WO2024085143A1 - Peptide antigénique dérivé de la nucléocapside, acide nucléique, vecteur, composition pharmaceutique, complexe peptide antigénique/hla et procédé de détection des lymphocytes t - Google Patents

Peptide antigénique dérivé de la nucléocapside, acide nucléique, vecteur, composition pharmaceutique, complexe peptide antigénique/hla et procédé de détection des lymphocytes t Download PDF

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WO2024085143A1
WO2024085143A1 PCT/JP2023/037529 JP2023037529W WO2024085143A1 WO 2024085143 A1 WO2024085143 A1 WO 2024085143A1 JP 2023037529 W JP2023037529 W JP 2023037529W WO 2024085143 A1 WO2024085143 A1 WO 2024085143A1
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hla
cells
peptide
cov
amino acid
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Japanese (ja)
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千尋 本園
貴将 上野
由比古 後藤
裕幸 岸
洋 浜名
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国立大学法人 熊本大学
国立大学法人富山大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor

Definitions

  • the present disclosure relates to nucleocapsid-derived antigenic peptides, nucleic acids, vectors, pharmaceutical compositions, prevention methods, HLA/antigenic peptide complexes, and methods for detecting T cells.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • the currently widely used mRNA vaccine for the spike protein of SARS-CoV-2 is a vaccine that aims to induce neutralizing antibodies that directly bind to and inhibit the virus.
  • the spike protein is prone to mutations, and the omicron-type SARS-CoV-2 has more than 30 mutations compared to the original SARS-CoV-2. Mutations allow the virus to evade the immune system from neutralizing antibodies. For this reason, there are concerns that the efficacy of an mRNA vaccine for the spike protein of SARS-CoV-2 may be reduced in terms of preventing infection and suppressing the rate of severe illness (Non-Patent Documents 1-3).
  • the present disclosure therefore aims to provide new peptides that can be used to induce immune responses against SARS-CoV-2.
  • the nucleocapsid-derived antigenic peptide of the present disclosure comprises the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (KAYNVTQAF); (P2) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, inserted, substituted, or added; (P3) A polypeptide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:1.
  • the nucleic acid of the present disclosure encodes the peptide of the present disclosure.
  • the vector of the present disclosure contains the nucleic acid of the present disclosure.
  • the cytotoxic T cell inducer and/or activator of the present disclosure includes the peptide, nucleic acid, and/or vector of the present disclosure.
  • the pharmaceutical composition of the present disclosure comprises the peptide, nucleic acid, vector, and/or cytotoxic T cell inducer and/or activator of the present disclosure and a pharma- ceutical acceptable carrier.
  • the prevention methods disclosed herein use the peptides, nucleic acids, vectors, and/or cytotoxic T cell inducers and/or activators disclosed herein.
  • the HLA/antigenic peptide complexes of the present disclosure include complexes of human leukocyte antigens (HLA) and antigenic peptides,
  • the antigenic peptides include the nucleocapsid-derived antigenic peptides of the present disclosure.
  • the method for detecting T cells includes a detection step of contacting a biological sample with an HLA/antigen peptide complex, thereby detecting T cells in the biological sample that bind to the HLA/antigen peptide complex,
  • the HLA/antigenic peptide complex is an HLA/antigenic peptide complex of the present disclosure.
  • the antigen peptide disclosed herein provides a new peptide that can be used to induce an immune response against SARS-CoV-2.
  • FIG. 1 is a schematic diagram of a method for detecting T cells specific to an antigen derived from SARS-CoV-2 from PBMCs collected from a patient who was infected with SARS-CoV-2 and recovered in Example 1.
  • FIG. 2 is a graph showing the results of flow cytometry in Example 1, in which overlapping peptides were added to PBMCs collected from patients who had recovered from SARS-CoV-2 infection, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • FIG. 3 is a graph showing the numerical values of the percentage of T cells expressing CD25 and CD137 from the results of flow cytometry in Example 1.
  • FIG. 1 is a schematic diagram of a method for detecting T cells specific to an antigen derived from SARS-CoV-2 from PBMCs collected from a patient who was infected with SARS-CoV-2 and recovered in Example 1.
  • FIG. 2 is a graph showing the results of flow cytometry in Example 1, in which overlapping peptides were added to PB
  • FIG. 4 is a schematic diagram of a method for detecting T cells specific to an antigen derived from SARS-CoV-2 after activating T cells of PBMCs collected from a patient who was infected with SARS-CoV-2 and recovered with inactivated SARS-CoV-2 virus in Example 1.
  • FIG. 5 is a graph showing the numerical values of the percentage of T cells expressing CD25 and CD137 from the results of flow cytometry in Example 1, in which overlapping peptides were added to PBMCs activated with inactivated SARS-CoV-2 virus.
  • FIG. 6 is a graph showing the results of flow cytometry in which overlapping peptides were added to B lymphocytes and the expression of IFN- ⁇ and CD8 on the cell surface was evaluated in Example 1.
  • FIG. 5 is a graph showing the numerical values of the percentage of T cells expressing CD25 and CD137 from the results of flow cytometry in Example 1, in which overlapping peptides were added to PBMCs activated with inactivated SARS-Co
  • FIG. 7 is a schematic diagram of the rapid TCR reconstitution method in Example 1.
  • FIG. 8 is a graph showing TCR recognition of antigen peptides in Example 1 as luciferase fluorescence intensity.
  • FIG. 9 is a graph showing the results of flow cytometry in Example 1 in which antigen peptides were added to PBMCs collected from patients who had recovered from infection with SARS-CoV-2 and possessed the HLA-A2402/B5202/C1202 alleles, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • FIG. 8 is a graph showing TCR recognition of antigen peptides in Example 1 as luciferase fluorescence intensity.
  • FIG. 9 is a graph showing the results of flow cytometry in Example 1 in which antigen peptides were added to PBMCs collected from patients who had recovered from infection with SARS-CoV-2 and possessed the HLA-A2402/B5202/C1202 alleles, and cells expressing CD25 and CD137
  • FIG. 10 is a graph showing the results of flow cytometry in Example 1, in which PBMCs were collected from patients who had recovered from infection with SARS-CoV-2 carrying the HLA-A2402/B5202/C1202 alleles and were activated with inactivated SARS-CoV-2 virus, to which peptides were added, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • FIG. 11 is a graph showing the results of the ELISPOT assay in Example 1.
  • FIG. 12 is a graph showing the results of the AIM assay in Example 1.
  • FIG. 13 is a graph showing the results of the MHC tetramer assay in Example 1.
  • FIG. 14 is a graph showing the results of the antiviral activity of KF9/C12-specific T cells or QI9/A24-specific T cells derived from patients who were infected with SARS-CoV-2 and had recovered, and who possess the HLA-C*1202 allele, against mutant strains and subspecies of the SARS-CoV-2 virus in Example 2.
  • FIG. 15 is a graph showing the results of the ELISPOT assay in Example 2.
  • FIG. 16 is a dot plot and a graph showing the results of the tetramer assay in Example 2.
  • FIG. 17 is a graph showing the results of flow cytometry in Example 2 in which KF9 tetramer was added to PBMCs collected from patients infected with SARS-CoV-2 and carrying the HLA-C*1202 allele, and cells expressing CCR7 and CD45RA on the cell surface were evaluated.
  • FIG. 18 is a graph showing the results of flow cytometry evaluating the increase and phenotype of KF9-specific T cells when peptides were added to PBMCs derived from healthy subjects in Example 2.
  • FIG. 19 is a graph showing the results of the AIM assay in Example 2.
  • FIG. 20 is a graph showing the results of flow cytometry in which KF9 peptide was added to B lymphocytes and the expression of IFN- ⁇ and CD8 on the cell surface was evaluated in Example 2.
  • peptide refers to a compound composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids, formed by condensing two or more amino acids through a peptide bond.
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Infection caused by SARS-CoV-2 is also called COVID-19.
  • SARS-CoV-2 is a virus classified in the Betacoronavirus genus.
  • SARS-CoV-2 is a virus that has a single-stranded positive-sense RNA genome.
  • nucleocapsid protein refers to a viral structural protein responsible for the transcription and replication of the viral genome within the viral envelope.
  • the NP is a protein that forms viral particles together with the spike protein (S), membrane protein (M), and envelope protein (E), and forms the capsid (shell) of SARS-CoV-2.
  • S spike protein
  • M membrane protein
  • E envelope protein
  • SEQ ID NO: 4 An example of the NP protein is a protein (SEQ ID NO: 4) consisting of the amino acid sequence registered in Genbank under accession number 43740575.
  • Nucleotide sequence encoding NP protein (SEQ ID NO: 5, including stop codon) 5'--3'
  • the term "immune response" refers to an antibody- or cell-mediated response exhibited by a vertebrate such as a human against an infectious agent or disease, which prevents or ameliorates infection, or alleviates at least one of the symptoms of a disease caused by the infection.
  • the immune response refers to at least one of the following: neutralization of an infectious agent (e.g., an immunogen, the same applies below); blocking, inhibiting, preventing, or inhibiting the entry of an infectious agent into a cell; blocking the replication of an infectious agent; and/or stimulating the production of antibodies that protect against infection and destruction of a host cell.
  • the immune response refers to an immune response mediated by, for example, T cells and/or other white blood cells against an infectious agent or disease.
  • the immune response refers to, for example, prevention or amelioration of infection or disease, or alleviation of at least one symptom of a disease caused by the infection, by a cell.
  • the immune response can also be referred to as, for example, a "protective response” or a "protective immune response”.
  • T cells refers to a type of white blood cell, classified as a lymphocyte, that expresses a T cell receptor (TCR).
  • TCR T cell receptor
  • Known examples of the T cells include CD4 positive (+) helper T cells and CD8+ cytotoxic T cells.
  • cytotoxic T lymphocytes are CD8+ T cells.
  • the cytotoxic T cells are known to have the function of attacking and killing foreign substances such as cells infected with viruses, bacteria, etc., and cancer cells.
  • the cytotoxic T cells are differentiated from naive CD8+ T cells, and specifically, when naive CD8+ T cells recognize specific antigen peptides of viruses, etc., presented by the class I major histocompatibility complex (MHC class I, HLA class I) of antigen-presenting cells, they become activated and differentiate into CTLs.
  • MHC class I, HLA class I class I major histocompatibility complex
  • the CTLs are known to induce apoptosis in infected cells and cancer cells.
  • positive (+) means that a higher signal is detected by an analytical method such as flow cytometry that utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen.
  • negative (-) means that a signal equal to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen is detected.
  • T cell receptor activation refers to the activation of an intracellular signaling pathway or a change to an activated state as a result of an antigen binding to the T cell receptor (TCR) expressed on the cell surface of a T cell. Activation of the intracellular signaling pathway induces T cell activation.
  • TCR T cell receptor
  • human leukocyte antigen means human MHC (major histocompatibility complex).
  • the HLA has the function of presenting antigens to T cells.
  • the HLA is broadly divided into two groups: class I antigens and class II antigens.
  • the class I antigens are known to have subtypes such as HLA-A, B, C, D, E, F, and G.
  • the class I antigens form complexes with human ⁇ 2 microglobulin protein and present peptides such as antigen peptides.
  • the class II antigens are known to have subtypes such as HLA-DR, DQ, and DP. Alleles are known for each subtype of HLA. It is also known that there are more than thousands of combinations of the alleles.
  • nucleic acid refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides.
  • DNA deoxyribonucleotides
  • RNA ribonucleotides
  • nucleic acid refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the nucleic acid include genomic DNA, cDNA, and mRNA.
  • the nucleic acid may be, for example, single-stranded or double-stranded.
  • the nucleic acid may be interchangeably referred to as "polynucleotide” or “oligonucleotide.”
  • a "vector" expression vector means a recombinant plasmid or virus containing a nucleic acid to be delivered to a host cell in vitro or in vivo .
  • a pharma- ceutically acceptable carrier means a solvent and/or an additive that can be commonly used in the formulation technology of a pharmaceutical composition. It is preferable that the pharma-ceutically acceptable carrier is one that is almost or completely non-toxic to living organisms.
  • adjuvant refers to a compound that, when used in combination with a particular immunogen (antigen) in a formulation or composition, enhances, alters, or modifies the resulting immune response.
  • Enhancement, alteration, or modification of the immune response refers, for example, to strengthening, increasing, or enhancing the specificity of at least one of an antibody response and a cellular immune response. Enhancement, alteration, or modification of the immune response may also refer to the reduction, reduction, or suppression of a particular antigen-specific immune response.
  • a "vaccine” refers to, for example, a preparation or composition of an attenuated or inactivated (killed) infectious agent or an antigenic determinant (epitope, e.g., peptide, glycan, lipid, etc.) from the infectious agent, which is used to induce humoral immunity (antibody production) or cellular immunity against the pathogen.
  • the vaccine may refer to, for example, a preparation or composition administered to provide immunity against SARS-CoV-2, or protection against COVID-19 caused by SARS-CoV-2.
  • the “vaccine” may also refer to, for example, a suspension or solution of an immunogen that, when administered to a subject, results in an immune response, i.e., immunity that prevents or reduces the severity of disease resulting from infection.
  • the vaccine may also be referred to, for example, as a "vaccine composition” or a "vaccine formulation.”
  • an “effective dose” refers to an amount of an immunogen sufficient to prevent and/or ameliorate an infection, for example by inducing an immune response.
  • the “effective dose” may also refer to an amount of an immunogen sufficient to reduce at least one symptom of an infection or disease resulting from an infection.
  • the “effective dose” may also refer to an amount of an immunogen sufficient to delay or minimize, reduce, or inhibit the onset of an infection or disease.
  • the “effective dose” may also refer to an amount of an immunogen sufficient to provide a therapeutic benefit in the treatment or management of an infection or disease.
  • the “effective dose” may also refer to an amount of an immunogen sufficient to provide a therapeutic benefit in the treatment or management of an infection or disease, alone or in combination with other therapies.
  • the “effective dose” may also be an amount of an immunogen sufficient to enhance an autoimmune response in a subject (e.g., a human) to subsequent exposure to SARS2-CoV-2 or a disease resulting from SARS2-CoV-2.
  • the “effective dose” may also be a dose that prevents SARS2-CoV-2 and/or reduces the severity of symptoms resulting from SARS2-CoV-2.
  • the “effective dose” can also be referred to as, for example, an "effective amount” or a "therapeutically effective amount.”
  • subject refers to an animal or a cell, tissue, or organ derived from an animal.
  • the subject is used to mean, in particular, a human.
  • the animal refers to a human or a non-human animal. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.
  • prevention means reducing the possibility of onset of a disease or pathological condition, suppressing, delaying, or stopping the onset of a disease or pathological condition, suppressing, reducing, delaying, or stopping the progression of a pathological condition, or suppressing, reducing, delaying, or stopping the worsening of the condition.
  • the "prevention” may be, for example, treatment of a subject (patient) who develops the target disease, or treatment of an animal model of the target disease.
  • aggravation means that the degree (severity) of a disease or condition worsens.
  • isolated means identified and separated and/or recovered from a component in its natural state.
  • the “isolation” can be achieved, for example, by obtaining at least one purification step.
  • the present disclosure discloses a nucleocapsid-derived antigenic peptide of Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) in a vaccine.
  • the antigenic peptide of the present disclosure comprises the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (KAYNVTQAF); (P2) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been deleted, inserted, substituted, or added; (P3) A polypeptide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:1.
  • T cell responses could be induced by using antigen peptides derived from nucleocapsid protein, based on the reactivity of peripheral blood mononuclear cells from SARS-CoV-2 recovered patients to SARS-CoV-2 antigens.
  • the inventors then discovered that a cellular immune response of T cells could be induced by using a specific antigen peptide derived from the nucleocapsid of SARS-CoV-2 as the active ingredient of a vaccine, leading to the establishment of the present disclosure.
  • the antigen peptide of the present disclosure can induce, for example, a cellular immune response against SARS-CoV-2.
  • nucleocapsid protein is less prone to mutation than spike protein.
  • the antigen peptide of the present disclosure is expected to maintain effectiveness, such as suppressing the rate of severe illness, compared to existing spike protein mRNA vaccines.
  • the antigen peptide of the present disclosure may be, for example, an isolated antigen peptide or an antigen peptide mixed with other substances (e.g., peptides or proteins).
  • the antigen peptide of the present disclosure preferably has immune response-inducing activity, and more preferably has CTL-inducing activity.
  • the CTL-inducing activity can be evaluated based on the proportion of CD25+CD137+ cells (CTL cells) in CD3+CD8+ cells, for example, with reference to Example 1(2) described below.
  • the antigen peptide of the present disclosure has a proportion of CD25+CD137+ cells (CTL cells) in CD3+CD8+ cells of, for example, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • CTL cells CD25+CD137+ cells
  • antigen peptide in the antigen peptide (P2), "one or several” may be within a range in which (P2) functions as an antigen peptide having immune response-inducing activity.
  • amino acid sequence of (P1) “one or several" in (P2) may be, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2.
  • the numerical range of the number discloses, for example, all positive integers within that range.
  • the substitution is preferably a conservative substitution.
  • the conservative substitution means a substitution of an amino acid residue with an amino acid residue having a similar side chain.
  • Examples of the conservative substitution include substitution between amino acid residues having a basic side chain such as lysine, arginine, histidine, etc.; substitution between amino acid residues having an acidic side chain such as aspartic acid, glutamic acid, etc.; substitution between amino acid residues having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.; substitution between amino acid residues having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.; substitution between amino acid residues having a ⁇ -branched side chain such as threonine, valine, isoleucine, etc.
  • the “identity” of the antigen peptide (P3) may be within a range in which the (P3) functions as an antigen peptide having immune response-inducing activity.
  • the “identity” of the (P3) is, for example, 70%, 75%, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the (P1).
  • the “identity” can be calculated, for example, by using the default parameters of the homology algorithm BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) of the National Center for Biotechnology Information (NCBI) (hereinafter the same).
  • the antigen peptide of the present disclosure may be, for example, a polypeptide consisting of an amino acid sequence in which one or several amino acids have been added to at least one of the C-terminus and N-terminus of the amino acid sequence of SEQ ID NO: 1.
  • the one or several is within the range in which the antigen peptide of the present disclosure functions as, for example, an antigen peptide having immune response-inducing activity.
  • the one or several is, for example, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.
  • the peptide (P2) or (P3) may be, for example, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or 3.
  • SEQ ID NO: 2 KRTATKAYNVTQAFG
  • SEQ ID NO: 3 TKAYNVTQAFGRRGP
  • the antigen peptide of the present disclosure preferably has TCR activation activity in a T cell receptor activation test.
  • the TCR is, for example, TCR-01, TCR-08, or TCR-22.
  • the TCR-01 is, for example, a TCR comprising an ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 6 below, and a ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 7 below.
  • the TCR-08 is, for example, a TCR comprising an ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 below, and a ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 9 below.
  • the TCR-22 is, for example, a TCR comprising an ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 10 below, and a ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 below.
  • the underlined amino acid sequences correspond to CDR1 to CDR3, respectively, from the N-terminus.
  • TCR-01 ⁇ chain variable domain TRAV29/DV5*01, TRAJ48*01 (SEQ ID NO: 6) MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYT NSMFDY FLWYKKYPAEGPTFLIS ISSIKDK NEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYF CAASARIFGNEKLTF GTGTRLTIIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS
  • TCR-01 ⁇ chain variable domain TRBV6-5*01, TRBJ1-6*01, TRBD1*01 (SEQ ID NO: 7) MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQD MNHEY MSWYRQDPGMGLRLIHY SVGAGI TDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYF CASSYFTPILGAKDNSPLHF GNGTRLTVTEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGP
  • TCR-08 alpha chain variable domain TRAV27*01, TRAJ40*01 (SEQ ID NO: 8) MVLKFSVSILWIQLAWVSTQLLEQSPQFLSIQEGENLTVYCNSS SVFSS LQWYRQEPGEGPVLLVT VVTGGEV KKLKRLTFQFGDARKDSSLHITAAQPGDTGLYL CAGPGTYKYIF GTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS
  • TCR-08 beta chain variable domain TRBV13*01, TRBJ2-1*01, TRBD2*02 (SEQ ID NO: 9) MLSPDLPDSAWNTRLLCRVMLCLLGAGSVAAGVIQSPRHLIKEKRETATLKCYPI PRHDT VYWYQQGPGQDPQFLIS FYEKMQ SDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYF CASSLGLAGDFSYNEQFF GPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGP
  • TCR-22 alpha chain variable domain TRAV5*01, TRAJ29*01 (SEQ ID NO: 10) MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYT DSSSTY LYWYKQEPGAGLQLLTY IFSNMDM KQDQRLTVLLNKKDKHLSLRIADTQTGDSAIYF CAEKEGNTPLVF GKGTRLSVIANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS
  • TCR-22 beta chain variable domain TRBV12-4*01, TRBJ1-1*01, TRBD2*02 (SEQ ID NO: 11) MGSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPI SGHDY LFWYRQTMMRGLELLIY FNNNVP IDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYF CASSPGGSPGGAFF GQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFGSGATNFSLLKQAGDVEENPGP
  • the T cell receptor activation test may be, for example, a flow cytometry method, an ELISPOT assay, a T cell proliferation assay, a T cell cytotoxicity assay, a T cell activation assay, or a tetramer assay.
  • the T cell activation assay may be performed, for example, by expressing an artificially introduced luciferase gene when the TCR recognizes an antigen and activates an intracellular signal transduction pathway; see the luciferase assay in Example 1 (5) below.
  • markers receptors, receptor ligands, etc.
  • cytokines, etc. expressed in activated T cells are labeled with fluorescent dye-labeled antibodies, etc., and the activation of the T cell receptor may be examined using a flow cytometer.
  • the antigen peptide of the present disclosure preferably has binding affinity to, for example, human leukocyte antigen (HLA).
  • HLA human leukocyte antigen
  • the HLA is preferably, for example, HLA-C*1202 and/or HLA-C*1402.
  • the antigen peptide of the present disclosure has binding affinity to a complex of the HLA-C*1202 and/or HLA-C*1402 and human ⁇ 2 microglobulin protein.
  • the binding affinity of the HLA can be evaluated, for example, by using as an index whether the antigen peptide can be recovered from HLA/ ⁇ 2 microglobulin after coexistence of a cell expressing the HLA and human ⁇ 2 microglobulin protein with the antigen peptide of the present disclosure.
  • the HLA-C*1202 is, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 18 below.
  • the HLA-C*1402 is, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 35 below.
  • the human ⁇ 2 microglobulin protein is, for example, a protein consisting of the amino acid sequence of SEQ ID NO: 19 below.
  • HLA-C*1202 (SEQ ID NO: 18) MRVMAPRTLILLLSGALALTETWACSHSMRYFYTAVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQADRVSLRNLRGYYNQSEAGSHTLQRMYGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQWRAYLEGTCVEWLRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEPSSQPTIPIVGIVAGLAVLAVLAVLGAVMAVVMCRRKSSGGKGGSCSQAASSNSAQGSDESLIACKA
  • HLA-C*1402 (SEQ ID NO:35) MRVMAPRTLILLLSGALALTETWACSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRGEPRAPWVEQEGPEYWDRETQKYKRQAQTDRVSLRNLRGYYNQSEAGSHTLQWMFGCDLGPDGRLLRGYDQSAYDGKDYIALNEDLRSWTAADTAAQITQRKWEAAREAEQRRAYLEGTCVEWLRRRYLENGKETLQRAEHPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQWDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLTLRWEPSSQPTIPIVGIVAGLAVLAVLAVLGAVVAVVMCRRKSSGGKGGSCSQAASSNSAQGSDESLITCKA
  • Nucleotide sequence encoding human ⁇ 2 microglobulin protein (SEQ ID NO:21, including stop codon) 5'-ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGCCTGGAGGCTATGATTCAAAGAACTCCAAAATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAATTGAAAAAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA-3'
  • the present disclosure provides a nucleic acid encoding the antigenic peptide.
  • the nucleic acid of the present disclosure encodes the antigenic peptide of the present disclosure.
  • the nucleic acid may be composed of deoxynucleotide residues, ribonucleotide residues, or both.
  • the nucleic acid may be composed of natural nucleic acid residues, non-natural nucleic acid residues, or both.
  • Specific examples of the nucleic acid include DNA, RNA, and/or DNA/RNA composed of natural and/or non-natural nucleic acid residues.
  • Examples of the non-natural nucleic acid residues include modified nucleotide residues or modified ribonucleotide residues in which the base, sugar residue, or sugar phosphate backbone of the nucleotide residue is modified.
  • examples of the non-natural nucleic acid residue include cEt (constrained ethyl bicyclic nucleic acid acid, Ionis Pharmaceuticals), LNA (trademark, Locked Nucleic Acid), ENA (registered trademark, 2'-O,4'-C-Ethylene bridged Nucleic Acid), and the like.
  • the nucleic acid may have a 5' cap at the 5' end, for example.
  • the nucleic acid may be a single-stranded nucleic acid molecule or a double-stranded nucleic acid molecule.
  • the nucleic acid of the present disclosure can be designed by substituting the corresponding codons based on the amino acid sequence of the antigen peptide of the present disclosure.
  • the base sequence of the nucleic acid of the present disclosure may be, for example, codon-optimized.
  • nucleic acids and antigen peptides can be synthesized, for example, by genetic engineering techniques or organic synthesis techniques, and can also be referred to as synthetic DNA such as cDNA, or synthetic RNA.
  • the nucleic acid of the present disclosure encodes the antigenic peptide, and therefore can be suitably used for synthesizing the antigenic peptide.
  • the nucleic acid of the present disclosure encodes the antigenic peptide, and therefore can be used to induce cellular immunity against the antigenic peptide when used as an active ingredient of a vaccine.
  • the present disclosure provides an expression vector that can be used to synthesize or express an antigen peptide.
  • the expression vector of the present disclosure includes the nucleic acid of the present disclosure.
  • the antigen peptide of the present disclosure can be suitably produced by genetic engineering techniques.
  • the peptide of the present disclosure can be expressed in vivo .
  • the expression vector of the present disclosure is, for example, an expression vector in which the nucleic acid of the present disclosure is inserted.
  • the expression vector can also be said to be, for example, a vector in which the nucleic acid is functionally linked.
  • the expression vector refers to, for example, a nucleic acid molecule that can transport an inserted gene into a target such as a cell.
  • the expression vector may contain, for example, a polynucleotide encoding an antigenic peptide so as to be capable of expressing the antigenic peptide encoded by the polynucleotide of the nucleic acid of the present disclosure, and its configuration is not particularly limited.
  • the expression vector can be prepared, for example, by inserting a polynucleotide encoding an antigen peptide, i.e., the nucleic acid of the present disclosure, into a backbone vector (hereinafter also referred to as a "basic vector").
  • a backbone vector hereinafter also referred to as a "basic vector”.
  • the type of the expression vector is not particularly limited and can be appropriately determined, for example, depending on the type of the host. Specifically, when the expression vector is synthesized by a genetic engineering technique, the expression vector is first synthesized by, for example, designing and synthesizing a nucleic acid encoding the antigen peptide.
  • the design and synthesis can be performed, for example, by a PCR method using a vector containing a nucleic acid encoding the antigen peptide as a template and primers designed to synthesize a desired nucleic acid region.
  • the obtained nucleic acid can then be linked to an appropriate vector to obtain a recombinant vector for protein expression (expression vector), and the recombinant vector can be introduced into a host so that the target gene can be expressed to obtain a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)).
  • the host used for transformation is not particularly limited as long as it can express the target gene, and examples thereof include non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells.
  • non-human hosts such as microorganisms, animal cells, insect cells, or cultured cells thereof, isolated human cells or cultured cells thereof, and mammalian cells.
  • the prokaryotic organisms include bacteria such as Escherichia coli and Pseudomonas putida .
  • Examples of the eukaryotic organisms include yeasts such as Saccharomyces cerevisiae .
  • Examples of the animal cells include COS cells and CHO cells, and examples of the insect cells include Sf9 and Sf21.
  • the expression vector may be a viral vector or a non-viral vector.
  • the expression vector may be, for example, a binary vector.
  • the expression vector may be, for example, pETDuet-1, pQE-80L, or pUCP26Km.
  • the expression vector may be, for example, pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, or pUC8.
  • the expression vector When transforming yeast, the expression vector may be, for example, pYepSec1, pMFa, or pYES2.
  • the expression vector may be, for example, pAc or pVL.
  • the expression vector may be, for example, pCDM8 or pMT2PC.
  • the expression vector preferably has a regulatory sequence that regulates, for example, the expression of the polynucleotide encoding the antigenic peptide and the expression of the antigenic peptide of the present disclosure encoded by the polynucleotide of the antigenic peptide.
  • the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori).
  • the arrangement of the regulatory sequence in the expression vector is not particularly limited.
  • the regulatory sequence may be arranged, for example, so long as it can functionally regulate the expression of the polynucleotide encoding the antigenic peptide and the expression of the antigenic peptide encoded thereby, and may be arranged based on a known method.
  • the regulatory sequence may utilize a sequence that is already included in the expression vector, or the regulatory sequence may be further inserted into the expression vector, or the regulatory sequence included in the basic vector may be replaced with another regulatory sequence.
  • the method of introducing the expression vector into the host is not particularly limited and can be performed by a known method.
  • the introduction method can be appropriately set depending on, for example, the type of the host.
  • Examples of the introduction method include an introduction method using a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, a method mediated by Agrobacterium, a protoplast method, and the like.
  • a gene gun such as a particle gun, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct
  • liposome examples include lipofectamine and cationic liposome
  • introduction aid examples include atelocollagen, nanoparticles, and polymers.
  • the host is a microorganism
  • a method mediated by, for example, E. coli or Ps. putida is preferable.
  • the polynucleotide of the antigen peptide of the present disclosure may be introduced into the host by, for example, an expression vector of the present disclosure.
  • the production method of the present disclosure involves culturing the transformant and collecting the antigen peptide from the culture.
  • the culture may be a culture supernatant, or a transformant such as cultured cells or cultured bacterial cells, or a processed or disrupted product thereof.
  • the production method of the present disclosure extracts the antigen peptide by disrupting the host.
  • the production method of the present disclosure uses the culture solution as is or removes the host by centrifugation or the like.
  • the production method of the present disclosure can isolate or purify the antigen peptide by using, for example, a general biochemical method used for isolating and purifying a protein, specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography; etc., alone or in appropriate combination.
  • a general biochemical method used for isolating and purifying a protein specifically, concentration using an ultrafiltration membrane; salting out such as ammonium sulfate precipitation; chromatography using various columns such as gel filtration, ion exchange chromatography, and affinity chromatography; etc., alone or in appropriate combination.
  • CTL inducer/activator ⁇ CTL inducer/activator>
  • the present disclosure provides an agent for inducing and/or activating CTLs.
  • the agent for inducing and/or activating CTLs is characterized by including the antigen peptide, nucleic acid, and/or vector of the present disclosure.
  • the agent for inducing and/or activating CTLs of the present disclosure is characterized by including an antigen peptide, nucleic acid, and/or vector as an active ingredient, and other configurations and conditions are not particularly limited. Since the agent for inducing and/or activating CTLs of the present disclosure includes an antigen peptide, nucleic acid, and/or vector as an active ingredient, naive T cells are activated, and differentiation induction and activation into cytotoxic T cells can be demonstrated.
  • the present disclosure provides a pharmaceutical composition that can be used as a vaccine.
  • the pharmaceutical composition is characterized by comprising the antigen peptide, nucleic acid, vector, and/or cytotoxic T cell inducer and/or activator of the present disclosure, and a pharma- ceutical acceptable carrier.
  • the pharmaceutical composition of the present disclosure is characterized by comprising an antigen peptide, nucleic acid, vector, and/or cytotoxic T cell inducer and/or activator (hereinafter, collectively referred to as "active ingredient”) as an active ingredient, and other configurations and conditions are not particularly limited.
  • the pharmaceutical composition of the present disclosure comprises an antigen peptide, nucleic acid, vector, and/or cytotoxic T cell inducer and/or activator as an active ingredient, it can induce cellular immunity against SARS-CoV-2 when used as an active ingredient of a vaccine.
  • the nucleocapsid is less susceptible to mutations than the spike protein, the pharmaceutical composition of the present disclosure contains an antigen peptide, a nucleic acid, a vector, and/or an inducer and/or activator of cytotoxic T cells as an active ingredient, and is therefore expected to maintain efficacy, such as suppressing the rate of severe illness, when used as an active ingredient in a vaccine.
  • the pharmaceutical composition of the present disclosure when administered to a subject, can induce cellular immunity against an antigen peptide or a nucleic acid encoding the same. Therefore, the pharmaceutical composition of the present disclosure can also be called a vaccine, a vaccine composition, or a vaccine preparation.
  • the pharmaceutical composition disclosed herein is, for example, a vaccine for use in preventing infection or aggravation of an infectious disease caused by SARS-CoV-2.
  • the pharmaceutical composition of the present disclosure may further contain a pharma- ceutically acceptable carrier.
  • the carrier may be a suspending agent, solubilizing agent, stabilizer, isotonicity agent, preservative, adsorption inhibitor, surfactant, diluent, vehicle, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc., for administering the active ingredient, and may be added appropriately within a range that does not interfere with the effects of the present disclosure.
  • the suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, powdered tragacanth, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, etc.
  • the solution adjuvant is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, castor oil fatty acid ethyl ester, etc.
  • the stabilizer is not particularly limited, and examples include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metasulfate, etc.
  • the isotonicity agent is not particularly limited, and examples include D-mannitol, sorbitol, etc.
  • the preservative is not particularly limited, and examples thereof include methyl parahydroxybenzoate, ethyl parahydroxybenzoate, sorbic acid, phenol, cresol, and chlorocresol.
  • the adsorption inhibitor is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide propylene oxide copolymer, hydroxypropyl cellulose, methyl cellulose, hydrogenated castor oil, polyethylene glycol, etc.
  • the sulfur-containing reducing agent is not particularly limited, and examples include those having a sulfhydryl group, such as N-acetylcysteine, N-acetylhomocysteine, thioxanthate, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atoms.
  • a sulfhydryl group such as N-acetylcysteine, N-acetylhomocysteine, thioxanthate, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, and thioalkanoic acids having 1 to 7 carbon atom
  • the antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallate, propyl gallate, or chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, and sodium metaphosphate.
  • EDTA sodium ethylenediaminetetraacetate
  • the pharmaceutical composition of the present disclosure may further contain, as appropriate, commonly added ingredients such as inorganic salts, such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate; organic salts, such as sodium citrate, potassium citrate, and sodium acetate; and sugars, such as glucose.
  • inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, and sodium bicarbonate
  • organic salts such as sodium citrate, potassium citrate, and sodium acetate
  • sugars such as glucose.
  • the pharmaceutical composition of the present disclosure may further include an adjuvant (immunostimulant), etc.
  • the adjuvant include Toll-like receptor stimulants such as CpG oligonucleotide (CpG ODN) and lipopolysaccharide (LPS); aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum chloride; interferons such as interferon (IFN- ⁇ ); inflammatory cytokines such as TNF- ⁇ ; interleukins such as IL-1, IL-2, IL-3, IL-4, IL-12, and IL-13; growth factors such as granulocyte macrophage (GM) colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF); cytokines such as Flt3 ligand, B7-1, and B7-2; chemokines; squalene-containing adjuvants such as squalane or squalene; complete or incomplete Freund's adju
  • LPS Lipid Precipient
  • MPL Monophosphoryl lipid A
  • squalene-containing adjuvant is AddaVax (trademark) or MF-59 (registered trademark), which is an oil-in-water emulsion of squalene.
  • the pharmaceutical composition of the present disclosure may be used, for example, in vitro or in vivo .
  • the pharmaceutical composition of the present disclosure may be used, for example, as a research reagent or as a pharmaceutical. In the former case, the pharmaceutical composition of the present disclosure may be called a test reagent or test kit.
  • the subject of administration of the pharmaceutical composition of the present disclosure is not particularly limited.
  • the subject may be, for example, the above-mentioned examples.
  • the pharmaceutical composition of the present disclosure is used in vitro
  • the subject of administration may be, for example, a cell, a tissue, an organ, etc.
  • the cell may be, for example, a cell or cultured cell taken from a living body
  • the tissue or organ may be, for example, a tissue (living tissue) or an organ taken from a living body.
  • the cell may be, for example, an immune cell such as a T cell, a B cell, a NK cell, or a dendritic cell.
  • the subject to which the composition is administered may be a healthy individual not infected with SARS-CoV-2, a person who may be infected with SARS-CoV-2, or a patient infected with SARS-CoV-2, but is preferably a subject for whom prevention of infection with SARS-CoV-2 or prevention of aggravation is desired.
  • the subject to which the composition is administered preferably has HLA-C*1202 antigen gene or HLA-C*1402 as an MHC (HLA) class I molecule in homozygous form, or at least one of HLA-C*1202 antigen gene and HLA-C*1402 as an MHC (HLA) class I molecule in heterozygous form.
  • HLA HLA
  • HLA MHC
  • the conditions for use (administration conditions) of the pharmaceutical composition of the present disclosure are not particularly limited, and the administration form, administration timing, dosage, etc. can be appropriately set depending on, for example, the type of active ingredient in the pharmaceutical composition, the type of subject to administration, etc.
  • the pharmaceutical composition of the present disclosure can be administered, for example, by intracerebral administration, intrathecal administration, intramuscular administration, subcutaneous administration, or intravenous administration.
  • intramuscular or subcutaneous administration by injection or infusion is preferred, since this allows safe and stable administration regardless of the skill of the administerer.
  • the dosage of the pharmaceutical composition of the present disclosure is an amount capable of inducing an immune response, particularly cellular immunity, in the subject to which it is administered, i.e., an effective dose.
  • the dosage can be appropriately determined depending on, for example, the age, weight, symptoms, etc., of the subject to which it is administered.
  • the pharmaceutical composition of the present disclosure may be administered once or multiple times.
  • the multiple times may be, for example, two, three, four, five or more times.
  • the number of administrations may be appropriately determined while checking the preventive effect on the subject of administration.
  • the administration interval may be appropriately determined while checking the preventive effect on the subject of administration, and may be, for example, once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.
  • the pharmaceutical composition of the present disclosure may prevent or reduce at least one symptom caused by SARS-CoV-2 infection in a subject to which the composition is administered.
  • Symptoms of SARS-CoV-2 infection include, for example, rhinorrhea, sore throat, headache, hoarseness, cough, sputum, fever, rales, wheezing, dyspnea, and pneumonia.
  • the pharmaceutical composition of the present disclosure may prevent or reduce at least one symptom associated with SARS-CoV-2 infection.
  • the reduction in the symptom may be evaluated subjectively or objectively, for example, by self-assessment by the subject to which the composition is administered; by a physician; by a quality of life (QOL) evaluation; or by evaluation of the delay in progression of SARS-CoV-2 infection or symptoms of SARS-CoV-2 infection, or the reduction in the severity of symptoms of SARS-CoV-2 infection.
  • the objective evaluation may be an evaluation by an animal or an evaluation by a human.
  • the present disclosure provides a method for preventing infection or aggravation of an infectious disease caused by SARS-CoV-2.
  • the method for preventing infection or aggravation of an infectious disease caused by SARS-CoV-2 of the present disclosure is characterized by using the antigen peptide, nucleic acid, vector, and/or an inducer and/or activator of cytotoxic T cells of the present disclosure for a subject, and other configurations and conditions are not particularly limited.
  • the preventive method of the present disclosure includes the antigen peptide of the present disclosure or a nucleic acid encoding the same as an active ingredient, and therefore, when treated on a subject, it may be possible to induce cellular immunity against SARS-CoV-2.
  • the preventive method of the present disclosure includes the antigen peptide of the present disclosure or a nucleic acid encoding the same as an active ingredient, and therefore, when used as an active ingredient of a vaccine, it is expected that the effectiveness of the method, such as suppression of the rate of aggravation, will be maintained.
  • the prevention method of the present disclosure can, for example, induce cellular immunity against SARS-CoV-2, and therefore can also be referred to as, for example, a vaccination method against SARS2-Cov-2, a method for inducing protective immunity, a method for stimulating an immune response, or a method for inducing an immune response.
  • the active ingredient is administered to a subject, and by inducing cellular immunity in the subject, the subject can be given immunity against SARS2-Cov-2.
  • the preventive method of the present disclosure includes, for example, a step of administering the active ingredient to the subject.
  • cellular immunity against an antigen peptide is induced in the subject, and a cellular immune response against the antigen peptide by T cells or the like against SARS2-Cov-2 occurs.
  • the administration step is carried out once or multiple times.
  • the administration conditions in the administration step can be referenced from the explanation of the pharmaceutical composition of the present disclosure.
  • the present disclosure discloses an HLA/antigenic peptide complex that can be used to detect T cells that can respond to SARS-CoV-2 infection.
  • the HLA/antigenic peptide complex of the present disclosure comprises a complex of a human leukocyte antigen (HLA) and an antigenic peptide, the antigenic peptide comprising a nucleocapsid-derived antigenic peptide of the present disclosure.
  • HLA human leukocyte antigen
  • the HLA/antigenic peptide complex of the present disclosure allows the detection of T cells that can respond to SARS-CoV-2.
  • the HLA is preferably HLA-C*1202 and/or HLA-C*1402.
  • the HLA is a complex of the HLA ⁇ chain (H chain) and ⁇ 2 microglobulin.
  • the HLA/antigen peptide complex may be a monomer, or may form an oligomer in which two or more of the HLA/antigen peptide complexes are assembled.
  • the oligomer include a dimer, trimer, and tetramer.
  • the oligomer can be prepared, for example, by a general method, for example, by using an ⁇ chain with a biotinylated C-terminus, ⁇ 2 microglobulin, and avidin.
  • the present disclosure discloses a method for detecting T cells capable of responding to SARS-CoV-2 infection.
  • the method for detecting T cells of the present disclosure includes a detection step of detecting T cells that bind to an HLA/antigenic peptide complex in a biological sample by contacting the biological sample with the HLA/antigenic peptide complex, the HLA/antigenic peptide complex being an HLA/antigenic peptide complex of the present disclosure.
  • T cells capable of responding to SARS-CoV-2 infection can be detected.
  • the living body is, for example, a human or non-human animal (e.g., cow, pig, sheep, mouse, rat, rabbit, horse, monkey, etc.).
  • the biological sample is, for example, a body fluid, cell, tissue, organ, etc.
  • the sample may be, for example, a liquid or a solid.
  • the detection method of the present disclosure preferably includes mixing the solid sample with a liquid to prepare a liquid sample prior to the detection step.
  • the detection step includes, for example, a complex formation step of contacting the biological sample with an HLA/antigen peptide complex (first complex) to form a second complex between the first complex and a TCR on a T cell that recognizes the HLA/antigen peptide complex in the biological sample, and a complex detection step of detecting the formation of the second complex.
  • a complex formation step of contacting the biological sample with an HLA/antigen peptide complex (first complex) to form a second complex between the first complex and a TCR on a T cell that recognizes the HLA/antigen peptide complex in the biological sample
  • a complex detection step of detecting the formation of the second complex.
  • the method of contacting the biological sample with the first complex is not particularly limited, and is preferably performed in a liquid, for example.
  • the contact may be performed, for example, by mixing the biological sample with the first complex.
  • the liquid include water, physiological saline, and buffer solutions.
  • the contact conditions in the complex formation step can be set, for example, as general conditions under which binding between TCR and peptide/MHC (HLA) complex occurs.
  • the complex detection process by detecting the presence or absence of binding between the first complex and TCR, it is possible to, for example, analyze (qualitatively) the presence or absence of T cells specific to the first complex in the biological sample, and by detecting the degree of binding between the two (amount of binding), it is possible to, for example, analyze (quantitatively) the amount of T cells specific to the first complex in the biological sample.
  • the binding between the first complex and the TCR cannot be detected, it can be determined that no T cell specific to the first complex is present in the biological sample, and if the binding is detected, it can be determined that T cell specific to the first complex is present in the biological sample.
  • the method for detecting the binding between the first complex and the TCR is not particularly limited.
  • the method may be a conventional method for detecting binding between substances or between a substance and a cell using a first complex labeled with a fluorescent label or the like.
  • the present disclosure relates to the use of an antigenic peptide, a nucleic acid, a vector, a pharmaceutical composition, and/or an agent for inducing and/or activating a cytotoxic T cell of the present disclosure for use in a method for preventing infection or aggravation of an infectious disease caused by SARS-CoV-2.
  • the present disclosure relates to the use of an antigenic peptide, a nucleic acid, a vector, a pharmaceutical composition, and/or an agent for inducing and/or activating a cytotoxic T cell of the present disclosure for use in the manufacture of a vaccine for use in suppressing infection or aggravation of an infectious disease caused by SARS-CoV-2.
  • an antigenic peptide, a nucleic acid, a vector, a pharmaceutical composition, and/or an agent for inducing and/or activating a cytotoxic T cell of the present disclosure may be described, for example, by referring to the explanations of the antigenic peptide, the nucleic acid, the vector, the agent for inducing and/or activating a CTL, the pharmaceutical composition, and the method for prevention of the present disclosure.
  • Example 1 We confirmed that an antigenic peptide derived from the nucleocapsid of SARS-CoV-2 can induce T cell activation.
  • PBMCs Peripheral blood mononuclear cells
  • SARS-CoV-2 serum-derived mononuclear cells
  • the PBMCs were obtained by density gradient centrifugation using peripheral blood and Ficoll-PaqueTM Plus (GE Healthcare Life Sciences, Cat No: 17-1440-03). After the centrifugation, the PBMCs were stored in liquid nitrogen until use.
  • Detection of T cells specific to antigens derived from SARS-CoV-2 was performed. The detection method was performed as shown in FIG. 1.
  • overlapping peptides of spike protein or nucleocapsid protein (hereinafter also referred to as "nucleocapsid") derived from SARS-CoV-2 are added to PBMCs collected from patients who have recovered from SARS-CoV-2 infection. After the addition, the cells are cultured for 10 to 14 days, and after the culture, detection of T cells specific to antigens derived from SARS-CoV-2 is performed.
  • overlapping peptides derived from SARS-CoV-2 adjusted to 100 nmol/l were added to the PBMCs prepared in Example 1(1) and pulsed.
  • the overlapping peptides were designed based on the amino acid sequences of the SARS-CoV-2 nucleocapsid protein (SEQ ID NO: 4) and spike protein (SEQ ID NO: 22) by shifting the peptides by 4 amino acids from the N-terminus to the C-terminus so that they overlap by 11 amino acids, and were prepared by synthesizing each of the designed peptides and then mixing them so that each peptide was equimolar (PepTivator SARS-CoV-2 Prot_S Complete, research grade Miltenyi biotec, Cat No: 130-127-951, 6 nmol).
  • the amino acid sequence of SARS-CoV-2 was referenced to the amino acid sequence registered under NCBI Gene accession number: 43740568. After the pulse, the cells were cultured for 14 days in RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 11875101) containing 10% fetal bovine serum (FBS) and 30 U/ml recombinant human IL-2 (Peprotec, Cat No: 200-02). After the culture, antibody staining of surface molecules was performed.
  • the antibodies used were CD3 antibody FITC (UCHT1, 100-fold dilution, Biolegend), CD8 antibody APCcy7 (RPA-T8, 100-fold dilution, Biolegend), CD14 antibody-PerCP/Cy5.5 (HCD14, 100-fold dilution, Biolegend), CD19 antibody-PerCP/Cy5.5 (HIB19, 100-fold dilution, Biolegend), CD25 antibody-PEcy7 (M-A251, 50-fold dilution, Biolegend), and CD137 antibody-APC (4B4-1, 50-fold dilution, Biolegend).
  • CD3 antibody FITC UCHT1, 100-fold dilution, Biolegend
  • CD8 antibody APCcy7 RPA-T8, 100-fold dilution, Biolegend
  • CD14 antibody-PerCP/Cy5.5 HCD14, 100-fold dilution, Biolegend
  • CD19 antibody-PerCP/Cy5.5 HBD19, 100-fold dilution
  • 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added to stain dead cells. After the addition, the mixture was incubated on ice for 20 minutes. After the incubation, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No: 09154-85). After the fixation, the expression levels of surface molecules were measured by flow cytometry using FACS Canto II (BD Biosciences). The data obtained by the flow cytometry were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The negative control was performed in the same manner, except that no overlapping peptide was added. The results are shown in Figures 2 and 3.
  • Figure 2 is a graph showing the results of flow cytometry in which overlapping peptide was added to PBMCs collected from a patient who had recovered from SARS-CoV-2 infection, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • the horizontal axis shows the expression level of CD25 after gating on CD3+
  • the vertical axis shows the expression level of CD137 after gating on CD3+.
  • the graph on the left shows the results when overlapping peptide was not added
  • the graph on the right shows the results when overlapping peptide was added.
  • T cell activity was increased when overlapping peptide was added compared to when overlapping peptide was not added.
  • Figure 3 is a graph showing the numerical values of the percentage of T cells expressing CD25 and CD137 from the results of flow cytometry.
  • the horizontal axis shows the type of protein from which the overlapping peptides were derived, and the vertical axis shows the percentage (%) of CD25+CD137+ T cells.
  • the left graph shows the percentage (%) of CD25+CD137+ T cells after gating on CD3+CD8+, and the right graph shows the percentage (%) of CD25+CD137+ T cells after gating on CD3+CD4+.
  • T cell activation is induced when antigen peptides derived from SARS-CoV-2 are added after activating T cells with inactivated SARS-CoV-2 virus.
  • the study was performed as shown in FIG. 4.
  • PBMCs collected from patients who had recovered from SARS-CoV-2 infection were infected with inactivated SARS-CoV-2 virus (Reference 1) and cultured for 12 to 14 days. After the culture, spike protein, membrane protein (SEQ ID NO: 24), or overlapping peptide of nucleocapsid derived from SARS-CoV-2 was added and re-stimulation was performed.
  • the overlapping peptide of the membrane protein was designed and prepared in the same manner as the overlapping peptide of the nucleocapsid.
  • Reference 1 Jureka AS, Silvas JA, Basler CF. Propagation, Inactivation, and Safety Testing of SARS-CoV-2. Viruses. 2020 Jun 6;12(6):622. doi: 10.3390/v12060622. PMID: 32517266; PMCID: PMC7354523.
  • Amino acid sequence of membrane protein (SEQ ID NO:24) MADSNGTITVEELKKLLEQWNLVIGFLFLTWICLLQFAYANRNRFLYIIKLIFLWLLWPVTLACFVLAAVYRINWITGGIAIAMACLVGLMWLSYFIASFRLFARTRSMWSFNPETNILLNVPLHGTILTRPLLESELVIGAVILRGHLRIAGHHLGRCDIKDLPKEITVATSRTLSYYKLGASQRVAGDSGFAAYSRYRIGNYKLNTDHSSSSDNIALLVQ
  • the cells were cultured for 24 hours, and T cells specific to the antigen derived from SARS-CoV-2 were detected. Specifically, the inactivated virus was added at 6 ⁇ 10 2 PFU/well to the PBMCs prepared in Example 1(1). After the addition, the cells were cultured for 14 days using RPMI1640 medium (Thermo Fisher Scientific, Cat No: 11875101) containing 10% fetal bovine serum (FBS) and 30 U/ml recombinant human IL-2 (Peprotec, Cat No: 200-02). After the culture, spike protein, membrane protein, or overlapping peptide of the nucleocapsid derived from SARS-CoV-2 adjusted to 100 nmol/l was added and pulsed.
  • RPMI1640 medium Thermo Fisher Scientific, Cat No: 11875101
  • FBS fetal bovine serum
  • Peprotec U/ml recombinant human IL-2
  • the cells were cultured for 24 hours in RPMI1640 medium (Thermo Fisher Scientific, Cat No: 11875101) containing 10% fetal bovine serum (FBS) and 30 U/ml recombinant human IL-2 (Peprotec, Cat No: 200-02).
  • FBS fetal bovine serum
  • Peprotec Cat No: 200-02
  • antibody staining of surface molecules was performed.
  • the antibodies used were the CD3 antibody-FITC, the CD8 antibody-APCcy7, the CD14 antibody-PerCP/Cy5.5, the CD19 antibody-PerCP/Cy5.5, the CD25 antibody-PEcy7, and the CD137 antibody-APC.
  • 7-AAD was added to stain dead cells. After the addition, the cells were incubated on ice for 20 minutes.
  • the PBMCs were fixed with 1% paraformaldehyde. After the fixation, the expression levels of surface molecules were measured by flow cytometry using a FACS Canto II. The data obtained by the flow cytometry were analyzed using FACS Diva v9.0 and FlowJo software v10. A negative control was performed in the same manner, except that no overlapping peptide was added. The results are shown in FIG.
  • Figure 5 is a graph showing the percentage (%) of T cells expressing CD25 and CD137, which was calculated from the results of flow cytometry after adding overlapping peptides to PBMCs activated with inactivated SARS-CoV-2 virus.
  • the horizontal axis shows the type of origin of the overlapping peptide
  • the vertical axis shows the percentage (%) of CD25+CD137+ T cells.
  • the left graph shows the percentage (%) of CD25+CD137+ T cells after gating on CD3+CD8+
  • the right graph shows the percentage (%) of CD25+CD137+ T cells after gating on CD3+CD4+.
  • overlapping peptides derived from nucleocapsid were found to induce a higher percentage of both cytotoxic T cells and helper T cells than the overlapping peptides derived from spike protein or membrane protein.
  • HLA alleles Since it was found that overlapping peptides derived from SARS-CoV-2 nucleocapsid induce T cell activation, we investigated which HLA alleles the antigen is presented to. Specifically, we treated B lymphocyte cells (C1R cells, provided by Kumamoto University), C1R-A2402 cells, which are C1R cells expressing HLA-A*2402, C1R-B5201 cells, which are C1R cells expressing HLA-B*5201, and C1R-C1202 cells, which are C1R cells expressing HLA-C*1202 (all provided by Dr.
  • C1R cells provided by Kumamoto University
  • C1R-A2402 cells which are C1R cells expressing HLA-A*2402
  • C1R-B5201 cells which are C1R cells expressing HLA-B*5201
  • C1R-C1202 cells which are C1R cells expressing HLA-C*1202 (all provided by Dr.
  • Each cell was prepared by introducing an expression vector containing a nucleic acid encoding HLA-A*2402, HLA-B*5201, or HLA-C*1202 (see sequences below) and an expression vector containing a nucleic acid encoding human ⁇ 2 microglobulin protein (sequence number 19) into C1R cells using a viral vector.
  • HLA-A*2402 MAVMAPRTLVLLLSGALALTQTWAGSHSMRYFSTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDEETGKVKAHSQTDRENLRIALRYYNQSEAGSHTLQMMFGCDVGSDGRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQITKRKWEAAHVAEQQRAYLEGTCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQPTVPIVGIIAGLVLLGAVITGAVVAAVMWRRNSSDRKGGSYSQAASSDSAQGSDVSLTACKV
  • HLA-B*5201 MLVTAPRTVLLLLWGAVALTETWAGSHSMRYFYTAMSRPGRGEPRFIAVGYVDDTQFVRFDSDAASPRTEPRAPWIEQEGPEYWDRETQISKTNTQTYRENLRIALRYYNQSEAGSHTWQTMYGCDVGPDGRLLRGHNQYAYDGKDYIALNEDLSSWTAADTAAQITQRKWEAAREAEQLRAYLEGLCVEWLRRHLENGKETLQRADPPKTHVTHHPVSDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDRTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWEPSSQSTIPIVGIVAGLAVLAVVVIGAVVATVMCRRKSSGGKGGSYSQAASSDSAQGSDVSLTA
  • C1R cells, C1R-A2402 cells, C1R-B5202 cells, or C1R-C1202 cells were pulsed with overlapping peptides of SARS-CoV-2 nucleocapsid adjusted to 100 nmol/l. After pulsing, the cells were cultured with the PBMCs of Example 1 (1) for 24 hours using RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 11875101) containing 10% FBS. After the culture, antibody staining of surface molecules was performed.
  • the antibodies used were the CD3 antibody-FITC, the CD8 antibody-APCcy7, the CD14 antibody-PerCP/Cy5.5, the CD19 antibody-PerCP/Cy5.5, the CD25 antibody-PEcy7, and the CD137 antibody-APC. 7-AAD was also added to stain dead cells. After the addition, the cells were incubated on ice for 20 minutes. After the incubation, the PBMCs from Example 1(1) were fixed with 1% paraformaldehyde.
  • the cells were permeabilized with a membrane permeabilization reagent (a Cytofix/Cytoperm Fixation/Permeabilization solution kit, BD Biosciences, Cat No: 554714) and stained with a PE-labeled anti-IFN ⁇ antibody (4S.B3, BD Biosciences, 100-fold dilution).
  • a membrane permeabilization reagent a Cytofix/Cytoperm Fixation/Permeabilization solution kit, BD Biosciences, Cat No: 554714
  • PE-labeled anti-IFN ⁇ antibody 4S.B3, BD Biosciences, 100-fold dilution
  • the expression levels of surface molecules were measured by flow cytometry using a FACS Canto II.
  • the data obtained by the flow cytometry were analyzed using FACS Diva v9.0 and FlowJo software v10.
  • the negative control was performed in the same manner, except that no overlapping peptide was added. The results are shown in Figure 6.
  • Figure 6 is a graph showing the results of flow cytometry in which overlapping peptides were added to B lymphocyte cells (C1R cells) and the expression of IFN- ⁇ and CD8 in T cells was evaluated.
  • the horizontal axis shows the expression level of CD8, and the vertical axis shows the expression level of IFN- ⁇ in a dot plot.
  • the horizontal axis shows the type of cell, and the vertical axis shows the percentage (%) of IFN- ⁇ +CD8+ T cells.
  • the cDNA of TCR ⁇ and the cDNA of TCR ⁇ are introduced by electroporation into luciferase gene-expressing TCR-deficient Jurkat cells to perform TCR reconstitution.
  • TCR expressed by TCR reconstitution recognizes the antigen presented to the HLA of the target cells and the activation of the intracellular signal transduction pathway is induced, luciferase is expressed. Therefore, by detecting luciferase, it is possible to judge whether or not the antigen has been recognized by the TCR.
  • A549-ACE2/C1202 cells which are pulmonary type II alveolar epithelial cells (A549 cells) stably expressing human ACE2 and HLA-C*1202-IRES (internal ribosome entry site)-NGFR, were prepared by retroviral transduction.
  • A549 cells KF9, FF9, SI9, LL9, FM9, or NL9-A549/C1202 cells
  • expressing each antigen peptide and HLA-C*1202-IRES internal ribosome entry site
  • the TCR ⁇ -P2A-TCR ⁇ -P2A-BlaR plasmid was introduced into luciferase gene-expressing TCR-deficient Jurkat cells (Jurkat ⁇ -Luc cells) by electroporation using a PiggyBac vector (SBI, Cat No: PB530A-2).
  • the electroporation was performed using the Neon® Transfection System (Thermo Fisher Scientific) under the conditions of 1200v, 5ms, and 5 pulses.
  • Jurkat ⁇ -Luc cells stably expressing TCR were selected using RPMI medium containing 10 ⁇ g/ml blasticidin for 10 to 14 days.
  • the A549-ACE2/C1202 cells, or KF9, FF9, SI9, LL9, FM9, or NL9-A549/C1202 cells were added to the TCR-expressing Jurkat ⁇ -Luc cells at a ratio of 2:1, and cultured together.
  • the culture was performed at 37° C. for 6 hours using RPMI1640 medium (Thermo Fisher Scientific, Cat No: 11875101) containing 10% FBS.
  • luciferase production was measured using a luminescent substrate (Promega, Cat No: E2510).
  • the measurement was performed using a CentroXS3 plate reader (Berthold Technologies).
  • KF9 KAYNVTQAF (SEQ ID NO: 1)
  • FF9 FAPSASAFF (SEQ ID NO: 30)
  • SI9 SAFFGMSRI (SEQ ID NO:31)
  • LL9 LTYTGAIKL (SEQ ID NO: 32)
  • FM9 FSKQLQQSM (SEQ ID NO: 33)
  • NL9 NTASWFTAL (SEQ ID NO: 34)
  • Figure 8 is a graph showing TCR recognition of antigen peptides as luciferase fluorescence intensity.
  • the horizontal axis indicates the type of antigen peptide, and the vertical axis indicates the sensitivity of TCR to the antigen peptide.
  • the addition of KF9 antigen peptide showed significantly higher sensitivity of TCR to antigen peptide than the addition of no antigen peptide or the addition of FF9, SI9, LL9, FM9, or NL9 antigen peptides.
  • the KF9 antigen peptide showed high sensitivity to TCR-01, TCR-08, and TCR-22, but was particularly sensitive to TCR-01.
  • Figure 9 is a graph showing the results of flow cytometry in which antigen peptides were added to PBMCs collected from a patient who had recovered from infection with SARS-CoV-2 and possessed the HLA-A2402/B5202/C1202 alleles, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • the horizontal axis indicates the expression level of CD25
  • the vertical axis indicates the expression level of CD137.
  • the upper row shows, from left to right, the results when no antigen peptide was added, when an overlapping peptide derived from a spike protein was added, when an overlapping peptide derived from a nucleocapsid was added, and when an overlapping peptide derived from a membrane protein was added.
  • the lower row shows the results when KF9 was added. As shown in Figure 9, it was found that T cell activity was increased when an overlapping peptide derived from a nucleocapsid was added compared to when no antigen peptide was added, when an overlapping peptide derived from a spike protein was added, and when an overlapping peptide derived from a membrane protein was added.
  • Figure 10 is a graph showing the results of flow cytometry in which peptides were added to PBMCs activated with inactivated SARS-CoV-2 virus collected from a patient who had recovered from infection with SARS-CoV-2 carrying the HLA-A2402/B5202/C1202 alleles, and cells expressing CD25 and CD137 on the cell surface were evaluated.
  • the horizontal axis shows the expression level of CD25
  • the vertical axis shows the expression level of CD137.
  • the upper row shows, from left to right, the results when overlapping peptides derived from spike protein were added, when overlapping peptides derived from nucleocapsid were added, and when overlapping peptides derived from membrane protein were added.
  • the lower row shows the results when KF9 was added.
  • T cell activation was induced when overlapping peptides derived from nucleocapsid were added, compared to when overlapping peptides derived from spike protein were added and when overlapping peptides derived from membrane protein were added.
  • KF9 induced T cell activation in the same way as the addition of the overlapping peptide derived from the nucleocapsid.
  • the CM was a mixture of 87 ml of RPMI, 10 ml of FBS, 1 ml of MEM Non-Essential Amino Acids Solution (Gibco, Cat No: 11140-50), 1 ml of Sodium Pyruvate (Gibco, Cat No: 11360-70), and 1 ml of Penicillin-Streptomycin Solution (Fujifilm Wako Pure Chemical Industries, Cat No: 168-23191). After the incubation, 10 ⁇ l of overlapping peptides of each nucleocapsid protein was added to each well. Next, cells were prepared.
  • PBMCs were obtained from patients infected with SARS-CoV-2 carrying the HLA-A2402/B5202/C1202 alleles and recovered, using the same method as in Example (1).
  • PBMCs were suspended using 3 ml of 10% CM, and 3.6 x 10 6 PBMCs were suspended in 1.8 ml of 10% CM to adjust the concentration to 2 x 10 6 cells/ml.
  • 210 ⁇ l of PBMCs were dispensed into one row (8 wells) of 96U. After the dispensing, 50 ⁇ l (1 x 10 5 cells) was transferred from 96U to an ELISPOT plate (4 rows per donor) and incubated overnight (20 hours or more).
  • the ELISPOT plate was washed with 200 ⁇ l of PBS containing 0.05% TweenTM-20, and the washing was performed a total of six times.
  • anti-human IFN- ⁇ mAb 7-B6-1, biotinylated MABTECH, Cat No: 3420-6-250
  • 100 ⁇ l of the diluted solution was dispensed into each well. After the dispensing, incubation was performed at room temperature for 1 to 2 hours.
  • the ELISPOT plate was washed with 200 ⁇ l of PBS containing 0.05% TweenTM-20, and the washing was performed six times in total.
  • streptavidin-ALP was diluted (2000-fold dilution) with 10 ml of PBS containing 5 ⁇ l of mAb stock and 50 ⁇ l of FBS (0.5% FBS/PBS), and 100 ⁇ l of the diluted solution was dispensed into each well. After the dispensing, incubation was performed at room temperature for 1 hour. After the incubation, the ELISPOT plate was washed with 200 ⁇ l of PBS containing 0.05% TweenTM-20, and the washing was performed a total of 6 times.
  • the substrate buffer was diluted 25 times (400 ⁇ l of substrate buffer was added with 9.6 ml of MQ water), and 100 ⁇ l of substrate A and 100 ⁇ l of substrate B were added. After the addition, 100 ⁇ l of the solution was added to each well. After the addition, incubation was performed under conditions of 20 to 40 minutes at room temperature in the dark. After the incubation, substrate was added, and both sides of the membrane were washed with water to stop the reaction. Thereafter, all wells were filled with water, and the plate was left standing for 10 minutes or more in the dark. After the standing, the water was removed from the wells, and the plate was rinsed with water several times. After the rinsing, the specimen was vigorously turned over so that no moisture remained, and dried overnight in a dark place. After drying, analysis was performed using ImmunoSpot (manufactured by CTL). The results are shown in FIG.
  • Figure 11 is a graph showing the results of the ELISPOT method.
  • the horizontal axis indicates the type of peptide derived from the SARS-CoV-2 nucleocapsid (the position on the N-terminal side of the 15 amino acid residues), and the vertical axis indicates the spot forming unit (SFU).
  • SFU spot forming unit
  • the cells were cultured for 14 days using 30 U/ml recombinant human IL-2 (Peprotec, Cat No: 200-02) and 10% FBS-containing RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02). After the culture, the in vitro expanded CD8+ T cells were restimulated in the presence or absence of peptide. The cells were then cultured at 37°C for 24 hours, washed, and surface stained with antibodies.
  • the antibodies used were CD3 FITC (UCHT1, 100-fold dilution, Biolegend), CD8 APCcy7 (RPA-T8, 100-fold dilution, Biolegend), CD14 PerCP/Cy5.5 (HCD14, 100-fold dilution, Biolegend), CD19 PerCP/Cy5.5 (HIB19, 10-fold dilution, Biolegend), CD25 PEcy7 (M-A251, 50-fold dilution, Biolegend), and CD137 APC (4B4-1, 50-fold dilution, Biolegend).
  • 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404 was added to stain dead cells.
  • the mixture was incubated on ice for 20 minutes. After the incubation, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No: 09154-85). After the fixation, the expression levels of surface molecules were measured by flow cytometry using FACS Canto II (BD Biosciences). The data obtained by the flow cytometry were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The negative control was performed in the same manner, except that no overlapping peptide was added. The results are shown in FIG. 12.
  • Figure 12 is a graph showing the results of the AIM assay.
  • the horizontal axis indicates whether HLA-C*1202 is positive or negative and whether or not peptide KF9 (KAYNVTQAF) was added, and the vertical axis indicates the percentage of CD25+ and CD137+ in CD8 T cells.
  • the percentage of CD25+ and CD137+ in CD8 T cells was high in cases of HLA-C*1202 positivity and addition of KF9 (KAYNVTQAF).
  • the PBMCs were collected from HLA-C*1202-positive SARS-CoV-2 recovered individuals, HLA-C*1202-negative SARS-CoV-2 recovered individuals, HLA-C*1202-positive SARS-CoV-2 negative individuals, and HLA-C*1202-negative SARS-CoV-2 negative individuals. Then, 0.1 ⁇ g of tetramer was added to the PBMCs, and the mixture was incubated at room temperature for 30 minutes. After the incubation, the mixture was washed with PBS containing 2% FCS. After the washing, the surface was stained with an antibody. The antibody used was CD8 APCcy7 (RPA-T8, 100-fold dilution, Biolegend).
  • 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was added to stain dead cells. After the addition, the mixture was incubated on and off ice for 20 minutes. After the incubation, the mixture was washed twice with PBS containing 2% FCS. After the washing, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No: 09154-85). After the fixation, the expression levels of surface molecules were measured by flow cytometry using a FACS Canto II (BD Biosciences). The data obtained by the flow cytometry were analyzed using FACS Diva v9.0 (BD Biosciences) and FlowJo software v10 (Tree Star). The results are shown in FIG.
  • Figure 13 is a graph showing the results of the MHC tetramer assay.
  • the horizontal axis indicates whether HLA-C*1202 is positive or negative, and whether SARS-CoV-2 recovered or negative, and the vertical axis indicates the percentage (%) of KF9 tetramers in CD8 T cells.
  • the percentage of KF9 tetramers in CD8 T cells was high in HLA-C*1202 positive and SARS-CoV-2 recovered individuals.
  • Example 2 We confirmed that an antigenic peptide derived from the nucleocapsid of SARS-CoV-2 can induce T cell activation.
  • A549 cells expressing human ACE2/HLA-C*1202 or human ACE2/HLA-A*2402 were infected with SARS-CoV-2 virus (Wuhan strain, prototype) or SARS-CoV-2 virus mutants, Omicron strains BA.1, BA.2, or BA.5, at an MOI of 0.1, at 37° C., for 120 minutes. After the infection, the A549 cells were washed.
  • PBMCs were collected from two patients (DC6 and GV69) who had recovered from SARS-CoV-2 infection carrying the HLA-C*1202 allele, and obtained in the same manner as in Example 1 (1) above.
  • C1R cells expressing HLA-C*1202 or HLA-A*2402 and irradiated with 110 Gy of X-rays, and antigen peptides were added to the T cells induced with the antigen peptides in the same manner as in Example 1 (2) above, except that the antigen peptide of KF9 or the antigen peptide of QI9 (QYIKWPWYI (SEQ ID NO: 37)) was used as the antigen.
  • the QI9 antigen peptide is an antigen peptide derived from the spike protein.
  • the KF9/C12-specific T cells or the QI9/A24-specific T cells were co-cultured with the A549 cells (T: target cells) at an E:T ratio of 2:1, 1:1, or 0:1 (none). 72 hours after the start of the culture, the culture supernatant was collected and the amount of viral RNA was measured by real-time RT-PCR.
  • the PCR reagent used was One-Step PrimeScriptTM III RT-qPCR Mix (Takara Bio, Cat No: RR600B), and the PCR device used was the LightCycler® 96 System (Roche Diagnostics).
  • the primers used were the following primer/probe set for detecting the new coronavirus (Primer/Probe N2 (2019-nCoV) (Takara Bio, Cat No: XD0008)).
  • the copy number of viral RNA was standardized using Positive Control RNA Mix (2019-nCoV) (Takara Bio, Cat No: XA0142).
  • the relative viral copy number was calculated by normalizing the viral RNA copy number obtained in A549 cells not co-cultured with T cells to 1. These results are shown in Figure 14.
  • Primer-probe set for detecting the new coronavirus NIID_2019-nCOV_N_ forward (SEQ ID NO: 38) 5'-AAATTTTGGGGACCAGGAAC-3' NIID_2019-nCOV_N_ reverse (SEQ ID NO:39) 5'-TGGCAGCTGTGTAGGTCAAC-3' NIID_2019-nCoV_N_ probe (SEQ ID NO: 40) 5'-FAM-ATGTCGCGCATTGGCATGGA-BHQ-3'
  • Figure 14 is a graph showing the results of the antiviral activity of KF9/C12-specific T cells or QI9/A24-specific T cells derived from patients who had recovered from SARS-CoV-2 infection carrying the HLA-C*1202 allele against mutant and subspecies SARS-CoV-2 virus.
  • the horizontal axis indicates the E:T ratio
  • the vertical axis indicates the relative virus copy number.
  • the horizontal axis indicates the type of T cell
  • the vertical axis indicates the relative virus copy number. Note that Figure 14(B) shows the results when the E:T ratio is 1:1.
  • KF9/C12-specific T cells and QI9/A24-specific T cells derived from patients DC6 and GV69 suppressed the replication of the prototype SARS-CoV-2 virus and all of the mutant Omicron strains BA.1, BA.2, and BA.5.
  • FIG. 14(B) when the E:T ratio was 1:1, KF9/C12-specific T cells were found to be able to suppress viral replication more effectively than QI9/A24-specific T cells.
  • Figure 15 is a graph showing the results of the ELISPOT method.
  • the top row shows the results for spot-forming units
  • the middle row shows the percentage (%) of seven subjects who responded with 10 or more spots (responders)
  • the bottom row shows a schematic diagram of the SARS-CoV-2 virus.
  • NTD indicates the N-terminal region
  • CTD indicates the C-terminal region.
  • the number of responders was high in 65-66, which contains the amino acid sequence of KF9.
  • Figure 16 shows dot plots and graphs showing the results of the tetramer assay.
  • the horizontal axis shows the mean fluorescence intensity of CD8, and the vertical axis shows the mean fluorescence intensity of tetramer.
  • the horizontal axis shows the number of months since infection, and the vertical axis shows the percentage (%) of KF9 tetramer+ and CD8+ T cells.
  • T cells differentiate into memory T cells capable of rapid immune response upon reinfection.
  • naive T cells CCR7+CD45RA+
  • the memory T cells are subdivided into memory stem cell T cells, central memory T cells (TCM, CCR7+CD45RA-), effector memory T cells (TEM, CCR7-CD45RA-), and terminally differentiated effector memory T cells (TEMRA, CCR7-CD45RA+).
  • PBMCs were obtained from a patient infected with SARS-CoV-2 carrying the HLA-C*1202 allele.
  • the phenotype of T cells was observed by staining of the surface antigens in the same manner as in Example 1(2) above, except that KF9 was used as the antigen and ccr7-bv510 antibody (Biolegend, 25-fold dilution, Cat No: 353232) and CD45RA-APC antibody (Biolegend, 100-fold dilution, Cat No: 304112) were used as the antibodies.
  • Figure 17 is a graph showing the results of flow cytometry in which KF9 tetramer was added to PBMCs collected from a patient infected with SARS-CoV-2 carrying the HLA-C*1202 allele, and cells expressing CCR7 and CD45RA on the cell surface were evaluated.
  • the horizontal axis shows the expression level of CCR7
  • the vertical axis shows the expression level of CD45RA.
  • the horizontal axis shows the number of months since infection
  • the vertical axis shows the expression rate (%).
  • DMSO containing Cell Trace Violets staining solution was diluted 5-fold with PBS to prepare 1 mmol/ ⁇ l.
  • PBMCs were obtained from healthy subjects in the same manner as in Example 1 (1). The PBMCs were adjusted to 1 ⁇ 10 6 cells/ml. 10 ⁇ l of the prepared Cell Trace Violets staining solution was added to 10 ml of the PBMCs. After the addition, the PBMCs were suspended in 200 ⁇ l of 10% FBS-containing RPMI 1640 medium (Thermo Fisher Scientific, Cat No: 200-02). 100 nmol/l of SARS-CoV-2 nucleocapsid-derived overlapping peptide or KF9 was added and suspended.
  • FBS-containing RPMI 1640 medium Thermo Fisher Scientific, Cat No: 200-02
  • the PBMCs were cultured at 37° C. for 7 days. After the culture, the PBMCs were washed, stained with KF9 tetramer, and then the surface was stained with an antibody.
  • the antibodies used in the proliferation test were CD3 FITC (UCHT1, 100-fold dilution, Biolegend), CD8 APCcy7 (RPA-T8, 100-fold dilution, Biolegend), CD14 PerCP/Cy5.5 (HCD14, 100-fold dilution, Biolegend), and CD19 PerCP/Cy5.5 (HIB19, 10-fold dilution, Biolegend), and in the phenotype test were ccr7-bv510 antibody (Biolegend, 25-fold dilution, Cat No: 353232) and CD45RA-APC antibody (Biolegend, 100-fold dilution, Cat No: 304112).
  • 7-aminoactinomycin D (7-AAD, Biolegend, Cat No: 420404) was also added to stain dead cells. After the addition, the mixture was incubated on ice for 20 minutes. After the incubation, the PBMCs were fixed with 1% paraformaldehyde (Nacalai Tesque, Cat No: 09154-85). After the fixation, the expression level of surface molecules was measured by flow cytometry using Cytek Notrhern Lights (Cytec Japan). The data obtained by the flow cytometry was analyzed using FlowJo software v10 (Tree Star). The negative control (No peptide) was performed in the same manner except that no peptide was added. These results are shown in FIG. 18.
  • FIG. 18 is a graph showing the results of flow cytometry evaluating the increase and phenotype of KF9-specific T cells when peptides were added to PBMCs derived from healthy subjects.
  • the horizontal axis indicates the mean fluorescence intensity of CTV
  • the vertical axis indicates the mean fluorescence intensity of tetramer.
  • the horizontal axis indicates the type of peptide
  • the vertical axis indicates the percentage (%) of CD8+ T cells with low CTV fluorescence intensity and positive for KF9 tetramer.
  • FIG. 18(C) the horizontal axis indicates the expression level of CCR7
  • the vertical axis indicates the expression level of CD45RA.
  • the horizontal axis indicates the type of T cells, and the vertical axis indicates the percentage (%) of KF9 tetramer-positive cells in CD8+ T cells.
  • the horizontal axis indicates the type of T cells, and the vertical axis indicates the percentage (%) of KF9 tetramer-positive cells in CD8+ T cells.
  • nucleocapsid-derived overlapping peptide Nuc OLP
  • KF9 KF9
  • Figure 19 is a graph showing the results of the AIM assay.
  • the horizontal axis indicates whether HLA-C*1402 is positive or negative and whether or not peptide KF9 was added, and the vertical axis indicates the percentage (%) of CD25+ and CD137+ positive cells in CD8 T cells.
  • the percentage of CD25+ and CD137+ positive cells in CD8 T cells was high when HLA-C*1402 was positive and KF9 was added. From these results, it was found that peptide KF9 derived from the nucleocapsid of SARS-CoV-2 is presented to HLA-C*14:02 and induces T cell activation.
  • the expression level of surface molecules was measured in the same manner as in Example 1(4), except that C1R-B5101 cells, which are C1R cells expressing HLA-B*5101, and C1R-C1402 cells, which are C1R cells expressing HLA-C*1402, were used as the cells, and the nucleocapsid-derived KF9 peptide (1 nmol/l, 10 nmol/l, 100 nmol/l, 1000 nmol/l) was used as the antigen.
  • the negative control was performed in the same manner, except that the KF9 peptide was not added.
  • Figure 20 is a graph showing the results of flow cytometry in which KF9 peptide was added to B lymphocytes (C1R cells) and the expression of IFN- ⁇ and CD8 in T cells was evaluated.
  • the horizontal axis shows the expression level of CD8, and the vertical axis shows the expression level of IFN- ⁇ in a dot plot.
  • the proportion of IFN- ⁇ + T cells was increased in C1R-C1402 cells compared to C1R-B5101 cells, indicating that the T cells were activated. From the above, it was found that the nucleocapsid-derived peptide KF9 is presented to HLA-C*1402, inducing the activation of T cells that recognize it.
  • Nucleocapsid-derived antigen peptide> (Appendix 1) A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid-derived antigenic peptide comprising the following polypeptide (P1), (P2), or (P3): (P1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 (KAYNVTQAF); (P2) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 in which 1 to 6 amino acids have been deleted, inserted, substituted, or added; (P3) A polypeptide consisting of an amino acid sequence having 80% or more identity to the amino acid sequence of SEQ ID NO:1.
  • the antibody has TCR-01, TCR-08, or TCR-22 activation activity;
  • the TCR-01 comprises an ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO:6 and a ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO:7;
  • the TCR-08 comprises an ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 8 and a ⁇ chain variable domain comprising the amino acid sequence of SEQ ID NO: 9; 3.
  • Appendix 4 The peptide according to any one of Appendix 1 to 3, wherein (P2) is a polypeptide consisting of an amino acid sequence in which 1 or 2 amino acids have been added to at least one of the C-terminus and N-terminus of the amino acid sequence of SEQ ID NO: 1.
  • Appendix 5 A peptide described in any one of appendix 1 to 4, which is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 or 3.
  • Appendix 6 6.
  • HLA human leukocyte antigen
  • Appendix 7 The peptide of claim 6, wherein the HLA is HLA-C*1202 and/or HLA-C*1402.
  • ⁇ Nucleic Acid> (Appendix 8) A nucleic acid encoding a peptide according to any one of claims 1 to 7.
  • ⁇ Vector> Appendix 9) A vector comprising the nucleic acid described in Appendix 8.
  • ⁇ CTL inducer/activator> (Appendix 10) An agent for inducing and/or activating cytotoxic T cells, comprising a peptide described in any one of appendices 1 to 7, a nucleic acid described in appendix 8, and/or a vector described in appendix 9.
  • ⁇ HLA/antigen peptide complex> (Appendix 11) comprising a complex of a human leukocyte antigen (HLA) and an antigenic peptide, The HLA/antigenic peptide complex, wherein the antigenic peptide comprises a peptide according to any one of claims 1 to 7.
  • (Appendix 12) The HLA/antigen peptide complex of claim 11, wherein the HLA is HLA-C*1202 and/or HLA-C*1402.
  • ⁇ Pharmaceutical Composition> (Appendix 13) A pharmaceutical composition comprising a peptide according to any of appendices 1 to 7, a nucleic acid according to appendices 8, a vector according to appendices 9, an inducer and/or activator of cytotoxic T cells according to appendices 10, and/or an HLA/antigenic peptide complex according to appendices 11 or 12, and a pharma- ceutical acceptable carrier.
  • Appendix 15 The pharmaceutical composition according to claim 13 or 14, which is a vaccine for use in preventing infection or aggravation of infectious disease caused by SARS-CoV-2.
  • Preventive measures> (Appendix 16) A method for preventing infection or aggravation of an infectious disease caused by SARS-CoV-2 using a peptide according to any one of appendices 1 to 7, a nucleic acid according to appendices 8, a vector according to appendices 9, an inducer and/or activator of cytotoxic T cells according to appendices 10, and/or an HLA/antigen peptide complex according to appendices 11 or 12.
  • Appendix 17 The method of claim 16, comprising an administration step of administering to a subject the peptide, the nucleic acid, the vector, the cytotoxic T cell inducer and/or activator, and/or the HLA/antigen peptide complex of claim 11 or 12.
  • ⁇ Use> Appendix 18
  • the biological sample is selected from the group consisting of blood and peripheral blood mononuclear cells.
  • the antigen peptide of the present disclosure can induce, for example, a cellular immune response against SARS-CoV-2. Therefore, the antigen peptide of the present disclosure can prevent, for example, SARS-CoV-2 infection and aggravation. Therefore, the present disclosure is extremely useful, for example, in the medical field.

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Abstract

La présente invention a pour but de procurer un nouveau peptide pouvant être utilisé pour induire une réponse immunitaire au SARS-CoV-2.　Un peptide antigénique dérivé de nucléocapside selon la présente invention comprend le polypeptide suivant (P1), (P2) ou (P3) : (P1) un polypeptide comprenant une séquence d'acides aminés représentée par SEQ ID NO : 1 (KAYNVTQAF) ; (P2) un polypeptide comprenant une séquence d'acides aminés obtenue par délétion, insertion, substitution ou ajout de 1 à 6 acides aminés dans la séquence d'acides aminés représentée par SEQ ID NO : 1 ; et (P3) un polypeptide comprenant une séquence d'acides aminés ayant au moins 80 % d'identité avec la séquence d'acides aminés représentée par SEQ ID NO : 1.
PCT/JP2023/037529 2022-10-17 2023-10-17 Peptide antigénique dérivé de la nucléocapside, acide nucléique, vecteur, composition pharmaceutique, complexe peptide antigénique/hla et procédé de détection des lymphocytes t WO2024085143A1 (fr)

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Citations (5)

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WO2021163371A1 (fr) * 2020-02-12 2021-08-19 La Jolla Institute For Immunology Nouveaux épitopes de lymphocytes t du coronavirus et utilisations associées
WO2021195108A1 (fr) * 2020-03-24 2021-09-30 Cue Biopharma, Inc. Polypeptides modulateurs de lymphocytes t et leurs méthodes d'utilisation
JP2022017865A (ja) * 2020-07-14 2022-01-26 学校法人 埼玉医科大学 ＳＡＲＳ－ＣｏＶ－２の細胞傷害性Ｔ細胞エピトープペプチド及びその利用
WO2022148455A1 (fr) * 2021-01-11 2022-07-14 The Hong Kong University Of Science And Technology Identification et utilisations de séquences peptidiques d'épitopes de lymphocytes t et b de sars-cov-2
WO2022149549A1 (fr) * 2021-01-05 2022-07-14 オンコセラピー・サイエンス株式会社 Peptide dérivé d'une protéine du ｓａｒｓ－ｃｏｖ－２, et vaccin contenant celui-ci

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WO2021163371A1 (fr) * 2020-02-12 2021-08-19 La Jolla Institute For Immunology Nouveaux épitopes de lymphocytes t du coronavirus et utilisations associées
WO2021195108A1 (fr) * 2020-03-24 2021-09-30 Cue Biopharma, Inc. Polypeptides modulateurs de lymphocytes t et leurs méthodes d'utilisation
JP2022017865A (ja) * 2020-07-14 2022-01-26 学校法人 埼玉医科大学 ＳＡＲＳ－ＣｏＶ－２の細胞傷害性Ｔ細胞エピトープペプチド及びその利用
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