Methods of inducing immunity against sars-cov-2

Abstract

The present disclosure relates to peptides that induce immunity against SARS-CoV-2. The present disclosure additionally relates to compositions containing the peptides and inducing immunity against SARS-CoV-2.

Description

Technical Field

[0001] This invention relates to a method for inducing immunity against SARS-CoV-2, a peptide for the method, and an immunogenic composition containing the peptide. Background Technology

[0002] SARS-CoV-2 is the coronavirus that caused the 2020 pandemic. On January 7, 2020, the World Health Organization (WHO) temporarily named the virus 2019-nCoV. On February 11 of the same year, the International Committee on Taxonomy of Viruses (ICTV) officially named the virus SARS-CoV-2. The WHO named the disease caused by this novel coronavirus COVID-19. The ICTV classifies SARS-CoV-2 as belonging to the genus β-coronaviruses, and is the same species as SARS-CoV (or its sister strain). The complete genome sequence of SARS-CoV-2 was accessed in GenBank under the accession number MN908947.3 at the National Center for Biotechnology Information (NCBI).

[0003] Viral particles (virus particles) have a diameter of approximately 50 nm to approximately 200 nm and, like typical coronaviruses, contain spike proteins, nucleocapsid proteins, membrane proteins, envelope proteins, and viral genomic RNA. The nucleocapsid protein forms a complex with the RNA, surrounding the lipid-bound spike protein, membrane proteins, and envelope proteins, thus forming the viral particle's envelope. It is generally believed that the spike protein, located on the outermost surface of the envelope, binds to the ACE2 receptor on the cell surface, promoting infection of cells. There are individuals infected with SARS-CoV-2 who do not exhibit symptoms; these are called asymptomatic carriers. It has been suggested that the virus carried by asymptomatic carriers may infect other people. It has been noted that infection with SARS-CoV-2 can cause a decrease or loss of smell and / or taste. SARS-CoV-2 sometimes causes severe acute respiratory syndrome (SACS). Major symptoms of SACS include fever around 40°C, cough, and shortness of breath. A significant complication is pneumonia.

[0004] Vaccines against SARS-CoV-2 are under development. Currently, there is a need to develop diverse vaccines. For SARS-CoV-2, target peptides that induce immune responses are being sought through sequence homology and bioinformatics approaches (Non-Patent Literature 1).

[0005] Existing technical documents

[0006] Non-patent literature

[0007] Non-patent literature 1: Grifoni et al., Cell host & Microbe, 27(4):671-680, 2020 Summary of the Invention

[0008] The present invention provides a method for inducing immunity against SARS-CoV-2, a peptide for the method, and an immunogenic composition containing the peptide.

[0009] More specifically, the present invention provides the following invention.

[0010] (1) A peptide, which is a partial peptide of the spike protein of SARS-CoV-2, containing the amino acid sequence described in sequence number 4, or a partial peptide of the spike protein at the position corresponding to the sequence, and having a length of 8 to 30 amino acids.

[0011] (2) The peptide according to (1) above has a continuous amino acid sequence of 8 to 20 amino acids in the region of position 1204 to 1226 of the amino acid sequence recorded in sequence number 8, or an amino acid sequence of a portion of the spike protein of SARS-CoV-2 at the position corresponding to the sequence.

[0012] (3) The peptide according to (1) or (2) above is composed of the amino acid sequence recorded in sequence number 4.

[0013] (4) The peptide according to (1) or (2) above has the amino acid sequence recorded in any one of sequence numbers 10 to 12 and has a length of 15 to 18 amino acids.

[0014] (5) An immunogenic composition comprising any one of (1) to (4) above.

[0015] (6) A pharmaceutical composition for inducing immunity against SARS-CoV-2, comprising any one of the peptides described in (1) to (4) above.

[0016] (7) A method for inducing immunity against SARS-CoV-2 in a subject, comprising administering the peptide of any one of (1) to (4) above, or the composition of (5) or (6) above, to the subject.

[0017] (8) According to the method described in (7) above, the object is an object that has been infected with SARS-CoV-2.

[0018] (9) According to the method described in (7) above, the object is an object that is at risk of being infected with SARS-CoV-2.

[0019] (10) According to the method described in (7) above, wherein the object is an object at risk of being infected with SARS-CoV-2. Attached Figure Description

[0020] Figure 1 This study presents an analysis of antigen-specific T cell generation from PBMCs of five healthy donors (HD1–HD5). Cytokine production in PBMCs was analyzed using FACS analysis. PBMCs were cultured for 21 days in the presence of the relevant peptides, and IFN-γ production was evaluated. T cell-induced IFN-γ production was assessed by intracellular staining analysis after 16 hours of culture in the presence of GolgiPlug (BD) and the relevant peptides.

[0021] Figure 2 This study demonstrates the cytotoxicity of T cells against HLA-A24+ target cells expressing the Cov2-S antigen. Cultured PBMCs from HD5 were co-cultured with the peptides, and harvested after 3 weeks. The cytotoxicity of T cells against peptide-peptide ...

[0022] Figure 3 This study presents an analysis of antigen-specific T cell generation from PBMCs of four healthy volunteers (HV1–5). Cytokine production in PBMCs was analyzed using FACS analysis. PBMCs were cultured for 21 days in the presence of the relevant peptides, and IFN-γ production was evaluated. T cell-induced IFN-γ production was assessed by intracellular staining analysis after 16 hours of culture in the presence of GolgiPlug (BD) and the relevant peptides.

[0023] Figure 4 To be Figure 3 The graph is obtained by visualizing the data. Detailed Implementation

[0024] In this specification, "object" refers to vertebrates, which can include mammals such as humans, and mammals that can be infected with SARS-CoV-2 (cats, ferrets, bats, and pangolins). Objects can be those infected with SARS-CoV-2, asymptomatic carriers of the pathogen infected with SARS-CoV-2, or those infected with SARS-CoV-2 and exhibiting COVID-19 symptoms. Objects can be those with the potential (risk) of being infected with SARS-CoV-2, or those with the potential (risk) of being infected with SARS-CoV-2. Objects can be children (e.g., toddlers (1–6 years old), school children (6–12 years old), adolescents (12 years old and above), and adults (20 years old and above). Adults can be those aged 30 years and older, 40 years and older, 50 years and older, 60 years and older, or 70 years and older.

[0025] In this specification, SARS-CoV-2 is the coronavirus that caused the 2020 pandemic. On January 7, 2020, the World Health Organization (WHO) provisionally named the virus 2019-nCoV. Additionally, on February 11 of the same year, the International Committee on Taxonomy of Viruses (ICTV) officially named the virus SARS-CoV-2. Coronaviruses can cause severe respiratory illnesses ranging from the common cold to severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS). The WHO named the disease caused by this novel coronavirus COVID-19. The ICTV classifies SARS-CoV-2 as belonging to the genus β-coronaviruses, the same species as SARS-CoV (or its sister strain). The complete genome sequence of SARS-CoV-2 is accessed in GenBank under the accession number MN908947.3 at the National Center for Biotechnology Information (NCBI). Viral particles have a diameter of approximately 50 nm to approximately 200 nm and, like typical coronaviruses, contain spike proteins, nucleocapsid proteins, membrane proteins, envelope proteins, and viral genomic RNA. The nucleocapsid protein forms a complex with RNA, surrounding it with lipid-binding spike proteins, membrane proteins, and envelope proteins, thus forming the viral particle envelope. It is generally believed that the spike protein, located on the outermost surface of the envelope, binds to the ACE2 receptor on the cell surface, promoting infection of cells. There are individuals infected with SARS-CoV-2 who do not show symptoms; these are called asymptomatic carriers. It has been suggested that the virus carried by asymptomatic carriers may infect other people. It has been noted that SARS-CoV-2 infection can cause a decrease or loss of smell and / or taste. SARS-CoV-2 sometimes causes severe acute respiratory syndrome (SARS). The main symptoms of SARS-CoV-2 include fever around 40°C, cough, and shortness of breath. A significant complication is pneumonia. The presence or absence of SARS-CoV-2 infection is primarily determined by PCR testing. This PCR test assesses the presence of the SARS-CoV-2 gene in the body by detecting the amplification of SARS-CoV-2-specific bands. Treatments for SARS-CoV-2 include antiviral drugs (e.g., remdesivir), steroidal anti-inflammatory drugs (e.g., dexamethasone), and inhibitors of inflammatory cytokines (e.g., IL-6 inhibitors, such as anti-IL-6 antibodies, TNF-α inhibitors, such as etanercept).

[0026] In this specification, "spike protein" can refer to the protein encoded by positions 21563 to 25384 of the SARS-CoV-2 genome, which is accessed in GenBank under accession number MN908947.3 and registered with the National Center for Biotechnology Information (NCBI). The spike protein of SARS-CoV-2 has the amino acid sequence number 8. The spike protein is also known as the S protein.

[0027] In this specification, "peptide" refers to a polymer of amino acids. Polymers are generally unbranched. "Partial peptide" refers to a specific portion of a peptide. As long as the peptide and partial peptide are exposed on a surface, they can be linked to other proteins, lipids, or non-electrolyte hydrophilic polymers (e.g., polyethylene glycol). Peptides and partial peptides can be manufactured from nucleic acids encoding the peptide. Alternatively, peptides and partial peptides can be chemically synthesized. Peptides and partial peptides can also be isolated, concentrated, or purified. Isolation refers to separating the peptide and partial peptide from at least other components; purification refers to selectively separating the peptide and partial peptide at least partially; concentration refers to increasing the concentration of the peptide and partial peptide.

[0028] In this specification, a "composition" is a mixture of one or more ingredients. A composition may, for example, contain a portion of a peptide and an aqueous solvent (e.g., water). The composition may also contain pharmaceutically acceptable excipients. In this specification, an immunogenic composition is a composition that, when administered to a subject, is capable of evoking an immune response in that subject. Immunogenic compositions can be used to induce an immune response in a subject. Because immunogenic compositions can evoke an immune response in a subject, they can therefore be used as vaccines. For example, the immunogenic compositions of the present invention can be used to induce an immune response against SARS-CoV-2, or can be used as a vaccine against SARS-CoV-2.

[0029] In this instruction manual, "treatment" includes preventative treatment and therapeutic treatment. Treatment can be performed on symptomatic patients or asymptomatic carriers of the pathogen. Therapeutic treatment can be performed on those already infected with the virus, while preventative treatment can be performed to prevent future infection, to delay the onset of COVID-19 symptoms due to future infection, or to alleviate existing COVID-19 symptoms.

[0030] In this specification, "human leukocyte antigen" (HLA) refers to the major histocompatibility complex (MHC). HLA is involved in the presentation of antigens to immune cells and is known to have a wide variety of allele polymorphisms. The most frequently detected allele in Japanese people is A*24:02, which is carried by approximately 36% of the Japanese population. Peptides presented to HLA can be useful as peptide vaccines; for example, peptides presented to A*24:02 can be useful as peptide vaccines in the Japanese population.

[0031] This disclosure provides a partial peptide of the spike protein of SARS-CoV-2. The partial peptide of the spike protein of SARS-CoV-2 contains the amino acid sequence described in Serial Number 4 or a partial peptide of the spike protein (e.g., a natural variant thereof) at the position corresponding to that sequence.

[0032] In one embodiment, the spike protein of SARS-CoV-2 is the spike protein registered with the National Center for Biotechnology Information (NCBI) in the United States under GenBank accession number MN908947.3, or the spike protein of SARS-CoV-2 having an amino acid sequence that is homologous to it by more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0033] This disclosure further provides a partial peptide of the SARS-CoV-2 spike protein, which contains the amino acid sequence described in Serial Number 4, or a partial peptide of the spike protein at the position corresponding to that sequence, and has a length of 8 to 30 amino acids (preferably 8 to 10 amino acids, 15 to 18 amino acids, 8 to 20 amino acids, 8 to 11 amino acids, 9 amino acids, or 10 amino acids, more preferably 9 amino acids). Here, the partial peptide having the amino acid sequence of the spike protein at the position corresponding to that sequence refers to the amino acid sequence arranged at the corresponding position in a natural variant of the SARS-CoV-2 spike protein when compared with the above-mentioned SARS-CoV-2 spike protein.

[0034] This disclosure further provides a peptide having a continuous amino acid sequence of 8 to 20 amino acids (e.g., 8 to 10 amino acids, 9 amino acids, or 15 to 18 amino acids) contained in the region at positions 1204 to 1226 of the amino acid sequence described in Serial No. 8. In a preferred embodiment, this peptide contains the amino acid sequence described in Serial No. 4.

[0035] This disclosure further provides a composition comprising two or more peptides selected from the group consisting of a continuous amino acid sequence of 8 to 20 amino acids (e.g., 8 to 10 amino acids, 9 amino acids, or 15 to 18 amino acids) selected from the region containing the amino acid sequence at positions 1204 to 1226 of the amino acid sequence described in Serial No. 8. This composition can provide diverse epitopes to TCRs (T cell receptors) expressed on T cells, thereby inducing (or activating) a diverse population of T cells with diverse antigen specificity.

[0036] Peptides of 8–10 amino acids (preferably 9 amino acids) can be directly presented as antigens by HLA class I, activating CD8 single-positive T cells. Conversely, peptides of 15–18 amino acids, after being ingested into cells, are broken down and can be presented as antigens by either HLA class I or II, activating both CD4 and CD8 single-positive T cells. Peptides of 8–10 amino acids (preferably 9 amino acids) do not undergo the process of ingestion into cells, breakdown, and antigen presentation to HLA, but can be directly presented as antigens by HLA, thus enabling immediate immune activation. In contrast, peptides of 15–18 amino acids can activate both CD4 and CD8 single-positive T cells. Those skilled in the art can appropriately select the amino acid length according to the purpose and use it as a vaccine.

[0037] Additionally, according to this disclosure, T cells activated by the peptides of the present invention can be provided. According to this disclosure, a pharmaceutical composition is provided containing T cells activated by the peptides of the present invention. The pharmaceutical composition containing T cells activated by the peptides of the present invention can, for example, be administered to a subject infected with coronaviruses such as SARS-CoV-2, enabling treatment of the infection in that subject. The pharmaceutical composition containing T cells activated by the peptides of the present invention has solution conditions suitable for cell preparation. The pharmaceutical composition containing T cells activated by the peptides of the present invention may further contain pharmaceutically acceptable excipients. Activation can be performed by culturing the peptides, antigen-presenting cells, and T cells (e.g., naive T cells, central memory T cells, effector memory T cells, or terminal effector T cells). In this case, the culture can be performed in the presence of cytokines (e.g., in the presence of IL-2). The T cells can also be further activated in the presence of brevidin A and / or monensin.

[0038] In this disclosure, partial peptides of the SARS-CoV-2 spike protein can be scored using NetMHCpan-4.0 (www.cbs.dtu.dk / services / NetMHCpan-4.0) at the default settings (where A*24:02) with scores of 0.1 or higher, 0.2 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, or 0.7 or higher.

[0039] In this disclosure, a partial peptide of the spike protein of SARS-CoV-2 is, for example, a partial peptide of the spike protein of SARS-CoV-2 containing the amino acid sequence described in Serial Number 4, or a partial peptide of the spike protein at the position corresponding to that sequence. This partial peptide is a peptide having a length of 8 to 30 amino acids (preferably 8 to 10 amino acids, 15 to 18 amino acids, 8 to 20 amino acids, 8 to 11 amino acids, or 9 amino acids, or 10 amino acids, more preferably 9 amino acids). In NetMHCpan-4.0 (www.cbs.dtu.dk / services / NetMHCpan-4.0), under the default settings (where A*24:02), the score can be 0.1 or higher, 0.2 or higher, 0.3 or higher, 0.4 or higher, 0.5 or higher, 0.6 or higher, or 0.7 or higher.

[0040] In this disclosure, a partial peptide of the spike protein of SARS-CoV-2 is, for example, a partial peptide of the spike protein of SARS-CoV-2 that has an amino acid sequence that is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more homologous to the amino acid sequence described in Serial No. 4, and has a score of 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 0.6 or more, or 0.7 or more in NetMHCpan-4.0 (www.cbs.dtu.dk / services / NetMHCpan-4.0) under the default setting (where A*24:02).

[0041] The peptides disclosed herein can be readily presented as antigens by HLA in humans with HLA type A*24:02. Furthermore, this antigen presentation can induce a peptide-specific immune response. Therefore, the peptides disclosed herein can be used to induce a peptide-specific immune response.

[0042] The peptides disclosed herein can induce peptide-specific T-cell immunity. Therefore, the peptides disclosed herein can be used to induce peptide-specific T-cell immunity.

[0043] The peptides disclosed herein can induce an immune response against SARS-CoV-2. Therefore, the peptides disclosed herein can be used to induce an immune response against SARS-CoV-2.

[0044] The peptides disclosed herein can induce T-cell immunity against SARS-CoV-2. Therefore, the peptides disclosed herein can be used to induce T-cell immunity against SARS-CoV-2.

[0045] The peptides disclosed herein can be used as vaccines against SARS-CoV-2.

[0046] The peptides disclosed herein may be included in immunogenic compositions. Therefore, according to this disclosure, an immunogenic composition containing the peptides disclosed herein is provided. The immunogenic composition may further contain an immunomodulator (adjuvant). Examples of adjuvants include settling adjuvants and oily adjuvants. Examples of settling adjuvants include sodium hydroxide; aluminum compound adjuvants, such as potassium aluminum sulfate, aluminum phosphate, aluminum hydroxide, calcium phosphate, alum, pethidine, and carboxyvinyl polymers. Examples of oily adjuvants include liquid paraffin, lanolin, and Freund's adjuvant. Examples of adjuvants include incomplete Freund's adjuvant and complete Freund's adjuvant. Other adjuvants may also be Toll-like receptor (TLR) ligands or antigen-presenting cells. A TLR ligand is a molecule that binds to at least one TLR and causes TLR activation, i.e., activation of TLR-mediated cell signaling. TLR ligands can be, for example, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 ligands, and can be TLR7 agonists, TLR8 agonists, or TLR9 agonists. Examples include double-stranded RNA, muramyl dipeptide (MDP), threonyl-muramyl dipeptide (t-MDP), OM-174, flagellin, single-stranded RNA, oligonucleotides (ORN), imidazoquinones (e.g., imiquimod (R-837)), lipopolysaccharides (LPS) and their derivatives (e.g., triacylated lipopeptides such as Pam3CSK4, arabinomannan). Lipopolysaccharides such as polysaccharides and lipomannan, yeast polysaccharides, monophospholipid A, FSL-1, loxoribin, brompirimidine, ss polyuridine (sspolyU), rasimod ((R-848)), CpG oligodeoxynucleotides (CpG-ODN), CpG pathogen-associated molecular patterns (PAMP), polyinosinic-cytosine (PolyIC), polyinosinic-polycytosine-poly-L-lysine (Poly-ICLC), nucleic acids containing unmethylated CpG islands, DNA originating from viruses or bacteria, molecules containing thiophosphates (e.g., thiophosphate nucleoside analogs), nucleic acids with a thiophosphate backbone, STING ligands, and granzyme A, etc. Antigen-presenting cells include dendritic cells, macrophages, and B cells. In one manner, the immunogenic composition is administered to humans.

[0047] The immunogenic composition is sterile. In one embodiment, the immunogenic composition does not contain pyrogens. The immunogenic composition can be adjusted to a pH of 6–8 (e.g., pH 7–7.4). The immunogenic composition can be formulated for injection. It can also be formulated for inhalation administration. The immunogenic composition may further contain pharmaceutically acceptable excipients. Examples of pharmaceutically acceptable excipients include water, physiological saline, pH buffers, isotonic agents, preservatives, and antioxidants. The immunogenic composition can be administered, for example, non-oral, such as intramuscular or intravenous administration.

[0048] According to this disclosure, a method for inducing antigen-specific immunity in a subject is provided, comprising the step of administering an effective amount of the peptide or immunogenic composition of this disclosure to the subject. According to this disclosure, a method for inducing antigen-specific T-cell immunity in a subject is provided, comprising the step of administering an effective amount of the peptide or immunogenic composition of this disclosure to the subject. According to this disclosure, a method for inducing antigen-specific cytotoxic T cells in a subject is provided, comprising the step of administering an effective amount of the peptide or immunogenic composition of this disclosure to the subject. According to this disclosure, a method for inducing immunity against SARS-CoV-2 in a subject is provided, comprising the step of administering an effective amount of the peptide or immunogenic composition of this disclosure to the subject.

[0049] According to this disclosure, the use of the aforementioned peptides or immunogenic compositions of this disclosure in the manufacture of a medicament for inducing antigen-specific immunity in a subject is provided. According to this disclosure, the use of the aforementioned peptides or immunogenic compositions of this disclosure in the manufacture of a medicament for inducing antigen-specific T-cell immunity in a subject is provided. According to this disclosure, the use of the aforementioned peptides or immunogenic compositions of this disclosure in the manufacture of a medicament for inducing antigen-specific cytotoxic T cells in a subject is provided. According to this disclosure, the use of the aforementioned peptides or immunogenic compositions of this disclosure in the manufacture of a medicament for inducing immunity against SARS-CoV-2 in a subject is provided.

[0050] Whether antigen-specific immunity was induced in a subject can be determined by an increase in the production of INF-γ in cytotoxic T cells in the subject's peripheral blood. A significant increase in INF-γ production in cytotoxic T cells indicates that antigen-specific immunity has been induced in the subject. For example, the production of INF-γ in cytotoxic T cells can reach 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times or more compared to pre-drug administration. Therefore, an increase in INF-γ production in cytotoxic T cells at such rates indicates that antigen-specific immunity has been induced in the subject. Cytotoxic T cells can be cultured in the presence of IL-2 and then used for the determinations described above. Cytotoxic T cells can be isolated by flow cytometry using methods known to those skilled in the art. Cytotoxic T cells can be isolated, for example, by indicators of CD3 and CD8 expression.

[0051] According to this disclosure, a T-cell receptor (TCR) containing TRA (α chain) as described in any one of No. 1 to No. 12 of Table 3 and TRB (β chain) as described in any one of No. 1 to No. 12 of Table 3 is provided. According to this disclosure, a TCR containing TRA and TRB as described in any one of No. 1 to No. 12 of Table 3 is also provided. According to this disclosure, a T-cell receptor (TCR) containing TRA and TRB as described in any one of No. 1 to No. 21 of Table 4 is provided. According to this disclosure, a TCR containing TRA and TRB as described in any one of No. 1 to No. 21 of Table 4 is also provided.

[0052] According to this disclosure, cells expressing one or more TCRs selected from the group consisting of the aforementioned TCRs are provided on their cell surface. According to this disclosure, a pharmaceutical composition is provided containing cells expressing one or more TCRs selected from the group consisting of the aforementioned TCRs on their cell surface. These cells may be, for example, immune cells, or cells selected from the group consisting of T cells, NK cells, NKT cells, γδT cells, and MAIT cells. Furthermore, this pharmaceutical composition can be used to treat infections caused by coronaviruses such as SARS-CoV-2. According to this disclosure, T cells activated in response to the aforementioned peptides (e.g., T cells producing INF-γ or TNF-α) are also provided. Such T cells can be induced by any one or more of the aforementioned peptides. According to this disclosure, a pharmaceutical composition is further provided containing T cells activated in response to the aforementioned peptides. These pharmaceutical compositions can be used to treat infections caused by coronaviruses such as SARS-CoV-2. According to this disclosure, a pharmaceutical composition is further provided containing cells (particularly immune cells) having TCRs activated in response to the aforementioned peptides. These pharmaceutical compositions can be used to treat infections caused by coronaviruses such as SARS-CoV-2.

[0053] The peptides or immunogenic compositions disclosed herein can be administered to a subject once or multiple times. Administration may, for example, be performed once weekly.

[0054] Example

[0055] Example 1: Synthesis of the peptide and immune induction against the peptide

[0056] 1. Materials and Methods

[0057] 1.1 Human Samples and Preparation

[0058] PBMCs were obtained from five healthy donors (HD1 to HD5) at RIKEN through Ficoll-paque. TM PLUS (GE Helmets) density centrifugation was used for separation. PBMCs were washed twice with PBS and then stored in liquid nitrogen until use.

[0059] 1.2. Reagents and Antibodies

[0060] Human IL-2 (hIL-3) was purchased from Shionogi & Co., Ltd. The following monoclonal antibodies (mAbs) were purchased from Biolegend: anti-human mAb; PE / Cy7 conjugated anti-CD3, PerCP-Cy5.5 conjugated anti-CD4, FITC conjugated anti-CD8, and APC conjugated anti-IFN-g mAb. Topo-Pro-3 and carboxyfluorescein succinimide (CFSE) were purchased from Thermo Fisher Scientific.

[0061] 1.3. T cell culture

[0062] PBMCs were cultured in RPMI supplemented with 10% FBS and 100 U / ml hIL-2, and stimulated with the peptides listed in Table 1 below (10 μM, GeneScript). Restimulation was performed weekly with the same peptide over 21 days. It is believed that peptide stimulation can surviv T cells with binding affinity to the peptide. The peptides listed in Table 1 are predicted to be HLA-A*24:02-restricted peptides present in the nucleocapsid or spike protein of SARS-CoV-2. Prediction was performed using NetMHCpan 4.0. The peptides in Table 1 were designed to be 8–10 amino acid long and, upon stimulation of T cells with HLA class I binding, can surviv CD8 single-positive T cells.

[0063] [Table 1]

[0064] Table 1: Amino acid sequences and sources of the peptides used

[0065]

[0066] 1.4. Flow cytometry

[0067] As mentioned above, T cells stimulated with peptides do not possess the ability to produce IFN-γ at this time. Therefore, to perform intracellular IFN-γ staining, cells were cultured for 16 hours with brevidin A and monensin under conditions of both presence and absence of various peptides, attempting to induce T cells to produce IFN-γ. It is believed that through this experimental procedure, T cells with peptide-specific TCRs will produce IFN-γ. After surface staining with surface antigens CD3, CD4, and CD8, the cells were fixed, and intracellular cytokine staining was performed using BD Cytofix / Cytoperm. TM (BD) was used for permeation. Data were analyzed using FlowJo software (Tree Star).

[0068] 1.5. Cell-killing analysis

[0069] Three weeks later, CTL strains were collected as effector cells. Cell-cytotoxicity analysis of T cells was performed using the following target cells. For A24-CIR cells labeled with CFSE, a 2-hour shock with the specified peptide or DMSO was performed, followed by two washes. Effector cells were mixed with 1 × 10⁻⁶ cells at effector / target cell ratios of 12.5, 25, and 50. 4Target cells were cultured together for 6 hours. After 6 hours, just before analysis, cells were stained with To-PRO3 to identify dead cells. Active target cell death (SD) was determined by labeling individually cultured target cells. As a positive control for total cytotoxicity (TD), labeled target cells were permeabilized with BDCytofix / Cytoperm reagent (BD Pharmingent). Specific lysis was calculated using the following formula: (sample - SD / TD - SD) × 100. Cells were analyzed by flow cytometry.

[0070] 2. Results

[0071] Flow cytometry was used to confirm whether the antigenic peptides listed in Table 1 induced intracellular TNF-γ expression in CD8 single-positive T cells. Unstimulated PBMCs were used as a negative control. Data were compared between peptide stimulation and non-stimulation for each healthy donor (HD1–HD5). Results are as follows: Figure 1 As shown. Figure 1 As shown, the peptide of pet#4 exhibits strong IFN-γ induction in CD8 single-positive T cells (cytotoxic T cells).

[0072] In the presence of IL-2, PBMCs from healthy HD-5 donors were shocked with pep#4 peptide to obtain PBMC cultures. Cytotoxic T cells (CTLs) were then isolated from the obtained PBMC cultures. CFSE-labeled A24-CIR cells were shocked with pep#4 peptide, presenting the peptide to HLA on the surface of C1R cells. The resulting A24-C1R cells were then treated with these CTLs to confirm whether antigen-specific CTLs were induced. As a negative control, A24-C1R cells not shocked with pep#4 peptide were used. E / T represents the ratio of CTL number to A24-C1R cell number. Results are as follows: Figure 2 As shown. Figure 2 As shown, CTLs exhibited antigen-specific killing activity against A24-C1R cells induced by pep#4 peptide. This indicates that pep#4 peptide induces antigen-specific CTLs.

[0073] Example 2: Synthesis of a 15-amino acid peptide and T cell induction

[0074] Using peptide #4 (9 amino acids in length) from Table 1 as the center, four peptides with a length of 15 amino acids were synthesized. The amino acid sequences of the four peptides are shown below.

[0075]

[0076] As described above, the 15mer-1 peptide is a continuous 15mer peptide formed by removing two amino acids from the C-terminus of nine amino acids and extending it by eight amino acids to the N-terminus. The 15mer-2 to -4 peptides are continuous 15mer peptides formed by moving four amino acids at a time towards the C-terminus.

[0077] [Table 2]

[0078] Table 2: Positions of four peptides with 15 amino acid lengths

[0079] 15mer-1 LQELGKYEQYIKWPW (Serial Number 9) 1200-1214 15mer-2 GKYEQYIKWPWYIWL (Serial Number 10) 1204-1218 15mer-3 QYIKWPWYIWLGFIA (Serial Number 11) 1208-1222 15mer-4 WPWYIWLGFIAGLIA (Serial Number 12) 1212-1226

[0080] * The number recorded in the position column indicates the position in the amino acid sequence of sequence number 8.

[0081] Similar to Example 1, cells were cultured for 21 days in the presence of IL-2 and peptides, and further stimulated with peptides in the presence of brevidin and monensin to investigate INF-γ production. Specifically, PBMCs from four healthy HLA-A2402 (HV) individuals were added with a mixture of 15-mer peptides at a concentration of 10 μg / ml and cultured in the presence of IL-2. T cells with binding affinity to the contacted peptides survived. Autologous PBMCs were weekly shocked with the 15-mer peptide mixture at 40 Gy and added to the culture as feeder cells. The 15-mer peptide mixture was further added on days 14 and 21. The resulting cultures were then shocked with individual 15-mer peptides and cultured for 16 hours in the presence of brevidin and monensin to induce INF-γ production. Intracellular cytokine (anti-IFN-g-APC, anti-TNF-α-PE) staining was performed to analyze CD8T cells that produce peptide-specific cytokines.

[0082] The results are as follows Figure 3 and 4 As shown. Figure 3 As shown, CD8 single-positive T cells respond to restimulation induced by 15mer-2, 15mer-3, and 15mer-4, resulting in increased production of INF-γ and TNF-α. This indicates that the cytotoxicity of T cells is enhanced by peptides. Figure 4 Yes Figure 3 The graph is a representation of the proportions of INF-γ-positive and TNF-α-positive CD8 T cells. Among 15-mer peptides, the 15-mer-3 peptide best stimulates T cells.

[0083] Then, the TCR repertoire of the obtained T cells was investigated.

[0084] For CTL strains established by restimulating autologous PBMCs with peptide #4 or the 15mer peptide in Table 1 2–3 times, anti-human CD107a-BV421 was added in the presence or absence of peptide #4 (10 μM) in Table 1, and the cells were cultured for 6 hours. After staining with anti-human CD8-PE and Aqua, peptide-specific CD8+CD107a+ cells were sorted into 96-well round-bottom plates at a rate of 1 cell / well using FACS Aria. For the 15mer peptide, anti-human CD107a-BV421 was added in the presence or absence of the peptide mixture (10 μg / ml of each peptide) and the cells were cultured for 16 hours. After staining with anti-human CD8-PE and Aqua, peptide-specific CD8+CD107a+ cells were sorted into 96-well round-bottom plates at a rate of 1 cell / well using FACS Aria. The TCR of T cells in each well was analyzed. The results are shown in Tables 3 and 4.

[0085] [Table 3]

[0086] Table 3: TCR library of T cells obtained by stimulation with peptide #4 of 9mer from Table 1

[0087]

[0088] [Table 4]

[0089] Table 4: TCR repertoire of T cells obtained by stimulation with a 15-mer peptide mixture

[0090]

[0091] As shown in Tables 3 and 4, T cells surviving via a 9-mer peptide exhibited 12 diversity in their TCR α and β genes, while those surviving via a 15-mer peptide mixture showed 21 diversity. As inferred, epitope diversity increases the diversity of TCRs possessed by activated T cells. This implies that peptide mixtures can induce T cells with diverse antigen specificity, thereby enabling diverse T cells to attack foreign invaders such as viruses. This suggests that even if a portion of the virus mutates, T cells with broader diversity can sustainably attack the virus. Furthermore, the aforementioned TCR data suggest that TCR gene-introduced cells (T cells, NK cells, NKT cells, γδT cells, MAIT cells) could also be used as therapeutics to kill SARS-CoV-2 infected cells.

[0092] Theoretically, the 15-mer peptide can be presented by both HLA class I and II, activating both CD4-positive and CD8-positive T cells. In this embodiment, CD8-positive T cells were confirmed, and the 15-mer peptide produced the production of three cytokines capable of activating CD8-positive T cells. Therefore, it can be expected that the 15-mer peptide will also activate CD4-positive T cells in the same way.

[0093] In addition, the 15-mer peptide is taken up into the cell and fragmented, presented to HLA as a partial peptide of approximately 9 mer. It is believed that this creates diversity in the possible production patterns of the 9-mer partial peptide, which acts on the more diverse TCRs, leading to T cell activation. sequence list <110> National Research Institute of Physics and Chemistry <120> Methods for inducing immunity against SARS-CoV-2 <130> PR13-9019WO <150> JP2020-139794 <151> 2020-08-21 <150> JP2020-203123 <151> 2020-12-08 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 9 <212> PRT <213> Artificial sequence <220> <223> pep#1 <400> 1 Asn Phe Lys Asp Gln Val Ile Leu Leu 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220> <223> pep#2 <400> 2 Val Tyr Ser Thr Gly Ser Asn Val Phe 1 5 <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> pep#3 <400> 3 Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe 1 5 10 <210> 4 <211> 9 <212> PRT <213> Artificial sequence <220> <223> pep#4 <400> 4 Gln Tyr Ile Lys Trp Pro Trp Tyr Ile 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <220> <223> pep#5 <400> 5 Tyr Asn Tyr Leu Tyr Arg Leu Phe 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial sequence <220> <223> pep#6 <400> 6 Tyr Phe Pro Leu Gln Ser Tyr Gly Phe 1 5 <210> 7 <211> 10 <212> PRT <213> Artificial sequence <220> <223> pep#7 <400> 7 Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe 1 5 10 <210> 8 <211> 1273 <212> PRT <213> SARS-CoV-2 <400> 8 Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val 1 5 10 15 Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe 20 25 30 Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu 35 40 45 His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp 50 55 60 Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp 65 70 75 80 Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu 85 90 95 Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser 100 105 110 Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile 115 120 125 Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr 130 135 140 Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr 145 150 155 160 Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu 165 170 175 Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe 180 185 190 Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr 195 200 205 Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu 210 215 220 Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr 225 230 235 240 Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser 245 250 255 Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro 260 265 270 Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala 275 280 285 Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys 290 295 300 Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val 305 310 315 320 Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys 325 330 335 Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala 340 345 350 Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu 355 360 365 Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro 370 375 380 Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe 385 390 395 400 Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly 405 410 415 Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys 420 425 430 Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn 435 440 445 Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe 450 455 460 Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys 465 470 475 480 Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly 485 490 495 Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val 500 505 510 Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys 515 520 525 Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn 530 535 540 Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu 545 550 555 560 Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val 565 570 575 Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe 580 585 590 Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val 595 600 605 Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile 610 615 620 His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser 625 630 635 640 Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val 645 650 655 Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala 660 665 670 Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala 675 680 685 Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser 690 695 700 Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile 705 710 715 720 Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val 725 730 735 Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu 740 745 750 Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr 755 760 765 Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln 770 775 780 Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe 785 790 795 800 Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser 805 810 815 Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly 820 825 830 Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp 835 840 845 Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu 850 855 860 Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly 865 870 875 880 Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile 885 890 895 Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr 900 905 910 Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn 915 920 925 Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala 930 935 940 Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn 945 950 955 960 Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val 965 970 975 Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln 980 985 990 Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val 995 1000 1005 Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn 1010 1015 1020 Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys 1025 1030 1035 Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro 1040 1045 1050 Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val 1055 1060 1065 Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His 1070 1075 1080 Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn 1085 1090 1095 Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln 1100 1105 1110 Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val 1115 1120 1125 Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro 1130 1135 1140 Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn 1145 1150 1155 His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn 1160 1165 1170 Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu 1175 1180 1185 Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu 1190 1195 1200 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1205 1210 1215 Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met 1220 1225 1230 Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys 1235 1240 1245 Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro 1250 1255 1260 Val Leu Lys Gly Val Lys Leu His Tyr Thr 1265 1270 <210> 9 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 15mer-1 <400> 9 Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 15mer-2 <400> 10 Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu 1 5 10 15 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 15mer-3 <400> 11 Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala 1 5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 15mer-4 <400> 12 Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala 1 5 10 15

Claims

1. The use of a peptide in the manufacture of an immunogenic composition for inducing cytotoxic T-cell immunity against SARS-CoV-2 in a subject, said peptide being a partial peptide of the spike protein of SARS-CoV-2, said peptide comprising the amino acid sequence described in sequence number 4 or 11, and capable of inducing specific T-cell immunity against the spike protein.

2. The application according to claim 1, wherein, The subjects are those who have been infected with SARS-CoV-2.

3. The application according to claim 1, wherein, The target group consists of individuals who are at risk of being infected with SARS-CoV-2.

4. The application according to claim 1, wherein, The target group consists of individuals at risk of SARS-CoV-2 infection.

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