Chicken antibody against corona virus variant

Abstract

The purpose of the present invention is to provide a new molecule neutralizing bonding between ACE2 and the RBD of the spike protein of a corona virus variant. The present invention provides a chicken antibody binding to the spike protein of a corona virus variant.

Description

Chicken antibodies against mutant coronaviruses

[0001] The present invention relates to chicken antibodies against mutant coronaviruses and uses thereof.

[0002] The novel coronavirus disease (COVID-19) that broke out in Wuhan, China in December 2019, rapidly spread worldwide, causing numerous deaths and severe damage to the global economy. SARS-CoV-2, the virus that causes COVID-19, is classified as a betacoronavirus and has a single-stranded RNA genome. The surface of the virus particle contains a protruding protein called the spike protein. This spike protein interacts with angiotensin-converting enzyme 2 (ACE2) expressed on the surface of human cells, establishing direct contact between the virus particle and the human cell (Non-Patent Document 1). COVID-19 can cause severe respiratory distress, and the global death toll exceeded 1.9 million by the end of 2020. However, no effective treatment has yet been found to be a panacea. Furthermore, as the global pandemic continues, mutations in the SARS-CoV-2 spike protein and altered amino acid sequences have been reported, and these mutations are expected to continue to increase.

[0003] Wrapp et al., Science 367, 1260-1263 (2020)

[0004] In this situation, it is desirable to develop new substances that neutralize the binding of the spike protein of mutant coronaviruses to angiotensin-converting enzyme 2 (ACE2).

[0005] As a result of intensive research to solve the above problems, the present inventors have succeeded in developing a novel chicken antibody that binds to the spike protein of a mutant coronavirus and can neutralize the binding of the receptor binding domain (RBD) of the spike protein of a mutant coronavirus to ACE2, thereby completing the present invention.

[0006] [1] A chicken antibody that binds to the spike protein of a mutant coronavirus. [2] The chicken antibody of [1] above, which is capable of neutralizing the binding of the receptor-binding domain (RBD) of the spike protein of a mutant coronavirus to angiotensin-converting enzyme 2 (ACE2). [3] The chicken antibody of [1] or [2] above, wherein the mutant coronavirus infects cells via ACE2. [4] The chicken antibody of any of [1] to [3] above, which further binds to the spike protein of a wild-type coronavirus. [5] The chicken antibody of any of [1] to [4] above, which is a polyclonal antibody. [6] A composition for treating or preventing coronavirus infection, comprising the chicken antibody of any of [1] to [5] above. [7] The composition of [6] above, wherein the coronavirus infection is a disease or symptom caused by infection with a coronavirus that infects cells via ACE2. [8] The composition according to [7] above, wherein the disease or symptom is at least one selected from the group consisting of respiratory disease, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms. [9] An inhibitor of the binding between the RBD of the spike protein of a mutant coronavirus and ACE2, comprising the chicken antibody according to any of [1] to [5] above.

[10] A reagent or kit comprising the chicken antibody according to any of [1] to [5] above.

[11] A method for treating or preventing coronavirus infection, comprising the step of administering to a subject a therapeutically effective amount of the chicken antibody according to any one of [1] to [5] above or a composition comprising the same.

[12] The chicken antibody according to any of [1] to [5] above or a composition comprising the same, for use in the treatment or prevention of coronavirus infection.

[13] Use of the chicken antibody according to any of [1] to [5] above in the manufacture of a medicament for treating or preventing coronavirus infection.

[0007] The present invention makes it possible to neutralize the binding of the RBD of the spike protein of a mutant coronavirus to ACE2.

[0008] 1 shows the results of evaluating the reactivity of antibodies of the present invention against the receptor binding domain (RBD) of spike protein of various mutant types of SARS-CoV-2. FIG. 2 shows the results of a test to inhibit the binding of various mutant types of RBD to ACE2 using antibodies of the present invention. FIG. 3 shows the results of a test to inhibit the binding of various mutant types of RBD to ACE2 using antibodies of the present invention. FIG. 4 shows the results of a test to inhibit the binding of various mutant types of RBD to ACE2 using antibodies of the present invention. FIG. 5 shows the results of a neutralization test of antibodies of the present invention against pseudoviruses. FIG. 6 shows the results of a neutralization test of antibodies of the present invention against pseudoviruses. FIG. 7 shows the results of a neutralization test of antibodies of the present invention against live viruses of various mutant types of SARS-CoV-2. FIG. 8 shows the results of a neutralization test of antibodies of the present invention against live viruses of various mutant types of SARS-CoV-2. FIG. 9 shows the results of a comparative test of chicken antibodies and chicken plasma with plasma derived from other animals.

[0009] The present invention will be described in detail below. The following embodiments are examples for explaining the present invention and are not intended to limit the present invention to these embodiments. The present invention can be implemented in various forms without departing from the gist of the invention. This specification also includes the contents described in the specifications and drawings of the Japanese patent application (Patent Application No. 2021-125338) filed on July 30, 2021, the Japanese patent application (Patent Application No. 2021-200190) filed on December 9, 2021, and the Japanese patent application (Patent Application No. 2022-024653) filed on February 21, 2022, which are the basis for the priority claim of this application.

[0010] 1. Overview Although the global death toll from COVID-19 exceeded 1.9 million by the end of 2020, no therapeutic drug has yet demonstrated a promising therapeutic effect. While compounds such as favipiravir and remdesivir are currently being considered as potential treatments for COVID-19, these drugs were originally developed to treat infectious diseases such as influenza and Ebola hemorrhagic fever, and it is unclear whether they are optimal for COVID-19. Antibody drugs such as tocilizumab and ravulizumab are also considered potential treatments. However, they do not directly recognize the virus; rather, they target IL-6R and complement (C5), which are biological factors involved in the immune system, and therefore act indirectly. Furthermore, as the global pandemic continues, mutations in the SARS-CoV-2 spike protein have been reported, resulting in altered amino acid sequences. This trend is expected to continue. In this context, the development of new drugs that can be used to treat or prevent infections caused by these mutant coronaviruses is urgently needed. As a result of extensive research into antibodies that bind to the spike protein of mutant coronaviruses, the inventors have succeeded in developing a novel chicken antibody that can neutralize the binding of the RBD of the spike protein of mutant coronaviruses to ACE2.

[0011] 2. Receptor Binding Domain (RBD) of Coronavirus Spike Protein The antibodies of the present invention can bind to the spike protein of a mutant coronavirus, for example, its receptor binding domain (RBD), and neutralize the binding of the RBD of the spike protein of the mutant coronavirus to angiotensin-converting enzyme 2 (ACE2). The RBD is present in the coronavirus spike protein (hereinafter also referred to as the "S protein") and is a domain that binds to ACE2, which is present in the cell membrane of human cells. Specifically, the coronaviruses SARS-CoV-1 and SARS-CoV-2 can infect human cells by binding the RBD of their S protein to ACE2.

[0012] ACE2 is mainly expressed in cells of the lungs, digestive system, heart, blood vessels, eyes, kidneys, cerebral cortex, amygdala, brainstem, medulla oblongata, etc. Therefore, by neutralizing the binding of the RBD of the coronavirus S protein to ACE2 expressed in these organs, the present invention can inhibit coronavirus infection of cells in these organs, contributing to the treatment or prevention of various diseases and symptoms associated with coronavirus infection, such as respiratory diseases.

[0013] As an example of the nucleotide sequence and amino acid sequence of the coronavirus S protein in the present invention, the nucleotide sequence and amino acid sequence of the wild-type SARS-CoV-2 S protein are shown as SEQ ID NOs: 1 and 2, respectively. The nucleotide sequence of the wild-type SARS-CoV-2 genome and the amino acid sequence of the S protein (SEQ ID NO: 2) are registered in the NCBI (National Center for Biotechnology Information) database (https: / / www.ncbi.nlm.nih.gov / ) under the following accession numbers: SARS-CoV-2 genome nucleotide sequence: NC_045512 SARS-CoV-2 S protein amino acid sequence: YP_009724390 In other words, the wild-type SARS-CoV-2 S protein is a protein containing the amino acid sequence shown in SEQ ID NO: 2. In addition, the nucleotide sequence encoding the wild-type SARS-CoV-2 S protein is disclosed on the webpage showing the nucleotide sequence of the above-mentioned SARS-CoV-2 genome in the NCBI database (NCBI accession number: NC_045512).

[0014] As mentioned above, coronavirus S proteins can mutate. However, the antibodies of the present invention can bind not only to wild-type coronaviruses but also to mutant coronavirus spike proteins with various mutations, and can neutralize the binding of the RBD of mutant coronavirus spike proteins to angiotensin-converting enzyme 2 (ACE2). The mutant coronavirus S proteins of the present invention include the following proteins (a) and (b): (a) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 2, and having ACE2-binding activity; and (b) a protein comprising an amino acid sequence with 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and having ACE2-binding activity.

[0015] In the present invention, examples of "an amino acid sequence in which one or more amino acids are deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 2" include: (i) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1) are deleted from the amino acid sequence shown in SEQ ID NO: 2; (ii) an amino acid sequence in which 1 to 40 amino acids (e.g., 1 to 39, 1 to 38, 1 to 37, 1 to 36, 1 to 35, 1 to 34, 1 to 33, 1 to 32, 1 to 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22, 1 an amino acid sequence in which the following amino acids are substituted with other amino acids: 1-21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 2, 1); (iii) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) have been inserted into the amino acid sequence shown in SEQ ID NO: 2; (iv) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) have been added to the amino acid sequence shown in SEQ ID NO: 2; and (v) an amino acid sequence mutated by a combination of the above (i) to (iv).

[0016] In the present invention, the presence or absence of "ACE2-binding activity" can be measured using known methods, for example, immunological techniques such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), and ELISA (enzyme-linked immunosorbent assay) (e.g., ELISA using polypeptides, cell ELISA, etc.), or pull-down assays. Furthermore, "ACE2-binding activity" means having at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more of the activity of a protein consisting of the amino acid sequence shown in SEQ ID NO: 2, taken as 100.

[0017] Furthermore, the S protein of the mutant coronavirus of the present invention includes a protein that contains an amino acid sequence with 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and has ACE2-binding activity. Such proteins also include those that contain an amino acid sequence with approximately 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 2 and have ACE2-binding activity. Sequence identity can be determined using homology searches such as FASTA, BLAST, and PSI-BLAST on online homology search sites, such as the DNA Data Bank of Japan (DDBJ). BLAST searches can also be performed at the National Center for Biotechnology Information (NCBI).

[0018] To prepare a protein having the above mutation, a mutation can be introduced into DNA encoding the protein using a site-directed mutagenesis method such as the Kunkel method or the Gapped duplex method, using a mutation introduction kit such as QuikChange TM Site-Directed Mutagenesis Kit (Stratagene), GeneTailor TMMutations can be introduced into DNA encoding a polypeptide of interest using overlap extension PCR (R. Higuchi, et al., Nucleic Acids Res. 1988 Aug. 11; 16(15): 7351-7367).

[0019] As described above, the antibodies of the present invention can bind to the S proteins of mutant coronaviruses having various mutations and neutralize the binding of the RBD of the S proteins of mutant coronaviruses to ACE2. Therefore, the mutant coronavirus S protein of the present invention is not limited to, and may include, for example, the following, counting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2: a mutation in the 5th amino acid, leucine (L), a mutation in the 13th amino acid, serine (S), a mutation in the 19th amino acid, threonine (T), a mutation in the 24th amino acid, leucine (L), a mutation in the 25th amino acid, proline (P), a mutation in the 26th amino acid, a mutation in the 27th amino acid, alanine (A), a mutation in the 67th amino acid, a mutation in the 69th amino acid, histidine (H), a mutation in the 70th amino acid, valine (V), a mutation in the 75th amino acid, glycine (G), a mutation in the 76th amino acid, threonine (T), a mutation in the 80th amino acid, aspartic acid (D), a mutation in the 95th amino acid, A mutation in the 142nd amino acid, glycine (G), A mutation in the 143rd amino acid, valine (V), A mutation in the 144th amino acid, tyrosine (Y), A mutation in the 145th amino acid, tyrosine (Y), A mutation in the 152nd amino acid, tryptophan (W), A mutation in the 154th amino acid, glutamic acid (E), A mutation in the 156th amino acid, glutamic acid (E), A mutation in the 157th amino acid, phenylalanine (F), A mutation in the 158th amino acid, arginine (R), A mutation in the 211th amino acid, asparagine (N), A mutation in the 212th amino acid, leucine (L), A mutation in the 213th amino acid, valine (V), A mutation in the 214th amino acid, arginine (R), A mutation in the 242nd amino acid,Mutation at amino acid alanine (A) at position 243, Mutation at amino acid leucine (L) at position 244, Mutation at amino acid serine (S) at position 247, Mutation at amino acid aspartic acid (D) at position 253, Mutation at amino acid glycine (G) at position 339, Mutation at amino acid arginine (R) at position 346, Mutation at amino acid serine (S) at position 371, Mutation at amino acid serine (S) at position 373, Mutation at amino acid serine (S) at position 375, Mutation at amino acid threonine (T) at position 376, Mutation at amino acid aspartic acid (D) at position 405, Mutation at amino acid arginine (R) at position 408, Mutation at amino acid lysine (K) at position 417, Mutation at amino acid asparagine (N) at position 439, Mutation at amino acid asparagine (N) at position 440, Mutation in glycine (G), the 446th amino acid; Mutation in leucine (L), the 452nd amino acid; Mutation in tyrosine (Y), the 453rd amino acid; Mutation in serine (S), the 477th amino acid; Mutation in threonine (T), the 478th amino acid; Mutation in glutamic acid (E), the 484th amino acid; Mutation in phenylalanine (F), the 486th amino acid; Mutation in phenylalanine (F), the 490th amino acid; Mutation in glutamine (Q), the 493th amino acid; Mutation in glycine (G), the 496th amino acid; Mutation in glutamine (Q), the 498th amino acid; Mutation in asparagine (N), the 501st amino acid; Mutation in tyrosine (Y), the 505th amino acid; Mutation in threonine (T), the 547th amino acid; Mutation in alanine (A), the 570th amino acid; Mutation in the 614th amino acid, aspartic acid (D), Mutation in the 655th amino acid, histidine (H), Mutation in the 677th amino acid, glutamine (Q),A mutation at amino acid asparagine (N) at position 679, A mutation at amino acid proline (P) at position 681, A mutation at amino acid alanine (A) at position 701, A mutation at amino acid serine (S) at position 704, A mutation at amino acid asparagine (N) at position 764, A mutation at amino acid threonine (T) at position 791, A mutation at amino acid aspartic acid (D) at position 796, A mutation at amino acid asparagine (N) at position 856, A mutation at amino acid threonine (T) at position 859, A mutation at amino acid phenylalanine (F) at position 888, A mutation at amino acid aspartic acid (D) at position 950, A mutation at amino acid glutamine (Q) at position 954, A mutation at amino acid asparagine (N) at position 969, A mutation at amino acid leucine (L) at position 981, A mutation at amino acid glutamine (Q) at position 1071, and and a mutation at valine (V), which is the amino acid at position 1176. Examples of the "mutation" include deletions, substitutions, and insertions.

[0020] More specifically, the S protein of the mutant coronavirus of the present invention may have, for example, the following amino acid sequences counting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2: a substitution of leucine (L) at the 5th amino acid with phenylalanine (F) (L5F), a substitution of serine (S) at the 13th amino acid with isoleucine (I) (S13I), a substitution of threonine (T) at the 19th amino acid with arginine (R) or isoleucine (I) (T19R or T19I), a substitution of leucine (L) at the 24th amino acid with serine (S) (L24S), a deletion of leucine (L) at the 24th amino acid, a deletion of proline (P) at the 25th amino acid, a deletion of proline (P) at the 26th amino acid, a deletion of alanine (A) at the 27th amino acid, a substitution of alanine (A) at the 27th amino acid with serine (S) (A27S), Substitution of alanine (A) to valine (V) at amino acid 67 (A67V), deletion of histidine (H) at amino acid 69, deletion of valine (V) at amino acid 70, substitution of glycine (G) to valine (V) at amino acid 75 (G75V), substitution of threonine (T) to isoleucine (I) at amino acid 76 (T76I), substitution of aspartic acid (D) to glycine (G) at amino acid 80 (D80G), substitution of threonine (T) to isoleucine (I) at amino acid 95 (T95I), substitution of glycine (G) to aspartic acid (D) at amino acid 142 (G142D), deletion of valine (V) at amino acid 143, deletion of tyrosine (Y) at amino acid 144, deletion of tyrosine (Y) at amino acid 145, Substitution of tryptophan (W) at amino acid position 152 with cysteine ​​(C) (W152C), substitution of glutamic acid (E) at amino acid position 154 with lysine (K) (E154K), deletion of glutamic acid (E) at amino acid position 156, deletion of phenylalanine (F) at amino acid position 157 or substitution of phenylalanine (F) with serine (S) (F157S),Deletion of arginine (R) at amino acid 158 or substitution of arginine (R) with glycine (G) (R158G), deletion of asparagine (N) at amino acid 211, substitution of leucine (L) with isoleucine (I) at amino acid 212 (L212I), substitution of valine (V) with glycine (G) at amino acid 213 (V213G), insertion of three amino acids (glutamic acid (E), proline (P), and glutamic acid (E)) immediately after arginine (R) at amino acid 214 (ins214EPE), deletion of leucine (L) at amino acid 242, deletion of alanine (A) at amino acid 243, deletion of leucine (L) at amino acid 244, deletion of serine (S) at amino acid 247, Deletion of aspartic acid (D) at amino acid position 253, or substitution of aspartic acid (D) with glycine (G) (D253G), substitution of glycine (G) with aspartic acid (D) at amino acid position 339 (G339D), substitution of arginine (R) with lysine (K) at amino acid position 346 (R346K), substitution of serine (S) with phenylalanine (F) or leucine (L) at amino acid position 371 (S371F, S371L), substitution of serine (S) with proline (P) at amino acid position 373 (S373P), substitution of serine (S) with phenylalanine (F) at amino acid position 375 (S375F), substitution of threonine (T) with alanine (A) at amino acid position 376 (T376A), Substitution of aspartic acid (D) to asparagine (N) at amino acid position 405 (D405N), substitution of arginine (R) to serine (S) at amino acid position 408 (R408S), substitution of lysine (K) to asparagine (N) or threonine (T) at amino acid position 417 (K417N or K417T), substitution of asparagine (N) to lysine (K) at amino acid position 439 (N439K), substitution of asparagine (N) to lysine (K) at amino acid position 440 (N440K), substitution of glycine (G) to serine (S) at amino acid position 446 (G446S),substitution of leucine (L) to arginine (R) or glutamine (Q) at amino acid position 452 (L452R or L452Q), substitution of tyrosine (Y) to phenylalanine (F) at amino acid position 453 (Y453F), substitution of serine (S) to asparagine (N) at amino acid position 477 (S477N), substitution of threonine (T) to lysine (K) at amino acid position 478 (T478K), substitution of glutamic acid (E) to lysine (K), glutamine (Q), or alanine (A) at amino acid position 484 (E484K, E484Q, or E484A), substitution of phenylalanine (F) to valine (V) at amino acid position 486 (F486V), substitution of phenylalanine (F) to serine (S) at amino acid position 490 (F490S), Substitution of glutamine (Q) at amino acid position 493 with lysine (K) or arginine (R) (Q493K, Q493R), substitution of glycine (G) at amino acid position 496 with serine (S) (G496S), substitution of glutamine (Q) with arginine (R) at amino acid position 498 (Q498R), substitution of asparagine (N) at amino acid position 501 with tyrosine (Y) (N501Y), substitution of tyrosine (Y) at amino acid position 505 with histidine (H) (Y505H), substitution of threonine (T) at amino acid position 547 with lysine (K) (T547K), substitution of alanine (A) at amino acid position 570 with aspartic acid (D) (A570D), substitution of aspartic acid (D) at amino acid position 614 with glycine (G) (D614G), Substitution of histidine (H) to tyrosine (Y) at amino acid 655 (H655Y), substitution of glutamine (Q) to histidine (H) at amino acid 677 (Q677H), substitution of asparagine (N) to lysine (K) at amino acid 679 (N679K), substitution of proline (P) to arginine (R) or histidine (H) at amino acid 681 (P681R, P681H), substitution of alanine (A) to valine (V) at amino acid 701 (A701V),Substitution of serine (S) to leucine (L) at amino acid position 704 (S704L), substitution of asparagine (N) to lysine (K) at amino acid position 764 (N764K), substitution of threonine (T) to isoleucine (I) at amino acid position 791 (T791I), substitution of aspartic acid (D) to tyrosine (Y) at amino acid position 796 (D796Y), substitution of asparagine (N) to lysine (K) at amino acid position 856 (N856K), substitution of threonine (T) to asparagine (N) at amino acid position 859 (T859N), substitution of phenylalanine (F) to leucine (L) at amino acid position 888 (F888L), substitution of aspartic acid (D) to asparagine (N) at amino acid position 950 (D950N), Examples include, but are not limited to, those containing at least one mutation selected from the group consisting of a glutamine (Q) to histidine (H) substitution at amino acid position 954 (Q954H), an asparagine (N) to lysine (K) substitution at amino acid position 969 (N969K), a leucine (L) to phenylalanine (F) substitution at amino acid position 981 (L981F), a glutamine (Q) to histidine (H) substitution at amino acid position 1071 (Q1071H), and a valine (V) to phenylalanine (F) substitution at amino acid position 1176 (V1176F). As used herein, "at the amino acid position N" means "at the amino acid position N."

[0021] More specifically, the S protein of the mutant coronavirus of the present invention may have, for example, the following amino acid substitutions, counting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2: a substitution of glycine (G) at the 339th amino acid with aspartic acid (D) (G339D), a substitution of arginine (R) at the 346th amino acid with lysine (K) (R346K), a substitution of serine (S) at the 371st amino acid with phenylalanine (F) or leucine (L) (S371F, S371L), a substitution of serine (S) at the 373rd amino acid with proline (P) (S373P), a substitution of serine (S) at the 375th amino acid with phenylalanine (F) (S375F), a substitution of aspartic acid (D) at the 405th amino acid with asparagine (N) (D405N), substitution of lysine (K) to asparagine (N) or threonine (T) at amino acid position 417 (K417N or K417T), substitution of asparagine (N) to lysine (K) at amino acid position 440 (N440K), substitution of glycine (G) to serine (S) at amino acid position 446 (G446S), substitution of lysine (K) to asparagine (N) or threonine (T) at amino acid position 417 (K417N or K417T), substitution of leucine (L) to arginine (R) or glutamine (Q) at amino acid position 452 (L452R or L452Q), substitution of serine (S) to asparagine (N) at amino acid position 477 (S477N), substitution of threonine (T) to lysine (K) at amino acid position 478 (T478K), Substitution of glutamic acid (E) at amino acid position 484 with lysine (K), glutamine (Q), or alanine (A) (E484K, E484Q, or E484A), and substitution of phenylalanine (F) at amino acid position 486 with valine (V) (F486V), substitution of phenylalanine (F) at amino acid position 490 with serine (S) (F490S), substitution of glutamine (Q) at amino acid position 493 with lysine (K) or arginine (R) (Q493K, Q493R), substitution of glycine (G) at amino acid position 496 with serine (S) (G496S),Examples of such mutations include, but are not limited to, those containing at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all of the mutations selected from the group consisting of: a glutamine (Q) to arginine (R) substitution at amino acid 498 (Q498R); a substitution of asparagine (N) to tyrosine (Y) at amino acid 501 (N501Y); and a tyrosine (Y) to histidine (H) substitution at amino acid 505 (Y505H).

[0022] Examples of mutant coronaviruses of the present invention include, but are not limited to, mutant strains of the B.1.1.7 lineage (alpha strain), mutant strains of the B.1.351 lineage (beta strain), mutant strains of the P.1 lineage (gamma strain), mutant strains of the B.1.617.2 lineage, AY.1 lineage, or AY.2 lineage (delta strain), mutant strains of the B.1.427 / B.1.429 lineage (epsilon strain), mutant strains of the P.2 lineage (zeta strain), mutant strains of the B.1.525 lineage (eta strain), mutant strains of the P.3 lineage (theta strain), mutant strains of the B.1.526 lineage (iota strain), mutant strains of the B.1.617.1 lineage (kappa strain), mutant strains of the C.37 lineage (lambda strain), mutant strains of the B.1.621 lineage (mu strain), and mutant strains of the B.1.1.529 lineage (omicron strain). Sublineages of the B.1.1.529 mutant strain include the BA.1, BA.2, BA.3, BA.4, and BA.5 mutant strains. The designations of the above strains follow the PANGO lineage, and the strain names are based on the WHO designation.B.1.1.7 lineage variants (alpha strains) contain at least N501Y as the main mutation, B.1.351 lineage variants (beta strains) contain at least K417N, E484K and N501Y as the main mutations, P.1 lineage variants (gamma strains) contain at least K417T, E484K and N501Y as the main mutations, B.1.617.2, AY.1 or AY.2 lineage variants (delta strains) contain at least L452R and T478K as the main mutations, B.1.427 / B.1.429 lineage variants (epsilon strains) contain at least L452R as the main mutation, P.2 lineage variants (zeta strains) contain at least E484K as the main mutation, and B.1.525 lineage variants (eta strains) contain at least E484K as the main mutation. The P.3 lineage variant (theta strain) contains at least E484K and N501Y as major mutations, the B.1.526 lineage variant (iota strain) contains at least E484K as major mutations, the B.1.617.1 lineage variant (kappa strain) contains at least L452R and E484Q as major mutations, and the C.37 lineage variant (lambda strain) contains at least L4 The B.1.1.529 lineage variants (Omicron strains) contain at least G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y and Y505H as major mutations. Mutant strains of the BA.1 lineage, a sublineage of the B.1.1.529 lineage, contain at least the following major mutations: G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, and Y505H, while mutant strains of the BA.2 lineage contain at least the following major mutations: G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H.

[0023] As an example of the nucleotide sequence and amino acid sequence of the RBD in the present invention, the nucleotide sequence and amino acid sequence of the RBD of wild-type SARS-CoV-2 are shown in SEQ ID NOs: 3 and 4, respectively. That is, in the present invention, the RBD of wild-type SARS-CoV-2 is a protein comprising the amino acid sequence shown in SEQ ID NO: 4.

[0024] In the present invention, spike proteins of mutant coronaviruses include spike proteins having a mutation in the RBD. The RBD of the S protein of mutant coronaviruses in the present invention includes the following proteins (a) and (b): (a) a protein comprising an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, or added in the amino acid sequence shown in SEQ ID NO: 4, and having ACE2-binding activity; (b) a protein comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 4, and having ACE2-binding activity.

[0025] In the present invention, the term "protein comprising the amino acid sequence shown in SEQ ID NO:4" includes a protein consisting of the amino acid sequence shown in SEQ ID NO:4. Furthermore, examples of "an amino acid sequence in which one or more amino acids have been deleted, substituted, inserted or added in the amino acid sequence shown in SEQ ID NO: 4" include: (i) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 1) have been deleted from the amino acid sequence shown in SEQ ID NO: 4; (ii) an amino acid sequence in which 1 to 20 amino acids (e.g., 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2, 1) have been substituted with other amino acids in the amino acid sequence shown in SEQ ID NO: 4; and (iii) (iv) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) have been added to the amino acid sequence shown in SEQ ID NO: 4; (v) an amino acid sequence in which 1 to 10 amino acids (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) have been inserted into the amino acid sequence shown in SEQ ID NO: 4; and (v) an amino acid sequence mutated by a combination of the above (i) to (iv).

[0026] In the present invention, the presence or absence of "ACE2-binding activity" can be measured using known methods, for example, immunological techniques such as immunoprecipitation, Western blotting, EIA (enzyme immunoassay), and ELISA (enzyme-linked immunosorbent assay) (e.g., ELISA using polypeptides, cell ELISA, etc.), or pull-down assays. Furthermore, "ACE2-binding activity" means having at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, and preferably 90% or more of the activity of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 4, defined as 100.

[0027] Furthermore, the RBD of the S protein of a mutant coronavirus of the present invention includes a polypeptide comprising an amino acid sequence having 80% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having ACE2-binding activity. Such polypeptides also include those comprising an amino acid sequence having about 80% or more, about 81% or more, about 82% or more, about 83% or more, about 84% or more, 85% or more, about 86% or more, about 87% or more, about 88% or more, about 89% or more, 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 4 and having ACE2-binding activity. Sequence identity can be determined using homology searches such as FASTA, BLAST, and PSI-BLAST on online homology search sites, such as the DNA Data Bank of Japan (DDBJ). BLAST searches can also be performed at the National Center for Biotechnology Information (NCBI).

[0028] The RBD of the S protein of a mutant coronavirus of the present invention is not limited to, and can be, for example, the following, counting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2: glycine (G) at the 339th amino acid, arginine (R) at the 346th amino acid, serine (S) at the 371st amino acid, serine (S) at the 373rd amino acid, serine (S) at the 375th amino acid, threonine (T) at the 376th amino acid, aspartic acid (D) at the 405th amino acid, arginine (R) at the 408th amino acid, lysine (K) at the 417th amino acid, asparagine (N) at the 439th amino acid, asparagine (N) at the 440th amino acid, glycine (G) at the 446th amino acid, leucine (L) at the 452nd amino acid, tyrosine (Y) at the 453rd amino acid, serine (S) at the 477th amino acid, threonine (T) at the 478th amino acid, The amino acid sequence may include at least one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, or all of the following mutations in amino acids corresponding to amino acids selected from the group consisting of glutamic acid (E) at amino acid position 484, phenylalanine (F) at amino acid position 486, phenylalanine (F) at amino acid position 490, glutamine (Q) at amino acid position 493, glycine (G) at amino acid position 496, glutamine (Q) at amino acid position 498, asparagine (N) at amino acid position 501, tyrosine (Y) at amino acid position 505, and threonine (T) at amino acid position 547.

[0029] More specifically, the RBD of the S protein of a mutant coronavirus in the present invention is not limited to, and examples thereof include, counting from the N-terminus of the amino acid sequence shown in SEQ ID NO: 2, a substitution of glycine (G) at the 339th amino acid with aspartic acid (D) (G339D), a substitution of arginine (R) at the 346th amino acid with lysine (K) (R346K), a substitution of serine (S) at the 371st amino acid with phenylalanine (F) or leucine (L) (S371F, S371L), a substitution of serine (S) at the 373rd amino acid with proline (P) (S373P), a substitution of serine (S) at the 375th amino acid with phenylalanine (F) (S375F), a substitution of threonine (T) at the 376th amino acid with alanine (A) (T376A), Substitution of aspartic acid (D) to asparagine (N) at amino acid position 405 (D405N), substitution of arginine (R) to serine (S) at amino acid position 408 (R408S), substitution of lysine (K) to asparagine (N) or threonine (T) at amino acid position 417 (K417N or K417T), substitution of asparagine (N) to lysine (K) at amino acid position 439 (N439K), substitution of asparagine (N) to lysine (K) at amino acid position 440 (N440K), substitution of glycine (G) to serine (S) at amino acid position 446 (G446S), substitution of leucine (L) to arginine (R) or glutamine (Q) at amino acid position 452 (L452R or L452Q), Substitution of tyrosine (Y) to phenylalanine (F) at amino acid position 453 (Y453F), substitution of serine (S) to asparagine (N) at amino acid position 477 (S477N), substitution of threonine (T) to lysine (K) at amino acid position 478 (T478K), substitution of glutamic acid (E) to lysine (K), glutamine (Q), or alanine (A) at amino acid position 484 (E484K, E484Q, or E484A), substitution of phenylalanine (F) to valine (V) at amino acid position 486 (F486V),Substitution of phenylalanine (F) to serine (S) at amino acid position 490 (F490S), substitution of glutamine (Q) to lysine (K) or arginine (R) at amino acid position 493 (Q493K, Q493R), substitution of glycine (G) to serine (S) at amino acid position 496 (G496S), substitution of glutamine (Q) to arginine (R) at amino acid position 498 (Q498R), substitution of asparagine (N) to tyrosine (Y) at amino acid position 501 (N501Y), substitution of tyrosine (Y) to histidine (H) at amino acid position 505 (Y505H), and substitution of threonine (T) to lysine (K) at amino acid position 547 (T547K), and wherein the amino acid sequence is selected from the group consisting of:

[0030] Here, "corresponding amino acid" refers to an amino acid in the amino acid sequence of the RBD of a wild-type or mutant coronavirus S protein that corresponds to an amino acid at a specific position, counting from the N-terminus, of the amino acid sequence shown in SEQ ID NO: 2. For example, in the case of a wild-type RBD, the asparagine (N) at the 501st amino acid, counting from the N-terminus, of the amino acid sequence shown in SEQ ID NO: 2 corresponds to the asparagine (N) at the 183rd amino acid, counting from the N-terminus, of the amino acid sequence shown in SEQ ID NO: 4. The same applies to mutant RBDs. Herein, the position and type of amino acid of a mutation in the RBD are represented by the corresponding position and type of amino acid, counting from the N-terminus, of the amino acid sequence shown in SEQ ID NO: 2. For example, if the RBD has a mutation from asparagine (N) to tyrosine (Y) at the 501st amino acid, counting from the N-terminus, of the amino acid sequence shown in SEQ ID NO: 2, the mutation in the RBD is represented as N501Y. Furthermore, herein, the term "amino acid corresponding to" can be omitted when referring to a mutation or a mutated amino acid.

[0031] In one aspect, the chicken antibody of the present invention can bind to at least one or all of an RBD or an S protein comprising the same selected from the group consisting of the following RBDs or S proteins comprising the same (i) to (xii): (i) an RBD comprising at least N501Y as a major mutation or an S protein comprising the same; (ii) an RBD comprising at least K417N, E484K, and N501Y as major mutations or an S protein comprising the same; (iii) an RBD comprising at least K417T, E484K, and N501Y as major mutations or an S protein comprising the same; (iv) an RBD comprising at least L452R and T478K as major mutations or an S protein comprising the same; (v) an RBD comprising at least L452R as a major mutation or an S protein comprising the same; (vi) an RBD comprising at least E484K as a major mutation or an S protein comprising the same; (vii) an RBD comprising at least E484K and N501Y as major mutations or an S protein comprising the same; (viii) (ix) an RBD comprising at least L452R and E484Q as major mutations or an S protein comprising the same; (x) an RBD comprising at least R346K, E484K, and N501Y as major mutations or an S protein comprising the same; (xi) an RBD comprising at least G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, and Y505H as major mutations or an S protein comprising the same; and (xii) An RBD or an S protein containing the same, which contains at least G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, and Y505H as major mutations.

[0032] The method for introducing a mutation into DNA encoding the protein in order to prepare the protein having the above mutation is as described above.

[0033] The ACE2 to which the RBD of the S protein of a mutant coronavirus of the present invention binds may be derived from any mammal. Examples of such mammals include mice, rats, rabbits, cats, dogs, goats, monkeys, and humans, and preferably, but not limited to, mice, rats, cats, dogs, and humans. Examples of the nucleotide and amino acid sequences of these ACE2s are shown in SEQ ID NOs: 5 and 6, respectively, but the nucleotide and amino acid sequences of the ACE2s are not limited thereto. The nucleotide and amino acid sequences of human ACE2 are registered in the NCBI (National Center for Biotechnology Information) database (https: / / www.ncbi.nlm.nih.gov / ) under the following accession numbers:

[0034] Base sequence of DNA encoding human ACE2: NM_001371415 (SEQ ID NO: 5) Amino acid sequence of human ACE2: NP_001358344.1 (SEQ ID NO: 6)

[0035] Those skilled in the art can identify the region of RBD to which the antibody of the present invention binds (the region containing the epitope to which the antibody of the present invention binds) by known methods, for example, by preparing multiple full-length or partial polypeptides of RBD and examining which RBD polypeptide the antibody of the present invention binds to. Furthermore, epitopes with three-dimensional (steric) structures can also be identified by using polypeptides with three-dimensional structures. Polypeptides with three-dimensional structures can be prepared, for example, by expressing RBD in mammalian cells.

[0036] The epitope to which the antibody of the present invention binds and the region containing the epitope are not limited, as long as the antibody of the present invention can neutralize the binding of the RBD of the coronavirus S protein to ACE2. Furthermore, based on the description herein, a person skilled in the art can determine whether a test antibody can neutralize the binding of the RBD of the coronavirus S protein to ACE2 without specifying the epitope of the test antibody. In other words, based on the description herein, a person skilled in the art does not need to specify the epitope of the test antibody to determine whether the test antibody can neutralize the binding of the RBD of the coronavirus S protein to ACE2.

[0037] 3. Antibodies of the Present Invention The antibodies of the present invention are chicken antibodies that bind to the spike (S) protein of a mutant coronavirus. Chicken antibodies that bind to the S protein of a mutant coronavirus of the present invention preferably include chicken antibodies that bind to the RBD of the S protein of a mutant coronavirus of the present invention.

[0038] Chicken antibodies include IgY, IgM, and IgA. Of these, IgY, which functionally corresponds to mammalian IgG, selectively migrates to egg yolk. Therefore, chicken antibodies can be easily purified in large quantities from egg yolk and provided at low cost. Furthermore, chicken antibodies purified from eggs, which are a food source, are safe for intranasal, buccal, and oral administration, and can be used in therapeutic or preventive methods using nebulizers or sprays. Other advantages of IgY include: (i) high safety because it does not activate mammalian complement; (ii) it does not bind to mammalian Fc receptors; and (iii) it does not cross-react with mammalian IgG. Furthermore, chickens can recognize biomolecules highly conserved among mammals as foreign substances, so chicken antibodies have an advantage over mammalian antibodies in terms of reactivity with such biomolecules. Furthermore, unlike mouse or human antibodies, the chicken antibodies of the present invention do not exhibit reduced binding inhibitory activity against omicron-type mutants. In other words, the chicken antibodies of the present invention have (can maintain) neutralizing activity against the binding of the RBD of the spike protein of a wider variety of mutant coronaviruses to ACE2, compared to antibodies obtained by immunization of other species.

[0039] The antibodies of the present invention may be either polyclonal or monoclonal. In the present invention, "binding to spike (S) protein" or "binding to the RBD of spike (S) protein" refers to forming a reversible non-covalent bond with the S protein or the RBD of the S protein through hydrogen bonds, hydrophobic interactions, electrostatic forces, van der Waals forces, or the like. In the present invention, "binding" includes "specific binding." In the present invention, "specific binding" refers to recognizing an epitope in a target protein or polypeptide and selectively or preferentially binding to a protein or polypeptide having that epitope compared with proteins or polypeptides not having that epitope. Antibodies of the present invention also include antigen-binding fragments of monoclonal antibodies. Examples of "antigen-binding fragments" in the present invention include, but are not limited to, single-chain Fv (scFv), sc(Fv)2, Fab, Fab', diabodies (dsFv), F(ab')2, and multispecific antibodies. The antibody-binding fragment can be obtained by known methods such as genetic engineering techniques, papain digestion, pepsin digestion, etc. In one aspect of the present invention, the antibody of the present invention is an isolated antibody.

[0040] The antibodies of the present invention can neutralize the binding of the RBD of the S protein of a mutant coronavirus to ACE2. Furthermore, the antibodies of the present invention can neutralize coronaviruses (have neutralizing activity against coronaviruses). In the present invention, coronaviruses refer to coronaviruses that infect cells via ACE2. Examples of such coronaviruses include SARS-CoV-1 and SARS-CoV-2, with SARS-CoV-2 being preferred. These coronaviruses infect cells by the RBD of their S protein binding to ACE2. As used herein, "coronaviruses" includes both wild-type and mutant coronaviruses. In the present invention, "neutralizing" refers to inhibiting the interaction (e.g., binding) between the RBD of the coronavirus S protein and ACE2 or inhibiting the activity of the coronavirus S protein. In the present invention, "neutralizing coronaviruses" refers to suppressing or inhibiting the ability of coronaviruses to bind to or invade cells. Whether a test antibody inhibits the interaction between the RBD of the coronavirus S protein and ACE2 can be examined, for example, by detecting the His-tagged RBD bound to ACE2 using a fluorescently labeled antibody capable of binding to the His tag (fluorescently labeled anti-His tag antibody). Specifically, the test antibody is reacted with the His-tagged RBD, and the reaction product is then added to ACE2-expressing cells. If the test antibody binds to the His-tagged RBD and inhibits the binding of the His-tagged RBD to ACE2 on ACE2-expressing cells, the fluorescent signal of the fluorescently labeled anti-His tag antibody bound to the His-tagged RBD will not be detected. On the other hand, if the test antibody does not inhibit the binding of the His-tagged RBD to ACE2 on ACE2-expressing cells, the His-tagged RBD will bind to ACE2 on ACE2-expressing cells, and the fluorescent signal of the fluorescently labeled anti-His tag antibody bound to the His-tagged RBD will be detected.Alternatively, whether a test antibody inhibits the interaction between the RBD of the coronavirus S protein and ACE2 can be examined by detecting RBD bound to ACE2 using a mouse anti-SARS-CoV-2 Spike antibody and a labeled secondary antibody (mouse anti-IgG-Fc antibody) that binds to this antibody, as described in Example 3 herein. Specifically, the test antibody is reacted with the RBD, and the reaction product is then added to ACE2-expressing cells. If the test antibody inhibits the binding of the RBD to ACE2 on ACE2-expressing cells by binding to the RBD, the fluorescent signal of the fluorescently labeled secondary antibody bound to the RBD via the mouse anti-SARS-CoV-2 Spike antibody will not be detected. On the other hand, if the test antibody does not inhibit the binding of the RBD to ACE2 on ACE2-expressing cells, the RBD will bind to ACE2 on ACE2-expressing cells, and the fluorescent signal of the fluorescently labeled secondary antibody bound to the RBD via the mouse anti-SARS-CoV-2 Spike antibody will be detected. Thus, if no RBD fluorescent signal is detected by the test antibody, it can be determined that the test antibody inhibits the interaction between RBD and ACE2. Furthermore, whether the test antibody can neutralize coronavirus can be confirmed, for example, by mixing the test antibody and coronavirus at a predetermined ratio, contacting the mixture with cultured cells, and observing the presence or absence of morphological changes in the cultured cells (cytopathic effect: CPE). If no morphological changes are observed in the cultured cells, it can be confirmed that the test antibody has neutralizing activity against coronavirus at the antibody concentration used in the test (e.g., Example 7).

[0041] The method for producing the antibodies of the present invention is described below. (1) Antigen Preparation: The immunogen for producing the antibodies of the present invention or the antigen used in the reactivity test of the antibodies of the present invention is not limited, and for example, the full-length coronavirus S protein or a partial polypeptide thereof can be used. The partial polypeptide of the coronavirus S protein is not limited, and for example, a polypeptide containing the RBD of the coronavirus S protein can be used. Examples of "polypeptides containing the RBD of the coronavirus S protein" include, for example, the RBD-containing polypeptides described in "2." above. Polypeptides containing the RBD of the coronavirus S protein can be obtained, for example, by expressing a polynucleotide encoding a polypeptide containing the RBD of the coronavirus S protein in mammalian cells (HEK293 cells, CHO cells). That is, immunogens for producing the antibodies of the present invention or antigens used in the reactivity test of the antibodies of the present invention include polypeptides containing the RBD of the coronavirus S protein expressed in mammalian cells. Furthermore, the polynucleotide encoding a polypeptide containing the RBD of the coronavirus S protein to be introduced into a mammal can be appropriately codon-optimized to be suitable for expression in mammalian cells. Furthermore, a polypeptide containing the RBD of a coronavirus S protein can be synthesized by a person skilled in the art by specifying its amino acid sequence using known protein synthesis methods such as the solid-phase method or a commercially available protein synthesizer. The synthesized peptide can be conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH) or thyroglobulin and used as an immunogen. However, methods for producing immunogens for producing antibodies of the present invention are not limited to these methods. In the present invention, examples of the RBD of a coronavirus S protein include the RBD of the S protein of SARS-CoV-1 and SARS-CoV-2, with the RBD of the S protein of SARS-CoV-2 being preferred.Furthermore, when a mutation occurs in the S protein (e.g., RBD) of a coronavirus that causes a disease to be treated, the mutant polypeptide can be obtained by expressing a nucleic acid encoding the polypeptide in the mammalian cells described above or by synthesizing the polypeptide using the techniques described above. The mutant polypeptide obtained in this manner can be used as an antigen / immunogen. However, the immunogen used to obtain an antibody that binds to the spike protein of a mutant coronavirus does not necessarily have to be a mutant polypeptide; a wild-type polypeptide can also be used.

[0042] (2) Preparation of Polyclonal Antibodies Egg-laying chickens, such as Julia Light, Boris Brown, and White Leghorn, can be used as immunized animals. Immunization is performed by administering the antigen polypeptide prepared in (1) above to chickens together with an adjuvant. Examples of adjuvants include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant. Immunization is typically performed by intramuscular, intravenous, or subcutaneous injection. The interval between immunizations is not particularly limited; immunizations are typically performed at intervals of several days to several weeks (e.g., 1-3 weeks, preferably 1-2 weeks) or several months (e.g., approximately 1-6 months, preferably approximately 1, 2, 3, or 4 months), for a total of 2-10 immunizations, preferably 2-5 immunizations (e.g., 4 immunizations). Those skilled in the art can determine the interval and number of immunizations based on the antibody titer to be obtained. Those skilled in the art can appropriately set the interval for egg collection depending on the experimental design, and for example, eggs can be collected once every 1 to 2 weeks (e.g., every 2 weeks) and the antibody titer in the egg yolk can be measured. ELISA can be used to measure the antibody titer.

[0043] (3) Antibody Purification. The eggshells of the collected eggs are cracked and the yolks are separated. A buffer solution is added to the egg yolks and stirred thoroughly, followed by the addition of polyethylene glycol to dissolve them. After centrifuging to remove lipid components, the supernatant is added with polyethylene glycol and stirred to dissolve. Further centrifugation is performed to precipitate proteins, including chicken antibodies. The precipitate is then added with buffer and dissolved. Polyethylene glycol is then added and stirred thoroughly to dissolve them. The chicken antibodies are precipitated by centrifugation, and the precipitate is washed with ice-cold 50% ethanol to remove residual polyethylene glycol. The washed chicken antibodies are then dissolved in a buffer solution. Known buffer solutions, such as PBS and TBS, can be used. The concentration of the purified chicken antibodies is determined by measuring the absorbance at 280 nm using a spectrophotometer and calculating the concentration based on the molar extinction coefficient of the chicken antibody (1.33) and the Lambert-Beer law. In this manner, purified chicken polyclonal antibodies can be obtained.

[0044] Specifically, the present invention provides a method for producing a chicken polyclonal antibody, comprising the following steps (a) and (b): (a) immunizing one or more chickens with a polypeptide containing the RBD of the coronavirus S protein, and (b) purifying a chicken polyclonal antibody from eggs laid by the one or more chickens immunized in step (a). The number of chickens immunized in step (a) may be, for example, 1 or more, 2 or more, 10 or more, 50 or more, or 100 or more, and may be, for example, 1,000 or less, 900 or less, 800 or less, 500 or less, 400 or less, 300 or less, 200 or less, 100 or less, 50 or less, 10 or less, or 2 or less, but is not limited to these. In one embodiment, step (b) can further include the steps of randomly selecting multiple eggs from those laid by one or more immunized chickens and purifying chicken polyclonal antibodies from the selected eggs. Furthermore, step (b) can include the steps of mixing the yolks of the selected eggs and purifying chicken polyclonal antibodies from the mixed yolks. The number of eggs selected is not limited and can be, for example, 1 or more, 2 or more, 10 or more, 50 or more, or 100 or more, and 200 or less, 150 or less, 100 or less, 50 or less, 10 or less, or 2 or less. The "polypeptide containing the RBD of the coronavirus S protein" used for immunization in step (a) can be a polypeptide containing the RBD described in "2." above. Furthermore, the "polypeptide containing the RBD of the coronavirus S protein" used for immunization in step (a) can be a polypeptide containing the RBD of the coronavirus S protein expressed in mammalian cells.

[0045] (4) Preparation of Monoclonal Antibodies Chicken monoclonal antibodies can be prepared by known methods, such as cell fusion (S. Nishinaka et al., Int Arch Allergy Appl Immunol. 1989;89(4):416-9) or phage display (JP 2001-238676 A).

[0046] (5) Production of Recombinant Antibodies One embodiment of the antibody of the present invention is a recombinant antibody. Examples of recombinant antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0047] A chimeric antibody is an antibody produced by linking immunoglobulin gene fragments from different animal species. In the present invention, an example of a chimeric antibody is a human chimeric antibody, but the type of animal from which the variable and constant regions of the chimeric antibody are derived is not limited. An example of a human chimeric antibody is an antibody in which the variable region of a chicken antibody is linked (conjugated) to the constant region of a human-derived antibody. Chimeric antibodies can be easily constructed using genetic recombination techniques.

[0048] In the present invention, a technique known as CDR grafting can be used to prepare humanized antibodies. CDR grafting is a method in which complementarity-determining regions (CDRs) from the variable regions of a chicken antibody are grafted onto human variable regions to produce reshaped variable regions in which the framework regions (FRs) are human and the CDRs are chicken-derived. These humanized reshaped human variable regions are then linked to human constant regions.

[0049] In the present invention, fully human antibodies can be produced, for example, using mammals capable of producing human antibodies in accordance with known techniques (WO96 / 9634096, WO98 / 24893, etc.).

[0050] In the present invention, chimeric antibodies and humanized antibodies can be produced using hybridomas or DNA or RNA extracted from the hybridomas as raw materials in accordance with the above-mentioned known methods. Furthermore, proteins to which the antibodies of the present invention are fused can be produced by fusing the variable regions of the antibodies with other proteins using known genetic recombination methods. Furthermore, such fusion proteins can be produced by crosslinking the monoclonal antibody with other proteins using a crosslinker.

[0051] (6) Preparation of Antibody-Binding Fragments Antigen-binding fragments of the antibodies of the present invention bind to the S protein of mutant coronaviruses. An antigen-binding fragment of an antibody refers to a polypeptide containing a portion of the antigen-binding portion of the antibody of the present invention. Examples of antigen-binding fragments include, but are not limited to, single-chain Fv (scFv), sc(Fv)2, Fab, Fab', diabodies (dsFv), F(ab')2, and multispecific antibodies. The antibody-binding fragments can be obtained by known methods, such as genetic engineering, papain digestion, or pepsin digestion.

[0052] (7) Binding Affinity The affinity of an antibody for an antigen can generally be expressed as the equilibrium dissociation constant (KD), and a lower KD value indicates a higher affinity of the antibody. Antibody affinity can be measured using known instruments and methods (e.g., Biacore®-3000 (GE Healthcare), ProteON XPR36 (Bio-Rad), etc.).

[0053] 4. Compositions The compositions of the present invention contain the antibodies described above in "3. Antibodies of the Present Invention" as an active ingredient and are used to treat or prevent coronavirus infection. The present invention also includes pharmaceutical compositions containing the antibodies described above in "3. Antibodies of the Present Invention" as an active ingredient and used to treat or prevent coronavirus infection. Coronavirus infections that are the subject of the compositions of the present invention refer to diseases or symptoms caused by coronavirus infection. Examples of such coronavirus infections include diseases or symptoms caused by infection with coronaviruses that infect cells via ACE2. Examples of "coronaviruses that infect cells via ACE2" include, but are not limited to, SARS-CoV-1 and SARS-CoV-2, with SARS-CoV-2 being preferred. Examples of "diseases or symptoms caused by infection with coronaviruses that infect cells via ACE2" include, for example, diseases or symptoms related to organs or tissues where ACE2 is primarily expressed, such as the lungs, digestive system, heart, blood vessels, eyes, kidneys, cerebral cortex, amygdala, brainstem, and medulla oblongata, as well as diseases or symptoms caused by immune responses. Such diseases or symptoms include, but are not limited to, respiratory diseases, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms.The compositions containing the antibodies of the present invention inhibit infection by coronaviruses that infect cells via ACE2, and are therefore effective in treating or preventing various diseases or symptoms associated with coronavirus infection.

[0054] In the present invention, "treatment" refers to the alleviation of disease symptoms by contacting (e.g., administering) an antibody of the present invention or a composition containing the antibody to a subject after the onset of disease, compared to the absence of contact, but does not necessarily mean the complete suppression of disease symptoms. In the present invention, "disease onset" includes not only the physical manifestation of disease symptoms, but also a positive result in a test for coronavirus infection (e.g., PCR test) even in the absence of symptoms. Furthermore, in the present invention, "treatment" of coronavirus infection includes the suppression or inhibition of the worsening of coronavirus infection. In the present invention, "prevention" refers to the suppression or inhibition of the onset or the suppression or inhibition of the worsening of coronavirus infection, but does not necessarily mean the complete suppression of onset. In the present invention, "prevention" includes the alleviation of disease symptoms after the onset of disease, compared to the absence of contact, by contacting (e.g., administering) a composition of the present invention to a subject before the onset of disease.

[0055] The compositions of the present invention can contain a pharmaceutically acceptable carrier in addition to an antibody that binds to ACE2. The term "pharmaceutically acceptable carrier" refers to any carrier (e.g., liposome, lipid vesicle, micelle), diluent, excipient, wetting agent, buffer, suspending agent, lubricant, adjuvant, emulsifier, disintegrating agent, absorbent, preservative, surfactant, colorant, flavoring agent, or sweetener that is suitable for the composition.

[0056] The antibodies and compositions of the present invention can be in the form of aerosols, lyophilized products, tablets, hard capsules, soft capsules, granules, powders, pills, syrups, suppositories, patches, ointments, creams, eye drops, injections, and the like. When the antibodies and compositions of the present invention are used as aerosols, they can be administered (sprayed) using, for example, a nebulizer or spray. This allows the antibodies of the present invention to inhibit coronavirus infection of the nasal mucosa or oral mucosa. Liquid preparations such as injections may also be in the form of a powder (e.g., a lyophilized powder) to be reconstituted immediately before use, to be dissolved in physiological saline or the like.

[0057] The antibodies and compositions of the present invention can be applied to (e.g., carried by) products (protective products, e.g., masks, filters, sprays, etc.) or devices used to prevent coronavirus infections in order to prevent coronavirus infections.

[0058] The antibodies and compositions of the present invention can be administered locally or systemically by any means known to those skilled in the art. The compositions of the present invention can be administered either orally or parenterally, and parenterally, for example, by intranasal administration, oral administration, or intramuscular administration. The compositions of the present invention can be administered in dosage forms suitable for these administration routes.

[0059] The dosage of the antibodies and compositions of the present invention varies depending on factors such as the subject's age, weight, health condition, sex, symptoms, animal species, administration route, frequency of administration, and dosage form, and specific administration procedures can be determined by those skilled in the art. The dosage of the antibodies of the present invention for treating coronavirus infection is, for example, but not limited to, 0.1 mg to 100 mg per kg of subject body weight per day, preferably 0.1 mg to 15 mg per kg. The dosage can be one to five times per day.

[0060] The timing of administration of the antibodies and compositions of the present invention can be determined appropriately depending on the symptoms, and multiple doses can be administered simultaneously or separately at intervals. Furthermore, the compositions of the present invention can be administered to a subject before or after the onset of disease.

[0061] The composition of the present invention can be administered to mammals as subjects, including, for example, mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, goats, pigs, sheep, cows, horses, monkeys, and humans.

[0062] 5. Method for Treating or Preventing Coronavirus Infection In the present invention, coronavirus infection can be treated or prevented by administering to a subject a chicken antibody that binds to the S protein of a mutant coronavirus or a composition containing the same. That is, the present invention provides a method for treating or preventing coronavirus infection, comprising administering to a subject a therapeutically effective amount of the antibody of the present invention or a composition containing the antibody. The therapeutically effective amount of the antibody of the present invention or a composition containing the antibody varies depending on factors such as the subject's age, weight, health condition, sex, symptoms, route of administration, number of administrations, and dosage form. Those skilled in the art can easily determine the therapeutically effective amount required for treating or preventing coronavirus infection. In the present invention, the "subject" includes subjects in need of treatment or prevention of coronavirus infection. Furthermore, mammals that can be treated or prevented as "subjects" are as described above. In the method for treating or preventing coronavirus infection of the present invention, the terms "coronavirus infection," "treatment," and "prevention" are as described above. Furthermore, the dosage form, route of administration, dosage, and timing of administration of the antibody of the present invention or a composition containing the antibody are also as described above.

[0063] 6. Inhibitors of Binding Between the RBD of the S Protein of a Mutant Coronavirus and ACE2 The antibodies of the present invention neutralize (inhibit) the binding between the RBD of the S protein of a mutant coronavirus and ACE2, thereby inhibiting coronavirus infection caused by this binding. Specifically, the present invention provides an inhibitor of binding between the RBD of the S protein of a mutant coronavirus and ACE2, or a composition for inhibiting binding between the RBD of the S protein of a mutant coronavirus and ACE2, comprising the antibody described above in "3. Antibodies of the Present Invention" as an active ingredient. The inhibitors of the present invention can be used as test reagents or for the treatment or prevention of mammals. The dosage form, additives, administration route, administration target, dosage, etc. can be appropriately selected in accordance with the description above in "4. Compositions." However, the inhibitors of the present invention may contain only the antibodies of the present invention.

[0064] 7. Uses of Antibodies The antibodies of the present invention can be used in methods for treating or preventing coronavirus infections or in the manufacture of medicaments for treating or preventing coronavirus infections. That is, the present invention provides the antibodies of the present invention for use in methods for treating or preventing coronavirus infections. The present invention also provides the antibodies of the present invention for use in the manufacture of medicaments for treating or preventing coronavirus infections. Furthermore, the present invention provides the antibodies of the present invention for use in the manufacture of inhibitors of binding between the S protein of a mutant coronavirus and ACE2.

[0065] 8. Combination Therapy The composition of the present invention can be used for combination administration with at least one other therapeutic agent, such as remdesivir, dexamethasone, favipiravir, nafamostat, camostat, ivermectin, tricizumab, or baricitinib.

[0066] The combined administration of the composition of the present invention and at least one other therapeutic agent is expected to produce more effective results than when each agent is used alone. The term "combined administration" as used herein refers to simultaneous or separate administration of the composition of the present invention and at least one other therapeutic agent. "Simultaneous administration" refers to administration at the same timing within a single administration schedule, and does not necessarily require the administration times to be exactly the same. "Separate administration" refers to administration at different timings within a single administration schedule. The administration forms, administration routes, and administration targets of the composition and other therapeutic agents used in the combination therapy of the present invention are not particularly limited and can be appropriately selected in accordance with the descriptions in "4. Composition" above. Furthermore, the administration forms or dosages of the drugs used in combination may differ from each other and can be appropriately adjusted depending on the combination. When the composition of the present invention is used in combination with other therapeutic agents, the dosage can be appropriately reduced. Therefore, in combining the composition of the present invention with another therapeutic agent, the following combinations can be used: (i) an effective amount of the composition of the present invention and an effective amount of the other therapeutic agent, (ii) an effective amount of the composition of the present invention and an ineffective amount of the other therapeutic agent, (iii) an ineffective amount of the composition of the present invention and an effective amount of the other therapeutic agent, and (iv) an ineffective amount of the composition of the present invention and an ineffective amount of the other therapeutic agent. Even if one or both of the composition and the other therapeutic agent are used in an ineffective amount, they can be administered in combination in such a manner as long as a pharmacological effect can be exerted by the combination.

[0067] 9. Reagents and Kits The antibody of the present invention can be included in a reagent or kit. That is, the present invention provides a reagent and kit comprising the antibody of the present invention. The reagent and kit of the present invention can be used, for example, as a reagent or kit for testing coronaviruses. In the reagents and kits of the present invention, the antibody of the present invention may be made easier to handle by methods such as freezing, and then used as is or mixed with known pharmaceutically acceptable carriers such as excipients, bulking agents, binders, and lubricants, and known additives (including buffers, isotonicity agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc.). In addition to the antibody of the present invention, the kit of the present invention can also include a buffer solution, an enzyme solution, a secondary antibody, a diluent solution, instructions for use, etc.

[0068] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0069] In this example, the SARS-CoV-2 spike protein RBD polypeptide (hereinafter referred to as "RBD polypeptide"), which contains the amino acid sequence shown in SEQ ID NO: 8 and was expressed in mammalian cells (HEK293 cells or CHO cells), was used as the immunogen. The polynucleotide encoding the RBD polypeptide (SEQ ID NO: 7) was codon-optimized for expression in mammalian cells.

[0070] (2) Immunization: Egg-laying chickens, such as Julia Light and Boris Brown, were used as immunized animals. Immunization was carried out at the Itochu Feed Mill Research Institute by administering 100 μg of the RBD polypeptide described in (1) above, together with an adjuvant, into the breast muscle of each chicken. The immunogen was administered four times, every two weeks. Eggs were collected once a week, and the antibody titer in the egg yolk was examined.

[0071] (3) Purification of Chicken Antibodies. The eggshell surface was disinfected with sodium hypochlorite and then washed with water. After wiping off any moisture from the eggshell, the shell was cracked and the yolk was removed. The egg yolk was washed with water to remove the albumen. A four-fold volume of PBS(-) was added to the egg yolk and stirred thoroughly, and polyethylene glycol 6000 was added to a final concentration of 3.5% to dissolve. After centrifugation and removal of lipid components, polyethylene glycol 6000 was added to the supernatant to a final concentration of 12%, stirred, and dissolved. Proteins containing chicken antibodies were precipitated by centrifugation, and PBS(-) was added to the precipitate and dissolved. Polyethylene glycol 6000 was added to a final concentration of 12%, stirred, and dissolved. The chicken antibodies were precipitated by centrifugation, and the precipitate was washed with ice-cold 50% ethanol to remove residual polyethylene glycol. After washing, the chicken antibodies were dissolved in PBS(-). The concentration of the purified chicken antibody was determined by measuring the absorbance at 280 nm using a spectrophotometer and calculating the concentration based on the molar extinction coefficient of the chicken antibody (1.33) and the Lambert-Beer law. As a result, chicken polyclonal antibodies were successfully produced.

[0072] Binding test between chicken antibodies and mutant RBD polypeptides In this example, the following mutant RBD polypeptides were used: alpha (having N501Y) (SEQ ID NO: 10), beta (having K417N, E484K, and N501Y) (SEQ ID NO: 12), gamma (theta) (having E484K and N501Y) (SEQ ID NO: 14), epsilon (having L452R) (SEQ ID NO: 16), kappa (having L452R and E484Q) (SEQ ID NO: 18), and eta (having E484K) (SEQ ID NO: 20). Wild-type (WT) RBD polypeptide (SEQ ID NO: 8) was also used. Mutant RBD polypeptides were prepared by overlap extension PCR (R. Higuchi, et al., Nucleic Acids Res. 1988 Aug 11; 16(15): 7351-7367) to construct DNAs encoding various RBD polypeptides and expressing them in mammalian cells (HEK293 cells or CHO cells). Wild-type (WT) RBD polypeptides were also prepared by the same method. The nucleotide sequences of the polynucleotides encoding the wild-type, alpha, beta, gamma (theta), epsilon, kappa, and eta RBD polypeptides are shown in SEQ ID NOs: 7, 9, 11, 13, 15, 17, and 19, respectively. Each mutant RBD polypeptide was diluted to 1 μg / mL in carbonate buffer, and 100 μL was added to each well of an ELISA plate (MaxiSorp, Nunc). The plate was incubated at 37°C for 1 hour to immobilize the antigen. After washing three times with 300 μL of PBS-T (D-PBS(-) containing 0.05% Tween 20) using a microplate washer, 300 μL of blocking buffer (PBS-T containing 5% skim milk) was added to each well of the ELISA plate and the plate was left to stand at 37°C for 1 hour. After washing three times with 300 μL of PBS-T using a microplate washer, 100 μL of chicken antibody diluted in dilution buffer (D-PBS(-) containing 2% skim milk and 0.2% Tween 20) was added to each well of the ELISA plate and the plate was left to stand at 37°C for 1 hour.The plate was washed three times with 300 μL of PBS-T using a microplate washer. 100 μL of secondary antibody (Rabbit anti-Chicken IgG (IgY)-heavy and light chain cross-adsorbed antibody HRP conjugated, Bethyl, A30-207P) diluted 1:10,000 with dilution buffer was added to each well of the ELISA plate and incubated at 37°C for 30 minutes. The plate was washed five times with 300 μL of PBS-T using a microplate washer. 100 μL of OPD solution was added to each well of the ELISA plate and incubated at room temperature while monitoring color development. 100 μL of 3N sulfuric acid solution was added to each well to stop the color reaction. The absorbance at 490 nm was measured using an absorbance meter.

[0073] The reactivity of the chicken antibodies of the present invention with each mutant RBD polypeptide was evaluated, and the results are shown in Figure 1. Two chicken antibody lots, #I2 and #I22, obtained from two chickens (strain: Julia Light), both bound to all mutant RBD polypeptides. These results demonstrated that the chicken antibodies of the present invention bind to all mutant RBD polypeptides and the S proteins of the mutant coronaviruses containing these mutant RBDs.

[0074] Inhibition Test of Binding of Mutant RBD Polypeptides to Human ACE2 In this example, the inhibitory (neutralizing) activity of the chicken antibodies of the present invention against the binding of mutant RBD polypeptides to human ACE2 was tested. The mutant RBD polypeptides used were the same as those used in Example 2, i.e., alpha, beta, gamma (theta), epsilon, kappa, and eta RBD polypeptides. Wild-type (WT) RBD polypeptides were also used. The binding inhibition test in this example was performed as follows: First, 100 μL of a cell suspension of mouse 4T1 cells stably expressing human ACE2 was seeded into each well of a 96-well cell culture plate at 10,000 cells per well and cultured at 37°C in the presence of 5% CO2 for 2 days. Each mutant RBD polypeptide was added to Blocking Buffer (D-PBS(-) containing 3% BSA and 0.2% FCS) to a final concentration of 5 nM. Chicken antibodies were diluted with this Blocking Buffer containing mutant RBD polypeptides to prepare chicken antibody solutions. The medium was removed from the 96-well plate by decantation, and 30 μL of the prepared chicken antibody solution was added to each well and incubated at 4°C for 1 hour. The chicken antibody solution was removed from the 96-well plate by decantation, and each well was washed twice with 250 μL of D-PBS(-). Mouse anti-SARS-CoV-2 Spike antibody (clone S28) was diluted in Blocking Buffer to a concentration of 1 μg / mL. 30 μL of the S28 antibody solution was added to each well and incubated at 4°C for 1 hour. The S28 antibody solution was removed by decantation, and each well was washed twice with 250 μL of D-PBS(-). 50 μL of 10% formalin solution was added to each well of a 96-well plate and allowed to stand at room temperature for 10 minutes to fix the cells. The 10% formalin solution was then decanted, and each well was washed twice with 250 μL of wash buffer (D-PBS(-) containing 0.05% Tween 20). 50 μL of 3% hydrogen peroxide solution was added to each well of the 96-well plate and allowed to stand at room temperature for 5 minutes to inactivate endogenous peroxidase.The 3% hydrogen peroxide solution was removed by decantation, and each well was washed three times with 250 μL of washing buffer (D-PBS(-) containing 0.05% Tween 20). 50 μL of secondary antibody (Mouse IgG-Fc Fragment cross-adsorbed antibody HRP conjugated, Bethyl, A90-231P) solution diluted 1:10,000 with blocking buffer was added to each well of the 96-well plate and incubated in the dark at room temperature for 1 hour. The secondary antibody solution was removed by decantation, and each well was washed three times with 250 μL of washing buffer (D-PBS(-) containing 0.05% Tween 20). 100 μL of OPD solution was added to each well of the 96-well plate and incubated at room temperature while monitoring color development. 100 μL of 3N sulfuric acid solution was added to each well to stop the color development. The absorbance at 490 nm was measured using an absorbance meter to detect the RBD polypeptide of the SARS-CoV-2 spike protein bound to ACE2 on the cell membrane. In this example, the inhibition rate of the binding of mutant RBD polypeptides to human ACE2 by the antibodies of the present invention was calculated. In calculating the inhibition rate, wells containing only the RBD polypeptides were used as positive controls, and wells containing only the antibodies of the present invention were used as negative controls.

[0075] The results of the binding inhibition test of mutant RBD polypeptides with human ACE2 using chicken antibodies are shown in Figure 2. When either of the two chicken antibody lots, #I2 and #I22, was used, no RBD polypeptides bound to ACE2 were detected at an antibody concentration of 125 μg / ml. These results demonstrated that the chicken antibodies of the present invention had 100% inhibitory activity against the binding of all mutant RBD polypeptides to ACE2. Furthermore, the IC of the antibodies of the present invention in competitive binding to the above mutant RBD polypeptides (inhibition of binding of mutant RBD polypeptides to human ACE2) was 50 The IC values ​​are shown in Table 1. Table 1 shows the IC values ​​using wild-type and mutant RBD polypeptides. 50The values ​​were 9.02-22.55 μg / mL. These results indicate that the chicken antibodies of the present invention have equivalent neutralizing activity against all mutant RBD polypeptides. In Table 1 and Figures 1 and 2, "WT" represents the wild-type RBD polypeptide, "Alpha" represents the RBD polypeptide of the alpha mutant, "Beta" represents the RBD polypeptide of the beta mutant, "Gamma" represents the RBD polypeptide of the gamma (theta) mutant, "Epsilon" represents the RBD polypeptide of the epsilon mutant, "Kappa" represents the RBD polypeptide of the kappa mutant, and "Eta" represents the RBD polypeptide of the eta mutant. In Figures 1 and 2, "IgY conc." represents the concentration of the antibody of the present invention.

[0076]

[0077] In other words, the results of this example demonstrated that the antibodies of the present invention can bind to the spike protein of a mutant coronavirus and neutralize the binding of the RBD of the spike protein of a mutant coronavirus to ACE2.

[0078] Inhibition Test of Binding of Mutant RBD Polypeptides to Human ACE2 In this Example, similar to Example 3, the inhibitory (neutralizing) activity of chicken antibodies of the present invention against the binding of mutant RBD polypeptides to human ACE2 was tested. As mutant RBD polypeptides, in addition to the alpha, beta, gamma (referred to as "theta" in this Example), epsilon, kappa, and eta RBD polypeptides used in Examples 2 and 3, a delta RBD polypeptide (having L452R and T478K) (SEQ ID NO: 22) was used. The wild-type (WT) RBD polypeptide used in Examples 2 and 3 was also used. Furthermore, the chicken antibody of the present invention, lot #122, used in Examples 2 and 3, was used. The method for the binding inhibition test of the mutant RBD polypeptides to human ACE2 was the same as in Example 3. Furthermore, in this Example, the inhibition rate of the antibodies of the present invention against the binding of mutant RBD polypeptides to human ACE2 was calculated. In calculating the inhibition rate, wells to which only each RBD polypeptide was added were used as positive controls, and wells to which only the antibody of the present invention was added were used as negative controls.

[0079] The results are shown in Table 2 and Figure 3. The inhibitory effect of the antibodies of the present invention on the binding of mutant RBD polypeptides to human ACE2 increased in an antibody concentration-dependent manner. Furthermore, at antibody concentrations of 60 μg / ml or higher, the antibodies of the present invention inhibited the binding of all mutant RBD polypeptides to ACE2 by 96% or more. More specifically, at antibody concentrations of 60 μg / ml or higher, the chicken antibodies used in this example inhibited the binding of epsilon RBD polypeptides to human ACE2 by 96%, eta and theta RBD polypeptides by 99%, and alpha, beta, and delta RBD polypeptides by 100% or more. In Table 2, Figure 3, and Table 3 described below, "WT" represents the wild-type RBD polypeptide, "Alpha" represents the RBD polypeptide of an alpha mutant, "Beta" represents the RBD polypeptide of a beta mutant, "Delta" represents the RBD polypeptide of a delta mutant, "Epsilon" represents the RBD polypeptide of an epsilon mutant, "Eta" represents the RBD polypeptide of an eta mutant, "Theta" represents the RBD polypeptide of a theta mutant, and "Kappa" represents the RBD polypeptide of a kappa mutant. Also, in Figure 3, "IgY Conc." represents the concentration of the antibody of the present invention.

[0080] These results demonstrated that the antibodies of the present invention have extremely high inhibitory activity against the binding of all mutant RBD polypeptides to human ACE2.

[0081] Furthermore, the IC of the antibody of the present invention in competitive binding to the mutant RBD polypeptide (inhibition of binding of the mutant RBD polypeptide to human ACE2) 50 The IC values ​​are shown in Table 3. Table 3 shows the IC values ​​using wild-type and mutant RBD polypeptides. 50 The values ​​were 11.34-29.08 μg / mL. These results indicate that the chicken antibodies of the present invention have equivalent neutralizing activity against all mutant RBD polypeptides.

[0082]

[0083] In other words, the results of this example demonstrated that the antibodies of the present invention can bind to the spike protein of a mutant coronavirus and neutralize the binding of the RBD of the spike protein of a mutant coronavirus to ACE2.

[0084] Inhibition Test of Binding of Mutant RBD Polypeptides to Human ACE2 In this example, similar to Examples 3 and 4, the inhibitory (neutralizing) activity of chicken antibodies of the present invention against binding of mutant RBD polypeptides to human ACE2 was tested. The mutant RBD polypeptides used were gamma (having K417T, L452R, and T478K) (SEQ ID NO: 24), mu (having R346K, E484K, and N501Y) (SEQ ID NO: 28), and omicron (having G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, and Y505H) (SEQ ID NO: 30). The wild-type (WT) RBD polypeptide used in Examples 2 to 4 was also used. The chicken antibodies of the present invention used were the chicken antibody lot #12 used in Example 2 and the chicken antibody lot #122 used in Examples 2 to 4. The same method as in Examples 3 and 4 was used to test for inhibition of binding between the mutant RBD polypeptide and human ACE2. In this Example, the inhibition rate of the antibodies of the present invention against the binding between the mutant RBD polypeptide and human ACE2 was calculated. In calculating the inhibition rate, wells to which only the RBD polypeptide was added were used as positive controls, and wells to which only the antibodies of the present invention were added were used as negative controls.

[0085] The results are shown in Figure 4. The inhibitory activity of the antibodies of the present invention against the binding of mutant RBD polypeptides to human ACE2 increased in an antibody concentration-dependent manner. In Figure 4 and Table 4 described below, "WT" represents wild-type RBD polypeptide, "Gamma" represents the RBD polypeptide of the gamma mutant strain, "Mu" represents the RBD polypeptide of the mu mutant strain, and "Omicron" represents the RBD polypeptide of the omicron mutant strain. Also in Figure 4, "IgY Conc." represents the concentration of the antibodies of the present invention. These results demonstrate that the antibodies of the present invention have extremely high inhibitory activity against the binding of all mutant RBD polypeptides to human ACE2.

[0086] Furthermore, the IC of the antibody of the present invention in competitive binding to the mutant RBD polypeptide (inhibition of binding of the mutant RBD polypeptide to human ACE2) 50 The IC values ​​are shown in Table 4. Table 4 shows the IC values ​​using wild-type and mutant RBD polypeptides. 50 The values ​​were 19.47-35.50 μg / mL when chicken antibody lot #I2 was used, and 7.93-17.83 μg / mL when chicken antibody lot #I22 was used.

[0087] This result indicates that the chicken antibodies of the present invention have equivalent neutralizing activity against all mutant RBD polypeptides.

[0088] In other words, the results of this example demonstrated that the antibodies of the present invention can bind to the spike protein of a mutant coronavirus and neutralize the binding of the RBD of the spike protein of a mutant coronavirus to ACE2.

[0089] Neutralization test against mutant pseudovirus In this example, the chicken antibody of the present invention was tested to determine whether it has inhibitory activity against the entry of mutant pseudovirus into human cells (whether it has neutralizing activity against mutant pseudovirus).

[0090] 1. Preparation of Various Mutant Pseudoviruses In this example, pseudoviruses were prepared using a polynucleotide encoding a full-length S protein in which the wild-type RBD was replaced with a mutant RBD. The mutant RBDs used were polynucleotides encoding the following RBDs: alpha (having N501Y) (SEQ ID NO: 10), beta (having K417N, E484K, and N501Y) (SEQ ID NO: 12), gamma (having K417T, E484K, and N501Y) (SEQ ID NO: 24), delta (having L452R and T478K) (SEQ ID NO: 22), eta (having E484K) (SEQ ID NO: 20), theta (having E484K and N501Y) (SEQ ID NO: 14), epsilon (having L452R) (SEQ ID NO: 16), kappa (having L452R and E484Q) (SEQ ID NO: 18), lambda (having L452Q and F490S) (SEQ ID NO: 26), and mu (having R346K, E484K, and N501Y) (SEQ ID NO: 28). A polynucleotide encoding the full-length S protein (SEQ ID NO: 2 (SEQ ID NO: 32)) containing the wild-type RBD was also used. The nucleotide sequence (3,822 bp) encoding the wild-type full-length S protein was obtained from the SARS-CoV-2 genome sequence (NCBI accession number: NC_045512), and this nucleotide sequence was codon-optimized for expression in human cells. DNA containing the nucleotide sequence of the codon-optimized full-length S protein (SEQ ID NO: 31) was artificially synthesized and inserted into a mammalian expression vector. The amino acid sequence encoded by the nucleotide sequence of the codon-optimized full-length S protein is shown in SEQ ID NO: 32. This amino acid sequence is identical to the amino acid sequence of SEQ ID NO: 2.

[0091] DNA encoding various mutant RBDs was generated by overlap extension PCR (R Higuchi, et al., Nucleic Acids Res. 1988 Aug 11; 16(15): 7351-7367). The RBD-encoding portion of the wild-type full-length S protein DNA was replaced with DNA encoding mutant RBDs to generate DNA encoding S proteins with mutant RBDs only.

[0092] The nucleotide sequences of polynucleotides encoding wild-type, alpha, beta, gamma, delta, epsilon, kappa, eta, lambda, and mu RBD polypeptides are set forth in SEQ ID NOs: 7, 9, 11, 23, 21, 15, 17, 19, 25, and 27, respectively. These polynucleotide sequences have been codon-optimized for expression in human cells.

[0093] Pseudoviruses expressing each S protein were generated using human 293T cells and a lentiviral vector system. In this example, to replace the lentiviral envelope protein with various mutant S proteins, plasmids carrying DNA encoding various mutant S proteins were transfected into human 293T cells. To evaluate the entry of pseudoviruses into cells, a plasmid carrying DNA encoding a luciferase protein was also transfected into human 293T cells. Forty-eight hours after plasmid transfection into human 293T cells, the culture supernatant was collected and centrifuged. This supernatant was collected as a pseudovirus solution and stored at -80°C until use.

[0094] 2. Titration of various pseudoviruses. A cell suspension of human 293T cells stably expressing human ACE2 was seeded in each well of a 96-well cell culture plate at 100 μL, with 10,000 cells per well, and cultured at 37°C in the presence of 5% CO2 for 24 hours. 25 μL of each pseudovirus solution was added dropwise per well and cultured for another 24 hours. After removing the culture supernatant, the pseudovirus was titrated using ONE-Glo TM The luminescence signal (Counts Per Signal: CPS) in each well was measured using EX Luciferase Assay System (Promega) and ARVO SX (PerkinElmer).

[0095] 3. Pseudovirus Neutralization Test Using Chicken Antibodies. A cell suspension of human 293T cells stably expressing human ACE2 was seeded into each well of a 96-well cell culture plate at 100 μL, with 10,000 cells per well, and cultured at 37°C and 5% CO2 for 24 hours. Based on the pseudovirus titration results, the volume of each virus was adjusted to ensure the same CPS for each pseudovirus. 25 μL of each pseudovirus was mixed with 25 μL of 300 nM (54 μg / mL) chicken antibody (IgY) solution. After allowing to stand at room temperature for 5 minutes, 50 μL of the pseudovirus and IgY mixture was added dropwise to each well and cultured at 37°C and 5% CO2 for 24 hours. After removing the culture supernatant, the ONE-Glo TM The luminescence signal (Counts Per Signal: CPS) of each well was measured using the EX Luciferase Assay System (Promega) and ARVO SX (PerkinElmer). The inhibition rate (%) of pseudovirus entry into cells was calculated based on the CPS values ​​of wells without and with chicken antibody solution.

[0096] 4. Results The results of evaluating the reactivity of the chicken antibodies of the present invention to each mutant pseudovirus are shown in Table 5 and Figure 5. When both chicken antibodies from lots #I2 and #I22 were used, a 60 to 100% inhibition rate was observed against each mutant pseudovirus at a chicken antibody concentration (final concentration) of 50 nM (9 μg / mL). In Table 5 and Figure 5, "WT" represents the wild-type RBD polypeptide, "Alpha" represents the RBD polypeptide of an alpha mutant, "Beta" represents the RBD polypeptide of a beta mutant, "Gamma" represents the RBD polypeptide of a gamma mutant, "Delta" represents the RBD polypeptide of a delta mutant, "Eta" represents the RBD polypeptide of an eta mutant, "Theta" represents the RBD polypeptide of a theta mutant, "Kappa" represents the RBD polypeptide of a kappa mutant, "Epsilon" represents the RBD polypeptide of an epsilon mutant, "Lambda" represents the RBD polypeptide of a lambda mutant, and "Mu" represents the RBD polypeptide of a mu mutant.

[0097] These results indicate that the chicken antibody of the present invention significantly inhibits the entry of all mutant pseudoviruses into human cells, that is, it has high neutralizing activity against all mutant pseudoviruses.

[0098] Neutralization test against mutant pseudovirus In this example, similar to Example 6, the chicken antibody of the present invention was tested to determine whether it has inhibitory activity against the entry of mutant pseudovirus into human cells (whether it has neutralizing activity against mutant pseudovirus).

[0099] 1. Construction of Various Mutant Pseudoviruses In this example, in addition to the polynucleotides encoding the wild-type and delta-type RBDs used in Example 6, polynucleotides encoding the Omicron-type (B.1.1.529 / BA.1) RBDs (A67V, deletion (Δ)H69-V70, T95I, G142D, ΔV143-Y145, ΔN211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, The polynucleotides used were those encoding the full-length S protein (SEQ ID NO: 34) of the Omicron-type (BA.2) strain (which has T19I, ΔL24-P26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K). As described above, in this example, the Omicron-type pseudovirus was produced using a polynucleotide encoding a full-length S protein with mutations in all regions of the S protein, not just the RBD, unlike the production of other mutant pseudoviruses.

[0100] The nucleotide sequence encoding the full-length S protein of Omicron type (B.1.1.529 / BA.1) was obtained by PCR amplification from pLV-SpikeV11 (Invivogen), and the nucleotide sequence encoding the full-length S protein of Omicron type (BA.2) was obtained by PCR amplification from pLV-SpikeV12 (Invivogen).

[0101] The nucleotide sequences of polynucleotides encoding the full-length S proteins of Omicron-type (B.1.1.529 / BA.1) and Omicron-type (BA.2) are shown in SEQ ID NOs: 33 and 35, respectively. The amino acid sequences of the RBDs of Omicron-type (B.1.1.529 / BA.1) and Omicron-type (BA.2) are shown in SEQ ID NOs: 38 and 40, respectively. In addition, the nucleotide sequences of polynucleotides encoding the RBDs of Omicron-type (B.1.1.529 / BA.1) and Omicron-type (BA.2) are shown in SEQ ID NOs: 37 and 39, respectively.

[0102] Pseudoviruses expressing each S protein were produced by the same method as described in Example 6.

[0103] 2. Titration of Various Pseudoviruses Using a method similar to that described in Example 6, titration of Omicron-type pseudoviruses was carried out.

[0104] 3. Pseudovirus Neutralization Test Using Chicken Antibodies. A cell suspension of human 293T cells stably expressing human ACE2 was seeded into each well of a 96-well cell culture plate at 10,000 cells per well (100 μL). The cells were cultured at 37°C and 5% CO2 for 24 hours. Based on the pseudovirus titration results, the volume of each virus was adjusted to ensure the same CPS. 25 μL of each pseudovirus was mixed with 25 μL of either a 300 nM (54 μg / mL) chicken antibody (IgY) solution (final concentration: 50 nM) or a 600 nM (108 μg / mL) chicken antibody (IgY) solution (final concentration: 100 nM). After leaving the plate at room temperature for 5 minutes, 50 μL of the mixture of pseudovirus and IgY was added dropwise to each well and cultured at 37°C in the presence of 5% CO2 for 24 hours. The chicken antibody of lot #12 used in Example 6 was used as the chicken antibody of the present invention. After removing the culture supernatant, ONE-Glo TMThe luminescence signal (Counts Per Signal: CPS) of each well was measured using the EX Luciferase Assay System (Promega) and ARVO SX (PerkinElmer). The inhibition rate (%) of pseudovirus entry into cells was calculated based on the CPS values ​​of wells without and with chicken antibody solution.

[0105] 4. Results The reactivity of the chicken antibodies of the present invention against Omicron-type pseudoviruses was evaluated, and the results are shown in Table 6 and Figure 6. When the #I2 chicken antibody was used at a final concentration of 50 nM (9 μg / mL), it exhibited 63.6% and 77.2% inhibition rates against the BA.1 and BA.2 Omicron-type pseudoviruses, respectively. At a final concentration of 100 nM (18 μg / mL), it exhibited 77.6% and 87.9% inhibition rates against the BA.1 and BA.2 Omicron-type pseudoviruses, respectively. In Table 6 and Figure 6, "WT" represents the wild-type full-length S protein, "Delta" represents the full-length S protein in which only the RBD has been replaced with a delta type, "Omicron(BA.1)" represents the full-length S protein of the Omicron-type (B.1.1.529 / BA.1) mutant strain, and "Omicron(BA.2)" represents the full-length S protein of the Omicron-type (BA.2) mutant strain.

[0106] These results and the results of Example 6 demonstrate that the chicken antibody of the present invention significantly inhibits the entry of all mutant pseudoviruses, including the Omicron type, into human cells, i.e., has high neutralizing activity against all mutant pseudoviruses.

[0107] Neutralization Test Against Live Virus 1. Production of Chicken Antibodies In this example, the same RBD polypeptide as in Example 1 was used as the immunogen. Boris Brown, a breed of egg-laying chickens, was used as the immunized animal. Immunization was performed by administering 10-25 μg of the RBD polypeptide per chicken together with an adjuvant into the breast muscle of the chicken. Specifically, the immunogen was administered three times every two weeks, with the fourth administration administered approximately one month after the third administration and the fifth administration administered approximately three months after the fourth administration. A total of five times (25 μg / chicken for the first time, 10 μg / chicken for the second through fourth administrations, and 25 μg / chicken for the fifth administration) were administered. To obtain chicken polyclonal antibodies on a larger scale than in Example 1, the above immunization was performed on several hundred chickens. Approximately 100 eggs were randomly selected from the eggs of several hundred immunized chickens, and these approximately 100 eggs constituted one lot. Egg yolks from each of the multiple lots were pooled (mixed), and chicken polyclonal antibodies were purified in the same manner as in Example 1. In this way, multiple lots of purified chicken polyclonal antibodies were obtained.

[0108] 2. Test for inhibiting binding between RBD polypeptide and human ACE2 The multiple lots of chicken antibodies obtained in 1 above were subjected to a test for inhibiting binding between wild-type (WT) RBD polypeptide and human ACE2 in the same manner as in Example 3, and their binding inhibitory activity was confirmed.

[0109] 3. Neutralization test against live virus (1) Chicken antibodies used and test summary In this neutralization test, several chicken antibody lots (hereinafter referred to as "IgY1" to "IgY5") and the chicken antibody lot #122 (hereinafter referred to as "IgY6") used in Examples 2 to 6 were used as representative examples from among the lots confirmed to have binding inhibitory activity in 2 above. The test method for this neutralization test was based on the "COVID-19 Serological Test Manual" of the National Institute of Infectious Diseases. Specifically, dilutions of the chicken antibody lots IgY1 to IgY6 (120-fold dilution for IgY1 to IgY5, 180-fold dilution for IgY6) and 100 TCID 50The mixture was mixed with an equal volume of SARS-CoV-2 suspension adjusted to a concentration of 50 μL / 50 μL, and incubated at 37°C for 1 hour, after which the neutralizing antibody titers of IgY1 to IgY6 were measured.

[0110] (2) Viruses Used In this example, "live virus" refers to a virus that can infect and replicate in living cells. In this example, the following virus strains were used as live SARS-CoV-2 viruses: (i) SARS-CoV-2 JPY / JPY / TY / WK-521 wild-type strain (ii) SARS-CoV-2 QHN001 (VOC-202012 / 01 lineage strain) alpha mutant strain (iii) SARS-CoV-2 TY8-612 (B.1.351 lineage strain) beta mutant strain (iv) SARS-CoV-2 TY7-503 (501Y.V3, P.1 lineage strain) gamma mutant strain (v) SARS-CoV-2 TY11-927 (B.1.617.2 lineage strain) delta mutant strain In addition, VeroE6 / TMPRSS2 cells (JCRB1819) were used in this example.

[0111] (3) Preparation of virus suspension The SARS-CoV-2 virus suspension was prepared as follows: (i) VeroE6 / TMPRSS2 cells were cultured in monolayer in tissue culture flasks using cell growth medium [Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418) (1 mg / mL)]. (ii) After cell culture, the cell growth medium was removed from the flasks, and various SARS-CoV-2 strains were inoculated. Cell maintenance medium [Dulbecco's Modified Eagle's Medium (Nacalai Tesque, Inc.) supplemented with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418) (1 mg / mL)] was added and the cells were cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. (iii) After 3 days of culture, cell morphology was observed using an inverted phase-contrast microscope to confirm the occurrence of morphological changes (cytopathic effect: CPE). The culture medium was then centrifuged (3500 rpm / min, 10 min), and the resulting supernatant was used as the test virus suspension stock solution.

[0112] (4) Measurement of Neutralizing Antibody Titers. VeroE6 / TMPRSS2 cells were grown in monolayers in tissue culture microplates (96-well flat-bottom) using cell growth medium. The cells were then washed with cell maintenance medium and the cell maintenance medium was removed. Next, 100 μL of serially diluted IgY1 to IgY6 solutions (1.5x) was inoculated into four wells and cultured at 37°C ± 1°C in a carbon dioxide incubator (5% CO2 concentration) for 3 days. After incubation, the presence or absence of cytopathic effect (CPE) was observed using an inverted phase-contrast microscope. The supernatant was removed, and the cells were fixed and washed with water. The fixed cells were stained and washed with water, and the highest dilution factor at which CPE was suppressed in two or more wells was calculated. The antibody concentration required for neutralization was calculated from the highest dilution factor and the antibody amount, and this was used as the neutralizing antibody titer. This test was repeated twice. As a result, IgY1 to IgY6 all exhibited neutralizing activity against all of the live viruses listed above. Among these, the neutralizing antibody titers (μg / ml) for IgY2 and IgY4 are shown in Table 7 and FIG. 7 as representative examples.

[0113] These results demonstrated that the chicken antibodies of the present invention have neutralizing activity against live SARS-CoV-2 mutant viruses, and are therefore highly effective in treating or preventing coronavirus infections caused by wild-type or mutant SARS-CoV-2 strains.

[0114] Neutralization Test Against Live Virus 1. Preparation of Chicken Antibody The chicken antibody used in this example was prepared by the same method as that described in Example 8.

[0115] 2. Test for inhibiting binding between RBD polypeptide and human ACE2 The multiple lots of chicken antibodies obtained in 1 above were subjected to a test for inhibiting binding between wild-type (WT) RBD polypeptide and human ACE2 in the same manner as in Example 3, and their binding inhibitory activity was confirmed.

[0116] 3. Neutralization test against live virus (1) Chicken antibodies used and test summary In this neutralization test, several chicken antibody lots (hereinafter referred to as "IgY7" to "IgY10") and the chicken antibody lot #122 (IgY6) used in Examples 2 to 8 were used as representative examples from among the lots confirmed to have binding inhibitory activity in 2 above. The test method for this neutralization test was based on the "COVID-19 Serological Test Manual" of the National Institute of Infectious Diseases. Specifically, diluted solutions (150-fold dilution) of the above chicken antibody lots IgY6 to IgY10 and 100 TCID 50 The antibody titers of IgY6 to IgY10 were measured after mixing an equal volume of the SARS-CoV-2 suspension with a concentration adjusted to 50 μL / 50 μL and incubating at 37°C for 1 hour.

[0117] (2) Viruses Used In this example, "live virus" refers to a virus that can infect and replicate in living cells. In this example, the following virus strains were used as live SARS-CoV-2 viruses: (i) SARS-CoV-2 JPY / JPY / TY / WK-521 wild-type strain; (ii) SARS-CoV-2 TY11-927 (B.1.617.2 lineage strain) delta mutant strain; and (iii) SARS-CoV-2 TY38-873 (BA.1 lineage strain) omicron mutant strain. VeroE6 / TMPRSS2 cells (JCRB1819) were used in this example.

[0118] (3) Preparation of virus suspension The SARS-CoV-2 virus suspension was prepared as follows: (i) VeroE6 / TMPRSS2 cells were cultured in monolayer in tissue culture flasks using cell growth medium [Dulbecco's modified Eagle's medium (Nacalai Tesque) supplemented with 10% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418) (1 mg / mL)]. (ii) After cell culture, the cell growth medium was removed from the flasks, and various SARS-CoV-2 strains were inoculated. Cell maintenance medium [Dulbecco's Modified Eagle's Medium (Nacalai Tesque, Inc.) supplemented with 2% fetal bovine serum, penicillin (100 U / mL), streptomycin (100 μg / mL), and geneticin (G418) (1 mg / mL)] was added and the cells were cultured for 3 days in a carbon dioxide incubator (CO2 concentration: 5%) at 37°C ± 1°C. (iii) After 3 days of culture, cell morphology was observed using an inverted phase-contrast microscope to confirm the occurrence of morphological changes (cytopathic effect: CPE). The culture medium was then centrifuged (3500 rpm / min, 10 min), and the resulting supernatant was used as the test virus suspension stock solution.

[0119] (4) Measurement of Neutralizing Antibody Titers. VeroE6 / TMPRSS2 cells were grown in monolayers in tissue culture microplates (96-well flat-bottom) using cell growth medium. The cells were then washed with cell maintenance medium and the cell maintenance medium was removed. Next, 100 μL of serially diluted IgY7 to IgY11 solutions (1.5x) was inoculated into four wells and cultured at 37°C ± 1°C in a carbon dioxide incubator (5% CO2) for 3 days. After incubation, the presence or absence of cytopathic effect (CPE) was observed using an inverted phase-contrast microscope. The supernatant was removed, and the cells were fixed and washed with water. The fixed cells were stained and washed with water, and the highest dilution factor at which CPE was suppressed in two or more wells was calculated. The antibody concentration required for neutralization was calculated from the highest dilution factor and the antibody amount, and this was used as the neutralizing antibody titer. This test was repeated twice. As a result, IgY6 to IgY10 all exhibited neutralizing activity against all of the live viruses listed above. As a representative example, the neutralizing antibody titer (μg / ml) for IgY6 (lot #122) is shown in Table 8 and FIG.

[0120]

[0121] These results and those of Example 8 demonstrate that the chicken antibodies of the present invention have neutralizing activity against live SARS-CoV-2 mutant strains, including the Omicron strain, and are therefore highly effective in treating or preventing coronavirus infections caused by wild-type or mutant strains of SARS-CoV-2.

[0122] Superiority of Chicken Antibodies over Antibodies Derived from Other Animal Species In this example, we tested whether chicken antibodies exhibit superior inhibitory activity against the binding of mutant RBD polypeptides to human ACE2 compared with antibodies derived from other animal species. Specifically, in this example, (i) chicken antibodies of the present invention, (ii) plasma from chickens immunized with RBD polypeptides, (iii) mouse plasma, and (iv) plasma from vaccinated individuals and individuals infected before the Omicron epidemic were used to test the inhibitory (neutralizing) activity of these antibodies or plasma against the binding of mutant RBD polypeptides to human ACE2, as in Examples 3 to 5. In this test, the IC50 values ​​of the antibodies or plasma (i) to (iv) above against the wild-type RBD polypeptide were set to "1," and the ratios of the IC50 values ​​of the antibodies or plasma against the mutant RBD polypeptides were calculated and compared. The IC50 value of plasma indicates the dilution ratio at which 50% inhibitory activity is observed, and therefore, a higher dilution ratio, i.e., a higher IC50 value, indicates higher inhibitory activity. For the chicken antibodies of the present invention described in (i) above, IC50 values ​​were calculated based on the dilution ratio, as with plasma. Delta and omicron (BA.1) RBD polypeptides were used as mutant RBD polypeptides. The wild-type (WT) RBD polypeptide used in Examples 2 to 5 was also used. Ten different lots of chicken antibodies purified by the same method as in Example 8 were used as chicken antibodies of the present invention. Plasma obtained by immunization with RBD polypeptides was obtained from 13 mice and 12 chickens. Human plasma samples were obtained from 15 individuals. The binding inhibition test between the mutant RBD polypeptide and human ACE2 was performed using the same method as described in Examples 3 to 5. Mouse plasma and chicken plasma were used by serially diluting the 64-fold diluted solution in two-fold increments. Human plasma was used by serially diluting the 2-fold diluted solution in two-fold increments. In addition, in this example, the inhibition rates of the antibodies of the present invention and plasma against the binding of mutant RBD polypeptides to human ACE2 were calculated. In calculating the inhibition rate, wells to which only each RBD polypeptide was added were used as positive controls, and wells to which only the antibody of the present invention or plasma was added were used as negative controls.

[0123] Mouse and human plasma showed comparable inhibitory activity against the delta RBD polypeptide to that of the wild-type, but tended to exhibit reduced inhibitory activity against the omicron RBD polypeptide. In contrast, chicken IgY and chicken plasma tended to exhibit comparable inhibitory activity against the delta and omicron RBD polypeptides compared to the wild-type.

[0124] The average results for each sample are shown in Figure 9 and Table 9. Chicken antibody (IgY) and chicken plasma showed comparable inhibitory activity against the delta and omicron (BA.1) RBD polypeptides compared to the wild-type. On the other hand, mouse and human plasma showed reduced activity against the omicron RBD polypeptide. Table 9 shows the average relative IC50 values ​​of each antibody or plasma against the delta and omicron (BA.1) RBD polypeptides, with the IC50 value of each antibody or plasma against the wild-type RBD polypeptide set at "1."

[0125]

[0126] The IC50 values ​​for the delta and Omicron forms were 0.95 and 0.97, respectively, using chicken IgY antibodies, respectively. The IC50 values ​​for the delta and Omicron forms were 1.03 and 1.16, respectively, using chicken plasma. The IC50 values ​​for the delta and Omicron forms were 1.03 and 0.74, respectively, using mouse plasma. The IC50 values ​​for the delta and Omicron forms were 1.02 and 0.68, respectively, using human plasma. The delta form, which contains two mutations in the RBD polypeptide, showed no decrease in inhibitory activity in chicken, mouse, or human. However, the Omicron form, which contains 15 mutations, showed decreased inhibitory activity in both mouse and human.

[0127] These results indicate that the chicken antibodies of the present invention have (can maintain) neutralizing activity against the binding of the spike protein RBD of a wider variety of mutant coronaviruses to ACE2, compared to antibodies obtained by immunization of other species.

[0128] The present invention provides a novel chicken antibody capable of binding to the spike protein of a mutant coronavirus.

[0129] SEQ ID NOs: 7 to 40: synthetic DNA or synthetic peptide

Claims

1. A chicken antibody that binds to the spike protein of a mutant coronavirus.

2. The chicken antibody of claim 1, which is capable of neutralizing the binding of the receptor binding domain (RBD) of the spike protein of a mutant coronavirus to angiotensin-converting enzyme 2 (ACE2).

3. The chicken antibody of claim 1, wherein the mutant coronavirus infects cells via ACE2.

4. The chicken antibody of claim 1, which further binds to the spike protein of a wild-type coronavirus.

5. The chicken antibody of claim 1, which is a polyclonal antibody.

6. A composition for treating or preventing coronavirus infection, comprising the chicken antibody according to any one of claims 1 to 5.

7. The composition of claim 6, wherein the coronavirus infection is a disease or symptom caused by infection with a coronavirus that infects cells via ACE2.

8. 8. The composition of claim 7, wherein the disease or symptom is at least one selected from the group consisting of respiratory disease, fever, fatigue, chills, pain, taste or smell disorders, rash, gastrointestinal symptoms, speech disorders, cognitive disorders, and cardiovascular symptoms.

9. An inhibitor of the binding between the RBD of the spike protein of a mutant coronavirus and ACE2, comprising the chicken antibody described in any one of claims 1 to 5.

10. A reagent or kit comprising the chicken antibody according to any one of claims 1 to 5.

11. 6. The chicken antibody according to claim 1, or a composition comprising the same, for use in treating or preventing coronavirus infection.

12. Use of the chicken antibody according to any one of claims 1 to 5 in the manufacture of a medicament for treating or preventing coronavirus infection.