WO2022216028A1 - Vaccine composition for preventing severe acute respiratory syndrome coronavirus type 2 infectious disease, having improved neutralization potency - Google Patents

Abstract

The present invention relates to a vaccine composition for preventing severe acute respiratory syndrome coronavirus type 2 infectious disease. The present invention enables mass-production in animal cells by using an RBD domain mutant of a spike protein of severe acute respiratory syndrome coronavirus type 2, can effectively induce antigen production as an antigen, and can exhibit excellent effects in disease prevention as a vaccine.

Description

Neutralizing capacity improved vaccine composition for preventing type 2 severe acute respiratory syndrome coronavirus infection disease

The present invention relates to a receptor binding domain (RBD) mutant protein antigen of the type 2 severe acute respiratory syndrome coronavirus 2 spike region and uses thereof, and more specifically, the mutant protein antigen and a product comprising the same as an active ingredient It relates to a vaccine composition for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease.

Coronavirus disease-19 (COVID-19) is a respiratory infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a novel single-stranded RNA beta-coronavirus newly discovered in Wuhan, China on December 23, 2019, and was named SARS-CoV-2 by the International Viral Classification Committee in January 2020. The route of SARS-CoV-2 infection has not yet been clearly identified, but according to some studies, it is reported that it is transmitted from bats to humans.

The genome of SARS-CoV-2 is non-segmented and about 30 kb in size encodes 10 proteins. The ten proteins consist of one replicating polyprotein (open reading frames 1ab), four structural proteins (Spike, envelope, membrane and nucleocapsid), and five non-structural proteins ( open reading frames 3a, 6, 7a, 8 and 10). Consists of.

SARS-CoV-2 is contagious from person to person and has a fairly high mortality rate, making it a big problem both at home and abroad. As of March 16, 2020, there were 167,511 cases, of which 13,908 deaths were reported to the WHO, and the number of deaths is rapidly increasing even now.

In Korea, the first SARS-CoV-2 infection was confirmed in a 36-year-old woman who arrived from China in January 2019, and by March 17, 2020, there were 8,320 SARS-CoV-2 infections. was confirmed, and 286,716 people were quarantined.

After its first discovery in China in 2019, SARS-CoV-2, which had a high transmission rate as infections occurred in more than 160 countries such as Korea, Japan, and Italy in 2020, There is a problem accompanied by complications such as acute renal failure, and it is causing a big problem due to the high mortality rate and contagiousness.

On the other hand, the genome of SARS-CoV-2 is non-segmented and has a size of about 30 kb and encodes 10 proteins. The ten proteins consist of one replicating polyprotein (open reading frames 1ab), four structural proteins (spike, envelope, membrane and nucleocapsid), and five non-structural proteins ( open reading frames 3a, 6, 7a, 8 and 10). Consists of.

Among them, the receptor binding domain (RBD) of the spike protein binds to the receptor of the host cell, angiotensin-converting enzyme 2 (ACE2), and mediates the entry into the cell. It has been known as an epitope that can be produced. In the case of SARS-CoV-2, many groups are developing vaccine candidates using the receptor binding domain, and when the full-length protein of the spike protein and the receptor binding domain are used as vaccine antigens, the formation of neutralizing antibodies and vaccine efficacy are very different. looks like

The strategy of avoiding immune response of viruses is known through HIV, influenza virus, etc., and occurs through regulation of glycosylation of glycoproteins in the viral envelope. As an amino acid motif of a viral glycoprotein, N-linked glycosylation at the asparagine position of Asn-X-Ser/Thr is produced after transcription into a protein, and may be changed into various forms in the cell. In addition, when a virus infects a host cell, the virus's glycosylation system is used to control the glycan of the viral glycoprotein, thereby affecting the stability, antigenicity, and infectivity of the virus. will give

According to previous studies, glycosylation was artificially introduced (T579N) to the antibody-binding epitope in the receptor-binding domain vaccine antigen of the MERS virus, thereby becoming a glycosylation shield and inducing more neutralizing antibodies to the neutralizing epitope. Neutralizing ability was increased 3-4 times. (Nat. Comm 2016)

As such, it is very urgently necessary for the development of an effective vaccine to introduce a glycan shild to the antibody epitope to produce a vaccine with a protein antigen of other coronavirus as well as the existing MERS virus. do.

The present researchers tried to develop a vaccine with improved neutralization ability based on the Receptor binding domain (RBD) recombinant protein in the spike region of the virus as a type 2 severe acute respiratory syndrome coronavirus vaccine.

As a result, the present inventors completed the present invention by preparing a neutralizing ability improved vaccine composition for preventing SARS-CoV-2 infection by using a variant of the receptor binding domain protein of the SARS-CoV-2 spike as an antigen protein.

Therefore, it is an object of the present invention, type 2 severe acute respiratory syndrome comprising an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein To provide a receptor binding domain mutant protein of the respiratory syndrome coronavirus spike protein or a fragment thereof.

Another object of the present invention is to provide a gene encoding the mutant protein or a fragment thereof, a recombinant vector containing the gene, and a host cell transformed with the recombinant vector.

Another object of the present invention is to provide a vaccine composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection, comprising the mutant protein or a fragment thereof.

Another object of the present invention is to provide a vaccine composition for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease, comprising the mutant protein or a fragment thereof.

Another object of the present invention is to provide a method for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection disease, comprising administering a vaccine composition comprising the mutant protein or fragment thereof to a subject in need thereof is to do

Another object of the present invention is to provide a method for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection, comprising administering to a subject a vaccine composition comprising the mutant protein or a fragment thereof.

Another object of the present invention is to provide a use of the mutant protein or a fragment thereof in the manufacture of a medicament for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease.

Another object of the present invention is to provide a use of the mutant protein or a fragment thereof in the manufacture of a medicament for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection.

Another object of the present invention is to provide a use for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease of a vaccine composition comprising the mutant protein or a fragment thereof.

Another object of the present invention is to provide a use for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection of a vaccine composition comprising the mutant protein or a fragment thereof.

In order to achieve the above object, the present invention provides a second comprising an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein. Provided is a receptor binding domain mutant protein of type severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.

Spike's receptor binding domain protein (receptor binding domain, RBD) binds to ACE2 (angiotensin-converting enzyme 2), a receptor of a host cell, mediates entry into the cell, and can be used as a main target for neutralizing antibodies of the virus. That is, it can be used as an epitope capable of producing a neutralizing antibody.

The nomenclature of residues according to the present invention is based on reference to the full-length sequence of the type 2 severe acute respiratory syndrome coronavirus spike protein.

The type 2 severe acute respiratory syndrome coronavirus spike protein and/or the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein according to the present invention may refer to the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 refers to the full-length sequence of the type 2 severe acute respiratory syndrome coronavirus spike protein or its receptor binding domain protein.

The type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is an RBD protein having a stable secondary structure through structural analysis, and may be a fragment of an RBD protein. For example, a fragment according to the present invention may have approximately 200 to 300 contiguous amino acid sequence of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein.

For example, it may have a contiguous amino acid sequence of 300 or less. For example, it may include the 323 to 532th amino acid sequence of the amino acid sequence of SEQ ID NO: 1.

In addition, having an amino acid sequence selected between an amino acid position selected from the group consisting of 280 to 323 of the amino acid sequence of SEQ ID NO: 1 and an amino acid position selected from the group consisting of 532 to 570 of the amino acid sequence of SEQ ID NO: 1 it could be Any integer between these numbers is also recited herein. For example, the selection of 281 and 533 in the above selection means having the sequence of amino acids 281 to 533 of SEQ ID NO: 1.

280 to 570, more preferably 290 to 560, even more preferably 300 to 550, and even more preferably 310 to 540 amino acids of the amino acid sequence of SEQ ID NO: 1. According to a specific embodiment of the present invention, the fragment of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein has the amino acid sequence at positions 323 to 532, and this sequence is shown in SEQ ID NO: 2.

as well as polypeptides whose amino acids are substituted by conservative substitutions, having 80 to 99%, 85 to 99%, preferably 90 to 99% sequence homology thereto, for example 91%, 92%, It may include all polypeptides having 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence homology. The same applies also to the corresponding polynucleotide.

“Percent (%) sequence homology” means, after aligning sequences and introducing gaps, amino acids in a reference polypeptide, without taking into account any conservative substitutions as part of sequence identity, to achieve maximum percent sequence identity if necessary. It is defined as the percentage of amino acid residues in the candidate sequence that are identical to the residues. Alignment for purposes of determining percent amino acid homology can be accomplished by a variety of methods that are within the skill of the art, eg, using publicly available computer software programs, eg, in Current Protocols in Molecular Biology (Ausubel). et al., eds., 1987), and using the BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximal alignment over the entire length of the sequences being compared.

That is, the fragment of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein according to the present invention may include an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520, This may include additional conservative substitutions.

As used herein, the term "conservative substitution" means that an amino acid of one class is replaced with another amino acid of the same class. Conservative substitutions do not alter the structure, function, or both of the polypeptide. Classes of amino acids for purposes of conservative substitutions include hydrophobic (eg, Met, Ala, Val, Leu), neutral hydrophilic (eg, Cys, Ser, Thr), acidic (eg, Asp, Glu). , basic (eg Asn, Gin, His, Lys, Arg), conformational disrupters (eg Gly, Pro) and aromatic (eg Trp, Tyr, Phe) .

Accordingly, in the present invention, a sequence having 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence of SEQ ID NO: 1 is combined with type 2 severe acute respiratory syndrome coronavirus spike receptor The domain protein may include an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 mentioned above.

In addition, a sequence comprising the 323 to 532th amino acid sequence of the amino acid sequence of SEQ ID NO: 1 and having 95%, 96%, 97%, 98%, 99% or more homology to the sequence is treated with type 2 severe acute Respiratory syndrome coronavirus spike receptor binding domain protein may include an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 mentioned above.

In addition, as the 280 to 570, more preferably 290 to 560, even more preferably 300 to 550, even more preferably 310 to 540 amino acids of the amino acid sequence of SEQ ID NO: 1, 95%, 96%, 97 of the sequence Any one selected from the group consisting of residues 372, 518 and 520 mentioned above with a sequence having %, 98%, 99% or more homology as a type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein It may include amino acid substitutions at more than one residue.

In addition, a sequence having 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence of SEQ ID NO: 2 as a type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein It may include amino acid substitution at any one or more residues selected from the group consisting of the above-mentioned 372, 518 and 520th residues.

According to a specific embodiment of the present invention, the 372th amino acid of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is A. Preferably, the mutation at amino acid 372 is T (ie, A372T).

According to a specific embodiment of the present invention, the 518th amino acid of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is L. Preferably, the mutation at the 518th amino acid is N (ie, L518N).

According to a specific embodiment of the present invention, the 520th amino acid of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is A. Preferably, the mutation at the 520th amino acid is T (ie, A520T).

That is, according to the present invention, type 2 severe acute respiratory syndrome comprising an amino acid substitution at any one or more residues selected from the group consisting of A372T, L518N and A520T in the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein A receptor binding domain variant protein of a coronavirus spike protein or fragment thereof is provided.

That is, the mutation may be A372T, L518N, A520T, A372T/L518N, A372T/A520T, L518N/A520T, and A372T/L518N/A520T.

More specifically, the mutation/substitution is preferably included in a fragment of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein, more specifically, the fragment is 280 to 570 of the amino acid sequence of SEQ ID NO: 1, more preferably It may be 290 to 560, more preferably 300 to 550, even more preferably 310 to 540 amino acids, and more specifically, the amino acid sequence of SEQ ID NO: 2.

The selected amino acid sequence has a difference in that not only the structural stability is very excellent compared to other regions of the RBD, but also the effect as an antigen is remarkably excellent compared to other regions.

A preferred mutant protein fragment according to the present invention may be any one or more selected from the group consisting of SEQ ID NOs: 3 to 9.

Specifically, SEQ ID NOs: 3 to 9 show the following mutations:

SEQ ID NO: 3 (A372T), SEQ ID NO: 4 (L518N), SEQ ID NO: 5 (A520T), SEQ ID NO: 6 (A372T/L518N), SEQ ID NO: 7 (A372T/A520T), SEQ ID NO: 8 (L518N/A520T), or the sequence No. 9 (A372T/L518N/A520T)

The mutant protein or fragment thereof according to the present invention is very easy to produce because it has a smaller size than the full-length sequence, and by including the mutant sequence, the ability to generate neutralizing antibodies is significantly superior to that of the wild type.

That is, in general, protein antigens have a problem of low immunogenicity, but the mutant protein or fragment thereof according to the present invention is excellent in both stability and immunogenicity, and can be usefully used as a vaccine because of its high expression level in host cells. . In particular, by artificially introducing and controlling one or more (eg, one or two) glycosylation, the ability to generate neutralizing antibodies is greatly enhanced.

If necessary, the mutant protein or fragment thereof according to the present invention may be fused with an Fc fragment (Fc antibody fragment). The Fc fragment includes a CH2 domain and/or a CH3 domain derived from an antibody Fc fragment, and refers to a portion capable of binding to an antigen-presenting cell (APC). The Fc fragment may be one selected from the group consisting of IgG, IgA, IgD, IgE and IgM, and preferably may be an Fc fragment derived from IgG. The IgG may be further divided into IgG1, IgG2, IgG3 and IgG4, and the Fc fragment of the present invention may most preferably be an Fc fragment derived from IgG1. An 'Fc fragment' is a fragment obtained when an immunoglobulin (Ig) molecule is digested with papain. The variable region (VL) and constant region (CL) of the light chain, and the variable region (VH) and heavy chain constant region 1 (CH1) of the heavy chain This is the removed area. That is, the Fc fragment refers to a dimer of two CH2-CH3 domain chains, and the two chains form a dimer structure by a disulfide bond. In addition, the Fc fragment may include all or part of a hinge region peptide in the heavy chain constant region. In addition, it may be an extended Fc fragment including a part or all of the heavy chain constant region 1 (CH1) and/or the light chain constant region 1 (CL1) as long as it has substantially the same or improved effect as the native type. It may also be a fragment in which some fairly long amino acid sequences corresponding to CH2 and/or CH3 have been removed.

In another aspect, the present invention relates to a gene encoding the mutant protein or fragment thereof, a recombinant vector containing the same, and a host cell transformed with the recombinant vector.

In a specific embodiment of the present invention, the gene may be characterized in that it is represented by any one of nucleotide sequences of SEQ ID NOs: 12 to 18 (SEQ ID NO: 12 (A372T), SEQ ID NO: 13 (L518N), SEQ ID NO: 14 (A520T), SEQ ID NO: 15 (A372T/L518N), SEQ ID NO: 16 (A372T/A520T), SEQ ID NO: 17 (L518N/A520T), or SEQ ID NO: 18 (A372T/L518N/A520T)).

As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. A vector can be a plasmid, a phage particle or simply a potential genomic insert. Upon transformation into an appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since a plasmid is currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) an origin of replication that allows efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure including an antibiotic resistance gene that allows selection and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

In the present invention, the term "recombinant vector" generally refers to a double-stranded DNA fragment as a recombinant carrier into which a heterologous DNA fragment is inserted. Here, heterologous DNA refers to heterologous DNA, which is DNA not found naturally in a host cell. A recombinant vector, once in the host cell, can replicate independently of the host chromosomal DNA and several copies of the vector and its inserted (heterologous) DNA can be produced.

In one embodiment of the present invention, the modified pcDNA3.4 vector prepared by modifying the pcDNA3.4 vector is cut using BamHI and XhoI restriction enzymes, and then the nucleotide sequence of the fragment of the mutant protein according to the present invention is amplified by PCR. By inserting one fragment, an expression vector loaded with a polynucleotide encoding a mutant protein fragment of the receptor expression domain of the type 2 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein was prepared.

More specifically, the nucleotide sequence prepared by attaching a restriction enzyme position and a signal sequence to the nucleotide sequence of any one of SEQ ID NOs: 12 to 18 may be loaded into an expression vector.

Exemplary nucleotide sequences and amino acid sequences of the signal sequence are shown in SEQ ID NOs: 19 and 20.

In addition, the fragment base and amino acid sequence of the mutant protein including the restriction enzyme site sequence and the signal sequence are shown in SEQ ID NOs: 21 to 26.

After ligation, the gene or the recombinant vector is transformed or transfected into a host cell. A variety of different techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transformation” or “transfection” such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection or lipofection may be used.

As is well known in the art, in order to increase the expression level of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

In the present invention, the term "transformation" refers to introducing DNA into a host so that the DNA can be replicated as an extrachromosomal factor or by chromosomal integration completion. Of course, it should be understood that not all vectors function equally well in expressing the gene sequences of the present invention. Likewise, not all hosts function equally against the same expression system. However, one of ordinary skill in the art can make an appropriate selection among various vectors, expression control sequences and hosts without departing from the scope of the present invention without undue experimental burden. For example, in selecting a vector, the host must be considered, since the vector must be replicated in it. The copy number of the vector, its ability to control the copy number and the expression of other proteins encoded by the vector must also be considered.

In the present invention, the transformed host cell is preferably selected from the group consisting of animal cells, plant cells, yeast, E. coli and insect cells, but is not limited thereto.

Specifically, in the present invention, if the microorganism used as the transformed host cell is non-toxic or attenuated when applied to a living body, Gram-negative bacteria E. coli, Salmonella typhi , Salmonella Typhimyrium ( Salmonella typhimurium ), Vibrio cholerae ( Vibrio cholerae ), Mycobacterium bovis ( Micobacterium bovis ), Shigella ( Shigella ), etc. can be used, and Gram positive bacteria Bacillus ( Bacillus ), lactose Bacillus ( Lactobacillus ), Lactococcus ( Lactococcus ), Staphylococcus ( Staphylococcus ), Listeria monocytogenes ( Listeria monocytogenes ), Streptococcus ( Streptococcus ) Any one can be used, and preferably, lactic acid bacteria that are edible microorganisms can be used, but is not limited thereto.

The lactic acid bacteria may include the genus Lactobacillus sp., the genus Streptococcus and the genus Bifido bacterium , and representatively, the genus Lactobacillus is Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus plantarum ( Lactobacillus plantarum ), Lactobacillus fermentum ( Lactobacillus fermentum ), Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), Lactobacillus johnsny ( Lactobacillus johnsonii ), Lactobacillus reuteri ( Lactobacillus johnsonii ), Lactobacillus reuteri ( Lactobacillus bulgaricus ) and Lactobacillus casei ( Lactobacillus casei ); The genus Streptococcus includes Streptococcus thermophiles ; The genera of Bifidobacterium include Bifido bacterium infantis, Bifido bacterium bifidum , Bifido bacterium longum , Bifido bacterium breve ), Bifido bacterium lactis ( Bifido bacterium lactis ) and Bifido bacterium adolescentis ( Bifido bacterium adolescentis ) and the like can be used.

Fungi such as Aspergillus sp., Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces sp. and Neurospora It may be a eukaryotic cell, such as yeast, such as Neurospora crassa , other lower eukaryotic cells, and cells of higher eukaryotes, such as cells from insects.

It may be of plant or mammalian origin. Preferably, monkey kidney cells 7 (COS7: monkey kidney cells) cells, NSO cells, SP2/0, Chinese hamster ovary (CHO: chinese hamster ovary) cells, W138, baby hamster kidney (BHK: baby hamster kidney) cells, MDCK, myeloma cell lines, HuT78 cells and HEK293 cells may be used, and more preferably, CHO cells may be used, but the present invention is not limited thereto.

In another aspect, the present invention relates to a method for producing an antigen of type 2 severe acute respiratory syndrome coronavirus by culturing the host cell.

When a recombinant vector capable of expressing the antigen of the type 2 severe acute respiratory syndrome coronavirus is introduced into a host cell, the antibody is produced for a period sufficient to allow the antigen to be expressed in the host cell, or more preferably, the host cell is cultured. It can be prepared by culturing the host cells for a period sufficient to allow the antigen to be secreted into the culture medium.

In some cases, the expressed antigen can be isolated from the host cell and purified to uniformity. Isolation or purification of the antigen may be performed by a separation or purification method used in conventional proteins, for example, chromatography. The chromatography may include, for example, affinity chromatography including a protein A column, a protein G column, ion exchange chromatography, or hydrophobic chromatography. In addition to the above chromatography, the antibody can be separated and purified by further combining filtration, ultrafiltration, salting out, dialysis, and the like.

In another aspect, the present invention is a type 2 comprising an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein It relates to a vaccine composition for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease, comprising a receptor binding domain mutant protein of the severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.

More specifically, the type 2 severe acute respiratory syndrome coronavirus is a type 2 severe acute respiratory syndrome coronavirus and its mutants (eg, alpha (B.1.1.7), beta (B1.351), gamma ( P1), delta (B.1.617.2), omicron (B.1.1.529)).

More specifically, wild-type type 2 severe acute respiratory syndrome coronavirus. Alpha (B.1.1.7) type 2 severe acute respiratory syndrome coronavirus, beta (B1.351) type 2 severe acute respiratory syndrome coronavirus, gamma (P1) type 2 severe acute respiratory syndrome coronavirus, delta ( B.1.617.2) Severe acute respiratory syndrome coronavirus type 2 and/or Omicron (B.1.1.529) type 2 severe acute respiratory syndrome coronavirus.

The vaccine composition according to the present invention focuses the immune response on a neutralizing epitope, so that it is excellent for variants with changes in viral properties such as transmission power, pathogenicity, and immune evasion by amino acid substitution of the binding site (Receptor binding domian, RBD). indicates vaccine efficacy.

The properties of each variant mentioned above are as well known in the art, and the properties of these variants are fully understandable by those skilled in the art. Some of the contents can be sufficiently applied.

In another aspect, the present invention is a type 2 comprising an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein Severe Acute Respiratory Syndrome Type 2 Severe Acute Respiratory Syndrome Coronavirus Infectious Disease, comprising administering to a subject in need thereof a vaccine composition comprising a receptor binding domain mutant protein or fragment thereof It's about treatment.

In another aspect, the present invention relates to the use of a vaccine composition comprising the mutant protein or a fragment thereof for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease.

The vaccine composition of the present invention induces a high titer of neutralizing antibodies. The vaccine composition of the present invention induces a higher titer of neutralizing antibodies compared to other SARS-CoV-2 derived proteins. Accordingly, the vaccine composition of the present invention exhibits an effective neutralizing antibody response. In addition, by selecting and using a structurally stable protein as an antigen protein, the effect of the vaccine can be stably exhibited. That is, it has the advantage of inducing a strong host antibody response or protective immunity.

The disease caused by the type 2 severe acute respiratory syndrome coronavirus infection may be a respiratory disease. The type 2 severe acute respiratory syndrome coronavirus-infected disease may show symptoms after, for example, an incubation period of 2 to 14 days after viral infection. These symptoms include, for example, high fever, cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (kidney failure, renal failure, etc.), septic shock, and in severe cases death. Any symptoms caused by

In the present invention, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject that has never been diagnosed as possessing a disease or disease, but is likely to be afflicted with such disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or symptom; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. The composition of the present invention suppresses the development of symptoms, eliminates them, or alleviates them by activating the immune response to the virus in an individual suffering from a disease caused by type 2 severe acute respiratory syndrome coronavirus infection. Accordingly, the composition of the present invention may be a therapeutic composition by itself, or may be administered together with other pharmacological ingredients to be applied as a therapeutic adjuvant for the disease.

Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

The term “immune response” as used herein refers to a cell-mediated (T-cell) immune response and/or an antibody (B-cell) response. Preferably, it is an antibody (B-cell) reaction following antigen treatment.

In the present invention, the vaccine composition may be characterized in that it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

That is, the vaccine composition of the present invention may include one or more adjuvants or excipients or carriers suitable for constructing the vaccine composition in addition to the antigen mutant protein or fragment thereof.

The adjuvant that may be included in the composition of the present invention refers to a substance that enhances the immune response of an injected animal, and a number of different adjuvants are known to those skilled in the art. The adjuvant includes Freund's complete and incomplete immune enhancers, vitamin E, nonionic blocking polymer, muramyl dipeptide, Quil A, mineral and non-mineral oils and Carbopol, water-in-oil type emulsion adjuvants, and the like, but is limited thereto. it's not going to be

Examples of excipients that may be included in the vaccine composition of the present invention include aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, and the like.

The carrier that may be included in the vaccine composition of the present invention may be a pharmaceutically acceptable carrier, and examples of the pharmaceutically acceptable carrier include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like.

The vaccine composition of the present invention may further comprise preservatives and other additives such as, for example, antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. The preservatives include formalin, thimerosal, neomycin, polymyxin B and amphotericin B, and the like. The vaccine composition of the present invention may comprise one or more suitable emulsifiers, such as Span or Tween. In addition, the vaccine composition of the present invention may include a protective agent, and any protective agent known in the art can be used without limitation, which may include lactose (Lactose; LPGG) or trehalose (Trehalose; TPGG), However, the present invention is not limited thereto.

In an embodiment of the present invention, the vaccine composition is preferably administered 1 to 3 times at intervals of 1 to 3 weeks, and more preferably inoculated twice at intervals of 2 weeks.

In an embodiment of the present invention, the vaccine composition may be formulated using methods known in the art to enable rapid, sustained or delayed release of the active ingredient upon administration to a mammal. Formulations include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

In an embodiment of the present invention, the vaccine composition may be administered by one or more routes selected from the group consisting of intramuscular, intravenous, subcutaneous, intradermal and intranasal. have.

In an embodiment of the present invention, the vaccine composition may be administered through a method of applying a physical stimulus to increase delivery efficiency during administration, examples of which include an electroporation method, a gene gun method, and a jet method. There is a jet injection method and the like.

The vaccine composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, carriers for parenteral administration such as water, suitable oils, saline, aqueous glucose and glycol, and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. In addition, the vaccine composition according to the present invention can be used as a suspending agent, solubilizer, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, excipient, pH adjuster, analgesic agent, buffer, if necessary depending on the administration method or formulation. , antioxidants and the like may be included as appropriate.

The dosage of the vaccine composition to a patient depends on many factors, including the patient's height, body surface area, age, the particular compound being administered, sex, time and route of administration, general health, and other drugs being administered concurrently.

In addition, the vaccine composition of the present invention is administered in a therapeutically effective amount.

In the present invention, a therapeutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level includes the subject type and severity, age, sex, drug activity, drug Sensitivity to, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The dosage of the vaccine composition of the present invention is in the range of 0.0001 to 1,000 mg/kg (body weight) per day, but may vary depending on the type of individual, and is not limited thereto.

Meanwhile, the dosage may be appropriately adjusted according to the age, sex and condition of the patient.

In another aspect, the present invention relates to a method for treating or preventing a disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of the vaccine composition.

The optimal dosage of a vaccine composition of the present invention can be ascertained by standard studies involving observation of an appropriate immune response in a subject. After the initial vaccination, the subject may undergo one or several booster immunizations at appropriate intervals.

Suitable dosages of the vaccine composition of the present invention vary depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity of the patient. and may be determined by a person skilled in the art in consideration of the above matters.

The present invention provides a vaccine composition for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection, comprising the mutant protein or a fragment thereof.

The present invention provides a vaccine composition for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease, comprising the mutant protein or a fragment thereof.

The present invention provides a vaccine composition comprising the mutant protein or a fragment thereof for use in the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection.

The present invention provides a vaccine composition comprising the mutant protein or a fragment thereof for use in the prevention or treatment of a type 2 severe acute respiratory syndrome coronavirus infection disease.

The present invention, the mutant protein or a fragment thereof; Or it provides a method for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection disease, comprising administering a vaccine composition comprising the mutant protein or fragment thereof to a subject in need thereof.

The present invention, the mutant protein or a fragment thereof; Or it provides a method for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection, comprising administering to a subject a vaccine composition comprising the mutant protein or fragment thereof.

The present invention provides the use of the mutant protein or a fragment thereof in the manufacture of a medicament for the prevention or treatment of a type 2 severe acute respiratory syndrome coronavirus infection disease.

The present invention provides the use of the mutant protein or a fragment thereof in the manufacture of a medicament for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection.

The present invention, the mutant protein or a fragment thereof; Or the use of a vaccine composition comprising the mutant protein or a fragment thereof for preventing or treating a type 2 severe acute respiratory syndrome coronavirus infection disease.

The present invention, the mutant protein or a fragment thereof; Or it provides a use for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection of a vaccine composition comprising the mutant protein or a fragment thereof.

Another object of the present invention is to inject a receptor binding domain mutant protein of the spike protein of type 2 severe acute respiratory syndrome coronavirus or a fragment thereof into animals other than humans; and

(b) to provide a method for producing a monoclonal antibody that specifically binds to type 2 severe acute respiratory syndrome coronavirus, comprising the step of recovering the antibody from the animal.

The introduction of the protein or fragment thereof can be carried out by intradermal, subcutaneous or intramuscular injection.

The term "antibody" is a component produced by antigen stimulation in the immune system, and is a protein that specifically binds to a specific antigen and circulates in the lymph and blood to cause an antigen-antibody reaction. Antigen-antibody reactions have high specificity for each antigen. This means that when antibodies are made by B cells of lymphocytes, antibodies produced by specific antigens do not react with other antigens in principle. This high specificity is used for tests such as immunity, allergy, and determination of the type and type of various diseases and infections.

In this case, the animal may be a mammal other than a human. Examples include, but are not limited to, rats, mice, hamsters, pigs, rabbits, horses, donkeys, goats, sheep, guinea pigs, llamas, and the like. For example, mice are preferably used to make monoclonal antibodies.

In one embodiment, neutralizing antibodies made in a non-human mammal can be converted to humanized antibodies. In this case, the method of the present invention may further comprise the step of humanizing the neutralizing antibody produced in the non-human mammal.

The neutralizing antibody of the present invention can be used to prevent or treat diseases caused by viral infection in mammals. The neutralizing antibody is used in an amount sufficient to neutralize the virus.

In addition, the present invention provides an antibody or fragment thereof that specifically binds to a receptor binding domain protein of SARS-CoV-2 spike protein or a fragment thereof.

The receptor binding domain mutant protein of SARS-CoV-2 spike protein or a fragment thereof according to the present invention has a stable protein structure and high neutralizing ability, and has a remarkably excellent effect as a vaccine through excellent protective ability against SARS-CoV-2 indicates In addition, by optimizing the optimized sequence, it can be efficiently produced at an excellent protein expression level with a reduced production cost.

In addition, by minimizing the immune-dominant non-neutralizing epitope, the antibody-defendant effect is minimized and the immune response is concentrated on the neutralizing epitope to show the optimal effect as a vaccine.

1 is a diagram illustrating an animal cell expression vector pcDNA3.4-SARS-CoV-RBD for the expression of the receptor binding domain protein (including variants) of the SARS-CoV-2 spike.

2 shows the results of confirming the expression of the receptor binding domain protein of the SARS-CoV-2 spike by SDS-PAGE and Western blot.

3 is a diagram showing the neutralizing antibody-forming effect of the SARS-CoV-2 receptor binding domain protein through an ELISA experiment using a mouse (wild type).

4 is a diagram showing the results of confirming the neutralizing antibody formation effect using wild-type and mutant proteins through an ELISA experiment.

5 shows the results of confirming the neutralizing antibody titer using the antiserum prepared in Example 2.

6 shows the results of confirming the neutralizing antibody titer against wild-type SARS-CoV-2 using wild-type and mutant proteins in hACE2 transgenic mice.

7 shows the results of confirming the virus neutralizing ability of wild-type and mutant proteins according to virus challenge inoculation in hACE2 transgenic mice.

8 shows the results of confirming that the neutralizing antibody produced through this vaccination competitively inhibits the binding of a commercially available monoclonal neutralizing antibody to RBD through Competition ELISA.

9 shows the results of confirming the neutralizing antibody ability against wild-type SAS-CoV-2 through a surrogate virus neutralization experiment.

10 shows the results of confirming the neutralizing antibody titer against beta-mutant SARS-CoV2 using wild-type and mutant proteins in hACE2 transgenic mice.

11 shows the results of confirming the neutralizing antibody titer against delta-mutated SARS-CoV-2 using wild-type and mutant proteins in hACE2 transgenic mice.

12 shows the results of confirming whether or not a cellular immune response is generated by the antigen component.

13 shows the results of confirming the survival rate of mice after wild-type SARS-CoV-2 challenge inoculation.

14 shows the results of confirming the survival rate of mice after inoculation with beta mutant SARS-CoV-2 challenge.

15 shows the results of confirming the level of virus in the lungs on days 2, 4 and 7 after wild-type SARS-CoV-2 challenge inoculation.

Figure 16 shows the results of confirming the level of virus in the lungs on the 2nd, 4th and 7th days after the beta mutant SARS-CoV-2 challenge inoculation.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of receptor binding domain protein variants of SARS-CoV-2 spike protein

To establish the sequence of the receptor binding protein (hereinafter, RBD) of the SARS-CoV-2 spike protein that can be used as an antigen for vaccine development, a protein structure analysis server (XtalPred-RF, RaptorX) based on structure prediction was used. was used to confirm the structure of the RBD protein.

The amino acid sequence of the RBD protein having a stable secondary structure was determined through the above structural analysis, and the following antigens were prepared based on this.

The RBD protein (SEQ ID NO: 2, 210 amino acids) of SARS-CoV-2 used in the experiment was prepared using the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleotide sequence (SEQ ID NO: 11).

Specifically, based on the RBD full-length protein (SEQ ID NO: 1, nucleotide sequence SEQ ID NO: 10), 323 to 532 amino acid sequences (SEQ ID NO: 2, total 210 amino acids, nucleotide sequence SEQ ID NO: 11) were used.

That is, the nucleotide sequence prepared by codon optimization of the fragment of the RBD protein of SARS-CoV-2 selected for the preparation of the antigen protein for the CHO cell line is shown in SEQ ID NO: 11 (WT).

A372T (SEQ ID NO: 3), L518N and A520T (SEQ ID NO: 8), and A372T, L518N, A520T (SEQ ID NO: 9) amino acids containing all these substitutions in the 323 to 532 amino acid sequence (SEQ ID NO: 2, total 210 amino acids) The nucleotide sequence for this polypeptide was codon-optimized for the CHO cell line and synthesized through Bioneer, and each codon-optimized sequence thereof is shown in SEQ ID NO: 12, 17 or 18, respectively.

A fragment of the SARS-CoV-2 RBD protein having the above-mentioned mutations/substitutions was prepared using a modified pcDNA3.4 vector. To this end, the sequence was designed by adding the above-mentioned sequence signal sequence and restriction enzyme site position. The corresponding amino acid sequences used in this Example are shown in SEQ ID NOs: 21, 23, and 25, and the base sequences are shown in SEQ ID NOs: 22, 24 and 26.

Specifically, a recombinant plasmid was prepared by subcloning the BamHI and XhoI sites of the modified pcDNA3.4 vector. As such, a schematic diagram of the prepared recombinant plasmid is shown in FIG. 1 .

The recombinant plasmid was transfected into a CHO cell line (Chinese hamster ovary) using the Expi CHO transfection kit. RBD protein was inoculated into 6.0 X 10 ⁶ cells/ml of CHO cell line cells and cultured for 12 days. The culture supernatant was first purified by nickel-affinity chromatography after removing the medium components using a semi-permeable membrane.

Thereafter, the protein was purified once more by HiPrep 16/60 Superdex 200 gel-filtration chromatography using 2X PBS buffer to obtain RBD protein. The results are shown in FIG. 2 .

Figure 2 shows the results of purification of the RBD vaccine through SDS-PAGE and Western blot. In FIG. 2, lane 1 indicates WT RBD, lane 2 indicates A372T, lane 3 indicates L518N-A520T, and lane 4 indicates DM (A372T-L518N-A520T). As can be seen in the figure, it was confirmed that the purification of the SARS-CoV-2 RBD protein was all done well.

The 220 amino acid sequences of the exemplary RBD protein variant thus prepared are shown in SEQ ID NO: 3, 8, or 9, and it was confirmed whether antibodies against SARS-CoV-2 were generated using the prepared RBD protein as an antigen.

Example 2. Confirmation of SARS-CoV-2 antibody production of SARS-CoV-2 RBD protein mutant

In order to determine whether the RBD protein mutant antigen prepared in Example 1 effectively induces antibody production, its antibody production effect was confirmed.

First, before injection of the purified RBD protein mutant antigen into mice, pre-immune serum was collected and used as a negative control.

For primary immunization, 10 μg of antigen was mixed with ISA 51 adjuvant and injected intramuscularly, and 2 weeks later, it was mixed with ISA 51 for boosting. After 2 weeks, the primary blood was collected and an ELISA test was performed, and 2 weeks after boosting, blood was collected from mice and the final serum was separated to perform an ELISA experiment. In the ELISA test, 100ng of antigen was used per well, and immune serum was 1:100 to 1:500 to 1:1,000 to 1:5,000 to 1:10,000 to 1:50,000 1:100,000 to 1:500,000 in 1X PBS. It was diluted, and for secondary confirmation, anti-mouse IgG-HRP (ABC5001) was diluted at a ratio of 1:5,000 and used. By detecting color development at an optical density of 450 nm, it was confirmed whether immune antibody was preferentially generated, which is shown in FIGS. 3 and 4 .

As shown in FIG. 3 , the wild-type RBD protein exhibited an excellent immune antibody-forming effect without any difference in all mice used in the experiment.

In addition, as can be seen in FIG. 4 , it was confirmed that both A372T and L518N-A520T can effectively induce antibodies in the subject.

In particular, based on the results of confirming that the RBD mutant protein of the present invention can effectively induce antibodies in an individual by acting as an antigen, the SARS-CoV-2 RBD protein can be used as a vaccine for SARS-CoV-2 prevention. It was also confirmed that

Example 3. Confirmation of SARS-CoV-2 Neutralizing Antibody Generation and Comparison of Neutralization Ability of SARS-CoV-2 RBD Protein Variants

SARS-CoV-2 (BetaCoV/ South Korea/ KUMC01/2020) virus was distributed and RBD protein mutant antisera generated in Example 2 was administered at a certain ratio (1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640, 1:1,280), each virus was mixed with 100 TCID50, treated with Vero E6 cells for 1 hour, then removed, and cytoplasmic after 72 hours in fresh culture medium The value of the dilution ratio just before the effect (Cytopathic effect, CPE) appeared was read and evaluated as neutralizing ability, and is shown in FIG. 5 .

As can be seen in FIG. 5 , when comparing the RBD mutant protein prepared in Example 2 with the wild type, the mutant antigens of the RBD protein of the present invention induce neutralizing antibodies 3 to 4 times more effectively than the wild type in the subject. confirmed that it can be done. From these results, it was confirmed that the SARS-CoV-2 RBD mutant protein according to the present invention can be used as an improved vaccine for SARS-CoV-2 prevention.

In addition, similar to the experimental method, neutralizing ability was evaluated using antisera from SARS-CoV-2 infected mice transfected with human ACE2, and the results are shown in FIG. 6 .

As shown in FIG. 6 , when using the triple mutations A372T, L518N, and A520T according to the present invention, excellent neutralizing antibody-forming ability was exhibited compared to using wild-type RBD.

Example 4. Confirmation of prophylactic effect following challenge inoculation of SARS-CoV-2 RBD protein mutant (using SARS-CoV-2 infected mice transfected with human ACE2)

Using human ACE2-transfected SARS-CoV-2 infected mice, the mutant antigen or wild-type antigen of the RBD protein of the present invention was first immunized with 10 ug/dose (50:50, ISA51) per animal, and after 2 weeks Secondary immunization was performed, and efficacy was evaluated through a virus challenge inoculation experiment two weeks later. The virus titer was measured 2 days and 7 days after challenge inoculation, and the results are shown in FIG. 7 .

As can be seen in FIG. 7 , when the triple mutations A372T, L518N, and A520T according to the present invention were treated, the virus proliferation was greatly reduced compared to that of the wild type. That is, treatment with the triple mutations A372T, L518N, and A520T according to the present invention significantly reduced virus proliferation on day 7 compared to WT RBD.

Based on these results, it was confirmed that the antigen composition according to the present invention exhibits an excellent preventive effect.

Example 5. Confirmation of binding inhibitory efficacy on RBD between a commercial neutralizing antibody and a neutralizing antibody formed by the vaccine

Through Competition ELISA, the ability of neutralizing antibodies generated through this vaccine to inhibit binding between commercial neutralizing antibodies and RBD was confirmed.

Specifically, RBD was coated with 100 ng/well of a general ELISA, and blocking with 2% BSA blocking buffer was performed at 37° C. for 1 hour, and the mouse serum obtained earlier was diluted with PBS (10, 20-fold dilution) on the plate. After treatment, the reaction was carried out at 37°C for 1 hour. After completion of the above reaction, the plate was washed twice with PBS-T buffer and then treated with 0.1 ug/ml of SARS-CoV-2 neutralizing rabbit mAb at 37°C for 1 hour. Next, after washing 3 times with PBS-T buffer, detection was performed with an HRP-conjugated anti-rabbit IgG antibody. Finally, the plate was washed 5 times with PBS-T, treated with TMB solution, and visualized at 37° C. for 6 minutes. Then, the reaction was terminated by treatment with stop solution, and absorbance was measured.

Absorbance for inhibition was calculated using the following equation.

Equation 1:

The results are shown in FIG. 8 .

As can be seen from FIG. 8 , when serum samples from mice treated with A372T, L518N-A520T and A372T-L518N-A520T (Double) were used, it was confirmed that they exhibit significantly superior inhibitory ability compared to that of WT.

Example 6. Confirmation of neutralization ability according to Surrogate virus neutralization experiment (including WT, beta and delta variants)

The neutralizing ability of the antibody according to the present invention was confirmed through a surrogate virus neutralization experiment.

Specifically, a Ni plate was coated with hACE2 at 250 ng/well and incubated at 4 °C for 20 hours.

Then, blocking was performed at 25° C. for 1 hour with 1% BSA blocking buffer. Next, mouse serum was diluted with PBS, mixed with 100 ng/50 ul SARS-CoV-2 RBD, and reacted at 25° C. for 1 hour. Next, after washing the plate once with PBS, 100 ul of a mixture of mouse serum and SARS-CoV-2 RBD was treated at 25° C. for 1 hour. Then, the plate was washed 3 times with PBS buffer, and detection was performed at 25° C. for one hour with HRP-conjugated streptavidin. Finally, the plate was washed 4 times with PBS-T, treated with TMB solution at 25°C for 10 minutes to visualize, and then the reaction was terminated by treatment with stop solution. Then the absorbance was measured.

Absorbance for inhibition was calculated using the following equation.

ceremony:

The results are shown in FIG. 9 .

As can be seen in FIG. 9 , it was confirmed that when a serum sample of a mouse treated with A372T-L518N-A520T (Double) according to the present invention was used, it exhibited significantly superior neutralizing efficacy compared to that of WT.

Example 7. Confirmation of neutralization ability according to Live virus neutralization experiment (beta and delta mutations)

An experiment was performed similarly to Example 3, and the neutralizing antibody production and neutralizing ability of the SARS-CoV-2 RBD protein mutants were confirmed and compared to the SARS-CoV-2 mutants.

Specifically, an experiment was performed on A372T-L518N-A520T (Double).

The results are shown in FIGS. 10 and 11 .

Figure 10 shows the results for the beta variant, Figure 11 shows the results for the delta variant.

As can be seen from each figure, when using the A372T, L518N, and A520T triple mutations (Double) according to the present invention, it exhibited excellent neutralizing antibody-forming ability compared to using the wild-type RBD.

Example 8. Cellular Immune Response Confirmation

In order to confirm the cellular immune response, a solid-phase enzyme-linked immunospot (ELISPOT) analysis was performed using a commercially available kit (BD bioscience). First, a 96-well polyvinylidene difluoride-supported plate was pre-coated with anti-mouse IFN-γ at 4°C overnight. Then, after discarding the coating antibody and washing once with blocking buffer. Plates were blocked at 37° C. for 2 hours. Next, the blocking buffer was discarded and antigen diluted in complete medium was added to the Elispot plate, and ConA (2 μg/mL) was used as a positive control. Thereafter, mouse splenocytes were distributed to each well at a predetermined density (1 x 10 ⁶ cell/well), and the plate was incubated at 37° C. for 72 hours. Next, cells were removed and plates were detected with Biotinylated Anti-mouse IFN-γ (1:250 dilution) and Biotinylated Anti-mouse IFN-γ detected by HRP-conjugated streptavidin. Pigmented spots representing epitope-specific IFN-producing T cells were visualized using an AEC substrate kit (BD bioscience) and staining was stopped by washing with distilled water. Pigmented spots were calculated using an automatic ELISPOT reader.

The results are shown in FIG. 12 .

As can be seen from FIG. 12 , it was confirmed that the receptor binding domain mutant protein of the spike protein according to the present invention does not cause a cellular immune response (T cell immune response), so that it has a high potential for use as a vaccine.

Example 9. Confirmation of prophylactic effect following challenge inoculation of SARS-CoV-2 RBD protein mutant (using SARS-CoV-2 infected mice transfected with human ACE2)

(1) Check the survival rate

Similar to Example 4, using SARS-CoV-2 infected mice transfected with human ACE2, the mutant antigen or wild-type antigen of the RBD protein of the present invention was first administered at 10 ug/dose (50:50, ISA51) per mouse. After immunization, a second immunization was performed two weeks later. Then, 3 weeks after vaccination, 10 ⁶ TCID ₅₀ , 30 μL/mouse of SARS-CoV-2 (WT virus or Beta mutant) was intranasally infected. Thereafter, the change in body weight and the survival rate were observed every day for 2 weeks, and if the body weight decreased by 20% or more, it was treated as death.

The survival rates confirmed for 2 weeks after infection with wild-type virus or beta mutants are shown in FIGS. 13 and 14, respectively.

13 shows the preventive effect on WT virus infection. Specifically, according to the results of FIG. 13 , compared to the WT antigen, all of the mutant antigens exhibited excellent mouse survival rates.

In addition, as can be seen in FIG. 14 , all of the mutant antigens exhibited excellent mouse survival rates as compared to the WT antigen for the beta mutant.

Through the above results, it was confirmed that the mutant antigen according to the present invention exhibits an excellent effect as a vaccine against SARS-CoV-2.

(2) Confirmation of lung virus titer

To quantify the viral titer of the lungs after SARS-CoV-2 infection of vaccinated mice, 10 ⁶ TCID50 lungs were obtained 2, 4, and 7 days after SARS-CoV-2 infection, homogenized, and total RNA was extracted. The extracted total RNA was analyzed using the known Taqman probe real-time RT-PCR method using specific primers targeting the RNA-dependent RNA polymerase protein (RdRp) gene of the SARS-CoV-2 genome. was measured.

The above results are shown in FIGS. 15 and 16 .

As can be seen through FIGS. 15 and 16, in the experimental group treated with A372T-L518N-A520T (Double), the number of SARS-CoV-2 in the lung tissue for both wild-type SARS-CoV-2 as well as beta variants was greatly reduced.

That is, it was confirmed that the mutant antigen according to the present invention exhibits a very excellent effect in terms of virus reduction as well as survival rate.

Above, a specific part of the present invention has been described in detail, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

Claims (23)

1. A type 2 severe acute respiratory syndrome coronavirus spike protein comprising an amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520 of the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein. of a receptor binding domain mutant protein or fragment thereof.
2. The method of claim 1, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein has 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence of SEQ ID NO: 1. The sequence, a receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.
3. The method of claim 1, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein comprises the amino acid sequence of positions 323 to 532 of the amino acid sequence of SEQ ID NO: 1, 95%, 96%, 97% of the amino acid sequence. , a sequence having 98%, 99% or more homology, a receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.
4. The method of claim 1, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein has 95%, 96%, 97%, 98%, 99% or more homology to the amino acid sequence of SEQ ID NO: 2 The sequence, a receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.
5. The method of claim 1, wherein the amino acid substitution at any one or more residues selected from the group consisting of residues 372, 518 and 520, comprises any one or more amino acid substitutions selected from the group consisting of A372T, L518N and A520T, A receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof.
6. The method of claim 5, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is a sequence comprising the amino acid sequence 323 to 532 of the amino acid sequence of SEQ ID NO: 1, the type 2 severe acute respiratory syndrome coronavirus spike A receptor binding domain mutant protein of a protein or a fragment thereof.
7. The method of claim 6, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein is an amino acid position selected from the group consisting of 280 to 323 of the amino acid sequence of SEQ ID NO: 1 and 532 to 532 of the amino acid sequence of SEQ ID NO: 1 A receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein, or a fragment thereof, having an amino acid sequence selected between amino acid positions selected from the group consisting of 570.
8. According to claim 5, wherein the amino acid substitution is any one selected from the group consisting of A372T, L518N, A520T, A372T / L518N, A372T / A520T, L518N / A520T and A372T / L518N / A520T, type 2 severe acute respiratory syndrome corona A receptor binding domain mutant protein of a viral spike protein or a fragment thereof.
9. The receptor binding domain mutation of the type 2 severe acute respiratory syndrome coronavirus spike protein according to claim 6, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein has a sequence of 300 or less consecutive amino acids. protein or fragment thereof.
10. The method according to claim 5, wherein the type 2 severe acute respiratory syndrome coronavirus spike receptor binding domain protein consists of the amino acid sequence of SEQ ID NO: 2, the receptor binding domain mutant protein of the type 2 severe acute respiratory syndrome coronavirus spike protein or fragment of it.
11. According to claim 1, wherein the receptor binding domain mutant protein of the type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof is any one selected from the group consisting of SEQ ID NOs: 3 to 9, type 2 severe acute respiratory syndrome A receptor binding domain variant protein of a coronavirus spike protein or a fragment thereof.
12. A gene encoding a receptor binding domain mutant protein of the type 2 severe acute respiratory syndrome coronavirus spike protein according to any one of claims 1 to 11 or a fragment thereof.
13. The gene according to claim 12, wherein the gene is any one selected from the group consisting of SEQ ID NOs: 12 to 18.
14. A recombinant vector containing the gene of claim 12 .
15. A host cell transformed with the recombinant vector of claim 14 .
16. For preventing or treating type 2 severe acute respiratory syndrome coronavirus infection comprising a receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof according to any one of claims 1 to 11 vaccine composition.
17. 17. The vaccine composition of claim 16, wherein the vaccine composition further comprises an adjuvant, excipient or carrier.
18. 18. The method of claim 17, wherein the adjuvant is Freund's complete and incomplete adjuvant; vitamin E; nonionic blocking polymers; muramyl dipeptide; Quil A; mineral and non-mineral oils; And Carbopol (Carbopol) any one or more selected from the group consisting of, a vaccine composition.
19. 17. The method of claim 16, wherein the type 2 severe acute respiratory syndrome coronavirus is a wild-type type 2 severe acute respiratory syndrome coronavirus. Alpha (B.1.1.7) type 2 severe acute respiratory syndrome coronavirus, beta (B1.351) type 2 severe acute respiratory syndrome coronavirus, gamma (P1) type 2 severe acute respiratory syndrome coronavirus, delta ( B.1.617.2) At least one selected from the group consisting of type 2 severe acute respiratory syndrome coronavirus and Omicron (B.1.1.529) type 2 severe acute respiratory syndrome coronavirus, a vaccine composition.
20. A receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein according to any one of claims 1 to 11 for use in the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection or a fragment thereof A vaccine composition comprising a.
21. A type 2 severe acute respiratory syndrome comprising administering to a subject a receptor binding domain mutant protein of type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof according to any one of claims 1 to 11 How to prevent or treat coronavirus infection.
22. The receptor binding domain mutant protein of the type 2 severe acute respiratory syndrome coronavirus spike protein according to any one of claims 1 to 11 in the manufacture of a medicament for the prevention or treatment of type 2 severe acute respiratory syndrome coronavirus infection or the use of fragments thereof.
23. Use of the receptor binding domain mutant protein of the type 2 severe acute respiratory syndrome coronavirus spike protein or a fragment thereof according to any one of claims 1 to 11 for preventing or treating type 2 severe acute respiratory syndrome coronavirus infection.

Images