 1:0.3-10, 1:0.3-7, 1:0.3-5, 1:0.3-3 .5, 1:0.3 to 3, 1:0.3 to 2.5, 1:0.3 to 2.1, 1:0.3 to 2, 1:0.3 to to 1.5, 1:0.3 to 1, 1:0.3 to 0.7, 1:0.3 to 0.5, 1:0.5 to 100, 1: 0.5-70, 1:0.5-50, 1:0.5-30, 1:0.5-10, 1:0.5-7, 1: 0.5 to 5, 1:0.5 to 3.5, 1:0.5 to 3, 1:0.5 to 2.5, 1:0.5 to 2.1 , 1:0.5-2, 1:0.5-1.5, 1:0.5-1, 1:0.5-0.7, 1:0.7-1 00, 1:0.7-70, 1:0.7-50, 1:0.7-30, 1:0.7-10, 1:0.7- to 7, 1:0.7 to 5, 1:0.7 to 3.5, 1:0.7 to 3, 1:0.7 to 2.5, 1:0.7 to The molar ratio may be, but is not limited to, 1:0.1 to 1, 1:0.1 to 0.7, 1:0.1 to 0.7, 1:0.1 to 0.5 (moles of hepatitis B virus surface antigen:moles of SARS-CoV-2 RBD).

[0046] The linker may be selected from cross-linker compounds having a functional group capable of linking proteins. In one example, the chemical linker may be a compound having one or more functional groups selected from the group consisting of oxygen-, nitrogen-, or sulfur-containing functional groups, such as carbonyl, imide, ester, thiol, and sulfonyl. Examples of such functional groups include glutaraldehyde, SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate), SMPB (succinimidyl 4-(p-maleimido-phenyl)butyrate), Sulfo-SMPB, Sulfo-EMCS (sulfo-(N-ε-maleimidocaproyl-oxysulfosuccinimide ester)), and Sulfo-GMBS (sulfo-(N-γ-maleimidobutyryl-oxysuccinimide ester)], SM[PEG]n(NHS-PEGn-Maliemide), AMAS(N-(α-Maleimidoacetoxy)-succinimide ester), BMPS(N-(β-Maleimidopropyloxy)succinimide ester), EMCA(N-ε-Maleimidocaproic acid), EMCS(N-(ε-Maleimidocaproyloxy)succinimide ester), GMBS(N-(γ-Maleimidobutyryloxy)succinimide ester), LC-SMCC(Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxy-(6-amidocaproate)), MBS(m-Maleimidobenzoyl-N-hydroxysuccinimide ester), SBAP(Succinimdyl 3-(bromoacetamido)propionate), SIA(N-succinimidyl iodoacetate), SIAB(N-Succinimidyl(4-iodoacetyl)aminobenzoate), Sulfo-KMUS(N-(κ-Maleimidoundecanoyloxy)sulfosuccinimideester), Sulfo-MBS (m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester), SUlfo-SIAB (Sulfosuccinimidyl(4-iodo-acetyl)aminobenzoate), and the like, but are not limited thereto.

[0047] In one embodiment, glutaraldehyde is a cross-linking agent containing two aldehyde groups as functional groups, which can react with amino groups of a protein to form an amide bond (peptide bond). The amino group that forms the amide bond with glutaraldehyde can be the N-terminal amino group (NH2-) of the protein or the amino group of a lysine within the protein. For example, one aldehyde group of glutaraldehyde can bind to the N-terminal amino group or the amino group of a lysine located in the middle of the amino acid sequence of a hepatitis B virus surface antigen, and the other aldehyde group can bind to the N-terminal amino group or the amino group of a lysine located in the middle of the amino acid sequence of a SARS-CoV-2 RBD, thereby linking the SARS-CoV-2 RBD and the hepatitis B virus surface antigen. In the vaccine compositions provided herein, the glutaraldehyde as a linker may be present in an amount of about 0.04 to about 1% (w / v), about 0.04 to about 0.8% (w / v), about 0.04 to about 0.5% (w / v), about 0.04 to about 0.3% (w / v), about 0.04 to about 0.1% (w / v), about 0.06 to about 1% (w / v), about 0.06 to about 0.8% (w / v), about 0.06 to about 0.5% (w / v), about 0.06 to about 0.3% (w / v), about It may be contained or used (added) in an amount of, but is not limited to, 0.06 to about 0.1% (w / v), about 0.08 to about 1% (w / v), about 0.08 to about 0.8% (w / v), about 0.08 to about 0.5% (w / ), about 0.08 to about 0.3% (w / v), about 0.08 to about 0.1% (w / v), about 0.1 to about 1% (w / v), about 0.1 to about 0.8% (w / v), about 0.1 to about 0.5% (w / v), or about 0.1 to about 0.3% (w / v).

[0048] In another embodiment, the SMPH is a bifunctional cross-linker containing an NHS ester group and a maleimide group as functional groups. The SMPH may link a lysine located in the middle of the amino acid sequence of one protein to a free cysteine ​​of another protein. In one example, the SMPH may link a lysine located in the middle of the amino acid sequence of hepatitis B virus surface antigen to a free cysteine ​​of SARS-CoV-2 RBD. In the vaccine compositions provided herein, the SMPH as a linker may be contained or used (added) in an amount of, but is not limited to, 2 to 40 moles, 2 to 35 moles, 2 to 30 moles, 2 to 25 moles, 2 to 20 moles, 5 to 40 moles, 5 to 35 moles, 5 to 30 moles, 5 to 25 moles, 5 to 20 moles, 7 to 40 moles, 7 to 35 moles, 7 to 30 moles, 7 to 25 moles, 7 to 20 moles, 10 to 40 moles, 10 to 35 moles, 10 to 30 moles, 10 to 25 moles, or 10 to 20 moles per mole of hepatitis B virus surface antigen.

[0049] virus-like particles In the conjugate, the SARS-CoV-2 RBD can be linked to a hepatitis B virus surface antigen in the form of a virus-like particle (VLP) with or without a linker, or the hepatitis B virus surface antigen linked to the SARS-CoV-2 RBD with or without a linker can self-assemble to form a VLP in which the SARS-CoV-2 RBD is located (exposed) on the particle surface. Thus, another example provides a virus-like particle containing the SARS-CoV-2 RBD-HBsAg conjugate.

[0050] In one specific example, the SARS-CoV-2 RBD and the hepatitis B virus surface antigen can be expressed alone in suitable host cells (e.g., yeast, E. coli, mammalian cells such as CHO), or the SARS-CoV-2 RBD-encoding gene and the hepatitis B virus surface antigen-encoding gene can be expressed together and produced / purified as VLPs.

[0051] For example, the virus-like particle may be The hepatitis B virus surface antigen exposed on the surface of the virus-like particle formed (self-assembled) and the SARS-CoV-2 RBD are linked with or without a linker, It may be formed (self-assembled) by the hepatitis B virus surface antigen in a conjugate formed by linking the hepatitis B virus surface antigen and the SARS-CoV-2 RBD with or without a linker.

[0052] Virus-like particles are molecules that closely resemble viruses, but lack the viral genetic material and are therefore not infectious. Hepatitis B virus surface antigens can form virus-like particles through self-assembly.

[0053] Pharmaceutical uses such as vaccine compositions Another example provides a vaccine composition against coronavirus comprising the conjugate and / or virus-like particle, which may additionally comprise an immune enhancer.

[0054] Another example provides a method of immunizing against coronavirus comprising administering the conjugate and / or virus-like particle, or vaccine composition, to an individual in need thereof.

[0055] Another example provides a pharmaceutical composition for the prevention of coronavirus infection or diseases and / or symptoms associated with infection, comprising said conjugate and / or virus-like particle, or vaccine composition.

[0056] Another example provides a method for preventing coronavirus infection or disease and / or symptoms associated with infection, comprising administering the conjugate and / or virus-like particle, or vaccine composition to an individual in need thereof.

[0057] Another example provides a pharmaceutical composition for treating coronavirus infection or a disease and / or symptom associated with infection, comprising an antibody that binds to the conjugate and / or virus-like particle, or blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered, wherein the antibody can be isolated from blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered.

[0058] Another example provides a method for treating coronavirus infection or a disease and / or symptom associated with infection, comprising administering to a patient in need of treatment an antibody that binds to the conjugate and / or virus-like particle, or blood or serum isolated from an individual to whom the conjugate and / or virus-like particle has been administered, wherein the antibody can be isolated from blood or serum isolated from the individual to whom the conjugate and / or virus-like particle has been administered.

[0059] The coronavirus may be the SARS-CoV-2 virus.

[0060] The content of the conjugate and / or virus-like particle contained as an antigen in the vaccine composition is 0.0001 mg / mL to 1 mg / mL, 0.0001 mg / mL to 0.9 mg / mL, 0.0001 mg / mL to 0.8 mg / mL, 0.0001 mg / mL to 0.7 mg / mL, 0.0001 mg / mL to 0.6 mg / mL, 0.0001 mg / mL to 0.5 mg / mL, 0.0001 mg / mL to 0.4 mg / mL, 0.0001 mg / mL to 0.3 mg / mL, 0.0001 mg / mL to 0.2 mg / mL, 0.0005 ...6 mg / mL, 0.0001 mg / mL to 0.5 mg / mL, 0.0001 mg / mL to 0.6 mg / mL, 0.0001 mg / mL to 0.7 mg / mL, 0.0001 mg / mL to 0.8 mg / mL, 0.0001 mg / mL to 0.9 mg / mL, 0.0001 mg / mL to 0.9 mg / mL, 0.0001 mg / mL to 0.8 mg / mL, 0.0001 mg / mL to 0.7 mg / mL, 0.0001 mg / mL to 0.6 mL to 1 mg / mL, 0.0005 mg / mL to 0.9 mg / mL, 0.0005 mg / mL to 0.8 mg / mL, 0.0005 mg / mL to 0.7 mg / mL, 0.0005 mg / mL to 0.6 mg / mL, 0.0005 mg / mL0.5 mg / mL, 0.000 5mg / mL to 0.4mg / mL, 0.0005mg / mL to 0.3mg / mL, 0.0005mg / mL to 0.2mg / mL, 0.001mg / mL to 1mg / mL, 0.001mg / mL to 0.9mg / mL, 0.001mg / mL to 0.8mg / mL, 0.0 01mg / mL to 0.7mg / mL, 0.001mg / mL to 0.6mg / mL, 0.001mg / mL to 0.5mg / mL, 0.001mg / mL to 0.4mg / mL, 0.001mg / mL to 0.3mg / mL, 0.001mg / mL to 0.2mg / mL, 0 .004mg / mL to 1mg / mL, 0.004mg / mL to 0.9mg / mL, 0.004mg / mL to 0.8mg / mL, 0.004mg / mL to 0.7mg / mL, 0.004mg / mL to 0.6mg / mL, 0.004mg / mL to 0.5mg / mL, 0. 004mg / mL to 0.4mg / mL, 0.004mg / mL to 0.3mg / mL, 0.004mg / mL to 0.2mg / mL, 0.008mg / mL to 1mg / mL, 0.008mg / mL to 0.9mg / mL, 0.008mg / mL to 0.8mg / mL, 0.0 08mg / mL to 0.7mg / mL, 0.008mg / mL to 0.6mg / mL, 0.008mg / mL to 0.5mg / mL, 0.008mg / mL to 0.4mg / mL, 0.008mg / mL to 0.3mg / mL, 0.008mg / mL to 0.2mg / mL, 0.01mg / mL to 1mg / mL, 0.01mg / mL to 0.9mg / , 0.01mg / mL to 0.8mg / mL, 0.01mg / mL to 0. 7mg / mL, 0.01mg / mL to 0.6mg / mL, 0.01mg / mL to 0.5mg / mL, 0.01mg / mL to 0.4mg / mL, 0 .01mg / mL to 0.3mg / mL, 0.01mg / mL to 0.2mg / mL, 0.05mg / mL to 1mg / mL, 0.05mg / mL to ~0.9mg / mL, 0.05mg / mL~0.8mg / mL, 0.05mg / mL~0.7mg / mL, 0.05mg / mL~0.6mg / m The concentration may be, but is not limited to, 0.05 mg / mL to 0.5 mg / mL, 0.05 mg / mL to 0.4 mg / mL, 0.05 mg / mL to 0.3 mg / mL, 0.05 mg / mL to 0.2 mg / mL, 0.1 mg / mL to 1 mg / mL, 0.1 mg / mL to 0.9 mg / mL, 0.1 mg / mL to 0.8 mg / mL, 0.1 mg / mL to 0.7 mg / mL, 0.1 mg / mL to 0.6 mg / mL, 0.1 mg / mL to 0.5 mg / mL, 0.1 mg / mL to 0.4 mg / mL, 0.1 mg / mL to 0.3 mg / mL, or 0.1 mg / mL to 0.2 mg / mL.

[0061] The coronavirus vaccine composition may further comprise one or more selected from the group consisting of an efficacy enhancer, a preservative, a buffer, a surfactant, a carrier, a penetrant (isotonicity agent), an antioxidant, and a stabilizer.

[0062] As used herein, the term "immune enhancer (adjuvant, efficacy enhancer, or immunopotentiator)" refers to a substance used to increase the immunogenicity of the immunogenic composition of the present invention. Such immunopotentiators are sometimes provided to enhance immune responses and are well known to those skilled in the art.

[0063] In the present invention, the term "antiseptic (or preservative)" refers to a substance that has antiviral and / or antibacterial properties that inhibit the growth of microorganisms in the vaccine composition, and may be, for example, one or more (one, two, or three) selected from the group consisting of thimerosal, phenoxyethanol (2-phenoxyethanol), formaldehyde, etc., but is not limited thereto, and all common preservatives used in the art may be used. For example, the preservative may include phenoxyethanol (2-phenoxyethanol), formaldehyde, or a mixture thereof, in which case, The content of phenoxyethanol in the vaccine composition is, based on the entire vaccine composition, 0.5 mg / mL to 20 mg / mL, 0.5 mg / mL to 18 mg / mL, 0.5 mg / mL to 15 mg / mL, 0.5 mg / mL to 12 mg / mL, 0.5 mg / mL to 10 mg / mL, 1 mg / mL to 20 mg / mL, 1 mg / mL to 18 mg / mL, 1 mg / mL to 15 mg / mL, 1 mg / mL to 12 mg / mL, 1 mg / mL to 10 mg / mL, 3 mg / mL to 20 mg / mL, 3 mg / mL to 18 mg / mL, 3 mg / mL to 1 It may be 5 mg / mL, 3 mg / mL to 12 mg / mL, 3 mg / mL to 10 mg / mL, 3.75 mg / mL to 20 mg / mL, 3.75 mg / mL to 18 mg / mL, 3.75 mg / mL to 15 mg / mL, 3.75 mg / mL to 12 mg / mL, or 3.75 mg / mL to 10 mg / mL, 4 mg / mL to 20 mg / mL, 4 mg / mL to 18 mg / mL, 4 mg / mL to 15 mg / mL, 4 mg / mL to 12 mg / mL, 4 mg / mL to 10 mg / mL, 4 mg / mL to 8 mg / mL, or 4 mg / mL to 6 mg / mL.

[0064] The formaldehyde content in the vaccine composition may be 1 μg / mL to 200 μg / mL, 1 μg / mL to 180 μg / mL, 1 μg / mL to 150 μg / mL, 1 μg / mL to 120 μg / mL, 1 μg / mL to 100 μg / mL, or 1 μg / mL to 80 μg / mL based on the entire vaccine composition.

[0065] The vaccine composition may also contain one or more physiologically acceptable buffers. For example, when the vaccine composition is an infusion or injection, the buffer may have a buffering capacity of pH 4.0 to 10.0, specifically pH 5.0 to 9.0, more specifically pH 6.0 to 8.0. The buffer may be one or more selected from the group consisting of TRIS, acetate, glutamate, lactate, malate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate, and triethanolamine buffers.

[0066] Particularly when the vaccine composition of the present invention is intended for parenteral administration, the buffer can be selected from buffers suitable for USP. For example, the buffer can be one or more selected from the group consisting of monobasic acids such as acetic acid, benzoic acid, gluconic acid, glucaric acid, and lactic acid; dibasic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polybasic acids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0067] The vaccine compositions provided herein can also additionally comprise a non-ionic surfactant. Examples of such additional nonionic surfactants include, but are not limited to, one or more selected from the group consisting of polyoxyethylene sorbitan ester compounds (polysorbate 20 and polysorbate 80), copolymers of ethylene oxide (EO), propylene oxide (PO), and butylene oxide (BO) (e.g., DOWFAX™); octoxynols with different numbers of repeating ethoxy (oxy-1,2-ethanediyl) groups, particularly octoxynol-9 (Triton-100); ethylphenoxypolyethoxyethanol (IGEPAL CA-630 / NP-40); phospholipids such as lecithin; nonylphenol ethoxylates such as the NP series; polyoxyethylene fatty acid ethers (Brij surfactants) derived from lauryl, cetyl, stearyl, and oleyl alcohols, particularly triethylene glycol monolauryl ether (Brij 30); and sorbitan ethers known as SPANs, particularly sorbitan trioleate (Span 85) and sorbitan monolaurate. Specifically, the polyoxyethylene sorbitan ester compounds (polysorbate 20 and polysorbate 80) may be present in an amount of, but not limited to, 0.001% (w / v) to 20% (w / v), for example, 0.001% (w / v) to 10% (w / v), particularly 0.001% (w / v) to 1% (w / v) or about 0.5% (w / v).

[0068] Physiologically acceptable carriers used in liquid formulations include aqueous or non-aqueous solvents, suspensions, emulsions, and oils. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and ethyl oleate. Aqueous carriers include water, alcohol / aqueous solvents, emulsions or suspensions, physiological saline, and buffer solutions. Examples of oils include vegetable or animal oils, synthetic oils such as peanut oil, soybean oil, olive oil, sunflower oil, cod liver oil, and marine oil, and lipids derived from milk or eggs. The vaccine composition of the present invention can be isotonic, hypertonic, or preserved. Pharmaceutical compositions administered by infusion or injection are generally isotonic, but are not limited to this. On the other hand, isotonicity or hypertonicity is advantageous for preserving the composition. If the vaccine composition is hypertonic, it can be diluted to isotonicity before administration. The isotonicity agent used for dilution can be an ionic isotonicity agent such as a salt or a non-ionic isotonicity agent such as a carbohydrate. Ionic tonicity agents include, but are not limited to, sodium chloride, calcium chloride, potassium chloride, magnesium chloride, etc. Non-ionic tonicity agents include, but are not limited to, sorbitol, glycerol, etc.

[0069] The vaccine compositions provided herein can contain an antioxidant. Exemplary reducing agents include mercaptopropionylglycine, N-acetylcysteine, β-mercaptoethylamine, glutathione, ascorbic acid and its salts, sulfite or sodium metabisulfite, or similar species. Additionally, antioxidants can also include, but are not limited to, natural antioxidants such as vitamins E, C, lecithin, xanthine, β-carotene, and substances such as zinc and selenium.

[0070] The vaccine compositions provided herein may contain additional substances such as stabilizers, wetting agents, emulsifiers, dispersing agents, and monosaccharides and polysaccharides. They may also contain immunostimulatory molecules to improve vaccine efficacy. Such molecules may enhance immune responses, induce inflammation, or may be specific lymphocytes or cytokines. Cytokines may include, but are not limited to, interleukin (IL)-1, IL-2, IL-3, IL-4, IL-12, IL-13, granulocyte-macrophage colony-stimulating factor (GMCSF), macrophage inflammatory factor, etc.

[0071] The coronavirus vaccine composition may be formulated as a single dose for single administration or as multiple doses (multiple doses) for multiple administration. As used herein, "multiple doses" may refer to a vaccine volume that can be administered to an individual more than once (e.g., two or more times) or to two or more individuals in a single or more than one dose (e.g., two or more times).

[0072] Optimal amounts of components for a particular vaccine can be ascertained through standard studies involving observation of appropriate immune responses in subjects. For example, results from animal studies can be extrapolated to determine vaccination doses for humans. Alternatively, one skilled in the art can empirically determine the dosage as needed.

[0073] The vaccine composition may further comprise an immunopotentiator, which may be one or more pharmaceutically acceptable components capable of enhancing the immune response (e.g., neutralizing antibody titer, binding antibody titer, etc.) of a recipient of the vaccine.

[0074] For example, the immune enhancer may be (1) Aluminum ion-containing adjuvants, including aluminum hydroxide (Al(OH)3), aluminum phosphate (AlPO4), amorphous aluminum hydroxyphosphate sulfate (AAHS), alum, etc. (Alhydrogel®, Rehydragel®, Adju-Phos®, etc.); (2) calcium ion-containing adjuvants, such as calcium phosphate (CaPO4); (3) Biocompatible oil-based (containing) emulsion adjuvants (oil-in-water or water-in-oil emulsions; ASO3, MF59, AddaS03™, AddaVax™, ISCOMATRIX™, Freund's Adjuvant, etc.) such as squalene, cholesterol, and mineral oil (e.g., paraffin); (4) Liposome-containing adjuvants such as virosomes, lipid-containing adjuvants such as monophosphoryl lipid A (MPL) (ASO4, AS018, etc.), (5) Adjuvants containing plant extracts or plant extract-derived compounds (e.g., saponin, etc.) such as Quillaja plants (e.g., Quillaja saponaria Molina), beans, Polygala senega, etc. (e.g., AS018, Quil A, Abisco-100, ISCOMATRIX (registered trademark), Matrix M (trade name), etc.); (6) nucleic acid-containing adjuvants such as CpG motifs (e.g., CpG1018); (7) Adjuvants containing bacteria or viruses, such as Mycobacterium bacteria (e.g., Freund's Adjuvant); (8) Peptide- or protein-containing adjuvants such as MDP (muramyl dipeptide), flagellin, and HSP (heat shock proteins); (9) cytokine-containing adjuvants such as interferon (e.g., rIFN) and GM-CSF; (10) Biocompatible polymer-containing adjuvants, such as anionic polymers (e.g., Carbopol) It may be one or more selected from the group consisting of, but is not limited to, the above.

[0075] The content of the immune enhancer in the vaccine composition provided herein may be appropriately adjusted depending on the type of immune enhancer, the manufacturer's formulation, the method of administration, the recipient (age, weight, medical condition, etc.), and / or the desired effect.

[0076] For example, in the case of an aluminum ion-containing adjuvant, the content in the vaccine composition may be 0.1 to 10 mg / ml, 0.1 to 7.5 mg / ml, 0.1 to 5 mg / ml, 0.1 to 2.5 mg / ml, 0.1 to 1 mg / ml, 0.3 to 10 mg / ml, 0.3 to 7.5 mg / ml, 0.3 to 5 mg / ml, 0.3 to 2.5 mg / ml, 0.3 to 1 mg / ml, 0.5 to The concentration may be, but is not limited to, 10 mg / ml, 0.5 to 7.5 mg / ml, 0.5 to 5 mg / ml, 0.5 to 2.5 mg / ml, 0.5 to 1 mg / ml, 0.7 to 10 mg / ml, 0.7 to 7.5 mg / ml, 0.7 to 5 mg / ml, 0.7 to 2.5 mg / ml, 0.7 to 1 mg / ml, 1 to 10 mg / ml, 1 to 7.5 mg / ml, 1 to 5 mg / ml, or 1 to 2.5 mg / ml.

[0077] In another example, the biocompatible oil-based emulsion adjuvant AS03 or AddaS03 is composed of 1.8% (v / v) polysorbate 80, 5% (v / v) squalene, 5% (v / v) α-tocopherol, and PBS (NaCl 121 mM, KCl 2.38 mM, NaHPO 7.14 mM, KHPO 1.3 mM), and the concentration may be 0.01X to 1X or 0.1X to 0.5X based on this (1X standard), but is not limited to this and may be appropriately adjusted according to the manufacturer's formulation. Another biocompatible oil-based emulsion adjuvant, MF59 or AddaVax (trademark), is a submicron emulsion containing 5% (v / v) squalene oil, 0.5% (w / v) polysorbate 80, 0.5% (w / v) sorbitan trioleate, and 10 mM sodium citrate buffer. Based on this (1X), the concentration can be 0.01X to 1X, or 0.1X to 0.5X, but is not limited thereto and can be appropriately adjusted according to the manufacturer's prescription.

[0078] The vaccine compositions according to the invention may be for administration in a pharmaceutically effective amount to an individual in need of prevention of infection by coronavirus and / or development of disease and / or symptoms due to coronavirus infection (e.g., at risk of infection by coronavirus and / or development of disease / symptoms due to coronavirus infection) for the prevention of infection by coronavirus and / or development of disease and / or symptoms due to coronavirus infection.

[0079] As used herein, the term "prevention" may refer to any action of administering the vaccine composition of the present invention to suppress or delay infection by the coronavirus and / or the onset of diseases / symptoms caused by coronavirus infection. The term "pharmaceutically effective amount" may refer to the dosage required to induce antibodies to a degree that can significantly reduce the probability of coronavirus infection or the severity of infection. The term "administration" refers to introducing a predetermined substance into an individual by any appropriate method. The vaccine composition may be administered by injection, for example, subcutaneous injection, intravenous injection, intramuscular injection, etc.

[0080] For example, the dose of the vaccine composition may be 0.1 to 1000 μg, 0.1 to 900 μg, 0.1 to 800 μg, 0.1 to 700 μg, 0.1 to 600 μg, 0.1 to 500 μg, 0.1 to 400 μg, 0.1 to 350 μg, 0.1 to 300 μg, 0.1 to 250 μg, 0.1 to 200 μg, 0.1 to 150 μg, 0.1 to 100 μg, 0.1 to 80 μg, 0.1 to 60 μg, 0.1 to 40 μg, 0.1 to 20 μg, or 0.1 to 300 μg, based on the weight of the antigen (the conjugate and / or virus-like particle). μg, 0.5-1000μg, 0.5-900μg, 0.5-800μg, 0.5-700μg, 0.5-600μg , 0.5-500μg, 0.5-400μg, 0.5-350μg, 0.5-300μg, 0.5-250μg, 0.5 200μg, 0.5-150μg, 0.5-100μg, 0.5-80μg, 0.5-60μg, 0.5-40μg , 0.5-20μg, 1-1000μg, 1-900μg, 1-800μg, 1-700μg, 1-600μg, 1- to 500μg, 1 to 400μg, 1 to 350μg, 1 to 300μg, 1 to 250μg, 1 to 200μg, 1 to 150 μg, 1-100μg, 1-80μg, 1-60μg1-40μg, 1-20μg, 5-1000μg, 5-900 μg, 5-800μg, 5-700μg, 5-600μg, 5-500μg, 5-400μg, 5-350μg, 5- to 300 μg, 5 to 250 μg, 5 to 200 μg, 5 to 150 μg, 5 to 100 μg, 5 to 80 μg, 5 to 60 μg, The amount may be approximately 5 to 40 μg, 5 to 20 μg, 10 to 1000 μg, 10 to 900 μg, 10 to 800 μg, 10 to 700 μg, 10 to 600 μg, 10 to 500 μg, 10 to 400 μg, 10 to 350 μg, 10 to 300 μg, 10 to 250 μg, 10 to 200 μg, 10 to 150 μg, 10 to 100 μg, 10 to 80 μg, 10 to 60 μg, 10 to 40 μg, or 10 to 20 μg, but is not limited to these amounts and can be appropriately adjusted taking into account the condition of the recipient, the desired effect, etc.

[0081] The "individual" to which the conjugate, virus-like particle, vaccine composition or pharmaceutical composition of the present invention is administered may refer to a living organism that can be infected with a pathogen, or a cell, tissue or culture thereof isolated therefrom, and the organism may be a higher vertebrate, more specifically a mammal such as a human, but is not particularly limited thereto.

[0082] Production of conjugates and / or virus-like particles and / or vaccine compositions Another example provides a nucleic acid molecule comprising a combination of a SARS-CoV-2 RBD-encoding polynucleotide and a Hepatitis B virus surface antigen-encoding polynucleotide.

[0083] Another example provides a recombinant vector comprising the nucleic acid molecule, which can be used as an expression vector in a host cell.

[0084] Another example provides a recombinant cell comprising the nucleic acid molecule or recombinant vector. The recombinant cell may be obtained by introducing the recombinant vector into a host cell.

[0085] Another example provides a method for producing a conjugate comprising SARS-CoV-2 RBD and Hepatitis B virus surface antigen, a virus-like particle comprising the conjugate, or a vaccine composition against coronavirus comprising the same, comprising expressing a nucleic acid molecule encoding SARS-CoV-2 RBD and Hepatitis B virus surface antigen in a host cell.

[0086] The method may further include a step of adding a linker to the SARS-CoV-2 RBD and the HBV surface antigen expressed after the expression step to link the SARS-CoV-2 RBD and the HBV surface antigen.

[0087] The SARS-CoV-2 RBD and the hepatitis B virus surface antigen can be expressed in the same host cell through separate vectors or in separate host cells, and can be produced in a conjugate form via a chemical linker (crosslinker).

[0088] When the SARS-CoV-2 RBD and the Hepatitis B virus surface antigen are each expressed through separate vectors, the hepatitis B virus surface antigen forms a virus-like particle, and the SARS-CoV-2 RBD is linked to the hepatitis B virus surface antigen on the surface of the formed virus-like particle with or without a linker to form a conjugate and / or to form a virus-like particle containing the conjugate; The SARS-CoV-2 RBD and the HBV surface antigen are linked with or without a linker to form a conjugate, and the HBV surface antigens in the multiple conjugates formed can form virus-like particles.

[0089] In one example, the conjugate or virus-like particle can be obtained by simultaneously reacting the previously described hepatitis B virus surface antigen, SARS-CoV-2 RBD, and linker, or the conjugate or virus-like particle can be obtained by sequentially reacting the previously described hepatitis B virus surface antigen, linker, and SARS-CoV-2 RBD (e.g., the conjugate or virus-like particle can be obtained by first reacting the previously described hepatitis B virus surface antigen and linker, and then reacting SARS-CoV-2 RBD). In this case, the reaction time may be 10 to 120 minutes, 10 to 90 minutes, 30 to 120 minutes, or 30 to 90 minutes, and the reaction temperature may be 2 to 40°C, 2 to 35°C, 2 to 30°C, 10 to 40°C, 10 to 35°C, 10 to 30°C, 15 to 40°C, 15 to 35°C, 15 to 30°C, 20 to 40°C, 20 to 35°C, or 20 to 30°C, but is not limited thereto.

[0090] The SARS-CoV-2 RBD, hepatitis B virus surface antigen, linker, and their ratios and contents are as described above.

[0091] The host cell may be one or more selected from the group consisting of yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, etc.), Escherichia coli, and animal cells (e.g., CHO (Chinese Hamster Ovary) cells, HEK293 cells, Vero cells, Per.C6 cells, etc.), but is not limited thereto.

[0092] Vectors that can be used as recombinant vectors (expression vectors) herein are not particularly limited as long as they are replicable in host cells and can be selected from any commonly used vector. Examples of commonly used vectors include naturally occurring or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage or cosmid vectors, and pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pCDNA, and pET can be used as plasmid vectors. Specific examples include, but are not limited to, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC, and pcDNA3.1 vectors.

[0093] The vector may further include a selection marker for verifying the presence or absence of insertion into the chromosome. The selection marker is used to select cells transformed with the vector, i.e., to verify the presence or absence of insertion of the polynucleotide, and can be selected from genes that confer a selectable phenotype, such as drug resistance, auxotrophy, resistance to cytotoxic agents, or expression of a surface protein. In an environment treated with a selective agent, only cells expressing the selection marker survive or exhibit other phenotypes, allowing the selection of transformed cells.

[0094] The proteins provided herein may not necessarily be naturally occurring, but may be recombinantly or chemically synthesized. When the proteins are recombinantly produced, they may be in a form to which a conventional signal peptide, cleavage site, tag, etc. is attached for purification purposes. Thus, in a non-limiting example, the proteins provided herein may be in a form that additionally contains one or more selected from the group consisting of a signal peptide, cleavage site, tag (e.g., His tag, GST (glutathione-S-transferase) tag, MBP (maltose binding protein) tag, etc.) that can be commonly used in the recombinant production of proteins, or in a purified form in which these have been removed. [Effects of the Invention]

[0095] The conjugates provided herein or virus-like particles formed therefrom have immunogenic effects against coronavirus (SARS-CoV-2 (COVID-19)) in vivo and can be usefully applied in the prevention of coronavirus (SARS-CoV-2 (COVID-19)) infection. [Brief explanation of the drawings]

[0096] [Figure 1] FIG. 1 is a schematic diagram illustrating the process for producing a SARS-CoV-2 RBD-HBsAg VLP conjugate according to one embodiment. [Figure 2] 1 shows a restriction enzyme map of the pCB-RBD Vector. [Figure 3] These are the results of purification through IEC (Ion Exchange Column Chromatography) and HIC (Hydrophobic Interaction Chromatography) to confirm the production of SARS-CoV-2 RBD. [Figure 4a]1 is a graph showing the dynamic light scattering (DLS) results of a SARS-CoV-2 RBD-HBsAg VLP conjugate (Sample 1) conjugated using glutaraldehyde (GA) as a linker (crosslinking agent) in comparison with HBsAg VLP. [Figure 4b] 1 is a graph showing the DLS results of a SARS-CoV-2 RBD-HBsAg VLP conjugate (Sample 5) conjugated using SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate) as a linker (crosslinker) compared to HBsAg VLP. [Figure 5] 1 is a graph showing the results of an ACE2 binding assay (in vitro) of the SARS-CoV-2 RBD-HBsAg VLP conjugate prepared in the Examples herein. [Figure 6] TEM image of a SARS-CoV-2 RBD-HBsAg VLP conjugate (sample 4) according to one embodiment. [Figure 7] 1 is a graph showing RBD-binding antibody titers of a vaccine formulation comprising a SARS-CoV-2 RBD-HBsAg VLP conjugate according to one embodiment. [Figure 8] 1 is a graph showing neutralizing antibody titers of a vaccine formulation comprising a SARS-CoV-2 RBD-HBsAg VLP conjugate according to one embodiment. [Figure 9] FIG. 1 is a schematic diagram showing a mouse immunization and efficacy testing schedule using a vaccine comprising a SARS-CoV-2 RBD-HBsAg VLP conjugate according to one embodiment. [Figure 10] 1 is a graph showing neutralizing antibody titers of a vaccine comprising a SARS-CoV-2 RBD-HBsAg VLP conjugate according to one embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0097] The present invention will be described in more detail below with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0098] Example 1: Preparation of SARS-CoV-2 RBD-HBsAg VLP conjugate SARS-CoV-2 (COVID-19) has a spike protein (sequence number 1, GenBank: QHD43416) on the surface of the virus, and the RBD (receptor binding domain; sequence numbers 2, 3, or 4) of the spike protein binds to receptors on human cells and enters the cells, so the RBD can be used as an antigen for SARS-CoV-2 vaccines.

[0099] Hepatitis B virus (HBV) middle S protein (SEQ ID NO: 14, UniprotKB: D0ESR0) can be expressed as a recombinant protein in yeast and used in the production of a hepatitis prevention vaccine in the form of a virus-like particle (VLP).

[0100] In this example, we attempt to produce a SARS-CoV-2 RBD-HBsAg VLP conjugate by chemically conjugating a VLP consisting of the antigen HBsAg (SEQ ID NO: 8), which is a C-terminal fragment of HBV middle S protein, with the antigen RBD of SARS-CoV-2 (wild-type (SEQ ID NO: 2) or mutant (SEQ ID NO: 3 or 4)) (see Figure 1).

[0101] More specifically, the receptor binding domain (RBD; SEQ ID NO: 2), which is the amino acid sequence region from R319 to F541 of the spike protein (GenBank: QHD43416.1; SEQ ID NO: 1) on the surface of the SARS-CoV-2 (COVID-19) virus, or its variants (SEQ ID NO: 3 or 4) was linked to the hepatitis B surface antigen (HBsAg; SEQ ID NO: 8) via a linker to prepare an RBD-HBsAg VLP conjugate.

[0102] The amino acid sequences used to prepare the RBD-HBsAg VLP conjugate are summarized in Table 3 below. [Table 3]

[0103] 1.1. Preparation of SARS-CoV-2 RBD The nucleic acid sequence (SEQ ID NO: 15) of the wild-type (original) SARS-CoV-2 RBD (SEQ ID NO: 2)-encoding gene (RBD_original) was synthesized and prepared. The RBD PCR product was obtained using the prepared RBD_original as a template and primers RBD2_GS_F and RBD2_R listed in Table 4 below. The South African variant RBD (RBD_ZA; SEQ ID NO: 4) was obtained by overlap PCR using RBD_Original as a template and primers RBD2-GS_F, MutI_ZA_R, MutI_F, MutI_R, Mut_II_F, and RBD2_R listed in Table 4. The Brazilian variant RBD (RBD_BR) was obtained by overlap PCR using RBD_Original as a template and primers RBD2-GS_F, MutI_Brazil_R, MutI_F, MutI_R, Mut_II_F, and RBD2_R listed in Table 4. [Table 4]

[0104] The linearized pCB vector was obtained by PCR using the pCB-CVRBD2-His vector (see Figure 2) as a template and the primers Xslip_F and SP_R_RBD2_OL in Table 4. The pCB vector has a GS selection marker and expresses the antigen (RBD) protein under the control of a promoter (KR103836) that combines the CMV promoter and the CHO beta-actin promoter, and the antigen protein is secreted extracellularly under the control of a signal peptide (KR2092225).

[0105] The RBD PCR products (RBD, RBD_ZA, RBD_BR) obtained above were cloned into the linearized pCB vector using the EZ Fusion Cloning Kit (Enzynomics, EZ015) to obtain the expression vectors pCB-RBD, pCB-RBD_ZA, and pCB-RBD_BR, respectively (see Figure 2).

[0106] The prepared expression vectors, pCB-RBD (RBD_ZA, RBD_BR), were introduced into CHO cells (HD-BIOP3, Horizon Discovery) and cultured in CD FortiCHO™ medium (Thermo Fisher Scientific) for 14 days (Fed-Batch Culture: Feedings 3, 5, 7, 9, and 11) to produce RBD (wild-type and mutant types).

[0107] The produced RBD was purified through IEC (ion exchange column chromatography) and HIC (hydrophobic interaction chromatography), etc. The purification results are shown in Figure 3.

[0108] 1.2. Preparation of Hepatitis B Virus Surface Antigen (HBsAg) VLPs HBsAg VLPs were prepared from a stock solution of HBsAg (SEQ ID NO: 8), an antigen that is a C-terminal fragment of HBV middle S protein, expressed in yeast. Specific methods are described in KR0177298, KR0159716, KR0194247, KR0251015, etc., and HBsAg prepared by the methods described in these documents forms HBsAg VLPs through self-assembly.

[0109] 1.3. Linker-mediated conjugation of SARS-CoV-2 RBD and HBsAg VLP The SARS-CoV-2 RBD prepared in Example 1.1 and the HBsAg VLP prepared in Example 1.2 were conjugated using a linker: glutaraldehyde (GA) and SMPH (succinimidyl-6-((b-maleimidopropionamido)hexanoate).

[0110] Glutaraldehyde (GA) reacts with an amino group to form an amide bond (peptide bond). The amino group may be the N-terminus NH2 of a protein or the NH2 of a lysine group within a protein. Conjugation using glutaraldehyde was carried out as follows, with reference to Table 5 below. [Table 5]

[0111] More specifically, Samples 1 and 2 were prepared by reacting HBsAg VLP and SARS-CoV-2 RBD with 0.1% (w / v) glutaraldehyde at room temperature for 30 minutes to achieve the desired conjugation ratio, while Sample A was prepared by reacting HBsAg VLP with 0.2% glutaraldehyde at room temperature for 60 minutes, followed by SARS-CoV-2 RBD for 30 minutes.

[0112] SMPH was used as a bifunctional cross-linker for conjugation between the lysine of yeast-derived HBsAg VLP (Example 1.2) and the free cysteine ​​of RBD (Example 1.1) expressed and purified in CHO. Conjugation was carried out using the NHS ester group and maleimide group contained in SMPH as shown in Table 6. [Table 6]

[0113] More specifically, HBsAg VLPs were activated by treatment with SMPH (10X to 20X molar excess relative to HBsAg) and reacted with SARS-CoV-2-RBD at the reaction ratio for 4 hours.

[0114] Example 2: Confirmation of SARS-CoV-2 RBD-HBsAg VLP conjugate formation As a result of the conjugation of SARS-CoV-2 RBD and HBsAg VLP using the chemical linker (crosslinker) described above, the formation of a VLP-type conjugate was confirmed by DLS (Dynamic Light Scattering; Zetasizer Pro (Malvern)) and TEM (Transmission Electron Microscopy; JEM 1400Flash - 120kV accelerating voltage).

[0115] SARS-CoV-2 RBD is known to bind to the extracellular domain of human Angiotensin Converting Enzyme 2 (ACE2-ED). To determine whether the SARS-CoV-2 RBD is displayed on the surface of the VLP-form conjugate and binds to ACE2-ED, we performed an ACE2 binding assay to confirm whether the SARS-CoV-2 RBD-HBsAg VLP conjugate binds to ACE2-ED. The ACE2 binding assay was performed using an Octet® Qke (Sartorius; Sensor: Protein A sensor (FORTEBIO); Ligand: ACE2-Fc (LG Chem); Analytical concentration: 100 μg / mL) according to the manufacturer's instructions.

[0116] The results obtained from the above analysis are shown in Table 7 and Figure 4a (DLS:GA conjugate; Sample 1), Figure 4b (DLS:SMPH conjugate; Sample 5), Figure 5 (ACE2 binding assay), and Figure 6b (TEM image of RBD-HBsAg VLP conjugate (Sample 4)). [Table 7]

[0117] As shown above, the DLS results (higher values ​​indicate larger particle size) confirmed that the conjugates had larger particle sizes than HBsAg VLPs, indicating that RBD was conjugated to the surface of HBsAg VLPs. The larger the difference in particle size (values ​​in parentheses) from HBsAg VLPs, the greater the amount of RBD conjugated. Furthermore, the ACE2 binding activity results indicate the amount of RBD conjugated (exposed) to the surface (higher ACE2 binding activity values ​​indicate greater amounts of RBD exposed on the surface). In the case of HepB ELISA, the more RBD conjugated to the surface, the lower the level of HBsAg exposure, resulting in a relatively low value. These results confirmed that RBD-HBsAg VLP conjugates successfully formed and maintained good ACE2 binding activity in both cases where conjugation was performed using GA as a linker (crosslinker) and SMPH.

[0118] Example 3: Immunogenicity of SARS-CoV-2 RBD-HBsAg VLP conjugates 1 The immunogenicity of the SARS-CoV-2 RBD-HBsAg VLP conjugate prepared in Example 1 was confirmed in animal experiments by preparing a vaccine formulation using the purified conjugate and an immunopotentiator (aluminum hydroxide). The vaccine formulation used in the test was prepared with the following composition: [Table 8]

[0119] For comparison, SARS-CoV-2 RBD (wild type; SEQ ID NO: 2; Example 1.1) was prepared.

[0120] The prepared formulation was administered to animals in a single or repeated dose of 100 μL each, and blood samples were collected to confirm the production of antibodies against the conjugated RBD-HBsAg VLP using methods such as ELISA. In addition, the virus prevention effect was examined by measuring neutralizing antibody titers in an in vitro neutralization assay using serum collected from animals immunized with the SARS-CoV-2 RBD-HBsAg VLP conjugate and SARS-CoV-2 or a SARS-CoV-2 pseudovirus.

[0121] More specifically, Balb / c mice (F, 6 weeks old, 17-19 g, 5 mice per group) were immunized at weeks 0 and 2 (see Table 9), and blood was collected at week 3 for analysis of binding and neutralizing antibody titers (see Figure 9). Binding antibody titers were analyzed using the Gyros system, and neutralizing antibody titers were measured using the FRNT (Focus Reduction Neutralization Test) method to determine neutralizing capacity against wild-type SARS-CoV-2 virus (Wuhan strain, NCCP 43326).

[0122] The results obtained are shown in Table 9, Figure 7 (RBD-binding antibody titer), and Figure 8 (neutralizing antibody titer). [Table 9]

[0123] As can be seen from the above results, the SARS-CoV-2 RBD-HBsAg VLP conjugate according to the present invention exhibited significantly higher binding antibody titers and neutralizing antibody titers than SARS-CoV-2 RBD. Based on these results, it is expected that the SARS-CoV-2 RBD-HBsAg VLP conjugate has an excellent in vivo antibody production rate and can therefore be advantageously used as a vaccine against coronaviruses, particularly SARS-CoV-2 (COVID-19) virus.

[0124] Example 4: Immunogenicity of SARS-CoV-2 RBD-HBsAg VLP conjugates 2 To confirm the immunogenicity of the SARS-CoV-2 RBD-HBsAg VLP conjugate (Sample A in Table 5) prepared in Example 1 depending on the type of immune enhancer, vaccine formulations were prepared using various immune enhancers and confirmed in animal experiments. The vaccine formulations used in the animal tests were prepared with the following composition. [Table 10]

[0125] For comparison, SARS-CoV-2 RBD (wild type; SEQ ID NO: 2; Example 1.1) was prepared (dosage form 7).

[0126] The above-prepared formulations 7 to 10 were administered to animals in a single or repeated dose of 100 μL each, and blood samples were collected to determine whether antibodies against the conjugated RBD-HBsAg VLPs were produced by ELISA or other methods.

[0127] More specifically, Balb / c mice (F, 6 weeks old, 10 mice per group) were immunized at weeks 0 and 2 (see Table 11), and blood was collected at week 3 for analysis of neutralizing antibody titers (see Figure 9). The neutralizing antibody titers were measured using a microneutralization assay to determine the neutralizing ability against wild-type SARS-CoV-2 virus (Wuhan strain, NCCP 43326).

[0128] The results obtained are shown in Table 11 and Figure 10 (mean neutralizing antibody titers): [Table 11]

[0129] As shown in Table 11 and Figure 10, the SARS-CoV-2 RBD-HBsAg VLP conjugate according to the present invention produced significantly higher neutralizing antibody titers compared to SARS-CoV-2 RBD when used in combination with all types of immune enhancing agents tested.

[0130] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and equivalent concepts thereof, rather than the above detailed description.

Claims

1. a receptor binding domain (RBD) of the spike protein of SARS-CoV-2 (SARS-CoV-2 RBD), a hepatitis B surface antigen (HBsAg), and a bifunctional chemical cross-linker; SARS-CoV-2 RBD-HBsAg conjugate, wherein the chemical cross-linking agent is glutaraldehyde.

2. 2. The SARS-CoV-2 RBD-HBsAg conjugate of claim 1, wherein the SARS-CoV-2 RBD comprises an amino acid sequence selected from SEQ ID NOs: 2 to 11.

3. 2. The SARS-CoV-2 RBD-HBsAg conjugate of claim 1, wherein the hepatitis B virus surface antigen comprises an amino acid sequence selected from SEQ ID NOs: 12 to 14.

4. A virus-like particle (VLP) comprising the SARS-CoV-2 RBD-HBsAg conjugate of any one of claims 1 to 3.

5. A vaccine composition against SARS-CoV-2 virus, comprising the SARS-CoV-2 RBD-HBsAg conjugate of any one of claims 1 to 3, or a virus-like particle comprising said SARS-CoV-2 RBD-HBsAg conjugate.

6. 6. The vaccine composition of claim 5, additionally comprising an immunopotentiator.

7. 7. The vaccine composition of claim 6, wherein the immune enhancer is at least one selected from the group consisting of aluminum ion-containing adjuvants, calcium ion-containing adjuvants, biocompatible oil-based emulsion adjuvants, liposome-containing adjuvants, lipid-containing adjuvants, nucleic acid-containing adjuvants, peptide- or protein-containing adjuvants, plant extract-containing adjuvants, saponin-derived adjuvants, nucleic acid-containing adjuvants, bacteria- or virus-containing adjuvants, cytokine-containing adjuvants, and biocompatible polymer-containing adjuvants.

8. A pharmaceutical composition for preventing SARS-CoV-2 virus infection, comprising the SARS-CoV-2 RBD-HBsAg conjugate according to any one of claims 1 to 3, or a virus-like particle containing the SARS-CoV-2 RBD-HBsAg conjugate.

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