Multi-antigenic peptides against coronavirus and immunostimulatory compositions containing the same

Abstract

The present application provides a vaccine against coronavirus. According to the present application, there are provided a peptide consisting of the amino acid sequence recited in SEQ ID NO: 1, or a partial peptide of the spike protein of coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO: 1, and a polyantigenic peptide containing a plurality of any of these peptides.

Description

Technical Field

[0001] This invention relates to multi-antigenic peptides against coronaviruses and immune-activating compositions containing them, particularly vaccines against coronaviruses. Background Technology

[0002] It is previously known that multiantigen peptides (MAPs) can induce antibody increases in vivo without the aid of T cells (Non-Patent Document 1). It is known that MAPs can induce autoantibodies (anti-IgE antibodies) that are difficult to induce in vivo (Patent Document 1). Furthermore, it has been disclosed that panviral MAPs are prepared by using a peptide portion of Ebola hemorrhagic fever virus using this method, and mice are immunized with the MAPs, thereby inducing target antibodies in the serum (Patent Document 2). Similarly, it has been disclosed that panviral MAPs are prepared using a partial peptide of hemagglutinin from influenza virus, and mice are immunized with the MAPs, thereby inducing target antibodies in the serum (Patent Document 3).

[0003] Existing technical documents

[0004] Patent documents

[0005] Patent Document 1: US2017-0158738A

[0006] Patent Document 2: US2019-0276495A

[0007] Patent Document 3: US2019-0337989A

[0008] Non-patent literature

[0009] Non-patent literature 1: Saravanan, P., et al., Acta Virol., (48) 39-45, 2004 Summary of the Invention

[0010] This invention provides a multi-antigenic peptide against coronaviruses and immune-activating compositions containing the same, particularly vaccines against coronaviruses. According to the invention, a peptide comprising the amino acid sequence described in Serial No. 1, or a partial peptide of the spike protein of a coronavirus comprising an amino acid sequence corresponding to the amino acid sequence of Serial No. 1 (a peptide comprising an amino acid sequence of the spike protein of a coronavirus corresponding to an amino acid sequence of 11 to 21 amino acids in length containing the amino acid sequence described in Serial No. 1), and a multi-antigenic peptide containing any two or more of these peptides are provided.

[0011] The inventors have discovered that a multiantigen peptide (MAP) comprising a peptide of the spike protein of a coronavirus, either a peptide consisting of the amino acid sequence described in Serial No. 1 or a partial peptide consisting of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1, induces antigen-specific IgM. This antigen-specific IgM can be maintained in the blood for a long period. It has also been found that the aforementioned MAP induces memory immunity. Furthermore, it has been discovered that the aforementioned MAP directly stimulates antibody production from marginal zone B cells and B1B cells, inducing T cell-independent antibody production. The inventors have found that this multiantigen peptide broadly induces antibody production against various coronaviruses. Furthermore, the inventors have found that, as an example of a highly pathogenic coronavirus, in an infection experiment against feline infectious peritonitis virus (FIPV) in cats, administering the multiantigen peptide of the present invention early after the onset of illness (in most cases, the initial symptom is high fever) (e.g., up to the 3rd day after the onset of high fever) can prevent the severity of infection and has a recovery effect.

[0012] The present invention provides the following invention.

[0013] [1] A peptide consisting of a continuous amino acid sequence of 11 to 21 amino acids containing the amino acid sequence of Serial No. 1, which is part of the amino acid sequence recorded as Serial No. 3, or a partial peptide of the spike protein of a coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of 11 to 21 amino acids (or a peptide consisting of an amino acid sequence of the spike protein of a coronavirus corresponding to the amino acid sequence of 11 to 21 amino acids).

[0014] [2] The peptide described in [1] above is a peptide composed of the amino acid sequence recorded in Serial No. 1, or a partial peptide of the spike protein of coronavirus composed of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1 (or a peptide composed of an amino acid sequence of the spike protein of coronavirus corresponding to the amino acid sequence of 11 to 21 amino acids).

[0015] [3] According to the peptide described in [2] above, wherein the amino acid sequence corresponding to sequence number 1 has any one of the following: addition, insertion, substitution and deletion of 1 base relative to the amino acid sequence recorded in sequence number 1.

[0016] [4] According to the peptide described in [2] or [3] above, wherein the amino acid sequence corresponding to sequence number 1 is a peptide composed of the amino acid sequence recorded in sequence number 2.

[0017] [5] A multiantigenic peptide containing any one of the peptides described in [1] to [4] above.

[0018] [6] The multiantigen peptide according to [5] above contains four or more peptides of any one of [1] to [4] above or a peptide having the amino acid sequence described in sequence number 7.

[0019] [7] A vaccine against a coronavirus containing the multiantigenic peptide described above [6].

[0020] [8] The vaccine described above [7] does not contain adjuvants.

[0021] [9] The vaccine described in [6] or [7] above is used in combination with α-galactosylceramide and not in combination with other adjuvants.

[0022]

[10] The vaccine according to any one of [7] to [9] above, wherein the coronavirus is SARS-CoV-2 or a mutant virus of SARS-CoV-2.

[0023]

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

[0024] [1A] A peptide consisting of a continuous amino acid sequence of 11 to 21 amino acids containing the amino acid sequence described in Serial No. 1, or a partial peptide of the spike protein of a coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of the 11 to 21 amino acids (or a peptide consisting of an amino acid sequence of the spike protein of a coronavirus corresponding to the amino acid sequence of the 11 to 21 amino acids).

[0025] [2A] The peptide described in [1A] above is a peptide consisting of the amino acid sequence recorded in Serial No. 1, or a partial peptide of the spike protein of a coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1 (or a peptide consisting of an amino acid sequence of the spike protein of a coronavirus corresponding to the amino acid sequence of 11 to 21 amino acids).

[0026] [3A] According to the peptide described in [2A] above, wherein the amino acid sequence corresponding to sequence number 1 has any one of the following: addition, insertion, substitution and deletion of 1 base relative to the amino acid sequence recorded in sequence number 1.

[0027] [4A] The peptide according to [2A] or [3A] above, wherein the amino acid sequence corresponding to sequence number 1 is a peptide composed of the amino acid sequence recorded in sequence number 2.

[0028] [5A] The partial peptide described in [1A] or [2A] above is a part of the amino acid sequence recorded in Serial No. 3.

[0029] [6A] A multiantigenic peptide containing any one of the peptides described in [1A] to [5A] above.

[0030] [7A] The multiantigen peptide according to [6A] above comprises four or more peptides of any one of [1A] to [5A] above or a peptide having the amino acid sequence described in sequence number 7.

[0031] [8A] The multiantigen peptide according to [6A] or [7A] above, wherein the multiantigen peptide contains a dendritic polymer backbone and a peptide, the peptide being any one of the peptides described in 1 to 5 above and attached to the very end of the dendritic polymer backbone.

[0032] [9A] According to the multiantigen peptide described in [8A] above, the backbone of the dendritic polymer is linked to the peptide by means of a linker.

[0033] [10A] According to the multiantigen peptide described in [8A] or [9A] above, in the backbone of the dendritic polymer, lysine is bonded to the two amino groups of lysine to form the first-generation branch, and the peptide is linked to the four amino groups of lysine forming the first-generation branch by means of a linker or without a linker.

[0034] [11A] According to the multiantigen peptide described in [8A] or [9A] above, in the backbone of the dendritic polymer, lysine is bonded to the two amino groups of lysine with peptide bonds forming the first generation branch, and the lysine forming the second generation branch is bonded to the four amino groups of the first generation lysine formed therefrom with peptide bonds, and the peptide is linked to the five, six, seven or eight amino groups of the lysine forming the second generation branch therefrom by means of a linker or without a linker.

[0035] [12A] The multiantigenic peptide according to any one of [6A] to [10A] above has the following formula (VI),

[0036]

[0037] {Here, R is a linker peptide or peptide, and the peptide is any one of the peptides described in [1A] to [5A] above, R} 2 The group can be hydrogen, an OH group, a substituted or unsubstituted lower alkyl group, an amino group, an amino acid (especially 3-aminopropionic acid (β-alanine)), a halogen, or a peptide, with the amino acid linked to the above molecules via an amide bond.

[0038] [13A] A vaccine against a coronavirus containing any of the above-mentioned [6A] to [12A] multiantigenic peptides.

[0039] [14A] The vaccine described in [13A] above does not contain adjuvants.

[0040] [15A] The vaccine described in [13A] or [14A] above is used in combination with α-galactosylceramide and not in combination with other adjuvants.

[0041] [16A] The vaccine according to any one of [13A] to [15A] above, wherein the coronavirus is SARS-CoV-2 or a mutant virus thereof.

[0042] [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).

[0043] [18A] A pharmaceutical composition comprising the polyantigen peptide described in any one of [6A] to [12A] above.

[0044] [19A] The pharmaceutical composition described in [18A] above is used to activate immunity against coronaviruses in a subject.

[0045] [20A] The pharmaceutical composition according to [18A] or

[19] above is used to induce antigen-specific immunity against coronavirus in a subject.

[0046] [21A] The pharmaceutical composition according to any one of [18A] to [20A] above is used to induce antigen-specific IgM antibodies against coronaviruses in a subject.

[0047] [22A] The pharmaceutical composition according to any one of [18A] to [21A] above is used to induce memory immunity against coronavirus in a subject.

[0048] [23A] The pharmaceutical composition according to any one of [18A] to [22A] above is used in subjects for the prevention and / or treatment of infections caused by coronaviruses.

[0049] [24A] The use of any of the above [6A] to [12A] multiantigen peptides in the manufacture of a medicament for activating immunity against coronaviruses in a subject.

[0050] [25A] The use of any of the above [6A] to [12A] multiantigen peptides in the manufacture of a medicament for inducing antigen-specific immunity against coronaviruses in a subject.

[0051] [26A] The use of the multiantigen peptide described in any one of [6A] to [12A] above in the manufacture of a medicament for inducing antigen-specific IgM antibodies against coronaviruses in a subject.

[0052] [27A] The use of any of the above [6A] to [12A] multiantigen peptides in the manufacture of a medicament for inducing memory immunity against coronaviruses in a subject.

[0053] [28A] The use of the multiantigenic peptide described in any one of [6A] to [12A] above in the manufacture of a medicament for the prevention and / or treatment of infections caused by coronaviruses in subjects.

[0054] [29A] A method of administering a peptide to a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition described in [18A] above.

[0055] [30A] A method for activating immunity against a coronavirus in a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition described in [18A] above.

[0056] [31A] A method for inducing antigen-specific immunity against a coronavirus in a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition described in [18A] above.

[0057] [32A] A method for inducing antigen-specific IgM antibodies against coronaviruses in a subject, comprising the step of administering an effective amount of the pharmaceutical composition described in [18A] above to the subject.

[0058] [33A] A method for inducing memory immunity against a coronavirus in a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition described in [18A] above.

[0059] [34A] A method for preventing and / or treating an infection caused by a coronavirus in a subject, comprising the step of administering to the subject an effective amount of the pharmaceutical composition described in [18A] above.

[0060] [35A] The pharmaceutical composition according to any one of

[19] to

[23] above, wherein the subject is a subject not infected with coronavirus.

[0061] [36A] The pharmaceutical composition according to any one of

[19] to

[23] above, wherein the object is an object infected with coronavirus.

[0062] [37A] The pharmaceutical composition according to any one of

[19] to

[23] above, wherein the subject is a subject who has developed an infection caused by a coronavirus.

[0063] [38A] The application according to any one of

[24] to

[28] above, wherein the object is an object that is not infected with coronavirus.

[0064] [39A] The application according to any one of

[24] to

[28] above, wherein the object is an object infected with coronavirus.

[0065] [40A] The application according to any one of

[24] to

[28] above, wherein the object is an object that has experienced an infection caused by a coronavirus.

[0066] [41A] The method according to any one of

[29] to

[34] above, wherein the object is an object not infected with coronavirus.

[0067] [42A] The method according to any one of

[29] to

[34] above, wherein the object is an object infected with coronavirus.

[0068] [43A] The method according to any one of

[29] to

[34] above, wherein the object is an object that has developed an infection caused by a coronavirus.

[0069] [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).

[0070] [45A] In any of the above

[24] to

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

[0071] [46A] The method described in any one of

[29] to

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

[0072] The multiantigen peptide of the present invention is advantageous in inducing antibody production against coronaviruses. It is also advantageous in establishing memory immunity after administration and in the fact that viral infection further enhances antibody production. Furthermore, the multiantigen peptide of the present invention does not stimulate helper T cells or follicular B cells to produce antibodies, but rather stimulates marginal zone B cells and BB cells in a T cell-independent manner, which is advantageous in preventing antibody-dependent enhancement. The multiantigen peptide of the present invention is also advantageous in being effective against highly pathogenic coronaviruses. Finally, the multiantigen peptide of the present invention is advantageous in being effective in both prophylactic and therapeutic treatments. Attached Figure Description

[0073] Figure 1This diagram illustrates the immune activation mechanisms of existing types of viral vaccines (i.e., inactivated viral vaccines, recombinant protein vaccines, DNA vaccines, and RNA vaccines). Existing vaccines require adjuvants for immune activation, which, when used in combination with adjuvants, induce phagocytosis of the vaccine onto macrophages, antigen presentation to helper T cells, and activation of helper T cells. Activated helper T cells stimulate follicular B cells expressing B cell receptors with affinity for viral antigens, leading to T cell-dependent production of IgG from follicular B cells. The blood concentration of IgM produced by follicular B cells transiently increases and disappears after approximately two weeks, while IgG remains persistently in the blood, predominantly in the form of IgG receptors bound to macrophages. Therefore, IgG produced by follicular B cells during viral infection causes antibody-dependent enhancement (ADE), promoting viral infection and potentially causing severe symptoms.

[0074] Figure 2 A schematic diagram illustrating the molecular structure of the coronavirus vaccine (i.e., the multiantigen peptide) of the present invention and the stimulation of marginal zone B cells and B1B cells by the multiantigen peptide is shown. Marginal zone B cells and B1B cells produce IgM and IgG upon stimulation by the multiantigen peptide without the aid of helper T cells. Thus, IgM, produced in a T cell-independent manner, persists in the blood for a long time, and IgG does not include the subtype that binds to the IgG receptor. This suggests that the coronavirus vaccine of the present invention, which induces the production of IgM and IgG in marginal zone B cells and B1B cells rather than follicular B cells, can provide long-term viral defense without inducing antibody-dependent enhancement.

[0075] Figure 3 This image shows a partial alignment of the amino acid sequences of the spike proteins of SARS (SARS-CoV) which circulated in 2003, feline infectious peritonitis virus (FIPV), and SARS (SARS-CoV-2) which began circulating in 2019. The peptides of this invention were designed for the coated portions.

[0076] Figure 4 An experimental protocol simulating the administration of the multiantigenic peptide (CoV-MAP) of the present invention and subsequent administration of inactivated virus for infection is shown.

[0077] Figure 5 Showing the display via Figure 4 The results of an ELISA experiment show the amount of IgM antibodies against canine coronavirus in serum obtained from the drug administration experiment.

[0078] Figure 6 Showing the display via Figure 4 The results of an ELISA experiment show the amount of antibody against canine coronavirus IgG in serum obtained from the drug administration experiment.

[0079] Figure 7aAn experimental protocol simulating the administration of the multiantigenic peptide (CoV-MAP) of the present invention and subsequent administration of inactivated virus to the infected organism is shown. CoV-MAP was administered at two doses (200 μg / mouse / inoculation).

[0080] Figure 7b To show the passage Figure 7a The results of the ELISA experiment showed the amount of IgG antibodies against porcine coronavirus (PDE) in the serum.

[0081] Figure 8a To demonstrate the results of ELISA (D-body peptide immobilization) of serum IgM in mice administered D-CoV-β-MAP, a peptide composed of D-body amino acids that forms CoV-MAP.

[0082] Figure 8b To demonstrate the results of ELISA (L-body peptide immobilization) of serum IgM in mice administered with D-CoV-β-MAP, a peptide composed of D-body amino acids that forms CoV-MAP.

[0083] Figure 8c To demonstrate the results of ELISA (D-body peptide immobilization) of serum IgG in mice administered D-CoV-β-MAP, which is composed of D-body amino acids that form the CoV-MAP peptide.

[0084] Figure 8d To demonstrate the results of ELISA (L-body peptide immobilization) of serum IgG in mice administered D-CoV-β-MAP, which is composed of D-body amino acids that form the CoV-MAP peptide.

[0085] Figure 9 This describes a dosing regimen for booster immunization with canine coronavirus antigen following three intravenous administrations of the 8-valent CoV-MAP peptide. Cont-MAP, a partial peptide containing influenza virus, served as the first negative control. MAP was administered intravenously at 200 μg / mouse in three divided doses (days 0, 7, and 25). In addition to MAP, α-GalCer, a CD1d ligand, was administered intravenously at a dose of 0.1 μg / mouse in the second dose. The booster immunization was administered intraperitoneally. As the second negative control, an immunogen obtained by binding a peptide (short chain peptide) having the amino acid sequence described in sequence number 8 to keyhole hemocyanin (KHL) was mixed with ALUM (aluminum hydroxide adjuvant) and administered intraperitoneally at a dose of 100 μg / mouse.

[0086] Figure 10 Showing based on Figure 9 The results of the experiment on the immunization procedure. Figure 10The figures show the amount of IgM produced in mouse serum against the aforementioned short-chain peptides after a series of immunization procedures. The vertical axis represents the amount of IgM produced using optical density (OD450 value). The horizontal axis represents the number of days since the first administration.

[0087] Figure 11 Showing based on Figure 9 The results of the experiment on the immunization procedure. Figure 11 The table shows the amount of IgM against canine coronavirus antigen produced in mouse serum after a series of immunization procedures. The vertical axis represents the amount of IgM produced using optical density (OD450 value). The horizontal axis represents the number of days since the first dose.

[0088] Figure 12 Showing based on Figure 9 The results of the experiment on the immunization procedure. Figure 12 The table shows the amount of IgM produced in mouse serum against porcine epidemic diarrhea virus (PDE) antigen after a series of immunization procedures. The vertical axis represents the amount of IgM produced using optical density (OD450 value). The horizontal axis represents the number of days since the first administration.

[0089] Figure 13 Showing based on Figure 9 The results of the experiment on the immunization procedure. Figure 13 The table shows the production of IgM against SARS-CoV-2 spike protein antigen, MERS spike protein antigen, and SARS-CoV-2 spike protein (S2) in mouse serum after a series of immunization procedures. The vertical axis represents the IgM production level using optical density (OD450 value). The horizontal axis represents the number of days since the first administration.

[0090] Figure 14 Showing with Figure 9 Results when the same regimen was used to administer the quadrivalent CoV-MAP peptide. Figure 14 The figures show the amount of IgM produced in mouse serum after a series of immunization procedures for each short-chain peptide, porcine epidemic diarrhea virus antigen, and canine coronavirus antigen.

[0091] Figure 15 Showing with Figure 9 The same regimen was used to induce the production of various IgMs in animals with MAP containing the No. 2 peptide (a peptide with the amino acid sequence described in sequence number 8, but whose constituent amino acids are all D-body peptides).

[0092] Figure 16 Shown to be with Figure 9 The same regimen was used to induce the production of various IgMs in animals with MAP containing the No. 3 peptide (a peptide with the amino acid sequence described in sequence number 8, but in which the amino acids constituting it are D-bodies in italics).

[0093] Figure 17 Shown to be with Figure 9 The same regimen was used to induce the production of various IgMs in animals with MAP containing the No. 4 peptide (a peptide with the amino acid sequence described in sequence number 8, but in which the amino acids constituting it are D-bodies in italics).

[0094] Figure 18 Shown to be with Figure 9 The same regimen was used to induce the production of various IgMs in animals with MAP containing the No. 5 peptide (a peptide with the amino acid sequence described in sequence number 2, but in which the amino acids constituting it are D-bodies in italics).

[0095] Figure 19 Shown to be with Figure 9 The same regimen was used to induce the production of various IgMs in animals with MAP containing the No. 6 peptide (a peptide with the amino acid sequence described in sequence number 1).

[0096] Figure 20 The results for peptides No.1 through No.6 are shown in comparison.

[0097] Figure 21 This demonstrates that the level of IgM induced by the CoV-MAP peptide (No.1 peptide) can be maintained for a long period of time. Figure 21 Serum IgM levels against various antigens on day 137 of administration are shown as a ratio to pre-treatment IgM levels.

[0098] Figure 22 The results of the ELISPOT assay, showing the long-term maintenance of IgM levels induced by the CoV-MAP peptide, are shown. The vertical axis represents the number of spleen cells producing IgM antibodies against the peptide. JEV-MAP is a negative control, representing an octavalent MAP containing a portion of the E protein of Japanese encephalitis virus.

[0099] Figure 23 The uptake of CoV-MAP into macrophages is shown. An anti-FLAG-tagged monoclonal antibody was used as a positive control. Serum from untreated mice was used as a negative control.

[0100] Figure 24 Showing comparisons before and after booster immunization to... Figure 9 Results of the production of various IgGs in animals treated with the same regimen for CoV-MAP.

[0101] Figure 25 Showing comparisons before and after booster immunization to... Figure 9 Results of the production of various IgGs in animals treated with the same regimen for CoV-MAP.

[0102] Figure 26 The resulting IgG subclasses are shown.

[0103] Figure 27 This experiment demonstrates the pre-administration and post-infection administration of CoV-MAP in feline infectious peritonitis virus (FIPV) infection experiments.

[0104] Figure 28 This demonstrates that the vaccine is effective against FIPV infection and exhibits antipyretic effects in the CoV-MAP treatment group. Detailed Implementation

[0105] In this specification, "object" refers to animals with an immune system, such as vertebrates, including mammals, fish, birds, amphibians, reptiles, such as humans, chimpanzees, gorillas, orangutans, monkeys, marmosets and bonobos, pigs, rats, mice, cattle, sheep, goats, horses, cats and dogs, and other tetrapods (such as carnivores, even-toed ungulates, odd-toed ungulates and rodents), and bats and other animals of the order Bats.

[0106] In this specification, "coronavirus" refers to a virus belonging to the family Coronaviridae in the order Nidovirales. Coronaviruses have two or more spike proteins (S proteins) on their envelope structure on the surface of the viral particle, and are named coronaviruses because their appearance under an electron microscope resembles the crown of the sun. Known to cause respiratory infections such as the common cold in humans, SARS coronavirus (SARS-CoV), MERS coronavirus (MERS-CoV), and the 2019 novel coronavirus (SARS-CoV-2) are known to be fatal. Other known lethal viruses include mouse hepatitis virus (MHV) and feline infectious peritonitis virus (FIPV). As of April 2020, no vaccines or antiviral drugs have been developed for the prevention or treatment of human coronavirus infection. Coronaviruses bind to the surface of target cells by binding their spike proteins, exposed on the envelope, to angiotensin-converting enzyme 2 (ACE2), a cell surface molecule, and are then ingested into the cell via endocytosis, thereby infecting the cell. Examples of coronaviruses include coronaviruses from the subfamily Coronaviridae, alpha coronaviruses (e.g., canine coronavirus, alpha coronavirus 1, human coronavirus 229E, human coronavirus NL63, porcine epidemic diarrhea virus), beta coronaviruses (e.g., subgenus Embecovirus, subgenus Sarbecovirus, subgenus Merbecovirus, subgenus Nobecovirus, such as human enterocoronavirus 4408, human coronavirus OC43, mouse coronavirus, human coronavirus HKU1), SARS-related coronaviruses (e.g., SARS coronavirus (SARS-CoV), the novel coronavirus of 2019 (SARS-CoV)). V-2), MERS coronavirus, equine coronavirus), gamma coronavirus (e.g., avian coronavirus, beluga whale coronavirus SW1), and delta coronavirus (e.g., bulbul coronavirus HKU11, weaver bird coronavirus HKU13, thrush coronavirus HKU12). Additionally, mutant strains of these can be listed (especially mutant viruses with sequence number 1 or the amino acid sequence corresponding to sequence number 1, such as mutant viruses with an amino acid sequence having 1 to 2 (preferably 1) amino acid additions, insertions, deletions, or substitutions (e.g., conserved substitutions: substitutions between acidic amino acids, substitutions between basic amino acids, and substitutions between hydrophobic amino acids, etc.) relative to the amino acid sequence of sequence number 1 or 2).

[0107] In this instruction manual, "adjuvant" refers to a substance that activates the immune system. Aluminum hydroxide and aluminum phosphate are commonly used as adjuvants.

[0108] In this specification, "α-galactosylceramide" refers to a glycosphingolipid that can be isolated from the sponge *Agelasmauritianus*, a type of sponge. α-galactosylceramide has the following structure.

[0109]

[0110] α-Galactosylceramide is a ligand that binds to CD1d and can activate NKT cells.

[0111] In this specification, "multi-antigen peptide" (MAP) refers to a molecule capable of multiple presentation of peptides having a specific amino acid sequence. A MAP may have a structure in which peptides are linked to side chains of a backbone with repeating structures. The backbone and peptides can be linked by a linker. Examples of MAPs include those with a dendritic polymer backbone. Examples of MAPs with a dendritic polymer backbone include those containing lysine (Lys) as a core, with lysine residues further linked to the core by peptide bonds to increase the number of branches, and having a 2% affinity relative to the number of branches n. n Molecules containing up to one amino group and the peptides linked to it (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).

[0112] In this specification, "dendritic polymer" refers to a molecule having a structure that branches regularly from a center (core). A dendritic polymer consists of a central molecule called the core and side chain portions called branches. Each arm of the molecule that forms the branching core has two or more arms, and these branches are connected to the branching molecules. Such branching molecules with two or more arms connect to form a dendritic polymer. The term "generation" is used to indicate the number of consecutive branches from the core in a dendritic polymer. The central molecule provides the 0th generation branch (G0), the branch at the root of the branch provides the 1st generation branch (G1), the branch further forming from this branch provides the 2nd generation branch (G2), and this process repeats to provide the nth generation branch (Gn).

[0113] In this specification, "lower alkyl group" refers to an alkyl group having 1 to 4 carbon atoms, for example. Examples of lower alkyl groups include straight-chain or branched alkyl groups. Examples of straight-chain alkyl groups include methyl, ethyl, n-propyl, and n-butyl. Examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, and tert-butyl. In this specification, substituted lower alkyl groups can be lower alkyl groups substituted with any substituent. Substituents can 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).

[0114] In this specification, "a partial peptide of the coronavirus spike protein consisting of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1" refers to a peptide that is a partial peptide of the coronavirus spike protein, consisting of a sequence that corresponds to the amino acid sequence of Serial No. 1 by alignment. Similarly, "a peptide consisting of an amino acid sequence of the coronavirus spike protein corresponding to the amino acid sequence of Serial No. 1" also refers to a peptide consisting of an amino acid sequence of the coronavirus spike protein, consisting of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1. For example, in Serial No. 1, amino acid numbers 798–808 in GenBank accession number AAP30030.1 are common amino acid sequences in SARS-CoV-2 and SARS-CoV. The amino acid sequence of Serial No. 1 has the LLF sequence (leucine-leucine-phenylalanine) formed by the three amino acids in the coronavirus spike protein. The LLF sequence is generally considered essential for coronavirus infection of animal cells. A peptide may contain more than one D-somatic amino acid.

[0115] According to the present invention, a peptide consisting of the amino acid sequence recorded in Serial No. 1, or a partial peptide of the spike protein of a coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of Serial No. 1 (or, a peptide having an amino acid sequence of the spike protein of a coronavirus corresponding to the amino acid sequence of Serial No. 1) may be provided.

[0116] According to the present invention, a peptide consisting of a continuous amino acid sequence of 11 to 21 amino acids containing the amino acid sequence of Serial Number 1, which is part of the amino acid sequence described in Serial Number 3, is also provided; or a partial peptide of the spike protein of a coronavirus consisting of an amino acid sequence corresponding to the amino acid sequence of 11 to 21 amino acids. The length of 11 to 21 amino acids refers to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20 amino acids, as will be stated hereinafter in this specification.

[0117] According to the present invention, a peptide comprising a continuous amino acid sequence of 11 to 21 amino acids of the spike protein of feline infectious peritonitis virus (FIPV) and containing the amino acid sequence described in Serial No. 2 is also provided. According to the present invention, a peptide having an amino acid sequence of the spike protein of FIPV corresponding to the above-described peptide is also provided.

[0118] According to the present invention, a peptide comprising a continuous amino acid sequence of 11 to 21 amino acids of the spike protein of porcine epidemic diarrhea virus (PEDV) and containing the amino acid sequence described in Serial No. 6 is also provided. According to the present invention, a peptide having an amino acid sequence of the PEDV spike protein corresponding to the above-described peptide is also provided.

[0119] According to the present invention, a peptide comprising a continuous amino acid sequence of 11 to 21 amino acids of the spike protein of canine coronavirus, and containing an amino acid sequence corresponding to the amino acid sequence described in Serial No. 1, is also provided. According to the present invention, a peptide having an amino acid sequence of the spike protein of PEDV corresponding to the above-described peptide is also provided.

[0120] In this specification, these peptides are sometimes referred to as the peptides of the present invention.

[0121] The peptides of this invention are isolated or purified. The peptides of this invention can be artificially synthesized.

[0122] The peptide composed of the amino acid sequence of sequence number 1 is 11 amino acids in length. By integrating this peptide into a multiantigenic peptide, coronavirus-specific antibodies can be induced. The amino acid sequence of sequence number 1 is widely conserved throughout the Coronaviridae family. In contrast, peptides containing this amino acid sequence and its successor amino acid sequences can exhibit high specificity for particular coronaviruses.

[0123] A partial peptide of the coronavirus spike protein, consisting of an amino acid sequence corresponding to the amino acid sequence of Serial Number 1, is as defined above. In one embodiment, the amino acid sequence of the partial peptide corresponding to Serial Number 1 may have any one of the following: addition, insertion, substitution, and deletion of a single base relative to the amino acid sequence described in Serial Number 1. Alternatively, in another embodiment, the partial peptide may have the amino acid sequence described in Serial Number 7: SX1IEDLLFX2KV {here, X1 is F, A, or V, and X2 is D or N}. Furthermore, in a preferred embodiment, the partial peptide may have a sequence consisting of the amino acid sequences described in Serial Number 2 or 6. Examples of substitutions include, for instance, 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 Serial Number 2 is a partial peptide of the spike protein of feline infectious peritonitis virus (FIPV), having a sequence corresponding to the peptide consisting of the amino acid sequence of Serial Number 1.

[0124] According to the present invention, a multi-antigen peptide containing the peptide of the present invention (hereinafter sometimes referred to as "the multi-antigen peptide of the present invention") is provided. In the multi-antigen peptide, the peptide of the present invention is attached to the terminal portion of a dendritic polymer backbone. This terminal portion can be connected to the peptide of the present invention via a linker. The connection can be a covalent bond. The linker is not particularly limited as long as it does not significantly hinder peptide presentation; chemically stable linkers such as polyethylene glycol, flexible linkers, etc. (e.g., non-peptide linkers or peptide linkers) can be used. A chemically stable linker can be stable at least under physiological conditions. Peptide linkers can be used as linkers. When using peptide linkers, flexible linkers such as GS linkers and linkers having secondary structures such as α-helical structures or β-sheet structures can be used. The multi-antigen peptide of the present invention may contain one or more peptides of the present invention. 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 peptide of the present invention is preferably isolated or purified.

[0125] In one embodiment of the present invention, the multi-antigen peptide is a multi-antigen peptide as follows:

[0126] The backbone contains a dendritic polymer and the multi-antigenic peptide of the present invention. The core of the dendritic polymer is lysine. Taking this lysine as the 0th generation, the following condition (condition 1) is satisfied when k is any natural number from 1 to n: the peptide of the present invention is attached to the amino group of the lysine of the nth generation.

[0127] (Requirement 1) Lysine of the kth generation is peptide bonded to the amino group of the amino group of the k-1th generation lysine.

[0128] 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, this embodiment of the present invention provides a 4-valent to 2-valent... n+1 Multivalent antigenic peptides.

[0129] In one embodiment of the present invention, the polyantigen peptide of the present invention may be the following polyantigen peptide (i.e., a tetravalent polyantigen peptide): containing a dendritic polymer backbone and the polyantigen peptide of the present invention, wherein the core of the dendritic polymer is lysine, and lysine forming first-generation branches is peptide-bonded to the two amino groups of the lysine, and the peptide of the present invention is respectively linked to the amino groups of the lysine forming the first-generation branches, thereby comprising four of the above-mentioned peptides.

[0130] In one embodiment of the present invention, the polyantigen peptide of the present invention is a polyantigen peptide (i.e., a 4-valent to 8-valent polyantigen peptide): containing a dendritic polymer backbone and the polyantigen peptide of the present invention, wherein the core of the dendritic polymer is lysine, and lysine forming a first-generation branch is peptide-bonded to the two amino groups of the lysine, lysine forming a second-generation branch is peptide-bonded to the amino groups of the lysine forming the first-generation branch, and the peptide of the present invention is attached to the amino groups of the lysine forming the second-generation branch, thereby containing 4 to 8 of the above-mentioned peptides.

[0131] In a preferred embodiment, the multi-antigen peptide of the present invention can 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. Corresponding to the algebraic number n of the branching, tetravalent to octavalent is provided. n+1 MAP-2 n+1 ).

[0132]

[0133] These multiantigenic peptides can be synthesized by introducing peptides into a dendritic backbone immobilized in resin. More specifically, in the case of MAP-4, for example, a multiantigenic peptide having the structure of formula (III) can be prepared by introducing peptides into an amino group that forms the backbone of the aforementioned dendritic polymer, consisting of a core lysine and two lysines forming the first-generation branch. The core lysine can be immobilized in resin.

[0134]

[0135] {In the formula, the lines represent peptide bonds formed by the aminobutyl and amino groups from lysine residues.}

[0136] Alternatively, MAP-8 can be prepared, for example, by introducing an amino group into a backbone consisting of a core lysine, two lysines forming the first-generation branch, and four lysines forming the second-generation branch, thus creating a multi-antigenic peptide having the structure of formula (IV). The core lysine can be immobilized in a resin.

[0137]

[0138] {In the formula, the lines represent peptide bonds formed by the aminobutyl and amino groups from lysine residues.}

[0139] In a preferred embodiment, the multi-antigen peptide of the present invention may have the structure of formula (III) or (IV) {here, the peptide is the peptide of the present invention}. That is, in one embodiment, the multi-antigen peptide of the present invention may be a peptide in which two lysine residues forming a first-generation branch are linked by peptide bonds to the amino-butyl and amino groups of the core lysine residue, and peptides are linked to the four amino groups of the lysine residues in the backbone of the formed dendritic polymer by peptide bonds or other chemically stable bonds. In another embodiment, the multi-antigen peptide of the present invention may be a peptide in which two lysine residues forming a first-generation branch are linked by peptide bonds to the amino-butyl and amino groups of the core lysine residue, and four lysine residues forming a second-generation branch are linked by peptide bonds to the amino-butyl and amino groups of each of these two lysine residues, and peptides are linked to the eight amino groups of the lysine residues in the backbone of the formed dendritic polymer by peptide bonds or other chemically stable bonds. Such peptides can be prepared as follows: using methods well known to those skilled in the art, a dendritic polymer backbone is formed by polymerizing Lys at two amino groups on a solid-state Lys core via the Fmoc method. The peptide is then further polymerized with the amino groups of lysine residues in the resulting dendritic polymer backbone. Alternatively, an azide group can be introduced into the amino groups of lysine residues in the resulting dendritic polymer backbone, and the peptide can be attached to the dendritic polymer backbone via click chemistry.

[0140] In a preferred embodiment, the multiantigen peptide of the present invention may have the structure of the following formula (VI).

[0141]

[0142] {Here, R stands for -connector-peptide or -peptide, and the peptide may contain the amino acid sequence of the peptide of the present invention, R} 2 It can be hydrogen, OH group, substituted or unsubstituted lower alkyl group, amino group, amino acid (e.g., 3-aminopropionic acid (β-alanine), i.e., -NH-C2H4-COOH), halogen, or peptide, and the amino acid can be linked to the above molecules via an amide bond. It should be noted that MAP-8 can have the following structure: two compounds having the structure of formula (VI) are formed by linking the carboxyl group of the 0th generation lysine in formula (VI) to the amino group of another lysine. Similarly, MAP-2 with an nth generation branch can be specified. n+1 Each has its own structure.

[0143]

[0144] {Here, R 2 As defined above.

[0145] In the above, the connector can be polyethylene glycol (PEG). In a preferred embodiment, the PEG can be, for example, a PEG with a degree of polymerization of 2 to 30, a PEG with a degree of polymerization of 5 to 20, or a PEG with a degree of polymerization of 10 to 15.

[0146] In the above description, the peptides are composed of, or comprise, the peptides of the present invention and the linker peptides. The linker peptides can be, for example, flexible linkers or peptides forming β-sheet structures.

[0147] Compounds having the structure of formula (VI) above can be obtained by reacting a peptide or peptide-connector compound with the ethynyl group (-C≡CH) of a compound having the structure of formula (VII) below using click chemistry. More specifically, they can be obtained by reacting a peptide-azido group or peptide-connector-azido group with an azido group at the end with a compound having the structure of formula (VII) below in the presence of monovalent copper ions. Peptide-azido groups and peptide-connector-azido 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 azido group, or by condensing a connector with an azido group and a carboxyl group at each end.

[0148]

[0149] {Here, R 2 As defined above.

[0150] The compound of formula (VII) is a synthetic intermediate of the compound of formula (VI). For the amino group of the lysine providing the first-generation branch, from the viewpoint of reaction convenience, it is attached to an artificial amino acid with an ethynyl group on the side chain via a peptide bond, thereby setting a reactive ethynyl group at the end (e.g., see WO2015190555A). It should be noted that the synthetic intermediate of MAP-8 can have the following structure: a structure formed by attaching two compounds having the structure of formula (VII) to the amino group of other lysines via the carboxyl group of the 0th-generation lysine in formula (VII). Similarly, MAP-2 with the nth-generation branch can be specified. n+1 The structures of the synthetic intermediates.

[0151] The multi-antigenic peptide of the present invention can be prepared, for example, according to the method described in WO2018 / 084247A. The lysine backbone of MAP can be prepared, for example, by polymerizing lysine with two amino groups protected by a 9-fluorenylmethoxycarbonyl group (Fmoc group) using the Fmoc method. The peptide can be further extended relative to the amino groups of the lysine using the Fmoc method. The Fmoc method can be appropriately implemented by those skilled in the art using well-known techniques. The Fmoc method can be carried out on a solid-phase support. Wang resin, HMPA resin, HMBA resin, and NovaSyn TGT resin, etc., can be used as solid-phase supports. The method of synthesizing peptides on a solid-phase support is called solid-phase synthesis and is well known to those skilled in the art. Thus, those skilled in the art can suitably prepare the multi-antigenic peptide of the present invention using conventional methods.

[0152] In a preferred embodiment, the multi-antigen peptide of the present invention can be a multiple homoantigen peptide. In a multiple homoantigen peptide, all antigen peptides contain the same amino acid sequence.

[0153] In another preferred embodiment, the multi-antigen peptide of the present invention may contain two or more peptides with different amino acid sequences as antigen peptides.

[0154] In this invention, the multi-antigen peptide can be administered to a subject to induce immunity (particularly antigen-specific immunity). In this invention, the multi-antigen peptide can be administered to a subject to induce the production of antigen-specific antibodies. Furthermore, in this invention, the multi-antigen peptide can be administered to a subject to induce antibodies against coronaviruses.

[0155] In this invention, a vaccine containing the polyantigenic peptide of this invention is provided (sometimes referred to as "the vaccine of this invention").

[0156] The vaccine of this invention is broadly effective against coronaviruses. The vaccine of this invention is effective against SARS-CoV-2. The vaccine of this invention is effective against feline infectious peritonitis virus (FIPV).

[0157] The vaccine of the present invention can be used without combining it with adjuvants that stimulate helper T cells (or T cell-dependent immunity) (e.g., aluminum salts, such as aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, such as incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, sedimenting adjuvants), preferably without combination. The vaccine of the present invention may be adjuvant-free (e.g., aluminum salts, such as aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, such as incomplete Freund's adjuvant, complete Freund's adjuvant, liquid paraffin, lanolin, sedimenting adjuvants), preferably without adjuvants. Preferably, the vaccine of the present invention can be used in combination with agents that do not stimulate helper T cells (T cell-dependent immunity), such as α-galactosylceramide.

[0158] The vaccine of the present invention may contain the multiantigenic peptide of the present invention and a pharmaceutically acceptable carrier and / or excipient. The vaccine of the present invention may be a non-oral administration vaccine (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation administration). Therefore, as pharmaceutically acceptable carriers and / or excipients, carriers and / or excipients suitable for non-oral administration (e.g., intravenous, intradermal, subcutaneous, intramuscular, intraperitoneal, nasal, mucosal, or inhalation administration) may be used. Examples of pharmaceutically acceptable carriers and / or excipients include, for example, salts, buffers, pH adjusters, isotonic agents, preservatives, and water.

[0159] According to the present invention, a vaccine may be a composition for generating antibodies against coronaviruses or establishing immunity against coronaviruses in a subject.

[0160] According to the present invention, a multi-antigenic peptide is provided for generating antibodies against coronaviruses in a subject.

[0161] According to the present invention, the use of the peptide of the present invention in the manufacture of vaccines is provided. According to the present invention, the use of the multi-antigen peptide of the present invention in the manufacture of vaccines is provided. The vaccine can be used to generate antibodies against coronaviruses or to establish immunity against coronaviruses in a subject.

[0162] According to the present invention, a method for administering an antigenic peptide to a subject is provided, comprising the step of administering the multiantigen peptide of the present invention to the subject. According to the present invention, a method for activating antigen-specific immunity in a subject is provided, comprising the step of administering an effective amount of the multiantigen peptide of the present invention to the subject. According to the present invention, a method for activating immunity against a coronavirus in a subject is provided, comprising the step of administering an effective amount of the multiantigen peptide of the present invention to the subject. According to the present invention, a multiantigen peptide of the present invention for use in these methods, or a pharmaceutical composition containing the multiantigen peptide of the present invention for use in these methods, is provided. According to the present invention, the use of the multiantigen peptide of the present invention in the manufacture of a pharmaceutical for use in these methods is provided.

[0163] According to the present invention, a method for administering a peptide to a subject is provided, comprising the step of administering the multiantigen peptide of the present invention to the subject according to a dosing schedule. According to the present invention, the subject may be a subject uninfected with coronavirus, a subject infected with coronavirus (which may be asymptomatic), or a subject who has developed an infection caused by coronavirus. The method or multiantigen peptide of the present invention can prevent infection caused by coronavirus in subjects uninfected with coronavirus, can delay the onset of infection caused by coronavirus, can suppress the onset or cure the infection, and, if the disease has already occurred, can alleviate symptoms, delay the progression of symptoms (e.g., inhibit severe illness), stop the progression, or reduce symptoms. The method or multiantigen peptide of the present invention can delay the onset of symptoms, inhibit the onset, or cure the infection in subjects infected with coronavirus (e.g., subjects before the onset of illness), and, if the disease has already occurred, can alleviate symptoms, delay the progression of symptoms (e.g., inhibit severe illness), stop the progression, or reduce symptoms. The method or multiantigen peptide of the present invention can delay the progression of symptoms (e.g., inhibit severe illness), stop the progression, or reduce symptoms in subjects suffering from an infection caused by coronavirus. The multi-antigen peptide of the present invention primarily induces IgM, thus the time from administration to antibody induction is short, and it is effective even when administered to subjects infected with coronaviruses or suffering from infectious diseases.

[0164] According to the present invention, a method for preventing the onset of symptoms in a subject infected with a coronavirus can be provided, comprising administering an effective amount of the multiantigenic peptide of the present invention to the subject. The subject infected with a coronavirus can, in one manner, be a subject pre-infectious. The subject infected with a coronavirus can be a subject confirmed to be infected with a coronavirus by examination (e.g., PCR test or antibody test). The subject infected with a coronavirus can be a subject confirmed to be infected with a coronavirus by examination (e.g., PCR test or antibody test) and pre-infectious. Thus, for the subject, the onset of symptoms can be delayed, the onset of symptoms suppressed, or the infectious state cured.

[0165] According to the present invention, a method is further provided, comprising the steps of administering an effective amount of the multiantigen peptide of the present invention to a subject before the onset of an infection caused by a coronavirus, and further comprising the steps of administering an effective amount of the multiantigen peptide of the present invention to the subject after the onset of the infection. According to the present invention, a method for administering a peptide to a subject is further provided, comprising the steps of administering the multiantigen peptide of the present invention to the subject according to a dosing schedule, the dosing schedule including the steps of administering the multiantigen peptide of the present invention to the subject (e.g., 1 to 3 times) before infection or at a stage considered to be pre-infection. According to the present invention, a method for administering a peptide to a subject is further provided, comprising the steps of administering the multiantigen peptide of the present invention to the subject according to a dosing schedule, the dosing schedule including the steps of administering the multiantigen peptide of the present invention to the subject 1 to 3 times before infection or at a stage considered to be pre-infection, and the steps of administering the multiantigen peptide of the present invention more than once after infection or at a stage considered to be post-infection. According to the present invention, a method for administering a peptide to a subject is provided, comprising the steps of administering the multiantigen peptide of the present invention to the subject according to a dosing schedule, the dosing schedule including the steps of administering the multiantigen peptide of the present invention 3 times before infection or at a stage considered to be pre-infection, and the steps of administering the multiantigen peptide of the present invention more than once after infection or at a stage considered to be post-infection. In this invention, by administering an additional dose after the onset of illness, the progression of symptoms can be delayed (e.g., inhibiting severe illness), the progression can be stopped, or symptoms can be alleviated in the aforementioned subjects. Regarding administration, the multiantigen peptide can be administered at a dosage of, for example, 1 mg / kg to 3 mg / kg, or for example, 1.5 mg / kg, for a single dose. Preferably, administration can be performed on different days. Preferably, administration can be performed at intervals of daily, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 8-13 days, 2 weeks, 15-20 days, 3 weeks, or more. In this method, the subject can be, for example, a human subject. In this method, the subject can be, for example, a feline subject. In this method, the subject can be, for example, a canine subject. In this method, the subject can be, for example, a swine subject. In this method, it is possible to prevent and / or treat infections caused by coronaviruses in the subjects.

[0166] Specifically, as shown in the examples described later, when CoV-mMAP, which is the multi-antigenic peptide of the present invention, was administered to a cat before infection according to the above-described dosing schedule, the infection symptoms of feline infectious peritonitis virus (FIP), which has a 100% mortality rate after onset, were recovered. We consider this a noteworthy effect. According to the present invention, a pharmaceutical composition containing the multi-antigenic peptide of the present invention is provided for administration according to the above-described dosing schedule. The above-described pharmaceutical composition can be a pharmaceutical composition for the prevention and / or treatment of coronavirus infection. According to the present invention, the use of the multi-antigenic peptide of the present invention in the manufacture of a medicament for administration according to the above-described dosing schedule is provided. The above-described medicament can be a medicament for the prevention and / or treatment of coronavirus infection.

[0167] According to the present invention, a method for preventing and / or treating infections caused by coronaviruses in subjects with such need is provided, comprising the step of administering the multiantigenic peptide of the present invention to the subject. According to the present invention, a pharmaceutical composition containing the multiantigenic peptide of the present invention is provided for a method of preventing and / or treating infections caused by coronaviruses in subjects with such need. According to the present invention, the use of the multiantigenic peptide of the present invention in a medicament for manufacturing a method of preventing and / or treating infections caused by coronaviruses in subjects with such need is provided.

[0168] According to the present invention, a method for treating a coronavirus infection in a subject is provided, comprising administering an effective amount of the multiantigen peptide of the present invention to the subject. In one manner, the subject suffering from coronavirus infection may be a subject exhibiting fever. The multiantigen peptide of the present invention primarily induces IgM, thus the time from administration to antibody induction is short, and it is effective even when administered to subjects in the early stages of infection. Therefore, the subject may, for example, exhibit any of the following symptoms: olfactory abnormalities, gustatory abnormalities, and fever, such as early symptoms.

[0169] According to the present invention, a method for preventing the development of severe coronavirus infection in a subject is provided, comprising administering an effective amount of the multiantigen peptide of the present invention to the subject. In one manner, the subject suffering from coronavirus infection may be a subject with fever. The multiantigen peptide of the present invention primarily induces IgM, thus the time from administration to antibody induction is short, and it is effective even when administered to an already infected subject (e.g., a subject in the initial stage of infection).

[0170] According to the present invention, the coronavirus can be a highly pathogenic coronavirus. For example, the coronavirus can be a coronavirus selected from the group consisting of MERS coronavirus, SARS coronavirus (e.g., SARS-CoV, SARS-CoV-2, etc.), feline infectious peritonitis virus (FIPV), and porcine epidemic diarrhea virus (PDE).

[0171] According to the present invention, the target can be a human or a non-human mammal. According to the present invention, the pharmaceutical composition and the drug can be administered to humans or non-human mammals.

[0172] Example

[0173] Example 1: Synthesis of antigenic peptides

[0174] As an antigenic peptide used to make vaccines against coronaviruses, it is a partial peptide of the spike protein, which is the surface protein of SARS-CoV, SARS-CoV-2, and feline infectious peritonitis virus (FIPV).

[0175] Specifically, the spike proteins of the three viruses were subjected to multiple alignment to generate a peptide with an 11-component amino acid sequence (Sequence No. 1) (see reference). Figure 3 ).

[0176] [Table 1]

[0177] Table 1: Amino acid sequence information of the antigenic peptide.

[0178]

[0179] * The numbers recorded in the amino acid sequence represent the amino acid numbers of the terminal amino acids in the spike protein.

[0180] As shown in Table 1, the 11 consecutive amino acid sequence segments showed high homology in SARS-CoV, SARS-CoV-2, feline infectious peritonitis virus (FIPV), and porcine epidemic diarrhea virus (PEDV).

[0181] A peptide with the amino acid sequence of sequence number 1 was artificially synthesized using conventional methods, and a 4-valent multiantigen peptide (MAP-4) with the structure shown in formula (I) and an 8-valent multiantigen peptide (MAP-8) with the structure shown in formula (II) were prepared.

[0182]

[0183] To achieve efficient synthesis on resin, these multiantigenic peptides were synthesized by introducing peptides into a dendritic backbone immobilized on the resin. More specifically, in the case of MAP-4, a resin-introduced peptide containing a dendritic backbone having the structure of formula (III) below was synthesized.

[0184]

[0185] In addition, in the case of MAP-8, a resin-introduced peptide having the structure of the following formula (IV) is used.

[0186]

[0187] In the above formula, lysine (Lys) is an amino acid with two amino groups. By linking the carboxyl groups of two lysines to each amino group via peptide bonds, a dendritic backbone is formed with the two terminal lysines having a total of four amino groups. By introducing a peptide into this lysine amino group using a linker, a tetravalent multiantigen peptide (MAP-4) can be prepared.

[0188] In the case of MAP-8, the terminal lysine residues are further branched into two, resulting in a resin-introducing peptide containing eight Fmoc protecting groups on a Lys residue. By linking the four amino groups of the lysine residues in the aforementioned dendritic backbone with a total of four amino groups to the carboxyl groups of four lysine residues via peptide bonds, a dendritic backbone with a total of eight amino groups on the terminal four lysine residues is formed. By introducing peptides into this lysine residue using a linker, tetravalent or octavalent multiantigen peptides can be prepared.

[0189] Peptides can be synthesized using the Fmoc method according to conventional procedures. Specifically, after swelling the resin and removing the N-terminal amino acid protecting group using a deprotecting agent, the peptide is synthesized by condensing the next amino acid using a coupling reagent and an amino acid derivative. As coupling reagents, 1-[bis(dimethylamino)methylene]-1H-benzotriazole 3-oxide hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIEA) are used. For the peptide, it is cleaved from the resin using a TFA mixture and precipitated with cold tert-butyl methyl ether, then washed and recovered. After drying, the ether is removed, the peptide is dissolved in a 50% aqueous acetonitrile solution, and then freeze-dried.

[0190] When introducing the above peptide, a linker is introduced at the C-terminus of the peptide. In this embodiment, polyethylene glycol (PEG) is used as the linker. The polyethylene glycol is PEG with a degree of polymerization of 12 (also referred to as "PEG(12)").

[0191] The more specific structure of MAP-4 is shown below.

[0192]

[0193] Here, R is a PEG(12)-peptide, R 2 As defined above.

[0194] The MAP-4 and MAP-8 mentioned above can be made by referring to WO2018062217A, for example.

[0195] Then, the obtained peptides were removed from the resin using 0.1% 2,2,2-trifluoroacetic acid and acetone, and the free MAP-4 and MAP-8 were purified by high performance liquid chromatography to obtain MAP-4 and MAP-8.

[0196] Example 2: Drug administration experiment

[0197] The multi-antigen peptides (MAP-4 and MAP-8; collectively referred to as CoV-MAP) linked with the amino acid sequence of sequence number 1 prepared in Example 1 were processed according to... Figure 4 The administration protocol was as follows in mice. Specifically, for Balb / c mice, CoV-MAP was administered via tail vein on days 0, 7, and 25 at a dose of 100 μg / mouse / inoculation or 200 μg / mouse / inoculation. On day 7, 0.1 μg of α-galactosylceramide (α-GalCer) was administered via tail vein. Then, on day 32, inactivated canine coronavirus was administered intraperitoneally. Vanguard (Ministry of Agriculture, Forestry and Fisheries Directive 25, Animal Drug No. 2225, Zoetis Japan Co., Ltd.; hereinafter the same) was used as the inactivated canine coronavirus.

[0198] Serum was recovered on day 0 (before administration), days 26–31 (after 3 administrations of CoV-MAP), and day 39 (one week after administration of inactivated canine coronavirus).

[0199] Antibody titers in serum were determined. Specifically, inactivated canine coronavirus was immobilized in 96-well plates. As a background control, wells immobilized with ovalbumin-bound ALUM instead of inactivated canine coronavirus were measured.

[0200] As the assay system, an ELISA method based on a peroxidase-based colorimetric system was used. The average OD value of two wells under the same conditions was calculated. The value obtained by subtracting the OD value of the background control from the OD value against canine inactivated coronavirus was calculated. In addition, to confirm the specificity of the reaction, mouse serum administered with influenza virus MAP (influenza-MAP 200 μg; MAP prepared separately according to WO2018084247A with the amino acid sequence described in Serial No. 5) was used instead of CoV-MAP to determine the reactivity to the coronavirus peptide.

[0201] The results of the above CoV-MAP drug administration experiment are as follows: Figure 5 and 6 As shown. Figure 5 This shows the changes in serum IgM antibody titer. Figure 6 The changes in serum IgG antibody titer are shown.

[0202] like Figure 5 As shown, serum IgM antibody titers increased in serum after three doses of CoV-MAP, and also increased or remained elevated after administration of inactivated canine coronavirus. On the other hand, no increase in antibody titers against canine coronavirus was observed in mice administered MAP containing influenza virus.

[0203] In addition, such as Figure 6 As shown, 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 MAP (metastatic influenza virus).

[0204] Example 3: Dosing Experiment of Canine Coronavirus Vaccine

[0205] The CoV-MAP (200 μg / mouse / inoculation) prepared as in Example 1 was used as follows: Figure 7a Mice were administered the drug on days 0, 7, and 25, as described in the literature. On day 7, 0.1 μg of α-galactosylceramide was administered via the tail vein. As a control, 2E5-MAP was administered instead of CoV-MAP. 2E5-MAP is a MAP containing a partial peptide of the hemagglutinin of the influenza virus, containing a peptide with an amino acid sequence portion shared by H3 belonging to group 1 and H1 belonging to group 2. Specifically, 2E5-MAP contains eight DGWYGFRHQNSEGTGQAADLKSTQA (serial number 5). Then, mimicking viral infection, 50 μL of Vanguard, an inactivated vaccine against canine coronavirus, was administered intraperitoneally on day 32.

[0206] 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. The ELISA used a system with immobilized porcine epidemic diarrhea virus (PED) antigen.

[0207] The results are as follows Figure 7b As shown. Figure 7b As shown, significant IgG induction was observed after Vanguard administration in a system with PED antigen immobilized. The PED antigen was an inactivated vaccine obtained by heating the Nippon Seikei live PED vaccine (which will be used as a porcine epidemic diarrhea vaccine) at 60°C for 10 hours.

[0208] Next, using the amino acid sequence of sequence number 1, all amino acids were replaced with D-molecules, and peptides forming β-sheet structures were linked before and after the C-terminus of these amino acids to form peptide linkers (sequence number 4). Otherwise, MAP-8 with the same structure as described above was prepared. The resulting CoV-mMAP was administered via tail vein at a dose of 200 μg / mouse / inoculation. Figure 8a and 8b The second combination used 0.1 μg / mouse of α-galactosylceramide. ELISA was performed using a plate with an immobilized structure containing a D-body peptide, where all amino acids of the peptide consisting of the amino acid sequence of sequence number 1 are D-body peptides.

[0209] The results are as follows Figures 8a-8d As shown. Figures 8a-8d As shown, the D-body CoV-mMAP also induces both IgM and IgG. This indicates that the amino acid sequence can be the D-body. Furthermore, antibody induction was not inhibited even when the backbone of the antigenic peptide and the multi-antigenic peptide was linked using peptide linkers (e.g., linkers capable of forming β-sheet structures).

[0210] according to Figure 9 The dosing regimen shown was used to administer CoV-mMAP, followed by booster immunization with canine coronavirus vaccine antigen. As a negative control (Cont-MAP), an octavalent MAP containing a partial peptide of the influenza virus was used. Additionally, an antigen, not a MAP but a general peptide vaccine, containing the amino acid sequence described by sequence number 8 linked to keyhole hemocyanin, was prepared, mixed with ALUM, and administered intraperitoneally. Serum was obtained on days 0, 14, 52, 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.

[0211] First, the vaccine efficacy was evaluated using serum IgM levels bound to the peptide used as the antigen. Mouse serum (1 / 200 dilution) was reacted overnight at 4°C with a BSA fusion antigen peptide immobilized on an ELISA plate. Detection was performed using standard methods, namely, binding to a biotin-labeled anti-mouse IgM polyclonal antibody (SouthernBiotech) used as the detection antibody, followed by further reaction of peroxidase-labeled streptavidin with the detection antibody. Results are as follows: Figure 10 As shown. Figure 10 As shown, a significant increase in serum IgM levels was observed in the CoV-mMAP treatment group after two immunizations. Furthermore, a significant increase in serum IgM levels was also observed in the CoV-mMAP treatment group following a booster immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. Additionally, in the group administered a conventional peptide vaccine of existing types, a transient increase in serum IgM levels was observed after the second immunization, but no increase in serum IgM levels was observed due to booster immunization. These results indicate that conventional peptide vaccines of existing types are ineffective in establishing IgM-generating memory immunity.

[0212] Then, the efficacy of the vaccine was evaluated using serum IgM levels that 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.). 10 μg / mL of the antigen was immobilized on an ELISA plate, exposed to serum, and serum IgM levels were measured in the same manner as described above. Results are as follows: Figure 11 As shown. Figure 11 As shown, a significant increase in serum IgM levels was observed in the CoV-mMAP treatment group after two immunizations. Furthermore, a booster immunization in the CoV-mMAP treatment group resulted in a significant increase in serum IgM levels. In contrast, no significant increase in serum IgM levels was observed in the negative control group. Additionally, in the group administered a conventional peptide vaccine of existing types, an increase in serum IgM levels was observed after the second immunization, but no increase in serum IgM levels due to booster immunization was observed. These results indicate that conventional peptide vaccines of existing types are ineffective in establishing IgM-generating memory immunity.

[0213] Furthermore, the efficacy of the porcine epidemic diarrhea virus (PED) vaccine was evaluated using serum IgM levels bound to the live vaccine (attenuated porcine epidemic diarrhea virus P-5V strain, Nippon Seikei Corporation). The live vaccine was inactivated by incubation at 60°C for 10 hours, immobilized on an ELISA plate, and then exposed to serum. Serum IgM levels were measured in the same manner as described above. Results are as follows... Figure 12 As shown. Figure 12 As shown, a significant increase in serum IgM levels was observed in the CoV-mMAP treatment group after two immunizations. Furthermore, a significant increase in serum IgM levels was also observed in the CoV-mMAP treatment group after booster immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. Additionally, in the groups treated with conventional peptide vaccines of existing types, the increase in serum IgM levels after the second immunization was weak, and no increase in serum IgM levels was observed after booster immunization. These results indicate that conventional peptide vaccines of existing types are difficult to establish IgM-generating memory immunity, and therefore cannot induce IgM responses to peptides, canine coronavirus antigens, and PED virus antigens.

[0214] Furthermore, the efficacy of the vaccine was evaluated using serum IgM levels targeting the full-length SARS-CoV-2 spike protein, the MERS spike protein, and the SARS-CoV-2 spike protein (S2 subunit), respectively. Recombinant MERS spike protein (SinoBiological), recombinant SARS-CoV-2 spike protein (Invitrogen), and its recombinant S2 subunit protein (RayBiotech) were immobilized at 5 μg / ml and then contacted with serum in the same manner as described above. Serum IgM levels were measured in the same way as described above. Results are as follows... Figure 13 As shown. Figure 13As shown, a significant increase in serum IgM levels was observed in the CoV-mMAP treatment group after two immunizations. Furthermore, a significant increase in serum IgM levels was also observed in the CoV-mMAP treatment group after booster immunization. In contrast, no significant increase in serum IgM levels was observed in the negative control group. These results indicate that CoV-mMAP induces IgM responses similar to coronaviruses that exhibit high pathogenicity in humans.

[0215] The quadrivalent CoV-mMAP was prepared in the same manner as the octavalent CoV-mMAP. Following the same... Figure 9 Mice were administered the same dosing regimen intravenously with quadrivalent CoV-mMAP, followed by booster immunization with canine coronavirus vaccine antigen administered intraperitoneally. The BSA-linked antigenic peptide, PED virus vaccine antigen, and canine coronavirus vaccine antigen were immobilized on an ELISA plate in the same manner as described above, and then contacted with serum in the same way. Serum IgM levels were measured in the same manner as described above. Results are as follows... Figure 14 As shown. Figure 14 As shown, after two immunizations, a significant increase in serum IgM levels was observed in the CoV-mMAP treatment group. This indicates that CoV-mMAP is also effective when it is a tetravalent peptide.

[0216] 8-valent CoV-mMAPs with various modified peptides were prepared. The peptides used are shown in Table 2 below.

[0217] [Table 2]

[0218] Table 2: Peptides used for MAP during the experiment

[0219]

[0220] In Table 2, italics and underlines indicate amino acids of the D body, and bold text indicates amino acids that are different from peptide No.1 (serial number 8).

[0221] These CoV-mMAPs are arranged according to Figure 9 The dosing regimen shown was administered to mice, and serum was obtained at 2 weeks, 4 weeks, 5–6 weeks, and 137 days. Additionally, PED virus vaccine and canine coronavirus vaccine were immobilized on ELISA plates and contacted with serum in the same manner as described above, and serum IgM levels were measured in the same way. Results are as follows... Figures 15-20 As shown. Figures 15-20 As shown, MAPs containing any peptide all induced an increase in virus-specific serum IgM levels. This indicates that even changing the amino acids used in CoV-mMAP to the D-type, or changing one amino acid to several amino acids, does not hinder the induction of target IgM; therefore, CoV-MAP could be a powerful vaccine capable of responding to amino acid variations.

[0222] In addition, serum IgM levels were investigated 137 days later. The serum IgM level before CoV-mMAP administration was set as 1, and serum IgM levels were calculated as relative values. Results are as follows... Figure 21 As shown. Figure 21 As shown, an increase in serum IgM levels specific to PED virus vaccine and canine coronavirus vaccine was observed. This indicates that CoV-mMAP-induced IgM can persist in the blood for more than 4 months and can be maintained long-term.

[0223] Then, the effect on memory immunity was confirmed. To detect cells producing memory IgM, mice were similarly immunized with CoV-mMAP, and spleen cells from day 133 of immunization were used to detect cells producing IgM against the peptide (SAIEDLLFNKV) by ELISPOT assay. JEV-MAP (an octavalent MAP containing a portion of the E protein amino acid sequence of Japanese encephalitis virus) was used as a negative control. The IgM was generated in ELISPOT plates immobilized with peptide-BSA at 5 μg / ml at 5 × 10⁻⁶ mcg / ml. 5 After inoculating spleen cells at a density of [number] cells / ml, they were cultured at 37°C and 5% CO2 for 22 hours. After washing, biotin-labeled anti-IgM antibody (Southern Biotech) was added, and the cells were reacted at 37°C for 1 hour. Then, streptavidin-labeled peroxidase was added, and the spots were developed using standard methods. The number of spots was counted using an ELISPOT analyzer (KS ELISPOT), and the average value of two wells was calculated for each individual. The number of cells producing anti-peptide IgM in the CoV-mMAP group was significantly higher than that in the JEV-MAP group. This indicates that CoV-mMAP-induced IgM-producing cells can serve as long-term memory cells in vivo.

[0224] Macrophages express Fc receptors on their cell surface, which bind to IgG antibodies against antigens, allowing them to take up IgG along with the antigen. It is generally believed that the amount of antigen taken up by macrophages reflects the amount of virus taken up. If a virus capable of infecting macrophages is taken up by macrophages with the aid of antibodies, the presence of antibodies promotes viral infection into macrophages, leading to antibody-dependent enhancement (ADE). A FLAG-tagged recombinant protein was prepared using an *E. coli* recombinant protein expression system, with bovine serum albumin (BSA) bound to a peptide (SAIEDLLFNKV; SEQ ID NO. 8). The prepared artificial viral peptide, namely peptide-FLAG-BSA (concentration 5 μg / ml), was mixed with serum (concentration 10%) and reacted at 37°C for 1 hour. This mixture was then added to the culture medium of mouse macrophage cell line P388-D1 (inoculated with 50,000 cells) and cultured at 37°C in the presence of 5% CO2 for 24 hours to induce uptake. For the peptide-FLAG-BSA taken up into cells, anti-FLAG monoclonal antibody (BioLegend) was labeled with PE (Phycoerythrin). After cell permeation treatment, the results were detected by flow cytometry using FACS CantoII (Becton Dickinson). The percentage of all viable cells was used to indicate the final uptake of peptide-FLAG-BSA. Results are as follows: Figure 23 As shown. Figure 23 As shown, it can be confirmed that when anti-FLAG mouse monoclonal antibody was used instead of mouse serum, almost all cells (99.5%) took up the antibody (positive control). On the other hand, in the case of untreated normal mouse serum, only 2.1% of positive cells were detected, indicating that most cells did not take up IgM-bound artificial viral peptides (negative control). Under these conditions, when CoV-mMAP-immunized mouse serum (a mixture of serum from days 28 and 32 before booster immunization) was used, only 3.5% of cells took up the artificial viral peptide, which was not significantly different from that in normal mouse serum. Therefore, it can be concluded that even if IgM induced by CoV-mMAP immunization binds to the virus, it will not be taken up by macrophages in the form of immune complexes.

[0225] CoV-mMAP according to Figure 9 Mice were administered the drug as described above, and serum samples were obtained on day 25 (before booster immunization) and day 60 (after booster immunization). The antigen-specific serum IgG levels of each serum sample were then measured. The antigenic peptide, canine coronavirus vaccine antigen, and porcine epidemic diarrhea virus antigen were immobilized on an ELISA plate as described above, contacted with serum, and serum IgG levels were measured in the same manner as above. Results are as follows... Figure 24 As shown. Figure 24As shown, an increase in serum IgG levels against each antigen was observed after booster immunization.

[0226] Similarly, serum IgG levels binding to the SARS-CoV-2 spike protein (full-length), MERS spike protein, and SARS-CoV-2 spike protein (S2 protein) were evaluated. Results are as follows... Figure 25 As shown. Figure 25 As shown, an increase in serum IgG levels against each antigen was observed after booster immunization. Subclasses of IgG were analyzed here.

[0227] As a comparison, serum from mice immunized with the KLH-binding peptide along with ALUM (containing KLH-peptide-induced IgG) was used. The KLH-peptide induces all IgG subclasses, but in CoV-mMAP-immunized mice, only IgG1 was elevated in all mice in the serum boosted with canine coronavirus antigen (CoV-mMAP-IgG), with almost no detectable uptake of IgG2a and IgG2b by macrophages. Therefore, while CoV-mMAP immunization may induce IgG during viral infection, it does not induce antibody-dependent enhancement-related IgG subclasses, and thus, unlike conventional vaccines, does not pose a risk of antibody-dependent enhancement.

[0228] The efficacy of CoV-mMAP against feline infectious peritonitis virus (FIPV) infection was investigated. Four experimental cats (approximately 5 months old) were prepared as a pre-administered CoV-mMAP group and a no-treatment group, respectively. The cats were infected with highly pathogenic coronaviruses (FIPV) with a 100% lethality rate after the onset of disease to verify the efficacy of CoV-mMAP (refer to...). Figure 27 CoV-mMAP was administered intravenously at a dose of 1.5 mg / kg on days 0, 7, and 21, with α-galactosylceramide 0.7 μg / kg administered intravenously concurrently on day 7. Regarding viral exposure, the formation of 3 × 10⁻⁶ cells was confirmed using CRFK cells. 5 10 ml of culture medium was used to treat the plaque at a PFU / ml level. The first administration was orally. For surviving cats, the second administration was administered intraperitoneally after three consecutive subcutaneous injections of prednisolone at an immunosuppressive dose of 2 mg / kg. In cats that developed symptoms, except for one cat that developed symptoms after the first administration, CoV-mMAP and α-galactosylceramide were administered intravenously on day 3 (early administration), days 13–15 (late administration), and day 7. Results are as follows... Figure 28As shown. A temperature above 39.5°C is defined as high fever, and the onset of FIP is defined as a period of high fever confirmed for more than 3 consecutive days. In the CoV-mMAP treatment group, No. 5, who developed symptoms, did not receive additional CoV-mMAP, and therefore euthanized due to persistent high fever (see cross symbol). Individuals who received additional CoV-mMAP after onset showed improvement in high fever. In particular, No. 7 in the CoV-mMAP group, who received early additional administration (on the 3rd day of onset), recovered to normal body temperature before viral exposure and was completely cured. FIPV is known to have the highest pathogenicity among coronaviruses, with almost 100% mortality in infected cats. The multi-antigen peptide of this invention has significant technical implications in preventing FIPV infection and providing therapeutic effects. In the CoV-mMAP treatment group, No. 3, who received late-term additional administration, although developing a low-grade fever, did not resolve other symptoms such as ascites, and therefore was euthanized. In the untreated group, No. 4 and No. 6 temporarily developed low-grade fever due to CoV-mMAP administration, but were euthanized due to a relapse into high fever. The results are summarized in Table 3.

[0229] [Table 3]

[0230] Table 3: Summary of individual symptoms, etc.

[0231]

[0232] [Inspection]

[0233] SARS-CoV-2 infects macrophages in addition to airway mucosal cells, leading to systemic inflammation caused by a cytokine storm and resulting in multi-organ failure. This can be explained by the increase in IgG subclasses (IgG2 in mice and IgG1 in humans) that have high affinity for IgG receptors on macrophages. Figure 1 Antibody-dependent enhancement (ADE) is common in dengue, Zika, and Ebola hemorrhagic fever viruses that infect macrophages, and existing inactivated vaccines using viral proteins cannot avoid this problem. This is because viral proteins stimulate T-cell-mediated immune responses to produce antibodies, thus (T-cell dependent) inducing high-affinity IgG receptors, thereby inducing ADE. Therefore, to avoid ADE, it is possible to avoid it by inducing antibodies that do not bind to IgG receptors, that is, by directly stimulating B cells without the aid of T cells (T-cell independent) (Saravanan, P., et al., Acta Virol., (48) 39-45, 2004). Technically, vaccines based on multi-antigenic peptides (MAPs) are equivalent to this specific approach.

[0234] Figure 2The structure of a polyantigen peptide presenting four antigenic peptides (i.e., tetravalent) is shown, and the polyantigen peptide directly stimulates marginal zone B cells and B1B cells without the aid of helper T cells, producing IgG subclasses (IgG1 and IgG3 in the mouse case) that do not bind to IgM and IgG receptors. It can be assumed that the polyantigen peptide directly stimulates marginal zone B cells and B1B cells, forming clusters with B cell receptors expressed on their surface, thereby stimulating B cells and stimulating the production of antigen-specific antibodies.

[0235] In WO2018 / 084247A, mice were administered tetravalent multiantigen peptides (MAP-4) and octavalent multiantigen peptides (MAP-8) containing partial peptides of hemagglutinin from influenza virus as antigens. The mice administered these peptides showed increased IgG and IgM levels.

[0236] Serum antibody levels were determined based on IgG subtypes. Subtypes that do not bind to the IgG receptor, namely IgG1 and IgG3, were induced, while IgG2a, which binds to the IgG receptor, was not induced. This indicates that in animals immunized with MAP, direct stimulation of marginal zone B cells and B1B cells induces only antibodies of the type that do not bind to the IgG receptor, and that the induction of this antibody production is independent of T cells. Furthermore, it is known that four antigenic peptides linked to multiple antigenic peptides are sufficient to stimulate marginal zone B cells and B1B cells.

[0237] In this result, the B cells stimulated by MAP were marginal zone B cells and B1B cells present on the mucosa and serous membrane, which are different from the follicular B cells that produce antibodies in a T cell-dependent manner. This is consistent with the observation that the antibodies produced by these B cells are mainly antibodies that do not bind to the IgG receptor (IgM and IgG3 in the mouse case) or IgG subclasses with low binding ability to the IgG receptor (IgG1 in the mouse case) (Bennett, KM., et al., BMC Biotechnol, (15) 71, 2015). Figure 2 ).

[0238] In addition, IgM produced through T cell-dependent stimulation disappears from the blood in about 7 to 10 days, while IgM produced independently of T cells persists in the blood for a long time (about 90 days in mice) (Dintzis, HM, et al., Proc. Natl. Acad. Sci. USA, (73) 3671-5, 1974), which can be expected to provide a certain degree of long-term viral defense.

[0239] Based on the above results, ADE can be avoided by inducing antibodies that do not bind to IgG receptors, that is, by directly stimulating B cells without the aid of T cells (T cell independent) (Saravanan, P., et al., Acta Virol., (48) 39-45, 2004). Technically, it is expected that ADE can be avoided by using vaccines with multiple antigenic peptides.

[0240] The peptides of this invention have a widely conserved amino acid sequence across all coronaviruses in the Coronaviridae family. Therefore, the peptides of this invention can produce antibodies effective against all coronaviruses. Furthermore, the widespread conserved sequence across all coronaviruses implies that it is a sequence conserved even with repeated natural mutations, meaning that the multiantigen peptides of this invention are mutation-compatible MAPs (mMAPs) and are effective against future coronaviruses.

[0241] In this invention, a short-chain peptide of 11 amino acids was used to induce antibody production against coronaviruses in vivo. This 11-amino acid short-chain peptide corresponds to a widely conserved amino acid sequence in all coronaviruses, and the fact that this short-chain peptide is sufficient to induce antibody production against coronaviruses suggests the potential of pan-coronavirus vaccines, which is significant. In fact, the multi-antigenic peptide of this invention promoted the production of antibodies effective against both porcine and canine coronaviruses, which is significant in the field of zoonotic infectious diseases.

[0242] Furthermore, unlike recombinant proteins that require culture processes and vaccines that use vectors, MAPs, which can be produced through chemical synthesis, can be produced rapidly and in large quantities. This makes it the most suitable method for vaccines of infectious diseases that require urgent countermeasures, such as those against COVID-19.

[0243] Description of sequence lists

[0244] Serial No. 1: An example of the amino acid sequence of a partial peptide of the SARS-CoV-2 spike protein.

[0245] Serial No. 2: The amino acid sequence of a partial peptide of the FIPV spike protein having an amino acid sequence corresponding to that of Serial No. 1.

[0246] Serial number 3: An example of the amino acid sequence of a partial peptide of the SARS-CoV-2 spike protein containing the amino acid sequence surrounding the amino acid sequence of Serial number 1.

[0247] Serial number 4: An example of the amino acid sequence of an antigenic peptide (partial peptide of the SARS-CoV-2 spike protein) integrated into a β-sheet peptide sequence.

[0248] Serial number 5: Amino acid sequence of a partial peptide of the hemagglutinin of influenza virus.

[0249] Serial number 6: The amino acid sequence of a partial peptide of the PEDV spike protein having an amino acid sequence corresponding to that of Serial number 1.

[0250] Serial number 7: Example of an antigenic peptide

[0251] Serial number 8: Example of an antigenic peptide. sequence list <110> National Research Institute of Physics and Chemistry Animal Allergy Clinical Testing Co., Ltd. <120> Multiantigen peptides targeting coronaviruses and immune-activating compositions containing them <130> PR13-9017WO <150> JP2020-89340 <151> 2020-05-22 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial sequence <220> <223> SARS-CoV-2 antigenic peptide <400> 1 Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial sequence <220> <223> FIPV antigen peptide <400> 2 Ser Ala Ile Glu Asp Leu Leu Phe Asp Lys Val 1 5 10 <210> 3 <211> 31 <212> PRT <213> Artificial sequence <220> <223> SARS-CoV-2 antigenic peptide L <400> 3 Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu 1 5 10 15 Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln 20 25 30 <210> 4 <211> 25 <212> PRT <213> Artificial sequence <220> <223> SARS-CoV-2 antigenic peptide integrated into β-sheet peptides <400> 4 Asp Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Gly Lys Gly 1 5 10 15 Ser Ile Asp Thr Ser Ala Lys Phe Ser 20 25 <210> 5 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Influenza virus antigen peptides <400> 5 Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln 1 5 10 15 Ala Ala Asp Leu Lys Ser Thr Gln Ala 20 25 <210> 6 <211> 11 <212> PRT <213> Artificial sequence <220> <223> PEDV antigen peptide <400> 6 Ser Val Ile Glu Asp Leu Leu Phe Asn Lys Val 1 5 10 <210> 7 <211> 11 <212> PRT <213> Artificial sequence <220> <223> antigenic peptide <220> <221> MISC_FEATURE <222> (2)..(2) <223> X = V, A, or F <220> <221> MISC_FEATURE <222> (9)..(9) <223> X = D or N <400> 7 Ser Xaa Ile Glu Asp Leu Leu Phe Xaa Lys Val 1 5 10 <210> 8 <211> 11 <212> PRT <213> Artificial sequence <220> <223> antigenic peptide <400> 8 Ser Ala Ile Glu Asp Leu Leu Phe Asn Lys Val 1 5 10

Claims

1. A multi-antigenic peptide comprising a peptide consisting of four or eight consecutive amino acid sequences of 11 amino acid lengths, comprising the amino acid sequence described in Serial No. 1, or an 11 amino acid sequence comprising an amino acid sequence of a fragment of a coronavirus spike protein corresponding to the 11 amino acid sequence, said fragment comprising an amino acid sequence selected from the group consisting of Serial Nos. 2, 6, and 8. Multiantigen peptides contain a dendritic polymer backbone and peptides, with the peptides attached to the very ends of the dendritic polymer backbone. In the backbone of dendritic polymers, lysine is bonded to the two amino groups of lysine to form the first-generation branch via peptide bonds. The peptide is then linked to the four amino groups of the lysine forming the first-generation branch, either via a linker or without a linker. In the backbone of dendritic polymers, lysine has two amino groups that are bonded to the lysine that forms the first-generation branch via peptide bonds. The lysine that forms the second-generation branch is bonded to the four amino groups of the first-generation lysine that forms the second-generation branch via peptide bonds. Peptides are linked to the eight amino groups of the lysine that forms the second-generation branch, either with or without a linker. The structure of the multiantigenic peptide is shown in formula (VI) or formula (VIII) below. Here, R stands for -linker-peptide or -peptide, which is a peptide consisting of the amino acid sequence described by sequence numbers 1, 2, 6, or 8. 2 It can be hydrogen, OH group, substituted or unsubstituted lower alkyl, amino, amino acid, or halogen, with the amino acid linked to the molecule via an amide bond. It also has the ability to induce antibodies against this peptide.

2. The multiantigen peptide according to claim 1, wherein, The amino acid sequence of the fragment of the coronavirus spike protein corresponding to the 11-amino acid sequence is a peptide composed of the amino acid sequence recorded in Serial No.

2.

3. The multiantigen peptide according to claim 1, wherein, The amino acid sequence of the fragment of the coronavirus spike protein corresponding to the 11-amino acid sequence is a peptide composed of the amino acid sequence recorded in Serial No.

8.

4. The multiantigen peptide according to claim 1, comprising 4 or 8 consecutive amino acid sequences of 11 amino acid lengths consisting of the amino acid sequence described in Serial Number 1.

5. The use of the multiantigen peptide of claim 1 in the manufacture of a vaccine against SARS-related coronaviruses, wherein the multiantigen peptide comprises 4 or 8 peptides of 11 amino acid length consisting of amino acid sequences of fragments of the spike protein of SARS-related coronaviruses corresponding to the 11-amino acid sequence.

6. The use of the multiantigen peptide of claim 1 in the manufacture of a vaccine against canine coronavirus, wherein the multiantigen peptide comprises 4 or 8 peptides of 11 amino acid length consisting of an amino acid sequence of a fragment of the spike protein of canine coronavirus corresponding to the 11-amino acid sequence.

7. The use of the multiantigen peptide of claim 1 in the manufacture of a vaccine against porcine epidemic diarrhea virus (PED), wherein the multiantigen peptide comprises 4 or 8 peptides of 11 amino acid length consisting of amino acid sequences of fragments of the spike protein of porcine epidemic diarrhea virus (PED) corresponding to the 11 amino acid sequence.

8. The use of the multiantigen peptide of claim 1 in the manufacture of a vaccine against feline infectious peritonitis virus (FIPV), said multiantigen peptide comprising 4 or 8 peptides of 11 amino acid length consisting of an amino acid sequence of a fragment of the spike protein of feline infectious peritonitis virus (FIPV) corresponding to the 11 amino acid sequence.

9. The application according to any one of claims 5 to 8, wherein, The vaccine does not contain adjuvants.

10. A pharmaceutical composition comprising the polyantigenic peptide according to any one of claims 1 to 4.

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