Multiple antigen peptides against coronavirus and immunostimulatory compositions containing the same

Abstract

The present invention provides a vaccine against a coronavirus. Specifically, the present invention provides a peptide containing the amino acid sequence set forth in SEQ ID NO: 1 or a partial peptide of the spike protein of a coronavirus containing an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, and a multiple antigenic peptide containing a plurality of either of these peptides.

Description

[Technical Field]

[0001] This invention relates to a multi-antigen peptide for coronaviruses and an immunostimulatory composition containing the same, particularly to a vaccine for coronaviruses. [Background technology]

[0002] It has long been known that antibody levels can be induced in vivo without the involvement of T cells by multiple antigen peptides (MAPs) (Non-Patent Document 1). It has been shown that MAPs can induce autoantibodies (anti-IgE antibodies) that are difficult to induce in vivo (Patent Document 1). Furthermore, it has been disclosed that by using this method, a panviral MAP can be created by producing a peptide portion of the Ebola hemorrhagic fever virus, and the target antibody can be induced in the serum of mice by immunizing them (Patent Document 2). Similarly, it has been disclosed that a panviral MAP can be created using a partial peptide of hemagglutinin from the influenza virus, and the target antibody can be induced in the serum of mice by immunizing them (Patent Document 3). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] US2017-0158738A [Patent Document 2] US2019-0276495A [Patent Document 3] US2019-0337989A [Non-patent literature]

[0004] [Non-Patent Document 1] Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004 [Overview of the project]

[0005] The present invention provides a multi-antigen peptide against coronavirus and an immunostimulatory composition containing the same, particularly a vaccine against coronavirus. According to the present invention, the present invention provides a peptide consisting of the amino acid sequence described in SEQ ID NO: 1, or a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 (a peptide consisting of an amino acid sequence of the coronavirus spike protein corresponding to an amino acid sequence of 11 to 21 amino acids including the amino acid sequence described in SEQ ID NO: 1), and a multi-antigen peptide containing any multiple of these peptides.

[0006] The inventors of this invention have found that a multi-antigen peptide (MAP) having a peptide consisting of the amino acid sequence described in SEQ ID NO: 1, or a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, induced antigen-specific IgM. They also found that this antigen-specific IgM could be maintained in the blood for a long period of time. Furthermore, they revealed that the MAP also induces memory immunity. Moreover, they found that the MAP can directly stimulate antibody production from marginal zone B cells and B1B cells, and induce T cell-independent antibody production. The inventors have found that this multi-antigen peptide induces antibody production against a wide range of coronaviruses. Furthermore, in infection experiments with feline infectious peritonitis virus (FIPV) in cats as an example of a highly pathogenic coronavirus, the inventors found that administering the multi-antigen peptide of this invention early after the onset of symptoms (for example, within 3 days after confirmation of high fever) (in most cases, the initial symptom is high fever) prevented the infection from becoming severe and also had the effect of promoting recovery.

[0007] This invention provides the following: [1] A peptide consisting of a part of the amino acid sequence described in Sequence ID No. 3, comprising a continuous amino acid sequence of 11 to 21 amino acids in length including the amino acid sequence described in Sequence ID No. 1, or a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the 11 to 21 amino acid sequence (or a peptide consisting of the amino acid sequence of the coronavirus spike protein corresponding to the 11 to 21 amino acid sequence). [2] A peptide as described in [1] above, comprising the amino acid sequence described in SEQ ID NO: 1, or a partial peptide of the coronavirus spike protein comprising the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 (or a peptide comprising the amino acid sequence of the coronavirus spike protein corresponding to the 11-21 amino acid sequence). [3] The peptide according to [2] above, wherein the amino acid sequence corresponding to SEQ ID NO: 1 has one of the following: addition, insertion, substitution, or deletion of a single base to the amino acid sequence described in SEQ ID NO: 1. [4] The peptide according to [2] or [3] above, wherein the amino acid sequence corresponding to SEQ ID NO: 1 is a peptide consisting of the amino acid sequence described in SEQ ID NO: 2. [5] A multi-antigen peptide containing any of the peptides described in [1] to [4] above. [6] The multi-antigen peptide according to [5] above, comprising four or more peptides having the amino acid sequence described in any of [1] to [4] above or the sequence number 7 above. [7] A vaccine against coronavirus, comprising the multi-antigen peptide described in [6] above. [8] The vaccine described in [7] above, which does not contain an adjuvant. [9] The vaccine described in [6] or [7] above, used in combination with alpha-galactosylceramide and not in combination with other adjuvants.

[10] A vaccine as described in any of the above [7]-[9], wherein the coronavirus is SARS-CoV-2 or a variant of SARS-CoV-2.

[11] A method for activating immunity against coronavirus in a subject in need, comprising administering to the subject an effective amount of the multi-antigen peptide described in [5] or [6] above.

[0008] [1A] A peptide consisting of a continuous amino acid sequence of 11 to 21 amino acids in length, including the amino acid sequence described in Sequence ID No. 1, or a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the 11 to 21 amino acid sequence (or a peptide consisting of the amino acid sequence of the coronavirus spike protein corresponding to the 11 to 21 amino acid sequence). [2A] A peptide as described in [1A] above, comprising the amino acid sequence described in SEQ ID NO: 1, or a partial peptide of the coronavirus spike protein comprising the amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 (or a peptide comprising the amino acid sequence of the coronavirus spike protein corresponding to the 11-21 amino acid sequence). [3A] The peptide according to [2A], wherein the amino acid sequence corresponding to Sequence ID No. 1 has one of the following: addition, insertion, substitution, or deletion of one base to the amino acid sequence described in Sequence ID No. 1. [4A] The peptide according to [2A] or [3A], wherein the amino acid sequence corresponding to Sequence ID No. 1 is a peptide consisting of the amino acid sequence described in Sequence ID No. 2. [5A] A partial peptide as described in [1A] or [2A] above, which is part of the amino acid sequence described in Sequence ID No. 3. [6A] A multi-antigen peptide containing any of the peptides described in [1A] to [5A] above. [7A] A multi-antigen peptide according to [6A] above, comprising four or more peptides having the amino acid sequence described in any of [1A] to [5A] above or the sequence number 7 above. [8A] The multi-antigen peptide according to [6A] or [7A] above, wherein the multi-antigen peptide comprises a dendritic polymer backbone and a peptide, the peptide being the peptide described in any one of claims 1 to 5 and linked to the terminal end of the dendritic polymer backbone. [9A] The multi-antigen peptide described in [8A] above, wherein a dendritic polymer backbone and a peptide are linked via a linker. [10A] The dendritic polymer skeleton is a lysine molecule, and a lysine molecule that forms a first-generation branch is peptide-bonded to each of the two amino groups of the lysine molecule, and a peptide is linked to each of the four amino groups of the lysine molecule that forms the first-generation branch, with or without a linker. This is the multiple antigen peptide described in [8A] or [9A] above. [11A] The dendritic polymer skeleton is a lysine molecule, to which a lysine molecule forming a first-generation branch is peptide-bonded to each of the two amino groups of the lysine molecule, to which a lysine molecule forming a first-generation branch is peptide-bonded to each of the four amino groups of the resulting first-generation lysine molecule, to which a lysine molecule forming a second-generation branch is peptide-bonded to each of the five, six, seven, or eight amino groups of the resulting second-generation branching lysine molecule, and to which a peptide is linked, with or without the linker, as described in [8A] or [9A] above. [12A] A multi-antigen peptide having the following formula (VI), as described in any of [6A] to "10A" above: TIFF0007854660000001.tif116170{where R is a -linker-peptide or -peptide, and the peptide is one of the peptides described in any of [1A] to [5A] above, R 2 These are hydrogen, an OH group, a substituted or unsubstituted lower alkyl group, an amino group, an amino acid (especially 3-aminopropanoic acid (β-alanine)), a halogen, or a peptide, and the amino acid is linked to the above molecule via an amide bond. [13A] A vaccine against coronavirus containing the multi-antigen peptide described in any of [6A] to [12A] above. [14A] The vaccine according to [13A] above, which does not contain an adjuvant. [15A] The vaccine according to [13A] or [14A] above, which is used in combination with α-galactosylceramide and not in combination with other adjuvants. [16A] The vaccine according to any one of [13A] to [15A] above, wherein the coronavirus is SARS-CoV-2 or a mutant virus thereof. [17A] The vaccine according to any one of [13A] to [15A] above, wherein the coronavirus is one or more coronaviruses selected from porcine epidemic diarrhea virus (PED), canine coronavirus, and feline infectious peritonitis virus (FIPV). [18A] A pharmaceutical composition comprising the multivalent antigen peptide according to any one of [6A] to [12A] above. [19A] The pharmaceutical composition according to [18A] above, which is used for activating immunity against coronavirus in a subject. [20A] The pharmaceutical composition according to [18A] or

[19] above, which is used for inducing antigen-specific immunity against coronavirus in a subject. [21A] The pharmaceutical composition according to any one of [18A] to [20A] above, which is used for inducing antigen-specific IgM antibodies against coronavirus in a subject. [22A] The pharmaceutical composition according to any one of [18A] to [21A] above, which is used for inducing memory immunity against coronavirus in a subject. [23A] The pharmaceutical composition according to any one of [18A] to [22A] above, which is used for preventing and / or treating an infectious disease caused by coronavirus in a subject. [24A] Use of the multivalent antigen peptide according to any one of [6A] to [12A] above in the manufacture of a medicament for activating immunity against coronavirus in a subject. [25A] Use of the multivalent antigen peptide according to any one of [6A] to [12A] above in the manufacture of a medicament for inducing antigen-specific immunity against coronavirus in a subject. Use of the multiple antigen peptide according to any one of [6A] to [12A] in the manufacture of a medicament for inducing an antigen-specific IgM antibody against the coronavirus in a subject. Use of the multiple antigen peptide according to any one of [6A] to [12A] in the manufacture of a medicament for inducing memory immunity against the coronavirus in a subject. Use of the multiple antigen peptide according to any one of [6A] to [12A] in the manufacture of a medicament for preventing and / or treating an infection caused by the coronavirus in a subject. [29A] A method for administering a peptide to a subject, The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. [30A] A method for activating immunity against the coronavirus in a subject, The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. [31A] A method for inducing antigen-specific immunity against the coronavirus in a subject, The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. [32A] A method for inducing an antigen-specific IgM antibody against the coronavirus in a subject, [[ID=I9]] The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. [33A] A method for inducing memory immunity against the coronavirus in a subject, The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. [34A] A method for preventing and / or treating an infection caused by the coronavirus in a subject, [[ID=I8]]The method comprising administering an effective amount of the pharmaceutical composition according to [18A] to the subject. <9000108>

[0009] [35A] A pharmaceutical composition according to any of

[19] to

[23] above, wherein the subject is not infected with the coronavirus. [36A] A pharmaceutical composition according to any of

[19] to

[23] above, wherein the subject is a subject infected with the coronavirus. [37A] A pharmaceutical composition according to any of

[19] to

[23] above, wherein the subject is a subject who has developed an infection caused by the coronavirus. [38A] Use as described in any of

[24] to

[28] above, wherein the subject is not infected with the coronavirus. [39A] Use as described in any of

[24] to

[28] above, wherein the subject is infected with the coronavirus. [40A] Use as described in any of

[24] to

[28] above, wherein the subject is a person who has developed an infection caused by the coronavirus. [41A] The method described in any of the above

[29] to

[34] , wherein the subject is not infected with the coronavirus. [42A] The method described in any of the above

[29] to

[34] , wherein the subject is infected with the coronavirus. [43A] The method described in any of the above

[29] to

[34] , wherein the subject is a person who has developed an infection caused by the coronavirus. [44A] The pharmaceutical composition according to any one of

[19] to

[23] above, wherein the coronavirus is one or more coronaviruses selected from porcine epidemic diarrhea virus (PED), canine coronavirus, and feline infectious peritonitis virus (FIPV). [45A] Uses described in any of the above

[24] -

[28] , wherein the coronavirus is one or more coronaviruses selected from porcine epidemic diarrhea virus (PED), canine coronavirus, and feline infectious peritonitis virus (FIPV). [46A] The method according to any of the above

[29] -

[34] , wherein the coronavirus is one or more coronaviruses selected from porcine epidemic diarrhea virus (PED), canine coronavirus, and feline infectious peritonitis virus (FIPV).

[0010] The multi-antigen peptide of the present invention is advantageous in that it induces antibody production against coronavirus. The multi-antigen peptide of the present invention may be advantageous in that memory immunity is established after administration, and antibody production is further enhanced by viral infection. The multi-antigen peptide of the present invention is also advantageous in that it stimulates marginal zone B cells and B1B cells to produce antibodies in a T cell-independent manner, rather than on helper T cells or follicular B cells, and the produced antibodies are thought to prevent antibody-dependent enhancement of infection. The multi-antigen peptide of the present invention may be advantageous in that it is also effective against highly pathogenic coronaviruses. The multi-antigen peptide of the present invention may be advantageous in that it is effective in both prophylactic and therapeutic measures. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 illustrates the mechanism of immunostimulation by conventional viral vaccines (i.e., inactivated viral vaccines, recombinant protein vaccines, DNA vaccines, and RNA vaccines). Conventional vaccines require adjuvants for immunostimulation. When used in combination with adjuvants, the vaccine is phagocytosed by macrophages, antigens are presented to helper T cells, and helper T cells are activated. Activated helper T cells stimulate follicular B cells that express B cell receptors with affinity for viral antigens, causing IgG production from follicular B cells in a T cell-dependent manner. IgM produced by follicular B cells causes a transient increase in blood concentration but disappears within about two weeks, while IgG remains in the blood for a long period, but mainly consists of subtypes that bind to IgG receptors on macrophages. Therefore, IgG produced by follicular B cells can cause antibody-dependent enhancement (ADE) during viral infection, promoting viral infection and potentially leading to severe symptoms. [Figure 2]Figure 2 shows a schematic diagram of the molecular structure of the coronavirus vaccine of the present invention (i.e., the multi-antigen peptide) and illustrates that the multi-antigen peptide stimulates marginal zone B cells and B1B cells. Marginal zone B cells and B1B cells produce IgM and IgG independently of helper T cells upon stimulation by the multi-antigen peptide. The IgM produced in this T-cell-independent manner remains in the bloodstream for a long period, and the IgG does not contain subtypes that bind to the IgG receptor. Therefore, the coronavirus vaccine of the present invention, which induces IgM and IgG production in marginal zone B cells and B1B cells rather than follicular B cells, is suggested to provide long-term viral protection without causing antibody-dependent enhancement of infection. [Figure 3] Figure 3 shows a portion of the amino acid sequence alignment of the spike proteins of SARS (SARS-CoV), which was prevalent in 2003, feline infectious peritonitis virus (FIPV), and SARS (SARS-CoV-2), which began to spread in 2019. The peptide of the present invention was designed for the shaded area. [Figure 4] Figure 4 shows an experimental scheme for the administration of the multi-antigen peptide (CoV-MAP) of the present invention and subsequent administration of inactivated virus simulating infection. [Figure 5] Figure 5 shows the results of an ELISA experiment, which reveals the amount of IgM antibodies against canine coronavirus in serum obtained from the administration experiment shown in Figure 4. [Figure 6] Figure 6 shows the results of an ELISA experiment, which reveals the amount of IgG antibodies against canine coronavirus in serum obtained from the administration experiment shown in Figure 4. [Figure 7a] Figure 7a shows an experimental scheme for the administration of the multi-antigen peptide (CoV-MAP) of the present invention and subsequent administration of inactivated virus simulating infection. CoV-MAP was administered in two doses (200 μg / mouse / inoculation). [Figure 7b] Figure 7b shows the ELISA results indicating the amount of IgG antibodies against porcine coronavirus (PDE) in serum obtained from the experiment in Figure 7a. [Figure 8a]Figure 8a shows the results of an ELISA (immobilized D-peptide) in mice administered D-CoV-β-MAP, which is a peptide of CoV-MAP composed of D-amino acids. [Figure 8b] Figure 8b shows the results of an ELISA (L-peptide immobilized) in mice administered D-CoV-β-MAP, which is a peptide of CoV-MAP composed of D-amino acids. [Figure 8c] Figure 8c shows the results of an ELISA (D-peptide immobilized) of serum IgG levels in mice administered D-CoV-β-MAP, which is a peptide of CoV-MAP composed of D-amino acids. [Figure 8d] Figure 8d shows the results of an ELISA (L-peptide immobilized) of serum IgG levels in mice administered D-CoV-β-MAP, which is a peptide of CoV-MAP composed of D-amino acids. [Figure 9] Figure 9 shows the administration scheme in which an octavalent CoV-MAP peptide is administered intravenously three times, followed by canine coronavirus antigen boost immunization. Cont-MAP is the first negative control and is a MAP containing a partial peptide of the influenza virus. MAP was administered intravenously in three divided doses (days 0, 7, and 25) at a dose of 200 μg / mouse. In the second dose, in addition to MAP, α-GalCer, a CD1d ligand, was administered intravenously at a dose of 0.1 μg / mouse. Boost immunization was administered intraperitoneally. As a second negative control, a peptide (short-chain peptide) having the amino acid sequence described in Sequence ID No. 8 was conjugated to keyhole limpet hemocyanin (KHL) and mixed with alum, administered intraperitoneally at a dose of 100 μg / mouse. [Figure 10] Figure 10 shows the results of the experiment using the immunization program shown in Figure 9. Figure 10 shows the amount of IgM produced in mouse serum in response to the short-chain peptides after a series of immunization programs. The vertical axis represents the amount of IgM produced as optical density (OD450 value). The horizontal axis represents the number of days elapsed since the first administration. [Figure 11]Figure 11 shows the results of the experiment using the immunization program described in Figure 9. Figure 11 shows the amount of IgM produced in mouse serum in response to canine coronavirus antigen after a series of immunization programs. The vertical axis represents the amount of IgM produced as optical density (OD450 value). The horizontal axis represents the number of days elapsed since the first dose. [Figure 12] Figure 12 shows the results of the experiment using the immunization program described in Figure 9. Figure 12 shows the amount of IgM produced in mouse serum in response to the porcine epidemic diarrhea virus (PDE) antigen after a series of immunization programs. The vertical axis represents IgM production as optical density (OD450 value). The horizontal axis represents the number of days elapsed since the first dose. [Figure 13] Figure 13 shows the results of the experiment using the immunization program described in Figure 9. Figure 13 shows the production of IgM in mouse serum after a series of immunization programs, in response to SARS-CoV-2 spike protein antigen, MERS spike protein antigen, and SARS-CoV-2 spike protein (S2). The vertical axis represents IgM production as optical density (OD450 value). The horizontal axis represents the number of days elapsed since the first dose. [Figure 14] Figure 14 shows the results when quadrivalent CoV-MAP peptides were administered using the same scheme as in Figure 9. Figure 14 shows the levels of IgM production in mouse serum after a series of immunization programs for short-chain peptides, porcine epidemic diarrhea virus antigen, and canine coronavirus antigen, respectively. [Figure 15] Figure 15 shows the production of various IgM molecules in animals administered MAP containing the octvalent No. 2 peptide (a peptide having the amino acid sequence described in SEQ ID NO: 8, but with all constituent amino acids being D-isomers) using the same scheme as in Figure 9. [Figure 16] Figure 16 shows the production of various IgMs in animals administered MAP containing the octvalent No. 3 peptide (a peptide having the amino acid sequence described in SEQ ID NO: 8, but with the italicized amino acid being the D-form) using the same scheme as in Figure 9. [Figure 17]Figure 17 shows the production of various IgMs in animals administered MAP containing the octavalent No. 4 peptide (a peptide having the amino acid sequence described in SEQ ID NO: 8, but with the italicized amino acid being the D-form) using the same scheme as in Figure 9. [Figure 18] Figure 18 shows the production of various IgMs in animals administered MAP containing the octvalent No. 5 peptide (a peptide having the amino acid sequence described in SEQ ID NO: 2, but with the italicized amino acid being the D-form) using the same scheme as in Figure 9. [Figure 19] Figure 19 shows the production of various IgM molecules in animals administered MAP containing the octavalent No. 6 peptide (a peptide having the amino acid sequence described in SEQ ID NO: 1) using the same scheme as in Figure 9. [Figure 20] Figure 20 shows a comparison of the results for peptides No. 1 through No. 6. [Figure 21] Figure 21 shows that the amount of IgM induced by the CoV-MAP peptide (peptide No. 1) is maintained over the long term. In Figure 21, the serum IgM levels for various antigens on day 137 of administration are shown as a ratio to the IgM level before treatment. [Figure 22] Figure 22 shows the results of the ELISPOT method, demonstrating that IgM levels induced by the CoV-MAP peptide are maintained over the long term. The vertical axis represents the number of cells in the spleen cells that produce IgM antibodies against the peptide. JEV-MAP is a negative control and is an octavalent MAP containing a peptide of the Japanese encephalitis virus E protein. [Figure 23] Figure 23 shows the amount of CoV-MAP taken up by macrophages. Anti-FLAG tagged monoclonal antibodies were used as positive controls. Serum from untreated mice was used as a negative control. [Figure 24] Figure 24 shows the results of comparing the production of various IgGs before and after boost immunization in animals administered CoV-MAP using the same scheme as in Figure 9. [Figure 25]Figure 25 shows the results of comparing the production of various IgGs before and after boost immunization in animals administered CoV-MAP using the same scheme as in Figure 9. [Figure 26] Figure 26 shows the subclasses of IgG produced. [Figure 27] Figure 27 shows experiments involving pre- and post-symptomatic administration of CoV-MAP in feline infectious peritonitis virus (FIPV) infection experiments. [Figure 28] Figure 28 shows that the CoV-MAP administration group exhibited a vaccine effect against FIPV infection and showed an antipyretic effect. Detailed description of the invention

[0012] In this specification, “subject” means an animal having an immune system, and may be, for example, a vertebrate, such as mammals, fish, birds, amphibians, reptiles, such as primates such as humans, chimpanzees, gorillas, orangutans, monkeys, marmosets and bonobos, tetrapods such as pigs, rats, mice, cattle, sheep, goats, horses, cats and dogs (e.g., carnivores, artiodactyls, odd-toed ungulates and rodents), and animals of the order Chiroptera, such as bats.

[0013] In this specification, "coronavirus" refers to a virus belonging to the family Coronaviridae in the order Nidovirales. Coronaviruses are named as such because they have multiple spikes (S proteins) on the envelope structure of the surface of the virus particle, and their image observed under an electron microscope resembles the sun's corona. In humans, they are known to cause respiratory infections such as the common cold, and SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), and the 2019 novel coronavirus (SARS-CoV-2) are lethal. Lethal viruses such as mouse hepatitis virus (MHV) and feline infectious peritonitis virus (FIPV) are also known. As of April 2020, no vaccines or antiviral drugs to prevent or treat human coronavirus infection have been developed. Coronaviruses bind to the surface of target cells by binding of the spike protein exposed on the envelope surface to the cell surface molecule angiotensin-converting enzyme 2 (ACE2), and then infect cells by being taken into the cell by endocytosis. Examples of coronaviruses include coronaviruses of the Coronavirinae subfamily, alphacoronaviruses (e.g., canine coronavirus, alphacoronavirus 1, human coronavirus 229E, human coronavirus NL63, porcine epidemic diarrhea virus), betacoronaviruses (e.g., Embecovirus subgenus, Salvecovirus subgenus, Merbecovirus subgenus, Novecovirus subgenus, e.g., human enteric coronavirus 4408, human coronavirus OC43, mouse coronavirus, human coronavirus HKU1, SARS-related coronaviruses (e.g., SARS coronavirus (SARS-CoV), 2019 novel coronavirus (SARS-CoV-2), MERS coronavirus, equine coronavirus), gammacoronaviruses (e.g., avian coronavirus, beluga whale coronavirus SW1), and deltacoronaviruses (e.g., bulbul coronavirus HKU11, munia coronavirus HKU13, thrush coronavirus HKU12).Also included are mutant virus strains (in particular, mutant viruses having an amino acid sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 1, for example, mutant viruses having an amino acid sequence with the addition, insertion, deletion, or substitution of one to two (preferably one) amino acids to the amino acid sequence of SEQ ID NO: 1 or 2 (for example, conservative substitutions: substitutions between acidic amino acids, substitutions between basic amino acids, and substitutions between hydrophobic amino acids, etc.)).

[0014] In this specification, "adjuvant" refers to a substance that stimulates the immune system. Aluminum hydroxide and aluminum phosphate are commonly used as adjuvants.

[0015] In this specification, "α-galactosylceramide" is a sphingoglycolipid that can be isolated from Agelas mauritianus, a type of sponge. α-galactosylceramide has the following structure.

[0016] [ka]

[0017] Alpha-galactosylceramide is a ligand that binds to CD1d and activates NKT cells.

[0018] In this specification, "multi-antigen peptide" (MAP) refers to a molecule that enables the multiple presentation of peptides having a specific amino acid sequence. A multi-antigen peptide may have a structure in which peptides are linked to the side chains of a repeating backbone. The backbone and peptides may be linked via a linker. Examples of multi-antigen peptides include those having a dendritic polymer backbone. An example of a multi-antigen peptide having a dendritic polymer backbone is a molecule that contains lysine (Lys) as a core, and the number of branches is increased by further linking of lysine to the core via peptide bonds, with n being the number of branches and the amino group and the peptide linked thereto being 2 nExamples include those having up to n elements {where n is a natural number greater than or equal to 2} (Francis, JP, et al., Immunology, 1991: 73; 249, Schott, ME, et al., Cell. Immuno. 1996: 174: 199-209, Tam, JP Proc. Natl. Acad. Sci. 1988: 85; 5409-5413).

[0019] In this specification, a "dendritic polymer" (dendrimer) refers to a molecule having a structure that branches regularly from a core. A dendritic polymer consists of a central molecule called a core and side chain portions called dendrons. A dendritic polymer has multiple arms, and each arm of the core molecule, which forms the nucleus (core) that forms the branch, is linked to molecules that have multiple arms and form branches. A dendrimer is formed by the linkage of such multi-armed branch-forming molecules. In a dendritic polymer, the number of consecutive branches from the core in the dendron portion is expressed as a generation. The central molecule provides the 0th generation branch (G0), the root branch of the dendron provides the 1st generation branch (G1), further branches from that branch provide the 2nd generation branch (G2), and so on, until the nth generation branch (Gn) is provided.

[0020] In this specification, "lower alkyl group" means, for example, an alkyl group having 1 to 4 carbon atoms. Examples of lower alkyl groups include linear or branched alkyl groups. Examples of linear alkyl groups include methyl, ethyl, n-propyl, and n-butyl groups. Examples of branched alkyl groups include isopropyl, isobutyl, s-butyl, and t-butyl groups. In this specification, a substituted lower alkyl group may be a lower alkyl group substituted with any substituent. Substituents may be, for example, substituents selected from the group consisting of hydroxyl, carboxyl, amino, nitro, thiol, oxo, and halogens (e.g., F, Cl, and Br) (e.g., 1 to 3 substituents).

[0021] In this specification, "a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1" means a partial peptide of the coronavirus spike protein, which, by alignment, consists of a sequence corresponding to the amino acid sequence of SEQ ID NO: 1. Similarly, "a peptide consisting of the amino acid sequence of the coronavirus spike protein corresponding to the amino acid sequence of SEQ ID NO: 1" also means a peptide consisting of the amino acid sequence of the coronavirus spike protein, which consists of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1. For example, in SEQ ID NO: 1, amino acid numbers 798-808 of GenBank registration number: AAP30030.1 is an amino acid sequence common to SARS-CoV-2 and SARS-CoV. The amino acid sequence of SEQ ID NO: 1 contains an amino acid sequence (LLF) consisting of three amino acids: leucine-leucine-phenylalanine, which is part of the coronavirus spike protein. The LLF sequence is considered to be an essential sequence for coronavirus infection of animal cells. The peptide may contain one or more D-amino acids.

[0022] According to the present invention, A peptide consisting of the amino acid sequence described in SEQ ID NO: 1, or a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 (or a peptide having the amino acid sequence of the coronavirus spike protein corresponding to the amino acid sequence of SEQ ID NO: 1) is provided. According to the present invention, A peptide is provided which is a part of the amino acid sequence described in Sequence ID No. 3 and comprises a continuous amino acid sequence of 11 to 21 amino acids in length, including the amino acid sequence described in Sequence ID No. 1, or a partial peptide of the coronavirus spike protein comprising an amino acid sequence corresponding to the 11 to 21 amino acid sequence. 11 to 21 amino acid lengths means 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 amino acids in length, and the same applies hereinafter in this specification.

[0023] According to the present invention, A peptide comprising a continuous amino acid sequence of 11 to 21 amino acids in length from the spike protein of feline infectious peritonitis virus (FIPV) may be provided, comprising the amino acid sequence described in Sequence ID No. 2. The present invention further provides a peptide having the above peptide and the corresponding amino acid sequence of the FIPV spike protein.

[0024] According to the present invention, A peptide comprising a continuous amino acid sequence of 11 to 21 amino acids in length from the spike protein of porcine epidemic diarrhea virus (PEDV) can be provided, comprising the amino acid sequence described in Sequence ID No. 6. The present invention further provides a peptide having the amino acid sequence of the PEDV spike protein corresponding to the above peptide.

[0025] According to the present invention, A peptide comprising a continuous amino acid sequence of 11 to 21 amino acids in length from the spike protein of canine coronavirus can be provided, the peptide containing an amino acid sequence corresponding to the amino acid sequence described in Sequence ID No. 1. The present invention further provides a peptide having the amino acid sequence of the spike protein of PEDV corresponding to the above peptide. In this specification, these peptides may be referred to as the peptides of the present invention below.

[0026] The peptides of the present invention are isolated or purified. The peptides of the present invention may be artificially synthesized.

[0027] The peptide consisting of the amino acid sequence of SEQ ID NO: 1 is 11 amino acids long. By incorporating the peptide of SEQ ID NO: 1 into a multi-antigen peptide, it can induce antibodies specific to coronaviruses. The amino acid sequence of SEQ ID NO: 1 is widely conserved across all coronaviruses. In contrast, peptides containing this amino acid sequence and subsequent amino acid sequences may exhibit high specificity to certain coronaviruses.

[0028] A partial peptide of the coronavirus spike protein, consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, is defined as described above. In some embodiments, the amino acid sequence corresponding to SEQ ID NO: 1 may have one of the following: addition, insertion, substitution, or deletion of a single base relative to the amino acid sequence described in SEQ ID NO: 1. In other embodiments, the partial peptide may have the amino acid sequence described in SEQ ID NO: 7: SX1IEDLLFX2KV {where X1 is F, A, or V, and X2 is D or N}. In some preferred embodiments, the partial peptide may have a sequence consisting of the amino acid sequence described in SEQ ID NO: 2 or 6. Examples of substitutions include conserved substitutions (e.g., substitutions between acidic amino acids, substitutions between basic amino acids, and substitutions between hydrophobic amino acids). The amino acid sequence described in SEQ ID NO: 2 is a partial peptide of the feline infectious peritonitis virus (FIPV) spike protein, having a sequence corresponding to the peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0029] The present invention provides a multi-antigen peptide containing the peptide of the present invention (hereinafter sometimes referred to as "the multi-antigen peptide of the present invention"). In the multi-antigen peptide, the peptide of the present invention is linked to the terminal end of a dendritic polymer backbone. The terminal end and the peptide of the present invention may be linked via a linker. The linkage may be a covalent bond. The linker is not particularly limited as long as it does not significantly hinder the presentation of the peptide, but a chemically stable linker such as polyethylene glycol or a flexible linker (for example, a non-peptide linker or a peptide linker) can be used. A chemically stable linker may be stable at least under physiological conditions. A peptide linker may be used as the linker. When a peptide linker is used, a flexible linker such as a GS linker and a linker having a secondary structure such as an α-helix structure or a β-sheet structure may be used. In the multi-antigen peptide of the present invention, one or more peptides of the present invention may be included. In a preferred embodiment, the multi-antigen peptide of the present invention may contain four or more peptides of the present invention. In a preferred embodiment, the multi-antigen peptide of the present invention may contain eight or more peptides of the present invention. The multi-antigen peptides of the present invention are preferably isolated or purified.

[0030] In one aspect of the present invention, the multi-antigen peptide of the present invention is A multi-antigen peptide comprising a dendritic polymer skeleton and the multi-antigen peptide of the present invention, wherein the core of the dendritic polymer is lysine, and the following (Requirement 1) is satisfied for any natural number k from 1 to n, with the peptide of the present invention linked to each amino group of the nth generation lysine: (Requirement 1) Each amino group of the k-1 generation lysine is peptide-bonded to a k-generation lysine molecule. In the above embodiment, n is 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 2, 3, or 4, and more preferably 2 or 3. That is, in this embodiment of the present invention, tetravalent to 2 n+1 Multiple antigen peptides of varying titers may be provided.

[0031] In one aspect of the present invention, the multi-antigen peptide of the present invention is The present invention may be a multi-antigen peptide (i.e., a tetravalent multi-antigen peptide) comprising a dendritic polymer backbone and the multi-antigen peptide of the present invention, wherein the core of the dendritic polymer is lysine, and each of the two amino groups of the lysine is peptide-bonded to a lysine molecule that forms a first-generation branch, and each of the amino groups of the lysine molecules that form the first-generation branch is linked to the peptide of the present invention, and the present invention may be comprising four of the aforementioned peptides.

[0032] In one aspect of the present invention, the multi-antigen peptide of the present invention is A multi-antigen peptide (i.e., a tetravalent to octavalent multi-antigen peptide) comprising a dendritic polymer backbone and the multi-antigen peptide of the present invention, wherein the core of the dendritic polymer is lysine, each of the two amino groups of the lysine is peptide-bonded to a lysine molecule that forms a first-generation branch, each of the amino groups of the lysine molecule that forms the first-generation branch is peptide-bonded to a lysine molecule that forms a second-generation branch, and each of the amino groups of the lysine molecule that forms the second generation is linked to the peptide of the present invention, and comprising 4 to 8 of the aforementioned peptides.

[0033] In a preferred embodiment, the multi-antigen peptide of the present invention may be a tetravalent multi-antigen peptide (MAP-4) having the structure shown in formula (I) below, or an octavalent multi-antigen peptide (MAP-8) having the structure shown in formula (II) below. Depending on the number of branching generations n, the number of tetravalents can range from 2. n+1 Multiple antigen peptides of valence (MAP-2) n+1 ) will be provided.

[0034] [ka]

[0035] [ka]

[0036] These multiple antigen peptides can be synthesized by introducing peptides into a dendritic skeleton immobilized on a resin. More specifically, with respect to MAP-4, for example, a multiple antigen peptide having the structure of formula (III) can be produced by introducing peptides into the amino groups of a skeleton having a core lysine and two lysine groups forming the first generation of branching as the skeleton of the dendritic polymer. The core lysine may be immobilized on the resin.

[0037] [ka] {In the formula, the lines represent peptide bonds formed by the aminobutyl group and amino group from the lysine residue.}

[0038] Furthermore, MAP-8 can be used to produce a multi-antigen peptide having the structure of formula (IV) below by introducing a peptide to the amino groups of a skeleton of the dendritic polymer, which has a core lysine, two lysine molecules forming the first generation of branching, and four lysine molecules forming the second generation of branching. The core lysine may be immobilized on the resin.

[0039] [ka] {In the formula, the lines represent peptide bonds formed by the aminobutyl group and amino group from the lysine residue.}

[0040] In a preferred embodiment, the multi-antigen peptide of the present invention may have the structure of formula (III) or (IV) {wherein the peptide is the peptide of the present invention}. That is, in a preferred embodiment, the multi-antigen peptide of the present invention may be a peptide in which a peptide is linked by peptide bonds or other chemically stable bonds to the four amino groups of a lysine molecule that forms a dendritic polymer skeleton, in which two lysine molecules forming a first-generation branch are linked by peptide bonds to the aminobutyl group and amino group of a core lysine molecule. In a preferred embodiment, the multi-antigen peptide of the present invention may be a peptide in which a peptide is linked by peptide bonds or other chemically stable bonds to the eight amino groups of a lysine molecule that forms a dendritic polymer skeleton, in which two lysine molecules forming a first-generation branch are linked by peptide bonds to the aminobutyl group and amino group of a core lysine molecule, and four lysine molecules forming a second-generation branch are linked by peptide bonds to the aminobutyl group and amino group of each of the two lysine molecules. Such peptides can be produced by using Lys, which has been immobilized on a solid phase by a method well known to those skilled in the art, as a core, and polymerizing Lys to the two amino groups of Lys by the Fmoc method to create a dendritic polymer skeleton, and then further polymerizing the peptide to the amino groups of lysine on the resulting dendritic polymer skeleton. Alternatively, azide groups can be introduced to the amino groups of lysine on the resulting dendritic polymer skeleton, and the peptide can be linked to the dendritic polymer skeleton by click chemistry.

[0041] In one preferred embodiment, the multi-antigen peptide of the present invention may have the structure of formula (VI) below. [ka]

[0042] {Here, R is a linker-peptide or a peptide, and the peptide may include the amino acid sequence of the peptide of the present invention, R 2This can be hydrogen, an OH group, a substituted or unsubstituted lower alkyl group, an amino group, an amino acid (e.g., 3-aminopropanoic acid (β-alanine), i.e., -NH-C2H4-COOH), a halogen, or a peptide, and the amino acid may be linked to the above molecule via an amide bond. Note that MAP-8 may have a structure in which two compounds having the structure of formula (VI) are linked to the amino groups of further lysines via the carboxyl groups of the 0th generation lysine in formula (VI). Similarly, MAP-2 having nth generation branching n+1 Each structure can be defined. [ka] {Here, R 2 This is as defined above.

[0043] In the above, the linker may be polyethylene glycol (PEG). In some preferred cases, the PEG may be, for example, PEG with a degree of polymerization of 2 to 30, PEG with a degree of polymerization of 5 to 20, or PEG with a degree of polymerization of 10 to 15.

[0044] In the above, the peptide consists of the peptide of the present invention, or comprises the peptide of the present invention and a linker peptide. The linker peptide may be, for example, a flexible linker or a peptide that forms a β-sheet structure.

[0045] Compounds having the structure of formula (VI) above can be obtained by reacting a peptide or peptide-linker compound with the acetylene group (-C≡CH) of a compound having the structure of formula (VII) below by click chemistry. More specifically, they can be obtained by reacting a compound having the structure of formula (VII) below with a peptide-azide group or a peptide-linker-azide group having an azide group at its terminus in the presence of monovalent copper ions. Peptide-azide groups and peptide-linker-azide groups can be obtained by known methods. For example, they can be obtained by converting the N-terminus of a peptide obtained by known methods to an azide group, or by condensing a linker moiety having an azide group and a carboxyl group at both ends, respectively. [Chemical formula] {Here, R 2 is as defined above.}

[0046] The compound of formula (VII) is an intermediate in the synthesis of the compound of formula (VI). For the amino group of lysine that provides the first-generation branch, for the convenience of the reaction, an artificial amino acid having an acetylene group in the side chain is linked by a peptide bond to provide a reactive acetylene group at the end (see, for example, WO2015190555A). The synthesis intermediate of MAP-8 may have a structure in which two compounds having the structure of formula (VII) are linked to each amino group of further lysine via the carboxyl group of the 0-generation lysine in formula (VII). Similarly, the structure of each synthesis intermediate of MAP-2 having an n-generation branch n+1 can be defined.

[0047] The multiple antigen peptide of the present invention can be produced, for example, according to the method described in WO2018 / 084247A. The lysine backbone of MAP can be produced, for example, by polymerizing lysine in which two amino groups are protected by a 9-fluorenylmethyloxycarbonyl group (Fmoc group) by the Fmoc method. The peptide can be further extended with respect to the amino group of lysine by the Fmoc method. The Fmoc method can be appropriately implemented using well-known techniques by those skilled in the art. The Fmoc method can be implemented on a solid-phase support. As the solid-phase support, Wang resin, HMPA resin, HMBA resin, NovaSyn TGT resin, etc. can be used. The method of synthesizing a peptide on a solid-phase support is called solid-phase synthesis and is well-known to those skilled in the art. Thus, the multiple antigen peptide of the present invention can be appropriately produced by those skilled in the art using conventional methods.

[0048] In a preferred embodiment, the multiple antigen peptide of the present invention can be a multiple homologous antigen peptide. In a multiple homologous antigen peptide, all antigen peptides have the same amino acid sequence.

[0049] In another preferred embodiment, the multi-antigen peptide of the present invention may include peptides having two or more different amino acid sequences as antigen peptides.

[0050] In this invention, the multi-antigen peptide can be administered to a target to induce immunity (particularly antigen-specific immunity) in that target. In this invention, the multi-antigen peptide can be administered to a target to induce antigen-specific antibody production. Furthermore, in this invention, the multi-antigen peptide can be administered to a target to induce antibodies against coronavirus.

[0051] The present invention provides a vaccine containing the multi-antigen peptide of the present invention (sometimes referred to as "the vaccine of the present invention").

[0052] The vaccine of the present invention may be broadly effective against coronaviruses. The vaccine of the present invention may be effective against SARS-CoV-2. The vaccine of the present invention may be effective against feline infectious peritonitis virus (FIPV).

[0053] The vaccine of the present invention does not need to be used in combination with an adjuvant that stimulates helper T cells (or T cell-dependent immunity) (e.g., aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, precipitated adjuvant), and preferably does not need to be used in combination. The vaccine of the present invention does not need to contain an adjuvant (e.g., aluminum salts, e.g., aluminum hydroxide, aluminum phosphate, aluminum potassium sulfate, e.g., incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, precipitated adjuvant), and preferably does not need to contain one. However, the vaccine of the present invention can preferably be used in combination with an activator that does not stimulate helper T cells (T cell-dependent immunity), such as α-galactosylceramide.

[0054] The vaccine of the present invention may comprise the multi-antigen peptide of the present invention and a pharmaceutically acceptable carrier and / or excipient. The vaccine of the present invention may be administered parenterally (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation). Therefore, a pharmaceutically acceptable carrier and / or excipient suitable for parenteral administration (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation) can be used. Examples of pharmaceutically acceptable carriers and / or excipients include salts, buffers, pH adjusters, isotonic agents, preservatives, and water.

[0055] According to the present invention, the vaccine may be a composition used to induce antibody production against coronavirus in a subject, or to establish immunity against coronavirus.

[0056] The present invention provides a multi-antigen peptide for use in producing antibodies against coronavirus in a subject.

[0057] The present invention provides for the use of the peptide of the present invention in the manufacture of a vaccine. The present invention provides for the use of the multi-antigen peptide of the present invention in the manufacture of a vaccine. The vaccine may be used to induce antibody production against coronavirus in a subject, or to establish immunity against coronavirus.

[0058] The present invention provides a method for administering an antigen peptide to a subject, comprising administering the multi-antigen peptide of the present invention to the subject. The present invention provides a method for activating antigen-specific immunity in a subject, comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject. The present invention provides a method for activating immunity against coronavirus in a subject, comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject. The present invention provides the multi-antigen peptide of the present invention for use in these methods, or a pharmaceutical composition comprising the multi-antigen peptide of the present invention for use in these methods. The present invention provides the use of the multi-antigen peptide of the present invention in the manufacture of a pharmaceutical for use in these methods.

[0059] The present invention provides a method for administering a peptide to a subject, comprising administering the multi-antigen peptide of the present invention to the subject according to an administration plan. According to the present invention, the subject may be a subject that is not infected with coronavirus, a subject that is infected with coronavirus (the subject may be asymptomatic), or a subject that has developed an infection caused by coronavirus. In subjects that are not infected with coronavirus, the method or multi-antigen peptide of the present invention may prevent infection by coronavirus, delay the onset of an infection caused by coronavirus, inhibit the onset of an infection, or cure the infection, and if the infection has developed, it may alleviate the symptoms, slow the progression of the symptoms (e.g., inhibit the worsening of the condition), stop the progression, or reduce the symptoms. In subjects that are infected with coronavirus (e.g., pre-symptomatic subjects), the method or multi-antigen peptide of the present invention may delay the onset of symptoms, inhibit the onset of the condition, or cure the infection, and if the infection has developed, it may alleviate the symptoms, slow the progression of the symptoms (e.g., inhibit the worsening of the condition), stop the progression, or reduce the symptoms. The method or multi-antigen peptide of the present invention may slow the progression of symptoms (e.g., inhibit the worsening of symptoms), halt the progression, or alleviate symptoms in subjects infected with coronavirus. Because the multi-antigen peptide of the present invention primarily induces IgM, the time from administration to antibody induction is short, and it may be effective when administered to subjects infected with coronavirus and subjects suffering from the infection.

[0060] According to the present invention, A method for preventing the onset of symptoms in a person infected with the coronavirus, A method comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject. This may be provided. A subject infected with coronavirus may, in some aspects, be a subject before the onset of symptoms of the infection. A subject infected with coronavirus may be a subject determined to be infected with coronavirus by testing (e.g., PCR test or antibody test). A subject infected with coronavirus may be a subject determined to be infected with coronavirus by testing (e.g., PCR test or antibody test) and also before the onset of symptoms of the infection. This may allow the subject to delay the onset of symptoms, inhibit the onset of symptoms, or cure the infection.

[0061] The present invention also provides a method comprising administering an effective amount of the multi-antigen peptide of the present invention to a subject before the onset of an infectious disease caused by a coronavirus, and further comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject after the onset of the disease. The present invention further provides a method for administering a peptide to a subject, comprising administering the multi-antigen peptide of the present invention to the subject according to an administration plan, the administration plan comprising administering the multi-antigen peptide of the present invention to the subject before infection or at a stage considered to be pre-infection (e.g., 1 to 3 administrations). The present invention also provides a method for administering a peptide to a subject, comprising administering the multi-antigen peptide of the present invention to the subject according to an administration plan, the administration plan comprising administering the multi-antigen peptide of the present invention to the subject 1 to 3 times at a stage before infection or at a stage considered to be pre-infection, and administering the multi-antigen peptide of the present invention to the subject once or more at a stage after infection or at a stage considered to be post-infection. The present invention provides a method for administering a peptide to a subject, comprising administering the multi-antigen peptide of the present invention to the subject according to an administration plan, wherein the administration plan comprises administering the multi-antigen peptide of the present invention to the subject three times at a pre-infection or pre-infection stage, and administering the multi-antigen peptide of the present invention to the subject one or more times at a post-infection or post-infection stage. In the present invention, additional administration after the onset of symptoms can slow the progression of symptoms (e.g., inhibit the worsening of symptoms), halt the progression, or alleviate symptoms in the subject. The administration may involve administering the multi-antigen peptide in doses of, for example, 1 mg / kg to 3 mg / kg, or for example, 1.5 mg / kg per dose. In a preferred embodiment, the administration may be carried out on different days. In a preferred embodiment, the administration may be daily, every other day, every two days, every three days, every four days, every five days, every six days, every week, every 8 to 13 days, every two weeks, every 15 to 20 days, every three weeks, or at longer intervals. In this embodiment, for example, the object may be a human object. In this embodiment, for example, the object may be a cat object. In this embodiment, the object may be a dog object. In this embodiment, the object may be a pig object.In this embodiment, coronavirus infections can be prevented and / or treated in the subject.

[0062] In particular, as shown in the examples described later, when CoV-mMAP, a multi-antigen peptide of the present invention, was administered to cats before infection according to the above administration plan, it restored symptoms caused by feline infectious peritonitis virus (FIP) infection, which has a 100% mortality rate after the onset of symptoms. This is considered a noteworthy effect. According to the present invention, a pharmaceutical composition containing the multi-antigen peptide of the present invention is provided for administration according to the above administration plan. The pharmaceutical composition may be for the prevention and / or treatment of coronavirus infection. According to the present invention, the use of the multi-antigen peptide of the present invention in the manufacture of a pharmaceutical for administration according to the above administration plan is provided. The pharmaceutical may be for the prevention and / or treatment of coronavirus infection.

[0063] The present invention provides a method for preventing and / or treating coronavirus infections in a target in need, comprising administering the multi-antigen peptide of the present invention to the target. The present invention provides a pharmaceutical composition comprising the multi-antigen peptide of the present invention for use in a method for preventing and / or treating coronavirus infections in a target in need. The present invention provides the use of the multi-antigen peptide of the present invention in the manufacture of a pharmaceutical for use in a method for preventing and / or treating coronavirus infections in a target in need.

[0064] According to the present invention, A method for treating a person infected with coronavirus infection, A method comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject. This can be provided. Subjects infected with coronavirus infection may, in some embodiments, be subjects with fever. Because the multi-antigen peptide of the present invention primarily induces IgM, the time from administration to antibody induction is short, and it may be effective even when administered to subjects with initial infection. Therefore, for example, a subject may have any of the following symptoms: olfactory abnormalities, gustatory abnormalities, and fever, for example, initial symptoms.

[0065] According to the present invention, A method for preventing severe illness in individuals infected with coronavirus infection, A method comprising administering an effective amount of the multi-antigen peptide of the present invention to the subject. This may be provided. Subjects infected with coronavirus infection may, in some embodiments, be subjects with fever. Because the multiantigen peptide of the present invention primarily induces IgM, the time from administration to antibody induction is short, and it may be effective even when administered to infected subjects (e.g., subjects with initial infection).

[0066] According to the present invention, the coronavirus may be a highly pathogenic coronavirus. The coronavirus may be a coronavirus selected from the group consisting of, for example, MERS coronavirus, SARS coronavirus (e.g., SARS-CoV, SARS-CoV-2, etc.), feline infectious peritonitis virus (FIPV), and porcine epidemic diarrhea virus (PDE).

[0067] According to the present invention, the target may be human or a non-human mammal. According to the present invention, the pharmaceutical composition and pharmaceutical of the present invention may be administered to human or non-human mammals. [Examples]

[0068] Example 1: Synthesis of antigen peptide For the creation of a vaccine against coronavirus, we used partial peptides of the spike protein, a surface protein of SARS-CoV, SARS-CoV-2, and feline infectious peritonitis virus (FIPV), as antigenic peptides. Specifically, the spike proteins of three viruses were subjected to multiple alignment to produce a peptide (SEQ ID NO: 1) with a sequence of 11 consecutive amino acids (see Figure 3).

[0069] [Table 1] *The numbers listed in the amino acid sequence represent the amino acid numbers of each terminal amino acid in the spike protein.

[0070] As shown in Table 1, the 11 consecutive amino acid sequences showed high homology with SARS-CoV, SARS-CoV-2, feline infectious peritonitis virus (FIPV), and porcine epidemic diarrhea virus (PEDV).

[0071] A peptide having the amino acid sequence of Sequence ID No. 1 was artificially synthesized using conventional methods to produce a tetravalent multi-antigen peptide (MAP-4) with the structure shown in formula (I) below and an octavalent multi-antigen peptide (MAP-8) with the structure shown in formula (II) below.

[0072] [ka]

[0073] [ka]

[0074] These multiple antigen peptides were synthesized by introducing the peptides into a dendritic skeleton immobilized on a resin, for the purpose of efficient synthesis on the resin. More specifically, for MAP-4, the peptide was introduced into a resin having a dendritic skeleton with the structure of formula (III) below.

[0075] [ka]

[0076] Furthermore, MAP-8 was introduced into a resin having the structure of formula (IV) below.

[0077] [ka]

[0078] In the above formula, lysine (Lys) is an amino acid having two amino groups. When the carboxyl groups of two lysine atoms are linked to each amino group by peptide bonds, the two terminal lysine atoms form a dendritic skeleton with a total of four amino groups. By introducing a peptide to the amino groups of this lysine via a linker, a tetravalent multi-antigen peptide (MAP-4) can be produced.

[0079] Furthermore, MAP-8 was created by further branching each terminal lysine (Lys) residue into two, and then introducing peptides into a resin containing lysine residues with eight Fmoc protecting groups. When the carboxyl groups of four lysine residues are linked to each of the four amino groups of the lysine residues in the dendritic skeleton with a total of four amino groups via peptide bonds, the four terminal lysine residues form a dendritic skeleton with a total of eight amino groups. By introducing peptides to the amino groups of these lysine residues via linkers, tetravalent or octavalent multi-antigen peptides can be produced.

[0080] The peptides were synthesized according to a conventional method using the Fmoc method. Specifically, the resin was swollen, the amino acid protecting group at the N-terminus was removed with a deprotecting agent, and then the following amino acids were condensed using a coupling reagent and an amino acid derivative to synthesize the peptides. As coupling reagents, 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxidehexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) were used. The peptides were cleaved from the resin with a TFA cocktail reagent, precipitated with cold t-butyl methyl ether, washed, and recovered. After drying, the ether was removed and the peptides were dissolved in a 50% aqueous acetonitrile solution, and then freeze-dried.

[0081] When introducing the above peptide, a linker was introduced at the C-terminus of the peptide. In this example, polyethylene glycol (PEG) was used as the linker. The polyethylene glycol was PEG with a degree of polymerization of 12 (also written as "PEG(12)").

[0082] The more specific structure of MAP-4 was as follows:

[0083] [ka]

[0084] {Here, R is a -PEG(12)-peptide, and R 2 This is as defined above.

[0085] The above MAP-4 and MAP-8 can be prepared, for example, by referring to WO2018062217A.

[0086] The obtained peptides were then released from the resin using 0.1% 2,2,2-trifluoroacetic acid and acetone. Subsequently, the liberated MAP-4 and MAP-8 were purified by high-performance liquid chromatography to obtain MAP-4 and MAP-8.

[0087] Example 2: Dosage experiment A multi-antigen peptide (MAP-4 and MAP-8; collectively referred to as CoV-MAP) containing the amino acid sequence of SEQ ID NO: 1, prepared in Example 1, was administered to mice according to the scheme shown in Figure 4. Specifically, CoV-MAP was administered to Balb / c mice via tail vein at doses of 100 μg / mouse / inoculation or 200 μg / mouse / inoculation on days 0, 7, and 25. On day 7, 0.1 μg of α-galactosylceramide (α-GalCer) was administered via tail vein. Subsequently, on day 32, inactivated canine coronavirus was administered intraperitoneally. Vanguard (Ministry of Agriculture, Forestry and Fisheries Directive 25 Animal Drug No. 2225, Zoetis Japan; hereinafter the same) was used as the inactivated canine coronavirus. Serum samples were collected on day 0 (before administration), days 26-31 (after three administrations of CoV-MAP), and day 39 (one week after administration of inactivated canine coronavirus).

[0088] Antibody titers in serum were measured. Specifically, inactivated canine coronavirus was immobilized on a 96-well plate. As a background control, wells immobilized with ovalbumin-conjugated alum instead of inactivated canine coronavirus were measured.

[0089] The ELISA method using a peroxidase-based colorimetric system was used for measurement. The average of the OD values ​​of two wells under identical conditions was calculated. The OD value for inactivated canine coronavirus was calculated by subtracting the OD value of the background control from the OD value for inactivated canine coronavirus. In addition, to confirm the specificity of the reaction, the reactivity to coronavirus peptides was measured using mouse serum administered with influenza virus MAP instead of CoV-MAP (influenza-MAP 200 μg; MAP with the amino acid sequence described in SEQ ID NO: 5 was prepared separately according to WO2018084247A).

[0090] The results of the above CoV-MAP administration experiment are shown in Figures 5 and 6. Figure 5 shows the changes in serum IgM antibody titers, and Figure 6 shows the changes in serum IgG antibody titers.

[0091] As shown in Figure 5, serum IgM antibody titers increased in serum after three administrations of CoV-MAP and remained elevated or maintained after administration of inactivated canine coronavirus. On the other hand, no increase in antibody titers against canine coronavirus was observed in mice administered with influenza virus MAP.

[0092] Furthermore, as shown in Figure 6, an increase in serum IgG antibody titers was observed one week after administration of canine coronavirus. On the other hand, no increase in antibody titers against canine coronavirus was observed in mice administered with influenza virus MAP.

[0093] Example 3: Experiment on administering canine coronavirus vaccine CoV-MAP (200 μg / mouse / inoculation), prepared according to Example 1, was administered to mice on days 0, 7, and 25 as shown in Figure 7a. On day 7, 0.1 μg of α-galactosylceramide was administered via tail vein. As a control, 2E5-MAP was administered instead of CoV-MAP. 2E5-MAP is a MAP containing a partial hemagglutinin peptide of the influenza virus, and contains a peptide set in the amino acid sequence portion common to H3 belonging to group 1 and H1 belonging to group 2. Specifically, 2E5-MAP was a MAP containing eight DGWYGFRHQNSEGTGQAADLKSTQA (Sequence ID 5). Subsequently, to simulate viral infection, 50 μL of Vanguard, an inactivated canine coronavirus vaccine, was administered intraperitoneally on day 32.

[0094] Serum was collected on day 28 (before viral infection) and day 60 (after viral infection), and antibody titers in the serum were confirmed using ELISA. For ELISA, a system with porcine epidemic diarrhea virus (PED) antigen immobilized was used.

[0095] The results are shown in Figure 7b. As shown in Figure 7b, significant IgG induction was observed after Vanguard administration in the system with immobilized PED antigen. As the PED antigen, we used the Nisseiken PED live vaccine, a porcine epidemic diarrhea vaccine, which was inactivated by heating at 60°C for 10 hours.

[0096] Next, MAP-8 was prepared with the same configuration as above, except that all amino acids in the amino acid sequence of SEQ ID NO: 1 were replaced with D-isomers, and a peptide (SEQ ID NO: 4) was used in which peptides forming a β-sheet structure as a peptidolytic linker were linked to the C-terminus of these amino acids. The obtained CoV-mMAP was administered via tail vein at a dose of 200 μg / mouse / inoculation (triangles in Figures 8a and 8b), and for the second dose, 0.1 μg / mouse of α-galactosylceramide was used in combination. ELISA was performed using a plate on which the D-isomer peptide, in which all amino acids of the peptide consisting of the amino acid sequence of SEQ ID NO: 1 were immobilized.

[0097] The results are shown in Figures 8a-8d. As shown in Figures 8a-8d, CoV-mMAP with the D-isomer also induced both IgM and IgG. Therefore, it was shown that the amino acid sequence can be that of the D-isomer. Furthermore, linking the antigen peptide and the backbone of the multi-antigen peptide with a peptidic linker (for example, a linker that can form a β-sheet structure) did not inhibit antibody induction.

[0098] CoV-mMAP was administered according to the administration scheme shown in Figure 9, followed by boost immunization with canine coronavirus vaccine antigen. An octavalent MAP containing a partial peptide of influenza virus was used as a negative control (Cont-MAP). Alternatively, a general peptide vaccine was prepared by linking the amino acid sequence described in Sequence ID No. 8 to keyhole limpet hemocyanin, which was mixed with Alam and administered intraperitoneally. Serum was collected on days 0, 14, 25, 32, 39, and 46, and serum IgM levels were measured. Vaccine efficacy was evaluated based on the amounts of peptide-specific IgM and virus antigen-specific IgM in the serum.

[0099] First, the vaccine effect was evaluated using serum IgM levels bound to the peptide used as the antigen as an indicator. Mouse serum (1 / 200 dilution) was reacted overnight at 4°C with the BSA fusion antigen peptide immobilized on an ELISA plate. A biotin-labeled anti-mouse IgM polyclonal antibody (Southern Biotech) was conjugated as the detection antibody, and detection was performed according to a standard method in which the detection antibody was further reacted with peroxidase-labeled streptavidin. The results are shown in Figure 10. As shown in Figure 10, a significant increase in serum IgM levels was observed in the CoV-mMAP administered group after two immunizations. Furthermore, a significant increase in serum IgM levels was also observed in the CoV-mMAP administered group after boost immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. In addition, in the group administered a conventional peptide vaccine, a transient increase in serum IgM levels was observed after the second immunization, but no increase in serum IgM levels was observed after boost immunization. This suggests that conventional peptide vaccines do not readily induce IgM-producing memory immunity.

[0100] Next, the vaccine effect was evaluated using serum IgM levels bound to the canine coronavirus vaccine antigen (feline kidney cell culture inactivated canine coronavirus NL-18 strain liquid vaccine, product name Vanguard Plus CV, Zoetis Japan Co., Ltd.). The antigen was immobilized on an ELISA plate at a concentration of 10 μg / mL, and serum was brought into contact with it to measure serum IgM levels in the same manner as described above. The results are shown in Figure 11. As shown in Figure 11, a significant increase in serum IgM levels was observed in the CoV-mMAP administration group after two immunizations. Furthermore, a significant increase in serum IgM levels was also observed in the CoV-mMAP administration group after boost immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. In addition, in the group administered a conventional peptide vaccine, an increase in serum IgM levels was observed after the second immunization, but no increase in serum IgM levels was observed after boost immunization. This suggests that conventional peptide vaccines do not easily induce IgM production memory immunity.

[0101] Furthermore, vaccine efficacy was evaluated using serum IgM levels bound to the porcine epidemic diarrhea virus (PED) vaccine (attenuated porcine epidemic diarrhea virus P-5V strain, Nisseiken Co., Ltd.) as an indicator. The live vaccine was inactivated by incubation at 60°C for 10 hours, immobilized on an ELISA plate, and serum IgM levels were measured in the same manner as described above after contact with serum. The results are shown in Figure 12. As shown in Figure 12, a significant increase in serum IgM levels was observed in the CoV-mMAP administration group after two immunizations. A significant increase in serum IgM levels was also observed in the CoV-mMAP administration group after boost immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. In addition, in the group administered a conventional peptide vaccine, the increase in serum IgM levels after the second immunization was weak, and no increase in serum IgM levels was observed at all with boost immunization. This suggests that conventional peptide vaccines are ineffective in establishing IgM-producing memory immunity, and furthermore, they cannot induce IgM that commonly responds to peptides, canine coronavirus antigens, and PED virus antigens.

[0102] Furthermore, the vaccine efficacy was evaluated using serum IgM levels as indicators for SARS-CoV-2 spike protein (full length), MERS spike protein, and SARS-CoV-2 spike protein (S2 subunit). MERS recombinant spike protein (Sino Biological), SARS-CoV-2 recombinant spike protein (Invitrogen), and its recombinant S2 subunit protein (Ray Biotech) were immobilized at 5 μg / ml, and then brought into contact with serum in the same manner as described above, and serum IgM levels were measured in the same manner as described above. The results are shown in Figure 13. As shown in Figure 13, a significant increase in serum IgM levels was observed in the CoV-mMAP administration group after two immunizations. A significant increase in serum IgM levels was also observed in the CoV-mMAP administration group after boost immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. These findings indicate that CoV-mMAP induces IgM that responds even to coronaviruses that exhibit high pathogenicity in humans.

[0103] A quadrivalent CoV-mMAP was prepared, similar to the octavalent CoV-mMAP. Following the same administration scheme as in Figure 9, the quadrivalent CoV-mMAP was administered intravenously to mice, followed by boost immunization with canine coronavirus vaccine antigen administered intraperitoneally. The BSA-linked antigen peptide, PED virus vaccine antigen, and canine coronavirus vaccine antigen were immobilized on ELISA plates as described above, and serum IgM levels were measured in contact with serum as before. The results are shown in Figure 14. As shown in Figure 14, a significant increase in serum IgM levels was observed in the CoV-mMAP-administered group after two immunizations. This indicates that CoV-mMAP is effective even in its quadrivalent peptide form.

[0104] We created octavalent CoV-mMAPs presenting peptides with various modifications. The peptides used are shown in Table 2 below.

[0105] [Table 2]

[0106] In Table 2, italics and underlines indicate D-isomer amino acids, and bold text indicates amino acids that differ from those of peptide No. 1 (SEQ ID NO: 8).

[0107] These CoV-mMAPs were administered to mice according to the administration scheme shown in Figure 9, and serum was collected at 2 weeks, 4 weeks, 5-6 weeks, and 137 days. PED virus vaccine and canine coronavirus vaccine were also immobilized on ELISA plates and brought into contact with serum in the same manner as above, and serum IgM levels were measured in the same manner as before. The results are shown in Figures 15-20. As shown in Figures 15-20, all MAPs containing the peptides induced an increase in virus-specific serum IgM levels. This indicates that even if the amino acids used in CoV-mMAP were modified to the D-isomer, or if the change was only one to a few amino acids, it did not hinder the induction of the target IgM, demonstrating that CoV-MAP can serve as a robust vaccine against amino acid changes.

[0108] Furthermore, serum IgM levels were examined after 137 days. Serum IgM levels were calculated as relative values, with the serum IgM level before CoV-mMAP administration set to 1. The results are shown in Figure 21. As shown in Figure 21, a specific increase in serum IgM levels was observed for both the PED virus vaccine and the canine coronavirus vaccine. This indicates that IgM induced by CoV-mMAP remains in the blood for more than 4 months and is maintained over the long term.

[0109] Next, we confirmed the effect on memory immunity. To detect memory IgM-producing cells, mice were similarly immunized with CoV-mMAP, and IgM-producing cells against the peptide (SAIEDLLFNKV) were detected using the ELISPOT method in spleen cells 133 days after the start of immunization. JEV-MAP (an octavalent MAP containing a portion of the amino acid sequence of the E protein of Japanese encephalitis virus) was used as a negative control. 5 × 10⁶ spleen cells were placed on an ELISPOT plate immobilized with peptide-BSA at 5 micrograms / ml. 5 After seeding at a concentration of cells / ml, the cells were cultured at 37°C in the presence of 5% CO2 for 22 hours. After washing, biotin-labeled anti-IgM antibody (Southern Biotech) was added and reacted at 37°C for 1 hour. Then, streptavidin-labeled peroxidase was added and spots were colored according to a standard method. The number of spots was measured using an ELISPOT reader (Carl Zeiss KS ELISPOT), and the average value for 2 wells per individual was calculated. The number of anti-peptide IgM-producing cells in the CoV-mMAP group was significantly higher than in the JEV-MAP group. This suggests that IgM-producing cells induced by CoV-mMAP may exist in vivo as long-term memory cells.

[0110] Macrophages express Fc receptors on their cell surface, which bind to IgG against antigens, and take up IgG along with the antigen. The amount of antigen taken up by macrophages is thought to reflect the amount of virus taken up. When a virus capable of infecting macrophages is taken up by macrophages via antibodies, the presence of antibodies promotes viral infection of macrophages, which can cause antibody-dependent enhancement of infection (ADE). A FLAG-tagged recombinant protein was created by conjugating bovine serum albumin (BSA) to the peptide (SAIEDLLFNKV; SEQ ID NO: 8) using a recombinant protein expression system in E. coli. The prepared artificial viral peptide, peptide-FLAG-BSA (concentration 5 μg / ml), was mixed with serum (concentration 10%) and reacted at 37°C for 1 hour. Then, it was added to the culture medium of mouse macrophage cell line P388-D1 cells (seeding number 50,000 cells) and cultured at 37°C in the presence of 5% CO2 for 24 hours to allow uptake. The peptide-FLAG-BSA taken up into cells was detected by flow cytometry using FACS Canto II (Becton Dickinson) after cell permeabilization treatment with PE (Phycoerythrin)-labeled anti-FLAG monoclonal antibody (BioLegend). The percentage of cells that ultimately took up the peptide-FLAG-BSA was expressed as the percentage of total viable cells. The results are shown in Figure 23. As shown in Figure 23, when anti-FLAG mouse monoclonal antibody was used instead of mouse serum, it was confirmed that most cells (99.5%) took up the peptide (positive control). On the other hand, only 2.1% of cells were detected as positive in untreated normal mouse serum, indicating that most cells did not take up the IgM-bound artificial viral peptide (negative control). Under these conditions, when CoV-mMAP immunized mouse serum (pooled serum from Day 28 and Day 32 before boost immunization) was used, only 3.5% of cells took up the artificial viral peptide, and no significant difference was observed compared to normal mouse serum. This suggests that IgM induced by CoV-mMAP immunization, even if it binds to the virus, is not taken up by macrophages as an immune complex.

[0111] CoV-mMAP was administered to mice according to the scheme shown in Figure 9, and serum was obtained on day 25 (before boost immunization) and day 60 (after boost immunization). Antigen-specific serum IgG levels were then measured in each serum sample. Antigen peptides, canine coronavirus vaccine antigen, and porcine epidemic diarrhea virus antigen were immobilized on ELISA plates as described above, contacted with serum, and serum IgG levels were measured in the same manner as described above. The results are shown in Figure 24. As shown in Figure 24, an increase in serum IgG levels for each antigen was observed after boost immunization.

[0112] Similarly, serum IgG levels bound to the SARS-CoV-2 spike protein (full length), MERS spike protein, and SARS-CoV-2 spike protein (S2 protein) were evaluated. The results are shown in Figure 25. As shown in Figure 25, an increase in serum IgG levels for each antigen was observed after boost immunization. Therefore, IgG subclasses were analyzed.

[0113] For comparison, we used mouse serum immunized with KLH-binding peptide along with Alam (containing KLH-peptide-induced IgG). While all IgG subclasses were induced by the KLH-peptide, in CoV-mMAP-immunized mice, only IgG1 subclass of serum IgG (CoV-mMAP-IgG) after canine coronavirus antigen boosting was elevated in all mice, and IgG2a and IgG2b, which are taken up by macrophages, were hardly detected. From this, we found that although CoV-mMAP immunization may induce IgG during viral infection, it does not induce IgG subclasses involved in antibody-dependent enhancement, and therefore, unlike conventional vaccines, there is no risk of antibody-dependent enhancement.

[0114] The effect of CoV-mMAP on feline infectious peritonitis virus (FIPV) infection was investigated. Four experimental cats (approximately 5 months old) were prepared in two groups: one pre-treated with CoV-mMAP and the other untreated. The experimental cats were infected with FIPV, a highly pathogenic coronavirus with a 100% fatality rate after symptom onset, and the effect of CoV-mMAP was examined (see Figure 27). CoV-mMAP was administered intravenously at a dose of 1.5 mg / kg on days 0, 7, and 21, and α-galactosylceramide 0.7 μg / kg was administered intravenously simultaneously on day 7. Viral exposure was measured in CRFK cells at a dose of 3 × 10⁶. 5 Using 10 ml of culture medium at the time of confirmation of plaque formation at PFU / ml, the first dose was administered orally. For surviving cats, the second dose was administered as prednisolone at an immunosuppressive dose of 2 mg / kg subcutaneously for 3 consecutive days, followed by intraperitoneal administration. Of the cats that developed symptoms, all but one that developed symptoms after the first dose received intravenous administration of CoV-mMAP and α-galactosylceramide on day 3 (early administration) and days 13-15 (late administration), similar to Day 7. The results are shown in Figure 28. High fever was defined as 39.5°C or higher, and FIP was considered to have developed when high fever was confirmed for 3 consecutive days or more. Cat No. 5, which developed symptoms in the CoV-mMAP administration group, did not receive additional CoV-mMAP and its high fever did not subside, so it was euthanized (see cross mark). All individuals that received additional CoV-mMAP after the onset of symptoms showed improvement in high fever. In particular, cat No. 7, which received an early additional dose (administered on the third day after symptom onset) in the CoV-mMAP group, returned to its normal body temperature before viral exposure and was completely cured. FIPV is known to be the most pathogenic of all coronaviruses, and almost 100% of cats that develop the disease die. The multi-antigen peptide of the present invention has great technical significance in that it prevents the onset of infection caused by FIPV and also provides a therapeutic effect. Cat No. 3, which received a late additional dose in the CoV-mMAP administration group, had a reduced fever, but other symptoms such as ascites did not subside, so it was euthanized. Cats No. 4 and No. 6 in the untreated group both had a reduced fever after CoV-mMAP administration, but then developed a high fever again, so they were euthanized. The results are also summarized in Table 3.

[0115] [Table 3]

[0116] [Consideration] SARS-CoV-2 infects not only respiratory tract mucosal cells but also macrophages, causing systemic inflammation due to cytokine storms and leading to multiple organ failure. This can be explained by an increase in IgG subclasses (IgG2 in mice, IgG1 in humans) that have high affinity for IgG receptors on macrophages (Figure 1). Antibody-dependent enhancement (ADE) is well known for viruses that infect macrophages, such as dengue fever, Zika fever, and Ebola hemorrhagic fever, and conventional inactivated vaccines using viral proteins cannot avoid it. This is because viral proteins stimulate an immune response mediated by T cells to produce antibodies (T cell-dependent), inducing high-affinity IgG on IgG receptors and thus inducing ADE. Therefore, to avoid ADE, it is possible to induce antibodies that do not bind to IgG receptors, that is, to directly stimulate B cells without involving T cells (T cell-independent) (Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004), and technically, vaccines using multiple antigen peptides (MAPs) are the specific method for achieving this.

[0117] Figure 2 shows the structure of a multi-antigen peptide that presents four antigen peptides (i.e., a quadrivalent peptide) and how the multi-antigen peptide directly stimulates marginal zone B cells and B1B cells without the involvement of helper T cells, producing IgM and subclasses of IgG that do not bind to IgG receptors (IgG1 and IgG3 in mice). It is thought that the multi-antigen peptide directly stimulates marginal zone B cells and B1B cells, causing clusters to form on the B cell receptors expressed on their surface, thereby stimulating B cells to produce antigen-specific antibodies.

[0118] In WO2018 / 084247A, mice were administered quadrivalent multiantigen peptides (MAP-4) and octavalent multiantigen peptides (MAP-8) containing a partial peptide of influenza virus hemagglutinin as an antigen. IgG and IgM levels increased in the mice after administration.

[0119] When serum antibody levels were measured by IgG subtype, IgG1 and IgG3, which do not bind to the IgG receptor, were induced, while IgG2a, which does bind to the IgG receptor, was not. This suggests that in animals immunostimulated with MAP, marginal zone B cells and B1B cells are directly stimulated, thereby inducing only antibodies that do not bind to the IgG receptor, and that this antibody production induction is T cell-independent. Furthermore, it has been shown that four antigen peptides linked to a multi-antigen peptide are sufficient to stimulate marginal zone B cells and B1B cells.

[0120] These results indicate that the B cells stimulated by MAP are marginal zone B cells and B1B cells present in the mucosa and serosa, and are different from the follicular B cells that produce antibodies in a T cell-dependent manner as described above. The antibodies produced by these B cells are mainly antibodies that do not bind to the IgG receptor (IgM and IgG3 in mice), or IgG subclasses with low binding ability (IgG1 in mice) (Bennett, KM., et. al., BMC Biotechnol, (15)71, 2015), which is consistent with the finding (Figure 2).

[0121] Furthermore, while IgM produced in response to T cell-dependent stimulation disappears from the bloodstream after about 7-10 days, IgM produced independently of T cells remains in the bloodstream for a longer period (approximately 90 days in mice) (Dintzis, HM, et. al., Proc. Natl. Acad. Sci.USA, (73) 3671-5, 1974), and can be expected to provide viral protection over a relatively long period.

[0122] Based on the results above, avoiding ADE is possible by inducing antibodies that do not bind to the IgG receptor, that is, by directly stimulating B cells without involving T cells (T cell-independent) (Saravanan, P., et. al., Acta Virol., (48) 39-45, 2004), and it is expected that ADE can be avoided technically by vaccines using multiple antigen peptides.

[0123] The peptide of the present invention has an amino acid sequence that is widely conserved across coronaviruses of the Coronaviridae family. Therefore, the peptide of the present invention can induce the production of antibodies effective against coronaviruses in general. Furthermore, the fact that it is widely conserved across coronaviruses suggests that the sequence has been preserved despite repeated natural mutations. In other words, the multi-antigen peptide of the present invention is a mutation-compatible multi-antigen peptide (mMAP) and is suggested to be effective against coronaviruses that may be produced in the future.

[0124] In this invention, we have successfully induced antibody production against coronavirus in vivo using an 11-amino acid short-chain peptide. This 11-amino acid short-chain peptide corresponds to a widely conserved amino acid sequence portion in coronaviruses in general, and the discovery that this short-chain peptide was sufficient to produce antibodies against coronavirus is significant as it suggests the potential of a pan-coronavirus vaccine. In fact, the multi-antigen peptide of this invention also promotes the production of antibodies effective against swine coronavirus and canine coronavirus, which is significant from a zoonotic disease perspective.

[0125] Furthermore, unlike vaccines that use recombinant proteins or vectors requiring a culture process, MAPs, which can be produced through chemical synthesis, can be rapidly mass-produced. This makes them an ideal method for vaccines against pandemic infectious diseases requiring urgent measures, such as those related to COVID-19.

[0126] Explanation of the sequence list Sequence ID 1: An example of the amino acid sequence of a partial peptide of the SARS-CoV-2 spike protein. SEQ ID NO: 2: Amino acid sequence of a partial peptide of the FIPV spike protein having an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 Sequence ID 3: An example of the amino acid sequence of a partial peptide of the SARS-CoV-2 spike protein, including the amino acid sequence surrounding the amino acid sequence of Sequence ID 1. Sequence ID 4: An example of the amino acid sequence of an antigen peptide (a partial peptide of the SARS-CoV-2 spike protein) incorporated into a β-sheet peptide sequence. Sequence ID 5: Amino acid sequence of a partial hemagglutinin peptide of the influenza virus SEQ ID NO: 6: Amino acid sequence of a partial peptide of the PEDV spike protein having an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1 SEQ ID NO: 7: Example of an antigenic peptide SEQ ID NO: 8: Example of an antigenic peptide

Claims

1. A multi-antigen peptide comprising four to eight peptides consisting of a continuous 11-amino acid sequence of amino acids corresponding to the amino acid sequence described in Sequence ID No. 1, or a peptide consisting of a 11-amino acid sequence of the coronavirus spike protein corresponding to the 11-amino acid sequence, and having antibody-inducing ability against said peptide, wherein the multi-antigen peptide comprises a dendritic polymer backbone and one of the peptides, the peptide being linked to the terminal end of the dendritic polymer backbone.

2. The multi-antigen peptide according to claim 1, wherein the 11-amino acid sequence of the coronavirus spike protein corresponding to the 11-amino acid sequence has a one-amino acid substitution with respect to the amino acid sequence described in SEQ ID NO:

1.

3. The multi-antigen peptide according to claim 1 or 2, wherein the 11-amino acid sequence of the coronavirus spike protein corresponding to the 11-amino acid sequence is the amino acid sequence described in SEQ ID NO:

7.

4. The multi-antigen peptide according to claim 2 or 3, wherein the 11-amino acid sequence of the coronavirus spike protein corresponding to the 11-amino acid sequence is a peptide consisting of the amino acid sequence described in SEQ ID NO:

2.

5. The multi-antigen peptide according to claim 2 or 3, wherein the 11-amino acid sequence of the coronavirus spike protein corresponding to the 11-amino acid sequence is a peptide consisting of the amino acid sequence described in SEQ ID NO:

8.

6. The multi-antigen peptide according to claim 1, comprising four or more peptides consisting of a continuous amino acid sequence of 11 amino acids in length, which is the amino acid sequence described in Sequence ID No.

1.

7. The multi-antigen peptide according to claim 6, comprising eight or more 11-amino acid peptides having the amino acid sequence described in any one of claims 1 to 5 or SEQ ID NO:

7.

8. A multi-antigen peptide according to any one of claims 1 to 7, wherein a dendritic polymer skeleton and a peptide are linked via a linker.

9. The multiple antigen peptide according to any one of claims 1 to 8, wherein the dendritic polymer skeleton is composed of lysine, and lysine that forms first-generation branches is peptide-bonded to each of the two amino groups of lysine, and peptides are linked to each of the four amino groups of the lysine that forms the first-generation branches, with or without the linker.

10. The dendritic polymer skeleton is a lysine molecule, to which lysine molecules forming first-generation branches are peptide-bonded to each of the two amino groups of the lysine molecule, to which lysine molecules forming second-generation branches are peptide-bonded to each of the four amino groups of the resulting first-generation lysine molecule, and to which peptide molecules are linked, with or without a linker, to each of the five, six or more, seven or more, or eight amino groups of the resulting second-generation branching lysine molecule.

11. A multi-antigen peptide according to any one of claims 6 to 9, having the following formula (VI): {Here, R is a -linker-peptide or a -peptide, and the peptide is the peptide described in any one of claims 1 to 5, R ２ These are hydrogen, an OH group, a substituted or unsubstituted lower alkyl group, an amino group, an amino acid (especially 3-aminopropanoic acid (β-alanine)), a halogen, or a peptide, and the amino acid is linked to the above molecule via an amide bond.

12. A vaccine against coronavirus comprising the multi-antigen peptide according to any one of claims 6 to 11.

13. The vaccine according to claim 12, which does not contain an adjuvant.

14. The vaccine according to claim 12 or 13, which is used in combination with α-galactosylceramide and not in combination with other adjuvants.

15. The vaccine according to any one of claims 12 to 14, wherein the coronavirus is SARS-CoV-2 or a variant virus.

16. The vaccine according to any one of claims 12 to 14, wherein the coronavirus is one or more coronaviruses selected from porcine epidemic diarrhea virus (PED), canine coronavirus, and feline infectious peritonitis virus (FIPV).

17. A pharmaceutical composition comprising the multi-antigen peptide described in any one of claims 1 to 11.

18. A pharmaceutical composition according to claim 17, for use in activating immunity against coronavirus in a target.

19. A pharmaceutical composition according to claim 17 or 18, for use in inducing antigen-specific immunity against coronavirus in a subject.

20. A pharmaceutical composition according to any one of claims 17 to 19, for use in inducing antigen-specific IgM antibodies against coronavirus in a target.

21. A pharmaceutical composition according to any one of claims 17 to 20, for use in inducing memory immunity against coronavirus in a subject.

22. A pharmaceutical composition according to any one of claims 17 to 21, for use in preventing and / or treating an infection caused by coronavirus in a subject.

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